Novel nannocystin A analogues as anticancer therapeutics: Synthesis, biological evaluations and structure–activity relationship studies

Article abstract of DOI:10.1016/j.ejmech.2019.03.011

Nannocystin A is a novel 21-membered macrocyclic lactam that targets eukaryotic translation elongation factor 1α (eEF1A) and displays potent antiproliferative activities. Herein, a series of nannocystin A analogues were synthesized and their structure–activity relationship (SAR) were established based on the MTT assay and western blotting analysis. The SAR enabled us to identify a structurally simplified nannocystin A analogue LQ18, which exhibited potent antiproliferative activities with IC₅₀ values ranging from 4.3 to 48 nM against the tested cell lines, and inhibited eEF1A1 expression in A549 cell line. LQ18 arrested cell cycle at G2 phase and induced A549 cell apoptosis via up-regulation of caspase-3, caspase-9 and bax protein expressions in a dose-dependent manner, while it significantly decreased the bcl-2 expression. Collectively, these data demonstrated that LQ18 could be a promising lead for the development of structurally novel eEF1A1 inhibitor for cancer treatment.

Full text of DOI:10.1016/j.ejmech.2019.03.011

Accepted Manuscript
Novel nannocystin A analogues as anticancer therapeutics: Synthesis, biological
evaluations and structure–activity relationship studies
Qiang Liu, Xiaohong Yang, Jie Ji, Shao-Lin Zhang, Yun He
PII:
S0223-5234(19)30222-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.03.011
EJMECH 11181
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 January 2019
Revised Date: 22 February 2019
Accepted Date: 5 March 2019
Please cite this article as: Q. Liu, X. Yang, J. Ji, S.-L. Zhang, Y. He, Novel nannocystin A analogues as
anticancer therapeutics: Synthesis, biological evaluations and structure–activity relationship studies,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.03.011.
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ACCEPTED MANUSCRIPT
Graphical Abstract
A structurally simplified nannocystin A analogue LQ18 exhibited potent antiproliferative
activities with IC50 values ranging from 4.3 to 48 nM against tested cancer cell lines, and inhibited
eEF1A1 expression in A549 cell line.

ACCEPTED MANUSCRIPT
Title page
Title:
Novel nannocystin A analogues as anticancer therapeutics: synthesis, biological evaluations and
structure–activity relationship studies
Author names and affiliations:
Qiang Liu#, Xiaohong Yang#, Jie Ji, Shao-Lin Zhang*, Yun He*
School of Pharmaceutical Sciences, Chongqing Key Laboratory of Natural Product Synthesis and
Drug Research, Chongqing University, Chongqing, 401331, P. R. China
* Corresponding address:
Tel.: +86-23-65678465; E-mail: zhangsl@cqu.edu.cn; yun.he@cqu.edu.cn
#: These authors contributed equally.
Abstract:
Nannocystin A is a novel 21-membered macrocyclic lactam that targets eukaryotic translation
elongation factor 1α (eEF1A) and displays potent antiproliferative activities. Herein, a series of
nannocystin A analogues were synthesized and their structure–activity relationship (SAR) were
established based on the MTT assay and western blotting analysis. The SAR enabled us to identify
a structurally simplified nannocystin A analogue LQ18, which exhibited potent antiproliferative
activities with IC50 values ranging from 4.3 to 48 nM against the tested cell lines, and inhibited
eEF1A1 expression in A549 cell line. LQ18 arrested cell cycle at G2 phase and induced A549 cell
apoptosis via up-regulation of caspase-3, caspase-9 and bax protein expressions in a
dose-dependent manner, while it significantly decreased the bcl-2 expression. Collectively, these
data demonstrated that LQ18 could be a promising lead for the development of structurally novel
eEF1A1 inhibitor for cancer treatment.
Keywords: Nannocystin A, anticancer, SAR study
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1. Introduction
Natural products are the most proliferative source of leads and / or drug candidates in drug
development [1]. Natural products from plants, microbes, and animals, as well as synthetic or
semi-synthetic compounds have continued to advance into clinical trials, particularly in anticancer
and antimicrobial fields [2, 3]. An analysis of the new drugs approved by the US Food and Drug
Administration (FDA) between 1981 and 2010 suggested that 34% of those drugs were natural
products or direct derivatives from natural products [4]. Thus drug discovery based on natural
products is becoming increasingly popular in recent years.
In 2015, Brönstrup et. al. [5] and Hoepfner et. al. [6] independently disclosed the isolation of
nannocystin A from the myxobaterium Nannocystis sp. Its relative and absolute configurations
were then confirmed by extensive NMR spectroscopic analysis and furtherer established by a
single-crystal X-ray analysis [6], which suggest that nannocystin A (Fig. 1) is a 21-membered
macrocycle containing both peptide and polyketide fragments, and possesses nine stereogenic
centers in the carbon backbone.
Fig. 1 Chemical structure of nannocystin A
Nannocystin A potently inhibited various cancer cell lines at nanomolar concentrations, the IC50
values are 1.0 and 12 nM for PC3 and HL60 cells, respectively [5]. Its antiproliferative activities
probably resulted from the inhibition of eukaryotic translation elongation factor 1-α 1 (eEF1A1),
which regulates the ribosomal protein synthesis and involves in cytoskeletal organization and
other physiological activities [7, 8]. In addition, eEF1A1 is associated with the development and
progression of various human cancers [9, 10] and has thus been proposed as a target for anticancer
therapy [11]. However, no drug based on this target has been successfully marketed. Moreover, the
overexpression or depletion of eEF1A1 has been shown to influence the rate of cancer cell
apoptosis. Collectively, these studies suggested that eEF1A1 was an attractive anticancer target,
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the inhibition of eEF1A1 protein could provide a promising approach for killing or, at least,
greatly reducing the growth of cancer cells.
Previously, our laboratory [12] and others completed the total synthesis of nannocystin A
[13-15], and some nannocystin A analogues were also disclosed [16, 17]. However, the
systematical modification of nannocystin A and its detailed SAR studies are limited. Continuing
our efforts in discovering novel therapeutics based on natural and designed products [18-23],
herein we report the optimization, biological evaluation, and SAR analysis of a series of novel
nannocystin A analogues.
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2. Results and discussion
2.1 Chemistry
The retrosynthetic analysis of nannocystin A was outlined in Scheme 1, which could be
assembled from three main fragments, A, B and C, respectively. The structures of each fragment
used in our study were listed in Table 1, which were individually synthesized and optimized. With
the diverse set of fragments in hand, nannocystin A analogues were conveniently assembled
(Table 2).
Scheme 1 Retrosynthetic analysis for the construction of nannocystin A
Table 1 Structures of fragments A, B and C
Fragment A
Fragment B
O
NH
O
NH2
O
NH
O
NH2
H
H
H
H
N
N
N
N
O
O
O
O
Cl
O
Cl
O
Cl
O
Cl
O
TBSO
TBSO
TBSO
TBSO
C1
C4
C2
C3
Cl
Cl
Cl
Cl
Fragment C
O
NH
O
NH
H
O
NH
H
H
O
NH
N
H
N
N
O
O
N
O
O
O
O
O
O
O
O
C8, R = H
C9,
R
TBSO
C5
TBSO
C7
C6
R = OH
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As shown in Scheme 2, the synthesis of fragments A1, A2, A4 started from known alcohol 2
[24], which was oxidized via PCC, then followed by Witting reaction to afford olefin 3 in 70%
yield [25]. Deprotection of the TBS group in 3 with TBAF gave rise to alcohol 4 in 95% yield.
Subsequent esterification with acids 5, 6 or 7 respectively provided the corresponding esters in
68-80% yields. After treating with TFA or TBAF, amine fragments A1, A2 and A4 were isolated
in 80-90% yields. Amines A3 and A5 were synthesized from known alcohol 8, which was
prepared according to the literature procedures [26].
Scheme 2 Synthesis of fragments A1-5
Fragment B was synthesized via a multiple step reaction as outlined in Scheme 3. Acid B1 was
prepared from aldehyde 9 [27]. Oxidation of allylic alcohol 11 gave rise to the corresponding
carboxylic acid B2 in a 90% yield [13]. Ester 12 could also be prepared from 9 by the protocol we
used in the total synthesis of aetheramides [28]. Subsequent hydrolysis of ethyl ester 12 in the
presence of lithium hydroxide in THF/H2O furnished the carboxylic acid B3 in 86% yield.
Alcohol 13 was synthesized according to the Kobayashi’ protocol [29] from aldehyde 10 [30].
Methylation with Meerwein’s salt (Me3OBF4) and proton sponge proceeded successfully to give
the corresponding ether in 80% yield, and subsequent removal of Evans chiral auxiliary provided
acid B4 in 85% yield.
