Divinylsulfonamides enable the construction of homogeneous antibody–drug conjugates

Article abstract of DOI:10.1016/j.bmc.2020.115793

Methods that site-specifically attach payloads to an antibody with controlled DAR (Drug-Antibody Ratio) are highly desirable for the generation of homogeneous antibody-drug conjugates (ADCs). We describe the use of N-phenyl-divinylsulfonamide scaffold as

Full text of DOI:10.1016/j.bmc.2020.115793

Bioorg. Med. Chem. 28 (2020) 115793
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Bioorganic & Medicinal Chemistry
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Divinylsulfonamides enable the construction of homogeneous
antibody–drug conjugates
,
b
,
,
,
b
,
,
,
,*
Rong Huanga 1, Yao Shenga 1, Ding Weia c, Wenwen Lua b, Zili Xua b, Hongli Chena
,
,
*
Biao Jianga
a Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, 393 Middle Huaxia Road, Pudong, Shanghai 201210, China
b University of Chinese Academy of Sciences, 19A Yuquan Road, Shijingshan District, Beijing 100049, China
c Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Methods that site-specifically attach payloads to an antibody with controlled DAR (Drug-Antibody Ratio) are
highly desirable for the generation of homogeneous antibody-drug conjugates (ADCs). We describe the use of N-
phenyl-divinylsulfonamide scaffold as a linker platform to site-specifically construct homogeneous DAR four
ADCs through a disulfide re-bridging approach. Several monomethyl auristatin E (MMAE)-linkers were syn-
thesized and the drug-linkers that contain electron-donating groups on the phenyl of the linker showed high
stability. Her2-targeted MMAE-linker-herceptin and EGFR targeted MMAE-linker-cetuximab conjugates were
prepared. The conjugates demonstrated high efficacy and selectivity for killing target-positive cancer cells in
vitro. The EGFR-targeted conjugates also showed significant antitumor activities in vivo.
Divinylsulfonamides
Antibody-drug conjugates
Selectivity
1. Introduction
can be re-connected by a chemical linker, which allows the insertion of
one drug per disulfide bond and thus DAR 4 ADCs can be achieved.8
Antibody–drug conjugates (ADCs) combine the targeting ability of
antibodies with the efficacy of the therapeutic payloads, demonstrating
considerable promise for more targeted and precise therapeutics.1–2
While the concept of ADCs is relatively straightforward, the develop-
ment of functional and effective ADCs is remarkably challenging. In the
past several years, a growing number of discontinuations of pre-clinical
and clinical ADCs slows the development of ADCs.3 However, the fail-
ures and setbacks did not limit the development of biotechnology and
chemical conjugation methods for ADCs. With the approval of three
ADCs (Polivy, Padcev and Enhertu) in 2019, Trodelvy and Blenrep in
2020, there are currently nine ADCs have received approval by the FDA.
ADCs are worth more attention to reach their true potential.4
Many efforts have been made to develop chemical linkers for disulfide
re-bridging. Bissulfone reagents represent the earliest developed linkers
to re-connect disulfide bonds.9–11 Then substituted maleimides and
pyridazinediones have been developed to generate homogeneous ADCs
using the reduction re-bridging strategy.12–14 Later, alternatively new
chemical entities have also been reported, such as dichloro- or dibromo-
reagents,15–17 thiol-yne coupling linkers,18 divinylpyrimidine19 and 3-
bromo-5-methylene pyrrolones.20
Recently, we have developed divinylsulfonamides as specific linkers
in the field of disulfide bonds stapling for peptides (Fig. 1A).21 And we
have also reported the use of bis(vinylsulfonyl)piperazines as efficient
linkers to achieving controlled DAR two ADCs (Fig. 1B).22 Whereas a
drug loading of four has been demonstrated to significantly increase the
therapeutic index for MMAE-based ADCs,11,23 in this study, we describe
an investigation in the development and application of divinylsulfona-
mides for the preparation of MMAE-based DAR 4 ADCs (Fig. 1C).
Linker technology and conjugation approach can strongly influence
the drug-antibody ratio (DAR), pharmacokinetics, safety and also the
ADC efficacy.5 Chemical methods that site-specifically attach payloads
to an antibody with controlled DAR are highly desirable for the gener-
ation of homogeneous ADCs.6–7 Recently, reduction re-bridging strategy
has emerged as a promising chemical approach: the four interchain di-
sulfide bonds in IgG1 can be reduced and the resulting cysteine residues
* Corresponding authors.
E-mail addresses: chenhl@shanghaitech.edu.cn (H. Chen), jiangbiao@shanghaitech.edu.cn (B. Jiang).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bmc.2020.115793
Received 31 July 2020; Received in revised form 23 September 2020; Accepted 24 September 2020
Available online 6 October 2020
0968-0896/© 2020 Elsevier Ltd. All rights reserved.

R. Huang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115793
2. Results and discussion
According to the previous study,21 N-phenyl-divinylsulfonamide was
employed as a scaffold with variant linkers attached to the phenyl for
disulfide re-bridging. Three linker molecules that contained an alkyne as
a handle to link a drug via click chemistry were designed and synthe-
sized. As shown in scheme 1, p-aminophenol (1) was protected by Boc to
give the product 2, which was followed by the reaction with 3-bromo-
prop-1-yne to provide an alkyne derivative 3. The Boc group was
removed and the resulting product 4 was treated with 2-chloroethane-1-
sulfonyl chloride (5) to obtain the ether linker 6. Meanwhile, phenyl-
amide and benzamide linkers 10 and 13 were also prepared. Commer-
cially available tert-butyl (4-aminophenyl)carbamate (7) was reacted
with hex-5-ynoic acid to afford compound 8, followed by deprotection
to give compound 9. Finally, the phenylamide linker 10 was prepared by
the reaction of 9 with the reagent 5 (Scheme 2). Benzamide linker (13)
was synthesized from 4-aminobenzoic acid (11) which was reacted with
prop-2-yn-1-amine, followed by the introduction of divinylsulfonamide
under the same conditions as those used for the preparation of 6 and 10
(Scheme 3).
