Synthesis and evaluation of azalamellarin N and its A-ring-modified analogues as non-covalent inhibitors of the EGFR T790M/L858R mutant

Article abstract of DOI:10.1016/j.bmc.2021.116039

Azalamellarin N, a synthetic lactam congener of the marine natural product lamellarin N, and its A-ring-modified analogues were synthesized and evaluated as potent and non-covalent inhibitors of the drug-resistant epidermal growth factor receptor T790M/L8

Full text of DOI:10.1016/j.bmc.2021.116039

Bioorg. Med. Chem. 34 (2021) 116039
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of azalamellarin N and its A-ring-modified
analogues as non-covalent inhibitors of the EGFR T790M/L858R mutant
a
,
*
b
b
b
Tsutomu Fukuda , Mizuho Anzai , Akane Nakahara , Kentaro Yamashita ,
c
c
d
d
d
Kazuaki Matsukura , Fumito Ishibashi , Yusuke Oku , Naoyuki Nishiya , Yoshimasa Uehara ,
b
Masatomo Iwao
a
Environmental Protection Center, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
b
Division of Chemistry and Materials Science, Graduate School of Engineering, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
c
Division of Marine Life Science and Biochemistry, Graduate School of Fisheries and Environmental Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-
8
d
521, Japan
Department of Integrated Information for Pharmaceutical Sciences, Iwate Medical University School of Pharmacy, 1-1-1 Idaidori, Yahaba-cho, Shiwa-gun, Iwate 028-
694, Japan
3
A R T I C L E I N F O
A B S T R A C T
Keywords:
Azalamellarin N, a synthetic lactam congener of the marine natural product lamellarin N, and its A-ring-modified
analogues were synthesized and evaluated as potent and non-covalent inhibitors of the drug-resistant epidermal
growth factor receptor T790M/L858R mutant. An in vitro tyrosine kinase assay indicated that the inhibitory
activities of the synthetic azalamellarin analogues were higher than those of the corresponding lamellarins. The
azalamellarin analogue bearing two 3-(dimethylamino)propoxy groups at C20- and C21-positions exhibited the
highest activity and selectivity against the mutant kinase [IC50 (T790M/L858R) = 1.7 nM; IC50 (WT) = 4.6 nM].
The inhibitory activity was attributed to the hydrogen bonding interaction between the lactam NH group of the
B-ring and carbonyl group of a methionine residue.
EGFR tyrosine kinase inhibitors (EGFR-TKIs)
EGFR T790M/L858R mutant
Azalamellarin N
A-ring-modified azalamellarin analogues
1
. Introduction
EGFR-TKIs.5,6 To overcome the acquired drug resistance, 4-anilinoqui-
7
8
nazoline-based irreversible inhibitors, such as afatinib, dacomitinib,
9
Activating mutations in the epidermal growth factor receptor
and neratinib , were developed by introducing an appropriate Michael
acceptor, which could form a covalent bond with the SH group of the
Cys797 residue. Although these second-generation EGFR-TKIs could
effectively inhibit EGFR with activating mutations as well as those with
the T790M resistance mutation, they caused serious side effects due to a
total loss of selectivity between the wild-type EGFR (EGFR WT) and
(
EGFR), a member of the ErbB family of membrane tyrosine kinases, are
1
responsible for causing a subset of non-small cell lung cancer (NSCLC).
Although several activating mutations have been identified, the most
common mutations in NSCLC patients are the L858R point mutation and
the exon 19 deletions in the kinase domain.2 Cancer cells having such
activating mutations are highly dependent on EGFR signaling for sur-
vival. Several EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been
mutant EGFR.1
0–12
Recently, pyrimidine-based irreversible inhibitors,
such as WZ4002,13 rociletinib, and osimertinib , have been devel-
oped to target EGFRs with the T790M mutation. The affinities of these
third-generation inhibitors for EGFR mutants, including T790M, are
much higher than those for EGFR WT, and they exhibit a promising
response to resistant NSCLC in clinical evaluations. Osimertinib was
approved for the treatment of NSCLC patients harboring EGFR T790M
mutation-positive tumors and, more recently, as a first-line therapy for
untreated EGFR mutant NSCLC.16 However, acquired resistance
emerged despite the remarkable success of osimertinib. One of the most
14
15
3
developed for treating the NSCLC patients. Initially, gefitinib and
4
erlotinib were found to be effective in NSCLC patients with activating
EGFR mutations. These first-generation EGFR-TKIs possess a 4-anilino-
quinazoline scaffold as a structural motif and act as competitive in-
hibitors of ATP that bind to the ATP binding pocket of the EGFR kinase
domain. However, drug resistance was observed within about one year
because of the T790M ‘gatekeeper’ mutation, which led to an increased
affinity to ATP and, hence, an increased resistance to the first-generation
*
Corresponding author.
E-mail address: t-fukuda@nagasaki-u.ac.jp (T. Fukuda).
https://doi.org/10.1016/j.bmc.2021.116039
Received 6 December 2020; Received in revised form 17 January 2021; Accepted 19 January 2021
Available online 23 January 2021
0
968-0896/© 2021 Elsevier Ltd. All rights reserved.

T. Fukuda et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116039
recognized mechanisms of osimertinib resistance and resistance to other
related irreversible inhibitors was the emergence of a tertiary point
mutation at Cys797, the site of covalent binding to serine (C797S).17
Therefore, to achieve the desired potency, inhibitors of the EGFR triple
mutants should be such that they do not rely on covalent bond formation
with Cys797.
A marine natural product, lamellarin N (1), and its analogues, inhibit
the catalytic activity of several protein kinases relevant to cancer and
neurodegenerative diseases. These kinases include cyclin-dependent
kinases (CDKs), glycogen synthase kinase-3 (GSK-3), Pim-1 proto-
oncogene serine/threonine kinase (PIM1), and dual-specificity tyrosine
phosphorylation regulated kinase 1A (DYRK1A).18,19 Recently, we
designed, synthesized, and examined A-ring-modified analogues of 1 as
non-covalent inhibitors of the EGFR T790M/L858R mutant and found
that several water-soluble ammonium- or guanidinium-tethered ana-
logues exhibited good inhibitory activities in the EGFR tyrosine kinase
Fig. 2. Structures of azalamellarins D (3) and N (5) and lamellarin D (4).
6–10 (Fig. 3).
2
. Results and discussion
2
0
assay. In particular, lamellarin-class analogue 2 bearing 3-(dimethy-
lamino)propoxy groups at C20- and C21-positions exhibited the most
potent and selective inhibition against the EGFR T790M/L858R mutant
2
.1. Synthesis
Recently, we successfully synthesized lamellarin N (1) and its A-ring-
[
IC50 (WT) = 31.8 nM; IC50 (T790M/L858R) = 8.9 nM] (Fig. 1). How-
modified analogues via cross-coupling of the common tricyclic inter-
ever, we thought that the inhibitory activity of 2 must be improved
mediate 11 (CDEF-ring of the lamellarin core) with various 2-
further, because the affinity of the EGFR T790M/L858R mutant to ATP
(
methoxymethoxy)phenylboronic acids bearing alkoxy functionalities at
6
is extremely high. We speculated that an additional hydrogen bonding
20,23
C4- and C5-positions.
Thus, we speculated that azalamellarin N and
or electrostatic interaction between an amino acid residue at the ATP
binding pocket of the mutant EGFR and A-ring-modified analogues of 1
could increase the affinity of the mutant EGFR to the analogues.
In 2010, Thasana and coworkers reported the synthesis and biolog-
ical evaluation of a series of newly designed azalamellarins in which the
lactone ring (B-ring) of lamellarins was substituted with a lactam ring.21
Although azalamellarin D (3) exhibited lower cytotoxicity than lamel-
larin D (4), it maintained a high level of activity at a submicromolar
range against several cancer cell lines such as HuCCA-1, A549, HepG2,
and MOLT-3 (Fig. 2). The lactam NH moiety of 3 was considered
responsible for its cytotoxicity, because N-allylated or N-propylated
azalamellarin analogues were much less cytotoxic than 3. Later,
Chittchang and coworkers synthesized lamellarins N (1) and D (4) and
azalamellarins N (5) and D (3) and evaluated their GSK-3β inhibitory
its A-ring-modified analogues 5–10 could also be accessed via a similar
route for the synthesis of A-ring-modified lamellarin N analogues. Cross-
coupling of 11 with 2-(tert-butoxycarbonylamino)phenylboronic acid
pinacol esters 12 would be the key step in this synthesis (Scheme 1).
The key common tricyclic intermediate 11 was synthesized in eight
steps from readily available 2,5-dibromo-1-(tert-butoxycarbonyl)-1H-
pyrrole (13) using a procedure established in our laboratories (Scheme
2
0,23
2
).
Three types of 2-(tert-butoxycarbonylamino)phenylboronic acid
pinacol esters 12a–c were synthesized via cross-coupling of 4,5-dia-
lkoxy-2-(tert-butoxycarbonylamino)phenyl bromides 14a–c with pina-
24
col borane (Scheme 3). Treatment of bromides 14a–c with 3.0 equiv of
2
pinacol borane in the presence of 5 mol% of PdCl (dppf) and 4.0 equiv of
Et
3
N under reflux in 1,4-dioxane gave 12a–c in good yields.
With the key common tricyclic intermediate 11 and arylboronic acid
2
2
activities (Fig. 2). Interestingly, replacing lactone ring B with a lactam
ring increased the GSK-3β inhibitory activity markedly (1 vs 5 and 4 vs
pinacol esters 12 in hand, we proceeded for the conversion to azala-
mellarin core (Scheme 4). Suzuki–Miyaura cross-coupling of 11 with
3
); azalamellarin N (5) was found to be more potent than azalamellarin
D (3). In addition, the inhibitory activity of the lamellarins against GSK-
1
2a–c in the presence of Pd(PPh
3
)
4
as a catalyst gave products 15a–c in
3
β was dependent on the ATP concentration. However, increasing the
good yields. Subsequent treatment of 15a–c with tetrabutylammonium
fluoride (TBAF) led to the removal of the N-Boc group and induced
ATP concentrations up to 1 mM did not affect the inhibitory activities of
the azalamellarins, suggesting that the lactam analogues could be ATP-
non-competitive inhibitors.
25
lactamization to give 16a–c in good yields.
Next, the O-isopropyl and/or O-benzyl groups of 16a–c were
Based on the abovementioned studies, we speculated that A-ring-
modified azalamellarin N analogues would exhibit more potent activ-
ities against EGFR tyrosine kinases than the corresponding A-ring-
modified lamellarin N analogues. In this paper, we report the synthesis
and evaluation of azalamellarin N (5) and its A-ring-modified analogues
removed (Scheme 5). Both the isopropyl and benzyl protecting groups in
1
6a and 16b were simultaneously removed upon treatment with BCl
3
in
dichloromethane (DCM) to afford fully deprotected azalamellarins 5 and
26
6
.
The spectroscopic data of compound 5 are in good agreement with
22
the previously reported data. Although we attempted the deprotection
of 16c, the desired product was not obtained. The O-benzyl groups of
1
6a and 16c could be selectively removed upon hydrogenolysis over Pd-
C using HCO NH as a hydrogen source, affording 17a and 17b in 99%
and 97% yields, respectively.
2
4
2
7
With the selectively debenzylated azalamellarins 17a and 17b in
hand, we proceeded for their conversion to 3-(dimethylamino)
Fig. 3. A-ring-modified azalamellarin N analogues designed to target EGFR
2
0
Fig. 1. Generation of lamellarin-class EGFR-TKI 2.
tyrosine kinases.
2

T. Fukuda et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116039
Scheme 1. Retrosynthetic analysis of azalamellarins.
