Novel antibody drug conjugates containing exatecan derivative-based cytotoxic payloads

Article abstract of DOI:10.1016/j.bmcl.2016.02.020

Trastuzumab conjugates consisting of exatecan derivatives were prepared and their biological activities and physicochemical properties were evaluated. The ADCs showed strong efficacy and a low aggregation rate. The exatecan derivatives were covalently connected via a peptidyl spacer (Gly-Gly-Phe-Gly), which is assumed to be stable in circulation, and were cleaved by lysosomal enzymes following ADC internalization into tumor tissue. These anti-HER2 ADCs exhibited a high potency, specifically against HER2-positive cancer cell lines in vitro. The ADCs, bearing exatecan derivatives which have more than two methylene chains, exhibited superior cytotoxicity. It was speculated that steric hindrance of the cleavable amide moiety could be involved in the drug release. The adequate alkyl lengths of exatecan derivatives (13, 14, 15) were from two to four in terms of aggregation rate. The ADC having a hydrophilic moiety showed good efficacy in a HER2-positive and Trastuzumab-resistant breast carcinoma cell model in mice.

Full text of DOI:10.1016/j.bmcl.2016.02.020

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel antibody drug conjugates containing exatecan
derivative-based cytotoxic payloads
⇑
Takashi Nakada , Takeshi Masuda, Hiroyuki Naito, Masao Yoshida, Shinji Ashida, Koji Morita,
Hideki Miyazaki, Yuji Kasuya, Yusuke Ogitani, Junko Yamaguchi, Yuki Abe, Takeshi Honda
Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Trastuzumab conjugates consisting of exatecan derivatives were prepared and their biological activities
and physicochemical properties were evaluated. The ADCs showed strong efficacy and a low aggregation
rate. The exatecan derivatives were covalently connected via a peptidyl spacer (Gly-Gly-Phe-Gly), which
is assumed to be stable in circulation, and were cleaved by lysosomal enzymes following ADC internal-
ization into tumor tissue. These anti-HER2 ADCs exhibited a high potency, specifically against HER2-pos-
itive cancer cell lines in vitro. The ADCs, bearing exatecan derivatives which have more than two
methylene chains, exhibited superior cytotoxicity. It was speculated that steric hindrance of the cleavable
amide moiety could be involved in the drug release. The adequate alkyl lengths of exatecan derivatives
(13, 14, 15) were from two to four in terms of aggregation rate. The ADC having a hydrophilic moiety
showed good efficacy in a HER2-positive and Trastuzumab-resistant breast carcinoma cell model in mice.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 9 November 2015
Revised 4 February 2016
Accepted 8 February 2016
Available online xxxx
Keywords:
Antibody drug conjugate
Camptothecin
Exatecan
Cytotoxic payload
Linker
Anti-HER2
Trastuzumab
Oncology
Cancer therapy
Antibody drug conjugates (ADCs) have been established over
the past several years and are now one of the most successful
and important strategies for treating patients with hematological
malignancies and solid tumors. At present, only three ADCs have
been launched; the first ADC (MylotargÒ, gemtuzumab ozogam-
icin, Pfizer) launched in 2001 for the treatment of patients with
acute myelogenous leukemia (AML). However, MylotargÒ was
withdrawn in 2010 due to both a lack of evidence confirming its
clinical benefits and safety concerns. Since then, two ADCs,
AdcetrisÒ (brentuximab vedotin, Seattle Genetics) and KadcylaÒ
(trastuzumab emtansine, Genetech and Roche), have been
launched. And currently, over 30 programs in clinical development
have been carried out in pursuit of ADC drug candidates.1
Camptothecin (CPT) has been demonstrated to be effective
against a broad spectrum of tumors. CPT’s target is human DNA
topoisomerase I (Topo I). CPT binds to complexes with Topo I
and DNA, and thereby is stabilized. This stabilized complex causes
apoptosis. Irinotecan hydrochloride (CPT-11, yakult) is a prodrug of
a potent CPT analog (SN-38) used for the treatment of patients
with various tumors. Exatecan methansulfonate (DX-8951f) is a
water soluble CPT which exhibits a stronger Topo I inhibitory
activity and antitumor activity than the other CPT analogs. Further-
more, exatecan is effective against P-glycoprotein (P-gp) mediated
multi-drug resistant cells.2
ADCs are composed of a carrier monoclonal antibody (mAb) and
cytotoxic drug payload. The ADCs discussed herein are ADCs com-
posed of a mAb with exatecan derivatives via a peptidyl linker, as
shown in Figure 1. It has been previously recognized that glycyl
glycyl phenylalanyl glycyl (GGFG) is selectively cleaved by lysoso-
mal enzymes (presumably cathepsins).3 GGFG is known to release
drugs into tumor tissue without releasing them into peripheral
circulation.