Scheme 1. Synthesis of Linker 6.
On the other hand, the derivative of monomethyl auristatin E
(MMAE), compound 14, containing a PEG moiety and an azide tag, was
prepared. The drug-linkers 15–17 were obtained by coupling com-
pounds 6, 10, 13 with 14 using the Cu(I)-catalyzed azide–alkyne
cycloaddition (CuAAC), respectively (Scheme 4).
Fig. 1. (A) Previous work: disulfide bonds stapling for peptides; (B) Previous work: reduction–rebridging approach for DAR 2 ADCs; (C) This work: reduction re-
bridging approach for DAR 4 ADCs.
2

R. Huang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115793
Scheme 2. Synthesis of Linker 10.
Scheme 3. Synthesis of Linker 13.
HER2-positive (SK-BR-3) and HER2-negative (MDA-MB-468, HCC827
and MCF7) cancer cell lines. The FDA-approved HER2 targeted ADC
trastuzumab emtansine (T-DM1), herceptin and MMAE were used as
controls (Fig. 3a-d). All four cell lines are sensitive to free MMAE with
high potencies. The ADCs 18–20 showed strong activities against the
HER2-positive cancer cell (SK-BR-3) with IC50 values of 14.0 nM, 4.8 nM
and 11.3 nM respectively, and their potencies were comparable with
that of T-DM1 (6.4 nM). Whilst, the conjugates demonstrated signifi-
cantly decreased activities in the antigen negative cell lines (>500 nM)
(Fig. 3e). These results supported the ADCs’ selectivity for killing HER2-
positive cancer cells.
Cetuximab that binds to epidermal growth factor receptor (EGFR)
was also used to the construction of EGFR-targeted ADCs. The conju-
gates 21–23 were obtained in a similar way with that of ADCs 18–20.
Likewise, the MS data showed that 21 and 22 were formed by disulfide
re-bridging approach, whereas 23 was a heterogeneous mixture
(Fig. S2). In vitro cytotoxicities of 21–23 were performed on HCC827
and NCI-H2228 cells (Fig. 4a-b). All the ADCs 21–23 showed strong
activities on HCC827 cancer cells(EGFR-positive), with even lower IC50
values (0.5 ± 0.1 nM, 0.5 ± 0.1 nM, 0.8 ± 0.1 nM) than that of free
MMAE (1.5 ± 0.2 nM). MMAE was also found to inhibit the proliferation
of EGFR-negative cells(NCI-H2228) with IC50 value (2.2 ± 0.1 nM).
When NCI-H2228 cells were incubated with ADCs 21–23, no toxicity
was observed in the study. (Fig. 4c).
Scheme 4. Synthesis of drug-linkers 15–17.
With the drug-linkers in hand, trastuzumab was employed for the
preparation of ADCs. The antibody was reduced by treatment with tris
(2-carboxyethyl)phosphine (TCEP) to release free thiols and the result-
ing mixture was subsequently incubated with the drug-linkers (15–17)
(Fig. 2A). The mass spectrometry (MS) analysis showed that for the
linkers 15 (MW = 1259.6, MW: molecular weight) and 16 (MW =
1314.7), the resulting ADCs 18 and 19 loaded four drug-linkers as the
major species (MW conjugate-MW antibody = 4*MW15 or 16) and a few DAR
two species were also observed (Fig. 2B). However, when the drug-
linker 17 that contains the electron-withdrawing group on the phenyl
of the linker reacted with the reduced antibody, it was tend to hydrolyze
to yield the loss of one vinylsulfonamide product 17–1 (MW = 1196.7)
and ethenesulfonic acid 17–2 (MW = 108.0). And 17-1 was attached to
antibody to form the conjugated mixture 20 with average DAR 3.9
(Fig. 2C). We also found that the drug-linkers 15 and 16 retained high
stability during long term storage (More than one year storage with
HPLC purity:96% and 95%, respectively), while 17 presented a risk of
instability and its HPLC purity was decreased to 80%, under the same
storage conditions with those of 15 and 16 (SI, Fig. S1). The conjugates
18–20 were analyzed by SEC-HPLC and the result showed that the ag-
gregation levels of all the ADCs were identical with the native antibody
(Fig. 2D).
The re-bridging conjugates 21 and 22 were further evaluated. Flow
cytometry analysis was used to monitor affinity and internalization of
the conjugates 21, 22 and cetuximab. ADCs 21 and 22 were proven to
have retained binding activities which were comparable to cetuximab in
both EGFR-positive (HCC827, Fig. 5a) and EGFR-negative (NCI-H2228,
Fig. 5b) cell lines. And the binding activities of all the molecules per-
formed in a dose-dependent manner (Fig. 5c). In addition, internaliza-
tion of the conjugates 21 and 22 in target cell was also comparable to
cetuximab (Fig. 6).
The ability of the conjugates 21 and 22 to inhibit tumor growth in
vivo was evaluated in a mouse xenograft study using HCC827 lung
cancer cells derived tumors. Conjugates 21 and 22 groups received four
doses of 20 mg/kg on days 0, 4, 8 and 12, injected intravenously. Tumor
growth were inhibited significantly both by 21 and 22 treatments
(Fig. 7a). Throughout the trial, the mice remained in good health and no
weight loss or other overt toxicity (Fig. 7b). Our results showed that
conjugates 21 and 22 are highly potent against EGFR+ tumor in vivo.