Scheme 4. Synthesis of O-protected azalamellarins 16a–c. Reagents and con-
ditions: (a) Pd(PPh
3
)
4
(11 mol%), 12 (1.7 equiv), Na
2
CO
3
(7.3 equiv), DME,
◦
water, 85 C, 24 h (15a: 91%, 15b: 88%, 15c: 93%); (b) TBAF (5.0 equiv), THF,
◦
6
5
C, 19 h (16a: 91%, 16b: 85%, 16c: 89%).
Scheme 2. Synthesis of tricyclic intermediate 11.
Scheme 3. Synthesis of arylboronic acid pinacol esters 12a–c. Reagents and
conditions: (a) Pd(dppf)Cl
2
⋅CH
2 2 3
Cl (5 mol%), pinacol borane (3.0 equiv), Et N
(
4.0 equiv), 1,4-dioxane, reflux, 1–1.5 h (12a: 67%, 12b: 84%, 12c: 76%).
propylated analogues (Scheme 6). When 17a was reacted with 2 equiv
of 3-(dimethylamino)propyl chloride hydrochloride in acetone in the
presence of 10 equiv of K
Treatment of 17b with 1.5 equiv of 3-(dimethylamino)propyl chloride
hydrochloride in acetone in the presence of 6 equiv of K CO afforded
0-O-alkylated 19, 21-O-alkylated 20, and 20-O,21-O-dialkylated 21 in
6%, 23%, and 4% yields, respectively, after chromatographic purifi-
2 3
CO , compound 18 was obtained in 59% yield.
2
3
2
5
cation. Increasing the amounts of the alkylating agent to 2.4 equiv
increased the yield of 21 to 28% and decreased the yield of 19 to 39%
yield. The O-isopropyl protecting group in 18, 19, 20, and 21 were
3
Scheme 5. Removal of O-protecting groups. Reagents and conditions: (a) BCl ,
DCM, ꢀ 78 ◦C, 0.5 h then rt, 3 h (5: 84%, 6: 83%); (b) HCO
2
4
NH , 10% Pd-C,
EtOAc, EtOH, reflux, 0.5–1 h (17a: 99%, 17b: 97%).
3
easily removed upon treatment with AlCl at room temperature for 3 d,
without affecting the 3-(dimethylamino)propoxy and methoxy groups.2
The deprotected azalamellarins 7, 8, 9, and 10 were purified using a
Sephadex LH-20 column and isolated as trifluoroacetate salts.
8
EGFR-TKIs, gefitinib and afatinib, were used as the positive controls.
The half-maximal inhibitory concentration (IC50) of the tested com-
pounds are listed in Table 1. The IC50 values of azalamellarin N (5) and
its 20-O-methyl analogue 6 for EGFR inhibition were in the sub-
micromolar range, whereas the corresponding lamellarin N analogues
were found to be inactive at concentrations lower than 1000 nM (entries
1 and 2).20 Introduction of a 3-(dimethylamino)propyl group into 20-O
position of 5 (i.e., compound 7) resulted in considerably increased
inhibitory activities (entry 3). The inhibitory activities of compound 8
2
.2. In vitro kinase assay
Kinase inhibitory activities of azalamellarins 5–10 were evaluated by
enzyme-linked immunosorbent assays using the recombinant kinase
domains of EGFR WT and EGFR T790M/L858R mutant.29 Approved
3

T. Fukuda et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116039
Scheme 6. Synthesis of A-ring-modified azalamellarin N analogues 7, 8, 9, and 10. Reagents and conditions: (a) Cl(CH
2
)
3
NMe
NMe
(5.0 equiv), acetone, reflux, 20 h (19: 39%, 21: 28%).
2
⋅HCl (2.0 equiv), K
2
CO
3
(10 equiv),
(6.0 equiv),
acetone, reflux, 20 h (59%); (b) (1) AlCl
3
, DCM, rt, 72 h, (2) TFA (7: quant, 8: 94%, 9: 95%, 10: 97%); (c) ClCH
2
CH
2
CH
2
2
⋅HCl (1.5 equiv), K
2
CO
3
acetone, reflux, 22 h (19: 56%, 20: 23%, 21: 4%); (d) Cl(CH
2
)
3
NMe
2
⋅HCl (2.4 equiv), K
2
CO
3
against EGFR T790M/L858R were higher than those against EGFR WT.
To verify whether azalamellarin compounds functioned as ATP-
competitive or non-competitive inhibitors, kinase inhibition rates were
determined using azalamellarins 5–10 (1 µM) and three ATP concen-
trations (10, 100, and 1,000 µM). The results are summarized in Table 2.
In the case of compounds 5 and 6, the inhibition rate against EGFR WT
and T790M/L858R mutant decreased proportionally to increasing ATP
concentrations. In contrast, in the case of compounds 7–10, the inhibi-
tion rate against EGFR T790M/L858R mutant at 100 µM ATP was
maintained compared with that against EGFR WT, which was
Table 1
Inhibitory activities of A-ring-modified azalamellarin N analogues 5–10 against
a.
EGFR WT and EGFR T790M/L858R mutant kinase domains.
Entry
Compound
X
Y
IC50 (nM)
WT
T790M/
L858R
1
2
3
5
6
7
OH
OMe
OMe
OMe
307.7
259
134.8
65.3
5.7
OMe
O(CH
TFA
O(CH
TFA
OH
2
2
)
3
NMe
NMe
2
2
⋅
⋅
15.5
4
5
6
8
)
3
OH
24.7
23.6
4.6
10.2
16.6
1.7
9
O(CH
TFA
2
)
)
3
NMe
NMe
2
2
⋅
⋅
Table 2
Effect of ATP concentrations on the inhibition potential of azalamellarin ana-
logues 5–10 against EGFR WT and EGFR T790M/L858R mutant kinase
10
O(CH
TFA
2
)
3
NMe
2
⋅
O(CH
TFA
2
3
a.
domains
7
8
gefitinib
afatinib
–
–
–
–
4.0
>1000
b
Compound
EGFR WT (% inhibition )
EGFR T790M/L858R (%
<1.0
3.8
b
inhibition )
a
IC50 values were determined at an ATP concentration of 10 µM.
ATP
10
ATP
(100
µM)
ATP
ATP
(10
ATP
(100
µM)
ATP
(
(1,000
µM)
(1,000
µM)
µM)
µM)
with 21-hydroxy and 20-[3-(dimethylamino)propoxy] groups were
slightly lower than those of 7 (entry 4). Transposition of the 20- and 21-
substituents (X and Y) in 8 had a minor effect on the activities (entry 5).
The activities of azalamellarin analogue 10 bearing 3-(dimethylamino)
propoxy groups at C20- and C21-positions were higher than those of the
corresponding monopropoxy azalamellarins and the corresponding
lamellarin analogue 2 (entry 6). Overall, the inhibitory activities of the
azalamellarins against EGFR were higher than those of the corre-
sponding lamellarins,2 indicating that the lactam NH group is an
essential structural unit to potentiate the kinase inhibitory activities.
Furthermore, inhibitory activities of all the azalamellarin analogues
5
79.4
71.5
83.3
83.2
84.9
87.7
18.1
14.1
19.4
26.9
27.8
33.2
4.2
0.6
4.7
7.4
9.1
12.8
88.9
92.7
99.1
99.0
99.1
97.8
18.9
37.8
69.9
77.5
91.5
84.9
5.5
6
16.9
c
7
0
c
8
0
9
14.3
13.3
10
a
Inhibition rates obtained using 1 µM of azalamellarin N analogues and three
concentrations of ATP (10, 100, and 1,000 µM).
0
b
Percentage (%) of inhibition is represented as the mean value of two inde-
pendent experiments.
c
In case of a negative value, it was considered to be zero.
4

T. Fukuda et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116039
significantly low; nonetheless, the inhibition rate of both EGFR WT and
T790M/L858R mutant decreased significantly at the highest ATP con-
centration used. Collectively, these results suggest that azalamellarin
compounds can inhibit both EGFR WT and T790M/L858R mutant in an
ATP-competitive manner.
2.3. Docking analysis
To rationalize the effects of B-ring moiety and A-ring-substituents X
and Y on the kinase inhibitory activity, docking simulations of com-
pounds 1, 5, 7, and 10 in the ATP-binding pocket of EGFR T790M/
L858R/V948R were performed using the published X-ray crystallo-
3
0
graphic data for the T790M/L858R/V948R kinase–gefitinib complex
PDB ID: 4I22]. An additional V948R mutation was introduced to
[
Fig. 4. Plausible binding modes of 1, 5, 7, and 10 in the ATP-binding pocket of EGFR T790M/L858R/V948R. (A) Side view from the entrance region (region
surrounding the ATP-binding pocket is shown by an electrostatic potential map). (B) Side view from the entrance region. (C) Top view from the N-terminal lobe.
5

T. Fukuda et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116039
prevent dimerization of the T790M/L858R kinase during crystallization.
This mutation had virtually no influence on the structure or activity of
the T790M/L858R kinase, except the changes observed in the mono-
meric state in solution. Gefitinib in the ATP-binding pocket of the kinase
was replaced with each ligand, and docking poses were predicted using
the Autodock Vina program.31,32 Plausible binding models and their
binding affinities are represented in Fig. 4. In the EGFR–lamellarin N (1)
complex model, the planar pentacyclic core (ABCDE-ring) of 1 occupied
the ATP-binding pocket in such a way that the A-ring was directed to the
solvent channel (entrance region) and the E-ring was oriented to the
specificity (back) pocket. The F-ring, which was perpendicularly con-
nected to the pentacyclic core, was situated at the ribose-binding site.
property. Indeed, lamellarin N (1), the parent compound of 5, is a
potent inhibitor of topoisomerase
kinases.1
I and a variety of protein
8,19,22
3. Conclusion
Azalamellarin N (5) and its A-ring-modified analogues 6–10 were
synthesized and evaluated as non-covalent inhibitors of the EGFR
T790M/L858R mutant. Analogues bearing 3-(dimethylamino) propoxy
group(s) at the C20- and/or C21-position(s) exhibited good inhibitory
activity against the WT and mutant EGFR, with IC50 values in the low,
nanomolar range. In addition, azalamellarins selectively inhibited the
EGFR T790M/L858R mutant over the EGFR WT and functioned as ATP-
competitive inhibitors. Docking studies revealed that the higher inhib-
itory activities of the azalamellarin N analogues compared to the cor-
responding lamellarin N analogues were attributable to the additional
hydrogen bonding interaction between the lactam NH group of the B-
–
The lactone carbonyl (C O) group of the B-ring formed a hydrogen
–
bond with the NH group of Met793 located in the hinge region. The
phenolic OH group at C8 also formed a hydrogen bond with the side
chain carboxylate group of Lys745 in the conserved catalytic salt bridge
(
Lys745–Asp855). Another phenolic OH at C13 of the F-ring was
–
–
–
directed downward and formed an additional hydrogen bond with C
O
ring and C
–
O of Met793. Moreover, azalamellarins 5, 7, and 10
of Arg841 in the A-loop. The predicted binding mode of 1 in this study
was substantially similar to that reported using a different docking
program.20 The binding mode of the EGFR–azalamellarin N (5) complex
model was similar to that of the corresponding EGFR–lamellarin N (1)
complex model. The lactam NH group of the B-ring formed an additional
hydrogen bond with C–
selectively inhibit the proliferation of EGFR T790M/L858R mutant cells
over EGFR WT cells. Further biological evaluations of these azala-
mellarins are currently in progress in our laboratories.