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I
I
I
I
R1
O
O
O
I
O
O
LiOH, THF/H2O
86%
O
O
O
O
ref 28
ref 13
ref 13
H
HO
B1
OH
O
HO
9 (R1 = H)
10 (R1 = CH3)
11
12
B3
1. DMP, CH2Cl2
2. NaClO2, NaH2PO4
2-methyl-2-butene
t-BuOH/H2O, 90%
ref 29
I
I
1. Proton sponge
Meerwein's salt
CH2Cl2, 80%
O
O
O
O
O
N
O
OH
2. LiOH, H2O2
THF/H2O, 85%
Bn
O
HO
HO
I
B2
B4
13
Scheme 3. Synthesis of fragments B1-4
Scheme 4. Synthesis of fragments C1-9
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The synthesis of fragment C started by preparing amines 15, 16 and 18. Amino esters 15 and 16
were prepared from β-iodo-D-alanine ester 14 by the Negishi reaction (Scheme 4) [28]. With
precursors 15, 16 and 18 in hand, fragment C was built via a two-step sequence. First,
condensation of amines 15, 16 and 18 with amino acids 17a-d under the condition of
HATU/DIPEA proceeded smoothly to afford the corresponding amides, which were treated with
TFA to remove of Boc group, yielding amines C1-9 in 60-80% yields.
Scheme 5 Synthesis of analogue LQ18
The synthesis of nannocystin A analogue LQ18 is shown in Scheme 5. Amine C6 and acid B2
were coupled under standard condensation conditions to produce the amide 19 in 55% yield.
Hydrolysis of methyl ester 19 with lithium hydroxide furnished the corresponding carboxylic acid,
which was coupled with amine fragment A2 using HATU/DIPEA to furnish iodide 20 directly.
Finally, analogue LQ18 was prepared by the intramolecular Heck reaction in 55% yield [15]. A
series of nannocystin A analogues were prepared following the same procedure as for LQ18 and
their structures are listed in Table 2. All nannocystin A analogues were characterized by 1H NMR,
13C NMR and HRMS spectra (see Supporting Information).
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Table 2 Structures of nannocystin A analogues
2.2 SAR studies
All nannocystin A analogues (LQ2-18) were evaluated for their antiproliferative activities on
A549 and HCT-116 cell lines, and some of them were tested for their effects on the expression of
the presumed target eEF1A1. The results of these biological evaluations were summarized in
Table 3 and Fig. 2.
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Fig. 2 The protein expression of eEF1A1 in A549 cells after treatment with nannocystin A and
analogues LQ2-6 and LQ18 was analyzed by Western blotting experiment. NP represents natural
product nannocystin A.
As shown in Tables 2 and 3, the replacement of the sec-butyl group in nannocystin A by a
isopropyl moiety (LQ2), or removal of a hydroxyl group from the isopropyl group in nannocystin
A (LQ6) led to 2 or 3-fold increases in antiproliferative activity on both A549 and HCT-116 cell
lines, while the removal of the (2R,3S)-epoxide in nannocystin A retained the anticancer activity
for LQ3 (IC50 = 10.1 nM against A549), which is in consistence with the previously report [6],
suggesting that the epoxide group is not involved in critical compound–protein interactions and
could be removed. Further the methyl group removal from the unsaturated lactam in LQ3 gave
LQ7, which displayed a decreased anticancer activity (IC50 = 40.5 nM against A549), suggesting
that the methyl group is important and should be retained for good activity. Removal of N-methyl
group in nannocystin A afforded LQ4 and LQ5, which demolished all activities on the tested cell
lines, indicating that the N-methyl is critical for its potency. In line with this observation, LQ2
completely inhibited the expression of eEF1A1, while LQ5 displayed little or no effect inhibiting
its expression (Fig. 2). Removal of the methyl group at C-2 of nannocystin A (LQ8) led to a
dramatic drop of anticancer activity, and deletion of all substitutes on benzyl moiety (LQ9) or the
chlorine atom (LQ12) from nannocystin A maintained their anticancer potency. Examination of
the docking model pose [6] suggested that a sub-pocket domain was near A375 of eEF1A which
could accommodate the dimethyl group of nannocystin A. Replacement the dimethyl group at
C-19 moiety of LQ11 with a larger isopropyl group (LQ13) led to loss of potency, which could be
explained by the collision of a bulkier side chain with the pocket. With these SAR information in
hand, we incorporated all preferential modifications into nannocystin (LQ10) with the aim to
further simplify its structure and improve potencies. To our disappointment, LQ10 was ineffective
on both cell lines. Finally, we optimized the substitutes on benzyl moiety of LQ10. A hydroxyl
group was incorporated onto the benzyl group yield LQ14, which strongly inhibited A549 cell
growth with an IC50 value of 35.9 nM. Incorporation a methoxy group into the benzyl group of
LQ14 generated LQ18 (IC50 = 20.2 nM against A549), which displayed a comparable
antiproliferative activity as nannocystin A. LQ18 exhibited the same level of anticancer activity as
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compared with nannocystin A, while it has a simplified chemical structure. Thus, we further
explored its biologicals impact on other cell lines.
Table 3 Antiproliferative activities of nannocystin A and its analogues LQ2-18
IC50 (nM) a
Compounds
A549
13.1 ± 0.3
3.5 ± 1.1
10.1 ± 2.5
557 ± 116
NI
HCT-116
17 ± 2.0
6.7 ± 1.1
16.7 ± 1.3
> 1000
Nannocystin A
LQ2
LQ3
LQ4
LQ5
NI
LQ6
3.2 ± 0.9
40.5 ± 10.1
90.3 ± 1.5
14.3 ± 0.6
> 100
10.5 ± 2.2
123.8 ± 27.2
78.2 ± 9.4
20.9 ± 4.1
> 100
LQ7
LQ8
LQ9
LQ10
LQ11
LQ12
LQ13
LQ14
LQ15
LQ17
LQ18
DOX b
21.6 ± 3.4
3.7 ± 0.71
> 100
42.1 ± 11.2
25.2 ± 2.6
> 100
35.9 ± 2.9
13.1 ± 3.6
> 100
59.7 ± 8.8
60.3 ± 13.3
> 100
20.2 ± 2.9
459 ± 56
48.5 ± 4.5
366 ± 43
a IC50 values were calculated from three independent experiments by using the MTT assay after 72
h treatment. The values were reported as the Mean ± SD. b DOX represents doxorubicin, a positive
control anticancer drug.
Then the anticancer activity of nannocystin A and LQ18 on other cancer cell line was measured.
As shown in Table 4, LQ18 displayed potent inhibitory effect on all tested cell lines, and the IC50
values ranged from 4.3 to 15.0 nM, similar to those of nannocystin A.
Table 4 Comparison of nannocystin A and LQ18 on the growth of different cell lines (IC50, nM) a
IC50 (nM)
Compounds
MCF-7
8.7 ± 0.5
15.0 ± 3.2
A375
SH-SY-5Y MDA-MB-231
Nannocystin A
2.4 ± 0.29
4.3 ± 0.70
2.1 ± 0.30
9.6 ± 0.6
7.5 ± 0.30
12.5 ± 2.6
LQ18
a
IC50 values were were reported as the Mean ± SD, which were derived from three independent
experimental measurements.
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2.3 LQ18 inhibited A549 cell colony formation
The growth inhibitory effect of LQ18 and nannocystin A against A549 was further measured by
colony formation assay. As shown in Fig. 3, LQ18 partially inhibited A549 cell colony formation
at 5 nM, while it prevented its formation at 10 and 20 nM. As a comparison, nannocystin A at 10
nM was used to inhibited A549 colony formation, which also displayed potent inhibitory potency,
as LQ18 did at the same concentration. These results demonstrated that LQ18 and nannocystin A
have comparable anticancer activities.
Fig. 3 LQ18 inhibited A549 cell colonies formation. Bar chart representation of the colonies
formed after LQ18 treatment. One colony was defined to be an aggregate of over 100 cells.
Nannocystin A was used as positive control. Data was shown as mean ± SD of three independent
experiments. *P < 0.05, versus control group.
2.4 LQ18 arrested cell cycle at G2
Since LQ18 showed the most promising anticancer potency, we conducted additional studies to
investigate its biological roles in A549 cells. We examined the effect of LQ18 on cell cycle
distribution using the propidium iodide (PI) staining kit. A549 cells were treated with LQ18 (30,
60, 90 nM), and nannocystin A (30 nM) or control for 24 h, then stained with PI and analyzed on a
flow cytometer.
As shown in Fig. 4, LQ18 led to a significant accumulation of cells at G2 phase from 30.02%
to 37.66% (30 nM), 40.80% (60 nM) and 57.73% (90 nM), accordingly. At the same time, it
reduced the cells at G1 phase from 57.41 to 37.81%, respectively. Meanwhile, we found that cell
cycle arrest at G2 phase induced by LQ18 was concentration-dependent, which might be one of
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the possible mechanisms for its cytotoxicity.
