Everolimus

159351-69-6[RN]
23,27-Epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone, 9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-(2-hydr oxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-, (3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,26R,27R,34aS)-
23,27-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone, 9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-, (3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-
42-O-(2-Hydroxyethyl)rapamycin

• Synonyms:RAD-001, SDZ-RAD, Afinitor
• ATC:L04AA18

Use:immunosuppressantChemical name:42-O-(2-hydroxyethyl)rapamycinFormula:C₅₃H₈₃NO₁₄

• MW:958.24 g/mol
• CAS-RN:159351-69-6

EverolimusCAS Registry Number: 159351-69-6CAS Name: 42-O-(2-Hydroxyethyl)rapamycinAdditional Names: 40-O-(2-hydroxyethyl)rapamycinManufacturers’ Codes: RAD-001; SDZ RADTrademarks: Certican (Novartis)Molecular Formula: C53H83NO14Molecular Weight: 958.22Percent Composition: C 66.43%, H 8.73%, N 1.46%, O 23.38%Literature References: Macrolide immunosuppressant; derivative of rapamycin, q.v. Inhibits cytokine-mediated lymphocyte proliferation. Prepn: S. Cottens, R. Sedrani, WO9409010; eidem, US5665772 (1994, 1997 both to Sandoz). Pharmacology: W. Schuler et al., Transplantation64, 36 (1997). Whole blood determn by LC/MS: N. Brignol et al., Rapid Commun. Mass Spectrom.15, 898 (2001); by HPLC: S. Baldelli et al., J. Chromatogr. B816, 99 (2005). Clinical pharmacokinetics in combination with cyclosporine: J. M. Kovarik et al., Clin. Pharmacol. Ther.69, 48 (2001). Clinical study in prevention of cardiac-allograft vasculopathy: H. J. Eisen et al.,N. Engl. J. Med.349, 847 (2003). Review: F. J. Dumont et al., Curr. Opin. Invest. Drugs2, 1220-1234 (2001); B. Nashan, Ther. Drug Monit.24, 53-58 (2002).Therap-Cat: Immunosuppressant.Keywords: Immunosuppressant.эверолимус[Russian][INN]إيفيروليموس[Arabic][INN]依维莫司[Chinese][INN]Trade Name：Certican^® / Zortress^® / Afinitor^®MOA：mTOR inhibitorIndication：Rejection of organ transplantation; Renal cell carcinoma; Advanced renal cell carcinoma (RCC); Advanced breast cancer; Pancreatic cancer; Renal angiomyolipoma; Tuberous sclerosis complex (TSC); Rejection in heart transplantation; Rejection of suppression renal transplantation; Subependymal giant cell astrocytoma; neuroendocrine tumors (NET); Advanced gastrointestinal tumorsStatus：ApprovedCompany：Novartis (Originator)Sales：$1,942 Million (Y2015);
$1,902 Million (Y2014);
$1,558 Million (Y2013);
$1,007 Million (Y2012);
$630 Million (Y2011);ATC Code：L04AA18Approved Countries or Area

• US
• EU
• JP
• CN
• SE

Approval Date	Approval Type	Trade Name	Indication	Dosage Form	Strength	Company	Review Classification
2012-08-29	New dosage form	Afinitor Disperz	Renal cell carcinoma , Advanced breast cancer, Pancreatic cancer, Renal angiomyolipoma, Tuberous sclerosis complex (TSC)	Tablet, For suspension	2 mg/3 mg/5 mg	Novartis	Priority
2010-04-20	New strength	Zortress	Advanced renal cell carcinoma (RCC)	Tablet	0.25 mg/0.5 mg/0.75 mg	Novartis
2009-03-30	Marketing approval	Afinitor	Advanced renal cell carcinoma (RCC)	Tablet	2.5 mg/5 mg/7.5 mg/10 mg	Novartis	Priority

Approval Date	Approval Type	Trade Name	Indication	Dosage Form	Strength	Company	Review Classification
2016-06-02	New indication	Afinitor	neuroendocrine tumors (NET), Advanced gastrointestinal tumors	Tablet		Novartis
2011-09-02	Marketing approval	Votubia	Advanced breast cancer, Renal cell carcinoma , Pancreatic cancer	Tablet	2.5 mg/5 mg/10 mg	Novartis	Orphan; Conditional Approval
2011-09-02	Marketing approval	Votubia	Advanced breast cancer, Renal cell carcinoma , Pancreatic cancer	Tablet, Orally disintegrating	2 mg/3 mg/5 mg	Novartis	Orphan; Conditional Approval
2009-08-03	Marketing approval	Afinitor	Advanced breast cancer, Renal cell carcinoma , Pancreatic cancer	Tablet	2.5 mg/5 mg/10 mg	Novartis

Approval Date	Approval Type	Trade Name	Indication	Dosage Form	Strength	Company	Review Classification
2011-12-22	New indication	Certican	Rejection of suppression renal transplantation	Tablet	0.25 mg/0.5 mg/0.75 mg	Novartis
2007-01-26	Marketing approval	Certican	Rejection in heart transplantation	Tablet	0.25 mg/0.5 mg/0.75 mg	Novartis

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Approval Date	Approval Type	Trade Name	Indication	Dosage Form	Strength	Company	Review Classification
2014-02-13	Marketing approval	飞尼妥/Afinitor	Advanced renal cell carcinoma (RCC), Subependymal giant cell astrocytoma	Tablet	2.5 mg	Novartis
2013-01-22	Marketing approval	飞尼妥/Afinitor	Advanced renal cell carcinoma (RCC), Subependymal giant cell astrocytoma	Tablet	10 mg	Novartis
2013-01-22	Marketing approval	飞尼妥/Afinitor	Advanced renal cell carcinoma (RCC), Subependymal giant cell astrocytoma	Tablet	5 mg	Novartis

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Approval Date	Approval Type	Trade Name	Indication	Dosage Form	Strength	Company	Review Classification
2003-07-18	Marketing approval	Certican	Rejection of organ transplantation, Renal cell carcinoma	Tablet	0.25 mg/0.5 mg/0.75 mg	Novartis

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Click to access afinitor-epar-public-assessment-report_en.pdf

Active Substance The active substance Everolimus is a hydroxyethyl derivative of rapamycin, which is a macrolide, isolated from the micro-organism Streptomyces hygroscopicus. The guideline, impurities in new active substances ICHQ 3A (R), does not apply to active substance of fermented origin. Everolimus (INN) or 42-O-(2-hydroxyethyl)-rapamycin (chemical name) or C5 3H8 3N O1 4 has been fully described. The molecule is amorphous and is stabilised with an antioxidant. Its physico-chemical properties including parameters such as solubility, pH, specific rotation, potential polymorphism and potential isomerism have been fully characterised. Everolimus is a white to faintly yellow amorphous powder. It is almost insoluble in water, is unstable at temperatures above 25 °C and is sensitive to light. In addition, possible isomerism has been investigated. Everolimus contains 15 asymmetric carbon atoms and 4 substituted double bonds. The configuration of the asymmetric carbon atoms and the double bonds is guaranteed by the microbial origin of Rapamycin. The configuration is not affected by the chemical synthesis. Polymorphism has been comprehensively discussed and it was demonstrated that the molecule domain remains amorphous.

Synthesis of Everolimus The manufacturing process consists of four main steps, (1) fermentation, (2) extraction of rapamycin from the fermentation broth, (3) chemical modification of rapamycin starting material, (4) purification of crude everolimus and stabilisation with BHT. The choice of the stabilizer has been sufficiently explained and justified by experimental results. Interactions products of Everolimus and the antioxidant were not detected, or were below detection limit. Rapamycin, obtained by a fermentation process, was used as the starting material. Reaction conditions and the necessary in-process controls are described in detail. Adequate specifications for starting materials and isolated intermediates and descriptions of the test procedures have been submitted. Control of the quality of solvents, reagents and auxiliary materials used in the synthesis has been adequately documented. It is stated by the manufacturer of rapamycin solution that no starting material of animal or human origin is used in the fermentation. Elucidation of structure and other characteristics The structure of Everolimus has been fully elucidated using several spectroscopic techniques such as ultraviolet absorption spectroscopy (UV), Infra-red spectroscopy (FT-IR), proton and carbon nuclear magnetic resonance spectroscopy (1 H and 13C NMR), mass spectroscopy, diffractometry (X-ray) and elemental analysis. Related substances An extensive discussion was presented on the related substances. The complex structure of Everolimus allows several possible degradation pathways to occur at various positions of the molecule. Everolimus alone is extremely sensitive to oxidation. By the addition of an antioxidant, the sensitivity to oxidation is significantly reduced (the antioxidant is known to react as a scavenger of peroxide radicals). It is assumed that oxidation of Everolimus proceeds via a radical mechanism. All the requirements set in the current testing instruction valid for Everolimus are justified on the basis of the results obtained during development and manufactured at the production scale.

fda

Click to access 022334s000_ChemR.pdf

Everolimus was first approved by Swiss Agency for therapeutic products，Swissmedic on July 18, 2003, then approved by Pharmaceuticals and Medicals Devices Agency of Japan (PMDA) on April 23, 2004, and approved by the U.S. Food and Drug Administration (FDA) on Mar 30, 2009, approved by European Medicine Agency (EMA) on Aug 3, 2009. It was developed and marketed as Certican^® by Novartis in SE.

Everolimus is an inhibitor of mammalian target of rapamycin (mTOR). It is indicated for the treatment of renal cell cancer and other tumours and currently used as an immunosuppressant to prevent rejection of organ transplants.

Certican^® is available as tablet for oral use, containing 0.25, 0.5 or 0.75 mg of free Everolimus. The recommended dose is 10 mg once daily with or without food for advanced HR+ breast cancer, advanced progressive neuroendocrine tumors, advanced renal cell carcinoma or renal angiomyolipoma with tuberous sclerosis complex.
Everolimus, also known as RAD001, is a derivative of the natural macrocyclic lactone sirolimus with immunosuppressant and anti-angiogenic properties. In cells, everolimus binds to the immunophilin FK Binding Protein-12 (FKBP-12) to generate an immunosuppressive complex that binds to and inhibits the activation of the mammalian Target of Rapamycin (mTOR), a key regulatory kinase. Inhibition of mTOR activation results in the inhibition of T lymphocyte activation and proliferation associated with antigen and cytokine (IL-2, IL-4, and IL-15) stimulation and the inhibition of antibody production.

Everolimus is a medication used as an immunosuppressant to prevent rejection of organ transplants and in the treatment of renal cell cancer and other tumours. Much research has also been conducted on everolimus and other mTOR inhibitors as targeted therapy for use in a number of cancers.^{[medical citation needed]}

It is the 40-O-(2-hydroxyethyl) derivative of sirolimus and works similarly to sirolimus as an inhibitor of mammalian target of rapamycin (mTOR).

It is marketed by Novartis under the trade names Zortress (USA) and Certican (European Union and other countries) in transplantation medicine, and as Afinitor (general tumours) and Votubia (tumours as a result of TSC) in oncology. Everolimus is also available from Biocon, with the brand name Evertor.

Medical uses

Everolimus is approved for various conditions:

• Advanced kidney cancer (US FDA approved in March 2009)^[3]
• Prevention of organ rejection after renal transplant(US FDA April 2010)^[4]
• Subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis (TS) in patients who are not suitable for surgical intervention (US FDA October 2010)^[5]
• Progressive or metastatic pancreatic neuroendocrine tumors not surgically removable (May 2011)^[6]
• Breast cancer in post-menopausal women with advanced hormone-receptor positive, HER2-negative type cancer, in conjunction with exemestane (US FDA July 2012)^[7]
• Prevention of organ rejection after liver transplant(Feb 2013)
• Progressive, well-differentiated non-functional, neuroendocrine tumors (NET) of gastrointestinal (GI) or lung origin with unresectable, locally advanced or metastatic disease (US FDA February 2016).^[8]
• Tuberous sclerosis complex-associated partial-onset seizures for adult and pediatric patients aged 2 years and older. (US FDA April 2018).^[9]

UK National Health Service

NHS England has been criticised for delays in deciding on a policy for the prescription of everolimus in the treatment of Tuberous Sclerosis. 20 doctors addressed a letter to the board in support of the charity Tuberous Scelerosis Association saying ” around 32 patients with critical need, whose doctors believe everolimus treatment is their best or only option, have no hope of access to funding. Most have been waiting many months. Approximately half of these patients are at imminent risk of a catastrophic event (renal bleed or kidney failure) with a high risk of preventable death.”^[10] In May 2015 it was reported that Luke Henry and Stephanie Rudwick, the parents of a child suffering from Tuberous Sclerosis were trying to sell their home in Brighton to raise £30,000 to pay for treatment for their daughter Bethany who has tumours on her brain, kidneys and liver and suffers from up to 50 epileptic fits a day.^[11]

Clinical trials

As of October 2010, Phase III trials are under way in gastric cancer, hepatocellular carcinoma, and lymphoma.^[12] The experimental use of everolimus in refractory chronic graft-versus-host disease was reported in 2012.^[13]

Interim phase III trial results in 2011 showed that adding Afinitor (everolimus) to exemestane therapy against advanced breast cancer can significantly improve progression-free survival compared with exemestane therapy alone.^[14]

A study published in 2012, shows that everolimus sensitivity varies between patients depending on their tumor genomes.^[15] A group of patients with advanced metastasic bladder carcinoma (NCT00805129) ^[16] treated with everolimus revealed a single patient who had a complete response to everolimus treatment for 26 months. The researchers sequenced the genome of this patient and compared it to different reference genomes and to other patients’ genomes. They found that mutations in TSC1 led to a lengthened duration of response to everolimus and to an increase in the time to cancer recurrence. The mutated TSC1 apparently had made these tumors vulnerable to treatment with everolimus.^{[medical citation needed]}

A phase 2a randomized, placebo-controlled everolimus clinical trial published in 2014 showed that everolimus improved the response to an influenza vaccine by 20% in healthy elderly volunteers.^[17] A phase 2a randomized, placebo-controlled clinical trial published in 2018 showed that everolimus in combination with dactolisib decreased the rate of reported infections in an elderly population.^[17]

Mechanism

Compared with the parent compound rapamycin, everolimus is more selective for the mTORC1 protein complex, with little impact on the mTORC2 complex.^[18] This can lead to a hyper-activation of the kinase AKT via inhibition on the mTORC1 negative feedback loop, while not inhibiting the mTORC2 positive feedback to AKT. This AKT elevation can lead to longer survival in some cell types.^{[medical citation needed]} Thus, everolimus has important effects on cell growth, cell proliferation and cell survival.

