Cryptophycin-55/52 based antibody-drug conjugates: Synthesis, efficacy, and mode of action studies

Article abstract of DOI:10.1016/j.ejmech.2020.112364

Cryptophycin-52 (CR52), a tubulin inhibitor, exhibits promising antitumor activity in vitro (picomolar level) and in mouse xenograft models. However, the narrow therapeutic window in clinical trials limits its further development. Antibody-drug conjugate (ADC), formed by coupling cytotoxic compound (payload) to an antibody via a linker, can deliver drug to tumor locations in a targeted manner by antibody, enhancing the therapeutic effects and reducing toxic and side effects. In this study, we aim to explore the possibility of CR52-based ADC for tumor targeted therapy. Due to the lack of a coupling site in CR52, its prodrug cryptophycin-55 (CR55) containing a free hydroxyl was synthesized and conjugated to the model antibody trastuzumab (anti-HER2 antibody drug approved by FDA for breast cancer therapy) via the linkers based on Mc-NHS and Mc-Val-Cit-PAB-PNP. The average drug-to-antibody ratios (DARs) of trastuzumab-CR55 conjugates (named T-L1-CR55, T-L2-CR55, and T-L3-CR55) were 3.50, 3.29, and 3.35, respectively. These conjugates exhibited potent cytotoxicity in HER2-positive tumor cell lines with IC₅₀ values at low nanomolar levels (0.58–1.19 nM). Further, they displayed significant antitumor activities at the doses of 10 mg/kg in established ovarian cancer (SKOV3) and gastric cancer (NCI–N87) xenograft models without overt toxicities. Finally, the drug releases were analyzed and the results indicated that T-L3-CR55 was able to effectively release CR55 and further epoxidized to CR52, which may be responsible for its best performance in antitumor activities. In conclusion, our results demonstrated that these conjugates have the potential for tumor targeted therapy, which provides insights to further research the CR55/CR52-based ADC for tumor therapy.

Full text of DOI:10.1016/j.ejmech.2020.112364

Journal Pre-proof
Cryptophycin-55/52 based antibody-drug conjugates: Synthesis, efficacy, and mode
of action studies
Qinhuai Lai, Mengdan Wu, Ruixue Wang, Weirong Lai, Yiran Tao, Ying Lu, Yuxi
Wang, Lin Yu, Ruirui Zhang, Yujia Peng, Xiaohua Jiang, Yuyin Fu, Xin Wang,
Zhixiong Zhang, Cuiyu Guo, Wei Liao, Yiwen Zhang, Tairan Kang, Hao Chen, Yuqin
Yao, Lantu Gou, Jinliang Yang
PII:
S0223-5234(20)30334-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.112364
EJMECH 112364
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 December 2019
Revised Date: 17 April 2020
Accepted Date: 18 April 2020
Please cite this article as: Q. Lai, M. Wu, R. Wang, W. Lai, Y. Tao, Y. Lu, Y. Wang, L. Yu, R. Zhang,
Y. Peng, X. Jiang, Y. Fu, X. Wang, Z. Zhang, C. Guo, W. Liao, Y. Zhang, T. Kang, H. Chen, Y. Yao,
L. Gou, J. Yang, Cryptophycin-55/52 based antibody-drug conjugates: Synthesis, efficacy, and mode
of action studies, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2020.112364.
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2020 Published by Elsevier Masson SAS.

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Cryptophycin-55/52 based antibody-drug conjugates: synthesis,
efficacy, and mode of action studies
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Qinhuai Lai , Mengdan Wu , Ruixue Wang , Weirong Lai , Yiran Tao , Ying Lu ,
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Yuxi Wang , Lin Yu , Ruirui Zhang , Yujia Peng , Xiaohua Jiang , Yuyin Fu , Xin
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Wang , Zhixiong Zhang , Cuiyu Guo , Wei Liao , Yiwen Zhang , Tairan Kang , Hao
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Chen , Yuqin Yao , Lantu Gou , Jinliang Yang
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State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation
Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, P.R.
China.
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West China School of Public Health and West China Fourth Hospital, Healthy Food
Evaluation Research Center/ Sichuan University, Chengdu, P.R. China.
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Department of Respiratory and Critical Care Medicine, West China Hospital, Sichuan
University, Chengdu, P.R. China.
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Department of Clinical Laboratory, Mianyang Central Hospital, Mianyang,
P.R.China.
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College of Pharmacy and Biological Engineering, Chengdu University, Chengdu, P.R.
China.
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The 32265 Army Hospital of PLA, Guangzhou, P.R. China.
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Corresponding author: Jinliang Yang, State Key Laboratory of Biotherapy and
Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital,
Sichuan University, Chengdu, Sichuan, 610041, 3-17 People Road, P.R. China, Tel: +
86 28 85502796; fax: +86 28 85502796; e-mail: jinliangyang@scu.edu.cn
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Abbreviations:
CR52, cryptophycin-52; CR55, cryptophycin-55; ADC, antibody-drug conjugate;
HER2, human epidermal growth factor receptor 2; DAR, drug-to-antibody ratio;
Mc-NHS, N-succinimidyl 6-maleimidohexanoate; Mc-Val-Cit-PAB-PNP,
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maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcohol p-nitrophenyl
carbonate; PMBCl, 4-methoxybenzylchloride; DMP, dess-martin periodinane;
HWE, horner-wadsworth-emmons; DIBAL-H, diisobutylaluminum hydride;
TBS, tert-butyldimethylsilyl; TBAF, tetrabutylammonium fluoride;
Boc, t-butyloxycarboryl; LDA, lithium diisopropylamide;
EDCI, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide;
DMAP, 4-dimethylaminopyridine; TFA, trifluoroethanoicacid;
HATU, O-(7-azabenzotriazol-1-yl)N,N,N',N'tetramethyluroniumhexafluorophosphate;
m-CPBA, 3-chloroperoxybenzoic acid; TMSCl, trimethylchlorosilane;
TCEP, tris (2-chloroethyl) phosphate; DTT, dithiothreitol;
MFI, maean fluorescence intensity; PI, propidium iodide; NAc, N-Acetyl-cysteine;
DIPEA, N,N-diisopropylethylamine; DTPA, diethylenetriaminepentaacetic acid.
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Abstract
Cryptophycin-52 (CR52), a tubulin inhibitor, exhibits promising antitumor
activity in vitro (picomolar level) and in mouse xenograft models. However, the
narrow therapeutic window in clinical trials limits its further development.
Antibody-drug conjugate (ADC), formed by coupling cytotoxic compound (payload)
to an antibody via a linker, can deliver drug to tumor locations in a targeted manner by
antibody, enhancing the therapeutic effects and reducing toxic and side effects. In this
study, we aim to explore the possibility of CR52-based ADC for tumor targeted
therapy. Due to the lack of a coupling site in CR52, its prodrug cryptophycin-55
(CR55) containing a free hydroxyl was synthesized and conjugated to the model
antibody trastuzumab (anti-HER2 antibody drug approved by FDA for breast cancer
therapy) via the linkers based on Mc-NHS and Mc-Val-Cit-PAB-PNP. The average
drug-to-antibody ratios (DARs) of trastuzumab-CR55 conjugates (named T-L1-CR55,
T-L2-CR55, and T-L3-CR55) were 3.50, 3.29, and 3.35, respectively. These
conjugates exhibited potent cytotoxicity in HER2-positive tumor cell lines with IC50
values at low nanomolar levels (0.58-1.19 nM). Further, they displayed significant
antitumor activities at the doses of 10 mg/kg in established ovarian cancer (SKOV3)
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and gastric cancer (NCI-N87) xenograft models without overt toxicities. Finally, the
drug releases were analyzed and the results indicated that T-L3-CR55 was able to
effectively release CR55 and further epoxidized to CR52, which may be responsible
for its best performance in antitumor activities. In conclusion, our results
demonstrated that these conjugates have the potential for tumor targeted therapy,
which provides insights to further research the CR55/CR52-based ADC for tumor
therapy.
