Syntheses and anti-pancreatic cancer activities of rakicidin A analogues

Accepted Manuscript Syntheses and anti-pancreatic cancer activities of rakicidin A analogues Jian Chen, Jingpei Li, Lingling Wu, Yan Geng, Jianming Yu, Chuanke Chong, Mengmeng Wang, Yuan Gao, Cuigai Bai, Yahui Ding, Quan Zhang, Yue Chen PII:

S0223-5234(18)30321-0

DOI:

10.1016/j.ejmech.2018.03.078

Reference:

EJMECH 10343

To appear in:

European Journal of Medicinal Chemistry

Received Date: 12 January 2018 Revised Date:

22 March 2018

Accepted Date: 27 March 2018

Please cite this article as: J. Chen, J. Li, L. Wu, Y. Geng, J. Yu, C. Chong, M. Wang, Y. Gao, C. Bai, Y. Ding, Q. Zhang, Y. Chen, Syntheses and anti-pancreatic cancer activities of rakicidin A analogues, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.078. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Graphical abstract

Syntheses and Anti-pancreatic Cancer Activities of Rakicidin A Analogues

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Jian Chena, Jingpei Lia,b, Lingling Wua, Yan Genga,b, Jianming Yua,b, Chuanke Chonga,b, Mengmeng Wangc, Yuan Gaoa,b, Cuigai Baib, Yahui Dinga,*, Quan Zhanga,*

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and Yue Chena,*

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Syntheses and Anti-pancreatic Cancer Activities of Rakicidin A Analogues

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Jian Chena, Jingpei Lia,b, Lingling Wua, Yan Genga,b, Jianming Yua,b, Chuanke Chonga,b, Mengmeng Wangc, Yuan Gaoa,b, Cuigai Baib, Yahui Dinga,*, Quan Zhanga,* and Yue

a

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Chena,*

The State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Tianjin

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Key Laboratory of Molecular Drug Research, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300350, People's Republic of China b

High-throughput Molecular Drug Discovery Center, Tianjin International Joint

Accendatech Company, Ltd., Tianjin 300384, People's Republic of China

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c

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Academy of BioMedicine, Tianjin 300457, People's Republic of China

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*Correspondence authors:

Y.C.: Tel./Fax +86 22 85358387, E-mail: [email protected]; Q.Z.:

Tel./Fax

+86

22

85358387,

E-mail:

[email protected]

or

[email protected]; Y.D.: Tel./Fax +86 22 85358387, E-mail: [email protected].

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Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignant tumor and resistant to most therapies. Pancreatic cancer stem cells (PCSCs) had critical role in

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regulating PDAC progression, metastasis, and drug resistance. Therefore, targeting PCSCs is considered to be a promising strategy for treatment of PDAC. However, there is no effective drug that can selectively ablate PCSCs. A series of twenty rakicidin A

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analogues were synthesized via a combinatorial strategy and evaluated for their antiPDAC activities, and the structure-activity relationship was also discussed. Compound

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32g was prepared in 14 linear steps with 5.05% overall yield, which is much more efficient than our previously reported total synthesis of rakicidin A (19 linear steps with 0.19% yield). In a highly metastatic pancreatic cancer cell line ASPC-1, compound 32g showed about 4 times higher potency (IC50 = 0.022 µM) than rakicidin A (IC50 = 0.082

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µM) at hypoxia condition, and 12 folds of hypoxia selectivity (IC50 = 0.27 µM at nomoxia condition). In contrast, the activity of adriamycin in the same hypoxic condition decreased. The percentage of PCSCs (with CD24+CD44+ESA+ biomarker), activity of

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ALDH, and the number of tumorspheres in PANC-1 cells were greatly reduced after treatment of 32g. More importantly, the tumor-initiating frequency was reduced by 19

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folds after the treatment of 32g, which is better than that of rakicidin A (reduction of 4.7 folds).

Keywords: Rakicidin A analogues; Pancreatic ductal adenocarcinoma; Pancreatic cancer stem cells; Combinatorial strategy; Total synthesis

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1. Introduction

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Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States, and is considered as one of the deadliest human cancers with 6% of 5-year survival rates.[1–3] PDAC is a highly complex and aggressive malignant tumor,

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presenting with early local invasion and metastasis, and is resistant to most therapies.[4] Cancer stem cells (CSCs) are presumed to be capable of unlimited self-renewal, and

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through asymmetric division, they give rise to further differentiated cells. Previous studies revealed that tumor recurrence or metastasis following anticancer treatment could be attributed to CSCs.[5–7] Pancreatic cancer stem cells (PCSCs) could self-renew, differentiate, and divide asymmetrically, like normal stem cells; the critical role of

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PCSCs in regulating pancreatic cancer progression, metastasis, and drug resistance has been proposed.[5,8,9] Therefore, targeting PCSCs, which are resistant to radiation and chemotherapy, is considered to be a promising strategy for treatment of PDAC. However,

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no effective drug can selectively eliminate PDAC, and there is urgent need for discovery of agents that can target PCSCs.

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Rakicidin A was isolated in 1995 as one member of a family of six macrocyclic

depsipeptides with congeners (A, B, C, D, E and F) that differed by the constitution of the lipophilic side chain and β-asparagine fragment.[10–13] Rakicidin A exhibited selective toxicity towards hypoxic cancer cells,[14,15] and was reported to induce cell death in tyrosine kinase inhibitor (TKI)-resistant chronic myelogenous leukemia (CML) stem celllike cells.[15] Its unique structure and interesting activities attracted the interest of

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pursuing its total synthesis and medicinal chemistry.[16-20] We reported the first total synthesis of rakicidin A with 19 linear steps in an overall yield of 0.19%[18] and studied

highly unstable at room temperature.[18,20]

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its preliminary structure-activity relationship.[19] However, rakicidin A was found to be

Previous studies revealed that the biological activities of rakicidin A was attributed to unique 4-amino-2,4-pentadienolate (APD) moiety,[19–21] which served as a good

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starting point for drug development. The APD moiety is found in few natural products, including BE-43547 family, vinylamycin and microtermolide A.[22–25] Poulsen and co-

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works accomplished the total synthesis of ent-BE-43547A1 and revealed that ent-BE43547A1 showed significant hypoxia-selectivity against PANC-1 cell line.[26] Recently, our group reported that BE-43547A2 could selectively target PCSCs.[27] Herein, we design and synthesize a series of rakicidin A analogues using an efficient

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combinatorial approach, and evaluate their anti-pancreatic activities. The structureactivity relationship (SAR) is also discussed. It is noteworthy that the most promising compound 32g showed high hypoxia selectivity. Moreover, 32g could reduce the

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percentage of PCSCs with biomarker of CD24+CD44+ESA+, the number of tumorspheres formed, and the tumor-initiating frequency in PANC-1, which demonstrated that 32g

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could selectively eliminate PCSCs.

2. Results and Discussion 2.1 Chemistry

2.1.1 Synthesis of rakicidin A analogues 1a–e

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Our preliminary SAR study have demonstrated that change of NH2 (rakicidin A) to OMe (methyl ester of rakicidin A (MERA)) enhanced or maintained its anticancer activity. Moreover, MERA was more stable than rakicidin A.[19] To further study the

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SAR, we designed analogues 1a–e (Scheme 1).

As shown in the retro-synthetic analysis (Scheme 1),[18] compounds 1 could be derived from alcohol 2. We envisioned that alcohol 2 may be prepared from phosphonate

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3, subunit 4, L-threo-β-hydroxyasparagine derivative 5 and polyketide fragment 6.

Synthesis of compounds 3 and 5 were shown in Scheme 2. Hydrolysis of ester 7 in the

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presence of LiOH gave acid 8, followed by coupling with 9a–e to afford phosphonate 3a–e. Synthesis of β-hydroxyasparagine derivative 5 was started with known compound 10.[28] Selective esterification of 10 yielded 11a–b. Protection with t-butyloxy carbonyl (Boc) and triethylsilyl (TES) provided 5a–b.[29,30]

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After all of the coupling partners were generated, fragment coupling was performed to synthesize compound 1 (Scheme 3). Acids 5a–b were coupled with diol 12[18] under EDCI and DMAP condition to give 13a–b. Treatment of 13a–b with LiHMDS induced

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migration of the ester bond and afforded the desired esters 14a–b in yields of 55%–65% based on recovery of starting material 13a–b.[18,31] Alcohols 14a–b were transformed

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into carboxylic acids 15a–b by sequential Dess−Martin periodinane (DMP) and Pinnick oxidations.[32] Subsequent EDCI/ HOBt–mediated coupling of carboxylic acids 15a–b with amino alcohol 4 produced amides 16a–b. Oxidation of the primary alcohol with DMP

afforded

aldehydes

17a–b,

which

were

subsequently subjected

to

a

Horne−Wadsworth−Emmons (HWE) olefination with phosphonates 3a–e to obtain compounds 18a–e.[33] The four protection groups in compounds 18a–e were then

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removed with TFA, and the resulting crude product was used directly in the next macrolactamization under HATU and DIPEA condition to produce 2a–e in yields of 13%–29% for four steps. Selective mesylation on the primary alcohol was performed,

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and finally, the resulting intermediates were eliminated under basic conditions to afford rakicidin A analogues 1a–e.

overall yields were only 0.24%–0.97%.

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In general, analogues 1a–e were totally synthesized with 18 linear steps. However, the

2.1.2 Synthesis of dehydroxy rakicidin A analogues 19a–d

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To evaluate the influence of hydroxyl group in rakicidin A for its anti-PDAC activity, we further designed rakicidin A analogues 19a–d with trim of hydroxyl group. As shown in Scheme 4, a convergent [2 + 2] strategy was applied for synthesis of analogues 19a–d. As shown in Scheme 5, oxidation of the primary alcohol 23 with DMP afforded

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aldehyde 26, which was subsequently subjected to a HWE olefination with phosphonate 3e to obtain compound 27. Removal of Fmoc in compound 27 gave fragment 21, which was used directly without purification in the next step. Compound 28 was synthesized

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following our previously reported procedure [18], which was then coupled with the known alkene 29 via cross-metathesis using Grubbs second generation catalyst, followed

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by hydrolyzation, reductive hydrogenation, and reaction with allyl bromide to provide 25. Coupling of compound 25 with 24a–d under optimized DIC−coupling condition generated the required esters 22a–d in yields of 82%−92% (Scheme 6). After deprotection of compounds 22a–d, the resulting acid were coupled with compound 21 to yield 31a–d. Treatment of compounds 31a–d with TFA, followed by macrolactamization under HATU and DIPEA condition to produce 20a–d in yields of 22%−26% for four

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steps. Mesylation of the resulting alcohol 20a–d followed by elimination with DBU to give rakicidin analogues 19a–d (15 linear steps, overall yields: 2.8%−4.3%). 2.1.3 Synthesis of rakicidin analogues 32a–h

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The yield of macrolactamization still deserved to be improved. We propose that the exposed hydroxyl group of cyclization precursor may reduce the yield of macrolactamization. The TBS protection group of fragment 31 was replaced by TBDPS.

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To further optimize the synthesis route of polyketide fragment, we designed to replace fragment 25 with fragment 36. Fragments 34 and 36 were used as common intermediates

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for synthesis of a series of rakicidin analogues 32a–h using a combinatorial strategy (Scheme 7).

Preparation of the fragments 34 and 36 was shown in Scheme 8. Synthesis of TBDPSprotected fragment 34 started with known TBDPS-protected serinol derivative 37.[34]

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Deprotection of compound 37 produced carboxylic acid 38, followed by coupling reaction with allyl-2-(methylamino)acetate to generate compound 39 in yield of 93% (Scheme 8). Compound 39 was then treated with TFA to remove Boc protection group,

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affording 34, which was used in the next step directly without further purification. Highly distereoselective aldol reaction between chiral aldehyde 42 and 44 yielded 45

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(Scheme 8). The aldol product 45 was hydrolyzed with LiOH in THF/H2O (1:1). The resulting carboxylic acids were then converted into ester 36. With common intermediates fragments 34 and 36 in hand, we posed to synthesize the designed rakicidin analogues 32a–h using a combinatorial strategy (Scheme 9). Coupling of compound 36 with the Boc-protected amino acid generated the esters 46a–h. Deprotection of 46a–h produced carboxylic acids which were then subjected to coupling

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reactions with 34 to generate the compounds 47a–h. After further deprotection of the allyl group and Boc group, the resulting compounds were subjected directly to macrolactamization reaction, and cyclic compounds 48a–h were synthesized in good

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yields (47%–59%) for three steps. Removal of TBDPS with TBAF produced primary alcohols 33a–h. The resulting hydroxyl group was activated by ethanesulfonyl chloride and eliminated with DBU to obtain rakicidin analogues 32a–h (14 linear steps, 5.1%–

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7.4% overall yield). It is important to note that 32g was stable in MeOH/ MeCN (1:1) with a concentration of 0.25 mg/mL at room temperature over 1 month by HPLC.

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2.1.4 Synthesis of demethylenerakicidin analogue 49

To further evaluate the importance of APD moiety for anti-PDAC activity. Demethylenerakicidin analogue 49 was designed. The retrosynthetic analysis of 49 was shown in Scheme 10.

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Deprotection of the compound 22a produced free carboxylic acid which was then subjected to coupling reaction with 2-aminoethanol (50) to generate the alcohol 51 (Scheme 11). Oxidation of the compound 51 with DMP, which was then subjected to a

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HWE olefination with phosphonate 3e to obtain compound 52.[33] The two protection groups of compound 52 were then removed with TFA, and the resulting crude product

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was used directly in the next macrolactamization under HATU and DIPEA condition to obtain desired compound 49. 2.2 Biological activity

2.2.1 Activities of the rakicidin analogues against pancreatic cancer cell lines The newly synthesized twenty analogues (1a–e, 19a–d, 32a–h, 33h, 48g, and 49) and previously synthesized MERA[19] were evaluated for their effects on viability of the

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pancreatic cancer cell lines PANC-1, ASPC-1 and PATU8988 (Table 1) at normoxia and hypoxia conditions, respectively. In addition, adriamycin (ADR) and gemcitabine (GEM)

43547A2, were included for comparison.

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was introduced as positive controls, and the natural products, rakicidin A and BE-

The natural product rakicidin A exhibited potent activity against the PANC-1 cells (IC50 = 0.22 µM at normoxia condition, IC50 = 0.042 µM at hypoxia condition, hypoxic

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selectivity index, HSI = 5.24). The anti-pancreatic cancer activity of MERA and 1e, which featured with replacement of the amide moiety in rakicidin A with ester fragment,

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was comparable to that of rakicidin A in both normoxia and hypoxia conditions, while the HSI (HSI = 2.98, 2.33, respectively) decreased. The inhibitory activities of analogues with substitution of the N-methyl in MERA with trifluoroethyl (1a) and n-hexyl (1b) fragments were greatly reduced in comparison with MERA. The result indicates that this

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position is not tolerated for modification. Introduction of methyl group to C-6 position gave 1c and 1d. 1c with R-configuration showed high activity with IC50 value of 0.043 µM, which was more potent than its diastereoisomers 1d (IC50 = 0.079 µM) with S-

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configuration at hypoxia condition for PANC-1. However, their activities were decreased for ASPC-1 and PATU8988 cells. The simplified rakicidin (19a) with removal of

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hydroxyl group at the C-3 position of L-threo-β-hydroxyaspartic acid ester moiety demonstrated comparable activity at hypoxia condition comparing with MERA for PANC-1. Analogues 19b–d with aspartic ester fragment all showed potent activities against PANC-1. Comparing the activities of 19a, 19b and 19c, with increasing of steric effect of aspartic ester fragment, the activity gradually reduced. Compound 19d showed comparable activity against PANC-1, ASPC-1 and PATU8988 with MERA and rakicidin

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A at hypoxia condition, which indicated that 19d may be used as a molecule probe for exploring the possible target(s) of rakicidins. Trim of hydroxyl group (32a) maintained anti-pancreatic cancer activity at both of normoxia and hypoxia conditions for all tested

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cancer cell lines. Employing several amino acids to replace aspartic ester moiety (32b–f) led to decreased anti-PDAC activity.

Replacement of the methylester of 32a with methylamide or cyclopropanylamide

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moiety in the β-Aspartic acid ester fragment gave 32h and 32g, respectively. Methylamide 32h and cyclopropanylamide 32g exhibited slightly more potent activity

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against PANC-1 cells comparing with 32a at normoxia and hypoxia conditions. To our surprise, the activities of 32h and 32g were significantly improved for ASPC-1 and PATU8988 cells at hypoxia condition. Especially, 32g exhibited very high potency against highly metastatic ASPC-1 cells with IC50 values of 0.022 µM at hypoxia

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condition, which were 16-fold more potent than those of 32a, moreover, the HSI value of 32g was higher than that of 32a for ASPC-1 (12.3 vs 2.8). It is noteworthy that 32g were more potent than natural product rakicidin A against ASPC-1 cells (IC50 = 0.022 µM vs

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0.082 µM).

To further explore the influence of APD moiety for anti-PDAC activity. 33g, 48g,

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and demethylenerakicidin 49 were evaluated for their potency against PANC-1. However, these analogues lost anti-pancreatic cancer activities (IC50 > 10 µM), which demonstrates that the conjugated APD moiety is essential for the anti-pancreatic cancer activity. 2.2.2 Anti-PCSCs activity of rakicidin analogues

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Compounds MERA, 19a, 19d, 32a, 32e, 32g, 32h, and RA were screened in the assay against CD24+CD44+ESA+ PCSCs in PANC-1. The clinically used drugs, taxol, gemcitabine (GEM), and adriamycin (ADR), were also evaluated for comparison.

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PANC-1 cells were treated with candidate compounds for 48 hours at the concentrations of 0.15 µM. As shown in Figure 2, the percentage of CD24+CD44+ESA+ pancreatic cancer stem cells decreased by 4.0, 3.4, 5.2, 2.4, 2.0-folds after treatment of

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rakicidin A (0.43%), 32g (0.50%), 32h (0.33%), 32a (0.70%) and MERA (0.83%), comparing with untreated control (1.7%), respectively. In contrast, ADR increased the

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percentage of CD24+CD44+ESA+ cells to 8.9%. The clinically used drugs, gemcitabine and taxol, which were used for treatment of pancreatic cancer, showed no apparent effect on the CD24+CD44+ESA+ cells.

2.2.3 Rakicidin analogue 32g reduced the activity of ALDH

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Aldehyde dehydrogenase (ALDH) activity was another important feature of cancer stem cells. Cancer stem cells showed high ALDH activity which could be detected by the Aldefluor enzymatic assay. As shown in Figure 3, the percentage of ALDH positive cells

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was greatly reduced by 2.26-fold compared with control group, which demonstrated that compound 32g inhibited human PCSCs by inhibiting the activity of ALDH.

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2.2.4 Tumorsphere formation assay For further exploring the effects of compounds rakicidin A, 32g, 32h, 32a, and

MERA on PCSCs, these compounds were evaluated in tumorsphere formation assay. PANC-1 cells were treated with these compounds, and then the number of tumorspheres was counted after 7 days. As shown in Figure 4, analogue 32g significantly reduced the number of tumorspheres by 8.7-fold comparing with untreated control.

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2.2.5 Tumor initiating assay Analogue 32g was selected for the limiting-dilution tumor-initiation assay with barb/c nude mice, which was considered as the most convincing assay for evaluation of

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the effect on cancer stem cells. As shown in Figure 5, rakicidin A and 32g treated groups resulted in greatly reduced tumor-initiating frequency. The tumor-initiating frequency (TIF) were reduced by 4.7 folds, 19 folds after the treatment of rakicidin A and 32g

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comparing to vehicle group, respectively. In contrast, clinically used drug, ADR (at a concentration of 0.5 µM), treated group showed 1.6 folds increase comparing with

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vehicle control. These results further confirmed that 32g could ablate tumorigenesis capability of PANC-1 cells in vivo and displayed superior selectivity to rakicidin A. 2.2.6 Safety and inhibitory activity in xenograft zebrafish model

The safety and inhibitory activity of compound 32b and 32g against PANC-1 cells

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in vivo were assayed with xenografted zebrafish model (Figure 6). Compounds 32b and 32g were safe to zebrafish even at the saturated concentration of 50 µM. The inhibition of cancer cells of compound 32g were 15%, 10%, 5%, 22% and 45% in concentration of

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0.005 µM, 0.05 µM, 0.5 µM, 5 µM and 50 µM, respectively. The inhibitory effects of 32g and 32b at concentrations of 50 µM were comparable with positive control gemcitabine

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at concentrations of 66.7 µM.

3. Conclusion

In conclusion, we designed and synthesized twenty rakicidin A analogues, and

evaluated their anti-PDAC activities, and the following SAR was concluded: (1) the conjugated APD moiety is essential; (2) amide moiety of β-hydroxyasparagine fragment

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is important for hypoxia selectivity; (3) N-methyl group is not tolerated for modifications with large substitute groups; (4) trim of hydroxyl group in β-hydroxyasparagine fragment

group resulted in comparable activity.

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maintain activity; (5) replacement of the terminal iso-propanyl group with n-propanyl

The most active simplified analogue 32g was prepared in 14 linear steps with 5.05% overall yield, which is much more efficient than our previously reported total synthesis of

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rakicidin A (19 linear steps with 0.19% yield). In highly metastatic pancreatic cancer cell line ASPC-1, compound 32g showed about 4 times higher potency (IC50 = 0.022 µM)

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than rakicidin A (IC50 = 0.082 µM) at hypoxia condition, and 12 folds of hypoxia selectivity (IC50 = 0.27 µM at normoxia condition). In contrast, the activity of ADR in the same hypoxic condition is lower than that in nomoxia condition (Table 1). More importantly, we found that 32g could selectively ablate PCSCs. The

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percentage of stem cell with CD24+CD44+ ESA+ biomarker was decreased by 3.4 folds after treatment of 32g (0.5%) comparing with untreated control (1.7%). In contrast, ADR increased the percentage of CD24+CD44+ESA+ cells to 8.9%. The clinically used drugs

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for pancreatic cancer, gemcitabine and taxol, exhibited no apparent effect (Figure 2). The number of tumorspheres in PANC-1 was reduced by 8.7-fold comparing untreated

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control, which was superior to that of natural product rakicidin A (4-fold reduction) (Figure 4). Tumor-initiation assay, the most convincing model for evaluation of the effect on CSCs, indicated that the TIF was significantly reduced by 19 folds after the treatment of 32g, which is better than that of rakicidin A (reduction of 4.7 folds). However, ADR treated group showed 1.6 folds increase (Figure 5).

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In summary, highly efficient and convergent routes were achieved for total syntheses of simplified rakicidin analogues. The most promising analogue 32g could selective ablation of PCSCs and was superior to the natural product rakicidin A and some

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clinically used drugs.

4. Experimental

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4.1 Chemistry

Unless otherwise mentioned, all reactions were carried out under an argon atmosphere

according

to

common

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with dry solvents under anhydrous conditions. The used solvents were purified and dried procedures.

Yields

refer

to

chromatographically

and

spectroscopically (1H NMR) homogeneous materials, unless otherwise stated. Reagents were purchased at the highest commercial quality and used without further purification,

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unless otherwise stated. Purity testing was done by means of analytical HPLC on a Shimadzu LD-20A system with an ODS-C18 column (4.6 × 150 mm, 5 µm) eluted at 1 to 1.3 mL/min with Milli-Q water and CH3CN. The purity of all tested compound

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were >95% by HPLC. FTIR spectra were obtained with Bruker Tensor 27 and Bruker Tensor II instrument. All IR samples were reported in wave numbers (cm-1). NMR

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spectra were recorded with a 400 MHz spectrometer using CDCl3, DMSO-d6 or CD3OD. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constants and integration. tert-Butyl-2-(2-(diethoxyphosphoryl)-N-(2,2,2-trifluoroethyl)acetamido)acetate (3a) To a solution of 8 (8.2 g, 41.8 mmol) and 9a (13.37 g, 62.7 mmol, 1.5 equiv) in DCM (66.0 mL) was added TEA (14.6 mL, 104.5 mmol, 2.5 equiv), EDCI (16.03 g, 83.6 mmol,

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2 equiv) and DMAP (0.51 g, 4.2 mmol, 0.1 equiv) subsequently. After being stirred at 20 ºC for 6 h, the reaction mixture was diluted with CH2Cl2 (600 mL), washed with 1% HCl (200 mL), saturated aqueous solution of NaHCO3 (200 mL), dried over Na2SO4 and

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concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (CH2Cl2 : MeOH = 100 : 1) to afford 3a (12.10g, 74%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 4.32 – 4.22 (m, 2H), 4.21 – 4.11 (m, 4H), 4.09

5.21H), 1.45 (s, 3.76H), 1.33 (t, J = 7.1 Hz, 3H);

13

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– 4.01 (m, 2H), 3.13 (d, J = 22.3 Hz, 0.84H), 3.03 (d, J = 22.0 Hz, 1.16H), 1.48 (s, C NMR (100 MHz, CDCl3) δ 167.8,

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167.1, 166.6, 166.5, 166.1, 166.0, 125.8, 125.7, 123.0, 122.9, 83.4, 82.3, 63.2, 63.1, 63.1, 51.8, 50.9, 50.6, 50.2, 49.9, 48.0, 47.6, 47.3, 47.0, 34.3, 34.2, 33.0, 32.9, 31.5, 30.3, 29.8, 28.1, 28.1, 16.4, 16.4, 16.3, 16.3; νmax(KBr): 2981, 2932, 1743, 1652, 1448, 1394, 1368, 1333, 1256, 1158, 1029, 968, 855 cm-1; HRMS (ESI) calcd for C14H25F3NNaO6P+ [M +

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Na]+: 414.1264, found, 414.1268.

tert-Butyl-2-(2-(diethoxyphosphoryl)-N-hexylacetamido)acetate (3b) The titled compound 3b was obtained following the general procedure described for 3a.