Then, the in vitro potencies of ADCs 18–20 were evaluated against
3

R. Huang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115793
A
B
C
S
S
1) TCEP, r.t., 2h
S
S
S
S
S S
S S
S
S
S S
S S
15 17
- , r.t., 12h
2)
Herceptin
Cetuximab
18
R= O:
O
O
19
22
NHCOCH2CH2:
20
O
S
S
Herceptin conjugates
CONH:
N
R
N
=
N
N
O
O
O
MMAE
O
21
R= O:
NHCOCH2CH2:
23
O
Cetuximab conjugates
CONH:
NH
O2S
O
N
17
NH
N
S
O
N
O
MMAE
OH
O
O
O
O
17-2
17-1
Fig. 2. Reaction of the drug-linkers 15–17 with herceptin or cetuximab and subsequent analysis. (A) Synthesis of herceptin-conjugated ADCs 18–20 and cetuximab-
conjugated ADCs 21–23. (B) MS analysis of ADCs 18–19. (C) The scheme for the hydrolysis of the drug-linker 17 and MS analysis of heavy and light chain of
herceptin and ADC 20. (D) SEC analysis of herceptin and 18–20.
3. Conclusions
platform will facilitate its use for the development of ADCs candidates.
4. Experimental section
In conclusion, N-phenyl-divinylsulfonamide linkages that contain
electron-donating groups on the phenyl of the linker have shown the
potencies to re-bridge the disulfide bonds to afford homogeneous DAR 4
ADCs. Linker-MMAE-ADCs demonstrated antigen-selective in vitro
cytotoxicity. The conjugates also shown significant antitumor activities
and were found to be well tolerated in vivo. We expect that this linker
4.1. General experimental details
All chemical reagents and solvents were of analytical grade, obtained
from commercial sources and used as supplied without further
4

R. Huang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115793
D
Fig. 2. (continued).
Fig. 3. In vitro potencies of ADCs 18–20, MMAE, Herceptin and T-DM1. Potent activities in (a) HER2 positive cell line SK-BR-3, (b) HER2 negative cell line HCC827,
(c) HER2 negative cell line MDA-MB-468, (d) HER2 negative cell line MCF7. (e) IC50 values (values = mean ± SD, n = 3) in both HER2 positive and negative
cell lines.
purification unless indicated. trastuzumab and cetuximab were pur-
chased from Shanghai huanyao biotechnology Co., Ltd. without further
purification.
Non-aqueous reactions were conducted under a stream of dry ni-
◦
trogen using oven dried glassware. Temperatures of 0 C were main-
tained using an ice-water bath. Room temperature (rt) refers to ambient
5

R. Huang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115793
Fig. 4. In vitro potencies of ADCs 21–23, MMAE and cetuximab. (a) Potent activities in EGFR positive cell line HCC827. (b) Potent activities in EGFR negative cell
line NCI-H2228. (c) IC50 values (values = mean ± SD, n = 3) in both positive and negative cell lines.
Fig. 5. Binding affinity of ADCs 21, 22 and cetuximab as analyzed by flow cytometry. (a) in HCC827 cells. (b) in NCI-H2228 cells. (c) dose-dependent experiments.
Fig. 6. Internalization of ADCs 21, 22 and cetuximab in HCC827 cells as analyzed by flow cytometry. (a) 21. (b) 22. (c) Cetuximab.
Fig. 7. In vivo efficacy of ADCs 21 and 22 against HCC827 subcutaneous tumor bearing models.
temperature. Yields refer to spectroscopically and chromatographically
pure compounds unless otherwise stated. Reactions were monitored by
thin layer chromatography (TLC) or liquid chromatography mass spec-
troscopy (LC-MS). TLC was purchased from Rushan Taiyang Desiccant
Co., Ltd. and visualized by quenching of UV fluorescence (λmax = 254
nm) or by staining with potassium permanganate. Flash chromatog-
raphy was carried out on silica gel (200–300 mesh).
High-resolution mass spectra (HRMS-ESI) were obtained on an
6

R. Huang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115793
ABsciex 4600 instrument. LC system: solvent A: 0.1% HCOOH on water;
solvent B: acetonitrile; column: Agela Technologies C18 column (2.1 ×
100 mm, 3 µm) at 30 ◦C; gradient: 0–3 min 5–100% B, 3–3.5 min 100%
B, 3.51–5 min 5% B at flow rate of 0.4 mL/min; detector: UV detection
(λmax = 220–254 nm). ESI refers to the electrospray ionisation tech-
nique. Analytical high performance liquid chromatography (HPLC) was
performed on SHIMADZU LC-30AD machine, using an Agela Technol-
ogies C18 column (2.1X100 mm, 3 µm). LC system: solvent A: 0.5% (v/v)
TFA in H2O; solvent B: acetonitrile at 30 ◦C; gradient: 0–10 min
10–100% B, 10–12 min 100% B at flow rate of 0.4 mL/min; detector: UV
detection (λmax = 220–254 nm). Proton and carbon nuclear magnetic
resonance (NMR) were recorded using an internal deuterium lock on
Bruker Avance 500 Cryo Ultrashield (500 MHz, 126 MHz). Tetrame-
thylsilane was used as an internal standard. In proton NMR, chemical
shifts (δH) are reported in parts per million (ppm), to the nearest 0.01
ppm and are referenced to the residual non-deuterated solvent peak
(CDCl3: 7.26, DMSO‑d6: 2.50, CD3OD: 3.31, D2O: 4.79). Coupling con-
stants (J) are reported in Hertz (Hz) to the nearest 0.1 Hz. Data are re-
ported as follows: chemical shift, multiplicity (s = singlet; d = doublet; t
= triplet; q = quartet; qn = quintet; sep = septet; m = multiplet; or as a
combination of these, e.g. dd, dt etc.), integration and coupling constant
(s). In carbon NMR, chemical shifts (δC) are quoted in ppm, to the
nearest 0.1 ppm, and are referenced to the residual non-deuterated
solvent peak (CDCl3: 77.16, DMSO‑d6, 39.52, CD3OD: 49.00). The
deuterated solvents employed were purchased from Energy Chemical.