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Keywords
Cryptophycin-52 (CR52), cryptophycin-55 (CR55), chemical synthesis,
antibody-drug conjugate (ADC), trastuzumab, antitumor.
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1. Introduction
As a class of 16-membered macrocyclic depsipeptides, cryptophycins were first
isolated from the cultivated cyanobacteria Nostoc species in 1990[1]. Among the
many analogues, cryptophycin-1 (CR1, Fig. 1A) was the first to be named,
synthesized, and evaluated. However, its in vivo antitumor activity is not significant
mainly due to the instability of macrolide in blood circulation[2, 3]. After further
structure modification, a similar analogue cryptophycin-52 (CR52, Fig. 1A) stood out.
It only possesses one more methyl substitution than CR1 in the α-position of β-alanine
in fragment C (Fig. 1A), resulting in the increase of steric hindrance and thereby
significantly improving the stability in vivo[2, 4, 5]. CR52 binds to the vincristine site
of β-tubulin, interferes with the formation of microtubules, arrests cell mitosis in
G2/M phase, and eventually induces cell apoptosis[6, 7]. Generally, CR52 exhibits
100 to 1000 fold increased in vitro antitumor activity compared to taxol or vinca
alkaloids[4]. Furthermore, it even shows strong activity against multi-drug resistant
(MDR) tumor cell lines[3]. However, the clinical studies of CR52 were terminated in
phase II due to dose-dependent toxicity and lack of significant efficacy[8-11].
Antibody-drug conjugate (ADC) is composed of antibody, linker, and payload
(cytotoxic compound). After being conjugated to an antibody via a linker, payload can
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be delivered into tumor cells in a targeted manner by virtue of the antibody's high
selectivity for tumor-associated antigen. Thus, ADC integrates the targeting ability of
antibody and the potent cytotoxicity of payload, possessing potential advantages in
enhancing the therapeutic effect and reducing toxic and side effects[12,13]. Currently,
there are seven ADCs approved by FDA, including gemtuzumab ozogamicin
(Mylotarg)[14], brentuximab vedotin (Adcetris)[15], ado-trastuzumab emtansine
(Kadcyla)[16], inotuzumab ozogamicin (Besponsa)[17], polatuzumab vedotin
(Polivy)[18], enfortumab vedotin (Padcev)[19], and trastuzumab deruxtecan
(Enhertu)[20], and about 80 ADCs are being developed at clinical trials[13, 21]. The
recent approvals of several new ADCs has encourage us to investigate alternative
cytotoxic compounds as candidates for ADC payloads.
Payload is the key component of ADC to exert cytotoxic effects, which meets the
following principal requirements: (1) highly cytotoxic, usually with the IC value at
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low nanomolar or picomolar level, (2) clear target and action mechanism, (3)
(potential) chemical group for coupling[22, 23]. Tubulin inhibitors such as
maytansinoids (DM1/DM4), auristatins (MMAE/MMAF), and tubulysins, and DNA
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interactive
agents
such
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calichomycins,
camptothecins
(SN38),
pyrrolobenzodiazepines (PBD), and duocarmycins, are frequently applied as ADC
payloads[12, 13, 22, 24]. Similarly, CR52 or its analogues also meet these standards
and have potential as payloads for ADC. In recent years, Genentech have carried out
the research of cryptothycin-based ADCs, in which the benzylamine analogue of
CR52 was applied as a payload[25, 26]. Their work inspires us to further explore
CR52-based ADC. Conjugating a payload to an antibody requires a suitable linker.
Therefore, the design of linker structure is one of the major challenges in ADC field,
which needs to be stable in systemic circulation after administration to prevent the
premature release of payload but can effectively release payload or its active
derivative once internalization into tumor cells[27, 28]. In many of the linkers, the
non-cleavable Mc-NHS and the cathepsin B cleavable Mc-Val-Cit-PAB-PNP (Fig. 1B)
have been widely applied[28-33]. The targeted cytotoxicity of payload depends on the
specificity of antibody. Currently, trastuzumab, the humanized monoclonal antibody
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targeting HER2 for breast cancer therapy[34], has been often served as the model
antibody in ADC research. Based on trastuzumab, the ADC drugs T-DM1 (Kadcyla)
and DS-8201a (Enhertu) were approved by FDA in 2013 and in late 2019[16, 20],
respectively. Besides, another trastuzumab-based ADC SYD985 is currently evaluated
in advanced clinical trials, with good prospects[35, 36].
In this study, we explored the potential of CR52-based ADC for tumor targeted
therapy. Due to that CR52 lacks a coupling site, its prodrug cryptophycin-55 (CR55,
Fig. 1A) that contains a free hydroxyl and can re-epoxidize to CR52 under
physiological condition[37], was developed as a payload to conjugate with
trastuzumab via the linkers based on Mc-NHS and Mc-Val-Cit-PAB-PNP (Fig. 1B).
Here, we report the preparation and evaluation of antitumor activities of
trastuzumab-CR55 conjugates (T-L1-CR55, T-L2-CR55, and T-L3-CR55, Fig. 1C).
These conjugates exhibited significant in vitro and in vivo activities against
HER2-positive tumor cells. Our research provides insights to develop the
CR55/CR52-based ADC as antitumor drugs.
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Fig. 1. The design of trastuzumab-CR55 conjugates. (A) The structures of cryptophycins (CR1,
CR52, and CR55). (B) The structures of Mc-NHS and Mc-Val-Cit-PBA-PNP. (C)The structures of
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trastuzumab-CR55 conjugates (T-L1-CR55, T-L2-CR55, and T-L3-CR55). The red parts represent
the joints between linkers and CR55, and the blue parts represent the Val-Cit dipeptide. The
arrows points represent the possible payload release pathways.
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2. Results and discussions
2.1. The design of linkers-CR55
In this study, the hydroxyl of CR55 was developed as the coupling site. In order
to explore the influences of the different joints (between linkers and CR55) and the
presence or absence of cathepsin B cleavable Val-Cit sequence on antitumor activities,
we designed three linkers-CR55 (L1-CR55, L2-CR55, and L3-CR55) based on
Mc-NHS and Mc-Val-Cit-PAB-PNP (Fig. 1B). Their joints are glycine ester (Gly),
glycine ester (Gly), and methyl (2-(methyl amino) ethyl) carbamate (MeNN),
respectively. Besides, the Val-Cit dipeptide consists in L2-CR55 and L3-CR55 while
dosen’t in L1-CR55 (Fig. 1C). Therefore, the payload releases of these conjugates
(T-L1-CR55, T-L2-CR55, and T-L3-CR55) may depend on the carboxylesterase,
cathepsin B and (or) carboxylesterase, and cathepsin B, respectively (Fig. 1C).