Fig. 4 LQ18 induced cell cycle arrest in A549 cells. Cells were treated with LQ18 for 30, 60, and
90 nM, and nannocystin A (30 nM) for 24ꢀh, and then fixed in ice cold 70% ethanol at 4ꢀ°C, then
stained with propidium iodide dye (50ꢀµg/mL), and the cell cycle analysis was evaluated via flow
cytometry (CytoFLEX, Beck- man, USA). The insert bar graph showed the percentages (%) of
cells in G1 and G2 phases, respectively.
2.5 LQ18 induced A549 cell apoptosis
To explore the mechanism of cells death, A549 cells were treated by LQ18 (30, 60, and 90 nM)
and nannocystin A (30 nM) for 12 h to induce cells apoptosis, which was then examined with
Annexin V-FITC/PI FACS assay. As shown in Fig. 5A, the percentages of apoptotic population in
A549 cells treated with LQ18 (30, 60, and 90 nM) for 12 h were 14.81, 23.58 and 31.77%,
respectively, suggesting that the induction of apoptosis in A549 cells follows a dose-dependent
manner, while for nannocystin A at 30 nM, the percentage was 26.99%. To verify this observation,
A549 cells were treated with LQ18 for 24 h and then stained with Hoechst 33342 dye. The cells
were then photographed by a confocal laser scanning microscopy. The A549 cells treated with
higher concentrations of LQ18 displayed stronger blue fluorescence (Fig. 5B), indicating an
increase of apoptosis caused by higher concentrations of LQ18.
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Fig. 5 LQ18 induced A549 cells apoptosis. (A) A549 cells were treated with various
concentrations of LQ18 as indicated for 12 h, then cells were stained with annexin V / PI, and
apoptosis was analyzed by using a flow cytometry. The insert bar graph represents statistics of
total apoptotic cell percentages from duplicate experiments; (B) Confocal laser scanning
microscopy analysis of nuclei fragmentation by Hoechst 33342 staining. Representative
photomicrographs of A549 cell line stained with Hoechst 33342 fluorescent dye after exposure to
LQ18 (30, 60 and 90 nM) or nannocystin A (30 nM) for 24 h. The control group cells exhibited
pale blue fluorescence, while the apoptotic cells exhibited strong blue fluorescence. Scale bars =
25 µm. *P < 0.05, versus control group.
Moreover, the percentages of apoptosis for A549 cells treated with LQ18 in 60 nM for 6, 12,
and 24 h were 5.42, 23.58, and 32.51%, respectively, suggesting that the induction of apoptosis in
A549 cells by LQ18 was time-dependent (Fig. 6A). To explore the apoptosis mechanism of A549
cells caused by LQ18, we examined the expressions of caspase-3, caspase-9, bax and bcl-2
proteins after LQ18 treatment (Fig. 6B). The expressions of caspase-3, caspase-9 and bax
increased in a dose-dependent manner after LQ18 treatment, whereas the bcl-2 expression was
obviously decreased.
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Fig. 6 LQ18 induced A549 cells apoptosis. (A) A549 cells were treated with various LQ18 at 60
nM for 6, 12 and 24 h, then cells were stained with Annexin V / PI, and apoptosis was analyzed by
using a flow cytometry; (B) The bar graph represents statistics of total apoptotic cell percentages
from duplicate experiments; (C) LQ18 induced caspases activation, down-regulation of Bcl-2 and
up-regulation of Bax in A549 cells. Cells were treated with various concentrations of LQ18 as
indicated for 24 h. *P < 0.05, versus control group.
3. Conclusion
We herein report the synthesis of a series of novel nannocystin A analogues and evaluation of
their anticancer activities on A549 and HCT116 cells. The effects of some nannocystin A analogs
on the eEF1A1 expression were also measured. Based on their antiproliferative activities against
multiple cancer cell lines and ability to inhibit eEF1A1 protein expression, we identified a novel
nannocystin A analogue LQ18, which exhibited potent antiproliferative activities with IC50 values
of 20.2, 4.3, and 9.6 nM against A549, A375, and SH-SY-5Y, respectively. LQ18 exhibited a
comparable anticancer activity as nannocystin A did, while it has a simplified chemical structure.
Further biological evaluations suggested that LQ18 arrested cell cycle at G2 phase and induce
A549 cell apoptosis via up-regulation of caspase-3, caspase-9 and bax protein expressions in a
dose-dependent manner, whereas the bcl-2 expression was significantly decreased. All these data
demonstrated that LQ18 could be a promising lead for the development of structurally novel
eEF1A1 inhibitor for the treatment of cancers.
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4. Experimental section
4.1 Materials and methods
All chemicals were purchased from commercial sources and used as received. For detailed
materials information and methods, please see Supporting Information.
4.2 Chemicals synthesis of intermediates
All the synthetic procedures of intermediates were listed in Supporting Information.
4.3 Chemical synthesis and spectra data of the target compounds LQ2-18
(3S,6R,9S,12E,15R,16E,18E,20R,21S)-6-(4-Hydroxy-3-methoxybenzyl)-3,9-diisopropyl-15-methox
y-10,12,16,20-tetramethyl-21-phenyl-1-oxa-4,7,10-triazacyclohenicosa-12,16,18-triene-2,5,8,11-t
etraone (LQ18)
To a solution of 20 (30 mg, 0.035 mmol, 1 equiv) in DMF (3 mL) was added Pd(OAc)2 (8 mg,
0.035 mmol, 1 equiv), Cs2CO3 (23 mg, 0.070 mmol, 2 equiv) and TEA (5 mg, 0.053 mmol, 1.5
equiv). The reaction was stirred at room temperature for 2 h, diluted with EtOAc (5 mL) and H2O
(5 mL), the mixtures were separated and the aqueous layer was extracted with EtOAc (5 mL × 3),
the combined organic layers were washed with brine, dried over anhydrous Na2SO4, concentrated
and purified with silica gel column chromatography (petroleum ether: EtOAc = 1:1) to afford
28
compound LQ18 (14 mg, 55%). [α]D = -64 (c 0.2, CHCl3); IR (KBr) νmax 3564, 3520, 3482,
1
1644, 700, 665, 607 cm-1; H NMR (600 MHz, CDCl3) δ 7.31 (t, J = 7.2 Hz, 2H), 7.27-7.24 (m,
4H), 7.22 (d, J = 7.8 Hz, 1H), 6.93 (d, J = 9.0 Hz, 1H), 6.78 (d, J = 8.4 Hz, 1H), 6.74 (s, 1H), 6.72
(d, J = 7.8 Hz, 1H), 6.53 (d, J = 9.0 Hz, 1H), 6.33-6.24 (m, 1H), 6.01 (s, 1H), 5.86 (d, J = 10.8 Hz,
1H), 5.77 (dd, J = 15.0, 4.8 Hz, 1H), 5.50 (s, 1H), 5.43 (t, J = 7.2 Hz, 1H), 4.61 (dd, J = 9.0, 3.6
Hz, 1H), 4.58-4.53 (m, 1H), 4.51 (d, J = 10.5 Hz, 1H), 3.84 (s, 3H), 3.55 (dd, J = 10.8, 3.0 Hz,
1H), 3.19 (s, 3H), 3.01 (dd, J = 13.8, 9.6 Hz, 1H), 2.86 (dd, J = 13.2, 5.4 Hz, 1H), 2.69 (s, 3H),
2.63-2.52 (m, 2H), 2.41-2.35 (m, 1H), 2.35-2.29 (m, 1H), 2.24-2.19 (m, 1H), 1.81 (s, 3H), 1.75
(s, 3H), 1.03 (d, J = 6.6 Hz, 3H), 0.95 (d, J = 6.6 Hz, 3H), 0.86 (d, J = 6.6 Hz, 3H), 0.67 (d, J =
6.6 Hz, 3H), 0.47 (d, J = 6.6 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 175.5 (C=O), 170.8 (C=O),
170.5 (C=O), 170.5 (C=O), 146.4, 144.4, 139.2, 136.3, 135.1, 133.4, 128.7, 128.4, 128.2, 127.7,
127.6, 125.8, 125.5, 122.1, 114.3, 111.7, 85.9, 78.0, 61.7, 56.0, 55.9, 55.8, 54.8, 42.3, 36.6, 32.2,
31.8, 31.4, 29.3, 24.9, 19.9, 19.0, 18.4, 16.2, 14.4, 11.1, 9.8.; HRMS (ESI): calcd for
C42H57N3O0Na+ [M+Na+] 754.4038, found 754.4030.