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Flash column chromatography eluent (petroleum ether : ethyl acetate = 4 : 1) to afford compound 3b (8.52 g, 56%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 4.25 – 4.07

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(m, 5H), 3.96 (s, 1H), 3.47 – 3.40 (m, 1.12H), 3.39 – 3.33 (m, 0.85H), 3.09 (d, J = 22.1 Hz, 1.13H), 3.00 (d, J = 21.8 Hz, 0.85H), 1.62 – 1.54 (m, 1H), 1.48 (s, 4.38H), 1.46 (s, 5.18H), 1.39 – 1.26 (m, 13H), 0.93 – 0.84 (m, 3H); 13C NMR (100 MHz, CDCl3) δ 168.8, 168.1, 165.3, 165.2, 82.7, 81.7, 62.8, 62.7, 51.4, 50.4, 48.9, 48.0, 34.5, 33.8, 33.2, 32.4, 31.7, 31.6, 28.7, 28.1, 28.1, 27.5, 26.6, 26.5, 22.6, 16.5, 16.4, 14.1, 14.1; νmax(KBr):2979,

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2932., 1743, 1652, 1446, 1394, 1368, 1256, 1158, 1028, 968, 853 cm-1; HRMS (ESI) calcd for C18H36NNaPO6Na+ [M + Na]+: 416.2178, found 416.2178. (S)-tert-Butyl-2-(2-(diethoxyphosphoryl)-N-methylacetamido)propanoate (3c)

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The titled compound 3c was obtained following the general procedure described for 3a. Flash column chromatography eluent (CH2Cl2 : MeOH = 100 : 1) to afford compound 3c (9.36g, 74%) as a yellow oil. [α]15D = − 32.6 (c = 2.0, CHCl3); 1H NMR (400 MHz,

SC

CDCl3) δ 5.14 (q, J = 7.3 Hz, 0.74H), 4.70 (q, J = 6.9 Hz, 0.26H), 4.33 − 4.09 (m, 4H), 3.34 − 2.81 (m, 5H), 1.49 − 1.44 (m, 10H), 1.39 − 1.32 (m, 8H);

13

C NMR (100 MHz,

M AN U

CDCl3) δ 170.7, 170.1, 165.3, 165.3, 165.2, 165.1, 82.3, 81.6, 62.9, 62.9, 62.7, 62.6, 62.6, 56.9, 53.1, 34.5, 34.4, 33.1, 33.1, 32.6, 29.0, 28.0, 28.0, 16.4, 16.4, 15.4, 14.5; νmax(KBr): 3434, 2981, 2936, 1733, 1649, 1479, 1449, 1395, 1370, 1333, 1255, 1157, 1094, 1052, 1028, 967, 847 cm-1; HRMS (ESI) calcd for C14H28NO6PNa+ [M + Na]+: 360.1546, found

TE D

360.1552.

(R)-tert-Butyl 2-(2-(diethoxyphosphoryl)-N-methylacetamido)propanoate (3d) The titled compound 3d was obtained following the general procedure described for 3a.

EP

Flash column chromatography eluent (CH2Cl2: MeOH = 100: 1) to afford compound 3d (9.61g, 76%) as a yellow oil. [α]20D = + 28.7 (c = 4.0, CHCl3); 1H NMR (400 MHz,

AC C

CDCl3) δ 5.07 (q, J = 7.3 Hz, 0.71H), 4.64 (q, J = 6.9 Hz, 0.29H), 4.25 − 4.01 (m, 4H), 3.28 − 2.73 (m, 5H), 1.42 − 1.37 (m, 10H), 1.31 − 1.25 (m, 8H);

13

C NMR (100 MHz,

CDCl3) δ 170.3, 169.8, 165.0, 164.9, 164.8, 164.8, 81.9, 81.2, 62.6, 62.5, 62.3, 62.2, 62.2, 56.6, 52.8, 34.1, 34.1, 32.8, 32.7, 32.3, 28.6, 27.7, 27.7, 16.1, 16.0, 15.0, 14.2; νmax (KBr): 3443, 2981, 2937, 1732, 1649, 1479, 1451, 1393, 1369, 1332, 1255, 1158, 1093, 1052,

16

ACCEPTED MANUSCRIPT

1028, 967, 848 cm-1; HRMS (ESI) calcd for C14H28NO6PNa+ [M + Na]+ : 360.1546, found 360.1550. (2S,3S)-2-((tert-Butoxycarbonyl)amino)-4-ethoxy-4-oxo-3-((triethylsilyl)oxy)butanoic

RI PT

acid (5b)

To a solution of 10 (8.68 g, 46.8 mmol, 1 equiv) in EtOH (220 mL) was added thionyl chloride (3.5 mL, 46.7 mmol, 1 equiv) at 0 oC. After stirred for 20 min, the reaction

SC

mixture was warmed to 20 oC and stirred for another 3 h. The solvent was evaporated to give a light yellow solid. This solid was dissolved in THF/water (V/V = 1:1, 220 mL).

M AN U

Triethylamine (19.5 mL, 140.3 mmol, 3 equiv) and di-tert-butyl dicarbonate (10.20 g, 66.7 mmol, 1.1 equiv) was then added to the reaction mixture. After stirred for 3 h, the reaction solution was extracted with ethyl acetate (50 mL) and then acidified to pH = 2 to 3 by 1 M HCl. The aqueous solution was extracted with ethyl acetate (3 × 100 mL). The

TE D

combined organic phases were washed with brine (50 mL), dried (Na2SO4) and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (CH2Cl2 : MeOH = 100 : 1 to 100 : 5) to obtain the acid as a

EP

yellow oil.

To a solution of above acid in CH2Cl2 (220 mL) was added imidazole (9.56 g，140.4

AC C

mmol, 3 equiv) and TESCl (17.64 g，117.0 mmol, 2.5 equiv) at 0 ºC. After stirred at 0 ºC for 3 h, EtOH (20 mL) was added to the reaction mixture, and the reaction mixture was then warmed to 20 ºC stirred for 10 min. The reaction mixture was acidified to pH = 3 by 1% HCl, extracted with CH2Cl2 (3 × 150 mL). The combined organic phases were dried (Na2SO4) and concentrated under reduced pressure. The crude product was purified by column chromatography (CH2Cl2 : MeOH = 100: 1) to obtain 5b (5.5 g, 45% for 3

17

ACCEPTED MANUSCRIPT

steps) as a colorless oil. [α]18D = + 2.3 (c = 2.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.30 (d, J = 9.2 Hz, 1H), 4.81 (d, J = 10.1 Hz, 2H), 4.28 – 4.16 (m, 2H), 1.43 (s, 9H), 1.28 (t, J = 7.1 Hz, 3H), 0.94 (dd, J = 9.5, 6.3 Hz, 9H), 0.66 – 0.59 (m, 6H) ; 13C NMR

RI PT

(100 MHz, CDCl3) δ 174.3, 170.5, 158.6, 80.9, 72.5, 61.7, 57.2, 28.6, 14.3, 6.7, 4.5; νmax (KBr): 3400, 2960, 2878, 1744, 1724, 1512, 1459, 1411, 1260, 1097, 1018, 861, 799, 686 cm-1; HRMS (ESI) calcd for C16H31NO7SiNa+ [M + Na]+: 414.1919, found 414.1924.

SC

(2S,3S)-4-Ethyl-1-((2R,3S,4R)-1-hydroxy-2,4,16-trimethylheptadecan-3-yl)-2-((tertbutoxycarbonyl)amino)-3-((triethylsilyl)oxy)succinate (14b)

M AN U

To a solution of compound 13b (1.9 g, 2.76 mmol, 1 equiv) in THF (26 mL) was added LiHMDS (6.0 mL, 6.0 mmol, 1 M in THF, 2.2 equiv) under argon atmosphere at – 40 ºC. After stirred for 1 h, the reaction mixture was quenched by addition of saturated aqueous NaHCO3 (40 mL), and extracted with extracted with ethyl acetate (3 × 60 mL). The

TE D

combined organic phases were dried (Na2SO4) and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (ethyl acetate: hexane = 1:15) to obtain 14b (1.23 g, 65%) as a colorless oil. [α]20D= – 22.4 (c = 1.0,

EP

CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.26 (d, J = 9.8 Hz, 1H), 4.87 – 4.83 (m, 2H), 4.73 (dd, J = 9.8, 2.3 Hz, 1H), 4.30 – 4.12 (m, 2H), 3.55 (dd, J = 11.8, 3.2 Hz, 1H), 3.43

AC C

(dd, J = 11.8, 3.6 Hz, 1H), 1.85 – 1.79 (m, 1H), 1.72 (m, 2H), 1.56 – 1.47 (m, 1H), 1.42 (s, 9H), 1.28 – 1.15 (m, 25H), 0.98 – 0.85 (m, 21H), 0.66 – 0.58 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 171.1, 170.0, 155.8, 80.5, 80.3, 71.8, 64.5, 61.7, 57.6, 39.2, 37.3, 34.3, 34.0, 32.1, 30.5, 30.1, 29.8, 29.5,28.4, 28.1, 27.6, 22.8, 14.5,14.2, 13.0, 6.9,4.8; νmax (KBr): 3450, 2961, 2877, 2855, 1760, 1727, 1495, 1413, 1367, 1260, 1167, 1063, 862, 797 cm-1; HRMS(ESI) calcd for C37H73NO8SiNa+[M + Na]+: 710.4998, found 710.5002.

18

ACCEPTED MANUSCRIPT

(2S,3S)-1-((2S,3S,4R)-1-(((R)-1-((tert-Butyldimethylsilyl)oxy)-3-hydroxypropan-2yl)amino)-2,4,16-trimethyl-1-oxoheptadecan-3-yl)-4-ethyl-2-((tertbutoxycarbonyl)amino)-3-((triethylsilyl)oxy)succinate (16b)

RI PT

To a solution of compound 14b (1.14 g, 1.69 mmol, 1 equiv) in CH2Cl2 (48 mL) was added DMP (0.86 g, 2.03 mmol, 1.2 equiv) at 20 ºC. After stirred for 2 h, the reaction mixture was quenched by addition of saturated aqueous solution of NaHCO3 (50 mL) and

SC

saturated aqueous Na2S2O3 (20 mL). The reaction mixture was stirred for 10 min, and extracted with extracted with CH2Cl2 (3 × 100 mL). The combined organic phases were

M AN U

dried (Na2SO4) and concentrated under reduced pressure.

The residue was dissolved in tert-butanol (25 mL). To resulting solution was added 2methyl-2-butene (7.9 mL, 75 mmol), followed by a solution of sodium chlorite (0.6 g, 6.7 mmol, 4 equiv) and monosodium phosphate (0.62 g, 5.2 mmol, 3 equiv) in 6 mL of water.

TE D

The reaction solution was stirred at 20 ºC for 10 h, diluted with saturated solution of NaHCO3 (100 mL), and extracted with hexane (3 × 100 mL). The combined organic phases were dried (Na2SO4), and concentrated under reduced pressure. The crude product

EP

was used without further purification.

To a solution of above acid (431 mg, 0.61 mmol, 1 equiv) and 4 (164 mg, 0.8 mmol, 1.3

AC C

equiv) in CH2Cl2 (6 mL) was added EDCI (235mg, 1.23 mmol, 2 equiv) and HOBt (125 mg, 0.92 mmol, 1.5 equiv), followed by addition of DIPEA (0.57 mL, 3.3 mmol, 5 equiv) under argon atmosphere at 20 ºC. The reaction mixture was stirred for 4 h, and then quenched with 1% HCl (100 mL). The aqueous phase was extracted with CH2Cl2 (3 × 80 mL). The combined organic phases were dried (Na2SO4) and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (ethyl

19

ACCEPTED MANUSCRIPT

acetate: hexane = 1: 50 to 1: 10) to obtain 16b (0.43 g, 69% for 3 steps) as a colorless oil. [α]20D = – 20.2 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 6.29 (d, J = 8.1 Hz, 1H), 5.26 (d, J = 9.8 Hz, 1H), 5.16 (dd, J = 9.6, 1.8 Hz, 1H)，4.98 (d, J = 1.7 Hz, 1H), 4.71

RI PT

(dd, J = 9.8, 1.8 Hz, 1H), 4.32 – 4.23 (m, 1H),4.17 – 4.09 (m, 1H), 4.05 – 3.98 (m, 1H), 3.79 – 3.71 (m, 2H), 3.68 – 3.63 (m, 3H) 2.35 – 2.26 (m, 1H), 1.76 – 1.72 (m, 1H), 1.54 – 1.47 (m, 1H), 1.44 (s, 9H), 1.26 – 1.08 (m, 30H), 0.93 (t, J = 7.9 Hz, 9H), 0.87 – 0.85 (m, 13

C NMR (100 MHz, CDCl3) δ

SC

19H), 0.66 – 0.58 (m, 6H), 0.05 (s, 3H) , 0.04 (s, 3H);

173.9, 171.2, 168.3, 156.6, 80.7, 80.5, 79.4, 71.2, 62.4, 61.6, 57.7, 53.0, 44.1, 39.2, 34.0,

M AN U

33.4, 32.0, 30.1, 29.9, 29.8, 29.5, 28.4, 28.1, 27.6, 27.5, 26.0, 22.8, 18.3, 14.2, 14.0, 12.5, 6.8, 4.7, -5.5; νmax (KBr): 3445, 2955, 2926, 2879, 1743, 1712, 1658, 1502, 1465, 1384, 1259, 1129, 1018, 838, 775 cm-1; HRMS (ESI) calcd for C46H92N2O10Si2Na+ [M + Na]+ : 911.6183, found 911.6188.

TE D

(S)-Methyl-2-hydroxy-2-((3S,11R,14S,15S,E)-11-(hydroxymethyl)-14-methyl-15-((R)-14methylpentadecan-2-yl)-2,5,8,13-tetraoxo-7-(2,2,2-trifluoroethyl)-1-oxa-4,7,12triazacyclopentadec-9-en-3-yl)acetate (2a)

EP

To a solution of 16a (0.2 g, 0.23 mmol, 1 equiv) in CH2Cl2 (4.6 mL) was added DMP (126 mg, 0.3 mmol, 1.3 equiv) at 20 ºC. After stirred for 1 h, the reaction mixture was

AC C

quenched by addition of saturated aqueous solution of NaHCO3 (12 mL) and saturated aqueous Na2S2O3 (6 mL). The reaction mixture was stirred for 10 min, and extracted with CH2Cl2 (3 × 30 mL). The combined organic phases were dried (Na2SO4) and concentrated under reduced pressure. The residue was dissolved in 15% ethyl acetate in hexane (50 mL), and the resulting mixture was filtered through a short pad of silica gel.

20

ACCEPTED MANUSCRIPT

The solvent was removed under reduced pressure to afford 17a (170 mg, 85%) as a colorless oil, which can used for the next step directly. To a solution of 17a (91.5 mg, 0.23 mmol, 1.2 equiv) in MeCN (0.75 mL) was added

RI PT

anhydrous LiCl (16.8 mg, 0.4 mmol, 2 equiv) and 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) (34.5 uL, 0.23 mmol, 1.2 equiv) at 0 ºC and stirred for 10min. Solution of 3a (0.17 g, 0.19 mmol, 1 equiv) in CH2Cl2 (1 mL) was then added to the reaction mixture.

SC

And the resulting solution was stirred for 40 min. Water (10 mL) was added to quench the reaction. And the aqueous phase was extracted with CH2Cl2 (3 × 30 mL). The

M AN U

combined organic phases were dried (Na2SO4) and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (ethyl acetate : hexane = 1 : 10) to obtain 18a (79 mg, 35%) as a colorless oil.

To a solution of 18a (0.26 g, 0.24 mmol, 1 equiv) in DCM (1.5 mL), Et3SiH (250 µL,

TE D

1.54 mmol, 6 equiv) was added at 0 ℃ , and a solution of TFA (1.76 mL) in DCM (1.2 mL) was dropwise added into the reaction mixture. It allowed to warm at 20 ℃ for 4 h. Then toluene (2.7 mL) was added and the solvent was removed by reduced pressure. The

EP

residue was purified by flash chromatography to obtain the crude product as a yellow solid. Then the above crude product was dissolved in THF (20 mL), the solution was

AC C

added slowly to a suspension of HATU (1.55 g, 3.56 mmol, 15 equiv) and DIPEA (1.42 mL, 7.1 mmol, 30 equiv) in THF (220 mL) over 8 h at 20 ℃ and then continued to be stirred another 12 h at 20 ℃. The solvent was removed under reduced pressure, diluted with MeOH / ethyl acetate (V/V = 2:1, 300 mL), and filtered through a celite pad. The filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (300 mL), washed with 1% HCl (80 mL), saturated aqueous solution of NaHCO3

21

ACCEPTED MANUSCRIPT

(60 mL), and brine (60 mL). The organic phase was dried over Na2SO4, concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (MeOH : CH2Cl2 = 1 : 30) to obtain the 2a (0.122 g, 70% for 2 steps) as a white

RI PT

solid. [α]18D = – 41.50 (c = 1.0, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.45 (d, J = 10.0 Hz, 1H), 7.82 (d, J = 8.9 Hz, 1H), 6.84 (d, J = 14.0 Hz, 1H), 5.98 (d, J = 5.0 Hz, 1H), 5.92 (d, J = 14.9 Hz, 1H), 5.11 (d, J = 10.5 Hz, 1H), 4.93 (t, J = 5.3 Hz, 1H), 4.88 (d, J =

SC

10.1 Hz, 1H), 4.63 – 4.51 (m, 1H), 4.45 (d, J = 18.1 Hz, 3H), 4.02 – 3.86 (m, 2H), 3.56 (s, 3H), 2.87 – 2.77 (m, 1H), 1.71 – 1.62 (m, 1H), 1.53 – 1.43 (m, 1H), 1.17 (br, 24H), 1.00

M AN U

(d, J = 6.7 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H), 0.83 (d, J = 6.6 Hz, 6H);

13

C NMR (100

MHz, DMSO-d6) δ 172.9, 171.6, 168.4, 167.0, 166.1, 145.6, 126.4, 123.6, 117.9, 78.0, 71.5, 63.0, 54.8, 51.9, 51.3, 51.2, 47.6, 47.3, 41.4, 38.5, 33.6, 33.0, 31.3, 30.5, 29.4, 29.2, 29.2, 29.1, 28.8, 27.4, 27.1, 26.9, 26.8, 22.6, 22.2, 15.9, 14.0, 13.2; νmax(KBr): 3422,

TE D

2926, 2854, 1744, 1700, 1647, 1538, 1468, 1421, 1384, 1271, 1154, 974, 898, 831, 669, 550 cm-1; HRMS (ESI) calcd for: C34H56F3N3NaO9+[M + Na]+:730.3861 found 730.3865. (S)-Methyl-2-((3S,11R,14S,15S,E)-7-hexyl-11-(hydroxymethyl)-14-methyl-15-((R)-14-

EP

methylpentadecan-2-yl)-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3-yl)2-hydroxyacetate (2b)

AC C

The titled compound 2b was obtained following the general procedure described for 2a. Flash column chromatography eluent (MeOH : CH2Cl2 = 1 : 30) to afford compound 2b (67 mg, 17% for 4 steps) as a white solid. [α]20D = – 42.67 (c = 1.0, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.38 (d, J = 9.8 Hz, 1H), 7.78 (d, J = 9.0 Hz, 1H), 6.72 (d, J = 14.3 Hz, 1H), 5.98 (d, J = 4.8 Hz, 1H), 5.90 (d, J = 14.6 Hz, 1H), 5.11 (d, J = 10.5 Hz, 1H), 4.94 – 4.83 (m, 2H), 4.51 – 4.30 (m, 3H), 3.65 (d, J = 18.4 Hz, 1H), 3.56 (s, 3H),

22

ACCEPTED MANUSCRIPT

3.49 – 3.40 (m, 1H), 3.24 – 3.15 (m, 1H), 2.82 (dd, J = 9.5, 7.0 Hz, 1H), 1.73 – 1.60 (m, 1H), 1.54 – 1.41 (m, 2H), 1.28 (br, 28H), 1.15 – 1.09 (m, 3H), 1.00 (d, J = 6.6 Hz, 3H), 0.93 (d, J = 6.5 Hz, 3H), 0.84 (d, J = 6.6 Hz, 9H);

13

C NMR (100 MHz, DMSO-d6) δ

RI PT

172.9, 171.7, 168.3, 167.9, 165.1, 143.3, 119.1, 78.1, 71.5, 63.2, 54.9, 51.8, 51.1, 50.6, 48.0, 41.5, 33.6, 33.0, 31.2, 29.4, 29.2, 29.1, 28.7, 27.6, 27.4, 27.0, 26.8, 26.0, 22.6, 22.1, 22.1, 15.9, 14.0, 13.2; νmax(KBr): 3435, 2921, 2850, 1740, 1712, 1693, 1645, 1536, 1463,

SC

1023, 802 cm-1; HRMS (ESI) calcd for C38H67N3O9Na+ [M + Na]+: 732.4775, found 732.4773.

M AN U

(S)-Methyl-2-hydroxy-2-((3S,6S,11R,14S,15S,E)-11-(hydroxymethyl)-6,7,14-trimethyl-15((R)-14-methylpentadecan-2-yl)-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9en-3-yl)acetate (2c)

The titled compound 2c was obtained following the general procedure described for 2a.

TE D

Flash column chromatography eluent (MeOH : CH2Cl2 = 1 : 30) to afford compound 2c (114 mg, 16% for 4 steps) as a white solid. [α]20D = – 10.5 (c = 0.25, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.29 (d, J = 9.6 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 6.79 (d, J =

EP

14.3 Hz, 1H), 6.11 (d, J = 14.6 Hz, 1H), 6.03 – 5.87 (m, 1H), 5.09 (d, J = 10.3 Hz, 1H), 4.99 – 4.86 (m, 1H), 4.78 (d, J = 8.9 Hz, 1H), 4.68 – 4.59 (m, 1H), 4.46 – 4.39 (m, 1H),

AC C

4.36 (s, 1H), 3.54 (s, 3H), 3.43 – 3.36 (m, 2H), 2.90 (s, 3H), 2.87 – 2.77 (m, 1H), 1.74 – 1.62 (m, 1H), 1.53 – 1.43 (m, 1H), 1.39 (d, J = 6.5 Hz, 3H), 1.25 (br m, 22H), 1.00 (d, J = 6.5 Hz, 3H), 0.93 (d, J = 6.3 Hz, 3H), 0.83 (d, J = 6.4 Hz, 6H);

13

C NMR (100 MHz,

DMSO-d6) δ 173.2, 172.2, 171.4, 167.8, 165.8, 144.7, 118.3, 78.2, 71.7, 63.0, 54.7, 54.0, 51.8, 51.6, 41.5, 38.5, 33.7, 33.0, 29.3, 29.2, 29.1, 28.9, 27.4, 27.0, 26.8, 22.5, 17.0, 15.8, 13.4; νmax (KBr): 3426, 2960, 2925, 2855, 1734, 1665, 1610, 1544, 1460, 1399, 1378,

23

ACCEPTED MANUSCRIPT

1262, 1097, 1024, 865, 804, 728, 702 cm-1; HRMS (MALDI) calcd for C34H59N3O9Na+ [M + Na]+ : 676.4144, found 676.4148. (S)-Methyl-2-hydroxy-2-((3S,6R,11R,14S,15S,E)-11-(hydroxymethyl)-6,7,14-trimethyl-

RI PT

15-((R)-14-methylpentadecan-2-yl)-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec9-en-3-yl)acetate (2d)

The titled compound 2d was obtained following the general procedure described for 2a.