Spectra were analyzed with MestReNova.
residue was dissolved in DCM (30 mL). The solution was cooled to 0 ◦C
with ice-bath followed trifluoroacetic acid (12 mL) added, and then the
reaction mixture was stirred for 1 h under ice-bath. The mixture was
concentrated under vacuum and residue was purified by column chro-
matography on a gradient form petroleum ether to 10% ethyl acetate/
0.5% triethylamine in petroleum ether to afford 1.0 g (6.8 mmol, 84%)
yield of 4 as red oil. 1H NMR (500 MHz, CDCl3) δ 6.80 (dd, J = 8.7, 1.6
Hz, 2H), 6.60 (dd, J = 8.8, 3.1 Hz, 2H), 4.58 (t, J = 2.4 Hz, 2H), 3.49 (s,
2H), 2.51 (d, J = 2.3 Hz, 1H) ppm. 13C NMR (126 MHz, CDCl3) δ 150.38,
140.99, 116.23, 116.09, 79.15, 75.27, 56.57 ppm. ESI-HRMS calcd for
C9H10NO [(M+H)+]: 148.0762, found:148.0715.
4.2.3. N-(4-(prop-2-yn-1-yloxy)phenyl)-N-(vinylsulfonyl)
ethenesulfonamide (6)
A stirred solution of 4-(prop-2-yn-1-yloxy)aniline (4) (180 mg, 1.22
mmol) and triethylamine (1.0 mL, 7.32 mmol) in DCM (15 mL) was
cooled to 0 ◦C with ice-bath and 2-chloroethanesµlfonyl chloride (288
µL, 2.69 mmol) was then injected slowly. The reaction mixture was
stirred at 0 ◦C for 20 min, and water (5 mL) was added. The product was
extracted with DCM (3 × 15 mL). The combined organic extracts were
washed with brine (10 mL) and dried over anhydrous Na2SO4. The
solvent was removed under vacuum and residue was purified by column
chromatography on a gradient form petroleum ether to 20% ethyl ace-
tate in petroleum ether to afford 252 mg (0.77 mmol, 64%) yield of 6 as
a slightly yellow solid. 1H NMR (500 MHz, CDCl3) δ 7.23–7.17 (m, 2H),
7.09–6.96 (m, 4H), 6.28 (d, J = 16.6 Hz, 2H), 6.14 (d, J = 9.9 Hz, 2H),
4.70 (d, J = 2.4 Hz, 2H), 2.56 (t, J = 2.4 Hz, 1H) ppm. 13C NMR (126
MHz, CDCl3) δ 159.16, 136.18, 132.24, 129.79, 126.81, 115.76, 77.97,
76.37, 56.17 ppm. ESI-HRMS calcd for C13H14NO5S2 [(M+H)+]:
328.0313, found:328.0352.
Protein MS was performed on an ABsciex 4600 using a GL Sciences
C4 column (2.1X150 mm, 5 µm). H2O with 0.1% formic acid (solvent A)
and acetonitrile (solvent B), were used as the mobile phase at a flow rate
of 0.4 mL/min. The gradient was programmed as follows: 0–2.5 min
15% B, 2.5–5 min 15–95% B, 5–6.5 min 95% B, 6.51–8.0 min 5% B. The
electrospray source was operated with a capillary voltage of 2.0 kV and a
cone voltage of 40 V. Nitrogen was used as the desolvation gas at a total
flow of 850 L/h. Total mass spectra were reconstructed from the ion
series using the MaxEnt algorithm preinstalled on PeakView2.2 software
(Version1.7.1 from AnalystTF) according to the manufacturer’s in-
structions. Antibody samples were deglycosylated with Endo S prior to
MS analysis. Size-exclusion chromatography (SEC) was performed using
Agilent technologies 1260 Infinity. Mobile phase is Phosphate Buffered
Saline (PBS (pH 7.4)). LC conditions: TSKgel G3000SWXL column: 7.8 ×
300 mm, 5 µm, column temperature: 40 ◦C, λ = 280 nm, gradient: 0–20
min 100% PBS (pH 7.4), flow rate: 1 mL/min.
4.2.4. Tert-butyl (4-(hex-5-ynamido)phenyl)carbamate (8)
Tert-butyl (4-aminophenyl)carbamate (2.291 g, 11 mmol), hex-5-
ynoic acid (1.1213 g, 10 mmol), HOBt (1.633 g, 12 mmol), EDCl
(2.30 g, 12.0 mmol) and N’N-diisopropylethylamine (4.958 mL, 30
mmol) were dissolved in DCM (30 mL). The reaction mixture was stirred
at room temperature overnight followed that water (100 mL) and DCM
(3 × 50 mL) were added to extract the product. The combined organic
extracts were washed sequentially with saturated NaHCO3 (20 mL),
NH4Cl (20 mL) and brine (20 mL) and dried over anhydrous Na2SO4. The
solvent was removed under vacuum and residue was purified by column
chromatography on a gradient form petroleum ether to 50% ethyl ace-
tate in petroleum ether to afford 2.11 g (7 mmol, 70%) yield of 8 as a
yellow solid. 1H NMR (500 MHz, CD3OD) δ 7.47–7.40 (m, 2H), 7.33 (d,
J = 8.8 Hz, 2H), 2.50–2.44 (m, 2H), 2.28 (d, J = 2.6 Hz, 1H), 2.27–2.24
(m, 2H), 1.87 (p, J = 7.0 Hz, 2H), 1.51 (s, 9H). 13C NMR (126 MHz,
CD3OD) δ 172.15, 153.99, 135.41, 133.37, 120.48, 118.81, 82.77,
79.39, 68.90, 35.15, 27.35, 24.38, 17.30. ESI-HRMS Calculated for
4.2. Chemical synthesis
4.2.1. Tert-butyl (4-hydroxyphenyl)carbamate (2)
A solution of 4-aminophenol (2.0 g, 18.3 mmol), di-tert-butyl
dicarbonate (5.0 mL, 22 mmol) and triethylamine (5.0 mL, 36.6 mmol)
in THF (50 mL) was stirred for overnight at room temperature. The
mixture was concentrated under vacuum and the residue was purified by
column chromatography to afford 3.27 g (15.6 mmol, 96%) yield of 2 as
a white solid. 1H NMR (500 MHz, DMSO‑d6) δ 9.04 (s, 1H), 8.99 (s, 1H),
7.22 (d, J = 7.1 Hz, 2H), 6.71–6.58 (m, 2H), 1.45 (s, 9H) ppm. 13C NMR
(126 MHz, DMSO‑d6) δ 153.06, 152.56, 131.08, 119.99, 115.07, 78.45,
28.24 ppm. ESI-HRMS calcd for C11H16NO3 [(M + H)+]: 210.1130,
found:210.1154.
