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

Article abstract of DOI:10.1021/jm400719y

The total synthesis of the optically active (aR)- and (aS)-16- methyllamellarins N (3a and 3b) was achieved via resolution on HPLC chiral stationary phase. The kinase inhibitory activities of both enantiomers were evaluated on eight protein kinases releva

Full text of DOI:10.1021/jm400719y

Article
pubs.acs.org/jmc
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
Kenyu Yoshida,† Ryosuke Itoyama,† Masashi Yamahira,‡ Junji Tanaka,§ Nadege Loaec,∥,⊥ Olivier Lozach,∥
̀
̈
Emilie Durieu,∥,⊥ Tsutomu Fukuda,† Fumito Ishibashi,‡ Laurent Meijer,∥,⊥ and Masatomo Iwao*,†
†Division of Chemistry and Materials Sciences, Graduate School of Engineering, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki
852-8521, Japan
‡Division of Marine Life Science and Biochemistry, Graduate School of Fisheries Science and Environmental Studies, Nagasaki
University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
§Institute for Materials Chemistry and Engineering, Kyushu University, Kasuga Koen 6-1, Kasuga 816-8580, Japan
∥Protein Phosphorylation and Human Disease Group, Station Biologique, CNRS, 29680 Roscoff, Bretagne, France
⊥ManRos Therapeutics, Centre de Perharidy, 29680 Roscoff, Bretagne, France
S
* Supporting Information
ABSTRACT: The total synthesis of the optically active (aR)- and
(aS)-16-methyllamellarins N (3a and 3b) was achieved via
resolution on HPLC chiral stationary phase. The kinase inhibitory
activities of both enantiomers were evaluated on eight protein
kinases relevant to cancer and neurodegenerative diseases (CDK1/
cyclin B, CDK2/cyclin A, CDK5/p25, GSK-3α/β, PIM1,
DYRK1A, CLK3, and CK1). Isomer (aR)-3b exhibited potent
but nonselective inhibition on all protein kinases except CK1,
while (aS)-3a selectively inhibited only GSK-3α/β, PIM1, and
DYRK1A. The different inhibition profiles of (aS)-3a and (aR)-3b
were elucidated by docking simulation studies. Although parental
lamellarin N (2) inhibited the action of topoisomerase I, both
(aS)-3a and (aR)-3b showed no inhibition of this enzyme. The phenotypic cytotoxic activities of 2, (aS)-3a, and (aR)-3b on
three cancer cell lines (HeLa, SH-SY5Y, and IMR32) changed according to their topoisomerase I and protein kinase inhibitory
activities.
activities and potential development in various therapeutic
areas.4,5 For example, Quesada reported in 1996 that the
triacetates of lamellarins D, N, and K showed potent
cytotoxicity on P-glycoprotein-mediated MDR cancer cell
lines as well as their respective parental cell lines at low
nanomolar concentrations.6 In addition, Faulkner reported in
1999 that lamellarin α 20-sulfate and related lamellarin sulfates
inhibit HIV-1 integrase and display anti-HIV-1 activity.7−9
In 1997, we achieved the first total synthesis of lamellarin-
class marine alkaloids (lamellarins D and H) by N-ylide
mediated cyclization.10 Using this method, we prepared a range
of differentially substituted non-natural lamellarin D analogues
and evaluated their cytotoxic activities on a HeLa cell line.11
The SAR study indicated that the hydroxyl groups at the C8
and C20 positions of lamellarin D (1) are essential for its
potent cytotoxicity, whereas the hydroxyl group at C14 is less
important.11 A more comprehensive SAR study conducted by
INTRODUCTION
■
Lamellarins are unique marine polycyclic alkaloids sharing a
common 14-phenyl-6H-[1]benzopyrano[4′,3′:4,5]pyrrolo[2,1-
a]isoquinolin-6-one ring system.1−3 Approximately 40 lamellar-
ins have been isolated from a variety of marine organisms such
as mollusks, ascidians, and sponges. These lamellarins differ in
the number and position of the OH and OMe groups on their
common scaffold (Figure 1). Lamellarins have received
considerable attention owing to their interesting biological
Received: May 14, 2013
Figure 1. Structures of lamellarins D and N.
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm400719y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Ruchirawat and Ploypradith using a variety of cancer cell lines
supports our results.12 Concerning the mechanism of action,
Bailly demonstrated that a major molecular target of 1 in cancer
cells is DNA topoisomerase I.13 They proposed a lamellarin D−
DNA−topoisomerase I ternary complex model by docking
simulation studies.14 This model is in perfect agreement with
our SAR study. Bailly also showed 1 could induce apoptosis of
cancer cells through direct inhibition of mitochondrial function
at micromolar concentrations.15,16 Recently, we have reported
that lamellarin N (2) strongly inhibits several protein kinases
relevant to cancer and neurodegenerative diseases (Down
syndrome, Alzheimer’s disease, etc.) such as CDKs, GSK-3,
PIM1, and DYRK1A.17 A good correlation was observed
between the effects of lamellarins on protein kinases and their
action on cell death, suggesting that inhibition of specific
kinases may contribute to the potent cytotoxicity of
lamellarins.17
From a structural viewpoint, lamellarins are interesting
because they possess axial chirality (atropisomerism) caused
by restricted rotation around the C1−C11 single bond.18
However, with the exception of lamellarin S,19 all naturally
occurring lamellarins isolated so far are optically inactive. In
general, biological effects of enantiomers (optical isomers) are
not identical owing to the chiral nature of the biological