(1R,4S,7R,10S,13S,14R,15E,17E,19R,21S)-7-(3,5-Dichloro-4-hydroxybenzyl)-10-(2-hydroxypropa
n-2-yl)-4-isopropyl-19-methoxy-1,3,14,18-tetramethyl-13-phenyl-12,22-dioxa-3,6,9-triazabicyclo[
19.1.0]docosa-15,17-diene-2,5,8,11-tetraone (LQ2)
28
LQ2 was obtained following the procedure for LQ18 in 53% yield. [α]D = -17.5 (c 0.08,
1
CHCl3); IR (KBr) νmax 3447, 3416, 1639, 1619, 762, 619 cm-1; H NMR (600 MHz, CDCl3) δ
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7.44-7.29 (m, 5H), 7.07 (s, 2H), 6.60 (d, J = 6.6 Hz, 1H), 6.46 (d, J = 7.8 Hz, 1H), 6.33 (dd, J =
15.0, 10.8 Hz, 1H), 6.15 (d, J = 10.8 Hz, 1H), 5.93 (d, J = 2.4 Hz, 1H), 5.85 (dd, J = 15.0, 5.4 Hz,
1H), 4.62 (q, J = 7.2 Hz, 1H), 4.58 (d, J = 7.8 Hz, 1H), 4.39 (d, J = 11.4 Hz, 1H), 3.69 (dd, J = 9.0,
2.4 Hz, 1H), 3.20 (s, 3H), 3.13 (s, 3H), 3.07 (dd, J = 8.4, 3.6 Hz, 1H), 2.94 (dd, J = 14.4, 7.2 Hz,
1H), 2.76-2.68 (m, 1H), 2.63 (dd, J = 13.8, 6.6 Hz, 1H), 2.35-2.25 (m, 2H), 2.09-2.02 (m, 1H),
1.73 (s, 3H), 1.53 (s, 3H), 1.20 (s, 3H), 1.17 (s, 3H), 1.05 (d, J = 6.6 Hz, 3H), 0.86 (d, J = 6.6 Hz,
3H), 0.79 (d, J = 6.6 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 171.0 (C=O), 170.2 (C=O), 169.5
(C=O), 169.0 (C=O), 146.8, 138.0, 135.7, 134.1, 129.6, 129.1, 128.3, 128.1, 126.8, 126.4, 126.3,
121.0, 83.9, 82.1, 80.3, 72.1, 65.5, 62.5, 61.8, 60.6, 59.9, 58.6, 56.6, 56.1, 54.9, 53.9, 42.3, 41.8,
37.7, 31.5, 29.8, 29.7, 27.0, 26.5, 25.7, 19.2, 18.4, 15.6, 11.8; HRMS (ESI): calcd for
C41H53Cl2N3O9Na+ [M+Na+] 824.3051, found 824.3061.
(3S,6R,9S,12E,15R,16E,18E,20R,21S)-9-((S)-Sec-butyl)-6-(3,5-dichloro-4-hydroxybenzyl)-3-(2-hy
droxypropan-2-yl)-15-methoxy-10,12,16,20-tetramethyl-21-phenyl-1-oxa-4,7,10-triazacyclohenico
sa-12,16,18-triene-2,5,8,11-tetraone (LQ3)
28
LQ3 was obtained following the procedure for LQ18 in 50% yield. [α]D = -62.5 (c 0.08,
CHCl3); 1H NMR (600 MHz, CDCl3) δ 7.38-7.29 (m, 5H), 7.02 (s, 2H), 6.41 (d, J = 6.6 Hz, 1H),
6.32 (dd, J = 15.0, 11.4 Hz, 1H), 5.86 (s, 1H), 5.85 (d, J = 9.6 Hz, 1H), 5.72 (s, 1H), 5.54 (dd, J =
15.0, 6.6 Hz, 1H), 5.47 (t, J = 7.2 Hz, 1H), 4.63 (q, J = 8.4 Hz, 1H), 4.57 (d, J = 9.0 Hz, 1H), 4.33
(d, J = 10.8 Hz, 1H), 3.66 (dd, J = 7.8, 3.0 Hz, 1H), 3.23 (s, 3H), 2.95 (dd, J = 13.8, 7.2 Hz, 1H),
2.82 (s, 3H), 2.81-2.77 (m, 1H), 2.73 (dd, J = 14.4, 6.6 Hz, 1H), 2.55-2.47 (m, 1H), 2.46-2.38 (m,
1H), 2.13-2.04 (m, 1H), 1.80 (s, 3H), 1.71 (s, 3H), 1.48-1.40 (m, 1H), 1.05 (s, 3H), 1.04 (d, J =
8.4 Hz, 3H), 0.94 (t, J = 7.2 Hz, 3H), 0.85 (d, J = 6.0 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ
176.2 (C=O), 170.8 (C=O), 170.1 (C=O), 169.2 (C=O), 146.7, 137.7, 134.6, 134.3, 133.2, 129.7,
129.0, 127.9, 127.8, 127.1, 126.9, 126.7, 121.0, 84.8, 80.4, 72.2, 61.8, 60.7, 56.3, 53.7, 41.7, 35.9,
32.6, 31.4, 31.2, 29.7, 26.9, 26.6, 25.4, 15.9, 14.3, 13.0, 12.8, 10.6.
(1R,4S,7R,10S,13S,14R,15E,17E,19R,21S)-4-((S)-Sec-butyl)-7-(3,5-dichloro-4-hydroxybenzyl)-10-
(2-hydroxypropan-2-yl)-19-methoxy-1,14,18-trimethyl-13-phenyl-12,22-dioxa-3,6,9-triazabicyclo[
19.1.0]docosa-15,17-diene-2,5,8,11-tetraone (LQ4)
LQ4 was obtained following the procedure for LQ18 in 58% yield. [α]D28 = 12 (c 0.1, CHCl3);
1
IR (KBr) νmax 3512, 3447, 1649, 1632, 684 cm-1; H NMR (600 MHz, CDCl3) δ 7.44-7.23 (m,
5H), 7.22-7.13 (m, 1H), 7.09 (s, 2H), 6.98 (d, J = 8.0 Hz, 1H), 6.76 (d, J = 9.2 Hz, 1H), 6.40-6.25
(m, 2H), 6.13-6.01 (m, 2H), 5.98 (s, 1H), 5.77 (dd, J = 15.2, 7.6 Hz, 1H), 4.73 (q, J = 7.6 Hz, 1H),
4.50 (d, J = 8.0 Hz, 1H), 4.21 (dd, J = 8.8, 6.4 Hz, 1H), 3.74 (d, J = 5.6 Hz, 1H), 3.22 (s, 3H), 2.97
(dd, J = 14.4, 6.8 Hz, 1H), 2.84 (d, J = 5.6 Hz, 1H), 2.82-2.72 (m, 2H), 2.72-2.63 (m, 1H),
2.00-1.95 (m, 1H), 1.90-1.77 (m, 2H), 1.69 (s, 3H), 1.50 (s, 3H), 1.22 (s, 3H), 1.13 (s, 3H), 1.00
(d, J = 6.8 Hz, 3H), 0.82 (t, J = 7.2 Hz, 3H), 0.77 (d, J = 6.4 Hz, 3H); 13C NMR (150 MHz, CDCl3)
16

ACCEPTED MANUSCRIPT
δ 171.4 (C=O), 170.9 (C=O), 170.3 (C=O), 170.1 (C=O), 147.0, 137.8, 135.8, 133.9, 129.5, 128.9,
128.2, 128.0, 127.2, 126.8, 126.4, 121.3, 83.1, 80.0, 71.5, 60.6, 60.2, 60.1, 57.2, 56.3, 53.8, 42.6,
37.2, 36.9, 31.4, 29.7, 27.0, 26.5, 24.7, 15.2, 13.7, 13.0, 12.5, 11.4; HRMS (ESI): calcd for
C41H53Cl2N3O9Na+ [M+Na+] 824.3051, found 824.3057.
(1R,4S,7R,10S,13S,14R,15E,17E,19R,21S)-7-(3,5-Dichloro-4-hydroxybenzyl)-10-(2-hydroxypropa
n-2-yl)-4-isopropyl-19-methoxy-1,14,18-trimethyl-13-phenyl-12,22-dioxa-3,6,9-triazabicyclo[19.1
.0]docosa-15,17-diene-2,5,8,11-tetraone (LQ5)
LQ5 was obtained following the procedure for LQ18 in 58% yield. [α]D28 = 20 (c 0.15, CHCl3);
1
IR (KBr) νmax 3511, 3445, 1627, 1652, 1285, 770 cm-1; H NMR (600 MHz, CDCl3) δ 7.38-7.25
(m, 5H), 7.10 (s, 2H), 6.76 (d, J = 9.0 Hz, 1H), 6.61 (d, J = 7.8 Hz, 1H), 6.46-6.35 (m, 1H), 6.31
(dd, J = 15.0, 10.8 Hz, 1H), 6.06 (d, J = 10.8 Hz, 1H), 6.00 (s, 1H), 5.78 (dd, J = 15.0, 7.2 Hz, 1H),
4.77 (q, J = 7.8 Hz, 1H), 4.55 (d, J = 8.4 Hz, 1H), 4.18 (dd, J = 9.0, 6.0 Hz, 1H), 3.74 (dd, J = 9.0,
3.0 Hz, 1H), 3.20 (s, 3H), 3.18-3.14 (m, 1H), 2.95 (dd, J = 13.8, 6.6 Hz, 1H), 2.84 (t, J = 6.0 Hz,
1H), 2.79 (dd, J = 14.4, 7.8 Hz, 1H), 2.71-2.64 (m, 1H), 2.05-1.95 (m, 2H), 1.93-1.86 (m, 1H),
1.85-1.78 (m, 1H), 1.67 (s, 3H), 1.49 (s, 3H), 1.21 (s, 3H), 1.11 (s, 3H), 0.99 (d, J = 6.6 Hz, 3H),
0.80 (d, J = 6.6 Hz, 3H), 0.76 (d, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 171.5 (C=O),
170.8 (C=O), 170.5 (C=O), 170.2 (C=O), 147.1, 138.0, 135.9, 133.8, 129.5, 129.0, 128.2, 127.9,
127.8, 126.6, 126.3, 121.4, 83.3, 79.8, 71.7, 60.4, 60.1, 60.1, 57.7, 56.2, 53.8, 42.6, 37.2, 31.5,
30.9, 29.7, 26.8, 26.4, 19.0, 17.5, 13.6, 12.7, 12.0; HRMS (ESI): calcd for C40H51Cl2N3O9Na+
[M+Na+] 810.2895, found 810.2891.