C
17H23N2O3 [M+H]+:303.1709, Found:303.1720.
4.2.5. N-(4-aminophenyl)hex-5-ynamide (9)
Tert-butyl (4-(hex-5-ynamido)phenyl)carbamate (8) (2.11 g,7 mmol)
was dissolved in DCM (20 mL). The solution was cooled to 0 ◦C with ice-
bath followed by trifluoroacetic acid (4 mL) was added, and then the
reaction mixture was stirred for 6 h under ice-bath. The mixture was
concentrated under vacuum and the residue was purified by column
chromatography on a gradient form petroleum ether to 30% ethyl ace-
tate (0.5% triethylamine in petroleum 1ether) to afford 1.23 g (6.08
mmol, 87%) yield of 9 as yellow solid. H NMR (500 MHz, CD3OD) δ
7.28–7.22 (m, 2H), 6.72–6.63 (m, 2H), 2.48–2.38 (m, 2H), 2.29–2.20
(m, 3H), 1.91–1.81 (m, 2H). 13C NMR (126 MHz, CD3OD) δ 172.04,
144.16, 129.26, 121.99, 115.33, 83.00, 69.05, 35.15, 24.54, 17.40. ESI-
HRMS Calculated for C12H15N2O [M+H]+:203.1184, Found:203.1151.
4.2.2. 4-(prop-2-yn-1-yloxy)aniline (4)
Tert-butyl (4-hydroxypHenyl)carbamate (2) (1.7 g, 8.1 mmol), 3-
bromopropyne (0.84 mL, 9.7 mmol) and potassium carbonate (3.3 g,
24.3 mmol) were dissolved in DMF (30 mL). The reaction mixture was
stirred at room temperature for overnight followed that water (600 mL)
and ethyl acetate (3 × 50 mL) were added to extract the product. The
combined organic extracts were washed sequentially with saturated
NaHCO3 (20 mL), NH4Cl (20 mL) and brine (20 mL) and dried over
anhydrous Na2SO4. The solvent was removed under vacuum and the
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R. Huang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115793
4.2.6. N-(4-(N-(vinylsulfonyl)vinylsulfonamido)phenyl)hex-5-ynamide
(10)
over 30 min) to afford 111 mg (0.12 mmol, 85%) yield of 14 as a white
solid. ESI-HRMS Calculated for C47H81N8O11 [M+H]+:993.6025,
Found:993.6059.
A stirred solution of N-(4-aminophenyl)hex-5-ynamide (9) (1.23 g,
6.08 mmol) and triethylamine (2.53 mL, 18.25 mmol) in DCM (20 mL)
◦
was cooled to 0 C with ice-bath and 2-chloroethanesµlfonyl chloride
4.2.10. 6-PEG3-MMAE(15)
(1.97 mL, 18.25 mmol) was then injected slowly. The reaction mixture
was stirred at 0 ◦C for 1 h, and water (100 mL) was added. The product
was extracted with DCM (3 × 20 mL). The combined organic extracts
were washed with brine (10 mL) and dried over anhydrous Na2SO4. The
solvent was removed under vacuum and residue was purified by column
chromatography on a gradient form petroleum ether to 30% ethyl ace-
tate in petroleum ether to afford 1.91 g (5 mmol, 82%) yield of 10 as a
yellow solid. 1H NMR (500 MHz, CD3OD) δ 7.68–7.61 (m, 2H),
7.25–7.19 (m, 2H), 7.11 (dd, J = 16.3, 10.0 Hz, 2H), 6.25 (dd, J = 7.8,
0.6 Hz, 2H), 6.22 (d, J = 0.6 Hz, 2H), 2.53 (t, J = 7.5 Hz, 2H), 2.32–2.24
(m, 3H), 1.93–1.84 (m, 2H). 13C NMR (126 MHz, CD3OD) δ 172.54,
140.50, 136.23, 131.36, 129.25, 128.94, 119.89, 82.71, 68.93, 35.20,
N3-PEG3-MMAE (14) (37 mg, 0.0397 mmol), 6 (15.58 mg, 0.0476
mmol), Na ascorbate (8.38 mg, 0.0476 mmol) and CuSO4 (7.57 mg,
0.0476 mmol) were dissolved in tBuOH/H2O/DMF (1/1/1) (6 mL). The
reaction mixture was stirred at room temperature for 2 h. Upon
completion, the resulting crude mixture was purified by HPLC
(10–100% CH3CN/H2O over 30 min) to afford 14.6 mg (0.01158 mmol,
29%) yield of 15 as a white solid. ESI-HRMS Calculated for
C
60H94N9O16S2 [M+H]+:1260.6260, Found:1260.6296. The product
was analyzed by HPLC for purity of 98%. 1H NMR (500 MHz, CD3OD) δ
8.21–8.14 (m, 1H), 7.99–7.94 (m, 1H), 7.41–7.35 (m, 2H), 7.35–7.27
(m, 2H), 7.24–7.19 (m, 3H), 7.15–7.05 (m, 4H), 6.23 (ddd, J = 11.7, 3.9,
2.0 Hz, 4H), 5.21 (s, 2H), 4.81–4.47 (m, 5H), 4.46–4.00 (m, 4H),
4.00–3.79 (m, 3H), 3.75–3.52 (m, 9H), 3.45–3.33 (m, 6H), 3.30–3.25
(m, 3H), 3.11 (d, J = 3.2 Hz, 1H), 3.02–2.89 (m, 4H), 2.56–2.42 (m, 2H),
2.38–1.21 (m, 10H), 1.21–1.10 (m, 6H), 1.06–0.79 (m, 19H). 13C NMR
(126 MHz, CD3OD) δ 175.71, 175.42, 172.71, 171.89, 171.75, 171.56,
161.13, 144.31, 144.09, 143.89, 137.65, 133.60, 130.59, 129.53,
129.23, 128.62, 128.38, 128.18, 128.08, 127.89, 126.44, 126.33,
116.48, 86.70, 83.51, 77.54, 77.25, 71.73, 71.55, 71.51, 71.44, 71.42,
70.61, 70.30, 63.68, 63.57, 62.68, 61.99, 61.50, 60.78, 60.59, 58.62,
58.35, 56.31, 56.11, 51.52, 51.43, 50.74, 48.07, 45.88, 45.53, 38.31,
36.94, 33.91, 31.85, 31.64, 30.38, 27.70, 27.61, 26.99, 26.60, 25.84,
25.64, 24.45, 19.76, 19.14, 18.97, 18.79, 18.72, 16.94, 16.31, 15.94,
15.83, 14.99, 10.90.