Human epidermal growth factor receptor 2 (HER2) is a very
effective therapeutic target for breast cancer patients.
Trastuzumab (HerceptinÒ, Genetech and Roche) is an anti-HER2
antibody and is used for breast cancer HER2-positive patients in
combination with the chemotherapy drugs, anthracycline and tax-
ane. Trastuzumab is the carrier mAb of trastuzumab emtansine.
Therefore, the tumor specificity of this mAb, trastuzumab, is suit-
able for being the ADC’s carrier.
The synthesis of ADC (1) was carried out via the route shown in
Scheme 1. Commercially available tert-butoxycarbonyl glycyl glycyl
phenylalanyl glycine (BOC-GGFG-OH) was dissolved in dichloro-
methane. N-Hydroxysuccinimide (HO-Su) and 1-(3-dimethy-
laminopropyl)-3-ethylcarbodiimide hydrochloride (WSCI) was
⇑
Corresponding author. Tel.: +81 3 3492 3131; fax: +81 3 5740 3641.
E-mail address: nakada.takashi.g6@daiichisankyo.co.jp (T. Nakada).
http://dx.doi.org/10.1016/j.bmcl.2016.02.020
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Nakada, T.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.02.020

2
T. Nakada et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
O
O
O
H
H
N
ADC (1): X = none,
Y = CH2
N
N
Y
N
H
X
NH
S
ADC (2): X = NH2-CH2-(C=O), Y = CH2
ADC (3): X = NH2-(CH2)2-(C=O), Y = CH2
ADC (4): X = NH2-(CH2)3-(C=O), Y = CH2
ADC (5): X = NH2-(CH2)4-(C=O), Y = CH2
ADC (6): X = NH2-(CH2)5-(C=O), Y = CH2
ADC (7): X = NH2-(CH2)3-(C=O), Y = (C=O)-NH-(CH2CH2O)2
n: DAR
O
O
O
O
N
F
N
O
O
OH
n
Figure 1. The structure of ADCs.
O
H
N
O
O
O
O
H2
N
H
N
H
N
N
O
O
NH2
H
N
N
H
N
N
O
F
O
N
O
1) Boc-GGFG-OH, WSCI,
Et3N / DMF
O
O
F
O
O
H
O
N
H
O
O
O
NH
TFA
10
N
NH
F
N
O
2) TFA / CH2Cl2
Et3N / DMF
O
O
N
N
N
MsOH
N
OH
O
O
O
9
DX-8951f (8)
OH
O
OH
O
mc-GGFG-DX (11)
O
O
H
N
H
N
N
S
N
H
O
O
O
O
N
H
O
conjugation
NH
Trastuzumab
O
N
F
N
O
OH
O
n
ADC (1)
Scheme 1. The synthesis of ADC (1).