mTORC1 inhibition by everolimus has been shown to normalize tumor blood vessels, to increase tumor-infiltrating lymphocytes, and to improve adoptive cell transfer therapy.^[19]

Additionally, mTORC2 is believed to play an important role in glucose metabolism and the immune system, suggesting that selective inhibition of mTORC1 by drugs such as everolimus could achieve many of the benefits of rapamycin without the associated glucose intolerance and immunosuppression.^[18]

TSC1 and TSC2, the genes involved in tuberous sclerosis, act as tumor suppressor genes by regulating mTORC1 activity. Thus, either the loss or inactivation of one of these genes lead to the activation of mTORC1.^[20]

Everolimus binds to its protein receptor FKBP12, which directly interacts with mTORC1, inhibiting its downstream signaling. As a consequence, mRNAs that code for proteins implicated in the cell cycle and in the glycolysis process are impaired or altered, and tumor growth is inhibited.^[20]

Adverse reactions

A trial using 10 mg/day in patients with NETs of GI or lung origin reported “Everolimus was discontinued for adverse reactions in 29% of patients and dose reduction or delay was required in 70% of everolimus-treated patients. Serious adverse reactions occurred in 42% of everolimus-treated patients and included 3 fatal events (cardiac failure, respiratory failure, and septic shock). The most common adverse reactions (incidence greater than or equal to 30%) were stomatitis, infections, diarrhea, peripheral edema, fatigue and rash. The most common blood abnormalities found (incidence greater than or equal to 50%) were anemia, hypercholesterolemia, lymphopenia, elevated aspartate transaminase (AST) and fasting hyperglycemia.”.^[8]

Role in heart transplantation

Everolimus may have a role in heart transplantation, as it has been shown to reduce chronic allograft vasculopathy in such transplants. It also may have a similar role to sirolimus in kidney and other transplants.^[21]

Role in liver transplantation

Although, sirolimus had generated fears over use of m-TOR inhibitors in liver transplantation recipients, due to possible early hepatic artery thrombosis and graft loss, use of everolimus in the setting of liver transplantation is promising. Jeng et al.,^[22] in their study of 43 patients, concluded the safety of everolimus in the early phase after living donor liver transplantation. In their study, no hepatic artery thrombosis or wound infection was noted. Also, a possible role of everolimus in reducing the recurrence of hepatocellular carcinoma after liver transplantation was correlated. A target trough level of 3 ng/mL at 3 months was shown to be beneficial in recipients with pre-transplant renal dysfunction. In their study, 6 of 9 renal failure patients showed significant recovery of renal function, whereas 3 showed further deterioration, one of whom required hemodialysis.^[23] Recently published report by Thorat et al. showed a positive impact on hepatocellular carcinoma (HCC) when everolimus was used as primary immunosuppression starting as early as first week after living donor liver transplantation (LDLT) surgery.^[24] In their retrospective and prospective analysis at China Medical University Hospital in Taiwan, the study cohort (n=66) was divided in two groups depending upon the postoperative immunosuppression. Group A: HCC patients that received Everolimus + Tacrolimus based immunosuppressive regimen (n=37). Group B: HCC patients that received standard Tacrolimus based immunosuppressive regimen without everolimus (n=29). The target trough level for EVR was 3 to 5 ng/ml while for TAC it was 8–10 ng/ml. The 1-year, 3-year and 4-year overall survival achieved for Group A patients (Everolimus group) was 94.95%, 86.48% and 86.48%, respectively while for Group B patients it was 82.75%, 68.96%, and 62.06%, respectively (p=0.0217). The first 12-month report of ongoing Everolimus multicenter prospective trial in LDLT (H2307 trial), Jeng LB et al. have shown a 0% recurrence of HCC in everolimus group at 12 months.^[25] Jeng LB concluded that an early introduction of everolimus + reduced tacrolimus was non-inferior to standard tacrolimus in terms of efficacy and renal function at 12 months, with HCC recurrence only in tacrolimus control patients.

Use in vascular stents

Everolimus is used in drug-eluting coronary stents as an immunosuppressant to prevent restenosis. Abbott Vascular produce an everolimus-eluting stent (EES) called Xience Alpine. It utilizes the Multi-Link Vision cobalt chromium stent platform and Novartis’ everolimus. The product is widely available globally including the US, the European Union, and Asia-Pacific (APAC) countries. Boston Scientific also market EESes, recent offerings being Promus Elite and Synergy.^{[citation needed]}

Use in aging

Inhibition of mTOR, the molecular target of everolimus, extends the lifespan of model organisms including mice,^[26] and mTOR inhibition has been suggested as an anti-aging therapy. Everolimus was used in a clinical trial by Novartis, and short-term treatment was shown to enhance the response to the influenza vaccine in the elderly, possible by reversing immunosenescence.^[27] Everolimus treatment of mice results in reduced metabolic side effects compared to sirolimus.^[18]Route 1

Reference：1. US5665772A.

2. Drug. Future 1999, 24, 22-29.Route 2

Reference：1. WO2014203185A1.Route 3

Reference：1. WO2012103959A1.Route 4

Reference：1. CN102731527A.

SYN

Synthetic Reference

Wang, Feng. Everolimus intermediate and preparation method thereof. Assignee Shanghai Institute of Pharmaceutical Industry, Peop. Rep. China; China State Institute of Pharmaceutical Industry. CN 109776570. (2019).

SYN 2

Synthetic Reference

Polymer compositions containing a macrocyclic triene compound; Shulze, John E.; Betts, Ronald E.; Savage, Douglas R.; Assignee Sun Bow Co., Ltd., Bermuda; Sun Biomedical Ltd. 2003; Patent Information; Nov 06, 2003; WO 2003090684 A2

SYN 3

Synthetic Reference

Wang, Feng. Everolimus intermediate and preparation method thereof. Assignee Shanghai Institute of Pharmaceutical Industry, Peop. Rep. China; China State Institute of Pharmaceutical Industry. CN 109776570. (2019).

SYN 4

Synthetic Reference

Zabudkin, Oleksandr; Schickaneder, Christian; Matviienko, Iaroslav; Sypchenko, Volodymyr. Method for the synthesis of rapamycin derivatives. Assignee Synbias Pharma AG, Switz. EP 3109250. (2016).

SYN 5

Synthetic Reference

Lu, Shiyong; Zhang, Xiaotian; Chen, Haohan; Ye, Weidong. Preparation of sirolimus 40-ether derivative. Assignee Zhejiang Medicine Co., Ltd. Xinchang Pharmaceutical Factory, Peop. Rep. China. CN 105237549. (2016).

SYN 6

Synthetic Reference

Seo, Jeong U.; Ham, Yun Beom; Kang, Heung Mo; Lee, Gwang Mu; Kim, In Gyu; Kim, Jeong Jin; Park, Ji Su. Preparation of everolimus and synthetic intermediate thereof. Assignee CKD Bio Corp., S. Korea. KR 1529963 (2015).

SYN

EP 0663916; EP 0867438; JP 1996502266; JP 1999240884; US 5665772; WO 9409010

Alkylation of rapamycin (I) with 2-(tert-butyldimethylsilyloxy)ethyl triflate (II) by means of 2,6-lutidine in hot toluene gives the silylated target compound (III), which is deprotected by means of 1N HCl in methanol.

SYN

J Label Compd Radiopharm 1999,42(1),29

The compound has been obtained biosynthetically by an optimized fermentation process using Streptomyces hygroscopicus mutant RSH 1701 with a complex culture medium were [14C]-labeled (1R,3R,4R)-2,3-dichydroxycyclo-hexanecarboxylic acid (I) and [14C]-labeled (S)-pipecolic acid (II) have been added. This fermentation process yielded [14C]-labeled rapamycin (III), which was finally selectively O-alkylated at the C-40 position with monosilylated ethylene glycol triflate in DMSO/dimethoxyethane.

SYN

The reaction of the labeled acylated (+)-bornane-10,2-sultam (IV) with triethyl phosphite gives the phosphonate (V), which is treated with paraformaldehyde, galvinoxyl and K2CO3 yielding the acrylate derivative (VI). The cyclization of (VI) with butadiene (VII) by means of diethylaluminum chloride and galvinoxyl (as radical scavenger) affords the cyclohexene-carboxamide derivative (VIII), which is hydrolyzed with LiOH in THF/water giving the (1R)-3-cyclohexenecarboxylic acid (IX). The oxidation of (IX) with m-chloroperbenzoic acid and triethylamine in CCl4 yielded regioselectively the hydroxylactone (X), which is finally hydrolyzed with HCl to the labeled intermediate (I).

SYN

The reaction of the labeled acylated (-)-bornane-10,2-sultam (XI) with benzophenone imine (XII) gives the glycylsultam derivative (XIII), which is alkylated with 4-iodobutyl chloride (XIV) by means of butyllithium and DMPU in THF yielding intermediate (XV). The selective hydrolysis of (XV) with HCl affords the omega-chloro-L-norleucine derivative (XVI), which is cyclized by means of tetrabutylammonium fluoride and DIEA in hot acetonitrile giving the (2S)-piperidyl derivative (XVII). Finally, this compound is hydrolyzed with LiOH in THF/water to the labeled intermediate (II).

clipRapamycin is a known macrolide antibiotic produced by Streptomvces hvgroscopicus. having the structure depicted in Formula A:

See, e.g., McAlpine, J.B., et al., J. Antibiotics (1991) 44: 688; Schreiber, S.L., et al., J. Am. Chem. Soc. (1991) J_13: ⁷⁴³³‘- US Patent No. 3 929 992. Rapamycin is an extremely potent immunosuppressant and has also been shown to have antitumor and antifungal activity. Its utility as a pharmaceutical, however, is restricted by its very low and variable bioavailabiiity as well as its high toxicity. Moreover, rapamycin is highly insoluble, making it difficult to formulate stable galenic compositions.

Everolimus, 40-O-(2-hydroxyethyl)-rapamycin of formula (1) is a synthetic derivative of rapamycin (sirolimus) of formula (2), which is produced by a certain bacteria strain and is also pharmaceutically active.

(1)                                                                                                               (2)

Everolimus is marketed under the brand name Certican for the prevention of rejection episodes following heart and kidney transplantation, and under the brand name Afinitor for treatment of advanced kidney cancer.

Due to its complicated macrolide chemical structure, everolimus is, similarly as the parent rapamycin, an extremely unstable compound. It is sensitive, in particular, towards oxidation, including aerial oxidation. It is also unstable at temperatures higher than 25°C and at alkaline pH.

Everolimus and a process of making it have been disclosed in WO 94/09010

Synthesis

Alkylation of rapamycin (I) with 2-(tert-butyldimethylsilyloxy)ethyl triflate (II) by means of 2,6-lutidine in hot toluene gives the silylated target compound (III), which is deprotected by means of 1N HCl in methanol (1). (Scheme 21042401a) Manufacturer Novartis AG (CH). References 1. Cottens, S., Sedrani, R. (Sandoz-Refindungen VmbH; Sandoz-Patent GmbH; Sandoz Ltd.). O-Alkylated rapamycin derivatives and their use, particularly as immunosuppressants. EP 663916, EP 867438, JP 96502266, US 5665772, WO 9409010.EP 0663916; EP 0867438; JP 1996502266; JP 1999240884; US 5665772; WO 9409010

…………..

SYNTHESIS

https://www.google.com/patents/WO2012103960A1

(US 5,665,772, EP 663916). The process principle is shown in the scheme below, wherein the abbreviation RAP-OH has been used as an abbreviation for the rapamycin structure of formula (2) above, L is a leaving group and P is a trisubstituted silyl group serving as a OH- protective group.

RAP-OH + L-CH₂-CH₂-0-P — –> RAP-O-CH2-CH2-O-P — – > RAP-O-CH2-CH2-OH

(2)                                                 (4)                                                                 (1)

Specifically, the L- group is a trifluoromethanesulfonate (triflate) group and the protective group P- is typically a tert-butyldimethylsilyloxy- group. Accordingly, the known useful reagent within the above general formula (3) for making everolimus from rapamycin is 2-(tert-butyldimethylsilyloxy)ethyl triflate of formula (3 A):

According to a known synthetic procedure disclosed in Example 8 of WO 94/09010 and in Example 1 of US application 2003/0125800, rapamycin (2) reacts in hot toluene and in the presence of 2,6-lutidine with a molar excess of the compound (3 A), which is charged in several portions, to form the t-butyldimethylsilyl-protected everolimus (4A). This compound is isolated and deprotected by means of IN aqueous HC1 in methanol. Crude everolimus is then purified by column chromatography. Yields were not reported.

(2)                                       (3A)                              (4A)                                (1)

In an article of Moenius et al. (J. Labelled Cpd. Radiopharm. 43, 113-120 (2000)), which used the above process for making C14-labelled and tritiated everolimus, a diphenyl- tert.butylsilyloxy -protective group was used as the alkylation agent of formula (3B).

Only 8% yield of the corresponding compound (4B)

and 21% yield of the compound (1) have been reported.

Little is known about the compounds of the general formula (3) and methods of their preparation. The synthesis of the compound (3 A) was disclosed in Example 1 of US application 2003/0125800. It should be noted that specification of the reaction solvent in the key step B of this synthesis was omitted in the disclosure; however, the data about isolation of the product allow for estimation that such solvent is dichloromethane. Similarly also a second article of Moenius et al. (J. Labelled Cpd. Radiopharm.42, 29-41 (1999)) teaches that dichloromethane is the solvent in the reaction.

It appears that the compounds of formula (3) are very reactive, and thus also very unstable compounds. This is reflected by the fact that the yields of the reaction with rapamycine are very low and the compound (3) is charged in high molar extent. Methods how to monitor the reactivity and/or improve the stability of compounds of general formula (3), however, do not exist.

Thus, it would be useful to improve both processes of making compounds of formula (3) and, as well, processes of their application in chemical synthesis.

xample 6: 40-O-[2-((2,3-dimethylbut-2-yl)dimethylsilyloxy)ethyl]rapamycin

In a 100 mL flask, Rapamycin (6 g, 6.56 mmol) was dissolved in dimethoxyethane (4.2 ml) and toluene (24 ml) to give a white suspension and the temperature was raised to 70°C. After 20 min, N,N-diisopropylethylamine (4.56 ml, 27.6 mmol) and 2-((2,3-dimethylbutan-2- yl)dimethylsilyloxy)ethyl trifluoromethanesulfonate (8.83 g, 26.3 mmol) were added in 2 portions with a 2 hr interval at 70°C. The mixture was stirred overnight at room temperature, then diluted with EtOAc (40 ml) and washed with sat. NaHC0₃ (30 ml) and brine (30 ml). The organic layer was dried with Na₂S0₄, filtered and concentrated. The cmde product was chromatographed on a silica gel column (EtOAc/heptane 1/1 ; yield 4.47 g).