24.11,
17.23.
ESI-HRMS
Calculated
for
C16H19N2O5S2
[M+H]+:383.0735, Found:383.0748.
4.2.7. 4-amino-N-(prop-2-yn-1-yl)benzamide (12)
4-aminobenzoic acid (1.50854 g, 11 mmol), propargylamine (0.686
mL, 10 mmol), HOBt (1.633 g, 12 mmol), EDCl (2.30 g, 12.0 mmol) and
N’N-diisopropylethylamine (4.958 mL, 30 mmol) were dissolved in THF
(30 mL). The reaction mixture was stirred at room temperature over-
night. The mixture was concentrated under vacuum and the residue was
dissolved with DCM (30 mL) and water (100 mL) and DCM (3 × 50 mL)
were added to extract the product. The combined organic extracts were
washed sequentially with saturated NaHCO3 (20 mL), NH4Cl (20 mL)
and brine (20 mL) and dried over anhydrous Na2SO4. The solvent was
removed under vacuum and residue was purified by column chroma-
tography on a gradient form petroleum ether to 50% ethyl acetate in
petroleum ether to afford 1 g (5.74 mmol, 57%) yield of 12 as a slightly
yellow solid. 1H NMR (500 MHz, DMSO‑d6) δ 8.42 (t, J = 5.1 Hz, 1H),
7.58 (d, J = 8.5 Hz, 2H), 6.54 (d, J = 8.5 Hz, 2H), 5.65 (s, 2H), 3.99 (dd,
J = 5.4, 2.2 Hz, 2H), 3.04 (s, 1H). 13C NMR (126 MHz, DMSO‑d6) δ
166.42, 152.31, 129.32, 120.93, 113.03, 82.45, 72.83, 28.70. ESI-HRMS
Calculated for C10H11N2O [M+H]+:175.0871, Found:175.0852.
4.2.11. 10-PEG3-MMAE (16)
N3-PEG3-MMAE (14) (37 mg, 0.0397 mmol), 10 (18.21 mg, 0.0476
mmol), Na ascorbate (8.38 mg, 0.0476 mmol) and CuSO4 (7.57 mg,
0.0476 mmol) were dissolved in tBuOH/H2O/DMF (1/1/1) (6 mL). The
reaction mixture was stirred at room temperature for 2 h. Upon
completion, the resulting crude mixture was purified by HPLC
(10–100% MeCN/H2O over 30 min) to afford 6.1 mg (0.0463 mmol,
11%) yield of 16 as a white solid. ESI-HRMS Calculated for
C63H99N10O16S2 [M + H]+:1315.6682, Found:1315.6658. The product
was analyzed by HPLC for purity of 98%. 1H NMR (500 MHz, CD3OD) δ
8.01–7.92 (m, 1H), 7.89–7.81 (m, 1H), 7.65 (dt, J = 8.7, 1.1 Hz, 2H),
7.41–7.35 (m, 2H), 7.36–7.27 (m, 2H), 7.25–7.17 (m, 3H), 7.12 (ddd, J
= 16.7, 9.8, 1.2 Hz, 2H), 6.28–6.20 (m, 4H), 4.80–4.47 (m, 5H),
4.47–4.01 (m, 4H), 3.97–3.80 (m, 3H), 3.79–3.50 (m, 9H), 3.46–3.33
(m, 6H), 3.30–3.25 (m, 3H), 3.11 (d, J = 3.9 Hz, 1H), 3.04–2.89 (m, 3H),
2.78 (t, J = 7.5 Hz, 2H), 2.57–2.38 (m, 4H), 2.37–1.21 (m, 11H),
1.21–1.09 (m, 6H), 1.06–0.74 (m, 19H). 13C NMR (126 MHz, CD3OD) δ
175.69, 175.41, 173.99, 172.73, 171.88, 171.73, 171.56, 148.11,
144.08, 143.88, 141.93, 137.63, 132.76, 130.68, 130.29, 129.53,
129.23, 128.62, 128.38, 128.08, 127.89, 124.32, 121.21, 86.67, 83.48,
77.53, 77.25, 71.73, 71.50, 71.42, 70.61, 70.37, 63.65, 61.99, 61.51,
60.77, 60.59, 58.61, 58.35, 57.75, 56.31, 56.11, 51.40, 51.30, 50.73,
48.07, 45.87, 45.52, 38.29, 37.15, 33.62, 33.10, 31.84, 30.36, 27.71,
27.62, 26.98, 26.58, 26.41, 25.84, 25.75, 25.63, 24.45, 19.75, 19.16,
18.98, 18.72, 16.95, 16.31, 15.97, 15.83, 15.00, 10.90.