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2.2. Chemistry
2.2.1. The synthesis of CR55 and CR52
According to the structure features, CR55 and CR52 can be divided into four
constituent units named fragments A, B, C, and D, connecting by lactone and lactam
bonds. Fragment A is the octanoic acid analogue with four continuous chiral centers
and β-epoxide, while fragments B, C, and D are derived from D-tyrosine, β-alanine,
and L-α-leucine, respectively (Fig. 1A). In our synthesis process, the precursors of the
four fragments were synthesized separately, and then connected in the order of B, C,
D, A, and B to form a macrolide, followed by the epoxidation of styrene, the
chlorohydrination of epoxide, and the re-epoxidation to obtain the target compounds.
Fragment A is the difficulty of the total synthesis of cryptophycins. We refered to
the work of Isao Shimizu in part to synthesize the fragment A precursor[38]. One of
the hydroxyl of the starting material 1,3-propanediol (A1) was protected by PMB,
while the other was oxidized to aldehyde by DMP, followed by HWE reaction to
extend the carbon chain. The resulting trans-α,β-unsaturated carboxylate (A4) was
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reduced by DIBAL-H to obtain allyl alcohol (A5). Then, the key Sharpless's
epoxidation was conducted to produce highly selective β-epoxy ethanol (A6),
followed by DMP oxidation and Wittig reaction to synthesize the cis-styrene (A8).
After that, the β-epoxide was opened by palladium catalyst to synthesize the
secondary alcohol (A9), which realized the constructions of the two continuous chiral
centers. After the PMB deprotection, TBS protection/deprotection, and DMP
oxidation, the second extension of carbon chain was achieved by HEW reaction to
construct α,β-unsaturated carboxylic acid tert-butyl ester (A14). Finally, fragment A
precursor (A15) was synthesized after the TBS deprotection by TBAF (Scheme 1).
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Scheme 1. The synthesis of fragment A precursor. Reagents and conditions: (1a) PMBCl, KOH,
DMSO, 0℃ -rt; (1b) DMP, NaHCO ,CH Cl , rt; (1c) triethyl 2-phosphonopropionate, DBU, LiCl,
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CH CN, rt; (1d) DIBAL-H, Et O, 0℃ ; (1e) Ti(Oi-Pr) , L-(+)-DET, TBHP, CH Cl , -25℃ ; (1f)
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Ph PPhCH Br, NaN(TMS) , HMPA, CH Cl , 0℃ -rt; (1g) Pd (dba) ·CHCl , n-Bu P, HCOOH,
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Et N, 1,4-dioxane, rt; (1h) AlCl , CH Cl , rt; (1i) TBSOTf, 2,6-lutidine, 0℃ ; (1j) p-TsOH·Py,
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MeOH, rt; (1k) tert-butyl diethylphosphonoacetate, DBU, LiCl, CH CN, rt; (1l) TBAF, THF, rt.
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By contrast, fragment B, C, and D were easier to be synthesized. Starting from
D-tyrosine, fragment B precursor (B5) was obtained by four steps reactions including
chlorination in the ortho position of phenol, Boc protection of amino, methoxylation
using (CH ) SO , and ester hydrolysis by NaOH. Fragment C precursor (C4) is an
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analogue of β-alanine, and the key step is the introduction of α-dimethyl under the
action of LDA/MeI after the carboxyl and amino protections. Fragment D precursor
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(D2) was obtained from L-α-leucic acid after carboxyl protection by allyl bromide.
After that, the three precursors were connected in the order of B, C, and D through the
amide and ester condensations, and then the allyl was reduced by Pd(PPh ) to get the
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fragment BCD precursor (BCD-2) (Scheme 2).
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Scheme 2. The synthesis of fragment BCD precursor. Reagents and conditions: (2a) sulfuryl
chloride AcOH, rt; (2b) (Boc) O, Et N, 1,4-dioxane, H O; (2c) (CH ) SO , K CO , CH CN, 80℃ ;
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(2d) NaOH (1 M), MeOH, rt; (2e) LDA, MeI, THF, -78℃ -rt; (2f) 1 M HCl in 1,4-dioxane, CH Cl ,
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+
-
rt; (2g) Na CO , allyl bromide, Bu N Cl , H O, CH Cl , rt; (2h) EDCI·HCl, HOBt, DIPEA,
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CH Cl , rt; (2i) D2, EDCI·HCl, DMAP, CH Cl , rt; (2j) morpholine, Pd(PPh ) , THF, rt.
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With the obtainings of fragments A and BCD precursors, the ring closure process
began as shown in Scheme 3. Firstly, they were connected through the ester
condensation under the action of EDCI/DMAP. Then, the protections of tert-butyl and
Boc were simultaneously removed by TFA, followed by the amide condensation by
HATU to produce the macrolide. After that, The styrene (CR51) was epoxidized by
m-CPBA to generate the mixed epoxides, consisting of CR52 and its isomer (CR53),
which were difficult to separate by chromatography. In this study, we conducted a
further chlorohydrination by TMSCl at -60 ℃ to obtain the mixed chlorohydrins,
which were easily separated by preparative TLC to get the target compound CR55.
Finally, CR52 was obtained from CR55 after the epoxidation in the presence of
K CO at room temperature (Scheme 3).
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Scheme 3. The synthesis of target compounds CR55 and CR52. Reagents and conditions: (3a)
EDCI·HCl, DMAP, CH Cl , 35℃ ; (3b) 1) TFA, CH Cl , rt; 2) HATU, DIPEA, CH Cl , rt; (3c)
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m-CPBA, CH Cl , rt; (3d) TMSCl, CHCl , -60℃ ; (3e) K CO , CH CN, rt.
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2.2.2. The synthesis of linkers-CR55
The synthesis of linkers-CR55 is outlined in Scheme 4. After the esterification of
CR55 with bocglycine under the action of EDCI/DMAP and the deprotection of Boc
by TFA, Mc-NHS and Mc-Val-Cit-PAB-PNP were attatched to generate the target
compounds L1-CR55 (Mc-Gly-CR55) and L2-CR55 (Mc-Val-Cit-PAB-Gly-CR55),
respectively. In addition, CR55 was also reacted with p-nitrophenyl chloroformate in
the presence of DMAP to activate the hydroxyl, followed by the introduction of
tert-butyl methyl (2-(methyl amino) ethyl) carbamate. Then, Mc-Val-Cit-PAB-PNP
was attached after the Boc deprotection to generate the target compound L3-CR55
(Mc-Val-Cit-PAB-MeNN-CR55).
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Scheme 4. The synthesis of linkers-CR55 (L1-CR55, L2-CR55, and L3-CR55). Reagents and
conditions: (4a) Bocglycine, EDCI·HCl, DMAP, CH Cl , 35℃ ; (4b) 1) TFA, CH Cl , rt; 2)
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Mc-NHS, DIPEA, DMF, rt; (4c) 1) TFA, CH Cl , rt; 2) Mc-Val-Cit-PAB-PNP, DIPEA, DMF, rt;
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(4d) p-nitrophenyl chloroformate, DMAP, CH Cl , rt; (4e) tert-butyl methyl (2-(methyl amino)
2
2
ethyl) carbamate, CH Cl , rt.