added. To this mixture, was added DX-8951f (8) and triethylamine
solution in N,N0-dimethylformamide (DMF). The Boc-GGFG-exate-
can was deprotected by trifluoroacetic acid (TFA), followed by
condensation with 6-maleimidohexanoic acid N-hydroxysuccin-
imide ester (mc-OSu) in DMF to obtain the maleimide precursor
drug linker (mc-GGFG-exatecan, 11).4
The maleimide precursor drug linker (11) and mAb conjugation
method used a conventional conjugated cysteine (Cys) strategy.5 A
disulfide group with hinges of mAb was reduced with tris(2-car-
boxyethyl)phosphine hydrochloride (TCEP HCl). And the precursor
drug linker (11) was added to the reduced antibody. The drug to
antibody ratio (DAR) was able to control an amount of the reducing
agent, as shown in Table 1. The reaction mixture was purified using
size exclusion chromatography (SEC). The DAR5,6 and aggregation
rate of the obtained product were measured.7
Exatecan derivatives were prepared by condensation and suc-
cessive deprotection as shown in Scheme 2,8 and these compounds
were used as starting materials to obtain other ADCs (2)–(7) via
similar synthetic routes of ADC (1).4
These prepared ADCs were shown to express in vitro cytotoxic-
ities in a HER2-positive cell line, KPL-4, as shown in Table 1. GI50
was the concentration for 50% of maximal inhibition of cell prolif-
eration. The KPL-4 cells used were a HER2-positive human breast
cancer cell line. The KPL-4 was provided by Junichi Kurebayashi
from Kawasaki Medical School in Japan. MCF7 cells used were a
HER2-negative breast cancer cell line. The cell line was obtained
from the European Collection of Cell Cultures (ECACC). None of
the ADCs could kill the MCF7 cell line, GI50 > 100 nM. These results
suggest that the anti-HER2 ADCs showed HER2 selective cytotoxi-
city. Though all of the ADCs (1)–(7) showed strong cytotoxicities
in vitro, the ADCs (1) and (2) had a decreased cytotoxicity com-
pared with that of other ADCs. It was speculated that degrading
enzymes in tumor cells might not work efficiently because of the
steric hindrance of the released drug moiety. The cleavable amide
position (C-terminal amide of GGFG tetrapeptide) between the
released drug and cleavable peptide linker was important for the
affinity of the enzyme. Therefore, the cleavable amide position
was changed away from the drug by adding some methylene
chains between the drug and cleavable peptide. In particular, the
drugs released from the ADCs (3)–(6) were changed to alkylamine
derivatives with methylene alkyl chains from exatecan. These
ADCs showed potent in vitro activity. However, the ADC (6) indi-
cated more aggregates, possibly due to its hydrophobicity. The
ADCs (3)–(5) had less aggregates than ADC (6). The alkyl length
of exatecan derivatives influenced the aggregation rate. Having
an alkyl length less than hexylene’s is preferred from the viewpoint
of an ADC’s physicochemical properties. Therefore, an alkyl length
from two to four seemed the best choice in this case. Moreover, a
hydrophilic moiety could be added in the linker portion. ADC
(7a) which has diethylene glycol showed less of an aggregation
rate than ADC (4). Even a high drug load ADC (7b, DAR 6.2) had
an acceptable low aggregation rate, and greater efficacy in vitro.
The in vivo efficacy of the ADC (7b) was compared with trastu-
zumab in mice bearing JIMT-1,
a
HER2-positive and
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T. Nakada et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Table 1
Drug linker structure, conjugation condition, and in vitro efficacy of ADCs
ADC
entry
Drug linker structure
Y
Conjugation condition
Characterization
Aggregation rate
In vitro
X
TCEP
Drug linker
(equiv)
Yield
(%)
DAR
KPL-4 GI50
(nM)
(equiv)
(%)
1
2
3
4
5
6
7a
7b
None
ANHACH2A(C@O)A
A(CH2)3A
A(CH2)3A
A(CH2)3A
A(CH2)3A
A(CH2)3A
A(CH2)3A
AC(@O)ANHA(CH2ACH2AO)2A
AC(@O)ANHA(CH2ACH2AO)2A
4.6
2.3
4.6
4.6
4.6
4.6
2.3
4.6
9.2
4.6
9.2
9.2
9.2
9.2
4.6
9.2
55
83
62
75
59
61
85
86
3.4
3.2
3.8
2.6
3.4
2.5
3.6
6.2
26
3
2
3
4
20
1
1
0.33
0.39
0.07
0.05
0.07
0.11
0.12
0.04
ANHA(CH2)2A(C@O)A
ANHA(CH2)3A(C@O)A
ANHA(CH2)4A(C@O)A
ANHA(CH2)5A(C@O)A
ANHA(CH2)3A(C@O)A
ANHA(CH2)3A(C@O)A
H2N
O
1)
BocHN
OH
NH2
n
NH
n
O
O
N
O
WSCI, Et3N / DMF
N
F
N
F
N
O
2) TFA / CH2Cl2
O
TFA
n = 0 (12), 1 (13), 2 (14), 3 (15), and 4 (16)
Scheme 2. The synthesis of exatecan derivatives.
