EA045993B1 - RAPAMYCIN ANALOGUES AND THEIR APPLICATIONS - Google Patents

Description

Technical field

The present invention relates to compounds and methods suitable for modulating mTORCl activity. The present invention also provides pharmaceutically acceptable compositions containing the compounds of the present invention and methods of using such compositions in the treatment of various disorders.

State of the art

mTOR complex 1 (mTORC1) mediates positive regulation of cell growth and proliferation by stimulating many anabolic processes, including protein, lipid, and organelle biosynthesis, and by limiting catabolic processes, such as autophagy. Most knowledge about mTORC1 function has been gained using the bacterial macrolide rapamycin. Once rapamycin enters the cell, it binds to the 12-kDa FK506-binding protein (FKBP12) and interacts with the FKBP12-panamycin-binding domain (FRB) of mTOR, thereby inhibiting mTORC1 functions (Guertin, D. A. & Sabatini, D. M. Cancer Cell 12( 1): 9-22 (2007)). In contrast to its action on mTORC1, FKBP12-rapamycin cannot physically interact with or specifically inhibit the mTOR complex 2 (mTORC2) (Janinto, E. et al., Nat. Cell Bio. 6(11): 1122-8 (2004 ); Sarbassov, D. D. et al., Curr. Biol. 14(14): 1296-302 (2004)). Based on these observations, mTORC1 and mTORC2 were respectively characterized as rapamycin-sensitive and rapamycin-insensitive complexes. However, this system is likely not entirely accurate, since chronic treatment with rapamycin can in some cases inhibit mTORC2 activity by blocking its assembly (Sarbassov, D.D. et al., Mol. Cell, 22(2): 159-68 (2006)). In addition, recent reports suggest that important functions of mTORC1 are not amenable to inhibition by rapamycin (Choo, A.Y. et al., Proc. Natl. Acad. Sci., 105(45): 17414-9 (2008); Feldman, M.E. et al. ., PLoS Biol., 7(2):e38 (2009); Garcia Martinez, J. M. et al., Biochem J., 421(1): 29-42 (2009); Thoreen, C. C. et al., J. Biol. Chem., 284(12): 8023-32 (2009)). Thus, selective inhibition of mTORC1 would allow the treatment of diseases in which there is dysregulation of protein synthesis and cellular metabolism. In addition, a detailed understanding of how mTORC1 activation pathways can be regulated will reveal new strategies for regulating abnormal processes occurring in disease by modulating mTORC1 activity across its range of functions. A large number of diseases are associated with abnormal cellular responses triggered by the events described above. These diseases include, but are not limited to, autoimmune diseases, inflammatory diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cancer, cardiovascular diseases, allergies and asthma, Alzheimer's disease and hormone-related diseases. Mechanistic target of rapamycin complex 1 (mTORC1) is a master growth regulator that senses various environmental signals such as growth factors, cellular stresses, and nutrient and energy levels. When activated, mTORC1 phosphorylates substrates, which stimulates anabolic processes such as mRNA translation and lipid synthesis and limits catabolic processes such as autophagy. Dysregulation of mTORC1 occurs in a wide range of diseases, including, but not limited to, diabetes, epilepsy, neurodegeneration, immune response, suppressed skeletal muscle growth, and cancer (Howell, J.J. et al., Biochem. Soc. Trans., 41: 906-12 (2013); Kim, S. G. et al., Molecular and cells, 35(6): 463-73 (2013); Laplante, M. & Sabatini, D. M., Cell, 149(2): 274-93 (2012)) .

Rapamycin was originally discovered as an antifungal metabolite produced by Streptomyces hygroscopicus from an Easter Island soil sample. Subsequently, rapamycin was found to have immunosuppressive and antiproliferative properties in mammalian cells, increasing interest in determining its mechanism of action. Rapamycin has been shown to be a potent inhibitor of S6K1 phosphorylation. At the same time, target of rapamycin (TOR) was identified in yeast and animal cells. Rapamycin forms a gain-of-function complex with the 12 kDa FK506-binding protein (FKBP12), and this complex binds and specifically acts as mammalian allosteric TOR inhibitor complex 1 (mTOR, also known as mechanistic TOR) (mTORC1).

Biochemical and genetic analysis of mTOR has shown that it is present in two functionally distinct complexes. The mTORC1 core components consist of mTOR, the mLST8 (mammalian lethal with sec-13 protein 8) protein, and the regulation-associated protein TOR (Raptor). Additional components include the DEP domain-containing mTOR-interacting protein (DEPTOR) and the 40-kDa proline-rich Akt substrate (PRAS40).

The mTOR core complex 2 (mTORC2) consists of mTOR, the rapamycin-insensitive companion of mTOR (Rictor), stress-activated protein kinase-activated protein 1 (mSIN1), and mLST8. The proteins protor 1/2 (protein observed with Rictor 1/2) and DEPTOR are additional regulatory components. S6 kinase 1 (S6K1) and eukaryotic inhibitory factor eIF4E binding protein 1 (4E-BP1) are two well-characterized substrates of mTORC1, and AKT is a well-characterized substrate of mTORC2 (Li, J. et al., Cell Met., 19(3) ):373-9 (2014)).

Because FKBP12-rapamycin does not bind to mTORC2, it was initially thought that rapamycin

- 1 045993 inhibits only mTORCl (Sarbassov, D.D. et al., Curr. Biol., 14(14): 1296-302 (2004)). However, in 2006, rapamycin was shown to inhibit mTORC2 assembly and function and inhibit pAkt (Sarbassov, D.D. et al., Molecular Cell, 22(2): 159-68 (2006)). The effects of rapamycin on phosphorylation of S473 Akt (mTORC2 substrate) and T389 S6K1 (mTORC1 substrate) were compared in multiple cell lines. For PC3, HEK-293T, HeLa, and H460 cells, treatment with rapamycin for 1 or 24 h inhibited S6K1 phosphorylation, consistent with inhibition of mTORC1. Selective inhibition of S6K1 by rapamycin should result in increased Akt phosphorylation, and indeed this has been reported in HeLa cells. However, in PC3 cells, the drug strongly reduced Akt phosphorylation, suggesting that rapamycin is not selective for this cell line. Partial inhibition of pAKT is observed for HEK293T cells. In approximately one third of the cell lines tested, rapamycin caused strong or partial inhibition of Akt phosphorylation, while in the remaining cases the drug either had no effect or increased Akt phosphorylation. Inhibition of pAKT at 24 h was also observed in primary and nontransformed cell lines, including endothelial and muscle cells. Rapamycin has also been shown to inhibit pAkt in vivo, as mice treated with the drug daily for 1 week had decreased Akt phosphorylation in the thymus, adipose tissue, heart, and lung.

These data showed that inhibition of Akt phosphorylation by rapamycin occurs in all of these cases and occurs in normal cell lines, cancer cell lines, and also in vivo.

Sarbassov et al concluded that rapamycin and its analogues (CCI 779, RAD001, also known as Everolimus, AP23573) can inhibit mTORC2 function in various cell lines and tissues. Rapamycin-mediated inhibition of Akt may help explain the side effects of this drug. For example, rapamycin potently inhibits Akt phosphorylation in adipose tissue, a tissue type in which insulin-stimulated Akt activity plays an important role in suppressing lipolysis. Inhibition of Akt by rapamycin in adipocytes may maintain high levels of lipolysis even in the presence of insulin, causing free fatty acids to accumulate in plasma. This may be used by the liver to produce triglycerides, providing a molecular mechanism for the hyperlipidemia that is often observed in patients treated with rapamycin.

Pereira et al (Mol Cell Endocrinol., 355(1): 96–105 (2012)) examined the effects of rapamycin on glucose uptake and insulin-related signaling proteins in adipocytes obtained from adipose tissue biopsies from human donors. At a therapeutic concentration (0.01 μM), rapamycin decreased Ser473 phosphorylation of AKT (PKB) and decreased glucose uptake in human adipocytes through disruption of insulin signaling.

Lamming et al (Science, 335(6076): 1638-1643 (2012)) showed that rapamycin disrupted mTORC2 in vivo, and that mTORC2 was required to suppress insulin-mediated suppression of gluconeogenesis in the liver.

Similar results have been found for humans. Di Paolo et al published similar findings in humans (JASN, 17(8): 2236–2244 (2006)). The primary objective of this study was to determine the effect of long-term exposure to rapamycin on AKT activation, given its critical role in the regulation of cell growth and survival, as well as in the response of cells to nutrients and growth factors. The study authors found that mTOR inhibition was associated with a marked suppression of basal and insulin-induced AKT phosphorylation. ACT is primarily responsible for many of the metabolic actions of insulin, and therefore the study authors concluded that suppression of ACT activation was significantly correlated with increased insulin resistance in kidney transplant patients. A review by Kennedy et al recently described the role of mTORC1 and mTORC2 in metabolism and aging (Cell Metab., 23(6): 990–1003 (2016)).

Surprisingly, the proposed compounds were found to inhibit mTORC1 but not mTORC2 (as determined by their effect on pAKT) over long periods of time (eg, 8, 24, 30 and 48 hours). This new activity is associated with the presence of a fairly large group at the C-7 position of rapamycin and its analogues. Small substituents at this position, such as OMe, as in the case of rapamycin, OEt, OBn do not provide selectivity for mTORC2 within 24 hours. Medium-length groups, such as OCH2CH2OH or OCH ₂ CH ₂ CH ₂ OH, show partial selectivity for to mTORC2 for 24 h but still show some level of inhibition. In comparison, larger groups, such as those of the present invention (eg, I-19), provide strong selectivity for mTORC2 on pAKT.

The location of the substituent is also important to achieve the observed selectivity. The introduction of substituents at a more distal position, for example at position 43, does not provide the unique selectivity profile claimed in this application.

For purposes of clarity, the structure of rapamycin is presented below, with positions C-7 and C-43 indicated.

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Some embodiments of the present invention provide novel rapamycin analogs that are potent inhibitors of mTORC1, as determined by their effect on pS6K. Unlike rapamycin and everolimus, these compounds do not inhibit pAKT over long periods of time (eg, 24 and 48 hours). These compounds also demonstrate increased solubility and improved pharmacokinetic properties compared to rapamycin.

The activity of the compounds used in the present invention as an mTORC1 inhibitor can be assessed in vitro, in vivo or in a cell line. In vitro studies include studies that measure mTORC1 inhibition. Detailed conditions for evaluating the compound used in the present invention as an mTORC1 inhibitor are well known to one of ordinary skill in the art. Such methods are described in detail in Liu et al., Cancer Research, 73(8): 2574-86 (2013) and Liu et al., J. Biological Chemistry 287(13): 9742-52 (2012).

Brief description of the invention

In the present invention, it has been found that the compounds of the present invention and pharmaceutically acceptable compositions thereof are effective as mTORC1 inhibitors. The present invention relates to compounds of general formula II

II or its pharmaceutically acceptable salts, where

R ¹ selected from or formula P-0

P-0 where / ⁵ means the site of attachment at the C-7-hydroxyl position;

each Z is independently -O-, -S- or -SO2-; n ranges from 2 to 300; And

R represents hydrogen or a C _1-6 aliphatic group, optionally substituted with halogen.

The compounds of the present invention and pharmaceutically acceptable compositions thereof are suitable for use in the treatment of a variety of diseases, disorders or conditions associated with mTORC1. Such diseases, disorders or conditions include the diseases, disorders or conditions described herein.

Brief description of drawings

In fig. 1 shows a comparison of two Western blots obtained after treatment of PC3 cells with rapamycin or a compound of the present invention (I-29) for 24 and 48 hours. The staining demonstrates strong inhibition of the mTORC1 pathway by both rapamycin and I-29. for both time points. In contrast, the mTORC2 pathway, which was inhibited by rapamycin at both 24 and 48 h, was not inhibited by I-29, as indicated by the lack of inhibition of Akt phosphorylation.

In fig. 2 shows a comparison of three Western blots obtained after treating PC3 cells with rapamycin or a compound of the present invention (I-29) for 30, 15 or 5 minutes. Staining demonstrates time-dependent inhibition of the mTORC1 pathway for both rapamycin and

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I-29. In fig. 3 shows a Western blot obtained after treatment of PC3 cells with rapamycin, temsirolimus, everolimus, ridaforolimus or a compound of the present invention (I118) for 24 hours. The staining shows strong inhibition of the mTORC1 pathway for all compounds and a moderate concentration-dependent inhibition of mTORC1 by compound I- 118. Importantly, rapamycin, temsirolimus, everolimus, and ridaforolimus demonstrated dose-dependent inhibition of the mTORC2 pathway, and I-118 did not inhibit the mTORC2 pathway, as indicated by the lack of inhibition of Akt phosphorylation. In fig. 4 shows a Western blot obtained after treating PC3 cells with rapamycin or the compounds of the present invention (I-106, I-113 and I-118) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for each of the compounds tested and no inhibition of phosphorylation 4E-BP1. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 5 shows a Western blot obtained after treating PC3 cells with rapamycin or the compounds of the present invention (I-117, I-102, I-103 and I-39) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for each of the compounds tested. and lack of inhibition of 4E-BP1 phosphorylation. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 6 shows a Western blot obtained after treating PC3 cells with rapamycin or the compounds of the present invention (I-117, I-99, I-100 and I-101) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway by rapamycin, I- 117, I-100 and I-101, and significant concentration-dependent inhibition of the mTORC1 pathway for I-99. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 7 shows a Western blot obtained after treating PC3 cells with rapamycin or a compound of the present invention (I-117) for 24 hours. The staining demonstrates strong inhibition of the mTOCR1 pathway by both compounds tested. It is important to note that the compound of the present invention does not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 8 shows a Western blot obtained after treatment of PC3 cells with rapamycin or the compounds of the present invention (I-39, I-101 and I-99) for 24 hours. The staining shows strong mTORC1 inhibition for rapamycin and I-39 and a significant dependence on mTORC1 pathway inhibition concentrations for I-101 and I-99. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 9 shows a Western blot obtained after treating PC3 cells with rapamycin or the compounds of the present invention (I-98 and I-97) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for rapamycin and I-98 and moderate concentration-dependent inhibition of the pathway mTORC1 for I-101 and I-99. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 10 shows a Western blot obtained after treatment of PC3 cells with rapamycin or the compounds of the present invention (I-96 and I-100) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for all compounds tested. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 11 shows a Western blot obtained after treating PC3 cells with rapamycin, everolimus or a compound of the present invention (I-7) for 90 minutes. Staining demonstrates strong inhibition of the mTORC1 pathway for rapamycin and everolimus and moderate concentration-dependent inhibition of the mTORC1 pathway for I-7. It is important to note that the compound of the present invention does not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 12 shows a Western blot obtained after treatment of PC3 cells with rapamycin, everolimus, or a compound of the present invention (I-7) for 24 hours. The staining shows strong inhibition of the mTORC1 pathway for rapamycin and everolimus and moderate concentration-dependent inhibition of the mTORC1 pathway for I- 7. It is important to note that the compound of the present invention does not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 13 shows two Western blots obtained after treatment of Jurkat cells with everolimus or the compounds of the present invention (I-29 and I-117) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for all compounds tested. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 14 shows Western blots obtained after treatment of tuberous sclerosis (TSC2) negative (TSC -/-) MEF cells with everolimus or a compound of the present invention (I-29, I-7 and I-117) for 90 min. Staining demonstrates strong inhibition of the mTORC1 pathway for everolimus, I-29, and I-117 and significant concentration-dependent inhibition of the mTORC1 pathway for I-7. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 15 shows two Western blots obtained after treatment of tuberous sclerosis type 2 (TSC2) positive (TSC+/+) MEF cells with everolimus or the compounds of the present invention (I-29, I-7 and I-117) for 90 minutes . Staining demonstrates strong mTORC1 pathway inhibition for everolimus and I-29, moderate concentration-dependent mTORC1 pathway inhibition for I-117, and significant concentration-dependent mTORC1 pathway inhibition for I7. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 16 shows two Western blots obtained after treatment of TSC -/- MEF cells with everolimus or the compounds of the present invention (I-29, I-7 and I-117) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway by everolimus, I -29 and I-117 and significant concentration-dependent inhibition of the mTORC1 pathway for I-7. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 17 shows two Western blots obtained after treatment of TSC +/+ MEF cells with everolimus or the compounds of the present invention (I-29 and I-7) for 24 hours. The staining shows strong inhibition of the mTORC1 pathway by everolimus, moderate concentration-dependent inhibition mTORC1 pathway for I-29 and low concentration-dependent inhibition of the mTORC1 pathway for I-7. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 18 shows a Western blot obtained after treatment of TSC −/− MEF cells with everolimus, rapamycin, or compounds of the present invention (I-2 and I-92) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for everolimus, rapamycin, and I-92. 2 and moderate concentration-dependent inhibition of the mTORC1 pathway for I-92. In fig. 19 shows a Western blot obtained after treatment of TSC +/+ MEF cells with everolimus, rapamycin, or compounds of the present invention (I-2 and I-92) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for everolimus and rapamycin and a moderate - dependent concentration-dependent inhibition of the mTORC1 pathway for I-2 and I-92. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 20 shows a Western blot obtained after treatment of TSC +/+ MEF cells with everolimus or the compounds of the present invention (I-20, I-29 and I-36) for 24 hours. The staining demonstrates a strong inhibition of the mTORC1 pathway by everolimus and a moderate - dependent concentration-dependent inhibition of the mTORC1 pathway for I-20, I-29 and I-36. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 21 shows a Western blot obtained after treatment of TSC +/+ MEF cells with everolimus or the compounds of the present invention (I-35, I-7 and I-26) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway by everolimus and low dependence concentration-dependent inhibition of the mTORC1 pathway for I-35, I-7 and I-26. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 22 shows a Western blot obtained after treatment of TSC +/+ MEF cells with everolimus or the compounds of the present invention (I-9, I-97 and I-98) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway by everolimus, moderately dependent concentration-dependent inhibition of the mTORC1 pathway for I-9, I-97 and I-98. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 23 shows a Western blot obtained after treatment of TSC +/+ MEF cells with everolimus or compounds of the present invention (I-91) for 90 minutes. Staining demonstrates strong inhibition of the mTORC1 pathway for rapamycin and I-91. Interestingly, I-91 shows some inhibition of mTORC2, as indicated by inhibition of Akt phosphorylation.

In fig. 24 shows a Western blot obtained after treatment of TSC +/+ MEF cells with everolimus or compounds of the present invention (I-91) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for rapamycin and I-91. Interestingly, I-91 shows some inhibition of mTORC2, as indicated by inhibition of Akt phosphorylation.

In fig. 25 shows a Western blot obtained after treatment of TSC +/+ MEF cells with everolimus or compounds of the present invention (I-105) for 90 minutes. Coloring de

- 5 045993 demonstrates potent inhibition of the mTORCl pathway for rapamycin and I-105. It is important to note that the compound of the present invention does not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 26 shows a Western blot obtained after treatment of TSC +/+ MEF cells with everolimus or compounds of the present invention (I-105) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for rapamycin and I-105. It is important to note that the compound of the present invention does not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 27 presented! Western blots obtained after treatment of PC3 cells with everolimus or the compounds of the present invention (I-2 and I-92) for 24 hours. Staining demonstrates strong inhibition of the mTORC1 pathway for each compound tested and no inhibition of 4E-BP1 phosphorylation. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 28 shows a Western blot obtained after treatment of TSC +/+ MEF cells with everolimus or a compound of the present invention (I-105) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for each of the compounds tested and no inhibition of 4E-BP1 phosphorylation. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 29 shows a Western blot obtained after treatment of TSC +/+ MEF cells with everolimus or the compounds of the present invention (I-90 and I-110) for 24 hours. The staining shows strong inhibition of the mTORC1 pathway for everolimus and I-90 and a moderate - dependent concentration-dependent inhibition of the mTORC1 pathway for I-110. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 30 shows a Western blot obtained after treatment of TSC +/+ MEF cells with everolimus or a compound of the present invention (I-115) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for everolimus and moderate concentration-dependent inhibition of the mTORC1 pathway for I- 115. It is important to note that the compound of the present invention does not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 31 shows a Western blot obtained after treatment of wild-type MEF cells with everolimus or the compounds of the present invention (I-105, I-117, I-29 and I-90) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway by everolimus and moderate inhibition of the mTORC1 pathway for I-105, I-117, I-29 and I-90. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 32 shows a Western blot obtained after treatment of wild-type MEF cells with everolimus or the compounds of the present invention (I-105, I-117, I-29 and I-90) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for each of studied compounds. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 33 shows a Western blot obtained after treating wild-type MEF cells with everolimus or the compounds of the present invention (I-85 and I-83) for 90 minutes. The staining demonstrates strong inhibition of the mTORC1 pathway for each of the compounds tested. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 34 shows a Western blot obtained after treating wild-type MEF cells with everolimus or the compounds of the present invention (I-85 and I-83) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for each of the compounds tested. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 35 shows a Western blot obtained after treatment of PC3 cells with everolimus or the compounds of the present invention (I-85 and I-83) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway by each of the compounds tested.

In fig. 36 shows a Western blot obtained after treatment of PC3 cells and wild-type MEF cells with everolimus or a compound of the present invention (I-115) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for each of the compounds tested in PC3 cells, strong inhibition of the pathway mTORC1 for everolimus in wild-type MEF cells and moderate concentration-dependent inhibition of the mTORC1 pathway for I-115 in WT MEF cells. It is important to note that the compound of the present invention does not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 37 shows a Western blot obtained after treatment of PC3 cells with everolimus, a compound of the present invention (I-117), short PEGs, everolimus in combination with

- 6 045993 short PEG or I-117 in combination with short PEG for 24 hours. Staining demonstrates strong inhibition of the mTORC1 pathway for everolimus and I-117, alone and in combination with short PEG. Short PEGs themselves did not demonstrate inhibition of mTORC1 or mTORC2. It is important to note that the compound of the present invention does not inhibit mTORC2, either alone or in combination with short PEG, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 38 shows a Western blot obtained after treating PC3 cells with everolimus or the compounds of the present invention (I-71, I-73 and I-75) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for all compounds tested. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 39 shows a Western blot obtained after treating PC3 cells with everolimus or the compounds of the present invention (I-85 and I-83) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for all compounds tested. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 40 shows a Western blot obtained after treatment of PC3 cells with everolimus or compounds of the present invention (I-65) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for both compounds tested. It is important to note that the compound of the present invention does not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 41 shows a Western blot obtained after treatment of PC3 cells with everolimus or the compounds of the present invention (I-5, I-106 and I-102) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for all compounds tested. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 42 shows a Western blot obtained after treatment of PC3 cells with everolimus or the compounds of the present invention (I-75, I-71 and I-62) for 24 hours. The staining demonstrates strong inhibition of the mTORC1 pathway for all compounds tested. It is important to note that the compounds of the present invention do not inhibit mTORC2, as evidenced by the lack of inhibition of Akt phosphorylation.

In fig. 43 shows the time course of glucose tolerance and insulin sensitivity test results for lean C57Bl/6 mice.

In fig. 44 shows the results of an intraperitoneal glucose tolerance test for lean C57Bl/6 mice treated chronically with rapamycin, I-29, or vehicle. ***p<0.001; ****P<0.0001; one-way analysis of variance; all bars mean mean and SD.

In fig. 45 shows the area under the curve (AUC) for glucose clearance in lean C57Bl/6 mice chronically treated with rapamycin, I-29, or vehicle. ***P<0.001; Tcriterion; and all bars mean mean and SD.

In fig. 46 shows the results of Sirius red staining of kidney tissue in the AKI/CKD mouse model.

In fig. 47 shows the percentage area of renal tissue fibrosis in the AKI/CKD mouse model following treatment with everolimus, I-29, I-117, or vehicle. *P=0.02 compared with vehicle, T-test.

In fig. 48 shows collagen I mRNA expression in a mouse model of AKI/CKD following treatment with vehicle or I-29. **P<0.01, ***P<0.001 compared with sham; ''"''P<0.01 compared to vehicle.

In fig. 49 shows collagen III mRNA expression in a mouse model of AKI/CKD following treatment with vehicle or I-29. **P<0.01 vs. sham; ^T P < 0.05 versus vehicle.

In fig. 50 shows fibronectin mRNA expression in a mouse model of AKI/CKD following treatment with vehicle or I-29. **P<0.01 vs. sham; ^T P < 0.05 versus vehicle.

In fig. 51 shows the area of kidney tissue infiltrated by macrophages in the AKI/CKD mouse model following treatment with vehicle or I-29. *P<0.05, ***P<0.001 compared with sham; 1”G<0.05 compared to the carrier.

In fig. 52 shows the percentage inhibition of IFN-γ production in the allogeneic mixed lymphocyte response (MLR) study following treatment with rapamycin, everolimus, I-29, or I-117.

Detailed description of some implementation options

1. General description of some embodiments of the present invention.

Although the present invention relates only to specific compounds of formula II, as indicated above, the inventors of the present invention have described, prepared and tested a large number of compounds, which in the context of the present invention are considered additional (possible) compounds not covered by the present invention, but which may be mentioned for clarity of description.

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In this context, a compound of formula I may be mentioned

or a pharmaceutically acceptable salt thereof, wherein ring A is a monovalent derivative of rapamycin or an analogue thereof (ie, rapalog);

where R ¹ is attached to it at the C-7 position of the hydroxyl of rapamycin or its analogue;

R ¹ is an optionally substituted straight or branched saturated or unsaturated monovalent C3.30 hydrocarbon chain, where one or more methylene units of R ¹ are optionally and independently replaced by -N(R)-, -N(R)C(O)-, -C(O)N(R)-, -N(R)S(O)2-, -S(O)2N(R)-, -О, -C(O)-, -OC(O)- , -C(O)O-, -S-, -S(O)-, ^(O)2- or -P(O)(R)2, or a 6-18 membered saturated or partially unsaturated heterocyclic ring, containing 1-6 heteroatoms independently selected from nitrogen, oxygen or sulfur; or

R ¹ is selected from the formula P-0

P-0 where means the place of connection to ring A;

each Z is independently -O-, -S-, -NR- or -SO2-; n is from about 2 to about 300; and each R independently represents hydrogen or an optionally substituted C _1-6 aliphatic group.

Also mentioned may be a compound of formula I other than compounds selected from

2. Connections and definitions.

The compounds of the present invention include those generally described herein and further illustrated by the classes, subclasses and types disclosed herein. Unless otherwise specified, the following definitions shall apply throughout this document. For the purposes of the present invention, chemical elements are identified in

- 8 045993 according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, ^75th Ed. In addition, the general principles of organic chemistry are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito: 1999, and March's Advanced Organic Chemistry, ^5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons , New York: 2001, the entire contents of said references are incorporated herein by reference.

The term aliphatic or aliphatic group as used herein means a straight-chain (i.e., straight-chain) or branched substituted or unsubstituted hydrocarbon chain that is fully saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is fully saturated or which contains one or more unsaturation units, but is not aromatic (also referred to herein as carbocycle, cycloaliphatic or cycloalkyl), in which or which has one attachment site to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, the aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, the aliphatic groups contain 1-4 aliphatic carbon atoms. In other embodiments, the aliphatic groups contain 1-3 aliphatic carbon atoms, and in other embodiments, the aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, cycloaliphatic (or carbocycle or cycloalkyl) refers to a monocyclic C ₃ -C ₆ hydrocarbon that is fully saturated or that contains one or more units of unsaturation but is not aromatic, having one attachment site to the rest of the molecule . Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

The term heteroatom means one or more of oxygen, sulfur, nitrogen, phosphorus or silicon (including any oxidized form of nitrogen, sulfur, phosphorus or silicon; the quaternized form of any basic nitrogen or a substituted heterocyclic ring nitrogen, e.g. N (as in 3,4-dihydro -2Hpyrrolil), NH (as in pyrrolidinyl) or NR+ (as in N-substituted pyrrolidinyl)).

The term unsaturated means that the fragment contains one or more units of unsaturation.

The term bivalent C _1-8 (or C _1-6 ) saturated or unsaturated, straight or branched hydrocarbon chain refers to bivalent alkylene, alkenylene and alkynylene chains that are straight or branched as defined herein. The term alkylene refers to a divalent alkyl group. An alkylene chain is a polymethylene group, i.e. -(CH ₂ ) _n - where n is a positive integer, preferably 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 2 to 3. The substituted alkylene chain is a polymethylene group, in which one or more methylene hydrogen atoms are replaced by a substituent. Suitable substituents include those described below for the substituted aliphatic group.

The term alkenylene refers to a divalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms have been replaced by a substituent. Suitable substituents include those described below for the substituted aliphatic group. The term halogen means F, Cl, Br or I.

The term aryl, used alone or as part of a larger moiety such as aralkyl, aralkoxy or aryloxyalkyl, refers to monocyclic or bicyclic ring systems containing a total of five to fourteen ring units, wherein at least one ring in the said system is aromatic, and each ring in said system contains from 3 to 7 ring elements. The term aryl may be used interchangeably with the term aryl ring. In some embodiments, aryl refers to an aromatic ring system that includes, but is not limited to, phenyl, biphenyl, naphthyl, anthracyl, and the like, which may contain one or more substituents. Also included within the scope of the term aryl as used herein is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthymidyl, phenanthridinyl or tetrahydronaphthyl and the like. The terms heteroaryl and heteroar-, used alone or as part of a larger moiety such as heteroaralkyl or heteroaralkoxy, refer to groups containing from 5 to 10 ring atoms, preferably 5, 6 or 9 ring atoms; containing 6, 10 or 14 π electrons distributed in a cyclic structure; and containing, in addition to carbon atoms, from one to five heteroatoms. The term heteroatom refers to nitrogen, oxygen or sulfur and includes any oxidized form of nitrogen or sulfur and any quaternized form of basic nitrogen. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, tria

- 9 045993 zolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl and pteridinyl. The terms heteroaryl and heteroar- as used herein also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic or heterocyclyl rings, the radical or attachment site being on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl and pyrido [2,3-b]-1,4-oxazin-3(4H)-one. The heteroaryl group may be mono- or bicyclic. The term heteroaryl may be used interchangeably with the terms heteroaryl ring, heteroaryl group, or heteroaromatic, all of which include rings that are optionally substituted. The term heteroaralkyl refers to an alkyl group substituted with heteroaryl, wherein the alkyl and heteroaryl moieties are independently optionally substituted.

The terms heterocycle, heterocyclyl, heterocyclic radical and heterocyclic ring are used interchangeably and refer to a stable 5-7 membered monocyclic or 710 membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated and contains, in addition to carbon atoms, one or more heteroatoms, preferably from one to four heteroatoms as defined above. When used in reference to the ring atom of a heterocycle, the term nitrogen includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring containing 0-3 heteroatoms selected from oxygen, sulfur, or nitrogen, said nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl ) or +NR (as in N-substituted pyrrolidinyl). The heterocyclic ring may be connected to its side group at any heteroatom or carbon atom that provides a stable structure, and any of the ring atoms may be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinil, morpholinil and quinuclidinil. The terms heterocycle, heterocyclyl, heterocyclyl ring, heterocyclic group, heterocyclic moiety and heterocyclic radical are used interchangeably herein and also include groups in which the heterocyclyl ring is fused to one or more aryl, heteroaryl or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl or tetrahydroquinolinyl. The heterocyclyl group may be mono- or bicyclic. The term heterocyclylalkyl refers to an alkyl group substituted with heterocyclyl, wherein the alkyl and heterocyclyl moieties are independently optionally substituted.

The term partially unsaturated refers to a ring moiety that contains at least one double or triple bond. The term partially unsaturated is intended to include rings containing multiple centers of unsaturation, but the term is not intended to include aryl or heteroaryl moieties as defined herein.

As described herein, the compounds may contain optionally substituted moieties. In general, the term substituted, whether optionally preceded by a term or not, means that one or more hydrogen atoms of the specified moiety have been replaced by a suitable substituent. Unless otherwise indicated, an optionally substituted group may contain a suitable substituent at each substitutable position of the specified group, and when more than one position in any particular structure can be substituted by more than one substituent selected from a particular group, the specified substituent may be the same or different in every position. The combinations of substituents provided in the present invention are preferably combinations that provide stable or chemically feasible compounds. The term stable as used herein refers to compounds that are substantially unchanged by conditions to enable their production, detection, and in some embodiments, their isolation, purification, and use for one or more of the purposes disclosed herein.

Suitable monovalent substituents on the substituted carbon atom of the optionally substituted group are independently halogen; -(CH ₂ ) _0-4 R°; -(CH ₂ ) _0-4 OR°; -O(CH ₂ ) _0-4 R ^o , -O(CH ₂ ) _0-4 C(O)OR°; -(CH ₂ ) _0-4 CH(OR°) ₂ ; -(CH ₂ ) _0-4 SR°; -(CH ₂ ) _0-4 Ph, which may be substituted by R°; -(CH ₂ ) ₀₄ O(CH ₂ ) _0-1 Ph, which can be substituted with R°; -CH=CHPh, which may be substituted with R°; -(CH ₂ ) _0-4 O(CH ₂ ) _0-1 -pyridyl, which can be substituted with R°; -NO ₂ ; -CN; -N ₃ ; -(CH ₂ ) _0-4 N(R°) ₂ ; -(CH2)0-4N(R°)C(O)R°; -N(R°)C(S)R°; -(CH2)0-4N(R°)C(O)NR°2; -N(R°)C(S)NR°2; -(CH2)0-4N(R°)C(O)OR°; N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; -(CH2)0-4C(O)R°; -C(S)R°; -(CH2)o-4C(0)OR°; -(CH2)o-4C(0)SR°; -№)o-4C(O)OSiR°3; -№)o-40C(0)R°; -oc(0)(CH2)o-4SR-sc(S)SR°;

- 10 045993

-(CH2)o-4SC(O)R°; -(CH2)o—4C(O)NR°2; -C(S)NR°2; -C(S)SR°; -SC(S)SR°, -(CH2)o—4OC(O)NR°2;

-C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)R°; -C(NOR°)R°; -(CH2)o—4SSR°; -(CH2)o—4S(O)2R°;

-(CH2)o-4S(O)2OR°; -(CH2)o-4OS(O)2R°; -S(O)2NR°2; —№)o—4S(O)R°; -N(R°)S(O^NR°2; -N(R°)S(O^R°; -N(OR°)R°; -C(NH)NR° ₂ ; -P(O) 2R°; -P(O)R°2; -OP(O)R°2; -OP(O)(OR°)2; SiR° ₃ ; -(C _1-4 straight or branched alkylene) O-H (P°) ₂ or -(C _1-4 straight or branched alkylene)C.'(O)O-H(P°) ₂ wherein each R° may be substituted as defined below and is independently hydrogen, C _1-6 aliphatic group, -CH ₂ Ph, -O(CH ₂ ) _0-1 Ph, -CH2- (5-6 membered heteroaryl ring) or 5-6 membered saturated, partially unsaturated or aryl ring containing 0- 4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or, regardless of the above definition, two independent R° variants together with the intervening atom(s) form a 312-membered saturated, partially unsaturated or aryl mono- or bicyclic ring containing 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R° (or a ring formed by two independent variants of R° together with intervening atoms) are independently halogen, -(CH2)o-2R·, -(halogen R), -( _CH2 )o- ₂ OH, -(CH ₂ )o- ₂ OR·, -(C1I2) 2C1 l(OR·).·; -O(halogen R), -CN, -N3, -(CH2)o-2C(0)R·, -(CH2)o-2C(0)0H, -(CH2)o-2C(0)0R· , -(CH2)o-2SR·, -(CH2)o-2SH, -(CH2)o-2NH2, -(CH2)o-2NHR·, -(CH ₂ ) _o-2 NR\ -NO2, -SiR \ -OSiR\ -C(O)SR·, -(C _1-4 straight or branched alkylene)C(O)OR· or -SSR·, where each R· is unsubstituted or, if preceded by a halogen, R· substituted with only one or more halogens and independently selected from a C _1-4 aliphatic group, -CH ₂ Ph, -O(CH ₂ ) _0-1 Ph or a 5-6 membered saturated, partially unsaturated or aryl ring containing 0-4 heteroatom independently selected from nitrogen, oxygen or sulfur. Suitable divalent substituents on the saturated carbon atom R° include =O u=S.

Suitable divalent substituents on the saturated carbon atom of an optionally substituted group include the following: =O, =S, =NNR* ₂ , =NNHC(O)R*, =NNHC(O)OR*, =NNHS(O) ₂ R*, = NR*, =NOR*, -O(C(R* ₂ )) _2-3 O- or -S(C(R* ₂ )) _2-3 S-, where R* in each independent case is selected from hydrogen, C _1-6 aliphatic group, which may be substituted as defined below, or an unsubstituted 5-6 membered saturated, partially unsaturated or aryl ring containing 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. Suitable divalent substituents that are bonded to the vicinal substituted carbon atoms of an optionally substituted group include: -O(CR*2)2-3O-, wherein R* is in each independent instance selected from hydrogen, a C1-6 aliphatic group which may be substituted, as defined below, or an unsubstituted 5-6 membered saturated, partially unsaturated or aryl ring containing 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

Suitable substituents on the aliphatic group R* include halogen, -R·, -(halogen R·), -OH, -O R·, -O(halogen R·), -CN, -C(O)OH, -C( O)OR·, -NH2, -NHR·, -NR* ₂ or -NO2, where each R· is unsubstituted or, if preceded by a halogen, R· is substituted with only one or more halogens and independently represents C _1-4 an aliphatic group, -CH ₂ Ph, -O(CH ₂ ) _0-1 Ph, or a 5-6 membered saturated, partially unsaturated or aryl ring containing 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. Suitable substituents on the substituted nitrogen atom of an optionally substituted group include -Rt, -NRt ₂ , -C(O)Rt, -C(O)ORt, -C(O)C(O)Rt, -C(O)CH2C(O )Rt, -S(O) ₂ Rt, -S(O) ₂ NRt ₂ , -C(S)NRt2, -C(NH)NRt ₂ , or -N(Rt)S(O)2Rt; where each Rt independently represents hydrogen, a C _1-6 aliphatic group, which may be substituted as defined below, unsubstituted -OPh or an unsubstituted 5-6 membered saturated, partially unsaturated or aryl ring containing 0-4 heteroatoms, independently selected from nitrogen, oxygen or sulfur, or, regardless of the above definition, two independent variants of Rt together with the intervening atom(s) form an unsubstituted 3-12 membered saturated, partially unsaturated or aryl mono- or bicyclic ring containing 0 -4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

Suitable substituents on the aliphatic group Rt are independently halogen, -R·, -(halogen R·), -OH, -OR·, -O(halogen R·), -CN, -C(O)OH, -C( O)OR*, -NH2, -NHR·, -NR* ₂ or -NO2, wherein each R· is unsubstituted or, if preceded by a halogen, R· is substituted with only one or more halogens, and is independently C _{1- 4} aliphatic group, -CH ₂ Ph, -O(CH ₂ ) _0-1 Ph or a 5-6 membered saturated, partially unsaturated or aryl ring containing 04 heteroatoms independently selected from nitrogen, oxygen or sulfur.

The term pharmaceutically acceptable salt refers to those salts which, in the opinion of those skilled in the medical field, are suitable for use in contact with tissues of humans and lower animals without undesirable toxicity, irritation, allergic reaction, or the like, and having an adequate benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al describe pharmacists in detail

- 11 045993 commercially acceptable salts in J. Pharmaceutical Sciences, 1977, 66, 1-19, and the contents of the said publication are incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are amino salts prepared using inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or using organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycero phosphate, gluconate, hemisulfate, heptanoate 3 -phenylpropionate, phosphate, pivalate , propionate, stearate, succinate, sulfate, tartrate, thiocyanate, n-toluenesulfonate, undecanoate, valerate salts and the like.

Salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium and N ⁺ (C _1-4 alkyl) ₄ salts. Typical alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. Other pharmaceutically acceptable salts, as appropriate, include salts containing non-toxic ammonium, quaternary ammonium cations and amine cations prepared using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and arylsulfonate.

It is also intended that, unless otherwise indicated, the structures presented herein include all isomeric (eg, enantiomeric, diastereomeric, and geometric (or conformational)) forms of the stated structures; for example, R- and S-configurations for each center of asymmetry, Z- and E-isomers according to the position of the substituents relative to the double bond, and conformational Z- and E-isomers. Thus, individual stereochemical isomers, as well as mixtures of enantiomers, diastereomers and geometric (or conformational) isomers of the compounds of the present invention are included within the scope of the present invention. Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are included within the scope of the present invention. In addition, it is also intended that, unless otherwise indicated, the structures presented herein also include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the structures of the present invention involving the replacement of hydrogen by deuterium or tritium or the replacement of carbon by ¹³ C- or ¹⁴ C-rich carbon are within the scope of the present invention. Such compounds are suitable for use, for example, as analytical tools, as probes in biological research or as therapeutic agents according to the present invention.

The terms measurable affinity and measurably inhibit as used herein mean a measurable change in mTORC1 activity when comparing a sample containing a compound of the present invention, or a composition thereof, and mTORC1, and an equivalent sample containing mTORC1 in the absence of the compound or composition thereof.

3. Description of individual implementation options.

As described above, a compound of formula I may be mentioned

t or a pharmaceutically acceptable salt thereof, wherein ring A is a monovalent derivative of rapamycin or an analogue thereof (i.e., rapalog); where R ¹ is attached to it at the C-7 position of the hydroxyl of rapamycin or its analogue; And

R ¹ is an optionally substituted straight or branched saturated or unsaturated monovalent C _3-30 hydrocarbon chain, wherein one or more methylene units of R are optionally and independently replaced by -N(R)-, -N(R)C(O)-, -C(O)N(R)-, -N(R)S(O) ₂ -, -S(O) ₂ N(R)-, -O-, -C(O)-, -OC(O )-, -C(O)O-, -S-, -S(O)-, -S(O) ₂ - or -P(O)(R) ₂ ; or a 6-18 membered saturated or partially unsaturated heterocyclic ring containing 1-6 heteroatoms independently selected from nitrogen, oxygen or sulfur; or

R ¹ is selected from the formula P-0

- 12 045993

where means the place of attachment to ring A;

each Z is independently -O-, -S-, -NR- or -SO2-; n is from about 2 to about 300; and each R independently represents hydrogen or an optionally substituted C1.6 aliphatic group.

It is obvious that the term rapamycin and its structures given in the present description are intended to include rapamycin and its analogs. For purposes of clarity, Formula II is shown below with the R ¹ moiety attached at the C-7-hydroxyl position. Accordingly, a compound of formula II may be mentioned

II or a pharmaceutically acceptable salt thereof, wherein

R ¹ is an optionally substituted straight or branched saturated or unsaturated monovalent C3-30 hydrocarbon chain, wherein one or more methylene units of R ¹ are optionally and independently replaced by -N(R)-, -N(R)C(O)-, -C(O)N(R)-, -N(R)S(O)2-, -S(O)2N(R)-, -O-, -C(O)-, -OC(O) -, -C(O)O-, -S-, -S(O)-, -S(O)2- or -P(O)(R)2; or a 6-18 membered saturated or partially unsaturated heterocyclic ring containing 1-6 heteroatoms independently selected from nitrogen, oxygen or sulfur; or

R ¹ is selected from the formula P-0

WzW

P-0 where denotes the site of attachment at the C-7-hydroxyl position;

each Z is independently -O-, -S-, -NR- or -SO2-; n is from about 2 to about 300; and each R independently represents hydrogen or an optionally substituted C _1-6 aliphatic group. For purposes of clarity, a compound of formula II-a or II-b may be mentioned

n-b or a pharmaceutically acceptable salt thereof, wherein each R ¹ is as defined above and

- 13 045993 is described herein by class and subclass. It is apparent that the term rapamycin and its structure as used herein are intended to include rapamycin and its analogues. Accordingly, in some embodiments, Ring A is rapamycin. In some embodiments, Ring A is everolimus. In some embodiments, ring A is temsirolimus. In some embodiments, ring A is ridaforolimus. In some embodiments, ring A is umirolimus.

The above rapamycin analogs (ie, rapalogues) are presented by way of example and are not intended to limit the present invention. As defined above, R ¹ is an optionally substituted straight or branched saturated or unsaturated monovalent C3-30 hydrocarbon chain, wherein one or more methylene units of R ¹ are optionally and independently replaced by -N(R)-, -N(R)C( O)-, -C(O)N(R)-, -N(R)S(O)2-, -S(O)2N(R)-, -O-, -C(O)-, - OC(O)-, -C(O)O-, -S-, -S(O)-, or -S(O)2-, or 6-18 membered saturated or partially unsaturated heterocyclic ring containing 1- 6 heteroatoms independently selected from nitrogen, oxygen or sulfur. In some embodiments, R ¹ is an optionally substituted straight or branched saturated or unsaturated monovalent C3-30 hydrocarbon chain, wherein one or more methylene units of R ¹ are optionally and independently replaced by -N(R)-, -N(R)C( O)-, -C(O)N(R)-, -N(R)S(O)2-, -S(O) ₂ N(R)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -S-, -S(O)-, -S(O) ₂ - or -P(O)(R) ₂ . In some embodiments, R ¹ is an optionally substituted branched saturated monovalent hydrocarbon chain, wherein one or more methylene units of R ¹ are optionally and independently replaced by -N(R)-, -N(R)C(O)-, -C( O)N(R)-, -N(R)S(O)2-, -S(O)2N(R)-, -O-, -C(O)-, -OC(O)-, - C(O)O-, -S-, -S(O)-, -S(O)2- or -P(O)(R)2. In some embodiments, R ¹ is an optionally substituted straight-chain unsaturated monovalent hydrocarbon chain, wherein one or more methylene units of R ¹ are optionally and independently replaced by -N(R)-, -N(R)C(O)-, -C( O)N(R)-, -N(R)S(O)2-, -S(O)2N(R)-, -O-, -C(O)-, -OC(O)-, - C(O)OS-, -S(O)-, -S(O)2- or -P(O)(R)2. In some embodiments, R ¹ is an optionally substituted branched unsaturated monovalent hydrocarbon chain, wherein one or more methylene units of R ¹ are optionally and independently replaced by -N(R)-, -N(R)C(O)-, -C( O)N(R)-, -N(R)S(O) ₂ -, -S(O) ₂ N(R)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -S-, -S(O)-, -S(O) ₂ - or -P(O)(R) ₂ . In some embodiments, R ¹ is an optionally substituted straight-chain saturated monovalent hydrocarbon chain, wherein one or more methylene units of R ¹ are optionally and independently replaced by -O-.

In some embodiments, R ¹ is

In some embodiments, R ¹ is In some embodiments, R ¹ is In some embodiments, R ¹ is In some embodiments, R ¹ is In some embodiments, R ¹ is

In some embodiments, R ¹ is In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

- 14 045993

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

- 15 045993

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ has the formula P-0

where means the place of attachment to ring A;

each Z is independently -O-, -S-, -NR- or -SO2-;

n is from about 2 to about 300; and each R independently represents hydrogen or an optionally substituted C _1-6 aliphatic group.

In some embodiments, n is from about 2 to about 10; from about 10 to about 20; from about 20 to about 30; from about 30 to about 40; from about 40 to about 50; from about 50 to about 60; from about 60 to about 70; from about 70 to about

80; from about 80 to about 90; from about 90 to about 100; from about 110 to about 120;

from about 120 to about 130; from about 140 to about 150; from about 150 to about 160;

from about 170 to about 180; from about 180 to about 190; from about 190 to about 200;

from 200 to about 210; from about 210 to about 220; from about 220 to about 230; from about 230 to about 240; from about 240 to about 250; from about 250 to about 260; from about 260 to about 270; from about 270 to about 280; from about 280 to about 290, or from about 290 to about 300.

In some embodiments, R ¹ is

where n and R are as described herein.

In some embodiments, R ¹ is

" "1 " .

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

- 16 045993

In some embodiments, R ¹ is In some embodiments, R ¹ is In some embodiments, R ¹ is In some embodiments, R ¹ is In some embodiments, R ¹ is In some embodiments, R ¹ is In some implementation options R ¹ represents

In some embodiments, R ¹ is In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

---ο-ο- ^ΟΗ

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

- 17 045993

In some embodiments, R ¹ is

In some embodiments, R ¹ is

------ ... . . .... .. .. _____ .

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

L^ _o ^ ^OMe

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

„,

In some embodiments, R ¹ is

In some embodiments, R ¹ is

Χρ^ _ο ·)ς^ ^ΟΜβ

In some embodiments, R ¹

In some embodiments, R ¹

In some embodiments, R ¹

In some embodiments, R ¹

In some embodiments, R ¹

In some embodiments, R ¹

In some embodiments, R ¹

In some embodiments, R ¹

In some embodiments, R ¹

In some embodiments, R ¹

In some embodiments, R ¹ _i _ ^o^ ^ome represents represents ^ΟΜθ represents .......

represents represents g - ''H^O'£> ^OMe represents ^{v 90} .

d.

represents

- . L^o^ ^OMe represents

6%^0-^^ОМе represents ¹²⁰ · represents

- „ A^ Oi ₄ ^ ^OMe represents

In some embodiments, R ¹ is

In some embodiments, R ¹ is

------ ... . . .... .. .. _____ .

In some embodiments, R ¹ is „ „, . ^o^° ^mv

In some embodiments, R ¹ is

In some embodiments, R ¹ is ob-° ^M '

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

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In some embodiments, R ¹ is

In some embodiments, R ¹ is ²⁴⁰ .

In some embodiments, R ¹ is

In some embodiments, R ¹ is „ „1, „ 0 · '"- _; ^ΟΜ “

In some embodiments, R is "____ .. . . .... .. "1 _____ . ^/ Ch-^O^° ^Me

In some embodiments, R ¹ is

In some embodiments, R ¹ is

X/ /ОМе °'29О ' ^SS ^^°^300^ ^OMe

In some embodiments, R ¹ is ³⁰⁰

In some embodiments, R ¹ is

Ό' . - ,. ⁿ . In some embodiments, R ¹ is „ ..... ___________ .... . - . 'si' ⁰

In some embodiments, R is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

He

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

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In some embodiments, R ¹ is he oX_on

In some embodiments, R ¹ is

In some embodiments, R ¹ is

In some embodiments, R ¹ is

CN

In some embodiments, R ¹ is

In some embodiments, R ¹ is a 6-18 membered saturated or partially unsaturated heterocyclic ring containing 1-6 heteroatoms independently selected from nitrogen, oxygen or sulfur.

In some embodiments, R ¹ is

In some embodiments, R ¹ is

.0 Oh.

In some embodiments, R ¹ is

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In some embodiments, R ¹ is

In some embodiments, R ¹ is selected from the options shown in table. 1 below.

In some embodiments, R independently represents hydrogen or an optionally substituted group selected from a C _1-6 aliphatic group. In some embodiments, R is hydrogen. In some embodiments, R is methyl. In some embodiments, R is ethyl. In some implementations of R, the representation

It's a cut. In some embodiments, R is

In some embodiments, R is selected from the options shown in table. 1 below.

In some embodiments, a compound of formula III may be mentioned

In some embodiments, a compound of formula III-a or III-b may be mentioned

In some embodiments, a compound of Formula IV may be mentioned

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or a pharmaceutically acceptable salt thereof, wherein: R ¹ is as described herein.

In some embodiments, a compound of formula IV-a or IV-b may be mentioned

or a pharmaceutically acceptable salt thereof, wherein: R ¹ is as described herein. In some embodiments, a compound of formula V may be mentioned

or a pharmaceutically acceptable salt thereof, wherein: R ¹ is as described herein.

In some embodiments, a compound of formula V-a or V-b may be mentioned

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or a pharmaceutically acceptable salt thereof, wherein: R ¹ is as described herein. In some embodiments, a compound of formula VI may be mentioned

or a pharmaceutically acceptable salt thereof, wherein: R ¹ is as described herein.

In some embodiments, a compound of formula VI-a or VI-b may be mentioned

VI-b or a pharmaceutically acceptable salt thereof, wherein: R ¹ is as described herein. In some embodiments, a compound of formula VII may be mentioned

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or a pharmaceutically acceptable salt thereof, wherein: R ¹ is as described herein.

In some embodiments, a compound of formula VII-a or VII-b may be mentioned

or a pharmaceutically acceptable salt thereof, wherein: R ¹ is as described herein. In some embodiments, a compound of formula VIII may be mentioned

or a pharmaceutically acceptable salt thereof, wherein: R ¹ is as described herein.

In some embodiments, a compound of formula VIII-a or VIII-b may be mentioned

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or a pharmaceutically acceptable salt thereof, wherein: R ¹ is as described herein.

In some embodiments, a compound of formula IX-a, IX-b, IX-c, IX-d, IX-e, IX-f, or IX-g may be mentioned.

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or a pharmaceutically acceptable salt thereof, wherein: R ¹ is as described herein.

In some embodiments, a compound of formula X-a, X-b, X-c, X-d, X-e, X-f, or X-g may be mentioned.

- 27 045993

- 28 045993 he is about

X-f he's about

Xg or a pharmaceutically acceptable salt thereof, wherein: R ¹ is as described herein.

In some embodiments, a compound of formula XI-a, XI-b, XI-c, XI-d, XI-e, XI-f, or XI-g may be mentioned

IT

XI-a

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or a pharmaceutically acceptable salt thereof, wherein: P-0 is as described herein. In some embodiments, a compound of formula XII may be mentioned

or a pharmaceutically acceptable salt thereof, wherein: R and n are as described herein.

In some embodiments, a compound of Formula XII-a or XII-b may be mentioned.

or a pharmaceutically acceptable salt thereof, wherein: R and n are as described herein. In some embodiments, a compound of formula XIII may be mentioned

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or a pharmaceutically acceptable salt thereof, wherein: R and n are as described herein.

In some embodiments, a compound of formula XIII-a or XIII-b may be mentioned

or a pharmaceutically acceptable salt thereof, wherein: R and n are as described herein. In some embodiments, a compound of formula XIV may be mentioned

or a pharmaceutically acceptable salt thereof.