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2.3. Preparation and charaterization of trastuzumab-CR55 conjugates
Two stages were needed for coupling CR55 to trastuzumab. The first is the
reduction of interchain disulfide bonds of trastuzumab by TCEP to make the thiols
free. And another is the coupling reaction by adding 4-fold molar excess of
linkers-CR55 to initiate the michael addition between the thiol and maleimide to
generate three novel trastuzumab-CR55 conjugates (T-L1-CR55, T-L2-CR55, and
T-L3-CR55) (Fig. 2A).
After preparation, these conjugates were detected by SDS-PAGE. As shown in
Fig. 2B, there were multiple bands under the non-reductive conditions, which were
caused by partial reduction of interchain disulfide bonds, and is consistent with the
bands of TCEP-treated trastuzumab. However, when they were completely reduced
into light chains and heavy chains, all the bands are cleaner and clearer, indicating that
the physicochemical properties of trastuzumab did not change significantly after
1
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43
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coupling with linkers-CR55. In addition, these conjugates were analysized by LC/MS
technology after deglycosylation by PNGase F and complete reduction by DTT, and
demonstrated that their average drug-to-antibody ratios (DARs) were 3.50, 3.29, and
3.35, respectively. T-L1-CR55 was took as an example to analyze and calculate the
DAR. As shown in Fig. 3, the molecular weight differences of adjacent heavy chains
and light chains peaks are about 956 Da, which is consistent with the molecular
weight of L1-CR55 (955.9 Da), indicating that L1-CR55 was successfully coupled to
trastuzumab, and each additional 956 Da means one molecular of L1-CR55 to be
conjugated (Fig. 3).
2
2
46
47
2
2
2
48
49
50
Fig. 2. The preparations of trastuzumab-CR55 conjugates. (A) The preparation diagram of
trastuzumab-CR55 conjugates. (B) The SDS-PAGE analysis of trastuzumab-CR55 conjugates.
1
1

2
2
2
2
2
2
51
52
53
54
55
56
Fig. 3. The molecular weight distributions of trastuzumab-CR55 conjugates (taking
T-L1-CR55 as example). The light chains and heavy chains were analyzed separately by LC/MS,
and each additional 956 Da means one molecular of L1-CR55 to be conjugated to antibody. The
DARs of light chains and heavy chains are caculated as 0.49 and 3.01, respectively, and the
average DAR of the whole T-L1-CR55 is 3.50.
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2.4. The targeting and internalization of trastuzumab-CR55 conjugates
Flow cytometry was applied to detect the targeting of trastuzumab-CR55
conjugates (T-L1-CR55, T-L2-CR55, and T-L3-CR55) against human ovarian
carcinoma cell line SKOV3 (HER2 3+), human gastric cancer cell line NCI-N87
(HER2 3+), and human breast carcinoma cell line MDA-MB-231 (HER2 0-1+). As
shown in Fig. 4A, these conjugates possessed similar targeting as trastuzumab. All of
them highly targeted against SKOV3 and NCI-N87 cells with the ratios of mean
fluorescence intensity (rMFI) at the ranges of 85.4-90.4 (SKOV3) and 152.6-168.4
(NCI-N87), which were consistent with their high HER2 expression. However, the
targeting against MDA-MB-231 cell were significantly reduced (rMFI=1.7-3.2) due to
the weak HER2 expression. These results suggested that the targeting of trastuzumab
had no significant change after coupling with linkers-CR55.
Meanwhile, their internalizations were also detected by flow cytometry.
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HER2-positive SKOV3 and NCI-N87 cells were employed for the experiment. The
primary antibodies (trastuzumab-CR55 conjugates and trastuzumab) were incubated
for a certain time (0 h, 3 h, and 6 h) at 37 ℃ , followed by the incubation of secondary
antibody (goat anti-human IgG/FITC) at 4 ℃ . The results showed that the conjugates
and trastuzumab possessed similar internalization levels. As shown in Fig. 4B, the
curve shifts slightly from rightmost to left with the extension of the internalization
time. They showed the internalization rates of 11.0%-19.4% at 3 h and 24.3%-29.9%
at 6 h in SKOV3 cells. As for NCI-N87 cells, the internalization rates were
15.4%-25.9% at 3 h and 34.2%-38.4% at 6 h. In general, the internalization level of
NCI-N87 cell was higher than that of SKOV3 cell, which is related to their HER2
expression. These results suggested that the internalization of trastuzumab did not
significantly alter after linkers-CR55 were coupled.
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81
82
1
3

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83
84
85
86
87
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90
Fig. 4. The targeting and internalization of trastuzumab-CR55 conjugates detected by flow
cytometry. (A) The targeting of trastuzumab-CR55 conjugates. The red and green line
respectively represents PBS control and the conjugates (or trastuzumab). The ratios of mean
fluorescence intensity (rMFI) are attatched on the diagrams. (B) The internalization of
trastuzumab-CR55 conjugates. The red, green, blue, and brown line represents PBS control,
internalization at 0 h, 3 h, and 6 h, respectively. The internalization rate (%) are attatched on the
diagrams.
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2.5. The in vitro antitumor activities of trastuzumab-CR55 conjugates
CCK-8 assay was used to detect the in vitro antitumor activities of
trastuzumab-CR55 conjugates against SKOV3 (HER2 3+), NCI-N87 (HER2 3+), and
MDA-MB-231 (HER2 0-1+). These conjugates exhibited potent antiproliferative
activities against HER2-positive SKOV3 and NCI-N87 cells, with IC50 at a low
nanomolar level ranging from 0.58-1.19 nM. While for HER2-negative or weakly
positive MDA-MB-231 cells, their activities were significantly reduced, with IC50
values of about 200-300 nM. Trastuzumab did not show antitumor effect in the
concentration ranges, with IC values greater than 1000 nM (Table 1).
5
0
Meanwhile, payloads (CR55 and CR52) and linkers-CR55 (L1-CR55, L2-CR55,
and L3-CR55) were also evaluated. Both CR55 and CR52 exhibited strong
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cytotoxicity with IC values at the picomolar level (IC = 0.021-0.053 nM). However,
50 50
when CR55 was derivatized by linkers, the activities of resulting linkers-CR55 were
significantly reduced by about 1000 times, with IC values increasing to nanomolar
50
level (IC = 17.4-40.7 nM) (Table 1). Chemical structure analysis indicated that the
5
0
decreases in in vitro activities of linkers-CR55 may be attributed to their much lower
membrane permeability. However, when coupled to trastuzumab, they can enter tumor
cells mediated by antibody targeting and internalization, which significantly increases
the concentrations of payloads in HER2-positive cells and thereby exerts potent
antitumor activities. These results indicated that there was no intercellular difference
in antitumor activities between payloads and linker-CR55, while the activities of the
conjugates were related to the HER2 expression of tumor cells.