MsOH
OH
O
OH O
DX-8951f (8)
presents a significant challenge for ADCs. Design of camptothecin
derivatives and implementation of a high polarity moiety is an
ideal solution to overcome this. ADCs bearing exatecan derivatives
of camptothecin analogs show a high potency for cancer therapy.
References and notes
1. Hamilton, G. S. Biologicals 2015, 43, 318.
2. Mitsui, I.; Kumazawa, E.; Hirota, Y.; Aonuma, M.; Sugimori, M.; Ohsuki, S.; Uoto,
K.; Ejima, A.; Terasawa, H.; Sato, K. Jpn. J. Cancer Res. 1995, 86, 776.
3. Inoue, K.; Kumazawa, E.; Kuga, H.; Susaki, H.; Masubuchi, N. Adv. Exp. Med. Biol.
2003, 519, 145.
4. Nuclear magnetic resonance (NMR) and liquid chromatography–mass (LC–MS)
analysis. LC–MS ionization mode was electrospray ionization (ESI) or
atmospheric pressure chemical ionization (APCI). For precursor drug linker
(11) of ADC (1); 1H NMR (400 MHz, DMSO-d6) d/ppm: 0.87 (3H, t, J = 7.4 Hz),
1.15–1.21 (2H, m), 1.41–1.50 (4H, m), 1.80–1.90 (2H, m), 2.07–2.12 (4H, m),
2.17–2.23 (1H, m), 2.35–2.40 (1H, m), 2.41 (3H, s), 2.73–2.81 (1H, m), 2.98 (1H,
dd, J = 13.7, 4.6 Hz), 3.15–3.20 (2H, m),3.53 (1H, dd, J = 16.6, 5.7 Hz), 3.62–3.77
(5H, m), 4.39–4.45 (1H, m), 5.22 (1H, d, J = 18.9 Hz),5.27 (1H, d, J = 18.9 Hz),5.39
(1H, d, J = 16.0 Hz), 5.44 (1H, d, J = 16.0 Hz), 5.55–5.60 (1H, m), 6.53 (1H, s), 6.98
(2H, s), 7.13–7.24 (5H, m), 7.32 (1H, s), 7.81 (1H, d, J = 10.3 Hz), 7.95–8.00 (1H,
m), 8.05–8.09 (2H, m), 8.28–8.31 (1H, m), 8.41 (1H, d, J = 8.6 Hz). MS (APCI) m/z:
947 (M+H)+. For precursor drug linker of ADC (2); 1H NMR (400 MHz, DMSO-d6)
d/ppm: 0.86 (3H, t, J = 7.2 Hz), 1.18–1.19 (2H, m), 1.45–1.48 (4H, m), 1.83–1.85
(2H, m), 2.12–2.17 (4H, m), 2.39 (3H, s), 2.68 (1H, dd, J = 24.4, 14.7 Hz), 2.83–
2.87 (1H, m), 3.17–3.78 (12H, m), 4.42–4.45 (1H, m), 5.23 (2H, s), 5.41 (2H, s),
5.58–5.60 (1H, m), 6.53 (1H, s), 6.99 (2H, s), 7.15–7.29 (6H, m), 7.76 (1H, d,
J = 10.9 Hz), 7.97–8.00 (1H, m), 8.09–8.12 (3H, m), 8.25–8.28 (1H, m), 8.44 (1H,
d, J = 8.2 Hz). MS (APCI) m/z: 1004 (M+H)+. For precursor drug linker of ADC (3);
1H NMR (400 MHz, DMSO-d6) d/ppm: 0.86 (3H, t, J = 7.2 Hz), 1.12–1.22 (2H, m),
1.39–1.51 (4H, m), 1.79–1.91 (2H, m), 2.02–2.20 (2H, m), 2.07 (2H, t, J = 7.4 Hz),
2.30–2.42 (4H, m), 2.40 (3H, s), 2.78 (1H, dd, J = 14.1, 9.4 Hz), 3.02 (1H, dd,
J = 14.7, 4.9 Hz), 3.12–3.21 (2H, m), 3.26–3.42 (2H, m), 3.50–3.80 (6H, m), 4.40–
4.51 (1H, m), 5.19 (1H, d, J = 19.6 Hz), 5.26 (1H, d, J = 19.2 Hz), 5.42 (2H, brs),
5.51–5.62 (1H, m), 6.53 (1H, s), 6.99 (2H, s), 7.13–7.28 (5H, m), 7.31 (1H, s),