4.2.8. N-(prop-2-yn-1-yl)-4-(N-(vinylsulfonyl)vinylsulfonamido)
benzamide (13)
A stirred solution of 4-amino-N-(prop-2-yn-1-yl)benzamide (12)
(1.0 g, 5.74 mmol) and triethylamine (2.386 mL, 17.22 mmol) in DCM
◦
(20 mL) was cooled to 0 C with ice-bath and 2-chloroethanesµlfonyl
chloride (1.86 mL, 17.22 mmol) was then injected slowly. The reaction
mixture was stirred at 0 ◦C for 1 h, and water (100 mL) was added. The
product was extracted with DCM (3 × 20 mL). The combined organic
extracts were washed with brine (10 mL) and dried over anhydrous
Na2SO4. The solvent was removed under vacuum and residue was pu-
rified by column chromatography on a gradient form petroleum ether to
50% ethyl acetate in petroleum ether to afford 1.51 g (4.25 mmol, 74%)
yield of 13 as a yellow solid. 1H NMR (500 MHz, DMSO‑d6) δ 9.08 (t, J =
5.5 Hz, 1H), 7.96–7.88 (m, 2H), 7.51–7.41 (m, 2H), 7.27 (dd, J = 16.3,
9.8 Hz, 2H), 6.40 (dd, J = 9.8, 1.0 Hz, 2H), 6.27 (dd, J = 16.3, 1.0 Hz,
2H), 4.07 (dd, J = 5.5, 2.5 Hz, 2H), 3.14 (t, J = 2.5 Hz, 1H). 13C NMR
(126 MHz, DMSO‑d6) δ 165.51, 136.54, 136.31, 136.02, 132.01, 131.53,
129.00, 81.50, 73.50, 29.09. ESI-HRMS Calculated for C14H15N2O5S2
[M+H]+:355.0422 Found:355.0451.
4.2.12. 13-PEG3-MMAE (17)
N3-PEG3-MMAE (14) (30 mg, 0.032 mmol), 13 (13.82 mg, 0.039
mmol), Na ascorbate (6.87 mg, 0.039 mmol) and CuSO4 (6.2 mg, 0.039
mmol) were dissolved in tBuOH/H2O/DMF (1/1/1) (6 mL). The reaction
mixture was stirred at room temperature for 2 h. Upon completion, the
resulting crude mixture was purified by HPLC (10–100% MeCN/H2O
over 30 min) to afford 17.5 mg (0.0136 mmol, 42%) yield of 17 as a
4.2.9. N3-PEG3-MMAE (14)
Monomethyl auristatin E (100.0 mg, 0.14 mmol), 2-(2-(2-(2-azi-
doethoxy)ethoxy)ethoxy)acetic acid (34.9 mg, 0.15 mmol), HOAt (37.9
mg, 0.28 mmol), EDCl (53.5 mg, 0.28 mmol) and N’N-diisopropyle-
thylamine (140.6 mg, 1.39 mmol) were dissolved in DMF (5 mL). The
reaction mixture was stirred at room temperature overnight. The
resulting crude mixture was purified by HPLC (10–100% MeCN/H2O
white
solid.
ESI-HRMS
Calculated
for
C63H99N10O16S2
[M+H]+:1287.6369, Found:1287.6390. The product was analyzed by
HPLC for purity of 94%. 1H NMR (500 MHz, CD3OD) δ 8.03–7.96 (m,
8

R. Huang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115793
1H), 7.92 (ddd, J = 8.4, 4.3, 1.9 Hz, 3H), 7.43–7.36 (m, 4H), 7.31 (dt, J
= 14.0, 7.6 Hz, 2H), 7.24–7.19 (m, 1H), 7.15 (ddd, J = 16.5, 9.8, 1.3 Hz,
2H), 6.30–6.20 (m, 4H), 4.80–4.46 (m, 7H), 4.31–4.13 (m, 3H),
3.90–3.84 (m, 2H), 3.78–3.50 (m, 9H), 3.45–3.33 (m, 6H), 3.30–3.25
(m, 3H), 3.11 (d, J = 4.3 Hz, 1H), 3.02–2.90 (m, 3H), 2.60–1.21 (m,
13H), 1.21–1.10 (m, 6H), 1.07–0.81 (m, 19H). 13C NMR (126 MHz,
CD3OD) δ 175.70, 175.41, 175.07, 172.74, 171.87, 171.74, 171.59,
168.69, 146.14, 144.08, 143.88, 138.23, 137.51, 137.20, 132.43,
131.11, 129.58, 129.54, 129.24, 128.62, 128.39, 128.08, 127.90,
125.24, 86.66, 83.48, 78.87, 77.53, 77.25, 71.83, 71.69, 71.50, 71.40,
71.37, 71.08, 70.53, 70.33, 63.64, 63.54, 61.99, 61.51, 60.77, 60.58,
58.61, 58.35, 56.34, 56.13, 51.42, 48.07, 45.86, 45.52, 38.27, 36.36,
33.62, 31.83, 30.34, 27.73, 27.65, 26.98, 26.58, 25.84, 25.63, 24.46,
19.75, 19.71, 19.36, 19.17, 18.99, 18.74, 16.95, 16.32, 15.97, 15.82,
15.01, 10.90.
(Cambridge, UK). DAPI was purchased from Cell Signaling Technology
(Boston, USA). HCC827 and NCI-H2228 cells (2 × 105 cells/tube) were
collected and incubated with varying concentrations of ADCs and
cetuximab at 4 ◦C for 30 min. After being washed with FACE solution
(1% BSA in PBS, pH 7.4), the cells were stained with Goat Anti-Human
◦
IgG H&L (FITC) (1:200) at 4 C for 30 min. After being washed with
FACE solution, the cells were stained with DAPI (1 μg/mL) in PBS. The
fluorescent signals in individual samples were detected by CytoFLEX
flow cytometer (Beckman Coulter, Brea, USA) and analyzed using the
FlowJo software.