3
3
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14
Table 1. The in vitro antitumor activities of trastuzumab-CR55 conjugates, payloads, and
linkers-CR55
IC (nM)
5
0
Drugs
SKOV3
HER2 3+)
NCI-N87
(HER2 3+)
MDA-MB-231
(HER2 0-1+)
(
CR52
CR55
0.028 ± 0.008
0.021 ± 0.003
18.2 ± 2.7
19.5 ± 2.9
25.6 ± 4.2
0.85 ± 0.36
`1.19 ± 0.43
0.64 ± 0.18
>1000
0.053 ± 0.02
0.036 ± 0.007
29.6 ± 4.4
33.3 ± 4.7
40.7 ± 9.1
1.04 ± 0.32
0.88 ± 0.23
0.58 ± 0.20
>1000
0.040 ± 0.005
0.045 ± 0.014
28.9 ± 5.3
17.4 ± 1.1
35.7 ± 6.5
272 ± 88
L1-CR55
L2-CR55
L3-CR55
T-L1-CR55
T-L2-CR55
T-L3-CR55
trastuzumab
306 ± 93
233 ± 66
>1000
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15
16
17
18
19
20
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2.6.Trastuzumab-CR55 conjugates inhibit cell cycle and induce apoptosis
The cell cycle arrests and apoptosis induced by trastuzumab-CR55 conjugates
(T-L1-CR55, T-L2-CR55, and T-L3-CR55) in SKOV3 cell were detected by flow
cytometry.
The content distributions of cellular DNA were analyzed by propidine iodide (PI)
single staining method after the exposure of cells to these conjugates for 24 hours. As
shown in Fig. 5A, 1 nM of T-L3-CR55 obviously arrested the cell cycle, with G2/M
phase cells at the percentage of 41.15%, while T-L1-CR55 and T-L2-CR55 did not,
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with the percentages only at 23.46% and 18.84%, respectively, equivalent to the
negative control (17.78%). But when the concentration increased to 5 nM, the cell
cycle arrests enhanced significantly, with the percentages at 56.52%, 62.26%, and
71.39%, respectively (Fig. 5A).
Meanwhile, the apoptosis assay was also conducted using Annexin V-FITC and
PI double staining, and the cells were treated with these conjugates for 48 hours.
-
-
+
-
+
+
-
+
Annexin V /PI , Annexin V /PI , Annexin V /PI , and Annexin V /PI are represented
as living cells, early apoptotic cells, late apoptotic cells, and necrotic cells,
respectively[39, 40]. As shown in Fig. 5B, conjugates at both concentrations of 1 nM
+
and 5 nM induced apoptosis. The percentages of apoptotic cells (Annexin-V ) at 1 nM
were 17.61%, 23.46%, and 30.87%, respectively, while negative control was only
4.91%. And when the concentration inceased to 5 nM, the apoptosis was much more
obvious, with the percentages at 47.32%, 58.42%, and 59.64%, respectively (Fig. 5B).
1
6

3
3
36
37
Fig. 5. Trastuzumab-CR55 conjugates induce cell cycle arrest and apoptosis detected by flow
cytometry. (A) Cell cycle was arrested at G2/M phase by trastuzumab-CR55 conjugates. SKOV3
cell was treated with these conjugates at the concentrations of 1 nM and 5 nM for 24 h, and then
collected, stained with PI, and finally analyzed by flow cytometry. (B) Trastuzumab-CR55
conjugates induced cell apoptosis. SKOV3 cell was treated with these conjugates at the
concentration of 1 nM and 5 nM for 48 h, and then collected, stained with Annexin V-FITC and PI,
and finally analyzed by flow cytometry.
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39
40
41
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43
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2.7. The in vivo antitumor activities of trastuzumab-CR55 conjugates
The subcutaneous tumor-bearing models of SKOV3 and NCI-N87 in BALB/c
nude mice were established to preliminarily evaluated the in vivo antitumor activities
of trastuzumab-CR55 conjugates (T-L1-CR55, T-L2-CR55, and T-L3-CR55). When
3
tumor volumes reached to about 200 mm , the mice were randomly assigned to five
groups and administered with conjugates (10 mg/kg, 5 mg/kg, and 1 mg/kg),
trastuzumab (10 mg/kg), or equal volume of normal saline by tail vein injection once
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every three days for three times.
The results showed that the conjugates at the doses of 10 mg/kg and 5 mg/kg
significantly delayed tumor growth, while dosed at 1 mg/kg did not, only slightly
better than trastuzumab (10 mg/kg) (Fig. 6). Generally, T-L3-CR55 exhibited better in
vivo antitumor activities than T-L1-CR55 and T-L2-CR55. T-L3-CR55 dosed at 10
mg/kg can keep the tumor from growing for nearly four weeks in both models.
However, when given the same dose of T-L1-CR55 or T-L2-CR55, the tumor volumes
increased gradually after drug withdrawn in SKOV3 model, or tumor growth was
inhibited for only two weeks in NCI-N87 model (Fig. 6). Further, we calculated that
the dose of CR55/CR52 equivalent to 10 mg/kg of the conjugates was about 0.15
mg/kg, which was much lower than that of CR55/CR52 or other cryptophycin
analogues that exhibited similar in vivo antitumor activities when administered
directly in several xenograft models (usually greater than 10 mg/kg)[37, 41, 42],
indicating an effective targeted delivery of CR55/CR52 based on ADC technology.
During the whole treatments, no significant changes of mice body weight were
observed (Fig. 6), and H&E staining proved that there were no observable pathologic
changes in vital organs, including heart, liver, spleen, lung, and kidney, after
treatment with the conjugates dosed at 10 mg/kg (Fig. 7), which suggested that our
conjugates did not show obvious toxic and side effects at given doses.
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70
71
Fig. 6. The antitumor activities of trastuzumab-CR55 conjugates against SKOV3 and
NCI-N87 subcutaneous tumor bearing models. From left to right respectively shows the
antitumor activities of T-L1-CR55, T-L2-CR55, and T-L3-CR55 against SKOV3 model (A) and
NCI-N87 model (B) in vivo. The arrow indicates the time points of administration.
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79
Fig. 7. Representative images of H&E-stained vital organs of the mice. No obvious changes
were observed in these organs, including heart, liver, spleen, lung, and kidney, after treatment with
T-L1-CR55, T-L2-CR55, and T-L3-CR55 at the dose of 10 mg/kg once every two days for three
times, and the normal saline was used as control. Magnification, × 400.
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2.8. The payload release processes of trastuzumab-CR55 conjugates
To verify the release of CR55 from its glycine ester catalyzed by
carboxylesterase and the epoxidation from CR55 to CR52, we simulated the processes
by incubating glyCR55 and CR55 in mouse plasma at 37 ℃ , and then sampled at
indicated time pionts and detected by HPLC. As shown in the Fig. 8A, CR55 was
gradually released from glyCR55 by carboxylesterase-catalyzed hydrolysis and
further epoxidized to CR52. The percentages of glyCR55, CR55, and CR52 were
11.52%, 10.42%, and 78.06% after incubation for seven days, respectively.
Meanwhile, CR55 was also incubated with mouse plasma and completely epoxidized
to CR52 on the seventh day (Fig. 8B). However, the epoxidation of CR55 in PBS with
pH 7.0, pH 6.5, and pH 5.0 gradually weakened to undetectable (Fig. 8C), indicating
that the epoxidation requires the diffusion of CR55 from the acidic lysosome to the
cytoplasm or extracellular matrix.