7.74–7.84 (2H, m), 8.01 (1H, t, J = 5.3 Hz), 8.06 (1H, t, J = 5.7 Hz), 8.14 (1H, d,
J = 8.2 Hz), 8.25 (1H, t, J = 5.7 Hz), 8.53 (1H, d, J = 8.6 Hz). MS (ESI) m/z: 1018 (M
+H)+. For precursor drug linker of ADC (4); 1H NMR (400 MHz, DMSO-d6) d/
ppm:0.87 (3H, t, J = 7.3 Hz), 1.12–1.22 (2H, m), 1.40–1.51 (4H, m), 1.66–1.76
(2H, m), 1.80–1.91 (2H, m), 2.05–2.21 (6H, m), 2.39 (3H, s), 2.79 (1H, dd, J = 14.0,
9.8 Hz), 2.98–3.21 (5H, m), 3.55–3.77 (8H, m), 4.41–4.48 (1H, m), 5.15 (1H, d,
J = 18.9 Hz), 5.24 (1H, d, J = 18.9 Hz), 5.40 (1H, d, J = 17.1 Hz), 5.44 (1H, d,
J = 17.1 Hz), 5.54–5.60 (1H, m), 6.53 (1H, s), 6.99 (2H, s), 7.20–7.27 (5H, m), 7.30
(1H, s), 7.70 (1H, t, J = 5.5 Hz), 7.80 (1H, d, J = 11.0 Hz), 8.03 (1H, t, J = 5.8 Hz),
8.08 (1H, t, J = 5.5 Hz), 8.14 (1H, d, J = 7.9 Hz), 8.25 (1H, t, J = 6.1 Hz), 8.46 (1H, d,
Figure 2. The in vitro efficacy of ADCs in JIMT-1 cells at an average starting tumor
volume 150 mm3 after 12 days.
trastuzumab-resistant breast carcinoma cell line. JIMT-1 was
obtained from the Deutshe Sammlung von Mikroorganismen und
Zellkulturen GmbH (DSMZ). Mice were treated with a single i.v.
injection of 10 mg/kg ADC (7b), or trastuzumab. Trastuzumab
had no effect on tumor growth. In contrast, the mice treated with
the ADC (7b) achieved tumor regressions, as shown in Figure 2.
These novel ADCs using exatecan derivatives having alkyl
amine release the drug efficiently. Furthermore, adding a hydro-
philic group to the C-terminus of the peptide moiety was effective
in reducing aggregation rates. The ADC (7b), which has a maximum
8 DAR, showed an acceptable aggregation rate and high efficacy
in vitro and in vivo. No change in mice body weight was observed
by the ADC treatment indicating that the ADC was well tolerated.
Camptothecins are an especially compelling drug class for ADCs
because they can be produced synthetically and modified to give
analogs. However, aqueous insolubility of the camptothecins
J = 8.5 Hz). MS (APCI) m/z: 1032 (M+H)+. For precursor drug linker of ADC (5); 1
NMR (400 MHz, DMSO-d6) d/ppm: 0.87 (3H, t, J = 7.4 Hz), 1.13–1.21 (2H, m),
H
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T. Nakada et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
1.36–1.52 (6H, m), 1.53–1.65 (2H, m), 1.79–1.92 (2H, m), 2.05–2.15 (4H, m),
(1 mg/mL) injection; ADC (1) in phosphate buffered saline (pH 7.5), ADC (2),
(7a), and (7b) in acetate buffer red 5% sorbitol (pH 5.5), and ADC (3)–(6) in
phosphate buffered saline (pH 6.0).
