Biocatalyzed Synthesis of Statins: A Sustainable Strategy for the Preparation of Valuable Drugs

Pilar Hoyos; Vittorio Pace; Andrés Alcántara

doi:10.3390/catal9030260

Hoyos, Pilar;Pace, Vittorio;Alcántara, Andrés;

2019-03-14 00:00:00

catalysts Review Biocatalyzed Synthesis of Statins: A Sustainable Strategy for the Preparation of Valuable Drugs 1 2 1 , Pilar Hoyos , Vittorio Pace and Andrés R. Alcántara * Department of Chemistry in Pharmaceutical Sciences, Faculty of Pharmacy, Complutense University of Madrid, Campus de Moncloa, E-28040 Madrid, Spain; [email protected] Department of Pharmaceutical Chemistry, Faculty of Life Sciences, Althanstrasse 14, A-1090 Vienna, Austria; [email protected] * Correspondence: [email protected]; Tel.: +34-91-394-1823 Received: 25 February 2019; Accepted: 9 March 2019; Published: 14 March 2019 Abstract: Statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are the largest selling class of drugs prescribed for the pharmacological treatment of hypercholesterolemia and dyslipidaemia. Statins also possess other therapeutic effects, called pleiotropic, because the blockade of the conversion of HMG-CoA to (R)-mevalonate produces a concomitant inhibition of the biosynthesis of numerous isoprenoid metabolites (e.g., geranylgeranyl pyrophosphate (GGPP) or farnesyl pyrophosphate (FPP)). Thus, the prenylation of several cell signalling proteins (small GTPase family members: Ras, Rac, and Rho) is hampered, so that these molecular switches, controlling multiple pathways and cell functions (maintenance of cell shape, motility, factor secretion, differentiation, and proliferation) are regulated, leading to beneﬁcial effects in cardiovascular health, regulation of the immune system, anti-inﬂammatory and immunosuppressive properties, prevention and treatment of sepsis, treatment of autoimmune diseases, osteoporosis, kidney and neurological disorders, or even in cancer therapy. Thus, there is a growing interest in developing more sustainable protocols for preparation of statins, and the introduction of biocatalyzed steps into the synthetic pathways is highly advantageous—synthetic routes are conducted under mild reaction conditions, at ambient temperature, and can use water as a reaction medium in many cases. Furthermore, their high selectivity avoids the need for functional group activation and protection/deprotection steps usually required in traditional organic synthesis. Therefore, biocatalysis provides shorter processes, produces less waste, and reduces manufacturing costs and environmental impact. In this review, we will comment on the pleiotropic effects of statins and will illustrate some biotransformations nowadays implemented for statin synthesis. Keywords: biocatalysis; biotransformations; statins; pleiotropic effects 1. Introduction It is very well known that raised cholesterol levels increase the risks of heart disease and stroke. Globally, a third of ischaemic heart disease is attributable to high cholesterol and, according to the World Health Organization, raised cholesterol is estimated to cause 2.6 million deaths (4.5% of total) and 29.7 million disability adjusted life years (DALYS) [1]. In this sense, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, commonly known as statins (Figure 1), are the largest selling class of drugs prescribed for the pharmacological treatment of hypercholesterolemia and dyslipidaemia [2,3], and it has been also reported that since their introduction in 1987, the lives of millions of people have been extended through statin therapy and, more importantly, quality of life has been drastically improved [4]. Catalysts 2019, 9, 260; doi:10.3390/catal9030260 www.mdpi.com/journal/catalysts Catalysts 2019, 9, 260 2 of 32 Catalysts 2019, 9, x FOR PEER REVIEW 2 of 32 Figure Figure 1. 1. Some Some Statins, Statins, inhibit inhibitors ors of 3-hydrox of 3-hydroxy-3-methylglutaryl y-3-methylglutaryl c coenzyme oenzyme A (HMG-CoA) A (HMG-CoA) r red eductase. uctase. Since the discovery of the ﬁrst statins from natural sources, mevastatin (Figure 1, 1, also named Since the discovery of the first statins from natural sources, mevastatin (Figure 1, 1, also named compactin, from the fungi Penicillium citrinum [5] and Penicillium brevicompactum [6]), lovastatin compactin, from the fungi Penicillium citrinum [5] and Penicillium brevicompactum [6]), lovastatin (Figure 1, 2, Mevinolin, found in Aspergillus terreus [7] and food such as oyster mushrooms [8] or (Figure 1, 2, Mevinolin, found in Aspergillus terreus [7] and food such as oyster mushrooms [8] or red red yeast rice [9]), simvastatin (Figure 1, 3, Mevacor, also isolated from Aspergillus terreus [10]), and yeast rice [9]), simvastatin (Figure 1, 3, Mevacor, also isolated from Aspergillus terreus [10]), and pravastatin (Figure 1, 4, initially known as CS-514, originally identiﬁed in the bacterium Nocardia pravastatin (Figure 1, 4, initially known as CS-514, originally identified in the bacterium Nocardia autotrophica [11]), synthetic more potent compounds (Figure 1, 5–9), also known as superstatins, autotrophica [11]), synthetic more potent compounds (Figure 1, 5–9), also known as superstatins, were were introduced into the drug market [12,13]. As can be observed, the common structure of these introduced into the drug market [12,13]. As can be observed, the common structure of these compounds is formed by a central core of different heterocyclic aromatic rings containing nitrogen, compounds is formed by a central core of different heterocyclic aromatic rings containing nitrogen, and a lateral chain derived from (3R,5R)-3,5-dihydroxyheptanoic acid. and a lateral chain derived from (3R,5R)-3,5-dihydroxyheptanoic acid. The economic impact of statins on the drug market is enormous. For instance, simvastatin The economic impact of statins on the drug market is enormous. For instance, simvastatin was was originally developed by Merck under the brand name Zocor™; in 2005, Zocor™ was Merck’s originally developed by Merck under the brand name Zocor™; in 2005, Zocor™ was Merck’s best- best-selling drug and the second-largest selling statin in the world (more than US$3 million only in selling drug and the second-largest selling statin in the world (more than US$3 million only in USA, USA, according to different reports [14–21]). In 2006, Zocor™ went off patent, and the annual sales according to different reports [14–21]). In 2006, Zocor™ went off patent, and the annual sales drastically dropped; anyhow, from that moment, generic simvastatin became the most-prescribed drastically dropped; anyhow, from that moment, generic simvastatin became the most-prescribed statin in the world between 2010 and 2015 [19,20,22]. On the other hand, atorvastatin (Figure 1, ATC statin in the world between 2010 and 2015 [19,20,22]. On the other hand, atorvastatin (Figure 1, ATC (Anatomical Therapeutic Chemical) classiﬁcation system, according to World Health Organization) (Anatomical Therapeutic Chemical) classification system, according to World Health Organization) Code C10AA05, DrugBank Code DB01076, 5) is the greatest blockbuster drug in pharmaceutical history, Code C10AA05, DrugBank Code DB01076, 5) is the greatest blockbuster drug in pharmaceutical and the best known representative of superstatins, receiving this name because of its pronounced history, and the best known representative of superstatins, receiving this name because of its ability to reduce low-density lipoprotein cholesterol levels and increase high-density lipoprotein pronounced ability to reduce low-density lipoprotein cholesterol levels and increase high-density cholesterol compared with other existing agents [13]. It was ﬁrst synthesized in 1985 by Bruce Roth lipoprotein cholesterol compared with other existing agents [13]. It was first synthesized in 1985 by of Parke-Davis Warner-Lambert Company (now Pﬁzer), which commercialized it under the name of Bruce Roth of Parke-Davis Warner-Lambert Company (now Pfizer), which commercialized it under the name of Lipitor™. Since it was approved in 1996, sales have exceeded US$125 billion, and the drug has topped the list of best-selling branded pharmaceuticals in the world for nearly a decade. Catalysts 2019, 9, 260 3 of 32 Lipitor™. Since it was approved in 1996, sales have exceeded US$125 billion, and the drug has topped the list of best-selling branded pharmaceuticals in the world for nearly a decade. When Pﬁzer ’s patent on Lipitor™ expired in USA by the end of 2011 and in Europe in mid-2012, generic atorvastatin from other companies became available, and it is still being widely sold (US$2.16 billion in sales, standing as the year ’s fourth-best-selling cardiovascular drug, with analysts predicting sales of US$1.85 billion in 2024 [23]). Finally, rosuvastatin (Figure 1, 9, ATC Code C10AA07, DrugBank Code DB01098) was marketed as calcium salt in 2003 by AstraZeneca under the name of Crestor™. Like atorvastatin, rosuvastatin is also a superstatin; the initial patent on rosuvastatin synthesis (purely chemical) was developed by Shionogi Research Laboratories [24] and later sold to AstraZeneca. This patent expired in June 2016, but anyhow, it still can be considered a blockbuster drug, by looking at the great volume of sales of Crestor™ (around U$2.7 billion in 2017, and US$727 million for the ﬁrst half of 2018 [23]). A recent study [25] points toward global sales of statins of US$1 trillion by 2020, thus pharmaceutical companies are still interested in developing new synthetic strategies for putting these drugs on the market. Thus, it is undoubtedly clear that the statin market involves a huge amount of money. Furthermore, the importance of this type of drug is even higher because of their new therapeutic uses that are recently becoming more and more recognized, which will be commented on in Section 2. Finally, as the absolute conﬁguration of statins plays a crucial role in the activity of these compounds, the enormous potential of an enantioselective biocatalytic process for the sustainable synthesis of chiral building blocks involved in statin preparative procedures will further be commented on in Section 3. 2. New Therapeutic Effects of Statins As mentioned before, these drugs act by reversibly and competitively inhibiting the bioreduction of S-3-hydroxy-3-methylglutaryl-coA (HMG-CoA), the rate-limiting step of the mevalonate pathway in cholesterol biosynthesis (Figure 2), because of the chemical similitude with mevalonyl-CoA, the intermediate obtained after the ﬁrst reduction of HMG-CoA. Furthermore, there is extensive recent evidence suggesting that statins are more than simple lipid-lowering drugs [3,26]; in fact, a large amount of up-to-date experimental data have conﬁrmed that statins may exert many different potentially beneﬁcial therapeutic effects, by several mechanisms not essentially related to cholesterol metabolism. These so-called pleiotropic effects [27] could be attributed to their ability to prevent the conversion of HMG-CoA to R-mevalonate, which results in the concomitant inhibition of the downstream biosynthesis of cholesterol, as well as of numerous isoprenoid metabolites, such as geranylgeranyl pyrophosphate (GGPP) or farnesyl pyrophosphate (FPP), as shown in Figure 2. These molecules are well-known key intermediates for prenylation of several cell signalling proteins (such as small GTPase family members: Ras, Rac, Rho, Rab), which act as molecular switches controlling multiple pathways and cell functions (maintenance of cell shape, motility, factor secretion, differentiation, and proliferation), so that they can be inhibited by statin treatment [28]. For instance, when Ras and Rho isoprenylation is inhibited, there is a concurrent accumulation of inactive forms of both proteins in cytoplasm and an inhibition of these signalling molecules [29]. Certainly, it has been reported that small G-proteins like Rho and Rac inﬂuence endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) availability [30]. Rho negatively regulates eNOS expression, while Rac activates nicotinamide dinucleotide phosphate (NADPH)-oxidase and the correspondent superoxide production, which in turn inactivates NO. If statins block both Rho and Rac GTPase activity via inhibition of geranylgeranylation, this leads to eNOS upregulation [31,32]. Remarkably, some beneﬁcial effects of statins were displayed before cholesterol levels were reduced [30], and it can be assumed that those effects, dependent on the enhancement of eNOS expression and/or activity, result in a decline of platelet activation, attenuation of adhesion molecules expression, decrease of inﬂammatory cytokine production, and increase of reactive oxygen species (ROS) [33]. Therefore, pleiotropic effects of statins include the reduction of haemostasis by reducing platelet activation and the pro-coagulation cascade; the increase of ﬁbrinolysis and the anticoagulation cascade; the improvement Catalysts 2019, 9, 260 4 of 32 of endothelial function; the increase of NO bioavailability; as well as antioxidant, immune modulatory, and anti-inﬂammatory activities and stabilization of atherosclerotic plaques [27,29,34–37]. Thus, the Catalysts 2019, 9, x FOR PEER REVIEW 4 of 32 therapeutic effects of statins are nowadays present in areas such as cardiovascular health, regulation of cardiovascular health, regulation of the immune system, anti-inflammatory and immunosuppressive the immune system, anti-inﬂammatory and immunosuppressive properties, prevention and treatment properties, prevention and treatment of sepsis, treatment of autoimmune diseases, osteoporosis, of sepsis, treatment of autoimmune diseases, osteoporosis, kidney and neurological disorders, and kidney and neurological disorders, and even in cancer therapy; some of these therapeutic areas will even in cancer therapy; some of these therapeutic areas will be commented on. be commented on. Figure Figure 2. 2. Meva Mevalonate lonate pathway. pathway. 2.1. Cardiovascular Effects 2.1. Cardiovascular Effects Aside from the main mechanism of action of lowering cholesterol levels, statins are also useful Aside from the main mechanism of action of lowering cholesterol levels, statins are also useful in the treatment of some other cardiovascular disorders, including acute coronary syndrome, heart in the treatment of some other cardiovascular disorders, including acute coronary syndrome, heart failure, cardiac arrhythmias, aortic stenosis, peripheral arterial disease, cerebrovascular disease, and failure, cardiac arrhythmias, aortic stenosis, peripheral arterial disease, cerebrovascular disease, and essential essential h hyperten ypertension, sion, as as r recently rev ecently reviewed iewed (see p (see apapers pers by M byihos Mihos et al. et[3 al. 8], [Oe 38sterle et al. ], Oesterle[39], and et al. [39 ], and referenc references es thether rein). In ein).fact, In chroni fact, chr c admi onic nist administr ration ofation statins ofis be statins lieved t is believed o produce what to produce is known as what is PIC (“pre-ischemic conditioning”), protecting the myocardium during ischemic insult and injury known as PIC (“pre-ischemic conditioning”), protecting the myocardium during ischemic insult and [40], as a consequence of an increase in nitric oxide availability and immunomodulation; thus, statins injury [40], as a consequence of an increase in nitric oxide availability and immunomodulation; thus, increase the production of nitric oxide and blunt the formation of superoxide radicals via the statins increase the production of nitric oxide and blunt the formation of superoxide radicals via the upregulation of eNOS and stabilization of its mRNA, leading to an improved vascular function and upregulation of eNOS and stabilization of its mRNA, leading to an improved vascular function and a a reduction in vascular inflammation [34]. In this sense, recent studies show the effectiveness of reduction in vascular inﬂammation [34]. In this sense, recent studies show the effectiveness of statins’ statins’ cardiovascular primary prevention [41], also for elderly people [42], and point towards the cardiovascular primary prevention [41], also for elderly people [42], and point towards the special special benefits of fluvastatin [43]. beneﬁts of ﬂuvastatin [43]. 2.2. Immunomodulatory Effects Catalysts 2019, 9, 260 5 of 32 2.2. Immunomodulatory Effects The main objectives of autoimmune therapies are to re-establish immunological homeostasis and reduce autoimmune damages. Different studies are increasingly suggesting that an imbalance between Th17 and Treg cells, as well as the incorrect release of potent pro-inﬂammatory mediators by Th17 cells, are crucial for the pathogenesis of a number of autoimmune disorders [44]. Thus, a new immunotherapeutic strategy could be based on increasing Treg or inhibiting Th17 differentiation/effector functions. In this respect, statins show an outstanding potential, especially considering the increasing evidence that they might inhibit Th17 differentiation/effector functions and conversely promote Treg differentiation/suppressive function selectively in the setting of autoimmune diseases [44]. Small GTPases have been centrally implicated in regulating the development and functions of T and B lymphocytes as well as of dendritic cells (DC) [45,46]. Thus, as a consequence of the inhibition of GTPases prenylation, statin-based therapy can be a potential alternative for the treatment of autoimmune diseases [44,47,48]. In fact, positive effects of statin treatment have been reported in numerous autoimmune diseases such as multiple sclerosis [49,50], systemic lupus erythematosus [51–53], autoimmune myocarditis [54–56], or rheumatoid arthritis [44,57–59]. 2.3. Neurological Disorders This is probably one of the most attractive therapeutic areas in which the use of statins introduces interesting advances. Pleiotropic effects of statins via GTPases inhibition might have potential therapeutic implications in many neurological disorders, as the current connection between neurodegenerative diseases and vascular risk factors is becoming more and more evident [30,60]; therefore, statin treatment could display beneﬁcial effects in neurological disorders such as stroke, Alzheimer ’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), primary brain tumours, or depression. 