Example 7: 40-O-(2-hydroxyethyl)-rapamycin [everolimus]

In a 100 mL flask, 40-O-[2-((2,3-dimethylbut-2-yl)dimethylsilyloxy)ethyl]rapamycin (4.47 g, 4.06 mmol) was dissolved in methanol (20 ml) to give a colorless solution. At 0°C, IN aqueous hydrochloric acid (2.0 ml, 2.0 mmol) was added and the mixture was stirred for 90 min. The reaction was followed by TLC (ethyl acetate/n-heptane 3 :2) and HPLC. Then 20 ml of saturated aqueous NaHC03 were added, followed by 20 ml of brine and 80 ml of ethyl acetate. The phases were separated and the organic layer was washed with saturated aqueous NaCl until pH 6/7. The organic layer was dried by Na₂S0₄, filtered and concentrated to yield 3.3 g of the product.

……………………….

SYNTHESIS

https://www.google.co.in/patents/WO1994009010A1

Example 8: 40-O-(2-Hydroxy)ethyl-rapamycin

a) 40-O-[2-(t-Butyldimethylsilyl)oxy]ethyl-rapamycin

A solution of 9.14 g (10 mmol) of rapamycin and 4.70 mL (40 mmol) of 2,6-lutidine in 30 mL of toluene is warmed to 60°C and a solution of 6.17 g (20 mmol) of 2-(t-butyldimethylsilyl)oxyethyl triflate and 2.35 mL (20 mmol) of 2,6-lutidine in 20 mL of toluene is added. This mixture is stirred for 1.5h. Then two batches of a solution of 3.08 g (10 mmol) of triflate and 1.2 mL (10 mmol) of 2,6-lutidine in 10 mL of toluene are added in a 1.5h interval. After addition of the last batch, stirring is continued at 60°C for 2h and the resulting brown suspension is filtered. The filtrate is diluted with ethyl acetate and washed with aq. sodium bicarbonate and brine. The organic solution is dried over anhydrous sodium sulfate, filtered and concentrated. The residue is purified by column chromatography on silica gel (40:60 hexane-ethyl acetate) to afford 40-O-[2-(t-butyldimethylsilyl)oxy]ethyl-rapamycin as a white solid: ¹H NMR (CDCl₃) δ 0.06 (6H, s), 0.72 (1H, dd), 0.90 (9H, s), 1.65 (3H, s), 1.75 (3H, s), 3.02 (1H, m), 3.63 (3H, m), 3.72 (3H, m); MS (FAB) m/z 1094 ([M+Na]⁺), 1022 ([M-(OCH₃+H₂O)]⁺).

b) 40-O-(2-Hydroxy)ethyl-rapamycin

To a stirred, cooled (0°C) solution of 4.5 g (4.2 mmol) of 40-O-[2-(t-butyldimethylsilyl)oxy]ethyl-rapamycin in 20 mL of methanol is added 2 mL of IN HCl. This solution is stirred for 2h and neutralized with aq. sodium bicarbonate. The mixture is extracted with three portions of ethyl acetate. The organic solution is washed with aq.

sodium bicarbonate and brine, dried over anhydrous sodium sulfate, filtered and

concentrated. Purification by column chromatography on silica gel (ethyl acetate) gave the title compound as a white solid:¹H NMR (CDCl₃) δ 0.72 (1H, dd), 1.65 (3H, s), 1.75 (3H, s), 3.13 (5H, s and m), 3.52-3.91 (8H, m); MS (FAB) m/z 980 ([M+Na]⁺), 926 ([M-OCH₃]⁺), 908 ([M-(OCH₃+H₂O)]⁺), 890 ([M-(OCH₃+2H₂O)]⁺), 876 ([M-(2CH₃OH+OH)]⁺), 858 ([M-(OCH₃+CH₃OH+2H₂O)]⁺).

MBA (rel. IC50) 2.2

IL-6 dep. prol. (rel. IC50) 2.8

MLR (rel. IC50) 3.4

…………………..

synthesis

Everolimus (Everolimus) was synthesized by the Sirolimus (sirolimus, also known as rapamycin Rapamycin) ether from. Sirolimus is from the soil bacterium Streptomyces hygroscopicus isolated metabolites. Activation end sirolimus (triflate, Tf) the other end of the protection (t-butyldimethylsilyl, TBS) of ethylene glycol 1 reaction of 2 , because the hydroxyl group 42 hydroxyl site over the 31-bit resistance is small, so the reaction only occurs in 42. Compound 2under acidic conditions TBS protection is removed everolimus.

PATENT

https://patents.google.com/patent/WO2016020664A1/en

Everolimus (RAD-001) is the 40-O- 2-hydroxyethyl)-rapamycin of formula (I),

It is a derivative of sirolimus of formula III),

and works similarly to sirolimus as an inhibitor of mammalian target of rapamycin (mTOR). Everolimus is currently used as an immunosuppressant to prevent rejection of organ transplants and treatment of renal cell cancer and other tumours. It is marketed by Novartis under the tradenames Zortress™ (USA) and Certican™ (Europe and other countries) in transplantation medicine, and Afinitor™ in oncology.

Trisubstituted silyloxyethyltrifluoromethane sulfonates (triflates) of the general formula (IV),

wherein R₂, R₃ are independently a straight or branched alkyl group, for example C^-Cw alkyl, and/or an aryl group, for example a phenyl group, are important intermediates useful in the synthesis of everolimus.

Everolimus and its process for manufacture using the intermediate 2-(t-butyldimethyl silyl) oxyethyl triflate of formula (IVA),

was first described in US Patent Number 5,665,772. The overall reaction is depicted in Scheme I.

Sche

Everolimus (I)

For the synthesis, firstly sirolimus of formula (III) and 2-(t-butyldimethylsilyl)oxyethyl triflate of formula (IVA) are reacted in the presence of 2,6-Lutidine in toluene at around 60°C to obtain the corresponding 40-O-[2-(t-butyldimethylsilyl)oxy]ethyl rapamycin of formula (I la), which is then deprotected in aqueous hydrochloric acid and converted into crude everolimus [40-O-(2- Hydroxy)ethyl rapamycin] of formula (I). However, this process results in the formation of impure everolimus, which requires purification by column chromatography. The process results in very poor overall yield and purity and thereby the process is not suitable for the commercial scale production of everolimus.

Moenius et al. (I. Labelled Cpd. Radiopharm. 43, 1 13-120 (2000) have disclosed a process to prepare C-14 labelled everolimus using the diphenyltert-butylsilyloxy-protective group of formula (IV B),

as the alkylation agent. The overall yield reported was 25%. International patent application, publication number WO 2012/103960 discloses the preparation of everolimus using the alkylating agent 2-((2,3-dimethylbut-2-yl)dimethylsilyloxy)ethyl triflate of formula (IVC),

wherein the overall yield reported is 52.54%. The process involves a derivatization method based on the reaction of the triflate (IV) with a derivatization agent, which preferably is a secondary aromatic amine, typically N-methylaniline.

International patent application, publication number WO 2012/103959 also discloses the preparation of everolimus using the alkylating agent of formula (IVC). The process is based on a reaction of rapamycin with the compound of formula (IVC) in the presence of a base (such as an aliphatic tertiary amine) to form 40-O-2-(t-hexyldimethylsiloxy)ethylrapamycin, which is subsequently deprotected under acidic conditions to obtain everolimus. European Patent Number 1518517B discloses a process for the preparation of everolimus which employs the triflate compound of formula (IVA), 2-(t-butyldimethyl silyl) oxyethyl triflate. The disclosed process for preparing the compound of formula (IVA) involves a flash chromatography purification step. The compounds of formula (IV) are key intermediates in the synthesis of everolimus. However, they are highly reactive and also very unstable, and their use often results in decomposition during reaction with sirolimus. This is reflected by the fact that the yields of the reaction with sirolimus are very low and the compounds of formula (IV) are charged in high molar extent. Thus it is desirable to develop a process to stabilize compounds of formula (IV) without loss of reactivity

 Example 1 :

Step 1 : Preparation of protected everolimus (TBS-everoismus) of formula (Ma) using metal salt, wherein “Pg” is t-butyldimethylsilyl t-butyldimethylsilyloxy ethanol, of formula (VA) (2.8g, 0.016mol) was dissolved in dichloromethane (DCM) (3 vol) and to this 2,6-Lutidine (3.50 g, 0.0327 mol) was added and the mixture was cooled to -40°C. Thereafter, trifluoromethane sulfonic anhydride (3.59ml, 0.021 mol) was added drop-wise. The mixture was maintained at -40°C for 30 minutes. Sirolimus (0.5g, 0.00054mol) was taken in another flask and dissolved in DCM (1 ml). To this sirolimus solution, silver acetate (0.018g, 0.000109mol) was added and cooled to -40°C. The earlier cooled triflate solution was transferred in 3 lots to the sirolimus solution maintaining temperature at -40°C. The reaction mixture was stirred at -40°C further for 15min before which it was slowly warmed to 0°C and further to RT. The reaction mixture was then warmed to 40°C and maintained at this temperature for 3 hours. The reaction was monitored by TLC. On completion of reaction, the reaction mixture was diluted with DCM and washed with water and brine. The organic layer was dried over anhydrous sodium sulphate and solvent was removed by vacuum distillation to obtain the title compound, which was directly used in the next step. HPLC product purity: 60%-85%.

Step 2: Preparation of everolimus of formula (I) Protected everolimus of formula (I la) obtained in step 1 was dissolved in methanol (10 volumes) and chilled to 0-5° C. To this solution was added drop wise, a solution of 1 N HCI. The pH of the reaction was maintained between 1-3. The temperature of the reaction mixture was raised to 25° C and stirred for 1 hour. After completion of reaction, the reaction mixture was diluted with water (15 volumes) and extracted in ethyl acetate (2X20 volumes). The organic layers were combined and washed with brine, dried over sodium sulphate. The organic layer was distilled off under reduced pressure at 30-35° C, to obtain a crude everolimus (0.8 g). The crude everolimus was further purified by preparative HPLC to yield everolimus of purity >99%.

Example 2:

Step 1 : Preparation of TBS-everoiimus of formula (Ma) without using metal salt, wherein “Pg” is t-butyldimethylsilyl t-butyldimethylsilyloxy ethanol, of formula (VA) (2.8g, 0.016mol) was dissolved in DCM (3 vol) and to this 2,6-Lutidine (3.50 g, 0.0327 mol) was added and the mixture was cooled to -40°C. Thereafter, trifluoromethane sulfonic anhydride (3.59ml, 0.021 mol) was added drop-wise. The mixture was maintained at -40°C for 30 minutes. Sirolimus (0.5g, 0.00054mol) was taken in another flask and dissolved in DCM (1 ml). The solution was cooled to -40°C. The earlier cooled triflate solution was transferred in 3 lots to the sirolimus solution maintaining temperature at -40°C. The reaction mixture was stirred at -40°C further for 15min before which it was slowly warmed to 0°C and further to RT. The reaction mixture was then warmed to 40°C and maintained at this temperature for 3 hours. On completion of reaction, the reaction mixture was diluted with DCM and washed with water and brine. The organic layer was dried over anhydrous sodium sulphate and solvent was removed by vacuum distillation to obtain the title compound, which was directly used in next step. HPLC purity: 10%-20%.

Step 2: Preparation of everolimus of formula (I)

Protected everolimus of formula (I la) obtained in step 1 was dissolved in methanol (10 volumes) and chilled to 0-5° C. To this solution was added drop wise, a solution of 1 N HCI. The pH of the reaction was maintained between 1-3. The temperature of the reaction mixture was raised to 25° C and stirred for 1 hour. After completion of reaction, the reaction mixture was diluted with water (15 volumes) and extracted in ethyl acetate (2X20 volumes). The organic layers were combined and washed with brine, dried over sodium sulphate. The organic layer was distilled off under reduced pressure at 30-35° C, to obtain a crude everolimus which was further purified by preparative HPLC. Example 3:

Preparation of crude Everolimus

Step 1 : Preparation of TBS-ethylene glycol of formula (Va)

Ethylene glycol (1.5L, 26.58 mol) and TBDMS-CI (485g, 3.21 mol) were mixed together with stirring and cooled to 0°C. Triethyl amine (679 ml, 4.83 mol) was then added at 0°C in 30-45 minutes. After addition, the reaction was stirred for 12 hours at 25-30°C for the desired conversion. After completion of reaction, the layers were separated and the organic layer (containing TBS- ethylene glycol) was washed with water (1 L.x2) and brine solution (1 L). The organic layer was then subjected to high vacuum distillation to afford 350g of pure product.

Step 2: Preparation of TBS-glycol-Triflate of formula (IVa)

The reaction was carried out under a nitrogen atmosphere. TBS- ethylene glycol prepared as per step 1 (85.10g, 0.48 mol) and 2, 6-Lutidine (84.28ml, 0.72 mol) were stirred in n-heptane (425ml) to give a clear solution which was then cooled to -15 to – 25°C. Trif!uoromethanesulfonic anhydride (Tf₂0) (99.74 ml, 0.590 mol) was added drop-wise over a period of 45 minutes to the n-heptane solution (white precipitate starts to form immediately) while maintaining the reaction at -15 to – 25°C. The reaction mixture was kept at temperature between -15 to -25°C for 2 hours. The precipitate generated was filtered off. The filtrate was then evaporated up to ~2 volumes with respect to TBS-ethyiene glycol (~200 ml).