4.7. Flow cytometry for internalization of ADCs
HCC827 cell (2 × 105 cells/tube) was collected and incubated with
ADCs and cetuximab in duplicate at 4 ◦C for 30 min. After being washed
with FACE solution, the samples were incubated with PBS at 4 ◦C and 37
◦C for 3 h. And then the samples were incubated with Goat Anti-Human
4.3. Antibody-drug conjugates
◦
IgG H&L (FITC) (1:200) at 4 C for 30 min. After being washed with
To a solution of Trastuzumab (100 μL, 20 μM, 3 mg/mL) in PBS (137
FACE solution, the cells were stained with DAPI (1 μg/mL) in PBS. The
mM NaCl, 2.67 mM KCl, 10 Mm Na2HPO4, 2 mM KH2PO4, pH 7.2–7.4)
was added tris(2-carboxyethyl)phosphine (TCEP, 10 eq.,2 µL, 10 mM
stock solution in H2O, pH 7.07 was adjusted by NaOH and H3PO4). The
mixture was vortexed and incubated at 37 ◦C for 1 h. The solutions of
compounds 15–17 (10 mM in DMSO, 20 eq., 4 µL) was added respec-
tively and the reaction mixture incubated at 37 ◦C for 12 h. The excess
reagents were removed by repeated diafiltration into PBS using an
Zeba™ Spin Desalting Columns (Thermo, 7K MWCO, 0.5 mL). The
resulting conjugates were characterized by MS and SEC analysis.
fluorescent signals in individual samples were detected by CytoFLEX
flow cytometer (Beckman Coulter, Brea, USA) and analyzed using the
FlowJo software. The percentage of internalization was determined
using the following formula: percentage of internalization = (fluores-
cence intensity of cells at 4 ◦C – fluorescence intensity of cells at 37 ◦C)/
fluorescence intensity of cells at 4 ◦C × 100%.
4.8. In vivo efficacy study
All experimental protocols were approved by Animal Ethics Com-
mittee of ShanghaiTech University. All procedures in efficacy study
were conducted according to the Animal Welfare Act and Regulations
published by the US National Institutes of Health. Five-week-old Nod-
Scid male mice were purchased from GemPharmatech Co., Ltd (Nanjing,
China). After acclimatization for one week, healthy mice were subcu-
taneously implanted with 5 × 106 HCC827 cells. Fourteen days after
implantation the mice were divided into three groups (n = 8 each): 21,
22 and PBS as control. All the groups received four doses of 20 mg/kg on
days 0, 4, 8 and 12, injected intravenously. Tumor volume and body-
weight were measured at regular intervals. Tumor volumes were
calculated with the formula: (mm3) = (length × width2)/2.
4.4. Cell lines and culture
Cancer cell lines SK-BR-3, MCF7, MDA-MB-468, HCC827 and NCI-
H2228 were obtained from American Type Culture Collection (ATCC,
Manassas, VA, USA). Fetal bovine serum (FBS), RPMI 1640 medium,
DMEM medium, MEM medium, Penicillin-Streptomycin (PS), non-
essential amino acid (NEAA) were purchased from Gibco Thermofisher
Scientific (Waltham, MA. USA). Recombinant human insulin was pur-
chased from Sigma Chemical (St. Louis, MO, USA). SK-BR-3 and MDA-
MB-468 cells were cultured in DMEM medium with 10% FBS and 1%
PS; HCC827 and NCI-H2228 cells were cultured in RPMI 1640 medium
with 10% FBS and 1% PS; MCF7 cell was cultured in MEM medium with
The animals were housed (4 mice/cage) in a specific pathogen-free
(SPF) animal laboratory of the National Center for Protein Science
Shanghai (Shanghai, China) under standard laboratory conditions
10% FBS, 1% PS, 1% NEAA and 10 μg/mL insulin; all cells were cultured
at 37 ◦C in a humidified incubator with 5% CO2.
◦
(adequate fresh air exchange, temperature 20–24 C and relative hu-
4.5. Cytotoxicity assay
midity 40–70%). A 12-h light/dark automatic cycle of artificial illumi-
nation was used. All animals were provided sterile drinking water.
Cytotoxicity assay of ADCs 18–20 was performed on SK-BR-3, MCF7,
MDA-MB-468 and HCC827 and NCI-H2228 cells; ADCs 21–23 was
performed on HCC827 and NCI-H2228 cells. Briefly, cells (5 × 103 cells/
Declaration of Competing Interest
well) were cultured in 96-well plates with 100 μL complete medium, and
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
24 h later the cells were treated in triplicate with varying concentrations
of ADCs, MMAE, T-DM1, herceptin or cetuximab for 72 h. The cells
cultured in medium alone served as the control and medium alone
served as the blank. Cell viability was determined using Cell Counting
Kit-8 (CCK-8) kit according to the manufacturer’s instructions. The
absorbent optical density (OD) values at 450 nm were measured in a
microplate reader (SpectraMax i3, MD, USA). The inhibition rate of cell
growth in individual wells was determined using the following formula:
growth inhibition rate = (OD value of control ꢀ OD value of dose)/(OD
value of control ꢀ OD value of blank) × 100%. The half maximal
inhibitory concentrations (IC50) of the compounds for each cell were
calculated using the Prism 7 software.
Acknowledgements
We thank Cunliang Zhao at Jing Medicine for his help to support this
research, Dr. Wei Huang and Dr. Feng Tang at Shanghai Institute of
Materia Medica (SIMM), Chinese Academy of Sciences for providing
Endo-S. This work was supported by the grant from China Postdoctoral
Science Foundation (2019M651608).
Appendix A. Supplementary material
4.6. Flow cytometry for affinity of ADCs
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2020.115793.
Goat Anti-Human IgG H&L (FITC) was purchased from Abcam
9

R. Huang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115793
13 Schumacher FF, Nunes JP, Maruani A, et al. Next generation maleimides enable the
controlled assembly of antibody-drug conjugates via native disulfide bond bridging.
Org Biomol Chem. 2014;12:7261–7269.
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10