2.19 (2H, s), 2.40 (3H, s), 2.79 (1H, dd, J = 13.7, 10.2 Hz), 2.98–3.10 (3H, m), 3.12–
3.21 (2H, m), 3.29–3.37 (2H, m), 3.53–3.79 (6H, m), 4.41–4.50 (1H, m), 5.16 (1H,
d, J = 18.8 Hz), 5.23 (1H, d, J = 18.8 Hz), 5.43 (2H, s), 5.52–5.60 (1H, m), 6.53 (1H,
s), 6.99 (2H, s), 7.12–7.28 (5H, m), 7.31 (1H, s), 7.63 (1H, t, J = 5.7 Hz), 7.80 (1H, d,
J = 10.6 Hz), 8.02 (1H, t, J = 5.9 Hz), 8.08 (1H, t, J = 5.7 Hz), 8.12 (1H, d, J = 7.8 Hz),
8.24(1H, t, J = 5.7 Hz), 8.45 (1H, d, J = 8.6 Hz). MS (ESI) m/z: 1046 (M+H)+. For
precursor drug linker of ADC (6); 1H NMR (400 MHz, CD3OD) d/ppm: 0.99 (3H, t,
J = 7.4 Hz), 1.27 (2H, td, J = 11.6, 6.1 Hz), 1.38–1.44 (2H, m), 1.50–1.63 (6H, m),
1.65–1.80 (2H, m), 1.89–1.98 (2H, m), 2.17–2.25 (3H, m), 2.26–2.36 (3H, m),
2.40 (3H, s), 2.95 (1H, dd, J = 14.3, 9.2 Hz), 3.12 (1H, dd, J = 13.7, 5.7 Hz), 3.15–
3.25 (4H, m), 3.44 (2H, t, J = 7.2 Hz), 3.65 (1H, d, J = 17.2 Hz), 3.76 (1H, d,
J = 17.2 Hz), 3.79––3.86 (4H, m), 4.43 (1H, dd, J = 8.9, 6.0 Hz), 5.10 (1H, d,
J = 18.9 Hz), 5.25 (1H, d, J = 18.9 Hz), 5.35 (1H, d, J = 16.6 Hz), 5.56 (1H, d,
J = 16.0 Hz), 5.60–5.64 (1H, m), 6.76 (2H, s), 7.12–7.24 (6H, m), 7.58 (1H, s), 7.60
(1H, d, J = 10.9 Hz), 7.68 (1H, t, J = 5.7 Hz). MS (ESI) m/z: 1060 (M+H)+. For
precursor drug linker of ADC (7); 1H NMR (400 MHz, DMSO-d6) d: 0.85 (3H, t,
J = 7.4 Hz), 1.65–1.74 (2H, m), 1.77–1.90 (2H, m) 2.07–2.19 (4H, m), 2.30 (2H, t,
J = 7.2 Hz), 2.33–2.36 (2H, m), 2.38 (3H, s), 2.76 (1H, dd, J = 13.7, 9.8 Hz), 2.96–
3.18 (9H, m), 3.42–3.44 (4H, m), 3.53–3.76 (10H, m), 4.43 (1H, td, J = 8.6, 4.7 Hz),
5.14 (1H, d, J = 18.8 Hz), 5.23 (1H, d, J = 18.8 Hz), 5.38 (1H, d, J = 17.2 Hz), 5.42
(1H, d, J = 17.2 Hz), 5.52–5.58 (1H, m), 6.52 (1H, s), 6.98 (2H, s), 7.12–7.17 (1H,
m), 7.18–7.25 (4H, m), 7.29 (1H, s), 7.69 (1H, t, J = 5.5 Hz), 7.78 (1H, d,
J = 11.3 Hz), 7.98–8.03 (2H, m), 8.11 (1H, d, J = 7.8 Hz), 8.16 (1H, t, J = 5.7 Hz),
8.23 (1H, t, J = 5.9 Hz), 8.44(1H, d, J = 9.0 Hz). MS (APCI) m/z: 1149 (M+H)+.