Step 3: Preparation of TBS-evero!imus of formula (Ha)

30g of sirolimus (0,0328 mo!) and toluene (150m!) were stirred together and the temperature was slowly raised to 60-65°C. At this temperature, a first portion of TBS-g!yco!-triflate prepared as per step 2 (100ml) and 2,6-Lutidine (1 1.45ml, 0.086 moles) were added and stirred for 40 min. Further, a second portion of TBS- glycol-triflate (50mi) and 2, 6-Lutidine (19.45ml, 0.138 mol) were added and the reaction was stirred for another 40 min. This was followed by a third portion of TBS- glycol- triflate (50m!) and 2, 6-Lutidine (19.45ml, 0.138 mol), after which the reaction was stirred for further 90 minutes. The reaction was monitored through HPLC to check the conversion of Sirolimus to TBS-everolimus after each addition of TBS-glycol-trifiate. After completion of the reaction, the reaction mixture was diluted with n-heptane (150mi), cooled to room temperature and stirred for another 60 minutes. The precipitated solids were filtered off and the filtrate was washed with deionized water (450 ml x4) followed by brine solution (450ml). The filtrate was subsequently distilled off to afford TBS-everolimus (60-65g) with 60-70% conversion from sirolimus.

Step 4: Preparation of everolimus of formula (I)

TBS-everolimus (65g) obtained in step 3 was dissolved in 300 mi methanol and cooled to 0°C. 1 N HCI was then added to the methanol solution (pH adjusted to 2-3) and stirred for 2 h. After completion of reaction, toluene (360m!) and deionized wafer (360mi) were added to the reaction mixture and the aqueous layer was separated. The organic layer was washed with brine solution (360ml). The organic layer was concentrated to obtain crude everolimus (39g) with an assay content of 30-35%, HPLC purity of 60-65%.

The crude everolimus purified by chromatography to achieve purity more than 99 %.

Patent

Publication numberPriority datePublication dateAssigneeTitleUS5665772A *1992-10-091997-09-09Sandoz Ltd.O-alkylated rapamycin derivatives and their use, particularly as immunosuppressantsEP1518517A2 *2002-04-242005-03-30Sun Biomedical, Ltd.Drug-delivery endovascular stent and method for treating restenosisWO2012103960A12011-02-042012-08-09Synthon BvProcess for making trisubstituted silyloxyethyl triflatesCN102786534A2012-05-252012-11-21上海现代制药股份有限公司Preparation method of everolimusCN103788114A *2012-10-312014-05-14江苏汉邦科技有限公司Preparation method for everolimusEP3166950A12014-08-042017-05-17Cipla LimitedProcess for the synthesis of everolimus and intermediates thereof 

CN107417718A *2017-08-182017-12-01常州兰陵制药有限公司The preparation method of everolimus intermediateUS9938297B22014-08-042018-04-10Cipia LimitedProcess for the synthesis of everolimus and intermediates thereofCN108676014A *2018-06-152018-10-19国药集团川抗制药有限公司The method for purifying the method for everolimus intermediate and preparing everolimus 

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References

• a WO 9 409 010 (Sandoz-Erfindungen; 28.4.1994; GB-prior. 9.10.1992).
• b US 6 277 983 (American Home Products; 21.8.2001; USA-prior. 27.9.2000).
•  US 6 384 046 (Novartis; 7.5.2002; GB-prior. 27.3.1996).
•  US 20 040 115 (Univ. of Pennsylvania; 15.1.2004; USA-prior. 9.7.2002).
• fermentation of rapamycin (sirolimus):
  • Chen, Y. et al.: Process Biochemistry (Oxford, U. K.) (PBCHE5) 34, 4, 383 (1999).
  • The Merck Index, 14th Ed., 666 (3907) (Rahway 2006).
  • US 3 929 992 (Ayerst McKenna & Harrison Ltd.; 30.12.1975; USA-prior. 29.9.1972).
  • WO 9 418 207 (Sandoz-Erfindungen; 18.8.1994; GB-prior. 2.2.1993).
  • EP 638 125 (Pfizer; 17.4.1996; J-prior. 27.4.1992).
  • US 6 313 264 (American Home Products; 6.11.2001; USA-prior. 8.3.1994).

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https://doi.org/10.1039/C7MD00474EIssue 1, 2018

• 
  MedChemComm

Ascomycins and rapamycins The ascomycin tacrolimus (44, FK-506) and the two rapamycins sirolimus (45, rapamycin) and everolimus (46) are macrolides that contain 21- and 29-membered macrocyclic rings, respectively (Figure 7).[3] Their MWs range from just over 800 Da for tacrolimus (44) to >900 Da for sirolimus (45) and everolimus (46) and they have >10 HBAs. Like other natural product derived drugs in bRo5 space, they are above average complexity (SMCM 119–134) due to their 14–15 chiral centres. All three are immunosuppressants that are mainly used to prevent rejection of transplanted organs. They bind to overlapping, but slightly different parts of a shallow pocket at the surface of the immunophilin FK506 binding protein (FKBP12, Figure 8 A). Whereas tacrolimus (44) only binds in the pocket on FKBP12 (Figure 8 B),[67] sirolimus (45) and everolimus (46) promote binding of mammalian target of rapamycin (mTOR) so that they bind in a groove formed by FKBP12 and mTOR (Figure 8 C).[68] The complex between tacrolimus (44) and FKBP12 inhibits calcineurin, which results in reduced production of interleukin-2 and inactivation of T cells. Formation of the ternary complexes between FKBP12, sirolimus (45) [or everolimus (46)] and mTOR inhibits mTOR, which arrests growth of T lymphocytes by reducing their sensitivity to interleukin 2. Both tacrolimus (44) and sirolimus (45) have low (15–20 %) and variable bioavailabilities, whereas the bioavailability of everolimus (46) has been increased somewhat as compared to sirolimus (45).[3] Tacrolimus (44) was isolated from Streptomyces tsukubaensis in 1987,[69, 70] while sirolimus (45) was first identified from a Streptomycete strain found in a soil sample from Easter Island.[71] Later it was also isolated from fermentation of another Streptomycete strain.[72, 73] Both drugs are now produced through fermentation.[74, 75] Sirolimus suffers from low bioavailability as well as toxicity, and semi-synthetic derivatives were therefore prepared to minimise these issues. This led to the discovery of everolimus (46), synthesised by selective alkylation of one of the two secondary hydroxyl groups of sirolimus (45) with 2-(tert-butyldimethylsilyl)oxyethyltriflate followed by silyl ether deprotection with HCl (Scheme 8).[76, 77]

Figure 7. Structures of the ascomycin tacrolimus (44) and the rapamycins sirolimus (45) and everolimus (46) that are used mainly to prevent rejection of organ transplants.

[67] G. D. Van Duyne, R. F. Standaert, P. A. Karplus, S. L. Schreiber, J. Clardy, Science 1991, 252, 839 – 842. [68] A. M. Marz, A.-K. Fabian, C. Kozany, A. Bracher, F. Hausch, Mol. Cell. Biol. 2013, 33, 1357 – 1367.

[69] T. Kino, H. Hatanaka, M. Hashimoto, M. Nishiyama, T. Goto, M. Okuhara, M. Kohsaka, H. Aoki, H. Imanaka, J. Antibiot. 1987, 40, 1249 – 1255. [70] H. Tanaka, A. Kuroda, H. Marusawa, H. Hatanaka, T. Kino, T. Goto, M. Hashimoto, T. Taga, J. Am. Chem. Soc. 1987, 109, 5031 – 5033. [71] C. Vzina, A. Kudelski, S. N. Sehgal, J. Antibiot. 1975, 28, 721 – 726. [72] S. N. Sehgal, H. Baker, C. Vzina, J. Antibiot. 1975, 28, 727 – 732. [73] S. N. Sehgal, T. M. Blazekovic, C. Vzina, 1975, US3929992A. [74] C. Barreiro, M. Mart nez-Castro, Appl. Microbiol. Biotechnol. 2014, 98, 497 – 507. [75] S. R. Park, Y. J. Yoo, Y.-H. Ban, Y. J. Yoon, J. Antibiot. 2010, 63, 434 – 441. [76] F. Navarro, S. Petit, G. Stone, 2007, US20020032213A1. [77] S. Cottens, R. Sedrani, 1997, US5665772A.

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Ferreting out why some cancer drugs struggle to shrink tumors

Study shows how stopping one enzyme could help drugs treat an important class of cancers more effectively

by Stu Borman

JUNE 27, 2018 | APPEARED IN VOLUME 96, ISSUE 27

In several types of cancer, including most cases of breast cancer, a cell-signaling network called the PI3K pathway is overactive. Drug designers have tried to quiet this pathway to kill cancer, but they haven’t had much success and, more frustratingly, haven’t understood why the problem is so hard to solve.

“There have been more than 200 clinical trials with experimental drugs that target the PI3K pathway, and probably more than $1 billion invested,” says Sourav Bandyopadhyay of the University of California, San Francisco. Just a handful of drugs have been approved by the U.S. FDA and one, Novartis’s Afinitor (everolimus), deters cancer growth but doesn’t shrink tumors, and it prolongs patient survival only a few months.

Bandyopadhyay, his UCSF colleague John D. Gordan, and coworkers used a proteomics approach to ferret out why previous attempts to target the PI3K pathway have had limited success and, using that information, devised and tested a possible fix (Nat. Chem. Biol. 2018, DOI: 10.1038/s41589-018-0081-9).

The stubborn pathway involves a series of kinases—enzymes that modify other proteins by adding phosphate groups—starting with one called PI3K. Overactivation of the pathway produces the transcription factor MYC, which turns on protein synthesis and can spark cancer growth.

The UCSF team used kinase-affinity beads and tandem mass spectrometry to survey all kinases active in breast cancer cells before and after treatment with a variety of cancer drugs. The team studied this so-called kinome to look for kinases associated with the cells’ tendency to resist drug treatments.

The researchers found that a kinase called AURKA undermines everolimus and other pathway-targeted drugs by reversing their effects. While the drugs try to turn off the PI3K pathway, AURKA, activated separately by other pathways, keeps the PI3K pathway turned on. To add insult to injury, MYC boosts AURKA production, maintaining a plentiful supply of the drug spoiler.

When the researchers coadministered everolimus with the AURKA inhibitor MLN8237, also called alisertib, everolimus could inhibit the PI3K pathway as it was designed to do, without interference. The combination treatment killed most types of cancer cells in culture and shrank tumors in mice with breast cancer, whereas everolimus alone permitted slow tumor growth to continue.

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19. ^ Wang S, Raybuck A, Shiuan E, Jin J (2020). “Selective inhibition of mTORC1 in tumor vessels increases antitumor immunity”. JCI Insight. 5 (15): e139237. doi:10.1172/jci.insight.139237. PMC 7455083. PMID 32759497.
20. ^ Jump up to:^a b “Archived copy”. Archived from the original on 8 March 2014. Retrieved 26 February 2014.
21. ^ Eisen HJ, Tuzcu EM, Dorent R, Kobashigawa J, Mancini D, Valantine-von Kaeppler HA, Starling RC, Sørensen K, Hummel M, Lind JM, Abeywickrama KH, Bernhardt P (August 2003). “Everolimus for the prevention of allograft rejection and vasculopathy in cardiac-transplant recipients”. The New England Journal of Medicine. 349 (9): 847–58. doi:10.1056/NEJMoa022171. PMID 12944570.
22. ^ Jeng LB, Thorat A, Hsieh YW, Yang HR, Yeh CC, Chen TH, Hsu SC, Hsu CH (April 2014). “Experience of using everolimus in the early stage of living donor liver transplantation”. Transplantation Proceedings. 46 (3): 744–8. doi:10.1016/j.transproceed.2013.11.068. PMID 24767339.
23. ^ Jeng L, Thorat A, Yang H, Yeh C-C, Chen T-H, Hsu S-C. Impact of Everolimus On the Hepatocellular Carcinoma Recurrence After Living Donor Liver Transplantation When Used in Early Stage: A Single Center Prospective Study [abstract]. Am J Transplant. 2015; 15 (suppl 3). http://www.atcmeetingabstracts.com/abstract/impact-of-everolimus-on-the-hepatocellular-carcinoma-recurrence-after-living-donor-liver-transplantation-when-used-in-early-stage-a-single-center-prospective-study/. Accessed 1 September 2015.
24. ^ Thorat A, Jeng LB, Yang HR, Yeh CC, Hsu SC, Chen TH, Poon KS (November 2017). “Assessing the role of everolimus in reducing hepatocellular carcinoma recurrence after living donor liver transplantation for patients within the UCSF criteria: re-inventing the role of mammalian target of rapamycin inhibitors”. Annals of Hepato-Biliary-Pancreatic Surgery. 21 (4): 205–211. doi:10.14701/ahbps.2017.21.4.205. PMC 5736740. PMID 29264583.
25. ^ Jeng LB, Lee SG, Soin AS, Lee WC, Suh KS, Joo DJ, Uemoto S, Joh J, Yoshizumi T, Yang HR, Song GW, Lopez P, Kochuparampil J, Sips C, Kaneko S, Levy G (December 2017). “Efficacy and safety of everolimus with reduced tacrolimus in living-donor liver transplant recipients: 12-month results of a randomized multicenter study”. American Journal of Transplantation. 18 (6): 1435–1446. doi:10.1111/ajt.14623. PMID 29237235.
26. ^ Harrison DE, Strong R, Sharp ZD, Nelson JF, Astle CM, Flurkey K, Nadon NL, Wilkinson JE, Frenkel K, Carter CS, Pahor M, Javors MA, Fernandez E, Miller RA (July 2009). “Rapamycin fed late in life extends lifespan in genetically heterogeneous mice”. Nature. 460 (7253): 392–5. Bibcode:2009Natur.460..392H. doi:10.1038/nature08221. PMC 2786175. PMID 19587680.
27. ^ Mannick JB, Del Giudice G, Lattanzi M, Valiante NM, Praestgaard J, Huang B, Lonetto MA, Maecker HT, Kovarik J, Carson S, Glass DJ, Klickstein LB (December 2014). “mTOR inhibition improves immune function in the elderly”. Science Translational Medicine. 6 (268): 268ra179. doi:10.1126/scitranslmed.3009892. PMID 25540326. S2CID 206685475.

Further reading

• Sedrani R, Cottens S, Kallen J, Schuler W (August 1998). “Chemical modification of rapamycin: the discovery of SDZ RAD”. Transplantation Proceedings. 30 (5): 2192–4. doi:10.1016/S0041-1345(98)00587-9. PMID 9723437.

External links

• “Everolimus”. Drug Information Portal. U.S. National Library of Medicine.