4. Experimental section
–
O of Met793. This could explain the higher
inhibitory activity and affinity energy of 5 compared to that of 1. Each 3-
dimethylamino)propoxy group at the C20-position of 7 and 10 was
embedded in the negatively charged small pocket in the entrance region
4.1. Synthesis
(
Melting points were determined using the Yanagimoto micro melting
point apparatus and are uncorrected. IR spectra were obtained with a
Thermo Nicolet Nexus 670 NT FT-IR instrument and are reported in
–
surrounded by stem chain C O of Phe795 and side chain carboxylate
–
groups of both Asp800 and Glu804. The ammonium group was involved
in hydrogen bonding and/or ionic interactions with the side-chain
carboxylate group of Glu804. The higher activities of 10 compared to
that of 7 implied that 3-(dimethylamino)propoxy group at the C21-
position of 10 could form an additional hydrogen bond with Asp800.
Although compounds 7 and 10 exhibited remarkably higher inhibitory
activity than compounds 1 and 5, their affinity energy was estimated to
be lower than those of 1 and 5. One possible reason for this poor cor-
relation between activity and affinity may be attributed to the fact that 1
and 5 are neutral compounds, whereas compounds 7 and 10 are posi-
tively charged owing to their 3-(dimethylamino)propoxy group(s). Since
the scoring function used in the Autodock Vina program does not
explicitly consider the hydrogen atoms or partial charges for atoms,31 it
may be difficult to compare the affinity energies between uncharged and
charged compounds.
ꢀ
1
terms of the absorption frequency (cm ). NMR spectra were recorded
1
on a JEOL JNM-AL400 instrument (400 MHz for H and 100 MHz for
1
3
1
C) or a Varian NMR System 500PS SN instrument (500 MHz for H and
126 MHz for 13C). Chemical shifts for H NMR are expressed in parts per
1
million (ppm) relative to CDCl3 (tetramethylsilane, δ 0.0 ppm) and
1
DMSO‑d (DMSO, δ 2.50 ppm) internal standards. H NMR spectral data
6
are reported as follows: chemical shift (δ ppm), multiplicity (s = singlet,
d = doublet, dd = double doublet, t = triplet, sep = septet, m =
multiplet, br s = broad singlet), coupling constant (Hz), and integration.
Chemical shifts for 13C NMR are expressed in ppm relative to CDCl
3
(tetramethylsilane, δ 0.0 ppm) and DMSO‑d6 (DMSO‑d , δ 39.52 ppm)
6
internal standards. High-resolution mass spectra (HRMS) were recorded
on a JEOL JMS-T100TD (direct analysis in real-time mass spectrometry,
DARTMS) instrument or a JEOL JMS-700 N (fast atom bombardment
mass spectrometry, FABMS) instrument. Column chromatography was
performed using silica gel 60 N (63–210 µm, Kanto Chemical Co., Inc.),
Chromatorex Diol MB100–75/200 (Fuji Silysia Chemical Ltd.), or
Sephadex LH-20 (GE Healthcare Life Sciences). Flash chromatography
was performed using Chromatorex NH-DM2035 (Fuji Silysia Chemical
Ltd.).
2
.4. Proliferation assay
Lastly, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay was performed to determine the effects of azala-
mellarin analogues 5, 7, and 10 on the proliferation of two kinds of
NSCLC cell lines, i.e., A549 and NCI-H1975 harboring the EGFR WT and
T790M/L858R mutation.29 The IC50 values of the tested compounds are
summarized in Table 3. All three compounds exhibited lower IC50 values
in NCI-H1975 than in A549 cells, indicating that they selectively
inhibited EGFR T790M/L858R rather than the WT genotype cells.
Additionally, compound 5 showed markedly more potent cytotoxicity
than compounds 7 and 10, which may be caused by its multi-targeting
4
.2. Synthesis of arylboronic acid pinacol esters 12a–c
4
1
.2.1. tert-Butyl N-[5-(benzyloxy)-4-methoxy-2-(4,4,5,5-tetramethyl-
,3,2-dioxaborolan-2-yl)phenyl]carbamate (12a)
Under an argon atmosphere, a mixture of 14a (1.01 g, 2.46 mmol),
pinacol borane (1.06 mL, 7.37 mmol), Pd(dppf)Cl
.123 mmol), triethylamine (1.37 mL, 9.83 mmol), and 1,4-dioxane (14
mL) was refluxed for 1.5 h. After cooling to room temperature, the
mixture was added saturated aqueous NH Cl and evaporated. The
product was extracted with DCM and the extract was washed with water
and brine, dried over Na SO , and evaporated. The residue was purified
2
⋅CH
2 2
Cl (100 mg,
0
Table 3
Cytotoxicity of azalamellarin N analogues 5, 7, and 10 against A549 and NCI-
4
H1975 cells.
2
4
Compound
IC50 (µM)
by column chromatography over silica gel 60 N (hexane–ethyl acetate =
A549
NCI-H1975
7
:1) to give 12a as a colorless solid (756 mg, 67%). Recrystallization
◦
5
7
0.0155
1.04
0.0019
0.35
from Et
O–hexane gave colorless powder. Mp 128.5–129.5 C. IR (KBr):
2
ꢀ 1
1
3
367, 1725, 1613, 1529, 1365, 1311, 1237, 1169 cm . H NMR (400
1
0
2.04
1.36
MHz, CDCl ): δ 1.35 (s, 12H), 1.52 (s, 9H), 3.86 (s, 3H), 5.19 (s, 2H),
3
6

T. Fukuda et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116039
7
8
5
1
4
.18 (s, 1H), 7.27–7.32 (m, 1H), 7.33–7.39 (m, 2H), 7.47–7.51 (m, 2H),
122.7, 123.0, 123.0, 123.6, 124.5, 127.7, 127.9, 127.9, 128.0, 128.3,
129.9, 129.9, 130.0, 130.4, 131.0, 131.1, 137.0, 144.2, 144.3, 146.3,
.02 (br s, 1H), 8.63 (br s, 1H). 13C NMR (100 MHz, CDCl
): δ 24.8, 28.4,
3
6.4, 70.4, 79.5, 83.9, 103.5, 118.3, 127.8, 127.8, 128.4, 136.7, 140.6,
146.4, 146.4, 146.4, 147.0, 147.1, 149.0, 149.1, 149.4, 149.4, 153.0,
+
+
43.9, 151.8, 153.2. HRDARTMS m/z. Calcd for C25
H34BNO
6
(M ):
153.3, 161.6. HRDARTMS m/z. Calcd for C47
H
53
N
2
O
10 [(M+H) ]:
55.2479. Found: 455.2485.
805.3700. Found: 805.3715.
4
.2.2. tert-Butyl N-[4,5-dimethoxy-2-(4,4,5,5-tetramethyl-1,3,2-
4.3.2. Methyl 2-[2-(tert-butoxycarbonylamino)-4,5-dimethoxyphenyl]-8-
isopropoxy-1-(3-isopropoxy-4-methoxyphenyl)-9-methoxy-pyrrolo[2,1-a]
isoquinoline-3-carboxylate (15b)
dioxaborolan-2-yl)phenyl]carbamate (12b)
According to the procedure described for the preparation of 12a, 14b
(
(
7.18 g, 21.6 mmol), pinacol borane (9.40 mL, 65.4 mmol), and Pd
According to the procedure described for the preparation of 15a, 11
(100 mg, 0.180 mmol) and 12b (114 mg, 0.301 mmol) were reacted.
After purification by column chromatography over silica gel 60 N
(hexane–ethyl acetate = 3:1–1:1), 15b was obtained as a pale yellow
semisolid (115 mg, 88%). IR (KBr): 3420, 1726, 1686, 1533, 1486,
dppf)Cl
2
⋅CH
2 2
Cl (885 mg, 1.08 mmol) were reacted for 1 h. After pu-
rification by column chromatography over silica gel 60 N (hexane–ethyl
acetate = 5:1), 12b was obtained as a colorless solid (6.88 g, 84%).
Recrystallization from Et
2
O–hexane gave colorless powder. Mp
◦
ꢀ 1
1
ꢀ 1
1
9
0–91 C. IR (KBr): 3369, 1727, 1532, 1363, 1166, 1139 cm . H NMR
1375, 1208, 1158 cm . H NMR (500 MHz, DMSO‑d
6
): δ 1.02 (d, J =
(
400 MHz, CDCl
3
): δ 1.35 (s, 12H), 1.52 (s, 9H), 3.88 (s, 3H), 3.94 (s,
6.1 Hz, 1.72H), 1.09 (d, J = 6.1 Hz, 1.72H), 1.11 (d, J = 6.1 Hz, 1.28H),
1.16 (d, J = 6.1 Hz, 1.28H), 1.30 (d, J = 6.1 Hz, 6H), 1.37 (s, 9H), 3.29
(s, 1.28H), 3.29 (s, 1.72H), 3.48 (s, 1.72H), 3.53 (s, 1.28H), 3.53 (s,
1.72H), 3.56 (s, 1.28H), 3.69 (s, 1.72H), 3.70 (s, 1.28H), 3.71 (s, 1.28H),
3.74 (s, 1.72H), 4.26 (sep, J = 6.1 Hz, 0.57H), 4.43 (sep, J = 6.1 Hz,
1
3
3
H), 7.15 (s, 1H), 7.93 (br s, 1H), 8.67 (br s, 1H). C NMR (100 MHz,
CDCl
3
): δ 24.9, 28.4, 55.8, 56.1, 79.6, 84.0, 101.9, 117.3, 140.8, 143.5,
+
1
52.4, 153.2. HRDARTMS m/z. Calcd for C19
H30BNO
6
(M ): 379.2166.
Found: 379.2169.
0
.43H), 4.70 (sep, J = 6.1 Hz, 1H), 6.52 (s, 0.57H), 6.71 (s, 0.43H),
4
.2.3. tert-Butyl N-[4,5-bis(benzyloxy)-2-(4,4,5,5-tetramethyl-1,3,2-
6.85–7.17 (m, 4H), 7.18 (d, J = 7.6 Hz, 0.57H), 7.18 (d, J = 7.6 Hz,
0.43H), 7.34 (s, 1H), 7.35 (br s, 0.43H), 7.44 (br s, 0.57H), 7.60 (br s,
0.43H), 7.85 (s, 0.57H), 9.20 (d, J = 7.6 Hz, 0.57H), 9.23 (d, J = 7.6 Hz,
dioxaborolan-2-yl)phenyl]carbamate (12c)
According to the procedure described for the preparation of 12a, 14c
(
(
2.00 g, 4.13 mmol), pinacol borane (1.79 mL, 12.4 mmol), and Pd
dppf)Cl Cl (169 mg, 0.206 mmol) were reacted for 1 h. After
0.43H). 13C NMR (126 MHz, DMSO‑d
): δ 21.6, 21.7, 21.7, 21.7, 21.8,
6
2
⋅CH
2
2
21.8, 21.8, 25.0, 28.1, 50.8, 50.9, 54.4, 54.4, 55.2, 55.3, 55.4, 55.5,
55.5, 55.6, 69.8, 70.1, 70.1, 73.5, 78.5, 78.5, 104.8, 106.4, 107.4, 110.2,
110.3, 111.7, 112.0, 112.1, 112.3, 112.3, 112.4, 112.4, 114.6, 114.8,
118.8, 118.8, 118.9, 118.9, 118.9, 119.0, 119.7, 119.8, 122.7, 122.7,
123.0, 123.0, 123.6, 124.5, 127.7, 127.9, 128.1, 129.9, 129.9, 130.0,
130.3, 130.4, 131.1, 131.1, 134.0, 143.8, 143.9, 146.4, 146.4, 147.1,
purification by column chromatography over silica gel 60 N (hex-
ane–ethyl acetate = 7:1), 12c was obtained as a colorless solid (1.66 g,
7
6%). Recrystallization from Et
2
O–hexane gave colorless powder. Mp
◦
1
50.5–151.5 C. IR (KBr): 3354, 1714, 1611, 1533, 1372, 1238, 1140
ꢀ
1 1
cm . H NMR (400 MHz, CDCl
3
): δ 1.35 (s, 12H), 1.53 (s, 9H), 5.07 (s,
2
7
H), 5.19 (s, 2H), 7.27–7.38 (m, 6H), 7.33 (s, 1H), 7.42–7.46 (m, 2H),
147.1, 147.1, 147.2, 149.0, 149.1, 149.4, 153.0, 153.3, 161.6.