(1R,4S,7R,10S,13S,14R,15E,17E,19R,21S)-4-((S)-Sec-butyl)-7-(3,5-dichloro-4-hydroxybenzyl)-10-
isopropyl-19-methoxy-1,3,14,18-tetramethyl-13-phenyl-12,22-dioxa-3,6,9-triazabicyclo[19.1.0]do
cosa-15,17-diene-2,5,8,11-tetraone (LQ6)
LQ6 was obtained following the procedure for LQ18 in 56% yield. [α]D28 = -39 (c 0.2, CHCl3);
1
IR (KBr) νmax 3466, 3425, 1627, 1590, 1248, 740 cm-1; H NMR (600 MHz, CDCl3) δ 7.38-7.18
(m, 5H), 7.12 (s, 2H), 6.89 (d, J = 7.2 Hz, 1H), 6.37-6.27 (m, 1H), 6.12 (s, 1H), 6.09 (d, J = 10.8
Hz, 1H), 6.06-6.00 (m, 1H), 6.01 (d, J = 8.4 Hz, 1H), 5.88 (dd, J = 15.0, 4.2 Hz, 1H), 4.71 (dd, J
= 8.4, 3.0 Hz, 1H), 4.54 (q, J = 6.0 Hz, 1H), 4.50 (d, J = 11.4 Hz, 1H), 3.65 (dd, J = 10.8, 2.4 Hz,
1H), 3.16 (s, 3H), 3.08 (s, 3H), 2.96-2.80 (m, 3H), 2.69-2.61 (m, 1H), 2.27-2.07 (m, 3H),
1.94-1.84 (m, 1H), 1.75 (s, 3H), 1.55-1.46 (m, 1H), 1.51 (s, 3H), 1.36-1.29 (m, 1H), 1.03 (d, J =
7.2 Hz, 3H), 1.02-0.96 (m, 1H), 0.88 (t, J = 4.2 Hz, 3H), 0.84 (d, J = 3.0 Hz, 3H), 0.83 (d, J = 2.4
Hz, 3H), 0.60 (d, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 170.9 (C=O), 170.7 (C=O),
169.8 (C=O), 168.5 (C=O), 147.0, 139.0, 136.3, 134.0, 129.7, 129.7, 129.1, 128.3, 127.7, 125.8,
125.7, 121.1, 84.7, 78.5, 61.6, 60.5, 58.5, 56.4, 55.6, 54.4, 42.1, 38.7, 31.7, 31.1, 29.8, 24.3, 19.0,
16.4, 15.5, 15.4, 10.7, 10.4, 9.5; HRMS (ESI): calcd for C42H55Cl2N3O8Na+ [M+Na+] 822.3258,
found 822.3263.
17

ACCEPTED MANUSCRIPT
(3S,6R,9S,12E,15R,16E,18E,20R,21S)-9-((S)-Sec-butyl)-6-(3,5-dichloro-4-hydroxybenzyl)-3-(2-hy
droxypropan-2-yl)-15-methoxy-10,16,20-trimethyl-21-phenyl-1-oxa-4,7,10-triazacyclohenicosa-12
,16,18-triene-2,5,8,11-tetraone (LQ7)
LQ7 was obtained following the procedure for LQ18 in 60% yield. [α]D28 = -50 (c 0.15, CHCl3);
IR (KBr) νmax 3693, 3401, 1724, 1659, 1201, 761 cm-1; 1H NMR (600 MHz, CDCl3) δ 7.48 (t, J =
7.8 Hz, 2H), 7.48 (t, J = 7.2 Hz, 1H), 7.31 (d, J = 7.2 Hz, 2H), 6.90 (s, 2H), 6.82-6.73 (m, 1H),
6.26 (dd, J = 15.0, 10.8 Hz, 1H), 6.20-6.12 (m, 2H), 6.03 (d, J = 11.4 Hz, 1H), 5.80 (d, J = 3.6 Hz,
1H), 5.82-5.75 (m, 1H), 5.51 (dd, J = 15.0, 9.0 Hz, 1H), 4.63-4.54 (m, 2H), 4.32 (d, J = 8.4 Hz,
1H), 3.76-3.69 (m, 1H), 3.28 (s, 3H), 2.98 (s, 3H), 2.84-2.76 (m, 1H), 2.74-2.66 (m, 1H),
2.45-2.36 (m, 1H), 2.07 (s, 1H), 2.04-1.95 (m, 1H), 1.84-1.74 (m, 1H), 1.68 (s, 3H), 1.68-1.62
(m, 3H), 1.33 (s, 3H), 1.17 (s, 3H), 0.99 (d, J = 7.2 Hz, 3H), 0.97-0.92 (m, 1H), 0.85 (t, J = 7.2 Hz,
3H), 0.64 (d, J = 6.0 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 170.6 (C=O), 170.4 (C=O), 168.7
(C=O), 168.1 (C=O), 146.6, 141.5, 136.3, 134.4, 133.7, 130.2, 128.5, 128.4, 128.2, 128.0, 126.8,
125.0, 122.8, 120.9, 83.4, 80.4, 70.7, 61.5, 60.9, 56.5, 53.3, 41.9, 35.6, 35.4, 31.6, 30.6, 29.7, 27.3,
27.0, 24.5, 15.3, 15.2, 14.4, 10.5; HRMS (ESI): calcd for C41H53Cl2N3O8Na+ [M+Na+] 808.3102,
found 808.3121.
(1R,4S,7R,10S,13S,15E,17E,19R,21S)-4-((S)-Sec-butyl)-7-(3,5-dichloro-4-hydroxybenzyl)-10-(2-h
ydroxypropan-2-yl)-19-methoxy-1,3,18-trimethyl-13-phenyl-12,22-dioxa-3,6,9-triazabicyclo[19.1.
0]docosa-15,17-diene-2,5,8,11-tetraone (LQ8)
LQ8 was obtained following the procedure for LQ18 in 60% yield. [α]D28 = -24 (c 0.1, CHCl3);
IR (KBr) νmax 3618, 3534, 3334, 1737, 1664, 1384, 965, 743 cm-1; 1H NMR (600 MHz, CDCl3) δ
7.47-7.29 (m, 5H), 7.17 (s, 2H), 6.89 (d, J = 6.6 Hz, 1H), 6.47-6.38 (m, 1H), 6.22 (d, J = 8.4 Hz,
1H), 6.07 (d, J = 10.8 Hz, 1H), 5.90 (d, J = 11.4 Hz, 1H), 5.82 (s, 1H), 5.71-5.62 (m, 1H),
4.64-4.52 (m, 2H), 4.49 (d, J = 11.4 Hz, 1H), 3.66 (dd, J = 11.4, 3.0 Hz, 1H), 3.17 (s, 3H), 3.09 (s,
3H), 2.98-2.91 (m, 2H), 2.91-2.81 (m, 2H), 2.58 (d, J = 10.8 Hz, 1H), 2.20-2.07 (m, 2H),
2.06-1.97 (m, 2H), 1.76 (s, 3H), 1.55-1.49 (m, 1H), 1.51 (s, 3H), 1.34-1.31 (m, 1H), 1.04-0.99
(m, 1H), 1.02 (s, 3H), 0.89 (t, J = 7.2 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H), 0.80 (s, 3H); 13C NMR
(150 MHz, CDCl3) δ 170.7 (C=O), 170.2 (C=O), 170.1 (C=O), 168.6 (C=O), 146.9, 139.1, 134.5,
129.8, 129.5, 129.4, 129.3, 129.2, 128.9, 128.7, 127.1, 121.0, 84.8, 72.9, 61.7, 60.5, 59.8, 58.6,
55.7, 54.2, 39.8, 38.6, 31.7, 31.2, 29.8, 29.7, 27.2, 25.4, 24.3, 15.5, 15.4, 10.5, 10.4.; HRMS (ESI):
calcd for C41H53Cl2N3O9Na+ [M+Na+] 824.3051, found 824.3057.