Clinical data
Pronunciation	Everolimus /ˌɛvəˈroʊləməs/
Trade names	Afinitor, Zortress
Other names	42-O-(2-hydroxyethyl)rapamycin, RAD001
AHFS/Drugs.com	Monograph
MedlinePlus	a609032
License data	EU EMA: by INNUS DailyMed: EverolimusUS FDA: Everolimus
Pregnancy category	AU: C[1]
Routes of administration	By mouth
ATC code	L01EG02 (WHO) L04AA18 (WHO)
Legal status
Legal status	US: ℞-onlyEU: Rx-onlyIn general: ℞ (Prescription only)
Pharmacokinetic data
Elimination half-life	~30 hours[2]
Identifiers
showIUPAC name
CAS Number	159351-69-6
PubChem CID	6442177
DrugBank	DB01590
ChemSpider	21106307
UNII	9HW64Q8G6G
KEGG	D02714
ChEMBL	ChEMBL1908360
CompTox Dashboard (EPA)	DTXSID0040599
ECHA InfoCard	100.149.896
Chemical and physical data
Formula	C53H83NO14
Molar mass	958.240 g·mol−1
3D model (JSmol)	Interactive image
hideSMILESOCCO[C@@H]1CC[C@H](C[C@H]1OC)C[C@@H](C)[C@@H]4CC(=O)[C@H](C)/C=C(\C)[C@@H](O)[C@@H](OC)C(=O)[C@H](C)C[C@H](C)\C=C\C=C\C=C(/C)[C@@H](OC)C[C@@H]2CC[C@@H](C)[C@@](O)(O2)C(=O)C(=O)N3CCCC[C@H]3C(=O)O4
hideInChIInChI=1S/C53H83NO14/c1-32-16-12-11-13-17-33(2)44(63-8)30-40-21-19-38(7)53(62,68-40)50(59)51(60)54-23-15-14-18-41(54)52(61)67-45(35(4)28-39-20-22-43(66-25-24-55)46(29-39)64-9)31-42(56)34(3)27-37(6)48(58)49(65-10)47(57)36(5)26-32/h11-13,16-17,27,32,34-36,38-41,43-46,48-49,55,58,62H,14-15,18-26,28-31H2,1-10H3/b13-11+,16-12+,33-17+,37-27+/t32-,34-,35-,36-,38-,39+,40+,41+,43-,44+,45+,46-,48-,49+,53-/m1/s1 Key:HKVAMNSJSFKALM-GKUWKFKPSA-N

////////////////  RAD-001,  SDZ RAD, Certican, Novartis, Immunosuppressant, Everolimus, Afinitor, эверолимус , إيفيروليموس , 依维莫司 , 

Everolimus

159351-69-6[RN]
23,27-Epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone, 9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-(2-hydr oxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-, (3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,26R,27R,34aS)-
23,27-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone, 9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-, (3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-
42-O-(2-Hydroxyethyl)rapamycin

• Synonyms:RAD-001, SDZ-RAD, Afinitor
• ATC:L04AA18

Use:immunosuppressantChemical name:42-O-(2-hydroxyethyl)rapamycinFormula:C₅₃H₈₃NO₁₄

• MW:958.24 g/mol
• CAS-RN:159351-69-6

EverolimusCAS Registry Number: 159351-69-6CAS Name: 42-O-(2-Hydroxyethyl)rapamycinAdditional Names: 40-O-(2-hydroxyethyl)rapamycinManufacturers’ Codes: RAD-001; SDZ RADTrademarks: Certican (Novartis)Molecular Formula: C53H83NO14Molecular Weight: 958.22Percent Composition: C 66.43%, H 8.73%, N 1.46%, O 23.38%Literature References: Macrolide immunosuppressant; derivative of rapamycin, q.v. Inhibits cytokine-mediated lymphocyte proliferation. Prepn: S. Cottens, R. Sedrani, WO9409010; eidem, US5665772 (1994, 1997 both to Sandoz). Pharmacology: W. Schuler et al., Transplantation64, 36 (1997). Whole blood determn by LC/MS: N. Brignol et al., Rapid Commun. Mass Spectrom.15, 898 (2001); by HPLC: S. Baldelli et al., J. Chromatogr. B816, 99 (2005). Clinical pharmacokinetics in combination with cyclosporine: J. M. Kovarik et al., Clin. Pharmacol. Ther.69, 48 (2001). Clinical study in prevention of cardiac-allograft vasculopathy: H. J. Eisen et al.,N. Engl. J. Med.349, 847 (2003). Review: F. J. Dumont et al., Curr. Opin. Invest. Drugs2, 1220-1234 (2001); B. Nashan, Ther. Drug Monit.24, 53-58 (2002).Therap-Cat: Immunosuppressant.Keywords: Immunosuppressant.эверолимус[Russian][INN]إيفيروليموس[Arabic][INN]依维莫司[Chinese][INN]Trade Name：Certican^® / Zortress^® / Afinitor^®MOA：mTOR inhibitorIndication：Rejection of organ transplantation; Renal cell carcinoma; Advanced renal cell carcinoma (RCC); Advanced breast cancer; Pancreatic cancer; Renal angiomyolipoma; Tuberous sclerosis complex (TSC); Rejection in heart transplantation; Rejection of suppression renal transplantation; Subependymal giant cell astrocytoma; neuroendocrine tumors (NET); Advanced gastrointestinal tumorsStatus：ApprovedCompany：Novartis (Originator)Sales：$1,942 Million (Y2015);
$1,902 Million (Y2014);
$1,558 Million (Y2013);
$1,007 Million (Y2012);
$630 Million (Y2011);ATC Code：L04AA18Approved Countries or Area

• US
• EU
• JP
• CN
• SE

Approval Date	Approval Type	Trade Name	Indication	Dosage Form	Strength	Company	Review Classification
2012-08-29	New dosage form	Afinitor Disperz	Renal cell carcinoma , Advanced breast cancer, Pancreatic cancer, Renal angiomyolipoma, Tuberous sclerosis complex (TSC)	Tablet, For suspension	2 mg/3 mg/5 mg	Novartis	Priority
2010-04-20	New strength	Zortress	Advanced renal cell carcinoma (RCC)	Tablet	0.25 mg/0.5 mg/0.75 mg	Novartis
2009-03-30	Marketing approval	Afinitor	Advanced renal cell carcinoma (RCC)	Tablet	2.5 mg/5 mg/7.5 mg/10 mg	Novartis	Priority

Approval Date	Approval Type	Trade Name	Indication	Dosage Form	Strength	Company	Review Classification
2016-06-02	New indication	Afinitor	neuroendocrine tumors (NET), Advanced gastrointestinal tumors	Tablet		Novartis
2011-09-02	Marketing approval	Votubia	Advanced breast cancer, Renal cell carcinoma , Pancreatic cancer	Tablet	2.5 mg/5 mg/10 mg	Novartis	Orphan; Conditional Approval
2011-09-02	Marketing approval	Votubia	Advanced breast cancer, Renal cell carcinoma , Pancreatic cancer	Tablet, Orally disintegrating	2 mg/3 mg/5 mg	Novartis	Orphan; Conditional Approval
2009-08-03	Marketing approval	Afinitor	Advanced breast cancer, Renal cell carcinoma , Pancreatic cancer	Tablet	2.5 mg/5 mg/10 mg	Novartis

Approval Date	Approval Type	Trade Name	Indication	Dosage Form	Strength	Company	Review Classification
2011-12-22	New indication	Certican	Rejection of suppression renal transplantation	Tablet	0.25 mg/0.5 mg/0.75 mg	Novartis
2007-01-26	Marketing approval	Certican	Rejection in heart transplantation	Tablet	0.25 mg/0.5 mg/0.75 mg	Novartis

More

Approval Date	Approval Type	Trade Name	Indication	Dosage Form	Strength	Company	Review Classification
2014-02-13	Marketing approval	飞尼妥/Afinitor	Advanced renal cell carcinoma (RCC), Subependymal giant cell astrocytoma	Tablet	2.5 mg	Novartis
2013-01-22	Marketing approval	飞尼妥/Afinitor	Advanced renal cell carcinoma (RCC), Subependymal giant cell astrocytoma	Tablet	10 mg	Novartis
2013-01-22	Marketing approval	飞尼妥/Afinitor	Advanced renal cell carcinoma (RCC), Subependymal giant cell astrocytoma	Tablet	5 mg	Novartis

More

Approval Date	Approval Type	Trade Name	Indication	Dosage Form	Strength	Company	Review Classification
2003-07-18	Marketing approval	Certican	Rejection of organ transplantation, Renal cell carcinoma	Tablet	0.25 mg/0.5 mg/0.75 mg	Novartis

clip

Click to access afinitor-epar-public-assessment-report_en.pdf

Active Substance The active substance Everolimus is a hydroxyethyl derivative of rapamycin, which is a macrolide, isolated from the micro-organism Streptomyces hygroscopicus. The guideline, impurities in new active substances ICHQ 3A (R), does not apply to active substance of fermented origin. Everolimus (INN) or 42-O-(2-hydroxyethyl)-rapamycin (chemical name) or C5 3H8 3N O1 4 has been fully described. The molecule is amorphous and is stabilised with an antioxidant. Its physico-chemical properties including parameters such as solubility, pH, specific rotation, potential polymorphism and potential isomerism have been fully characterised. Everolimus is a white to faintly yellow amorphous powder. It is almost insoluble in water, is unstable at temperatures above 25 °C and is sensitive to light. In addition, possible isomerism has been investigated. Everolimus contains 15 asymmetric carbon atoms and 4 substituted double bonds. The configuration of the asymmetric carbon atoms and the double bonds is guaranteed by the microbial origin of Rapamycin. The configuration is not affected by the chemical synthesis. Polymorphism has been comprehensively discussed and it was demonstrated that the molecule domain remains amorphous.

Synthesis of Everolimus The manufacturing process consists of four main steps, (1) fermentation, (2) extraction of rapamycin from the fermentation broth, (3) chemical modification of rapamycin starting material, (4) purification of crude everolimus and stabilisation with BHT. The choice of the stabilizer has been sufficiently explained and justified by experimental results. Interactions products of Everolimus and the antioxidant were not detected, or were below detection limit. Rapamycin, obtained by a fermentation process, was used as the starting material. Reaction conditions and the necessary in-process controls are described in detail. Adequate specifications for starting materials and isolated intermediates and descriptions of the test procedures have been submitted. Control of the quality of solvents, reagents and auxiliary materials used in the synthesis has been adequately documented. It is stated by the manufacturer of rapamycin solution that no starting material of animal or human origin is used in the fermentation. Elucidation of structure and other characteristics The structure of Everolimus has been fully elucidated using several spectroscopic techniques such as ultraviolet absorption spectroscopy (UV), Infra-red spectroscopy (FT-IR), proton and carbon nuclear magnetic resonance spectroscopy (1 H and 13C NMR), mass spectroscopy, diffractometry (X-ray) and elemental analysis. Related substances An extensive discussion was presented on the related substances. The complex structure of Everolimus allows several possible degradation pathways to occur at various positions of the molecule. Everolimus alone is extremely sensitive to oxidation. By the addition of an antioxidant, the sensitivity to oxidation is significantly reduced (the antioxidant is known to react as a scavenger of peroxide radicals). It is assumed that oxidation of Everolimus proceeds via a radical mechanism. All the requirements set in the current testing instruction valid for Everolimus are justified on the basis of the results obtained during development and manufactured at the production scale.

fda

Click to access 022334s000_ChemR.pdf

Everolimus was first approved by Swiss Agency for therapeutic products，Swissmedic on July 18, 2003, then approved by Pharmaceuticals and Medicals Devices Agency of Japan (PMDA) on April 23, 2004, and approved by the U.S. Food and Drug Administration (FDA) on Mar 30, 2009, approved by European Medicine Agency (EMA) on Aug 3, 2009. It was developed and marketed as Certican^® by Novartis in SE.

Everolimus is an inhibitor of mammalian target of rapamycin (mTOR). It is indicated for the treatment of renal cell cancer and other tumours and currently used as an immunosuppressant to prevent rejection of organ transplants.

Certican^® is available as tablet for oral use, containing 0.25, 0.5 or 0.75 mg of free Everolimus. The recommended dose is 10 mg once daily with or without food for advanced HR+ breast cancer, advanced progressive neuroendocrine tumors, advanced renal cell carcinoma or renal angiomyolipoma with tuberous sclerosis complex.
Everolimus, also known as RAD001, is a derivative of the natural macrocyclic lactone sirolimus with immunosuppressant and anti-angiogenic properties. In cells, everolimus binds to the immunophilin FK Binding Protein-12 (FKBP-12) to generate an immunosuppressive complex that binds to and inhibits the activation of the mammalian Target of Rapamycin (mTOR), a key regulatory kinase. Inhibition of mTOR activation results in the inhibition of T lymphocyte activation and proliferation associated with antigen and cytokine (IL-2, IL-4, and IL-15) stimulation and the inhibition of antibody production.

Everolimus is a medication used as an immunosuppressant to prevent rejection of organ transplants and in the treatment of renal cell cancer and other tumours. Much research has also been conducted on everolimus and other mTOR inhibitors as targeted therapy for use in a number of cancers.^{[medical citation needed]}

It is the 40-O-(2-hydroxyethyl) derivative of sirolimus and works similarly to sirolimus as an inhibitor of mammalian target of rapamycin (mTOR).

It is marketed by Novartis under the trade names Zortress (USA) and Certican (European Union and other countries) in transplantation medicine, and as Afinitor (general tumours) and Votubia (tumours as a result of TSC) in oncology. Everolimus is also available from Biocon, with the brand name Evertor.