SC

Flash column chromatography eluent (MeOH : CH2Cl2 = 1 : 30) to afford compound 2d (121 mg, 13% for 4 steps) as a white solid. [α]21D = – 57.0 (c = 0.1, DMSO);1H NMR

M AN U

(400 MHz, DMSO-d6) δ 8.30 (d, J = 9.9 Hz, 1H), 7.71 (d, J = 8.6 Hz, 1H), 6.78 (dd, J = 14.8, 2.5 Hz, 1H), 6.11 (d, J = 14.8 Hz, 1H), 6.04 – 5.81 (m, 1H), 5.08 (d, J = 10.5 Hz, 1H), 5.02 – 4.84 (m, 1H), 4.77 (dd, J = 9.9, 2.2 Hz, 1H), 4.69 – 4.56 (m, 1H), 4.45 – 4.38 (m, 1H), 4.35 (d, J = 2.3 Hz, 1H), 3.53 (s, 3H), 3.42 – 3.35 (m, 2H), 2.89 (s, 3H), 2.86 –

TE D

2.77 (m, 1H), 1.72 – 1.63 (m, 1H), 1.52 – 1.41 (m, 1H), 1.38 (d, J = 6.9 Hz, 3H), 1.24 (br, 22H), 0.99 (d, J = 6.9 Hz, 3H), 0.92 (d, J = 6.7 Hz, 3H), 0.82 (d, J = 6.6 Hz, 6H);

13

C

NMR (100 MHz, DMSO-d6) δ 173.2, 172.3, 171.5, 167.9, 165.8, 144.7, 118.3, 78.2, 71.7,

EP

63.0, 54.7, 54.0, 51.9, 51.6, 41.5, 38.5, 33.7, 33.0, 29.3, 29.3, 29.1, 29.1, 28.9, 27.4, 27.0, 26.8, 22.5, 17.0, 15.8, 13.4; νmax (KBr): 3314, 3054, 2925, 2853, 1741,1665, 1609, 1541,

AC C

1462, 1394, 1334, 1262, 1113, 979, 898, 851, 812 cm-1; HRMS (MALDI) calcd for C34H59N3O9Na+ [M + Na]+ : 676.4144, found 676.4148. (S)-Ethyl-2-hydroxy-2-((3S,11R,14S,15S,E)-11-(hydroxymethyl)-7,14-dimethyl-15-((R)14-methylpentadecan-2-yl)-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3yl)acetate (2e)

24

ACCEPTED MANUSCRIPT

The titled compound 2e was obtained following the general procedure described for 2a. Flash column chromatography eluent (MeOH : CH2Cl2 = 1 : 30) to afford compound 2e (156 mg, 29% for 4 steps) as a white solid. [α]20D = – 40.5 (c = 0.13, DMSO); 1H NMR

RI PT

(400 MHz, DMSO-d6) δ 8.43 (d, J = 10.0 Hz, 1H), 7.83 (d, J = 9.0 Hz, 1H), 6.73 (dd, J = 14.9, 2.7 Hz, 1H), 5.95 (dd, J = 14.9, 3.7 Hz, 2H), 5.12 (d, J = 10.7 Hz, 1H), 4.91 (dd, J = 10.7, 4.8 Hz, 2H), 4.41 – 4.45 (m, 3H), 4.06 – 4.02 (m, 2H), 3.66 (d, J = 18.3 Hz, 1H),

SC

2.91 (s, 3H), 2.85 – 2.80 (m, 1H), 1.69 – 1.64 (m, 1H), 1.49 (m, 1H), 1.35 – 1.24 (m, 20H), 1.13 (t, J = 7.1 Hz, 6H), 1.01 (d, J = 7.0 Hz, 3H), 0.91 (d, J = 12.0 Hz, 3H), 0.84 (d, 13

C NMR (400 MHz, DMSO-d6) δ173.0, 171.1, 168.4, 167.9, 165.7,

M AN U

J = 6.6 Hz, 6H);

143.7, 118.5, 71.7, 63.1, 60.6, 54.7, 52.1, 51.1, 36.4, 33.7, 33.0, 31.4, 29.4, 29.3, 29.1, 27.4, 27.1, 26.8, 22.6, 22.2, 16.0, 13.9, 13.3; νmax (KBr): 3459, 3383, 2959, 2925, 2854, 1740, 1709,1693, 1671, 1489, 1260, 829, 772 cm-1; HRMS (ESI) calcd for

TE D

C34H59N3O9Na+ [M + Na]+: 676.4144, found 676.4148.

(S)-Methyl-2-hydroxy-2-((3S,14S,15S,E)-14-methyl-11-methylene-15-((R)-14methylpentadecan-2-yl)-2,5,8,13-tetraoxo-7-(2,2,2-trifluoroethyl)-1-oxa-4,7,12-

EP

triazacyclopentadec-9-en-3-yl)acetate (1a)

To a solution of 2a (122.0 mg, 0.172mmol, 1 equiv) in THF (8 mL) was added

AC C

triethylamine (63.79 µL, 0.459 mmol, 2.5 equiv) and methanesulfonyl chloride (26.68 uL, 0.344 mmol, 2 equiv) at 0 ºC. After stirred for 2 h, the reaction solution was quenched by addition of water (0.1 mL). Dried with Na2SO4 (10 g) over 0.5 h and concentrated under reduced pressure. The resulting crude product was dissolved in THF (10 mL). To the resulting solution was added DBU (258 µL, 1.72 mmol, 10 equiv) at 20 ºC. After stirred for 2 h, the reaction was quenched by addition of 1 % HCl (30 mL). The aqueous phase

25

ACCEPTED MANUSCRIPT

was extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with NaHCO3 (10 mL) and brine (10 mL), dried (Na2SO4) and concentrated under reduced pressure. The crude product was purified by column chromatography on silica

RI PT

gel (MeOH : CH2Cl2 = 1 : 20) to obtain the 1a (60 mg, 50 % for 2 steps) as a white solid. [α]20D = – 34.8 (c = 1.0, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.91 (s, 1H), 8.45 (d, J = 9.6 Hz, 1H), 6.90 (d, J = 14.9 Hz, 1H), 6.17 (d, J = 14.6 Hz, 1H), 5.99 (s, 1H), 5.45 (s,

SC

1H), 5.38 (s, 1H), 5.08 (d, J = 9.8 Hz, 1H), 4.86 (d, J = 8.6 Hz, 1H), 4.62 – 4.36 (m, 3H), 4.08 – 3.97 (m, 1H), 3.92 (d, J = 17.8 Hz, 1H), 3.53 (s, 3H), 2.95 – 2.81 (m, 1H), 1.71 –

M AN U

1.62 (m, 1H), 1.49 – 1.43 (m, 1H), 1.16 (br, 24H), 1.01 (d, J = 5.7 Hz, 3H), 0.92 (d, J = 5.6 Hz, 3H), 0.81 (d, J = 6.1 Hz, 6H); 13C NMR (101 MHz, DMSO-d6) δ 172.4, 171.6, 168.2, 167.0, 166.5, 139.8, 137.3,129.2, 126.4, 123.6, 120.8, 118.0, 117.4, 78.5, 71.5, 54.9, 54.9, 51.9, 51.7, 47.6, 47.3, 41.8, 38.5, 33.7, 32.9, 31.4, 29.8, 29.4, 29.2, 29.1, 27.5,

TE D

27.0, 26.9, 22.6, 22.2, 15.0, 14.0, 13.0; νmax (KBr): 3382, 2971, 2926, 2855, 1745, 1700, 1656, 1530, 1467, 1384, 1273, 1212, 1145, 1106, 1050, 974, 881, 830, 677, 636, 551 cm1

; HRMS (ESI) calcd for C34H54F3N3NaO8+[M + Na]+: 712.3755, found 712.3758.

EP

(S)-Methyl-2-((3S,14S,15S,E)-7-hexyl-14-methyl-11-methylene-15-((R)-14methylpentadecan-2-yl)-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3-yl)-

AC C

2-hydroxyacetate (1b)

The titled compound 1b was obtained following the general procedure described for 1a. Flash column chromatography eluent (MeOH : CH2Cl2 = 1 : 20) to afford compound 1b (70 mg, 80% for 2 steps) as a white solid. [α]20D = – 110.2 (c = 1.0, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.87 (s, 1H), 8.37 (d, J = 9.8 Hz, 1H), 6.78 (d, J = 15.0 Hz, 1H), 6.14 (d, J = 15.0 Hz, 1H), 5.99 (d, J = 5.4 Hz, 1H), 5.37 (s, 1H), 5.33 (s, 1H), 5.07 (d, J =

26

ACCEPTED MANUSCRIPT

10.3 Hz, 1H), 4.84 (d, J = 9.9 Hz, 1H), 4.48 – 4.33 (m, 2H), 3.66 (d, J = 18.0 Hz, 1H), 3.52 (s, 3H), 3.44 – 3.36 (m, 1H), 3.31 – 3.20 (m, 1H), 2.95 – 2.83 (m, 1H), 1.71 – 1.63 (m, 1H), 1.53 – 1.42 (m, 2H), 1.20 (br, 28H), 1.12 – 1.06 (m, 3H), 1.00 (d, J = 6.8 Hz,

RI PT

3H), 0.92 (d, J = 6.7 Hz, 3H), 0.81 (d, J = 6.6 Hz, 9H); 13C NMR (100 MHz, DMSO-d6) δ 172.4, 171.7, 168.2, 168.0, 165.4, 138.3, 137.6, 119.3, 116.1, 78.5, 71.5, 54.9, 51.8, 50.9, 48.0, 41.8, 38.5, 33.6, 32.9, 31.2, 29.4, 29.2, 29.1, 27.6, 27.4, 27.0, 26.9, 26.0, 22.6,

SC

22.1, 15.0, 14.0, 13.1; νmax(KBr): 3443, 2926, 2855, 1726, 1694, 1674, 1646, 1612, 1469, 975, 800 cm-1; HRMS (ESI) calcd for C38H65NO8Na+ [M+Na]+: 714.4669, found,

M AN U

714.4670.

(S)-Methyl-2-hydroxy-2-((3S,6S,14S,15S,E)-6,7,14-trimethyl-11-methylene-15-((R)-14methylpentadecan-2-yl)-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3yl)acetate (1c)

TE D

The titled compound 1c was obtained following the general procedure described for 1a. Flash column chromatography eluent (MeOH : CH2Cl2 = 1 : 20) to afford compound 1c (40 mg, 40% for 2 steps) as a white solid. [α]23D = – 87.0 (c = 0.1, DMSO); 1H NMR

EP

(400 MHz, DMSO-d6) δ 8.90 (s, 1H), 8.28 (d, J = 9.5 Hz, 1H), 6.91 (d, J = 14.9 Hz, 1H), 6.39 (d, J = 14.9 Hz, 1H), 5.96 (s, 1H), 5.41 (s, 1H), 5.39 (s, 1H), 5.06 (d, J = 10.2 Hz,

AC C

1H), 4.77 (d, J = 9.6 Hz, 1H), 4.73 – 4.63 (m, 1H), 4.35 (s, 1H), 3.54 (s, 3H), 3.00 – 2.85 (m, 4H), 1.70 (br, 1H), 1.52 – 1.44 (m, 1H), 1.41 (d, J = 6.8 Hz, 3H), 1.37 – 1.06 (m, 21H), 1.03 (d, J = 6.7 Hz, 3H), 0.94 (d, J = 6.6 Hz, 3H), 0.82 (d, J = 6.5 Hz, 6H);

13

C

NMR (100 MHz, DMSO-d6) δ 172.7, 172.4, 171.4, 167.8, 166.1, 139.5, 138.1, 118.4, 116.9, 78.6, 71.6, 54.9, 54.3, 51.9, 41.8, 38.5, 33.8, 32.8, 29.3, 29.2, 29.1, 29.1, 29.0, 29.0, 27.4, 26.9, 26.8, 22.5, 16.8, 15.1, 13.2; νmax (KBr): 3460, 3396, 3279, 2959, 2926, 2854,

27

ACCEPTED MANUSCRIPT

1737, 1700, 1680, 1646, 1610, 1521, 1461, 1433, 1395, 1261, 1208, 1105, 1020, 979, 861, 804 cm-1; HRMS (MALDI) calcd for C34H57N3O8Na+ [M + Na]+ : 658.4038, found 658.4045.

RI PT

(S)-methyl-2-hydroxy-2-((3S,6R,14S,15S,E)-6,7,14-trimethyl-11-methylene-15-((R)-14methylpentadecan-2-yl)-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3yl)acetate (1d)

SC

The titled compound 1d was obtained following the general procedure described for 1a. Flash column chromatography eluent (MeOH : CH2Cl2 = 1 : 20) to afford compound 1d

M AN U

(45 mg, 45% for 2 steps) as a white solid. [α]21D = – 117.4 (c = 0.1, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.89 (s, 1H), 8.27 (d, J = 9.3 Hz, 1H), 6.90 (d, J = 14.8 Hz, 1H), 6.38 (d, J = 15.1 Hz, 1H), 6.00 (s, 1H), 5.40 (s, 1H), 5.38 (s, 1H), 5.05 (d, J = 9.8 Hz, 1H), 4.76 (d, J = 8.6 Hz, 1H), 4.67 (d, J = 6.1 Hz, 1H), 4.34 (s, 1H), 3.52 (s, 3H), 3.00 – 2.82

TE D

(m, 4H), 1.69 (d, J = 6.6 Hz, 1H), 1.51 – 1.44 (m, 1H), 1.40 (d, J = 6.1 Hz, 3H), 1.16 (br, J = 39.4 Hz, 21H), 1.02 (d, J = 6.2 Hz, 3H), 0.93 (d, J = 5.9 Hz, 3H), 0.81 (d, J = 6.2 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 172.7, 172.5, 171.4, 167.8, 166.1, 139.5, 138.1,

EP

118.4, 116.9, 78.6, 71.7, 54.9, 54.3, 51.9, 41.8, 38.5, 33.8, 32.8, 29.3, 29.2, 29.1, 29.1, 29.0, 27.4, 26.9, 26.8, 22.5, 16.8, 15.1, 13.2; νmax (KBr): 3493, 3461, 3389, 3283, 2925,

AC C

2853, 2719, 1736, 1680, 1649, 1610, 1520, 1462, 1334, 1259, 1208, 1112, 1011, 978, 901, 859,813 cm-1; HRMS (MALDI) calcd for C34H57N3O8Na+ [M + Na]+ : 658.4038, found 658.4042.

(S)-Ethyl-2-((3S,14S,15S,E)-7,14-dimethyl-11-methylene-15-((R)-14-methylpentadecan2-yl)-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3-yl)-2-hydroxyacetate (1e)

28

ACCEPTED MANUSCRIPT

The titled compound 1e was obtained following the general procedure described for 1a. Flash column chromatography eluent (MeOH: CH2Cl2 = 1: 20) to afford compound 1e (30 mg, 45% for 2 steps) as a white solid. [α]20D = – 117.8 (c = 0.17, DMSO); 1H NMR

RI PT

(400 MHz, DMSO-d6) δ 8.93 (s, 1H), 8.40 (d, J = 9.9 Hz, 1H), 6.85 (d, J = 15.0 Hz, 1H), 6.19 (d, J = 15.0 Hz, 1H), 5.99 (d, J = 5.4 Hz, 1H), 5.43 (s, 1H), 5.33 (s, 1H), 5.11 (d, J = 10.3 Hz, 1H), 4.89 (d, J = 9.9 Hz, 1H), 4.50 – 4.42 (m, 2H), 4.03 (q, J = 6.7 Hz, 1H),

SC

3.69 (d, J = 18.2 Hz, 1H), 2.95 – 2.87 (m, 4H), 1.72 – 1.67 (m, 1H), 1.52 – 1.45 (m, 1H), 1.33 – 1.19 (br, 19H), 1.04 (d, J = 6.9 Hz, 6H), 0.94 (d, J = 6.8 Hz, 3H), 0.84 (d, J = 6.6 13

C NMR (400 MHz, DMSO-d6) δ172.4, 171.0, 168.5, 167.8, 166.2, 138.6,

M AN U

Hz, 6H);

137.7, 118.6, 117.1 , 78.4, 71.5, 60.7, 54.9, 51.8, 41.7, 38.5, 36.5, 33.7, 32.8, 29.3, 29.2, 27.4, 27.0, 26.8, 26.1, 22.5, 22.1,15.2, 14.0, 13.9,13.1; νmax(KBr): 3383, 2961, 2923, 2854, 2054, 1944, 1694, 1673, 1527, 1456, 1377, 1261, 1097, 862, 799 cm-1; HRMS (ESI)

TE D

calcd for C34H57N3O8Na+ [M + Na]+: 658.4038, found 658.4038. (R,E)-tert-Butyl-2-(4-((tert-butoxycarbonyl)amino)-5-((tert-butyldimethylsilyl)oxy)-Nmethylpent-2-enamido)acetate (27)

EP

To a solution of 23 (5.75 g, 13.46 mmol, 1 equiv) in CH2Cl2 (67.3 mL) was added DMP (7.42 g, 17.50 mmol, 1.3 equiv) at 20 ºC. After stirred for 3 h, the reaction mixture was

AC C

quenched by addition of saturated aqueous solution of NaHCO3 (100 mL) and saturated aqueous Na2S2O3 (50 mL). The reaction mixture was stirred for 10 min, and extracted with CH2Cl2 (3 × 100 mL). The combined organic phases were dried (Na2SO4) and concentrated under reduced pressure. The residue purified through a short pad of silica gel. The solvent was removed under reduced pressure to afford 26 (5.09 g, 89%) as a colorless oil, which can used for the next step directly.

29

ACCEPTED MANUSCRIPT

To a solution of 3e (2.53 g, 0.19 mmol, 1.2 equiv) in MeCN (15 mL) was added anhydrous LiCl (523 mg, 12.34 mmol, 2 equiv) and DBU (1.1 mL, 7.4 mmol, 1.2 equiv) at 0 ºC and stirred for 10min. Solution of 26 (2.63 g, 6.17 mmol, 1 equiv) in CH2Cl2 (1

RI PT

mL) was then added to the reaction mixture. And the resulting solution was stirred for 45 min. Water (100 mL) was added to quench the reaction. And the aqueous phase was extracted with CH2Cl2 (3 × 100 mL). The combined organic phases were dried (Na2SO4)

SC

and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (ethyl acetate: hexane = 1:5 to 1:3) to obtain 27 (2.1 g, 57%)

M AN U

as a colorless oil. [α]24D = − 5.3 (c = 0.5, CHCl3); 1H NMR (400 MHz, DMSO-d6) δ 7.88 (d, J = 7.5 Hz, 2H), 7.69 (t, J = 7.5 Hz, 2H), 7.57 – 7.45 (m, 1H), 7.40 (t, J = 7.4 Hz, 2H), 7.31 (t, J = 7.4 Hz, 2H), 6.70 – 6.30 (m, 2H), 4.36 – 4.06 (m, 5H), 3.99 (s, 1H), 3.60 – 3.44 (m, 2H), 3.04 (s, 2H), 2.84 (s, 1H), 1.41 – 1.36 (m, 9H), 0.84 – 0.81 (m, 9H), 0.03 – 13

C NMR (100 MHz, DMSO-d6) δ 168.5, 168.3, 166.1, 165.5, 155.7,

TE D

-0.02 (m, 6H);

143.8, 142.6, 141.7, 140.8, 127.6, 127.0, 125.2, 121.4, 121.0, 120.1, 81.4, 80.8, 79.2, 65.5, 64.6, 64.4, 54.0, 51.5, 50.0, 46.7, 36.2, 34.5, 27.7, 27.6, 25.7, 17.9, -5.4; νmax(neat): 3294,

EP

3042, 2856, 1722, 1665, 1623, 1450, 1278, 1251, 1228, 1117, 836, 739 cm-1; HRMS (MALDI) calcd for C33H46N2NaO6Si+ [M + Na]+: 617.3023, found 617.3020.

AC C

(2S,3S,4R)-Allyl-3-hydroxy-2,4,16-trimethylheptadecanoate(25) To a solution of 28 (15.55g, 42.13 mmol, 1 equiv) and alkene 29 (23 g, 126.4 mmol, 3 equiv) in CH2Cl2 (25 mL) was added Grubbs 2nd generation catalyst (0.72 g, 0.84 mmol, 0.02 equiv)under argon atmosphere at 40 oC. After stirred and argon is bubbled through the solution for 10 h, the reaction mixture was concentrated under reduced pressure. The

30

ACCEPTED MANUSCRIPT

residue was purified by flash chromatography on silica gel (petroleum ether: ethyl acetate = 8: 1 to 6:1) to afford 30 (17.68 g, 80%) as a colorless oil. To a solution of 30 (8.73 g, 16.69 mmol, 1 equiv) in THF/ MeOH/ H2O (60 mL/ 20 mL/

RI PT

20 mL) was added LiOH•H2O (2.8 g, 66.74 mmol, 4 equiv) at room temperature. After being stirred for 4 h at this temperature, the reaction mixture acidified to pH = 3.0 with aqueous 10% NaHSO4 and extracted with ethyl acetate (2 × 300 mL). The solvent was

SC

evaporated, and the resulting mixture was directly used for the next step without further purification.

M AN U

The above colorless oil was dissolved in MeOH (100 mL), and 10% Pd/C was added Hydrogen was used to exchange internal atmosphere for three times. The reaction was allowed to stir under hydrogen atmosphere at 20 oC for 20 h. The resulting mixture was filtered through a short pad of silica gel, concentrated under reduced pressure. To a

TE D

solution of crude acid in DMF (25.0 mL) was added K2CO3 (6.92 g, 50.07 mmol, 3 equiv) and allyl bromide (5.05 g, 41.73 mmol, 2.5 equiv) at room temperature. The mixture was stirred at room temperature for 10 h, and then H2O (100 mL) was added, and the resultant

EP

mixture was extracted with ethyl acetate (3 × 150 mL). The organic phase was washed by brine (100 mL × 2). Dried over MgSO4, filtered and concentrated under reduced pressure.

AC C

The residue was purified with column chromatography on silica gel (petroleum ether: ethyl acetate = 50:1) to afford compound 25 (11.18 g, 55% for 3 steps) as a colorless oil. [α]22D = − 42.6 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.97 – 5.86 (m, 1H), 5.33 (d, J = 17.2 Hz, 1H), 5.24 (d, J = 10.4 Hz, 1H), 4.61 (d, J = 5.7 Hz, 2H), 3.63 – 3.57 (m, 1H), 2.71 – 2.61 (m, 1H), 2.42 (d, J = 6.3 Hz, 1H), 1.61 – 1.45 (m, 2H), 1.25 (s, 20H), 1.18 – 1.11 (m, 5H), 0.88 – 0.83 (m, 9H); 13C NMR (100 MHz, CDCl3) δ 176.3, 132.1,

31

ACCEPTED MANUSCRIPT

118.6, 76.1, 65.4, 43.3, 39.2, 35.1, 34.1, 30.1, 30.0, 29.9, 29.8, 29.8, 28.1, 27.6, 27.4, 22.8, 14.6, 13.0; νmax(neat): 3520, 3085, 2923, 2853, 1721, 1649, 1382, 1338, 1263, 1169, 1106,

369.3363, found 369.3358.

RI PT

1034, 983, 929, 738, 613, 450 cm-1; HRMS (ESI) calcd for C23H45O3+ [M + H]+:

(S)-1-((2S,3S,4R)-1-(Allyloxy)-2,4,16-trimethyl-1-oxoheptadecan-3-yl)-4-methyl-2-((tertbutoxycarbonyl)amino)succinate (22a)

SC

To a solution of acid 24a (2.16 g, 8.75 mmol, 2.5 equiv) and 25 (1.29 g, 3.50mmol, 1 equiv) in CH2Cl2 (10 mL) was added DMAP (128 mg, 1.05 mmol, 0.3 equiv) and DIC

M AN U

(1.33 g, 10.5 mmol, 3 equiv) under argon atmosphere at 0 ºC. The reaction mixture was stirred for 1.5 h, and diluted with CH2Cl2 (50 mL) then quenched with H2O (100 mL). The aqueous phase was extracted with CH2Cl2 (3 × 50 mL). And the combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The residue

TE D

was purified by column chromatography on silica gel (petroleum ether/ethyl acetate =19: 1 to 9 : 1) to obtain compound 22a (1.93 g, 92%) as colorless oil. [α]22D = − 38.7 (c = 1.5, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.96 – 5.82 (m, 1H), 5.46 (d, J = 8.6 Hz, 1H),

EP

5.30 (d, J = 16.5 Hz, 1H), 5.21 (d, J = 10.4 Hz, 1H), 5.13 (dd, J = 8.2, 3.9 Hz, 1H), 4.69 – 4.40 (m, 3H), 3.67 (s, 2H), 2.95 – 2.87 (m, 1H), 2.84 – 2.70 (m, 2H), 1.79 – 1.68 (m, 1H),

AC C

1.53 – 1.45 (m, 1H), 1.42 (s, 9H), 1.31 – 1.19 (m, 18H), 1.12 (d, J = 7.1 Hz, 5H), 0.87 – 0.82 (m, 9H);

13

C NMR (100 MHz, CDCl3) δ 173.4, 171.4, 170.4, 155.4, 132.3, 118.5,

80.0, 78.8, 65.5, 52.0, 50.1, 42.0, 39.2, 36.5, 34.2, 33.5, 30.1, 29.8, 29.8, 29.8, 29.7, 28.4, 28.1, 27.5, 27.2, 22.8, 14.0, 13.6; νmax(neat): 3436, 3378, 3085, 2924, 2854, 1732, 1649, 1496, 1458, 1439, 1366, 1338, 1243, 1207, 1162, 1042, 1025, 990, 934, 861, 811, 778,

32

ACCEPTED MANUSCRIPT

760, 736, 555, 463 cm-1; HRMS (ESI) calcd for C33H60NO8+ [M + H]+:598.4313, found 598.4315.

butoxycarbonyl)amino)succinate (22b)

RI PT

(S)-1-((2S,3S,4R)-1-(Allyloxy)-2,4,16-trimethyl-1-oxoheptadecan-3-yl)-4-ethyl-2-((tert-

The titled compound 22b was obtained following the procedure described for 22a. Flash column chromatography (petroleum ether : ethyl acetate = 9 : 1); yield: 90%; colorless oil;

SC

[α]21D = − 41.4 (c = 1.3, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.89 (M, 1H), 5.46 (d, J = 8.0 Hz, 1H), 5.29 (d, J = 17.2 Hz, 1H), 5.20 (d, J = 10.3 Hz, 1H), 5.15 – 5.08 (m, 1H),

M AN U

4.53 (s, 3H), 4.18 – 4.04 (m, 2H), 2.94 – 2.84 (m, 1H), 2.82 – 2.67 (m, 2H), 1.73 (s, 1H), 1.53 – 1.46 (m, 1H), 1.41 (s, 9H), 1.35 – 1.16 (m, 22H), 1.12 (d, J = 5.3 Hz, 6H), 0.88 – 0.80 (m, 9H);

13

C NMR (100 MHz, CDCl3) δ 173.4, 171.0, 170.5, 155.4, 132.3, 118.5,

70.0, 78.8, 65.5, 61.0, 50.1, 42.0, 39.2, 36.6, 34.2, 33.4, 30.0, 29.8, 29.8, 29.7, 28.4, 28.1,

TE D

27.5, 27.2, 22.8, 14.2, 14.0, 13.6; νmax(neat): 3436, 3371, 3085, 2924, 2854, 1723, 1650, 1462, 1367, 1243, 1161, 1022, 976, 939, 856, 779, 760, 737, 553, 464, 434 cm-1; HRMS (MALDI) calcd for C34H61NNaO8+ [M + Na]+:642.4289, found 642.4293.