Further, the N-acetylcysteine-enclosed linkers-CR55 (NAc-linkers-CR55),
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serving as the surrogates of trastuzumab-CR55 conjugates, were also incubated with
mouse plasma. As shown in the Fig. 8D, the percentages of NAc-L1-CR55,
NAc-L2-CR55, and NAc-L3-CR55 remained at 86.99%, 87.01%, and 89.10% five
days later, respectively, indicating a good plasma stability. Obviously, we failed to
detect CR55 released from NAc-L3-CR55 that only contains the cathepsin B
cleavable Val-Cit sequence, which may be attributed to that the required enzyme is
not in plasma but mainly in endolysosomal vesicles[43]. However, there was also no
detection of CR55 released from NAc-L1-CR55 or NAc-L2-CR55 that contain the
glycine ester, which was different from glyCR55 described above and possibly due to
the change of chemical environment around glycine ester after the derivatization of
glyCR55 by Mc-NHS or Mc-Val-Cit-PAB-PNP, resulting in the significant reduction
of carboxylesterase hydrolysis ability.
As the payload release process of cathepsin B cleavable Val-Cit based linker that
has been widely applied in ADC is already validated[28-31]. Therefore, combined
with the above results, the intracellular processes of these conjugates can be inferred
as follows (Fig. 8E). After internalizing into lysosome, the conjugates will be
degraded to cysteine adducts (Cys-linkers-CR55) by protease. Among them,
Cys-L3-CR55 and Cys-L2-CR55 can effectively release CR55 and glyCR55,
respectively, based on the cathepsin B cleavable trigger. While T-L1-CR55 may only
remain in the stage of Cys-L1-CR55 like a non-cleavable ADC. For T-L2-CR55, a
further release of CR55 from glyCR55 is still required, but the degree depends on the
activity and substrate selectivity of carboxylesterase in the lysosome [44, 45].
Subsequently, the newly released CR55 diffuses from lysosome to cytoplasm or
extracellular matrix and ultimately epoxidize to CR52, playing the antitumor activities
(including bystander effect). However, most of the charged Cys-L1-CR55 may mainly
rely on the lysosomal transport pathway to reach the cytoplasm before it can play an
antitumor role [46, 47]. In conclusion, these results indicated that only T-L3-CR55 can
effectively release CR55/CR52, which may be associated with its better antitumor
activities than the two others.
2
1

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24
25
26
27
28
29
30
31
32
Fig. 8. The payload release processes of trastuzumab-CR55 conjugates. (A) The release of
CR55 from glyCR55 and futher epoxidation to CR52. glyCR55 was incubated in mouse plasma at
37 ℃ for seven days. (b) The epoxidation of CR55 to CR52. CR55 was incubated in mouse plasma
at 37 ℃ for seven days. (C) The epoxidation of CR55 to CR52. CR55 was incubated in PBS with
pH 7.0, 6.5, and 5.0 at 37 ℃ for three days, and the ability of epoxidation was gradully decreased.
(D) The stability of NAc-enclosed linkers-CR55 (NAc-linkers-CR55) serving as the surogates of
trastuzumab-CR55 conjugates in mouse plasma at 37 ℃ for 5 days. (E) The payload release
processes of trastuzumab-CR55 conjugates.
4
4
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4
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4
33
34
35
36
37
38
3. Conclusions
The potent antitumor activity makes cryptophycins candidate payloads for ADC
research. In this study, we developed cryptophycin-55 (CR55), the prodrug of
cryptophycin-52 (CR52), to conjugate with the model antibody trastuzumab via the
linkers based on Mc-NHS and Mc-Val-Cit-PAB-PNP, and proved that the
trastuzumab-CR55 conjugates (T-L1-CR55, T-L2-CR55, T-L3-CR55) showed
2
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4
4
4
4
39
40
41
42
significant antitumor activities in HER2-positive xenograft models. Among them,
T-L3-CR55 performed better than the two others, which may be associated with its
effective payload release. Our results indicated that the CR55/CR52-based ADCs have
the potentials for tumor targeted therapy and are worthy for further research.
4
43
4. Experimental section
4
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45
46
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50
51
52
53
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4.1. General chemistry
All the chemicals and reagents were commercially available without further
purification. TLC was performed on 0.20 mm silica gel 60 F254 plates (Qingdao
1
13
Haiyang Chemical, China). H NMR and C NMR spectra were recorded on a Bruker
Avance 400 spectrometer (Bruker Company, Germany), using TMS as an internal
standard and CDCl , DMSO-d , and CD OD as solvents. The chemical shifts are
3
6
3
given as parts per million (ppm) relative to TMS. The mass spectral data were
collected on a Q-TOF Premier mass spectrometer (Micromass, Manchester, UK). The
purities of compounds CR55, CR52, L1-CR55, L2-CR55, and L3-CR55 were
determined by reverse-phase HPLC (Xbridge column, C18, 4.6 mm × 150 mm, 5 μm)
using a Water 2695 series LC system.
4
55
4.2. The synthesis of CR55 and CR52
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(1) Compound A2
To the stirring solution of compound A1 (48.6 g, 638.5 mmol) in 225 mL DMSO
at 0 ℃ was added KOH (35.8 g, 638.5 mmol), followed by PMBCl (50.0 g, 319.3
mmol) over a period of about 30 minutes. The mixture was stirred at room
temperature and monitored by TLC. After 5 hours, 250 mL diethyl ether was added,
and the mixture was stirred at 0 ℃ again, followed by the addition of 125 mL
hydrochloric acid (5 M) to quench the reaction. The aqueous phase was separated
from the mixture and extracted with diethyl ether (200 mL × 2). The combined
organic phase (diethyl ether) was successively washed with brine (300 mL × 1), dried
over Na SO , filtered, and concentrated. Purification by column chromatography on
2
4
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3

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silica gel (ethyl acetate/petroleum ether = 1/3 to 1/5) gave the compound A2 as a light
1
yellow oil (40.9 g) in 65.3% yield. H NMR (400 MHz, CDCl ) δ 7.25 (d, J = 8.6 Hz,
3
2H), 6.88 (d, J = 8.6 Hz, 2H), 4.45 (s, 2H), 3.80 (s, 3H), 3.76 (t, J = 5.7 Hz, 2H), 3.63
1
3
(t, J = 5.8 Hz, 2H), 2.38 (s, 1H), 1.84 (p, J = 5.8 Hz, 2H). C NMR (101 MHz, CDCl )
3
δ 159.2, 130.2, 129.3, 113.8, 72.9, 69.0, 61.8, 55.3, 32.1. HRMS (ESI, m/z) calcd for
+
C H O [M+Na] 219.0992, found 219.0998.