Medical uses

Everolimus is approved for various conditions:

• Advanced kidney cancer (US FDA approved in March 2009)^[3]
• Prevention of organ rejection after renal transplant(US FDA April 2010)^[4]
• Subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis (TS) in patients who are not suitable for surgical intervention (US FDA October 2010)^[5]
• Progressive or metastatic pancreatic neuroendocrine tumors not surgically removable (May 2011)^[6]
• Breast cancer in post-menopausal women with advanced hormone-receptor positive, HER2-negative type cancer, in conjunction with exemestane (US FDA July 2012)^[7]
• Prevention of organ rejection after liver transplant(Feb 2013)
• Progressive, well-differentiated non-functional, neuroendocrine tumors (NET) of gastrointestinal (GI) or lung origin with unresectable, locally advanced or metastatic disease (US FDA February 2016).^[8]
• Tuberous sclerosis complex-associated partial-onset seizures for adult and pediatric patients aged 2 years and older. (US FDA April 2018).^[9]

UK National Health Service

NHS England has been criticised for delays in deciding on a policy for the prescription of everolimus in the treatment of Tuberous Sclerosis. 20 doctors addressed a letter to the board in support of the charity Tuberous Scelerosis Association saying ” around 32 patients with critical need, whose doctors believe everolimus treatment is their best or only option, have no hope of access to funding. Most have been waiting many months. Approximately half of these patients are at imminent risk of a catastrophic event (renal bleed or kidney failure) with a high risk of preventable death.”^[10] In May 2015 it was reported that Luke Henry and Stephanie Rudwick, the parents of a child suffering from Tuberous Sclerosis were trying to sell their home in Brighton to raise £30,000 to pay for treatment for their daughter Bethany who has tumours on her brain, kidneys and liver and suffers from up to 50 epileptic fits a day.^[11]

Clinical trials

As of October 2010, Phase III trials are under way in gastric cancer, hepatocellular carcinoma, and lymphoma.^[12] The experimental use of everolimus in refractory chronic graft-versus-host disease was reported in 2012.^[13]

Interim phase III trial results in 2011 showed that adding Afinitor (everolimus) to exemestane therapy against advanced breast cancer can significantly improve progression-free survival compared with exemestane therapy alone.^[14]

A study published in 2012, shows that everolimus sensitivity varies between patients depending on their tumor genomes.^[15] A group of patients with advanced metastasic bladder carcinoma (NCT00805129) ^[16] treated with everolimus revealed a single patient who had a complete response to everolimus treatment for 26 months. The researchers sequenced the genome of this patient and compared it to different reference genomes and to other patients’ genomes. They found that mutations in TSC1 led to a lengthened duration of response to everolimus and to an increase in the time to cancer recurrence. The mutated TSC1 apparently had made these tumors vulnerable to treatment with everolimus.^{[medical citation needed]}

A phase 2a randomized, placebo-controlled everolimus clinical trial published in 2014 showed that everolimus improved the response to an influenza vaccine by 20% in healthy elderly volunteers.^[17] A phase 2a randomized, placebo-controlled clinical trial published in 2018 showed that everolimus in combination with dactolisib decreased the rate of reported infections in an elderly population.^[17]

Mechanism

Compared with the parent compound rapamycin, everolimus is more selective for the mTORC1 protein complex, with little impact on the mTORC2 complex.^[18] This can lead to a hyper-activation of the kinase AKT via inhibition on the mTORC1 negative feedback loop, while not inhibiting the mTORC2 positive feedback to AKT. This AKT elevation can lead to longer survival in some cell types.^{[medical citation needed]} Thus, everolimus has important effects on cell growth, cell proliferation and cell survival.

mTORC1 inhibition by everolimus has been shown to normalize tumor blood vessels, to increase tumor-infiltrating lymphocytes, and to improve adoptive cell transfer therapy.^[19]

Additionally, mTORC2 is believed to play an important role in glucose metabolism and the immune system, suggesting that selective inhibition of mTORC1 by drugs such as everolimus could achieve many of the benefits of rapamycin without the associated glucose intolerance and immunosuppression.^[18]

TSC1 and TSC2, the genes involved in tuberous sclerosis, act as tumor suppressor genes by regulating mTORC1 activity. Thus, either the loss or inactivation of one of these genes lead to the activation of mTORC1.^[20]

Everolimus binds to its protein receptor FKBP12, which directly interacts with mTORC1, inhibiting its downstream signaling. As a consequence, mRNAs that code for proteins implicated in the cell cycle and in the glycolysis process are impaired or altered, and tumor growth is inhibited.^[20]

Adverse reactions

A trial using 10 mg/day in patients with NETs of GI or lung origin reported “Everolimus was discontinued for adverse reactions in 29% of patients and dose reduction or delay was required in 70% of everolimus-treated patients. Serious adverse reactions occurred in 42% of everolimus-treated patients and included 3 fatal events (cardiac failure, respiratory failure, and septic shock). The most common adverse reactions (incidence greater than or equal to 30%) were stomatitis, infections, diarrhea, peripheral edema, fatigue and rash. The most common blood abnormalities found (incidence greater than or equal to 50%) were anemia, hypercholesterolemia, lymphopenia, elevated aspartate transaminase (AST) and fasting hyperglycemia.”.^[8]

Role in heart transplantation

Everolimus may have a role in heart transplantation, as it has been shown to reduce chronic allograft vasculopathy in such transplants. It also may have a similar role to sirolimus in kidney and other transplants.^[21]

Role in liver transplantation

Although, sirolimus had generated fears over use of m-TOR inhibitors in liver transplantation recipients, due to possible early hepatic artery thrombosis and graft loss, use of everolimus in the setting of liver transplantation is promising. Jeng et al.,^[22] in their study of 43 patients, concluded the safety of everolimus in the early phase after living donor liver transplantation. In their study, no hepatic artery thrombosis or wound infection was noted. Also, a possible role of everolimus in reducing the recurrence of hepatocellular carcinoma after liver transplantation was correlated. A target trough level of 3 ng/mL at 3 months was shown to be beneficial in recipients with pre-transplant renal dysfunction. In their study, 6 of 9 renal failure patients showed significant recovery of renal function, whereas 3 showed further deterioration, one of whom required hemodialysis.^[23] Recently published report by Thorat et al. showed a positive impact on hepatocellular carcinoma (HCC) when everolimus was used as primary immunosuppression starting as early as first week after living donor liver transplantation (LDLT) surgery.^[24] In their retrospective and prospective analysis at China Medical University Hospital in Taiwan, the study cohort (n=66) was divided in two groups depending upon the postoperative immunosuppression. Group A: HCC patients that received Everolimus + Tacrolimus based immunosuppressive regimen (n=37). Group B: HCC patients that received standard Tacrolimus based immunosuppressive regimen without everolimus (n=29). The target trough level for EVR was 3 to 5 ng/ml while for TAC it was 8–10 ng/ml. The 1-year, 3-year and 4-year overall survival achieved for Group A patients (Everolimus group) was 94.95%, 86.48% and 86.48%, respectively while for Group B patients it was 82.75%, 68.96%, and 62.06%, respectively (p=0.0217). The first 12-month report of ongoing Everolimus multicenter prospective trial in LDLT (H2307 trial), Jeng LB et al. have shown a 0% recurrence of HCC in everolimus group at 12 months.^[25] Jeng LB concluded that an early introduction of everolimus + reduced tacrolimus was non-inferior to standard tacrolimus in terms of efficacy and renal function at 12 months, with HCC recurrence only in tacrolimus control patients.

Use in vascular stents

Everolimus is used in drug-eluting coronary stents as an immunosuppressant to prevent restenosis. Abbott Vascular produce an everolimus-eluting stent (EES) called Xience Alpine. It utilizes the Multi-Link Vision cobalt chromium stent platform and Novartis’ everolimus. The product is widely available globally including the US, the European Union, and Asia-Pacific (APAC) countries. Boston Scientific also market EESes, recent offerings being Promus Elite and Synergy.^{[citation needed]}

Use in aging

Inhibition of mTOR, the molecular target of everolimus, extends the lifespan of model organisms including mice,^[26] and mTOR inhibition has been suggested as an anti-aging therapy. Everolimus was used in a clinical trial by Novartis, and short-term treatment was shown to enhance the response to the influenza vaccine in the elderly, possible by reversing immunosenescence.^[27] Everolimus treatment of mice results in reduced metabolic side effects compared to sirolimus.^[18]Route 1

Reference：1. US5665772A.

2. Drug. Future 1999, 24, 22-29.Route 2

Reference：1. WO2014203185A1.Route 3

Reference：1. WO2012103959A1.Route 4

Reference：1. CN102731527A.

SYN

Synthetic Reference

Wang, Feng. Everolimus intermediate and preparation method thereof. Assignee Shanghai Institute of Pharmaceutical Industry, Peop. Rep. China; China State Institute of Pharmaceutical Industry. CN 109776570. (2019).

SYN 2

Synthetic Reference

Polymer compositions containing a macrocyclic triene compound; Shulze, John E.; Betts, Ronald E.; Savage, Douglas R.; Assignee Sun Bow Co., Ltd., Bermuda; Sun Biomedical Ltd. 2003; Patent Information; Nov 06, 2003; WO 2003090684 A2

SYN 3

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Wang, Feng. Everolimus intermediate and preparation method thereof. Assignee Shanghai Institute of Pharmaceutical Industry, Peop. Rep. China; China State Institute of Pharmaceutical Industry. CN 109776570. (2019).

SYN 4

Synthetic Reference

Zabudkin, Oleksandr; Schickaneder, Christian; Matviienko, Iaroslav; Sypchenko, Volodymyr. Method for the synthesis of rapamycin derivatives. Assignee Synbias Pharma AG, Switz. EP 3109250. (2016).

SYN 5

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Lu, Shiyong; Zhang, Xiaotian; Chen, Haohan; Ye, Weidong. Preparation of sirolimus 40-ether derivative. Assignee Zhejiang Medicine Co., Ltd. Xinchang Pharmaceutical Factory, Peop. Rep. China. CN 105237549. (2016).

SYN 6

Synthetic Reference

Seo, Jeong U.; Ham, Yun Beom; Kang, Heung Mo; Lee, Gwang Mu; Kim, In Gyu; Kim, Jeong Jin; Park, Ji Su. Preparation of everolimus and synthetic intermediate thereof. Assignee CKD Bio Corp., S. Korea. KR 1529963 (2015).

SYN

EP 0663916; EP 0867438; JP 1996502266; JP 1999240884; US 5665772; WO 9409010

Alkylation of rapamycin (I) with 2-(tert-butyldimethylsilyloxy)ethyl triflate (II) by means of 2,6-lutidine in hot toluene gives the silylated target compound (III), which is deprotected by means of 1N HCl in methanol.

SYN

J Label Compd Radiopharm 1999,42(1),29

The compound has been obtained biosynthetically by an optimized fermentation process using Streptomyces hygroscopicus mutant RSH 1701 with a complex culture medium were [14C]-labeled (1R,3R,4R)-2,3-dichydroxycyclo-hexanecarboxylic acid (I) and [14C]-labeled (S)-pipecolic acid (II) have been added. This fermentation process yielded [14C]-labeled rapamycin (III), which was finally selectively O-alkylated at the C-40 position with monosilylated ethylene glycol triflate in DMSO/dimethoxyethane.

SYN

The reaction of the labeled acylated (+)-bornane-10,2-sultam (IV) with triethyl phosphite gives the phosphonate (V), which is treated with paraformaldehyde, galvinoxyl and K2CO3 yielding the acrylate derivative (VI). The cyclization of (VI) with butadiene (VII) by means of diethylaluminum chloride and galvinoxyl (as radical scavenger) affords the cyclohexene-carboxamide derivative (VIII), which is hydrolyzed with LiOH in THF/water giving the (1R)-3-cyclohexenecarboxylic acid (IX). The oxidation of (IX) with m-chloroperbenzoic acid and triethylamine in CCl4 yielded regioselectively the hydroxylactone (X), which is finally hydrolyzed with HCl to the labeled intermediate (I).

SYN

The reaction of the labeled acylated (-)-bornane-10,2-sultam (XI) with benzophenone imine (XII) gives the glycylsultam derivative (XIII), which is alkylated with 4-iodobutyl chloride (XIV) by means of butyllithium and DMPU in THF yielding intermediate (XV). The selective hydrolysis of (XV) with HCl affords the omega-chloro-L-norleucine derivative (XVI), which is cyclized by means of tetrabutylammonium fluoride and DIEA in hot acetonitrile giving the (2S)-piperidyl derivative (XVII). Finally, this compound is hydrolyzed with LiOH in THF/water to the labeled intermediate (II).

clipRapamycin is a known macrolide antibiotic produced by Streptomvces hvgroscopicus. having the structure depicted in Formula A:

See, e.g., McAlpine, J.B., et al., J. Antibiotics (1991) 44: 688; Schreiber, S.L., et al., J. Am. Chem. Soc. (1991) J_13: ⁷⁴³³‘- US Patent No. 3 929 992. Rapamycin is an extremely potent immunosuppressant and has also been shown to have antitumor and antifungal activity. Its utility as a pharmaceutical, however, is restricted by its very low and variable bioavailabiiity as well as its high toxicity. Moreover, rapamycin is highly insoluble, making it difficult to formulate stable galenic compositions.

Everolimus, 40-O-(2-hydroxyethyl)-rapamycin of formula (1) is a synthetic derivative of rapamycin (sirolimus) of formula (2), which is produced by a certain bacteria strain and is also pharmaceutically active.

(1)                                                                                                               (2)

Everolimus is marketed under the brand name Certican for the prevention of rejection episodes following heart and kidney transplantation, and under the brand name Afinitor for treatment of advanced kidney cancer.

Due to its complicated macrolide chemical structure, everolimus is, similarly as the parent rapamycin, an extremely unstable compound. It is sensitive, in particular, towards oxidation, including aerial oxidation. It is also unstable at temperatures higher than 25°C and at alkaline pH.

Everolimus and a process of making it have been disclosed in WO 94/09010

Synthesis

Alkylation of rapamycin (I) with 2-(tert-butyldimethylsilyloxy)ethyl triflate (II) by means of 2,6-lutidine in hot toluene gives the silylated target compound (III), which is deprotected by means of 1N HCl in methanol (1). (Scheme 21042401a) Manufacturer Novartis AG (CH). References 1. Cottens, S., Sedrani, R. (Sandoz-Refindungen VmbH; Sandoz-Patent GmbH; Sandoz Ltd.). O-Alkylated rapamycin derivatives and their use, particularly as immunosuppressants. EP 663916, EP 867438, JP 96502266, US 5665772, WO 9409010.EP 0663916; EP 0867438; JP 1996502266; JP 1999240884; US 5665772; WO 9409010

…………..

SYNTHESIS

https://www.google.com/patents/WO2012103960A1

(US 5,665,772, EP 663916). The process principle is shown in the scheme below, wherein the abbreviation RAP-OH has been used as an abbreviation for the rapamycin structure of formula (2) above, L is a leaving group and P is a trisubstituted silyl group serving as a OH- protective group.

RAP-OH + L-CH₂-CH₂-0-P — –> RAP-O-CH2-CH2-O-P — – > RAP-O-CH2-CH2-OH

(2)                                                 (4)                                                                 (1)

Specifically, the L- group is a trifluoromethanesulfonate (triflate) group and the protective group P- is typically a tert-butyldimethylsilyloxy- group. Accordingly, the known useful reagent within the above general formula (3) for making everolimus from rapamycin is 2-(tert-butyldimethylsilyloxy)ethyl triflate of formula (3 A):

According to a known synthetic procedure disclosed in Example 8 of WO 94/09010 and in Example 1 of US application 2003/0125800, rapamycin (2) reacts in hot toluene and in the presence of 2,6-lutidine with a molar excess of the compound (3 A), which is charged in several portions, to form the t-butyldimethylsilyl-protected everolimus (4A). This compound is isolated and deprotected by means of IN aqueous HC1 in methanol. Crude everolimus is then purified by column chromatography. Yields were not reported.