1
3
+
.47–7.51 (m, 2H), 8.05 (br s, 1H), 8.66 (br s, 1H). C NMR (100 MHz,
): δ 24.8, 28.4, 70.4, 72.4, 79.6, 84.0, 103.9, 123.0, 127.6, 127.6,
HRDARTMS m/z. Calcd for C41
H
49
N
2
O10 [(M+H) ]: 729.3387. Found:
CDCl
3
729.3358.
1
27.7, 128.3, 128.4, 136.9, 137.7, 141.5, 143.1, 153.1, 153.2.
+
HRDARTMS m/z. Calcd for C31
H38BNO
6
(M ): 531.2792. Found:
4.3.3. Methyl 2-[4,5-bis(benzyloxy)-2-(tert-butoxycarbonylamino)
phenyl]-8-isopropoxy-1-(3-isopropoxy-4-methoxyphenyl)-9-methoxy-
pyrrolo[2,1-a]isoquinoline-3-carboxylate (15c)
5
4
4
31.2773.
.3. Synthesis of O-protected azalamellarins 16a–c
According to the procedure described for the preparation of 15a, 11
(
200 mg, 0.359 mmol) and 12c (321 mg, 0.603 mmol) were reacted.
.3.1. Methyl 2-[4-(benzyloxy)-2-(tert-butoxycarbonylamino)-5-
After purification by column chromatography over silica gel 60 N
(hexane–ethyl acetate = 3:1), 15c was obtained as a pale yellow semi-
solid (294 mg, 93%). IR (KBr): 3418, 1726, 1685, 1507, 1486, 1372,
methoxyphenyl]-8-isopropoxy-1-(3-isopropoxy-4-methoxyphenyl)-9-
methoxy-pyrrolo[2,1-a]isoquinoline-3-carboxylate (15a)
ꢀ
1 1
Under an argon atmosphere, a mixture of 11 (100 mg, 0.180 mmol),
1224, 1155 cm . H NMR (500 MHz, DMSO‑d
6
): δ 1.03 (d, J = 6.0 Hz,
1
2a (137 mg, 0.302 mmol), Pd(PPh
3
)
4
(23.2 mg, 20.1 µmol), Na
2
CO
3
1.65H), 1.09 (d, J = 6.0 Hz, 1.35H), 1.10 (d, J = 6.0 Hz, 1.65H), 1.15 (d,
J = 6.0 Hz, 1.35H), 1.30 (d, J = 6.0 Hz, 6H), 1.37 (s, 4.95H), 1.37 (s,
4.05H), 3.30 (s, 3H), 3.48 (s, 1.35H), 3.51 (s, 1.65H), 3.71 (s, 1.35H),
3.72 (s, 1.65H), 4.28 (sep, J = 6.0 Hz, 0.55H), 4.45 (sep, J = 6.0 Hz,
0.45H), 4.70 (sep, J = 6.0 Hz, 1H), 4.72 (d, J = 12.2 Hz, 0.55H), 4.78 (d,
J = 12.0 Hz, 0.45H), 4.88 (d, J = 12.2 Hz, 0.55H), 4.96 (d, J = 12.0 Hz,
0.45H), 4.97 (d, J = 11.7 Hz, 0.45H), 4.99 (d, J = 11.7 Hz, 0.55H), 5.03
(d, J = 11.7 Hz, 1H), 6.67 (s, 0.55H), 6.87–7.01 (m, 4.45H), 7.18 (d, J =
7.6 Hz, 0.55H), 7.19 (d, J = 7.6 Hz, 0.45H), 7.28–7.39 (m, 10H),
7.42–7.46 (m, 2H), 7.65 (s, 0.45H), 7.84 (s, 0.55H), 9.20 (d, J = 7.6 Hz,
(
141 mg, 1.33 mmol), DME (5.0 mL), and degassed water (0.5 mL) was
◦
heated in a sealed tube at 85 C for 24 h. After cooling to room tem-
perature, the mixture was evaporated and the product was extracted
with DCM. The extract was washed with water and brine, dried over
2 4
Na SO , and evaporated. The residue was purified by column chroma-
tography over silica gel 60 N (hexane–ethyl acetate = 3:1–1:2) to give
1
5a as a pale yellow semisolid (131 mg, 91%). IR (KBr): 3421, 1725,
ꢀ 1
1
1
685, 1533, 1486, 1373, 1156, 1122 cm
): δ 1.02 (d, J = 6.1 Hz, 1.67H), 1.10 (d, J = 6.1 Hz, 1.67H),
.13 (d, J = 6.1 Hz, 1.33H), 1.17 (d, J = 6.1 Hz, 1.33H), 1.30 (d, J = 6.1
Hz, 6H), 1.37 (s, 9H), 3.29 (s, 1.33H), 3.30 (s, 1.67H), 3.49 (s, 1.67H),
.
H NMR (500 MHz,
DMSO‑d
6
1
3
1
0.55H), 9.23 (d, J = 7.6 Hz, 0.45H). C NMR (126 MHz, DMSO‑d
6
): δ
21.6, 21.7, 21.7, 21.7, 21.8, 21.8, 21.8, 28.1, 28.1, 50.8, 50.9, 54.4,
54.4, 55.6, 55.6, 69.9, 70.1, 70.1, 70.2, 70.3, 70.8, 70.8, 78.6, 78.6,
104.8, 108.8, 109.5, 110.2, 110.3, 111.8, 112.1, 112.1, 112.3, 112.4,
117.6, 117.8, 118.7, 118.8, 118.8, 118.9, 118.9, 119.9, 120.4, 122.7,
122.7, 123.0, 123.1, 123.7, 124.4, 127.4, 127.4, 127.6, 127.7, 127.8,
127.8, 128.3, 128.3, 128.3, 129.9, 130.7, 130.8, 130.9, 131.0, 137.1,
137.2, 137.3, 137.4, 143.4, 143.5, 146.4, 146.4, 147.1, 147.1, 147.1,
3
1
.53 (s, 1.33H), 3.54 (s, 1.67H), 3.57 (s, 1.33H), 3.72 (s, 1.33H), 3.74 (s,
.67H), 4.28 (sep, J = 6.1 Hz, 0.56H), 4.44 (sep, J = 6.1 Hz, 0.44H), 4.71
(
sep, J = 6.1 Hz, 1H), 4.92–5.01 (m, 2H), 6.56 (s, 0.56H), 6.75 (s,
0
.44H), 6.86–7.10 (m, 4H), 7.18 (d, J = 7.6 Hz, 0.56H), 7.19 (d, J = 7.6
Hz, 0.44H), 7.23–7.47 (m, 7H), 7.64 (s, 0.44H), 7.89 (s, 0.56H), 9.21 (d,
1
3
J = 7.6 Hz, 0.56H), 9.23 (d, J = 7.6 Hz, 0.44H). C NMR (126 MHz,
DMSO‑d ): δ 21.5, 21.7, 21.7, 21.7, 21.7, 21.7, 21.8, 21.8, 28.1, 50.8,
0.9, 54.3, 54.4, 55.5, 55.5, 55.6, 59.7, 69.8, 70.0, 70.1, 70.1, 78.5,
149.1, 149.1, 149.5, 152.9, 153.2, 161.6, 161.6. HRDARTMS m/z. Calcd
6
+
5
7
1
for C53
H
57
2
N O
10 [(M+H) ]: 881.4013. Found: 881.4040.
8.5, 104.8, 108.2, 109.1, 110.2, 110.3, 111.7, 112.0, 112.1, 112.3,
12.4, 114.8, 115.1, 118.8, 118.8, 118.8, 118.9, 119.7, 120.4, 122.7,
7

T. Fukuda et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116039
4
2
.3.4. 3-(Benzyloxy)-11-isopropoxy-14-(3-isopropoxy-4-methoxyphenyl)-
,12-dimethoxy-benzo[7,8]indolizino[3,2-c]quinolin-6(5H)-one (16a)
Under an argon atmosphere, a mixture of 15a (600 mg, 0.745 mmol),
(5.0 mL), was added dropwise a solution of BCl
3
in heptane (1.0 M,
0.445 mL, 0.445 mmol) under an argon atmosphere. The mixture was
◦
◦
stirred at ꢀ 78 C for 30 min and 0 C for 3 h. The reaction was quenched
by adding saturated aqueous NaHCO and the DCM was removed in
TBAF (1.0 M solution in THF, 3.70 mL, 3.70 mmol), and THF (30 mL)
3
◦
was heated in a sealed tube at 65 C for 19 h. After cooling to room
vacuo. The precipitated solid was filtered, washed with water, and dried
in vacuo. After purification by column chromatography over silica gel 60
N (ethyl acetate to acetone), 5 was obtained as a colorless powder (18.6
temperature, the reaction mixture was quenched with water and evap-
orated. The precipitate thus formed was collected by filtration, washed
with water and MeOH, and dried under reduced pressure to give 16a as
◦
22
◦
mg, 84%). Mp > 300 C (sealed capillary) [lit. Mp > 290 C]. IR (KBr):
3301, 1648, 1606, 1428, 1274, 1215 cm 1. 1H NMR (500 MHz,
ꢀ
a
pale brown powder (458 mg, 91%). Recrystallization from
◦
DCM–hexane gave a pale brown powder. Mp 292–295 C. IR (KBr):
1
DMSO‑d ): δ 3.36 (s, 3H), 3.39 (s, 3H), 3.86 (s, 3H), 6.84 (s, 1H), 6.89 (s,
6
ꢀ
1 1
652, 1489, 1430, 1262, 1222, 1192 cm . H NMR (500 MHz, CDCl
3
):
1H), 6.98–7.03 (m, 3H), 7.13 (s, 1H), 7.15 (s, 1H), 7.23 (d, J = 8.2 Hz,
1H), 9.33 (s, 1H), 9.37 (d, J = 7.4 Hz, 1H), 9.49 (s, 1H), 9.71 (s, 1H),
δ 1.34 (d, J = 6.1 Hz, 3H), 1.34 (d, J = 6.1 Hz, 3H), 1.43 (d, J = 6.1 Hz,
3
4
6
1
5
1
1
1
6
H), 1.44 (d, J = 6.1 Hz, 3H), 3.42 (s, 3H), 3.46 (s, 3H), 3.93 (s, 3H),
.53 (sep, J = 6.1 Hz, 1H), 4.68 (sep, J = 6.1 Hz, 1H), 5.22 (s, 2H),
.76–6.88 (m, 2H), 7.00–7.30 (m, 9H), 7.43–7.47 (m, 2H), 9.44 (br s,
11.27 (s, 1H). 13C NMR (126 MHz, DMSO‑d
) δ 54.5, 54.8, 56.2, 102.3,
6
105.4, 105.9, 108.5, 109.7, 110.4, 111.6, 112.1, 113.7, 117.7, 118.5,
122.3, 122.6, 123.9, 127.7, 128.8, 131.2, 131.4, 143.4, 147.1, 147.4,
H), 11.05 (br s, 1H). 13C NMR (126 MHz, CDCl
): δ 21.9, 22.0, 22.0,
147.7, 148.0, 155.4. HRDARTMS m/z Calcd for C28
H
[(M+H) ]:
+
3
23
N
2
O
7
5.1, 55.3, 56.4, 70.8, 71.1, 71.2, 101.3, 105.7, 105.8, 110.5, 110.6,
10.7, 112.6, 112.9, 118.4, 119.3, 123.7, 124.2, 124.5, 127.4, 128.0,
499.1505. Found: 499.1502. These physical and spectroscopic data are
in good agreement with those previously reported.22
28.6, 128.6, 129.4, 130.6, 132.3, 136.6, 145.2, 147.8, 148.1, 148.2,
+
49.8, 150.0, 156.5. HRDARTMS m/z. Calcd for C41
H
41
2
N O
7
[(M+H) ]:
4.4.2. 11-Hydroxy-14-(3-hydroxy-4-methoxyphenyl)-2,3,12-trimethoxy-
benzo[7,8]indolizino[3,2-c]quinolin-6(5H)-one (6)
73.2914. Found: 673.2939.
According to the procedure described for the preparation of 5, 16b
4
.3.5. 11-Isopropoxy-14-(3-isopropoxy-4-methoxyphenyl)-2,3,12-
(20.0 mg, 33.5 µmol) and a solution of BCl
3
in heptane (1.0 M, 0.235 mL,
trimethoxy-benzo[7,8]indolizino[3,2-c]quinolin-6(5H)-one (16b)
According to the procedure described for the preparation of 16a, 15b
0.235 mmol) were reacted. After purification by column chromatog-
raphy over silica gel 60 N (acetone), 6 was obtained as a colorless
◦
(
(
620 mg, 0.850 mmol) was reacted to give 16b as a pale yellow powder
430 mg, 85%). Recrystallization from DCM–hexane gave a colorless
powder (14.2 mg, 83%). Mp > 300 C (sealed capillary). IR (KBr): 3409,
ꢀ 1
1
1652, 1604, 1492, 1431, 1271, 1205 cm
.