2.3.1. Stroke The risk factors for cerebrovascular disease are well known and largely variable and, in this sense, reduction of serum cholesterol levels could be highly beneﬁcial for reducing the hazard of suffering a cerebrovascular accident (CVA), also named stroke [30]. Anyhow, an indubitable link between high cholesterol level and stroke risk is difﬁcult to establish, because controversial data from several clinical studies have been published in the literature, some of them ﬁnding no relationship between cholesterol and stroke [61,62], while in some other cases, the beneﬁcial effects are indeed observed [63,64]. A possible explanation for these discrepancies could be based on the fact that stroke can be either ischemic or haemorrhagic, and there are evidences supporting an association between elevated cholesterol and increased risk of ischemic stroke, but also showing a relationship between low cholesterol levels and increased risk of haemorrhagic stroke [30]. So, while disagreements are still present on the usefulness of statins in the primary prevention of acute stroke, there is a wide consensus on the positive aspects of statins treatment in secondary prevention after stroke or transient ischemic attack for diminishing the menace of suffering a new stroke [65–68]. Even in haemorrhagic stroke, some data from recent studies suggest that statin therapy could improve the outcome after spontaneous intra-cerebral haemorrhage and statin therapy should be not discontinued [69–71]. In any case, the most feasible explanation for reduction in clinical events reported for patients treated with statins is the stabilization of atherosclerotic plaques, which are generated by lipids deposition and migration and proliferation of vascular smooth muscle cells (see report from Malﬁtano et al. [30] and references cited therein for a more detailed explanation). 2.3.2. Alzheimer ’s Disease (AD) AD is a chronic neurodegenerative syndrome caused by the appearance of brain senile plaques composed of aggregated forms of -amyloid peptide (A ), and it is the most common cause of Catalysts 2019, 9, 260 6 of 32 dementia in elderly people, with a new case globally occurring every seven seconds [72]. Emerging evidence suggests a link between cholesterol and AD [37,72–76], and extensive studies have been published stressing the therapeutic utility of pleiotropic effects of statins, showing a dose-dependent beneﬁcial effect on cognition, memory, and neuroprotection [72] by different mechanisms, such as altering the properties of plasma membrane by a reduction in cholesterol levels and a modulation of secretase activities, thus decreasing amyloid precursor protein (APP) processing [77], or by altering neuronal activity via modiﬁcation of GTPases prenylation [28,74,78]. On the other hand, a possible effect of statins in cholinergic neurotransmission has been also described; in fact, simvastatin inhibits acetyl cholinesterase (AChE) activity in rats [79] and prevents the blockade caused by AChE inhibitors at 7-nicotinic AChE receptors [80], thus increasing cholinergic neurotransmission. In this sense, Ghodke et al. [81] reported that statins treatment for 4 months, but not for 15 days, showed noteworthy enhancement in mice memory function, whereas a high cholesterol diet showed signiﬁcant diminishing of memory. However, long-term statin treatment showed a signiﬁcant decrease in serum cholesterol level as well as brain AChE level. Moreover, a high cholesterol diet showed a signiﬁcant decrease in memory function with an increase in serum cholesterol level as well as brain AChE level. Thus, they concluded that there was no direct correlation between brain cholesterol level, as well as HMG-CoA activity with memory function regulation, although there is tangible link between plasma cholesterol level and AChE level, and long-standing plasma cholesterol alteration may be essential to regulate memory function through the AChE modulated pathway. Finally, a simvastatin-related rise of butyryl cholinesterase (BuChE) activity in mice brain, which may be a potential adverse effect in patients with AD, has been recently reported [82]. Another feasible mechanism for explaining statins’ neuroprotective effect considers an activation of the heme oxygenase/biliverdin reductase (HO/BVR-A) system [37]. Statins can also be active in AD treatment because of their protecting effect against glutamate toxicity over primary cortical neurons [83,84]. Low-dose administration of statins avoids aberrant neuronal entry into mitosis [85], promotes anti-apoptotic pathways [86], and impairs inﬂammation [87], although higher doses of statins have been shown to induce toxic effects [88]. Recently, some studies point towards the utility of simvastatin administration in the improving of hippocampus-dependent spatial memory in mice, due to an activation of Akt (protein kinase B), via a depletion of FPP and inhibition of farnesylation [89,90]. This same group has recently shown how simvastatin administration potentiates the contribution of N-methyl D-aspartate receptor (NMDAR) to synaptic transmission, by increasing the surface distribution of the GluN2B subunit of the NMDAR without affecting cellular cholesterol content [91]. The inﬂuence of statins in these ionotropic glutamate-receptors, and the succeeding utility of these drugs on treatment of AD and other mental disorders, is undoubtedly a very attractive and innovative research ﬁeld [91,92]. Lamentably, although most evidence consistently conﬁrms how statins do afford neuroprotection and improve disease pathology in animal models [93,94], results are rather controversial or even disappointing in human trials [72,95–98], thus a very careful study design and analysis will be essential in the future [95]. 2.3.3. Parkinson’s Desease (PD) PD, the second most common chronic neurodegenerative disorder in adults over the age of 65 years [99], is a progressive neurodegenerative disorder characterised by the presence of intracellular protein aggregates (Lewy bodies) and the loss of dopaminergic neurons from the pars compacta component of the substantia nigra in the midbrain; PD-related clinical manifestations of dopamine deﬁciency (gait, tremor, rigidity, and bradykinesia) are the most archetypical symptoms of this disease. There are several studies showing that some statins (simvastatin, but neither atorvastatin nor lovastatin) may reduce the incidence of PD in patients aged over 65 years [100]. Compared with discontinuation of statins, continuation of lipophilic statin use has been associated with a reduced risk of PD, particularly in the elderly [101]; nevertheless, in patients with existing PD, 10-day treatment of simvastatin Catalysts 2019, 9, 260 7 of 32 (40 mg/day) showed no signiﬁcant effects on dyskinesia, functional impairment, or involuntary movement [102]. As inﬂammation is accepted to be a main contributor to the PD aetiology, the anti-inﬂammatory action of statins could be a rational explanation for their activity [30]; in fact, simvastatin has been reported useful for reversing the loss of striatal dopamine activity and the production of nitrosylated free radicals, thus inducing neuro-protection [103,104], by decreasing the release of inﬂammatory mediators from microglia. Also, some studies in rats have shown that simvastatin can protect against loss of NMDA receptors produced by 6-hydroxydopamine (6-OHDA) [105]; also using the 6-OHDA model in rats, Wang et al. [106] recently described the beneﬁcial effect of simvastatin in reducing abnormal involuntary movements known as L-DOPA-induced dyskinesia, commonly observed in patients chronically treated with L-DOPA. Finally, simvastatin and pravastatin can decrease the dopaminergic neuronal loss induced by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) via inhibition of p21(Ras)-induced NF-B (nuclear factor kappa-light-chain-enhancer of activated B cells) [107]. Anyhow, as mentioned in the previous paragraph, more deﬁnitive evidence from prospective and clinical studies is required before drawing any conclusions about statins efﬁcacy for treatment of PD. 2.3.4. Depression As well as for the previous neurological disorders discussed so far, there are reported discrepancies about statins’ effect in depression, with some studies reporting positive effects of statins in reducing depression and depression-like symptoms in animals [108–115] or humans [116,117], while some others stated no relationships [118–120]. These divergences require more detailed studies, also for elucidating the possible mechanism of the positive effects, which, in some cases, have been associated with a modulation of NMDA receptors [121] or peroxisome proliferator-activated receptor gamma (PPAR- ) receptors, by NO inhibition [122]. 2.3.5. Epilepsy In several studies, a reduced risk of developing epilepsy after age 50 has been reported [123–128], and the mechanism for this neuroprotective effect has been associated with a decrease in the association of subunit 1 of NMDA receptors to lipid rafts [129], as well as inhibition of calcium-dependent calpain activation, ROCK inhibition, the activation of the PI3K pathway, and increased APP cleavage [124], or the increased expression level of eNOS [130]; a recent publication by Scicchitano et al. [131] summarises the currently available data concerning statin effects in modulating epileptic seizure activity (sometimes adversely) and epileptogenesis in different experimental models, as well as in clinical studies [123,132]. 2.4. Cancer There are many studies dealing with the potential antitumor efﬁcacy of statins, reporting effects in different cancer cell lines, as well as the possible risks of cancer development caused by statins treatment and the results of different clinical trials [133–138]. Once again, the molecular mechanisms explaining statins’ effects are quite different, and clinical trials are not reporting conclusive results; in fact, although some large scale meta-analyses seem to indicate that statins do not have signiﬁcant effects on cancer incidence [133,139,140], in some other cases, some beneﬁcial effects associated with statins’ administration in the treatment of different cancers have been described [141–144]. What is really clear is that there is not just one mechanism explaining the anticancer activity of statins, because depending on the type and dosages of statin used, the type of cancer cells, and the time of exposure of cells to statins, different effects leading to cell-cycle arrest, induction of apoptosis, or changes in molecular pathways are reported [138]. Concerning cell modiﬁcations, a common scheme is followed, starting with an arrest of cells in the G1 [145,146] or S-phase [147], and this inhibition of cell-cycle progression is mediated by cyclins (such as cyclin D1 [148]), cyclin-dependent kinases WAF1/CIP1 (CDKs, such as p21 [148], p27 [149], CD4 [148], or p53 [150,151]), and inhibitors of CDKs [145]. Catalysts 2019, 9, 260 8 of 32 Simultaneously, inhibition of G-protein prenylation is produced, leading to the arrest of proliferation and/or induction of apoptosis in cancer cells [152,153], by an increase in caspases activity [147,154,155]; henceforward, inhibition of prenylation is a promising way to impede progression of cancer (see the recent review of Matusewicz et al. [138] and cites therein). On the other hand, it has been also reported that a substantial reduction in the amount of cholesterol leads to a reduction in the content of membrane lipid rafts in the cell membrane, altering cell signalling [156,157], and loosing membrane integrity; in this sense, it is known that the membrane of breast cancer and prostate cancer cells has higher level of cholesterol and lipid rafts, so these cells are more susceptible to apoptosis promoted by statins compared with normal cells [158,159]. In another feasible mechanism, statins are associated with inhibition of phosphorylation of caveolin-1 (Cav-1), the integral membrane protein that binds and transport cholesterol, which promotes tumour cell survival and resistance to chemotherapy by different mechanisms [160]. Anyhow, an exhaustive recompilation of all other mechanisms proposed to explain the action of statins in cancer treatment would be out of the scope of this manuscript and can be found in recent reviews [135,136,138,161]. However, once again, while the correlation between data obtained in vitro with those other ones reported in animal models is very high, clinical trials are not that irrefutable in their conclusions, and more detailed studies are demanded. 3. Biocatalyzed Synthesis of Statins As previously indicated in the Introduction, the use of biocatalyzed steps for preparing homochiral synthons useful for the synthesis of statins is a smart strategy for gaining both efﬁciency and sustainability. In this section, we will present some examples. 3.1. Simvastatin Lovastatin (Mevacor 2, ATC Code C10AA02, DrugBank Code DB00227, Figure 1) is a naturally-occurring fungal polyketide produced by Aspergillus terreus [162], while simvastatin (3, ATC Code C10AA01, DrugBank Code DB00641, Figure 1) is a semisynthetic analogue of 2 and is more effective in treating hypercholesterolemia, because of the fact that the substitution of the -methylbutyrate side chain with -dimethylbutyrate signiﬁcantly increases the inhibitory properties of 2, while lowering undesirable side effects [10]. Because of the economic importance of simvastatin, as mentioned in the Introduction, various multistep syntheses of 3 starting from 2 have been described; thus, a widely used process (route #1) starts with the hydrolysis of the C8 ester in 2 to yield the triol Monacolin J 10, followed by selective silylation of the C13 alcohol to yield 11, esteriﬁcation of C8 alcohol with dimethylbutyryl chloride to furnish 12, and deprotection of C13 alcohol to ﬁnally yield 3 [163] (Figure 3). In another option, namely route #2 [164], lovastatin 2 was treated with n-butylamine and TBSCl to obtain 13, which was alkylated with another methyl group to furnish 14, and ﬁnally transformed into 3 by hydrolysis and lactonization. Both multistep processes shown in Figure 3 were laborious, thus contributing to simvastatin being nearly ﬁve times more expensive than lovastatin [165]. Some enzymatic transformations using lipases and esterases were investigated as alternatives to chemical hydrolysis leading to Monacolin J 10 [166,167]. However, the requirement of regioselective esteriﬁcation of the C8 alcohol invariably involves protection of other reactive alcohol groups in 10, and generally leads to lowered overall yield. Therefore, a speciﬁc reagent that is able to selectively acylate C8 of 10 is important for the efﬁcient synthesis of simvastatin 3 and additional statin analogues. In this sense, Tang and co-workers [22] described an acyltransferase (LovD) able to catalyse the last step of lovastatin biosynthesis, as shown in Figure 4, by transferring a 2,2-dimethylbutyryl acyl group from dimethylbutyryl-S-methylmercaptopropionate (DMB-SMMP, 16) regioselectively to the C8 hydroxyl of Monacolin J 10, the immediate biosynthetic precursor of simvastatin. The reaction proceeds via a ping-pong mechanism, and LovD is inhibited by Monacolin J at moderate substrate concentrations. LovD displayed broad substrate speciﬁcity toward the acyl carrier, the acyl substrate, and the decalin Catalysts 2019, 9, 260 9 of 32 core of the acyl acceptor. This same group developed a one-step, whole-cell biocatalyzed process for the synthesis of Simvastatin from Monacolin J using an Escherichia coli strain overexpressing LovD, leading Catalysts 2019, 9, x FOR PEER REVIEW 9 of 32 to >99% conversion of monacolin J to simvastatin without the use of any chemical protection steps [165]. Catalysts 2019, 9, x FOR PEER REVIEW 9 of 32 The process was scaled up for gram-scale synthesis of simvastatin, also showing that simvastatin simvastatin, also showing that simvastatin synthesized via this method could be readily purified synthesized via this method could be readily puriﬁed from the fermentation broth with >90% recovery from simvastatin, the fer also show mentation b ingr that simv oth with ast >9a0% r tin se ycov ntheery sized v and ia t >9h8% is m peutrit hod coul y, as dd b eteerm reined adil b y pyu hi rifgh- ied and >98% purity, as determined by high-performance liquid chromatography. performanc from the fer e lim qu ent id at chrom ion br aot toh wit graphh y . >9 0% recovery and >98% purity, as determined by high- performance liquid chromatography. Figure 3. Chemical transformations of lovastatin 2 into simvastatin 3. Figure 3. Chemical transformations of lovastatin 2 into simvastatin 3. Figure 3. Chemical transformations of lovastatin 2 into simvastatin 3. Figure 4. Biocatalyzed transformations of lovastatin 2 into simvastatin 3. Figure 4. Biocatalyzed transformations of lovastatin 2 into simvastatin 3. Figure 4. Biocatalyzed transformations of lovastatin 2 into simvastatin 3. Codexis improved not only the enzyme (previously modified used directed evolution at lab scale in an Codexis impr E. coli-based oved not only the en biocatalytic platfo zym rm [ e1 (prev 68]) biu ou t al sly so p modified rocess chem used d istriyrected to enab ev le olution a large at -sclab ale Codexis improved not only the enzyme (previously modiﬁed used directed evolution at lab scale in an simvastatin E. coli manu -b fact ased ur b in io g process catalytic, p by latcarry forming o [168]) u b t nine it ut also p erat roc ion ess s of in v chemist itrr o e y tv oolut enab ion, c le a rla ea rge ting 2 -scale 16 scale in an E. coli-based biocatalytic platform [168]) but also process chemistry to enable a large-scale libr simva arie stsa and tin manu screenin factg 6 urin 1,g process 779 variant , by s tcarry o develop ing ou at LovD va nine iterria ation nt wi s of in v th imiproved a tro evolut cti ion, c vity, i rea n-process ting 216 simvastatin manufacturing process, by carrying out nine iterations of in vitro evolution, creating 216 stability, and tolerance to product inhibition. The approximately 1000-fold improved enzyme and the libraries and screening 61,779 variants to develop a LovD variant with improved activity, in-process st ability, and tolerance to product inhibition. The approximately 1000-fold improved enzyme and the Catalysts 2019, 9, 260 10 of 32 libraries and screening 61,779 variants to develop a LovD variant with improved activity, in-process stability, and tolerance to product inhibition. The approximately 1000-fold improved enzyme and the new process pushed the reaction to completion at high substrate loading and minimized the amounts of acyl donor and of solvents for extraction and product separation. This process possesses many Catalysts 2019, 9, x FOR PEER REVIEW 10 of 32 advantageous characteristics from a Green Chemistry point of view: Catalyst new proces is s pr pu oduced shed the re efﬁciently action tofr com ompr let enewable ion at high feedstock. substrate loading and minimized the amounts of acyl donor and of solvents for extraction and product separation. This process possesses many Reduced use of toxic and hazardous substances like tert-butyl dimethyl silane chloride, methyl advantageous characteristics from a Green Chemistry point of view: iodide, and n-butyl lithium. • Catalyst is produced efficiently from renewable feedstock. Improved energy efﬁciency as the reaction is run at ambient temperature and at near • Reduced use of toxic and hazardous substances like tert-butyl dimethyl silane chloride, methyl atmospheric pressure. iodide, and n-butyl lithium. Reduction in solvent use because of the aqueous nature of the reaction conditions. • Improved energy efficiency as the reaction is run at ambient temperature and at near The only by-product (methyl 3-mercaptopropionic acid) is recycled. atmospheric pressure. The major waste streams generated are biodegraded in bio treatment facilities. • Reduction in solvent use because of the aqueous nature of the reaction conditions. Codexis’ process can produce simvastatin with yields of 97%, signiﬁcant when compared with • The only by-product (methyl 3-mercaptopropionic acid) is recycled. • The major waste streams generated are biodegraded in bio treatment facilities. <70% with other manufacturing routes. • Codexis’ process can produce simvastatin with yields of 97%, significant when compared with For these reasons, Codexis and Prof. Tang obtained the U.S. Environmental Protection Agency’s <70% with other manufacturing routes. Green Chemistry Presidential Award in 2012 [169], inside the category of Greener Synthetic Pathway. For these reasons, Codexis and Prof. Tang obtained the U.S. Environmental Protection Agency’s Recently, identiﬁcation of the complete biosynthetic pathway leading to monacolin J has been Green Chemistry Presidential Award in 2012 [169], inside the category of Greener Synthetic Pathway. reported [170]. Recently, identification of the complete biosynthetic pathway leading to monacolin J has been reported [170]. 