EP

(S)-1-((2S,3S,4R)-1-(allyloxy)-2,4,16-trimethyl-1-oxoheptadecan-3-yl)-4-isopropyl-2((tert-butoxycarbonyl)amino)succinate(22c)

AC C

The titled compound 22c was obtained following the procedure described for 22a. Flash column chromatography (petroleum ether : ethyl acetate = 9 : 1); yield: 86%; colorless oil; [α]21D = − 40.8(c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.99 – 5.81 (m, 1H), 5.47 (d, J = 8.6 Hz, 1H), 5.31 (d, J = 17.1 Hz, 1H), 5.22 (d, J = 10.3 Hz, 1H), 5.17 – 5.11 (m, 1H), 5.03 – 4.93 (m, 1H), 4.60 – 4.46 (m, 3H), 2.91 – 2.67 (m, 3H), 1.74 (d, J = 3.7 Hz, 1H), 1.55 – 1.47 (m, 1H), 1.43 (s, 9H), 1.33 – 1.18 (m, 25H), 1.13 (d, J = 6.1 Hz, 6H),

33

ACCEPTED MANUSCRIPT

0.90 – 0.81 (m, 9H);

13

C NMR (100 MHz, CDCl3) δ 173.4, 170.6, 170.6, 155.4, 132.3,

118.6, 79.9, 78.7, 68.6, 65.5, 50.2, 42.0, 39.2, 36.9, 34.2, 33.4, 30.1, 29.9, 29.8, 29.7, 28.4, 28.1, 27.5, 27.2, 22.8, 21.9, 14.0, 13.7; νmax(neat): 3436, 3370, 3085, 2977, 2925, 2854,

RI PT

1722, 1650, 1495, 1367, 1338, 1243, 1207, 1165, 1107, 1043, 1025, 979, 930, 861, 807, 779, 722, 557, 462, 413 cm-1; HRMS (MALDI) calcd for C35H63NNaO8+ [M + Na]+: 648.4446, found 648.4450.

2-((tert-butoxycarbonyl)amino)succinate(22d)

SC

(S)-1-((2S,3S,4R)-1-(allyloxy)-2,4,16-trimethyl-1-oxoheptadecan-3-yl)-4-prop-2-yn-1-yl-

M AN U

The titled compound 22d was obtained following the procedure described for 22a. Flash column chromatography (petroleum ether : ethyl acetate = 9 : 1); yield: 82%; colorless oil; [α]21D = − 45.2 (c = 2.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.98 – 5.80 (m, 1H), 5.46 (d, J = 7.8 Hz, 1H), 5.30 (d, J = 17.1 Hz, 1H), 5.21 (d, J = 10.3 Hz, 1H), 5.16 – 5.10 (m,

TE D

1H), 4.53 (s, 3H), 3.67 (s, 3H), 2.96 – 2.84 (m, 1H), 2.84 – 2.70 (m, 2H), 1.79 – 1.67 (m, 1H), 1.54 – 1.46 (m, 1H), 1.42 (s, 9H), 1.37 – 1.17 (m, 19H), 1.12 (d, J = 5.9 Hz, 6H), 0.89 – 0.79 (m, 9H);

13

C NMR (100 MHz, CDCl3) δ 173.4, 171.4, 170.4, 155.4, 132.3,

EP

118.5, 80.0, 78.8, 65.5, 52.0, 50.1, 42.0, 39.2, 36.5, 34.2, 33.5, 30.0, 29.8, 29.8, 29.7, 28.4, 28.1, 27.5, 27.2, 22.8, 14.0, 13.6; νmax(neat): 3436, 3376, 3085, 2924, 2854, 1732, 1650,

AC C

1496, 1458, 1439, 1366, 1244, 1208, 1162, 1042, 1026, 990, 934, 860, 812, 779, 760, 737, 557, 463 cm-1; HRMS (ESI) calcd for C35H58NO8-[M − H]-: 620.4162, found 620.4160. Methyl-2-((3S,11R,14S,15S,E)-11-(hydroxymethyl)-7,14-dimethyl-15-((R)-14methylpentadecan-2-yl)-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3yl)acetate (20a)

34

ACCEPTED MANUSCRIPT

To a solution of 22a (2.25 g, 3.76 mmol, 1 equiv) in anhydrous THF (37.6 mL), Pd(PPh3)4 (870 mg, 0.75 mmol, 0.2 equiv) and N-methylaniline (816µL, 7.53 mmol, 2 equiv) were added. The reaction mixture was stirred for 1 h at room temperature, and

RI PT

diluted with ethyl acetate (200 mL). The organic phase was washed by 1 % HCl (2 × 60 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was purified bycolumn chromatography on silica gel (petroleum ether: ethyl acetate = 20: 1 to

SC

1: 1) to afford the acid.

The obtained acid above and amine 21 (2.10 g, 5.64 mmol, 1.5 equiv) was dissolved in

M AN U

anhydrous CH2Cl2 (15 mL), HOBt (762 mg, 5.64 mmol, 1.5 equiv) and EDCI (1.44 g, 7.52 mmol, 2 equiv) were added successively. The reaction mixture was stirred for 18 h and the solvent was removed. The residue was diluted with CH2Cl2 (100 mL) and washed successively with 1 % HCl, saturated aqueous NaHCO3, brine, dried over Na2SO4 and

TE D

filtrated. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 5 : 1) to obtain compound 31a (1.82 g, 53 % for 2 steps) as a colorless oil.

EP

The compound 21a (0.89 g, 0.97 mmol) was dissolved in CH2Cl2 (12 mL) and TFA (6 mL), the reaction mixture was stirred at room temperature for 3 h, and then the solvent

AC C

was removed under reduced pressure to afford the crude amino acid. Then the above crude amino acid was dissolved in THF (20 mL), the solution was added slowly to a suspension of HATU (5.53 g, 14.55 mmol, 15 equiv) and DIPEA (5.08 mL, 29.1mmol, 30 equiv) in THF (950 ml) over 8h at 40℃ and then continued to stirred another 12h at 25℃. The solvent was removed under reduced pressure, diluted with MeOH/ ethyl acetate (V/V = 2: 1, 300 mL), and filtered through a celite pad. The filtrate

35

ACCEPTED MANUSCRIPT

was concentrated under reduced pressure, and then the residue was dissolved in ethyl acetate (500 mL) and washed successively with 1 % HCl, saturated aqueous NaHCO3, brine, dried over Na2SO4. The solution was filtrated and concentrated under reduced

RI PT

pressure. The residue was purified by column chromatography on silica gel (CH2Cl2: MeOH = 100: 1 to 100: 4) to obtain the cyclic peptide 20a (270 mg, 45% for 2 steps) as a white powder. [α]19D = − 131.4(c = 0.2, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.50

SC

(d, J = 8.9 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 6.74 (d, J = 15.0 Hz, 1H), 5.94 (d, J = 14.9 Hz, 1H), 5.13 (d, J = 10.4 Hz, 1H), 4.77 – 4.67 (m, 1H), 4.46 – 4.36 (m, 1H), 4.31 (d, J =

M AN U

18.0 Hz, 1H), 3.80 (d, J = 18.0 Hz, 1H), 3.60 (s, 3H), 2.95 (s, 3H), 2.86 – 2.76 (m, 1H), 2.75 – 2.62 (m, 2H), 1.74 – 1.64 (m, 1H), 1.54 – 1.43 (m, 1H), 1.28 (br, J = 38.8 Hz, 20H), 1.16 – 1.09 (m, 2H), 1.01 (d, J = 6.7 Hz, 4H), 0.93 (d, J = 6.5 Hz, 3H), 0.84 (d, J = 6.6 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 173.0, 169.7, 169.1, 167.9, 165.7, 143.7,

TE D

118.5, 77.4, 63.0, 52.1, 51.8, 51.6, 49.0, 41.5, 38.5, 37.5, 36.5, 33.6, 33.3, 29.4, 29.2, 29.1, 29.1, 29.0, 27.4, 26.9, 26.8, 22.6, 16.0, 13.1; νmax(neat): 3638, 3524, 3448, 3372, 3254, 3210, 3084, 2921, 2852, 1741, 1720, 1668, 1646, 1617, 1575, 1538, 1482, 1439, 1402,

EP

1383, 1356, 1316, 1284, 1220, 1151, 1126, 1090, 1057, 1027, 993, 841, 767, 737, 700, 631, 557, 442 cm-1; HRMS (ESI) calcd for C33H58N3O8+ [M + H]+:624.4218, found

AC C

624.4219.

Ethyl-2-((3S,11R,14S,15S,E)-11-(hydroxymethyl)-7,14-dimethyl-15-((R)-14methylpentadecan-2-yl)-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3yl)acetate (20b)

The titled compound 20b was obtained following the procedure described for 20a. Flash column chromatography (CH2Cl2: MeOH = 100: 2 to 100: 8); yield: 22% for 4 steps;

36

ACCEPTED MANUSCRIPT

white powder; [α]23D = − 145.1(c = 0.25, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 9.0 Hz, 1H), 7.75 (d, J = 8.7 Hz, 1H), 6.69 (dd, J = 14.9, 2.4 Hz, 1H), 5.88 (d, J = 14.6 Hz, 1H), 5.08 (d, J = 10.5 Hz, 1H), 4.87 (t, J = 5.4 Hz, 1H), 4.68 (dd, J = 14.7, 7.4

RI PT

Hz, 1H), 4.40 – 4.33 (m, 1H), 4.26 (d, J = 18.0 Hz, 1H), 4.05 – 3.96 (m, 2H), 3.73 (d, J = 18.0 Hz, 1H), 2.89 (s, 3H), 2.80 – 2.73 (m, 1H), 2.66 – 2.57 (m, 2H), 1.68 – 1.59 (m, 1H), 1.49 – 1.38 (m, 1H), 1.33 – 1.04 (m, 27H), 0.96 (d, J = 6.9 Hz, 3H), 0.88 (d, J = 6.7 Hz, 13

C NMR (100 MHz, DMSO-d6) δ 173.0, 169.2, 169.1,

SC

3H), 0.78 (d, J = 6.6 Hz, 6H);

167.8, 165.7, 143.7, 118.5, 77.3, 63.0, 60.5, 52.1, 51.6, 49.1, 41.5, 38.5, 37.8, 36.4, 33.6,

M AN U

33.4, 29.4, 29.2, 29.1, 29.1, 29.0, 27.4, 26.9, 26.8, 22.6, 15.9, 14.0, 13.1; νmax(neat): 3373, 2922, 2852, 1738, 1719, 1669, 1647, 1617, 1316, 1219, 1150, 1124, 1090, 1058, 1023, 972, 558 cm-1; HRMS (MALDI) calcd for C34H59N3NaO8+ [M + Na]+:660.4194, found 660.4198.

TE D

Isopropyl-2-((3S,11R,14S,15S,E)-11-(hydroxymethyl)-7,14-dimethyl-15-((R)-14methylpentadecan-2-yl)-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3yl)acetate (20c)

EP

The titled compound 20c was obtained following the procedure described for 20a. Flash column chromatography (CH2Cl2: MeOH = 100: 2 to 100: 8); yield: 26% for 4 steps;

AC C

white powder; [α]23D = − 134.9 (c = 0.4, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J = 9.1 Hz, 1H), 7.83 (d, J = 8.7 Hz, 1H), 6.75 (dd, J = 14.8, 2.3 Hz, 1H), 5.94 (d, J = 14.7 Hz, 1H), 5.14 (d, J = 10.5 Hz, 1H), 4.98 – 4.83 (m, 2H), 4.74 (dd, J = 15.5, 6.8 Hz, 1H), 4.46 – 4.37 (m, 1H), 4.31 (d, J = 18.1 Hz, 1H), 3.78 (d, J = 18.1 Hz, 1H), 3.43 – 3.37 (m, 2H), 2.95 (s, 3H), 2.86 – 2.77 (m, 1H), 2.64 (d, J = 6.7 Hz, 2H), 1.73 – 1.65 (m, 1H), 1.55 – 1.43 (m, 1H), 1.36 – 1.09 (m, 28H), 1.01 (d, J = 6.8 Hz, 3H), 0.94 (d, J = 6.7

37

ACCEPTED MANUSCRIPT

Hz, 3H), 0.85 (d, J = 6.6 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 173.0, 169.1, 168.7, 167.7, 165.6, 143.7, 118.5, 77.2, 67.9, 63.0, 52.1, 51.5, 49.1, 41.4, 38.5, 38.1, 36.4, 33.6, 33.3, 29.3, 29.19, 29.0, 27.4, 26.9, 26.8, 22.5, 21.5, 21.4, 15.9, 13.1; νmax(neat): 3386,

for C35H61N3NaO8+ [M + Na]+:674.4351, found 674.4355.

RI PT

2851, 1721, 1648, 1482, 1376, 1216, 1109, 960, 722, 559 cm-1; HRMS (MALDI) calcd

Prop-2-yn-1-yl-2-((3S,11R,14S,15S,E)-11-(hydroxymethyl)-7,14-dimethyl-15-((R)-14-

SC

methylpentadecan-2-yl)-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3yl)acetate (20d)

M AN U

The titled compound 20d was obtained following the procedure described for 20a. Flash column chromatography (CH2Cl2 : MeOH = 100 : 2 to 100 : 8); yield: 20% for 4 steps; white powder; [α]18D = − 128.4 (c = 0.16, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.52 (d, J = 9.1 Hz, 1H), 7.79 (d, J = 8.7 Hz, 1H), 6.74 (dd, J = 14.9, 2.8 Hz, 1H), 5.93

TE D

(dd, J = 15.0, 2.0 Hz, 1H), 5.13 (d, J = 10.1 Hz, 1H), 4.90 (t, J = 5.6 Hz, 1H), 4.73 (dd, J = 15.7, 6.7 Hz, 1H), 4.69 (d, J = 2.4 Hz, 2H), 4.46 – 4.37 (m, 1H), 4.30 (d, J = 18.1 Hz, 1H), 3.77 (d, J = 18.1 Hz, 1H), 3.58 (t, J = 2.3 Hz, 1H), 3.44 – 3.35 (m, 3H), 2.94 (s, 3H),

EP

2.81 (dd, J = 10.4, 7.2 Hz, 1H), 2.75 (d, J = 6.7 Hz, 2H), 1.75 – 1.64 (m, 1H), 1.55 – 1.43 (m, 1H), 1.24 (br s, 20H), 1.17 – 1.10 (m, 2H), 1.01 (d, J = 7.0 Hz, 3H), 0.93 (d, J = 6.8

AC C

Hz, 3H), 0.84 (d, J = 6.6 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 172.9, 168.9, 168.6, 167.8, 165.6, 143.7, 118.5, 78.0, 77.9, 77.4, 63.0, 52.2, 51.5, 48.9, 41.4, 38.5, 37.4, 36.4, 33.6, 33.3, 29.3, 29.2, 29.1, 29.1, 29.0, 27.4, 26.9, 26.8, 22.5, 15.9, 13.1; νmax(neat): 3369, 2920, 2850, 1724, 1699, 1670, 1649, 1612, 1251, 985, 843, 558 cm-1; HRMS (MALDI) calcd for C35H57N3NaO8+ [M + Na]+:670.4038, found 670.4042.

38

ACCEPTED MANUSCRIPT

Methyl-2-((3S,14S,15S,E)-7,14-dimethyl-11-methylene-15-((R)-14-methylpentadecan-2yl)-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3-yl)acetate (19a) To a solution of obtained solid 20a (187.0 mg, 0.30mmol, 1 equiv) in THF (8 mL), then

RI PT

triethylamine (84.6µL, 0.6mmol, 2 equiv) and methanesulfonyl chloride (35.2µL, 0.12 mmol, 1.5 equiv) were added at 0 ºC. After stirred for 30 min, the reaction solution was quenched by addition of water (0.1 mL), dried over Na2SO4, filtered and concentrated

SC

under reduced pressure. The resulting crude product was dissolved in THF (16 mL). To the resulting solution was added DBU (0.46g, 3.0 mmol, 10 equiv) at 20 ºC. After stirred

M AN U

for 2 h, the reaction mixture was diluted with ethyl acetate/THF (200 mL/50 mL). The organic phases were washed with 1 % NaHSO4 (20 mL), saturated aqueous NaHCO3 (3 × 10 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 100: 1 to 100: 4)

1

TE D

to obtain 19a (100 mg, 55% for 2 steps) as a white solid. [α]20D = − 46.4 (c = 0.5, DMSO); H NMR (400 MHz, DMSO-d6) δ 8.93 (s, 1H), 8.48 (d, J = 8.9 Hz, 1H), 6.90 (d, J = 15.0

Hz, 1H), 6.15 (d, J = 15.0 Hz, 1H), 5.44 (s, 1H), 5.34 (s, 1H), 5.08 (d, J = 9.0 Hz, 1H),

EP

4.71 (dd, J = 14.6, 7.0 Hz, 1H), 4.34 (d, J = 17.9 Hz, 1H), 3.84 (d, J = 17.9 Hz, 1H), 3.59 (s, 3H), 2.98 (s, 3H), 2.93 – 2.84 (m, 1H), 2.72 (qd, J = 16.1, 6.4 Hz, 2H), 1.81 – 1.67 (m,

AC C

1H), 1.58 – 1.42 (m, 1H), 1.23 (s, 20H), 1.16 – 1.09 (m, 2H), 1.05 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H), 0.84 (d, J = 6.6 Hz, 6H);

13

C NMR (101 MHz, DMSO-d6) δ

172.3, 169.7, 168.9, 167.9, 165.9, 138.7, 138.2, 118.7, 116.9, 77.8, 52.3, 51.7, 48.9, 41.8, 38.5, 37.4, 36.5, 33.7, 33.1, 29.3, 29.2, 29.1, 29.0, 28.9, 27.4, 26.8, 22.5, 15.2, 13.1; νmax(neat): 3372, 2922, 2852, 1732, 1693, 1672, 1648, 1613, 1533, 1516, 1255, 975, 844,

39

ACCEPTED MANUSCRIPT

623 cm-1; HRMS (MALDI) calcd for C33H56N3NaO7+ [M + Na]+:628.3932, found 628.3938. Ethyl-2-((3S,14S,15S,E)-7,14-dimethyl-11-methylene-15-((R)-14-methylpentadecan-2-yl)-

RI PT

2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3-yl)acetate (19b)

The titled compound 19b was obtained following the procedure described for 19a. Flash column chromatography (CH2Cl2: MeOH = 100: 1 to 100: 4); yield: 60% for 2 steps;

SC

white powder; [α]23D = − 45.3 (c = 0.1, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.89 (s, 1H), 8.44 (d, J = 8.9 Hz, 1H), 6.83 (d, J = 15.0 Hz, 1H), 6.10 (d, J = 15.0 Hz, 1H),

M AN U

5.38 (s, 1H), 5.28 (s, 1H), 5.02 (d, J = 10.1 Hz, 1H), 4.66 (dd, J = 15.1, 6.8 Hz, 1H), 4.29 (d, J = 17.9 Hz, 1H), 3.99 (q, J = 7.0 Hz, 2H), 3.76 (d, J = 17.9 Hz, 1H), 2.92 (s, 3H), 2.82 (dt, J = 14.3, 6.9 Hz, 1H), 2.70 – 2.56 (m, 2H), 1.71 – 1.63 (m, 1H), 1.48 – 1.36 (m, 1H), 1.21 – 1.06 (m, 25H), 0.99 (d, J = 6.9 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H), 0.77 (d, J =

TE D

6.6 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 172.5, 169.3, 169.0, 167.9, 166.0, 138.8, 138.2, 118.7, 117.1, 77.8, 60.4, 52.3, 49.0, 41.8, 38.5, 37.7, 36.6, 33.8, 33.2, 29.4, 29.2, 29.1, 29.07, 29.0, 27.4, 26.8, 22.6, 15.2, 14.0, 13.1; νmax(neat): 3366, 2954, 2922, 2852,

EP

1738, 1723, 1673, 1658, 1612, 1552, 1293, 1281, 844, 555 cm-1; HRMS (MALDI) calcd for C34H57N3NaO7+[M + Na]+:642.4089, found 642.4093.

AC C

Isopropyl-2-((3S,14S,15S,E)-7,14-dimethyl-11-methylene-15-((R)-14-methylpentadecan2-yl)-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3-yl)acetate (19c) The titled compound 19c was obtained following the procedure described for 19a. Flash column chromatography (CH2Cl2: MeOH = 100: 1 to 100: 4); yield: 62% for 2 steps; white powder; [α]20D = − 35.2 (c = 0.1, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.93 (s, 1H), 8.50 (d, J = 9.1 Hz, 1H), 6.88 (d, J = 15.0 Hz, 1H), 6.13 (d, J = 15.0 Hz, 1H),

40

ACCEPTED MANUSCRIPT

5.43 (s, 1H), 5.32 (s, 1H), 5.07 (d, J = 9.2 Hz, 1H), 4.93 – 4.81 (m, 1H), 4.77 – 4.64 (m, 1H), 4.33 (d, J = 17.8 Hz, 1H), 3.79 (d, J = 17.9 Hz, 1H), 2.96 (s, 3H), 2.90 – 2.81 (m, 1H), 2.69 – 2.60 (m, 2H), 1.78 – 1.61 (m, 1H), 1.54 – 1.41 (m, 1H), 1.16 (br, 28H), 1.03 13

C NMR (100

RI PT

(d, J = 6.9 Hz, 3H), 0.93 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.6 Hz, 6H);

MHz, DMSO-d6) δ 172.4, 169.0, 168.8, 167.8, 165.9, 138.8, 138.1, 118.6, 117.1, 77.7, 67.9, 52.3, 49.0, 41.8, 38.5, 38.0, 36.6, 33.7, 33.2, 29.3, 29.2, 29.1, 29.1, 29.0, 27.4, 26.9,

SC

26.8, 22.6, 21.5, 21.5, 15.2, 13.1; νmax(neat): 3378, 2925, 1734, 1648, 1467, 1384, 1218,

Na]+:656.4245, found 656.4248.

M AN U

1023, 920, 803, 705, 584, 407 cm-1; HRMS (MALDI) calcd for C35H59N3NaO7+ [M +

Prop-2-yn-1-yl-2-((3S,14S,15S,E)-7,14-dimethyl-11-methylene-15-((R)-14methylpentadecan-2-yl)-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3yl)acetate (19d)

TE D

The titled compound 19d was obtained following the procedure described for 19a. Flash column chromatography (CH2Cl2: MeOH = 100: 1 to 100: 4); yield: 55% for 2 steps; white powder; [α]21D = − 37.6 (c = 0.12, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.92

EP

(s, 1H), 8.52 (d, J = 9.0 Hz, 1H), 6.91 (d, J = 14.9 Hz, 1H), 6.15 (d, J = 15.0 Hz, 1H), 5.45 (s, 1H), 5.35 (s, 1H), 5.09 (d, J = 9.8 Hz, 1H), 4.74 (dd, J = 15.4, 6.7 Hz, 1H), 4.69

AC C

(d, J = 2.3 Hz, 1H), 4.34 (d, J = 17.9 Hz, 1H), 3.83 (d, J = 17.9 Hz, 1H), 3.60 – 3.56 (m, 1H), 2.99 (s, 3H), 2.92 – 2.85 (m, 1H), 2.80 – 2.73 (m, 2H), 1.79 – 1.68 (m, 1H), 1.56 – 1.45 (m, 1H), 1.27 – 1.15 (m, 22H), 1.06 (d, J = 7.0 Hz, 3H), 0.96 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 6.6 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 172.4, 168.9, 168.7, 167.9, 165.9, 138.7, 138.2, 118.7, 117.0, 78.0, 77.9, 52.3, 52.1, 48.9, 41.8, 38.5, 37.3, 36.5, 33.7, 33.2, 29.3, 29.2, 29.1, 29.1, 29.0, 27.4, 26.8, 22.5, 15.2, 13.1; νmax(neat): 3372, 2292,

41

ACCEPTED MANUSCRIPT

2852, 1727, 1694, 1674, 1649, 1613, 1252, 977, 556, 529cm-1; HRMS (MALDI) calcd for C35H55N3NaO7+ [M + Na]+:652.3932, found 652.3938. (R,E)-Allyl-2-(4-((tert-butoxycarbonyl)amino)-5-((tert-butyldiphenylsilyl)oxy)-N-

RI PT

methylpent-2-enamido)acetate (39)

To a solution of obtained compound 37 (35.70 g, 70.00 mmol, 1 equiv) in anhydrous THF (350 mL), Pd(PPh3)4 (16.19 g, 14.00 mmol, 0.2 equiv) and N-methylaniline (15 g,

SC

140.00 mmol, 2 equiv) were added. The reaction mixture was stirred for 1.5 h at room temperature, and diluted with ethyl acetate (500 mL). The organic phase was washed by

M AN U

1% HCl (2 × 120 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 10: 1 to 1: 1) to afford the acid 38 (30.25 g, 92%).