(1R,4S,7R,10S,13S,14R,15E,17E,19R,21S)-7-Benzyl-4-((S)-sec-butyl)-10-(2-hydroxypropan-2-yl)-
19-methoxy-1,3,14,18-tetramethyl-13-phenyl-12,22-dioxa-3,6,9-triazabicyclo[19.1.0]docosa-15,1
7-diene-2,5,8,11-tetraone (LQ9)
28
LQ9 was obtained following the procedure for LQ18 in 56% yield. [α]D = -16.6 (c 0.15,
CHCl3); IR (KBr) νmax 3581, 3465, 1738, 1651, 1246, 1046, 739 cm-1; 1H NMR (600 MHz, CDCl3)
18

ACCEPTED MANUSCRIPT
δ 7.40-7.28 (m, 5H), 7.27-7.04 (m, 5H), 6.67 (s, 1H), 6.38-6.27 (m, 1H), 6.19 (d, J = 7.8 Hz, 1H),
6.14 (d, J = 10.8 Hz, 1H), 5.94 (s, 1H), 5.86 (dd, J = 15.0, 5.4 Hz, 1H), 4.72-4.63 (m, 1H), 4.54 (d,
J = 8.4 Hz, 1H), 4.47 (d, J = 11.4 Hz, 1H), 3.72-3.66 (m, 1H), 3.20 (s, 3H), 3.12 (s, 3H), 3.05 (d, J
= 6.6 Hz, 1H), 2.96 (dd, J = 13.8, 7.8 Hz, 1H), 2.85 (dd, J = 13.8, 6.6 Hz, 1H), 2.75-2.66 (m, 1H),
2.17-2.01 (m, 3H), 1.74 (s, 3H), 1.65-1.59 (m, 1H), 1.52 (s, 3H), 1.38-1.28 (m, 2H), 1.08 (s, 3H),
1.06 (d, J = 6.6 Hz, 3H), 1.03-0.96 (m, 1H), 0.88 (t, J = 7.2 Hz, 3H), 0.86-0.78 (m, 1H), 0.68 (d, J
= 6.6 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 170.8 (C=O), 170.6 (C=O) 169.6 (C=O), 169.0
(C=O), 138.2, 136.5, 135.9, 134.1, 129.1, 128.8, 128.5, 128.2, 128.0, 127.2, 126.8, 126.3, 84.2,
80.3, 72.2, 61.8, 60.7, 60.5, 58.6, 56.0, 54.4, 41.8, 39.3, 31.5, 29.9, 29.7, 26.7, 26.6, 24.4, 15.6,
15.2, 11.5, 11.3, 10.4; HRMS (ESI): calcd for C42H57N3O8Na+ [M+Na+] 754.4038, found
754.4068.
(3S,6R,9S,12E,15R,16E,18E,20R,21S)-6-Benzyl-3,9-diisopropyl-15-methoxy-10,12,16,20-tetramet
hyl-21-phenyl-1-oxa-4,7,10-triazacyclohenicosa-12,16,18-triene-2,5,8,11-tetraone (LQ10)
LQ10 was obtained following the procedure for LQ18 in 65% yield. [α]D28 = -63 (c 0.1, CHCl3);
1
IR (KBr) νmax 3679, 3309, 3022, 1742, 1686, 1216, 759 cm-1; H NMR (600 MHz, CDCl3) δ
7.40-7.11 (m, 10H), 6.93 (d, J = 8.4 Hz, 1H), 6.48 (d, J = 9.0 Hz, 1H), 6.29 (dd, J = 15.0, 12.0 Hz,
1H), 6.09 (s, 1H), 5.86 (d, J = 10.8 Hz, 1H), 5.77 (dd, J = 15.6, 4.8 Hz, 1H), 5.43 (t, J = 7.2 Hz,
1H), 4.67-4.55 (m, 2H), 4.51 (d, J = 11.4 Hz, 1H), 3.55 (dd, J = 10.8, 2.4 Hz, 1H), 3.19 (s, 3H),
3.09 (dd, J = 13.2, 9.6 Hz, 1H), 2.92 (dd, J = 13.8, 6.0 Hz, 1H), 2.68 (s, 3H), 2.64-2.52 (m, 2H),
2.42-2.35 (m, 1H), 2.35-2.27 (m, 1H), 2.25-2.15 (m, 1H), 1.82 (s, 3H), 1.75 (s, 3H), 1.03 (d, J =
6.6 Hz, 3H), 0.93 (d, J = 6.6 Hz, 3H), 0.86 (d, J = 6.6 Hz, 3H), 0.62 (d, J = 6.6 Hz, 3H), 0.45 (d, J
= 6.6 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 175.5 (C=O), 170.8 (C=O), 170.5 (C=O), 170.4
(C=O), 139.3, 136.6, 136.3, 135.1, 133.4, 129.2, 129.2, 128.7, 128.5, 128.2, 127.6, 126.8, 125.8,
125.5, 85.9, 78.0, 61.6, 56.0, 55.8, 54.6, 42.3, 36.9, 32.1, 31.8, 31.3, 24.8, 19.8, 19.0, 18.4, 16.2,
14.4, 11.1, 9.7; HRMS (ESI): calcd for C41H55N3O6Na+ [M+Na+] 708.3983, found 708.3993.
(3S,6R,9S,12E,15R,16E,18E,20R,21S)-6-(3,5-Dichloro-4-hydroxybenzyl)-3,9-diisopropyl-15-meth
oxy-10,12,16,20-tetramethyl-21-phenyl-1-oxa-4,7,10-triazacyclohenicosa-12,16,18-triene-2,5,8,11
-tetraone (LQ11)
LQ11 was obtained following the procedure for LQ18 in 64% yield. [α]D28 = -21 (c 0.1, CHCl3);
1
IR (KBr) νmax 3682, 3657, 3295, 2927, 1741, 1688, 1213, 1148, 760 cm-1; H NMR (600 MHz,
CDCl3) δ 7.40-7.20 (m, 5H), 7.15 (s, 1H), 6.97 (d, J = 8.4 Hz, 1H), 6.62 (d, J = 9.0 Hz, 1H), 6.30
(dd, J = 15.6, 10.8 Hz, 1H), 6.10 (s, 1H), 5.85 (d, J = 10.8 Hz, 1H), 5.77 (dd, J = 15.6, 5.4 Hz, 1H),
5.47-5.37 (m, 1H), 4.62 (dd, J = 8.4, 3.0 Hz, 1H), 4.56-4.47 (m, 2H), 3.56 (dd, J = 10.8, 3.0 Hz,
1H), 3.19 (s, 3H), 3.05 (dd, J = 13.2, 9.6 Hz, 1H), 2.77 (dd, J = 13.8, 5.4 Hz, 1H), 2.67 (s, 3H),
2.64-2.51 (m, 2H), 2.42-2.29 (m, 2H), 2.29-2.21 (m, 1H), 1.80 (s, 3H), 1.76 (s, 3H), 1.73-1.64
(m, 2H), 1.04 (d, J = 7.2 Hz, 3H), 0.97 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H), 0.69 (d, J =
19

ACCEPTED MANUSCRIPT
6.6 Hz, 3H), 0.50 (d, J = 6.6 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 175.7 (C=O), 171.7 (C=O),
170.7 (C=O), 169.4 (C=O), 146.7, 139.1, 136.2, 135.3, 133.3, 130.2, 129.5, 129.0, 128.7, 128.2,
127.9, 127.7, 125.9, 125.5, 120.9, 85.7, 78.1, 67.8, 61.8, 55.9, 54.3, 50.3, 42.3, 42.0, 35.4, 32.3,
31.8, 24.9, 24.7, 23.0, 21.8, 19.9, 18.5, 14.4, 11.3, 10.0; HRMS (ESI): calcd for
C41H53Cl2N3O7Na+ [M+Na+] 792.3153, found 792.3166.