H NMR (500 MHz,
◦
ꢀ 1
1
powder. Mp > 300 C. IR (KBr): 1646, 1510, 1434, 1265, 1224 cm . H
DMSO‑d ): δ 3.34 (s, 3H), 3.39 (s, 3H), 3.78 (s, 3H), 3.86 (s, 3H), 6.87 (s,
6
NMR (500 MHz, CDCl
3
): δ 1.35 (d, J = 6.1 Hz, 6H), 1.43 (d, J = 6.1 Hz,
1H), 6.98–7.02 (m, 3H), 7.04 (d, J = 7.4 Hz, 1H), 7.14 (s, 1H), 7.16 (s,
1H), 7.23 (d, J = 8.7 Hz, 1H), 9.34 (s, 1H), 9.38 (d, J = 7.4 Hz, 1H), 9.73
6
6
1
H), 3.45 (s, 3H), 3.47 (s, 3H), 3.96 (s, 3H), 3.99 (s, 3H), 4.54 (sep, J =
.1 Hz, 1H), 4.69 (sep, J = 6.1 Hz, 1H), 6.90 (s, 1H), 6.94 (d, J = 7.4 Hz,
H), 6.94 (s, 1H), 7.10 (s, 1H), 7.16 (d, J = 8.1 Hz, 1H), 7.17 (d, J = 1.7
(s, 1H), 11.34 (s, 1H). 13C NMR (126 MHz, DMSO‑d
): δ 54.5, 54.7, 55.4,
6
56.2, 99.1, 105.4, 105.5, 109.2, 109.9, 110.7, 111.6, 112.3, 113.7,
Hz, 1H), 7.20 (s, 1H), 7.22 (dd, J = 1.7 and 8.1 Hz, 1H), 9.59 (d, J = 7.4
117.7, 118.5, 122.3, 122.5, 123.9, 127.4, 128.7, 131.1, 131.3, 144.0,
Hz, 1H), 10.58 (br s, 1H). 13C NMR (126 MHz, CDCl
): δ 21.9, 22.0, 55.1,
5.3, 56.0, 56.4, 71.2, 71.2, 98.9, 105.6, 105.8, 110.5, 110.6, 110.8,
147.5, 147.7, 147.7, 148.1, 149.0, 155.3. HRDARTMS m/z Calcd for
3
+
5
1
1
C
29
H
25
N
2
O
7
[(M+H) ]: 513.1662. Found: 513.1676.
10.9, 112.8, 112.9, 118.4, 119.3, 123.6, 124.2, 124.3, 128.8, 129.4,
30.6, 132.6, 144.8, 147.9, 148.2, 149.3, 149.9, 150.0, 156.6.
4.4.3. 3-Hydroxy-11-isopropoxy-14-(3-isopropoxy-4-methoxyphenyl)-
2,12-dimethoxy-benzo[7,8]indolizino[3,2-c]quinolin-6(5H)-one (17a)
Under an argon atmosphere, ammonium formate (844 mg, 13.4
mmol) was added portionwise to a mixture of 16a (300 mg, 0.446
mmol), palladium carbon (Pd: 10%, 60.0 mg), ethyl acetate (45 mL),
and EtOH (45 mL) at room temperature and the mixture was refluxed for
0.5 h. After cooling to room temperature, the mixture was passed
through a pad of Celite. The filtrate was evaporated under reduced
pressure. The residue was purified by column chromatography over
silica gel 60 N (acetone) to give 17a as a pale yellow powder (258 mg,
99%). Recrystallization from DCM–hexane gave a pale brown powder.
+
HRDARTMS m/z. Calcd for C35
H
37
N
2
O
7
[(M+H) ]: 597.2601. Found:
5
97.2612.
4
.3.6. 2,3-Bis(benzyloxy)-11-isopropoxy-14-(3-isopropoxy-4-
methoxyphenyl)-12-methoxy-benzo[7,8]indolizino[3,2-c]quinolin-6(5H)-
one (16c)
According to the procedure described for the preparation of 16a, 15c
(
(
655 mg, 0.744 mmol) was reacted to give 16c as a pale yellow powder
498 mg, 89%). Recrystallization from DCM–hexane gave a pale yellow
◦
powder. Mp 281.5–282.5 C. IR (KBr): 1651, 1432, 1263, 1222, 1193
cm . H NMR (500 MHz, CDCl
ꢀ
1
1
◦
3
): δ 1.34 (d, J = 6.1 Hz, 3H), 1.36 (d, J
Mp 280–300 C (dec) (sealed capillary). IR (KBr): 3565, 3335, 1629,
ꢀ
1 1
=
6.1 Hz, 3H), 1.43 (d, J = 6.1 Hz, 3H), 1.43 (d, J = 6.1 Hz, 3H), 3.45 (s,
1490, 1460, 1432, 1257, 1220 cm . H NMR (500 MHz, DMSO‑d ):
6
3
4
1
H), 3.96 (s, 3H), 4.54 (sep, J = 6.1 Hz, 1H), 4.68 (sep, J = 6.1 Hz, 1H),
.73 (s, 2H), 5.23 (s, 2H), 6.85 (s, 1H), 6.92 (d, J = 7.4 Hz, 1H), 7.06 (s,
H), 7.08 (s, 1H), 7.13 (d, J = 8.3 Hz, 1H), 7.13 (d, J = 1.7 Hz, 1H), 7.16
δ1.22 (d, J = 6.0 Hz, 6H), 1.29 (d, J = 6.0 Hz, 6H), 3.33 (s, 3H), 3.33 (s,
3H), 3.85 (s, 3H), 4.57 (sep, J = 6.0 Hz, 1H), 4.71 (sep, J = 6.0 Hz, 1H),
6.76 (s, 1H), 6.90 (s, 1H), 7.08 (s, 1H), 7.10 (d, J = 7.5 Hz, 1H), 7.14 (d,
J = 2.0 Hz, 1H), 7.14 (dd, J = 2.0 and 8.6 Hz, 1H), 7.28 (d, J = 8.6 Hz,
1H), 7.35 (s, 1H), 9.42 (d, J = 7.5 Hz, 1H), 9.51 (s, 1H), 11.31 (s, 1H).
(
(
s, 1H), 7.17 (dd, J = 1.7 and 8.3 Hz, 1H), 7.26–7.38 (m, 8H), 7.45–7.48
13
m, 2H), 9.44 (br s, 1H), 9.53 (d, J = 7.4 Hz, 1H). C NMR (126 MHz,
): δ 21.9, 21.9, 22.0, 22.1, 55.2, 56.4, 70.7, 71.1, 71.2, 71.2, 101.9,
1
3
CDCl
3
6
C NMR (126 MHz, DMSO‑d ): δ21.7, 21.7, 21.8, 54.3, 54.8, 56.0, 70.1,
1
1
1
1
7
05.8, 109.2, 110.7, 110.8, 111.0, 111.1, 112.8, 112.8, 118.3, 119.3,
70.3, 102.4, 105.2, 105.8, 108.5, 110.0, 110.7, 110.8, 112.2, 113.7,
23.6, 124.2, 127.2, 127.3, 127.7, 128.0, 128.4, 128.4, 128.5, 128.6,
118.4, 118.5, 122.7, 123.7, 123.9, 127.8, 128.6, 131.0, 131.4, 143.4,
29.2, 130.7, 132.6, 136.8, 136.9, 144.5, 147.9, 148.3, 149.1, 149.9,
147.2, 147.2, 147.7, 149.3, 150.0, 155.5. HRFABMS m/z. Calcd for
+
+
50.1, 156.1. HRDARTMS m/z. Calcd for C47
H
45
2
N O
7
[(M+H) ]:
C
34
H
35
N
2
O
7
[(M+H) ]: 583.2444. Found: 583.2444.
49.3227. Found: 749.3221.
4
.4.4. 2,3-Dihydroxy-11-isopropoxy-14-(3-isopropoxy-4-methoxyphenyl)-
4
4
.4. Removal of O-protecting groups of 16a–c
12-methoxy-benzo[7,8]indolizino[3,2-c]quinolin-6(5H)-one (17b)
According to the procedure described for the preparation of 17a, 16c
(503 mg, 0.671 mmol), palladium carbon (Pd: 10%, 151 mg), ethyl
acetate (500 mL), and EtOH (250 mL) were reacted for 1 h. After puri-
fication by column chromatography over silica gel 60 N (acetone), 17b
.4.1. 3,11-Dihydroxy-14-(3-hydroxy-4-methoxyphenyl)-2,12-dimethoxy-
benzo[7,8]indolizino[3,2-c]quinolin-6(5H)-one (azalamellarin N, 5)
◦
To a cooled (ꢀ 78 C) solution of 16a (30.0 mg, 44.6 µmol) in DCM
8

T. Fukuda et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116039
was obtained as a pale yellow powder (371 mg, 97%). Recrystallization
111.6, 112.4, 113.7, 117.7, 118.5, 122.2, 122.5, 123.9, 127.3, 128.6,
◦
from acetone–hexane gave a pale brown powder. Mp > 300 C (sealed
131.0, 131.3, 144.1, 147.5, 147.7, 147.8, 147.8, 148.1, 155.3.
ꢀ 1
1
+
capillary). IR (KBr): 3550, 3385, 1655, 1490, 1436, 1249 cm . H NMR
HRDARTMS m/z. Calcd for C33
H
34
N
3
O
7
[(Mꢀ CF
3
COO) ]: 584.2397.
(
500 MHz, DMSO‑d
6
): δ1.20 (d, J = 6.1 Hz, 3H), 1.24 (d, J = 6.1 Hz, 3H),
Found: 584.2381.