3.2. Biocatalyzed Synthesis of the Lateral Chain of Superstatins 3.2. Biocatalyzed Synthesis of the Lateral Chain of Superstatins Different biocatalytic routes have been proposed and implemented at industrial scale for the stereoselective preparation of the lateral chain (bearing the stereocentres) of superstatins. Thus, we Different biocatalytic routes have been proposed and implemented at industrial scale for the would stereose use the lectiv preparation e preparation of atorvastatin of the lateral chain as a refer (beence aring the stereoce to illustratentres) o how dif f supe ferent rstatins. Thus, we biotransformations would use the preparation of atorvastatin as a reference to illustrate how different biotransformations can be included in the overall protocol. can be included in the overall protocol. The chemical synthesis of atorvastatin originally described by researchers at Warner-Lambert The chemical synthesis of atorvastatin originally described by researchers at Warner-Lambert Company [171], shown in Figure 5, started from a chiral building block, ethyl (R)-4-cyano-3- Company [171], shown in Figure 5, started from a chiral building block, ethyl (R)-4-cyano-3- hydroxybutyrate 18, also known as “hydroxynitrile” (HN), and the second stereogenic centre of hydroxybutyrate 18, also known as “hydroxynitrile” (HN), and the second stereogenic centre of 20 20 was obtained by diastereomeric induction, using cryogenic borohydride reduction of a boronate was obtained by diastereomeric induction, using cryogenic borohydride reduction of a boronate derivative of the 5-hydroxy-3-keto intermediate 19 derived from HN. derivative of the 5-hydroxy-3-keto intermediate 19 derived from HN. Figure 5. Chemical synthesis of atorvastatin 5. Figure 5. Chemical synthesis of atorvastatin 5. Catalysts 2019, 9, 260 11 of 32 Taking this procedure as model, different strategies for generating the desired chirality can be Catalysts 2019, 9, x FOR PEER REVIEW 11 of 32 envisaged from a biocatalytic retrosynthetic scheme [172], as depicted in Figure 6, in which purely chemical steps are denoted by a red C, while those syntheses feasible to be biocatalyzed are represented Taking this procedure as model, different strategies for generating the desired chirality can be by a blue BT and a number, corresponding to the type of biocatalyst used. Thus, route #1 creates the envisaged from a biocatalytic retrosynthetic scheme [172], as depicted in Figure 6, in which purely desired chirality by a stereoselective desymmetrization of dinitrile 25 using a nitrilase (BT-1), while chemical steps are denoted by a red C, while those syntheses feasible to be biocatalyzed are routerepresented #2 requires bthe y a b pr lue eparation BT and a n of u HN mber, corre 18 via asponding to th bioreductione t of ype ketoester of bioca27 tal,ys so t us a e ketor d. Teductase hus, rout( e BT-2) #1 creates the desired chirality by a stereoselective desymmetrization of dinitrile 25 using a nitrilase is the biocatalyst required for that aim. Anyhow, in this synthetic path, another bioreduction should be (BT-1), while route #2 requires the preparation of HN 18 via a bioreduction of ketoester 27, so a used for avoiding the previously mentioned borohydride reduction of intermediate 19, using another ketoreductase (BT-2) is the biocatalyst required for that aim. Anyhow, in this synthetic path, another ketoreductase (BT-3), so this can be considered route #3. Finally, if an aldolase (BT-4) is the enzyme bioreduction should be used for avoiding the previously mentioned borohydride reduction of selected, it is possible to envisage route #4 as an alternative through cyclic intermediate 28. These intermediate 19, using another ketoreductase (BT-3), so this can be considered route #3. Finally, if an different routes will be discussed in the following sections. aldolase (BT-4) is the enzyme selected, it is possible to envisage route #4 as an alternative through cyclic intermediate 28. These different routes will be discussed in the following sections. 3.2.1. Hydrolases as Catalysts for the Preparation of the Lateral Chain of Atorvastatin and Other Superstatins 3.2.1. Hydrolases as Catalysts for the Preparation of the Lateral Chain of Atorvastatin and Other Superstatins As shown in route #1, a nitrilase-catalyzed enzymatic desymmetrization of prochiral 3-hydroxyglutaronitrile 25 and subsequent esteriﬁcation of the resulting (R)-3-hydroxy-4-cyanobutyric As shown in route #1, a nitrilase-catalyzed enzymatic desymmetrization of prochiral 3- acid (R)-26 can lead to HN. The use of enzymatic protocols for hydrolysing nitriles is a green alternative hydroxyglutaronitrile 25 and subsequent esterification of the resulting (R)-3-hydroxy-4-cyanobutyric compar acid ed (R with )-26 ca chemical n lead tomethodologies HN. The use of en [173zym ], because atic protocols fo of the harsh r hydrroly eact sin ion g nconditions itriles is a gree requir n ed, alternative compared with chemical methodologies [173], because of the harsh reaction conditions demanding either strong mineral acids (e.g., hydrochloric or phosphoric acid) or bases (e.g., potassium required, demanding either strong mineral acids (e.g., hydrochloric or phosphoric acid) or bases (e.g., or sodium hydroxide) and relatively high reaction temperatures. Moreover, chemical procedures potassium or sodium hydroxide) and relatively high reaction temperatures. Moreover, chemical sometimes give low yields because of both unwanted by-product formation and the generation of procedures sometimes give low yields because of both unwanted by-product formation and the concentrated contaminating waste salt streams (e.g., 6 mol L ) when the acid or base is neutralized −1 generation of concentrated contaminating waste salt streams (e.g., 6 mol L ) when the acid or base is prior to disposal [174]. neutralized prior to disposal [174]. Figure 6. Biocatalytic retrosynthetic routes to atorvastatin. BT represents biotransformation step, Figure 6. Biocatalytic retrosynthetic routes to atorvastatin. BT represents biotransformation step, while while C stands for chemical processes. C stands for chemical processes. Catalysts 2019, 9, 260 12 of 32 Thus, researchers at Diversa described a wild type nitrilase enzyme that catalysed the desymmetrization of 25 at high substrate concentration (3M) at lab-scale reaction, with an enantiomeric excess (ee) of 88%. [175]. A mutant nitrilase, obtained by directed evolution using gene site saturation mutagenesis (GSSM), and showing Ala190His single mutation, resulted in an excellent biocatalyst; hence, after a 15 h reaction at 20 C, (R)-26 was isolated in 96% yield, with an excellent ee of 98.5% 1 1 and a volumetric productivity of 619 g L d [176]. Subsequently, Dow Chirotech, a subsidiary of Dow Chemical Company, developed the Diversa nitrilase further into a biocatalysis process [177] and used the Pfenex expression system (a Pseudomonas ﬂuorescens-based host expression system) to overproduce the enzyme. In this way, optimal reaction conditions for desymmetrization of 25 were as follows: 3 M (330 g L ), pH 7.5, 27 C, under 16 h reaction time. A conversion of 100% and 99% product ee was obtained, and the so-formed (R)-26 was consequently esteriﬁed to give HN. Overall, a highly efﬁcient three-stage synthesis of HN starting from low-cost epichlorohydrin (required to produce 25) was achieved with an overall yield of 23%, 98% ee, and 97% purity [177]. Recently, an enzymatic method has been described for the synthesis of ethyl (R)-3-hydroxyglutarate from HN using free and immobilized recombinant Escherichia coli BL21(DE3)pLysS harbouring a nitrilase gene from Arabidopsis thaliana (AtNIT2) [178]. The hydrolysis of HN proceeded with the freely suspended cells of 1 1 the biocatalyst under the optimized conditions of 1.5 mol L (235.5 g L ) substrate concentration and 6.0 wt % loading of wet cells at pH 8.0 and 25 C, with 100% conversion obtained in 4.5 h. Furthermore, immobilization of the whole cells enhanced their substrate tolerance, stability, and reusability. Under the optimized conditions (100 mmol L tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 8.0, 25 C), the immobilized biocatalyst could be reused for up to 16 batches, with a biocatalyst 1 1 1 productivity of 55.6 g gwet cells and a space–time productivity of 625.5 g L d . Hydrolases are also useful for preparing (S)-3-hydroxy butyrolactone (S)-32, another enantiopure intermediate to furnish HN (Figure 7). In fact, opening of (S)-32 with HBr/EtOH will yield the corresponding ethyl (S)-4-bromo-3-hydroxybutanoate ((S)-BHBE, (S)-33) [179], later transformed into HN via S 2 when treated with sodium (or potassium) bromide. Although (S)-32 can be produced from chiral pool raw materials (lactose or malic acid), it can be conveniently obtained by enzymatic hydrolysis of the racemic ethyl 4-chloro-3-hydroxybutanoate (CHBE, rac-29) in the aqueous phase [180]. The lipase stereoselectively hydrolysed only the (S)-enantiomer; however, the resulting acid (S)-30 is unstable, and it readily loses one HCl molecule to give the corresponding lactone of high enantiopurity (>99% ee). However, the enantiopurity of the lactone rapidly decreased when the process was operated at yields of more than 40%. The hydrolysis of the enantiopure benzoic ester of (S)-hydroxybutyrolactone (S)-31 has also been described using lipase from Candida rugosa (CRL) immobilized on amberlite XAD-7 as polymeric support, with ee of 99% [181]. This enzymatic hydrolysis was observed to be non-stereoselective in nature, because the enzymatic hydrolysis of the racemic benzoic ester yielded the racemic lactone, so that a chiral pool precursor (L-malic acid) for this process was necessary. Anyhow, this method has been scaled up to a ton scale, with an overall yield of over 80%, and a reaction time of 14 h [182]. Recently, a platform pathway for the production of 3-hydroxyacids has been described as an alternative biosynthetic route to generate the enantiopure lactone [183]. Catalysts 2019, 9, 260 13 of 32 Catalysts 2019, 9, x FOR PEER REVIEW 13 of 32 Catalysts 2019, 9, x FOR PEER REVIEW 13 of 32 Figure 7. Preparation of HN 18 starting from (S)-3-hydroxy butyrolactone (S)-32. Figure 7. Preparation of HN 18 starting from (S)-3-hydroxy butyrolactone (S)-32. Figure 7. Preparation of HN 18 starting from (S)-3-hydroxy butyrolactone (S)-32. More recently, new biocatalytic approaches employing hydrolases have been described for More recently, new biocatalytic approaches employing hydrolases have been described for More recently, new biocatalytic approaches employing hydrolases have been described for furnishing the lateral chain of superstatins. Actually, Figure 8 shows a synthetic scheme for preparing furnishing the lateral chain of superstatins. Actually, Figure 8 shows a synthetic scheme for preparing furnishing the lateral chain of superstatins. Actually, Figure 8 shows a synthetic scheme for preparing rosuvastatin 9. As can be seen, conjugated ketoester 36 is subsequently transformed into final calcium rosuvastatin 9. As can be seen, conjugated ketoester 36 is subsequently transformed into ﬁnal calcium rosuvastatin 9. As can be seen, conjugated ketoester 36 is subsequently transformed into final calcium rosuvastatin 9 by different steps (silyl ether cleavage, diastereoselective Narasaka-Prasad [184,185] rosuvastatin 9 by different steps (silyl ether cleavage, diastereoselective Narasaka-Prasad [184,185] rosuvastatin 9 by different steps (silyl ether cleavage, diastereoselective Narasaka-Prasad [184,185] syn-reduction using diethylmethoxy borane leading to ester 37, and finally ester hydrolysis and salt syn-reduction using diethylmethoxy borane leading to ester 37, and ﬁnally ester hydrolysis and salt syn-reduction using diethylmethoxy borane leading to ester 37, and finally ester hydrolysis and salt formation). formation). formation). Figure 8. Final steps in the chemical synthesis of rosuvastatin 9. Figure 8. Final steps in the chemical synthesis of rosuvastatin 9. Figure 8. Final steps in the chemical synthesis of rosuvastatin 9. Aldehyde 34 can be easily obtained [186], while the preparation of enantiopure ylide 35 is much more complicated. Thus, several examples can be found in the literature starting from racemic diethyl 3-hydroxyglutarate, which had to be previously transformed in an activated derivative to react with Catalysts 2019, 9, x FOR PEER REVIEW 14 of 32 Catalysts 2019, 9, x FOR PEER REVIEW 14 of 32 Aldehyde 34 can be easily obtained [186], while the preparation of enantiopure ylide 35 is much Catalysts 2019, 9, 260 14 of 32 Aldehyde 34 can be easily obtained [186], while the preparation of enantiopure ylide 35 is much more complicated. Thus, several examples can be found in the literature starting from racemic diethyl more complicated. Thus, several examples can be found in the literature starting from racemic diethyl 3-hydroxyglutarate, which had to be previously transformed in an activated derivative to react with 3-hydroxyglutarate, which had to be previously transformed in an activated derivative to react with the corresponding methyltriphenylphosphonium ylide to finally yield 35; although this route has the corresponding methyltriphenylphosphonium ylide to ﬁnally yield 35; although this route has been the corresponding methyltriphenylphosphonium ylide to finally yield 35; although this route has been described using an enzymatic desymmetrization step [187,188], different side reactions were described using an enzymatic desymmetrization step [187,188], different side reactions were observed been described using an enzymatic desymmetrization step [187,188], different side reactions were observed to decrease either the final yield or the enantiomeric excess. Recently, a bi-enzymatic to decrease either the ﬁnal yield or the enantiomeric excess. Recently, a bi-enzymatic process has observed to decrease either the final yield or the enantiomeric excess. Recently, a bi-enzymatic process has been described for obtaining enantiopure monoester (R)-40 (Figure 9), combining a been described for obtaining enantiopure monoester (R)-40 (Figure 9), combining a stereoselective process has been described for obtaining enantiopure monoester (R)-40 (Figure 9), combining a stereoselective hydrolysis of prochiral 38 to obtain (R)-39 with high yield and enantiopurity, and a hydrolysis of prochiral 38 to obtain (R)-39 with high yield and enantiopurity, and a subsequent removal stereoselective hydrolysis of prochiral 38 to obtain (R)-39 with high yield and enantiopurity, and a subsequent removal of the acetyl group with cephalosporin acetyl esterase [189]. of the acetyl group with cephalosporin acetyl esterase [189]. subsequent removal of the acetyl group with cephalosporin acetyl esterase [189]. Figure 9. Two-enzymatic system for synthesizing chiral intermediates for Rosuvastatin, as described Figure 9. Two-enzymatic system for synthesizing chiral intermediates for Rosuvastatin, as described Figure 9. Two-enzymatic system for synthesizing chiral intermediates for Rosuvastatin, as described by Metzner et al. [189]. by Metzner et al. [189]. by Metzner et al. [189]. Furthermore, these same authors have optimized the overall procedure, using a smart Furthermor Furthermore, these same authors h e, these same authors have optimized ave optimize the d the overall procedure, usin overall procedure, using a smartg a sm engineering art engineering approach with an enzyme recycling of chymotrypsin and immobilized cephalosporin C appr engineer oaching with approach with an enzyme recycling an enzyme r of chymotrypsin ecycling of ch and ymotrypsin immobilized and immo cephalosporin bilized cepha C acetyl losp esterase, orin C acetyl esterase, with excellent volumetric productivity, transferring this technology to Sandoz for its acetyl esterase, with excellent volumetric productivity, transferring this technology to Sandoz for its with excellent volumetric productivity, transferring this technology to Sandoz for its industrial industrial implementation [190]. industrial implementation [190]. implementation [190]. 3.2.2. Ketoreductases as Catalysts for the Preparation of the Lateral Chain of Atorvastatin and Other 3.2.2. Ketoreductases as Catalysts for the Preparation of the Lateral Chain of Atorvastatin and 3.2.2. Ketoreductases as Catalysts for the Preparation of the Lateral Chain of Atorvastatin and Other Superstatins Other Superstatins Superstatins As commented before, HN 18 was the starting point for the first synthesis of atorvastatin (Figure As commented before, HN 18 was the starting point for the ﬁrst synthesis of atorvastatin (Figure 5). As commented before, HN 18 was the starting point for the first synthesis of atorvastatin (Figure 5). For preparing HN, apart from the hydrolytic procedures described in Figure 7, some different For preparing HN, apart from the hydrolytic procedures described in Figure 7, some different purely 5). For preparing HN, apart from the hydrolytic procedures described in Figure 7, some different purely chemical methodologies have been also described [191], and are depicted in Figure 10. chemical methodologies have been also described [191], and are depicted in Figure 10. purely chemical methodologies have been also described [191], and are depicted in Figure 10. Figure 10. Different chemical methodologies for the preparation of HN 18. Figure Figure 10. 