The obtained acid 38 (19.20 g, 40.88 mmol, 1 equiv) and Allyl-2-(methylamino)acetate

TE D

(7.92 g, 61.32 mmol, 1.5 equiv) was dissolved in anhydrous CH2Cl2 (160 mL), HOBt (8.290g, 61.32 mmol, 1.5 equiv) and EDCI (15.67 g, 81.76 mmol, 2 equiv) were added successively. The reaction mixture was stirred for 12 h and the solvent was removed. The

EP

residue was diluted with ethyl acetate (600 mL) and washed successively with 1% HCl, saturated aqueous NaHCO3, brine, dried over Na2SO4 and filtrated. The filtrate was

AC C

concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 5:1) to obtain compound 39 (22.0 g, 93 %) as a colorless oil. [α]24D = + 72.0(c = 0.5, CHCl3); 1H NMR (400 MHz, DMSO-d6) δ 7.59 (s, 4H), 7.47 – 7.32 (m, 6H), 7.20 – 7.02 (m, 1H), 6.73 – 6.34 (m, 2H), 5.93 – 5.78 (m, 1H), 5.28 (d, J = 17.3 Hz, 1H), 5.18 (d, J = 10.5 Hz, 1H), 4.57 (s, 2H), 4.44 – 4.20 (m, 2H), 4.15 (s, 1H), 3.64 – 3.49 (m, 2H), 3.07 (s, 2H), 2.85 (s, 1H), 1.36 (s,

42

ACCEPTED MANUSCRIPT

9H), 0.98 (d, J = 10.5 Hz, 9H);

13

C NMR (101 MHz, DMSO-d6) δ 169.1, 169.0, 166.0,

165.7, 155.1, 143.1, 142.3, 135.1, 132.9, 132.3, 132.2, 129.9, 127.9, 121.1, 120.9, 118.0, 117.8, 79.2, 78.0, 65.6, 65.4, 65.1, 64.8, 53.5, 50.7, 49.3, 36.2, 34.4, 28.1, 26.6, 18.8;

RI PT

νmax(neat): 3308, 2931, 2858, 1747, 1710, 1665, 1623, 1168, 1109, 702, 504 cm-1; HRMS (ESI) calcd for C32H44N2NaO6Si+ [M + Na] +: 603.2866, found 603.2865.

1-((3aR,6R)-8,8-Dimethyl-2,2-dioxidotetrahydro-3H-3a,6-methanobenzo[c]isothiazol-

SC

1(4H)-yl)hexadecan-1-one (40)

To a flask was added palmitic acid (256.42 g, 1.0 mol, 1.2 equiv). 500 mL of oxalyl

M AN U

chloride and then DMF (10 mL) were added dropwise at 0°C. The reaction mixture was stirred for 4h at 0°C under N2. The solvent was removed under reduced pressure to afford the acid chloride as a light yellow oil. This oil residue was cooled to 0°C, and slowly added to a stirred solution of (D)-sultam (180.29 g, 840 mmol, 1 equiv) and NaH (40.03 g,

TE D

1.0 mol, 1.2 equiv) in 2.5 L anhydrous toluene at 0°C. The reaction mixture was allowed to warm to room temperature slowly and stirred overnight under nitrogen. TLC analysis of the reaction confirmed the formation of the new compound 40. After completion of the

EP

reaction, filtered through a pad of celite, water (2 L) was added and the aqueous layer was extracted with ethyl acetate (3 × 1 L) ,washed by 0.5 mol/L NaOH (2 × 500 mL).

AC C

Dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified with column chromatography on silica gel (petroleum ether: ethyl acetate = 10: 1) to afford compound 32 (304 g, 82% for 2 steps) as a white solid. 1H NMR (400 MHz, CDCl3) δ 3.88 – 3.83 (m, 1H), 3.45 (q, J = 13.8 Hz, 2H), 2.78 – 2.61 (m, 2H), 2.16 – 2.02 (m, 2H), 1.96 – 1.81 (m, 3H), 1.64 (dd, J = 15.1, 7.7 Hz, 3H), 1.45 – 1.19 (m, 27H), 1.15 (s, 3H), 0.96 (s, 3H), 0.90 – 0.83 (m, 3H); 13C NMR (10 MHz, CDCl3) δ 172.3, 65.4, 53.1,

43

ACCEPTED MANUSCRIPT

48.5, 47.9, 44.8, 38.7, 35.6, 33.0, 32.1, 29.8, 29.7, 29.6, 29.5, 29.4, 29.1, 26.6, 24.6, 22.8, 21.0, 20.0, 14.3; νmax(neat): 2918, 2850, 1695, 1330, 1271, 1210, 1132, 1055, 543, 533

(2R)-1-((3aR,6R)-8,8-dimethyl-2,2-dioxidotetrahydro-3H-3a,6-

RI PT

cm-1; HRMS (MALDI) calcd for C26H47NNaO3S+ [M + Na] +: 476.3169, found 476.3173.

methanobenzo[c]isothiazol-1(4H)-yl)-2-methylhexadecan-1-one (41)

To a solution of compound 40 (305.29 g, 690 mmol, 1 equiv) and HMPA (240.77 g, 1360

SC

mmol, 2 equiv) in THF (1500 mL) was added NaHMDS (430 mL, 860 mmol, 2 mol/L in THF, 1.2 equiv) under argon atmosphere at – 78 ºC. The reaction mixture was stirred for

M AN U

0.5 h and CH3I (240.77 g, 1700 mol, 2.5 equiv) was added for over 30 min. Stirred for another 3 h and quenched with NH4Cl (600 mL).Warmed to room temperature and the aqueous phase was extracted with ethyl acetate (3 × 500 mL). The combined organic phases were washed with H2O (2 × 400 mL) and brine (2 × 400 mL). Dried over Na2SO4,

TE D

filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether/ ethyl acetate =20:1) to obtain compound 41 (280.44 g, 90%) as a white solid. [α]23D = − 58.4 (c = 1.5, CHCl3); 1H NMR (400 MHz,

EP

CDCl3) δ 3.88 (t, J = 6.3 Hz, 1H), 3.45 (q, J = 13.8 Hz, 2H), 3.08 – 2.97 (m, 1H), 2.03 (d, J = 6.3 Hz, 2H), 1.96 – 1.81 (m, 3H), 1.80 – 1.69 (m, 1H), 1.45 – 1.20 (m, 28H), 1.17 (d,

AC C

J = 6.9 Hz, 3H), 1.14 (s, 3H), 0.95 (s, 3H), 0.88 – 0.83 (m, 3H);

13

C NMR (100 MHz,

CDCl3) δ 176.5, 65.2, 53.3, 48.3, 47.8, 44.7, 40.5, 38.6, 32.9, 32.8, 32.0, 29.8, 29.8, 29.7, 29.6, 29.5, 27.4, 26.5, 22.8, 20.9, 20.0, 19.1, 14.2; νmax(neat): 2920, 2849, 1679, 1460, 1328, 1249, 1165, 1134, 1114, 722, 681, 534, 512 cm-1; HRMS (MALDI) calcd for C27H49NNaO3S+ [M + Na] +: 490.3325, found 490.3328.

44

ACCEPTED MANUSCRIPT

(2S,3S,4R)-3-((tert-Butyldimethylsilyl)oxy)-1-((3aR,6R)-8,8-dimethyl-2,2dioxidohexahydro-1H-3a,6-methanobenzo[c]isothiazol-1-yl)-2,4-dimethyloctadecan-1one (45')

RI PT

To a solution of compound 41 (80.3 g, 177.0 mmol, 1 equiv) in CH2Cl2 (800 mL) was added DIBAL-H (220 mL, 220 mmol, 1 M in hexane, 1.25 equiv) under argon atmosphere at – 78 ºC. After stirred at – 78 ºC for 3 h, the reaction was quenched by

SC

addition of H2O (8.8 mL), 15% NaOH (8.8 mL) and H2O (26.4 mL). The resulting mixture was diluted with hexane (100 mL), and the aqueous layer was extracted with

M AN U

hexane (2 × 50 mL). The combined organic extracts were dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate =100:1) and the upper clear oil was used for the next step directly.

TE D

To a solution of (D)-Propionyl Sultam (60.22 g, 221.1 mmol, 1 equiv) in CH2Cl2 (300 mL) was added triethylamine (31.44 g, 310.76 mmol, 1.4 equiv) and tert-butyldimethyl silyltriflate (78.77 g, 298.18 mmol, 1.3 equiv). The reaction mixture was stirred at room

EP

temperature for 10 h. All of the volatile materials were evaporated and pentane (1000 mL) was then added. The suspension was stirred for 10 min and pentane was decanted under

AC C

argon, which was dissolved in CH2Cl2 (300 mL). To a solution of aldehyde 42 in CH2Cl2 (100 mL) at – 78 ºC was added dropwise BF3•Et2O (15.13 g, 105.17 mmol). After stirred for 30 min, the above obtained solution was added to the reaction mixture. The reaction was stirred at –78oC for 3 h before saturated aqueous NH4Cl (250 mL) was added. The aqueous phase was extracted with CH2Cl2 (2 × 300 mL). The combined organic phases were dried (Na2SO4) and

45

ACCEPTED MANUSCRIPT

concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate =10:1) to obtain 45 mixed with (D)-Propionyl Sultam as a white solid. To confirm its structure, the hydroxyl group

RI PT

is protected with tert-butyldimethylsilyl.

To a solution of compound 45 (0.53 g, 1 mmol) in CH2Cl2 (10 mL) was added 2,6lutidine (0.32 g, 3 mmol) and tert-butyldimethyl silyltriflate (0.53 g, 2 mmol)at –78 ºC.

SC

The reaction mixture was warmed to room temperature for 10 h. The reaction was quenched with saturated aqueous NH4Cl (20 mL) was added. The aqueous phase was

M AN U

extracted with CH2Cl2 (2 × 20 mL). The combined organic phases were dried (Na2SO4) and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate =20:1) to obtain 45' as a colorless oil. [α]21D = − 65.3 (c = 1.3, CHCl3); 1H NMR (400 MHz, CDCl3) δ 4.04 (d, J =

TE D

6.1 Hz, 1H), 3.88 (t, J = 6.2 Hz, 1H), 3.45 (q, J = 13.8 Hz, 2H), 3.35 (p, J = 6.6 Hz, 1H), 2.04 (d, J = 6.3 Hz, 2H), 1.95 – 1.81 (m, 3H), 1.52 – 1.44 (m, 1H), 1.44 – 1.20 (m, 30H), 1.18 (d, J = 6.8 Hz, 5H), 1.15 (s, 4H), 0.96 (s, 3H), 0.92 – 0.83 (m, 16H), 0.08 (s, 3H), 13

C NMR (100 MHz, CDCl3) δ 174.2, 75.5, 65.7, 53.3, 48.1, 47.8, 46.8,

EP

0.06 (s, 3H);

44.9, 38.8, 36.6, 35.6, 33.1, 32.1, 30.0, 29.8, 29.8, 29.5, 27.9, 26.6, 26.2, 22.8, 21.1, 20.0,

AC C

18.4, 14.8, 14.3, 12.2, -3.9, -4.8; νmax(neat): 2923, 2853, 1696, 1462, 1332, 1209, 836, 774, 536 cm-1; HRMS (MALDI) calcd for C36H69NNaO4SSi+ [M + Na] +: 662.4609, found 662.4612.

Allyl-(2S,3S,4R)-3-hydroxy-2,4-dimethyloctadecanoate (36) To a solution of 45 (120.78 g, 269 mmol) in THF/ MeOH/ H2O (180 mL/ 60 mL/ 60 mL) was added LiOH•H2O (19.55 g, 466 mmol) at room temperature. After being stirred for 4

46

ACCEPTED MANUSCRIPT

h at this temperature, the reaction mixture acidified to pH = 3.0 with aqueous 10% NaHSO4 and extracted with ethyl acetate (2 × 300 mL). The solvent was evaporated, and the resulting mixture was directly used for the next step without further purification.

RI PT

The above colorless oil was dissolved in DMF (250 mL) was added K2CO3 (71.35g, 510 mmol) and allyl bromide (50 mL, 590 mmol) at room temperature. The mixture was stirred at room temperature for 10 h, and then H2O (100 mL) was added, and the resultant

SC

mixture was extracted with ethyl acetate (3 × 150 mL). The organic phase was washed by brine (2 × 100 mL). Dried over MgSO4, filtered and concentrated under reduced pressure.

M AN U

The residue was purified with column chromatography on silica gel (petroleum ether: ethyl acetate = 50:1) to afford compound 36 (47.59 g, 48% for 4 steps) as a colorless oil. [α]20D = − 38.6 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.97 – 5.84 (m, 1H), 5.31 (d, J = 17.2 Hz, 1H), 5.22 (d, J = 10.4 Hz, 1H), 4.60 (d, J = 5.5 Hz, 2H), 3.59 (s, 1H),

TE D

2.65 (p, J = 7.3 Hz, 1H), 2.48 (d, J = 5.7 Hz, 1H), 1.60 – 1.50 (m, 1H), 1.45 – 1.36 (m, 1H), 1.29 – 1.21 (m, 26H), 1.15 (d, J = 7.4 Hz, 3H), 0.90 – 0.82 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 176.3, 132.1, 118.5, 76.1, 65.3, 43.3, 35.1, 34.0, 32.0, 30.0, 29.8, 29.5,

EP

27.4, 26.1, 22.8, 14.6, 14.2, 12.9; νmax(neat): 3529, 2922, 2853, 1720, 1459, 1167, 983, 538 cm-1; HRMS (ESI) calcd for C23H44NaO3+ [M + Na] +: 391.3183, found 391.3188.

AC C

1-((2S,3S,4R)-1-(Allyloxy)-2,4-dimethyl-1-oxooctadecan-3-yl)-4-methyl-(tertbutoxycarbonyl)-L-aspartate (46a) To a solution of acid Boc-Asp(OMe)-OH (1.85 g, 6.50 mmol, 2.5 equiv) and 36 (1.10 g, 3.00mmol, 1 equiv) in CH2Cl2 (6 mL) was added DMAP (110 mg, 0.90mmol, 0.3 equiv) and DIC (1.14 g, 9.00 mmol, 3 equiv) under argon atmosphere at 0 ºC. The reaction mixture was stirred for 1.5 h, and diluted with ethyl acetate (100 mL). The organic phases

47

ACCEPTED MANUSCRIPT

were washed with 1% NaHSO4 (20 mL), saturated aqueous NaHCO3 (20 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether: ethyl acetate =19:1 to 9:1) to obtain

RI PT

compound 46a (1.64 g, 88%) as colorless oil. [α]24D = − 41.8 (c = 3.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.98 – 5.82 (m, 1H), 5.47 (d, J = 8.4 Hz, 1H), 5.31 (d, J = 17.1 Hz, 1H), 5.22 (d, J = 10.4 Hz, 1H), 5.18 – 5.11 (m, 1H), 4.58 – 4.48 (m, 3H), 3.67 (s, 3H),

SC

2.96 – 2.87 (m, 1H), 2.84 – 2.71 (m, 2H), 1.80 – 1.68 (m, 1H), 1.42 (s, 10H), 1.34 – 1.19 (m, 26H), 1.13 (d, J = 6.7 Hz, 4H), 0.91 – 0.82 (m, 6H); 13C NMR (100 MHz, CDCl3) δ

M AN U

173.4, 171.4, 170.4, 155.4, 132.3, 118.5, 80.0, 78.9, 65.5, 52.0, 50.1, 42.1, 36.5, 34.2, 33.5, 32.0, 29.8, 29.8, 29.7, 29.5, 28.4, 27.2, 22.8, 14.2, 14.0, 13.6; νmax(neat): 2924, 2853, 1732, 1497, 1366, 1162, 1026, 989, 556 cm-1; HRMS (MALDI) calcd for C33H59NO8Na+ [M + Na] +: 620.4133, found 620.4138.

(46b)

TE D

(2S,3S,4R)-Allyl-3-(2-((tert-butoxycarbonyl)amino)acetoxy)-2,4-dimethyloctadecanoate

The titled compound 46b was obtained following the procedure described for 46a. Flash

EP

column chromatography (petroleum ether: ethyl acetate =19:1 to 9:1); yield: 86%; colorless oil; [α]21D = − 38.4 (c = 1.2, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.93 – 5.78

AC C

(m, 1H), 5.29 (d, J = 17.2 Hz, 1H), 5.21 (d, J = 10.3 Hz, 1H), 5.14 (d, J = 8.5 Hz, 1H), 5.00 (s, 1H), 4.51 (d, J = 4.0 Hz, 2H), 3.92 – 3.70 (m, 2H), 2.91 – 2.69 (m, 1H), 1.73 (s, 1H), 1.42 (s, 10H), 1.34 – 1.16 (m, 27H), 1.13 (d, J = 6.6 Hz, 4H), 0.92 – 0.78 (m, 6H); 13

C NMR (100 MHz, CDCl3) δ 173.7, 169.8, 155.6, 132.1, 118.7, 79.9, 78.4, 65.5, 42.4,

42.1, 34.0, 33.7, 32.0, 29.8, 29.7, 29.7, 29.4, 28.4, 27.2, 22.8, 14.2, 14.0, 13.2; νmax(neat):

48

ACCEPTED MANUSCRIPT

2934, 2854, 1720, 1505, 1457, 1367, 1163, 732 cm-1; HRMS (MALDI) calcd for C30H55NNaO6+ [M + Na] +: 548.3922, found 548.3925. (2S,3S,4R)-Allyl-3-(((S)-2-((tert-butoxycarbonyl)amino)propanoyl)oxy)-2,4-

RI PT

dimethyloctadecanoate (46c)

The titled compound 46c was obtained following the procedure described for 46a. Flash column chromatography (petroleum ether: ethyl acetate = 19:1 to 9:1); yield: 90%;

SC

colorless oil; [α]21D = − 43.9 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.93 – 5.80 (m, 1H), 5.29 (d, J = 17.2 Hz, 1H), 5.21 (d, J = 10.3 Hz, 1H), 5.13 (d, J = 8.3 Hz, 1H),

M AN U

5.07 (d, J = 4.6 Hz, 1H), 4.52 (s, 2H), 4.24 (s, 1H), 2.90 – 2.67 (m, 1H), 1.74 (s, 1H), 1.42 (s, 10H), 1.35 – 1.17 (m, 30H), 1.14 (d, J = 5.5 Hz, 3H), 0.89 – 0.78 (m, 6H).

13

C

NMR (100 MHz, CDCl3) δ 173.6, 172.7, 155.2, 132.1, 118.6, 79.8, 78.2, 65.4, 49.4, 42.2, 34.1, 33.6, 32.0, 29.8, 29.8, 29.7, 29.5, 28.4, 27.3, 22.8, 18.8, 14.2, 13.4; νmax(neat): 2923,

TE D

2853, 1741, 1716, 1498, 1455, 1162 cm-1; HRMS (MALDI) calcd for C31H57NNaO6+ [M + Na] +: 562.4078, found 562.4080.

(2S,3S,4R)-Allyl-3-(((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoyl)oxy)-2,4-

EP

dimethyloctadecanoate (46d)

The titled compound 46d was obtained following the procedure described for 46a. Flash

AC C

column chromatography (petroleum ether: ethyl acetate = 19:1 to 9:1); yield: 73%; colorless oil; [α]21D = − 40.2 (c = 1.5, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.94 – 5.80 (m, 1H), 5.30 (d, J = 17.0 Hz, 1H), 5.22 (d, J = 10.5 Hz, 1H), 5.17 (d, J = 7.5 Hz, 1H), 4.97 (d, J = 8.8 Hz, 1H), 4.52 (s, 2H), 4.21 – 4.12 (m, 1H), 2.88 – 2.76 (m, 1H), 2.07 (s, 1H), 1.74 (s, 1H), 1.42 (s, 9H), 1.32 – 1.20 (m, 26H), 1.14 (d, J = 5.4 Hz, 3H), 0.94 (d, J = 4.6 Hz, 3H), 0.89 – 0.81 (m, 9H); 13C NMR (100 MHz, CDCl3) δ 173.6, 171.8, 155.8,

49

ACCEPTED MANUSCRIPT

132.1, 118.5, 79.7, 78.3, 65.4, 58.7, 42.2, 34.3, 33.6, 32.0, 30.9, 29.8, 29.8, 29.7, 29.5, 28.4, 27.3, 22.8, 19.5, 17.2, 14.3, 14.2, 13.5; νmax(neat): 2924, 2854, 1741, 1721, 1496, 1458, 1156, 732 cm-1; HRMS (MALDI) calcd for C33H61NNaO6+ [M + Na] +: 590.4391,

RI PT

found 590.4395.

(2S,3S,4R)-Allyl-3-(((S)-2-((tert-butoxycarbonyl)amino)-3-phenylpropanoyl)oxy)-2,4dimethyloctadecanoate (46e)

SC

The titled compound 46e was obtained following the procedure described for 46a. Flash column chromatography (petroleum ether: ethyl acetate = 19:1 to 9:1); yield: 95%;

M AN U

colorless oil; [α]20D = − 43.7 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.32 – 7.16 (m, 1H), 5.94 – 5.82 (m, 1H), 5.30 (d, J = 17.0 Hz, 1H), 5.23 – 5.13 (m, 1H), 4.89 (d, J = 7.7 Hz, 1H), 4.53 (s, 1H), 3.12 (d, J = 13.4 Hz, 1H), 2.90 – 2.74 (m, 1H), 1.75 (s, 1H), 1.37 (s, 1H), 1.31 – 1.20 (m, 5H), 1.15 (d, J = 5.4 Hz, 1H), 0.92 – 0.79 (m, 1H);

13

C

TE D

NMR (100 MHz, CDCl3) δ 173.7, 171.6, 155.2, 136.5, 132.1, 129.5, 128.6, 126.9, 118.7, 79.8, 78.5, 65.5, 54.5, 42.2, 38.2, 34.1, 33.5, 32.0, 29.8, 29.8, 29.5, 28.4, 27.8, 27.3, 22.8, 14.2, 14.1, 13.5; νmax(neat): 2924, 2853, 1717, 1496, 1456, 1166, 733 cm-1; HRMS

EP

(MALDI) calcd for C37H61NNaO6+ [M + Na] +: 638.4391, found 638.4394. (S)-1-((2S,3S,4R)-1-(Allyloxy)-2,4-dimethyl-1-oxooctadecan-3-yl)-5-methyl-2-((tert-

AC C

butoxycarbonyl)amino)pentanedioate (46f) The titled compound 46f was obtained following the procedure described for 46a. Flash column chromatography (petroleum ether: ethyl acetate = 19:1 to 9:1); yield: 83%; colorless oil; [α]22D = − 41.7 (c = 0.8, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.95 – 5.82 (m, 1H), 4.54 (s, 2H), 4.27 (s, 1H), 3.66 (s, 3H), 2.91 – 2.76 (m, 1H), 2.45 – 2.34 (m, 2H), 2.20 – 2.06 (m, 1H), 1.89 – 1.79 (m, 1H), 1.74 (s, 1H), 1.43 (s, 10H), 1.33 – 1.19 (m,

50

ACCEPTED MANUSCRIPT

27H), 1.15 (d, J = 6.0 Hz, 3H), 0.91 – 0.83 (m, 6H);

13

C NMR (100 MHz, CDCl3) δ

173.7, 173.4, 171.6, 155.5, 132.2, 118.7, 80.0, 78.6, 65.6, 53.1, 51.9, 42.1, 34.1, 33.7, 32.1, 30.3, 29.8, 29.8, 29.8, 29.5, 28.4, 27.9, 27.3, 22.8, 14.3, 13.3; νmax(neat): 2934, 2853,

+

: 634.4289, found 634.4293.

RI PT

1739, 1717, 1501, 1455, 1164 cm-1; HRMS (MALDI) calcd for C34H61NNaO8+ [M + Na]

oxobutanoyl)oxy)-2,4-dimethyloctadecanoate (46g)

SC

(2S,3S,4R)-Allyl-3-(((S)-2-((tert-butoxycarbonyl)amino)-4-(cyclopropylamino)-4-

The titled compound 46g was obtained following the procedure described for 46a. Flash

M AN U

column chromatography (petroleum ether: ethyl acetate = 9:1); yield: 75%; colorless oil; [α]23D = − 43.5 (c = 1.2, CHCl3); 1H NMR (400 MHz, CDCl3) δ 6.01 – 5.69 (m, 2H), 5.30 (d, J = 17.5 Hz, 1H), 5.23 (d, J = 10.3 Hz, 1H), 5.16 – 5.01 (m, 1H), 4.55 (s, 2H), 4.39 (d, J = 7.5 Hz, 1H), 2.90 – 2.76 (m, 1H), 2.70 – 2.47 (m, 2H), 1.79 – 1.65 (m, 1H), 1.40 (s,

0.52 – 0.44 (m, 2H);

13

TE D

9H), 1.22 (br s, 26H), 1.14 (d, J = 7.1 Hz, 3H), 0.89 – 0.81 (m, 6H), 0.73 – 0.67 (m, 2H), C NMR (100 MHz, CDCl3) δ 174.2, 171.3, 170.9, 155.7, 132.1,

118.7, 79.8, 78.7, 65.7, 50.7, 41.9, 37.3, 34.4, 33.3, 32.0, 29.8, 29.8, 29.5, 28.4, 27.2, 22.8,

EP

22.7, 14.3, 14.2, 13.7, 6.4, 6.2; νmax(neat): 3350, 2923, 2853, 1738, 1717, 1651, 1249, 1165, 555cm-1; HRMS (MALDI) calcd for C35H62N2NaO7+ [M + Na] +: 645.4449, found

AC C

645.4452.