(1R,4S,7R,10S,13S,14R,15E,17E,19R,21S)-4-((S)-Sec-butyl)-7-(4-hydroxy-3-methoxybenzyl)-10-(2
-hydroxypropan-2-yl)-19-methoxy-1,3,14,18-tetramethyl-13-phenyl-12,22-dioxa-3,6,9-triazabicycl
o[19.1.0]docosa-15,17-diene-2,5,8,11-tetraone (LQ12)
28
LQ12 was obtained following the procedure for LQ18 in 52% yield. [α]D = -14.3 (c 0.1,
CHCl3); IR (KBr) νmax 3632, 3527, 3300, 1736, 1700, 1216, 759 cm-1; 1H NMR (600 MHz, CDCl3)
δ 7.40-7.23 (m, 5H), 6.77 (d, J = 7.8 Hz, 1H), 6.72 (s, 2H), 6.68 (d, J = 6.6 Hz, 1H), 6.64 (d, J =
7.8 Hz, 1H), 6.33 (dd, J = 13.8, 10.8 Hz, 1H), 6.19 (d, J = 8.4 Hz, 1H), 6.13 (d, J = 10.8 Hz, 1H),
5.93 (d, J = 3.6 Hz, 1H), 5.86 (d, J = 5.4 Hz, 1H), 5.84 (d, J = 5.4 Hz, 1H), 5.50 (s, 1H), 4.62 (q, J
= 7.2 Hz, 1H), 4.55 (d, J = 8.4 Hz, 1H), 4.48 (d, J = 11.4 Hz, 1H), 3.84 (s, 3H), 3.69 (dd, J = 9.6,
3.0 Hz, 1H), 3.20 (s, 3H), 3.13 (s, 3H), 3.05 (dd, J = 9.0, 3.0 Hz, 1H), 2.84 (d, J = 7.2 Hz, 1H),
2.74-2.65 (m, 1H), 2.22-2.15 (m, 1H), 2.15-2.05 (m, 2H), 1.75 (s, 3H), 1.65-1.61 (m, 3H), 1.11
(s, 3H), 1.07 (s, 3H), 1.06 (s, 3H), 0.89 (t, J = 7.2 Hz, 3H), 0.74 (d, J = 6.0 Hz, 3H); 13C NMR
(150 MHz, CDCl3) δ 170.8 (C=O), 170.6 (C=O), 169.5 (C=O), 169.0 (C=O), 146.7, 144.7, 138.1,
135.9, 134.1, 128.5, 128.2, 128.2, 128.0, 126.7, 126.3, 122.0, 114.4, 111.2, 84.2, 80.3, 72.2, 61.8,
60.7, 60.5, 58.6, 56.0, 55.9, 54.7, 41.8, 39.0, 31.6, 29.9, 29.7, 26.8, 26.4, 24.4, 22.7, 15.6, 15.3,
14.1, 11.5, 11.2, 10.5; HRMS (ESI): calcd for C43H59N3O10Na+ [M+Na+] 800.4093, found
800.4107.
(3S,6R,9S,12E,15R,16E,18E,20R,21S)-6-(3,5-Dichloro-4-hydroxybenzyl)-3-isobutyl-9-isopropyl-1
5-methoxy-10,12,16,20-tetramethyl-21-phenyl-1-oxa-4,7,10-triazacyclohenicosa-12,16,18-triene-
2,5,8,11-tetraone (LQ13)
28
LQ13 was obtained following the procedure for LQ18 in 62% yield. [α]D = -22.7 (c 0.1,
CHCl3); IR (KBr) νmax 3757, 3694, 3043, 1777, 1742, 1217, 761 cm-1; 1H NMR (600 MHz, CDCl3)
δ 7.33 (t, J = 7.2 Hz, 2H), 7.28 (d, J = 7.2 Hz, 1H), 7.22 (d, J = 7.2 Hz, 2H), 7.13 (s, 2H), 6.93 (d,
J = 8.4 Hz, 1H), 6.66 (d, J = 8.4 Hz, 1H), 6.30 (dd, J = 14.4, 10.8 Hz, 1H), 6.06 (s, 1H), 5.86 (d, J
= 10.2 Hz, 1H)., 5.84-5.80 (m, 1H), 5.77 (dd, J = 15.0, 4.8 Hz, 1H), 5.46 (t, J = 7.2 Hz, 1H),
4.64-4.54 (m, 1H), 4.54-4.43 (m, 2H), 3.57 (dd, J = 10.8, 2.4 Hz, 1H), 3.20 (s, 3H), 3.03 (dd, J =
13.8, 9.6 Hz, 1H), 2.77 (dd, J = 13.8, 5.4 Hz, 1H), 2.70 (s, 3H). 2.65-2.51 (m, 2H), 2.44-2.26 (m,
2H), 1.81 (s, 3H), 1.76 (s, 3H), 1.58-1.52 (m, 1H), 1.41-1.33 (m, 2H), 1.02 (d, J = 6.6 Hz, 3H),
0.96 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H), 0.85 (d, J = 6.6 Hz, 3H), 0.76 (d, J = 6.6 Hz,
3H); 13C NMR (150 MHz, CDCl3) δ 175.7 (C=O), 171.7 (C=O), 170.7 (C=O), 169.4 (C=O), 146.7,
20

ACCEPTED MANUSCRIPT
139.1, 136.2, 135.1, 133.3, 130.2, 129.5, 129.0, 128.5, 128.2, 128.0, 127.7, 125.9, 125.6, 120.9,
85.7, 78.1, 67.8, 61.8, 55.9, 54.3, 50.3, 42.3, 42.0, 35.4, 32.3, 31.8, 24.9, 24.7, 23.0, 21.8, 19.9,
18.5, 14.4, 11.3, 10.0; HRMS (ESI): calcd for C42H55Cl2N3O7Na+ [M+Na+] 806.3309, found
806.3332.
(3S,6R,9S,12E,15R,16E,18E,20R,21S)-6-(4-Hydroxybenzyl)-3,9-diisopropyl-15-methoxy-10,12,16,
20-tetramethyl-21-phenyl-1-oxa-4,7,10-triazacyclohenicosa-12,16,18-triene-2,5,8,11-tetraone
(LQ14)
28
LQ14 was obtained following the procedure for LQ18 in 64% yield. [α]D = -21.5 (c 0.19,
1
CHCl3); IR (KBr) νmax 3461, 3391, 1639, 1092, 738, 703 cm-1; H NMR (600 MHz, CDCl3) δ
7.37-7.18 (m, 5H), 7.06 (dd, J = 8.4, 2.4 Hz, 2H), 6.93 (dd, J = 8.4, 3.6 Hz, 1H), 6.71 (d, J = 8.4
Hz, 2H), 6.59 (dd, J = 9.0, 4.8 Hz, 1H), 6.30 (dd, J = 15.0, 10.8 Hz, 1H), 6.09 (s, 1H), 5.86 (d, J =
10.8 Hz, 1H), 5.78 (dd, J = 15.6, 4.8 Hz, 1H), 5.43 (t, J = 7.2 Hz, 1H), 4.62 (dd, J = 8.4, 3.0 Hz,
1H), 4.56 (q, J = 8.4, 1H), 4.51 (d, J = 11.4 Hz, 1H), 3.56 (dd, J = 10.2, 3.0 Hz, 1H), 3.20 (s, 3H),
3.01 (dd, J = 13.8, 9.0 Hz, 1H), 2.85 (dd, J = 13.8, 6.4 Hz, 1H), 2.69 (s, 3H). 2.64-2.52 (m, 2H),
2.43-2.35 (m, 1H), 2.35-2.27 (m, 1H), 2.27-2.18 (m, 1H), 1.81 (s, 3H), 1.75 (s, 3H), 1.03 (d, J =
6.8 Hz, 3H), 0.92 (d, J = 6.4 Hz, 3H), 0.85 (d, J = 6.6 Hz, 3H), 0.69 (d, J = 6.9 Hz, 3H), 0.51 (d, J
= 6.9 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 175.5 (C=O), 170.9 (C=O), 170.6 (C=O), 170.6
(C=O), 139.3, 136.3, 135.1, 133.3, 130.4, 130.4, 128.6, 128.2, 127.8, 127.8, 127.6, 125.8, 125.5,
115.4, 85.9, 78.0, 61.7, 56.1, 55.8, 54.7, 42.4, 36.0, 32.2, 31.8, 31.4, 24.9, 19.9, 19.1, 18.4, 16.3,
14.4, 11.2, 9.8; HRMS (ESI): calcd for C41H55N3O7Na+ [M+Na+] 724.3932, found 724.3925.
(3S,6R,9S,12E,15R,16E,18E,20R,21S)-6-(3,5-Dichloro-4-hydroxybenzyl)-16-ethyl-3,9-diisopropyl
-15-methoxy-10,12,20-trimethyl-21-phenyl-1-oxa-4,7,10-triazacyclohenicosa-12,16,18-triene-2,5,
8,11-tetraone (LQ15)
LQ15 was obtained following the procedure for LQ18 in 60% yield. [α]D28 = -90 (c 0.3, CHCl3);
IR (KBr) νmax 3680, 3078, 2903, 1750, 1708, 1384, 1242, 916 cm-1; 1H NMR (600 MHz, CDCl3) δ
7.40-7.19 (m, 5H), 7.12 (s, 2H), 6.90 (d, J = 8.4 Hz, 1H), 6.68 (d, J = 9.0 Hz, 1H), 6.33 (dd, J =
14.4, 10.8 Hz, 1H), 6.01 (s, 1H), 5.82 (d, J = 10.8 Hz, 1H), 5.84-5.75 (m, 1H), 5.70 (dd, J = 15.0,
6.0 Hz, 1H), 5.43 (t, J = 7.8 Hz, 1H), 4.60 (dd, J = 9.0, 3.6 Hz, 1H), 4.57-4.51 (m, 1H), 4.49 (d, J
= 11.4 Hz, 1H), 3.65 (dd, J = 9.6, 2.4 Hz, 1H), 3.26 (s, 1H), 3.05 (dd, J = 13.8, 9.6 Hz, 1H), 2.77
(dd, J = 13.2, 5.4 Hz, 1H), 2.67 (s, 3H), 2.64-2.56 (m, 2H), 2.48-2.38 (m, 1H), 2.36-2.28 (m, 1H),
2.28-2.20 (m, 1H), 2.21 (q, J = 7.8 Hz, 2H), 1.82 (s, 3H), 1.09 (t, J = 7.5 Hz, 3H), 1.00 (d, J = 6.8
Hz, 3H), 0.95 (d, J = 6.3 Hz, 3H), 0.86 (d, J = 6.5 Hz, 3H), 0.72 (d, J = 6.8 Hz, 3H), 0.56 (d, J =
6.8 Hz, 3H).; 13C NMR (150 MHz, CDCl3) δ 175.6 (C=O), 170.7 (C=O), 170.7 (C=O) , 170.1
(C=O), 146.6, 140.8, 138.8, 136.4, 132.9, 130.1, 129.0, 128.2, 128.1, 127.7, 127.6, 126.0, 125.7,
120.9, 84.4, 78.6, 61.9, 56.3, 56.2, 54.3, 42.5, 35.4, 32.2, 31.5, 31.2, 29.7, 24.9, 20.7, 19.9, 19.0,
21

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18.5, 16.5, 14.5, 14.4, 11.1; HRMS (ESI): calcd for C42H55Cl2N3O7Na+ [M+Na+] 806.3309, found
806.3336.