10. Diff Differ erent chemical ent chemical methodologies methodologiesfor the preparation of for the preparation ofHN HN 18 18 . . The ﬁrst synthetic protocols involved kinetic resolutions of prochiral 1,3-dichloropropan-2-ol 43 using microbes, and transformation to dihidroxyester (S)-45 and subsequently to bromohydrine Catalysts 2019, 9, 260 15 of 32 (S)-33 [179]. Later routes have involved asymmetric reduction of ethyl 4-chloroacetoacetate (COBE, 27), produced from diketene, to furnish ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE, (S)-46), using either chemical or biocatalytic reductions, as previously shown in route #2, Figure 6. Finally, the corresponding halohydrin ((S)-33 or (S)-46) could be converted to HN by treatment with cyanide. In this sense, the enzymatic asymmetric reduction of 4-bromo-3-oxobutyrate esters has hardly been investigated compared with the corresponding chlorine analogue, because of the lower reactivity and enantioselectivity of enzymes towards brominated compounds, although (S)-4-bromo-3-hydroxybutanoate esters would be better substrates for the ulterior cyanide treatment; anyhow, some examples can be found in the literature, starting from methyl 4-bromo-3-oxobutyrate (BAM), using Escherichia coli engineered cells containing a mutant -keto ester reductase (KER-L54Q) from Penicillium citrinum and a cofactor-regeneration enzyme such as glucose dehydrogenase (GDH) or Leifsonia sp. alcohol dehydrogenase (LSADH) [192,193]. Regarding chlorine containing oxoesters, the seminal paper of Patel et al. using glucose-, acetate-, or glycerol-grown cell (10% w/v) suspensions of Geotrichum candidum SC 5469 [194] to produce (S)-46 in reaction yield of 95% and optical purity of 96%, starting from 10 mg mL of 27, showed how the bio-reduction could be an interesting alternative to asymmetrical chemical reduction. Furthermore, the optical purity of (S)-46 was increased to >99% by heat treatment of cell suspensions (55 C for 30 min) prior to conducting bio-reduction at 28 C. Ye et al. [195] have reviewed a list of different yeast able to reduce 27 to furnish (S)-46, such as Candida etchellsii [196], Candida parapsilosis [197], Candida magnoliae [198], Saccharomycopsis lipolytica [196], or Candida macedoniensis [199], but in many cases, the stereoselectivity values obtained were not very high. Also, fungi as Aureobasidium pullulans CGMCC 1244 [200], Cylindrocarpon sclerotigenum IFO31855 [201], Penicillium oxalicum IFO 5748 [197], Botrytis allii IFO9430 [197], or Pichia stipitis CBS 6054 [202] can produce (S)-46 with a higher enantiomeric excess compared with yeasts. This same group, through genome database mining of this yeast Pichia stipitis, found two carbonyl reductases (PsCRI and PsCRII) leading to (S)-46 with >99% enantiomeric excess, which were subsequently characterized, cloned, and expressed in E. coli [195]. On the other hand, Cai et al. [203] described a substrate-coupled biocatalytic process based on the reactions catalyzed by an NADPH-dependent sorbose reductase (SOU1) from Candida albicans in which 27 was reduced to (S)-46, while NADPH was regenerated by the same enzyme via oxidation of sugar alcohols (sorbitol, mannitol, or xylitol). Optimization of COBE and sorbitol proportions yielded 2340 mM of (S)-46 starting from 2500 mM 27 with an enantiomeric excess was 99%. This substrate-coupled system maintained a stable pH and a robust intracellular NADPH circulation, so that pH adjustment and the addition of extra coenzymes were unnecessary, thus making this system very attractive. The bio-reduction of 27 and the scaling up of the process using Escherichia coli cells expressing a reductase (ScCR) from Streptomyces coelicolor to afford enantiopure (S)-46 has recently being described [204], at substrate loading of 100 g/L, while the concentration of coenzyme NAD was limited to 0.1 mM based on cost considerations, other reaction parameters were optimized as 25 C and pH 6.5, with a biocatalyst dose of 10 kU/L in the presence of isopropanol (1.5 equiv of 27) as co-substrate for regenerating NADH. The reaction was performed in a tolueneaqueous biphasic system (1:1, v/v), with agitation at the maximal linear rate of 0.88 m/s. Finally, the bio-reaction was performed on a pilot scale using a 50 L thermostatised stirred-tank-reactor, affording (S)-46 in 85.4% yield and 99.9% ee, and a total turnover number (TTN) of 6060 for the cofactor NAD . The speciﬁc production was calculated to be 36.8 g product/g dcw, which is the highest value reported to date among the whole-cell-mediated processes for producing (S)-46. Furthermore, from the point of view of sustainability, for this bio-reduction, the reaction and extraction solvent (toluene) was recycled with a loss of only 4.1%, so that the E factor (kg waste per kg product) for the process was determined as 1.8 if the process water was excluded, which was much lower than that value (2.3) obtained from the process using isolated ketoreductase, glucose dehydrogenase as the biocatalyst for cofactor regeneration, and glucose as the co-substrate [179]. The main contributors to the low E factor were the loss of the solvent toluene (46.1%), the use of excessive isopropanol, and Catalysts 2019, 9, 260 16 of 32 Catalysts 2019, 9, x FOR PEER REVIEW 16 of 32 the isop formation ropanol, of and t copr he form oduct at acetone ion of cop (combined roduct acet ca. one 35%). (com Ifbwater ined ca was . 35% also ). If wat included, er was then also inc theluded, E factor then the E factor would be 13.4. would be 13.4. Very recently, a recombinant Escherichia coli harbouring both the carbonyl reductase and glucose Very recently, a recombinant Escherichia coli harbouring both the carbonyl reductase and glucose dehydrogenase has been described [205]. The recombinant E. coli was cultured in a 500-L fermenter, dehydrogenase has been described [205]. The recombinant E. coli was cultured in a 500-L fermenter, and the biocatalytic process for the synthesis of (S)-46 in an aqueous-organic solvent system was and the biocatalytic process for the synthesis of (S)-46 in an aqueous-organic solvent system was constructed and optimized with a substrate fed-batch strategy. Concentration of 27 reached to 1.7 M, constructed and optimized with a substrate fed-batch strategy. Concentration of 27 reached to 1.7 M, and (S)-46 was obtained after a 4 h reaction in a 50-L reactor with yield of 97.2% and enantiomeric and (S)-46 was obtained after a 4 h reaction in a 50-L reactor with yield of 97.2% and enantiomeric excess excess (ee) of 99%. Finally, (S)-46 was extracted from the reaction mixture with 82% yield and 95% (ee) of 99%. Finally, (S)-46 was extracted from the reaction mixture with 82% yield and 95% purity. purity. Nevertheless, because of the great overall demand of HN required for atorvastin synthesis Nevertheless, because of the great overall demand of HN required for atorvastin synthesis (estimated to be in excess of 100 mT [179]), it is highly desirable to reduce the wastes and hazards (estimated to be in excess of 100 mT [179]), it is highly desirable to reduce the wastes and hazards involved in its manufacture, while reducing its cost and maintaining or, preferably, improving involved in its manufacture, while reducing its cost and maintaining or, preferably, improving its its quality. This has been successfully carried out on a multiton scale by Codexis by means of a quality. This has been successfully carried out on a multiton scale by Codexis by means of a three- three-enzyme two-step process, the detailed description of which is depicted in Figure 11. enzyme two-step process, the detailed description of which is depicted in Figure 11. Figure Figure 11. 11. Codexis Codexis sy synthesis nthesis of ofHN HN . . Hence, Hence, the the ﬁrst first step invo step involves lves the biocat the biocatalytic alytic reduct reduction ion of of 27,27 us ,in using g a kea tor ketor educeductase tase (KRE(KRED) D) in in com combination bination wit with h gl glucose ucose and and an NA an NADP-dependent DP-dependent gluc glucose ose de dehydr hydrogen ogenase ase (GDH (GDH) ) for c forocofactor factor regeneration, leading to (S)-46 in 96% isolated yield and >99.5% ee. In the second step, a halohydrin regeneration, leading to (S)-46 in 96% isolated yield and >99.5% ee. In the second step, a halohydrin dehalogenase (HHDH), an enzyme capable of catalysing the elimination of halides from vicinal dehalogenase (HHDH), an enzyme capable of catalysing the elimination of halides from vicinal haloalcohols, resulting in epoxide ring formation [206], was employed to catalyse a nucleophilic haloalcohols, resulting in epoxide ring formation [206], was employed to catalyse a nucleophilic substitution of chloride by cyanide, using HCN at neutral pH and ambient temperature. The substitution of chloride by cyanide, using HCN at neutral pH and ambient temperature. The efﬁciency efficiency and greenness of this protocol (Codexis was awarded the U.S. Environmental Protection and greenness of this protocol (Codexis was awarded the U.S. Environmental Protection Agency’s Agency’s Presidential Green Chemistry Challenge Award in 2006 for this work [207]) is based on the Presidential Green Chemistry Challenge Award in 2006 for this work [207]) is based on the fact that fact that all previous manufacturing routes to HN shown in Figure 10 involved, as the final step, a all previous manufacturing routes to HN shown in Figure 10 involved, as the ﬁnal step, a standard standard but troublesome SN2 substitution of halide with cyanide ion in alkaline solution (pH = 10) but troublesome S 2 substitution of halide with cyanide ion in alkaline solution (pH = 10) at high at high temperatures (80 °C), being this reaction substituted in the Codexis protocol. In fact, in the temperatures (80 C), being this reaction substituted in the Codexis protocol. In fact, in the S 2 SN2 chlorine substitution, both (S)-46 and HN are base-sensitive molecules, and extensive by-product chlorine substitution, both (S)-46 and HN are base-sensitive molecules, and extensive by-product formation is observed, leading to high E values [179]. Moreover, the product is a high-boiling oil, and formation is observed, leading to high E values [179]. Moreover, the product is a high-boiling oil, and a troublesome high-vacuum fractional distillation is required to recover HN, resulting in further yield a troublesome high-vacuum fractional distillation is required to recover HN, resulting in further yield losses and waste, and clearly contravening the first and sixth principles of Green Chemistry [208]. losses and waste, and clearly contravening the ﬁrst and sixth principles of Green Chemistry [208]. Thus, conducting the cyanation reaction under milder conditions at neutral pH, by employing the Thus, conducting the cyanation reaction under milder conditions at neutral pH, by employing the enzyme, HHDH, is the key step for increasing the greenness of the overall process. enzyme, HHDH, is the key step for increasing the greenness of the overall process. Coming back to the Codexis protocol, awkwardly, both the wild-type KRED and GDH as well Coming back to the Codexis protocol, awkwardly, both the wild-type KRED and GDH as well as HHDH displayed very low activities, so that in the first experiments, huge enzyme loadings were as requ HHDH ireddisplayed to obtain an very econ low omica activities, lly feas so ible re that ac intithe on ﬁrst rate, thus l experiments, eading to troub huge enzyme lesome em loadings ulsions, were requir which h ed toaobtain mpered the an economically subsequentfeasible downstream reaction proce rate, ssithus ng. Addi leading tiona tolltr y, severe product oublesome emulsions, inhibiti which on and poor stability under operating conditions were observed. To enable a practical large-scale hampered the subsequent downstream processing. Additionally, severe product inhibition and poor process, the three enzymes were optimized by in vitro enzyme evolution using gene shuffling stability under operating conditions were observed. To enable a practical large-scale process, the three technologies according to predefined criteria and process parameters, resulting in an overall process enzymes were optimized by in vitro enzyme evolution using gene shufﬂing technologies according to in which the volumetric productivity per mass catalyst load of the cyanation process was improved predeﬁned criteria and process parameters, resulting in an overall process in which the volumetric ~2500-fold, comprising a 14-fold reduction in reaction time, a 7-fold increase in substrate loading, a productivity per mass catalyst load of the cyanation process was improved ~2500-fold, comprising a 25-fold reduction in enzyme use, and a 50% improvement in isolated yield [179]. 14-fold reduction in reaction time, a 7-fold increase in substrate loading, a 25-fold reduction in enzyme use, and a 50% improvement in isolated yield [179]. Catalysts 2019, 9, 260 17 of 32 Catalysts 2019, 9, x FOR PEER REVIEW 17 of 32 Also using bio-reductions, some other strategies have been developed for the preparation of Also using bio-reductions, some other strategies have been developed for the preparation of chiral building blocks for statins synthesis. Thus, Figure 12 illustrates route #3, previously shown chiral building blocks for statins synthesis. Thus, Figure 12 illustrates route #3, previously shown in in Figure 6, depicting bioreduction of the corresponding 6-substitued-3,5-dioxohexanoates 48 to Figure 6, depicting bioreduction of the corresponding 6-substitued-3,5-dioxohexanoates 48 to furnish furnish (R or S)-49 (similar to (R)-19, Figure 6). As depicted in Figure 5, the homochiral intermediate (R or S)-49 (similar to (R)-19, Figure 6). As depicted in Figure 5, the homochiral intermediate (3R,5R)- (3R,5R)-20 was originally prepared by diastereoselective chemical reduction of (R)-19, using NaBH 20 was originally prepared by diastereoselective chemical reduction of (R)-19, using NaBH4 and 4 and MeOBEt and, so as to obtain a high diastereoselectivity (>99.5% de), an extremely low temperature MeOBEt2 and 2 , so as to obtain a high diastereoselectivity (>99.5% de), an extremely low temperature (90 C) and pyrophoric triethyl borane were demanded [209], with a concomitant extensive energy (−90 °C) and pyrophoric triethyl borane were demanded [209], with a concomitant extensive energy consumption and substantial amount of waste formation. Another alternative chemical route using consumption and substantial amount of waste formation. Another alternative chemical route using chlororuthenium(II) arene/ -amino alcohol as the catalyst for the reduction was described [210], chlororuthenium(II) arene/β-amino alcohol as the catalyst for the reduction was described [210], although the diastereoselectivity was insufﬁcient (80% de). although the diastereoselectivity was insufficient (80% de). Figure 12. Bio-reductions to produce chiral building blocks for statins. Figure 12. Bio-reductions to produce chiral building blocks for statins. Therefore, the use of a ketoreductase is highly desirable to develop green and sustainable Therefore, the use of a ketoreductase is highly desirable to develop green and sustainable bioreduction. This process has been described [211,212] using NADP(H)-dependent alcohol bioreduction. This process has been described [211,212] using NADP(H)-dependent alcohol dehydrogenase of Lactobacillus brevis. This enzyme was overexpressed in a recombinant E. coli and dehydrogenase of Lactobacillus brevis. This enzyme was overexpressed in a recombinant E. coli and the cell extracts were then employed for carrying out the biocatalytic reactions on a gram scale, to the cell extracts were then employed for carrying out the biocatalytic reactions on a gram scale, to reduce (S)-48a to give the corresponding (3S, 5R)-49a in >99.5% de and isolated yield of 72%, at reduce (S)-48a to give the corresponding (3S, 5R)-49a in >99.5% de and isolated yield of 72%, at 24 h. 24 h. Alcohol dehydrogenase itself recycles its cofactor by a substrate coupled methodology, by Alcohol dehydrogenase itself recycles its cofactor by a substrate coupled methodology, by oxidation oxidation of 2-propanol to acetone. This process was scaled up to 100 g [213] using a fed-batch of 2-propanol to acetone. This process was scaled up to 100 g [213] using a fed-batch reactor, with the reactor, with the conversion of more than 90% attained in a total reaction time of 24 h. For the conversion of more than 90% attained in a total reaction time of 24 h. For the same substrate, Liu and same substrate, Liu and co-workers have reported the use of a ketoreductase from Rhodosporidium co-workers have reported the use of a ketoreductase from Rhodosporidium toruloides, wild-type and toruloides, wild-type and genetically evolved, under different reaction conditions [214–217], while genetically evolved, under different reaction conditions [214–217], while Xu et al. used a Xu et al. used a ketoreductase from Klebsiella oxytoca [218]. On the other hand, for reducing (R)-19 (up−1to ketoreductase from Klebsiella oxytoca [218]. On the other hand, for reducing (R)-19 (up to 300 g L ), 300 g L ), the ketoreductase from L. brevis overexpressed in E. coli cells has also been employed [219], the ketoreductase from L. brevis overexpressed in E. coli cells has also been employed [219], coupled 1 1 coupled to glucose-GDH for cofactor recycling, yielding (R,R)-20 in >99.5% de and −1 351 −1 g L d to glucose-GDH for cofactor recycling, yielding (R,R)-20 in >99.5% de and 351 g L d space–time space–time yield under the optimized conditions. Very recently, the same group has evolved the yield under the optimized conditions. Very recently, the same group has evolved the ketoreductase ketoreductase in order to improve the activity and thermostability of the enzyme [220]; thus, by in order to improve the activity and thermostability of the enzyme [220]; thus, by coexpressing both coexpressing both the mutant ketoreductase and GDH, they describe the bioreduction of (R)-19 to the mutant ketoreductase and GDH, they describe the bioreduction of (R)-19 to (R,R)-20 at 40 °C in 1 1 (R,R)-20 at 40 C in only 6 h, leading to values of >99.5% de−1 and −1 1050 g L d space–time yield. only 6 h, leading to values of >99.5% de and 1050 g L d space–time yield. Other similar Other similar bioreductions have also been reported using ketoreductases from other sources, such as bioreductions have also been reported using ketoreductases from other sources, such as Rhodotorula Rhodotorula glutinis (whole cells [221]); engineered cells containing overexpressed NADPH-dependant glutinis (whole cells [221]); engineered cells containing overexpressed NADPH-dependant ketoreductase from Saccharomyces cerevisiae and GDH [222,223]; a wild-type ketoreductase from ketoreductase from Saccharomyces cerevisiae and GDH [222,223]; a wild-type ketoreductase from Kluyveromuces lactis XP1461 (NADH-dependant) expressed in E. coli [224], subsequently improved by Kluyveromuces lactis XP1461 (NADH-dependant) expressed in E. coli [224], subsequently improved by site-saturation mutagenesis [225]; or the ketoreductase from Candida albicans XP1463, also expressed in site-saturation mutagenesis [225]; or the ketoreductase from Candida albicans XP1463, also expressed E. coli cells [226]. in E. coli cells [226]. In a similar way, the double reduction of dioxoesters 50 (Figure 13) would directly lead to the In a similar way, the double reduction of dioxoesters 50 (Figure 13) would directly lead to the target dihydroxyester 51. For this purpose, whole cells of Lactobacillus keﬁr, which contain two different target dihydroxyester 51. For this purpose, whole cells of Lactobacillus kefir, which contain two types of alcohol dehydrogenase, are able to convert 50b into the dihydroxy ester (3R, 5S)-51a (99% ee different types of alcohol dehydrogenase, are able to convert 50b into the dihydroxy ester (3R, 5S)- in a total yield of 47.5% after 22 h, [227]) and the cofactor NADP(H) was regenerated by the usual 51a (99% ee in a total yield of 47.5% after 22 h, [227]) and the cofactor NADP(H) was regenerated by glucose metabolism of the cell. the usual glucose metabolism of the cell. Catalysts 2019, 9, 260 18 of 32 Catalysts 2019, 9, x FOR PEER REVIEW 18 of 32 Catalysts 2019, 9, x FOR PEER REVIEW 18 of 32 Figure 13. Figure 13. Bio- Bio-r redu eductions ctions of d of dioxoesters ioxoesters to pr to produ oduce ce chiral b chiral building uilding blo blocks cks for statins for statins. . Figure 13. Bio-reductions of dioxoesters to produce chiral building blocks for statins. The double bio-reduction has been also described using isolated enzymes, from Acinetobacter The double bio-reduction has been also described using isolated enzymes, from Acinetobacter The double bio-reduction has been also described using isolated enzymes, from Acinetobacter species; in fact, Patel et al. originally described the bio-reduction of 50b using both whole cells species; in fact, Patel et al. originally described the bio-reduction of 50b using both whole cells and species; in fact, Patel et al. originally described the bio-reduction of 50b using both whole cells and and cell extracts from Acinetobacter calcoaceticus [228], and some years later, they also cloned and cell extracts from Acinetobacter calcoaceticus [228], and some years later, they also cloned and cell extracts from Acinetobacter calcoaceticus [228], and some years later, they also cloned and overexpressed [229] the diketoreductase responsible for the double reduction, which was efﬁciently overexpressed [229] the diketoreductase responsible for the double reduction, which was efficiently overexpressed [229] the diketoreductase responsible for the double reduction, which was efficiently carried out with the engineered enzyme [230]. Similarly, a diketoreductase from Acinetobacter baylyi carried out with the engineered enzyme [230]. Similarly, a diketoreductase from Acinetobacter baylyi carried out with the engineered enzyme [230]. Similarly, a diketoreductase from Acinetobacter baylyi ATCC 33305 was cloned and heterogeneously expressed in Escherichia coli by Wu et al. [231], showing ATCC 33305 was cloned and heterogeneously expressed in Escherichia coli by Wu et al. [231], showing ATCC 33305 was cloned and heterogeneously expressed in Escherichia coli by Wu et al. [231], showing an excellent biocatalytic performance at substrate concentration around 100 g L [232] for the double −1 an excellent biocatalytic performance at substrate concentration around 100 g L [232] for the double −1 an excellent biocatalytic performance at substrate concentration around 100 g L [232] for the double reduction of 50a. Interestingly, the 3D structure of this enzyme was reported, and the details of the reduction of 50a. Interestingly, the 3D structure of this enzyme was reported, and the details of the reduction of 50a. Interestingly, the 3D structure of this enzyme was reported, and the details of the catalytic mechanism were explained [233–235]. catalytic mechanism were explained [233–235]. catalytic mechanism were explained [233–235]. 3.2.3. Aldolases for the Preparation of the Lateral Chain of Atorvastatin and Other Superstatins 3.2.3. Aldolases for the Preparation of the Lateral Chain of Atorvastatin and Other Superstatins 3.2.3. Aldolases for the Preparation of the Lateral Chain of Atorvastatin and Other Superstatins Aldolases can also be used in the preparation of chiral building blocks for statin synthesis. Aldolases can also be used in the preparation of chiral building blocks for statin synthesis. This Aldolases can also be used in the preparation of chiral building blocks for statin synthesis. This This would correspond to route #4 in Figure 6. In fact, Gijsen and Wong [236,237] ﬁrst described the would correspond to route #4 in Figure 6. In fact, Gijsen and Wong [236,237] first described the use would correspond to route #4 in Figure 6. In fact, Gijsen and Wong [236,237] first described the use use of 2-deoxy-D-ribose 5-phosphate aldolase (DERA) from E. coli in the preparation of intermediate of 2-d of 2-d ee oxy- oxy- D D-ribose 5-phosphate -ribose 5-phosphate aldo aldo lase (DE lase (DE RR A) fro A) fro mm E. coli E. coli in in the preparation the preparation of intermed of intermed iate iate 28, 28, 28, in a reaction mixture consisting of 133 mg of chloroacetaldehyde and 264 mg of acetaldehyde in a in a reaction mixture consisting of 133 mg of chloroacetaldehyde and 264 mg of acetaldehyde in a in a reaction mixture consisting of 133 mg of chloroacetaldehyde and 264 mg of acetaldehyde in a total reaction volume of 20 mL (Figure 14). The atorvastatin intermediate lactone (4R, 6S)-54 can be total reaction volume of 20 mL (Figure 14). The atorvastatin intermediate lactone (4R, 6S)-54 can be total reaction volume of 20 mL (Figure 14). The atorvastatin intermediate lactone (4R, 6S)-54 can be easily formed by oxidation of lactol 28. However, aldolase showed low afﬁnity to chloroacetaldehyde easily formed by oxidation of lactol 28. However, aldolase showed low affinity to chloroacetaldehyde easily formed by oxidation of lactol 28. However, aldolase showed low affinity to chloroacetaldehyde and was promptly inactivated at required aldehyde concentrations, so that a huge amount of aldolase and was promptly inactivated at required aldehyde concentrations, so that a huge amount of aldolase and was promptly inactivated at required aldehyde concentrations, so that a huge amount of aldolase was required. Furthermore, a very long reaction time of 6 days was required because of the reversible was required. Furthermore, a very long reaction time of 6 days was required because of the reversible was required. Furthermore, a very long reaction time of 6 days was required because of the reversible nature of aldol reactions, making this process unpractical for scaling up. nature of aldol reactions, making this process unpractical for scaling up. nature of aldol reactions, making this process unpractical for scaling up. Figure 14. Aldolase-catalysed synthesis of chiral building blocks for statins. Figure 14. Aldolase-catalysed synthesis of chiral building blocks for statins. Figure 14. Aldolase-catalysed synthesis of chiral building blocks for statins. Subsequent Subsequent st studies udies by by LiLiu u et al et. [ al. 238 [] de 238] scrib described ed a mut a ant mutant aldolas aldolase, e, leading t leading o an increas to an ed y incr iele d ased of (4R, 6S)-54 to 43%, in comparison with 25% for the wild type aldolase, although the other reaction yield of (4R, 6S)-54 to 43%, in comparison with 25% for the wild type aldolase, although the other Subsequent studies by Liu et al. [238] described a mutant aldolase, leading to an increased yield drawbacks were not overpassed. The process was markedly improved and scaled up by Greenberg reaction drawbacks were not overpassed. The process was markedly improved and scaled up by of (4R, 6S)-54 to 43%, in comparison with 25% for the wild type aldolase, although the other reaction et al. [239] of Diversa Corporation, by genetically modifying DERA by means of high throughput Greenberg et al. [239] of Diversa Corporation, by genetically modifying DERA by means of high drawbacks were not overpassed. The process was markedly improved and scaled up by Greenberg screenings of environmental DNA libraries, focussing on chloroacetaldehyde resistance and higher throughput screenings of environmental DNA libraries, focussing on chloroacetaldehyde resistance et al. [239] of Diversa Corporation, by genetically modifying DERA by means of high throughput productivity; in a second step, the process was further improved by using a fed-batch bioreactor, in and higher productivity; in a second step, the process was further improved by using a fed-batch screenings of environmental DNA libraries, focussing on chloroacetaldehyde resistance and higher order to avoid significant substrate inhibition. Thus, the final synthesis of (4R, 6S)-54 on a 100 g scale bioreactor, in order to avoid signiﬁcant substrate inhibition. Thus, the ﬁnal synthesis of (4R, 6S)-54 productivity; in a second step, the process was further improved by using a fed-batch bioreactor, in in a total reaction time of 3 h with an ee of >99.9% and a 10-fold reduction in catalyst load over the on a 100 g scale in a total reaction time of 3 h with an ee of >99.9% and a 10-fold reduction in catalyst order to avoid significant substrate inhibition. Thus, the final synthesis of (4R, 6S)-54 on a 100 g scale previous method [240]. More recently, the use of whole cells systems is being evaluated for this load over the previous method [240]. More recently, the use of whole cells systems is being evaluated in a total reaction time of 3 h with an ee of >99.9% and a 10-fold reduction in catalyst load over the process [241,242], as well as new strategies for improving DERA by genetic engineering [243]. Finally, previous method [240]. More recently, the use of whole cells systems is being evaluated for this process [241,242], as well as new strategies for improving DERA by genetic engineering [243]. Finally, Catalysts 2019, 9, 260 19 of 32 for this process [241,242], as well as new strategies for improving DERA by genetic engineering [243]. Finally, a simple basic hydrolysis of lactone (4R, 6S)-54 leads to the trihydroxyacid (3R, 5S)-55, which is the precursor [244] of the lateral chain of superstatins. On the other hand, scientists from Lek Pharmaceutical (a Sandoz company) have described the use of whole cells of Escherichia coli BL21 (DE3) overexpressing the native E. coli deoC DERA gene for production of chiral lactols such as 28 [241], with 1 1 excellent volumetric productivity (up to 50 g L h ), >80% yield, and >80% chromatographic purity with titers reaching 100 g L . This process is highly cost effective and environmentally friendly, and its sustainability is even improved if the oxidation of 28 to (4R, 6S)-54 is also catalysed with an enzyme, as this same group has reported using PQQ-dependent glucose dehydrogenases [245]. Ohshima and co-workers described the sequential aldol reactions depicted in Figure 14 using DERA isolated from thermophilic organisms, describing a relatively lower activity compared with the enzyme from E. coli, although this fact was compensated by a better synthetic yield caused by the increased acetaldehyde resistance shown by the thermophilic enzyme [246]. Shen and co-workers reported higher conversions when chloroacetaldehyde was used as the acceptor substrate, as compared with acetaldehyde [243], and thus the development of new DERAs from different microorganisms is an open research area, as reported in recent revisions [247,248]. In any case, compared with other chemical protocols, most pharmaceutical processes are performed on a smaller scale, with the production volume of 1000 to 10,000 tons per year and product concentration ranging between 50 and 100 g/L; hence, the main drawback is the transfer of the biocatalytic process from laboratory to a larger scale, especially with respect to retention times, which are greater on a larger scale (compared with those in the laboratory). A good example of industrial scale-up has been described by Rucigaj ˇ and Krajnc [242], who used acetoxyacetaldehyde and acetaldehyde as substrates, which are presented in an aldol reaction catalyzed by a crude DERA expressing culture lysate. By optimizing addition regimes of both reactants into a reaction mixture, the corresponding lactol was produced at near 77 g/L. The complete process was designed in a practical and economical manner and could be used further on an industrial scale. Another industrial scale, low temperature process was developed by DSM, leading to a ﬁnal product concentration of 100 g L [249]. 4. Prognosis and Conclusions It is easily foreseen that because our diet habits are becoming progressively unhealthier, with an increased uptake of fats and abandoning the traditional “Mediterranean diet”, hypercholesterolemia and dyslipidaemia will be typical maladies in Western society. Thus, statins would be gradually more present in our lives, being a very important piece of the global pharmaceutical market, either as branded or generic drugs. This fact, combined with the plethora of other pharmacological activities, called pleiotropic effects, that are being ascribed to statins, as revised in Section 2, makes us predict an ever-growing market for this type of drug. Anyway, more detailed and careful studies are demanded in order to be sure about the real efﬁciency of pleiotropic therapeutic effects of statins, by clearly identifying those patients who could be the best ones for responding to the desired effect of statins, and by establishing the most effective dose, duration of use, and statin drug entity required. Besides, more accurate clinical trials have to be conducted in order to evaluate the real effect upon the desired target, by designing more effective and truthful biomarkers. For statins’ preparation, new and more sustainable protocols would be demanded; in this context, the substitution of chemical by biocatalyzed processes will certainly help to gain sustainability, because of the well-known green features of biocatalysis—synthetic routes conducted under mild reaction conditions; at ambient temperature; using water as reaction medium in many cases; and, last but not least, avoiding functional group activation and protection/deprotection steps usually required in traditional organic synthesis. Thus, we also foresee a growing increase in the use of biocatalysis and biotransformations for the preparation of statins, mainly promoted by the enhancement of biocatalysts’ performance through chemical modiﬁcation and genetic engineering. Catalysts 2019, 9, 260 20 of 32 In another context, very recently, a new type of drug has emerged for dealing with those patients already using statins, but not reaching low-density lipoprotein cholesterol levels, rather by genetic and environmental factors or by pathological states—known as statin-resistance [250,251]. These drugs are the inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9), a hepatic protease that becomes attached to low-density lipoproteins receptors (LDLRs), causing them to remain inside liposomes and get destroyed [252]. Nowadays, there are two PCSK9 inhibitors commercialized, both of them ® ® approved in 2015: alirocumab (Praluent , from Sanoﬁ) and evolucomab (Repatha , from Amgen), both of them are used not as monotherapy, but are rather combined with a low cholesterol diet as well as with statins at maximally tolerated doses [253]. These two drugs are monoclonal antibodies, and their high price hampers their prior authorization practices and reduces their long-term adherence, so that the search for small molecules active as PCSK9 inhibitors is a “Holy Grail” in medicinal chemistry [254]. This situation leads us to think that (a) the statin market is not going to decrease, because they are going to be complemented (not substituted) with new drugs; and (b) as most of the new small molecules tested as PCSK9 inhibitors contain stereocenters in their structures [254], surely biocatalysis would become a very useful tool to facilitate more sustainable synthetic routes for their preparation. Author Contributions: All authors (P.H., V.P., and A.R.A.) contributed equally in the preparation of this manuscript. Funding: This research was partially funded by the Spanish Ministerio de Economia, Industria y Competitividad (MINECO), Project CTQ2015-66206-C2-1-R. Acknowledgments: Authors thank Pablo Domínguez de María (CEO of Sustainable Momentum) for the critical reading of the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. World Health Organization. Raised Cholesterol. Situation and Trends. Available online: https://www.who. int/gho/ncd/risk_factors/cholesterol_text/en/ (accessed on 15 February 2019). 2. Stein, E.A. The power of statins: Aggressive lipid lowering. Clin. Cardiol. 2003, 26, 25–31. [CrossRef] 3. Bifulco, M.; Endo, A. Statin: New life for an old drug. Pharmacol. Res. 2014, 88, 1–2. [CrossRef] 4. Stossel, T.P. The discovery of statins. Cell 2008, 134, 903–905. [CrossRef] 5. Endo, A.; Kuroda, M.; Tsujita, Y. ML-236A, ML-236B, and ML-236C, new inhibitors of cholesterogenesis produced by Penicillium citrinum. J. Antibiot. 1976, 29, 1346–1348. [CrossRef] 6. Brown, A.G.; Smale, T.C.; King, T.J.; Hasenkamp, R.; Thompson, R.H. Crystal and molecular-structure of compactin, a new antifungal metabolite from Penicillium brevicompactum. J. Chem. Soc. Perkin Trans. 1 1976, 1165–1173. [CrossRef] 7. Moore, R.N.; Bigam, G.; Chan, J.K.; Hogg, A.M.; Nakashima, T.T.; Vederas, J.C. Biosynthesis of the hypocholesterolemic agent mevinolin by Aspergillus terreus. Determination of the origin of carbon, hydrogen, and oxygen-atoms by C NMR and Mass Spectrometry. J. Am. Chem. Soc. 1985, 107, 3694–3701. [CrossRef] 8. Gunde-Cimerman, N.; Cimerman, A. Pleurotus fruiting bodies contain the inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase—Lovastatin. Exp. Mycol. 1995, 19, 1–6. [CrossRef] 9. Liu, J.; Zhang, J.; Shi, Y.; Grimsgaard, S.; Alraek, T.; Fonnebo, V. Chinese red yeast rice (Monascus purpureus) for primary hyperlipidemia: A meta-analysis of randomized controlled trials. Chin. Med. 2006, 1, 4. [CrossRef] 10. Mol, M.; Erkelens, D.W.; Leuven, J.A.G.; Schouten, J.A.; Stalenhoef, A.F.H. Simvastatin (MK-733)—A potent cholesterol-synthesis inhibitor in heterozygous familial hypercholesterolemia. Atherosclerosis 1988, 69, 131–137. [CrossRef] 11. Yoshino, G.; Kazumi, T.; Kasama, T.; Iwatani, I.; Iwai, M.; Inui, A.; Otsuki, M.; Baba, S. Effect of CS-514, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on lipoprotein and apolipoprotein in plasma of hypercholesterolemic diabetics. Diabetes Res. Clin. Pract. 