In some embodiments, a compound of formula XIV-a or XIV-b may be mentioned.

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or a pharmaceutically acceptable salt thereof.

In some embodiments, a compound of formula XV may be mentioned

or a pharmaceutically acceptable salt thereof.

In some embodiments, a compound of formula XV-a or XV-b may be mentioned

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XV-b or a pharmaceutically acceptable salt thereof.

Rapamycin is marketed under the brand name Rapamune® (generic name sirolimus) and is well known for its antiproliferative and immunosuppressive activities. Rapamycin is approved by the US Food and Drug Administration (FDA) to prevent graft failure and to coat stents to prevent restenosis. In addition to the stated benefits of rapamycin, it is well known that there are a number of serious side effects associated with the use of rapamycin. Such side effects include diabetes-like symptoms of decreased glucose tolerance and decreased insulin sensitivity. In addition, rapamycin has been reported to activate the Akt signaling pathway (including activation of Akt and ERK), thereby increasing a patient's risk of cancer.

As used herein, the phrase rapamycin alone is intended to compare a compound of the present invention with rapamycin or an analogue thereof as alternatives.

In some embodiments, a compound of Formula I, II, III, IV, V, VI, VII, VIII, XII, XIII, XIV, or XV is more effective than rapamycin alone. In some embodiments, a compound of formula II-a, III-a, IV-a, V-a, VI-a, VII-a, VIII-a, XII-a, XIII-a, XIV-a, or XV-a is more than more effective than rapamycin alone. In some embodiments, a compound of Formula II-b, III-b, IV-b, Vb, VI-b, VII-b, VIII-b, XII-b, XIII-b, XIV-b, or XV-b is more effective than rapamycin alone.

In some embodiments, a compound of formula IX-a, IX-b, IX-c, IX-d, IX-e, IX-f, or IX-g is more effective than rapamycin alone.

In some embodiments, a compound of formula X-a, X-b, X-c, X-d, X-e, X-f, or X-g is more effective than rapamycin alone.

In some embodiments, a compound of Formula XI-a, XI-b, XII-c, XI-d, XI-e, XI-f, or XI-g is more effective than rapamycin alone.

In some embodiments, a compound of formula II-a or II-b is more effective than rapamycin alone. In some embodiments, a compound of formula III-a or III-b is more effective than rapamycin alone. In some embodiments, a compound of formula IV-a or IV-b is more effective than rapamycin alone. In some embodiments, a compound of Formula Va or V-b is more effective than rapamycin alone. In some embodiments, a compound of formula VI-a or VI-b is more effective than rapamycin alone. In some embodiments, a compound of formula VII-a or VII-b is more effective than rapamycin alone. In some embodiments, a compound of formula VIII-a or VIII-b is more effective than rapamycin alone. In some embodiments, the provided compound of Formula XII-a or XII-b is more effective than rapamycin alone. In some embodiments, a compound of Formula XIII-a or XIII-b is more effective than rapamycin alone. In some embodiments, a compound of Formula XIVa or XIV-b is more effective than rapamycin alone. In some embodiments, a compound of formula XV-a or XV-b is more effective than rapamycin alone.

In some embodiments, a compound of Formula I, II, III, IV, V, VI, VII, VIII, XII, XIII, XIV, or XV, when administered to a patient, produces fewer and/or less severe side effects compared to administration of rapamycin.

In some embodiments, a compound of formula IX-a, IX-b, IX-c, IX-d, IX-e, IX-f, or IX-g, when administered to a patient, produces fewer and/or less severe side effects compared to administration rapamycin.

In some embodiments, a compound of formula X-a, X-b, X-c, X-d, X-e, X-f, or X-g, when administered to a patient, provides fewer and/or less severe side effects compared to administration of rapamycin.

In some embodiments, a compound of formula XI-a, XI-b, XI-c, XI-d, XI-e, XI-f, or XI-g, when administered to a patient, provides fewer and/or less severe side effects. compared with the administration of rapamycin.

Exemplary compounds are presented in table. 1 below.

Table 1. Illustrative Connections

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In some embodiments, the connection shown in the table above may be mentioned.

1, or a pharmaceutically acceptable salt thereof. It is obvious that the compound shown in the table above may also be mentioned in the present invention. 1, in the form of a racemic mixture of isomers at the C7 position, or a pharmaceutically acceptable salt thereof. In addition, it is obvious that the racemic mixtures of isomers of the compounds shown above in the table. 1, at the hydroxyl at position C7, can be separated into diastereomers by various methods, for example, using chiral chromatography.

4. Applications, formulation and administration.

Pharmaceutically acceptable compositions.

According to another embodiment, the present invention provides a composition containing a compound of the present invention or a pharmaceutically acceptable form thereof.

- 74 045993 derivative and pharmaceutically acceptable carrier, adjuvant or medium. The amount of compound in the composition of the present invention is such that it is effective to measurably inhibit mTORC1 in a biological sample or patient. In some embodiments, the amount of a compound in a composition of the present invention is such that it is effective to measurably inhibit mTORC1 in a biological sample or patient. In some embodiments, a composition of the present invention is formulated for administration to a patient in need of such a composition. In some embodiments, the composition of the present invention is formulated for oral administration to a patient. The term patient means an animal, preferably a mammal, and most preferably a human.

The term pharmaceutically acceptable carrier, adjuvant or vehicle refers to a non-toxic carrier, adjuvant or vehicle that does not interfere with the pharmacological activity of the compound being formulated with it. Pharmaceutically acceptable carriers, adjuvants or carriers that may be used in the composition of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffers such as phosphates , glycine, sorbic acid, potassium sorbate, partial glycerides of a mixture of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, substances based on cellulose, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin (wool fat).

The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or by an implantable reservoir. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion. Preferably the compositions are administered orally, intraperitoneally or intravenously. Sterile injectable forms of the compositions of the present invention may be an aqueous or oily suspension. These suspensions can be prepared in accordance with methods known in the art, using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic diluent or solvent suitable for parenteral administration, for example a solution in 1,3-butanediol. Acceptable carriers and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are usually used as a solvent or suspending medium.

For this purpose, any light (bland) fixed oil can be used, including synthetic mono or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives can be used to prepare injectable forms, examples being natural pharmaceutically acceptable oils such as olive oil or castor oil, in particular in their polyoxyethylated forms. These oily solutions or suspensions may also contain a long-chain alcohol solvent or dispersant, such as carboxymethylcellulose or similar dispersants, which are commonly used in pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as the Tween, Span, and other emulsifying agents or bioavailability enhancers that are commonly used to formulate pharmaceutically acceptable solid, liquid, or other dosage forms, may also be used to formulate the formulations.

The pharmaceutically acceptable compositions of the present invention can be administered orally in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions or solutions. For oral tablets, carriers typically include lactose and corn starch. Lubricants such as magnesium stearate are also commonly added. For oral administration in capsule form, suitable diluents include lactose and dried corn starch. When aqueous suspensions for oral administration are required, the active ingredient is combined with emulsifying and suspending agents. Certain sweetening, flavoring or coloring agents may also be added if desired. Alternatively, the pharmaceutically acceptable compositions of the present invention may be administered in the form of suppositories for rectal administration. They can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature and liquid at rectal temperature and will thus melt in the rectum, releasing the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. Pharmaceutically acceptable compositions according to the present invention can also be administered topically, particularly

- 75 045993 when the target for treatment includes areas or organs that are easily accessible for topical application, including diseases of the eyes, skin or lower gastrointestinal tract. Suitable topical formulations can be readily prepared for each of these areas or organs.

Topical administration to the lower gastrointestinal tract can be achieved using a rectal suppository formulation (see above) or a suitable enema formulation. Topical transdermal patches can also be used.

For topical use, the pharmaceutically acceptable compositions of the invention may be formulated as a suitable ointment containing the active ingredient suspended or dissolved in one or more vehicles.

Carriers for topical application of the compounds of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutically acceptable compositions provided may be formulated as a suitable lotion or cream containing the active ingredients suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. For ophthalmic use, the pharmaceutically acceptable compositions of the invention may be formulated as micronized suspensions in isotonic, sterilized pH-adjusted saline or as solutions in isotonic, sterilized pH-adjusted saline, with or without the addition of a preservative such as benzalkonium chloride. Alternatively, for ophthalmic use, pharmaceutically acceptable compositions may be formulated as an ointment such as petroleum jelly.

Pharmaceutically acceptable compositions of the present invention can also be administered by nasal aerosol or by inhalation. Such compositions are prepared according to techniques well known in the pharmaceutical formulation art and can be prepared as saline solutions using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons and/or other conventional solubilizing or dispersing agents.

Most preferably, the pharmaceutically acceptable compositions of the present invention are formulated for oral administration. Such formulations may be administered with or without food. In some embodiments, the pharmaceutically acceptable compositions of the present invention are administered without food. In other embodiments, the pharmaceutically acceptable compositions of the present invention are administered with food.

The amount of a compound of the present invention that can be combined with carrier substances to formulate a single dosage form will vary depending on the host being treated and the particular route of administration. Preferably, the proposed compositions should be prepared in such a way that a dose of 0.01-100 mg/kg body weight/day of inhibitor can be administered to a patient receiving said compositions. It should also be understood that the specific dosage and treatment regimen for any particular patient will depend on many different factors, including the potency of the particular compound used, age, body weight, general health, gender, diet, time of administration, rate of elimination, drug combination, opinion the attending physician and the severity of the specific disease being treated. The amount of a compound of the present invention in a composition will also depend on the particular compound in the composition.

Uses of Compounds and Pharmaceutically Acceptable Compositions As used herein, the terms treatment, treat and treater refer to achieving regression, alleviation, delay in onset or suppression of the progression of a disease or disorder or one or more symptoms thereof described herein. In some embodiments, treatment may be administered after the development of one or more symptoms. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a predisposed individual prior to the onset of symptoms (eg, based on a history of symptoms and/or genetic or other predisposition factors). Treatment may also be continued after symptoms have disappeared, for example to prevent or delay their recurrence.

The compounds of the invention are mTORC1 inhibitors and are therefore suitable for use in the treatment of one or more disorders associated with mTORC1 activity. Thus, a method for treating an mTORC1-mediated disorder may be mentioned, comprising the step of administering to a patient in need thereof a compound of the present invention or a pharmaceutically acceptable composition thereof.

The term mTORC1-mediated disorders, diseases and/or conditions means any

- 76 045993 disease or other harmful condition known to involve mTORCl. Accordingly, a method of treating or reducing the severity of one or more diseases known to involve mTORC1 may be mentioned. In some embodiments, the mTORC1-mediated disorder, disease, and/or condition is selected from the disorders, diseases, and/or conditions described in Matt Kaeberlin, Scientifica, vol. 2013, Article ID 849186.

The methods described herein include methods for treating cancer in a subject. The term treatment means reducing the severity or improving the condition of at least one symptom or clinical parameter of cancer. For example, treatment may result in a decrease in tumor size or rate of growth. Treatment does not necessarily mean curing cancer or eliminating its cause 100% of the time for all subjects.

The term cancer refers to cells that have the ability to grow autonomously, i.e. an abnormal status or condition characterized by rapid proliferative cell growth. This term is intended to include all types of cancerous growth or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathological type or stage of invasive action. The term tumor refers to cancer cells, such as a mass of cancer cells.

Cancers that can be treated or diagnosed using the methods described herein include malignancies in various organ systems, such as those affecting the lung, breast, thyroid, lymphoid tissue, gastrointestinal and genitourinary systems, as well as adenocarcinomas, which include malignancies such as most colon cancers, renal cell carcinoma, prostate and/or testicular cancers, non-small cell lung carcinoma, small bowel cancer and esophageal cancer.

In some embodiments, the described methods are used to treat or diagnose carcinoma in a subject. The term carcinoma is common in the art and refers to malignant tumors of epithelial or endocrine tissues, including respiratory carcinomas, gastrointestinal carcinomas, genitourinary carcinomas, ovarian carcinomas, mammary carcinomas, prostate carcinomas, endocrine carcinomas and melanomas. In some embodiments, said cancer is renal carcinoma or melanoma.

Examples of carcinomas include those arising from tissue of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcoma, such as carcinosarcoma, which includes malignant tumors consisting of carcinomatous and sarcomatous tissue. The term adenocarcinoma refers to carcinoma formed from glandular tissue or in which tumor cells form recognizable glandular structures.

The term sarcoma is generally accepted in the field and refers to malignant tumors of mesenchymal origin.

In some embodiments, the cancers that can be treated according to the described methods are cancers characterized by increased levels of mTORC1 or increased expression or activity of mTORC1 relative to normal tissues or other cancers of the same tissues; and methods known in the art and described herein can be used to determine these cancers. In some embodiments, the methods include obtaining a sample containing cancer cells, determining mTORC1 activity in the sample, and administering treatment as described herein (eg, with a provided mTORC1 inhibitor). In some embodiments, the cancer is a cancer provided herein in which there are elevated levels of mTORC1 activity. In some embodiments, a method of treating one or more disorders, diseases and/or conditions may be mentioned, wherein said disorder, disease or condition includes, but is not limited to, a cell proliferative disorder.

Cell proliferative disorders.

Methods and compositions for diagnosing and prognosticating cell proliferative disorders (eg, cancer) and treating these disorders by inhibiting mTORC1 activity. Cellular proliferative disorders described herein include, for example, cancer, obesity, and proliferation-dependent diseases. Such disorders can be diagnosed using methods known in the art.

Cancer.

Cancers include, but are not limited to, leukemias (eg, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (eg, Hodgkin's disease or non-Hodgkin's lymphoma), Waldenström's macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors such as

- 77 045993 sarcoma and carcinoma (for example, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, carcinoma of the colon th colon, pancreatic cancer, cancer breast, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, biliary carcinoma pathways, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial cell carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma and retinoblastoma). In some embodiments, said cancer is melanoma or breast cancer.

Fibrotic diseases.

Idiopathic pulmonary fibrosis (IPF). The PI3K pathway is activated in areas of fibrosis, the cardinal lesions of IPF. The mTOR kinase inhibitor GSK2126458 reduces PI3K signaling and functional responses in IPF-derived lung fibroblasts, and mTOR inhibition reduces collagen expression in IPF patient models. In the bleomycin model of pulmonary fibrosis, treatment with rapamycin induces an antifibrotic effect, and also rapamycin reduces the expression of smooth muscle actin and fibronectin by fibroblasts in vitro. In some embodiments, the method of inhibiting mTORC1 activity is used to treat idiopathic pulmonary fibrosis (IPF) (See Mercer, P.F. et al., Thorax., 71(8): 701-11 (2016); Patel, A.S., et al., PLoS One, 7(7): e41394 (2012)). Accordingly, in some embodiments, there may be mentioned a method of treating idiopathic pulmonary fibrosis (IPF) in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Kidney fibrosis. mTORC1 is activated in myofibroblasts, the main pathogenic cell type in renal fibrosis. Inhibition of mTOR by rapamycin in a mouse model of renal fibrosis (UUO) attenuated the expression of markers of fibrosis and tubulointerstitial damage.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat renal fibrosis (see Jiang, L., et al., J Am Soc Nephrol, 24(7): 1114-26 (2013); Wu, M. J. et al., Kidney International, 69(11): 2029-36 (2006); Chen, G. et al., PLoS One, 7(3): e33626 (2012); Liu, C. F. et al., Clin Invest Med, 37(34) : E142-53 (2014)). Accordingly, in some embodiments, there may be mentioned a method of treating renal fibrosis in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat scleroderma (see Mitra, A., et al., J Invest Dermatol. 135(11): 2873-6 (2015)). Accordingly, in some embodiments, there may be mentioned a method of treating scleroderma in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat hypertrophic scars and keloid disease (See Syed, F., et al., Am J Pathol. 181(5): 164258 (2012)). Accordingly, in some embodiments, there may be mentioned a method of treating hypertrophic scars and keloid disease in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat cardiac fibrosis (see Yano, T., et al., J Mol Cell Cardiol. 91: 6-9 (2016)). Accordingly, in some embodiments, there may be mentioned a method of treating cardiac fibrosis in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Other proliferative diseases.

Other proliferative diseases include, for example, obesity, benign prostatic hyperplasia, psoriasis, abnormal keratinization, lymphoproliferative disorders (eg, a disorder in which there is abnormal proliferation of cells in the lymphatic system), chronic rheumatoid arthritis, arteriosclerosis, restenosis, and diabetic retinopathy. Proliferative diseases, which are hereby incorporated by reference herein, include those described in US Pat. Nos. 5,639,600 and 7,087,648.

Other disorders.

Other disorders include lysosomal storage diseases including, but not limited to, Pompe disease, Gaucher disease, mucopolysaccharidosis, multiple sulfatase deficiency; neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, alpha-1 antitrypsin deficiency and spinal bulbar muscular atrophy.

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In some embodiments, a method of inhibiting mTORC1 activity is used in the treatment of asthma (see Hua, W., et al., Respirology, 20(7):1055-65 (2015)). Accordingly, in some embodiments, there may be mentioned a method of treating asthma in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat a lysosomal storage disease (see Sardiello, M., Annals of the New York Academy of Sciences, 1371(1): 3-14 (2016); Awad, O., et al ., Hum Mol Genet. 24(20): 5775-88 (2015); Spampanato, C., et al., EMBO Mol Med., 5(5): 691-706 (2013); Medina, D. L., et al. ., Dev Cell., 21(3): 421-30 (2011). Accordingly, in some embodiments, a method of treating a lysosomal storage disease in a patient in need thereof may be mentioned, comprising administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat Parkinson's disease (see Decressac, M., et al., Proc Natl Acad Sci U S A., 110(19):E1817-26 (2013)). Accordingly, in some embodiments, there may be mentioned a method of treating Parkinson's disease in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof. In some embodiments, a method of inhibiting mTORC1 activity is used to treat Alzheimer's disease (see Polito, V.A., et al., EMBO Mol Med. 6(9): 1142-60 (2014)). Accordingly, in some embodiments, there may be mentioned a method of treating Alzheimer's disease in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat Huntington's disease (see Tsunemi, T., et al., Sci Transl Med., 4(142): 142ra97 (2012)). Accordingly, in some embodiments, there may be mentioned a method of treating Huntington's disease in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof. In some embodiments, a method of inhibiting mTORC1 activity is used to treat alpha-1 antitrypsin deficiency (see Pastore, N. et al., EMBO Mol Med., 5(3): 397-412 (2013)). Accordingly, in some embodiments, there may be mentioned a method of treating alpha-1 antitrypsin deficiency in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof. In some embodiments, a method of inhibiting mTORC1 activity is used to treat spinal bulbar muscular atrophy (see Cortes, C. J., et al., Nat Neurosci., 17(9): 1180-9 (2014)). Accordingly, in some embodiments, there may be mentioned a method of treating spinal bulbar muscular atrophy in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

In some embodiments, a compound of the present invention binds to FKBP12 to form a complex. In some embodiments, the complex between a compound of the present invention and FKBP12 interacts with FK506, the rapamycin binding domain of mTOR.

In some embodiments, a compound of the present invention binds to FKBP12 and interferes with protein-protein interaction between FRAP and FKBP12. In some embodiments, the R moiety of a compound of the present invention interacts with both FRAP and FKBP12. The present invention provides compounds that are inhibitors of mTORC1 activity and have been demonstrated to selectively inhibit mTORC1 relative to mTORC2, as measured by inhibition of pS6K (an indicator of mTORC1 activity) and activation of pAKT (an indicator of mTORC2 activity). In some embodiments, the present compound inhibits mTORC1 selectively relative to mTORC2. In some embodiments, the present compound does not inhibit mTORC2 to a significant extent. In some embodiments, the present compound has a pAKT activation IC ₅₀ of >10 μM. In some embodiments, the present compound inhibits mTORC1 with >10-fold selectivity over mTORC2. In some embodiments, the present compound inhibits mTORC1 with >20-fold selectivity over mTORC2. In some embodiments, the present compound inhibits mTORC1 with >50-fold selectivity over mTORC2. In some embodiments, the present compound inhibits mTORC1 with >100-fold selectivity over mTORC2. In some embodiments, the present compound inhibits mTORC1 with >150-fold selectivity over mTORC2. In some embodiments, the present compound inhibits mTORC1 with >200-fold selectivity over mTORC2. In some embodiments, the present compound inhibits mTORC1 with >500-fold selectivity over mTORC2. In some embodiments, the present compound inhibits mTORC1 with >1000-fold selectivity over mTORC2.

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In some embodiments, the present compound inhibits mTORCl selectively over mTORC2 after prolonged treatment or exposure. In some embodiments, the present compound inhibits mTORC1 selectively over mTORC2 after approximately 24 hours of treatment or exposure. In some embodiments, the present compound inhibits mTORC1 selectively over mTORC2 after approximately 36 hours of treatment or exposure. In some embodiments, the present compound inhibits mTORC1 selectively over mTORC2 after approximately 48 hours of treatment or exposure. In some embodiments, the present compound inhibits mTORC1 selectively over mTORC2 after approximately 72 hours of treatment or exposure. In some embodiments, the present compound inhibits mTORC1 selectively over mTORC2 after approximately 96 hours of treatment or exposure. In some embodiments, the present compound inhibits mTORC1 selectively over mTORC2 after approximately 120 hours of treatment or exposure. In some embodiments, the present compound inhibits mTORC1 selectively over mTORC2 after approximately 144 hours of treatment or exposure. In some embodiments, the present compound inhibits mTORC1 selectively over mTORC2 after approximately one week of treatment or exposure. In some embodiments, the provided compound inhibits mTORC1 selectively over mTORC2 after more than about one week of treatment or exposure.

In some embodiments, the proposed compound is less immunosuppressive than existing rapalogues. In some embodiments, the present compound is less immunosuppressive than rapamycin. In some embodiments, the present compound is less immunosuppressive than everolimus. In some embodiments, the present compound is less immunosuppressive than temsirolimus. In some embodiments, the present compound is less immunosuppressive than ridaforolimus. In some embodiments, the present compound is less immunosuppressive than umirolimus. In some embodiments, the present compound inhibits the production of interferon gamma (IFN-γ) to a lesser extent than rapalogs. In some embodiments, the present compound inhibits IFN-γ production to a lesser extent than rapamycin. In some embodiments, the present compound inhibits IFN-γ production to a lesser extent than everolimus. In some embodiments, the present compound suppresses IFN-γ production to a lesser extent than temsirolimus. In some embodiments, the present compound inhibits IFN-γ production to a lesser extent than ridaforolimus. In some embodiments, the present compound inhibits IFN-γ production to a lesser extent than umirolimus.

In some embodiments, the present compound reduces the expression of fibrosis biomarkers in damaged tissue. In some embodiments, the present compound reduces the expression of collagen I (COLIA2) in damaged tissue. In some embodiments, the present compound reduces the expression of collagen III (COL3A1) in damaged tissue. In some embodiments, the present compound reduces the expression of fibronectin (FN1) in damaged tissue.

In some embodiments, the present compound reduces the ability of immune cells to invade damaged tissue. In some embodiments, the present compound reduces the ability of macrophage cells to invade damaged tissue.

In some embodiments, the present compound causes less glucose tolerance than rapalogs. In some embodiments, the present compound causes less glucose tolerance than rapamycin. In some embodiments, the present compound causes less glucose tolerance than everolimus. In some embodiments, the present compound causes less glucose tolerance than temsirolimus. In some embodiments, the present compound causes less glucose tolerance than ridaforolimus. In some embodiments, the present compound causes less glucose tolerance than umirolimus. In some embodiments, the present compound does not induce glucose tolerance to a significantly greater extent than placebo or vehicle.

Accordingly, in some embodiments, there may be mentioned a method of treating a disorder associated with mTORC1, comprising administering to a patient a compound that inhibits mTORC1, wherein the compound does not inhibit mTORC2. Such compounds may be used for indications where rapamycin and rapalogues have demonstrated benefit either in animal models or in human disease settings. Such indications include the following: Treatment of metabolic disease (obesity and insulin resistance in type 2 diabetes).

Inhibition of the mTORC1 pathway results in increased lifespan in yeast, flies and mice, and caloric restriction increases lifespan and insulin sensitivity. The underlying mechanism has been proposed to operate by regulating mTORC1 activation. Rapamycin-induced insulin resistance has been demonstrated to be mediated by mTORC2 inhibition, and a selective mTORC1 inhibitor has been suggested to improve

- 80 045993 insulin sensitivity and glucose homeostasis.

In some embodiments, the method of inhibiting mTORCl activity is used to treat a metabolic disease (obesity and insulin resistance in type 2 diabetes) (see Yu, Z., et al., J Gerontol A Biol Sci Med Sci, 70(4), 410-20 (2015); Fok, W.C., et al., Aging Cell 13 (2):311-9 (2014); Shum, M., et al., Diabetologia, 59(3):592-603 (2016); Lamming , D.W., et al., Science 335(6076): 163843 (2012)). Accordingly, in some embodiments, there may be mentioned a method of treating a metabolic disease (obesity and insulin resistance in type 2 diabetes) in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Neurofibromatosis.

Neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene. Its protein product, neurofibromin, acts as a tumor suppressor and ultimately induces constitutive positive regulation of mTOR. mTOR inhibitors have been demonstrated to reduce tumor size and induce antiproliferative effects in #1-associated plexiform neurofibroma.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat neurofibromatosis (See Franz, D.N., et al., Curr Neurol Neurosci Rep., 12(3): 294-301 (2012); Varin, J., et al., Oncotarget, 7: 35753-67 (2016)).

Accordingly, in some embodiments, there may be mentioned a method of treating neurofibromatosis in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Cardiomyopathy and skeletal muscle dystrophy, Emery-Dreyfus model of muscular dystrophy (LMNA ^- / ^- ).

LMNA mutations lead to several human diseases, including limb-girdle muscular dystrophy (LGMD1B), Emery-Dreyfus muscular dystrophy (EDMD2/3), dilated cardiomyopathy (DCM) and conduction system disease (CMD1A), lipodystrophy, Charcot-Marie-Tooth disease and syndrome Hutchinson-Gilford progeria (HGPS). Lmna ^−/− mice exhibit increased mTORC1 activity, and short-term rapamycin treatment of Lmna ^−/− mice results in decreased mTORC1 signaling, improved cardiac and skeletal muscle function, and increased survival by approximately 50%.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat cardiomyopathy and skeletal muscle dystrophy (see Ramos, F., et al., Sci Transl Med., 4(144): 144ra103 (2012); Bonne, G. & Quijano- Roy, S., Handb Clin Neurol., 113: 1367-76 (2013). Accordingly, in some embodiments, there may be mentioned a method of treating cardiomyopathy and skeletal muscle dystrophy in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Leigh syndrome.

Ndufs4 knockout (KO) mice are used as a model of Leigh syndrome and exhibit mTORC1 hyperactivation and metabolic defects. Treatment of mice with Ndufs4 AN with rapamycin increases lifespan and reduces the metabolic and neurological defects associated with this disease. In some embodiments, a method of inhibiting mTORC1 activity is used to treat Leigh syndrome (see Johnson, S.C., et al., Science, 342(6165): 1524-8 (2013)). Accordingly, in some embodiments, there may be mentioned a method of treating Leigh's syndrome in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Oncology.

Inhibition of mTOR using rapalogs has been demonstrated to have antitumor activity in mouse models of cancer and in patients with cancer. Examples of susceptible cancers include, but are not limited to, hepatocellular carcinoma, various types of breast cancer, mantle cell lymphomas, lung carcinoma, tuberous sclerosis and lymphangioleiomyomatosis. In some embodiments, a method of inhibiting mTORC1 activity is used to treat cancer and cancer-related disorders (see Ilagan, E. & Manning, B.D., Trends Cancer, 2(5): 241-51 (2016)). Accordingly, in some embodiments, there may be mentioned a method of treating cancer and oncological disorders in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Non-alcoholic steatohepatitis (NASH).

The present invention provides inhibitors that induce autophagy to clear destroyed cytoplasmic proteins, and the disease NASH is characterized by lipid accumulation, inflammation and fibrosis in the liver. Inhibition of the mTORC1 pathway induces autophagy and down-regulates SREBP-1 to reduce lipid biosynthesis to reduce lipid accumulation.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat non-alcoholic steatohepatitis (NASH) (See Puri, P. & Chandra, A., J Clin Exp Hepatol, 4(1): 51-9

- 81 045993 (2014)). Accordingly, in some embodiments, there may be mentioned a method of treating non-alcoholic steatohepatitis (NASH) in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Tuberous sclerosis (TSC) and lymphangioleiomyomatosis (LAM).

Dysregulation of mTOR is critical to the pathogenesis of the inherited disorder tuberous sclerosis complex (TSC) and the associated lung disease lymphangioleiomyomatosis (LAM). Both diseases are caused by mutations in TSC1 or TSC2, leading to inappropriate signaling activity downstream of mTORC1. Patients with TSC develop benign tumors in multiple organs, including the brain, while patients with LAM, mostly women, accumulate abnormal muscle-like cells in some organs or tissues, particularly the lungs, lymph nodes, and kidneys. The rapalogues, everolimus and sirolimus, are currently approved by the U.S. Food and Drug Administration (U.S. FDA) for the treatment of TSC and LAM, respectively.

In some embodiments, the method of inhibiting mTORC1 activity is used to treat tuberous sclerosis and lymphangioleiomyomatosis (See Wander, S.A., et al., J. Clin. Invest., 121(4): 1231-41 (2011); Taveira-DaSilva, A.M. & Moss, J., J. Clin Epidemiol., 7: 249-57 (2015)). Accordingly, in some embodiments, there may be mentioned a method of treating tuberous sclerosis and lymphangioleiomyomatosis in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Aging and age-related diseases.

Rapamycin inhibits the mammalian TORC1 complex, which regulates translation, and increases lifespan in various species, including mice. Rapamycin has been demonstrated to inhibit the proinflammatory phenotype of senescent cells. Because senescent cells accumulate with age, the senescence-associated secretory phenotype (SASP) can destroy tissue and contribute to the development of age-related pathologies, including cancer. Inhibition of mTOR suppressed the secretion of inflammatory cytokines by senescent cells. Rapamycin decreased levels of cytokines, including IL6, and suppressed the translation of the membrane-bound cytokine ILIA. Reduced ILIA attenuates the transcriptional activity of NF-κB, which controls SASP. Thus, mTORC1 inhibitors may attenuate age-related pathologies, including late-life cancer, by suppressing inflammation associated with aging.

In some embodiments, the method of inhibiting mTORC1 activity is used to treat aging and age-related diseases (see Laberge, R. M., et al., Nature Cell Biology, 17(8): 1049-61 (2015); Nacarelli, T., et al. , Free Radic Biol Med., 95: 133-54 (2016)). Accordingly, in some embodiments, there may be mentioned a method of treating aging and age-related diseases in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Diabetic nephropathy and kidney-related complications of type 1 diabetes and type 2 diabetes.

Diabetic nephropathy is a kidney-related complication of type 1 and type 2 diabetes that affects up to 40% of people with diabetes. High glucose levels cause the kidneys to filter blood too hard, which leads to kidney damage. Research suggests that in patients with diabetic neuropathy, the mTOR pathway is highly activated and may play a role in kidney pathology and dysfunction due to chronically high glucose levels. In addition, mTOR inhibition may attenuate hyperinsulinemia. In some embodiments, a method of inhibiting mTORC1 activity is used to treat diabetic nephropathy or kidney-related complications of type 1 diabetes and type 2 diabetes (see Mori, H., et al., Biochem. Res. Commun. 384(4): 471- 5 (2009)). Accordingly, in some embodiments, there may be mentioned a method of treating diabetic nephropathy or kidney-related complications of type 1 diabetes and type 2 diabetes in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Polycystic kidney disease.

Polycystic kidney disease (PKD) is characterized by the development and accumulation of destructive kidney cysts, which ultimately leads to kidney failure. PKD can be autosomal dominant (ADPKD) or autosomal recessive (ARPKD). Dysfunctional mTOR signaling pathway has been noted in ADPKD and ARPKD. Thus, normalization of the mTORC1 pathway may attenuate the development of cysts and the progression of this disease.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat PPD (see Torres, V.E., et al., Clin. J. Am. Soc. Nephrol. 5(7): 1312-29 (2010)). Accordingly, in some embodiments, there may be mentioned a method of treating PPD in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof. In some embodiments, PBP is autosomal dominant. In some embodiments, PKD is autosomal recessive.

Focal segmental glomerulosclerosis (FSGS) and other diseases associated with sclerosis

- 82 045993 kidneys.

FSGS is the most common primary glomerular disorder causing end-stage renal disease (ESRD) in the United States. As this disease progresses, there is a discrepancy between the podocytes in Bowman's capsule and the surface area of the glomerular basement membrane that they cover. Studies have shown that control of podocyte size is regulated by mTOR, and that activation of mTOR contributes to disease progression. In addition, knockdown experiments in mice demonstrated that constitutive activation of mTORC1 causes FSGS-like lesions. Thus, inhibition of mTORC1 may attenuate FSGS or other diseases associated with renal sclerosis by normalizing or increasing autophagic activity.

In some embodiments, the method of inhibiting mTORC1 activity is used to treat FSGS or other diseases associated with renal sclerosis (see Zschiedrich, S. et al., J. Am. Soc. Nephrol. 28(7): 2144-57 (2017) ). Accordingly, in some embodiments, there may be mentioned a method of treating FSGS or other diseases associated with renal sclerosis in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness, characterized by the death of photoreceptors in the macula. Possible mechanisms for the progression of RHD include oxidative stress, which leads to the accumulation of proteins and dysfunctional organelles, resulting in hypertrophy, dedifferentiation, and ultimately atrophy of the retinal pigment epithelium. mTOR is involved in the dedifferentiation of the retinal pigment epithelium. Thus, inhibition of mTORC1 may attenuate VJP by blocking hypertrophy and dedifferentiation.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat age-related macular degeneration (see Kolosova, N.G., et al., Am. J. Path. 181(2): 472-7 (2012) and Zhen, C & Vollrath, D., Aging 3(4): 346-47 (2011)). Accordingly, in some embodiments, there may be mentioned a method of treating age-related macular degeneration in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Diabetic macular edema.

Diabetic macular edema (DME) is the leading cause of blindness in people with diabetes, affecting approximately 35% of people with diabetes. Research suggests that the pathogenesis of DME is an inflammatory disease involving various cytokines and chemokines. Chronic inflammatory and oxidative stress may contribute to the progression of DME. Thus, mTORC1 inhibition may attenuate the symptoms and progression of DME by reducing the inflammatory response.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat DME (see Okamoto, T., et al., PLOS ONE, (11)(1): e0146517, https://doi.org/10.1371/ioumal.pone. 0146517 (2016)). Accordingly, in some embodiments, there may be mentioned a method of treating DME in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Diabetic retinopathy.

Diabetic retinopathy (DR) is a common eye disease, accounting for ~5% of blindness in adults, and is associated with chronic hyperglycemia and defects in insulin signaling pathways. Patients with DR suffer from persistent damage to blood vessels and retinal neurons due to inflammation, reactive oxygen species, and endoplasmic reticulum stress caused by chronic hyperglycemia. Importantly, rapamycin has been shown to block the action of insulin-induced hypoxia-inducible factor 1 (HIF-1) and retinal cell senescence and induce autophagy, and it could provide a beneficial effect in promoting apoptosis of developing blood vessel cells and preventing angiogenesis. Thus, mTORC1 inhibition may attenuate the symptoms and progression of DR by reducing inflammation and inhibiting pathogenic signaling pathways.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat DR (see Di Rosa, M., et al., Curr. Neuropharmacol. 14(8): 810-25 (2016)). Accordingly, in some embodiments, there may be mentioned a method of treating DR in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Glaucoma.

Glaucoma is a common optic neuropathy associated with aging and increased intraocular pressure and is a leading cause of irreversible blindness. Issle

- 83 045993 data indicate that mTOR-dependent dysregulation of autophagocytosis may be a factor in the progression of this disease. Thus, inhibition of mTORC1 may slow the progression of glaucoma or attenuate glaucoma by normalizing or enhancing autophagy.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat glaucoma (see Porter, K., et al., Biochim. Biophys. Acta. 1852(3): 379-85 (2014)). Accordingly, in some embodiments, there may be mentioned a method of treating glaucoma in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Restoration of immune function.

Inhibition of mTORC1 has been demonstrated to reduce programmed death receptor-1 (PD-1) expression in CD4+ and CD8' T cells. which stimulates T cell signaling. Thus, mTORCl inhibition may restore immune function by improving the adaptive immune response.

In some embodiments, a method of inhibiting mTORC1 activity is used to restore immune function (see Mannick, J.B., et al., Sci. Trans. Med. 6(268): ppra179 (2014)). Accordingly, in some embodiments, there may be mentioned a method of restoring immune function in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Treatment of respiratory and/or urinary tract infections.

Inhibition of mTORC1 may attenuate infections through up-regulation of antiviral gene expression and response. Thus, mTORC1 inhibition may enhance the patient's immune system's ability to defend against respiratory and/or urinary tract infections.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat respiratory and/or urinary tract infections (see Mannick, J.B., et al., Sci. Trans. Med. 10(449): eaaq1564 (2018)). Accordingly, in some embodiments, there may be mentioned a method of restoring immune function to a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Heart failure.

mTORC1 activity is important for cardiac hypertrophy in response to stress but may lead to cardiac dysfunction as a result of cardiac remodeling after myocardial infarction. Inhibition of mTORC1 reduces cardiac remodeling and heart failure in response to pressure overload. Thus, inhibition of mTORC1 may improve heart failure in patients with myocardial injury.

In some embodiments, the method of inhibiting mTORC1 activity is used to treat heart failure (Sciarretta, S. et al., Circ. Res. 122(3): 489-505 (2018)). Accordingly, in some embodiments, there may be mentioned a method of treating heart failure in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Osteoarthritis.

Osteoarthritis (OA) is a chronic degenerative disease that results in cartilage loss and joint inflammation. mTOR may play a significant role in collagen homeostasis and cartilage turnover and remodeling.

Thus, inhibition of mTORC1 may slow the progression of osteoarthritis or reduce its symptoms by normalizing cartilage turnover.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat osteoarthritis (see Pal, V., et al., Drugs R&D, 15(1): 27-36 (2017)). Accordingly, in some embodiments, there may be mentioned a method of treating osteoarthritis in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive, fatal disease associated with increased pulmonary vascular resistance. Proliferation and migration of pulmonary artery smooth muscle cells are involved in the progression of arterial wall thickening, which increases vasoconstriction. Thus, inhibition of mTORC1 may alleviate PAH by reducing vascular remodeling.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat PAH (see Ma, X., et al., Interact. Cardiovasc. Thorac. Surg. 25(2): 206-11 (2017)). Accordingly, in some embodiments, there may be mentioned a method of treating PAH in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Chronic obstructive pulmonary disease.

- 84 045993

Decreased autophagy leads to the accumulation of proteins and other cellular substances, which accelerates cellular aging in patients with chronic obstructive pulmonary disease (COPD). Thus, inhibition of mTORC1 may slow the progression of COPD or alleviate its symptoms by normalizing or enhancing autophagy.

In some embodiments, a method of inhibiting mTORC1 activity is used to treat COPD (see Fujii, S., et al., Oncoimmunology 1(5): 630-41 (2012)). Accordingly, in some embodiments, there may be mentioned a method of treating COPD in a patient in need thereof, comprising the step of administering to said patient a compound of the invention or a pharmaceutically acceptable salt thereof.

Additional therapeutic indications where mTORC inhibition may provide benefit include the following: cardiovascular disease (acute coronary syndrome), coronary occlusions using eluting stents, polycystic kidney disease and kidney disease associated with cyst formation or cystogenesis, neurofibromatosis, epilepsy associated with TSC1 and/or TSC2 mutations, polycystic liver disease, pachyonychia congenita, fragile X syndrome, Friedreich's ataxia, Peutz-Jeghers syndrome, eye disease including neovascular age-related macular degeneration, uveitis, diabetic macular edema, fibroblast growth including pulmonary fibrosis , renal failure/renal fibrosis, metabolic syndrome, immune system diseases including immune senescence, lupus nephritis, chronic immune thrombocytopenia, multiple sclerosis, cancer including lymphoma, tumors associated with TSC1/2 mutations, angiomyolipoma associated with TSC1/2 mutations , breast cancer, hepatocellular cancer, leukemia, glioma, adenoid cystic carcinoma, aging, autism and vascular rheumatoid arthritis. In some embodiments, the method of inhibiting mTORC1 activity is used to treat cardiovascular disease (acute coronary syndrome), coronary occlusions using eluting stents, polycystic kidney disease, neurofibromatosis, epilepsy associated with TSC1 and/or TSC2 mutations, polycystic liver disease, pachyonychia congenita , fragile X syndrome, Friedreich's ataxia, Peutz-Jeghers syndrome, eye diseases including neovascular age-related macular degeneration, uveitis, diabetic macular edema, fibroblast growth including pulmonary fibrosis, renal failure/renal fibrosis, metabolic syndrome, immune system diseases including immune senescence, lupus nephritis, chronic immune thrombocytopenia, multiple sclerosis, cancers including lymphoma, tumors associated with TSC1/2 mutations, angiomyolipoma associated with TSC1/2 mutations, breast cancer, hepatocellular carcinoma, leukemia, glioma, adenoid cystic carcinoma , aging, autism and vascular rheumatoid arthritis.

Accordingly, in some embodiments, a method for treating cardiovascular disease (acute coronary syndrome), coronary occlusions using eluting stents, polycystic kidney disease, neurofibromatosis, epilepsy associated with TSC1 and/or TSC2 mutations, polycystic liver disease, pachyonychia congenita may be mentioned. , fragile X syndrome, Friedreich's ataxia, Peutz-Jeghers syndrome, eye diseases including neovascular age-related macular degeneration, uveitis, diabetic macular edema, fibroblast growth including pulmonary fibrosis, renal failure/renal fibrosis, metabolic syndrome, immune system diseases including immune senescence, lupus nephritis, chronic immune thrombocytopenia, multiple sclerosis, cancers including lymphoma, tumors associated with TSC1/2 mutations, angiomyolipoma associated with TSC1/2 mutations, breast cancer, hepatocellular carcinoma, leukemia, glioma, adenoid cystic carcinoma , aging, autism and vascular rheumatoid arthritis in a patient in need thereof, comprising the step of administering to said patient the proposed compound or a pharmaceutically acceptable salt thereof.

The pharmaceutically acceptable compositions of the present invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (in the form of powders, ointments or drops), buccally, in the form of an oral or nasal spray or the like depending on severity of the infection being treated. In some embodiments, the compounds of the present invention may be administered orally or parenterally in an amount of from about 0.01 mg/kg to about 50 mg/kg, and preferably from about 1 mg/kg to about 25 mg/kg of the subject's body weight in day one or several times a day to obtain the desired therapeutic effect. Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, these liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate , propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (particularly cottonseed, peanut, corn, germ, olive, castor and sesame oils), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures of them. In addition to inert diluents, oral compositions also

- 85 045993 may contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and fragrance agents. Injectable preparations, for example sterile aqueous or oleaginous injectable suspensions, can be prepared by methods known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally acceptable diluent or solvent, for example a solution in 1,3-butanediol. Acceptable transfer media and solvents that may be used include water, USP Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fatty oils are traditionally used as a solvent or suspending medium. For this purpose, any light fatty oil (bland fixed oil), including synthetic mono or diglycerides, can be used. In addition, fatty acids such as oleic acid are used to prepare injection formulations.

Injectable formulations may be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

To prolong the effect of a compound of the present invention, it is often desirable to slow down the absorption of the compound by subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous substance with poor water solubility. The rate of absorption of the specified compound in this case depends on the rate of its dissolution, which, in turn, may depend on the crystal size and crystalline form. As an alternative, delayed absorption of a parenterally administered form of the compound is achieved by dissolving or suspending the compound in an oily vehicle. Depot injection forms are prepared by preparing microencapsulated matrices of the specified compound in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of the compound to the polymer and the nature of the particular polymer used, the rate of release of the compound can be adjusted. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot formulations are also prepared by incorporating the compound into liposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are preferably suppositories, which can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol or suppository wax, which are solid at ambient temperature but liquid at body temperature and thus melt in the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or calcium hydrogen phosphate, and/or a) fillers or extenders, such as starches, lactose, sucrose, glucose , mannitol and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose and gum arabic, c) humectants such as glycerin, d) leavening agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates and sodium carbonate, f) dissolution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, said dosage form may also contain buffering agents.

Solid compositions of this type can also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like. Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared using coatings and coatings, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may optionally contain opacifying agents and may also be formulated such that they release the active ingredient(s) only or predominantly in a specific part of the intestine, optionally in a delayed manner. Examples of encapsulating compositions that can be used include polymeric substances and waxes. Solid compositions of this type can also be used as fillers in soft and hard gelatin capsules using such auxiliary ingredients

- 86 045993 substances such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

Said active compounds may also be in microencapsulated form with one or more of the excipients mentioned above. Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared using coatings and shells, such as enteric coatings, controlled release coatings and other coatings well known in the art of pharmaceutical formulations. In such solid dosage forms, the active compound may be mixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also contain, as is common practice, additional substances other than inert diluents, such as tablet lubricants and other tableting excipients such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, these dosage forms may also contain buffering agents. They may optionally contain opacifying agents and may also be formulated such that they release the active ingredient(s) only or predominantly in a specific part of the intestine, optionally in a delayed manner. Examples of encapsulating compositions that may be used include polymeric materials and waxes.

Dosage forms for topical or transdermal administration of a compound of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. Said active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffers that may be required. Ophthalmic composition, ear drops and eye drops are also included within the scope of the present invention. In addition, the present invention involves the use of transdermal patches, which have the additional advantage of providing controlled delivery of the compound to the body. Such dosage forms can be prepared by dissolving or dispersing the said compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate can be controlled either by providing a rate-regulating membrane or by dispersing the compound in a polymer matrix or gel. The term biological sample includes, without limitation, cell cultures or extracts thereof; biopsy material obtained from the body of a mammal, or extracts thereof, and blood, saliva, urine, feces, semen, tears or other body fluids or extracts thereof.

In other embodiments, there may be mentioned a method of treating an mTORC1-mediated disorder in a patient in need thereof, comprising the step of administering to said patient a compound of the present invention or a pharmaceutically acceptable composition thereof. Such violations are described in detail herein.

Depending on the particular condition or disease being treated, additional therapeutic agents that are typically administered to treat said condition may also be present in the compositions of the present invention. As used herein, additional therapeutic agents that are typically administered to treat a particular disease or condition are known to be suitable for the disease or condition being treated.

The compound of the present invention can also be used to obtain benefits in combination with antiproliferative compounds. Such antiproliferative compounds include, but are not limited to, aromatase inhibitors; antiestrogens; topoisomerase I inhibitors; topoisomerase II inhibitors; compounds having activity against microtubules; alkylating compounds; histone deacetylase inhibitors; compounds that induce cell differentiation processes; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antitumor antimetabolites; platinum compounds; compounds targeting/reducing protein kinase or lipid kinase activity and other antiangiogenic compounds; compounds that target, reduce or inhibit the activity of protein phosphatase or lipid phosphatase; gonadorelin agonists; antiandrogens; methionine aminopeptidase inhibitors; matrix metalloproteinase inhibitors; bisphosphonates; biological response modifiers; antiproliferative antibodies; heparanase inhibitors; inhibitors of oncogenic Ras isoforms; telomerase inhibitors; proteasome inhibitors; compounds used in the treatment of hematological malignancies; compounds that target, reduce or inhibit Flt-3 activity; Hsp90 inhibitors such as 17-AAG (17-allylaminogeldanamycin, NSC330507), 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin, NSC707545), IPI-504, CNF1010, CNF2024, CNF1010 from Conforma Therapeutics; temozolomide (Temodal®); kinesin spindle protein inhibitors such as SB715992 or SB743921 from GlaxoSmithKline, or pentamidine/chlorpromazine from CombinatoRx; MEK inhibitors such as ARRY142886 from Array BioPharma, AZD6244 from AstraZeneca, PD181461 from Pfizer and leucovorin. The term aromatase inhibitor as used herein refers to a compound that inhibits the production of estrogens, such as the conversion of the substrates androstenedione and testosterone to es.

- 87 045993 throne and estradiol, respectively. The term includes, but is not limited to, steroids, in particular atamestane, exemestane and formestane, and in particular non-steroidal compounds, in particular aminoglutethimide, rogletimide, pyridoglutetimide, trilostane, testolactone, ketoconazole, vorozole, fadrozole, anastrozole and letrozole. Exemestane is marketed under the trade name Aromasin™. Formestane is marketed under the trade name Lentaron™. Fadrozole is marketed under the trade name Afema™. Anastrozole is marketed under the trade name Arimidex™. Letrozole is marketed under the trade names Femara™ or Femar™. Aminoglutethimide is marketed under the trade name Orimeten™. The combination according to the present invention containing a chemotherapeutic agent that is an aromatase inhibitor is particularly suitable for the treatment of hormone receptor-positive tumors such as breast tumors. The term antiestrogen refers to a compound that antagonizes the effect of estrogens at the estrogen receptor level. The term includes, but is not limited to, tamoxifen, fulvestrant, raloxifene and raloxifene hydrochloride. Tamoxifen is marketed under the trade name Nolvadex™. Raloxifene hydrochloride is marketed under the trade name Evista™. Fulvestrant is available for administration under the trade name Faslodex™. The combination of the present invention containing a chemotherapeutic agent that is an antiestrogen is particularly suitable for use in the treatment of estrogen receptor positive tumors, such as breast tumors.

The term antiandrogen refers to any substance that is capable of inhibiting the biological effects of androgenic hormones and includes, but is not limited to, bicalutamide (Casodex™). As used herein, the term gonadorelin agonist includes, but is not limited to, abarelix, goserelin, and goserelin acetate. Goserelin is available for administration under the trade name Zoladex™.

The term topoisomerase I inhibitor includes, but is not limited to, topotecan, hymatecan, irinotecan, camptothecin and its analogs, 9-nitrocamptothecin and the macromolecular camptothecin conjugate PNU-166148. Irinotecan is available for administration, for example, in the form in which it is marketed, for example, under the brand name Camptosar™. Topotecan is marketed under the trade name Hycamptin™.

The term topoisomerase II inhibitor includes, but is not limited to, anthracyclines such as doxorubicin (including a liposomal formulation such as Caelyx™), daunorubicin, epirubicin, idarubicin and nemorubicin, anthraquinones, mitoxantrone and losoxantrone, and the podophyllotoxins etoposide and teniposide. Etoposide is marketed under the trade name Etopophos™. Teniposide is marketed under the trade name VM 26-Bristol. Doxorubicin is marketed under the trade name Acriblastin™ or Adriamycin™. Epirubicin is marketed under the trade name Farmorubicin™. Idarubicin is marketed under the trade name Zavedos™. Mitoxantrone is marketed under the trade name Novantron.

The term microtubule-active agent refers to microtubule-stabilizing, microtubule-destabilizing, and microtubule polymerization inhibitor compounds, including, but not limited to, taxanes such as paclitaxel and docetaxel; vinca alkaloids such as vinblastine or vinblastine sulfate, vincristine or vincristine sulfate and vinorelbine; discodermolides; colchicine and epothilones and their derivatives. Paclitaxel is marketed under the trade name Taxol™. Docetaxel is marketed under the trade name Taxotere™. Vinblastine sulfate is marketed under the trade name Vinblastin R.P™. Vincristine sulfate is marketed under the trade name Farmistin™. The term alkylating agent includes, but is not limited to, cyclophosphamide, ifosfamide, melphalan or nitrosourea (BCNU or Gliadel). Cyclophosphamide is marketed under the trade name Cyclostin™. Ifosfamide is marketed under the trade name Holoxan™. The term histone deacetylase inhibitors or HDAC inhibitors refers to compounds that inhibit histone deacetylase and which have antiproliferative activity. These include, but are not limited to, suberoylanilide hydroxamic acid (SAHA).

The term antitumor antimetabolite includes, but is not limited to, 5-fluorouracil or 5-FU, capecitabine, gemcitabine, DNA demethylating compounds such as 5-azacytidine and decitabine, methotrexate and edatrexate, and folate antagonists such as pemetrexed. Capecitabine is marketed under the trade name Xeloda™. Gemcitabine is marketed under the trade name Gemzar™.