(2)                                       (3A)                              (4A)                                (1)

In an article of Moenius et al. (J. Labelled Cpd. Radiopharm. 43, 113-120 (2000)), which used the above process for making C14-labelled and tritiated everolimus, a diphenyl- tert.butylsilyloxy -protective group was used as the alkylation agent of formula (3B).

Only 8% yield of the corresponding compound (4B)

and 21% yield of the compound (1) have been reported.

Little is known about the compounds of the general formula (3) and methods of their preparation. The synthesis of the compound (3 A) was disclosed in Example 1 of US application 2003/0125800. It should be noted that specification of the reaction solvent in the key step B of this synthesis was omitted in the disclosure; however, the data about isolation of the product allow for estimation that such solvent is dichloromethane. Similarly also a second article of Moenius et al. (J. Labelled Cpd. Radiopharm.42, 29-41 (1999)) teaches that dichloromethane is the solvent in the reaction.

It appears that the compounds of formula (3) are very reactive, and thus also very unstable compounds. This is reflected by the fact that the yields of the reaction with rapamycine are very low and the compound (3) is charged in high molar extent. Methods how to monitor the reactivity and/or improve the stability of compounds of general formula (3), however, do not exist.

Thus, it would be useful to improve both processes of making compounds of formula (3) and, as well, processes of their application in chemical synthesis.

xample 6: 40-O-[2-((2,3-dimethylbut-2-yl)dimethylsilyloxy)ethyl]rapamycin

In a 100 mL flask, Rapamycin (6 g, 6.56 mmol) was dissolved in dimethoxyethane (4.2 ml) and toluene (24 ml) to give a white suspension and the temperature was raised to 70°C. After 20 min, N,N-diisopropylethylamine (4.56 ml, 27.6 mmol) and 2-((2,3-dimethylbutan-2- yl)dimethylsilyloxy)ethyl trifluoromethanesulfonate (8.83 g, 26.3 mmol) were added in 2 portions with a 2 hr interval at 70°C. The mixture was stirred overnight at room temperature, then diluted with EtOAc (40 ml) and washed with sat. NaHC0₃ (30 ml) and brine (30 ml). The organic layer was dried with Na₂S0₄, filtered and concentrated. The cmde product was chromatographed on a silica gel column (EtOAc/heptane 1/1 ; yield 4.47 g).

Example 7: 40-O-(2-hydroxyethyl)-rapamycin [everolimus]

In a 100 mL flask, 40-O-[2-((2,3-dimethylbut-2-yl)dimethylsilyloxy)ethyl]rapamycin (4.47 g, 4.06 mmol) was dissolved in methanol (20 ml) to give a colorless solution. At 0°C, IN aqueous hydrochloric acid (2.0 ml, 2.0 mmol) was added and the mixture was stirred for 90 min. The reaction was followed by TLC (ethyl acetate/n-heptane 3 :2) and HPLC. Then 20 ml of saturated aqueous NaHC03 were added, followed by 20 ml of brine and 80 ml of ethyl acetate. The phases were separated and the organic layer was washed with saturated aqueous NaCl until pH 6/7. The organic layer was dried by Na₂S0₄, filtered and concentrated to yield 3.3 g of the product.

……………………….

SYNTHESIS

https://www.google.co.in/patents/WO1994009010A1

Example 8: 40-O-(2-Hydroxy)ethyl-rapamycin

a) 40-O-[2-(t-Butyldimethylsilyl)oxy]ethyl-rapamycin

A solution of 9.14 g (10 mmol) of rapamycin and 4.70 mL (40 mmol) of 2,6-lutidine in 30 mL of toluene is warmed to 60°C and a solution of 6.17 g (20 mmol) of 2-(t-butyldimethylsilyl)oxyethyl triflate and 2.35 mL (20 mmol) of 2,6-lutidine in 20 mL of toluene is added. This mixture is stirred for 1.5h. Then two batches of a solution of 3.08 g (10 mmol) of triflate and 1.2 mL (10 mmol) of 2,6-lutidine in 10 mL of toluene are added in a 1.5h interval. After addition of the last batch, stirring is continued at 60°C for 2h and the resulting brown suspension is filtered. The filtrate is diluted with ethyl acetate and washed with aq. sodium bicarbonate and brine. The organic solution is dried over anhydrous sodium sulfate, filtered and concentrated. The residue is purified by column chromatography on silica gel (40:60 hexane-ethyl acetate) to afford 40-O-[2-(t-butyldimethylsilyl)oxy]ethyl-rapamycin as a white solid: ¹H NMR (CDCl₃) δ 0.06 (6H, s), 0.72 (1H, dd), 0.90 (9H, s), 1.65 (3H, s), 1.75 (3H, s), 3.02 (1H, m), 3.63 (3H, m), 3.72 (3H, m); MS (FAB) m/z 1094 ([M+Na]⁺), 1022 ([M-(OCH₃+H₂O)]⁺).

b) 40-O-(2-Hydroxy)ethyl-rapamycin

To a stirred, cooled (0°C) solution of 4.5 g (4.2 mmol) of 40-O-[2-(t-butyldimethylsilyl)oxy]ethyl-rapamycin in 20 mL of methanol is added 2 mL of IN HCl. This solution is stirred for 2h and neutralized with aq. sodium bicarbonate. The mixture is extracted with three portions of ethyl acetate. The organic solution is washed with aq.

sodium bicarbonate and brine, dried over anhydrous sodium sulfate, filtered and

concentrated. Purification by column chromatography on silica gel (ethyl acetate) gave the title compound as a white solid:¹H NMR (CDCl₃) δ 0.72 (1H, dd), 1.65 (3H, s), 1.75 (3H, s), 3.13 (5H, s and m), 3.52-3.91 (8H, m); MS (FAB) m/z 980 ([M+Na]⁺), 926 ([M-OCH₃]⁺), 908 ([M-(OCH₃+H₂O)]⁺), 890 ([M-(OCH₃+2H₂O)]⁺), 876 ([M-(2CH₃OH+OH)]⁺), 858 ([M-(OCH₃+CH₃OH+2H₂O)]⁺).

MBA (rel. IC50) 2.2

IL-6 dep. prol. (rel. IC50) 2.8

MLR (rel. IC50) 3.4

…………………..

synthesis

Everolimus (Everolimus) was synthesized by the Sirolimus (sirolimus, also known as rapamycin Rapamycin) ether from. Sirolimus is from the soil bacterium Streptomyces hygroscopicus isolated metabolites. Activation end sirolimus (triflate, Tf) the other end of the protection (t-butyldimethylsilyl, TBS) of ethylene glycol 1 reaction of 2 , because the hydroxyl group 42 hydroxyl site over the 31-bit resistance is small, so the reaction only occurs in 42. Compound 2under acidic conditions TBS protection is removed everolimus.

PATENT

https://patents.google.com/patent/WO2016020664A1/en

Everolimus (RAD-001) is the 40-O- 2-hydroxyethyl)-rapamycin of formula (I),

It is a derivative of sirolimus of formula III),

and works similarly to sirolimus as an inhibitor of mammalian target of rapamycin (mTOR). Everolimus is currently used as an immunosuppressant to prevent rejection of organ transplants and treatment of renal cell cancer and other tumours. It is marketed by Novartis under the tradenames Zortress™ (USA) and Certican™ (Europe and other countries) in transplantation medicine, and Afinitor™ in oncology.

Trisubstituted silyloxyethyltrifluoromethane sulfonates (triflates) of the general formula (IV),

wherein R₂, R₃ are independently a straight or branched alkyl group, for example C^-Cw alkyl, and/or an aryl group, for example a phenyl group, are important intermediates useful in the synthesis of everolimus.

Everolimus and its process for manufacture using the intermediate 2-(t-butyldimethyl silyl) oxyethyl triflate of formula (IVA),

was first described in US Patent Number 5,665,772. The overall reaction is depicted in Scheme I.

Sche

Everolimus (I)

For the synthesis, firstly sirolimus of formula (III) and 2-(t-butyldimethylsilyl)oxyethyl triflate of formula (IVA) are reacted in the presence of 2,6-Lutidine in toluene at around 60°C to obtain the corresponding 40-O-[2-(t-butyldimethylsilyl)oxy]ethyl rapamycin of formula (I la), which is then deprotected in aqueous hydrochloric acid and converted into crude everolimus [40-O-(2- Hydroxy)ethyl rapamycin] of formula (I). However, this process results in the formation of impure everolimus, which requires purification by column chromatography. The process results in very poor overall yield and purity and thereby the process is not suitable for the commercial scale production of everolimus.

Moenius et al. (I. Labelled Cpd. Radiopharm. 43, 1 13-120 (2000) have disclosed a process to prepare C-14 labelled everolimus using the diphenyltert-butylsilyloxy-protective group of formula (IV B),

as the alkylation agent. The overall yield reported was 25%. International patent application, publication number WO 2012/103960 discloses the preparation of everolimus using the alkylating agent 2-((2,3-dimethylbut-2-yl)dimethylsilyloxy)ethyl triflate of formula (IVC),

wherein the overall yield reported is 52.54%. The process involves a derivatization method based on the reaction of the triflate (IV) with a derivatization agent, which preferably is a secondary aromatic amine, typically N-methylaniline.

International patent application, publication number WO 2012/103959 also discloses the preparation of everolimus using the alkylating agent of formula (IVC). The process is based on a reaction of rapamycin with the compound of formula (IVC) in the presence of a base (such as an aliphatic tertiary amine) to form 40-O-2-(t-hexyldimethylsiloxy)ethylrapamycin, which is subsequently deprotected under acidic conditions to obtain everolimus. European Patent Number 1518517B discloses a process for the preparation of everolimus which employs the triflate compound of formula (IVA), 2-(t-butyldimethyl silyl) oxyethyl triflate. The disclosed process for preparing the compound of formula (IVA) involves a flash chromatography purification step. The compounds of formula (IV) are key intermediates in the synthesis of everolimus. However, they are highly reactive and also very unstable, and their use often results in decomposition during reaction with sirolimus. This is reflected by the fact that the yields of the reaction with sirolimus are very low and the compounds of formula (IV) are charged in high molar extent. Thus it is desirable to develop a process to stabilize compounds of formula (IV) without loss of reactivity

 Example 1 :

Step 1 : Preparation of protected everolimus (TBS-everoismus) of formula (Ma) using metal salt, wherein “Pg” is t-butyldimethylsilyl t-butyldimethylsilyloxy ethanol, of formula (VA) (2.8g, 0.016mol) was dissolved in dichloromethane (DCM) (3 vol) and to this 2,6-Lutidine (3.50 g, 0.0327 mol) was added and the mixture was cooled to -40°C. Thereafter, trifluoromethane sulfonic anhydride (3.59ml, 0.021 mol) was added drop-wise. The mixture was maintained at -40°C for 30 minutes. Sirolimus (0.5g, 0.00054mol) was taken in another flask and dissolved in DCM (1 ml). To this sirolimus solution, silver acetate (0.018g, 0.000109mol) was added and cooled to -40°C. The earlier cooled triflate solution was transferred in 3 lots to the sirolimus solution maintaining temperature at -40°C. The reaction mixture was stirred at -40°C further for 15min before which it was slowly warmed to 0°C and further to RT. The reaction mixture was then warmed to 40°C and maintained at this temperature for 3 hours. The reaction was monitored by TLC. On completion of reaction, the reaction mixture was diluted with DCM and washed with water and brine. The organic layer was dried over anhydrous sodium sulphate and solvent was removed by vacuum distillation to obtain the title compound, which was directly used in the next step. HPLC product purity: 60%-85%.

Step 2: Preparation of everolimus of formula (I) Protected everolimus of formula (I la) obtained in step 1 was dissolved in methanol (10 volumes) and chilled to 0-5° C. To this solution was added drop wise, a solution of 1 N HCI. The pH of the reaction was maintained between 1-3. The temperature of the reaction mixture was raised to 25° C and stirred for 1 hour. After completion of reaction, the reaction mixture was diluted with water (15 volumes) and extracted in ethyl acetate (2X20 volumes). The organic layers were combined and washed with brine, dried over sodium sulphate. The organic layer was distilled off under reduced pressure at 30-35° C, to obtain a crude everolimus (0.8 g). The crude everolimus was further purified by preparative HPLC to yield everolimus of purity >99%.

Example 2:

Step 1 : Preparation of TBS-everoiimus of formula (Ma) without using metal salt, wherein “Pg” is t-butyldimethylsilyl t-butyldimethylsilyloxy ethanol, of formula (VA) (2.8g, 0.016mol) was dissolved in DCM (3 vol) and to this 2,6-Lutidine (3.50 g, 0.0327 mol) was added and the mixture was cooled to -40°C. Thereafter, trifluoromethane sulfonic anhydride (3.59ml, 0.021 mol) was added drop-wise. The mixture was maintained at -40°C for 30 minutes. Sirolimus (0.5g, 0.00054mol) was taken in another flask and dissolved in DCM (1 ml). The solution was cooled to -40°C. The earlier cooled triflate solution was transferred in 3 lots to the sirolimus solution maintaining temperature at -40°C. The reaction mixture was stirred at -40°C further for 15min before which it was slowly warmed to 0°C and further to RT. The reaction mixture was then warmed to 40°C and maintained at this temperature for 3 hours. On completion of reaction, the reaction mixture was diluted with DCM and washed with water and brine. The organic layer was dried over anhydrous sodium sulphate and solvent was removed by vacuum distillation to obtain the title compound, which was directly used in next step. HPLC purity: 10%-20%.