1
1
2
1
1
7
1
1
.29 (d, J = 6.1 Hz, 6H), 3.31 (s, 3H), 3.88 (s, 3H), 4.56 (sep, J = 6.1 Hz,
H), 4.69 (sep, J = 6.1 Hz, 1H), 6.78 (s, 1H), 6.87 (s, 2H), 7.06–7.09 (m,
H), 7.08 (dd, J = 2.0 and 8.0 Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.33 (s,
H), 8.61 (br s, 1H), 9.45 (d, J = 7.4 Hz, 1H), 9.50 (br s, 1H), 11.22 (br s,
4.5.3. Treatment of 17b with 3-(dimethylamino)propyl chloride
hydrochloride
Method 1: Under an argon atmosphere, a mixture of 17b (371 mg,
0.653 mmol), 3-(dimethylamino)propyl chloride hydrochloride (155
1
3
6
H). C NMR (126 MHz, DMSO‑d ): δ21.7, 21.7, 21.8, 21.9, 54.3, 55.7,
0.1, 70.1, 102.1, 105.1, 109.0, 109.3, 110.3, 110.6, 110.7, 112.2,
2 3
mg, 0.979 mmol), K CO (541 mg, 3.92 mmol), and acetone (350 mL)
13.5, 118.2, 118.5, 122.7, 123.7, 123.8, 127.5, 128.5, 130.4, 131.1,
was refluxed for 22 h. After cooling to room temperature, the mixture
was passed through a pad of Celite. The filtrate was evaporated under
reduced pressure. The residue was purified by flash chromatography
over Chromatorex NH-DM2035 (DCM–MeOH = 99:1 to 97:3 to 9:1) to
give 19 as a pale brown solid (237 mg, 56%), 20 as a pale brown solid
(99.3 mg, 23%), and 21 as a pale brown solid (17.4 mg, 4%).
41.0, 146.4, 147.1, 147.6, 149.2, 149.9, 155.4. HRDARTMS m/z. Calcd
+
for C33
H
33
N
2
O
7
[(M+H) ]: 569.2288. Found: 569.2299.
4
.5. Synthesis of A-ring-modified azalamellarin N analogues 7, 8, 9, and
1
0
Method 2: Under an argon atmosphere, a mixture of 17b (50.0 mg,
87.9 µmol), 3-(dimethylamino)propyl chloride hydrochloride (33.4 mg,
4
.5.1. 3-[3-(Dimethylamino)propoxy]-11-isopropoxy-14-(3-isopropoxy-4-
methoxyphenyl)-2,12-dimethoxy-benzo[7,8]indolizino[3,2-c]quinolin-6
2 3
0.211 mmol), K CO (60.8 mg, 0.440 mmol), and acetone (50 mL) was
(
5H)-one (18)
refluxed for 20 h. After cooling to room temperature, the mixture was
passed through a pad of Celite. The filtrate was evaporated under
reduced pressure. The residue was purified by column chromatography
over Chromatorex Diol MB100–75/200 (DCM–MeOH = 20:1 to 10:1) to
give 19 as a pale brown solid (22.2 mg, 39%) and 21 as a pale brown
solid (17.9 mg, 28%).
Under an argon atmosphere, a mixture of 17a (117 mg, 0.201 mmol),
3
-(dimethylamino)propyl chloride hydrochloride (63.6 mg, 0.402
mmol), K CO (278 mg, 2.01 mmol), and acetone (25 mL) was refluxed
2
3
for 20 h. After cooling to room temperature, the mixture was passed
through a pad of Celite. The filtrate was evaporated under reduced
pressure. The residue was purified by column chromatography over
Chromatorex Diol MB100–75/200 (ethyl acetate to DCM–MeOH = 10:1
to 1:1) to give 18 as a pale brown solid (78.8 mg, 59%). Recrystallization
4.5.3.1. 3-[3-(Dimethylamino)propoxy]-2-hydroxy-11-isopropoxy-14-(3-
isopropoxy-4-methoxyphenyl)-12-methoxy-benzo[7,8]indolizino[3,2-c]qui-
nolin-6(5H)-one (19). Recrystallization from DCM–hexane gave a pale
◦
from DCM–hexane gave a pale brown powder. Mp 150–180 C (dec)
ꢀ 1
1
◦
(
sealed capillary). IR (KBr): 1652, 1452, 1430, 1260, 1223 cm
.
H
green powder. Mp 180–200 C (dec) (sealed capillary). IR (KBr): 1650,
ꢀ 1 1
NMR (500 MHz, CDCl
3
): δ1.35 (d, J = 6.1 Hz, 6H), 1.43 (d, J = 6.1 Hz,
1449, 1434, 1259, 1219, 1188 cm . H NMR (500 MHz, CDCl ): δ1.33
3
6
3
1
7
1
1
2
1
1
1
6
H), 2.08 (quin, J = 6.9 Hz, 2H), 2.28 (s, 6H), 2.51 (t, J = 7.2 Hz, 2H),
(d, J = 6.1 Hz, 3H), 1.38 (d, J = 6.1 Hz, 3H), 1.42 (d, J = 6.1 Hz, 3H),
.45 (s, 6H), 3.96 (s, 3H), 4.17 (t, J = 6.7 Hz, 2H), 4.54 (sep, J = 6.1 Hz,
H), 4.68 (sep, J = 6.1 Hz, 1H), 6.88 (s, 1H), 6.93 (d, J = 7.4 Hz, 1H),
.00 (s, 1H), 7.09 (s, 1H), 7.14 (d, J = 8.2 Hz, 1H), 7.16 (d, J = 1.7 Hz,
H), 7.19 (s, 1H), 7.21 (dd, J = 1.7 and 8.2 Hz, 1H), 9.59 (d, J = 7.4 Hz,
1.42 (d, J = 6.1 Hz, 3H), 1.93–2.01 (m, 2H), 2.32 (s, 6H), 2.59 (t, J = 5.6
Hz, 2H), 3.43 (s, 3H), 3.98 (s, 3H), 4.01–4.07 (m, 2H), 4.55 (sep, J = 6.1
Hz, 1H), 4.67 (sep, J = 6.1 Hz, 1H), 6.93 (d, J = 7.4 Hz, 1H), 6.99 (s, 1H),
7.03 (s, 1H), 7.04 (s, 1H), 7.07 (s, 1H), 7.10–7.18 (m, 3H), 9.60 (d, J =
H), 10.86 (br s, 1H). 13C NMR (126 MHz, CDCl
): δ22.0, 22.0, 22.0,
13
3
7.4 Hz, 1H), 9.89 (br s, 1H). C NMR (126 MHz, CDCl ): δ21.9, 21.9,
3
7.3, 45.5, 55.2, 55.4, 56.3, 56.5, 67.4, 71.2, 71.3, 100.4, 105.8, 105.9,
22.1, 26.4, 44.9, 55.1, 56.2, 57.4, 71.1, 71.1, 72.5, 105.8, 107.0, 110.7,
110.8, 111.0, 111.5, 112.9, 113.1, 118.4, 119.4, 123.7, 124.1, 124.2,
128.2, 129.0, 129.0, 132.6, 145.8, 147.3, 147.7, 148.2, 149.8, 150.2,
10.5, 110.7, 110.8, 110.9, 112.8, 112.9, 118.4, 119.4, 123.7, 124.2,
24.4, 128.9, 129.5, 130.7, 132.5, 145.1, 147.9, 148.2, 148.7, 149.9,
+
+
50.0, 156.7. HRDARTMS m/z. Calcd for C39
H
46
3
N O
7
[(M+H) ]:
156.3. HRDARTMS m/z. Calcd for C38H44N O [(M+H) ]: 654.3179.
3
7
68.3336. Found: 668.3352.
Found: 654.3175.
4
.5.2. Trifluoroacetic acid salt of 3-[3-(dimethylamino)propoxy]-11-hy-
4.5.3.2. 2-[3-(Dimethylamino)propoxy]-3-hydroxy-11-isopropoxy-14-(3-
isopropoxy-4-methoxyphenyl)-12-methoxy-benzo[7,8]indolizino[3,2-c]qui-
nolin-6(5H)-one (20). Recrystallization from DCM–hexane gave a pale
droxy-14-(3-hydroxy-4-methoxyphenyl)-2,12-dimethoxy-benzo[7,8]indoli-
zino[3,2-c]quinolin-6(5H)-one (7)
◦
Under an argon atmosphere, a nitrobenzene solution of AlCl
3
(1.0 M,
brown powder. Mp 170–200 C (dec) (sealed capillary). IR (KBr): 1650,
ꢀ 1 1
1
2
45 µL, 0.145 mmol) was added dropwise to a solution of 18 (15.2 mg,
2.8 µmol) in DCM (5.0 mL) at room temperature. After stirring for 72 h
1488, 1451, 1432, 1259, 1220 cm . H NMR (500 MHz, CDCl ): δ1.35
3
(d, J = 6.1 Hz, 3H), 1.38 (d, J = 6.1 Hz, 3H), 1.43 (d, J = 6.1 Hz, 6H),
at room temperature, a solution of NaHCO
3
(36.7 mg, 0.437 mmol) and
1.82–1.93 (m, 2H), 2.36 (s, 6H), 2.55–2.64 (m, 2H), 3.44 (s, 3H),
3.72–3.82 (m, 2H), 3.99 (s, 3H), 4.56 (sep, J = 6.1 Hz, 1H), 4.68 (sep, J
= 6.1 Hz, 1H), 6.88 (s, 1H), 6.93 (d, J = 7.4 Hz, 1H), 7.03 (s, 1H), 7.08 (s,
2H), 7.14 (d, J = 1.6 Hz, 1H), 7.14 (d, J = 8.2 Hz, 1H), 7.17 (dd, J = 1.6
Rochelle salt (123 mg, 0.437 mmol) in water (2.1 mL) was added. The
mixture was stirred for an additional 1 h and then evaporated. The
nitrobenzene was removed azeotropically with water under reduced
pressure. To the residue was added DCM (1.0 mL) and TFA (1.0 mL) and
then the mixture was evaporated. The residue was purified by column
chromatography over Sephadex LH-20 using following solvent systems
1
3
and 8.2 Hz, 1H), 9.57 (d, J = 7.4 Hz, 1H), 9.92 (br s, 1H). C NMR (126
MHz, CDCl ): δ21.9, 22.0, 22.0, 26.2, 44.9, 55.1, 56.3, 57.7, 71.1, 71.1,
3
73.4, 103.1, 105.7, 109.4, 110.6, 110.7, 110.8, 112.4, 112.8, 115.7,
(
water containing 0.1% TFA, water–MeOH = 1:1 containing 0.1% TFA,
118.3, 119.3, 123.9, 124.3, 124.3, 128.8, 129.4, 132.5, 133.9, 143.2,
and MeOH containing 0.1% TFA) to give 7 as a brown powder (15.8 mg,
quant). Mp 125–155 ◦C (dec) (sealed capillary). IR (KBr): 3128, 1681,
147.7, 148.1, 149.7, 149.9, 151.5, 156.6. HRFABMS m/z. Calcd for
+
C38H44N O [(M+H) ]: 654.3179. Found: 654.3178.