(2S,3S,4R)-Allyl-3-(((S)-2-((tert-butoxycarbonyl)amino)-4-(methylamino)-4oxobutanoyl)oxy)-2,4-dimethyloctadecanoate (46h) The titled compound 46h was obtained following the procedure described for 46a. Flash column chromatography (petroleum ether: ethyl acetate = 9:1); yield: 78%; colorless oil; [α]23D = − 45.8 (c = 2.3, CHCl3); 1H NMR (400 MHz, CDCl3) δ 6.00 – 5.74 (m, 2H), 5.31

51

ACCEPTED MANUSCRIPT

(d, J = 17.7 Hz, 1H), 5.23 (d, J = 10.1 Hz, 1H), 5.15 – 5.04 (m, 1H), 4.55 (s, 2H), 4.47 – 4.37 (m, 1H), 2.88 – 2.79 (m, 1H), 2.75 (d, J = 3.2 Hz, 3H), 2.69 – 2.57 (m, 1H), 1.78 – 1.65 (m, 1H), 1.40 (s, 9H), 1.22 (br, 26H), 1.13 (d, J = 6.5 Hz, 3H), 0.90 – 0.81 (m, 6H); 13

RI PT

C NMR (101 MHz, CDCl3) δ 174.2, 171.0, 170.5, 155.7, 132.1, 118.7, 79.8, 78.8, 65.6,

50.8, 41.9, 37.3, 34.4, 33.3, 32.0, 29.8, 29.8, 29.5, 28.4, 27.2, 26.3, 22.8, 14.2, 14.2, 13.7; νmax(neat): 3307, 2923, 2853, 1738, 1717, 1650, 1250, 1164, 557 cm-1; HRMS (MALDI)

SC

calcd for C33H60N2NaO7+ [M + Na] +: 619.4293, found 619.4298.

Methyl-2-((3S,11R,14S,15S,E)-11-(((tert-butyldiphenylsilyl)oxy)methyl)-15-((R)-

M AN U

hexadecan-2-yl)-7,14-dimethyl-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en3-yl)acetate (48a)

To a solution of obtained compound 46a (1.256 g, 2.10 mmol, 1 equiv) in anhydrous THF (21 mL), Pd(PPh3)4 (485 mg, 0.42 mmol, 0.2 equiv) and N-methylaniline (450 mg,

TE D

4.2 mmol, 2 equiv) were added. The reaction mixture was stirred for 1 h at room temperature, and diluted with ethyl acetate (200 mL). The organic phase was washed by 1% HCl (2 × 80 mL), dried over Na2SO4 and concentrated under reduced pressure. The

EP

residue was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 10: 1 to 1: 1) to afford the acid.

AC C

The obtained acid above and amine 34 (1.51 g, 3.15 mmol, 1.5 equiv) was dissolved in anhydrous CH2Cl2 (8.4 mL), HOBt (426 mg, 3.15 mmol, 1.5 equiv) and EDCI (805 mg, 4.20 mmol, 2 equiv) were added successively. The reaction mixture was stirred for 12 h and the solvent was removed. The residue was diluted with ethyl acetate (200 mL) and washed successively with 1% HCl, saturated aqueous NaHCO3, brine, dried over Na2SO4 and filtrated. The filtrate was concentrated under reduced pressure. The residue was

52

ACCEPTED MANUSCRIPT

purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 3:1) to obtain compound 47a (1.40 g, 65% for 2 steps) as a colorless oil. The compound 47a (1.40 g, 1.37 mmol, 1 equiv) and Pd(PPh3)4 (317 mg, 0.27 mmol, 0.2

RI PT

equiv) were dissolved in anhydrous THF (13.7 mL), and N-methylaniline (294 mg, 2.75 mmol, 2 equiv) was added. After stirred at room temperature for 1.5 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by column

SC

chromatography on silica gel (petroleum ether: ethyl acetate = 20: 1 to 1: 1) to afford the acid as pale yellow foam. The obtained acid was dissolved in CH2Cl2 (9 mL) and TFA

M AN U

(2.3 mL), the reaction mixture was stirred at room temperature for 3 h, and then the solvent was removed under reduced pressure to afford the crude amino acid. Then the above crude amino acid was dissolved in THF (20mL), the solution was added slowly to a suspension of HATU (6.50 g, 17.10 mmol, 15 equiv) and DIPEA(5.97 mL,

TE D

34.2 mmol, 30 equiv) in THF (1100 ml) over 8h at 40 ºC and then continued to stirred another 12h at 25 ºC. The solvent was removed under reduced pressure, diluted with MeOH /ethyl acetate (V/V = 1: 2, 300 mL), and filtered through a celite pad. The filtrate

EP

was concentrated under reduced pressure, and then the residue was dissolved in ethyl acetate (500 mL) and washed successively with 1% HCl, saturated aqueous NaHCO3,

AC C

brine, dried over Na2SO4. The solution was filtrated and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (CH2Cl2: MeOH = 100: 1 to 100: 4) to obtain the cyclic peptide 48a (715 mg, 59% for 3 steps) as a white powder. [α]22D = − 135.6 (c = 1.5, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.50 (d, J = 9.0 Hz, 1H), 7.98 (d, J = 8.9 Hz, 1H), 7.64 – 7.53 (m, 4H), 7.48 – 7.34 (m, 6H), 6.67 (dd, J = 14.9, 2.7 Hz, 1H), 5.97 (dd, J = 14.9, 1.8 Hz, 1H), 5.12 (d, J = 10.6 Hz, 1H),

53

ACCEPTED MANUSCRIPT

4.76 – 4.64 (m, 1H), 4.64 – 4.57 (m, 1H), 4.29 (d, J = 18.1 Hz, 1H), 3.81 (d, J = 18.1 Hz, 1H), 3.65 – 3.46 (m, 5H), 2.92 (s, 3H), 2.85 – 2.60 (m, 3H), 1.74 – 1.60 (m, 1H), 1.20 (br s, 26H), 0.98 (d, J = 7.0 Hz, 3H), 0.95 (s, 9H), 0.92 (d, J = 6.8 Hz, 3H), 0.82 (t, J = 6.7 13

C NMR (100 MHz, DMSO-d6) δ 173.0, 169.8, 169.1, 167.8, 165.6, 142.2,

RI PT

Hz, 3H);

135.1, 135.1, 132.8, 132.7, 130.0, 130.0, 127.9, 119.2, 77.4, 65.2, 52.1, 51.8, 51.2, 48.9, 41.5, 37.6, 36.5, 33.5, 33.3, 31.3, 29.2, 29.1, 29.1, 29.0, 28.7, 26.9, 26.6, 22.1, 18.8, 15.8,

SC

14.0, 13.1; νmax(neat): 3293, 2925, 2854, 1742, 1671, 1616, 1256, 1111, 702, 505 cm-1; HRMS (ESI) calcd for C49H75N3NaO8Si+ [M + Na] +: 884.5216, found 884.5220.

M AN U

(11R,14S,15S,E)-11-(((tert-Butyldiphenylsilyl)oxy)methyl)-15-((R)-hexadecan-2-yl)-7,14dimethyl-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (48b) The titled compound 48b was obtained following the procedure described for 48a. Flash column chromatography (CH2Cl2: MeOH = 100: 1 to 100: 4); yield: 48% for 5 steps;

TE D

white powder; [α]22D = − 145.7 (c = 1.3, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.53 – 8.38 (m, 1H), 8.01 (d, J = 8.8 Hz, 1H), 7.61 (t, J = 6.6 Hz, 4H), 7.50 – 7.36 (m, 6H), 6.70 (d, J = 14.9 Hz, 1H), 6.07 (d, J = 15.0 Hz, 1H), 4.62 (s, 1H), 4.27 (d, J = 18.1 Hz,

EP

1H), 3.95 (dd, J = 16.7, 7.6 Hz, 1H), 3.88 – 3.76 (m, 2H), 3.68 – 3.60 (m, 1H), 3.55 (t, J = 8.6 Hz, 1H), 2.91 (s, 3H), 2.85 – 2.74 (m, 1H), 1.69 (br, 1H), 1.22 (br, 26H), 1.03 –

AC C

0.95 (m, 12H), 0.89 (d, J = 6.5 Hz, 3H), 0.84 (t, J = 6.3 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 172.9, 168.1, 167.8, 165.6, 141.9, 135.1, 135.0, 132.8, 132.7, 129.9, 127.9, 119.8, 77.0, 65.2, 52.3, 51.3, 41.4, 40.8, 36.0, 33.6, 33.6, 31.3, 29.2, 29.0, 28.7, 26.8, 26.6, 22.1, 18.8, 15.7, 13.9, 13.1; νmax(neat): 3269, 2924, 2853, 1738, 1652, 1612, 1550, 1216, 1109, 702, 504cm-1; HRMS (MALDI) calcd for C46H71N3NaO6Si+ [M + Na] +: 812.5004, found 812.5008.

54

ACCEPTED MANUSCRIPT

(3S,11R,14S,15S,E)-11-(((tert-Butyldiphenylsilyl)oxy)methyl)-15-((R)-hexadecan-2-yl)3,7,14-trimethyl-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (48c) The titled compound 48c was obtained following the procedure described for 48a. Flash

RI PT

column chromatography (CH2Cl2: MeOH = 100: 1 to 100: 4); yield: 38% for 5 steps; white powder; [α]22D = − 142.3 (c = 0.8, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.50 (d, J = 8.9 Hz, 1H), 8.01 (d, J = 8.7 Hz, 1H), 7.58 (t, J = 6.4 Hz, 4H), 7.40 (dd, J = 11.7,

SC

6.6 Hz, 6H), 6.67 (dd, J = 15.0, 2.2 Hz, 1H), 6.04 (d, J = 14.9 Hz, 1H), 5.17 (d, J = 10.3 Hz, 1H), 4.69 – 4.54 (m, 1H), 4.47 – 4.29 (m, 2H), 4.10 (q, J = 5.2 Hz, 1H), 3.79 (d, J =

M AN U

17.9 Hz, 1H), 3.64 – 3.56 (m, 1H), 3.56 – 3.48 (m, 1H), 3.13 (d, J = 5.1 Hz, 4H), 2.91 (s, 3H), 2.86 – 2.77 (m, 1H), 1.71 – 1.60 (m, 1H), 1.30 – 1.11 (br, 29H), 1.00 – 0.93 (m, 12H), 0.89 (d, J = 6.6 Hz, 3H), 0.81 (t, J = 6.4 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 173.1, 170.6, 167.4, 165.4, 141.9, 135.1, 135.1, 132.8, 132.7, 129.9, 129.9, 127.9, 119.4,

TE D

76.2, 65.3, 52.1, 51.3, 48.6, 47.9, 41.5, 36.4, 33.6, 33.4, 31.3, 29.2, 29.0, 29.0, 29.0, 28.9, 28.7, 26.7, 26.6, 22.1, 19.4, 18.8, 15.6, 13.9, 13.0; νmax(neat): 3279, 2923, 2853, 1737, 1654, 1542, 1266, 1109, 701, 505 cm-1; HRMS (MALDI) calcd for C47H73N3NaO6Si+ [M

EP

+ Na] +: 826.5161, found 826.5164.

(3S,11R,14S,15S,E)-11-(((tert-Butyldiphenylsilyl)oxy)methyl)-15-((R)-hexadecan-2-yl)-3-

AC C

isopropyl-7,14-dimethyl-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (48d) The titled compound 48d was obtained following the procedure described for 48a. Flash column chromatography (CH2Cl2:MeOH = 100:1 to 100:4); yield: 38% for 5 steps; white powder; [α]21D = − 136.2 (c = 0.5, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.31 (d, J = 9.8 Hz, 1H), 8.00 (d, J = 8.8 Hz, 1H), 7.59 (t, J = 6.5 Hz, 4H), 7.49 – 7.33 (m, 6H), 6.68 (dd, J = 14.9, 2.5 Hz, 1H), 6.12 (d, J = 14.7 Hz, 1H), 5.21 (d, J = 10.4 Hz, 1H), 4.68

55

ACCEPTED MANUSCRIPT

– 4.56 (m, 1H), 4.50 (d, J = 17.8 Hz, 1H), 4.19 (dd, J = 9.5, 7.3 Hz, 1H), 3.82 (d, J = 17.7 Hz, 1H), 3.65 – 3.51 (m, 2H), 2.95 (s, 3H), 2.91 – 2.81 (m, 1H), 1.94 – 1.83 (m, 1H), 1.71 – 1.61 (m, 1H), 1.21 (br, 26H), 1.00 – 0.94 (m, 12H), 0.91 – 0.79 (m, 12H);

13

C NMR

RI PT

(100 MHz, DMSO-d6) δ 173.2, 168.9, 168.2, 165.3, 142.2, 135.1, 135.1, 132.8, 132.7, 130.0, 129.9, 127.9, 119.4, 76.5, 65.3, 57.5, 52.1, 51.3, 41.6, 36.6, 33.5, 33.4, 31.5, 31.3, 29.2, 29.0, 29.0, 28.9, 28.8, 28.7, 26.8, 26.6, 22.1, 19.3, 18.8, 18.4, 15.9, 13.9, 13.3;

SC

νmax(neat): 2920, 2851, 1713, 1689, 1650, 1275, 1111, 702, 505 cm-1; HRMS (MALDI) calcd for C49H77N3NaO6Si+ [M + Na] +: 854.5474, found 854.5478.

M AN U

(3S,11R,14S,15S,E)-3-Benzyl-11-(((tert-butyldiphenylsilyl)oxy)methyl)-15-((R)hexadecan-2-yl)-7,14-dimethyl-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (48e)

The titled compound 48e was obtained following the procedure described for 48a. Flash

TE D

column chromatography (CH2Cl2: MeOH = 100: 1 to 100: 4); yield: 37% for 5 steps; white powder; [α]22D = − 146.7 (c = 1.0, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 9.2 Hz, 1H), 7.90 (d, J = 8.6 Hz, 1H), 7.59 (t, J = 6.5 Hz, 4H), 7.48 – 7.34 (m, 6H),

EP

7.29 – 7.14 (m, 5H), 6.69 (dd, J = 14.9, 2.1 Hz, 1H), 5.99 (d, J = 14.8 Hz, 1H), 5.23 (d, J = 10.4 Hz, 1H), 4.69 – 4.52 (m, 2H), 4.30 (d, J = 18.0 Hz, 1H), 3.59 (td, J = 17.8, 11.5

AC C

Hz, 3H), 2.95 (dd, J = 13.5, 3.7 Hz, 1H), 2.89 – 2.72 (m, 5H), 1.75 – 1.59 (m, 1H), 1.18 (br, 26H), 1.00 (d, J = 6.9 Hz, 3H), 0.96 (s, 9H), 0.90 (d, J = 6.6 Hz, 3H), 0.81 (t, J = 6.5 Hz, 3H);

13

C NMR (100 MHz, DMSO-d6) δ 173.0, 169.4, 167.6, 165.3, 142.3, 136.8,

135.1, 135.0, 132.7, 132.6, 129.9, 129.9, 128.9, 128.2, 127.9, 126.6, 119.0, 76.6, 65.2, 53.6, 52.0, 51.3, 41.6, 36.4, 33.4, 31.3, 29.1, 29.0, 28.9, 28.7, 26.8, 26.5, 22.1, 18.8, 15.8,

56

ACCEPTED MANUSCRIPT

13.9, 13.0; νmax(neat): 3305, 2923, 2853, 1735, 1655, 1539, 1110, 700, 504 cm-1; HRMS (MALDI) calcd for C53H77N3NaO6Si+ [M + Na] +: 902.5474, found 902.5478. Methyl-3-((3S,11R,14S,15S,E)-11-(((tert-butyldiphenylsilyl)oxy)methyl)-15-((R)-

RI PT

hexadecan-2-yl)-7,14-dimethyl-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en3-yl)propanoate (48f)

The titled compound 48f was obtained following the procedure described for 48a. Flash

SC

column chromatography (CH2Cl2: MeOH = 100: 1 to 100: 4); yield: 47% for 5 steps; white powder; [α]22D = − 141.5 (c = 2.4, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.37

M AN U

(d, J = 9.3 Hz, 1H), 7.94 (d, J = 8.7 Hz, 1H), 7.60 (t, J = 6.7 Hz, 4H), 7.48 – 7.36 (m, 6H), 6.70 (d, J = 14.9 Hz, 1H), 6.00 (d, J = 14.9 Hz, 1H), 5.21 (d, J = 10.4 Hz, 1H), 4.68 – 4.57 (m, 1H), 4.41 – 4.32 (m, 1H), 4.27 (d, J = 17.9 Hz, 1H), 3.90 (d, J = 17.9 Hz, 1H), 3.68 – 3.51 (m, 5H), 2.94 (s, 3H), 2.84 – 2.72 (m, 1H), 2.46 – 2.29 (m, 2H), 1.99 – 1.87

TE D

(m, 1H), 1.87 – 1.74 (m, 1H), 1.73 – 1.63 (m, 1H), 1.22 (br, 26H), 1.00 (d, J = 6.9 Hz, 4H), 0.97 (s, 9H), 0.91 (d, J = 6.5 Hz, 3H), 0.83 (t, J = 6.4 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 173.0, 172.3, 169.6, 167.9, 165.4, 142.2, 135.1, 135.0, 132.8, 132.7, 129.9,

EP

129.9, 127.9, 119.1, 76.3, 65.2, 52.0, 51.4, 51.4, 51.2, 41.5, 36.4, 33.5, 33.4, 31.3, 29.6, 29.2, 29.0, 29.0, 28.9, 28.7, 28.4, 26.7, 26.5, 22.1, 18.8, 15.7, 13.9, 13.0; νmax(neat): 3284,

AC C

2923, 2853, 1738, 1652, 1542, 1247, 1108, 701, 505 cm-1; HRMS (MALDI) calcd for C50H77N3NaO8Si+ [M + Na] +: 898.5372, found 898.5375. 2-((3S,11R,14S,15S,E)-11-(((tert-Butyldiphenylsilyl)oxy)methyl)-15-((R)-hexadecan-2yl)-7,14-dimethyl-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3-yl)-Ncyclopropylacetamide (48g)

57

ACCEPTED MANUSCRIPT

The titled compound 48g was obtained following the procedure described for 48a. Flash column chromatography (CH2Cl2: MeOH = 100: 1 to 100: 4); yield: 36% for 5 steps; white powder; [α]22D = − 145.1 (c = 0.5, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.40

RI PT

(d, J = 9.1 Hz, 1H), 8.05 (d, J = 4.1 Hz, 1H), 7.98 (d, J = 8.9 Hz, 1H), 7.60 (t, J = 6.7 Hz, 4H), 7.48 – 7.34 (m, 6H), 6.67 (dd, J = 15.0, 2.6 Hz, 1H), 6.03 (d, J = 14.0 Hz, 1H), 5.13 (d, J = 10.4 Hz, 1H), 4.74 – 4.56 (m, 2H), 4.37 (d, J = 18.1 Hz, 1H), 3.73 (d, J = 18.0 Hz,

SC

1H), 3.63 – 3.57 (m, 1H), 3.56 – 3.48 (m, 1H), 2.92 (s, 3H), 2.87 – 2.76 (m, 1H), 2.61 – 2.53 (m, 1H), 2.37 (dd, J = 15.0, 4.4 Hz, 1H), 1.71 – 1.61 (m, 1H), 1.21 (br, 26H), 1.00 –

M AN U

0.90 (m, 15H), 0.82 (t, J = 6.6 Hz, 3H), 0.59 – 0.52 (m, 2H), 0.37 – 0.31 (m, 2H);

13

C

NMR (100 MHz, DMSO-d6) δ 173.0, 169.7, 169.1, 167.5, 165.5, 142.0, 135.1, 135.0, 132.8, 132.7, 129.9, 129.9, 127.9, 119.3, 76.8, 65.2, 54.9, 52.1, 51.2, 49.3, 41.6, 38.5, 36.4, 33.6, 33.3, 31.3, 29.2, 29.1, 28.7, 27.0, 26.5, 22.2, 22.1, 18.8, 15.8, 13.9, 13.1, 5.6,

TE D

5.4; νmax(neat): 3284, 2923, 2854, 1740, 1665, 1609, 1551, 1280, 1112, 701, 504 cm-1; HRMS (MALDI) calcd for C51H78N4NaO7Si+ [M + Na] +: 909.5532, found 909.5535. 2-((3S,11R,14S,15S,E)-11-(((tert-butyldiphenylsilyl)oxy)methyl)-15-((R)-hexadecan-2-

EP

yl)-7,14-dimethyl-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3-yl)-Nmethylacetamide (48h)

AC C

The titled compound 48h was obtained following the procedure described for 48a. Flash column chromatography (CH2Cl2: MeOH = 100: 1 to 100: 4); yield: 36% for 5 steps; white powder; [α]22D = − 139.6 (c = 0.3, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.31 (d, J = 8.9 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.90 – 7.85 (m, 1H), 7.61 (t, J = 6.7 Hz, 4H), 7.48 – 7.38 (m, 6H), 6.69 (dd, J = 14.9, 2.6 Hz, 1H), 6.01 (d, J = 14.8 Hz, 1H), 5.14 (d, J = 10.4 Hz, 1H), 4.74 – 4.58 (m, 2H), 4.34 (d, J = 18.1 Hz, 1H), 3.78 (d, J = 18.1 Hz, 1H),

58

ACCEPTED MANUSCRIPT

3.65 – 3.59 (m, 1H), 3.58 – 3.49 (m, 1H), 2.93 (s, 3H), 2.83 – 2.76 (m, 1H), 2.59 – 2.51 (m, 4H), 2.43 (dd, J = 15.1, 4.4 Hz, 1H), 1.73 – 1.62 (m, 1H), 1.22 (br, 26H), 1.02 – 0.92 (m, 15H), 0.84 (t, J = 6.6 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 173.0, 169.7, 168.5,

RI PT

167.5, 165.5, 142.0, 135.1, 135.1, 132.8, 132.7, 130.0, 129.9, 127.9, 119.3, 76.9, 65.2, 52.1, 51.1, 49.3, 41.6, 38.4, 36.4, 33.6, 33.2, 31.3, 29.2, 29.0, 28.7, 27.0, 26.6, 25.5, 22.1, 18.8, 15.8, 13.9, 13.1; νmax(neat): 3280, 2923, 2853, 1740, 1663, 1611, 1551, 1278, 1111,

SC

701, 504 cm-1; HRMS (MALDI) calcd for C49H76N4NaO7Si+ [M + Na] +: 883.5375, found 883.5378.

M AN U

Methyl-2-((3S,14S,15S,E)-15-((R)-hexadecan-2-yl)-7,14-dimethyl-11-methylene-2,5,8,13tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3-yl)acetate (32a) To a solution of compound 48a (186 mg, 0.22 mmol, 1 equiv) in THF (3 mL) were added HOAc (38 µL, 0.65 mmol) and TBAF (169 mg, 0.65 mmol). The mixture was stirred at

TE D

room temperature for 24 h, and then diluted with ethyl acetate (200 mL). The organic phase was washed with H2O (3 × 20 mL), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (CH2Cl2:MeOH = 100:3 to

EP

100:8) to obtain a white powder.

To a solution of obtained solid 33a (84 mg, 0.135mmol, 1 equiv) in THF (2.7 mL), then

AC C

triethylamine (47µL, 0.338mmol, 2.5 equiv) and ethanesulfonyl chloride (25µL, 0.270mmol, 2 equiv) were added at 0 ºC. After stirred for 30 min, the reaction solution was quenched by addition of water (0.1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting crude product was dissolved in THF (5.4 mL). To the resulting solution was added DBU (0.64g, 1.08 mmol, 8 equiv) at 20 ºC. After stirred for 2 h, the solution was dissolved in ethyl acetate/ THF (80 mL/ 20 mL) and

59

ACCEPTED MANUSCRIPT

washed successively with 1% HCl, saturated aqueous NaHCO3 (3 × 3 mL),brine, dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 100: 1 to 100: 6) to obtain

RI PT

32a (60 mg, 45% for 3 steps) as a white solid. [α]20D = − 45.6 (c = 0.3, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 8.93 (s, 1H), 8.48 (d, J = 8.6 Hz, 1H), 6.90 (d, J = 14.9 Hz, 1H), 6.16 (d, J = 15.3 Hz, 1H), 5.44 (s, 1H), 5.35 (s, 1H), 5.09 (d, J = 9.7 Hz, 1H), 4.80 – 4.65

SC

(m, 1H), 4.34 (d, J = 17.6 Hz, 1H), 3.85 (d, J = 17.7 Hz, 1H), 3.60 (s, 3H), 2.99 (s, 3H), 2.93 – 2.83 (m, 1H), 2.82 – 2.63 (m, 2H), 1.82 – 1.66 (m, 1H), 1.24 (br, 26H), 1.06 (d, J

M AN U

= 6.4 Hz, 3H), 0.95 (d, J = 6.1 Hz, 3H), 0.85 (d, J = 6.5 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 172.3, 169.8, 168.9, 167.9, 165.9, 138.7, 138.2, 118.7, 117.0, 77.8, 52.3, 51.7, 48.9, 41.8, 37.4, 36.5, 33.7, 33.1, 31.3, 29.2, 29.0, 28.9, 28.7, 26.8, 22.1, 15.2, 13.9, 13.1; νmax(neat): 3349, 2919, 2847, 1742, 1731, 1655, 1536, 1287, 1256, 962, 555 cm-1;

TE D

HRMS (MALDI) calcd for C33H55N3NaO7+ [M + Na] +: 628.3932, found 628.3937. (14S,15S,E)-15-((R)-Hexadecan-2-yl)-7,14-dimethyl-11-methylene-1-oxa-4,7,12triazacyclopentadec-9-ene-2,5,8,13-tetraone (32b)

EP

The titled compound 32b was obtained following the procedure described for 32a. Flash column chromatography (CH2Cl2: MeOH = 100: 1 to 100: 6); yield: 40% for 3 steps;

AC C

white powder; [α]20D = − 35.2 (c = 0.1, DMSO/ CHCl3=3:1); 1H NMR (400 MHz, DMSO-d6/ CDCl3=2:1) δ 8.68 (s, 1H), 8.44 – 8.33 (m, 1H), 6.91 (d, J = 15.1 Hz, 1H), 6.22 (d, J = 15.1 Hz, 1H), 5.45 (s, 1H), 5.35 (s, 1H), 5.08 (dd, J = 8.8, 3.3 Hz, 1H), 4.29 (d, J = 17.9 Hz, 1H), 4.02 (dd, J = 17.0, 7.3 Hz, 1H), 3.91 – 3.80 (m, 2H), 3.00 (s, 3H), 2.88 (p, J = 7.1 Hz, 1H), 1.76 – 1.65 (m, 1H), 1.23 (br, 26H), 1.10 (d, J = 7.0 Hz, 3H), 0.91 – 0.83 (m, 6H); 13C NMR (100 MHz, DMSO-d6/ CDCl3=2:1) δ 172.0, 167.9, 166.0,

60

ACCEPTED MANUSCRIPT

138.3, 137.8, 119.0, 115.9, 77.7, 52.6, 42.0, 40.7, 36.0, 34.0, 33.1, 31.2, 29.2, 29.0, 29.0, 28.7, 26.5, 22.0, 14.9, 13.8, 13.2; νmax(neat): 3330, 2917, 2848, 1732, 1675, 1657, 1639, 1484, 1341, 1284, 1220, 960, 559 cm-1; HRMS (MALDI) calcd for C30H51N3NaO5+ [M +

RI PT

Na] +: 556.3721, found 556.3725.