1986, 2, 179–181. [CrossRef] 12. Li, J.J. Triumph of the Heart: The Story of Statins; Oxford University Press: New York, NY, USA, 2009. 13. Casar, Z. Historic Overview and Recent Advances in the Synthesis of Super-statins. Curr. Org. Chem. 2010, 14, 816–845. [CrossRef] Catalysts 2019, 9, 260 21 of 32 14. Lindsley, C.W. The top prescription drugs of 2010 in the United States: Antipsychotics show strong growth. ACS Chem. Neurosci. 2011, 2, 276–277. [CrossRef] [PubMed] 15. Lindsley, C.W. The top prescription drugs of 2011 in the United States: Antipsychotics and antidepressants once again lead CNS therapeutics. ACS Chem. Neurosci. 2012, 3, 630–631. [CrossRef] 16. Lindsley, C.W. 2012 Trends and statistics for prescription medications in the United States: CNS therapeutics continue to hold leading positions. ACS Chem. Neurosci. 2013, 4, 1133–1135. [CrossRef] [PubMed] 17. Lindsley, C.W. The top prescription drugs of 2012 globally: Biologics dominate, but small molecule CNS drugs hold on to top spots. ACS Chem. Neurosci. 2013, 4, 905–907. [CrossRef] [PubMed] 18. Lindsley, C.W. 2013 Trends and statistics for prescription medications in the United States: CNS highest ranked and record number of prescriptions dispensed. ACS Chem. Neurosci. 2015, 6, 356–357. [CrossRef] [PubMed] 19. Aitken, M.; Kleinrock, M.; Lyle, J.; Nass, D.; Caskey, L. Medicines Use and Spending Shifts: A Review of the Use of Medicines in the U.S. in 2014; IMS Institute for Healthcare Informatics: Parsippany, NJ, USA, 2015. 20. Lindsley, C.W. 2014 Prescription medications in the United States: Tremendous growth, specialty/orphan drug expansion, and dispensed prescriptions continue to increase. ACS Chem. Neurosci. 2015, 6, 811–812. [CrossRef] [PubMed] 21. Lindsley, C.W. 2014 Global prescription medication statistics: Strong growth and CNS well represented. ACS Chem. Neurosci. 2015, 6, 505–506. [CrossRef] [PubMed] 22. Xie, X.K.; Watanabe, K.; Wojcicki, W.A.; Wang, C.C.C.; Tang, Y. Biosynthesis of lovastatin analogs with a broadly speciﬁc acyltransferase. Chem. Biol. 2006, 13, 1161–1169. [CrossRef] 23. FiercePharma. The Cardiovascular Scene to Shift by 2024 as Next-Generation Drugs like Eliquis and Xarelto Eclipse Stalwarts. Available online: https://www.ﬁercepharma.com/pharma/cardiovascular-landscape- from-2017-to-2024-featuring-eliquis-xarelto-and-more (accessed on 14 February 2019). 24. Watanabe, M.; Koike, H.; Ishiba, T.; Okada, T.; Seo, S.; Hirai, K. Synthesis and biological activity of methanesulfonamide pyrimidine- and N-methanesulfonyl pyrrole-substituted 3,5-dihydroxy-6-heptenoates, a novel series of HMG-CoA reductase inhibitors. Bioorg. Med. Chem. 1997, 5, 437–444. [CrossRef] 25. Demasi, M. Statin wars: Have we been misled about the evidence? A narrative review. Br. J. Sports Med. 2018, 52, 905–909. [CrossRef] [PubMed] 26. Ferri, N.; Corsini, A. Clinical evidence of statin therapy in non-dyslipidemic disorders. Pharmacol. Res. 2014, 88, 20–30. [CrossRef] [PubMed] 27. Wang, C.Y.; Liu, P.Y.; Liao, J.K. Pleiotropic effects of statin therapy: Molecular mechanisms and clinical results. Trends Mol. Med. 2008, 14, 37–44. [CrossRef] [PubMed] 28. Cordle, A.; Koenigsknecht-Talboo, J.; Wilkinson, B.; Limpert, A.; Landreth, G. Mechanisms of statin-mediated inhibition of small G-protein function. J. Biol. Chem. 2005, 280, 34202–34209. [CrossRef] [PubMed] 29. Liao, J.K.; Laufs, U. Pleiotropic effects of statins. Annu. Rev. Pharmacol. Toxicol. 2005, 45, 89–118. [CrossRef] [PubMed] 30. Malﬁtano, A.M.; Marasco, G.; Proto, M.C.; Laezza, C.; Gazzerro, P.; Bifulco, M. Statins in neurological disorders: An overview and update. Pharmacol. Res. 2014, 88, 74–83. [CrossRef] [PubMed] 31. John, S.; Delles, C.; Jacobi, J.; Schlaich, M.P.; Schneider, M.; Schmitz, G.; Schmieder, R.E. Rapid improvement of nitric oxide bioavailability after lipid-lowering therapy with cerivastatin within two weeks. J. Am. Coll. Cardiol. 2001, 37, 1351–1358. [CrossRef] 32. Cheng, W.H.; Ho, W.Y.; Chang, C.F.; Lu, P.J.; Cheng, P.W.; Yeh, T.C.; Hong, L.Z.; Sun, G.C.; Hsiao, M.; Tseng, C.J. Simvastatin induces a central hypotensive effect via Ras-mediated signalling to cause eNOS up-regulation. Br. J. Pharmacol. 2013, 170, 847–858. [CrossRef] [PubMed] 33. Mason, R.P.; Walter, M.F.; Jacob, R.F. Effects of HMG-CoA reductase inhibitors on endothelial function—Role of microdomains and oxidative stress. Circulation 2004, 109, 34–41. [CrossRef] [PubMed] 34. Mihos, C.G.; Salas, M.J.; Santana, O. The pleiotropic effects of the hydroxy-methyl-glutaryl-CoA reductase inhibitors in cardiovascular disease a comprehensive review. Cardiol. Rev. 2010, 18, 298–304. [CrossRef] [PubMed] 35. Yanuck, D.; Mihos, C.G.; Santana, O. Mechanisms and clinical evidence of the pleiotropic effects of the Hydroxy-Methyl-Glutaryl-CoA Reductase inhibitors in Central Nervous System disorders: A comprehensive review. Int. J. Neurosci. 2012, 122, 619–629. [CrossRef] Catalysts 2019, 9, 260 22 of 32 36. Mihos, C.G.; Artola, R.T.; Santana, O. The pleiotropic effects of the hydroxy-methyl-glutaryl-CoA reductase inhibitors in rheumatologic disorders: A comprehensive review. Rheumatol. Int. 2012, 32, 287–294. [CrossRef] 37. Barone, E.; Di Domenico, F.; Butterﬁeld, D.A. Statins more than cholesterol lowering agents in Alzheimer disease: Their pleiotropic functions as potential therapeutic targets. Biochem. Pharmacol. 2014, 88, 605–616. [CrossRef] [PubMed] 38. Mihos, C.G.; Pineda, A.M.; Santana, O. Cardiovascular effects of statins, beyond lipid-lowering properties. Pharmacol. Res. 2014, 88, 12–19. [CrossRef] 39. Oesterle, A.; Laufs, U.; Liao, J.K. Pleiotropic effects of statins on the cardiovascular system. Circ. Res. 2017, 120, 229–243. [CrossRef] 40. Cohen, M.V.; Yang, X.M.; Downey, J.M. Nitric oxide is a preconditioning mimetic and cardioprotectant and is the basis of many available infarct-sparing strategies. Cardiovasc. Res. 2006, 70, 231–239. [CrossRef] 41. Mills, E.J.; Wu, P.; Chong, G.; Ghement, I.; Singh, S.; Akl, E.A.; Eyawo, O.; Guyatt, G.; Berwanger, O.; Briel, M. Efﬁcacy and safety of statin treatment for cardiovascular disease: A network meta-analysis of 170 255 patients from 76 randomized trials. QJM-Int. J. Med. 2011, 104, 109–124. [CrossRef] [PubMed] 42. Teng, M.; Lin, L.; Zhao, Y.J.; Khoo, A.L.; Davis, B.R.; Yong, Q.W.; Yeo, T.C.; Lim, B.P. Statins for primary prevention of cardiovascular disease in elderly patients: Systematic review and meta-analysis. Drugs Aging 2015, 32, 649–661. [CrossRef] 43. Tervonen, T.; Naci, H.; van Valkenhoef, G.; Ades, A.E.; Angelis, A.; Hillege, H.L.; Postmus, D. Applying multiple criteria decision analysis to comparative beneﬁt-risk assessment: Choosing among statins in primary prevention. Med. Decis. Mak. 2015, 35, 859–871. [CrossRef] 44. Ulivieri, C.; Baldari, C.T. Statins: From cholesterol-lowering drugs to novel immunomodulators for the treatment of Th17-mediated autoimmune diseases. Pharmacol. Res. 2014, 88, 41–52. [CrossRef] 45. Scheele, J.S.; Marks, R.E.; Boss, G.R. Signaling by small GTPases in the immune system. Immunol. Rev. 2007, 218, 92–101. [CrossRef] 46. Greenwood, J.; Steinman, L.; Zamvil, S.S. Statin therapy and autoimmune disease: From protein prenylation to immunomodulation. Nat. Rev. Immunol. 2006, 6, 358–370. [CrossRef] 47. Chow, S.C. Immunomodulation by statins: Mechanisms and potential impact on autoimmune diseases. Arch. Immunol. Ther. Exp. (Warsz.) 2009, 57, 243–251. [CrossRef] [PubMed] 48. Khattri, S.; Zandman-Goddard, G. Statins and autoimmunity. Immunol. Res. 2013, 56, 348–357. [CrossRef] 49. Ciurleo, R.; Bramanti, P.; Marino, S. Role of statins in the treatment of multiple sclerosis. Pharmacol. Res. 2014, 87, 133–143. [CrossRef] 50. Pihl-Jensen, G.; Tsakiri, A.; Frederiksen, J.L. Statin treatment in multiple sclerosis: A systematic review and meta-analysis. CNS Drugs 2015, 29, 277–291. [CrossRef] [PubMed] 51. Soubrier, M.; Mathieu, S.; Hermet, M.; Makarawiez, C.; Bruckert, E. Do all lupus patients need statins? Jt. Bone Spine 2013, 80, 244–249. [CrossRef] [PubMed] 52. Yu, H.-H.; Chen, P.-C.; Yang, Y.-H.; Wang, L.-C.; Lee, J.-H.; Lin, Y.-T.; Chiang, B.-L. Statin reduces mortality and morbidity in systemic lupus erythematosus patients with hyperlipidemia: A nationwide population-based cohort study. Atherosclerosis 2015, 243, 11–18. [CrossRef] 53. Ruiz-Limon, P.; Barbarroja, N.; Perez-Sanchez, C.; Aguirre, M.A.; Bertolaccini, M.L.; Khamashta, M.A.; Rodriguez-Ariza, A.; Almaden, Y.; Segui, P.; Khraiwesh, H.; et al. Atherosclerosis and cardiovascular disease in systemic lupus erythematosus: Effects of in vivo statin treatment. Ann. Rheum. Dis. 2015, 74, 1450–1458. [CrossRef] [PubMed] 54. Liu, X.; Li, B.; Wang, W.; Zhang, C.; Zhang, M.; Zhang, Y.; Xia, Y.; Dong, Z.; Guo, Y.; An, F. Effects of HMG-CoA Reductase inhibitor on experimental autoimmune myocarditis. Cardiovasc. Drugs Ther. 2012, 26, 121–130. [CrossRef] 55. Lazzerini, P.E.; Capecchi, P.L.; Laghi-Pasini, F. Statins as a new therapeutic perspective in myocarditis and postmyocarditis dilated cardiomyopathy. Cardiovasc. Drugs Ther. 2013, 27, 365–369. [CrossRef] 56. Tajiri, K.; Shimojo, N.; Sakai, S.; Machino-Ohtsuka, T.; Imanaka-Yoshida, K.; Hiroe, M.; Tsujimura, Y.; Kimura, T.; Sato, A.; Yasutomi, Y.; et al. Pitavastatin regulates helper T-cell differentiation and ameliorates autoimmune myocarditis in mice. Cardiovasc. Drugs Ther. 2013, 27, 413–424. [CrossRef] 57. Lv, S.; Liu, Y.; Zou, Z.; Li, F.; Zhao, S.; Shi, R.; Bian, R.; Tian, H. The impact of statins therapy on disease activity and inﬂammatory factor in patients with rheumatoid arthritis: A meta-analysis. Clin. Exp. Rheumatol. 2015, 33, 69–76. Catalysts 2019, 9, 260 23 of 32 58. Tascilar, K.; Dell’Aniello, S.; Hudson, M.; Suissa, S. Statins and risk of rheumatoid arthritis: A nested case-control study. Arthritis Rheumatol. 2016, 68, 2603–2611. [CrossRef] 59. de Jong, H.J.I.; Tervaert, J.W.C.; Lalmohamed, A.; de Vries, F.; Vandebriel, R.J.; van Loveren, H.; Klungel, O.H.; van Staa, T.P. Pattern of risks of rheumatoid arthritis among patients using statins: A cohort study with the clinical practice research datalink. PLoS ONE 2018, 13, 1–17. [CrossRef] 60. McFarland, A.J.; Anoopkumar-Dukie, S.; Arora, D.S.; Grant, G.D.; McDermott, C.M.; Perkins, A.V.; Davey, A.K. Molecular mechanisms underlying the effects of statins in the central nervous system. Int. J. Mol. Sci. 2014, 15, 20607–20637. [CrossRef] 61. Qizilbash, N.; Lewington, S.; Duffy, S.; Peto, R.; Smith, T.; Spiegelhalter, D.; Iso, H.; Shimamoto, T.; Komachi, Y.; Iida, M.; et al. Cholesterol, diastolic blood pressure, and stroke: 13000 strokes in 450000 people in 45 prospective cohorts. Lancet 1995, 346, 1647–1653. 62. O’Brien, E.C.; Greiner, M.A.; Xian, Y.; Fonarow, G.C.; Olson, D.M.; Schwamm, L.H.; Bhatt, D.L.; Smith, E.E.; Maisch, L.; Hannah, D.; et al. Clinical effectiveness of statin therapy after ischemic stroke: Primary results from the statin therapeutic area of the Patient-centered Research into Outcomes Stroke Patients prefer and Effectiveness Research (PROSPER) study. Circulation 2015, 132, 1404–1413. [CrossRef] 63. Naci, H.; Brugts, J.J.; Fleurence, R.; Ades, A.E. Comparative effects of statins on major cerebrovascular events: A multiple-treatments meta-analysis of placebo-controlled and active-comparator trials. QJM-Int. J. Med. 2013, 106, 299–306. [CrossRef] 64. Markel, A. Statins and peripheral arterial disease. Int. Angiol. 2015, 34, 416–427. 65. Colivicchi, F.; Bassi, A.; Santini, M.; Caltagirone, C. Discontinuation of statin therapy and clinical outcome after ischemic stroke. Stroke 2007, 38, 2652–2657. [CrossRef] 66. Laloux, P. Risk and beneﬁt of statins in stroke secondary prevention. Curr. Vasc. Pharmacol. 2013, 11, 812–816. [CrossRef] 67. Song, B.; Wang, Y.L.; Zhao, X.Q.; Liu, L.P.; Wang, C.X.; Wang, A.X.; Du, W.L.; Wang, Y.J. Association between statin use and short-term outcome based on severity of ischemic stroke: A cohort study. PLoS ONE 2014, 9, 7. [CrossRef] 68. Gutierrez-Vargas, J.; Cespedes-Rubio, A.; Cardona-Gomez, G. Perspective of synaptic protection after post-infarction treatment with statins. J. Transl. Med. 2015, 13, 118. [CrossRef] 69. Bustamante, A.; Montaner, J. Statin therapy should not be discontinued in patients with intracerebral hemorrhage. Stroke 2013, 44, 2060–2061. [CrossRef] 70. Molina, C.A.; Selim, M.H. Continued statin treatment after acute intracranial hemorrhage ﬁghting ﬁre with ﬁre. Stroke 2013, 44, 2062–2063. [CrossRef] 71. Pan, Y.S.; Jing, J.; Wang, Y.L.; Zhao, X.Q.; Song, B.; Wang, W.J.; Wang, D.; Liu, G.F.; Liu, L.P.; Wang, C.X.; et al. Use of statin during hospitalization improves the outcome after intracerebral hemorrhage. CNS Neurosci. Ther. 2014, 20, 548–555. [CrossRef] 72. Anand, R.; Gill, K.D.; Mahdi, A.A. Therapeutics of Alzheimer ’s disease: Past, present and future. Neuropharmacology 2014, 76, 27–50. [CrossRef] 73. Silva, T.; Teixeira, J.; Remiao, F.; Borges, F. Alzheimer ’s disease, cholesterol, and statins: The junctions of important metabolic pathways. Angew. Chem. Int. Ed. 2013, 52, 1110–1121. [CrossRef] 74. Hottman, D.A.; Li, L. Protein prenylation and synaptic plasticity: Implications for Alzheimer ’s disease. Mol. Neurobiol. 2014, 50, 177–185. [CrossRef] 75. Mendoza-Oliva, A.; Zepeda, A.; Arias, C. The complex actions of statins in brain and their relevance for Alzheimer ’s disease treatment: An analytical review. Curr. Alzheimer Res. 2014, 11, 817–833. [CrossRef] [PubMed] 76. Wanamaker, B.L.; Swiger, K.J.; Blumenthal, R.S.; Martin, S.S. Cholesterol, statins, and dementia: What the cardiologist should know. Clin. Cardiol. 2015, 38, 243–250. [CrossRef] [PubMed] 77. Buxbaum, J.D.; Cullen, E.I.; Friedhoff, L.T. Pharmacological concentrations of the HMG-CoA reductase inhibitor lovastatin decrease the formation of the Alzheimer beta-amyloid peptide in vitro and in patients. Front. Biosci. 2002, 7, A50–A59. [CrossRef] [PubMed] 78. Li, L.; Zhang, W.; Cheng, S.W.; Cao, D.F.; Parent, M. Isoprenoids and related pharmacological interventions: Potential application in Alzheimer ’s disease. Mol. Neurobiol. 2012, 46, 64–77. [CrossRef] [PubMed] 79. Cibickova, L.; Palicka, V.; Cibicek, N.; Cermakova, E.; Micuda, S.; Bartosova, L.; Jun, D. Differential effects of statins and alendronate on cholinesterases in serum and brain of rats. Physiol. Res. 2007, 56, 765–770. Catalysts 2019, 9, 260 24 of 32 80. Mozayan, M.; Lee, T.J.F. Statins prevent cholinesterase inhibitor blockade of sympathetic alpha 7-nAChR-mediated currents in rat superior cervical ganglion neurons. Am. J. Physiol.-Heart Circul. Physiol. 2007, 293, H1737–H1744. [CrossRef] [PubMed] 81. Ghodke, R.M.; Tour, N.; Devi, K. Effects of statins and cholesterol on memory functions in mice. Metab. Brain Dis. 2012, 27, 443–451. [CrossRef] [PubMed] 82. Macan, M.; Vuksic, A.; Zunec, S.; Konjevoda, P.; Lovric, J.; Kelava, M.; Stambuk, N.; Vrkic, N.; Bradamante, V. Effects of simvastatin on malondialdehyde level and esterase activity in plasma and tissue of normolipidemic rats. Pharmacol. Rep. 2015, 67, 907–913. [CrossRef] [PubMed] 83. Bosel, J.; Gandor, F.; Harms, C.; Synowitz, M.; Harms, U.; Djoufack, P.C.; Megow, D.; Dirnagl, U.; Hortnagl, H.; Fink, K.B.; et al. Neuroprotective effects of atorvastatin against glutamate-induced excitotoxicity in primary cortical neurones. J. Neurochem. 2005, 92, 1386–1398. [CrossRef] [PubMed] 84. Krisanova, N.; Sivko, R.; Kasatkina, L.; Borisova, T. Neuroprotection by lowering cholesterol: A decrease in membrane cholesterol content reduces transporter-mediated glutamate release from brain nerve terminals. Biochim. Biophys. Acta-Mol. Basis Dis. 2012, 1822, 1553–1561. [CrossRef] 85. Sala, S.G.; Munoz, U.; Bartolome, F.; Bermejo, F.; Martin-Requero, A. HMG-CoA reductase inhibitor simvastatin inhibits cell cycle progression at the G(1)/S checkpoint in immortalized lymphocytes from Alzheimer ’s disease patients independently of cholesterol-lowering effects. J. Pharmacol. Exp. Ther. 2008, 324, 352–359. [CrossRef] [PubMed] 86. Merla, R.; Ye, Y.; Lin, Y.; Manickavasagam, S.; Huang, M.-H.; Perez-Polo, R.J.; Uretsky, B.F.; Birnbaum, Y. The central role of adenosine in statin-induced ERK1/2, Akt, and eNOS phosphorylation. Am. J. Physiol.-Heart Circul. Physiol. 2007, 293, H1918–H1928. [CrossRef] [PubMed] 87. Cordle, A.; Landreth, G. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors attenuate beta-amyloid-induced microglial inﬂammatory responses. J. Neurosci. 2005, 25, 299–307. [CrossRef] 88. Fonseca, A.C.R.G.; Resende, R.; Oliveira, C.R.; Pereira, C.M.F. Cholesterol and statins in Alzheimer ’s disease: Current controversies. Exp. Neurol. 2010, 223, 282–293. [CrossRef] [PubMed] 89. Mans, R.A.; McMahon, L.L.; Li, L. Simvastatin-mediated enhancement of long-term potentiation is driven by farnesyl-pyrophosphate depletion and inhibition of farnesylation. Neuroscience 2012, 202, 1–9. [CrossRef] 90. Mans, R.A.; Chowdhury, N.; Cao, D.; McMahon, L.L.; Li, L. Simvastatin enhances hippocampal long-term potentiation in C57BL/6 mice. Neuroscience 2010, 166, 435–444. [CrossRef] 91. Parent, M.; Hottman, D.A.; Cheng, S.W.; Zhang, W.; McMahon, L.L.; Yuan, L.L.; Li, L. Simvastatin treatment enhances NMDAR-mediated synaptic transmission by upregulating the surface distribution of the GluN2B subunit. Cell. Mol. Neurobiol. 2014, 34, 693–705. [CrossRef] 92. Wollmuth, L.P. Is cholesterol good or bad for your brain?—NMDARs have a say. J. Physiol.-Lond. 2015, 593, 2251–2252. [CrossRef] [PubMed] 93. Roy, A.; Jana, M.; Kundu, M.; Corbett, G.T.; Rangaswamy, S.B.; Mishra, R.K.; Luan, C.H.; Gonzalez, F.J.; Pahan, K. HMG-CoA Reductase Inhibitors bind to PPAR alpha to upregulate neurotrophin expression in the brain and improve memory in mice. Cell Metab. 2015, 22, 253–265. [CrossRef] [PubMed] 94. Jeong, J.H.; Yum, K.S.; Chang, J.Y.; Kim, M.; Ahn, J.Y.; Kim, S.; Lapchak, P.A.; Han, M.K. Dose-speciﬁc effect of simvastatin on hypoxia-induced HIF-1 alpha and BACE expression in Alzheimer ’s disease cybrid cells. BMC Neurol. 2015, 15, 7. [CrossRef] 95. Power, M.C.; Weuve, J.; Sharrett, A.R.; Blacker, D.; Gottesman, R.F. Statins, cognition, and dementia-systematic review and methodological commentary. Nat. Rev. Neurol. 2015, 11, 220–229. [CrossRef] [PubMed] 96. Strom, B.L.; Schinnar, R.; Karlawish, J.; Hennessy, S.; Teal, V.; Bilker, W.B. Statin therapy and risk of acute memory impairment. JAMA Intern. Med. 2015, 175, 1399–1405. [CrossRef] [PubMed] 97. Mendoza-Oliva, A.; Ferrera, P.; Fragoso-Medina, J.; Arias, C. Lovastatin differentially affects neuronal cholesterol and amyloid- production invivo and invitro. CNS Neurosci. Ther. 2015, 21, 631–641. [CrossRef] [PubMed] 98. Chuang, C.S.; Lin, C.L.; Lin, M.C.; Sung, F.C.; Kao, C.H. Decreased prevalence of dementia associated with statins: A national population-based study. Eur. J. Neurol. 