The term platinum compound includes, but is not limited to, carboplatin, cisplatin, cisplatin and oxaliplatin. Carboplatin can be administered, for example, in the form in which it is marketed, for example, under the trade name Carboplat™. Oxaliplatin is available for administration, for example, in the form in which it is marketed, for example, under the trade name Eloxatin™. As used herein, the term compounds that target/reduce protein kinase or lipid kinase activity; or protein phosphatase or lipid phosphatase activity; or other antiangiogenic

- 88 045993 compounds, includes, but is not limited to, protein tyrosine kinase inhibitors, and/or serine and/or threonine kinase inhibitors or lipid kinase inhibitors, such as a) compounds that target, reduce or inhibit the activity of platelet-derived growth factor receptors (PDGFR) , such as compounds that target, reduce or inhibit the activity of PDGFR, in particular compounds that inhibit the TRF receptor, such as an N-phenyl-2-pyrimidinamine derivative such as imatinib, SU101, SU6668 and GFB-111; b) compounds that target, reduce or inhibit the activity of fibroblast growth factor receptors (FGFR); c) compounds that target, reduce or inhibit insulin-like growth factor receptor I (IGF-IR) activity, such as compounds that target, reduce or inhibit IGF-IR activity, in particular compounds that inhibit IGF receptor I kinase activity , or antibodies that target the extracellular domain of IGF receptor I or its growth factors; d) compounds that target, reduce or inhibit the activity of members of the Trk receptor tyrosine kinase family, or ephrin B4 inhibitors; f) compounds that target, reduce or inhibit the activity of members of the AxI family of receptor tyrosine kinases; f) compounds that target, reduce or inhibit the activity of Ret receptor tyrosine kinase; g) compounds that target, reduce or inhibit Kit/SCFR receptor tyrosine kinase activity, such as imatinib; h) compounds that target, reduce or inhibit the activity of C-kit receptor tyrosine kinases that are part of the PDGFR family, such as compounds that target, reduce or inhibit the activity of members of the c-Kit receptor tyrosine kinase family, in particular compounds that inhibit c-Kit receptor, such as imatinib; i) compounds that target, reduce or inhibit the activity of c-Abl family members, their gene fusion products (e.g. BCR-Abl kinase) and mutant variants, such as compounds that target, reduce or inhibit the activity of c-Abl family members and products fusions of their genes, such as an N-phenyl-2-pyrimidinamine derivative such as imatinib or nilotinib (AMN107); PD180970; AG957; NSC 680410; PD173955 from ParkeDavis; or dasatinib (BMS-354825); j) compounds that target, reduce or inhibit the activity of members of the protein kinase C (PKC) family and the Raf family of serine/threonine kinases, members of the MEK, SRC, JAK/pan-JAK, FAK, PDK1, PKB/Akt, Ras/MAPK, PI3K families , SYK, TYK2, BTK and TEC and/or members of the cyclin-dependent kinase (CDK) family, including staurosporine derivatives such as midostaurin; examples of other compounds include UCN-01, safingol, BAY 43-9006, bryostatin 1, perifosine; ilmofosine; RO 318220 and RO 320432; GO 6976; Isis 3521; LY333531/LY379196; isoquinoline compounds; FTI; PD184352 or QAN697 (PI3K inhibitor) or AT7519 (CDK inhibitor); k) compounds that target, reduce or inhibit the activity of protein tyrosine kinase inhibitors, such as compounds that target, reduce or inhibit the activity of protein tyrosine kinase inhibitors, including imatinib mesylate (Gleevec™) or tyrphostin, such as Tyrphostin A23/RG-50810; AG 99; Tyrphostin AG 213; Tyrphostin AG 1748; Tyrphostin AG 490; Tyrphostin B44; (+) enantiomer of Tyrphostin B44; Tyrphostin AG 555; AG 494; Tyrphostin AG 556, AG957 and adaphostin, adamantyl ester (4-{[(2,5-dihydroxyphenyl)methyl]amino}benzoic acid; NSC 680410, adaphostin); l) compounds that target, reduce or inhibit the activity of members of the epidermal growth factor receptor tyrosine kinase family (EGFR1 ErbB2, ErbB3, ErbB4 as homo- or heterodimers) and mutant variants thereof, such as compounds that target, reduce or inhibit the activity of members the epidermal growth factor (EGF) receptor family, in particular, compounds, proteins or antibodies that inhibit members of the EGF receptor tyrosine kinase family, such as EGF receptor, ErbB2, ErbB3 and ErbB4, or bind to EGF or EGF-related ligands, CP 358774, ZD 1839, ZM 105180; trastuzumab (Herceptin™), cetuximab (Erbitux™), Iressa, Tarceva, OSI-774, Cl-1033, EKB-569, GW-2016, E1.1, E2.4, E2.5, E6.2, E6. 4, E2.11, E6.3 or E7.6.3 and 7H-pyrrolo[2,3L|pyrimidine; m) compounds that target, reduce or inhibit the activity of the c-Met receptor, such as compounds that target, reduce or inhibit the activity of c-Met, in particular compounds that inhibit the kinase activity of the c-Met receptor or antibodies that target the extracellular domain c-Met or HGF binding, n) compounds that target, reduce or inhibit the kinase activity of one or more members of the JAK family (JAK1/JAK2/JAK3/TYK2 and/or rap-JAK), including, but not limited to, PRT- 062070, SB-1578, baricitinib, pacritinib, momelotinib, VX-509, AZD-1480, TG-101348, tofacitinib and ruxolitinib; o) compounds aimed at reducing or inhibiting the kinase activity of PI3 kinase (PI3K), including, but not limited to, ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib, pictrelisib, PF-4691502, BYL-719, dactolisib, XL-147, XL-765 and idelalisib; And; and q) compounds that target, reduce or inhibit the signaling effects of hedgehog (Hh) or smoothed receptor (SMO) protein pathways, including, but not limited to, cyclopamine, vismodegib, itraconazole, erizmodegib and IPI-926 (saridegib).

The term PI3K inhibitor includes, but is not limited to, compounds having inhibitory activity against one or more enzymes of the phosphatidylinositol-3 family

- 89 045993 kinases, including, but not limited to, ΡΙ3Κα, ΡΙ3Κγ, ΡΙ3Κδ, ΡΙ3Κβ, PI3K-C2a, Р13К-С2в, PI3K-C2y, Vps34, ρ110-α, ρ110-β, ρ110- γ, ρ110-δ, ρ85-α, ρ85-β, ρ55-γ, p150, p101 and p87. Examples of PI3K inhibitors that can be used in the present invention include, but are not limited to, ATU027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib, picterlisib, PF-4691502, BYL -719, dactolisib, XL-147, XL-765 and idelalisib. The term Bcl-2 inhibitor includes, but is not limited to, compounds having inhibitory activity against B-cell lymphoma protein 2 (Bcl-2), including, but not limited to, ABT-199, ABT-731, ABT-737, apogosypol, panBcl-2 inhibitors Ascenta, curcumin (and its analogs), dual action Bcl-2/Bcl-xL inhibitors (Infinity Pharmaceuticals/Novartis Pharmaceuticals), Genasense (G3139), HA14-1 (and its analogs; see WO 2008/118802), navitoclax (and its analogues, see US patent No. 7390799), NH-1 (Shenayng Pharmaceutical University), obatoclax (and its analogues, see WO 2004/106328), S-001 (Gloria Pharmaceuticals), TW series connections (Univ. of Michigan) and venetoclax. In some embodiments, the Bcl-2 inhibitor is a small molecule drug. In some embodiments, the Bcl-2 inhibitor is a peptidomimetic. The term BTK inhibitor includes, but is not limited to, compounds having Bruton's tyrosine kinase (BTK) inhibitory activity, including, but not limited to, AVL-292 and ibrutinib.

The term SYK inhibitor includes, but is not limited to, compounds having spleen tyrosine kinase (SYK) inhibitory activity, including, but not limited to, PRT-062070, R-343, R-333, Excellair, PRT-062607 and fostamatinib.

Other examples of BTK inhibitory compounds and conditions that can be treated with such compounds in combination with the compounds of the present invention can be found in WO 2008/039218 and WO 2011/090760, the entire contents of which are incorporated herein by reference.

Other examples of SYK inhibitory compounds and conditions that can be treated with such compounds in combination with the compounds of the present invention can be found in WO 2003/063794, WO 2005/007623 and WO 2006/078846, the entire contents of which are incorporated herein by reference. .

Other examples of PI3K inhibitory compounds and conditions that can be treated with such compounds in combination with compounds of the present invention can be found in WO 2004/019973, WO 2004/089925, WO 2007/016176, US Patent No. 8138347, WO 2002/088112, WO 2007/084786, WO 2007/129161, WO 2006/122806, WO 2005/113554 and WO 2007/044729, the entire contents of which are incorporated herein by reference.

Other examples of JAK inhibitory compounds and conditions that can be treated with such compounds in combination with the compounds of the present invention can be found in WO 2009/114512, WO 2008/109943, WO 2007/053452, WO 2000/142246 and WO 2007/070514. the entire contents of said references are incorporated herein by reference.

Other antitumor compounds include compounds whose activity is mediated by a mechanism other than protein kinase or lipid kinase inhibition, such as thalidomide (Thalomid™) and TNP-470.

Examples of proteasome inhibitors suitable for use in combination with the compounds of the present invention include, but are not limited to, bortezomib, disulfiram, epigallocatechin-3-gallate (EGCG), salinosporamide A, carfilzomib, ONX-0912, CEP-18770 HMLN9708.

Compounds that target, reduce or inhibit the activity of protein phosphatase or lipid phosphatase are, for example, phosphatase 1, phosphatase 2A or CDC25 inhibitors, such as okadaic acid or a derivative thereof.

Compounds that induce cell differentiation processes include, but are not limited to, retinoic acid, α-γ- or δ-tocopherol, or α-γ- or δ-tocotrienol.

The term cyclooxygenase inhibitor includes, but is not limited to, Cox-2 inhibitors, 5-alkyl-2-arylaminophenylacetic acid and derivatives thereof, such as celecoxib (Celebrex™), rofecoxib (Vioxx™), etoricoxib, valdecoxib or 5-alkyl-2-arylaminophenylacetic acid , such as 5-methyl-2-(2'-chloro-6'-fluoroanilino)phenylacetic acid, lumiracoxib.

The term bisphosphonates includes, but is not limited to, etridonic, clodronic, tiludronic, pamidronic, alendronic, ibandronic, risedronic and zoledronic acids. Etridonic acid is marketed under the trade name Didronel™. Clodronic acid is marketed under the trade name Bonefos™. Tiludronic acid is marketed under the trade name Skelid™. Pamidronic acid is marketed under the trade name Aredia™. Alendronic acid is marketed under the trade name Fosamax™. Ibandronic acid is marketed under the trade name Bondranat™. Risedronic acid is marketed under the trade name Actonel™. Zoledronic acid is marketed under the trade name Zometa™. The term mTOR inhibitors refers to compounds that inhibit mammalian target of rapamycin (mTOR) and that have antiproliferative properties.

- 90 045993 activity, such as sirolimus (Rapamune), everolimus (Certican™), CCI-779 and ABT578. The term heparanase inhibitor refers to compounds that target, reduce or inhibit the degradation of heparin sulfate. The term includes, but is not limited to, PI-88. The term biological response modifier as used herein refers to lymphokine or interferons. The term inhibitor of oncogenic Ras isoforms, such as H-Ras, K-Ras or N-Ras, refers to compounds that target, reduce or inhibit the oncogenic activity of Ras; an example is a farnesyltransferase inhibitor such as L-744832, DK8G557 or R115777 (Zarnestra™). The term telomerase inhibitor refers to compounds that target, reduce or inhibit telomerase activity. Compounds that target, reduce or inhibit telomerase activity are, in particular, compounds that inhibit the telomerase receptor, such as telomestatin. The term methionine aminopeptidase inhibitor as used herein refers to compounds that target, reduce or inhibit the activity of methionine aminopeptidase. Compounds that target, reduce or inhibit methionine aminopeptidase activity include, but are not limited to, benhamide or a derivative thereof.

The term proteasome inhibitor refers to compounds that target, reduce or inhibit the activity of proteasomes. Compounds that target, reduce or inhibit proteasome activity include, but are not limited to, Bortezomib (Velcade™) and MLN 341. The term matrix metalloproteinase inhibitor or (MMP inhibitor) includes, but is not limited to, collagen inhibitors of the peptidomimetic class and non-peptidomimetics, tetracycline derivatives, for example, the hydroxamate inhibitor from the peptidomimetic class, batimastat and its analogue, which is orally bioavailable, marimastat (BB-2516), prinomastat (AG3340), metastat (NSC 683551), BMS-279251, BAY 12-9566, TAA211 , MMI270B or AAJ996.

The term compounds used in the treatment of hematological malignancies include, but are not limited to, FMS-like tyrosine kinase inhibitors, which are compounds that target, reduce or inhibit the activity of FMS-like tyrosine kinase receptors (Flt-3R); interferon, 1-β-O-arabinofuransylcytosine (ara-c) and bisulfan; and ALK inhibitors, which are compounds that target, reduce or inhibit anaplastic lymphoma kinase activity.

Compounds that target, reduce or inhibit the activity of FMS-like tyrosine kinase receptors (Flt-3R) are, in particular, compounds, proteins or antibodies that inhibit members of the Flt-3R receptor kinase family, such as PKC412, midostaurin, a staurosporine derivative, SU11248 HMLN518.

The term HSP90 inhibitors includes, but is not limited to, compounds that target, reduce or inhibit the ATPase activity inherent in HSP90; involved in the degradation of, targeting, reducing, or inhibiting HSP90-dependent proteins through the ubiquitinin-proteasome pathway. Compounds that target, reduce or inhibit the ATPase activity inherent in HSP90 are, in particular, compounds, proteins or antibodies that inhibit the ATPase activity of HSP90, such as 17-allylamino, 17-demethoxygeldanamycin (17AAG), a derivative of geldanamycin; other geldanamycin-related compounds; radicicol and HDAC inhibitors.

The term antiproliferative antibodies includes, but is not limited to, trastuzumab (Herceptin™), Trastuzumab-DM1, Erbitux, bevacizumab (Avastin™), rituximab (Rituxan®), PRO64553 (anti-CD40) and 2C4. Antibodies include intact monoclonal antibodies, polyclonal antibodies, polyspecific antibodies derived from at least 2 intact antibodies, and antibody fragments, provided they exhibit the desired biological activity.

For the treatment of acute myeloid leukemia (AML), the compounds of the present invention can be used in combination with standard treatments for leukemia, in particular in combination with treatments used for the treatment of AML. In particular, the compounds of the present invention can be administered in combination with, for example, farnesyltransferase inhibitors and/or other drugs suitable for use in the treatment of AML, such as daunorubicin, adriamycin, Ara-C, VP-16, teniposide, mitoxantrone, idarubicin , carboplatin and PKC412.

Other antileukemic compounds include, for example, Ara-C, a pyrimidine analogue, which is a 2'-alpha-hydroxyribose (arabinoside) derivative of deoxycytidine. Also included are the purine analogue of hypoxanthine, 6-mercaptopurine (6-MP), and fludarabine phosphate. Compounds that target, reduce, or inhibit histone deacetylase (HDAC) activity, such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA), inhibit the activity of enzymes known as histone deacetylases. Specific HDAC inhibitors include MS275, SAHA, FK228 (formerly FR901228), trichostatin A and the compounds disclosed in US Pat. No. 6,552,065, including, but not limited to, Nahydroxy-3-[4-[[[2-(2-methyl -III-indol-3-yl)ethyl]amino]methyl]phenyl]-2E2-propenamide or a pharmaceutically acceptable salt thereof and H-hydroxy-3-[4-[(2-hydroxyethyl){2-(1H

- 91 045993 indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide or a pharmaceutically acceptable salt thereof, in particular the lactate salt. As used herein, somatostatin receptor antagonists refer to compounds that target, act on, or inhibit the somatostatin receptor, such as octreotide and SOM230. Approaches based on tumor cell damage include approaches such as ionizing radiation. The term ionizing radiation, as referred to above and below, means ionizing radiation that occurs in the form of either electromagnetic radiation (such as X-rays and gamma rays) or particles (such as alpha and beta particles). Ionizing radiation has been proposed for, without limitation, radiation therapy and is known in the art. See Hellman, Principles of Radiation Therapy, Cancer, in Principles and Practice of Oncology, Devita et al., Eds., ^4th Edition, Vol. 1, pp. 248-275 (1993).

Also included are EDG binders and ribonucleotide reductase inhibitors. The term EDG binders refers to a class of immunosuppressants that modulate lymphocyte recycling, such as FTY720. The term ribonucleotide reductase inhibitors refers to pyrimidine or purine nucleoside analogues, including, but not limited to, fludarabine and/or cytosine arabinoside (ara-C), 6-thioguanine, 5-fluorouracil, cladribine, 6-mercaptopurine (particularly in combination with ara -C against ALL) and/or pentostatin. Ribonucleotide reductase inhibitors are, in particular, hydroxyurea or derivatives of 2-hydroxy-III-isoindole-1,3dione. Also included, in particular, are compounds, proteins or monoclonal antibodies against vascular endothelial growth factor (VEGF) such as 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or its pharmaceutically acceptable salt, 1-(4) succinate -chloranilino)-4-(4-pyridylmethyl)phthalazine; Angiostatin™; Endostatin™; anthranilic acid amides; ZD4190; ZD6474; SU5416; SU6668; bevacizumab; or anti-VEGF antibodies or anti-VEGF receptor antibodies such as rhuMAb and RHUFab, a VEGF aptamer such as Macugon; FLT-4 inhibitors, FLT-3 inhibitors, anti-VEGFR-2 IgGI antibody, angiozyme (RPI4610) and bevacizumab (Avastin™). Photodynamic therapy refers to a therapy that uses certain chemicals known as photosensitizing compounds to treat or prevent cancer. Examples of photodynamic therapy include treatment with compounds such as Visudyne™ and sodium porfimer.

Angiostatic steroids refer to compounds that block or inhibit angiogenesis, such as, for example, anecortave, triamcinolone, hydrocortisone, 11a-epihydrocortisol, cortexolone, 17a-hydroxyprogesterone, corticosterone, deoxycorticosterone, testosterone, estrone and dexamethasone. Implants containing corticosteroids include compounds such as fluocinolone and dexamethasone.

Other chemotherapeutic compounds include, but are not limited to, plant alkaloids, hormonal compounds and antagonists; biological response modifiers, preferably lymphokines or interferons; antisense oligonucleotides or derivatives of oligonucleotides; shRNA or siRNA; or other compounds or compounds with a different or unknown mechanism of action.

The structure of active compounds was identified by code numbers, nonproprietary names or trademarks, which can be obtained from the current edition of the standard manual The Merck Index or from databases such as Patents International (eg, IMS World Publications). The compound of the present invention can also be used in combination with known therapeutic processes, for example the administration of hormones or radiation. In some embodiments, the proposed compound is used as a radiosensitizer, in particular for the treatment of tumors that exhibit poor sensitivity to radiotherapy.

A compound of the present invention may be administered alone or in combination with one or more other therapeutic compounds, with possible combination therapy taking the form of fixed combinations or administration of a compound of the present invention and one or more other therapeutic compounds at intervals independently of each other or combined administration of fixed combinations and one or more other therapeutic compounds. The compound of the present invention can be administered as a substitute or additive, particularly for the treatment of a tumor in combination with chemotherapy, radiotherapy, immunotherapy, phototherapy, surgery or a combination thereof. Long-term therapy is also possible, an example being adjuvant therapy in the context of other treatment strategies as described above. Another possible treatment is maintenance therapy after tumor regression or chemoprophylactic therapy, for example in patients at risk. These additional agents may be administered separately from the compositions containing the compound of the present invention, as part of a multiple-dose regimen. Alternatively, these agents may be part of a single dosage form when mixed with a compound of the present invention to form a single composition. When administered as part of a multiple-dose regimen, the two active agents may be administered simultaneously, sequentially, or at intervals typically within five hours.

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The terms combination, combined and related terms refer to the simultaneous or sequential administration of therapeutic agents in accordance with the present invention. For example, a compound of the present invention may be administered with another therapeutic agent simultaneously or sequentially in a divided dose dosage form or together in a single dosage form. Accordingly, the present invention may include a single dosage form containing a compound of the present invention, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant or vehicle. The amount of the compound of the present invention and the additional therapeutic agent (in compositions that contain the additional therapeutic agent described above) that can be combined with carrier materials to form a single dosage form may vary depending on the host being treated. Or a specific route of administration. Preferably, the compositions of the present invention should be formulated such that a dose of 0.01-100 mg/kg body weight/day of a compound of the present invention can be administered. In compositions that contain an additional therapeutic agent, the additional therapeutic agent and the compound of the present invention may act synergistically. Thus, the amount of additional therapeutic agent in such compositions will be less than that required for monotherapy using only that therapeutic agent. In such compositions, a dose of 0.011000 μg/kg body weight/day of additional therapeutic agent may be administered.

The amount of additional therapeutic agent present in the composition of the present invention will be no more than would normally be administered in a composition containing said therapeutic agent as the sole active agent. Preferably, the amount of additional therapeutic agent in the compositions disclosed herein will be from about 50 to 100% of the amount typically present in a composition containing such agent when it is the only therapeutically active agent.

In some embodiments, the additional therapeutic agent administered in combination with a compound of the present invention is another mTOR inhibitor. In some embodiments, the additional mTOR inhibitor inhibits mTOR by binding to a catalytically active site of mTOR. Examples of such additional mTOR inhibitors include: dactolisib, 8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethylphenyl)-1,3dihydroimidazo[4,5-c ]quinolin-2-one (WO 2006/122806), vistusertib (AZD2014; WO 2009/153597); AZD8055 (WO 2009/153597; XL388 (US Patent Application Publication 2010/0305093); sapanisertib (sapanisertib; MLN0128; INK128; WO 2015/051043); DS3078; apitolisib (apitolisib; GDC0980; WO 20 08/070740); omipalisib ; GSK-2126458; WO 2008/14446); NVP-BGT226 (Chang, K.Y., et al., Clin. Cancer Res. 17(22): 7116-26 (2011)); voxtalisib (voxtalisib; XL765; SAR245409; WO 2007/044813); PF04691502 (WO 2008/032162); gedatolisib (gedatolisib; PF05212384; PKI-587; WO 2009/143313); SF1126 (WO 2004/089925); GSK1059615 (WO 20 07/136940); BI-860585; OSI 027 (WO 2007/061737); VS 5584 (WO 2010/114484); CC-223 (WO 2010/062571); DCBCI-0901 (Lee, Y.E., et al., Mol. Canc. Thera. 12(11 Suppl) : Abstract nr C270 (2013)):); LY3023414 (WO 2012/097039); P529 (WO 2007/133249); panulisib (panulisib; P7170; WO 2012/007926); DS-7423 (Kashiyama, T., et al., PLoS One 9(2): e87220 (2014)); PWT33567 mesylate (VCD-597; WO 2010/110685); ME-344 (NV-128; Navarro, P., et al., Cell Rep. 15(12):2705-18 (2016)); ABTL0812 (WO 2010/106211); WYE-132; EXEL-3885 (Eur J Cancer Suppl. 6(12): Abst 322 (2008)); EXEL-4431 (Eur J Cancer Suppl. 6(12): Abst 322 (2008)); AR-mTOR-26 (101st Annu Meet Am Assoc Cancer Res (AACR) (April 17-21, Washington, D.C.) 2010, Abst 4484); NV-128 (A.B. Alvero et al., Mol Cancer Ther. 10(8): 1385-93 (2011)); salinomycin (VS-507; Gupta, P.B., et al., Cell 138(4): 645-59 (2009)); BN-107; BN-108; WAY-600; WYE-687; WYE-354 (Yu, K., et al., Cancer Res. 69(15): 6232-40 (2009)); Ku-063794 (Garcia-Martinez, J.M., et al., Biochem. J. 421(1): 29-42 (2009)); torkinib (PP242; Apsel, V., et al., Nat. Chem. Biol. 4(11): 69I-99 (2008)); PP30; CZ415 (KEF); INK1069; EXEL-2044; EXEL-7518; SB2158; SB2280; AR-mTOR-1 (Wallace, E.M., et al., Mol. Canc. Thera. 8(12 Suppl): Abst. B267 (2009)).

Reference herein to any specific additional mTOR inhibitor also includes any pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, hydrates and polymorphs thereof.

The compounds of the present invention or pharmaceutical compositions thereof may also be included in compositions for coating implantable medical devices such as prostheses, prosthetic valves, vascular grafts, stents and catheters. Vascular stents, for example, are used to avoid restenosis (re-narrowing of the vessel wall after injury). However, patients with stents or other implantable devices are at risk for clot formation or platelet activation. Such undesirable effects can be prevented or reduced by precoating the device with a pharmaceutically acceptable composition containing a kinase inhibitor. Implantable devices coated with a compound of the present invention represent another embodiment of the invention.

- 93 045993

Examples

As presented in the examples below, in certain illustrative embodiments, compounds are prepared according to the following general procedures. It will be appreciated that while the general methods present the synthesis of individual compounds of the present invention, the following general methods and other methods known to one of ordinary skill in the art can be applied to all compounds, subclasses and species of each of these compounds described herein ( see also Luengo, J. I. et al., Chem. Biol., 2(7): 471-81 (1995); and Grinfeld, A. A. et al., Tet. Lett., 35(37): 683538 (1994)) .

List of abbreviations used in the experimental part

CH ₃ CN: acetonitrile;

DCE: dichloroethane;

DCM: dichloromethane;

DIPEA: N.N-diisopropylethylamine;

DMF: N,N-dimethylformamide;

DMSO: dimethyl sulfoxide;

ESI: electrospray ionization;

EtOAc: ethyl acetate;

EtOH: ethanol;

h: hours;

HBr: hydrogen bromide;

HF: hydrogen fluoride;

HND-8: acid ion exchange resin (eg Amberlyst);

H ₂ O: water;

HPLC: high performance liquid chromatography;

MeOH: methanol;

min: minutes;

ml: milliliters;

mM: millimolar;

mmol: millimoles;

MS: mass spectrometry;

N ₂ : nitrogen, gas;

NaHCO3: sodium bicarbonate;

NaI: sodium iodide;

NaN3: sodium azide;

NaOH: sodium hydroxide;

Na ₂ SO ₄ : sodium sulfate;

NH ₄ Cl: ammonium chloride;

NMR: nuclear magnetic resonance;

°C: degrees Celsius;

Rev. HPLC: preparative high performance liquid chromatography;

PPh ₃ : triphenylphosphine;

p-TsOH: p-toluenesulfonic acid;

RT: room temperature;

TEA: triethylamine;

TFA: trifluoroacetic acid;

THF: tetrahydrofuran.

Example 1. Synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy43-[2-[2-(2-hydroxyethoxy )ethoxy]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1methylethyl]-46-methoxy-34,35,36,37,47, 48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-28, I-29 and I- thirty)

- 94 045993

Synthesis circuit

THF, 20 C, 3 h

HE

- 95 045993

Methods and characteristics.

2-[2-(2-Hydroxyethoxy)ethoxy]ethanol (5 ml) was added to a solution of rapamycin (0.5 g, 0.547 mmol) and p-toluenesulfonic acid hydrate (0.52 g, 2.73 mmol) in THF ( 15 ml) at 25°C. The resulting mixture was stirred for 2 hours, then added to ice-cold saturated aqueous NaHCO ₃ and extracted with EtOAc (30 ml x 3 ). The organic layers were combined, then dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude material was purified by reverse phase chromatography (CH ₃ CN/pure water: 7:3) to give (21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R ,46R,55S)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4- hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26 (48)-tetraene49,50,51,52,53-pentone (I-28: 0.19 g, 33.7% yield) as a white solid. MS (EI+, m/z): 1054.4 [M+Na] ⁺ . 1.5 g of this material was sent for chiral separation, resulting in I29 (0.6 g) and I-30 (0.2 g).

Chiral separation method

Column

Column size Mobile phase Flow rate Wavelength Temperature

CHIRALPAK IC (IC00CD-TB016)

0.46 cm I.D. x 15 cm L

Hexane/EtOH = 60/40 (v/v)

1.0 ml/min

UV 254 nm °C

HPLC equipment:

Shimadzu LC-20AD CP-HPLC-05

I-29: ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.41 - 6.20 (m, 2H), 6.13 (dd, J=15.0, 10.3 Hz, 1H), 5, 92 (dd, J=32.7, 11.0 Hz, 1H), 5.51 (dd, J=15.1, 8.9 Hz, 1H), 5.41 (d, J=9.9 Hz , 1H), 5.27 (d, J=5.3 Hz, 1H), 5.13 (dd, J=26.5, 20.5 Hz, 1H), 4.85 (s, 1H), 4 ,19 (t, J=8.9 Hz, 1H), 3.92 (d, J=36.4 Hz, 1H), 3.80 - 3.51 (m, 12H), 3.50 - 3, 24 (m, 12H), 2.87-2.51 (m, 6H), 2.29 (t, J=34.7 Hz, 2H), 2.12 - 1.87 (m, 5H), 1 .84 - 1.66 (m, 13H), 1.53 - 1.15 (m, 9H), 1.15 - 0.77 (m, 18H), 0.65 (dt, J=20.2, 10.1 Hz, 1H).

Example 2. Synthesis of salt (21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-43-[2-[2-(2aminoethoxy)ethoxy]ethoxy] -45,55-dihydroxy-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37 ,47,48-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone formed with trifluoroacetic acid (I- 39)

- 96 045993

Synthesis scheme η ημ ^HBr О Br ^P ' ^TS0H HO^—° HCT^ ⁺ Rapamycin

20% THF, 23°C, 2 h

33%

Methods and characteristics.

Step 1: 2-(2-(2-bromoethoxy)ethoxy)ethanol.

Hydrogen bromide (86.21 g, 1.07 mmol, 115 mL) was added to a solution of 2-[2-(2-hydroxyethoxy)ethoxy]ethanol (100 g, 665.90 mmol) in toluene (1.15 L) and the resulting mixture was stirred at reflux for 18 hours, then the aqueous layer was discarded. The organic layer was washed with aqueous NaOH, concentrated in vacuo, then purified by silica gel chromatography (MeOH:DCM=1:20) to give 2-[2-(2-bromoethoxy)ethoxy]ethanol (20 g, yield 14%) in liquid form. ¹ H NMR (400 MHz, CDCl ₃ ) δ 3.83 (t, J=6.2 Hz, 2H), 3.77 - 3.72 (m, 2H), 3.69 (s, 4H), 3 .64 - 3.61 (m, 2H), 3.49 (t, J=6.1 Hz, 2H), 2.51 (t, J=6.1 Hz, 1H).

Stage 2: (21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-43-[2-[2-(2-bromoethoxy)ethoxy]ethoxy] -45,55-dihydroxy-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37 ,47,48-hexamethyl-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(47), 26(48)-tetraene-49,50,51,52,53-pentone.

2-[2-(2-Bromoethoxy)ethoxy]ethanol (0.12 g, 0.547 mmol, 2 ml) was added to a solution of rapamycin (0.5 g, 0.547 mmol) and p-toluenesulfonic acid hydrate (0.5 g, 2.73 mmol) in THF (7 ml) at room temperature and the resulting mixture was stirred for 2 hours. An aqueous solution of ice-cold NaHCO ₃ was then added and the mixture was extracted with EtOAc (30 ml x 3 ). The organic phase was then dried over Na ₂ SO ₄ , filtered and concentrated in vacuo.

The resulting crude material was purified by reverse phase chromatography (CH ₃ CN/4uci^ water=7:3) to give (21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S ,45R,46R,55S)-43-[2-[2-(2-bromoethoxy)ethoxy]ethoxy]-45,55-dihydroxy-44-[(1S)-2-[(1S,3R,4R)- 4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-65,66-dioxa-56azatricyclohexatriaconta-21,23,25(47),26 (48)-tetraene-49,50,51,52,53-pentone (0.2 g, 33.4% yield, 1H NMR showed rapamycin impurity) as a white solid. MS (EI ⁺ , m/z): 1116.4 [M+Na] ⁺ .

Step 3: (21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-43-[2-[2-(2-azidoethoxy) si]ethoxy] -45,55-dihydroxy-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46methoxy-34,35,36,37,47 ,48-hexamethyl-68,69-dioxa-59-azatricyclohexatriaconta-21,23,25(47),26(48)tetraene-49,50,51,52,53-pentone.

A solution of NaN3 (1.07 g, 16.44 mmol), NaI (0.33 g, 2.19 mmol) and (21E,23E,25E,26E,34R,35S,36R, 37R,39R,41S,44S, 45R,46R,55S)-43-[2-[2-(2-bromoethoxy)ethoxy]ethoxy]-45,55-dihydroxy-44-[(1S)-2[(1S,3R,4R)-4- hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48 )-tetraene-49,50,51,52,53-pentone (0.6 g, 0.548 mmol) in DMF (10 ml) was stirred at 60°C for 1.5 hours. The reaction mixture was then quenched with EtOAc (50 ml) and the mixture was washed with aqueous NH ₄ Cl (20 ml), dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The resulting crude material was purified using

- 97 045993 reverse phase chromatography (CI 1 ₃ C\ pure water = 4:1), resulting in (21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R ,46R,55S)-43-[2-[2-(2-αzidoethoxy)ethoxy]ethoxy]45,55-dihydroxy-44-[( 1S)-2-[( 1S,3R,4R)-4-hydroxy -3-methoxycyclohexyl]-1 -methylethyl]-46-methoxy34,35,36,37,47,48-hexamethyl-68,69-dioxa-59-azatricyclohexatriaconta-21,23,25(47),26(48) -tetraene49,50,51,52,53-pentone (0.3 g, 51.8% yield) as a light yellow solid. MS (EI ⁺ , m/z): 1079.4[M+Na] ⁺ .

Step 4: salt (21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-43-[2-[2-(2-aminoethoxy)ethoxy]ethoxy ]-45,55-dihydroxy-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36, 37,47,48-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone, formed from trifluoroacetic acid.

Triphenylphosphine (0.186 g, 0.7 mmol) was slowly added to the solution of (21E,23E,25E,26E,34R,35S, 36R,37R,39R,41S,44S,45R,46R,55S)-43-[2-[ 2-(2-αzidoethoxy) ethoxy]ethoxy]-45,55 -dihydroxy-44-[(^)2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46 -methoxy-34,35,36,37,47,48-hexamethyl68,69-dioxa-59-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53 -pentone (0.25 g, 0.24 mmol) in THF (5 ml) triphenylphosphine. The resulting solution was stirred at 60°C for 2 hours, then 0.05 ml of water was added and the resulting mixture was stirred at room temperature for 6 hours and then concentrated. The resulting crude material was purified by reverse phase chromatography (CH3CN/0.02% TFA in water (2:3) to give (21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S ,45R,46R,55S)-43-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]45,55-dihydroxy-44-[( 1S)-2-[( 1S,3R,4R)-4 -hydroxy-3-methoxycyclohexyl]-1 -methylethyl]-46-methoxy34,35,36,37,47,48-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26( 48)-tetraene 49,50,51,52,53-pentone (I-39: 0.035 g, 14% yield) as a white solid MS (EI ⁺ , m/z): 1031.4 [M+H ] ^{+ .1H} NMR (500 MHz, DMSO-06) δ 7.82 (s, 3H), 6.42 (dd, J=33.7, 19.3 Hz, 2H), 6.25 - 6, 09 (m, 2H), 5.46 (dd, J=14.7, 9.7 Hz, 1H), 5.28 (s, 1H), 5.08 (d, J=10.1 Hz, 1H ), 5.00 - 4.92 (m, 1H), 4.08 - 3.92 (m, 2H), 3.78 (d, J=11.6 Hz, 1H), 3.63 - 3, 38 (m, 16H), 3.36 - 3.08 (m, 12H), 2.99 (dd, J=22.3, 17.1 Hz, 2H), 2.87 2.73 (m, 2H ), 2.37 (dd, J=17.9, 8.4 Hz, 1H), 2.30-1.75(m, 4H), 1.7-1.49 (m, 15H), 1, 51 - 1.01 (m, 6H), 1.010.65 (m, 18H), 0.63 - 0.56 (m, 1H).

Example 3. Synthesis of (21E,23E,25E,26E,36R,37S,38R,39R,41R,43S,46S,47R,48R,57S)-47,57-dihydroxy45-[2-[2-[2-( 2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-46-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-48-methoxy-36.37 ,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone (I -36)

Synthesis circuit

p-TsOH

1-36

THF 23°C, 2 25%

Methods and characteristics.

2-[2-[2-(2-Hydroxyethoxy)ethoxy]ethoxy]ethanol (1.06 g, 5.47 mmol, 5 ml) was added to a solution of rapamycin (0.5 g, 0.547 mmol) and p-toluenesulfonic acid hydrate acid (0.21 g, 1.09 mmol) in THF (15 ml) at room temperature. The reaction mixture was stirred at room temperature for 2 hours, then added to ice-cold saturated aqueous _NaHCO3 and extracted with EtOAc (30 mlx3). The organic layers were combined and dried over Na ₂ SO ₄ , filtered and concentrated. The crude product was purified using reverse phase chromatography

- 98 045993 (C'l 1 ₃ C'\/pure water=3:2), resulting in (21E,23E,25E,26E,36R,37S,38R,39R,41R,43S, 46S,47R, 48R,57S)-47,57-gurugpoKCu-45-[2-[2-[2-(2-rugpoKC№TOKCu^TOKCu^TOKCu^TOKCu]-46-[(1S)-2[(1S,3R, 4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49 ),26(50)-tetraene-51,52,53,54,55-pentone (I-36: 0.15 g, 25.5% yield) as a white solid MS (EI ⁺ , m/ z): 1098.4[M+H] ⁺ .1H NMR (500 MHz,

CDCl ₃ ) δ 6.40 - 5.92 (m, 4H), 5.73 - 5.35 (m, 3H), 5.25 - 5.05 (m, 2H), 4.31 - 4.12 (m, 1H), 3.97 (dd, J=25.7, 6.3 Hz, 1H), 3.87 - 3.53 (m, 15H), 3.52 - 3.17 (m, 11H ), 2.99 - 2.46 (m, 6H), 2.36 - 1.93 (m, 9H), 1.90 - 1.54 (m, 13H), 1.52 -1.17 (m , 9H), 1.15 - 0.81 (m, 18H), 0.68 - 0.58 (m, 1H).

Example 4. Synthesis of (21E,23E,25E,26E,38R,39S,40R,41R,43R,45S,47R,48S,49R,50R,59S)-49,59-dihydroxy-47-[2-[2- [2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-48-[(1S)-2-[(1S,3R,4R)-4-hydroxy3-methoxycyclohexyl]-1-methylethyl]- 50-methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56, 57-pentone (I-35)

Synthesis circuit

Methods and characteristics.

2-[2-[2-[2-(2-Hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethanol (0.08 g, 0.33 mmol, 2 ml) was added to the rapamycin solution (0.3 g, 0.328 mmol) and p-toluenesulfonic acid hydrate (0.31 g, 1.64 mmol) in THF (6 ml) at room temperature. The resulting mixture was stirred at room temperature for 2 hours, then added to ice-cold saturated aqueous NaHCO ₃ and extracted with EtOAc (20 ml x 3 ). The organic layers were combined and then dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude material was purified by reverse phase chromatography (CH3CN/pure water: 3:2) to give (21E,23E,25E,26E,38R, 39S,40R,41R,43R,45S,47R,48S,49R ,50R,59S)-49,59-dihydroxy-47-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-48-[(1S)-2-[ (1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-50methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21, 23,25(51),26(52)tetraene-53,54,55,56,57-pentone (I-35: 0.06 g, 16.3% yield) as a white solid. MS (EI ⁺ , m/z): 1042.4[M+Na] ⁺ . ¹ H NMR (500 MHz, CDCl3) δ 6.42 - 5.81 (m, 4H), 5.58-4.81 (m, 4H), 4.31-4.11 (m, 1H), 4 .01 -3.51 (m, 22H), 3.49 - 3.13 (m, 11H), 3.01-2.43 (m, 6H), 2.29 (t, J=30.6 Hz , 2H), 2.15 - 1.88 (m, 7H), 1.76 - 1.55 (m, 12H), 1.51 -1.18 (m, 9H), 1.15 - 0.74 (m, 18H), 0.66 (dd, J=23.9, 12.0 Hz, 1H).

Example 5. Synthesis of (21E,23E,25E,26E,30R,31S,32R,33R,35R,37S,39S,40S,41R,42R,52S)-39-(2,3-dihydroxynpropoxy)-41,52- dihydroxy-40-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-42-methoxy-30,31,32,33,43,44- hexamethyl-64,65-dioxa-53-azatricyclohexatriaconta-21,23,25(43), 26(44)-tetraene-45,46,47,48,49-pentone (I-25)

- 99 045993

Synthesis scheme but but

pH p-TsOH

THF, 20°С, 2 h

Methods and characteristics.

Step 1: (24E,26E,28E,29E,32K,338,34K,35K,37K,398,418,428,44K,45K,558)-41-[(2,2-dimethyl-1,3dioxolan-4-yl)methoxy ]-44,55-dihydroxy-42-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1methylethyl]-45-methoxy-32,33,34,35, 46,47-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta24,26,28(46),29(47)-tetraene-48,49,50,51,52-pentone.

(2,2-Dimethyl-1,3-dioxolan-4-yl)methanol (1 ml) was added to a solution of rapamycin (0.2 g, 0.22 mmol) and p-toluenesulfonic acid hydrate (0.1 g, 0.547 mmol) in THF (3 ml) at room temperature. The resulting mixture was stirred at room temperature for 2 hours, then added to ice-cold saturated aqueous NaHCO ₃ and extracted with EtOAc (20 ml x 2 ). The organic layers were combined and then dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude product was purified by reverse phase chromatography (C^CN/pure water: 7:3) to give (24E,26E,28E,29E,32R,33S,34R,35R,37R,39S,41S,42S ,44R,45R,55S)-41-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]-44,55-dihydroxy-42-[(1S)-2[(1S,3R ,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-32,33,34,35,46,47-hexamethyl66,67-dioxa-56-azatricyclohexatriaconta-24,26,28( 46),29(47)-tetraene-48,49,50,51,52-pentone (0.07 g, 32% yield) as a white solid. MS (EI ⁺ , m/z): 1036.4[M+Na] ⁺ .

Stage 2: (21E,23E,25E,26E,30R,31S,32R,33R,35R,37S,39S,40S,41R,42R,52S)-39-(2,3-dihydroxypropoxy)-41,52-dihydroxy -40-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-42methoxy-30,31,32,33,43,44-hexamethyl-64 ,65-dioxa-53-azatricyclohexatriaconta-21,23,25(43),26(44)tetraene-45,46,47,48,49-pentone (I-25).

4-Methylbenzenesulfonic acid-pyridine (0.037 g, 0.148 mmol) was added to the solution of (24E,26E,28E,29E,32R,33S,34R,35R,37R,39S,41S,42S,44R,45R,55S)-41- [(2,2-dimethyl-1,3-dioxolαn-4yl)methoxy]-44,55-dihydroxy-42-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl ]-1-methylethyl]45-methoxy-32,33,34,35,46,47-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta24,26,28(46),29(47)-tetraene-48, 49,50,51,52-pentone (0.05 g, 0.05 mmol) in MeOH (2 ml). The mixture was stirred at room temperature for 18 hours. After the methanol had evaporated, the residue was neutralized with saturated aqueous NaHCO3 solution. The mixture was then extracted using EtOAc (10 mlx3). The organic layers were combined, dried over Na ₂ SO ₄ and concentrated with the resulting crude material, purified by reverse phase chromatography (CH3CN/pure water=1:1), resulting in (21E,23E,25E,26E,30R, 31S,32R,33R,35R,37S,39S,40S,41R,42R,52S) - 100 045993

39-(2,3-gurugpoKCunponoKCu)-41,52-gurugpoKCu-40-[(1S)-2-[(1S,3R,4R)-4-rugpoKCu-3-MeTOKcm,uKTOreKsil] -1 -methylethyl] - 42-methoxy-30,31,32,33,43,44-hexamethyl-64,65-dioxa-53-azatricyclohexatriaconta21,23,25(43),26(44)-tetraene-45,46,47,48, 49-pentone (I-25: 0.005 g, 10% yield) as a white solid. MS (EI ⁺ , m/z): 996.5[M+Na] ⁺ . ^1H NMR (500 MHz, CDC13) δ 6.21-5.97 (m, 4H), 5.77 - 4.78 (m, 5H), 4.53 -4.12 (m, 2H), 4 .05 - 3.11 (m, 19H), 3.07 - 2.88 (m, 2H), 2.65-2.5 (m, 4H), 2.39 - 1.91 (m, 7H) , 1.89 1.69 (m, 12H), 1.52 - 1.20 (m, 9H), 1.17 - 0.76 (m, 18H), 0.73 - 0.61 (m, 1H ).

Example 6. Synthesis of (21E,23E,25E,26E,42R,43S,44R,45R,47S,49S,51S,52S,53R,54R,63R)-53,63dihydroxy-51-[2-[2-[2 -[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-52-[(lR)-2-[(1S,3R,4R) -4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-54-methoxy-42,43,44,45,55, 56-hexamethyl-74,75-dioxa-64-azatricyclohexatriaconta-21,23,25(55 ),26(56)-tetraene-57,58,59,60,61pentone (I-24)

It

-—O, no-O ^λ Synthesis scheme he o noCh^y ud, _o Ch y > N ^><.0,,. ^^/=/ ]nO*T G I ---% %Х·· : 'Ό o ^H >U °^ _o ^ ^he 1-24 he o he o $ — o, l °\ [ ° I / HO'-CH J \ _p %. \ p-TsOH U— ⁷ 9 1 /° * H \ THF 23°C, 2 h 1 ν Ζ o A==/ o ^{? 19%} wT 'Yg o' - ²⁴

He

Methods and characteristics.

A solution of rapamycin (0.5 g, 547 mmol) and 2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (0.2 g, 0.547 mmol, 3 ml) and p-toluenesulfonic acid hydrate (0.52 g, 2.73 mmol) in THF (15 ml) were stirred at room temperature for 3 hours, then quenched with EtOAc (30 mlx3). The combined organic layers were washed with ice-cold saturated NaHCO ₃ solution and concentrated. The resulting crude material was purified by reverse phase chromatography (CI 13C'\pure water=7:3) to give (21E,23E,25E,26E,42R,43S,44R,45R,47S,49S,51S, 52S,53R,54R,63R)-53,63-dihydroxy-51-[2-[2-[2-[2-[2-[2[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]-52-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-54-methoxy-42,43,44,45 ,55,56-hexamethyl-74,75-dioxa-64azatricyclohexatriaconta-21,23,25(55),26(56)-tetraene-57,58,59,60,61-pentone (I-24: 0.13 g, 19%) in the form of a white solid. MS (EI ⁺ m/z): 1275.6[M+Na] ⁺ . 1H NMR (500 MHz, CDC13) δ 6.45 - 5.80 (m, 4H), 5.57 - 5.05 (m, 4H), 4.85 - 4.08 (m, 2H), 3, 90 - 3.50 (m, 34H), 3.47 - 3.24 (m, 13H), 2.97 - 2.44 (m, 7H), 2.39 - 2.06 (m, 2H), 2.03 -1.84 (m, 6H), 1.78 - 1.58 (m, 13H), 1.53 - 1.16 (m, 9H), 1.14 - 0.78 (m, 18H ), 0.670.53 (m, 1H).

Example 7 Synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,38S,41S,43S,45R,46R,55R)-45,55-dihydroxy42-[2-(2-hydroxyethoxy)ethoxy] -43-[(1R)-2-[(1S,2R,3R)-3-(2-hydroxyethoxy)-2-methoxycyclohexyl]-1methylethyl]-46-methoxy-34,35,36,37,47,48 -hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-23)

- 101 045993

Synthesis circuit

Methods and characteristics.

Step 1: 2-[tert-butyl(dimethyl)silyl]oxyethyl trifluoromethanesulfonate.

A mixture of 2-[tert-butyl(dimethyl)silyl]oxyethanol (4 g, 22.69 mmol) and DIPEA (3.81 g, 29.49 mmol, 5.14 ml) in DCM (50 ml) was cooled to 0° C under N2, then trifluoromethylsulfonyl trifluoromethanesulfonate (7.04 g, 24.95 mmol) was added and the resulting mixture was stirred at 0°C for 1 hour. The reaction mixture was diluted with EtOAc (200 ml) and then washed with saturated NaHCO ₃ solution (200 ml), water (200 ml), brine (200 ml), and then the organic layer was dried over Na ₂ SO ₄ , filtered and concentrated in vacuo to obtain 2-[tert

- 102 045993 butyl(dimethyl)silyl]oxyethyltrifluoromethanesulfonate (5.5 g, yield 78.6%) in the form of a brown oil. ^1H NMR (500 MHz, CDCl ₃ ) δ 4.50 - 4.42 (t, 2H), 3.85 - 3.80 (t, 2H), 0.84 - 0.78 (s, 9H), 0.00 (s, 6H).

Stage 2: (27E,29E,31E,32E,36R,37S,38R,39R,40S,43S,44S,45S,47R,48R,57R)-45-[(1R)-2-[(1S,2R, 3R)3-[2-[tert-butyl(dimethyl)silyl]oxyethoxy]-2-methoxycyclohexyl]-1-methylethyl]-47,57-dihydroxy44,48-dimethoxy-36,37,38,39,49,50 -hexamethyl-67,68-dioxa-59-azatricyclohexatriaconta-27,29,31 (49),32(50)-tetraene-51,52,53,54,55-pentone.

Rapamycin (2 g, 2.19 mmol) and DIPEA (2.26 g, 17.50 mmol, 3.05 ml) were dissolved in toluene (60 ml), then heated to 60°C. 2-[tert-Butyl(dimethyl)silyl]oxyethyl trifluoromethanesulfonate (5.40 g, 17.50 mmol) was then added under N2, then stirred at 60°C for 16 hours. The mixture was poured into ice-cold saturated NaHCO ₃ solution (100 ml) and then subjected to extraction using EtOAc (150 mlx3). The combined organic layers were washed with water and brine, then concentrated in vacuo. The residue was then purified by reverse phase chromatography (CH ₃ CN/4uc^ water=4:1) to give (27E,29E,31E,32E,36R,37S,38R,39R,40S,43S,44S, 45S,47R,48R,57R)-45-[(1R)-2-[(1S,2R,3R)-3-[2-[tert-butyl(dimethyl)silyl]oxyethoxy]-2-methoxycyclohexyl]-1 -methylethyl]-47,57-dihydroxy-44,48-dimethoxy-36,37,38,39,49,50-hexamethyl-67,68-dioxa-59-azatricyclohexatriaconta-27,29,31(49),32 (50)-tetraene-51,52,53,54,55-pentone (0.75 g, 32% yield) as a colorless oil. ESI-MS (EI+, m/z): 1095.5 [M+Na] ⁺ .

Stage 3: (22E,24E,26E,27E,31R,32S,33R,34R,35S,38S,39S,40S,42R,43R,52R)-42,52-dihydroxy-40[(1R)-2-[ ( 1 S,2R,3R)-3 -(2-hydroxyethoxy)-2-methoxycyclohexyl]-1-methylethyl]-39,43-dimethoxy31,32,33,34,44,45-hexamethyl-62,63-dioxa -53-azatricyclohexatriaconta-22,24,26(44),27(45)-tetraene46,47,48,49,50-pentone.

Solution of TEA-3No (4.65 g, 28.87 mmol) and (27E,29E,31E,32E,36R,37S,38R,39R,40S,43S,44S,45S,47R,48R,57R)-45 -[(1R)-2-[(1S,2R,3R)-3-[2-[tert-butyl(dimethyl)silyl]ethoxy]-2-methoxycyclohexyl]-1-methylethyl]-47,57-dihydroxy- 44,48-dimethoxy-36,37,38,39,49,50-hexamethyl-67,68-dioxa-59-azatricyclohexatriaconta-27,29,31(49),32(50)-tetraene-51,52, 53,54,55-pentone (3.05 g, 2.89 mmol) in THF (50 ml) was stirred at 20°C for 2 hours. The mixture was poured into ice-cold saturated NaHCO3 solution (100 ml), then extracted with using EtOAc (150 mlx3). The organic layers were combined and washed with water and brine, then concentrated in vacuo. The residue was purified by reverse phase chromatography (pure water: 7:3) to give (22E,24E,26E,27E,31R,32S,33R,34R,35S,38S,39S,40S,42R ,43R,52R)-42,52-dihydroxy-40-[(1R)-2-[(1S,2R, 3R)-3-(2-hydroxyethoxy)-2-methoxycyclohexyl]-1-methylethyl]-39, 43-dimethoxy-31,32,33,34,44,45-hexamethyl-62,63-dioxa-53-azatricyclohexatriaconta-22,24,26(44),27(45)-tetraene-46,47,48, 49,50-pentone (1.5 g, 54% yield) as a white solid. ESI-MS (EI ⁺ , m/z): 980.5 [M+Na] ⁺ .

Step 4: (21E,23E,25E,26E,34R,35S,36R,37R,38S,41S,43S,45R,46R,55R)-45,55-dihydroxy-42-[2-(2hydroxyethoxy)ethoxy] - 43-[(1R)-2-[(1 S,2R,3R)-3 -(2-hydroxyethoxy)-2-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47 ,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47), 26(48)-tetraene-49,50,51,52,53-pentone (I-23).

Mixture of (22E,24E,26E,27E,31R,32S,33R,34R,35S,38S,39S,40S,42R,43R,52R)-42,52-dihydroxy-40[(1R)-2-[( 1 S,2R,3R)-3 -(2-hydroxyethoxy)-2-methoxycyclohexyl]-1-methylethyl]-39,43-dimethoxy-31,32, 33,34,44,45-hexamethyl-62,63-dioxa -53-azatricyclohexatriaconta-22,24,26(44),27(45)-tetraene-46,47,48, 49,50-pentone (0.5 g, 0.52 mmol), 2-(2-hydroxyethoxy )ethanol (2.77 g, 26.09 mmol) and ptoluenesulfonic acid hydrate (0.54 g, 3.13 mmol) in THF (6 ml) were stirred at 20°C for 2 hours. The mixture was poured into an ice-cold saturated solution NaHCO3 (30 ml) and subjected to extraction using EtOAc (50 mlx3). The organic layers were combined and then washed with water and brine, then concentrated in vacuo. The residue was purified by reverse phase chromatography ^Η^Ν/4Нτλλ water=1:1), resulting in (21E,23E,25E,26E,34R,35S,36R,37R,38S,41S,43S, 45R,46R,55R)-45,55-dihydroxy-42-[2-(2-hydroxyethoxy)ethoxy]-43-[(1R)-2-[(1S,2R,3R)-3-(2-hydroxyethoxy )-2-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa56-azatricyclohexatriaconta-21,23,25(47),26(48 )-tetraene-49,50,51,52,53-pentone (I-23: 0.1 g, 19% yield) as a white solid. ESI-MS (EI ⁺ , m/z): 1054.4 [M+Na] ⁺ . ¹ H NMR (500 MHz, CDCl3) δ 6.63 - 5.86 (m, 4H), 5.70 - 5.00 (m, 4H), 4.87 - 4.15 (m, 2H), 4 .02 - 3.54 (m, 14H), 3.50 - 3.26 (m, 1H), 3.24 3.02 (m, 3H), 2.76 - 2.46 (m, 3H), 2.38 - 1.86 (m, 8H), 1.84 - 1.54 (m, 14H), 1.47 (m, 3H), 1.25 (m, 6H), 1.00 (m, 16H), 0.69 (dt, J=34.1, 12.1 Hz, 1H).