3 7
ꢀ 1 1
1
457, 1432, 1276, 1199 cm . H NMR (500 MHz, DMSO‑d ): δ2.14 (dt,
6
J = 6.0 and 15.3 Hz, 2H), 2.82 (s, 3H), 2.83 (s, 3H), 3.20–3.26 (m, 2H),
4
.5.3.3. 2,3-Bis[3-(dimethylamino)propoxy]-11-isopropoxy-14-(3-iso-
3
1
1
9
4
.36 (s, 3H), 3.39 (s, 3H), 3.86 (s, 3H), 4.05 (t, J = 5.9 Hz, 2H), 6.90 (s,
H), 6.98–7.02 (m, 3H), 7.05 (d, J = 7.5 Hz, 1H), 7.14 (s, 1H), 7.16 (s,
H), 7.23 (d, J = 8.7 Hz, 1H), 9.37 (d, J = 7.5 Hz, 1H), 9.60 (br s, 1H),
propoxy-4-methoxyphenyl)-12-methoxy-benzo[7,8]indolizino[3,2-c]quino-
lin-6(5H)-one (21). Recrystallization from DCM–hexane gave a brown
◦
needles. Mp 165–200 C (dec) (sealed capillary). IR (KBr): 3440, 1650,
.75 (br s, 2H), 11.37 (s, 1H). 13C NMR (126 MHz, DMSO‑d
6
): δ23.9,
ꢀ 1
1
1
434, 1260, 1223, 1199 cm . H NMR (500 MHz, CDCl
3
): δ1.33 (d, J =
2.4, 54.5, 54.8, 56.2, 65.8, 100.6, 105.4, 105.7, 109.8, 109.9, 110.8,
9

T. Fukuda et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116039
6
.1 Hz, 3H), 1.33 (d, J = 6.1 Hz, 3H), 1.43 (d, J = 6.1 Hz, 6H), 1.84
quin, J = 6.9 Hz, 2H), 2.06 (quin, J = 6.8 Hz, 2H), 2.29 (s, 6H), 2.32 (s,
H), 2.38–2.48 (m, 2H), 2.54–2.61 (m, 2H), 3.43 (s, 3H), 3.59 (t, J = 6.3
Hz, 2H), 3.95 (s, 3H), 4.08–4.20 (m, 2H), 4.52 (sep, J = 6.1 Hz, 1H), 4.68
sep, J = 6.1 Hz, 1H), 6.86 (s, 1H), 6.92 (s, 1H), 6.93 (d, J = 7.3 Hz, 1H),
.08 (d, J = 8.3 Hz, 1H), 7.09 (s, 1H), 7.12 (d, J = 1.6 Hz, 1H), 7.15 (dd,
J = 1.6 and 8.3 Hz, 1H), 7.16 (s, 1H), 9.54 (d, J = 7.3 Hz, 1H), 10.10 (br
s, 1H). 13C NMR (126 MHz, CDCl
): δ21.9, 21.9, 22.0, 22.1, 27.2, 27.3,
5.4, 45.4, 55.1, 56.3, 56.4, 66.8, 67.3, 71.2, 71.2, 100.7, 105.8, 107.7,
MeOH containing 0.1% TFA), 10 was obtained as a brown powder (23.1
◦
(
mg, 97%). Mp 160–200 C (dec) (sealed capillary). IR (KBr): 3413, 1683,
ꢀ 1 1
6
1432, 1275, 1201, 1130 cm . H NMR (500 MHz, DMSO‑d ): δ1.94 (dt,
6
J = 6.1 and 15.6 Hz, 2H), 2.15 (dt, J = 6.0 and 15.3 Hz, 2H), 2.81 (s,
3H), 2.82 (s, 3H), 2.83 (s, 3H), 2.83 (s, 3H), 3.05–3.13 (m, 2H),
3.19–3.26 (m, 2H), 3.39 (s, 3H), 3.55–3.67 (m, 2H), 3.90 (s, 3H), 4.06 (t,
J = 6.0 Hz, 2H), 6.89 (s, 1H), 6.99 (d, J = 2.0 Hz, 1H), 7.01 (dd, J = 2.0
and 8.0 Hz, 1H), 7.02 (s, 1H), 7.06 (d, J = 7.4 Hz, 1H), 7.15 (s, 2H), 7.25
(d, J = 8.0 Hz, 1H), 9.37 (d, J = 7.4 Hz, 1H), 9.42 (br s, 1H), 9.79 (br s,
(
7
3
4
1
1
1
7
10.6, 110.6, 110.7, 110.9, 112.7, 112.8, 118.5, 119.3, 123.6, 124.2,
3H), 11.40 (s, 1H). 13C NMR (126 MHz, DMSO‑d
): δ23.7, 23.9, 42.3,
6
24.3, 128.7, 129.3, 130.6, 132.5, 144.3, 147.8, 148.2, 149.1, 149.9,
42.4, 54.1, 54.4, 54.5, 56.1, 65.6, 65.8, 100.9, 105.4, 107.8, 109.8,
+
50.1, 156.3. HRDARTMS m/z. Calcd for C43
H
55
4
N O
7
[(M+H) ]:
109.9, 110.9, 111.6, 112.3, 113.6, 117.6, 118.4, 122.2, 122.5, 123.9,
39.4071. Found: 739.4045.
127.1, 128.6, 131.3, 131.5, 142.7, 147.5, 147.7, 147.7, 148.1, 148.1,
+
1
55.3. HRDARTMS m/z. Calcd for C37
H
43
4
N O
7
[(M ꢀ 2CF
3
COO ꢀ H) ]:
4
.5.4. Trifluoroacetic acid salt of 3-[3-(dimethylamino)propoxy]-2,11-
655.3132. Found: 655.3104.
dihydroxy-14-(3-hydroxy-4-methoxyphenyl)-12-methoxy-benzo[7,8]indo-
lizino[3,2-c]quinolin-6(5H)-one (8)
4.6. In vitro kinase assay
According to the procedure described for the preparation of 7, 19
(
14.3 mg, 21.9 µmol) and AlCl
3
(1.0 M solution in nitrobenzene, 162 µL,
Recombinant kinase domains (from amino residues 696 to the C-
terminus) of EGFR WT and EGFR T790M/L858R (Cell Signaling Tech-
nology) (100 ng) were preincubated with 1–10,000 nM of inhibitors in
0
.162 mmol) were reacted. After purification by column chromatog-
raphy over Sephadex LH-20 using following solvent systems (water
containing 0.1% TFA, water–MeOH = 1:1 containing 0.1% TFA, and
MeOH containing 0.1% TFA), 8 was obtained as a brown powder (14.1
25 µL of kinase reaction buffer (120 mM HEPES, pH 7.5, 10 mM MnCl ,
2
◦
6 µM Na VO , and 2.5 mM DTT) at 25 C for 30 min. Then, 25 µL ATP/
3
4
◦
mg, 94%). Mp 230–260 C (dec) (sealed capillary). IR (KBr): 3115, 1683,
1
substrate solution containing 20, 200, or 2,000 µM ATP and 6 µM poly
(Glu-Tyr) biotinylated peptide (Cell Signaling Technology) was added to
the preincubated samples. The kinase reaction was allowed to proceed at
ꢀ 1 1
436, 1277, 1205, 1130 cm . H NMR (500 MHz, DMSO‑d ): δ2.14 (dt,
6
J = 5.8 and 15.2 Hz, 2H), 2.82 (s, 3H), 2.83 (s, 3H), 3.27–3.33 (m, 2H),
◦
3
1
1
.37 (s, 3H), 3.90 (s, 3H), 4.06 (t, J = 5.7 Hz, 2H), 6.89 (s, 1H), 6.89 (s,
H), 6.92–6.96 (m, 2H), 6.98 (s, 1H), 7.02 (d, J = 7.5 Hz, 1H), 7.12 (s,
H), 7.22 (d, J = 8.0 Hz, 1H), 8.66 (br s, 1H), 9.35 (br s, 1H), 9.41 (d, J
25 C for 30 min and terminated by adding 50 µL of 50 mM of EDTA,
pH 8.0. Phosphorylation levels were quantified using ELISA, with
avidin-coated 96-well plates and an anti-phosphotyrosine antibody
(PY20). Unless otherwise mentioned, relative inhibitions were calcu-
=
7.5 Hz, 1H), 9.64 (br s, 2H), 11.29 (s, 1H). 13C NMR (126 MHz,
DMSO‑d
6
): δ23.8, 42.4, 54.4, 54.4, 55.9, 65.6, 100.3, 105.3, 109.4,
10.2, 110.4, 110.7, 111.6, 112.3, 113.6, 117.7, 118.3, 122.1, 122.5,
23.9, 126.9, 128.6, 130.1, 131.5, 141.6, 147.1, 147.3, 147.6, 147.7,
lated from at least three independent experiments and IC5 values were
0
estimated using the mean relative inhibition.29
1
1
1
48.0, 155.3. HRDARTMS m/z. Calcd for
C
32
H
32
N
3
O
7
[(M
ꢀ
4.7. Docking simulation
+
CF
3
COO) ]: 570.2240. Found: 570.2216.
Three-dimensional structures of 1, 5, 7, and 10 were built and
optimized using the PM3 method implemented in the Spartan 18
package (version 1.4.4).33 Crystal structure of the EGFR (T790M/
4
.5.5. Trifluoroacetic acid salt of 2-[3-(dimethylamino)propoxy]-3,11-
dihydroxy-14-(3-hydroxy-4-methoxyphenyl)-12-methoxy-benzo[7,8]indo-
lizino[3,2-c]quinolin-6(5H)-one (9)
3
0
L858R/V948R)–gefitinib complex (PDB ID: 4I22) was obtained from
the Protein Data Bank. Molecular docking of ligands into the EGFR ki-
nase domain was carried out using the AutoDock Vina program (version
1.1.2)31 after preparing the ligand and EGFR pdbqt files by the Auto-
Dock Tools program (version 1.5.6).34 For ligands, the Gasteiger charges
were calculated, and all nonpolar hydrogen atoms were merged with the
carbon atoms. For EGFR, hydrogen atoms were added to the structure,
and the Gasteiger charges were computed. Gefitinib and all the water
molecules were removed from the crystal structure of EGFR. The
nonpolar hydrogen atoms were merged with the carbon atoms. Leu718,
Val726, Lys745, Met790, Leu792, Cys797, Asp800, Glu804, Leu844,
Thr854, and Asp855 in EGFR were set as flexible residues. To ensure the
According to the procedure described for the preparation of 7, 20
(
10.8 mg, 16.5 µmol) and AlCl
3
(1.0 M solution in nitrobenzene, 122 µL,
0
.122 mmol) were reacted. After purification by column chromatog-
raphy over Sephadex LH-20 using following solvent systems (water
containing 0.1% TFA, water–MeOH = 1:1 containing 0.1% TFA, and
MeOH containing 0.1% TFA), 9 was obtained as a brown powder (10.7
◦
mg, 95%). Mp 165–195 C (dec) (sealed capillary). IR (KBr): 3392, 1683,
1
ꢀ
1 1
647, 1432, 1276, 1203, 1129 cm . H NMR (500 MHz, DMSO‑d ):
6
δ1.94 (dt, J = 5.9 and 15.3 Hz, 2H), 2.80 (s, 3H), 2.81 (s, 3H), 3.16 (dt, J
=
5.7 and 9.0 Hz, 2H), 3.39 (s, 3H), 3.55–3.65 (m, 2H), 3.89 (s, 3H),
6
8
.82 (s, 1H), 6.92 (s, 1H), 6.99 (d, J = 2.0 Hz, 1H), 7.00 (dd, J = 2.0 and
3
.0 Hz, 1H), 7.02 (d, J = 7.4 Hz, 1H), 7.13 (s, 1H), 7.14 (s, 1H), 7.24 (d,
inclusion of the ATP-binding site of the EGFR, a 24 × 22 × 22 Å grid
J = 8.0 Hz, 1H), 9.37 (d, J = 7.4 Hz, 1H), 9.55 (br s, 4H), 11.31 (s, 1H).
box with 1.000 Å grid spacing was used; the grid center coordinates
were placed at x = 12.097 Å, y = ꢀ 12.296 Å, and z = 10.985 Å. The
exhaustiveness of the global search and the maximum number of bind-
ing modes to be generated were set to 8 and 100. The maximum energy
difference between the best and worst binding modes was fixed at 5
kcal/mol. After five docking runs for each ligand, the obtained poses
were evaluated using the predicted binding affinity, and the plausible
low energy pose for each docked ligand in EGFR was visualized and
analyzed using PyMOL (version 2.3.4).35 These results are shown in
Fig. 4.