(3S,14S,15S,E)-15-((R)-Hexadecan-2-yl)-3,7,14-trimethyl-11-methylene-1-oxa-4,7,12triazacyclopentadec-9-ene-2,5,8,13-tetraone (32c)

SC

The titled compound 32c was obtained following the procedure described for 32a. Flash column chromatography (CH2Cl2: MeOH = 100: 1 to 100: 6); yield: 48% for 3 steps;

M AN U

white powder; [α]21D = − 32.9 (c = 0.1, DMSO: CHCl3=3:1); 1H NMR (400 MHz, DMSO-d6/ CDCl3=3:1) δ 8.96 (s, 1H), 8.46 (d, J = 8.9 Hz, 1H), 6.90 (d, J = 15.0 Hz, 1H), 6.27 (d, J = 15.0 Hz, 1H), 5.41 (s, 1H), 5.36 (s, 1H), 5.15 (d, J = 10.1 Hz, 1H), 4.53 – 4.44 (m, 1H), 4.41 (d, J = 17.6 Hz, 1H), 3.77 (d, J = 17.6 Hz, 1H), 3.02 (s, 3H), 2.96 –

TE D

2.86 (m, 1H), 1.78 – 1.68 (m, 1H), 1.31 (d, J = 7.1 Hz, 3H), 1.24 (s, 26H), 1.08 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 6.7 Hz, 3H), 0.86 (t, J = 6.7 Hz, 3H);

13

C NMR (101 MHz,

DMSO-d6/ CDCl3=3:1) δ 172.3, 169.7, 168.9, 167.9, 165.9, 138.7, 138.2, 118.7, 117.0,

EP

77.8, 52.3, 51.7, 48.9, 41.8, 38.5, 37.4, 36.5, 33.7, 33.1, 29.3, 29.2, 29.1, 29.0, 28.9, 27.4, 26.8, 22.5, 15.2, 13.1; νmax(neat): 3343, 2919, 2847, 1734, 1659, 1612, 1546, 1278, 1046,

AC C

560 cm-1; HRMS (MALDI) calcd for C31H53N3NaO5+ [M + Na] +: 570.3877, found 570.3880.

(3S,14S,15S,E)-15-((R)-Hexadecan-2-yl)-3-isopropyl-7,14-dimethyl-11-methylene-1-oxa4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (32d) The titled compound 32d was obtained following the procedure described for 32a. Flash column chromatography (CH2Cl2: MeOH = 100:1 to 100:6); yield: 45% for 3 steps; white

61

ACCEPTED MANUSCRIPT

powder; [α]22D = − 41.8 (c = 0.1, DMSO: CHCl3=3:1); 1H NMR (400 MHz, DMSO-d6: CDCl3=2:1) δ 8.77 (s, 1H), 8.06 (d, J = 9.8 Hz, 1H), 6.91 (d, J = 15.0 Hz, 1H), 6.29 (d, J = 15.0 Hz, 1H), 5.44 (s, 1H), 5.34 (s, 1H), 5.21 (d, J = 10.5 Hz, 1H), 4.51 (d, J = 17.5 Hz,

RI PT

1H), 4.26 (dd, J = 9.6, 7.4 Hz, 1H), 3.75 (d, J = 17.4 Hz, 1H), 3.06 (s, 3H), 2.96 – 2.85 (m, 1H), 2.00 – 1.88 (m, 1H), 1.79 – 1.68 (m, 1H), 1.24 (s, 26H), 1.10 (d, J = 7.0 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H), 0.90 (dd, J = 6.6, 1.3 Hz, 6H), 0.86 (t, J = 6.7 Hz, 3H);

13

C

SC

NMR (100 MHz, DMSO-d6: CDCl3=2:1) δ 172. 5, 168.4, 167.9, 165.6, 138.7, 137.9, 118.2, 116.2, 76.8, 57.6, 52.3, 41.9, 36.6, 33.4, 33.3, 31.4, 31.2, 29.1, 29.0, 28.9, 28.8,

M AN U

28.6, 26.8, 22.0, 19.1, 18.2, 15.1, 13.7, 13.0; νmax(neat): 3273, 2918, 2851, 1723, 1665, 1649, 1614, 1538, 1467, 1264, 983, 555 cm-1; HRMS (MALDI) calcd for C33H57N3NaO5+ [M + Na] +: 598.4190, found 598.4194.

(3S,14S,15S,E)-3-Benzyl-15-((R)-hexadecan-2-yl)-7,14-dimethyl-11-methylene-1-oxa-

TE D

4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (32e)

The titled compound 32e was obtained following the procedure described for 32a. Flash column chromatography (CH2Cl2: MeOH = 100: 1 to 100: 6); yield: 46% for 3 steps;

EP

white powder; [α]20D = − 32.7 (c = 0.1, DMSO: CHCl3=3:1); 1H NMR (400 MHz, DMSO-d6: CDCl3=3:1) δ 8.90 (s, 1H), 8.43 (d, J = 8.9 Hz, 1H), 7.27 – 7.16 (m, 5H), 6.90

AC C

(d, J = 14.9 Hz, 1H), 6.17 (d, J = 14.8 Hz, 1H), 5.36 (d, J = 4.9 Hz, 2H), 5.16 (d, J = 10.0 Hz, 1H), 4.67 – 4.57 (m, 1H), 4.36 (d, J = 17.5 Hz, 1H), 3.58 (d, J = 17.5 Hz, 1H), 3.00 – 2.95 (m, 1H), 2.94 (s, 3H), 2.91 – 2.84 (m, 2H), 1.75 – 1.64 (m, 1H), 1.21 (br, 26H), 1.07 (d, J = 6.6 Hz, 3H), 0.92 (d, J = 6.3 Hz, 3H), 0.86 – 0.81 (m, 3H); 13C NMR (100 MHz, DMSO-d6: CDCl3=3:1) δ 172.4, 169.1, 167.6, 165.5, 138.7, 138.3, 136.6, 128.8, 128.0, 126.4, 118.4, 116.8, 77.0, 53.5, 52.1, 41.7, 38.7, 36.5, 33.5, 33.3, 31.3, 29.1, 29.0, 29.0,

62

ACCEPTED MANUSCRIPT

28.9, 28.7, 26.8, 22.1, 15.1, 13.8, 12.9; νmax(neat): 3372, 2921, 2851, 1716, 1691, 1675, 1648, 1531, 1289, 1253, 975, 696, 551 cm-1; HRMS (MALDI) calcd for C37H57N3NaO5+ [M + Na] +: 646.4190, found 646.4193.

RI PT

Methyl-3-((3S,14S,15S,E)-15-((R)-hexadecan-2-yl)-7,14-dimethyl-11-methylene-2,5,8,13tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3-yl)propanoate (32f)

The titled compound 32f was obtained following the procedure described for 32a. Flash

SC

column chromatography (CH2Cl2: MeOH = 100: 1 to 100: 6); yield: 44% for 3 steps; white powder; [α]24D = − 43.8 (c = 0.1, DMSO: CHCl3=3:1); 1H NMR (400 MHz,

M AN U

DMSO-d6: CDCl3=3:1) δ 8.86 (s, 1H), 8.27 (d, J = 9.3 Hz, 1H), 6.89 (d, J = 15.1 Hz, 1H), 6.21 (d, J = 15.1 Hz, 1H), 5.40 (s, 1H), 5.36 (s, 1H), 5.16 (d, J = 10.0 Hz, 1H), 4.47 – 4.40 (m, 1H), 4.36 (d, J = 17.9 Hz, 1H), 3.84 (d, J = 17.6 Hz, 1H), 3.59 (s, 3H), 3.02 (s, 3H), 2.91 – 2.83 (m, 1H), 2.38 – 2.31 (m, 2H), 1.98 – 1.93 (m, 1H), 1.86 – 1.80 (m, 1H),

TE D

1.74 – 1.69 (m, 1H), 1.23 (br s, 26H), 1.07 (d, J = 6.8 Hz, 3H), 0.94 (d, J = 6.4 Hz, 3H), 0.85 (t, J = 6.2 Hz, 3H);

13

C NMR (100 MHz, DMSO-d6: CDCl3=3:1) δ 172.4, 172.1,

169.2, 167.9, 165.6, 138.6, 138.0, 118.3, 116.5, 76.8, 52.2, 51.3, 51.2, 41.7, 36.5, 33.5,

EP

33.4, 31.3, 29.6, 29.2, 29.0, 29.0, 28.9, 28.7, 28.4, 26.7, 22.1, 14.9, 13.8, 12.8; νmax(neat): 3371, 2922, 2851, 1742, 1695, 1671, 1652, 1287, 1254, 975, 551 cm-1; HRMS (MALDI)

AC C

calcd for C34H57N3NaO7+ [M + Na] +: 642.4089, found 642.4095. N-cyclopropyl-2-((3S,14S,15S,E)-15-((R)-hexadecan-2-yl)-7,14-dimethyl-11-methylene2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3-yl)acetamide (32g) The titled compound 32g was obtained following the procedure described for 32g. Flash column chromatography (CH2Cl2: MeOH = 100: 1 to 100: 6); yield: 43% for 3 steps; white powder; [α]18D = − 41.5 (c = 0.1, DMSO: CHCl3=3:1); 1H NMR (400 MHz,

63

ACCEPTED MANUSCRIPT

DMSO-d6: CDCl3=3:1) δ 8.83 (s, 1H), 8.21 (d, J = 8.2 Hz, 1H), 7.92 (d, J = 4.0 Hz, 1H), 6.84 (d, J = 15.0 Hz, 1H), 6.15 (d, J = 15.0 Hz, 1H), 5.35 (s, 1H), 5.32 (s, 1H), 5.04 (d, J = 9.8 Hz, 1H), 4.69 – 4.60 (m, 1H), 4.35 (d, J = 17.8 Hz, 1H), 3.72 (d, J = 17.8 Hz, 1H),

RI PT

2.96 (s, 3H), 2.90 – 2.80 (m, 1H), 2.57 – 2.51 (m, 1H), 2.50 – 2.35 (m, 2H), 1.72 – 1.62 (m, 1H), 1.19 (br, 26H), 1.02 (d, J = 6.9 Hz, 3H), 0.92 (d, J = 6.7 Hz, 3H), 0.81 (t, J = 6.7 Hz, 3H), 0.57 – 0.50 (m, 2H), 0.36 – 0.28 (m, 2H);

13

C NMR (100 MHz, DMSO-d6:

SC

CDCl3=3:1) δ 172.5, 169.4, 169.2, 167.6, 165.9, 138.5, 138.1, 118.7, 116.5, 77.5, 52.4, 49.3, 41.9, 38.4, 36.5, 33.8, 33.1, 31.3, 31.1, 29.8, 29.2, 29.0, 29.0, 28.7, 26.9, 22.1, 22.1,

M AN U

15.2, 13.9, 13.1, 5.6, 5.4; νmax(neat): 3305, 2921, 2852, 1716, 1688, 1650, 1614, 1537, 1279, 1252, 897, 548 cm-1; HRMS (MALDI) calcd for C35H58N4NaO6+ [M + Na] +: 653.4249, found 653.4250.

2-((3S,14S,15S,E)-15-((R)-hexadecan-2-yl)-7,14-dimethyl-11-methylene-2,5,8,13-

TE D

tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3-yl)-N-methylacetamide (32h) The titled compound 32h was obtained following the procedure described for 32a. Flash column chromatography (CH2Cl2: MeOH = 100: 1 to 100: 6); yield: 49% for 3 steps;

EP

white powder; [α]18D = − 48.6 (c = 0.2, MeOH: CHCl3=4:1); 1H NMR (400 MHz, MeOD-d4: CDCl3=4:1) δ 7.16 (d, J = 15.0 Hz, 1H), 6.30 (d, J = 15.1 Hz, 1H), 5.62 (s,

AC C

1H), 5.57 (s, 1H), 5.35 (d, J = 9.5 Hz, 1H), 4.99 – 4.93 (m, 1H), 4.54 (d, J = 17.7 Hz, 1H), 3.97 (d, J = 17.8 Hz, 1H), 3.22 (s, 3H), 3.07 – 2.96 (m, 1H), 2.87 (dd, J = 15.6, 6.4 Hz, 1H), 2.78 (s, 3H), 2.74 (dd, J = 15.6, 4.3 Hz, 1H), 1.99 – 1.84 (m, 1H), 1.36 (br, 26H), 1.30 (d, J = 6.9 Hz, 3H), 1.12 (d, J = 6.7 Hz, 3H), 0.97 (t, J = 6.6 Hz, 3H); 13C NMR (100 MHz, MeOD-d4: CDCl3=4:1) δ 172.5, 169.4, 169.2, 167.6, 165.9, 138.5, 138.1, 118.7, 116.5, 77.5, 52.4, 49.3, 41.9, 38.4, 36.5, 33.8, 33.1, 31.3, 31.1, 29.8, 29.2, 29.1, 29.0, 28.7,

64

ACCEPTED MANUSCRIPT

26.9, 22.1, 22.1, 15.2, 13.9, 13.1, 5.6, 5.4; νmax(neat): 3251, 2920, 2851, 1743, 1679, 1639, 1611, 1544, 1259, 1238, 990, 693, 530 cm-1; HRMS (MALDI) calcd for C33H56N4NaO6+ [M + Na] +: 627.4092, found 627.4098.

methyl-(tert-butoxycarbonyl)-L-aspartate (51)

RI PT

1-((2S,3S,4R)-1-((2-Hydroxyethyl)amino)-2,4,16-trimethyl-1-oxoheptadecan-3-yl)-4-

To a solution of 22a (1.23 g, 1.88 mmol, 1 equiv) in anhydrous THF (18.8 mL),

SC

Pd(PPh3)4 (435 mg, 0.38 mmol, 0.2 equiv) and N-methylaniline (407µL, 3.76 mmol, 2 equiv) were added. The reaction mixture was stirred for 1 h at room temperature, and

M AN U

diluted with ethyl acetate (150 mL). The organic phase was washed by 1% HCl (2 × 50 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was purified bycolumn chromatography on silica gel (petroleum ether: ethyl acetate = 20: 1 to 1: 1) to afford the acid (1.01 g, 89%).

TE D

To a solution of above acid (1.32 g, 2.37 mmol, 1 equiv) and 2-Aminoethanol(217 mg, 3.55 mmol, 1.5 equiv) in CH2Cl2 (20 mL) was added EDCI (0.90 g, 4.73 mmol,2 equiv) and HOBt (480 mg, 3.55 mmol, 1.5 equiv)at 0 ºC. The reaction mixture was warmed to

EP

room temperature and stirred for 4 h, and then quenched with 1% HCl (100 mL). The aqueous phase was extracted with CH2Cl2 (3 × 120 mL). The combined organic phases

AC C

were dried (Na2SO4) and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 3:1) to obtain 51 (1.15 g, 65%) as a colorless oil. [α]24D = − 321.5 (c = 0.5, CHCl3); 1H NMR (400 MHz, MeOD-d4) δ 7.94 (t, J = 5.5 Hz, 1H), 6.98 (d, J = 8.8 Hz, 1H), 5.10 (dd, J = 10.0, 1.5 Hz, 1H), 4.56 – 4.38 (m, 1H), 3.63 (s, 3H), 3.54 (t, J = 5.7 Hz, 2H), 3.25 – 3.15 (m, 2H), 2.87 – 2.71 (m, 1H), 2.70 – 2.54 (m, 2H), 1.77 – 1.66 (m, 1H), 1.53 – 1.43 (m,

65

ACCEPTED MANUSCRIPT

1H), 1.24 (br s, 20H), 1.14 – 1.09 (m, 2H), 1.03 (d, J = 6.9 Hz, 3H), 0.87 (d, J = 6.8 Hz, 3H), 0.83 (d, J = 6.6 Hz, 6H);

13

C NMR (100 MHz, MeOD-d4) δ 177.0, 172.5, 171.9,

157.7, 80.6, 79.2, 61.5, 52.4, 51.6, 44.1, 43.2, 40.3, 36.7, 35.0, 34.9, 31.1, 30.9, 30.9, 30.8,

RI PT

30.7, 29.2, 28.8, 28.6, 28.4, 23.1, 14.6, 13.4; νmax(neat): 3307, 2924, 2853, 1721, 1651, 1366, 1163, 543 cm-1; HRMS (MALDI) calcd for C32H60N2NaO8+ [M + Na] +: 623.4242, found 623.4245.

SC

Methyl-2-((3S,14S,15S,E)-7,14-dimethyl-15-((R)-14-methylpentadecan-2-yl)-2,5,8,13tetraoxo-1-oxa-4,7,12-triazacyclopentadec-9-en-3-yl)acetate (49)

M AN U

To a solution of 51 (1.00 g, 1.67 mmol, 1 equiv) in CH2Cl2 (20 mL) was added DessMartin periodinane (920 mg，2.17 mmol, 1.3 equiv) at 20 ºC. After stirred for 1 h, the reaction mixture was quenched by addition of saturated aqueous solution of NaHCO3 (40 mL) and saturated aqueous Na2S2O3 (20 mL). The reaction mixture was stirred for 10

TE D

min, and extracted with CH2Cl2 (3 × 100 mL). The combined organic phases were dried (Na2SO4) and concentrated under reduced pressure. The residue was dissolved in 15% ethyl acetate in hexane (50 mL), and the resulting mixture was filtered through a short

EP

pad of silica gel. The solvent was removed under reduced pressure to afford the aldehyde as a colorless oil, which can used for the next step directly.

AC C

To a solution of aldehyde in MeCN (3 mL) was added anhydrous LiCl (142 mg, 3.34 mmol, 2 equiv) and DBU (300 uL, 2.00 mmol, 1.2 equiv) at 0 ºC and stirred for 10min. Solution of 3e (0.65 g, 2.00 mmol, 1.2 equiv) in CH2Cl2 (15 mL) was then added to the reaction mixture. And the resulting solution was stirred for 40 min. Water (100 mL) was added to quench the reaction. And the aqueous phase was extracted with CH2Cl2 (3 × 100 mL). The combined organic phases were dried (Na2SO4) and concentrated under reduced

66

ACCEPTED MANUSCRIPT

pressure. The crude product was purified by column chromatography on silica gel (ethyl acetate: hexane = 1:10) to obtain 52 (874 mg, 70%) as a colorless oil. To a solution of 52 (0.87g, 1.1 mmol, 1 equiv) in DCM (12.5 mL), Et3SiH (1.06 mL, 6.6

RI PT

mmol, 6 equiv) was added at 0 ºC, and TFA (7.5 mL) was dropwise added into the reaction mixture. It allowed to warm at 20 ºC for 4h. Then toluene (7.5 mL) was added and the solvent was removed by reduced pressure. The residue was purified by flash

SC

chromatography to obtain the crude amino acid as a yellow solid. Then amino acid was dissolved in THF (20 mL), the solution was added slowly to a suspension of HATU (6.27

M AN U

g, 16.50 mmol, 15 equiv) and DIPEA(5.76 ml, 33 mmol, 30 equiv) in THF (1100 ml) over 8h at 37℃ and then continued to stirred another 12h at 20℃. The solvent was removed under reduced pressure, diluted with MeOH: ethyl acetate (V/V = 2:1, 300 mL), and filtered through a celite pad. The filtrate was concentrated under reduced pressure.

TE D

The residue was dissolved in ethyl acetate (500 mL), washed with 1% NaHSO4 (80 mL), saturated aqueous solution of NaHCO3 (60 mL), and brine (60 mL). The organic phase was dried over Na2SO4, concentrated under reduced pressure. The crude product was

EP

purified by column chromatography on silica gel (MeOH: CH2Cl2 = 1:30) to obtain the 49 (0.44 g, 65% for 2 steps) as a white solid. [α]26D = − 25.4 (c = 0.1, DMSO); 1H NMR

AC C

(400 MHz, DMSO) δ 8.51 (d, J = 8.9 Hz, 1H), 8.03 (dd, J = 7.8, 3.3 Hz, 1H), 6.62 (d, J = 14.9 Hz, 1H), 5.89 (d, J = 15.0 Hz, 1H), 5.13 (d, J = 10.2 Hz, 1H), 4.77 – 4.66 (m, 1H), 4.30 (d, J = 18.0 Hz, 1H), 4.18 (dd, J = 18.9, 8.3 Hz, 1H), 3.79 (d, J = 18.0 Hz, 1H), 3.60 (s, 3H), 3.44 – 3.35 (m, 1H), 2.94 (s, 3H), 2.80 – 2.66 (m, 3H), 1.79 – 1.65 (m, 1H), 1.49 (td, J = 13.3, 6.7 Hz, 1H), 1.24 (br s, 20H), 1.16 – 1.09 (m, 2H), 1.01 (d, J = 7.0 Hz, 3H), 0.92 (d, J = 6.7 Hz, 3H), 0.84 (d, J = 6.6 Hz, 6H); 13C NMR (101 MHz, DMSO) δ 173.1,

67

ACCEPTED MANUSCRIPT

169.7, 169.0, 167.9, 165.8, 142.5, 117.6, 77.3, 52.1, 51.8, 49.1, 41.5, 38.5, 37.5, 36.3, 33.6, 33.3, 29.3, 29.2, 29.1, 29.1, 29.0, 27.4, 26.9, 26.8, 22.6, 15.8, 13.2; νmax(neat): 3424,

C32H55N3NaO7+ [M + Na] +: 616.3932, found 616.3938. 4.2 Biological assay 4.2.1 Experimental Procedure for the Cytotoxicity Assay

RI PT

2924, 2853, 1734, 1671, 1656, 1541, 1267, 834, 557 cm-1; HRMS (MALDI) calcd for

SC

The human pancreatic cancer cell line PANC1, PATU8988T and ASPC-1 cells were purchased from American Type Culture Collection (ATCC, Rockville, MD). Cells were

M AN U

cultured in DMEM medium supplement with 10% fetal bovine serum at 37 °C in a 5% CO2 incubator.

As to MTT assay, PANC1, PATU8988T and ASPC-1 cells were seeded into 96-well plates respectively (4000 cells/well). After 6 hours, various concentrations of compounds

TE D

were added. Normoxic condition was obtained by incubation at 37 °C, 5% CO2. Hypoxic conditions were obtained by placing the 96-well plates in a modular incubator chamber (Billups-Rothenberg) and purging the chamber with 5% CO2, 1% O2 and 94% N2 gas

EP

mixture for 10 min. Then sealing the chamber subsequently and placing it in 37 °C incubator. After 72 hours, 20 µL MTT solution (5 mg/mL) was added into 96-well plates

AC C

and then incubated in 37 °C, 5% CO2 incubator for additional 4 hours. The supernatant was discarded and the formazan crystal was dissolved by DMSO to determine the optical density values at 570 nm. The IC50 were calculated by Graph Pad Prism 5. Each experiment was repeated for three times. 4.2.2 Flow cytometry analysis

68

ACCEPTED MANUSCRIPT

The PANC-1 cells were seeded in 24-well plates (5×104 cells/well/1 mL). After 6 hours, cells were treated with different concentrations of candidate compounds. After being treated for 48 hours, cells were collected and washed with cold PBS for three times. Then

RI PT

the cells were incubated with the directly conjugated antibodies anti-human CD44-APC, anti-human CD24-PE and anti-human ESA-FITC for 30 mins. Furthermore, the cells were washed with PBS and flow cytometry analysis was performed in 1 hour.

SC

4.2.3 ALDH activity analysis

Aldefluor assay was conducted by using the Aldefluor Assay Kit (STEMCELL

M AN U

Technologies). The PANC-1 cells were seeded in 24-well plates (5×104 cells/well), and medium volume was 1 mL. 6 hours later, cells were treated with various concentrations of compound 32g. After 72 hours, the living PANC-1 cells were collected and suspended with 500 µL assay buffer. Then 2.5 µL Aldefluor substrate was added and incubated for

TE D

60 min at 37 ˚C. The cells were treated with 2.5 µL Aldefluor substrate and 5 µL ALDH inhibitor diethylamino-benzaldehyde (DEAB) which was used as a negative control. ALDH assay was conducted following the manufacturer's instructions.