Example 8. Synthesis of (21E,23E,25E,26E,36R,37S,38R,39R,40S,43S,45S,47R,48R,57R)-47,57-dihydroxy44-[2-[2-(2-hydroxyethoxy )ethoxy]ethoxy]-45-[(1R)-2-[(1S,2R,3R)-3-(2-hydroxyethoxy)-2-methoxycyclohexyl]-1-methylethyl]-48-methoxy-36.37, 38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone (I- 32)

- 103 045993

Synthesis circuit

Methods and characteristics.

A mixture of everolimus (3.92 g, 26.09 mmol) and p-toluenesulfonic acid hydrate (0.45 g, 2.61 mmol) in THF (10 ml) was stirred at 20°C for 2 hours. The mixture was poured into ice-cold saturated NaHCO ₃ solution (30 ml) and subjected to extraction using EtOAc (50 mlx3). The organic layers were combined and then washed with water and brine, then concentrated in vacuo. The residue was purified by reverse phase chromatography (CH ₃ CN/pure water=1:1) to give (21E,23E,25E,26E,36R,37S,38R,39R,40S,43S,45S,47R, 48R,57R)-47,57-dihydroxy-44-[2-[2(2-hydroxyethoxy)ethoxy]ethoxy]-45-[(1R)-2-[(1S,2R,3R)-3-(2 -hydroxyethoxy)-2-methoxycyclohexyl]1-methylethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta21,23,25(49),26( 50)-tetraene-51,52,53,54,55-pentone (I-32: 0.155 g, 28% yield) as a white solid. ESI-MS (EI+, m/z): 1098.4 [M+Na]+. ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.49 - 5.83 (m, 4H), 5.67 5.35 (m, 2H), 5.33 - 5.01 (m, 2H), 4 .92 - 4.08 (m, 2H), 4.05 - 3.51 (m, 17H), 3.52 - 3.23 (m, 11H), 3.23 - 3.01 (m, 3H) , 2.66 (m, 4H), 2.40 - 1.95 (m, 5H), 1.95 - 1.55 (m, 17H), 1.52 - 1.13 (m, 9H), 1 .13-0.79 (m, 16H), 0.71 (dd, J=23.8, 11.9 Hz, 1H).

Example 9. Synthesis of (21E,23E,25E,26E,38R,39S,40R,41R,42S,45S,47S,49R,50R,59R)-49,59-dihydroxy46-[2-[2-[2-( 2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-47-[(1R)-2-[(1S,2R,3R)-3-(2-hydroxyethoxy)-2-methoxycyclohexyl]-1-methylethyl]-50-methoxy -38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56,57 -pentone (I-22)

- 104 045993

Synthesis circuit

Methods and characteristics.

A mixture of everolimus (0.5 g, 0.52 mmol), 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethanol (2.03 g, 10.44 mmol) and p-toluenesulfonic acid hydrate ( 0.54 g, 3.13 mmol) in THF (6 ml) was stirred at 20°C for 2 hours. The mixture was poured into ice-cold saturated NaHCO ₃ solution (30 ml) and extracted with EtOAc (50 mlx3). The combined organic layers were washed with water, brine, then concentrated in vacuo. The residue was purified by reverse phase chromatography (CH ₃ CN/pure water=1:1) to give (21E,23E,25E,26E, 38R,39S,40R,41R,42S,45S,47S,49R,50R, 59R)-49,59-dihydroxy-46-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-47-[(1R)-2-[(1S,2R,3R)- 3-(2-hydroxyethoxy)-2-methoxycyclohexyl]-1-methylethyl]-50methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25( 51),26(52)tetraene-53,54,55,56,57-pentone (1-22: 0.1 g, 17% yield) as a white solid. ESI-MS (EI+, m/z): 1043.4 [M+Na]+. ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.71 - 5.78 (m, 4H), 5.77 - 5.01 (m, 4H), 4.65 - 3.88 (m, 3H), 3.87 - 3.50 (m, 20H), 3.50 - 3.00 (m, 14H), 2.77 -1.95 (m, 11H), 1.89 - 1.53 (m, 13H ), 1.53 - 0.79 (m, 27H), 0.76 - 0.62 (m, 1H).

Example 10: Synthesis of (22E,24E,26E,27E,35R,36S,37R,38R,39S,42S,44S,46R,47R,56R)-46,56-dihydroxy-44-[(1R)-2-[ (1S,2R,3R)-3-hydroxy-2-methoxycyclohexyl]-1-methylethyl]-47-methoxy-43-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]-35,36,37, 38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone (I-27)

- 105 045993

Synthesis circuit

Methods and characteristics.

A mixture of rapamycin (1 g, 1.09 mmol), 2-[2-(2-methoxyethoxy)ethoxy]ethanol (8.98 g, 54.69 mmol) and p-toluenesulfonic acid hydrate (0.94 g, 5. 47 mmol) in THF (20 ml) was stirred at 20°C for 2 hours. The mixture was poured into ice-cold saturated NaHCO ₃ solution (50 ml) and extracted with EtOAc (100 mlx3). The combined organic layers were washed with water, brine, then concentrated in vacuo. The residue was purified by reverse phase chromatography (CII3CN pure water=4:1) to obtain (22E,24E,26E,27E,35R,36S,37R,38R, 39S,42S,44S,46R,47R,56R )-46,56-dihydroxy-44-[(1R)-2-[(1S,2R,3R)-3-hydroxy-2-methoxycyclohexyl]1-methylethyl]-47-methoxy-43-[2-[2 -(2-methoxyethoxy)ethoxy]ethoxy]-35,36,37,38,48,49-hexamethyl-66,67dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50 ,51,52,53,54-pentone (I-27: 0.16 g, 14% yield) as a white solid. ESI-MS (EI ⁺ , m/z): 1068.4 [M+Na]+. 1H NMR (500 MHz, CDC13) δ 6.50 - 5.81 (m, 4H), 5.73 - 5.05 (m, 4H), 4.85 - 3.98 (m, 3H), 3, 90 - 3.10 (m, 27H), 3.02 - 2.24 (m, 7H), 1.98 (m, 6H), 1.82 - 1.55 (m, 13H), 1.54 - 1.16 (m, 9H), 1.16 - 0.78 (m, 17H), 0.75 - 0.59 (m, 1H).

Example 11. Synthesis of (21E,23E,25E,26E,31R,32S,33R,34R,35S,38S,40S,42R,43R,52R)-42,52-dihydroxy-40-[(1R)-2-[ (1S,2R,3R)-3-hydroxy-2-methoxycyclohexyl]-1-methylethyl]-39-(3-hydroxypropoxy)-43methoxy-31,32,33,34,44,45-hexamethyl-63,64- dioxa-53-azatricyclohexatriaconta-21,23,25(44),26(45)tetraene-46,47,48,49,50-pentone (I-34)

- 106 045993

Synthesis circuit

Methods and characteristics.

A mixture of rapamycin (0.5 g, 0.55 mmol), propane-1,3-diol (13.13 g, 172.48 mmol, 12.5 ml), and p-toluenesulfonic acid hydrate (0.47 g, 2.74 mmol) in THF (37.5 ml) was stirred at 20°C for 2 hours. The mixture was poured into ice-cold saturated NaHCO ₃ solution (100 ml) and extracted with EtOAc (100 mlx3). The organic layers were combined and washed with water and brine, then concentrated in vacuo. The residue was purified by reverse phase chromatography (CH ₃ CN/pure water=7:3) to give (21E,23E,25E,26E,31R,32S,33R,34R,35S, 38S,40S,42R, 43R,52R)-42,52-dihydroxy-40-[(1R)-2-[(1S,2R,3R)-3-hydroxy-2-methoxycyclohexyl]-1methylethyl]-39-(3-hydroxypropoxy)-43 -methoxy-31,32,33,34,44,45-hexamethyl-63,64-dioxa-53-azatricyclohexatriaconta-21,23,25(44),26(45)-tetraene-46,47,48,49 .50-pentone (0.15 g, 29% yield) as a white solid. ESI-MS (EI+ m/z): 980.3 [M+Na]+. ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.58 - 5.84 (m, 4H), 5.72 - 4.83 (m, 4H), 4.65 - 4.06 (m, 2H), 4.03 - 3.63 (m, 5H), 3.62 - 3.05 (m, 12H), 3.03 - 2.40 (m, 6H), 2.42 - 1.91 (m, 7H ), 1.89 - 1.56 (m, 17H), 1.53 - 1.27 (m, 6H), 1.25 - 0.76 (m, 19H), 0.62 (m, 1H).

Example 12 Synthesis of (21E,23E,25E,26E,30R,31S,32R,33R,35R,37S,40S,41R,42R,51S)-41,51-dihydroxy-39-(2-hydroxyethoxy)-40- [(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-42methoxy-30,31,32,33,43,44-hexamethyl-62,63- dioxa-52-azatricyclohexatriaconta-21,23,25(43),26(44)tetraene-45,46,47,48,49-pentone (I-38)

- 107 045993

Synthesis circuit

Methods and characteristics.

p-toluenesulfonic acid hydrate (0.31 g, 1.64 mmol) was added to a mixture of rapamycin (0.5 g, 0.547 mmol) and ethylene glycol (3 ml) in THF (10 ml) at 10°C. The resulting mixture was stirred at 10°C for 17 hours, then the reaction mixture was diluted with EtOAc (100 ml) and the pH was adjusted to 9 using saturated aqueous NaHCO ₃ solution (about 50 ml). The organic layer was concentrated in vacuo, then the residue was purified by reverse phase chromatography (CI hCN/pure water=5.5:4.5). Next, the solvent was removed by lyophilization, resulting in (21E,23E,25E,26E,30K,318,32K,33K,35K,378,408,41K,42K,518)-41,51-dihydroxy-39-(2-hydroxyethoxy )-40-[(1S)-2-[(1S,37R,47R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-42-methoxy-30,31,32, 33,43,44- hexamethyl-62,63-dioxa-52-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone (0.05 g, 9%) in as a white solid. MS (EI ⁺ m/z): 966.3 [M+Na] ⁺ . ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.47-5.91 (m, 4H), 5.60-5.13 (m, 4H), 4.85-3.92 (m, 3H), 3.88-3.68 (m, 4H), 3.603.53 (m, 1H), 3.46-3.29 (m, 10H), 3.25-3.19 (m, 1H), 2, 97-2.84 (m, 2H), 2.76-2.53 (m, 4H), 2.35-1.83 (m, 8H), 1.80-1.64 (m, 12H), 1.53-1.16 (m, 9H), 1.14-0.82 (m, 18H), 0.67-0.57 (m, 1H).

Example 13: Synthesis of (21E,23E,25E,26E,40K,418,42K,43K,45K,478,508,51K,52K,618)-51,61-dihydroxy-49-[2-[2-[2-[ 2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-50-[(1S)-2-[(1S,3R,4R)-4hydroxy-3-methoxycyclohexyl]-1-methylethyl] -52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene-55,56,57,58 .59-pentone (I-33)

Synthesis circuit

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Methods and characteristics.

p-Toluenesulfonic acid hydrate (0.19 g, 1.02 mmol) was added to a mixture of rapamycin (0.31 g, 0.34 mmol) and hexaethylene glycol (2 ml) in THF (6 ml) at 15°C. The resulting mixture was stirred at 15°C for 17 hours, then the reaction mixture was diluted with EtOAc (200 ml) and the pH was adjusted to 9 using saturated aqueous NaHCO ₃ solution (about 100 ml). The organic layer was concentrated in vacuo. The residue was purified by reverse phase chromatography (C^CN/pure water=3:2). The solvent was then removed by lyophilization, resulting in (21E,23E,25E,26E,40E,418,42E,43E,45E,478,508,51E,52E,618)-51,61-dihydroxy-49-[2-[ 2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-50-[(^)-2-[(^,3K,4K)-4-hydroxy-3 -methoxycyclohexyl]-1-methylethyl]-52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta21,23,25(53),26(54)-tetraene -55,56,57,58,59-pentone (I-33: 0.21 g, 51% yield) as a white solid. MS (EI ⁺ , m/z): 1186.8 [M+Na] ⁺ . ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.39-5.94 (m, 4H), 5.55-5.14 (m, 4H), 4.93-3.75 (m, 2H), 3.73-3.71 (m, 2H), 3.67-3.55 (m, 22H), 3.48-3.44 (m, 1H), 3.41-3.16 (m, 12H ), 2.982.86 (m, 2H), 2.75-2.69 (m, 2H), 2.59-2.44 (m, 1H), 2.34-2.21 (m, 1H), 2.11-1.97 (m, 4H), 1.90 (s, 3H), 1.781.54 (m, 13H), 1.50-1.12 (m, 9H), 1.08-0, 83 (m, 18H), 0.71-0.62 (m, 1H).

Example 14: Synthesis of (21E,23E,25E,26E,32R,33S,34R,35R,37R,39S,42S,43R,44R,53S)-43,53-dihydroxy-41-[2-(2-hydroxyethoxy) ethoxy]-42-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-44-methoxy-32,33,34,35,45,46 -hexamethyl-64,65-dioxa-54-azatricyclohexatriaconta-21,23,25(45), 26(46)-tetraene-47,48,49,50,51-pentone (I-37)

Methods and characteristics.

p-Toluenesulfonic acid hydrate (0.187 g, 0.98 mmol) was added to a mixture of rapamycin (0.3 g, 0.33 mmol) and diethylene glycol (2 ml) in THF (6 ml) at 10°C. The resulting mixture was stirred at 10°C for 17 hours, then the reaction mixture was diluted with EtOAc (100 ml) and the pH was adjusted to 9 using saturated aqueous NaHCO ₃ solution (about 50 ml). The organic layer was concentrated in vacuo. The residue was purified using reverse phase (CH ₃ CN/pure water=3:2). Solvents were removed by lyophilization to give (21E,23E,25E,26E,32R,33S,34R, 35R,37R,39S,42S,43R,44R,53S)-43,53-dihydroxy-41-[2- (2-hydroxyethoxy)ethoxy]-42-[(1S)-2[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-44-methoxy-32,33,34,35 ,45,46-hexamethyl64,65-dioxa-54-azatricyclohexatriaconta-21,23,25(45),26(46)-tetraene-47,48,49,50,51-pentone (1-37:

0.126 g, yield 37%) as a white solid. MS (EI ⁺ , m/z): 1010.7 [M+Na] ⁺ . 1H NMR (500 MHz, CDCl ₃ ) δ 6.40-5.95 (m, 4H), 5.55-5.14 (m, 4H), 4.85-4.17 (m, 2H), 4 .05-3.57 (m, 10H), 3.53-3.15 (m, 12H), 2.97-2.89 (m, 2H), 2.76-2.48 (m, 4H) , 2.36-1.84 (m, 7H), 1.79-1.56 (m, 14H), 1.49-1.14 (m, 9H), 1.10

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0.81 (m, 18H), 0.70-0.61 (m, 1H).

Example 15. Synthesis of (21E,23E,25E,26E,31R,32S,33R,34R,36R,38S,41S,42R,43R,53S)-42,53-gurugpoKsi-40-[3-hydroxy-2-( hydroxymethyl)npropoxy]-41-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-43-methoxy-31,32,33,34,44 ,45-hexamethyl-65,66-dioxa-54-azatricyclohexatriaconta21,23,25(44),26(45)-tetraene-46,47,48,49,50-pentone (I-31) it o

he he

1-31

Synthesis circuit

he he

Methods and characteristics.

p-Toluenesulfonic acid hydrate (0.31 g, 1.64 mmol) was added to a mixture of rapamycin (0.5 g, 0.547 mmol) and 2-(hydroxymethyl)-propane-1,3-diol (0.58 g, 5 .47 mmol) in THF (10 ml) at 15°C. The resulting mixture was stirred at 15°C for 17 hours, then the reaction mixture was diluted with EtOAc (100 ml) and the pH was adjusted to 9 using saturated aqueous NaHCO ₃ solution (about 50 ml). The organic layer was concentrated in vacuo. The residue was purified by reverse phase chromatography (CH ₃ CN/pure water=3:2). The solvent was removed by lyophilization to give (21E,23E,25E,26E,31R,32S, 33R,34R,36R,38S,41S,42R,43R,53S)-42,53-dihydroxy-40[3-hydroxy -2-(hydroxymethyl) propoxy]-41-[(1S)-2-[(1 S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1 methylethyl]-43-methoxy-31,32,33 ,34,44,45-hexamethyl-65,66-dioxa-54-azatricyclohexatriaconta-21,23,25(44),26(45)-tetraene-46,47,48,49,50-pentone (I-31 : 0.054 g, yield 9%) as a white solid. MS (EI ⁺ , m/z): 1010.4 [M+Na] ⁺ . ^1H NMR (500 MHz, CDCl ₃ ) δ 6.37-5.96 (m, 4H), 5.56-5.15 (m, 4H), 4.86-4.17 (m, 2H), 3.85-3.47 (m, 10H), 2.96-2.91 (m, 2H), 2.75-2.58 (m, 3H), 2.35-2.22 (m, 3H ), 2.11-1.94 (m, 6H), 1.84-1.46 (m, 23H), 1.35-1.12 (m, 9H), 1.11-0.88 (m , 18H), 0.67-0.65 (m, 1H).

Example 16. Synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy-43-[2-[2-(2 -hydroxyethylsulfanyl)ethylsulfanyl]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36, 37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-21)

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Synthesis circuit

Methods and characteristics.

p-Toluenesulfonic acid hydrate (0.62 g, 3.28 mmol) was added to a mixture of rapamycin (1 g, 1.09 mmol) and 2-[2-(2-hydroxyethylsulfanyl)ethylsulfanyl]ethanol (1.99 g, 10 .94 mmol) in THF (20 ml) at 15°C. The resulting mixture was stirred at 15°C for 17 hours, then the reaction mixture was diluted with EtOAc (100 ml) and the pH was adjusted to 9 using saturated aqueous NaHCO ₃ solution (about 50 ml). The organic layer was concentrated in vacuo. The residue was purified by reverse phase chromatography (CI I3CN pure water=3:2). Solvents were removed by lyophilization to give (21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)45,55-dihydroxy-43-[2-[ 2-(2-hydroxyethylsulfanyl)ethylsulfanyl]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4hydroxy-3-methoxy-cyclohexyl]-1-methylethyl]-46-methoxy-34, 35,36,37,47,48-hexamethyl-66,67-dioxa56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I- 21: 0.15 g, 0.133 mmol, 12% yield) as a yellow solid. MS (EI ⁺ , m/z): 1086.4 [M+Na] ⁺ .

1H NMR (500 MHz, CDC13) δ 6.39-5.95 (m, 4H), 5.54-5.19 (m, 4H), 4.81-4.17 (m, 2H), 3, 96-3.73 (m, 4H), 3.59-3.14 (m, 12H), 2.96-2.55 (m, 14H), 2.35-1.87 (m, 6H), 1.81-1.59 (m, 13H), 1.53-1.13 (m, 11H), 1.16-0.84 (m, 18H), 0.71-0.63 (m, 1H ).

Example 17. Synthesis of (21E,23E,25E,26E,42K,438,44K,45K,478,498,518,528,53K,54K^)-53,63-dihydroxy-51-[2-[2- [2- [2- [ 2- [2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-52- [(1 R)-2[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]- 1-methylethyl]-54-methoxy-42,43,44,45,55,56-hexamethyl74,75-dioxa-64-azatricyclohexatriaconta-21,23,25(55),26(56)-tetraene-57,58 ,59,60,61-pentons (I-26):

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Synthesis circuit

Methods and characteristics.

p-Toluenesulfonic acid hydrate (0.52 g, 2.73 mmol) was slowly added to a solution of rapamycin (0.5 g, 0.55 mmol) and 2-[2-[2-[2-[2-[2- (2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (3.57 g, 10.94 mmol) in THF (10 ml). The resulting solution was stirred at 20°C for 17 hours and then concentrated. The residue was purified by reverse phase chromatography (CI 13 (pure water=7:3) to give (21E,23E,25E,26E,42R,43S,44R,45R,47S,49S,51S, 52S,53R,54R,63R)-53,63-dihydroxy-51-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]-52-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-54-methoxy42,43,44,45,55,56-hexamethyl -74,75-dioxa-64-azatricyclohexatriaconta-21,23,25(55),26(56)-tetraene 57,58,59,60,61-pentone (I-26: 0.16 g, 24% yield) as a white solid MS (EI ⁺ , m/z): 1230.6[M+Na] ⁺ .

1H NMR (500 MHz, CDCl ₃ ) δ 6.46 - 5.74 (m, 4H), 5.61 - 4.74 (m, 4H), 4.05-4.5 (m, 2H), 4 .02 - 3.51 (m, 35H), 3.43 - 3.16 (m, 14H), 2.99 - 2.42 (m, 8H), 2.4-1.6 (m, 7H) , 1.61-1.1 (m, 12H), 1.13 - 0.79 (m, 18H), 0.74 0.61 (m, 1H).

Example 19. Synthesis of (22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[ (1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-42-[R-2-(2-methoxyethoxy)ethoxy]-33,34,35,36,46 ,47-hexamethyl-64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46), 27(47)-tetraene-48,49,50,51,52-pentone (I-119) and (22E ,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R, 54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R )-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-42-^-2-(2-methoxyethoxy)ethoxy]-33,34,35,36,46,47-hexamethyl-64, 65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene-48,49,50,51,52-pentone (I-120)

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Synthesis circuit

Methods and characteristics.

Step 1: synthesis of (22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy43-[(1R)-2-[(1S ,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-42-[2-(2-methoxyethoxy)ethoxy]-33,34,35,36,46,47-hexamethyl -64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46), 27(47)-tetraene-48,49,50,51,52-pentone.

To a mixture of rapamycin (2 g, 2.19 mmol), 2-(2-methoxyethoxy)ethanol (4 ml) in THF (30 ml) was added HND-8 (240 mg, 2.19 mmol) and the reaction mixture was stirred at 50 °C for 4 hours under N2, then filtered and concentrated. The resulting crude product was purified by reverse phase chromatography (C18, CH ₃ CN: H ₂ O=3:1), resulting in (22E,24E,26E,27E, 33R,34S,35R,36R,38S,40S ,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45- methoxy-42-[2-(2-methoxyethoxy)ethoxy]-33,34,35,36,46,47-hexamethyl-64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27( 47)-tetraene-48,49,50,51,52-pentone (0.6 g, 27% yield) as a light yellow solid. ESI-MS (EI ⁺ , m/z): 1024.3 [M+Na] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.56 - 5.82 (m, 4H), 5.47 (ddd, J=37.4, 17.7, 9.4 Hz, 2H), 5.31 - 5.06 (m, 2H), 4.82 - 4.50 (m, 1H), 4.32 - 3.94 (m, 2H), 3.92 - 3.71 (m, 2H), 3, 70 - 3.44 (m, 8H), 3.43 - 3.26 (m, 12H), 3.20 (dd, J=27.0, 16.2 Hz, 1H), 3.00 -2, 22 (m, 7H), 2.18 - 1.56 (m, 19H), 1.54 - 1.25 (m, 7H), 1.24 - 0.81 (m, 19H), 0.67 ( dd, J=23.8, 11.9 Hz, 1H).

Step 2: synthesis of (22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy43-[(1R)-2-[(1S ,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-42-^-2-(2-methoxyethoxy)ethoxy]-33,34,35,36,46,47- hexamethyl-64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46), 27(47)-tetraene-48,49,50,51,52-pentone (I-119) and (22E,24E, 26E,27E,33R,34S,35R,36R,38S,40S,43S,44R, 45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4 -hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45methoxy-42-[8-2-(2-methoxyethoxy)ethoxy]-33,34,35,36,46,47-hexamethyl-64,65-dioxa- 55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene-48,49,50,51,52-pentone (I-120).

(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-Dihydroxy-43-[(1R)-2[(1S,3R, 4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-42-[2-(2-methoxyethoxy)ethoxy]-33,34,35,36,46,47-hexamethyl-64, 65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene-48,49,50,51,52-pentone (0.095 g, 0.095 mmol) was purified by preparative chiral HPLC, in resulting in (22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54dihydroxy-43-[(1R)-2-[(1S, 3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-42-[R-2(2-methoxyethoxy)ethoxy]-33,34,35,36,46,47-hexamethyl -64,65-dioxa-55-azatricyclohexatriaconta22,24,26(46),27(47)-tetraene-48,49,50,51,52-pentone (I-119: 13.2 mg, 14% yield) and (22E,24E,26E,27E, 33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S, 3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-42-^-2-(2-methoxyethoxy)ethoxy]-33,34,35,36,46,47-hexamethyl -64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene-48,49,50,51,52-pentone (I120: 18.1 mg, 19% yield) , both in the form of a white solid.

The chiral separation method is presented below

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Tool

Column

Column temperature

Mobile phase

Flow rate

Detection wavelength

Cycle time

Sample solution

Administration volume:

Gilson-281

CHIRALPAK IC 20^250 mm, 10 µm (Daicel) °C n-Hexane: Ethanol = 60:40 ml/min

214 nm min mg dissolved in 7 ml methanol

0.5 ml (load: 7.1 mg/administration)

I-119: ESI-MS (EI ⁺ , m/z): 1024.4 [M+Na] ⁺ . ^1H NMR (400 MHz, CDCl ₃ ) δ 6.42 - 5.92 (m, 4H), 5.75 5.05 (m, 4H), 4.49 (s, 1H), 4.28 (s , 1H), 4.15 (d, J=10.9 Hz, 1H), 3.99 (t, J=13.3 Hz, 1H), 3.88 - 3.73 (m, 1H), 3 .69 - 3.46 (m, 8H), 3.45 - 3.29 (m, 11H), 3.22 (dd, J=10.1, 6.5 Hz, 2H), 2.99 - 2 .77 (m, 3H), 2.68 (dt, J=28.5, 11.1 Hz, 3H), 2.61 -2.22 (m, 4H), 2.05 (ddd, J=21 .5, 15.3, 7.5 Hz, 5H), 1.88 - 1.65 (m, 12H), 1.53 - 1.30 (m, 7H), 1.17 - 0.78 (m , 19H), 0.73 - 0.57 (m, 1H).

I-120: ESI-MS (EI ⁺ , m/z): 1024.3 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 6.43 -6.18 (m, 2H), 6.13 (dd, J=15.0, 10.0 Hz, 1H), 5.91 (dd, J =31.6, 10.6 Hz, 1H), 5.56 - 5.36 (m, 2H), 5.27 (d, J=4.9 Hz, 1H), 5.16 (dt, J= 18.2, 9.1 Hz, 1H), 4.78 (s, 1H), 4.20 (dd, J=21.1, 11.1 Hz, 1H), 3.96-3.71 (m , 3H), 3.70 - 3.43 (m, 9H), 3.42 3.26 (m, 14H), 2.98-2.87 (m, 1H), 2.77 - 2.62 ( m, 3H), 2.58 (dd, J=16.9, 6.3 Hz, 1H), 2.34 (d, J=13.4 Hz, 2H), 2.15 - 1.84 (m , 5H), 1.83 - 1.64 (m, 7H), 1.34 (dddd, J=22.7, 19.4, 18.1, 9.1 Hz, 12H), 1.16 - 0 .80 (m, 19H), 0.66 (dt, J=16.8, 8.4 Hz, 1H).

Example 20. Synthesis of (23E,25E,27E,28E,32R,33S,34R,35R,36S,39S,41S,45R,46R,55R)-45,55-dihydroxy-41-[(1R)-2-[ (lS,2R,3R)-3-hydroxy-2-methoxycyclohexyl]-1-methylethyl]-40-[[(2S,5S)-5-(hydroxymethyl)-1,4-dioxan-2-yl]methoxy] -46-methoxy-32,33,34,35,47,48-hexamethyl-68,69-dioxa-56azatricyclohexatriaconta-23,25,27(47),28(48)-tetraene-49,50,51,52 .53-pentone (I-118)

Synthesis circuit

NaOH/ _H2O

0-90°С, 18 h 40%

Pd/C, MeOH _n BP ₃ E1 ₂ O, DCE

0-80°С, 18 h 86%

BF ₃ Et ₂ O, EDC

TosO°C-k.t 18 h

Methods and characteristics.

Step 1: synthesis of ^)-1-(benzyloxy)-3-chloropropan-2-ol.

A solution of (2R)-2-(chloromethyl)oxirane (1 g, 10.81 mmol) and phenylmethanol (2.34 g, 21.62 mmol) in EDC (10 ml) was stirred at room temperature for 1 h, then cooled up to 0°C, slowly

- 114 045993 boron trifluoride etherate (76.7 mg, 0.54 mmol) was added. The reaction mixture was allowed to warm to room temperature, stirred overnight and then refluxed for 2 hours. After cooling, the reaction mixture was diluted with 10% aqueous NaHCO ₃ and extracted with EtOAc (100 ml x 3 ). The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The resulting residue was purified by silica gel chromatography (PE: EtOAc=10:1) to obtain the desired product (1.8 g, 86% yield) as a colorless oil. ESI-MS (EI ⁺ , m/z): 218.1 [M+H2O] ⁺ . ^1H NMR (500 MHz, CDCl3) δ 7.367.27 (m, 5H), 4.64 (s, 2H), 3.99-3.95 (m, 1H), 3.64-3.55 (m , 4H), 2.75(d, J=6.0 Hz, 1H).

Step 2: synthesis of ^)-2-(^)-1-(benzyloxy)-3-chloropropan-2-yloxy)-3-hydroxypropyl 4-methylbenzenesulfonate.

A solution of ^)-1-benzyloxy-3-chloro-propan-2-ol (6 g, 29.9 mmol) and [^)-oxiran-2-yl]methyl-4methylbenzenesulfonate (6.83 g, 29.9 mmol ) in EDC (100 ml) was stirred at room temperature for 1 hour, then cooled to 0°C and boron trifluoride etherate (254.6 mg, 1.79 mmol) was added slowly. The reaction mixture was warmed to room temperature, stirred at room temperature overnight, refluxed for 2 hours, then cooled to room temperature. EtOAc (100 ml) and water (50 ml) were added. The layers were separated and the aqueous layer was further extracted using EtOAc (100 mlx2). The combined organic layers were then washed with water (2x50 ml) and brine (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The resulting residue was purified by silica gel chromatography (8% EtOAc in PE) to give [^)-2-[(^)-1-(benzyloxymethyl)-2-chloroethoxy]-3-hydroxypropyl]-4-methylbenzenesulfonate (4 .4 g, yield 34%) as a colorless oil. ESI-MS (EI ⁺ , m/z): 429.1 [M+H] ⁺ .

Step 3: synthesis of ((2R,5R)-5-(benzyloxymethyl)-1,4-dioxan-2-yl)methanol.

Solution of [(2S)-2-[(1S)-1-(benzyloxymethyl)-2-chloroethoxy]-3-hydroxypropyl]-4-methylbenzenesulfonate (0.78 g, 1.82 mmol), NaOH (0.22 g , 5.46 mmol) in H ₂ O (10 ml) stirred at room temperature for 2.5 hours, then heated at 90°C for 4 hours, cooled to room temperature and stirred overnight, then heated at 90 °C for another 2 hours. The reaction mixture was acidified with 1 N. aqueous HCl solution and subjected to extraction with DCM (100 mlx3). The combined organic layers were washed with aqueous NaHCO3, dried over anhydrous Na2SO4, filtered and concentrated to give [(2R,5R)-5-(benzyloxymethyl)-1,4-dioxan-2-yl]methanol (4.4 g , yield 41%) as a colorless oil. This material was used without further purification. ESI-MS (EI ⁺ , m/z): 239.1 [M+H] ⁺ .

Step 4: synthesis of ((2R,5R)-1,4-dioxane-2,5-diyl)dimethanol.

Pd/C (0.86 g) was added to a solution of [(2R,5R)-5-(benzyloxymethyl)-1,4-dioxan-2-yl]methanol (2.4 g, 10 mmol) in MeOH (30 ml) . The mixture was left to stir at room temperature overnight under a hydrogen cylinder, then filtered through a small pad of celite, rinsing with ethanol. The combined organic washes were concentrated under reduced pressure to give (2R,5R)-1,4-dioxane-2,5-diyl)dimethanol (1.2 g, 74% yield) as an oil. ESI-MS (EI ⁺ , m/z): 149.2 [M+H] ⁺ .

Step 5: synthesis of (23E,25E,27E,28E,32R,33S,34R,35R,36S,39S,41S,45R,46R,55R)-45,55-dihydroxy41-[(1R)-2-[(1S ,2R,3R)-3-hydroxy-2-methoxycyclohexyl]-1-methylethyl]-40-[[(2S,5S)-5-(hydroxymethyl)1,4-dioxan-2-yl]methoxy]-46- methoxy-32,33,34,35,47,48-hexamethyl-68,69-dioxa-56-azatricyclohexatriaconta-23,25,27(47),28(48)-tetraene-49,50,51,52, 53-pentone (I-118).

[(2R,5R)-5-(hydroxymethyl)-1,4 -dioxan-2-yl]methanol (0.81 g, 5.47 mmol) at 25°C. After stirring at room temperature for 2 hours, the reaction was quenched with cold aqueous NaHCO ₃ and extracted with EtOAc (50 ml x 2 ). The combined organic layers were then dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The residue was purified by reverse phase chromatography (C18, CH ₃ CN:H ₂ O=65:35) and then by silica gel chromatography (hexane:DCM:EtOAc:MeOH=8:8:3:1), in resulting in (23E,25E,27E,28E,32R,33S,34R,35R,36S,39S,41S,45R,46R,55R)-45,55-dihydroxy-41-[(1R)-2-[( 1S,2R,3R)3-hydroxy-2-methoxycyclohexyl]-1-methylethyl]-40-[[(2S,5S)-5-(hydroxymethyl)-1,4-dioxan-2-yl]methoxy]-46 -methoxy-32,33,34,35,47,48-hexamethyl-68,69-dioxa-56-azatricyclohexatriaconta-23,25, 27(47),28(48)-tetraene-49,50,51,52 .53-pentone (I-118: 80 mg, 14% yield) as a white solid. ESI-MS (EI ⁺ , m/z): 1052.9 [M+Na] ⁺ . ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.46 - 5.86 (m, 4H), 5.69 - 5.06 (m, 4H), 4.16 (ddd, J=56.1, 52 ,0, 39.5 Hz, 3H), 3.94 - 3.49 (m, 10H), 3.48 -3.11 (m, 12H), 3.08 - 2.46 (m, 7H), 2.40 1.93 (m, 7H), 1.73 (dd, J=16.9, 10.5 Hz, 13H), 1.52 - 1.17 (m, 8H), 1.16 - 0 .79 (m, 18H), 0.65 (d, J=17.5 Hz, 1H).

Example 21. Synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-(2- hydroxyethoxy)ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36 ,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconte

- 115 045993

21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-116) and (21E,23E,25E,26E,34R,35S,36R,37R, 39S,41S, 43S,44S,45R,46R,55R)-45,55-gurugpoKCu-43-[2-(2-rugpoKC№TOKCuLTOKCu]-44-[(1R)-2-[(1S,3R,4R )-4-(2hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67dioxa-56-azatricyclohexatriaconta-21,23,25( 47),26(48)-tetraene-49,50,51,52,53-pentone (I-117)

Synthesis circuit

Methods and characteristics.

Step 1: synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy43-[2-(2-hydroxyethoxy)ethoxy] -44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1methylethyl]-46-methoxy-34,35,36,37,47,48 -hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23, 25(47),26(48)-tetraene-49,50,51,52,53-pentone.

A solution of everolimus (1 g, 1.04 mmol) in THF (15 ml) was degassed with N2. Ptoluenesulfonic acid (0.895 g, 5.20 mmol) was added at 0°C, then 2-(2-hydroxyethoxy)ethanol (2.8 ml) was added. The resulting mixture was stirred at 0°C for 0.5 h in an N2 atmosphere, then at 25°C while

- 116 045993 duration 3 hours. The reaction mixture was poured into saturated NaHCO ₃ solution (40 ml), extracted with EtOAc (30 ml), washed with water (30 mlx2) and saline (40 ml), then dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The resulting residue was purified by normal phase chromatography on silica gel (MeOH: DCM=1:15) followed by reverse phase chromatography (C18, CH ₃ CN:H ₂ c)=7:3 to obtain (21E ,23E,25E,26E,34R,35S,36R,37R,39S,41S, 44S,45R,46R,55R)-45,55-dihydroxy-43-[2-(2-hydroxyethoxy)ethoxy]-44-[ (1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl -66,67-dioxa56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (0.43 g, 40% yield) as a solid light yellow color. ESI-MS (EI ⁺ , m/z): 1053.9 [M+Na] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.47 - 5.85 (m, 4H), 5.40 (ddd, J=99.7, 51.9, 29.1 Hz, 4H), 4, 80 (d, J=22.0 Hz, 1H), 4.23 (d, J=42.5 Hz, 1H), 4.05 - 3.54 (m, 13H), 3.52 - 3.01 (m, 14H), 2.67 (ddd, J=46.8, 27.3, 6.8 Hz, 4H), 2.17 (dd, J=82.0, 45.9 Hz, 6H), 1.70 (dt, J=21.0, 15.8 Hz, 12H), 1.34 (dd, J=105.2, 26.3 Hz, 11H), 1.15 -0.79 (m, 18H), 0.76 - 0.64 (m, 1H).

Step 2: synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55dihydroxy-43-[2-(2-hydroxyethoxy) ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37 ,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-116) and (21E,23E,25E,26E,34R,35S,36R,37R, 39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-(2-hydroxyethoxy)ethoxy] -44-[(1R)-2[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47, 48hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-117):

mg (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-(2hydroxyethoxy)ethoxy]-44- [(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48- hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone was purified by preparative chiral HPLC to obtain epimers : (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S, 43R,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-(2-hydroxyethoxy)ethoxy ]-44-[(1R)-2-[(1S,3R,4R)-4-(2hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47, 48-hexamethyl-66,67dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-116: 14 mg, 16% yield ) and (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-43-[2(2-hydroxyethoxy)ethoxy ]-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37, 47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47), 26(48)-tetraene-49,50,51,52,53-pentone (I-117: 15 mg , yield 17%), both as a white solid.

The chiral separation method is presented below

Column:

Column size:

Solution concentration: Introduction:

Mobile phase:

Flow rate:

Wavelength:

Temperature:

CHIRALPAK IC

5.0 cm I.D. x 25 cm L

11.5 mg/ml ml

Hexane/EUN=5 0/50(v/v) ml/min

UV 254 nm °C

I-116: ESI-MS (EI ⁺ , m/z): 1054.0 [M+Na] ⁺ . ¹ H NMR (500 MHz, CDCl3) δ 6.42 - 5.90 (m, 4H), 5.79 (ddd, J=51.6, 30.9, 16.6 Hz, 1H), 5.54 - 5.08 (m, 4H), 5.03 - 4.88 (m, 1H), 4.74 (d, J=61.6 Hz, 1H), 4.28 (dd, J=57.6 , 29.0 Hz, 2H), 3.99 (dd, J=26.5, 6.0 Hz, 1H), 3.89 - 3.55 (m, 12H), 3.54 - 2.96 ( m, 15H), 2.87 - 2.47 (m, 4H), 2.38 - 1.92 (m, 8H), 1.86-1.67 (m, 11H), 1.51 - 1, 30 (m, 6H), 1.14-0.80 (m, 18H), 0.76 - 0.64 (m, 1H). I-117: ESI-MS (EI ⁺ , m/z): 1053.9 [M+Na] ⁺ . ¹ H NMR (500 MHz, CDCl3) δ 6.41 - 6.22 (m, 2H), 6.13 (dd, J=15.1, 10.1 Hz, 1H), 5.94 (dd, J =22.3, 10.8 Hz, 1H), 5.52 (dt, J=18.4, 9.2 Hz, 1H), 5.41 (d, J=9.9 Hz, 1H), 5 .27 (d, J=5.3 Hz, 1H), 5.12 (dt, J=46.3, 5.6 Hz, 1H), 4.83 (s, 1H), 4.23-4, 14 (m, 1H), 3.91 - 3.52 (m, 15H), 3.49 - 3.25 (m, 12H),

3.23 - 3.03 (m, 3H), 2.94 - 2.80 (m, 1H), 2.65 (ddd, J=23.4, 16.9, 6.0 Hz, 3H), 2.39 - 2.15 (m, 2H), 2.16 - 1.85 (m, 5H), 1.82 - 1.64 (m, 10H), 1.47 (dd, J=26.8 , 15.9 Hz, 5H), 1.38 - 1.16 (m, 6H), 1.10 (d, J=6.8 Hz, 3H), 1.07 1.03 (m, 3H), 1.00 (t, J=6.8 Hz, 3H), 0.94 (d, J=6.6 Hz, 3H), 0.88 (ddd, J=34.0, 16.9, 5, 0 Hz, 6H), 0.71 (dd, J=23.9, 11.8 Hz, 1H).

Example 22. Synthesis of (21E,23E,25E,26E,38R,39S,40R,41R,43S,45S,47R,48S,49R,50R,59R)-49,59-dihydroxy-47-[2-[2- [2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-48-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)3-methoxycyclohexyl]-1-methylethyl]- 50-methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55, 56,57-pentone (I-114) and (21E,23E,25E,26E,38R, 39S,40R,41R,43S,45S,47S,48S,49R,50R,59R)-49,59-dihydroxy-47 -[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-48-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3- methoxycyclohexyl]-1-methylethyl]-50-methoxy-3 8,39,40,41,51,52-hexamethyl-7 0.71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52 )-tetraene53,54,55,56,57-pentone (I-115):

- 117 045993

1-114

1-115

Synthesis circuit

Methods and characteristics.

Step 1: synthesis of (21E,23E,25E,26E,38R,39S,40R,41R,43S,45S,48S,49R,50R,59R)-49,59-dihydroxy47-[2-[2-[2-( 2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-48-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-50-methoxy -38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56,57 -pentone: A mixture of everolimus (1 g, 1.04 mmol), 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethanol (4.05 g, 20.87 mmol) and p-toluenesulfonic acid ( 0.898 g, 5.22 mmol) in THF (20 ml) was stirred at 20°C for 2 hours. The mixture was then poured into cold saturated NaHCO ₃ solution (30 ml), extracted with EtOAc (50 ml x 3) and combined organic the layers were washed with water and brine, then dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The resulting residue was purified by reverse phase chromatography (C18, CH3CN:H2O=6:4), resulting in (21E,23E,25E,26E,38R,39S,40R, 41R,43S,45S,48S,49R, 50R,59R)-49,59-dihydroxy-47-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]48-[(1R)-2-[(1S,3R,4R) -4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-50-methoxy38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25 (51),26(52)-tetraene 53,54,55,56,57-pentone (0.25 g, 21% yield) as a white solid. ESI-MS (EI ⁺ , m/z): 1142.4 [M+Na] ⁺ . ^1H NMR (400 MHz, CDCl ₃ ) δ 6.41 - 5.85 (m, 4H), 5.78 (s, 1H), 5.60 - 4.98 (m, 4H), 4.22 ( t, J=27.1 Hz, 1H), 3.97 (dd, J=17.7, 6.4 Hz, 1H), 3.87 - 3.54 (m, 21H), 3.51 - 3 .03 (m, 15H), 2.72 - 2.45 (m, 3H), 2.28 (s, 6H), 2.16 - 1.96 (m, 4H), 1.89 - 1.56 (m, 10H), 1.51 - 1.17 (m, 8H), 1.17 - 0.81 (m, 18H), 0.78 - 0.62 (m, 1H).

Step 2: synthesis of (21E,23E,25E,26E,38R,39S,40R,41R,43S,45S,47R,48S,49R,50R,59R)-49,59dihydroxy-47-[2-[2-[2 -(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-48-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-50 -methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56,57 -pentone (I-114) and (21E,23E,25E, 26E,38R,39S,40R,41R,43S,45S,47S,48S,49R,50R,59R)-49,59-dihydroxy-47-[2 -[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-48-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]- 1-methylethyl]-50-methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51), 26(52)-tetraene-53 ,54,55,56,57-pentons (I-115):

1.5 g (21Ε,23Ε,25Ε,26Ε^^,40^4^^^^^^^)-49.59^^ροκ^-47-[2-[2-[2-(2hydroxyethoxy)ethoxy ]ethoxy]ethoxy]-48-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-50-methoxy-38.39, 40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta21,23,25(51),26(52)-tetraene-53,54,55,56,57-pentone was purified using preparative chiral

- 118 045993

HPLC and the resulting epimers were purified by silica gel chromatography (hexan:DCM:EULe:MeOH=8:8:3:1.2), resulting in (21E,23E,25E,26E,38R,39S,40R, 41R,

43S,45S,47R,48S,49R,50R,59R)-49,59-dihydroxy-47-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]48-[(1R)- 2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-50-methoxy

38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene

53,54,55,56,57-pentone (I-114: 300 mg, 20% yield) and (21E,23E,25E,26E,38R,39S,40R,41R,43S,45S,

47S,48S,49R,50R,59R)-49,59-dihydroxy-47-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-48-[(1R)-2

[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-50-methoxy-38,39,40,41,51,52hexamethyl-70,71-dioxa-60 -azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56,57pentone (I-115: 563 mg, 38% yield) as a white solid.

Conditions for chiral analysis

Column:

Column size:

Introduction:

Mobile phase:

Flow rate:

Wavelength:

Temperature:

HPLC equipment:

I-114: ESI-MS (EI ⁺ , m/z): 1142.5 [M+Na] ⁺ .

CHIRALPAK IC-3(IC30CE-NJ008)

0.46 cm I.D. χ 15 cm L

20.0 µl

Hexane/EUN=60/40(v/v)

1.0 ml/min

UV 254 nm °C

Shimadzu LC-20AT

1H NMR (500 MHz, CDCI3) δ 6.42 - 5.91 (m, 4H), 5.60

5.08 (m, 4H), 4.19 (dd, J=43.8, 32.6 Hz, 1H), 3.96 (dd, J=27.1, 6.3 Hz, 1H), 3 .85 - 3.54 (m, 21H), 3.53 - 3.01 (m, 12H), 2.93 - 2.80 (m, 1H), 2.75 - 2.45 (m, 3H) , 2.30 (d, J=12.1 Hz, 1H), 2.03 (dd, J=37.0, 32.8 Hz, 13H), 1.84 1.69 (m, 12H), 1 .50 - 1.18 (m, 5H), 1.16 - 0.82 (m, 18H), 0.71 (dt, J=23.8, 12.1 Hz, 1H).

I-115: ESI-MS (EI ⁺ , m/z): 1142.4 [M+Na] ⁺ . 1H NMR (500 MHz, CDCI3) δ 6.36 (dd, J=14.7, 10.9 Hz, 1H), 6.23 (dd, J=14.7, 10.6 Hz, 1H), 6 ,10 (dd, J=15.0, 10.5 Hz, 1H), 5.92 (dd, J=45.2, 10.5 Hz, 1H), 5.61 (s, 1H), 5, 43 (dd, J=15.6, 9.6 Hz, 2H), 5.21 (d, J=5.4 Hz, 1H), 5.10 (dd, J=9.9, 5.9 Hz , 1H), 4.20 (d, J=4.5 Hz, 1H), 4.10 3.95 (m, 1H), 3.84 (d, J=5.0 Hz, 1H), 3, 80-3.49 (m, 21H), 3.48-3.13 (m, 12H), 3.11 - 3.01 (m, 1H), 2.73-2.50 (m, 3H), 2.34 - 2.19 (m, 2H), 2.01 (ddd, J=62.0, 34.6, 28.2 Hz, 12H), 1.80 - 1.54 (m, 10H), 1.51 - 1.37 (m, 5H), 1.35 - 1.12 (m, 6H), 1.05 (dd, J=6.4, 5.0 Hz, 6H), 0.97 ( d, J=6.5 Hz, 3H), 0.93 (d, J=6.6 Hz, 3H), 0.91 - 0.82 (m, 6H),

0.74 - 0.67 (m, 1H).

Example 23. Synthesis of (21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[ (1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-42-(5-hydroxypentoxy)-45methoxy-33,34,35,36,46,47-hexamethyl-65,66- dioxa-55-azatricyclohexatriaconta-21,23,25(46),26(47)tetraene-48,49,50,51,52-pentone (I-113)

1-113

Synthesis circuit

He

Methods and characteristics.

Step 1: synthesis of (21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy43 - [(1R)-2-[( 1S ,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-42-(5-hydroxypentoxy)-45

- 119 045993 methoxy-33,34,35,36,46,47-hexamethyl-65,66-dioxa-55-azatricyclohexatriaconta-21,23,25(46),26(47)tetraene-48,49,50, 51,52-pentone (I-113).

To a solution of rapamycin (0.5 g, 0.547 mmol) in THF (10 ml) was added 4-methylbenzenesulfonic acid hydrate (0.52 g, 2.73 mmol) and pentane-1,5-diol (3 ml). The resulting solution was stirred at room temperature for 2 hours, then poured into cold aqueous NaHCO ₃ and extracted with EtOAc. The organic layer was concentrated and purified by reverse phase chromatography (C18, CH ₃ CN:H ₂ O yield 10% to 72%) to give (21E,23E,25E,26E,33R,34S,35R,36R ,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)4-hydroxy-3-methoxycyclohexyl]-1-methylethyl] -42-(5-hydroxypentoxy)-45-methoxy-33,34,35,36,46,47hexamethyl-65,66-dioxa-55-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene -48,49,50,51,52-pentone (I-113: 150 mg, 28% yield) as a white solid. ESI-MS (EI ⁺ , m/z): 1008.0 [M+Na] ⁺ . 1HNMR (500 MHz, CDCl ₃ ) δ 6.42 - 5.82 (m, 4H), 5.58 - 5.37 (m, 2H), 5.32 -5.02 (m, 2H), 4, 78 (t, J=25.9 Hz, 1H), 4.31 - 4.08 (m, 1H), 4.00 - 3.53 (m, 5H), 3.53 - 3.05 (m, 12H), 2.99 - 2.80 (m, 2H), 2.77 - 2.51 (m, 3H), 2.48 - 2.23 (m, 2H), 2.15 - 1.89 ( m, 4H), 1.89 - 1.16 (m, 32H), 1.15 - 0.78 (m, 18H), 0.65 (dt, J=24.1, 12.0 Hz, 1H) .

Example 24. Synthesis of (28E,30E,32E,33E,36R,37S,38R,39R,41S,43S,45R,46S,48R,49R,58R)-48,58dihydroxy-46-[(1R)-2-[ (1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-49-methoxy-36,37, 38,39,50,51-hexamethyl-45-(1,4,7,10 -tetraoxacyclododec-2-ylmethoxy)-72,73-dioxa-59-azatricyclohexatriaconta-28,30,32(50),33(51)-tetraene-52,53,54,55,56-pentone (I-111) and (28E,30E,32E,33E,

36R,37S,38R,39R,41S,43S,45S,46S,48R,49R,58R)-48,58-dihydroxy-46-[(1R)-2-[(1S,3R,4R)-4-hydroxy -3methoxycyclohexyl]-1-methylethyl]-49-methoxy-36,37,38,39,50,51-hexamethyl-45-(1,4,7,10-tetraoxacyclododec-2-ylmethoxy)-72,73-dioxa -59-azatricyclohexatriaconta-28,30,32(50),33(51)-tetraene52,53,54,55,56-pentone (I-112):

Chiral

Scheme for the synthesis of p-TsOH H ₂ O, THF

25°C, 2 hours

- 120 045993

Methods and characteristics.

Step 1: synthesis of (28B,30B,32B,33B,36R,37S,38R,39R,41S,43S,46S,48R,49R,58R)-48,58-dihydroxy46-[(1R)-2-[(1S ,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-49-methoxy-36,37,38,39,50,51hexamethyl-45-(1,4,7,10-tetraoxacyclododec-2 -ylmethoxy)-72,73-dioxa-59-azatricyclohexatriaconta28,30,32(50),33(51)-tetraene-52,53,54,55,56-pentone: To rapamycin solution (0.5 g, 0.547 mmol) and methylbenzenesulfonic acid 4-hydrate (0.471 g, 2.73 mmol) in THF (15 ml) was added 1,4,7,10tetraoxacyclododec-2-ylmethanol (2.25 g, 10.9 mmol) at 25°C. The resulting mixture was stirred at room temperature for 2 hours, then poured into ice-cold aqueous NaHCO ₃ and extracted with EtOAc. The organic layer was then dried over anhydrous Na ₂ SO _4; filtered and concentrated. The resulting residue was purified by reverse phase chromatography (C18, CH ₃ CN:H ₂ O=7:3), resulting in (28E,30E,32E,33E,36R,37S,38R,39R,41S,

43S,46S,48R,49R,58R)-48,58-dihydroxy-46-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1methylethyl]-49-methoxy -36,37,38,39,50,51-hexamethyl-45-(1,4,7,10-tetraoxacyclododec-2-ylmethoxy)72,73-dioxa-59-azatricyclohexatriaconta-28,30,32(50) ,33(51)-tetraene-52,53,54,55,56-pentone (70 mg, 11% yield) as a white solid. ESI-MS (EI ⁺ , m/z): 1110.5 [M+Na] ⁺ . 1H NMR (500 MHz, CDC13) δ 6.41 - 5.82 (m, 4H), 5.54 - 5.04 (m, 4H), 4.72 (d, J=22.1 Hz, 1H) , 4.35 - 4.09 (m, 1H), 3.92 3.51 (m, 18H), 3.48 - 3.03 (m, 14H), 2.99 - 2.51 (m, 5H ), 2.34 (d, J=13.4 Hz, 1H), 2.04 (d, J=62.6 Hz, 4H), 1.72 (ddd, J=43.8, 30.6, 28.9 Hz, 12H), 1.53 - 1.17 (m, 10H), 1.14 - 0.81 (m, 18H), 0.70 - 0.62 (m, 1H).