2015, 22, 912–918. [CrossRef] [PubMed] 99. Phani, S.; Loike, J.D.; Przedborski, S. Neurodegeneration and inﬂammation in Parkinson’s disease. Parkinsonism Relat. Disord. 2012, 18 (Suppl. S1), S207–S209. [CrossRef] Catalysts 2019, 9, 260 25 of 32 100. Wolozin, B.; Kellman, W.; Ruosseau, P.; Celesia, G.G.; Siegel, G. Decreased prevalence of Alzheimer disease associated with 3-hydroxy-3-methyglutaryl coenzyme A reductase inhibitors. Arch. Neurol. 2000, 57, 1439–1443. [CrossRef] 101. Lee, Y.-C.; Lin, C.-H.; Wu, R.-M.; Lin, M.-S.; Lin, J.-W.; Chang, C.-H.; Lai, M.-S. Discontinuation of statin therapy associates with Parkinson disease. A population-based study. Neurology 2013, 81, 410–416. [CrossRef] 102. Tison, F.; Negre-Pages, L.; Meissner, W.G.; Dupouy, S.; Li, Q.; Thiolat, M.-L.; Thiollier, T.; Galitzky, M.; Ory-Magne, F.; Milhet, A.; et al. Simvastatin decreases levodopa-induced dyskinesia in monkeys, but not in a randomized, placebo-controlled, multiple cross-over (“n-of-1”) exploratory trial of simvastatin against levodopa-induced dyskinesia in Parkinson’s disease patients. Parkinsonism Relat. Disord. 2013, 19, 416–421. [CrossRef] 103. Selley, M.L. Simvastatin prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced striatal dopamine depletion and protein tyrosine nitration in mice. Brain Res. 2005, 1037, 1–6. [CrossRef] 104. Xu, Y.Q.; Long, L.; Yan, J.Q.; Wei, L.; Pan, M.Q.; Gao, H.M.; Zhou, P.; Liu, M.; Zhu, C.S.; Tang, B.S.; et al. Simvastatin induces neuroprotection in 6-OHDA-lesioned PC12 via the PI3K/AKT/caspase 3 pathway and anti-inﬂammatory responses. CNS Neurosci. Ther. 2013, 19, 170–177. [CrossRef] 105. Yan, J.; Xu, Y.; Zhu, C.; Zhang, L.; Wu, A.; Yang, Y.; Xiong, Z.; Deng, C.; Huang, X.-F.; Yenari, M.A.; et al. Simvastatin prevents dopaminergic neurodegeneration in experimental parkinsonian models: The association with anti-inﬂammatory responses. PLoS ONE 2011, 6, e20945. [CrossRef] [PubMed] 106. Wang, T.; Cao, X.B.; Zhang, T.; Shi, Q.Q.; Chen, Z.B.; Tang, B.S. Effect of simvastatin on L-DOPA-induced abnormal involuntary movements of hemiparkinsonian rats. Neurol. Sci. 2015, 36, 1397–1402. [CrossRef] [PubMed] 107. Ghosh, A.; Roy, A.; Matras, J.; Brahmachari, S.; Gendelman, H.E.; Pahan, K. Simvastatin inhibits the activation of p21(Ras) and prevents the loss of dopaminergic neurons in a mouse model of Parkinson’s disease. J. Neurosci. 2009, 29, 13543–13556. [CrossRef] [PubMed] 108. Yang, C.C.; Jick, S.S.; Jick, H. Lipid-lowering drugs and the risk of depression and suicidal behavior. Arch. Intern. Med. 2003, 163, 1926–1932. [CrossRef] 109. Feng, L.; Tan, C.-H.; Merchant, R.A.; Ng, T.-P. Association between depressive symptoms and use of HMG-CoA reductase inhibitors (statins), corticosteroids and histamine H-2 receptor antagonists in community-dwelling older persons—Cross-sectional analysis of a population-based cohort. Drugs Aging 2008, 25, 795–805. [CrossRef] [PubMed] 110. Stafford, L.; Berk, M. The use of statins after a cardiac intervention is associated with reduced risk of subsequent depression: Proof of concept for the inﬂammatory and oxidative hypotheses of depression? J. Clin. Psychiatry 2011, 72, 1229–1235. [CrossRef] [PubMed] 111. Otte, C.; Zhao, S.; Whooley, M.A. Statin use and risk of depression in patients with coronary heart disease: Longitudinal data from the heart and soul study. J. Clin. Psychiatry 2012, 73, 610–615. [CrossRef] [PubMed] 112. Kim, J.-M.; Stewart, R.; Kang, H.-J.; Bae, K.-Y.; Kim, S.-W.; Shin, I.-S.; Kim, J.-T.; Park, M.-S.; Cho, K.-H.; Yoon, J.-S. A prospective study of statin use and poststroke depression. J. Clin. Psychopharmacol. 2014, 34, 72–79. [CrossRef] 113. Chuang, C.-S.; Yang, T.-Y.; Muo, C.-H.; Su, H.-L.; Sung, F.-C.; Kao, C.-H. Hyperlipidemia, statin use and the risk of developing depression: A nationwide retrospective cohort study. Gen. Hosp. Psych. 2014, 36, 497–501. [CrossRef] [PubMed] 114. Lin, P.-Y.; Chang, A.Y.W.; Lin, T.-K. Simvastatin treatment exerts antidepressant-like effect in rats exposed to chronic mild stress. Pharmacol. Biochem. Behav. 2014, 124, 174–179. [CrossRef] 115. ElBatsh, M.M. Antidepressant-like effect of simvastatin in diabetic rats. Can. J. Physiol. Pharmacol. 2015, 93, 649–656. [CrossRef] 116. Abbasi, S.H.; Mohammadinejad, P.; Shahmansouri, N.; Salehiomran, A.; Beglar, A.A.; Zeinoddini, A.; Forghani, S.; Akhondzadeh, S. Simvastatin versus atorvastatin for improving mild to moderate depression in post-coronary artery bypass graft patients: A double-blind, placebo-controlled, randomized trial. J. Affect. Disord. 2015, 183, 149–155. [CrossRef] 117. Gougol, A.; Zareh-Mohammadi, N.; Raheb, S.; Farokhnia, M.; Salimi, S.; Iranpour, N.; Yekehtaz, H.; Akhondzadeh, S. Simvastatin as an adjuvant therapy to ﬂuoxetine in patients with moderate to severe major depression: A double-blind placebo-controlled trial. J. Psychopharmacol. 2015, 29, 575–581. [CrossRef] [PubMed] Catalysts 2019, 9, 260 26 of 32 118. Agostini, J.V.; Tinetti, M.E.; Han, L.; McAvay, G.; Foody, J.M.; Concato, J. Effects of statin use on muscle strength, cognition, and depressive symptoms in older adults. J. Am. Geriatr. Soc. 2007, 55, 420–425. [CrossRef] [PubMed] 119. Feng, L.; Yap, K.B.; Kua, E.H.; Ng, T.P. Statin use and depressive symptoms in a prospective study of community-living older persons. Pharmacoepidemiol. Drug Saf. 2010, 19, 942–948. [CrossRef] 120. Al Badarin, F.J.; Spertus, J.A.; Gosch, K.L.; Buchanan, D.M.; Chan, P.S. Initiation of statin therapy after acute myocardial infarction is not associated with worsening depressive symptoms: Insights from the Prospective Registry Evaluating Outcomes After Myocardial Infarctions: Events and Recovery (PREMIER) and Translational Research Investigating Underlying Disparities in Acute Myocardial Infarction Patients’ Health Status (TRIUMPH) registries. Am. Heart J. 2013, 166, 879–886. [PubMed] 121. Ludka, F.K.; Zomkowski, A.D.E.; Cunha, M.P.; Dal-Cim, T.; Zeni, A.L.B.; Rodrigues, A.L.S.; Tasca, C.I. Acute atorvastatin treatment exerts antidepressant-like effect in mice via the L-arginine-nitric oxide-cyclic guanosine monophosphate pathway and increases BDNF levels. Eur. Neuropsychopharmacol. 2013, 23, 400–412. [CrossRef] [PubMed] 122. Shahsavarian, A.; Javadi, S.; Jahanabadi, S.; Khoshnoodi, M.; Shamsaee, J.; Shafaroodi, H.; Mehr, S.E.; Dehpour, A. Antidepressant-like effect of atorvastatin in the forced swimming test in mice: The role of PPAR-gamma receptor and nitric oxide pathway. Eur. J. Pharmacol. 2014, 745, 52–58. [CrossRef] 123. Citraro, R.; Chimirri, S.; Aiello, R.; Gallelli, L.; Trimboli, F.; Britti, D.; De Sarro, G.; Russo, E. Protective effects of some statins on epileptogenesis and depressive-like behavior in WAG/Rij rats, a genetic animal model of absence epilepsy. Epilepsia 2014, 55, 1284–1291. [CrossRef] 124. Etminan, M.; Samii, A.; Brophy, J.M. Statin use and risk of epilepsy A nested case-control study. Neurology 2010, 75, 1496–1500. [CrossRef] 125. Piermartiri, T.C.B.; Vandresen-Filho, S.; Herculano, B.d.A.; Martins, W.C.; Dal’Agnolo, D.; Stroeh, E.; Carqueja, C.L.; Boeck, C.R.; Tasca, C.I. Atorvastatin prevents hippocampal cell death due to quinolinic acid-induced seizures in mice by increasing Akt phosphorylation and glutamate uptake. Neurotox. Res. 2009, 16, 106–115. [CrossRef] [PubMed] 126. Ma, T.; Zhao, Y.; Kwak, Y.-D.; Yang, Z.; Thompson, R.; Luo, Z.; Xu, H.; Liao, F.-F. Statin’s excitoprotection is mediated by sAPP and the subsequent attenuation of calpain-induced truncation events, likely via Rho-ROCK signaling. J. Neurosci. 2009, 29, 11226–11236. [CrossRef] [PubMed] 127. Lee, J.-K.; Won, J.-S.; Singh, A.K.; Singh, I. Statin inhibits kainic acid-induced seizure and associated inﬂammation and hippocampal cell death. Neurosci. Lett. 2008, 440, 260–264. [CrossRef] [PubMed] 128. Siniscalchi, A.; Mintzer, S. Statins for poststroke seizures The ﬁrst antiepileptogenic agent? Neurology 2015, 85, 661–662. [CrossRef] [PubMed] 129. Ponce, J.; de la Ossa, N.P.; Hurtado, O.; Millan, M.; Arenillas, J.F.; Davalos, A.; Gasull, T. Simvastatin reduces the association of NMDA receptors to lipid rafts: A cholesterol-mediated effect in neuroprotection. Stroke 2008, 39, 1269–1275. [CrossRef] [PubMed] 130. Seker, F.B.; Kilic, U.; Caglayan, B.; Ethemoglu, M.S.; Caglayan, A.B.; Ekimci, N.; Demirci, S.; Dogan, A.; Oztezcan, S.; Sahin, F.; et al. HMG-CoA reductase inhibitor rosuvastatin improves abnormal brain electrical activity via mechanisms involving eNOs. Neuroscience 2015, 284, 349–359. [CrossRef] [PubMed] 131. Scicchitano, F.; Constanti, A.; Citraro, R.; De Sarro, G.; Russo, E. Statins and epilepsy: Preclinical studies, clinical trials and statin-anticonvulsant drug interactions. Curr. Drug Targets 2015, 16, 747–756. [CrossRef] 132. Sierra-Marcos, A.; Alvarez, V.; Faouzi, M.; Burnand, B.; Rossetti, A.O. Statins are associated with decreased mortality risk after status epilepticus. Eur. J. Neurol. 2015, 22, 402–405. [CrossRef] [PubMed] 133. Dale, K.M.; Coleman, C.I.; Henyan, N.N.; Kluger, J.; White, C.M. Statins and cancer risk—A meta-analysis. JAMA-J. Am. Med. Assoc. 2006, 295, 74–80. [CrossRef] [PubMed] 134. Caporaso, N.E. Statins and Cancer-Related Mortality—Let’s Work Together. N. Engl. J. Med. 2012, 367, 1848–1850. [CrossRef] [PubMed] 135. Osmak, M. Statins and cancer: Current and future prospects. Cancer Lett. 2012, 324, 1–12. [CrossRef] [PubMed] 136. Pisanti, S.; Picardi, P.; Ciaglia, E.; D’Alessandro, A.; Bifulco, M. Novel prospects of statins as therapeutic agents in cancer. Pharmacol. Res. 2014, 88, 84–98. [CrossRef] 137. Kubatka, P.; Kruzliak, P.; Rotrekl, V.; Jelinkova, S.; Mladosievicova, B. Statins in oncological research: From experimental studies to clinical practice. Crit. Rev. Oncol. Hematol. 2014, 92, 296–311. [CrossRef] Catalysts 2019, 9, 260 27 of 32 138. Matusewicz, L.; Meissner, J.; Toporkiewicz, M.; Sikorski, A.F. The effect of statins on cancer cells-review. Tumor Biol. 2015, 36, 4889–4904. [CrossRef] [PubMed] 139. Baigent, C.; Blackwell, L.; Emberson, J.; Holland, L.E.; Reith, C.; Bhala, N.; Peto, R.; Barnes, E.H.; Keech, A.; Simes, J.; et al. Efﬁcacy and safety of more intensive lowering of LDL cholesterol: A meta-analysis of data from 170000 participants in 26 randomised trials. Lancet 2010, 376, 1670–1681. [PubMed] 140. Mihaylova, B.; Emberson, J.; Blackwell, L.; Keech, A.; Simes, J.; Barnes, E.H.; Voysey, M.; Gray, A.; Collins, R.; Baigent, C.; et al. The effects of lowering LDL cholesterol with statin therapy in people at low risk of vascular disease: Meta-analysis of individual data from 27 randomised trials. Lancet 2012, 380, 581–590. [PubMed] 141. Shepherd, J.; Blauw, G.J.; Murphy, M.B.; Bollen, E.; Buckley, B.M.; Cobbe, S.M.; Ford, I.; Gaw, A.; Hyland, M.; Jukema, J.W.; et al. Pravastatin in elderly individuals at risk of vascular disease (PROSPER): A randomised controlled trial. Lancet 2002, 360, 1623–1630. [CrossRef] 142. Nielsen, S.F.; Nordestgaard, B.G.; Bojesen, S.E. Statin use and reduced cancer-related mortality. N. Engl. J. Med. 2012, 367, 1792–1802. [CrossRef] [PubMed] 143. Peng, Y.C.; Lin, C.L.; Hsu, W.Y.; Chang, C.S.; Yeh, H.Z.; Tung, C.F.; Wu, Y.L.; Sung, F.C.; Kao, C.H. Statins are associated with a reduced risk of cholangiocarcinoma: A population-based case-control study. Br. J. Clin. Pharmacol. 2015, 80, 755–761. [CrossRef] [PubMed] 144. Archibugi, L.; Maisonneuve, P.; Piciucchi, M.; Valente, R.; Delle Fave, G.; Capurso, G. Statins but not aspirin nor their combination have a chemopreventive effect on pancreatic cancer occurrence. Pancreas 2015, 44, 1359. 145. Sivaprasad, U.; Abbas, T.; Dutta, A. Differential efﬁcacy of 3-hydroxy-3-methylglutaryl CoA reductase inhibitors on the cell cycle of prostate cancer cells. Mol. Cancer Ther. 2006, 5, 2310–2316. [CrossRef] [PubMed] 146. Song, X.; Liu, B.-C.; Lu, X.-Y.; Yang, L.-L.; Zhai, Y.-J.; Eaton, A.F.; Thai, T.L.; Eaton, D.C.; Ma, H.-P.; Shen, B.-Z. Lovastatin inhibits human B lymphoma cell proliferation by reducing intracellular ROS and TRPC6 expression. Biochim. Biophys. Acta-Mol. Cell Res. 2014, 1843, 894–901. [CrossRef] [PubMed] 147. Tu, Y.-S.; Kang, X.-L.; Zhou, J.-G.; Lv, X.-F.; Tang, Y.-B.; Guan, Y.-Y. Involvement of Chk1-Cdc25A-cyclin A/CDk2 pathway in simvastatin induced S-phase cell cycle arrest and apoptosis in multiple myeloma cells. Eur. J. Pharmacol. 2011, 670, 356–364. [CrossRef] [PubMed] 148. Yu, X.; Luo, Y.; Zhou, Y.; Zhang, Q.; Wang, J.; Wei, N.; Mi, M.; Zhu, J.; Wang, B.; Chang, H.; et al. BRCA1 overexpression sensitizes cancer cells to lovastatin via regulation of cyclin D1-CDK4-p21(WAF1/CIP1) pathway: Analyses using a breast cancer cell line and tumoral xenograft model. Int. J. Oncol. 2008, 33, 555–563. [PubMed] 149. Rao, S.; Porter, D.C.; Chen, X.M.; Herliczek, T.; Lowe, M.; Keyomarsi, K. Lovastatin-mediated G(1) arrest is through inhibition of the proteasome, independent of hydroxymethyl glutaryl-CoA reductase. Proc. Natl. Acad. Sci. USA 1999, 96, 7797–7802. [CrossRef] [PubMed] 150. Horiguchi, A.; Sumitomo, M.; Asakuma, J.; Asano, T.; Asano, T.; Hayakawa, M. 3-Hydroxy-3-methylglutaryl- coenzyme A reductase inhibitor, ﬂuvastatin, as a novel agent for prophylaxis of renal cancer metastasis. Clin. Cancer Res. 2004, 10, 8648–8655. [CrossRef] 151. Denoyelle, C.; Vasse, M.; Korner, M.; Mishal, Z.; Ganne, F.; Vannier, J.P.; Soria, J.; Soria, C. Cerivastatin, an inhibitor of HMG-CoA reductase, inhibits the signaling pathways involved in the invasiveness and metastatic properties of highly invasive breast cancer cell lines: An in vitro study. Carcinogenesis 2001, 22, 1139–1148. [CrossRef] 152. Spampanato, C.; De Maria, S.; Sarnataro, M.; Giordano, E.; Zanfardino, M.; Baiano, S.; Carteni, M.; Morelli, F. Simvastatin inhibits cancer cell growth by inducing apoptosis correlated to activation of Bax and down-regulation of BCL-2 gene expression. Int. J. Oncol. 2012, 40, 935–941. [CrossRef] 153. Herrero-Martin, G.; Lopez-Rivas, A. Statins activate a mitochondria-operated pathway of apoptosis in breast tumor cells by a mechanism regulated by ErbB2 and dependent on the prenylation of proteins. FEBS Lett. 2008, 582, 2589–2594. [CrossRef] 154. Zhu, Y.; Casey, P.J.; Kumar, A.P.; Pervaiz, S. Deciphering the signaling networks underlying simvastatin-induced apoptosis in human cancer cells: Evidence for non-canonical activation of RhoA and Rac1 GTPases. Cell Death Dis. 2013, 4, e568. [CrossRef] 155. Miller, T.; Yang, F.; Wise, C.E.; Meng, F.; Priester, S.; Munshi, M.K.; Guerrier, M.; Dostal, D.E.; Glaser, S.S. Simvastatin stimulates apoptosis in cholangiocarcinoma by inhibition of Rac1 activity. Dig. Liver Dis. 2011, 43, 395–403. [CrossRef] [PubMed] Catalysts 2019, 9, 260 28 of 32 156. Simons, K.; Ehehalt, R. Cholesterol, lipid rafts, and disease. J. Clin. Investig. 2002, 110, 597–603. [CrossRef] [PubMed] 157. Gniadecki, R. Depletion of membrane cholesterol causes ligand-independent activation of Fas and apoptosis. Biochem. Biophys. Res. Commun. 2004, 320, 165–169. [CrossRef] [PubMed] 158. Zhuang, L.Y.; Kim, J.; Adam, R.M.; Solomon, K.R.; Freeman, M.R. Cholesterol targeting alters lipid raft composition and cell survival in prostate cancer cells and xenografts. J. Clin. Investig. 2005, 115, 959–968. [CrossRef] [PubMed] 159. Li, Y.C.; Park, M.J.; Ye, S.K.; Kim, C.W.; Kim, Y.N. Elevated levels of cholesterol-rich lipid rafts in cancer cells are correlated with apoptosis sensitivity induced by cholesterol-depleting agents. Am. J. Pathol. 2006, 168, 1107–1118. [CrossRef] [PubMed] 160. Boscher, C.; Nabi, I.R. CAVEOLIN-1: Role in Cell Signaling. In Caveolins and Caveolae: Roles in Signaling and Disease Mechanisms; Jasmin, J.F., Frank, P.G., Lisanti, M.P., Eds.; Springer: Berlin/Heidelberg, Germany, 2012; Volume 729, pp. 29–50. 161. Altwairgi, A.K. Statins are potential anticancerous agents (Review). Oncol. Rep. 2015, 33, 1019–1039. [CrossRef] [PubMed] 162. Barrios-Gonzalez, J.; Miranda, R.U. Biotechnological production and applications of statins. Appl. Microbiol. Biotechnol. 2010, 85, 869–883. [CrossRef] [PubMed] 163. Hoffman, W.F.; Alberts, A.W.; Anderson, P.S.; Chen, J.S.; Smith, R.L.; Willard, A.K. 3-hydroxy-3- methylglutaryl-coenzyme a reductase inhibitors. 4. Side-chain ester derivatives of mevinolin. J. Med. Chem. 1986, 29, 849–852. [CrossRef] [PubMed] 164. Askin, D.; Verhoeven, T.R.; Liu, T.M.H.; Shinkai, I. Synthesis of synvinolin—Extremely high conversion alkylation of an ester enolate. J. Org. Chem. 1991, 56, 4929–4932. [CrossRef] 165. Xie, X.K.; Tang, Y. Efﬁcient synthesis of simvastatin by use of whole-cell biocatalysis. Appl. Environ. Microbiol. 2007, 73, 2054–2060. [CrossRef] 166. Schimmel, T.G.; Borneman, W.S.; Conder, M.J. Puriﬁcation and characterization of a lovastatin esterase from Clonostachys compactiuscula. Appl. Environ. Microbiol. 1997, 63, 1307–1311. [PubMed] 167. Chen, L.C.; Lai, Y.K.; Wu, S.C.; Lin, C.C.; Guo, J.H. Production by Clonostachys compactiuscula of a lovastatin esterase that converts lovastatin to monacolin J. Enzyme Microb. Technol. 2006, 39, 1051–1059. [CrossRef] 168. Gao, X.; Xie, X.K.; Pashkov, I.; Sawaya, M.R.; Laidman, J.; Zhang, W.J.; Cacho, R.; Yeates, T.O.; Tang, Y. Directed Evolution and Structural Characterization of a Simvastatin Synthase. Chem. Biol. 2009, 16, 1064–1074. [CrossRef] 169. United States Environmental Protection Agency. Presidential Green Chemistry Challenge: 2012 Greener Synthetic Pathways Award. Available online: http://www2.epa.gov/green-chemistry/2012-greener- synthetic-pathways-award (accessed on 14 February 2019). 170. Xu, W.; Chooi, Y.H.; Choi, J.W.; Li, S.; Vederas, J.C.; Da Silva, N.A.; Tang, Y. LovG: The thioesterase required for dihydromonacolin L release and lovastatin nonaketide synthase turnover in lovastatin biosynthesis. Angew. Chem. Int. Ed. 2013, 52, 6472–6475. [CrossRef] [PubMed] 171. Butler, D.E.; Le, T.V.; Millar, A.; Nanninga, T.N. Process for the synthesis of (5R)-1,1-dimethylethyl-6-cyano-5- hydroxy-3-oxo-hexanoate. US Patent 5155251, 13 October 1992. 172. Turner, N.J.; O’Reilly, E. Biocatalytic retrosynthesis. Nat. Chem. Biol. 2013, 9, 285–288. [CrossRef] 173. Brady, D. Biocatalytic Hydrolysis of Nitriles. In Handbook of Green Chemistry; Wiley-VCH Verlag GmbH & Co. KGaA: Hoboken, NJ, USA, 2010; Volume 3, (Biocatalysis). 