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72
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87
(2) Compound A3
To the stirring solution of compound A2 (20.0 g, 101.9 mmol) in 300 mL CH Cl
2
2
at room temperature was added NaHCO (25.7 g, 305.8 mmol), followed by DMP
3
(64.8 g, 152.9 mmol) in batches. After 30 minutes, TLC showed that the reaction was
completed. Then, the mixture was filtered and the filtrate was set aside to produce
many floccules and filtered twice again. To the filtrate was added saturated NaHCO3
aqueous solution in batches (still produce many bubbles and floccules) untill no
bubbles. After filtration again, the aqueous phase was separated from the mixture and
extracted with ethyl acetate (200 mL × 2). The combined organic phase was
successively washed with brine (300 mL × 1), dried over Na SO , filtered, and
2
4
concentrated. Purification by column chromatography on silica gel (ethyl
acetate/petroleum ether = 1/8 to 1/5) gave the compound A3 as a light yellow oil (13.0
1
g) in 65.8% yield. H NMR (400 MHz, CDCl ) δ 9.77 (s, 1H), 7.24 (d, J = 8.6 Hz,
3
2H), 6.88 (d, J = 8.6 Hz, 2H), 4.45 (s, 2H), 3.79 (s, 3H), 3.79-3.73 (m, 2H), 2.71-2.62
1
3
(m, 2H). C NMR (101 MHz, CDCl ) δ 201.3, 159.3, 130.0, 129.4, 113.8, 72.9, 63.5,
3
+
55.3, 43.9. HRMS (ESI) calcd for C H O [M+Na] 217.0835, found 217.0839.
11
14
3
4
4
4
4
4
4
88
89
90
91
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93
(3) Compound A4
Triethyl 2-phosphonopropionate (61.3 g, 257.4 mmol), DBU (23.5 g, 154.5
mmol), and LiCl (7.6 g, 132.4 mmol) were respectively added to a 1000 mL
round-bottom flask, followed by 250 mL CH CN, and the resulting mixture was
3
stirred vigorously at room temperature. After 30 minutes, the solution of compound
A3 (25.0 g, 128.7 mmol) in CH CN (120 mL) was added dropwise into the above
3
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reaction mixture. After stirring at room temperature for 1 hour, TLC showed that the
reaction was completed. To the reaction mixture was added 200 mL saturated NH Cl
4
aqueous solution, and the aqueous phase was separated from the mixture and
extracted with ethyl acetate (150 mL × 3). The combined organic phase was
successively washed with brine (300 mL × 1), dried over Na SO , filtered, and
2
4
concentrated. Purification by column chromatography on silica gel (ethyl
acetate/petroleum ether = 1/6) gave the compound A4 as a light yellow oil (30.2 g) in
1
84.3% yield. H NMR (400 MHz, CDCl ) δ 7.26 (d, J = 8.6 Hz, 2H), 6.88 (d, J = 8.6
3
Hz, 2H), 6.77 (td, J = 7.2, 1.5 Hz, 1H), 4.46 (s, 2H), 4.19 (q, J = 7.1 Hz, 2H), 3.80 (s,
3H), 3.53 (t, J = 6.8 Hz, 2H), 2.48 (q, J = 7.0 Hz, 2H), 1.84 (s, 3H), 1.29 (t, J = 7.1 Hz,
1
3
3H). C NMR (101 MHz, CDCl ) δ 159.2, 138.3, 130.3, 129.4, 129.3, 113.8, 72.7,
3
+
68.3, 60.5, 55.3, 29.4, 14.3, 12.6. HRMS (ESI) calcd for C H O [M+Na] 301.1410,
1
6
22
4
found 301.1413.
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
(4) Compound A5
Compound A4 (20.0 g, 70.9 mmol) was added to a 500 mL double-mouth round
bottom flask, followed by 200 mL dry diethyl ether. Then, 150 mL DIBAL-H (1 M in
hexane) was added dropwise under N atmosphere at 0 ℃ . The resulting mixture was
2
stirred at room temperature for 2 hours and monitored by TLC. After completion, 100
mL MeOH and water were added dropwise at 0 ℃ , which resulted in producing a lot
of white solids. The mixture was filtered, and the aqueous phase was separated from
the filtrate and extracted with ethyl acetate (100 mL × 2). The combined organic
phase was successively washed with brine (200 mL × 1), dried over Na SO , filtered,
2
4
and concentrated. Purification by column chromatography on silica gel (ethyl
acetate/petroleum ether = 1/5) gave the compound A5 as a light yellow oil (15.1g) in
1
89.7% yield. H NMR (400 MHz, CDCl ) δ 7.28-7.24 (m, 2H), 6.88 (d, J = 8.6 Hz,
3
2H), 5.47-5.40 (m, 1H), 4.45 (s, 2H), 4.00 (s, 2H), 3.80 (s, 3H), 3.46 (t, J = 7.0 Hz,
1
3
2H), 2.36 (q, J = 6.8 Hz, 2H), 1.68 (s, 3H). C NMR (101 MHz, CDCl ) δ 159.2,
3
136.8, 130.5, 129.3, 121.8, 113.8, 72.5, 69.4, 68.5, 55.3, 28.3, 13.8. HRMS (ESI)
+
calcd for C H O [M+Na] 259.1305, found 259.1305.
1
4
20
3
2
5

5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
(5) Compound A6
Compound A5 (10.0 g, 42.4 mmol), Ti(Oi-Pr) (2.6 mL, 8.9 mmol), L-(+)-DET
4
(1.7 mL, 9.7 mmol), and 5Å molecular sieve (15 g) were added respectively to a 250
mL round bottom flask, followed by the injection of 100 mL CH Cl under N
2
2
2
atmosphere. After stirring at -25 ℃ for 30 minutes, TBHP (23.1 mL, 127.0 mmol) was
injected into above solution. The mixture was strred at this tempreature for more than
12 hours and monitored by TLC. After the reaction was completed, the mixture was
filtered, and the filtrate was successively washed with saturated NaF aqueous solution
(100 mL × 1), brine (200 mL × 1), dried over Na SO , filtered, and concentrated.
2
4
Purification by column chromatography on silica gel (ethyl acetate/petroleum ether =
1/4, 1/1 and 2/1) gave the compound A5 as a light yellow oil (9.0 g) in 83.9% yield.
1
H NMR (400 MHz, CDCl ) δ 7.26 (d, J = 8.5 Hz, 2H), 6.88 (d, J = 8.6 Hz, 2H), 4.46
3
(s, 2H), 3.80 (s, 3H), 3.70-3.54 (m, 4H), 3.16 (dd, J = 7.0, 5.2 Hz, 1H), 2.19-2.16 (m,
1
3
1H), 1.97-1.80 (m, 2H), 1.28 (s, 3H). C NMR (101 MHz, CDCl ) δ 159.2, 130.3,
3
129.2, 113.8, 72.8, 67.1, 65.5, 60.9, 58.0, 55.3, 29.0, 14.4. HRMS (ESI) calcd for
+
C H O [M+Na] 275.1254, found 275.1256.
14
20
4
5
5
5
5
5
5
5
5
5
5
5
5
5
39
40
41
42
43
44
45
46
47
48
49
50
51
(6) Compound A7
To the stirring solution of compound A6 (11.1 g, 44.0 mmol) in 200 mL CH Cl
2
2
at room temperature was added NaHCO (11.1 g, 132.0 mmol), followed by DMP
3
(28.0 g, 66.0 mmol) in batches. After 1 hour, TLC showed that the reaction was
completed,
The mixture was filtered, and the filtrate was set aside to produce many
floccules. The filtrate was filtered twice again and then saturated NaHCO aqueous
3
solution was added in batches (producing many bubbles and floccules) untill almost
no bubbles. After filtering again, the aqueous phase was separated from the filtrate
and extracted with ethyl acetate (100 mL × 2). The combined organic phase was
successively washed with brine (200 mL × 1), dried over Na SO , filtered, and
2
4
concentrated. Purification by column chromatography on silica gel (ethyl
acetate/petroleum ether = 1/5) gave the compound A7 as a light yellow oil (8.1 g) in
1
73.5% yield. H NMR (400 MHz, CDCl ) δ 8.86 (s, 1H), 7.25 (d, J = 8.5 Hz, 2H),
3
2
6

5
5
5
5
52
53
54
55
6.88 (d, J = 8.6 Hz, 2H), 4.47 (s, 2H), 3.81 (s, 3H), 3.64 (dd, J = 6.9, 5.4 Hz, 2H),
13
3.32 (dd, J = 6.7, 5.2 Hz, 1H), 2.02-1.85 (m, 2H), 1.40 (s, 3H). C NMR (101 MHz,
CDCl ) δ 199.9, 159.3, 130.1, 129.3, 113.9, 72.9, 66.5, 62.1, 57.9, 55.3, 28.8, 10.2.