Step 2: synthesis of (28B,30B,32B,33B,36R,37S,38R,39R,41S,43S,45S,46S,48R,49R,58R)-48,58-dihydroxy-46-[(1R)-2 -[(1 S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-49-methoxy-3 6,37,38, 39,50,51-hexamethyl-45-(1,4 ,7,10-tetraoxacyclododec-2-ylmethoxy)-72,73-dioxa-59-azatricyclohexatriaconta-28,30,32(50),33(51)-tetraene-52,53,54,55,56-pentone ( I-112) and (28E,30E,32E,33E,36R,

37S,38R,39R,41S,43S,45R,46S,48R,49R,58R)-48,58-dihydroxy-46-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3methoxycyclohexyl ]-1-methylethyl]-49-methoxy-36,37,38,39,50,51-hexamethyl-45-(1,4,7,10-tetraoxacyclododec-2-ylmethoxy)-72,73-dioxa-59 -azatricyclohexatriaconta-28,30,32(50),33(51)-tetraene52,53,54,55,56-pentone (I-111):

(28B,30B,32B,33B,36R,37S,38R,39R,41S,43S,46S,48R,49R,58R)-48,58-dihydroxy-46-[(1R)-2[(1S,3R, 4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-49-methoxy-36,37,38,39,50,51-hexamethyl45-(1,4,7,10-tetraoxacyclododec-2-ylmethoxy) -72,73-dioxa-59-azatricyclohexatriaconta28,30,32(50),33(51)-tetraene-52,53,54,55,56-pentone (170 mg) was purified by preparative chiral HPLC and the resulting epimers were purified using silica gel chromatography (hexane:DCM:VULs:MeOH=3:3:1:0.5), resulting in (28E,30E,32E,33E,36R,37S,38R,39R,41S, 43S, 45S,46S,48R,49R,58R)-48,58-dihydroxy-46-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]1-methylethyl]-49- methoxy-36,37,38,39,50,51-hexamethyl-45-(1,4,7,10-tetraoxacyclododec-2-ylmethoxy)72,73-dioxa-59-azatricyclohexatriaconta-28,30,32(50 ),33(51)-tetraene-52,53,54,55,56-pentone (I-112: 55 mg, 32% yield) and (28B,30B,32B,33B,36R,37S,38R,39R, 41S,43S,45R,46S,48R,49R,58R)-48,58-dihydroxy46-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl] -49-methoxy-36,37,38,39,50,51hexamethyl-45-(1,4,7,10-tetraoxacyclododec-2-ylmethoxy)-72,73-dioxa-59-azatricyclohexatriaconta28,30,32(50 ),33(51)-tetraene-52,53,54,55,56-pentone (I-111: 11 mg, 6% yield), both as a white solid.

Chiral analysis method

Column

Column Size Introduction

Mobile phase Flow rate Wavelength Temperature HPLC equipment

CHIRALPAK IC(IC00CE-OL002) 0.46 cm I.D. x 25 cm L 100.0 µl

Hexane/EUN=60/40 (v/v) 1.0 ml/min

UV 254 nm °C

Shimadzu LC-20AT CP-HPLC-06

I-112: ESI-MS (EI ⁺ , m/z): 1109.9 [M+Na] ⁺ . 1H NMR (500 MHz, CDCl ₃ ) δ 6.40 - 6.03 (m, 3H), 5.90 (dd, J=37.1, 9.8 Hz, 1H), 5.54 - 5.06 (m, 4H), 4.71 (d, J=22.4 Hz, 1H), 4.20 (t, J=20.9 Hz, 1H), 3.93 - 3.52 (m, 17H) , 3.50 - 3.24 (m, 11H), 3.14 - 3.02 (m, 1H), 2.93 (dt, J=31.2, 12.0 Hz, 1H), 2.63 (tdd, J=17.0, 14.2, 5.5 Hz, 3H), 2.37-2.18 (m, 2H), 1.94 (ddd, J=31.3, 24.3, 22.1 Hz, 5H), 1.71 (dt, J=22.5, 10.1 Hz, 11H), 1.51-1.17 (m, 13h), 1.15-0.81 (m , 18H), 0.67 (dd, J=23.7, 11.9 Hz, 1H).

I-111: 1H NMR (500 MHz, CDCl ₃ ) δ 6.46 - 5.78 (m, 4H), 5.75 - 5.14 (m, 4H), 4.58 (d, J=31, 9 Hz, 1H), 4.21 (d, J=66.8 Hz, 1H), 3.87 - 3.47 (m, 16H), 3.43 - 3.14 (m, 10H), 2, 94 (s, 1H), 2.80 - 2.54 (m, 3H), 2.26 (ddd, J=110.3, 80.5, 42.6 Hz, 6H), 1.81 - 1, 49 (m, 18H), 1.46 - 1.25 (m, 10H), 1.18 - 0.77 (m, 18H), 0.72 - 0.61 (m, 1H).

Example 25. Synthesis.

(21B,23B,25B,26B,36R,37S,38R,39R,41S,43S,45R,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-[2-(2hydroxyethoxy)ethoxy ]ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1methylethyl]-48-methoxy-36,37,38,39, 49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta- 121 045993

21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone (I-109) and (21E,23E,25E,26E,36R,37S,38R,39R,

41S.43S.45S.46S.47R.48R.57R)-47.57-giirngpoi<cn-45-[2-[2-(2-rngpoi<cinTOi<cn)jTOi<cii|'Hoi<cii|-46- [(1R)-2[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-48-methoxy-36,37,38,39,49,50hexamethyl-68 ,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55pentone (I-110)

Synthesis scheme

Methods and characteristics.

Stage 1: synthesis (21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy45-[2-[2-(2-hydroxyethoxy )ethoxy]ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-48-methoxy-36.37, 38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49), 26(50)-tetraene-51,52,53,54,55-pentone.

Everolimus (0.5 g, 0.522 mmol), 2-[2-(2-hydroxyethoxy)ethoxy]ethanol (3.92 g, 26.09 mmol) and p-toluenesulfonic acid monohydrate (0.45 g, 2 61 mmol) in THF (10 ml) was stirred at 20°C for 2 hours. The solution was then poured into ice-cold NaHCO ₃ solution (30 ml) and extracted with EtOAc (50 ml x 3). The combined organic c.igii was washed with water and spruce solution, and then dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The resulting residue was purified by reverse phase chromatography (C18, CH ₃ CN:H ₂ O=6.5:3.5) to give (21E,23E,25E,26E,36R,37S,38R, 39R,41S,

43S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-46-[(1R)-2[(1S,3R,4R )-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21, 23,25(49),26(50)-tetraene-51,52,53,54,55-pentone (0.155 g, 28%) as a white solid. ESI-MS (EI ⁺ , m/z): 1098.4 [M+Na] ⁺ . 1H NMR (500 MHz, CDC13) δ 6.42 - 5.87 (m, 4H), 5.63 - 5.34 (m, 2H), 5.32 - 5.00 (m, 2H), 4, 85 (s, 1H), 4.35 - 4.09 (m, 1H),

- 122 045993

4.05 - 3.49 (m, 18H), 3.49 - 3.01 (m, 14H), 2.66 (dddd, J=31.3, 24.8, 21.2, 13.0 Hz , 4H), 2.33 (d, J=12.0 Hz, 2H), 2.06 (dd, J=39.9, 10.6 Hz, 3H), 1.77 - 1.53 (m, 13H), 1.51 - 1.14 (m, 10H), 1.14 - 0.81 (m, 18H), 0.71 (dd, J=23.8, 11.9 Hz, 1H).

Stage 2: synthesis (21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,45S,46S,47R,48R,57R)-47.57dii1dro1<ci-45-[2-[2- (2-and1dro1<sieto1<si)is1<si]is1<si]-46-[(11<)-2-[(^,31<,41<)-4-(2-hydro1<sieto1<si) -3methoxycyclohexyl]-1-methylethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50) -tetraene-51,52,53,54,55-pentone (I-110) and (21E,23E,25E,26E,36R, 37S,38R,39R,41S,43S,45R,46S,47R,48R,57R )-47,57-dihydroxy-45-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy )-3-methoxycyclohexyl]-1-methylethyl]-48-methoxy36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50 )-tetraene51,52,53,54,55-pentone (I-109):

170 mg (21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-[2-(2hydroxyethoxy)ethoxy ]ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1methylethyl]-48-methoxy-36,37,38,39, 49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone was purified by preparative chiral HPLC and the resulting epimers purified by silica gel chromatography (hexane:DCM:EtOAc:MeOH=3:3:1:0.8) to give (21E,23E,25E,26E,36R,37S,38R,39R, 41S,43S ,45S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-46-[(1R)-2[(1S,3R, 4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-48-methoxy-36,37,38,39,49,50hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23 ,25(49),26(50)-tetraene-51,52,53,54,55pentone (I-110: 37 mg, 21% yield) and (21E,23E,25E,26E,36R,37S,38R, 39R,41S,43S,45R,46S,47R,48R,57R)47,57-dihydroxy-45-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-46-[(1R)-2-[ (1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa- 58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone (I-109: 33 mg, 19% yield), both as white solid colors.

Chiral analysis method

Column

Column size Introduction Mobile phase Flow rate Wavelength Temperature

CHIRALPAK IC(IC00CE-OL002) 0.46 cm I.D. x 25 cm L 30.0 µl

Hexane/EUN=60/40(v/v)

1.0 ml/min

UV 254 nm oC

HPLC Equipment

Shimadzu LC-20AT CP-HPLC-06

I-110: ESI-MS (EI+, m/z): 1098.0 [M+Na]+. ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.41 - 6.18 (m, 2H), 6.12 (dd, J=15.0, 10.3 Hz, 1H), 5.93 (dd, J=33.7, 10.6 Hz, 1H), 5.47 (ddd, J=33.0, 20.8, 9.5 Hz, 2H), 5.26 (d, J=5.6 Hz , 1H), 5.13 (dt, J=21.8, 10.8 Hz, 1H), 4.88 (s, 1H), 4.19 (t, J=9.3 Hz, 1H), 3 .94 - 3.52 (m, 19H), 3.49 - 3.25 (m, 12H), 3.24 - 3.02 (m, 3H), 2.76 (ddd, J=26.2, 16.6, 10.3 Hz, 3H), 2.57 (dd, J=17.0, 6.3 Hz, 1H), 2.29 (t, J=26.2 Hz, 2H), 2, 16 - 1.85 (m, 6H), 1.74 - 1.53 (m, 10H), 1.53 - 1.16 (m, 9H), 1.15 - 1.01 (m, 8H), 1.01 - 0.82 (m, 10H), 0.71 (dd, J=23.9, 12.0 Hz, 1H).

I-109: ESI-MS (EI ⁺ , m/z): 1098.0 [M+Na] ⁺ . 1H NMR (500 MHz, CDCl ₃ ) δ 6.43 - 5.90 (m, 4H), 5.62 5.02 (m, 5H), 4.24 (d, J=63.6 Hz, 1H) , 3.97 (dd, J=21.5, 6.8 Hz, 1H), 3.86 - 3.50 (m, 18H), 3.45 - 3.01 (m, 14H), 2.73 - 2.46 (m, 3H), 2.39 - 1.94 (m, 6H), 1.91-1.69 (m, 10H), 1.50-1.31 (m, 12H), 1 .16-0.85 (m, 18H), 0.69 (d, J=11.7 Hz, 1H).

Example 26. Synthesis of (30E,32E,34E,35E,38R,39S,40R,41R,43S,45S,47R,48S,50R,51R,60R)-50,60-dihydroxy-48-[(1R)-2 -[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-51-methoxy-38,39,40,41, 52,53 -hexamethyl-47-( 1,4,7 ,10,13 -pentaoxacyclopentadec-2-ylmethoxy)-7 5,76-dioxa-61-azatricyclohexatriaconta-30,32,34(52),35(53)-tetraene-54,55,56,57,58-pentone (I-107): (30E,32E,34E,35E,38R,39S, 40R,41R,43S,45S,47S,48S,50R,51R,60R)-50,60-dihydroxy-48-[(1R) -2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-51-methoxy-38,39,40,41,52,53-hexamethyl-47-(1,4 ,7,10,13-pentaoxacyclopentadec-2-ylmethoxy)-75,76-dioxa-61-azatricyclohexatriaconta-30,32,34(52),35(53)-tetraene54,55,56,57,58-pentone ( I-108)

- 123 045993

Synthesis circuit

Methods and characteristics.

Step 1: synthesis of (30E,32E,34E,35E,38R,39S,40R,41R,43S,45S,48S,50R,51R,60R)-50,60-dihydroxy48-[(1R)-2-[(1S ,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-51-methoxy-38,39,40,41,52,53hexamethyl-47-(1,4,7,10,13-pentaoxacyclopentadec -2-ylmethoxy)-75,76-dioxa-61-azatricyclohexatriaconta-30,32,34(52),35(53)-tetraene-54,55,56,57,58-pentone.

A solution of rapamycin (2 g, 2.19 mmol), 1,4,7,10,13-pentaoxacyclopentadec-2-ylmethanol (3.83 g, 15.31 mmol) and p-toluenesulfonic acid monohydrate (1.88 g, 10.94 mmol) in THF (10 ml) was stirred at 20°C for 2 hours. The mixture was then poured into ice-cold saturated NaHCO ₃ solution (50 ml) and extracted with EtOAc (100 ml x 3 ). The combined organic layers were washed with water and brine, then dried under vacuum and the resulting residue was purified using reverse phase chromatography (C18, CH ₃ CN:H ₂ O=8:2), resulting in (30E,32E,34E, 35E,38R,39S,40R,41R,43S,45S,48S,50R,51R,60R)-50,60-dihydroxy-48-[(1R)-2-[(1S,3R,4R)4-hydroxy- 3-methoxycyclohexyl] -1 -methylethyl] -51 -methoxy-3 8,39,40,41,52,53-hexamethyl-4 7 (1,4,7,10,13-pentaoxacyclopentadec-2-ylmethoxy)-75 ,76-dioxa-61-azatricyclohexatriaconta30,32,34(52),35(53)-tetraene-54,55,56,57,58-pentone (80 mg, 2.6% yield) as a white solid . ESI-MS (EI ⁺ , m/z): 1154.0 [M+Na] ⁺ . ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.16 (tdt, J=40.0, 33.8, 20.7 Hz, 4H), 5.54 - 5.03 (m, 4H), 4, 21 (t, J=22.5 Hz, 1H), 3.90 - 3.46 (m, 22H), 3.44 -3.04 (m, 12H), 2.74 (dddd, J=27, 8, 22.2, 13.7, 4.7 Hz, 5H), 2.37 - 1.56 (m, 22H), 1.50 -1.16 (m, 8H), 1.13-0, 81 (m, 18H), 0.67 (dd, J=23.8, 11.9 Hz, 1H).

Step 2: synthesis of (30E,32E,34E,35E,38R,39S,40R,41R,43S,45S,47S,48S,50R,51R,60R)-50,60-dihydroxy-48-[(1R)-2 -[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-51-methoxy-38,39,40,41, 52,53-hexamethyl-47-(1,4,7 ,10,13-pentaoxacyclopentadec-2-ylmethoxy)-75,76-dioxa-61-azatricyclohexatriaconta-30,32,34(52),35(53)-tetraene-54,55,56,57,58-pentone ( I-108) and (30E,32E,34E,35E,

38R,39S,40R,41R,43S,45S,47R,48S,50R,51R,60R)-50,60-dihydroxy-48-[(1R)-2-[(1S,3R,4R)-4-hydroxy -3

- 124 045993 methoxycyclohexyl]-1-methylethyl]-51-methoxy-38,39,40,41,52,53-hexamethyl-47-(1,4,7,10,13pentaoxacyclopentadec-2-ylmethoxy)-75,76 -dioxa-61-azatricyclohexatriaconta-30,32,34(52),35(53)tetraene-54,55,56,57,58-pentone (I-107):

130 mg (30Ε,32Ε,34Ε,35Ε,38Κ,398,40Κ,41Κ,438,458,488,50Κ,51Κ^)-50.60- _No. β^ροκ^-48-[(^)-2[(1S,3R ,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-51-methoxy-38,39,40,41,52,53-hexamethyl47-(1,4,7,10,13-pentaoxacyclopentadec-2 -ylmethoxy)-7 5,76-dioxa-61-azatricyclohexatriaconta30,32,34(52),35(53)-tetraene-54,55,56,57,58-pentone was purified by preparative chiral HPLC and the resulting epimers were purified using silica gel chromatography (hexane:DCM:EtOAc:MeOH=3:3:1:0.5), resulting in (30E,32E,34E,35E,38R,39S, 40R,41R,43S, 45S, 47S,48S,50R,51R,60R)-50,60-dihydroxy-48-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]1-methylethyl]-51- methoxy-38,39,40,41,52,53-hexamethyl-47-(1,4,7,10,13-pentaoxacyclopentadec-2ylmethoxy)-75,76-dioxa-61-azatricyclohexatriaconta-30,32,34( 52),35(53)-tetraene-54,55,56,57,58pentone (I-108: 18 mg, 13% yield) and (30E,32E,34E,35E,38R,39S,40R,41R,43S ,45S,47R,48S,50R,51R,60R)50,60-dihydroxy-48-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl] -51-methoxy38,39,40,41,52,53-hexamethyl-47-(1,4,7,10,13-pentaoxacyclopentadec-2-ylmethoxy)-75,76-dioxa-61azatricyclohexatriaconta-30,32,34 (52),35(53)-tetraene-54,55,56,57,58-pentone (I-107: 16 mg, 12% yield), both as white solids. Chiral analysis method

Column

Column size Introduction Mobile phase Flow rate Wavelength Temperature

CHIRALPAK IC(IC00CE-OL002) 0.46 cm I D. χ 25 cm L 100.0 µl

Hexane/EUN=60/40(v/v)

1.0 ml/min

UV 254 nm °C

HPLC equipment: Shimadzu LC-20AT CP-HPLC-06

I-108: ESI-MS (EI ⁺ , m/z): 1153.9 [M+Na] ⁺ . 1H NMR (500 MHz, CDCl ₃ ) δ 6.31 (dt, J=25.1, 14.5 Hz, 2H), 6.17 - 6.08 (m, 1H), 5.95 (s, 1H ), 5.55 - 5.37 (m, 2H), 5.26 (s, 1H), 5.16 (d, J=4.4 Hz, 1H), 4.71 (d, J=28, 0 Hz, 1H), 4.19 (s, 1H), 3.93 - 3.48 (m, 25H), 3.44 - 3.26 (m, 12H), 3.20 - 3.03 (m , 1H), 2.94 (dd, J=16.5, 7.7 Hz, 1H), 2.70 (dd, J=17.9, 12.1 Hz, 3H), 2.56 (d, J=17.0 Hz, 1H), 2.33 (d, J=12.0 Hz, 2H), 2.13 - 1.84 (m, 5H), 1.81 -1.67 (m, 8H ), 1.50 - 1.17 (m, 8H), 1.14-0.81 (m, 20H), 0.67 (dd, J=23.6, 11.9 Hz, 1H).

I-107: ESI-MS (EI ⁺ , m/z): 1153.9 [M+Na] ⁺ . ^1H NMR (500 MHz, CDCi ₃ ) δ 6.40-5.95 (m, 4H), 5.58-5.11 (m, 4H), 4.30 - 4.12 (m, 1H), 3.88-3.51 (m, 25H), 3.42-3.12 (m, 12H), 2.99 - 2.53 (m, 5H), 2.50 - 1.90 (m, 5H ), 1.83 - 1.65 (m, 14H), 1.44 - 1.30 (m, 8H), 1.12 - 0.76 (m, 18H), 0.74 - 0.62 (m , 1H).

Example 27: Synthesis of (21E,23E,25E,26E,31R,32S,33R,34R,36S,38S,41S,42R,43R,52R)-40-[bis(hydroxymethyl)phosphorylmethoxy]-42,52-dihydroxy- 41-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1methylethyl]-43-methoxy-31,32,33,34,44,45-hexamethyl-65, 66-dioxa-53-azatricyclohexatriaconta21,23,25(44),26(45)-tetraene-46,47,48,49,50-pentone (I-106)

Synthesis circuit

Ba(OH) ₂ , n ₂ o ₂

1-106

- 125 045993

Methods and characteristics.

Step 1: synthesis of bis(hydroxymethyl)phosphorylmethanol.

Ba(OH)2 (8.43 g, 49.23 mmol) was dissolved in 50 ml of distilled water at 60°C. Tetrakis(hydroxymethyl)phosphonium sulfate (20 g, 49.23 mmol) was added dropwise to the solution and this solution was then stirred at 60°C for 4 hours. BaSO ₄ was then removed by centrifugation and hydrogen peroxide (30 % solution, 98.45 mmol) and the resulting reaction mixture was stirred at room temperature for 5 hours. The mixture was washed with chloroform, then the desired product was obtained by removing water under reduced pressure (6.5 g, 94% yield) as an oil. IERMS (EI+, m/z): 141.1 [M+H]+. ¹ NMR (500 MHz, DMSO-06): δ 5.35 (bs, 3H), 3.97 (s, 6H).

Step 2: synthesis of (21E,23E,25E,26E,31R,32S,33R,34R,36S,38S,41S,42R,43R,52R)-40-[bis(hydroxymethyl)phosphorylmethoxy]-42,52-dihydroxy- 41-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1methylethyl]-43-methoxy-31,32,33,34,44,45-hexamethyl-65, 66-dioxa-53-azatricyclohexatriaconta21,23,25(44),26(45)-tetraene-46,47,48,49,50-pentone (I-106).

A mixture of rapamycin (0.5 g, 0.547 mmol), bis(hydroxymethyl)phosphorylmethanol (0.766 g, 5.47 mmol) and methylbenzenesulfonic acid 4-hydrate (0.52 g, 2.73 mmol) in THF (20 ml) was stirred at 25°C for 4 hours. EtOAc (100 ml) and water (50 ml) were added. The aqueous layer was extracted with EtOAc (100 mlx2) and the combined organic layers were washed with water (2x50 ml) and brine (50 ml), then dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The resulting residue was purified by reverse phase chromatography (C18, CH ₃ CN:H ₂ O=7:3), resulting in (21E,23E,25E,26E,31R,32S,33R,34R,36S,38S, 41S,42R,43R,52R)-40-[bis(hydroxymethyl)phosphorylmethoxy]-42,52-dihydroxy-41-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl ]-1methylethyl]-43-methoxy-31,32,33,34,44,45-hexamethyl-65,66-dioxa-53-azatricyclohexatriaconta21,23,25(44),26(45)-tetraene-46,47 ,48,49,50-pentone (I-106: 50 mg, 9% yield) as a white solid. ESI-MS (EI ⁺ , m/z): 1043.9 [M+Na]+. ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.47 - 5.91 (m, 4H), 5.58 5.08 (m, 4H), 4.51 - 3.63 (m, 12H), 3 .59 - 3.16 (m, 14H), 2.80 (d, J=138.1 Hz, 6H), 2.38-1.91 (m, 8H), 1.50 0.77 (m, 35H), 0.67 (d, J=9.0 Hz, 1H).

Example 28 Synthesis of (22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,44R,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2 -[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-44[2-(2-methoxyethoxy)ethoxy]-35,36,37, 38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone (I-104) and ( 22E,24E,26E,27E,35R,36S,37R,38R,40S,42S, 44S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S, 3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48, 49-hexamethyl-66,67dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone (I-105)

Synthesis circuit

- 126 045993

Methods and characteristics.

Step 1: Synthesis of (22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,45S,46R,47R,56R)-46,56-dihydroxy45-[(1R)-2-[( 1 S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-44-[2-(2methoxyethoxy)ethoxy]-35,36,37,38,48 ,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone:

A mixture of everolimus (5 g, 5.22 mmol) and 2-(2-methoxyethoxy)ethanol (15 ml) in THF (80 ml) was degassed with N2, then heated at 50°C. HND-8 (600 mg) was added and the resulting mixture was stirred at 50°C for 4 hours under N2, then filtered and diluted with EtOAc. After concentration, the residue was purified by reverse phase chromatography (C18, CH ₃ CN: H ₂ O yield 0% to 100%), resulting in (22E,24E,26E,27E,35R,36S,37R,38R, 40S,42S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]- 1-methylethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48,49-hexamethyl-66,67dioxa-57-azatricyclohexatriaconta-22,24,26( 48),27(49)-tetraene-50,51,52,53,54-pentone (1.5 g, 27% yield) as a white solid. ESI-MS (EI ⁺ , m/z): 1068.4 [M+Na] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.40 - 5.85 (m, 4H), 5.56 - 5.36 (m, 2H), 5.21 (ddd, J=16.0, 11 ,7, 5.6 Hz, 2H), 4.21 (dd, J=25.8, 15.5 Hz, 2H), 3.94 - 3.26 (m, 28H), 3.25 -3, 02 (m, 4H), 2.76 - 2.40 (m, 3H), 2.34 (d, J=13.3 Hz, 2H), 2.19 - 2.06 (m, 2H), 2 .01 - 1.67 (m, 13H), 1.54 - 1.30 (m, 7H), 1.15-0.81 (m, 18H), 0.72 (dd, J=23.1, 11.7 Hz, 1H).

Step 2: synthesis of (22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2 -[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-44[2-(2-methoxyethoxy)ethoxy]-35,36,37, 38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone (I-105) and ( 22E,24E,26E,27E,35R,36S,37R,38R,40S, 42S,44R,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S, 3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48, 49-hexamethyl-66,67dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone (I-104):

120 mg (22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2[(1S, 3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48, 49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone was purified using preparative chiral HPLC, resulting in what we got (22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,56R)-46,56-dihydroxy45-[(1 R)-2-[( 1 S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-44-[2-(2methoxyethoxy)ethoxy]-35,36,37,38,48 ,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone (I-105:17 mg, yield 20%) and (22E,24E,26E,27E,35R,36S, 37R,38R,40S,42S,44R,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)- 2-[(1S,3R,4R)-4-(2-hydroxyethoxy)3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37 ,38,48,49hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54pentone (I-104: 15 mg, yield 16%), both as a white solid.

Chiral separation method

- 127 045993

Column

Column size Sample solution Introduction Mobile phase Flow rate Wavelength Temperature

CHIRALPAK IC

2.5 cm I.D. x 25 cm L, 10 µm mg/ml in mobile phase ml

Hexane/EUN=5 0/50(v/v) ml/min

UV 254 nm °C

I-105: ESI-MS (EI ⁺ , m/z): 1068.4 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 6.42 - 6.19 (m, 2H), 6.13 (dd, J=15.1, 10.0 Hz, 1H), 5.91 (dd, J =33.0, 10.5 Hz, 1H), 5.56 - 5.38 (m, 2H), 5.27 (d, J=5.0 Hz, 1H), 5.15 (dt, J= 15.2, 7.6 Hz, 1H), 4.76 (s, 1H), 4.18 (d, J=5.6 Hz, 1H), 3.93 - 3.25 (m, ZON), 3.24 - 3.03 (m, 3H), 2.72 (dd, J=16.7, 5.6 Hz, 2H), 2.57 (dd, J=16.8, 6.5 Hz, 1H), 2.34 (d, J=13.9 Hz, 2H), 2.13 - 1.84 (m, 6H), 1.82 - 1.67 (m, 7H), 1.47 (dd , J=24.1, 16.7 Hz, 4H), 1.25 (ddd, J=24.1, 20.2, 10.0 Hz, 7H), 1.14 - 0.81 (m, 18H ), 0.72 (dd, J=23.9, 12.1 Hz, 1H).

I-104: ESI-MS (EI ⁺ , m/z): 1068.4 [M+Na] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.19 (m, 4H), 5.56 - 5.36 (m, 2H), 5.28 - 5.07 (m, 2H), 4.83 ( d, J=4.9 Hz, 4H), 4.28 (s, 1H), 4.21 - 4.09 (m, 1H), 4.04 - 3.51 (m, 15H), 3.46 3.29 (m, 11H), 3.27 - 2.91 (m, 5H), 2.76 - 2.42 (m, 3H), 2.31 (d, J=11.3 Hz, 2H) , 2.18 - 1.70 (m, 13H), 1.53 1.19 (m, 8H), 1.16 - 0.84 (m, 18H), 0.76 - 0.60 (m, 1H ).

Example 29. Synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-[2- (2-hydroxyethylsulfonyl)ethylsulfonyl]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36, 37,47,48-hexamethyl-70,71-dioxa-56azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-95) and ( 21E,23E,25E,26E, 34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethylsulfonyl)ethylsulfonyl] ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy34,35,36,37,47,48-hexamethyl -70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene49,50,51,52,53-pentone (I-102)

- 128 045993

Synthesis circuit

Methods and characteristics.

Step 1: synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy43-[2-[2-(2-hydroxyethylsulfanyl )ethylsulfanyl]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37, 47,48-hexamethyl-66,67-dioxa-56-azatricyclohex satriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone.

4-Methylbenzenesulfonic acid monohydrate (0. 62 g, 3.28 mmol) at 15°C. The resulting mixture was stirred at 15°C for 17 hours, then diluted with EtOAc (100 ml) and the pH adjusted to 9 using saturated aqueous NaHCO ₃ solution (about 50 ml). The organic layer was then concentrated and the residue was purified by reverse phase chromatography (C18, CH ₃ CN:H2O=6:4) to give (21E,23E,25E,26E,34R,35S, 36R,37R,39R,41S, 44S,45R,46R,55S)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethylsulfanyl)ethylsulfanyl]ethoxy]-44-[(1S)-2-[(1S,3R,4R) -4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35, 36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47 ),26(48)-tetraene 49,50,51,52,53-pentone (150 mg, 12% yield) as a yellow solid. ESI-MS (EI ⁺ , m/z): 1086.4 [M+Na] ⁺ . ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.39-5.95 (m, 4H), 5.54-5.19 (m, 4H), 4.81-4.17 (m, 2H), 3.963.73 (m, 4H), 3.59-3.14 (m, 12H), 2.96-2.55 (m, 14H), 2.35-1.87 (m, 6H), 1, 81-1.59 (m, 13H), 1.53-1.13 (m, 11H), 1.16-0.84 (m, 18H), 0.71-0.63 (m, 1H).

Step 2: synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy43-[2-[2-(2-hydroxyethylsulfonyl )ethylsulfonyl]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37, 47,48-hexamethyl-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-102).

To a solution of (21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy-43-[2[2-(2-hydroxyethylsulfanyl) ethylsulfanyl]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47 ,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (170 mg, 0.16 mmol ) in methanol (8 ml) Oxon (393 mg, 0.64 mmol) was added at 0°C. The resulting mixture was allowed to warm to 10°C and stirred for 5 hours. The reaction solution was filtered, then purified by reverse phase chromatography (C18, using 5-60% acetonitrile-water), resulting in (21E,23E,25E,26E ,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy-43-[2-[2-(2-hydroxy

- 129 045993 ethylsulfonyl)ethylsulfonyl]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1methylethyl]-46-methoxy-34,35,36, 37,47,48-hexamethyl-70,71-dioxa-56-azatricyclohexatriaconta21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-102, 30 mg , yield 17%) in the form of a white solid. ESI-MS (EI ⁺ , m/z): 1150.8 [M+Na] ⁺ . ^1H NMR (500 MHz, CDCl ₃ ) δ 6.40 - 5.90 (m, 4H), 5.57 5.08 (m, 5H), 4.14 (s, 4H), 3.68 (tdd , J=37.6, 33.2, 11.6 Hz, 11H), 3.48 - 3.13 (m, 20H), 2.95 (s, 2H), 2.68 (dd, J=36 ,4, 30.5 Hz, 5H), 2.37 - 1.70 (m, 12H), 1.31 (dd, J=78.6, 46.8 Hz, 7H), 1.13 - 0, 81 (m, 18H), 0.67 (d, J=11.9 Hz, 1H).

Step 3: synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-43 - [2- [2- (2-hydroxyethylsulfonyl)ethylsulfonyl]ethoxy]-44-[(1R)-2-[(1 S,3R,4R)-4-hydroxy-3methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36 ,37,47,48-hexamethyl-70,71-dioxa-56azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-95):

mg (21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy-43-[2-[2-(2hydroxyethylsulfonyl)ethylsulfonyl] ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48 -Hexamethyl-70,71-dioxa-56-azatricyclohexatriaconta21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone was purified by preparative chiral HPLC to give ( 21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)45,55-dihydroxy-43-[2-[2-(2-hydroxyethylsulfonyl)ethylsulfonyl ]ethoxy]-44-[(1R)-2-[(1 S,3R,4R)-4hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48 -hexamethyl-70,71-dioxa56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-95: 15 mg, yield

16%) in the form of a white solid.

Chiral analysis method

Column:

Column size:

Introduction:

Mobile phase:

Flow rate:

Wavelength: UV:

Temperature:

HPLC equipment:

CHIRALPAK IC(IC00CD-NA012) 0.46 cm ID. x 15 cm L 10.0 µl

EtOH=100%

0.5 ml/min

254 nm °C

Shimadzu LC-20AD CP-HPLC-06

I-95: ESI-MS (EI ⁺ , m/z): 1150.3 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 6.32 (td, J=24.8, 14.8 Hz, 2H), 6.13 (dd, J=14.9, 9.9 Hz, 1H), 5.98 (dd, J=22.1, 10.3 Hz, 1H), 5.56 - 5.31 (m, 2H), 5.26 (d, J=5.4 Hz, 1H), 5 ,14 (d, J=4.1 Hz, 1H), 4.86 (s, 1H), 4.16 (dd, J=11.9, 5.5 Hz, 3H), 3.92 - 3, 49 (m, 11H), 3.44 - 3.17 (m, 15H), 2.93 (dd, J=14.1, 5.5 Hz, 1H), 2.78 - 2.50 (m, 5H), 2.36 - 2.17 (m, 2H), 2.01 (ddd, J=21.5, 18.0, 9.0 Hz, 5H), 1.84 1.65 (m, 11H ), 1.49-1.16 (m, 12H), 1.14-0.82 (m, 14H), 0.66 (dd, J=23.8, 12.0 Hz, 1H).

Example 29: Synthesis of 3-[2,2-bis(2-cyanoethoxymethyl)-3-[[(21E,23E,25E,26E,41R,42S,43R,44R,46S,48S, 51S,52R,53R,62R )-52,62-dihydroxy-51-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-53-methoxy-41,42,43, 44,54,55-hexamethyl-56,57,58,59,60-pentaoxo-76,77-dioxa-67-azatricyclohexatriaconta-21,23,25(54),26(55)-tetraen-50-yl] oxy]propoxy]propanenitrile (I-101)

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Synthesis circuit

Methods and characteristics.

Step 1: synthesis of (1-methyl-2,6,7-trioxabicyclo[2,2.2]octan-4-yl)methanol: To a solution of 2,2bis(hydroxymethyl)propane-1,3-diol (20 g, 146, 9 mmol) and 4-methylbenzenesulfonic acid (0.25 g, 1.47 mmol) in toluene (200 ml) was added 1,1,1-triethoxyethane (27 ml, 146.9 mmol) under reflux. The reaction mixture was then stirred at 130°C until the solution became clear, a few drops of TEA were added and the reaction mixture was filtered while it was hot. The filtrate was cooled and then concentrated to give (1-methyl-2,6,7trioxabicyclo[2,2.2]octan-4-yl)methanol (20 g, 85% yield) as colorless crystals. 1H NMR (500

MHz, DMSO-de): δ 4.79 (t, J=5.3 Hz, 1H), 3.85 (s, 6H), 3.22 (d, J=5.3 Hz, 2H), 1 .29 (s, 3H).

Step 2: synthesis of 4-(benzyloxymethyl)-1-methyl-2,6,7-trioxabicyclo[2,2.2]octane.

Solid (1-methyl-2,6,7-trioxabicyclo[2,2.2]octan-4-yl)methanol (1.5 g, 9.37 mmol) was added to a stirred suspension of finely ground KOH (2.47 g, 44 mmol) and BnBr (1.86 g, 10.86 mmol) in DMSO (5 ml). The resulting solution was stirred at room temperature for 1 hour, then poured into ice water and extracted with EtOAc, dried, filtered and concentrated. The residue was purified by silica gel chromatography (BYLs:PE=1:10) to give 4-(benzyloxymethyl)-1-methyl-2,6,7-trioxabicyclo[2,2.2]octane (1.5 g, yield 64%) in the form of a light yellow solid. ESI-MS (EI ⁺ , m/z): 251.1 [M+H]+. 1H NMR (500 MHz, CDCl ₃ ) δ 7.36-7.25 (m, 5H), 4.45 (s, 2H), 4.01 (s, 6H), 3.19 (s, 2H), 1.45 (s, 3H).

Step 3: synthesis of 2-(benzyloxymethyl)-2-(hydroxymethyl)propane-1,3-diol.

A solution of 4-(benzyloxymethyl)-1-methyl-2,6,7-trioxabicyclo[2,2.2]octane (2 g, 8 mmol) and hydrogen chloride (2 ml, 2M in water) in THF (20 ml) was stirred at room temperature overnight, then concentrated and purified by reverse phase chromatography (C18, CH ₃ CN:H ₂ O=1:3), resulting in 2-(benzyloxymethyl)-2-(hydroxymethyl)propane-1 ,3-diol (1.3 g, 72% yield) as a thick yellow oil. ESI-MS (EI ⁺ , m/z): 227.1 [M+H] ⁺ . 1H NMR (500 MHz, CDCl ₃ ) δ 7.36-7.26 (m, 5H), 4.48 (s, 2H), 3.68 (s, 6H), 3.46 (s, 2H), 3.40 (bs, 3H).

Step 4: synthesis of 3-[2-(benzyloxymethyl)-3-(2-cyanoethoxy)-2-(2-cyanoethoxymethyl)propoxy]propanenitrile.

Acrylonitrile (11.49 g) was slowly added to a mixture of 2-(benzyloxymethyl)-2-(hydroxymethyl)propane-1,3-diol (4.9 g, 21.66 mmol) and KOH (98 mg, 1.75 mmol) , 216.56 mmol), ensuring that the internal reaction temperature does not exceed 30°C. The mixture was then stirred overnight at room temperature, neutralized with 1 N. aqueous HCl solution and subjected to extraction using EtOAc (200 ml). The combined organic layers were washed twice with water, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to give 3-[2(benzyloxymethyl)-3-(2-cyanoethoxy)-2-(2-cyanoethoxymethyl)propoxy]propanenitrile ( 7 g, yield 84%) as a yellow solid. ESI-MS (EI ⁺ , m/z): 386.3 [M+H] ⁺ .

Step 5: synthesis of 3-[2,2-bis(2-cyanoethoxymethyl)-3-hydroxy-propoxy]propanenitrile.

Pd/C (1 .59 g). The mixture was stirred at room

- 131 045993 temperature overnight in a hydrogen cylinder, then filtered through a layer of celite and washed with ethanol. The resulting solution was concentrated to give 3-[2,2-bis(2cyanoethoxymethyl)-3-hydroxypropoxy]propanenitrile (2.6 g, 68% yield) as a colorless oil. ESI-MS (EI+, m/z): 296.2 [M+H]+.

Step 6: synthesis of 3-[2,2-bis(2-cyanoethoxymethyl)-3-[[(21E,23E,25E,26E,41R,42S,43R,44R, 46S,48S,51S,52R,53R,62R )-52,62-dihydroxy-51-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]1-methylethyl]-53-methoxy-41,42,43,44 ,54,55-hexamethyl-56,57,58,59,60-pentaoxo-76,77-dioxa-67-azatricyclohexatriaconta-21,23,25(54),26(55)-tetraen-50-yl]oxy ]propoxy]propanenitrile (I-101).

To a solution of rapamycin (0.5 g, 0.547 mmol) in DCM (30 ml) was added 2,2,2-trifluoroacetic acid (2.4 ml) at -40°C. The reaction mixture was stirred at -40°C for 10 min, then 3-[2,2-bis(2-cyanoethoxymethyl)-3-hydroxypropoxy]propanenitrile (0.48 g, 1.64 mmol) was added. After stirring for an additional 1 hour, the reaction mixture was diluted with DCM (3 ml) and poured into cold aqueous NaHCO ₃ solution. The organic layer was washed with water and brine, dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by reverse phase chromatography (C18, CH ₃ CN:H ₂ O=8:2), resulting in 3-[2,2-bis(2-cyanoethoxymethyl)-3[[(21E,23E, 25E,26E,41R,42S,43R,44R,46S,48S,51S,52R,53R,62R)-52,62-dihydroxy-51-[(1R)-2[(1S,3R,4R)-4- hydroxy-3-methoxycyclohexyl]-1-methylethyl]-53-methoxy-41,42,43,44,54,55-hexamethyl56,57,58,59,60-pentaoxo-76,77-dioxa-67-azatricyclohexatriaconta- 21,23,25(54),26(55)-tetraen-50yl]oxy]propoxy]propanenitrile (I-101: 80 mg, 12% yield) as a white solid. IERMS (EI+, m/z): 1198.8 [M+Na]+. ¹ H NMR (500 MHz, CDCh) δ 6.42 -5.87 (m, 4H), 5.35 (ddd, J=120.9, 41.2, 32.5 Hz, 4H), 4.24 (dd, J=29.5, 16.4 Hz, 2H), 3.98 (d, J=4.2 Hz, 1H), 3.87 - 3.60 (m, 7H), 3.56 - 3.01 (m, 18H), 2.88 (d, J=59.1 Hz, 2H), 2.74 - 2.42 (m, 9H), 2.34 (s, 2H), 2.23 - 1.84 (m, 5H), 1.82 - 1.65 (m, 13H), 1.53 - 1.22 (m, 10H), 1.16 - 0.84 (m, 18H), 0 .72 - 0.61 (m, 1H).

Example 30. Synthesis of (21E,23E,25E,26E,37R,38S,39R,40R,42S,44S,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3-(2 -hydroxyethoxy)-2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4hydroxy-3-methoxycyclohexyl]-1-methylethyl]-49-methoxy- 37,38,39,40,50,51-hexamethyl-71,72-dioxa60-azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54,55,56-pentone ( I-100) and (21E,23E,25E, 26E,37R,38S,39R,40R,42S,44S,46R,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3 -(2-hydroxyethoxy)-2-(2hydroxyethoxymethyl)propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-49 -methoxy-37,38,39,40,50,51-hexamethyl-71,72-dioxa-60-azatricyclohexatriaconta-21,23, 25(50),26(51)-tetraene-52,53,54,55 ,56-pentone (I-64) and (21E,23E,25E,26E,37R,38S,39R,40R,42S,44S,

46S,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3-(2-hydroxyethoxy)-2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47-[( 1 R) -2-[(1 S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-49-methoxy-3 7,38,39, 40,50,51-hexamethyl-71,72- dioxa-60-azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54, 55,56-pentone (I-65)

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Synthesis circuit

Methods and characteristics.

Step 1: synthesis of 7-((2-(benzyloxy)ethoxy)methyl)-1,13-diphenyl-2,5,9,12-tetraoxatridecane.

To a solution of 2-(hydroxymethyl)propane-1,3-diol (2 g, 18.85 mmol) in THF (30 ml) at 0° C. was added sodium hydride (9.05 g, 376.93 mmol), and the reaction the mixture was heated to 50°C for 1 hour and then cooled to room temperature. Next, 2-bromoethoxymethylbenzene (40.54 g, 188.47 mmol) was added and the mixture was heated to 65°C for 17 hours. The reaction was quenched with ice water (50 ml), then the reaction mixture was extracted with EtOAc (50 mlx2). The combined organic layers were washed with brine (100 ml), dried, filtered, concentrated and then purified by silica gel chromatography (PE:EAAc=20:1) to give 2-[3-(2-benzyloxyethoxy)-2 (2-benzyloxyethoxymethyl)propoxy]ethoxymethylbenzene (5 g, 52% yield) as a colorless liquid.

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ESI-MS (EI+, m/z): 509.0 [M+H]+.

Step 2: synthesis of 2,2'-(2-((2-hydroxyethoxy)methyl)propane-1,3-diyl)bis(oxy)diethanol.

Pd/C (2.41 g) was added to a solution of 2-[3-(2-benzyloxyethoxy)-2-(2-benzyloxyethoxymethyl)propoxy]ethoxymethylbenzene (2 g, 3.93 mmol) in MeOH (20 ml). The mixture was stirred at room temperature overnight in a hydrogen cylinder. The reaction mixture was then filtered through a pad of celite and then washed with ethanol. The resulting solution was concentrated under reduced pressure to obtain 2,2'-((2-((2-hydroxyethoxy)methyl)propan-1,3-diyl)bis(oxy))bis(ethan-1-ol) ( 0.92 g, yield 98%). ¹ H NMR (500 MHz, CDCl ₃ ) δ 3.65 (dd, J=5.7, 3.4 Hz, 6H), 3.53 - 3.48 (m, 12H), 2.22-2, 14 (m, 1H).

Step 3: synthesis of (21E,23E,25E,26E,37R,38S,39R,40R,42S,44S,47S,48R,49R,59R)-48,59-dihydroxy46-[2-[3-(2-hydroxyethoxy )-2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-49-methoxy- 37,38,39,40,50,51-hexamethyl-71,72-dioxa-60azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54,55,56-pentone ( I-100).

2-[3-(2-hydroxyethoxy)-2-(2 -hydroxyethoxymethyl)propoxy]ethanol (1.3 g, 5.47 mmol). The mixture was stirred at 25°C for 2 hours, then poured into ice-cold aqueous NaHCO ₃ and extracted with EtOAc. The organic layer was dried, filtered and concentrated. The residue was purified by reverse phase chromatography (C18, CH ₃ CN:H ₂ O: 7:3) followed by silica gel chromatography (DCM:MeOH=15:1) to give (21E,23E,25E, 26E,37R,38S,39R,40R,42S,44S,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3-(2-hydroxyethoxy)-2-(2-hydroxyethoxymethyl) propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-49-methoxy-37,38,39,40,50 ,51-hexamethyl-71,72-dioxa-60-azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54,55,56-pentone (I-100, 150 mg, yield 25%) in the form of a white solid. ESI-MS (EI+, m/z): 1142.0 [M+Na]+. ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.21 (dddd, J=32.0, 27.8, 21.3, 10.2 Hz, 3H), 6.05 - 5.84 (m, 1H ), 5.57 -5.36 (m, 2H), 5.29 - 4.97 (m, 2H), 4.83 (s, 1H), 4.20 (dd, J=36.3, 30 ,2 Hz, 1H), 4.01 - 3.66 (m, 6h), 3.62 - 3.22 (m, 29H), 3.00 - 2.43 (m, 9H), 2.36 - 1.85 (m, 9H), 1.77 1.51 (m, 6H), 1.52 - 1.17 (m, 9H), 1.16-0.79 (m, 18H), 0.65 (dt, J=21.9, 11.0 Hz, 1H).

Step 4: synthesis of (21E,23E,25E,26E,37R,38S,39R,40R,42S,44S,46S,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3- (2-hydroxyethoxy)-2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4hydroxy-3-methoxycyclohexyl]-1-methylethyl]-49- methoxy-37,38,39,40,50,51-hexamethyl-71,72-dioxa60-azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54,55,56- penton (I-65) and (21E,23E,25E,26E,37R,38S,39R,40R,42S,44S,46R,47S,48R,49R,59R)-48,59-dihydroxy-46-[2- [3-(2hydroxyethoxy)-2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3methoxycyclohexyl]-1-methylethyl]-49 -methoxy-37,38,39,40,50,51-hexamethyl-71,72-dioxa-60azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54,55,56 -pentone (I-64):

140 mg (21E,23E,25E,26E,37R,38S,39R,40R,42S,44S,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3-(2hydroxyethoxy)- 2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3methoxycyclohexyl]-1-methylethyl]-49-methoxy-37.38, 39,40,50,51-hexamethyl-71,72-dioxa-60-azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54,55,56-pentone was purified using preparative chiral HPLC and the resulting epimers were purified by silica gel chromatography (hexane:DCM:EtOAc:MeOH=3:3:1:1), resulting in (21E,23E,25E,26E,37R,38S,39R, 40R ,42S,44S,46S,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3-(2-hydroxyethoxy)-2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47- [(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-49-methoxy-37,38,39,40,50,51-hexamethyl-71, 72-dioxa-60-azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54,55,56-pentone (I-65: 35.2 mg, 25% yield) and (21E,23E,25E,26E,37R,38S,39R,40R,42S, 44S,46R,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3-(2-hydroxyethoxy )-2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-49-methoxy- 37,38, 39,40,50,51-hexamethyl-71,72-dioxa-60-azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53, 54,55,56- pentone (I-64:16 mg, 11% yield), both as a white solid.

Chiral separation method

Column CHIRALPAK IC Column size 2.5 cm I.D. x 25 cm L Solution concentration 1.4 mg/ml Introduction 7 ml Mobile phase Hexane/EUN=60/40(v/v) Flow rate 30 ml/min Wavelength UV 254 nm Temperature 35°C

I-65: ESI-MS (EI+, m/z): 1142.4 [M+Na]+. ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.42 -6.18(m, 2H), 6.13 (dd, J=14.9, 10.1 Hz, 1H), 5.93 (dd, J=23.9, 11.0 Hz, 1H), 5.54 - 4.89 (m, 6H), 4.80 (s, 1H), 4.18 (d, J=6.0 Hz, 1H ), 3.90 (s, 1H), 3.80 - 3.66 (m, 5H), 3.63 - 3.48 (m, 11H), 3.47 - 3.21 (m, 11H), 2.99 - 2.47 (m, 6H), 2.38 - 2.15 (m,

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3H), 2.14 - 1.85 (m, 5H), 1.84 - 1.64 (m, 12H), 1.46 (dd, J=28.7, 18.7 Hz, 5H), 1 .35 - 1.15 (m, 7H), 1.15-0.81 (m, 18H), 0.66 (dd, J=23.7, 11.8 Hz, 1H).

I-64: ESI-MS (EI ⁺ , m/z): 1142.4 [M+Na] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.34 - 5.86 (m, 4H), 5.41 (ddd, J=54.0, 26.4, 18.2 Hz, 2H), 5, 16 (dd, J=9.6, 5.0 Hz, 2H), 4.88 (s, 1H), 4.20 (d, J=11.0 Hz, 1H), 4.14 - 4.02 (m, 1H), 3.93 (d, J=3.9 Hz, 1H), 3.87 - 3.72 (m, 1H), 3.64 (d, J=4.4 Hz, 4H) , 3.57 - 3.36 (m, 14H), 3.36 - 3.22 (m, 8H), 3.21 - 3.11 (m, 1H), 2.92-2.33 (m, 8H), 2.32-2.11 (m, 3H), 2.11 - 1.85 (m, 5H), 1.82-1.63 (m, 11H), 1.481.25 (m, 9H) , 1.09 - 0.72 (m, 18H), 0.66 - 0.50 (m, 1H).

Example 31. Synthesis of (21E,23E,25E,26E,48R,49S,50R,51R,53S,55S,58S,59R,60R,69R)-59,69-dihydroxy-57 -[2-[2-[2 -[2-[2-[2-[2-[2-[2-(2 - hydroxyethoxy)ethoxy]ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy]ethoxy]ethoxy] -58-[(1 R) -2-[( 1 S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-60-methoxy48,49,50,51,61,62-hexamethyl-80,81-dioxa-7 0-azatricyclohexatriaconta-21,23,25(61),26(62)-tetraene63,64,65,66,67-pentone (I-98)

Synthesis circuit

Methods and characteristics.

Step 1: synthesis of (21E,23E,25E,26E,48R,49S,50R,51R,53S,55S,58S,59R,60R,69R)-59,69-dihydroxy57-[2-[2-[2-[ 2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-58-[(1R)-2- [(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-60-methoxy-48,49,50, 51,61,62-hexamethyl-80,81-dioxa-70-azatricyclohexatriaconta -21,23,25(61),26(62)-tetraene-63,64,65, 66,67-pentone (I-98).

To a solution of rapamycin (0.5 g, 0.547 mmol) and 4-methylbenzenesulfonic acid (0.47 g, 2.73 mmol) in THF (15 ml) was added 2-[2-[2-[2-[2-[ 2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (2.51 g, 5.47 mmol) and the mixture was stirred at 25° C for 2 hours. The reaction mixture was then poured into cold aqueous NaHCO ₃ solution, which was then extracted using EtOAc. The organic layer was then dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by reverse phase chromatography (CI bcxpure water=7:3) to give (21E,23E,25E,26E,48R,49S,50R,51R,53S,55S,58S,59R,60R,69R )-59,69dihydroxy-57-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]-58-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-60methoxy-48,49,50,51 ,61,62-hexamethyl-80,81-dioxa-70-azatricyclohexatriaconta-21,23,25(61),26(62)tetraene-63,64,65,66,67-pentone (I-98: 115 mg , yield 15%) in the form of a thick oil. ESI-MS (EI ⁺ , m/z): 1362.9

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[M+Na]+. 1H NMR (500 MHz, CDCl ₃ ) δ 6.40 -5.83 (m, 4H), 5.55-5.35 (m, 2H), 5.32 - 5.03 (m, 2H), 4 .31 - 4.10 (m, 1H), 3.93 (dd, J=70.7, 6.3 Hz, 1H), 3.78 - 3.15 (m, 53H), 2.97 - 2 .41 (m, 5H), 2.32 (s, 2H), 2.15 - 1.55 (m, 18H), 1.52 - 1.16 (m, 10H), 1.14 - 0.81 (m, 18H), 0.73 - 0.58 (m, 1H).