174. Faber, K. Biotransformations of non-natural compounds: State of the art and future development. Pure Appl. Chem. 1997, 69, 1613–1632. [CrossRef] 175. DeSantis, G.; Zhu, Z.L.; Greenberg, W.A.; Wong, K.V.; Chaplin, J.; Hanson, S.R.; Farwell, B.; Nicholson, L.W.; Rand, C.L.; Weiner, D.P.; et al. An enzyme library approach to biocatalysis: Development of nitrilases for enantioselective production of carboxylic acid derivatives. J. Am. Chem. Soc. 2002, 124, 9024–9025. [CrossRef] [PubMed] 176. DeSantis, G.; Wong, K.; Farwell, B.; Chatman, K.; Zhu, Z.L.; Tomlinson, G.; Huang, H.J.; Tan, X.Q.; Bibbs, L.; Chen, P.; et al. Creation of a productive, highly enantioselective nitrilase through gene site saturation mutagenesis (GSSM). J. Am. Chem. Soc. 2003, 125, 11476–11477. [CrossRef] [PubMed] Catalysts 2019, 9, 260 29 of 32 177. Bergeron, S.; Chaplin, D.A.; Edwards, J.H.; Ellis, B.S.W.; Hill, C.L.; Holt-Tifﬁn, K.; Knight, J.R.; Mahoney, T.; Osborne, A.P.; Ruecroft, G. Nitrilase-catalysed desymmetrisation of 3-hydroxyglutaronitrile: Preparation of a statin side-chain intermediate. Org. Process Res. Dev. 2006, 10, 661–665. [CrossRef] 178. Yao, P.; Li, J.; Yuan, J.; Han, C.; Liu, X.; Feng, J.; Wu, Q.; Zhu, D. Enzymatic synthesis of a key intermediate for rosuvastatin by nitrilase-catalyzed hydrolysis of ethyl (R)-4-cyano-3-hydroxybutyate at high substrate concentration. ChemCatChem 2015, 7, 271–275. [CrossRef] 179. Ma, S.K.; Gruber, J.; Davis, C.; Newman, L.; Gray, D.; Wang, A.; Grate, J.; Huisman, G.W.; Sheldon, R.A. A green-by-design biocatalytic process for atorvastatin intermediate. Green Chem. 2010, 12, 81–86. [CrossRef] 180. Chung, S.; Hwang, Y. Stereoselective hydrolysis of racemic ethyl 4-chloro-3-hydroxybutyrate by a lipase. Biocatal. Biotransform. 2008, 26, 327–330. [CrossRef] 181. Lee, S.H.; Park, O.J.; Uh, H.S. A chemoenzymatic approach to the synthesis of enantiomerically pure (S)-3-hydroxy-gamma-butyrolactone. Appl. Microbiol. Biotechnol. 2008, 79, 355–362. [CrossRef] [PubMed] 182. Patel, J.M. Biocatalytic synthesis of atorvastatin intermediates. J. Mol. Catal. B-Enzym. 2009, 61, 123–128. [CrossRef] 183. Martin, C.H.; Dhamankar, H.; Tseng, H.C.; Sheppard, M.J.; Reisch, C.R.; Prather, K.L.J. A platform pathway for production of 3-hydroxyacids provides a biosynthetic route to 3-hydroxy-gamma-butyrolactone. Nat. Commun. 2013, 4, 1414. [CrossRef] [PubMed] 184. Narasaka, K.; Pai, F.C. Stereoselective reduction of beta-hydroxyketones to 1,3-diols highly selective 1,3-asymmetric induction via boron chelates. Tetrahedron 1984, 40, 2233–2238. [CrossRef] 185. Chen, K.M.; Hardtmann, G.E.; Prasad, K.; Repic, O.; Shapiro, M.J. 1,3-syn diastereoselective reduction of beta-hydroxyketones utilizing alkoxydialkylboranes. Tetrahedron Lett. 1987, 28, 155–158. [CrossRef] 186. Sterk, D.; Casar, Z.; Jukic, M.; Kosmrlj, J. Concise and highly efﬁcient approach to three key pyrimidine precursors for rosuvastatin synthesis. Tetrahedron 2012, 68, 2155–2160. [CrossRef] 187. Ohrlein, R.; Baisch, G. Chemo-enzymatic approach to statin side-chain building blocks. Adv. Synth. Catal. 2003, 345, 713–715. [CrossRef] 188. Santaniello, E.; Chiari, M.; Ferraboschi, P.; Trave, S. Enhanced and reversed enantioselectivity of enzymatic-hydrolysis by simple substrate modiﬁcations—The case of 3-hydroxyglutarate diesters. J. Org. Chem. 1988, 53, 1567–1569. [CrossRef] 189. Metzner, R.; Hummel, W.; Wetterich, F.; Konig, B.; Groger, H. Integrated biocatalysis in multistep drug synthesis without intermediate isolation: A de novo approach toward a rosuvastatin key building block. Org. Process Res. Dev. 2015, 19, 635–638. [CrossRef] 190. König, B.; Wetterich, F.; Gröger, H.; Metzner, R. Process for enantioselective synthesis of 3-hydroxy-glutaric acid monoesters and use thereof. WO 2014/140006, 18 September 2014. 191. You, Z.Y.; Liu, Z.Q.; Zheng, Y.G. Chemical and enzymatic approaches to the synthesis of optically pure ethyl (R)-4-cyano-3-hydroxybutanoate. Appl. Microbiol. Biotechnol. 2014, 98, 11–21. [CrossRef] 192. Asako, H.; Shimizu, M.; Itoh, N. Biocatalytic production of (S)-4-bromo-3-hydroxybutyrate and structurally related chemicals and their applications. Appl. Microbiol. Biotechnol. 2009, 84, 397–405. [CrossRef] 193. Asako, H.; Shimizu, M.; Makino, Y.; Itoh, N. Biocatalytic reduction system for the production of chiral methyl (R)/(S)-4-bromo-3-hydroxybutyrate. Tetrahedron Lett. 2010, 51, 2664–2666. [CrossRef] 194. Patel, R.N.; McNamee, C.G.; Banerjee, A.; Howell, J.M.; Robison, R.S.; Szarka, L.J. Stereoselective reduction of beta-keto-esters by Geotrichum candidum. Enzyme Microb. Technol. 1992, 14, 731–738. [CrossRef] 195. Ye, Q.; Ouyang, P.K.; Ying, H.J. A review-biosynthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoate ester: Recent advances and future perspectives. Appl. Microbiol. Biotechnol. 2011, 89, 513–522. [CrossRef] 196. Yasohara, Y.; Kizaki, N.; Hasegawa, J.; Takahashi, S.; Wada, M.; Kataoka, M.; Shimizu, S. Synthesis of optically active ethyl 4-chloro-3-hydroxybutanoate by microbial reduction. Appl. Microbiol. Biotechnol. 1999, 51, 847–851. [CrossRef] [PubMed] 197. Kita, K.; Kataoka, M.; Shimizu, S. Diversity of 4-chloroacetoacetate ethyl ester-reducing enzymes in yeasts and their application to chiral alcohol synthesis. J. Biosci. Bioeng. 1999, 88, 591–598. [CrossRef] 198. Wada, M.; Kataoka, M.; Kawabata, H.; Yasohara, Y.; Kizaki, N.; Hasegawa, J.; Shimizu, S. Puriﬁcation and characterization of NADPH-dependent carbonyl reductase, involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate, from Candida magnoliae. Biosci. Biotechnol. Biochem. 1998, 62, 280–285. [CrossRef] Catalysts 2019, 9, 260 30 of 32 199. Kataoka, M.; Doi, Y.; Sim, T.S.; Shimizu, S.; Yamada, H. A novel NADPH-dependent carbonyl reductase of Candida macedoniensis. Puriﬁcation and characterization. Arch. Biochem. Biophys. 1992, 294, 469–474. [CrossRef] 200. He, J.Y.; Sun, Z.H.; Ruan, W.Q.; Xu, Y. Biocatalytic synthesis of ethyl (S)-4-chloro-3-hydroxy-butanoate in an aqueous-organic solvent biphasic system using Aureobasidium pullulans CGMCC 1244. Process Biochem. 2006, 41, 244–249. [CrossRef] 201. Saratani, Y.; Uheda, E.; Yamamoto, H.; Nishimura, A.; Yoshizako, F. Stereoselective reduction of ethyl 4-chloro-3-oxobutanoate by fungi. Biosci. Biochem. Biophys. 2001, 65, 1676–1679. [CrossRef] 202. Ye, Q.; Yan, M.; Xu, L.; Cao, H.; Li, Z.; Chen, Y.; Li, S.; Ying, H. A novel carbonyl reductase from Pichia stipitis for the production of ethyl (S)-4-chloro-3-hydroxybutanoate. Biotechnol. Lett. 2009, 31, 537–542. [CrossRef] 203. Cai, P.; An, M.D.; Xu, L.; Xu, S.; Hao, N.; Li, Y.; Guo, K.; Yan, M. Development of a substrate-coupled biocatalytic process driven by an NADPH-dependent sorbose reductase from Candida albicans for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate. Biotechnol. Lett. 2012, 34, 2223–2227. [CrossRef] 204. Pan, J.; Zheng, G.-W.; Ye, Q.; Xu, J.-H. Optimization and scale-up of a bioreduction process for preparation of ethyl (S)-4-chloro-3-hydroxybutanoate. Org. Process Res. Dev. 2014, 18, 739–743. [CrossRef] 205. Liu, Z.-Q.; Ye, J.-J.; Shen, Z.-Y.; Hong, H.-B.; Yan, J.-B.; Lin, Y.; Chen, Z.-X.; Zheng, Y.-G.; Shen, Y.-C. Upscale production of ethyl (S)-4-chloro-3-hydroxybutanoate by using carbonyl reductase coupled with glucose dehydrogenase in aqueous-organic solvent system. Appl. Microbiol. Biotechnol. 2015, 99, 2119–2129. [CrossRef] 206. Schallmey, M.; Floor, R.J.; Hauer, B.; Breuer, M.; Jekel, P.A.; Wijma, H.J.; Dijkstra, B.W.; Janssen, D.B. Biocatalytic and structural properties of a highly engineered halohydrin dehalogenase. ChemBioChem 2013, 14, 870–881. [CrossRef] 207. Ritter, S.K. Going green keeps getting easier. Chem. Eng. News 2006, 84, 24–27. [CrossRef] 208. Li, C.-J.; Anastas, P.T. Green Chemistry: Present and future. Chem. Soc. Rev. 2012, 41, 1413–1414. [CrossRef] 209. Chen, X.F.; Xiong, F.J.; Chen, W.X.; He, Q.Q.; Chen, F.E. Asymmetric Synthesis of the HMG-CoA Reductase Inhibitor Atorvastatin Calcium: An Organocatalytic Anhydride Desymmetrization and Cyanide-Free Side Chain Elongation Approach. J. Org. Chem. 2014, 79, 2723–2728. [CrossRef] 210. Everaere, K.; Franceschini, N.; Mortreux, A.; Carpentier, J.F. Diastereoselective synthesis of syn-3,5-dihydroxyesters via ruthenium-catalyzed asymmetric transfer hydrogenation. Tetrahedron Lett. 2002, 43, 2569–2571. [CrossRef] 211. Wolberg, M.; Hummel, W.; Muller, M. Biocatalytic reduction of beta, delta-diketo esters: A highly stereoselective approach to all four stereoisomers of a chlorinated beta, delta-dihydroxy hexanoate. Chem. Eur. J. 2001, 7, 4562–4571. [CrossRef] 212. Wolberg, M.; Hummel, W.; Wandrey, C.; Muller, M. Highly regio- and enantioselective reduction of 3,5-dioxocarboxylates. Angew. Chem. Int. Ed. 2000, 39, 4306–4308. [CrossRef] 213. Wolberg, M.; Villela, M.; Bode, S.; Geilenkirchen, P.; Feldmann, R.; Liese, A.; Hummel, W.; Muller, M. Chemoenzymatic synthesis of the chiral side-chain of statins: Application of an alcohol dehydrogenase catalysed ketone reduction on a large scale. Bioproc. Biosyst. Eng. 2008, 31, 183–191. [CrossRef] 214. Liu, Z.Q.; Hu, Z.L.; Zhang, X.J.; Tang, X.L.; Cheng, F.; Xue, Y.P.; Wang, Y.J.; Wu, L.; Yao, D.K.; Zhou, Y.T.; et al. Large-scale synthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate by a stereoselective carbonyl reductase with high substrate concentration and product yield. Biotechnol. Progr. 2017, 33, 612–620. [CrossRef] [PubMed] 215. Liu, Z.Q.; Wu, L.; Zhang, X.J.; Xue, Y.P.; Zheng, Y.G. Directed evolution of carbonyl reductase from Rhodosporidium toruloides and its application in stereoselective synthesis of tert-butyl (3R,5S)-6-chloro- 3,5-dihydroxyhexanoate. J. Agric. Food Chem. 2017, 65, 3721–3729. [CrossRef] [PubMed] 216. Liu, Z.Q.; Yin, H.H.; Zhang, X.J.; Zhou, R.; Wang, Y.M.; Zheng, Y.G. Improvement of carbonyl reductase activity for the bioproduction of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. Bioorg. Chem. 2018, 80, 733–740. [CrossRef] [PubMed] 217. Liu, Z.Q.; Wu, L.; Zheng, L.; Wang, W.Z.; Zhang, X.J.; Jin, L.Q.; Zheng, Y.G. Biosynthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate by carbonyl reductase from Rhodosporidium toruloides in mono and biphasic media. Bioresour. Technol. 2018, 249, 161–167. [CrossRef] Catalysts 2019, 9, 260 31 of 32 218. Xu, T.T.; Wang, C.; Zhu, S.Z.; Zheng, G.J. Enzymatic preparation of optically pure t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate by a novel alcohol dehydrogenase discovered from Klebsiella oxytoca. Process Biochem. 2017, 57, 72–79. [CrossRef] 219. Gong, X.M.; Zheng, G.W.; Liu, Y.Y.; Xu, J.H. Identiﬁcation of a robust carbonyl reductase for diastereoselectively building syn-3,5-dihydroxy hexanoate: A bulky side chain of atorvastatin. Org. Process Res. Dev. 2017, 21, 1349–1354. [CrossRef] 220. Gong, X.M.; Qin, Z.; Li, F.L.; Zeng, B.B.; Zheng, G.W.; Xu, J.H. Development of an engineered ketoreductase with simultaneously improved thermostability and activity for making a bulky atorvastatin precursor. ACS Catal. 2019, 9, 147–153. [CrossRef] 221. Luo, X.; Wang, Y.-J.; Zheng, Y.-G. Improved stereoselective bioreduction of t-butyl 6-cyano-(5R)-hydroxy-3- oxohexanoate by Rhodotorula glutinis through heat treatment. Biotechnol. Appl. Biochem. 2016, 63, 795–804. [CrossRef] 222. Wu, X.; Gou, X.; Chen, Y. Enzymatic preparation of t-butyl-6-cyano-(3R,5R)-dihydroxyhexanoate by a whole-cell biocatalyst co-expressing carbonyl reductase and glucose dehydrogenase. Process Biochem. 2015, 50, 104–110. [CrossRef] 223. Wang, Y.J.; Chen, X.P.; Shen, W.; Liu, Z.Q.; Zheng, Y.G. Chiral diol t-butyl 6-cyano-(3R,5R)- dihydroxylhexanoate synthesis catalyzed by immobilized cells of carbonyl reductase and glucose dehydrogenase co-expression E. coli. Biochem. Eng. J. 2017, 128, 54–62. [CrossRef] 224. Luo, X.; Wang, Y.-J.; Zheng, Y.-G. Cloning and characterization of a NADH-dependent aldo-keto reductase from a newly isolated Kluyveromyces lactis XP1461. Enzyme Microb. Technol. 2015, 77, 68–77. [CrossRef] 225. Luo, X.; Wang, Y.-J.; Shen, W.; Zheng, Y.-G. Activity improvement of a Kluyveromyces lactis aldo-keto reductase KlAKR via rational design. J. Biotechnol. 2016, 224, 20–26. [CrossRef] 226. Wang, Y.-J.; Liu, X.-Q.; Luo, X.; Liu, Z.-Q.; Zheng, Y.-G. Cloning, expression and enzymatic characterization of an aldo-keto reductase from Candida albicans XP1463. J. Mol. Catal. B-Enzym. 2015, 122, 44–50. [CrossRef] 227. Pfruender, H.; Amidjojo, M.; Hang, F.; Weuster-Botz, D. Production of Lactobacillus keﬁr cells for asymmetric synthesis of a 3,5-dihydroxycarboxylate. Appl. Microbiol. Biotechnol. 2005, 67, 619–622. [CrossRef] 228. Patel, R.N.; Banerjee, A.; McNamee, C.G.; Brzozowski, D.; Hanson, R.L.; Szarka, L.J. Enantioselective microbial reduction of 3,5-dioxo-6-(benzyloxy) hexanoic acid, ethyl-ester. Enzyme Microb. Technol. 1993, 15, 1014–1021. [CrossRef] 229. Guo, Z.; Chen, Y.; Goswami, A.; Hanson, R.L.; Patel, R.N. Synthesis of ethyl and t-butyl (3R,5S)-dihydroxy-6-benzyloxy hexanoates via diastereo- and enantioselective microbial reduction. Tetrahedron Asymm. 2006, 17, 1589–1602. [CrossRef] 230. Goldberg, S.; Guo, Z.; Chen, S.; Goswami, A.; Patel, R.N. Synthesis of ethyl-(3R,5S)-dihydroxy-6-benzyloxyhexanoates via diastereo- and enantioselective microbial reduction: Cloning and expression of ketoreductase III from Acinetobacter sp. SC 13874. Enzyme Microb. Technol. 2008, 43, 544–549. [CrossRef] 231. Wu, X.; Liu, N.; He, Y.; Chen, Y. Cloning, expression, and characterization of a novel diketoreductase from Acinetobacter baylyi. Acta Biochim. Biophys. Sin. 2009, 41, 163–170. [CrossRef] [PubMed] 232. Chen, Y.; Chen, C.; Wu, X. Dicarbonyl reduction by single enzyme for the preparation of chiral diols. Chem. Soc. Rev. 2012, 41, 1742–1753. [CrossRef] 233. Lu, M.; Huang, Y.; White, M.A.; Wu, X.; Liu, N.; Cheng, X.; Chen, Y. Dual catalysis mode for the dicarbonyl reduction catalyzed by diketoreductase. Chem. Commun. 2012, 48, 11352–11354. [CrossRef] [PubMed] 234. Huang, Y.; Lu, Z.; Ma, M.; Liu, N.; Chen, Y. Functional roles of tryptophan residues in diketoreductase from Acinetobacter baylyi. BMB Rep. 2012, 45, 452–457. [CrossRef] [PubMed] 235. Huang, Y.; Lu, Z.; Liu, N.; Chen, Y. Identiﬁcation of important residues in diketoreductase from Acinetobacter baylyi by molecular modeling and site-directed mutagenesis. Biochimie 2012, 94, 471–478. [CrossRef] [PubMed] 236. Gijsen, H.J.M.; Wong, C.H. Sequential 3-substrate and 4-substrate aldol reactions catalyzed by aldolases. J. Am. Chem. Soc. 1995, 117, 7585–7591. [CrossRef] 237. Gijsen, H.J.M.; Wong, C.H. Unprecedented asymmetric aldol reactions with 3 aldehyde substrates catalyzed by 2-deoxyribose-5-phosphate aldolase. J. Am. Chem. Soc. 1994, 116, 8422–8423. [CrossRef] 238. Liu, J.J.; Hsu, C.C.; Wong, C.H. Sequential aldol condensation catalyzed by DERA mutant Ser238Asp and a formal total synthesis of atorvastatin. Tetrahedron Lett. 2004, 45, 2439–2441. [CrossRef] Catalysts 2019, 9, 260 32 of 32 239. Greenberg, W.A.; Varvak, A.; Hanson, S.R.; Wong, K.; Huang, H.J.; Chen, P.; Burk, M.J. Development of an efﬁcient, scalable, aldolase-catalyzed process for enantioselective synthesis of statin intermediates. Proc. Natl. Acad. Sci. USA 2004, 101, 5788–5793. [CrossRef] 240. Jennewein, S.; Schurmann, M.; Wolberg, M.; Hilker, I.; Luiten, R.; Wubbolts, M.; Mink, D. Directed evolution of an industrial biocatalyst: 2-deoxy-D-ribose 5-phosphate aldolase. Biotechnol. J. 2006, 1, 537–548. [CrossRef] 241. Oslaj, M.; Cluzeau, J.; Orkic, D.; Kopitar, G.; Mrak, P.; Casar, Z. A highly productive, whole-cell DERA chemoenzymatic process for production of key lactonized side-chain intermediates in statin synthesis. PLoS ONE 2013, 8, e62250. [CrossRef] 242. Rucigaj, ˇ A.; Krajnc, M. Optimization of a crude deoxyribose-5-phosphate aldolase lyzate-catalyzed process in synthesis of statin intermediates. Org. Process Res. Dev. 2013, 17, 854–862. [CrossRef] 243. You, Z.Y.; Liu, Z.Q.; Zheng, Y.G.; Shen, Y.C. Characterization and application of a newly synthesized 2-deoxyribose-5-phosphate aldolase. J. Ind. Microbiol. Biotechnol. 2013, 40, 29–39. [CrossRef] 244. Casar, Z.; Steinbucher, M.; Kosmrlj, J. Lactone pathway to statins utilizing the Wittig reaction. The synthesis of rosuvastatin. J. Org. Chem. 2010, 75, 6681–6684. [CrossRef] 245. Vajdic, T.; Oslaj, M.; Kopitar, G.; Mrak, P. Engineered, highly productive biosynthesis of artiﬁcial, lactonized statin side-chain building blocks: The hidden potential of Escherichia coli unleashed. Metab. Eng. 2014, 24, 160–172. [CrossRef] 246. Sakuraba, H.; Yoneda, K.; Yoshihara, K.; Satoh, K.; Kawakami, R.; Uto, Y.; Tsuge, H.; Takahashi, K.; Hori, H.; Ohshima, T. Sequential aldol condensation catalyzed by hyperthermophilic 2-deoxy-D-ribose-5-phosphate aldolase. Appl. Environ. Microbiol. 2007, 73, 7427–7434. [CrossRef] 247. Fei, H.; Zheng, C.C.; Liu, X.Y.; Li, Q. An industrially applied biocatalyst: 2-Deoxy-D-ribose-5-phosphate aldolase. Process Biochem. 2017, 63, 55–59. [CrossRef] 248. Haridas, M.; Abdelraheem, E.M.M.; Hanefeld, U. 2-Deoxy-D-ribose-5-phosphate aldolase (DERA): Applications and modiﬁcations. Appl. Microbiol. Biotechnol. 2018, 102, 9959–9971. [CrossRef] 249. Muller, M. Chemoenzymatic synthesis of building blocks for statin side chains. Angew. Chem. Int. Ed. 2005, 44, 362–365. [CrossRef] 250. Garattini, L.; Padula, A. Cholesterol-lowering drugs: Science and marketing. J. R. Soc. Med. 2017, 110, 57–64. [CrossRef] 251. Reiner, Z. Resistance and intolerance to statins. Nutr. Metab. Carbiovasc. Dis. 2014, 24, 1057–1066. [CrossRef] [PubMed] 252. Chaudhary, R.; Garg, J.; Shah, N.; Sumner, A. PCSK9 inhibitors: A new era of lipid lowering therapy. World J. Cardiol. 2017, 9, 76–91. [CrossRef] [PubMed] 253. Palmer, C. New directions in managing dyslipidemia. J. Nurse Pract. 2019, 15, 73–79. [CrossRef] 254. Xu, S.T.; Luo, S.S.; Zhu, Z.Y.; Xu, J.Y. Small molecules as inhibitors of PCSK9: Current status and future challenges. Eur. J. Med. Chem. 2019, 162, 212–233. [CrossRef] [PubMed] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png

Catalysts Unpaywall

http://www.deepdyve.com/lp/unpaywall/biocatalyzed-synthesis-of-statins-a-sustainable-strategy-for-the-d1p7HwWhCf

Biocatalyzed Synthesis of Statins: A Sustainable Strategy for the Preparation of Valuable Drugs

Loading next page...

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher: Unpaywall
ISSN: 2073-4344
DOI: 10.3390/catal9030260
Publisher site: See Article on Publisher Site

Abstract

Journal

Catalysts – Unpaywall

Published: Mar 14, 2019

Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Biocatalyzed Synthesis of Statins: A Sustainable Strategy for the Preparation of Valuable Drugs

Biocatalyzed Synthesis of Statins: A Sustainable Strategy for the Preparation of Valuable Drugs

Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Biocatalyzed Synthesis of Statins: A Sustainable Strategy for the Preparation of Valuable Drugs

Biocatalyzed Synthesis of Statins: A Sustainable Strategy for the Preparation of Valuable Drugs

References

Abstract

Journal

Recommended Articles

There are no references for this article.

Our policy towards the use of cookies