3
+
HRMS (ESI) calcd for C H O [M+Na] 273.1097, found 273.1098.
1
4
18
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
(7) Compound A8
To the stirring solution of Ph PPhCH Br (9.9 g, 22.7 mmol) in 30 mL THF was
3
2
added NaN(TMS) (9.1 mL, 18.2 mmol) dropwise under N atmosphere at 0 ℃ . After
2
2
that, the mixture was stirred for 1 hour before the addition of 20 mL HMPA. After
stirring additional 1 hour, the solution of compound A7 (3.8g, 15.2mmol) in THF (30
mL) was added. After stirring for additional 30 minutes, the reaction mixture was
stirred at room temperature for more than 12 hours and monitored by TLC. After
completion, 50 mL saturated NH Cl aqueous solution was added. The aqueous phase
4
was separated from the mixture and extracted with ethyl acetate (50 mL × 2). The
combined organic phase was successively washed with brine (100 mL × 1), dried over
Na SO , filtered, and concentrated. Purification by column chromatography on silica
2
4
gel (ethyl acetate/petroleum ether = 1/8) gave the compound A8 as a light yellow oil
1
(1.94 g) in 39.6% yield. H NMR (400 MHz, CDCl ) δ 7.30-7.27 (m, 4H), 7.25-7.22
3
(m, 2H), 7.22-7.20 (m, 1H), 6.86 (d, J = 8.6 Hz, 2H), 6.44 (d, J = 12.0 Hz, 1H), 5.86
(d, J = 12.0 Hz, 1H), 4.42 (s, 2H), 3.79 (s, 3H), 3.53 (t, J = 6.7 Hz, 2H), 2.90 (dd, J =
7.6, 4.4 Hz, 1H), 1.99 (ddd, J = 14.4, 7.2, 4.3 Hz, 1H), 1.75 (ddt, J = 14.0, 7.7, 6.1 Hz,
1
3
1H), 1.46 (s, 3H). C NMR (101 MHz, CDCl ) δ 159.2, 136.5, 132.1, 130.4, 129.3,
3
128.8, 128.3, 127.5, 113.8, 72.8, 67.2, 62.7, 58.9, 55.3, 29.3, 17.7. HRMS (ESI) calcd
+
for C H O [M+Na] 347.1618, found 347.1620.
2
1
24
3
5
5
5
5
5
75
76
77
78
79
(8) Compound A9
To the stirring solution of Pd (dba)CHCl (3.3 g, 13.2 mmol) and n-Bu P (1.6 mL,
2
3
3
6.4 mmol) in 100 mL 1,4-dioxane was added a mixed solution of HCOOH (12.1 mL,
319.3 mmol) and Et N(17.8 mL, 127.7 mmol) in 50 mL 1,4-dioxane. After stirring for
3
15 minutes, the solution of compound A8 (20.7 g, 63.9 mmol) in 100 mL 1,4-dioxane
2
7

5
5
5
5
5
5
5
5
5
5
5
5
5
5
80
81
82
83
84
85
86
87
88
89
90
91
92
93
was added, and the resulting dark brown mixture was stirred for 10 hours at room
temperature and monitored by TLC. Then, 200 mL saturated NH Cl aqueous solution
4
was added to quench the reaction. After seperating from the mixture, the aqueous
phase was extracted with ethyl acetate (100 mL × 3). The combined organic phase
was successively washed with brine (200 mL × 1), dried over Na SO , filtered, and
2
4
concentrated. Purification by column chromatography on silica gel (ethyl
acetate/petroleum ether = 1/15 and 1/10) gave the compound A9 as a light yellow oil
1
(16.3 g) in 78.3% yield. H NMR (400 MHz, CDCl ) δ 7.36 (d, J = 7.4 Hz, 2H), 7.29
3
(t, J = 7.6 Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H), 7.19 (s, 1H), 6.87 (d, J = 8.6 Hz, 2H),
6.42 (d, J = 16.0 Hz, 1H), 6.22 (dd, J = 16.0, 8.2 Hz, 1H), 4.45 (s, 2H), 3.80 (s, 3H),
3.77-3.67 (m, 2H), 3.63 (ddd, J = 9.3, 5.6, 3.5 Hz, 1H), 2.47-2.33 (m, 1H), 1.79-1.72
1
3
(m, 2H), 1.14 (d, J = 6.9 Hz, 3H). C NMR (101 MHz, CDCl ) δ 159.3, 137.5, 132.2,
3
130.7, 130.1, 129.4, 128.5, 127.1, 126.1, 113.9, 74.7, 73.0, 69.0, 55.3, 43.5, 33.9, 16.5.
+
HRMS (ESI) calcd for C H O [M+Na] 349.1744, found 349.1775.
2
1
26
3
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
(9) Compound A10
To the stirring solution of compound A9 (5.0 g, 15.3 mmol) in 100 mL CH Cl
2
2
was added AlCl (2.3 g, 16.9 mmol) at room temperature. After 15 minutes, TLC
3
showed that the reaction was completed. To the mxiture was added 50 mL saturated
NH Cl aqueous solution for quenching the reaction. The aqueous phase was seperated
4
from the mixture and extracted with ethyl acetate (50 mL × 3). The combined organic
phase was successively washed with brine (100 mL × 1), dried over Na SO , filtered,
2
4
and concentrated. Purification by column chromatography on silica gel (ethyl
acetate/petroleum ether = 1/2 and 2/1) gave the compound A10 as a light yellow oil
1
(2.6 g) in 83.0% yield. H NMR (400 MHz, CDCl ) δ 7.40-7.18 (m, 6H), 6.47 (d, J =
3
15.8 Hz, 1H), 6.13 (dd, J = 15.9, 8.5 Hz, 1H), 3.86 (dq, J = 7.7, 4.6, 3.9 Hz, 2H), 3.75
(ddt, J = 9.1, 6.2, 3.7 Hz, 1H), 2.45-2.39 (m, 1H), 2.38 (d, J = 6.3 Hz, 2H), 1.82-1.69
1
3
(m, 2H), 1.13 (d, J = 6.8 Hz, 3H). C NMR (101 MHz, CDCl ) δ 137.1, 131.7, 131.6,
3
+
128.6, 127.4, 126.2, 53.5, 44.1, 35.5, 16.5. HRMS (ESI) calcd for C H O [M+Na]
1
3
18
2
229.1199, found 229.1204.
2
8