Example 32: Synthesis of 2-[[(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,55R)-44,55dihydroxy-43-[(1R)-2 -[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-48,49, 50,51,52-pentaoxo-67,68-dioxa-57-azatricyclohexatriaconta22,24,26(46),27(47)-tetraen-42-yl]oxy]ethyl^-methylcarbamate (I-81) and 2- [[(22E,24E,26E,27E,33R,34S, 35R,36R,38S,40S,42R,43S,44R,45R,55R)-44,55-dihydroxy-43-[(1R)-2-[ (1S,3R,4R)-4-(2-hydroxyethoxy)3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-48,49,50,51 ,52-pentaoxo67,68-dioxa-57-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraen-42-yl]oxy]ethyl-Nmethylcarbamate (I-74) and 2-[[( 22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,42S,43S,44R,45R,55R)-44,55-dihydroxy-43[(1R)-2-[(1S,3R ,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36, 46,47-hexamethyl-48,49,50,51,52- pentaoxo-67,68-dioxa-57-azatricyclohexatriaconta-22,24,26(46), 27(47)-tetraen-42-yl]oxy]ethyl-Nmethylcarbamate (I-75)

Synthesis circuit

Methods and characteristics.

Step 1: synthesis of 2-[[(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,55R)-44,55-dihydroxy-43-[(1R) -2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy33,34,35,36,46,47-hexamethyl-48,49, 50,51,52-pentaoxo-67,68-dioxa-57-azatricyclohexatriaconta- 136 045993

22,24,26(46),27(47')-1etrae11-42-i..l|oxy|'ethi..lA'-methyl..lcarbamate (I-81): To degassed solution of everolimus (0, 5 g, 0.52 mmol) in THF (10 ml) at 0°C, p-toluenesulfonic acid (0.45 g, 2.61 mmol) and 2-hydroxyethyl-Nmethylcarbamate (2.80 ml) were added. The resulting mixture was stirred at 0°C for 0.5 h under N2, then heated to 23°C and stirred for 3 hours. The mixture was poured into saturated NaHCO ₃ solution (40 ml), which was extracted with EtOAc (30 ml ). The organic layer was washed with water (30 mlx2), brine (40 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The residue was purified by silica gel chromatography (DCM:MeOH=10:1) and then further purified by reverse phase chromatography (C18, CH3CN:H ₂ O=7:3) to give 2-[[( 22E,24E,26E,27E,33R,34S,35R,36R,38S, 40S,43S,44R,45R,55R)-44,55-dihydroxy-43-[(1R)-2-[(1S,3R, 4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-48,49,50,51,52-pentaoxo -67,68-dioxa57-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraen-42-yl]oxy]ethyl-^methylcarbamate (I-81: 100 mg, yield 18%) as a solid white substances. ESI-MS (EI ⁺ , m/z): 1067.4 [M+Na] ⁺ . 1H NMR (400 MHz, CDC13) δ 6.50-5.91 (m, 4H), 5.58 -4.97 (m, 4H), 4.70 (s, 1H), 4.51 (d, J=40.4 Hz, 1H), 4.33 - 4.02 (m, 3H), 3.93 - 3.62 (m, 6H), 3.61 - 3.00 (m, 13H), 2 .86 - 2.46 (m, 6H), 2.40 - 2.22 (m, 2H), 2.18-1.69 (m, 22H), 1.58-1.25 (m, 7H) , 1.24 - 0.79 (m, 18h), 0.79 - 0.62 (m, 1H).

Step 2: synthesis of 2-[[(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,42S,43S,44R,45R,55R)-44,55dihydroxy-43-[(1R) -2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-48, 49,50,51,52-pentaoxo-67,68-dioxa-57-azatricyclohexatriaconta22,24,26(46),27(47)-tetraen-42-yl]oxy]ethyl-Nmethylcarbamate (I-75) and 2-[[(22E,24E,26E,27E,33R, 34S,35R,36R,38S,40S,42R,43S,44R,45R,55R)-44,55-dihydroxy-43-[(1R)-2 -[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-48,49, 50,51,52pentaoxo-67,68-dioxa-57-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraen-42-yl]oxy]ethyl-No. methylcarbamate (I-74):

120 mg 2-[[(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,55R)-44,55-dihydroxy-43[(1R)-2- [(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35, 36,46,47-hexamethyl-48,49,50 ,51,52-pentaoxo-67,68-dioxa-57-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraen-42-yl]oxy]ethyl-Nmethylcarbamate was purified using preparative chiral HPLC and the resulting epimers were purified by silica gel chromatography (hexai:DCM:EEAc:MeOH=3:3:1:0.6), resulting in 2-[[(22E,24E,26E,27E,33R,34S,35R , 36R,38S,40S,42S,43S,44R,45R,55R)-44,55-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)- 3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-48,49,50,51,52-pentaoxo67,68-dioxa-57-azatricyclohexatriaconta-22, 24,26(46),27(47)-tetraen-42-yl]oxy]ethyl-N-methylcarbamate (I-75: 18 mg, 15% yield) and 2-[[(22E,24E,26E,27E ,33R,34S,35R,36R,38S,40S,42R,43S,44R,45R,55R)44,55-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-( 2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-45methoxy-33,34,35,36,46,47-hexamethyl-48,49,50,51,52-pentaoxo-67,68-dioxa-57 -azatricyclohexatriaconta-22,24,26(46),27(47)-tetraen-42-yl]oxy]ethyl-Nmethylcarbamate (I-74: 20 mg, 16% yield), both as a white solid.

Chiral separation method

Column

Column size

Solution concentration

Introduction

Mobile phase Flow rate Wavelength Temperature

CHIRALPAK IC

5.0 cm I.D. x 25 cm L

2.4 mg/ml ml

Hexane/E10H=60/40(v/v) ml/min

UV 254 nm °C

I-75: ESI-MS (EI ⁺ , m/z): 1067.3 [M+Na] ⁺ . 1H NMR (400 MHz, CDCI3) δ 6.33 (dt, J=24.4, 14.6 Hz, 2H), 6.13 (dd, J=15.0, 9.9 Hz, 1H), 5 .93 (dd, J=24.5, 10.6 Hz, 1H), 5.52 (dd, J=15.0, 8.8 Hz, 1H), 5.41 (d, J=10.2 Hz, 1H), 5.35 (t, J=4.7 Hz, 1H), 5.27 (d, J=5.3 Hz, 1H), 5.16 (d, J=4.9 Hz, 1H), 4.75 (s, 1H), 4.29 - 4.07 (m, 3H), 3.93 - 3.63 (m, 6H), 3.62 - 3.47 (m, 3H) , 3.46 - 3.25 (m, 11H), 3.23 - 3.01 (m, 3H), 2.79 (d, J=4.9 Hz, 3H), 2.74 2.62 ( m, 2H), 2.57 (dd, J=16.5, 6.4 Hz, 1H), 2.34 (d, J=12.7 Hz, 2H), 2.25 - 2.19 (m , 1H), 2.08 (s, 1H), 1.93 (dd, J=30.0, 22.7 Hz, 5H), 1.83 - 1.65 (m, 7H), 1.55 - 1.42 (m, 5H), 1.28 (s, 6H), 1.15 - 0.83 (m, 18H), 0.72 (dd, J=23.6, 12.0 Hz, 1H) .

I-74: ESI-MS (EI ⁺ , m/z): 1067.4 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 6.50-5.95 (m, 4H), 5.60 - 4.96 (m, 5H), 4.59 - 3.96 (m, 4H), 3 .95 - 3.66 (m, 6H), 3.64 - 2.97 (m, 15H), 2.95 - 2.65 (m, 6H), 2.59 (d, J=11.0 Hz , 1H), 2.51 - 1.95 (m, 5H), 1.78 (q, J=6.8 Hz, 12H), 1.55-1.29 (m, 11H), 1.15- 0.83 (m, 18H), 0.69 (dd, J=23.6, 11.5 Hz, 1H).

Example 33. Synthesis of (21E,23E,25E,26E,30R,31S,32R,33R,35S,37S,40S,41R,42R,55R)-41,55-dihydroxy-40-[(1R)-2-[ (1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-42-methoxy-30,31,32,33,43,44hexamethyl-39-[(2R,3R,4R,5R) -2,3,4,5,6-pentahydroxyhexoxy]-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49- penton (I-63) and (21E,23E,25E,26E,30R,31S,32R,33R,

- 137 045993

35S,37S,39R,40S,41R,42R,55R)-41,55-dihydroxy-40-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1- methylethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-39-[(2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexoxy]-70,71 -dioxa-56-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone (I-57) and (21E,23E,25E,26E,30R ,31S,32R,33R,35S,37S,39S,40S,41R,42R,55R)-41,55-gurugpoKCu-40[(1R)-2-[(1S,3R,4R)-4-hydroxy-3 -methoxycyclohexyl]-1-methylethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-39-[(2R,3R,4R,5R)-2,3,4,5,6- pentahydroxyhexoxy]-70,71-dioxa-56-azatricyclohexatriaconta-

1-57 1-58

Synthesis circuit

- 138 045993

Methods and characteristics.

Step 1: synthesis of (2R,3R,4R,5R)-2,3,4,5-tetrahydroxyhexane-1,6-diylbibenzoate: To a solution of (2R,3R,4R,5R)-hexane-1,2,3, 4,5,6-hexaol (10.0 g, 54.89 mmol, 1.0 eq.) in pyridine (25.39 g, 548.9 mmol, 10.0 eq.) was added at 0°C with benzyl chloride ( 7.72 g, 54.89 mmol, 1.0 eq.). The reaction mixture was then stirred at room temperature for 16 hours, diluted with H ₂ O (200 ml) and extracted with DCM (150 ml x 3 ). The combined organic layers were washed with brine (100 ml), dried over Na ₂ SO ₄ , filtered, concentrated, and purified by reverse phase chromatography (yield CH ₃ CN:H ₂ O=40%~60%), resulting in (2R,3R,4R,5R)-2,3,4,5tetrahydroxyhexane-1,6-diyldibenzoate (4.7 g, 22% yield) was obtained as a white solid.

ESI-MS (EI ⁺ , m/z): 391.1 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-de) δ 8.07 - 8.00 (m, 4H), 7.66 (t, J=7.4 Hz, 2H), 7.54 (t, J=7 .7 Hz, 4H), 5.09 (d, J=6.1 Hz, 2H), 4.60 - 4.48 (m, 4H), 4.28 (dd, J=11.2, 6, 2 Hz, 2H), 3.87 (dt, J=6.1, 5.3 Hz, 2H), 3.77 (t, J=8.6 Hz, 2H).

Step 2: synthesis ((4R,4'R,5R,5'R)-2,2,2',2'-tetramethyl-4,4'-bi(1,3-dioxolane)-5,5'- diyl)bis(methylene) dibenzoate and ((4R,4aR,8R,8aR)-2,2,6,6-tetramethyltetrahydro-[1,3]dioxino[5,4-d][1,3]dioxin-4 ,8diyl)bis(methylene) dibenzoate.

A solution of [(2R,3R,4R,5R)-6-benzoyloxy-2,3,4,5-tetrahydroxy-hexyl]benzoate (5 g, 12.81 mmol) and pTsOH (1.22 g, 6.40 mmol ) in 2,2-dimethoxypropane (50 ml) was stirred at 28°C for 10 hours. The resulting mixture was extracted with EtOAc (100 mlx3). The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The residue was purified by chromatography on silica gel (EAAc:PE=1:10 to 1:2), resulting in [(4R,5R)-5-[(4R,5R)-5(benzoyloxymethyl)-2,2- dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyl-1,3-dioxolan-4-yl]methyl benzoate (2.4 g, 40% yield) and [(4R,4aR,8R,8aR )-4-(benzoyloxymethyl)-2,2,6,6-tetramethyl-4,4a,8,8a-tetrahydro[1,3]dioxino[5,4^][1,3]dioxin-8-yl] methyl benzoate 1.2 g, yield 20%), both as a white solid. ESI-MS (EI ⁺ , m/z): 493.2 [M+Na] ⁺ .

[(4R,5R)-5-[(4R,5R)-5-(Benzoyloxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyl-1,3dioxolan-4 -yl]methyl benzoate: 1H NMR (400 MHz, DMS.'OL ₆ ): δ 7.98 (dd, J=11.6, 4.5 Hz, 4H), 7.67 (dd, J=11.7 , 4.3 Hz, 2H), 7.55 (t, J=7.7 Hz, 4H), 4.42 -4.33 (m, 4H), 4.08 - 3.94 (m, 4H) , 1.33 (s, 6H), 1.26 (s, 6H). ¹³ C NMR (101 MHz, DMSO<) δ 165.52, 133.44, 129.47, 129.15, 128.78, 100.61, 67.89, 67.60, 64.25, 24.24 , 23.45.

[(4R,4aR,8R,8aR)-4-(Benzoyloxymethyl)-2,2,6,6-tetramethyl-4,4a,8,8a-tetrahydro-[1,3]dioxino[5^][1, 3]dioxin-8-yl]methyl benzoate: 1H NMR (400 MHz, DMSO<): δ 8.03 - 7.92 (m, 4H), 7.67 (dd, J=10.6, 4.3 Hz , 2H), 7.54 (t, J=7.7 Hz, 4H), 4.57 (dd, J=11.1, 5.9 Hz, 2H), 4.53 - 4.44 (m, 4H), 4.39 (dd, J=11.5, 7.3 Hz, 2H), 1.43 (s, 6H), 1.29 (s, 6H). ¹³ C NMR (101 MHz, DMSO-de): δ 165.45, 133.42, 129.52, 129.17, 128.75, 108.39, 74.49, 73.77, 64.17, 27 .04, 25.27.

Step 3: synthesis ((4R,4'R,5R,5'R)-2,2,2',2'-tetramethyl-4,4'-bi(1,3-dioxolane)-5,5'- diyl)dimethanol.

A mixture of [(4R,5R)-5-[(4R,5R)-5-(benzoyloxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyl-1,3dioxolan- 4-yl]methyl benzoate (8.5 g, 18.07 mmol) and K ₂ CO ₃ (7.48 g, 54.20 mmol) in THF (25 ml) and CH3OH (25 ml) were stirred at 29°C in for 18 hours. The reaction mixture was concentrated and then purified by chromatography on silica gel (EAAc/PE=1:1), resulting in [(4R,5R)-5-[(4R,5r)5-(hydroxymethyl)- 2,2-dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyl-1,3-dioxolan-4-yl]methanol (3.2 g, 68% yield) as a white solid . ESI-MS (EI ⁺ , m/z): 285.1 [M+Na] ⁺ . 1H NMR (400 MHz, DMSO<): δ 4.73 (t, J=5.8 Hz, 2H), 3.71 (t, J=5.6 Hz, 2H), 3.55 - 3.35 (m, 6H), 1.29 (s, 6H), 1.23 (s, 6H).

Step 4: synthesis of (25E,27E,29E,30E,34R,35S,36R,37R,39S,41S,44S,49R,50R,61R)-49,61-dihydroxy44-[(1R)-2-[(1S ,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-43-[[(4R,5R)-5-[(4R,5R)-5(hydroxymethyl)-2,2-dimethyl- 1,3-dioxolan-4-yl]-2,2-dimethyl-1,3-dioxolan-4-yl]methoxy]-50-methoxy34,35,36,37,51,52-hexamethyl-72,73- dioxa-62-azatricyclohexatriaconta-25,27,29(51),30(52)-tetraene53,54,55,56,57-pentone.

To a solution of rapamycin (0.5 g, 0.547 mol) in DCM (16 ml) was added 2,2,2-trifluoroacetic acid (1.2 ml) at -40°C. After stirring for 10 min, [(4R,5R)-5-[(4R,5R)-5(hydroxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyl was added -1,3-dioxolan-4-yl]methanol (0.43 g, 1.64 mmol) and the reaction mixture was stirred at -30°C for 1.5 h, then diluted with DCM (10 ml) and poured into cold aqueous solution of NaHCO ₃ . The organic layer was washed with water and brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The resulting residue was purified by reverse phase chromatography (C18, CH ₃ CN/H ₂ O=7:3), resulting in (25E,27E,29E,30E,34R,35S,36R,37R,39S,41S, 44S,49R,50R,61R)-49,61-dihydroxy-44-[(1R)-2-[(1S,3R,4R)4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-43-[[ (4R,5R)-5-[(4R,5R)-5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyl-1,3-dioxolan- 4-yl]methoxy]-50-methoxy-34,35,36,37,51,52-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-25,27,29(51),30(52)-tetraene -53,54,55,56,57-pentone (150 mg, 24% yield) as a white solid. ESI-MS (EI ⁺ m/z): 1166.4 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 6.52 - 5.69 (m, 4H), 5.48 - 5.10 (m, 4H), 4.51 (d, J=40.1 Hz, 1H ), 4.23 (s, 1H), 3.94 (dd, J=26.3, 5.8 Hz, 4H), 3.52 - 3.12 (m, 13H), 2.98 -2, 45 (m, 8H), 2.41 - 2.17 (m, 2H), 2.00 (s, 3H), 1.76 (dt, J=29.0,

- 139 045993

14.4 Hz, 15H), 1.50 - 1.31 (m, 24H), 1.14 - 0.77 (m, 18H), 0.62 (d, J=12.1 Hz, 1H).

Step 5: synthesis of (21E,23E,25E,26E,30R,31S,32R,33R,35S,37S,40S,41R,42R,55R)-41,55-gurugpoKCu40-[(1R)-2-[(1S ,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-42-methoxy-30,31,32,33,43,44rexamethyl-39-[(2R,3R,4R,5R)-2 ,3,4,5,6-pentahydroxyrexoxy]-70,71-dioxa-56-azatricyclorexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone( I-63).

To a solution of (25E,27E,29E,30E,34R,35S,36R,37R,39S,41S,44S,49R,50R,61R)-49,61-dihydroxy-44[(1R)-2-[(1S, 3R,4R)-4-hydroxy-3-methoxycyclorexyl]-1-methylethyl]-43-[[(4R,5R)-5-[(4R,5R)-5-(hydroxymethyl)-2,2-dimethyl- 1,3-dioxolan-4-yl]-2,2-dimethyl-1,3-dioxolan-4-yl]methoxy]-50-methoxy-34.35, 36,37,51,52-hexamethyl-72, 73-dioxa-62-azatricyclohexatriaconta-25,27,29(51),30(52)-tetraene-53,54, 55,56,57-pentone (500 mg, 0.437 mmol) in 1,4-dioxane (6 ml) and H ₂ O (6 ml), Dowex 50W-X8 (1.0 g) was added and the resulting mixture was stirred at 50°C for 24 hours, then filtered and purified using reverse phase chromatography (C18, CH ₃ CN :H2O=4:6), resulting in (21E,23E,25E,26E,30R,31S,32R,33R,35S,37S,40S,41R,42R,55R)-41,55-dihydroxy-40- [(1R)-2-[(1S,3R,4R)4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-39[( 2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexoxy]-70,71-dioxa-56-azatricyclohexatriaconta-21,23, 25(43),26(44)-tetraene-45, 46,47,48,49-pentone (I-63: 70 mg, 15% yield) as a white solid. ESI-MS (EI ⁺ , m/z): 1086.4 [M+Na]+. ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.51 - 5.91 (m, 4H), 5.61 - 4.98 (m, 5H), 4.24 (d, J=22.2 Hz, 1H), 3.78 (dd, J=34.1, 28.6 Hz, 9H), 3.59 - 3.17 (m, 19H), 3.00 - 2.49 (m, 5H), 2 ,38 2.22 (m, 1h), 2.05 (d, J=33.7 Hz, 3H), 1.82 - 1.68 (m, 11H), 1.29 (ddd, J=46, 4, 34.9, 8.8 Hz, 12H), 1.09 - 0.77 (m, 18H), 0.70 - 0.56 (m, 1H).

Step 6: synthesis of (21E,23E,25E,26E,30R,31S,32R,33R,35S,37S,39S,40S,41R,42R,55R)-41,55dihydroxy-40-[(1R)-2-[ (1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-42-methoxy-30,31, 32,33,43,44-hexamethyl-39-[(2R,3R,4R, 5R)-2,3,4,5,6-pentahydroxyhexoxy]-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48, 49-pentone (I-58) and (21E,23E,25E,26E,30R,31S, 32R,33R,35S,37S,39R,40S,41R,42R,55R)-41,55-dihydroxy-40-[ (1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-39-[ (2R,3R,4R,5R)-2,3,4,5,6pentahydroxyhexoxy]-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene45,46,47 ,48,49-pentons (I-57).

mg (21E,23E,25E,26E,30R,31S,32R,33R,35S,37S,40S,41R,42R,55R)-41,55-dihydroxy-40-[(1R)-2[(1S,3R ,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl39-[(2R,3R,4R,5R)-2,3 ,4,5,6-pentahydroxyhexoxy]-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone was purified using preparative chiral HPLC and the resulting epimers were purified by silica gel chromatography (hexane:DCM:ESAAc:McOH=3:3:1:1), resulting in (21E,23E,25E,26E,30R,31S,32R,33R ,35S,37S,39S, 40S,41R,42R,55R)-41,55-dihydroxy-40-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1 -methylethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-39-[(2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexoxy]70,71 -dioxa-56-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone (I-58: 15 mg, 17%) and (21E,23E ,25E,26E,30R,31S,32R,33R,35S,37S,39R,40S,41R,42R,55R)-41,55-dihydroxy-40-[(1R)2-[(1S,3R,4R) -4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl39-[(2R,3R,4R,5R)-2,3,4, 5,6-pentahydroxyhexoxy]-70,71-dioxa-56-αzatricyclohexatriaconta-21,23, 25(43),26(44)-tetraene-45,46,47,48,49-pentone (I-57: 8 mg, yield 9%), both as a white solid.

Chiral Analysis Method Column Column Size Introduction Mobile Phase Flow Rate Wavelength Temperature

CHIRALPAK IC-3(IC30CE-NJ008) 0.46 cm I.D. x 25 cm L 50.0 µl

Hexane/EUN=50/50(v/v) 0.8 ml/min

UV 254 nm oC

HPLC equipment: Shimadzu LC-20AT CP-HPLC-06

I-58: ESI-MS (EI ⁺ , m/z): 1086.3 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl3) δ 6.45 - 5.93 (m, 4H), 5.59 - 4.88 (m, 5H), 4.23 (d, J=26.4 Hz, 1H) , 4.00 - 3.48 (m, 14H), 3.46 -3.23 (m, 11H), 2.93 (d, J=6.8 Hz, 2n), 2.79 - 2.49 (m, 3H), 2.38 - 1.84 (m, 8H), 1.66 (t, J=14.8 Hz, 9H), 1.50-1.17 (m, 11H), 1, 16-0.79 (m, 18H), 0.64 (dd, J=23.7, 11.8 Hz, 1H). I-57: ESI-MS (EI ⁺ , m/z): 1086.3 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl3) δ 6.49-5.98 (m, 4H), 5.52-5.11 (m, 5H), 4.32-4.23 (m, 1H), 4, 08 - 3.11 (m, 14H), 3.05 - 2.43 (m, 9H), 2.37 - 1.95 (m, 8H), 1.91 1.53 (m, 17H), 1 .46 - 0.54 (m, 30N).

Example 34. Synthesis of (21E,23E,25E,26E,40R,41S,42R,43R,45S,47S,50S,51R,52R,61R)-51,61-dihydroxy-49-[2-[2-(2 -hydroxyethoxy)ethoxy]ethoxy]-50-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxycyclohexyl]-1 -methylethyl]-52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene-55,56, 57,58,59-pentone (I-78) and (21E,23E, 25E,26E,40R,41S,42R,43R,45S,47S,49R,50S,51R,52R,61R)-51,61-dihydroxy -49-[2-[2-(2-hydroxyeth

- 140 045993 c)ethoxy]ethoxy]-50-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxycyclohexyl] -1-methylethyl]-52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene- 55,56,57,58,59-pentone (I-72) and (21E,23E,25E,26E,40R,41S,42R,43R,45S, 47S,49S,50S,51R,52R,61R)-51 ,61-dihydroxy-49-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-50-[(1R)-2[(1S,3R,4R)-4-[2-[2-(2 -hydroxyethoxy)ethoxy]ethoxy]-3-methoxycyclohexyl]-1-methylethyl]-52-methoxy40,41,42,43,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-21,23,25( 53),26(54)-tetraene 55,56,57,58,59-pentone (I-73)

- 141 045993

Synthesis circuit

Methods and characteristics.

Step 1: synthesis of 2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethanol.

Tert-Butylchlorodiphenylsilane (30 g, 109.2 mmol) was added to a solution of 2-(2-hydroxyethoxy)ethanol (50 g, 471.2 mmol) in pyridine (49.6 mL) at 0°C. The resulting solution was stirred at room temperature for 1 hour, then poured into water (300 ml) and extracted with EtOAc (300 mlx3). The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The resulting residue was purified by chromatography on silica gel (EAAc:PE=1:8), resulting in 2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethanol (29.9 g, 80%) in in the form of a colorless oil. ESI-MS (EI ⁺ , m/z): 367.2 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 7.74 - 7.64 (m, 4H), 7.46 - 7.33 (m, 6H), 3.81 (t, J=5.2 Hz, 2H ), 3.73 - 3.66 (m, 2H), 3.63 - 3.54 (m, 4H), 2.32 (d, J=3.5 Hz, 1H), 1.06 (s, 9H).

Step 2: synthesis of 2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethyl trifluoromethanesulfonate.

To a solution of 2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethanol (7.6 g, 22 mmol) and DIPEA (5.76 ml)

- 142 045993 trifluoromethylsulfonyl-1,1-difluoroethanesulfonate (3.92 ml) was added to DCM (50 ml) at 0° C. under N2 atmosphere. The mixture was then diluted with DCM (150 ml), washed with saturated NaHCO ₃ solution (150 ml), water (150 ml) and brine (150 ml). Next, the organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo, resulting in 2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethyl trifluoromethanesulfonate (10.21 g, 97% yield) as brown oils which were used without any additional purification. ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.69-7.66 (m, 4H), 7.43-7.36 (m, 6H), 4.58 (t, J=4.8 Hz, 2H), 3.83-3.80 (m, 4H), 3.64-3.61 (t, J=5.6 Hz, 2H), 1.05 (s, 9H).

Stage 3: synthesis (35E,37E,39E,40E,50R,51S,52R,53R,55S,57S,59S,60S,61R,62R,71R)-60-[(1R)-2[(1S,3R, 4R)-4-[2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethoxy]-3-methoxycyclohexyl]-1methylethyl]-61,71-dihydroxy-59,62-dimethoxy- 50,51,52,53,63,64-hexamethyl-81,82-dioxa-73-azatricyclohexatriaconta-35,37,39(63),40(64)-tetraene-65,66,67,68,69- penton.

A mixture of everolimus (2 g, 2.09 mmol), 2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethyltrifluoromethanesulfonate (9.95 g, 20.87 mmol) and Na-ethyl-N-isopropyl-propane- 2-amine (5.82 ml) in toluene (50 ml) was stirred at 60°C for 18 hours, then poured into ice-cold saturated NaHCO3 solution (60 ml).

The reaction mixture was extracted with EtOAc (40 ml) and the organic layer was washed with water (50 mlx3) and brine (50 ml), and then dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The residue was purified by silica gel chromatography (PE:EA=5:1 to 3:1) to give (35E,37E,39E,40E,50R,51S,52R,53R,55S,57S,59S,60S, 61R,62R,71R)-60[(1R)-2-[(1S,3R,4R)-4-[2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethoxy] -3-methoxycyclohexyl]-1-methylethyl]-61,71-dihydroxy-59,62-dimethoxy-50,51,52,53,63,64-hexamethyl-81,82-dioxa-73azatricyclohexatriaconta-35,37,39 (63),40(64)-tetraene-65,66,67,68,69-pentone (1.7 g, 63% yield) as a yellow solid. ESI-MS (EI ⁺ , m/z): 1307.5 [M+Na] ⁺ .

Step 4: synthesis of (22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2 -[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxycyclohexyl]-1-methylethyl]-44,47-dimethoxy-35,36,37, 38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24, 26(48),27(49)-tetraene-50,51,52,53,54-pentone.

To solution (35E,37E,39E,40E,50R,51S,52R,53R,55S,57S,59S,60S,61R,62R,71R)-60-[(1R)-2[(1S,3R,4R) -4-[2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethoxy]-3-methoxycyclohexyl]-1methylethyl]-61,71-dihydroxy-59,62-dimethoxy-50, 51,52,53,63,64-hexamethyl-81,82-dioxa-73-azatricyclohexatriaconta-35,37,39(63),40(64)-tetraene-65,66,67,68,69-pentone ( 322 mg, 0.251 mmol) to THF (10 ml) was added HF-pyridine (248.5 mg, 2.51 mmol). The resulting solution was stirred at room temperature for 3 hours, then poured into saturated aqueous NaHCO ₃ and extracted with EtOAc. The organic layer was concentrated and purified by silica gel chromatography (acetone:PE=1:3) to obtain (22E,24E,26E,27E,35R,36S,37R,38R,40S, 42S,44S,45S,46R ,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3- methoxycyclohexyl]-1-methylethyl]-44,47-dimethoxy-35,36,37,38,48,49-hexamethyl-66,67dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)- tetraene-50,51,52,53,54-pentone (114 mg, 44% yield) as a light yellow solid. ESI-MS (EI ⁺ , m/z): 1069.3 [M+Na] ⁺ .

Step 5: synthesis of (21E,23E,25E,26E,40R,41S,42R,43R,45S,47S,50S,51R,52R,61R)-51,61-dihydroxy49-[2-[2-(2-hydroxyethoxy )ethoxy]ethoxy]-50-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxycyclohexyl]-1-methylethyl ] -52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene-55,56, 57,58,59-pentone (I-78).

Mixture of (22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,56R)-46,56-dihydroxy-45[(1R)-2-[(1S ,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxycyclohexyl]-1-methylethyl]44,47-dimethoxy-35,36,37,38,48,49 -hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48), 27(49)-tetraene-50,51,52,53,54-pentone (0.95 g, 0.908 mmol), 2-[2-(2-hydroxyethoxy)ethoxy]ethanol (2 ml) and p-toluenesulfonic acid (0.78 g, 4.54 mmol) in THF (20 ml) were stirred at 20°C for 2 hours. The reaction the mixture was poured into ice-cold saturated aqueous NaHCO3 (30 ml) and extracted with EtOAc (50 mlx3).

The combined organic layers were washed with water and brine, then concentrated and purified by reverse phase chromatography (C18, CH ₃ CN:H ₂ O=6.5:1), resulting in (21E,23E,25E,26E, 40R,41S,42R,43R,45S,47S,50S,51R,52R,61R)-51,61-dihydroxy-49-[2-[2-(2hydroxyethoxy)ethoxy]ethoxy]-50-[(1R)- 2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxycyclohexyl]-1-methylethyl]-52-methoxy-40,41,42,43 ,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene-55,56,57,58,59-pentone (I-78: 0.317 g, yield 30%) in the form of a white solid. ESI-MS (EI ⁺ , m/z): 1186.4 [M+Na] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.39-5.95 (m, 4H), 5.59-5.34 (m, 2H), 5.26-5.09 (m, 2H), 4.81 (s, 1H), 4.29-4.15 (m, 1H), 4.00-3.49 (m, 28H), 3.48-3.34 (m, 9H), 3, 16-3.0 (m, 4H), 2.80-2.52 (m, 3H), 2.34-2.20 (m, 2H), 2.07-1.88 (m, 4H), 1.79-1.72 (m, 5H), l.66(s, 3H), 1.51-1.38 (m, 4H), 1.37-1.22 (m, 7H), 1. 21-0.84 (m, 20H), 0.76-0.64 (m, 1H).

Step 6: synthesis of (21E,23E,25E,26E,40R,41S,42R,43R,45S,47S,49S,50S,51R,52R,61R)-51,61-dihydroxy-49-[2-[2- (2-hydroxyethoxy)ethoxy]ethoxy]-50-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)eth

- 143 045993 si]ethoxy]-3-methoxycyclohexyl]-1-methylethyl]-52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa62-azatricyclohexatriaconta-21,23,25( 53),26(54)-tetraene-55,56,57,58,59-pentone (I-73) and (21E,23E,25E, 26E,40R,41S,42R,43R,45S,47S,49R, 50S,51R,52R,61R)-51,61-gurugpoKCu-49-[2-[2-(2-rugpoKCU3TOKCu)3TOKsi]ethoxy]-50-[(1R)-2-[(1S,3R,4R) -4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxycyclohexyl]-1methylethyl]-52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa -62-azatricyclohexatriaconta21,23,25(53),26(54)-tetraene-55,56,57,58,59-pentone (I-72):

330 mg (21E,23E,25E,26E,40R,41S,42R,43R,45S,47S,50S,51R,52R,61R)-51,61-gurugpoKCu-49-[2-[2-(2rugpoKCU3TOKCu)3TOKCu ]3TOKCu]-50-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-rugpoKCU3TOKCu)3TOKCu]3TOKCu]-3methoxycyclohexyl]-1-methylstil]-52- methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene-55,56,57,58, The 59-pentone was purified using the preparative yu chiral HPLC and the resulting 3-pimers were purified using chromatography on silica gel (reKsan:DCM:EAAc:MeOH = 3:3:1:1), resulting in crushing (21E,23E,25E,26E, 40R,41S,42R,43R,45S ,47S,49S,50S,51R,52R,61R)-51,61-gurugpoKCu-49-[2-[2-(2-rugpoKCU3TOKCu)3TOKCu]3TOKCu]-50-[ (1R)-2[(1S,3R,4R)-4-[2-[2-(2-hydroxystoxy)htoxy]htoxy]-3-methoxycyclorexyl]-1-methylstil]-52-methoxy40,41,42, 43,53,54-rexamethyl-72,73-dioxa-62-azatricyclorexatriaconta-21,23,25(53),26(54)-tetraene 55,56,57,58,59-pentone (I-73: 77 mg , ebixog 23%) and (21E,23E,25E,26E,40R,41S,42R,43R,45S,47S,49R,50S, 51R,52R,61R)-51,61-dihydroxy-49-[2-[ 2-(2-hydroxystoxy)ztoxy]ztoxy]-50-[(1R)-2-[(1S,3R,4R)-4-[2[2-(2-hydroxystoxy)ztoxy]ztoxy]-3-methoxycyclorexyl ]-1-methylstil]-52-methoxy-40,41,42,43,53,54rexamethyl-72,73-dioxa-62-azatricyclorexatriaconta-21,23,25(53),26(54)-tetraene-55 ,56,57,58,59pentone (I-72: 50 mg, ebixog 15%), both in the form of a white solid.

Cnocoo chiral division column

Column size

Solution Concentration Introduction

Mobile phase Flow rate Wavelength Temperature

CHIRALPAK IC

2.5 cm I D. x 25 cm L

6.5 mg/ml ml

Hexane/EUN=60/40 (v/v) ml/min

UV 254 nm °C

I-73: ESI-MS (EI ⁺ , m/z): 1186.6 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 6.42 - 6.06 (m, 3H), 5.92 (dd, J=29.5, 10.8 Hz, 1H), 5.55 - 5.37 (m, 2H), 5.26 (d, J=5.2 Hz, 1H), 5.14 (d, J=4.3 Hz, 1H), 4.80 (s, 1H), 4.23 (d, J=24.9 Hz, 1H), 3.90 (s, 1H), 3.81 - 3.22 (m, 36H), 3.19 - 2.99 (m, 3H), 2, 78 - 2.48 (m, 3H), 2.33 (d, J=12.4 Hz, 2H), 2.10 - 1.87 (m, 5H), 1.76 - 1.55 (m, 13H), 1.46 (s, 4H), 1.39 - 1.18 (m, 5H), 1.15 - 0.81 (m, 18H), 0.71 (dd, J=23.5, 11.7 Hz, 1H).

I-72: 1H NMR (400 MHz, CDCl ₃ ) δ 6.42 - 5.92 (m, 4H), 5.61 - 5.04 (m, 5H), 4.24 (d, J=53, 9 Hz, 2H), 3.99 (dd, J=13.8, 6.9 Hz, 1H), 3.83 - 2.90 (m, 36H), 2.75 - 2.46 (m, 3H ), 2.16 (ddd, J=109.4, 53.2, 24.9 Hz, 7H), 1.86 - 1.69 (m, 7H), 1.52 - 1.16 (m, 17H ), 1.15-0.80 (m, 18H), 0.66 (dd, J=23.9, 11.6 Hz, 1H).

Example 35. Synthesis of (21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[ (1S,3R,4R)-4-hydroxy-3-methoxycyclorexyl]-1-methylstil]-45-methoxy-33,34,35,36, 46,47-rexamethyl-42-[2-[2-(2 ,2,2-trifluoroztoxy)ztoxy]ztoxy]-65,66-dioxa-56-azatricyclorexatriaKOirra-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone ( I-91)

- 144 045993

Synthesis circuit

Methods and characteristics.

Step 1: Synthesis of 2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxymethylbenzene: To a suspension of sodium hydride (12.49 g, 520.5 mmol) in DMF (150 ml) was added 2-(2,2 ,2-trifluoroethoxy)ethanol (5 g, 34.7 mmol) in DMF (10 ml) under N ₂ at 0°C. The mixture was stirred for 1 hour, then 2-bromoethoxymethylbenzene (18.66 g, 86.75 mmol) was added dropwise. The mixture was stirred at room temperature for 20 hours, then quenched with water (50 ml) and extracted with EtOAc (80 ml). The organic layer was washed with water (50 mlx3), brine (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The residue was purified by silica gel chromatography (PE:EA=25:1 to 20:1) to give 2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxymethylbenzene (8.1 g, yield 84 %) in the form of a colorless liquid.

1H NMR (400 MHz, CDCl ₃ ): δ 7.37 - 7.26 (m, 5H), 4.57 (s, 2H), 3.90 (q, J=8.8 Hz, 2H), 3 .79 (dd, J=5.6, 3.5 Hz, 2H), 3.71 - 3.61 (m, 6H).

Step 2: synthesis of 2-[2-(2,2,2-trifluoroethoxy)ethoxy].

To a solution of 2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxymethylbenzene (0.5 g, 1.8 mmol) in CH ₃ OH (10 ml) was added Pd/C (0.43 g) and the reaction the mixture was stirred under H ₂ atmosphere at room temperature (20° C.) for 20 hours. Pd/C was removed by filtration and the resulting filtrate was concentrated and purified by silica gel chromatography (DCM:CH ₃ OH = 50:1), resulting in which gave 2-[2(2,2,2-trifluoroethoxy)ethoxy]ethanol (0.30 g, 89% yield) as a colorless oil. 1H NMR (400 MHz, CDCl ₃ ) δ 3.91 (q, J=8.7 Hz, 2H), 3.80 (dd, J=5.6, 3.4 Hz, 2H), 3.75 ( d, J=4.0 Hz, 2H), 3.70 (dd, J=5.5, 3.5 Hz, 2H), 3.62 (dd, J=5.2, 3.9 Hz, 2H ), 2.23 (t, J=5.7 Hz, 1H (OH)). ¹⁹ F NMR (376 MHz, CDCl3, (trifluoromethylbenzene as standard) S - 74.33 (t, J=8.8 Hz).

Step 3: synthesis of (21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy43-[(1R)-2-[(1S ,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47hexamethyl-42-[2-[2-(2,2,2 -trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone.

To degassed solution (22E,24E,26E,27E,29R,30S,31R,32R,34S,36S,38S,39S,40R,41R,50R)40,50-dihydroxy-39-[(1R)-2-[ (1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-38,41dimethoxy-29,30,31,32,42,43-hexamethyl-60,61-dioxa-51-azatricyclohexatriaconta- 22,24,26(42),27(43)tetraene-44,45,46,47,48-pentone (0.1 g, 0.11 mmol) in THF (5 ml) at 0°C was added p- toluenesulfonic acid (94 mg, 0.547 mmol) and 2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethanol (0.2 g, 1.09 mmol). The resulting mixture was stirred at 23°C for 5 hours under N ₂ atmosphere, then poured into saturated NaHCO ₃ solution (40 ml), and then subjected to extraction using EtOAc (30 ml). The organic layer was washed with water (30 mlx2), brine (20 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The residue was purified by reverse phase chromatography ( _C18 ,

CH ₃ CN:H ₂ O=0% to 65% yield, resulting in (21E,23E,25E,26E,33R,34S,35R,36R, 38S,40S,43S,44R,45R,54R)- 44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]1-methylethyl]-45-methoxy-33,34,35,36,46 ,47-hexamethyl-42-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)- tetraene-48,49,50,51,52-pentone (I-91: 30

- 145 045993 mg, yield 26%) in the form of a white solid.

ESI-MS (EI+, m/z): 1093.5 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 6.38-5.90 (m, 4H), 5.46-5.05 (m, 4H), 4.5-4.4 (m, 1H), 4 .21-4.11 (m, 2H), 3.91-3.52 (m, 6H), 3.34-3.25 (m, 8H), 3.07 (s, 1H), 2.84 -2.59 (m, 5H), 2.31-1.91 (m, 6H), 1.77-1.54 (m, 22H), 1.43-1.19 (m, 10H), 1 .04-0.80 (m, 16H), 0.60 (q, J=12 Hz, 1H).

Example 36. Synthesis of (23E,25E,27E,28E,36R,37S,38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy-48-methoxy-45-[2-( 2-methoxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methylethyl]-36,37,38, 39,49,50-hexamethyl-66,67-dioxa-58-azatricyclohexatriaconta-23,25, 27(49),28(50)-tetraene-51,52,53,54,55-pentone (I-92) and (23E,25E,27E,28E,36R,37S,38R,39R,41S,43S, 45S,46S,47R,48R,57R)-47,57-dihydroxy-48-methoxy-45-[2-(2 -methoxyethoxy)ethoxy]-46-[(1R)-2[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methylethyl]-36,37,38,39, 49,50-hexamethyl66,67-dioxa-58-azatricyclohexatriaconta-23,25,27(49),28(50)-tetraene-51,52,53,54,55-pentone (I-90) and (23E, 25E,27E,28E,36R,37S,38R,39R,41S,43S,45R,46S,47R,48R,57R)-47,57-dihydroxy-48-methoxy-45-[2(2-methoxyethoxy)ethoxy] -46-[(1 R)-2-[(1 S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1methylethyl]-36,37,38,39,49,50- hexamethyl-66,67-dioxa-58-azatricyclohexatriaconta-23,25,27(49), 28(50)-tetraene-51,52,53,54,55-pentone (I-89)

- 146 045993

Synthesis circuit

Methods and characteristics.

Step 1: Synthesis of 2-methoxyethyl trifluoromethanesulfonate: A solution of 2-methoxyethanol (3 g, 39.42 mmol) and DIPEA (10.30 ml, 59.14 mmol) in DCM (60 ml) was cooled to 0°C under N ₂ and trifluoromethanesulfonic acid anhydride (7.28 mL, 43.37 mmol) was added dropwise. The mixture was stirred at 0°C for 2 hours, then diluted with DCM (50 ml). The organic layer was washed with saturated NaHCO ₃ solution (50 ml), water (50 ml), brine (50 ml), then dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo to obtain 2methoxyethyl trifluoromethanesulfonate as a brown oil . It was used in the next step without further purification. ^1H NMR (400 MHz, CDCl ₃ ) δ 4.62 - 4.58 (t, J=4.4 Hz 2H), 3.70 - 3.65 (t, J=4.4 Hz, 2H), 3.39(s, 3H).

Step 2: synthesis of (23E,25E,27E,28E,32R,33S,34R,35R,37S,39S,41S,42S,43R,44R,53R)-43,53-dihydroxy-41,44-dimethoxy-42- [(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methylethyl]-32,33,34,35,45,46-hexamethyl-62 ,63-dioxa-54-azatricyclohexatriaconta-23,25,27(45),28(46)tetraene-47,48,49,50,51-pentone.

A mixture of rapamycin (2 g, 2.19 mmol), 2-methoxyethyltrifluoromethanesulfonate and U-ethyl-U-isopropylpropan-2-amine (6.48 ml, 37.19 mmol) in toluene (60 ml) was stirred at 58°C in for 18 hours, then diluted with EtOAc (100 ml), poured into ice-cold saturated NaHCO ₃ solution (150 ml), washed twice with ice water (250 ml), saline (200 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The crude product was purified by silica gel chromatography (acetone:PE=1:6) to give (23E,25E,27E,28E,32R,33S,34R,35R,37S,39S,41S,42S,43R,44R ,53R)-43,53-dihydroxy-41,44-dimethoxy-42-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1- methylethyl]-32,33,34,35,45,46-hexamethyl-62,63-dioxa-54-azatricyclohexatriaconta23,25,27(45),28(46)-tetraene-47,48,49,50,51 -pentone (0.53 g, 25% yield) as a light brown solid. ESI-MS (EI+, m/z): 994.5 [M+Na]+. 1H NMR (400 MHz, CDC13) δ 6.42-6.11 (m, 3H), 5.96 (m, 1H), 5.58-5.40 (m, 2H), 5.29-5, 14 (m, 2H), 4.80 (s, 1H), 4.18 (m, 1H), 3.75-3.64 (m, 5H), 5.59-3.50 (m, 3H) , 3.46-3.43 (m, 4H), 3.38-3.30 (m, 6H), 3.17-3.05 (m, 4H), 2.80-2.56 (m, 3H), 2.06-1.92 (m, 4H), 1.86-1.75 (m, 6H), 1.69-1.59 (m, 10H), 1.51-1.42 ( m, 5H), 1.31-1.15 (m, 8H), 1.11-1.04 (m, 6H), 1.00-0.83 (m, 10H), 0.72(q, J=12 Hz, 1H).

Step 3: synthesis of (23E,25E,27E,28E,36R,37S,38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy48-methoxy-45-[2-(2- methoxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclo

- 147 045993 hexyl]-1-methylethyl]-36,37,38,39,49,50-hexamethyl-66,67-dioxa-58-azatricyclohexatriaconta-23.25, 27(49),28(50)-tetraene -51,52,53,54,55-pentons (I-92):

Solution (23E,25E,27E,28E,32R,33S,34R,35R,37S,39S,41S,42S,43R,44R,53R)-43,53-gurugpoKCu-41,44dimethoxy-42-[(1R)- 2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methylethyl]-32,33,34,35, 45,46-hexamethyl-62,63-dioxa- 54-Azatricyclohexatriaconta-23,25,27(45),28(46)-tetraene-47,48,49,50,51-pentone (0.45 g, 0.463 mmol) in THF (25 ml) was degassed with N2, 4methylbenzenesulfonic acid (0.4 g, 2.31 mmol) was added at 0°C, then 2-(2-methoxyethoxy)ethanol (4 ml). The resulting mixture was stirred at 0°C for 0.5 h under N2, then at 25°C for 3 h. The mixture was poured into ice-cold saturated NaHCO ₃ solution (50 ml), extracted with EtOAc (80 mlx2), the organic layer was washed with water (100 ml), brine (100 ml) and concentrated in vacuo. The residue was purified by reverse phase chromatography (C18, CH ₃ CN:H ₂ O from 10%~75% yield), resulting in (23E,25E,27E,28E,36R,37S,38R,39R,41S, 43S,46S,47R,48R,57R)-47,57-dihydroxy-48-methoxy-45-[2-(2-methoxyethoxy)ethoxy]-46-[(1R)-2[(1S,3R,4R) -3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methylethyl]-36,37,38,39,49,50-hexamethyl66,67-dioxa-58-azatricyclohexatriaconta-23,25,27(49) ,28(50)-tetraene-51,52,53,54,55-pentone (I-92: 0.11 g, 22% yield) as a white solid. ESI-MS (EI ⁺ m/z): 1082.5 [M+Na] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.32-5.99 (m, 3H), 5.89-5.78 (m, 1H), 5.48-5.07 (m, 4H), 4.70-4.51 (m, 1H), 4.20-4.10 (m, 1H), 3.83-3.76 (m, 1h), 3.73-3.62 (m, 3H ), 3.57-5.43 (m, 8H), 3.41-3.37 (m, 4H), 3.35-3.21 (m, 12H), 3.14-3.97 (m , 3H), 2.68-2.45 (m, 3H), 2.24 (m, 2H), 1.99-1.81 (m, 4H), 1.68-1.52 (m, 15H ), 1.44-1.34 (m, 4H), 1.26-1.14 (m, 5H), 1.05-0.94 (m, 8H), 0.92-0.77 (m , 10H), 0.65 (q, J=12 Hz, 1H).

Step 4: synthesis of (23E,25E,27E,28E,36R,37S,38R,39R,41S,43S,45S,46S,47R,48R,57R)-47,57-dihydroxy-48-methoxy-45-[2 -(2-Methoxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methylethyl]-36.37, 38,39,49,50-hexamethyl-66,67-dioxa-58-azatricyclohexatriaconta23,25,27(49),28(50)-tetraene-51,52,53,54,55-pentone (I-90) and (23E,25E,27E,28E,36R,37S,38R,39R,

41S,43S,45R,46S,47R,48R,57R)-47,57-dihydroxy-48-methoxy-45-[2-(2-Methoxyethoxy)ethoxy]-46-[(1R)-2[(1S, 3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methylethyl]-36,37,38,39,49,50-hexamethyl66,67-dioxa-58-azatricyclohexatriaconta-23,25, 2 7(49),28(50)-tetraene-51,52,53,54,55-pentone (I-89):

1.16 g (23E,25E,27E,28E,36R,37S, 38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy-48-methoxy45-[2-(2- methoxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methylethyl]-36,37,38,39, 49,50-hexamethyl-66,67-dioxa-58-azatricyclohexatriaconta-23,25,27(49),28(50)tetraene-51,52,53,54,55-pentone was purified using preparative chiral HPLC and the resulting epimers were purified by silica gel chromatography (hexane:

DCM:EtOAc:MeOH=3:3:1:0.3), resulting in (23E,25E,27E,28E,36R,37S,38R, 39R,41S,43S,45S,46S,47R,48R, 57R)-47,57-dihydroxy-48-methoxy-45-[2-(2-methoxyethoxy)ethoxy]-46[(1 R)-2-[(1 S,3R,4R)-3 -methoxy-4 -(2-methoxyethoxy)cyclohexyl]-1-methylethyl] -36,37,3 8,39,49,50-hexamethyl-66,67-dioxa-58-azatricyclohexatriaconta-23,25,27(49),28( 50)-tetraene-51,52,53,54,55-pentone (I90: 340 mg, 28% yield) and (23E,25E,27E,28E,36R,37S,38R,39R,41S,43S,45R, 46S,47R,48R,57R)-47,57dihydroxy-48-methoxy-45-[2-(2-methoxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-3- methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methylethyl]-36,37,38,39,49,50-hexamethyl-66,67-dioxa-58-azatricyclohexatriaconta-23,25,27(49), 28(50)-tetraene-51,52,53,54,55-pentone (I-89: 135 mg, 11% yield), both as a white solid.

Chiral separation method Column Column size Solution concentration Introduction

Mobile phase Flow rate Wavelength Temperature

CHIRALPAK IC

2.5 cm I.D. x 25 cm L

2.5 mg/ml ml

Hexane/EUN=60/40 (v/v) ml/min

UV 254 nm °C

I-90: ESI-MS (EI ⁺ , m/z): 1082.4 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 6.40 -6.22 (m, 2H), 6.14 (dt, J=15.0, 9.8 Hz, 1H), 5.90 (dd, J =32.4, 10.7 Hz, 1H), 5.56 - 5.32 (m, 2H), 5.27 (d, J=5.0 Hz, 1H), 5.16 (d, J= 4.2

Hz, 1H), 4.77 (s, 1H), 4.18 (d, J=5.9 Hz, 1H), 3.87 (s, 1H), 3.80 - 3.24 (m, 29H ), 3.22 - 3.01 (m, 3H), 2.72 (dd, J=16.9, 5.8 Hz, 2H), 2.57 (dd, J=16.8, 6.4 Hz, 1H), 2.30 (t, J=21.1 Hz, 2H), 1.93 (ddd, J=26.1, 21.2, 9.8 Hz, 6H), 1.82 - 1 .64 (m, 8H), 1.50 (dd, J=22.1, 10.9 Hz, 6H), 1.37 - 1.15 (m, 6H), 1.15 - 0.82 (m , 18H), 0.71 (q, J=8.0, 20.0 Hz, 1H).

I-89: ESI-MS (EI ⁺ , m/z): 1082.4 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 6.41 - 5.93 (m, 4H), 5.59 - 5.37 (m, 2H), 5.20 (dd, J=23.8, 19, 2 Hz, 2H), 4.55 (d, J=10.4 Hz, 1H), 4.28 (s, 1H), 4.15 (d, J=10.2 Hz, 1H), 4.00 (d, J=3.7 Hz, 1H), 3.91 - 3.27 (m, 28H), 3.27 - 2.85 (m, 5H), 2.76 - 2.24 (m, 5H ), 2.18 - 1.55 (m, 14H), 1.54 - 1.20 (m, 10H), 1.16 -0.82 (m, 18H), 0.75 - 0.61 (m , 1H).

Example 37. Synthesis of (21E,23E,25E,26E,35R,36S,37R,38R,40S,42S,45S,46R,47R,56R)-46,56-dihydroxi-45-|(1R)-2- |(1 S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy- 148 045993

35,36,37,38,48,49-hexamethyl-44-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23, 25(48),26(49)-tetraene-50,51,52,53,54-pentone (I-86) and (21E,23E,25E,26E,35R,

36S,37R,38R,40S,42S,44S,45S,46R,47R,56R)-46,56-gurugpoKCu-45-[(1R)-2-[(1S,3R,4R)-4-(2- rugpoKCuethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-44-[2-[2-(2,2,2trifluoroethoxy)ethoxy]ethoxy] -67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(48),26(49)-tetraene50,51,52,53,54-pentone (I-85)

1-85

Synthesis circuit

1-84

- 149 045993

Methods and characteristics.