EP

4.2.4 Tumorsphere formation assay

Single-cell PANC-1 cell suspensions were plated into ultra-low attachment 6-well plates

AC C

(Corning, NY, USA) at a density of 2,000 cells/mL in Complete MammoCult Medium (Stem Cell Technologies). After 7 days, the number of tumorspheres was counted. Each experiment was repeated for three times. 4.2.5 Subcutaneous xenograft mouse model Barb/c nude mice assay PANC-1 cells were treated with compounds for 48 hours. The cells were collected and injected subcutaneously into 5-week-old female BALB/c nude mice (Chinese Academy

69

ACCEPTED MANUSCRIPT

of Sciences, Shanghai, China). The tumor formation rate was calculated after 7 days. All animal experiments were performed in accordance with guidelines approved by the ethics

4.2.6 Inhibitory activity in xenograft zebrafish model

RI PT

committee of Nankai University.

PANC-1 cells were transplanted into the zebrafish yolk of 2 dpf wild-type AB strain by microinjection. About 300 cells were transplanted into the zebrafish to establish the

SC

tumor transplantation model. Zebrafish injected with PANC-1 cells were placed at 35 °C to 3 dpf. The zebrafish were randomly assigned to 6-well plates and were administered

M AN U

with 32b and 32g. The positive control group was added 66.7 µM gemcitabine. The blank control group was added 10% aqueous castor oil solution. Every group had 30 zebrafish per well and administrated one time during experiment. The zebrafish were incubated at 35 °C for 2 days, and 15 zebrafish were randomly selected to observe, photographed, and

TE D

preserved in a fluorescence microscope. The images were collected using Nikon NISElements D 3.10 image analysis software to calculate the fluorescence intensity of cancer cells, respectively, to evaluate the fluorescence intensity. The cancer inhibition calculated

EP

as following formulation: Inhibition (%) = (1-S(sample)/S(blank control))× 100%. All animal experiments were performed in accordance with guidelines approved by the ethics

AC C

committee of Nankai University.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (NO. 81573282 to Y.C., NO. 81573308 to Q.Z.), The National Science Fund for Distinguished Young Scholars (NO. 81625021) to Y.C., Natural Science Foundation of Tianjin (NO. 70

ACCEPTED MANUSCRIPT

17JCQNJC13400) to Q.Z., Hundred Young Academic Leaders Program of Nankai University to Y.C., and the Ph.D. Candidate Research Innovation Fund of Nankai

RI PT

University to J.C..

Supplementary data

References

M AN U

[1] A. C. Society, Cancer Facts & Figures 2015.

SC

Copies of the NMR spectra of all new compounds.

[2] American Cancer Society Pancreatic Cancer overview. https://www.cancer.org/cancer/pancreatic-cancer.

[3] C. Hajj, K. A. Goodman, Pancreatic cancer and SBRT: A new potential option? Rep.

TE D

Pract. Oncol. Radiother. 20 (2015) 377–384.

[4] T. L. Fitzgerald, J. A. Mccubrey, Pancreatic cancer stem cells: Association with cell surface markers, prognosis, resistance, metastasis and treatment. Adv. Biol. Regul. 56

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(2014) 45–50.

[5] C. Li, D. G. Heidt, P. Dalerba, C. F. Burant, L. Zhang, V. Adsay, M. Wicha, M. F.

AC C

Clarke, D. M. Simeone, Identification of pancreatic cancer stem cells. Cancer Res. 130 (2007) 194–195.

[6] P. Wang, J. Zhang, L. Zhang, Z. Zhu, J. Fan, L. Chen, L. Zhuang, J. Luo, H. Chen. L. Liu, Z. Chen, Z. Meng, MicroRNA 23b regulates autophagy associated with radio resistance of pancreatic cancer cells. Gastroenterology 145 (2013) 1133–1143.

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[7] M. Maugeri-Saccà, P. Vigneri, R. De Maria, Cancer stem cells and chemosensitivity. Clin. Cancer Res. 17 (2011) 4942–4947. [8] J. A. Castellanos, N. B. Merchant, N. S. Nagathihalli, Emerging targets in pancreatic

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cancer: epithelial–mesenchymal transition and cancer stem cells. Onco. Targets Ther. 6 (2013) 1261–1267.

[9] J. Xia, C. Chen, Z. Chen, L. Miele, F. H. Sarkar, Z. Wang, Targeting pancreatic

SC

cancer stem cells for cancer therapy, Biochim. Biophys. Acta. 1826 (2012) 385–399.

[10] K. D. Mcbrien, R. L. Berry, S. E. Lowe, K. M. Neddermann, I. Bursuker, S. Huang,

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S. R. Klohr, J. E. Leet, Rakicidins, new cytotoxic lipopeptides from Micromonospora sp. fermentation, isolation nd characterization. J. Antibiot. 48 (1995) 1446–1452. [11] Y. Igarashi, R. Shimasaki, S. Miyanaga, N. Oku, H. Onaka, H. Sakurai, I. Saiki, S. Kitani, T. Nihira, W. Wimonsiravude, W. Panbangred, Rakicidin D, an inhibitor of tumor

TE D

cell invasion from marine-derived Streptomyces sp. J. Antibiot. 63 (2010) 563–565. [12] J. Hu, D. Wunderlich, I. Sattler, X. Feng, S. Grabley, R. Thiericke, Rakicidin C, a new cyclic depsipeptide from Streptomyces sp. Eur. J. Org. Chem. 19 (2000) 3353–3356.

EP

[13] S. Kitani, T. Ueguchi, Y. Igarashi, K. Leetanasaksakul, A. Thamchaipenet, T. Nihira, Rakicidin F, a new antibacterial cyclic depsipeptide from a marine sponge-derived

AC C

Streptomyces sp. J. Antibiot. 2017, DOI: 10.1038/ja.2017.92. [14] Y. Yamazaki, S. Kunimoto, D. Ikeda, Rakicidin A: A hypoxia-selective cytotoxin. Biol. Pharm. Bull. 30 (2007) 261–265. [15] M. Takeuchi, E. Ashihara, Y. Yamazaki, S. Kimura, Y. Nakagawa, R. Tanaka, H. Yao, R. Nagao, Y. Hayashi, H. Hirai, Maekawa, T. Rakicidin A effectively induces

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apoptosis in hypoxia adapted Bcr-Abl positive leukemic cells. Cancer Sci. 102 (2011) 591−596. [16] T. B. Poulsen, A concise route to the macrocyclic core of the rakicidins. Chem.

RI PT

Commun. 47 (2011) 12837–12839.

[17] L. L. Clement, M. Tsakos, E. S. Schaffert, C. Scavenius, J. J. Enghild, T. B. Poulsen, The amido-pentadienoate-functionality of the rakicidins is a thiol reactive electrophile-

SC

development of a general synthetic strategy. Chem. Commun. 51 (2015) 12427–12430.

[18] F. Sang, D. Li, X. Sun, X. Cao, L. Wang, J. Sun, B. Sun, L. Wu, G. Yang, X. Chu, J.

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Wang, C. Dong, Y. Geng, H. Jiang, H. Long, S. Chen, G. Wang, S. Zhang, Q. Zhang, Y. Chen, Total synthesis and determination of the absolute configuration of rakicidin A. J. Am. Chem. Soc. 136 (2014) 15787–15791.

[19] F. Sang, Y. Ding, J. Wang, B. Sun, J. Sun, Y. Geng, Z. Zhang, K. Ding, L.-L. Wu,

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J.-W. Liu, C. Ba, G. Yang, Q. Zhang, L.-Y. Li, Y. Chen, Structure-activity relationship study of rakicidins: Overcoming chronic myeloid leukemia resistance to imatinib with 4methylester-rakicidin A. J. Med. Chem. 59 (2016) 1184–1196.

EP

[20] M. Tsakos, L. L. Clement, E. S. Schaffert, F. N. Olsen, S. Rupiani, R. Djurhuus, W. Yu, K. M. Jacobsen, N. L. Villadsen, T. B. Poulsen, Total synthesis and biological

AC C

evaluation of rakicidin A and discovery of a simplified bioactive analogue. Angew. Chem., Int. Ed. 55 (2016) 030–1035. [21] N. Oku, S. Matoba, Y. M. Yamazaki, R. Shimasaki, S. Miyanaga, Y. Igarashi, Complete stereochemistry and preliminary structure-activity relationship of rakicidin A, a hypoxia-selective cytotoxin from Micromonospora sp. J. Nat. Prod. 77 (2014) 2561–2565.

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RI PT

[23] Z. Yang, G. Yang, M. Ma, J. Li, J. Liu, J. Wang, S. Jiang, Q. Zhang, Y. Chen, Total synthesis and determination of the absolute configuration of vinylamycin. Org. Lett. 17 (2015) 5725−5727.

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[24] G. Carr, M. Poulsen, J. L. Klassen, Y. Hou, T. P. Wyche, T. S. Bugni, C. R. Currie, J.

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Clardy, Microtermolides A and B from termite-associated Streptomyces sp. and structural revision of vinylamycin. Org. Lett. 14 (2012) 2822−2825.

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[26] N. L. Villadsen, K. M. Jacobsen, U. B.Keiding, E. T. Weibel, B. Christiansen, T. Vosegaard, M. Bjerring, F. Jensen, M. Johannsen, T. Tørring, T. B. Poulsen, Synthesis of ent-BE-43547A1 reveals a potent hypoxia-selective anticancer agent and uncovers the

EP

biosynthetic origin of the APD-CLD natural products. Nat. Chem. 9 (2017) 264−272.

AC C

[27] Y. Sun, Y. Ding, D. Li, R. Zhou, X. Su, J. Yang, X. Guo, C. Chong, J. Wang, W. Zhang, C. Bai, L. Wang, Y. Chen, Cyclic depsipeptide BE-43547A2: Synthesis and activity against pancreatic cancer stem cells. Angew. Chem., Int. Ed. 56 (2017) 14627– 14631.

[28] G. Cardillo, L. Gentilucci, A. Tolomelli, C. Tomasini, A practical method for the synthesis of β-amino α-hydroxy acids synthesis of enantiomerically pure hydroxyaspartic acid and isoserine. Synlett. 11 (1999) 1727–1730. 74

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[29] F. B. Charvillon, R. Amouroux, Synthesis of 3-hydroxylated analogues of D-aspartic acid β-hydroxamate. Synthetic Commun. 27 (1997) 395–403. [30] P. G. Mattingly, M. J. Miller, R. D. G. Cooper, B. W. Daughtery, Chiral synthesis of

RI PT

protected 3-amino-4-(alkoxycarbonyl)-2-azetidinones from β-hydroxyaspartic acid. J. Org. Chem. 48 (1983) 3556−3559.

[31] F. Sarabia, M. García-Castro, S. Chammaa, Synthesis of [13]-membered

SC

macrocyclic stevastelins via a transesterification reaction as the key step: total synthesis

M AN U

of stevastelin C3. Tetrahedron Lett. 46 (2005) 7695−7699.

[32] S. M. Bauer, R. W. Armstrong, Total synthesis of Motuporin (Nodularin-V). J. Am. Chem. Soc. 121 (1999) 6355–6366.

[33] M. A. Blanchette, W. Choy, J. T. Davis, A. P. Essenfeld, S. Masamune, W. R. Roush, T. Sakai, Horner-wadsworth-emmons reaction: Use of lithium chloride and an

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amine for base-sensitive compounds. Tetrahedron Lett. 25 (1984) 2183−2186. [34] J. Wang, B. Kuang, X. Guo, J. Liu, Y. Ding, J. Li, S. Jiang, Y. Liu, F. Bai, L. Li Q., Zhang, X. Zhu, B. Xia, C. Li, L. Wang, G. Yang, Y. Chen, Total syntheses and biological

AC C

EP

activities of vinylamycin analogs. J. Med. Chem. 60 (2017) 1189–1209.

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Scheme 1. Retrosynthetic analysis of compound 1. HO 11

NH

O

13

2

R

12

8

7 6

O

5

O 3

HO

11

R2

27

1

N R1

11 O O

HO 2 O R3

R3

R2

O

O

HN

4

O

O O P O

15 14

O O

2

O

6

1

HN

O

R1 N

TBSO Boc

Ot-Bu

HO

NH2

O

OH

H N

HO O O O

4

5

R3

6

AC C

EP

TE D

3

HO

11

TESO

M AN U

R1 N

NH

O

SC

10

RI PT

9

O

76

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Scheme 2. Preparation of fragments 3 and 5.

77

ACCEPTED MANUSCRIPT

Scheme 3. Completion of total synthesis of rakicidin A analogues 1a–e. HO Boc O

HO

OH OH

O

O

EDCI, DMAP

R3

O

TES

CH2Cl2

11

3

R

O

HN

LIHMDS

13a R3 = CH3 13b R3 = CH2CH3

OTBS

OTBS

HO

O

O O

EDCI, HOBt, CH2Cl2

R3

TESO

52%-69% for 3 steps

15

16

15a R3 = CH3 15b R3 = CH2CH3

MeCN, DCM

O

1

R

O

R

O HN

11

O O

HO

AC C

O 1a

40%-80% for 2 steps

R3

2a R1 = CH2CF3, R2 = H, R3 = CH3 2b R1 = CH2(CH2)4CH3, R2 = H, R3 = CH3 2c R1 = CH3, R2 = (S)-CH3, R3 = CH3 2d R1 = CH3, R2 = (R)-CH3, R3 = CH3 2e R1 = CH3, R2 = H, R3 = CH2CH3

TE D

O N

O

O O

O

NH

O

O

EP

F3C

HN

1a-e

2)DBU, THF

11

2

18a R1 = CH2CF3, R2 = H, R3 = CH3 18b R1 = CH2(CH2)4CH3, R2 = H, R3 = CH3 18c R1 = CH3, R2 = (S)-CH3, R3 = CH3 18d R1 = CH3, R2 = (R)-CH3, R3 = CH3 18e R1 = CH3, R2 = H, R3 = CH2CH3

N

O

HO

3

18

NH

O

13%-29% for 4 steps

O

O

R2

2)HATU, DIPEA, THF

11 O O

TESO

O

R N

1)MsCl, Et3N, THF

O

1

1)TFA, Et3SiH, CH2Cl2

BocHN

17a R3 = CH3 17b R3 = CH2CH3

NH

O

O

O

R3

O 17

OH

NH N

O O

TESO

M AN U

O R2

11

BocHN

16a R3 = CH3 16b R3 = CH2CH3

OTBS

3a-e, LiCl, DBU

R3

O

O

CH2Cl2

11

BocHN

O

NH

O

DMP O

11 O O

TESO

NH

NH2 4

O BocHN

O

HO

R3

O 14

SC

O HO

2)NaClO2, NaH2PO4, t-BuOH, H2O

TESO

14a R3 = CH3 14b R3 = CH2CH3

OTBS

1)DMP, CH2Cl2

O O

13

5a R3 = CH3 5b R3 = CH2CH3

11

BocHN

THF 55%-65%

11

Boc

12

5

O

OH O

O

70%-80%

TES

O

O

RI PT

NH

O

O

HN

O O

HO

N

11

NH

O

N O

HN

11 O O

HO

NH

O

O

O

N O

HN

11 O O

HO

NH

O

O

O O

O

HN

11 O O

HO

O

O

O

O

1b

1c

1d

1e

78

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Scheme 4. Retrosynthetic analysis of dehydroxy rakicidin A analogues 19a–d.

79

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Scheme 5. Preparation of the fragment 21 and polyketide fragment 25.

80

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Scheme 6. Total Synthesis of dehydroxy rakicidin analogues 19a–d.

81

ACCEPTED MANUSCRIPT

Scheme 7. Retrosynthetic analysis of compound 32. HO

10 8

O

12

O

O

3

N

14 13

13

1

HN

O

HN

O

2

O O

R5

13 O

R5 33

32

O O

N

TBDPSO NH2

Boc

O

O

OH

H N

O R5

O

RI PT

6 4

SC

N

NH

O

11

7

HO

13

34

36

EP

TE D

M AN U

35

AC C

5

NH

9

O

82

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Scheme 8. Preparation of the fragment 34 and polyketide fragment 36.

83

ACCEPTED MANUSCRIPT

Scheme 9. Total synthesis of rakicidin analogues 32a–h. TBDPSO

Boc

OH

H N

O HO

Boc

N O

13 O

O

2) 34, EDCI, HOBt, DCM

R5

73%-95%

O

1) Pd(PPh3)4, N-Methylaniline

O

H N

DIC/DMAP

36

NH

O

O R5

13

O

13 O

5

R

62%-88% for 2 steps

46

O

BocHN

RI PT

O

O

47

TBDPSO

HO

N

1) Pd(PPh3)4, N-Methylaniline 2) TFA, Et3SiH, CH2Cl2

O 13

HN

3) HATU, DIPEA, THF

N

TBAF.HOAc

O

NH

O

O

THF

O

O O

O

13

HN

O

5

R

R5

47%-59% for 3 steps 48

O

33

b. R5 =

O f. R5 =

5

e. R =

40%-49% for 3 steps

M AN U

a. R5 =

1) EtSO2Cl Et3N, THF 2) DBU,THF

c. R5 =

H

O

H N

O

O

O

O

13

HN

O 5

R

32

h. R5 =

H N

O

AC C

EP

TE D

O

N

d. R5 =

CH3

g. R5 =

NH

O

SC

NH

O

84

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Scheme 10. Retrosynthetic analysis of demethylenerakicidin analogue 49.

85

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Scheme 11. Total Synthesis of demethylenerakicidin analogue 49.

86

ACCEPTED MANUSCRIPT

RI PT

Table 1. Anti-pancreatic cancer activities of rakicidin analogues.a PANC-1b IC50d (µM)

Structures

hypoxia

normoxia

0.48±0.23

1.7±0.43

0.29

Gemcitabinef

–

0.40

1.7

0.24

BE-43547A2

–

0.65

0.015

43.33

0.22±0.11

0.042±0.004

of Rakicidin A(MERA)

5.24

HSI normoxia

hypoxia

2.0±1.02

0.78

N.A.

N.A.

N.A.

N.A.g

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

0.68±0.34

0.082±0.018

8.40

0.24±0.078

0.54±0.29

0.15±0.057

3.63

0.45±0.042

0.033±0.005 0

7.54

EP AC C

Methyl ester

1.6±0.86

TE D

–

IC50 (µM)

HSI

hypoxia

M AN U

normoxia Adriamycinf

RakicidinA

PATU8988b

IC50 (µM) HSIe

SC

Compounds

ASPC-1c

0.27±0.059

0.09±0.013

2.98

0.12±0.0070

3.91

1b

>50

1c

0.41±0.071

1d

0.66±0.28

0.48±0.014

1.5±0.41

1.72

N.A.

7.0±0.78

>7.11

0.043±0.002 8

9.53

0.18±0.026

2.64

0.38±0.16

0.12±0.061

3.25

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

1.2±0.31

0.20±0.0030

5.71

0.54±0.049

0.20±0.064

2.79

3.2±0.085

0.40±0.12

7.87

1.1±0.007

0.38±0.11

2.86

SC

2.5±0.014

2.33

5

M AN U

1a

0.067±0.003

TE D

0.16±0.049

AC C

EP

1e

RI PT

ACCEPTED MANUSCRIPT

0.079±0.003 5

8.34

ACCEPTED MANUSCRIPT

0.048±0.014

2.36

0.36±0.064

19b

0.18±0.018

0.074±0.013

2.43

0.28±0.064

19c

1.8±0.092

0.70±0.25

2.60

19d

0.15±0.02

0.066±0.002

49

21±0.76

0.59

0.36±0.23

0.073±0.022

5.03

M AN U

0.15±0.08

1.85

0.32±0.19

0.11±0.053

2.89

N.A.

N.A.

N.A.

N.A.

N.A.

0.26±0.049

0.12±0.058

2.23

0.30±0.13

0.11±0.0080

2.80

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

TE D

N.A.

AC C

EP

2.21

0.60±0.080

RI PT

0.11±0.023

SC

19a

28±0.88

0.76

0.058±0.030

1.98

0.98±0.062

2.80

0.64±0.12

0.34±0.20

1.87

32b

2.48±0.31

0.31±0.083

7.93

4.17±0.64

8.60

2.47±2.50

0.44±0.18

5.67

32c

1.1±0.49

0.27±0.071

4.04

32d

1.0±0.44

0.35±0.082

32e

0.43±0.13

RI PT

0.12±0.068

0.49±0.078

M AN U

32a

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

1.4±0.14

0.60±0.085

2.40

1.4±0.057

0.65±0.35

2.14

TE D

N.A.

AC C

EP

3.00

0.35±0.13

SC

ACCEPTED MANUSCRIPT

0.12±0.036

3.76

ACCEPTED MANUSCRIPT

32g

0.13±0.047

32h

0.17±0.051

0.038±0.001

48g

>10

>10

0.024±0.004

N.A.

5.25

0.27

4.44

N.A.

N.A.

N.A.

12.27

0.20±0.092

0.046±0.023

10.98

0.26±0.021

0.060±0.028

4.42

N.A.

N.A.

N.A.

N.A.

N.A.

0.022

0.50±0.035

N.A.

0.024±0.009 0

N.A.

8.30

TE D

0

3.54

RI PT

0.30±0.057

SC

1.1±0.43

M AN U

32f

AC C

EP

–

N.A.

ACCEPTED MANUSCRIPT

HO NH

O

33g

O

O 13

HN

>10

>10

–

RI PT

O

N

N.A.

N.A.

O O NH

N.A.

N.A.

N.A.

All values are the mean of three independent experiments and reported as Mean ± SD. bHuman pancreatic cancer cell line. cHuman metastasis of pancreatic adenocarcinoma cells. dHalf maximal inhibitory concentration. eHSI was calculated from: IC50 (normoxia)/IC50 (hypoxia).

EP

TE D

M AN U

Adriamycin and gemcitabine were used as positive controls. gN.A.: Not available.

AC C

f

SC

a

N.A.

RI PT

ACCEPTED MANUSCRIPT

Figure 1. Structures of rakicidin A–F, vinylamycin, microtermolide A, and

AC C

EP

TE D

M AN U

SC

BE-43547A1.

10

8.9

8

*

EM

*

1.4

0.83

G

**

0.43

SC

32 e

32 a

19 d

19 a

0.33

M ER A

* 0

1.6

0.50

D R

1.6 0.70

32 R h ak ic id in A

1.2

Ta xo l

1.9

1.7

32 g

2

A

4

RI PT

6

co nt ro l

Percentage of CD24+ CD44+ ESA+ cells

ACCEPTED MANUSCRIPT

M AN U

Figure 2. The percentage of CD24+CD44+ESA+ pancreatic cancer stem cells in PANC-1 cells after the treatment of tested compounds at a concentration of 0.15 µM for 48 hours. Clinically used drugs, taxol, ADR and GEM were used as positive

AC C

EP

0.01 vs control.

TE D

controls. The results represent the mean ±SEM, n = 3, *p < 0.05 vs control, **p <

RI PT

ACCEPTED MANUSCRIPT

Figure 3. The percentage of ALDH+ pancreatic cancer stem cells in PANC-1 cells

SC

after the treatment of tested compounds at a concentration of 0.2 µM for 72 hours. Clinically used drug ADR (at a concentration of 0.5 µM) was used as a positive

M AN U

control. The results represent the mean ±SEM, n = 3, *p < 0.05 vs control, **p <

AC C

EP

TE D

0.01 vs control.

50 35

40

35

20

25

20 *

10

22 16 *

4 ** 32 h ak ic id in A

SC

R

32 g

32 e

32 a

19 d

co n

19 a

0

Ta xo l

16 *

22 *

RI PT

30

M ER A

29

tr ol

Number of tumorspheres

ACCEPTED MANUSCRIPT

Figure 4. The number of tumorspheres in PANC-1 cells after the treatment of tested

M AN U

compounds at a concentration of 0.25 µM. The results represent the mean ±SEM, n

AC C

EP

TE D

= 3, *p < 0.05 vs control, **p < 0.01 vs control.

ACCEPTED MANUSCRIPT (a)

0.37

RI PT

control Rakicidin A 32g ADR

0.1

0.01 0

500

1000

2000

M AN U

Cell Dose（ 104）

1500

SC

Percentage of negative

1

EP

TE D

(b)

Figure 5. Analogue 32g reduced the tumor initiating frequency in a murine tumor

AC C

initiating assay. (a) Analysis of the limiting dilution experiment with Poisson statistics. Rakicidin A and ADR was used as positive control. (b) The limiting dilution assay showed that rakicidin A and 32g reduced the tumor initiating frequency (TIF). TIF refers to the average number of cells required to cause tumor initiation in the recipient cohort and was calculated using the L-Calc software. *p < 0.05 vs control, **p < 0.01 vs control, ***p < 0.01 vs control.

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55 *** 47

60

45

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26 14

22 15

6

20 -2 0

10 5

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Inhibition %

80

GEM 32b 32g

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0. 00 0. 5 05 0. 0 50 5. 0 0 50 00 .0 00

0. 00 0. 5 05 0. 0 50 5. 0 0 50 00 .0 00

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Figure 6. Inhibitory activity against PANC-1 cells in zebrafish xenograft model. (a) The results shown represent the mean ±SEM, n = 15, *p < 0.05 vs control, **p < 0.01 vs control, ***p < 0.001 vs control. (b) Representative images of zebrafish in zebrafish xenograft model.

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Highlights Compound 32g was totally synthesized in 14 linear steps with 5.05% overall yield.

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Compound 32g was 4 times more potent than rakicidin A at hypoxia against ASPC-1.

Compound 32g showed 12 folds of hypoxia selectivity in ASPC-1 cells.

The tumor-initiating frequency was reduced by 19 folds after the treatment of

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32g.

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Compound 32g selectively ablated pancreatic cancer stem cells.