1
3
C NMR (126 MHz, DMSO‑d ): δ23.8, 42.3, 42.4, 54.3, 54.5, 56.2, 65.5,
6
1
1
1
02.4, 105.4, 107.7, 108.5, 109.7, 110.5, 111.6, 112.1, 113.7, 117.7,
18.5, 122.3, 122.6, 123.9, 127.5, 128.8, 131.2, 131.8, 141.9, 147.4,
47.5, 147.7, 147.7, 148.0, 155.5. HRDARTMS m/z. Calcd for
+
C
32
H
32
3
N O
7
[(M ꢀ CF
3
COO) ]: 570.2240. Found: 570.2239.
4
.5.6. Trifluoroacetic acid salt of 2,3-bis[3-(dimethylamino)propoxy]-11-
hydroxy-14-(3-hydroxy-4-methoxyphenyl)-12-methoxy-benzo[7,8]
indolizino[3,2-c]quinolin-6(5H)-one (10)
According to the procedure described for the preparation of 7, 21
(
20.0 mg, 27.1 µmol) and AlCl
3
(1.0 M solution in nitrobenzene, 200 µL,
4.8. Proliferation assay
0
.200 mmol) were reacted. After purification by column chromatog-
4
raphy over Sephadex LH-20 using following solvent systems (water
A549 or NCI-H1975 cells (1 × 10 cells/well) were cultured in flat-
containing 0.1% TFA, water–MeOH = 1:1 containing 0.1% TFA, and
bottom 96-well plates in 150 µL of media containing various
1
0

T. Fukuda et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116039
concentrations of inhibitors for 72 h. Next, the inhibitor-treated cells
patients (pts) with NSCLC. J Clin Oncol. 2008;26(15_suppl):8027. https://doi.org/
1
0.1200/jco.2008.26.15_suppl.8027.
2
were incubated with 0.5 mg/mL MTT in a CO incubator for 4 h, and
1
2
Sequist LV, Besse B, Lynch TJ, et al. Neratinib, an irreversible pan-ErbB receptor
tyrosine kinase inhibitor: results of a phase II trial in patients with advanced
non–small-cell lung cancer. J Clin Oncol. 2010;28:3076–3083. https://doi.org/
then 100 µL of 20% SDS was added to each well to dissolve the purple
formazan product. The absorption was determined at 570 nm using a
spectrophotometer. The relative viability values were calculated from at
least three independent experiments. IC50 values were estimated using
the mean relative viability.29
1
0.1200/JCO.2009.27.9414.
1
1
1
1
1
3
4
5
6
7
Zhou W, Ercan D, Chen L, et al. Novel mutant-selective EGFR kinase inhibitors
against EGFR T790M. Nature. 2009;462:1070–1074. https://doi.org/10.1038/
nature08622.
Walter AO, Sjin RTT, Haringsma HJ, et al. Discovery of a mutant-selective covalent
inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC. Cancer
Discov. 2013;3:1404–1415. https://doi.org/10.1158/2159-8290.CD-13-0314.
Cross DAE, Ashton SE, Ghiorghiu S, et al. AZD9291, an irreversible EGFR TKI,
overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer
Discov. 2014;4:1046–1061. https://doi.org/10.1158/2159-8290.CD-14-0337.
Soria J-C, Ohe Y, Vansteenkiste J, et al. Osimertinib in untreated EGFR-mutated
advanced non–small-cell lung cancer. N Engl J Med. 2018;378:113–125. https://doi.
org/10.1056/NEJMoa1713137.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could influence the work re-
ported in this paper.
Thress KS, Paweletz CP, Felip E, et al. Acquired EGFR C797S mutation mediates
resistance to AZD9291 in non–small cell lung cancer harboring EGFR T790M. Nat
Med. 2015;21:560–562. https://doi.org/10.1038/nm.3854.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Numbers
18 Baunbæk D, Trinkler N, Ferandin Y, et al. Anticancer alkaloid lamellarins inhibit
protein kinases. Mar Drugs. 2008;6:514–527. https://doi.org/10.3390/md20080026.
2
6293028, 19K05715, and partially 16H06276 (AdAMS). The author (T.
1
9
Yoshida K, Itoyama R, Yamahira M, et al. Synthesis, resolution, and biological
evaluation of atropisomeric (aR)- and (aS)-16-methyllamellarins N: unique effects of
the axial chirality on the selectivity of protein kinases inhibition. J Med Chem. 2013;
F.) would like to thank the Foundation for Promotion of Cancer Research
in Japan and the Naito Foundation for financial support. Some of the
results presented herein were obtained using research equipment shared
in MEXT Project for promoting public utilization of advanced research
infrastructure (Program for supporting introduction of the new sharing
system) Grant Number JPMXS0422500320.
5
6:7289–7301. https://doi.org/10.1021/jm400719y.
2
0
Fukuda T, Umeki T, Tokushima K, et al. Design, synthesis, and evaluation of A-ring-
modified lamellarin N analogues as noncovalent inhibitors of the EGFR T790M/
L858R mutant. Bioorg Med Chem. 2017;25:6563–6580. https://doi.org/10.1016/j.
bmc.2017.10.030.
2
2
1
2
Boonya-udtayan S, Yotapan N, Woo C, Bruns CJ, Ruchirawat S, Thasana N. Synthesis
and biological activities of azalamellarins. Chem - An Asian J. 2010;5:2113–2123.
https://doi.org/10.1002/asia.201000237.
Theppawong A, Ploypradith P, Chuawong P, Ruchirawat S, Chittchang M. Facile and
divergent synthesis of lamellarins and lactam-containing derivatives with improved
drug likeness and biological activities. Chem - An Asian J. 2015;10:2631–2650.
https://doi.org/10.1002/asia.201500611.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116039.
2
2
3
4
Komatsubara M, Umeki T, Fukuda T, Iwao M. Modular synthesis of lamellarins via
regioselective assembly of 3,4,5-differentially arylated pyrrole-2-carboxylates. J Org
Chem. 2014;79:529–537. https://doi.org/10.1021/jo402181w.
References
Ikegashira K, Oka T, Hirashima S, et al. Discovery of conformationally constrained
tetracyclic compounds as potent hepatitis C virus NS5B RNA polymerase inhibitors.
J Med Chem. 2006;49(24):6950–6953. https://doi.org/10.1021/
jm061024510.1021/jm0610245.s001.
1
2
3
da Cunha Santos G, Shepherd FA, Tsao MS. EGFR mutations and lung cancer. Annu
Rev Pathol Mech Dis. 2011;6:49–69. https://doi.org/10.1146/annurev-pathol-
0
11110-130206.
Shigematsu H, Lin L, Takahashi T, et al. Clinical and biological features associated
with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer
Inst. 2005;97:339–346. https://doi.org/10.1093/jnci/dji055.
25 Jacquemard U, B ´e n ´e teau V, Lefoix M, Routier S, M ´e rour J-Y, Coudert G. Mild and
selective deprotection of carbamates with Bu NF. Tetrahedron. 2004;60(44):
4
1
0039–10047. https://doi.org/10.1016/j.tet.2004.07.071.
Cohen MH, Williams GA, Sridhara R, Chen G, Pazdur R. FDA drug approval
summary: gefitinib (ZD1839) (Iressa ®) tablets. Oncologist. 2003;8:303–306. https://
doi.org/10.1634/theoncologist.8-4-303.
26 Fujikawa N, Ohta T, Yamaguchi T, Fukuda T, Ishibashi F, Iwao M. Total synthesis of
lamellarins D, L, and N. Tetrahedron. 2006;62(4):594–604. https://doi.org/10.1016/
j.tet.2005.10.014.
4
5
Dowell J, Minna JD, Kirkpatrick P. Erlotinib hydrochloride. Nat Rev Drug Discov.
27 Kamiyama H, Kubo Y, Sato H, et al. Synthesis, structure–activity relationships, and
2
005;4:13–14. https://doi.org/10.1038/nrd1612.
mechanism of action of anti-HIV-1 lamellarin
α
20-sulfate analogues. Bioorg Med
Engelman JA, Janne PA. Mechanisms of acquired resistance to epidermal growth
factor receptor tyrosine kinase inhibitors in non–small cell lung cancer. Clin Cancer
Res. 2008;14:2895–2899. https://doi.org/10.1158/1078-0432.CCR-07-2248.
The T790M secondary mutation restore the ATP affinity of the mutant to near EGFR
WT levels (Km[ATP] values for EGFR WT, L858R, and T790M/L858R mutants are 5.2,
Chem. 2011;19(24):7541–7550. https://doi.org/10.1016/j.bmc.2011.10.030.
Banwell MG, Flynn BL, Stewart SG. Selective cleavage of isopropyl aryl ethers by
aluminum trichloride. J Org Chem. 1998;63(24):9139–9144. https://doi.org/
10.1021/jo9808526.
2
8
6
29 Nishiya N, Sakamoto Y, Oku Y, Nonaka T, Uehara Y. JAK3 inhibitor VI is a mutant
specific inhibitor for epidermal growth factor receptor with the gatekeeper mutation
T790M. World J Biol Chem. 2015;6:409–418. https://doi.org/10.4331/wjbc.v6.
i4.409.
1
48, and 8.4 µM). Please see: Yun C-H, Mengwasser KE, Toms AV, et al. The T790M
mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP
Proc Natl Acad Sci. 2008;105:2070–2075. https://doi.org/10.1073/
pnas.0709662105.
3
0
Gajiwala K, Feng J, Ferre R, et al. Insights into the aberrant activity of mutant EGFR
kinase domain and drug recognition. Structure. 2013;21(2):209–219. https://doi.
org/10.1016/j.str.2012.11.014.
7
8
Li D, Ambrogio L, Shimamura T, et al. BIBW2992, an irreversible EGFR/HER2
inhibitor highly effective in preclinical lung cancer models. Oncogene. 2008;27:
4
702–4711. https://doi.org/10.1038/onc.2008.109.
3
1
Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking
with a new scoring function, efficient optimization, and multithreading. J Comput
Chem. 2010;31:455–461. https://doi.org/10.1002/jcc.21334.
Engelman JA, Zejnullahu K, Gale C-M, et al. PF00299804, an irreversible pan-ERBB
inhibitor, is effective in lung cancer models with EGFR and ERBB2 mutations that are
resistant to gefitinib. Cancer Res. 2007;67:11924–11932. https://doi.org/10.1158/
32 Gaillard T. Evaluation of AutoDock and AutoDock Vina on the CASF-2013
benchmark. J Chem Inf Model. 2018;58(8):1697–1706. https://doi.org/10.1021/acs.
jcim.8b0031210.1021/acs.jcim.8b00312.s00110.1021/acs.jcim.8b00312.s002.
33 Spartan 18, version 1.4.4; Wavefunction, Inc. https://www.wavefun.com.
0
008-5472.CAN-07-1885.
9
Minami Y, Shimamura T, Shah K, et al. The major lung cancer-derived mutants of
ERBB2 are oncogenic and are associated with sensitivity to the irreversible EGFR/
ERBB2 inhibitor HKI-272. Oncogene. 2007;26:5023–5027. https://doi.org/10.1038/
sj.onc.1210292.
Kim Y, Ko J, Cui Z, et al. The EGFR T790M mutation in acquired resistance to an
irreversible second-generation EGFR inhibitor. Mol Cancer Ther. 2012;11:784–791.
https://doi.org/10.1158/1535-7163.MCT-11-0750.
3
4
Morris GM, Huey R, Lindstrom W, et al. AutoDock4 and AutoDockTools4: automated
docking with selective receptor flexibility. J Comput Chem. 2009;30(16):2785–2791.
https://doi.org/10.1002/jcc.v30:1610.1002/jcc.21256.
1
0
1
35 The PyMOL Molecular Graphics System, version 2.3.4; Schr o¨ dinger, LLC. https://
pymol.org.
1
Janne PA, Schellens JH, Engelman JA, et al. Preliminary activity and safety results
from a phase I clinical trial of PF-00299804, an irreversible pan-HER inhibitor, in
1
1