8. NMR and LC–MS analysis. Compound (12); 1H NMR (400 MHz, CD3OD) d/ppm:
0.96 (3H, t, J = 7.0 Hz), 1.89–1.91 (2H, m), 2.14–2.16 (1H, m), 2.30 (3H, s), 2.40–
2.42 (1H, m), 3.15–3.21 (2H, m), 3.79–3.86 (2H, m), 4.63–4.67 (1H, m), 5.00–5.05
(1H, m), 5.23 (1H, d, J = 16.0 Hz), 5.48 (1H, d, J = 16.0 Hz), 5.62–5.64 (1H, m),
7.40–7.45 (2H, m). MS (APCI) m/z: 493 (M+H)+. Compound (13); 1H NMR
(400 MHz, DMSO-d6) d/ppm: 0.87 (3H, t, J = 7.2 Hz), 1.86 (2H, dquin, J = 14.6,
7.2 Hz), 2.06–2.27 (1H, m), 2.41 (3H, s), 2.46–2.57 (2H, m), 3.08 (2H, t,
J = 6.8 Hz), 3.14–3.24 (2H, m), 5.22 (1H, d, J = 18.8 Hz), 5.29 (1H, d, J = 18.8 Hz),
5.43 (2H, s), 5.58 (1H, dt, J = 8.5, 4.5 Hz), 6.55 (1H, s), 7.32 (1H, s), 7.74 (3H, brs),
7.82 (1H, d, J = 11.0 Hz), 8.67 (1H, d, J = 8.6 Hz). MS (ESI) m/z: 507 (M+H)+.
Compound (14); 1H NMR (400 MHz, DMSO-d6) d/ppm: 0.87 (3H, t, J = 7.2 Hz),
1.79–1.92 (4H, m), 2.10–2.17 (2H, m), 2.27 (2H, t, J = 7.0 Hz), 2.40 (3H, s), 2.80–
2.86 (2H, m), 3.15–3.20 (2H, m), 5.15 (1H, d, J = 18.8 Hz), 5.26 (1H, d, J = 18.8 Hz),
5.42 (2H, s), 5.54–5.61 (1H, m), 6.55 (1H, s), 7.32 (1H, s), 7.72 (3H, brs), 7.82 (1H,
d, J = 11.0 Hz), 8.54 (1H, d, J = 8.6 Hz). MS (APCI) m/z: 521 (M+H)+. Compound
(15); 1H NMR (400 MHz, DMSO-d6) d/ppm: 0.88 (3H, t, J = 7.4 Hz), 1.52–1.71
(4H, m), 1.87 (2H, tt, J = 14.4, 6.9 Hz), 2.07–2.18 (2H, m), 2.22 (2H, t, J = 7.0 Hz),
2.40 (3H, s), 2.76–2.88 (2H, m), 3.13–3.22 (2H, m), 5.18 (1H, d, J = 18.8 Hz), 5.24
(1H, d, J = 18.8 Hz), 5.43 (2H, s), 5.53–5.61 (1H, m), 6.55 (1H, s),7.33 (1H, s), 7.65
(3H, brs), 7.81 (1H, d, J = 11.3 Hz), 8.49 (1H, d, J = 8.6 Hz). MS (ESI) m/z: 535 (M
+H)+. Compound (16); 1H NMR (400 MHz, DMSO-d6) d/ppm: 0.88 (3H, t,
J = 7.2 Hz), 1.31–1.41 (2H, m), 1.52–1.70 (4H, m), 1.80–1.94 (2H, m), 2.05–2.18
(2H, m), 2.21 (2H, t, J = 7.4 Hz), 2.40 (3H, s), 2.81 (2H, t, J = 7.4 Hz), 3.10–3.25
(2H, m), 3.33 (2H, brs), 5.18 (1H, d, J = 19.8 Hz), 5.22 (1H, d, J = 19.8 Hz), 5.41
(2H, d, J = 16.6 Hz), 5.45 (2H, d, J = 16.6 Hz), 5.53–5.60 (1H, m), 6.55 (1H, s), 7.32
(1H, s), 7.80 (1H, d, J = 10.9 Hz), 8.49 (1H, d, J = 9.2 Hz). MS (ESI) m/z: 549 (M
+H)+.
5. Lyon, R. P.; Meyer, D. L.; Setter, J. R.; Senter, P. D. Methods Enzymol. 2012, 502,
123.
6. DAR was calculated by UV absorption measurement of 280 nm and 370 nm.
7. Aggregation rate was measured by high performance liquid chromatography
(HPLC). Column; Tosoh TSK gel G3000SWXL (300 ꢀ 7.8 mm). Mobile phase;
0.5 mL/min 0.03 M phosphate buffer (0.2 M arginine, pH 6.8). Sample; 10 lL
Please cite this article in press as: Nakada, T.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.02.020