Step 1: Synthesis of 2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxymethylbenzene: To a suspension of sodium hydride (12.49 g, 520.5 mmol) in DMF (150 ml) was added 2-(2,2 ,2-trifluoroethoxy)ethanol (5 g, 34.7 mmol) in DMF (10 ml) under N ₂ at 0°C. The mixture was stirred at 0°C for 1 hour, then bromoethoxymethylbenzene (18.66 g, 86.75 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 20 hours. The mixture was quenched with water (50 ml) and subjected to extraction using EtOAc (80 ml). The organic layer was washed with water (50 mlx3), brine (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The residue was purified by silica gel chromatography (PE:EAAc=25:1 to 20:1) to give 2-[2-(2,2,2trifluoroethoxy)ethoxy]ethoxymethylbenzene (8.1 g, yield 83.9 %) in the form of a colorless liquid. 1H NMR (400 MHz, CDCl·/): δ 7.37 - 7.26 (m, 5H), 4.57 (s, 2H), 3.90 (q, J=8.8 Hz, 2H), 3.79 (dd, J=5.6, 3.5 Hz, 2H), 3.71 - 3.61 (m, 6H).

Step 2: synthesis of 2-[2-(2,2,2-trifluoroethoxy)ethoxy].

Pd/C (436.45 mg) was added to a solution of 2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxymethylbenzene (0.5 g, 1.80 mmol) in CH ₃ OH (10 ml). The resulting mixture was then stirred under H2 at room temperature for 20 hours, filtered and the filtrate was concentrated and purified by silica gel chromatography (DCM: _CH3OH =50:1) to give 2-[2-(2. 2,2trifluoroethoxy)ethoxy]ethanol (0.30 g, 89% yield) as a colorless oil. 1H NMR (400 MHz, CDCl ₃ ) δ 3.91 (q, J=8.7 Hz, 2H), 3.80 (dd, J=5.6, 3.4 Hz, 2H), 3.75 ( d, J=4.0 Hz, 2H), 3.70 (dd, J=5.5, 3.5 Hz, 2H), 3.62 (dd, J=5.2, 3.9 Hz, 2H ), 2.23 (t, J=5.7 Hz, 1H (OH)). ¹⁹ F NMR (376 MHz, CDCl ₃ , (trifluoromethyl)benzene as standard) 8 -74.33 (t, J=8.8 Hz).

Step 3: synthesis of (21E,23E,25E,26E,35R,36S,37R,38R,40S,42S,45S,46R,47R,56R)-46,56-dihydroxy45-[(1R)-2-[(1S ,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-35,36,37, 38,48,49-hexamethyl-44-[2-[2- (2,2,2-trifluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(48),26(49)-tetraene-50,51,52,53,54- penton (I-86).

A solution of everolimus (0.5 g, 0.52 mmol) in THF (5 ml) was degassed, p-toluenesulfonic acid (0.45 g, 2.61 mmol) was added at 0°C, then 2-[2-( 2,2,2-trifluoroethoxy)ethoxy]ethanol (0.98 g, 5.22 mmol). The resulting mixture was stirred at 0°C for 0.5 h under N2, then at 23°C for 6 h, poured into saturated NaHCO ₃ solution (40 ml) and extracted with EtOAc (30 ml). The organic layer was washed with water (30 mlx2), brine (40 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by reverse phase chromatography (C18, yield CH ₃ CN:H ₂ O from 0% to 70%), resulting in (21E,23E,25E,26E,35R,36S,37R,38R,40S, 42S,45S,46R,47R,56R)46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl ]-47methoxy-35,36,37,38,48,49-hexamethyl-44-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta- 21,23,25(48),26(49)-tetraene-50,51,52,53,54-pentone (I-86: 0.08 g, 14% yield) as a white solid. ESI-MS (EI ⁺ , m/z): 1136.5 [M+Na]+. 1H NMR (400 MHz, CDCl ₃ ): δ 6.44-5.88 (m, 4H), 5.73-5.06 (m, 4H), 4.52-4.32 (m, 1H), 4.22-4.12 (m, 1H), 3.91-3.81 (m, 2H), 3.71-3.51 (m, 6H), 3.42-3.21 (m, 16H ), 3.13-2.98 (m, 4H), 2.63-2.42 (m, 4H), 2.32-2.14 (m, 2H), 2.05-1.93 (m , 3H), 1.86-1.55 (m, 16H), 1.44-1.35 (m, 4H), 1.24-1.15 (m, 5H), 1.06-0.78 (m, 17H), 0.65-0.51 (m, 1H).

Step 4: synthesis of (21E,23E,25E,26E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2 -[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy35,36,37,38,48,49-hexamethyl-44-[2-[ 2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(48),26(49)-tetraene-50,51,52,53, 54-pentone (I-85):

100 mg (21E,23E,25E,26E,35R,36S,37R,38R,40S,42S,45S, 46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2[(1S, 3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-44-[2-[2-( 2,2,2-trifluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23, 25(48),26(49)-tetraene-50,51,52,53,54-pentone purified by preparative chiral HPLC to give (21E,23E,25E,26E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,56R)-46,56dihydroxy-45-[ (1R)-2-[(1 S,3R,4R)-4-(2-hydroxyethoxy)-3 -methoxycyclohexyl]-1 -methylethyl]-47 -methoxy-35,36,37,38,48,49- hexamethyl-44-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(48),26(49)-tetraene- 50,51,52,53,54-pentone (I-85: 14.3 mg, 14.3% yield) as a white solid.

Chiral separation method

Column CHIRALPAK IC Column size 5.0 cm ID. x 25 cm L Solution concentration 2.4 mg/ml Introduction 5 ml Mobile phase Hexane/EUN=70/30(v/v) Flow rate 30 ml/min Wavelength UV 254 nm

Temperature: 35°C

- 150 045993

I-85: ESI-MS (EI ⁺ , m/z): 1136.4 [M+Na] ⁺ . ¹ H NMR (400 MHz, CDCI3) δ 6.42 - 6.06 (m, 3H), 5.92 (dd, J=30.3, 10.3 Hz, 1H), 5.56 - 5.06 (m , 5H), 4.74 (s, 1H), 4.18 (d, J=5.7 Hz, 1H), 3.94 - 3.83 (m, 2H), 3.82 - 3.51 (m, 12H), 3.49 - 3.25 (m, 11H), 3.22 - 3.03 (m, 2H), 2.72 (dd, J=16.6, 5.5 Hz, 2H), 2 .57 (dd, J=17.0, 6.5 Hz, 1H), 2.34 (d, J=12.4 Hz, 2H), 2.25-2.18 (m, 1H), 2, 13 - 1.85 (m, 5H), 1.69 (dd, J=35.2, 8.9 Hz, 10H), 1.47 (dd, J=20.5, 13.6 Hz, 5H) , 1.26 (s, 7H), 1.15-0.81 (m, 18H), 0.71 (dd, J=23.9, 12.0 Hz, 1H).

Example 38 Synthesis of (21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[ (1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35, 36, 46,47-hexamethyl-42-[2-[2-(2 ,2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone ( I-91) and (21E,23E,25E,26E,33R,34S,35R,36R, 38S,40S,42S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)- 2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-[2 -(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52 -pentone (1

85) and (21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,42R,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2[ (1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl42-[2-[2-(2,2 ,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone

- 151 045993

Methods and characteristics.

Step 1: synthesis of 2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethanol.

2-[2-(2,2,2-Trifluoroethoxy)ethoxy]ethanol was the same as in Example 37.

Step 2: synthesis of (21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy43-[(1R)-2-[(1S ,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47hexamethyl-42-[2-[2-(2,2,2 -trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone (I-91).

A solution of rapamycin (0.5 g, 0.547 mmol) in DCM (20 ml) was degassed at -40°C and trifluoroacetic acid (1.67 ml) was added. After stirring for 10 minutes, 2-[2-(2,2,2trifluoroethoxy)ethoxy]ethanol (0.2 g, 1.09 mmol) was added. The mixture was stirred at -40°C for an additional 40 minutes, then poured into ice-cold NaHCO ₃ (60 ml aq), washed with water (20 ml), brine (20 ml), dried over MgSO ₄ , filtered and concentrated. The residue was purified by reverse phase chromatography (C18, CH ₃ CN:H ₂ O=65:35), resulting in (21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S ,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33 ,34,35,36,46,47-hexamethyl-42-[2-[2(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25( 46),26(47)tetraene-48,49,50,51,52-pentone (I-91: 85 mg, 15% yield) as a white solid. ESI-MS (EI+, m/z): 1092.4 [M+Na]+. ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.45 - 5.76 (m, 4H), 5.40 (ddd, J=24.9, 15.2, 8.0 Hz, 2H), 5, 25 - 4.99 (m, 2H), 4.57 - 4.01 (m, 3H), 3.98 - 3.45 (m, 7H), 3.43 - 2.99 (m, 11H), 2.95 - 2.37 (m, 6H), 2.26 (d, J=13.9 Hz, 2H), 2.08 - 1.76 (m, 6H), 1.75-1.52 ( m, 14 N) 1.48 - 1.10 (m, 10H), 1.07 - 0.74 (m, 18H), 0.60 (dd, J=23.5, 12.0 Hz, 1H) .

Step 3: synthesis of (21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,42R,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2 -[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36, 46,47-hexamethyl-42-[2-[2- (2,2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52- penton (I-126):

159 mg (21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2[(1S, 3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl42-[2-[2-(2,2,2- trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone was purified using preparative chiral HPLC , resulting in (21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,42S,43S,44R,45R,54R)-44,54-dihydroxy43-[(1R)-2- [(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47hexamethyl-42-[2-[2-(2, 2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone (I-125 : 48.5 mg, 30% yield) and (21E,23E,25E,26E, 33R,34S,35R,36R,38S,40S,42R,43S,44R,45R,54R)-44,54-dihydroxy-43 -[(1R)-2-[(1S,3R,4R)-4-hydroxy-3methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[ 2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50 , 51,52-pentone (I-126:43 mg, 27% yield) as a white solid.

Chiral separation method

Column

Column size

Solution Concentration Introduction

Mobile phase Flow rate Wavelength Temperature

CHIRALPAK IC

5.0 cm I.D. x 25 cm L

3.0 mg/ml ml

Hexane/EUN=70/30(v/v) ml/min

UV 254 nm °C

I-125: ESI-MS (EI ⁺ , m/z): 1092.4 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 6.36 - 6.13 (m, 2H), 6.06 (dd, J=15.0, 10.0 Hz, 1H), 5.85 (dd, J =29.4, 10.8 Hz, 1H), 5.49 - 5.37 (m, 1H), 5.33 (d, J=10.0 Hz, 1H), 5.20 (d, J= 4.6 Hz, 1H), 5.08 (t, J=11.7 Hz, 1H), 4.68 (s, 1H), 4.11 (d, J=5.8 Hz, 1H), 3 .94 - 3.64 (m, 7H), 3.63 - 3.46 (m, 5H), 3.42 - 3.18 (m, 12H), 2.93 - 2.79 (m, 2H) , 2.71-2.43 (m, 4H), 2.27 (d, J=11.8 Hz, 2H), 2.08-1.85 (m, 6H), 1.83 1.61 ( m, 11H), 1.48-1.21 (m, 8H), 1.08 - 0.74 (m, 18H), 0.65 - 0.54 (m, 1H).

I-126: ESI-MS (EI ⁺ , m/z): 1092.4 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 6.41 - 6.07 (m, 3H), 6.00 5.81 (m, 1H), 5.56 - 5.05 (m, 4H), 4, 75 (s, 1H), 4.18 (d, J=5.8 Hz, 1H), 3.98 - 3.52 (m, 11H), 3.50 - 3.22 (m, 12H), 2 .95 (d, J=8.6 Hz, 1H), 2.77 - 2.50 (m, 4H), 2.38 - 2.16 (m, 2H), 2.12 - 1.83 (m , 5H), 1.69 (dd, J=39.3, 11.0 Hz, 12H), 1.49 - 1.17 (m, 11H), 1.15-0.80 (m, 18H), 0.74 - 0.60 (m, 1H).

Example 39. Synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[ (1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34.35, 36,37,47,48-hexamethyl-43-[2-[ 2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I- 88) and (21E,23E,25E,26E,34R,35S,36R,37R, 39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2- [(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2- [2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pento

- 152 045993 on (1-82) and (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-44- [(1R)2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48hexamethyl-43 -[2-[2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-83)

Scheme for the synthesis of p-TsOH, THF

О °С-25 °C, 6 h

Pd/C, Ho, CH ₃ OH

BUT

Py(HF)g, DCM

-78°С~-50°С,2h II

39%

Methods and characteristics.

Step 1: synthesis of O-[2-(2-benzyloxyethoxy)ethyl]methylsulfanylmethanethioate.

- 153 045993

2-(2benzyloxyethoxy)ethanol (12 g, 61.2 mmol) and benzyl(triethyl) ammonium chloride (1.0 g, 4.87 mmol) were added to a 1000 mL two-neck round-bottom flask equipped with a magnetic stirrer. 50% aqueous sodium hydroxide solution (141 ml) was added through a dropping funnel. The mixture was then stirred for 10 min, CS ₂ (141 mL, 2.34 mol) followed by iodomethane (22.0 g, 154 mmol) was added dropwise. The mixture was stirred for 3 hours at room temperature. Water (100 ml) was added. The organic layer was removed and the aqueous phase was subjected to extraction with CH ₂ Cl ₂ (100 mlx3). The combined organic layers were washed with brine (2x100 ml), dried over MgSO ₄ filtered and concentrated. The residue was purified by silica gel chromatography (SAAc:PE=1:3) to give O-[2-(2-benzyloxyethoxy)ethyl]-methylsulfanylmethanethioate (16.8 g, 95% yield) as a yellow oil. ESI-MS (EI ⁺ , m/z): 308.9 [M+Na] ⁺ . ¹ H NMR (500 MHz, DMSO-de) δ 7.32 (dt, J=18.3, 6.8 Hz, 5H), 4.74 - 4.62 (m, 2H), 4.50 (s , 2H), 3.85 - 3.72 (m, 2H), 3.60 (ddd, J=8.7, 6.2, 3.6 Hz, 4H), 2.56 (s, 3H).

Step 2: Synthesis of 2-[2-(trifluoromethoxy)ethoxy]ethoxymethylbenzene: To a suspension of 1,3-dibromo-5,5dimethyl-imidazolidine-2,4-dione (29.95 g, 104.75 mmol) in DCM (150 ml) (HF) ₉ /Py pyridinium poly(hydrogen fluoride) (49.36 ml, 209.49 mmol) and O-[2-(2-benzyloxyethoxy)ethyl]methylsulfanylmethanethioate (10 g, 34.92 mmol) were added at -78 °C, and the mixture was stirred at -50°C for 2 hours. The mixture was poured into an aqueous solution of NaHCO ₃ and NaHSO ₃ , dried over anhydrous Na ₂ SO ₄ , filtered and concentrated, then purified by silica gel chromatography (PE:ESAc =25:1), resulting in a crude product. The crude product was then further purified by silica gel chromatography (PE:EEAc=100:1 to 50:1 to 40:1) to obtain 2-[2(trifluoromethoxy)ethoxy]ethoxymethylbenzene (3.6 g, yield 39 %) in the form of a colorless liquid. ESI-MS (EI ⁺ , m/z): 287.1 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 7.37-7.22 (m, 5H), 4.57 (s, 2H), 4.10 (t, J=4.8 Hz, 2H), 3, 75 (t, J=4.8 Hz, 2H), 3.72 - 3.62 (m, 4H).

Step 3: synthesis of 2-[2-(trifluoromethoxy)ethoxy]ethanol.

Pd/C (3.13 g) was added to a solution of 2-[2-(trifluoromethoxy)ethoxy]ethoxymethylbenzene (3.4 g, 12.87 mmol) in CH3OH (60 mL). The resulting mixture was then stirred under _H2 at room temperature for 20 hours, filtered, concentrated, then purified by silica gel chromatography (DCM: _CH3OH =50:1) to give 2-[2-(trifluoromethoxy )ethoxy]ethanol (1.87 g, 83% yield) as a colorless liquid. 1H NMR (400 MHz, CDCl ₃ ) δ 4.11 - 4.03 (m, 2H), 3.69 (dt, J=4.4, 2.3 Hz, 4H), 3.61-3.54 (m, 2H), 2.75 (t, J=5.9 Hz, 1H). ¹⁹ F NMR (376 MHz, CDCl ₃ ) δ -61.11 (s).

Step 4: Synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy44-[(1R)-2-[(1S ,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36, 37,47,48-hexamethyl-43-[2-[2- (trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-88) .

To a degassed solution of everolimus (0.5 g, 0.52 mmol) in THF (30 ml) was added ptoluenesulfonic acid (0.45 g, 2.61 mmol) at 0°C, followed by 2-[2-(trifluoromethoxy) ethoxy]ethanol (0.91 g, 5.22 mmol). The resulting mixture was stirred at 0°C for 0.5 hour under N ₂ atmosphere, then at 23°C for 6 hours. The reaction mixture was poured into saturated NaHCO ₃ solution (40 ml) and extracted with EtOAc (30 ml) . The organic layer was washed with water (30 mlx2), brine (20 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure at room temperature. The residue was purified by reverse phase chromatography (C18, CH ₃ CN:H ₂ O=0% to 70% yield), resulting in (21E,23E,25E,26E,34R,35S, 36R,37R,39S, 41S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3methoxycyclohexyl]-1-methylethyl] -46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25 (47),26(48)-tetraene-49,50,51, 52,53-pentone (I-88: 141 mg, 24% yield) as a white solid. ESI-MS (EI ⁺ , m/z): 1122.3 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 6.39-5.88 (m, 4H), 5.73-5.05 (m, 5H), 4.52-3.83 (m, 5H), 3.703 .50 (m, 6H), 3.43-3.21 (m, 8H), 3.12-2.93 (m, 4H), 2.80-2.44 (m, 4H), 2.31 -2.16 (m, 4H), 2.05-1.59 (m, 20H), 1.43-1.34 (m, 4H), 1.21-1.09 (m, 6H), 1 .01-0.78 (m, 17H), 0.62-0.51 (m, 1H).

Step 5: synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2 -[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy34,35,36,37,47,48-hexamethyl-43-[2-[ 2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I- 83) and (21E,23E,25E,26E,34R,35S, 36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2- [(1S,3R,4R)-4-(2-hydroxyethoxy)3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[ 2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51, 52,53-pentone (I- 82):

130 mg (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2[(1S, 3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48hexamethyl-43-[2-[2-(trifluoromethoxy) ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone was purified using preparative chiral HPLC, in resulting in (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R) -45.55

- 154 045993 dihydroxy-444(1R)-24(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47 ,48-hexamethyl-43-[2-[2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50 ,51,52,53-pentone (I-82: 19 mg, yield 14.6%) and (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R, 46R,55R)-45,55-dihydroxy-44-[(1R)-2[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy- 34,35,36,37,47,48hexamethyl-43-[2-[2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23, 25(47),26(48 )-tetraene-49,50,51,52,53-pentone (I-83: 12 mg, 9.2% yield), both as a white solid.

Chiral analysis method: Column

Column size Introduction Mobile phase Flow rate Wavelength Temperature

CHIRALPAK IC(IC00CE-OL002)

0.46 cm I.D. x 25 cm L

40.0 µl

Hexane/EtOH =60/40(v/v)

1.0 ml/min

UV 254 nm °C

HPLC equipment: Shimadzu LC-20AT CP-HPLC-07

I-82: ESI-MS (EI+, m/z): 1122.4 [M+Na] ⁺ . 1H NMR (500 MHz, CDC13) δ 6.43 - 5.83 (m, 4H), 5.57 - 5.13 (m, 5H), 4.31 - 4.04 (m, 3H), 3, 91 - 3.53 (m, 1H), 3.49 - 3.00 (m, 19H), 2.76 - 2.52 (m, 3H), 2.25 (dd, J=34.2, 26 ,6 Hz, 3H), 2.12 - 1.96 (m, 5H), 1.75 (dd, J=35.2, 24.7 Hz, 8H), 1.52 - 1.34 (m, 8H), 1.15 - 0.79 (m, 18H), 0.72 (d, J=12.1 Hz, 1H).

I-83: ESI-MS (EI ⁺ , m/z): 1122.4 [M+Na] ⁺ . ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.54 - 6.41 (m, 1H), 6.18 (tdd, J=29.7, 22.4, 12.7 Hz, 3H), 5, 82 - 5.68 (m, 1H), 5.46 - 5.30 (m, 2H), 5.19 (dd, J=25.5, 20.6 Hz, 2H), 4.62 - 4, 40 (m, 1H), 4.21 (d, J=18.4 Hz, 1H), 3.94 (dd, J=34.8, 4.5 Hz, 1H), 3.83 - 3.62 (m, 4H), 3.59 (d, J=3.3 Hz, 1H), 3.50 - 2.95 (m, 13H), 2.62 (dt, J=55.5, 38.6 Hz, 2H), 2.42 - 2.17 (m, 3H), 2.16 - 1.57 (m, 24H), 1.54 - 1.27 (m, 10H), 1.12 - 0, 80 (m, 18H), 0.71 - 0.62 (m, 1H).

Example 40: Synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[ (1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy34,35,36,37,47,48-hexamethyl-43-[2-[2- (1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51, 52,53-pentone (I-87) and (21 E,23E,25E,26E,34R, 35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy- 44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47, 48-hexamethyl-43-[2-[2(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta21,23,25(47),26(48) -tetraene-49,50,51,52,53-pentone (I-79) and (21E,23E,25E,26E,34R,35S,36R,37R,39S, 41S,43S,44S,45R,46R,55R )-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34, 35,36,37,47,48-hexamethyl-43-[2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21, 23,25(47),26(48)-tetraene-49,50,51,52, 53-pentone (I-80):

- 155 045993

Synthesis circuit

Methods and characteristics.

Step 1: Synthesis of 2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxymethylbenzene: Under nitrogen atmosphere, stirred silver trifluoromethanesulfonate (19.64 g, 76.44 mmol), lithium trifluoromethanesulfonate (3.97 g, 25.48 mmol), 1-(chloromethyl)-4-fluoro-1,4-diazoniabicyclo[2,2.2]octane-bis(tetrafluoroborate) (18.05 g, 50.96 mmol) and potassium fluoride (5 .92 g, 101.92 mmol). Then 2-(2benzyloxyethoxy)ethanol (5 g, 25.48 mmol), trimethyl(1,1,2,2,2-pentafluoroethyl)silane (14.69 g, 76.44 mmol), EtOAc ( 20 ml), trifluoromethylbenzene (20 ml) and 2-fluoropyridine (7.42 g, 76.44 mmol) under N2. The reaction mixture was stirred under N2 for 60 hours at 30°C, then filtered through a pad of silica (eluting with EtOAc). The filtrate was collected and concentrated. The residue was purified by chromatography on silica gel (PE:EAAc=25:1), resulting in

2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxymethylbenzene (2.5 g, 31% yield) as a light yellow liquid. 1H NMR (400 MHz, CDClj) δ 7.36 - 7.24 (m, 5H), 4.56 (s, 2H), 4.17 - 4.11 (m, 2H), 3.76 - 3, 72 (m, 2H), 3.71 - 3.65 (m, 2H), 3.65 -3.60 (m, 2H). ^19F NMR (376 MHz, CDClj) δ -90.77 (s), -86.02 (s).

Step 2: synthesis of 2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethanol.

Pd/C (0.58 g) was added to a solution of 2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxymethylbenzene (0.757 g, 2.41 mmol) in CH ₃ OH (10 ml). The mixture was stirred under H ₂ at room temperature for 20 hours, then filtered and concentrated. The residue was purified by silica gel chromatography (DCM: _CH3OH =50:1) to give 2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethanol (0.5 g, yield 93%) in the form of a colorless liquid. 1H NMR (400 MHz, CDCl ₃ ) δ 4.13 - 4.06 (m, 2H), 3.69 (dd, J=5.4, 4.1 Hz, 4H), 3.54 (dd, J =5.2, 3.9 Hz, 2H), 2.47 (s, 1H). ¹⁹ F NMR (376 MHz, CDCl ₃ ) δ 86.21 (d, J=1.2 Hz), -90.98 (d, J=1.2 Hz).

Step 3: synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy44-[(1R)-2-[(1S ,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36, 37,47,48-hexamethyl-43-[2-[2- (1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51, 52,53-pentone (I-87).

To a solution of everolimus (0.5 g, 0.52 mmol) in THF (10 ml) was added p-toluenesulfonic acid (0.45 g, 2.61 mmol) at 0°C, followed by 2-[2-(1 ,1,2,2,2-pentafluoroethoxy)ethoxy]ethanol (0.58 g, 2.61 mmol). The resulting mixture was stirred at 0°C for 0.5 h under N ₂ , then at 22°C for 6 h, poured into saturated NaHCO ₃ solution (40 ml) and extracted with EtOAc (30

- 156 045993 ml). The organic layer was washed with water (30 mlx2), brine (20 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure at room temperature. The residue was purified by reverse phase chromatography (C18, CH ₃ CN: H ₂ O=0% to 70% yield), resulting in (21E,23E,25E,26E,34R,35S,36R,37R,39S, 41S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1- methylethyl]-46-methoxy34,35,36,37,47,48-hexamethyl-43-[2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa -58-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-87: 40 mg, 7% yield) as a white solid . ESI-MS (EI+ m/z): 1172.3 [M+Na]+. 1H NMR (400 MHz, CDCl ₃ ) δ 6.455.95 (m, 4H), 5.79-5.13 (m, 5H), 4.59-4.17 (m, 3H), 3.98-3 .59 (m, 8H), 3.50-3.28 (m, 10H), 3.20-3.00 (m, 5H), 2.89-2.49 (m, 4H), 2.37 -2.22 (m, 4H), 2.10-1.61 (m, 20H), 1.51-1.23 (m, 8H), 1.06-0.85 (m, 16H), 0 .71-0.63 (m, 1H).

Step 4: synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2 -[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy34,35,36,37,47,48-hexamethyl-43-[2-[ 2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51, 52,53-pentone (I-80) and (21E,23E,25E, 26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-44 -[(1R)-2-[(1S,3R,4R)-4-(2hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl -43-[2-[2(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(47), 26(48)- tetraene-49,50,51,52,53-pentone (I-79):

mg (21E,23E,25E,26E, 34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2[(1S,3R ,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(1 ,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta21,23,25(47),26(48)-tetraene-49,50,51,52,53- the penton was sent for chiral resolution, resulting in (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-44[ (1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl -43-[2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(47),26(48) -tetraene-49,50,51,52,53-pentone (I-80: 7.2 mg, 7.5% yield) and (21E,23E,25E, 26E,34R,35S,36R,37R,39S, 41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2hydroxyethoxy)-3-methoxycyclohexyl]-1- methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68- dioxa-58-azatricyclohexatriaconta-21,23,25(47),

26(48)-tetraene-49,50,51,52,53-pentone (I-79: 5.1 mg, 5.3% yield) as a white solid.

Chiral separation method

Column:

Column size:

Solution concentration:

Introduction:

Mobile phase:

Flow rate:

Wavelength:

Temperature:

I-80: ESI-MS (EI ⁺ , m/z): 1150.3 [M+H]+. ^1H NMR

CHIRALPAK IC

5.0 cm I.D. x 25 cm L

0.79 mg/ml ml

Hexane/EUN=70/30(v/v) ml/min

UV 254 nm °C (400 MHz, CDCl ₃ ) δ 6.42 -5.80 (m, 4H), 5.58 - 5.05 (m, 5H), 4.74 (s, 1H), 4, 24 - 4.08 (m, 3H), 3.92 - 3.51 (m, 10H), 3.48-3.24 (m, 12H), 3.13 (ddd, J=24.2, 17 ,1, 11.3 Hz, 3H), 2.64 (ddd, J=23.4, 16.8,6.0 Hz, 3H), 2.42-2.15 (m, 3H), 2, 14-1.88 (m, 6H), 1.84-1.64 (m, 14H), 1.54-1.39 (m, 5H), 1.17 - 0.81 (m, 18H), 0.71 (dd, J=23.8, 12.1 Hz, 1H).

I-79: ESI-MS (EI ⁺ , m/z): 1172.3 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 6.42 - 5.90 (m, 4H), 5.54 - 5.03 (m, 6H), 4.31 - 3.96 (m, 5H), 3 .88 - 3.01 (m, 22H), 2.94 - 2.37 (m, 5H), 2.39 - 1.94 (m, 7H), 1.68 (dd, J=28.2, 19.2 Hz, 8H), 1.52 - 1.31 (m, 6H), 1.14 -1.00 (m, 6H), 0.97 - 0.62 (m, 19H).

Example 41 Synthesis of (21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-(2-hydroxyethoxy) ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxycyclohexyl]-1-methylethyl]-48-methoxy-36, 37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone ( I-76) and (21E,23E,25E,26E,36R,37S,

38R,39R,41S,43S,45S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-(2-hydroxyethoxy)ethoxy]-46-[(1R)-2[(1S, 3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxycyclohexyl]-1-methylethyl]-48-methoxy-36,37, 38,39,49,50-hexamethyl-68,69- dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52, 53,54,55-pentone (I-66) and (21E,23E,25E,26E,36R, 37S,38R,39R,41S,43S,45S,46S,47R,48R,57R)-47,57dihydroxy-45-[2-(2-hydroxyethoxy)ethoxy]-46-[(1R)-2-[(1S ,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3methoxycyclohexyl]-1-methylethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa -58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone (I-67)

- 157 045993

Synthesis circuit

- 158 045993

Methods and characteristics.

Step 1: synthesis of 2-[tert-butyl(diphenyl)silyl]oxyethyl trifluoromethanesulfonate.

A solution of 2-[tert-butyl(diphenyl)silyl]oxyethanol (4.3 g, 14.31 mmol) and DIPEA (2.77 g, 21.47 mmol) in DCM (40 ml) was cooled to 0°C under atmosphere N ₂ and trifluoromethanesulfonic acid anhydride (4.44 g, 15.74 mmol) was added dropwise.

The mixture was stirred at 0°C for 2 hours, then diluted with DCM (50 ml), washed with saturated NaHCO ₃ solution (50 ml), water (50 mlx3) and saline (50 ml). The organic layer was then dried over MgSO ₄ , filtered and concentrated in vacuo to give 2-[tert-butyl(diphenyl)silyl]oxyethyl trifluoromethanesulfonate (6.19 g, 99% yield) as a brown oil. It was used in the next step without any further purification. 1H NMR (400 MHz, CDCl ₃ ) δ 7.66 (dd, J=7.9, 1.5 Hz, 4H), 7.45-7.38 (m, 6H), 4.56 (t, J =4.4 Hz, 2H), 3.91 (t, J=4.4 Hz, 2H).

Stage 2: synthesis (35E,37E,39E,40E,48R,49S,50R,51R,53S,55S,57S,58S,59R,60R,69R)-58-[(1R)-1[(1S,3R, 4R)-4-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy-3-methoxycyclohexyl]-1-methylethyl]59,69-dihydroxy-57,60-dimethoxy-48,49,50 ,51,61,62-hexamethyl-79,80-dioxa-71-azatricyclohexatriaconta-35.3 7,39(61),40(62)-tetraene-63,64,65,66,67-pentone.

A solution of everolimus (1.5 g, 1.57 mmol), 2-[tert-butyl(diphenyl)silyl]oxyethyl trifluoromethanesulfonate and DIPEA (3.27 mL, 18.78 mmol) in toluene (20 mL) was stirred at 45°C for 18 hours. The mixture was then poured into ice-cold saturated NaHCO ₃ solution (50 ml), washed twice with ice water (60 ml), brine (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The mixture was purified by silica gel chromatography (PE:EAAc=5:1 to 2:1, then PE:acetone=4:1) to give (35E,37E,39E,40E,48R,49S,50R,51R ,53S,55S,57S,58S,59R,60R,69R)-58-[(1R)-2[(1S,3R,4R)-4-[2-[2-[tert-butyl(diphenyl)silyl] oxyethoxy]ethoxy]-3-methoxycyclohexyl]-1-methylethyl]-59,69-dihydroxy-57,60-dimethoxy-48,49,50,51,61,62-hexamethyl-79,80-dioxa-71-azatricyclohexatriaconta -35,37,39(61),40(62)-tetraene-63,64,65,66,67-pentone (1.15 g, 59% yield) as a brown solid. ESI-MS (EI ⁺ , m/z): 1263.4 [M+Na] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.62-7.60 (m, 4H), 7.35-7.28 (m, 6H), 6.35-5.80 (m, 4H), 5.51-5.09 (m, 4H), 4.75 (s, 1H), 4.37-4.02 (m, 2H), 3.87-3.49 (m, 11H), 3. 37-3.26 (m, 8H), 3.10-2.96 (m, 5H), 2.76-2.48 (m, 3H), 2.28-2.21 (m, 2H), 1.98-1.90 (m, 3H), 1.67-1.39 (m, 18H), 1.26-1.08 (m, 7H), 1.04-0.76 (m, 26H ), 0.64 (q, J=11.2 Hz, 1H).

Step 3: Synthesis of (22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,42S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2 -[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxycyclohexyl]-1-methylethyl]-42,45dimethoxy-33,34,35,36,46,47-hexamethyl -64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46), 27(47)-tetraene-48,49,50,51,52-pentone:

(35E,37E,39E,40E,48R,49S,50R,51R,53S,55S,57S,58S,59R,60R,69R)-58-[(1R)-2-[(1S,3R,4R)- 4-[2-[2[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]-3-methoxycyclohexyl]-1-methylethyl]-59,69-dihydroxy57,60-dimethoxy-48,49,50,51,61 ,62-hexamethyl-79,80-dioxa-71-azatricyclohexatriaconta-35,37,39(61),40(62)-tetraene-63,64,65,66,67-pentone (1.15 g, 0. 93 mmol) was dissolved in THF (10 ml). Pyridinium hydrofluoride (0.437 mL, 1.85 mmol) was added and the mixture was stirred for 3 hours at room temperature. The mixture was diluted with EtOAc (30 ml), washed with saturated NaHCO ₃ (aq, 40 ml x 2) until the pH was 10, then washed with water until neutral, saline (40 ml), dried over anhydrous Na2SO4 , filtered and concentrated. The residue was purified by silica gel chromatography (PE: acetone=4:1 to 2:1) to give (22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,42S,43S, 44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2[(1 S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxycyclohexyl]-1 -methylethyl]-42,45-dimethoxy33,34,35,36,46,47-hexamethyl-64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene48,49, 50,51,52-pentone (680 mg, 73% yield) as a white solid. ESI-MS (EI ⁺ m/z): 1024.3 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 6.35-5.80 (m, 4H), 5.51-5.09 (m, 4H), 4.75 (s, 1H), 4.37-4 .02 (m, 2H), 3.87-3.49 (m, 11H), 3.37-3.26 (m, 8H), 3.10-2.96 (m, 5H), 2.76 -2.48 (m, 3H), 2.28-2.21 (m, 2H), 1.981.90 (m, 3H), 1.67-1.39 (m, 18H), 1.26-1 .08 (m, 7H), 1.04-0.76 (m, 17H), 0.64 (q, J=11.2 Hz, 1H).

Step 4: Synthesis of (21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy45-[2-(2-hydroxyethoxy)ethoxy] -46-[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxycyclohexyl]-1-methylethyl]-48-methoxy-36.37, 38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone (I- 76).

To a solution of (22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,42S,43S,44R,45R,54R)-44,54-dihydroxy-43[(1R)-2-[( 1 S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxycyclohexyl]-1-methylethyl]-42,45-dimethoxy-33,34,35,36,46,47-hexamethyl -64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene48,49,50,51,52-pentone (0.65 g, 0.65 mmol) in THF ( 6 ml) under N2 atmosphere at 0°C, p-TsOH (0.56 g, 3.24 mmol) was added, followed by 2-(2-hydroxyethoxy)ethanol (1.38 g, 12.97 mmol). The resulting mixture was stirred at 0°C for 10 minutes, then at 20°C for 2 hours. The mixture was poured into saturated NaHCO3 solution (40 ml) and extracted with EtOAc (30 ml). The organic layer was washed with water (30 mlx2), brine (40 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure at room temperature. The residue was purified by silica gel chromatography (PE:EAAc=50% to 100% EtOAc, then to DCM: _CH3OH =95:5 to 90:10) and then purified by reverse phase chromatography ( C18, CH ₃ CN:H ₂ O=50:50), resulting in (21E,23E,25E,26E,36R,37S,38R,39R,41S,43S, 46S,47R,48R,57R)-47 ,57-dihydroxy-45-[2-(2hydroxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxycyclohexyl ]1-methylethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21, 23,25(49),26(50)-tetraene- 51,52,53,54,55-pentone (I-76: 140 mg, 20% yield) as a white solid. ESI-MS (EI ⁺ , m/z): 1098.5 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 6.31-5.80 (m, 4H), 5.46-4.74 (m, 5H), 4.21-4.11 (m, 2H), 3 .68-3.56 (m, 18H), 3.4-3.14 (m, 12H), 3.03-2.97 (m, 2H), 2.68-2.39 (m, 5H) , 2.28-2.25 (d, 2H), 1.73-1.55 (m, 18H), 1.40-1.14 (m, 8H), 1.03-0.79 (m, 16H), 0.64 (q, J=11.2 Hz, 1H).

Step 5: synthesis of ^IE^E^E^E^R^S^R^R^IS^S^S^S^R^R^R)^^^^roxy-45-[2-(2-hydroxyethoxy )ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxycyclohexyl]-1-methylethyl]-48-methoxy-36 ,3 7,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55- penton (I-67) and (21E,23E,25E,26E,36R,37S, 38K,39E,418,438,458,468,47E,48E,57E)-47,57-dihydroxy-45-[2-(2-hydroxyethoxy)ethoxy ]-46-[(1E)-2[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxycyclohexyl]-1-methylethyl]-48-methoxy-36.37, 38, 39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53, 54,55-pentone (I- 66):

140 mg (21E,23E,25E,26E,36R,37S,38R,39R,41S,43S, 46S,47R,48R,57R)-47,57-dihydroxy-45-[2-(2hydroxyethoxy)ethoxy]-46 -[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxycyclohexyl]1-methylethyl]-48-methoxy-36,37,38,39 ,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone was purified using preparative chiral HPLC and the resulting The epimers were repurified by silica gel chromatography (hexane:DCM:EtOAc:MeOH=3:3:1:1) to give (21E,23E,25E,26E,36R,37S,38R,39R, 41S,43S ,45S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-(2-hydroxyethoxy)ethoxy]-46-[(1R)-2[(1S,3R,4R)-4- [2-(2-hydroxyethoxy)ethoxy]-3-methoxycyclohexyl]-1-methylethyl]-48-methoxy36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23 ,25(49),26(50)-tetraene51,52,53,54,55-pentone (I-67: 25.3 mg, yield 18.1%) and (21E,23E,25E,26E,36R, 37S,38R,39R,41S,43S,45S, 46S,47R,48R,57R)-47,57-dihydroxy-45-[2-(2-hydroxyethoxy)ethoxy]-46[(1R)-2[(1S ,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxycyclohexyl]-1-methylethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl68,69-dioxa -58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone (I-66: 34 mg, 24.3% yield), both as white solid.

Chiral Analysis Method Column Column Size Introduction Mobile Phase Flow Rate Wavelength Temperature

CHIRALPAK IC(IC00CD-OL002)

0.46 cm I.D. x 25 cm L

100.0 µl

Hexane/EUN=60/40(v/v)

1.0 ml/min

UV 254 nm °C

HPLC equipment: Shimadzu LC-20AD CP-HPLC-08

I-67: ESI-MS (EI ⁺ , m/z): 1098.4 [M+Na] ⁺ . 1H NMR (400 MHz, CDC13) δ 6.49 - 6.19 (m, 2H,) 6.13 (dd, J=15.0, 9.8 Hz, 1H), 5.94 (dd, J= 24.3, 10.5 Hz, 1H), 5.58 - 5.45 (m, 1H), 5.41 (d, J=9.8 Hz, 1H), 5.37 - 5.24 (m , 1H), 5.13 (t, J=10.8 Hz, 1H), 4.80 (s, 1H), 4.19 (t, J=8.7 Hz, 1H), 3.92 - 3 .52 (m, 16H), 3.51 - 3.25 (m, 11H), 3.25 - 2.99 (m, 3H), 2.72 (dd, J=16.9, 5.6 Hz , 2H), 2.57 (dd, J=16.8, 6.3 Hz, 1H), 2.39 - 2.18 (m, 2H), 1.95 (ddd, J=29.6, 22 ,0, 10.2 Hz, 6H), 1.83 - 1.40 (m, 15H), 1.37 - 1.16 (m, 8H), 1.15 - 0.82 (m, 18H), 0.71 (dd, J=24.0, 12.0 Hz, 1H).

I-66: ESI-MS (EI ⁺ , m/z): 1098.4 [M+Na] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.45 - 5.92 (m, 4H), 5.52 - 4.75 (m, 5H), 4.31 - 3.92 (m, 3H), 3.88 - 3.54 (m, 16H), 3.51 - 3.13 (m, 13H), 3.07 (s, 2H), 2.87 - 2.42 (m, 4H), 2, 38 1.55 (m, 12H), 1.51 - 1.29 (m, 15H), 1.13 - 0.72 (m, 18H), 0.69 - 0.58 (m, 1H).

Example 42. Synthesis of (22Ε,24Ε,26Ε,27Ε,34Κ,358,36Ε,3^398,418,438,448,45Ε,46Ε^)-43-γ€κοοκοπ45,55-dihydroxy-44-[(1R)-2-[( 1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy34,35,36,37,47,48-hexamethyl-65,66-dioxa-56-azatricyclohexatriaconta-22,24 ,26(47),27(48)-tetraene49,50,51,52,53-pentone (I-103)

- 160 045993

Synthesis circuit

1-103

Methods and characteristics.

Step 1: synthesis of (22E,24E,26E,27E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-43-hexoxy45,55-dihydroxy-44-[(1R) -2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy34,35,36,37,47,48-hexamethyl-65,66-dioxa-56- azatricyclohexatriaconta-22,24,26(47),27(48)-tetraene49,50,51,52,53-pentone (I-103):

To a solution of rapamycin (0.5 g, 0.547 mmol) and hexan-1-ol (56 mg, 0.547 mmol) in THF (10 mL), 4-methylbenzenesulfonic acid hydrate (0.52 g, 2.73 mmol) was slowly added. The resulting solution was stirred at 20°C for 2 hours under N2, then the mixture was poured into ice-cold aqueous NaHCO ₃ and extracted with EtOAc (30 ml). The organic layer was dried, filtered and concentrated. The residue was then purified by reverse phase chromatography (C18, CH ₃ CN:H2O=78:22) to give (22E,24E,26E,27E,34R,35S,36R,37R,39S,

41S,43S,44S,45R,46R,55R)-43-hexoxy-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]- 1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-65,66-dioxa-56-azatricyclohexatriaconta-22,24,26(47),27(48)-tetraene-49 ,50,51,52,53-pentone (I-103: 72 mg, 13% yield) as a white solid. ESI-MS (EI ⁺ , m/z): 1006.0 [M+Na] ⁺ . ¹ H NMR(500 MHz, CDCl ₃ ) δ 6.44 - 5.85 (m, 4H), 5.60 - 5.07 (m, 4H), 4.77 (s, 1H), 4.29 - 3.98 (m, 2H), 3.76 -3.67 (m, 1H), 3.46 - 3.28 (m, 10H), 3.23 - 3.03 (m, 2H), 3, 02 - 2.77 (m, 2H), 2.69 (m, 3H), 2.36 (d, J=32.6 Hz, 2H), 2.17 - 1.90 (m, 4H), 1 .66 - 1.44 (m, 22H), 1.35 - 1.21 (m, 11H), 1.17 - 0.84 (m, 22H), 0.72-0.65 (m, 1H) .

Example 43. Synthesis of (21E,23E,25E,26E,44R,45S,46R,47R,49S,51S,54S,55R,56R,65R)-55,65-dihydroxy-53-[2-[2-[2 -[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-54-[(^)-2[(1S,3R,4R)-4-(2- hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-56-methoxy-44,45,46,47,57,58hexamethyl-76,77-dioxa-66-azatricyclohexatriaconta-21,23,25(57),26( 58)-tetraene-59,60,61,62,63pentone (I-99)

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Synthesis circuit

Methods and characteristics.

Step 1: synthesis of (21E,23E,25E,26E,44R,45S,46R,47R,49S,51S,54S,55R,56R,65R)-55,65-dihydroxy53-[2-[2-[2-[ 2-[2 - [2-(2-hydroxyethoxy) ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] -54-[(1R)-2[(1S,3R,4R)-4-(2-hydroxyethoxy) -3-methoxycyclohexyl]-1-methylethyl]-56-methoxy-44,45,46,47,57,58hexamethyl-76,77-dioxa-66-azatricyclohexatriaconta-21,23,25(57),26(58) -tetraene-59,60,61,62,63-pentone (I-99):

To a solution of everolimus (0.5 g, 0.52 mmol) and 2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (3 41 g, 10.44 mmol) 4methylbenzenesulfonic acid hydrate (0.52 g, 2.73 mmol) was slowly added to THF (15 mL). The resulting mixture was stirred at 22°C for 18 hours under N ₂ and then quenched with aqueous NaHCO ₃ (30 ml) and extracted with EtOAc (60 ml x 3 ). The combined organic layers were washed with water (50 ml), brine (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by reverse phase chromatography (C18, CH ₃ CN:H ₂ 0=70:30), resulting in (21E,23E,25E,26E,44R,45S,46R,47R,49S,51S,54S ,55R,56R,65R)-55,65-dihydroxy-53-[2-[2[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] -54-[(1R)-2-[(1S,3R,4R)-4-(2hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-5 6-methoxy-44,45,46,47,57, 58-hexamethyl-76,77dioxa-66-azatricyclohexatriaconta-21,23,25(57),26(58)-tetraene-59,60,61,62,63-pentone (I-99: 93 mg, 14% yield ) in the form of a white solid. ESI-MS (EI ⁺ , m/z): 1274.9 [M+Na] ⁺ . ^1H NMR (500 MHz, CDCl3) δ 6.42 - 5.82 (m, 4H), 5.64 - 5.06 (m, 4H), 4.81 (s, 1H), 4.30 - 4 .08 (m, 1H), 3.81 - 3.53 (m, 35H), 3.46 3.26 (m, 12H), 3.22 - 3.05 (m, 4H), 2.76 - 2.65 (m, 2H), 2.39 - 2.22 (m, 2H), 2.14 - 1.97 (m, 3H), 1.75 - 1.55 (m, 13H), 1, 52 -1.39 (m, 4H), 1.30 - 1.13 (m, 6H), 1.08 - 0.82 (m, 17H), 0.76 - 0.65 (m, 1H).

Example 44. Synthesis of PIE^E^E^E^R^S^R^R^IS^S^S^R^R^R)^^^^roxy-55-[2-[2-[2-[ 2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-56-[(1R)-2-[(1S,3R ,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-58-methoxy-46,47,48, 49,59,60-hexamethyl-78,79-dioxa-68-azatricyclohexatriaconta-21,23, 25(59),26(60)-tetraene-61,62,63, 64,65-pentone (I-97):

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Synthesis circuit

Methods and characteristics.

Step 1: synthesis of (21E,23E,25E,26E,46R,47S,48R,49R,51S,53S,56S,57R,58R,67R)-57,67-dihydroxy55-[2-[2-[2-[ 2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] 56-[(1R)-2-[(1S,3R, 4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-58-methoxy-46,47,48,49,59,60hexamethyl-78,79-dioxa-68-azatricyclohexatriaconta-21,23,25(59 ),26(60)-tetraene-61,62,63,64,65pentone (I-97).

To a solution of rapamycin (0.5 g, 0.547 mmol) in THF (15 ml) was added 4-methylbenzenesulfonic acid (0.47 g, 2.73 mmol) and nonaethylene glycol (2.27 g, 5.47 mmol) at 10° WITH. The reaction mixture was stirred at 30°C for 18 h under N2 and then quenched with aqueous _NaHCO3 and extracted with EtOAc (60 mLx3). The combined organic layers were washed with water (50 ml), brine (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The residue was purified by reverse phase chromatography (C18, CH ₃ CN:H2O=63:37) to give (21E,23E,25E,26E,46R,47S,48R,49R,51S,53S,56S,57R ,58R,67R)-57,67-dihydroxy55-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy ]ethoxy]ethoxy]ethoxy]56-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-58-methoxy-46,47,48,49 ,59,60hexamethyl-78,79-dioxa-68-azatricyclohexatriaconta-21,23,25(59),26(60)-tetraene-61,62,63,64,65-pentone (I-97: 111 mg, yield 16%) in the form of a white solid. ESI-MS (EI ⁺ , m/z): 1318.9 [M+Na] ⁺ . 1H NMR (500 MHz, CDCl ₃ ) δ 6.50 - 5.79 (m, 4H), 5.64 - 5.06 (m, 4H), 4.89 (d, J=53.5 Hz, 1H ), 4.51 3.94 (m, 2H), 3.75 - 3.18 (m, 46H), 3.02 - 2.86 (m, 2H), 2.82 - 2.61 (m, 3H), 2.39 - 2.18 (m, 2H), 2.20 - 1.91 (m, 6H), 1.78 - 1.54 (m, 16H), 1.51 - 1.19 ( m, 11H), 1.09 - 0.82 (m, 17H), 0.74 - 0.62 (m, 1H).

Example 45. Synthesis of (21E,23E,25E,26E,50R,51S,52R,53R,55S,57S,60S,61R,62R,71R)-61,71-dihydroxy-59-[2-[2-[2 -[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-60- [(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-62-methoxy-50,51,52,53,63,64-hexamethyl-82, 83-dioxa-72-azatricyclohexatriaconta-21,23,25(63),26(64)-tetraene-65,66,67,68,69-pentone (I-96):

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Synthesis circuit

Methods and characteristics.

Stage 1: synthesis of (21E,23E,25E,26E,50K518,52K53K558,578,608,61K62K71K)-61,71-dahydroxy59-[2-[2-[2-[2-[2-[2-[2-[ 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-60-[(1K)-2-[(^,3K,4K )-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-62-methoxy-50,51,52,53,63,64-hexamethyl-82,83-dioxa-72-azatricyclohexatriaconta-21,23,25( 63),26(64)-tetraene-65,66,67,68,69-pentone (I-96).

To a solution of rapamycin (0.2 g, 0.22 mmol) and 4-methylbenzenesulfonic acid (0.19 g, 1.09 mmol) in THF (6 ml) was added 2-[2-[2-[2-[2 -[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (1.10 g, 2.19 mmol) at 0°C and the reaction mixture was stirred at 20°C for 2 hours, then poured into saturated NaHCO ₃ solution (30 ml) and extracted with EtOAc (40 mlx3). The combined organic layers were washed with water (40 ml), brine (40 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The residue was purified by reverse phase chromatography (C18, CH ₃ CN:H ₂ O=51:49), resulting in (21Ε,23Ε,25Ε,26Ε,50Κ518,52Κ53Κ558,578,608,61Κ62Κ71Κ)-61,71- №γηαροικοη-59-[2-[2-[2-[2[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy ]ethoxy]ethoxy]ethoxy]ethoxy]-60-[(1R)-2-[(18,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-62-methoxy-50.51, 52, 53,63,64-hexamethyl-82,83-dioxa-72-azatricyclohexatriaconta-21,23,25(63),26(64)-tetraene-65,66,67, 68,69-pentone (I- 96: 61 mg, 20% yield) as a colorless oil. ESI-MS (EI ⁺ m/z): 1408.0 [M+Na] ⁺ . 1H NMR (500 MHz, CDC13) δ 6.41 - 5.84 (m, 4H), 5.61 - 5.08 (m, 4H), 4.79 (s, 1H), 4.32 - 4, 05 (m, 1H), 3.89 - 3.46 (m, 44H), 3.46 - 3.16 (m, 12H), 2.98 - 2.50 (m, 7H), 2.32 ( s, 2H), 2.15 - 1.89 (m, 4H), 1.82 - 1.68 (m, 8H), 1.56 1.16 (m, 14H), 1.12 - 0.82 (m, 18H), 0.74-0.56 (m, 1H).

Example 46. Synthesis of (21E,23E,25E,26E,33R,348,35R,36R,388,408,438,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(18,3R ,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy33,34,35,36,46,47-hexamethyl-42-[2-(2,2,2- trifluoroethoxy)ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone (I-77) and (21E ,23E,25E,26E,33R,34S,35R,36R,38S, 408,428,438,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(18,3R,4R)- 4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-(2,2,2-trifluoroethoxy) ethoxy]65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(21E, 23E,25E,26E,33R, 348,35R,36R,388,408,428,43R,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(18,3R,4R)-4(2-hydroxyethoxy)-3- methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2(2,2,2-trifluoroethoxy)ethoxy]-65,66-dioxa-56- azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene48,49,50,51,52-pentone

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Synthesis circuit

Methods and characteristics.

Step 1: synthesis of (21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy43-[(1R)-2-[(1S ,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35, 36,46,47-hexamethyl-42-[2-(2, 2,2-trifluoroethoxy)ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone (I-77).