(3S,6R,9S,12E,15R,16E,18E,21S)-6-Benzyl-3,9-diisopropyl-15-methoxy-10,12,16-trimethyl-21-ph
enyl-1-oxa-4,7,10-triazacyclohenicosa-12,16,18-triene-2,5,8,11-tetraone (LQ17)
LQ17 was obtained following the procedure for LQ18 in 70% yield. [α]D28 = -52 (c 0.3, CHCl3);
1
IR (KBr) νmax 3564, 3520, 3482, 1644, 700, 665, 607 cm-1; H NMR (600 MHz, CDCl3) δ
7.34-7.19 (m, 9H), 7.18-7.13 (m, 1H), 6.95 (t, J = 9.0 Hz, 1H), 6.48 (t, J = 7.8 Hz, 1H), 6.37 (dd,
J = 14.4, 11.4 Hz, 1H), 5.89 (d, J = 11.4 Hz, 1H), 5.82 (d, J = 10.8 Hz, 1H), 5.59-5.49 (m, 1H),
5.40 (t, J = 7.2 Hz, 1H), 4.62-4.55 (m, 1H), 4.53 (d, J = 11.4 Hz, 1H), 4.43 (dd, J = 8.4, 3.6 Hz,
1H), 3.54 (dd, J = 10.8, 2.4 Hz, 1H), 3.18 (s, 3H), 3.07 (dd, J = 13.2, 9.6 Hz, 1H), 2.91 (dd, J =
13.8, 6.0 Hz, 1H), 2.76-2.67 (m, 1H), 2.64 (s, 3H), 2.61-2.53 (m, 1H), 2.53-2.46 (m, 1H),
2.40-2.28 (m, 2H), 1.95-1.84 (m, 2H), 1.82 (s, 3H), 1.75 (s, 3H), 0.95 (d, J = 6.6 Hz, 1H), 0.86 (d,
J = 6.6 Hz, 1H), 0.45 (d, J = 6.6 Hz, 1H), 0.30 (d, J = 7.2 Hz, 1H).; 13C NMR (150 MHz, CDCl3)
δ 175.5 (C=O), 171.1 (C=O), 170.5 (C=O), 170.2 (C=O), 139.9, 136.6, 135.7, 133.6, 129.5, 129.2,
129.0, 128.6, 128.5, 128.5, 128.4, 127.1, 126.7, 126.5, 86.1, 75.8, 61.5, 55.9, 55.8, 54.8, 40.3, 36.8,
32.1, 31.9, 31.3, 24.7, 19.8, 18.5, 18.3, 16.3, 14.3, 10.5.; HRMS (ESI): calcd for C40H53N3O6Na+
[M+Na+] 694.3827, found 694.3812.
4.4 Cell viability
Cancer cell viability after the treatment of LQ2-18 and nannocystin A was assessed by MTT
assay following manufacturer’s suggestion. LQ2-18 and nannocystin A were dissolved in pure
DMSO to prepare 10 mM stocking solution and diluted with culture medium. Cells (A549,
HCT-116, MCF-7, A375, SH-SY-5Y, and MDA-MB-231) at a density of 5 × 103 per well were
seeded in 96-well plates and cultured overnight, which were then treated with either vehicle (1%
DMSO PBS buffer) or desired concentrations of LQ2-18 and nannocystin A (10, 1, 0.1, 0.01,
0.001, and 0.0001 µM) for 72 h at 37 °C. Then, culturing medium was discarded and 100 µL fresh
media containing 0.5 mg/mL MTT was added. After 4 h of incubation at 37 °C, the supernatant
was removed and 100 µL DMSO was added to each well with the aid of gentle shaking. The
absorbance of the each well was measured at 570 nm in a microplate reader (Bio-Rad
Laboratories). At last, the resultant OD570 nm values were expressed as IC50 values, which were
the mean values derived from three independent experiments.
4.5 Colony formation assay
A549 cells were seeded in 6-well plates, each well contained 2 mL medium with 500 A549 cells
and incubated for 24 h. Then LQ18 (5, 10 and 20 nM) and nannocystin A (10 nM) in fresh
medium were added into the corresponding well to treat cells for a continuous 3 days, then
replaced by 2 mL of drug-free medium for every 3 days. 15 days after the cell plating, cell
22

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colonies were fixed in 95% ethanol for 15 min and stained by 0.1% crystal violet (Sigma), and
dried for colonies counting.
4.6 Cell cycle
A549 cells at the density of 1 × 105 cells/well were seeded in 6-well plates and treated with
LQ18 (30, 60 and 90 nM) and nannocystin A (30 nM) for 24 h at 37 °C. Then, the cells were
harvested and fixed with 70% precooled ethanol. Cells were then treated with RNase A, and
stained by PI (Product #: C1052, Beyotime, Jiangsu, China). Finally, the suspended cell was
analyzed with a flow cytometer (Accuri C6, BD Biosciences), a minimum total of 10,000 events
were recorded.
4.7 Cell apoptosis
A549 cells at a density of 2 × 105 cells/well were seeded on each well of 6-well plates and
allowed to grow overnight. Then the cells were treated with LQ18 (30, 60 and 90 nM) and
nannocystin A (30 nM) for 12 h (dose-dependent experiment), or cells were treated with LQ18 (60
nM) for 6, 12, and 24 h (time-dependent experiment), while cells without treatment were used as
control group. The treated cells were trypsinized and washed with cold PBS for three times, then
centrifuged at 1200 rpm for 5 min. The supernatants were discarded, and the cells were stained by
an Annexin-V-fluorescein isothiocyanate (FITC) kit with the binding buffer for 15 min.
Subsequently, the cells were then labeled by PI and the apoptotic cells were measured by a flow
cytometer (Accuri C6), data were analyzed with BD Accuri C6 Plus.
4.8 Western blotting
After the treatment of A549 cells with LQ18 (30, 60 and 90 nM) and nannocystin A (30 nM) for
24 h, the total proteins from A549 cells were extracted and their concentrations were balanced to
the same level by using BCA protein assay reagent (Beyotime, Jiangsu, China), followed by 8 min
of protein denaturation with SDS loading buffer at 100 °C. Proteins (30 µg) were separated by
sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and transferred onto
polyvinylidene fluoride (PVDF) membranes, which were blocked with 5 mL of 5% fat-free dry
milk in 1 × Tris-buffered saline (TBS) containing 0.05% Tween 20 for 2 h. Then the membranes
were incubated with caspase-3 (Beyotime, no: AC030), caspase-9 (Beyotime, no: AC062), Bcl-2
(Beyotime, no: AB112), Bad (Beyotime, no: AB008) and β-actin (Beyotime, no: AF0003), and
eEF1A1 (proteintech) antibodies at 4 °C for overnight, followed by treatment of secondary
horseradish-peroxidase-conjugated anti-rabbitIgG (Beyotime, no: A0208) for 2 h. Membranes
were finally scanned in a ChemiDoc MP Imaging System (Bio-Rad) after 2 min incubation in
Clarity Western ECL Substrate (Bio-Rad).
4.9 Statistical analysis
Data were reported as means ± SD. Statistical analysis was performed using GraphPad Prism
23

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version. *p < 0.05 was considered as statistically significant.
Acknowledgments
We thank the financial support from National Natural Science Foundation of China (No.
21807008, 21572027).
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Highlights
► The SAR of nannocystin A analogues was established based on the MTT assay and Western
blotting analysis
► LQ18 exhibited potent antiproliferative activities with IC50 values ranging from 4.3 to 48 nM
against tested cancer cell lines
► LQ18 inhibited eEF1A expression in A549 cell line