Step 2: Preparation of everolimus of formula (I)

Protected everolimus of formula (I la) obtained in step 1 was dissolved in methanol (10 volumes) and chilled to 0-5° C. To this solution was added drop wise, a solution of 1 N HCI. The pH of the reaction was maintained between 1-3. The temperature of the reaction mixture was raised to 25° C and stirred for 1 hour. After completion of reaction, the reaction mixture was diluted with water (15 volumes) and extracted in ethyl acetate (2X20 volumes). The organic layers were combined and washed with brine, dried over sodium sulphate. The organic layer was distilled off under reduced pressure at 30-35° C, to obtain a crude everolimus which was further purified by preparative HPLC. Example 3:

Preparation of crude Everolimus

Step 1 : Preparation of TBS-ethylene glycol of formula (Va)

Ethylene glycol (1.5L, 26.58 mol) and TBDMS-CI (485g, 3.21 mol) were mixed together with stirring and cooled to 0°C. Triethyl amine (679 ml, 4.83 mol) was then added at 0°C in 30-45 minutes. After addition, the reaction was stirred for 12 hours at 25-30°C for the desired conversion. After completion of reaction, the layers were separated and the organic layer (containing TBS- ethylene glycol) was washed with water (1 L.x2) and brine solution (1 L). The organic layer was then subjected to high vacuum distillation to afford 350g of pure product.

Step 2: Preparation of TBS-glycol-Triflate of formula (IVa)

The reaction was carried out under a nitrogen atmosphere. TBS- ethylene glycol prepared as per step 1 (85.10g, 0.48 mol) and 2, 6-Lutidine (84.28ml, 0.72 mol) were stirred in n-heptane (425ml) to give a clear solution which was then cooled to -15 to – 25°C. Trif!uoromethanesulfonic anhydride (Tf₂0) (99.74 ml, 0.590 mol) was added drop-wise over a period of 45 minutes to the n-heptane solution (white precipitate starts to form immediately) while maintaining the reaction at -15 to – 25°C. The reaction mixture was kept at temperature between -15 to -25°C for 2 hours. The precipitate generated was filtered off. The filtrate was then evaporated up to ~2 volumes with respect to TBS-ethyiene glycol (~200 ml).

Step 3: Preparation of TBS-evero!imus of formula (Ha)

30g of sirolimus (0,0328 mo!) and toluene (150m!) were stirred together and the temperature was slowly raised to 60-65°C. At this temperature, a first portion of TBS-g!yco!-triflate prepared as per step 2 (100ml) and 2,6-Lutidine (1 1.45ml, 0.086 moles) were added and stirred for 40 min. Further, a second portion of TBS- glycol-triflate (50mi) and 2, 6-Lutidine (19.45ml, 0.138 mol) were added and the reaction was stirred for another 40 min. This was followed by a third portion of TBS- glycol- triflate (50m!) and 2, 6-Lutidine (19.45ml, 0.138 mol), after which the reaction was stirred for further 90 minutes. The reaction was monitored through HPLC to check the conversion of Sirolimus to TBS-everolimus after each addition of TBS-glycol-trifiate. After completion of the reaction, the reaction mixture was diluted with n-heptane (150mi), cooled to room temperature and stirred for another 60 minutes. The precipitated solids were filtered off and the filtrate was washed with deionized water (450 ml x4) followed by brine solution (450ml). The filtrate was subsequently distilled off to afford TBS-everolimus (60-65g) with 60-70% conversion from sirolimus.

Step 4: Preparation of everolimus of formula (I)

TBS-everolimus (65g) obtained in step 3 was dissolved in 300 mi methanol and cooled to 0°C. 1 N HCI was then added to the methanol solution (pH adjusted to 2-3) and stirred for 2 h. After completion of reaction, toluene (360m!) and deionized wafer (360mi) were added to the reaction mixture and the aqueous layer was separated. The organic layer was washed with brine solution (360ml). The organic layer was concentrated to obtain crude everolimus (39g) with an assay content of 30-35%, HPLC purity of 60-65%.

The crude everolimus purified by chromatography to achieve purity more than 99 %.

Patent

Publication numberPriority datePublication dateAssigneeTitleUS5665772A *1992-10-091997-09-09Sandoz Ltd.O-alkylated rapamycin derivatives and their use, particularly as immunosuppressantsEP1518517A2 *2002-04-242005-03-30Sun Biomedical, Ltd.Drug-delivery endovascular stent and method for treating restenosisWO2012103960A12011-02-042012-08-09Synthon BvProcess for making trisubstituted silyloxyethyl triflatesCN102786534A2012-05-252012-11-21上海现代制药股份有限公司Preparation method of everolimusCN103788114A *2012-10-312014-05-14江苏汉邦科技有限公司Preparation method for everolimusEP3166950A12014-08-042017-05-17Cipla LimitedProcess for the synthesis of everolimus and intermediates thereof 

CN107417718A *2017-08-182017-12-01常州兰陵制药有限公司The preparation method of everolimus intermediateUS9938297B22014-08-042018-04-10Cipia LimitedProcess for the synthesis of everolimus and intermediates thereofCN108676014A *2018-06-152018-10-19国药集团川抗制药有限公司The method for purifying the method for everolimus intermediate and preparing everolimus 

Enzymes

• Streptomyces hygroscopicus NRRL 5491 (ATCC 29253)

Synthesis Path

Trade Names

Country	Trade Name	Vendor	Annotation
D	Certican	Novartis ,2004
F	Certican	Novartis
I	Certican	Novartis
J	Certican	Novartis

Formulations

• tabl. 0.25 mg, 0.5 mg, 0.75 mg

References

• a WO 9 409 010 (Sandoz-Erfindungen; 28.4.1994; GB-prior. 9.10.1992).
• b US 6 277 983 (American Home Products; 21.8.2001; USA-prior. 27.9.2000).
•  US 6 384 046 (Novartis; 7.5.2002; GB-prior. 27.3.1996).
•  US 20 040 115 (Univ. of Pennsylvania; 15.1.2004; USA-prior. 9.7.2002).
• fermentation of rapamycin (sirolimus):
  • Chen, Y. et al.: Process Biochemistry (Oxford, U. K.) (PBCHE5) 34, 4, 383 (1999).
  • The Merck Index, 14th Ed., 666 (3907) (Rahway 2006).
  • US 3 929 992 (Ayerst McKenna & Harrison Ltd.; 30.12.1975; USA-prior. 29.9.1972).
  • WO 9 418 207 (Sandoz-Erfindungen; 18.8.1994; GB-prior. 2.2.1993).
  • EP 638 125 (Pfizer; 17.4.1996; J-prior. 27.4.1992).
  • US 6 313 264 (American Home Products; 6.11.2001; USA-prior. 8.3.1994).

clip

https://doi.org/10.1039/C7MD00474EIssue 1, 2018

• 
  MedChemComm

Ascomycins and rapamycins The ascomycin tacrolimus (44, FK-506) and the two rapamycins sirolimus (45, rapamycin) and everolimus (46) are macrolides that contain 21- and 29-membered macrocyclic rings, respectively (Figure 7).[3] Their MWs range from just over 800 Da for tacrolimus (44) to >900 Da for sirolimus (45) and everolimus (46) and they have >10 HBAs. Like other natural product derived drugs in bRo5 space, they are above average complexity (SMCM 119–134) due to their 14–15 chiral centres. All three are immunosuppressants that are mainly used to prevent rejection of transplanted organs. They bind to overlapping, but slightly different parts of a shallow pocket at the surface of the immunophilin FK506 binding protein (FKBP12, Figure 8 A). Whereas tacrolimus (44) only binds in the pocket on FKBP12 (Figure 8 B),[67] sirolimus (45) and everolimus (46) promote binding of mammalian target of rapamycin (mTOR) so that they bind in a groove formed by FKBP12 and mTOR (Figure 8 C).[68] The complex between tacrolimus (44) and FKBP12 inhibits calcineurin, which results in reduced production of interleukin-2 and inactivation of T cells. Formation of the ternary complexes between FKBP12, sirolimus (45) [or everolimus (46)] and mTOR inhibits mTOR, which arrests growth of T lymphocytes by reducing their sensitivity to interleukin 2. Both tacrolimus (44) and sirolimus (45) have low (15–20 %) and variable bioavailabilities, whereas the bioavailability of everolimus (46) has been increased somewhat as compared to sirolimus (45).[3] Tacrolimus (44) was isolated from Streptomyces tsukubaensis in 1987,[69, 70] while sirolimus (45) was first identified from a Streptomycete strain found in a soil sample from Easter Island.[71] Later it was also isolated from fermentation of another Streptomycete strain.[72, 73] Both drugs are now produced through fermentation.[74, 75] Sirolimus suffers from low bioavailability as well as toxicity, and semi-synthetic derivatives were therefore prepared to minimise these issues. This led to the discovery of everolimus (46), synthesised by selective alkylation of one of the two secondary hydroxyl groups of sirolimus (45) with 2-(tert-butyldimethylsilyl)oxyethyltriflate followed by silyl ether deprotection with HCl (Scheme 8).[76, 77]

Figure 7. Structures of the ascomycin tacrolimus (44) and the rapamycins sirolimus (45) and everolimus (46) that are used mainly to prevent rejection of organ transplants.

[67] G. D. Van Duyne, R. F. Standaert, P. A. Karplus, S. L. Schreiber, J. Clardy, Science 1991, 252, 839 – 842. [68] A. M. Marz, A.-K. Fabian, C. Kozany, A. Bracher, F. Hausch, Mol. Cell. Biol. 2013, 33, 1357 – 1367.

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Ferreting out why some cancer drugs struggle to shrink tumors

Study shows how stopping one enzyme could help drugs treat an important class of cancers more effectively

by Stu Borman

JUNE 27, 2018 | APPEARED IN VOLUME 96, ISSUE 27

In several types of cancer, including most cases of breast cancer, a cell-signaling network called the PI3K pathway is overactive. Drug designers have tried to quiet this pathway to kill cancer, but they haven’t had much success and, more frustratingly, haven’t understood why the problem is so hard to solve.

“There have been more than 200 clinical trials with experimental drugs that target the PI3K pathway, and probably more than $1 billion invested,” says Sourav Bandyopadhyay of the University of California, San Francisco. Just a handful of drugs have been approved by the U.S. FDA and one, Novartis’s Afinitor (everolimus), deters cancer growth but doesn’t shrink tumors, and it prolongs patient survival only a few months.

Bandyopadhyay, his UCSF colleague John D. Gordan, and coworkers used a proteomics approach to ferret out why previous attempts to target the PI3K pathway have had limited success and, using that information, devised and tested a possible fix (Nat. Chem. Biol. 2018, DOI: 10.1038/s41589-018-0081-9).

The stubborn pathway involves a series of kinases—enzymes that modify other proteins by adding phosphate groups—starting with one called PI3K. Overactivation of the pathway produces the transcription factor MYC, which turns on protein synthesis and can spark cancer growth.

The UCSF team used kinase-affinity beads and tandem mass spectrometry to survey all kinases active in breast cancer cells before and after treatment with a variety of cancer drugs. The team studied this so-called kinome to look for kinases associated with the cells’ tendency to resist drug treatments.

The researchers found that a kinase called AURKA undermines everolimus and other pathway-targeted drugs by reversing their effects. While the drugs try to turn off the PI3K pathway, AURKA, activated separately by other pathways, keeps the PI3K pathway turned on. To add insult to injury, MYC boosts AURKA production, maintaining a plentiful supply of the drug spoiler.

When the researchers coadministered everolimus with the AURKA inhibitor MLN8237, also called alisertib, everolimus could inhibit the PI3K pathway as it was designed to do, without interference. The combination treatment killed most types of cancer cells in culture and shrank tumors in mice with breast cancer, whereas everolimus alone permitted slow tumor growth to continue.

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Further reading

• Sedrani R, Cottens S, Kallen J, Schuler W (August 1998). “Chemical modification of rapamycin: the discovery of SDZ RAD”. Transplantation Proceedings. 30 (5): 2192–4. doi:10.1016/S0041-1345(98)00587-9. PMID 9723437.

External links

• “Everolimus”. Drug Information Portal. U.S. National Library of Medicine.

Clinical data
Pronunciation	Everolimus /ˌɛvəˈroʊləməs/
Trade names	Afinitor, Zortress
Other names	42-O-(2-hydroxyethyl)rapamycin, RAD001
AHFS/Drugs.com	Monograph
MedlinePlus	a609032
License data	EU EMA: by INNUS DailyMed: EverolimusUS FDA: Everolimus
Pregnancy category	AU: C[1]
Routes of administration	By mouth
ATC code	L01EG02 (WHO) L04AA18 (WHO)
Legal status
Legal status	US: ℞-onlyEU: Rx-onlyIn general: ℞ (Prescription only)
Pharmacokinetic data
Elimination half-life	~30 hours[2]
Identifiers
showIUPAC name
CAS Number	159351-69-6
PubChem CID	6442177
DrugBank	DB01590
ChemSpider	21106307
UNII	9HW64Q8G6G
KEGG	D02714
ChEMBL	ChEMBL1908360
CompTox Dashboard (EPA)	DTXSID0040599
ECHA InfoCard	100.149.896
Chemical and physical data
Formula	C53H83NO14
Molar mass	958.240 g·mol−1
3D model (JSmol)	Interactive image
hideSMILESOCCO[C@@H]1CC[C@H](C[C@H]1OC)C[C@@H](C)[C@@H]4CC(=O)[C@H](C)/C=C(\C)[C@@H](O)[C@@H](OC)C(=O)[C@H](C)C[C@H](C)\C=C\C=C\C=C(/C)[C@@H](OC)C[C@@H]2CC[C@@H](C)[C@@](O)(O2)C(=O)C(=O)N3CCCC[C@H]3C(=O)O4
hideInChIInChI=1S/C53H83NO14/c1-32-16-12-11-13-17-33(2)44(63-8)30-40-21-19-38(7)53(62,68-40)50(59)51(60)54-23-15-14-18-41(54)52(61)67-45(35(4)28-39-20-22-43(66-25-24-55)46(29-39)64-9)31-42(56)34(3)27-37(6)48(58)49(65-10)47(57)36(5)26-32/h11-13,16-17,27,32,34-36,38-41,43-46,48-49,55,58,62H,14-15,18-26,28-31H2,1-10H3/b13-11+,16-12+,33-17+,37-27+/t32-,34-,35-,36-,38-,39+,40+,41+,43-,44+,45+,46-,48-,49+,53-/m1/s1 Key:HKVAMNSJSFKALM-GKUWKFKPSA-N

////////////////  RAD-001,  SDZ RAD, Certican, Novartis, Immunosuppressant, Everolimus, Afinitor, эверолимус , إيفيروليموس , 依维莫司 , 

#RAD-001,  #SDZ RAD, #Certican, #Novartis, #Immunosuppressant, #Everolimus, #Afinitor, #эверолимус , #إيفيروليموس , #依维莫司 ,