A solution of rapamycin (0.3 g, 0.313 mmol) and 2-(2,2,2-trifluoroethoxy)ethanol (0.9 g, 6.26 mmol) in THF (9 ml) was cooled to 0°C and p- TsOH (0.27 g, 1.57 mmol). The resulting mixture was stirred at 35°C for 5 hours under N2, then poured into ice-cold _NaHCO3 and extracted with EtOAc (40 ml x 3). The combined organic layers were washed with water (30 ml), brine (30 ml), dried, filtered and concentrated. The residue was purified by reverse phase chromatography (C18, CH ₃ CN:H ₂ O=57:43), resulting in (21E,23E,25E,26E,33R,34S,35R, 36R,38S,40S,43S ,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]- 45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-(2,2,2-trifluoroethoxy)ethoxy]- 165 045993

65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone (I-77:

0.063 g, yield 19%) as a white solid. ESI-MS (EI ⁺ , m/z): 1092.3 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 6.55 - 5.88 (m, 4H), 5.72 - 5.03 (m, 4H), 4.78 (s, 1H), 4.63 - 4 .34 (m, 1H), 4.32 - 4.09 (m, 1H), 4.00 - 2.81 (m, 21H), 2.77 - 2.43 (m, 3H), 2.41 -2.17 (m, 2h), 2.18 -1.93 (m, 3H), 1.93 - 1.54 (m, 18H), 1.54 - 1.14 (m, 10H), 1 .13 - 0.79 (m, 16H), 0.78 - 0.63 (m, 1H).

Step 2: synthesis of (21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,42S,43S,44R,45R,54R)-44,54dihydroxy-43-[(1R)-2-[ (1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-( 2,2,2-trifluoroethoxy)ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone (I -71) and (21E,23E,25E,26E,33R,34S,35R, 36R,38S,40S,42S,43R,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2 -[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2 -(2,2,2-trifluoroethoxy)ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52pentone (I -70).

100 mg (21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-(1R)-2[(1 S, 3R,4R)-4-(2-hydroxyethoxy)-3 -methoxycyclohexyl]-1 -methylethyl]-45-methoxy-3 3,34,35,36,46,47-hexamethyl-42-[2-(2, 2,2-Trifluoroethoxy)ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46), 26(47)-tetraene-48,49,50,51,52-pentone was purified using preparative chiral HPLC and the resulting epimers were purified by silica gel chromatography (hexane:DCM:EtOAc:MeOH=3:3:1:0.3), resulting in (21E,23E,25E,26E,33R,34S,35R, 36R,38S,40S,42S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3 -methoxycyclohexyl]-1-methylethyl]-45-methoxy33,34,35,36,46,47-hexamethyl-42-[2-(2,2,2-trifluoroethoxy)ethoxy]-65,66-dioxa-56- azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone (I-71: 16 mg, 16% yield) and (21E,23E,25E,26E , 33R,34S,35R,36R,38S,40S,42S,43R,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4- (2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-(2,2,2trifluoroethoxy)ethoxy]-65 ,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50, 51,52-pentone (I-70: 8 mg, 8% yield) as white solid.

Chiral separation method: Column

Column size

Solution Concentration Introduction

Mobile phase Flow rate Wavelength Temperature

CHIRALPAK IC

2.5 cm I.D. x 25 cm L

2.0 mg/ml ml

Hexane/EUN=70/30(v/v) ml/min

UV 254 nm °C

I-71: ESI-MS (EI+, m/z): 1092.4 [M+Na]+. 1H NMR (400 MHz, CDC13) δ 6.41 -6.20 (m, 2H), 6.13 (dd, J=15.1, 9.7 Hz, 1H), 5.93 (dd, J= 22.9, 10.3 Hz, 1H), 5.58 - 5.45 (m, 1H), 5.41 (d, J=9.8 Hz, 1H), 5.27 (d, J=5 ,2 Hz, 1H), 5.19 - 5.03 (m, 1H), 4.78 (s, 1H), 4.19 (dd, J=13.9, 5.9 Hz, 1H), 3 .95 - 3.63 (m, 10H), 3.63 - 3.53 (m, 2H), 3.52 - 3.25 (m, 11H), 3.24 - 3.01 (m, 3H) , 2.72 (dd, J=16.8, 5.8 Hz, 2H), 2.58 (dd, J=16.8, 6.3 Hz, 1H), 2.31 (t, J=23 ,5 Hz, 2H), 2.15 - 1.40 (m, 18h), 1.27 (ddd, J=32.5, 16.2, 6.3 Hz, 8H), 1.15 -0, 81 (m, 18H), 0.70 (dt, J=17.8, 9.0 Hz, 1H).

I-70: ESI-MS (EI ⁺ , m/z): 1092.4 [M+Na] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.43 - 5.90 (m, 4H), 5.56 - 5.08 (m, 5H), 4.33 - 3.99 (m, 3H), 3.95 - 3.63 (m, 8H), 3.62 - 3.02 (m, 18H), 2.89 - 1.97 (m, 12H), 1.76 (dd, J=31.4 , 24.8 Hz, 8H), 1.40 (ddd, J=39.2, 29.5, 12.0 Hz, 9H), 1.14 - 0.79 (m, 18H), 0.76 - 0.61 (m, 1H).

Example 47. Synthesis of 4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,45S,46R,47R,57R)-46,57-dihydroxy-45-[(1R) -2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy35,36,37,38,48,49-hexamethyl-50,51, 52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta23,25,27(48),28(49)-tetraen-44-yl]oxy]-N^Ni-dimethylbutanamide (I-69) and 4-[[(23E,25E,27E,28E,35R,36S, 37R,38R,40S,42S,44R,45S,46R,47R,57R)-46,57-dihydroxy-45-[(1R)-2 -[(1S,3R,4R)-4-(2-hydroxyethoxy)3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-50,51,52 ,53,54-pentaoxo69,70-dioxa-58-azatricyclohexatriaconta-23,25,27(48),28(49)-tetraen-44-yl]oxy]-Y^-dimethylbutanamide (I-61) and 4- [[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,57R)-46,57-dihydroxy45-[(1R)-2-[(1S ,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-35,36,37, 38,48,49-hexamethyl-50,51,52,53, 54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta-23,25,27(48), 28(49)-tetraen-44-yl]oxy]-N^-dimethylbutanamide (I-62)

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Synthesis circuit

Chiral separation

Methods and characteristics.

Step 1: synthesis of 4-hydroxy-K,K-dimethylbutanamide:

A mixture of tetrahydrofuran-2-one (5 g, 58.1 mmol) and N-methylmethanamine (43.64 g, 290.4 mmol, 90 ml) was stirred at 10°C for 18 h, the solvent was removed and then lyophilized, resulting in 4-hydroxy-NiNi-dimethylbutanamide (6.9 g, 91% yield) as a colorless oil. ESI-MS (EI ⁺ m/z): 132.3 [M+H] ⁺ . ¹ H NMR (400 MHz, CDCh) δ 3.67 (t, J=5.8 Hz, 2H), 3.04 (s, 3H), 2.96 (s, 3H), 2.49 (t, J=6.9 Hz, 2H), 1.94 - 1.84 (m, 2H).

Step 2: synthesis of 4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,45S,46R,47R,57R)-46,57-dihydroxy-45-[(1R) -2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy- 167 045993

35,36,37,38,48,49-hexamethyl-50,51,52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta23,25,27(48),28(49)-tetraene- 44-yl]oxy]-N^Ni-dimethylbutanamide (1-69).

Ptoluenesulfonic acid (0.27 g, 1.57 mmol) and 4-hydroxy-U,U-dimethylbutanamide were added to a solution of everolimus (0.3 g, 0.313 mmol) in THF (9 ml) at 0°C under _N2 atmosphere. (0.82 g, 6.26 mmol). The mixture was heated to 35°C and stirred for 18 hours, then poured into ice-cold NaHCO ₃ and extracted with EtOAc (20 ml x 3 ). The combined organic layers were washed with water (30 ml), brine (30 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The residue was purified by reverse phase chromatography (C18, CH ₃ CN:H ₂ O=55:45), resulting in 4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S ,42S,45S,46R,47R,57R)-46,57dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl ]-47-methoxy-35,36,37,38,48,49-hexamethyl-50,51,52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta23,25,27(48),28 (49)-tetraen-44-yl]oxy]-N^Ni-dimethylbutanamide (I-69: 0.05 g, 15% yield) as a white solid. ESI-MS (EI+, m/z): 1079.4 [M+Na]+. ^1H NMR (400 MHz, CDCl ₃ ) δ 6.52 - 5.78 (m, 4H), 5.67 - 5.05 (m, 4H), 4.75 (s, 1H), 4.48 - 4.09 (m, 2H), 4.04 - 3.51 (m, 7H), 3.52 - 3.12 (m, 12H), 3.12 - 2.88 (m, 7H), 2, 82 - 2.27 (m, 6H), 2.22 - 1.53 (m, 23H), 1.54 - 1.13 (m, 10H), 1.12 - 0.80 (m, 15H), 0.79 - 0.61 (m, 1H).

Step 3: synthesis of 4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,57R)-46,57dihydroxy-45-[(1R) -2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-50, 51,52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta23,25,27(48),28(49)-tetraen-44-yl]oxy]-N^Ni-dimethylbutanamide (1-62 ) and 4-[[(23E,25E,27E,28E,35R,36S, 37R,38R,40S,42S,44R,45S,46R,47R,57R)-46,57-dihydroxy-45-[(1R) -2-[(1S,3R,4R)-4-(2-hydroxyethoxy)3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-3 5,36,37,38,48,49-hexamethyl-50, 51,52,53,54-co-69,70dioxa-58-azatricyclohexatriaconta-23,25,27(48), 28(49)-tetraen-44-yl]oxy]-No,Nodimethylbutanamide (1-61) :

120 mg 4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,45S,46R,47R,57R)-46,57-dihydroxy-45[(1R)-2- [(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-35,36,37,38, 48,49-hexamethyl-50,51,52 ,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta-23,25,27(48),28(49)-tetraen-44-yl]oxy]-N^Ni-dimethylbutanamide was purified using preparative chiral HPLC, resulting in 4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,57R)-46,57-dihydroxy-45- [(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-35,36,37,38,48,49- hexamethyl-50,51,52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta-23,25,27(48),28(49)-tetraen-44-yl]oxy]-N^Y -dimethylbutanamide (I-62: 34.3 mg, 29% yield) and 4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,44R,45S,46R,47R, 57R)-46,57-dihydroxy-45-[(1R)2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-35, 36,37,38,48,49hexamethyl-50,51,52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta-23,25,27(48),28(49)tetraen-44-yl ]oxy]-N,Ndimethylbutanamide (I-61: 24.2 mg, 20% yield), both as a white solid.

Chiral separation method:

Column

Column size

Solution Concentration Introduction

Mobile phase Flow rate Wavelength Temperature

CHIRALPAK IC 5.0 cm I.D. x 25 cm L 1 mg/ml 5 ml

EUN=100% ml/min

UV 254 nm °C

I-62: ESI-MS (EI+, m/z): 1079.4 [M+Na]+. ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.30 (tt, J=34.4, 17.0 Hz, 2H), 6.13 (dd, J=14.9, 10.0 Hz, 1h) , 5.91 (dd, J=28.8, 10.6 Hz, 1H), 5.51 (dd, J=15.0, 8.9 Hz, 1H), 5.45 - 5.37 (m , 1H), 5.27 (d, J=5.4 Hz, 1H), 5.17 (d, J=4.4 Hz, 1H), 4.75 (s, 1H), 4.19 (d , J=4.9 Hz, 1H), 3.88 (s, 1H), 3.82 - 3.64 (m, 5H), 3.62 - 3.52 (m, 2H), 3.47 - 3.25 (m, 11H), 3.24 - 3.14 (m, 2H), 3.10 (d, J=7.0 Hz, 1H), 3.01 (s, 3H), 2.94 (s, 3H), 2.71 (dd, J=16.7, 5.7 Hz, 2H), 2.55 (dd, J=16.8, 6.6 Hz, 1H), 2.44 - 2.25 (m, 4H), 2.14-1.63 (m, 17H), 1.33 (ddd, J=40.8, 27.4, 12.3 Hz, 11H), 1.14- 0.83 (m, 18H), 0.71 (dd, J=23.8, 11.9 Hz, 1H).

I-61: ESI-MS (EI+, m/z): 1079.3 [M+Na]+. 1H NMR (400 MHz, CDCl ₃ ) δ 6.43 -5.88 (m, 4H), 5.65 - 5.08 (m, 5H), 4.33 - 4.08 (m, 2H), 3 .94 - 3.52 (m, 6H), 3.49 - 3.31 (m, 8H), 3.30 - 3.12 (m, 8H), 3.09 - 2.81 (m, 8H) , 2.75 - 2.26 (m, 6H), 2.10 (d, J=63.9 Hz, 3H), 1.88-1.65 (m, 14H), 1.35 (dt, J =49.7, 11.3 Hz, 9H), 1.18-0.81 (m, 18H), 0.77 - 0.60 (m, 1H).

Example 48. Synthesis of 4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,45S,46R,47R,57R)-46,57-dihydroxy-45-[(1R) -2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy35,36,37,38,48,49-hexamethyl-50,51, 52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta23,25,27(48),28(49)-tetraen-44-yl]oxy]-N^Ni-dimethylbutanamide (I-68) and 4-[[(22E,24E,26E,27E,34R,35S, 36R,37R,39S,41S,43R,44S,45R,46R,56R)-45,56-dihydroxy-44-[(1R)-2 -[(1S,3R,4R)-4-(2-hydroxyethoxy)- 168 045993

3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-49,50,51,52,53-pentaoxo68,69-dioxa-58-azatricyclohexatriaconta-22, 24,26(47),27(48)-tetraen-43-yl]oxy]-K-methylbutanamide (I-59) and 4-[[(22E,24E,26E,27E,34R,35S,36R,37R ,39S,41S,43S,44S,45R,46R,56R)-45,56-dihydroxy-44-[(1R)2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl ]-1-methylethyl]-46-methoxy-34,35,36,37,47,48hexamethyl-49,50,51,52,53-pentaoxo-68,69-dioxa-58-azatricyclohexatriaconta-22,24,26 (47),27(48)tetraen-43-yl]oxy]-N-methylbutanamide (I-60)

Synthesis circuit

n

Chiral separation

Methods and characteristics.

Stage 1: synthesis of 4-hydroxy-H^dimethylbutanamide.

To a solution of methylamine (5.41 g, 174.24 mmol) in water (30 ml) was added dropwise tetrahydrofuran2-one (5 g, 58.08 mmol) at 0°C. The mixture was stirred at 5°C for 2 hours, then concentrated and lyophilized to give 4-hydroxy-Nmethylbutanamide (6.5 g, 95.5% yield) as a thick liquid.

ESI-MS (EI+, m/z): 118.3 [M+H] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.02 (s, 1H), 3.69 (t, J=5.7 Hz, 2H),

- 169 045993

2.81 (d, J=4.8 Hz, 3H), 2.36 (t, J=6.8 Hz, 2H), 1.88 (dt, J=12.2, 6.1 Hz, 2H ).

Step 2: synthesis of 4-[[(22E,24E,26E,27E,34R,35S,36R,37R,39S,41S,44S,45R,46R,56R)-45,56-gurugroxy-44-[(1R) -2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy34,35,36,37,47,48-hexamethyl-49,50, 51,52,53-pentaoxo-68,69-dioxa-58-azatricyclohexatriaconta22,24,26(47),27(48)-tetraen-43-yl]oxy]-N-methylbutanamide (I-68): Everolimus solution (1 g, 1.04 mmol) and 4-hydroxy-N-methylbutanamide (2.45 g, 20.87 mmol) in THF (30 ml) were cooled to 0°C under N2 and p-toluenesulfonic acid (0 .9 g, 5.22 mmol). The reaction mixture was heated to 35°C and stirred for 18 hours, then poured into saturated NaHCO ₃ solution (150 ml) and extracted with EtOAc (100 ml x 3 ). The combined organic layers were washed with water (80 ml), brine (80 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The residue was purified by reverse phase chromatography (C18, 80 g, CH3CN:H ₂ O=37:33), resulting in 4-[[(22E,24E,26E,27E,34R,35S,36R,37R, 39S,41S,44S,45R,46R,56R)-45,56-dihydroxy-44[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1 -methylethyl]-46-methoxy-34,35,36, 37,47,48-hexamethyl-49,50,51,52,53-pentaoxo-68,69-dioxa-58-azatricyclohexatriaconta-22,24, 26( 47),27(48)-tetraen-43-yl]oxy]-N-methylbutanamide (I-68: 0.14 g, 13% yield) as a white solid. ESI-MS (EI+, m/z): 1065.3 [M+Na]+. ^1H NMR (400 MHz, CDCl ₃ ) δ 6.50 - 5.85 (m, 4H), 5.78 4.96 (m, 5H), 4.78 (s, 1H), 4.33 - 4 .03 (m, 2H), 3.98 - 3.64 (m, 5H), 3.63 - 3.49 (m, 2H), 3.49 - 2.90 (m, 13H), 2.90 - 2.48 (m, 6H), 2.41 - 1.94 (m, 7H), 1.93 - 1.54 (m, 18h), 1.53-1.11 (m, 10H), 1 .11-0.80 (m, 16H), 0.780.54 (m, 1H).

Step 3: synthesis of 4-[[(22E,24E,26E,27E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,56R)-45,56-dihydroxy-44-[( 1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy34,35,36,37,47,48-hexamethyl-49, 50,51,52,53-pentaoxo-68,69-dioxa-58-azatricyclohexatriaconta22,24,26(47),27(48)-tetraen-43-yl]oxy]-N-methylbutanamide (I-60) and 4-[[(22E,24E,26E,27E,34R,35S,36R, 37R,39S,41S,43R,44S,45R,46R,56R)-45,56-dihydroxy-4-4[(1R)- 2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3methoxycyclohexyl]-1-mepshlethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-49,50,51 ,52,53-pentaoxo68,69-dioxa-58-azatricyclohexatriaconta-22,24,26(47), 27(48)-tetraen-43-yl]oxy]-N-methylbutanamide (1-59).

130 mg 4-[[(22E,24E,26E,27E,34R,35S,36R,37R,39S,41S,44S,45R,46R,56R)-45,56-dihydroxy-44[(1R)-2- [(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy34,35,36,37,47,48-hexamethyl-49,50,51,52 ,53-pentaoxo-68,69-dioxa-58-azatricyclohexatriaconta22,24,26(47),27(48)-tetraen-43-yl]oxy]-N-methylbutanamide was purified by preparative chiral HPLC and the resulting epimers were purified with using silica gel chromatography (hexane:DCM:EtOAc:MeOH=3:3:1:0.8), resulting in 4[[(22E,24E,26E,27E,34R,35S,36R,37R,39S, 41S,43S, 44S,45R,46R,56R)-45,56-dihydroxy-44-[(1R)-2[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1 -methylethyl]-46-methoxy-34,35,36,37,47,48hexamethyl-49,50,51,52,53-pentaoxo-68,69-dioxa-58-azatricyclohexatriaconta-22,24,26(47) ,27(48)tetraen-43-yl]oxy]-N-methylbutanamide (I-60: 25 mg, yield 19%) and 4[[(22E,24E,26E,27E,34R,35S,36R,37R, 39S,41S,43R,44S,45R,46R,56R)-45,56-dihydroxy-44-[(1R)-2[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl] -1-methylethyl]-46-methoxy-34,35,36,37, 47,48hexamethyl-49,50,51,52,53-pentaoxo-68,69-dioxa-58-azatricyclohexatriaconta-22,24,26( 47), 27(48)tetraen-43-yl]oxy]-N-methylbutanamide (I-59: 36 mg, 27% yield), both as a white solid.

Chiral analysis method: Column Column size Introduction Mobile phase Flow rate Wavelength Temperature

CHIRALPAK IC-3(IC30CE-NJ008) 0.46 cm I.D. *25 cm L 50.0 µl

Hexane/EUN=5 0/50(v/v)

1.0 ml/min

UV 254 nm °C

HPLC equipment: Shimadzu LC-20AT CP-HPLC-06

I-60: ESI-MS (EI ⁺ , m/z): 1065.4 [M+Na] ⁺ . 1H NMR (400 MHz, CDC13) δ 6.32 (ddd, J=31.4, 14.8, 10.2 Hz, 2H), 6.13 (dd, J=15.1, 9.9 Hz, 1H), 6.00 - 5.85 (m, 1H), 5.69 (s, 1H), 5.51 (dd, J=14.9, 9.0 Hz, 1H), 5.41 (d , J=9.9 Hz, 1H), 5.27 (d, J=5.7 Hz, 1H), 5.13 (dt, J=48.5, 24.3 Hz, 1H), 4.77 (s, 1H), 4.18 (d, J=5.7 Hz, 1H), 3.92 3.63 (m, 6H), 3.61 - 3.50 (m, 2H), 3.46 - 3.25 (m, 10H), 3.22 - 3.00 (m, 3H), 2.79 (dd, J=4.8, 2.2 Hz, 3H), 2.72 (dd, J =16.9, 5.5 Hz, 2H), 2.55 (dd, J=16.8, 6.5 Hz, 1H), 2.38 - 2.14 (m, 4H), 2.12- 1.91 (m, 4H), 1.89-1.62 (m, 15H), 1.52-1.11 (m, 13H), 1.10-1.01 (m, 6H), 1, 00-0.81 (m, 9H), 0.71 (dd, J=23.6, 12.1 Hz, 1H).

I-59: ESI-MS (EI ⁺ , m/z): 1065.4 [M+Na] ⁺ . 1H NMR (400 MHz, CDCl ₃ ) δ 6.37 - 5.81 (m, 4H), 5.66 (d, J=15.6 Hz, 1H), 5.60 - 4.89 (m, 4H ), 4.16 (ddd, J=31.9, 15.6, 5.8 Hz, 2H), 3.93 - 3.42 (m, 8H), 3.38 - 2.95 (m, 12H ), 2.89 - 2.39 (m, 7H), 2.37 - 1.89 (m, 9H), 1.83 - 1.60 (m, 13H), 1.45 - 1.05 (m , 11H), 1.03 - 0.73 (m, 18H), 0.62 (dd, J=23.9, 12.1 Hz, 1H).

Example 49 Synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihyde

- 170 045993 roxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]- 1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta21,23,25(47),26(48)-tetraene-49,50 ,51,52,53-pentons (I-28)

Synthesis circuit

Methods and characteristics.

Step 1: synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy43-[2-[2-(2-hydroxyethoxy )ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1methylethyl]-46-methoxy-34,35,36,37,47, 48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-28).

To a solution of rapamycin (0.5 g, 0.547 mmol) in THF (10 ml) at 20°C under N ₂ , 4-methylbenzenesulfonic acid hydrate (0.52 g, 2.73 mmol) and 2-[2- (2-hydroxyethoxy)ethoxy]ethanol (1.72 g, 11.49 mmol, 3 ml). The resulting solution was stirred for 2 hours, then poured into saturated NaHCO ₃ solution (80 ml) and extracted with EtOAc (60 ml x 3). The combined organic layers were washed with water (60 ml), brine (60 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The residue was purified by reverse phase chromatography (C-18, CH ₃ CN:H ₂ O=75:25), resulting in (21E,23E,25E,26E,34R, 35S,36R,37R,39S,41S,44S ,45R,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44[(1R)-2-[(1S,3R,4R)-4 -hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47), 26(48)-tetraene-49,50,51,52,53-pentone (I28: 125 mg, 22% yield) as a white solid. ESI-MS (EI ⁺ , m/z): 1054.5 [M+Na] ⁺ . ^1H NMR (500 MHz, CDCl ₃ ) δ 6.44 - 5.87 (m, 4H), 5.52 - 5.12 (m, 4H), 4.90 (s, 1H), 4.34 - 4.11 (m, 1H), 4.05 - 3.83 (m, 1H), 3.80 - 3.53 (m, 13H), 3.50 -3.22 (m, 12H), 3, 01 - 2.49 (m, 6H), 2.39 - 1.87 (m, 6H), 1.82 - 1.68 (m, 8H), 1.44 - 1.15 (m, 13H), 1.11-0.85 (m, 18H), 0.71-0.57 (m, 1H).

Example 50. Synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-[2- (2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methylethyl]-46-methoxy-34, 35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone ( I-30)

- 171 045993

Synthesis circuit

Methods and characteristics.

Step 1: synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-[2- (2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34.35, 36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-30) : 7.0 g of epimer mixture (Example 49; I-28) was purified by preparative chiral HPLC to give (21E,23E,25E, 26E,34R,35S,36R,37R,39S,41S,43R,44S ,45R,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)- 4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36, 37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47) ,26(48)-tetraene-49,50,51,52,53-pentone (I-30: 1.113 g, 16% yield) as a white solid. ESI-MS (EI ⁺ , m/z): 1054.4 [M+Na] ⁺ . ¹ H NMR (500 MHz, CDCls) δ 6.41 - 5.92 (m, 4H), 5.63 (ddd, J=23.1, 15.1, 8.2 Hz, 1H), 5.47 (dd, J=29.9, 10.3 Hz, 1H), 5.30 - 5.00 (m, 2H), 4.33 - 4.12 (m, 2H), 3.97 (dd, J =18.9, 6.6 Hz, 1H), 3.86 - 3.48 (m, 14H), 3.44 - 3.22 (m, 10H), 2.97 -2.88 (m, 1H ), 2.82 (s, 1H), 2.74 - 2.46 (m, 3H), 2.30 (d, J=14.3 Hz, 2H), 2.21 - 1.91 (m, 5H), 1.86 - 1.57 (m, 11H), 1.50 - 1.22 (m, 12H), 1.16 - 0.81 (m, 18H), 0.66 (dt, J= 23.8, 11.7 Hz, 1H).

Example 51. Synthesis of (22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,45S,46S,47R,56R)-46,56-dihydroxy45-[(1R)-2-[(1S ,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-44-[2-(2methoxyethoxy)ethoxy]-35,36,37,38,48,49 -hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone (I-121) he o

O.

1-121

- 172 045993

Synthesis circuit

Methods and characteristics.

Step 1: synthesis (22E,24E,26E,27E,31R,32S,33R,34R,36S,38S,40S,41S,42S,43R,52R ₂ ,42,52-dihydroxy-41-[(1R)-2 -[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-40,43-dimethoxy-31,32,33,34,44,45-hexamethyl-62, 63-dioxa-53-azatricyclohexatriaconta-22,24,26(44),27(45)-tetraene46,47,48,49,50-pentone.

To a solution of everolimus (1 g, 1.04 mmol) in DCM (50 ml) was added Ti(OiPr) ₄ (0.89 g, 3.13 mmol) dropwise at room temperature. The reaction mixture turned pale yellow. After 30 min, the solution was poured into a separatory funnel containing a heterogeneous mixture of 1 N. HCl (50 ml) and EtOAc (50 ml). The organic layer was washed successively with saturated aqueous NaHCO ₃ (30 ml), H ₂ O (50 ml), brine (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (hexane:acetone=2:1) to give (22E,24E,26E,27E,31R,32S,33R,34R,36S,38S,40S,41S,42S,43R, 52R)42,52-dihydroxy-41-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]40,43-dimethoxy-31 ,32,33,34,44,45-hexamethyl-62,63-dioxa-53-azatricyclohexatriaconta-22,24,26(44), 27(45)-tetraene-46,47,48,49,50-pentone (380 mg, 38% yield) as a light yellow solid. ¹ H NMR (400 MHz, DMSO-06): δ 6.44 (dt, J=13.9, 10.3 Hz, 2H), 6.33 - 6.06 (m, 3H), 5.54 - 4.85 (m, 5H),

4.48 (t, J=5.3 Hz, 1H), 4.10 - 3.91 (m, 2H), 3.89 - 3.79 (m, 1H), 3.62 (d, J= 11.5 Hz, 1H), 3.54 - 3.41 (m, 5H),

3.38 - 3.28 (m, 8H), 3.19 (dt, J=11.9, 7.6 Hz, 4H), 3.10 - 2.92 (m, 6H), 2.71 ( t, J=14.8 Hz, 1H), 2.43 - 1.78 (m,

6H), 1.75 - 1.44 (m, 10H), 1.38 - 0.90 (m, 14H), 0.89 -0.67 (m, 13H), 0.66 - 0.56 ( m, 1H).

Step 2: synthesis of (22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,45S,56R)-46,56-dihydroxy-45-[(1R)-2[(1S,3R, 4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48,49- hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48), 27(49)-tetraene-50,51,52,53,54-pentone (I-121).

HND-8 (35 mg) was added to a solution of 28-epi-everolimus (0.2 g, 0.208 mmol) and 2-(2-methoxyethoxy)ethanol (0.99 ml, 8.35 mmol) in sulfolane (5 ml) at 50°C in N2 atmosphere. The resulting solution was stirred at 50°C for 4 hours, filtered and diluted with water (30 ml) and EtOAc (30 ml). The organic layer was washed with water (10 mlx3), brine (20 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The residue was purified by reverse phase chromatography (C18, CH3CN:H2O=6.5:3.5) to give (22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,45S ,56R)46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-47methoxy-44-[ 2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene- 50,51,52,53,54-pentone (I-121: 60 mg, 27% yield) as a white solid. ESI-MS (EG+, m/z): 1068.1 [M+Na]+. ¹ H NMR (500 MHz, DMSO-d): δ 6.57 - 6.35 (m, 2H), 6.31 - 5.88 (m, 3H), 5.61 - 5.38 (m, 1H), 5.28 - 4.82 (m, 4H), 4.47 (t, J=5.3 Hz, 1H), 3.98 (dd, J=40.4, 6.9 Hz, 2H ), 3.88 - 3.73 (m, 2H), 3.59 - 3.40 (m, 12H), 3.30 (dd, J=12.1, 8.2 Hz, 4H), 3, 26 - 3.14 (m, 8H), 3.08 2.92 (m, 3H), 2.85 - 2.62 (m, 2H), 2.43 - 2.22 (m, 2H), 2 .19 - 1.82 (m, 6H), 1.79 - 1.45 (m, 9H), 1.32 (dd, J=58.1, 21.2 Hz, 5H), 1.19 - 0 .92 (m, 10H), 0.90 - 0.57 (m, 15H).

Example 52. Synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45S,46R,55R)-45,55-dihydroxy43-[2-[2-(2-hydroxyethoxy )ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1methylethyl]-46-methoxy-34,35,36,37,47, 48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta- 173 045993

21,23,25(47),26(48)-tetraene-49,50,51,52,53-pengon (I-127) and (21E,23E,25E,26E,34R,35S,36R,37R, 39S, 41S,43S,44S,45S,46R,55R)-45,55-gurugpoKCu-43-[2-[2-(2-rugpoKcmTOKCu^TOKCu^TOKCu]-44-[(1R)-2[(1S ,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl66,67-dioxa-56-azatricyclohexatriaconta-21,23, 25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-128) and (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S, 43R,44S,45S,46R,55R)-45,55-gurugpoKCu-43-[2-[2-(2-rugpoKsiethoxy)ethoxy]ethoxy]-44-[( 1 R)-2- [(1 S, 3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25 (47),26(48)tetraene-49,50,51,52,53-pentone (I-129) it o

He

1-127 he about he about

1-128 1-129

Synthesis scheme

he he oh he oh

Methods and characteristics.

Stage 1. Synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45S,46R,55R)-45,55-dihydroxy- 174 045993

43-[2-[2-(2-1hydroxyethoxy)ethoxy]ethoxy]-44-[(III)-2-[(^,31C4I)-4-hydroxy-3-\1ethoxy11.cyclohexyl]-1methylethyl]-46 -methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta21,23,25(47), 26(48)-tetraene-49,50,51,52,53 -pentone (I-127).

To a solution of 28-epi-rapamycin (0.2 g, 0.22 mmol; see example 52) and 2-[2-(2-hydroxyethoxy)ethoxy]ethanol (0.436 ml, 3.28 mmol) in sulfolane (5 ml) HND-8 (30 mg) was added and the mixture was stirred at 50°C for 5 hours. After cooling, the reaction mixture was diluted with EtOAc (50 ml), filtered, washed with water (50 mlx3) and brine (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The residue was purified by reverse phase chromatography (C18, CH ₃ CN:H ₂ O, 0 to 70% yield), resulting in (21E,23E,25E,26E,34R,35S,36R, 37R,39S, 41S,44S,45S,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2[(1S,3R,4R )-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47) ,26(48)-tetraene-49,50,51,52,53-pentone (I-127: 60 mg, 26% yield) as a light yellow solid. ESI-MS (EI ⁺ , m/z): 1054.4 [M+Na] ⁺ . ^1H NMR (400 MHz, DMSO-d^δ 6.61 - 6.32 (m, 2H), 6.30 - 5.98 (m, 3H), 5.65-5.38 (m, 1H) , 5.28-4.89 (m, 4H), 4.68 - 4.53 (m, 2H), 4.11-3.91 (m, 2H), 3.89 - 3.71 (m, 2H), 3.54 - 3.44 (m, 9H), 3.44 - 3.40 (m, 3H), 3.31 - 3.28 (m, 4H), 3.27 - 3.11 ( m, 6H), 2.86 - 2.66 (m, 3H), 2.20 - 1.82 (m, 7H), 1.80 - 1.58 (m, 13H), 1.42 - 1, 06 (m, 9H), 1.05 - 0.68 (m, 18H), 0.56 (dd, J=23.8, 12.0 Hz, 1H).

Step 2. Synthesis of (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45S,46R,55R)-45,55-dihydroxy-43-[2-[2- (2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]1-methylethyl]-46-methoxy-34,35,36 ,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-128) and (21E,23E,25E,26E,34R,35S,36R,37R,39S, 41S,43R,44S,45S,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy )ethoxy]ethoxy]-44-[(1R)-2[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47 ,48-hexamethyl66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-129):

130 mg (21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45S,46R,55R)-45,55-dihydroxy-43-[2-[2-(2hydroxyethoxy)ethoxy ]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]46-methoxy-34,35,36,37,47,48 -Hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47), 26(48)-tetraene-49,50,51,52,53-pentone was purified by preparative chiral HPLC and the resulting epimers were purified using silica gel chromatography (hexane:DCM:EtOAc:MeOH=3:3:1:0 to 3:3:1:0.8), resulting in (21E,23E,25E,26E,34R,35S, 36R,37R,39S,41S,43S,44S,45S,46R,55R)45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2 -[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56- azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone (I-128: 28 mg, yield 21.5%) and (21E,23E,25E ,26E,34R,35S,36R,37R,39S,41S,43R,44S,45S,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]- 44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-46methoxy-34,35,36,37,47,48-hexamethyl-66, 67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)tetraene-49,50,51,52,53-pentone (I-129: 22 mg, 16.9% yield), both in the form of a white solid.

Chiral separation method

Column

Column Size Introduction

Mobile phase Flow rate Wavelength Temperature HPLC equipment

CHIRALPAK IC(IC00CE-WF029) 0.46 cm I.D. x 25 cm L 10.0 µl

Hexane/EtOH =60/40(v/v)

1.0 ml/min

UV 254 nm °C

Shimadzu LC-20AT CP-HPLC-09

I-128: ESI-MS (EI ⁺ , m/z): 1054.2 [M+Na] ⁺ . ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.56 -6.22 (m, 2H), 6.14 (dd, J=15.0, 10.3 Hz, 1H), 6.00 (dd, J=17.9, 11.0 Hz, 1H), 5.61 - 5.46 (m, 1H), 5.46 - 5.32 (m, 1H), 5.29 - 5.12 (m, 2H), 4.70 (s, 1H), 4.15 - 4.04 (m, 1H), 3.99 -3.88 (m, 1H), 3.86 - 3.52 (m, 13H) , 3.51 - 3.25 (m, 11H), 3.01 2.60 (m, 5H), 2.52 (dd, J=16.7, 7.1 Hz, 2H), 2.39- 1.87 (m, 7H), 1.74 (dt, J=13.3, 8.7 Hz, 8H), 1.63 - 1.16 (m, 13H), 1.14 - 0.78 ( m, 18H), 0.67 (dd, J=23.8, 12.0 Hz, 1H).

I-129: ESI-MS (EI ⁺ , m/z): 1054.1 [M+Na] ⁺ . ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.46 - 5.89 (m, 4H), 5.60 4.93 (m, 5H), 4.15 (dd, J=50.5, 20, 8 Hz, 2H), 3.97 - 3.15 (m, 25H), 3.06 - 2.43 (m, 8H), 2.39 - 1.69 (m, 16H), 1.54 - 1 .19 (m, 10H), 1.13 - 0.79 (m, 18H), 0.66 (dd, J=23.6, 11.7 Hz, 1H).

Example 53 AlphaLISA Ultra pS6K1 Study.

Study protocol.

1. MCF-7 cells are seeded into a Corning 3701 plate and incubated for 20-24 hours. 12,000-16,000 cells are seeded in 36 μl of medium per well.

2. The culture medium is replaced with fresh medium and incubated for another 2 hours.

3. Add 12 µl (4x) to the cell plate using the HAMILTON system. The final concentration of DMSO is 0.5%. Incubate for 90 minutes.

4. Take 38 µl using the HAMILTON system, leaving 10 µl in each well.

- 175 045993

5. Add 10 µl of 2x lysis buffer using the HAMILTON system; the total volume in the wells is 20 µl. The cells are left to mix for 30 min. The plate is covered with a polymer film and the plate is kept at -80°C for analysis.

6. The cell lysate is thawed at room temperature and 10 μl of the lysate is transferred to an assay plate (Optiplate-384).

7. Add 5 µl of acceptor beads to the assay plate and incubate for 2 hours.

8. Add 5 µl of donor beads and incubate for 2 hours.

9. Calculate using the EnSpire Multimode Plate Reader.

Table 2. Key reagents/suppliers

Reagents/materials Provider Catalog no. Batch number MCF-7 ATCC HTB-22 5105360 DMEM Invitrogen 12430-054 1677193 FBS Invitrogen 10099-141 1660516 0.25% trypsin-EDTA Invitrogen 25200-072 1638603 Treated 3 84-well intramural for tissue culture Corning CLS3701 29214010 Corning 384-well storage plates Corning CLS3656 29514036 Thorin 1 Selleck S2827 01 Rapamycin SELLECK S1039 08 3 84-well microplate with white opaque wells OptiPlate-384 PerkinElmer 6007299 8210-14501 AlphaLISA SureFire Ultra р-р70 S6 Kinase (Thr389) Assay Kit PerkinElmer ALSU-PP70A10K U0381

Example 54 AlphaLISA Ultra pAKT Study.

Study protocol.

1. MCF-7 cells are seeded into a Corning 3701 plate and incubated for 20-24 hours. 16,000-20,000 cells are seeded in 36 μl of medium per well.

2. The culture medium is replaced with fresh medium and incubated for another 90 minutes.

3. Add 12 µl (4x) of the compound to the cell plate using the HAMILTON system. The final concentration of DMSO is 0.5%. Incubate for 2 hours.

4. Take 38 µl using the HAMILTON system, leaving 10 µl in each well.

5. Add 10 µl of 2x lysis buffer using the HAMILTON system; the total volume in the wells is 20 µl. The cells are left to mix for 30 min. The plate is covered with a polymer film and the plate is kept at -80°C for analysis.

6. The cell lysate is thawed at room temperature and 10 μl of the lysate is transferred to an assay plate (Optiplate-384).

7. Add 5 µl of acceptor beads to the assay plate and incubate for 2 hours.

8. Add 5 µl of donor beads and incubate for 2 hours.

9. Calculate using the EnSpire Multimode Plate Reader.

Table 3. Key Reagents/Suppliers

Reagents/materials Provider Catalog no. Batch number MCF-7 ATCC HTB-22 5105360 DMEM Invitrogen 12430-054 1677193 FBS Invitrogen 10099-141 1660516 0.25% trypsin-EDTA Invitrogen 25200-072 1638603 Treated 3 84-well tissue culture plate Corning CLS3701 29214010 3 Corning 84-well storage plates Corning CLS3656 29514036 Thorin 1 Selleck S2827 01 Rapamycin SELLECK S1039 08 3 84-well microplate with white opaque wells OptiPlate-384 PerkinElmer 6007299 8210-14501 AlphaLISA SureFire Ultra p-Akt 1/2/3 (Ser473) Assay Kits PerkinElmer ALSU-PAKTB10K U0329

Example 55 Western Blot Based Study of pS6K1 and pAKT at 24 and 48 hour time points.

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Study protocol.

1. PC3 cells are seeded into six-well plates at a rate of 500,000 cells per well and incubated for 20-24 hours.

2. Compounds are added to each cell of the plate. Incubate for 24-48 hours.

4. Place the plate on ice and remove the medium using a pump. The wells are washed with 1 ml of 1x PBS and then it is completely removed using a pump.

5. Add 110 µl of lysis buffer with 1% Triton and scrub each well vigorously.

6. Cell homogenates are transferred to 1.5 ml Eppendorf tubes on ice and centrifuged at 4°C for 10 minutes at 10,000 rpm.

7. Determine the protein concentration in the resulting cell lysates using the Bradford method and analyze the samples using Western blotting with 4-12% Bis/Tris gels with 1x MES buffer.

8. Gels are transferred to membranes at 50V for 100 min, blocked with Odyssey blocking buffer for 30 min and then incubated overnight with primary antibody (pS6K1 T389 Rabbit or pAkt S473 Rabbit) overnight at 4°C on a shaker.

9. Membranes are washed 3 times with TBS-T with a 5 minute incubation between each wash and then incubated with secondary antibody (LiCor IRDye 800 Donkey Anti Rabbit) for at least 30 minutes.

10. Membranes were washed 3 times with TBS-T with a 5-minute incubation between each wash.

11. The gels were then incubated for 5 min with PBS at room temperature and then imaged using Li-Cor.

Results for an exemplary Western blot are shown in FIG. 1. PC3 cells were treated with rapamycin (0.1 and 0.01 µM) or I-29 (1, 0.1, 0.01 and 0.001 µM) for 24 and 48 h. The blots clearly show a significant decrease in pS6K1 as in rapamycin, and in the case of I-29 after 24 and 48 hours, which is an indicator of inhibition of the mTORC1 pathway. Importantly, I-29 did not decrease levels at 24 or 48 h. In contrast, rapamycin showed inhibition of S6K1 ( ^S473 ) phosphorylation at both 24 and 48 h, an indicator of inhibition of the mTORC2 pathway. The results of additional illustrative Western blot analyzes and compounds thus evaluated are shown in FIG. 2-42. The Methods used were essentially the same as those described above. Compounds were evaluated in PC3 cells, Jurkat cells, wild-type mouse emryon fibroblast (MEF) cells, tuberous sclerosis type 2 (TSC2) negative (TSC -/-) MEF cells, and tuberous sclerosis type 2 (TSC2) positive (TSC + /+) MEF cells. Cells were incubated with compounds of the present invention for various periods of time (eg, 5, 15, 30, 90 minutes, 24 or 48 hours) and assessed in accordance with known techniques such as those described herein. In table 4 shows the inhibitory activity (IC ₅₀ ) of selected compounds of the present invention in the pS6K1 and pAKT assays and their solubility in 100 mM phosphate buffer (pH 7.4). The connection numbers correspond to the connection numbers in the table. 1.

Compounds of the present invention that selectively inhibited mTORC1 over mTORC2 and maintained their selectivity for at least 24 hours are indicated with the word YES in the mTORC1 selective at 24 hours column of the table. 4. Compounds that are not selective at 24 h are marked with the word FIET in the column selective for mTORC1 at 24 h in the table. 4. Compounds that partially retain their selectivity for inhibiting mTORC1 over mTORC2 are indicated by the word Partially in the column selective for mTORC1 after 24 hours of the table. 4. N/A means not determined.

Compounds designated + show solubility less than 30 μM (x < 30 μM). Compounds designated ++ exhibit solubility greater than or equal to 30 μM and less than 60 μM (30 μM < x < 60 μM). Compounds designated +++ demonstrate a solubility greater than or equal to 60 μM (60 μM<x). Compounds designated A exhibited an IC ₅₀ of less than 0.1 nM (x<0.1 nM). Compounds designated B exhibited IC50 greater than or equal to 0.1 nM and less than 1 nM (0.1 nM < x < 1.0 nM). Compounds designated C exhibited an IC ₅₀ greater than or equal to 1.0 nM and less than 10 nM (1.0 nM < x < 10 nM). Compounds designated D exhibited an IC ₅₀ greater than or equal to 10 nM and less than 100 nM (10 nM < x < 100 nM). Compounds designated E exhibited an _IC50 greater than or equal to 100 nM (100 nM<x).

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Table 4. Study results for individual compounds

pS6Kl V rACTv Selectively Connection number MCF7 MCF7 V Solubility in 90 after 90 minutes: mTORCl in water at pH 7.4 (µM) min: 1C ₅₀ in 24 hours IC50 1-1 IN E YES ++ 1-2 IN E YES ++ 1-3 IN E YES ++ 1-4 IN BUT YES BUT 1-5 But BUT YES BUT 1-6 With E YES But 1-7 But BUT YES But 1-8 With But YES But 1-9 But But YES But 1-10 A E YES But 1-11 WITH E YES But 1-12 BUT BUT YES But 1-13 With But YES But 1-14 But But YES But 1-15 With E YES ++ 1-16 V E YES ++ 1-17 V E YES ++ 1-19 A E YES ++ 1-20 IN E YES ++ 1-21 A E YES + 1-22 IN E YES But 1-23 A E YES But 1-24 A E YES + 1-25 IN E YES But 1-26 IN E YES +++ 1-27 IN E YES ++ 1-30 IN E YES +++ 1-31 IN E YES +++

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1-32 IN E YES +++ 1-33 WITH E YES +++ 1-34 IN E NO ++ 1-35 IN E YES +++ 1-36 WITH E YES +++ 1-37 WITH E YES ++ 1-38 A E NO ++ 1-39 WITH E YES BUT 1-29 A E YES ++ 1-57 A BUT YES But 1-58 IN But YES But 1-59 E BUT YES But 1-60 WITH But YES But 1-61 WITH But YES But 1-62 WITH But YES But 1-63 E But YES But 1-64 WITH But YES But 1-65 V But YES But 1-66 With But YES But 1-67 V But YES But 1-68 With But YES But 1-69 V But YES But 1-70 With But YES But 1-71 V But YES But 1-72 With But YES But 1-73 With But YES But 1-74 V But NO But 1-75 A But YES But 1-76 With But YES But 1-77 A But YES But 1-78 WITH But YES But 1-79 WITH But YES But

- 179 045993

1-80 IN But YES BUT 1-81 C But YES But 1-82 IN But YES But 1-83 H/0 But YES But 1-84 V But YES But 1-85 H/0 But YES But 1-86 A But YES But 1-87 A But YES But 1-88 A But YES But 1-89 C But YES But 1-90 But YES But 1-91 A But YES But 1-92 IN But YES But 1-28 H/0 But YES But 1-95 H/0 But YES But 1-96 V But YES But 1-97 c But YES But 1-98 c But YES But 1-99 V But YES But 1-100 c But YES But 1-101 V But YES But 1-102 D But YES But 1-103 IN But YES But 1-104 IN But YES But 1-105 A But YES But 1-106 IN But YES But 1-107 E But YES But 1-108 IN But YES But 1-109 WITH But YES But 1-110 IN But YES But 1-111 WITH But YES But

- 180 045993

1-112 IN But YES BUT 1-113 IN But YES But 1-114 WITH But YES But 1-115 IN But YES But 1-116 IN But YES But 1-117 A But YES But 1-118 WITH But YES But 1-119 A But YES But 1-120 A But YES But

Example 56. Pharmacokinetic properties.

The pharmacokinetic properties of the compounds of the present invention were evaluated in C57B1/6 mice and compared with those of rapamycin. Animals were fasted overnight before administration of I-29 or rapamycin (1 mg/kg intravenously, 10 mg/kg orally, or 2 mg/kg intraperitoneally). Animals were bled at specified time intervals for 48 hours after administration of the compound. Whole blood from each mouse was individually collected into polypropylene tubes and immediately centrifuged. Aliquots of separated plasma were quickly prepared for HPLC analysis. The results of pharmacokinetic studies are given in table. 5. Compound I-29 demonstrates improved oral bioavailability compared to rapamycin, as well as reduced clearance, increased half-life, increased Cmax and increased AUC compared to rapamycin.

Table 5. Comparison of pharmacokinetic properties of rapamycin and I-29 in mice

1-29 Rapamycin

1 mg/kg i.v. CL (l/h/kg) 0.054 0.276 V _ss (l/kg) 0.863 1.73 Final Τι/ ₂ (h) 14.7 8.35 AUCiast (h*ng/ml) 17591 3616 AUCinf (h*ng/ml) 18393 3627 MRT _inf (h) 15.9 6.27 10 mg/kg po Tschah ( ^h ) 0.25 3 S _tach (NG/ML) 3287 52.8 Final Τι/ ₂ (h) eleven BUT AUC _las t (h*ng/ml) 31236 292 AUCinf (h*ng/ml) 31726 BUT F (%) 17.2 0.8 2 mg/kg i.p. Tschah C ⁴ ) 0.25 0.25 Stakh (NG/ML) 2590 614 Final Τι/ ₂ (h) 12.6 4.8 AUCiast (h*ng/ml) 16607 2481 AUCinf (h*ng/ml) 16937 2544 F (%) 46 35.1

Example 57: Evaluation of the effect of long-term treatment with I-29 on glucose tolerance and insulin sensitivity in the C57Bl/6 mouse strain.

C57Bl/6 mice (n=12; 8 weeks old) were randomly assigned to groups and baseline measurements (body weight, fasting glucose and insulin) were performed four (4) days prior to compound or vehicle administration. Next, the animals were administered I-29 (10 mg/kg po), rapamycin (10 mg/kg i.p.) or vehicle (po or i.p.) for 19 days. On days 7 and 14, the animals were weighed and determined.

- 181 045993 fasting glucose and insulin levels were measured. The animals were fasted overnight, and on days 14 and 15, insulin levels were determined and an intraperitoneal glucose tolerance test (ipGTT) was performed. On day 19, animals were sacrificed one (1) hour after administration of compound or vehicle. Tissues were collected to evaluate compound content and pharmacodynamics. The results of the study depending on time are shown in Fig. 43.

The ipGTT results are shown in Fig. 44 and 45. In brief, chronic treatment with rapamycin for 15 days induced impaired glucose tolerance in C57Bl/6 mice, characterized by increased glucose levels compared to Vh R. On the other hand, I-29 does not cause impaired glucose tolerance .

Example 58: Evaluation of the effects of I-29, I-117 and everolimus in a mouse model of acute kidney disease/chronic kidney disease (AKI/CKD).

C57Bl/6 mice (n=15; male; 10 weeks old) were randomly assigned to groups. After 7 days of adaptation, IR or sham surgery was performed. Mice were allowed to recover for one (1) day and, starting from day 2, animals with IR surgery were allowed to recover. vehicle, everolimus (10 mg/kg po), I-29 (10 mg/kg po), or I-117 (10 mg/kg po) were administered. On day 9, one kidney was removed (Unx) or a simulated operation was performed. On day 29, the animals were sacrificed.

Histological assessment of the kidneys was carried out using the PAS reaction, Masson trichrome or Sirius red staining. The results of Sirius red staining are shown in Fig. 46 and 47 In brief, I-29 demonstrated a significant reduction in renal fibrosis compared with vehicle.

Kidney tissue mRNA was analyzed for markers of inflammation and fibrosis (qPCR for TGFe, collagen I, collagen III, CCTC-binding factor (CTCF), fibronectin (FN) and alpha-smooth muscle actin (α-SMA)).

Immunohistochemical analysis was performed to evaluate collagen I, collagen IV, α-SMA, 4-hydroxynonenal (4-HNE) and macrophage F4/80.

Plasma levels of the compound were determined, and renal tissues were evaluated for pharmacodynamics.

The results of the expression of markers of fibrosis and macrophage infiltration in the kidneys are shown in Fig. 48-51. In brief, I-29 significantly reduced the mRNA expression of collagen I, collagen III, and fibronectin. In addition, I-29 significantly reduced macrophage infiltration in the kidney.

Example 59. Assessment of the effect on the production of IFN-γ in the allogeneic reaction of mixed lymphocytes.

The effects of rapamycin, everolimus, I-29, and I-117 on IFN-γ production were assessed in an allogeneic mixed lymphocyte response (eg, Eleftheriadis, T. et al., Int. J. Mol. Med., 37(5): 1412- 20 (2016) https://doi.org/10.3892/ijmm.2016.2547). The IC ₅₀ values are given in the table below. 6 and FIG. 52 In brief, I-29 and I-117 did not inhibit IFN-γ production, whereas rapamycin and everolimus significantly inhibited IFN-γ production.

Table 6. IFN-γ inhibition

Rapamycin Everolimus 1-29 1-117 1C ₅₀ (nM) 2.4 15.2 >3000 >3000

Claims (4)

1. Compound of formula II -o. X Г\-\ 1 ^{0 but} ЧУ УЧ ' n I Гт ?< S ^ho ; I or a pharmaceutically acceptable salt thereof, where R ¹ is selected from or formula P-0 P-0 he o l' θχ H''> \ > \ G ⁿ T O. _R1 [ ^on ^OR - 182 045993 where means the place of attachment at the C-7-hydroxyl position; each Z is independently -O-, -S- or -SO2-; n ranges from 2 to 300; And R represents hydrogen or a C _1-6 aliphatic group, optionally substituted with halogen. 2. The connection according to claim 1, where R ¹ is selected from 3. The connection according to claim 1, where the specified connection is selected from it 1-8 It 1-9 It 1-13 - 183 045993 - 184 045993 - 185 045993 or a pharmaceutically acceptable salt thereof. 4. A pharmaceutical composition for the treatment of an mTORC-mediated disease, disorder or condition, comprising a compound according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier, adjuvant or vehicle.