receptors.20 Recently, the importance of axial chirality has been
recognized in drug discovery.21−24 In the case of lamellarins,
however, all biological data reported so far were obtained from
optically inactive compounds. Therefore, we decided to
produce optically active lamellarins to elucidate relationships
between their chiral structures and biological activities. In this
paper, we describe the first resolution of a lamellarin class of the
compound, 16-methyllamellarin N (3), and discuss the kinase
inhibitory activities of the separated enantiomers (aS)-3a and
(aR)-3b (Figure 2). Topoisomerase I inhibitory and cytotoxic
activities of these compounds are also described.
followed by dehydrogenation. Selective deprotection of the
methoxymethyl (MOM) group of 8e with HCl/MeOH
provided partially protected 8f.
After producing O-protected lamellarins N 8a−f, we
examined the chiral resolution of these compounds by
HPLC. Although we tested a large variety of chiral stationary
phases, the resolution of these compounds was unsuccessful.
Several representative HPLC profiles are shown in Figure 3.
These chromatographic patterns indicated that the separated
enantiomers were racemized in HPLC columns.28 Thus, the
rotational energy barrier around the C1−C11 single bond of
lamellarin was assumed to be lower than initially expected.18,29
Synthesis and Resolution of 16-Methyllamellarin N.
We then attempted to resolve more sterically congested 3. For
the synthesis of 3, Suzuki−Miyaura coupling of bromide 5 with
boronic acid 11 was first examined. Although we tested a
variety of conditions, the reaction was not successful in most
cases owing to preferential formation of debrominated
compound 4. The desired cross-coupling product 12 was
obtained in low yield only under Qiu’s conditions26 (Scheme
2). Insufficient formation of 12 is apparently due to severe
steric hindrance of cross-coupling partners 5 and 11. Thus, we
designed a new synthetic route in which the cross-coupling was
conducted at an earlier stage to avoid steric hindrance (Scheme
3). The known tricyclic lactone 1325 was alkylated with tosylate
14 to produce 15. Reaction of 15 with NBS produced 16, in
which the β-position of the pyrrole ring was brominated
regioselectively.25 As expected, the cross-coupling of the less
congested bromide 16 with boronic acid 11 smoothly
proceeded to give 17 in good yield. Reaction of 17 with NBS
produced bromide 18. At this time, the pendent aromatic ring
was brominated regioselectively.25 Intramolecular Pd-catalyzed
direct arylation30 of 18 provided pentacyclic lamellarin 12 in
good yield. DDQ dehydrogenation gave 5,6-unsaturated 19.
Treatment of 19 with BCl3 gave racemic 3.
O-Protected 16-methyllamellarin N 19 was then converted
to the optically active (aS)-3a and (aR)-3b (Scheme 4). As
shown in Figure 4, racemic 19 was cleanly separated into
optically pure 19a and 19b by HPLC using a CHIRALPAK IC
semipreparative column. The CD spectra of 19a and 19b,
shown in Figure 5, clearly indicate that these compounds are
enantiomers of each other. The separated enantiomers were
thermally stable and did not racemize even upon heating at 120
°C for 24 h in toluene. Selective deprotection of the isopropyl
groups of 19a and 19b with BCl3 produced optically active
(aS)-3a and (aR)-3b, respectively.
Figure 2. Enantiomers of axially chiral 16-methyllamellarins N.
The absolute configurations of optically active 16-methyl-
lamellarins N were then determined by X-ray crystallographic
analysis. Because the anomalous dispersion effect of a heavy
atom must be used for the determination of the absolute
configuration, enantiomer 19b was converted to its bromo
derivative 21b in two steps (Scheme 5). The X-ray crystallo-
graphic structure shown in Figure 6 clearly indicates that the
absolute configuration of 21b is aS. Thus, the absolute
configurations of 19a and 3a are aS, and those of 19b and
3b are aR.
Kinase Inhibitory Activity. With enantiomerically pure
(aS)-3a and (aR)-3b isolated, we evaluated the inhibitory
pattern of these compounds on several protein kinases relevant
to cancer and neurodegenerative diseases. The kinases tested
were CDK1, CDK2, CDK5,31 GSK-3α/β,32 PIM1,33,34
DYRK1A,35−38 CLK3,39,40 and CK1.41 The IC50 values of
2,17 (aS)-3a, and (aR)-3b for these kinases are summarized in
RESULTS AND DISCUSSION
■
Synthesis and Attempted Resolution of O-Protected
Lamellarins N. We first attempted to prepare enantiomerically
pure lamellarin N isomers via HPLC resolution using a chiral
stationary phase. Since 2 is insoluble in most chromatographic
solvents, we prepared different types of O-protected derivatives
8a−f (Scheme 1). The known pentacyclic compound 425 was
brominated with NBS to give 5. Suzuki−Miyaura coupling of 5
with boronic acid 6 under Qiu’s conditions26 [Pd(PPh3)4 and
CsF/Ag2O] provided 7 in good yield. DDQ dehydrogenation
of 7 yielded 8a. Deprotection of the isopropyl groups of 8a with
BCl3 led to 2.27 Tri-O-protected 8b−d were prepared from 2
using appropriate reagents (8b, TBSCl/imidazole; 8c, Ac2O/
py; 8d, Boc2O/DMAP). Differentially protected 8e was also
synthesized from 5 by a cross-coupling with pinacol borate 9
B
dx.doi.org/10.1021/jm400719y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of 8a−f
a
Reagents and conditions: (a) NBS, DMF, 0 °C, 89%; (b) 6, Pd(PPh3)4, CsF, Ag2O, DME, 85 °C, 80%; (c) DDQ, toluene, reflux, 97%; (d) BCl3,
DCM, −78 to 0 °C, 97%; (e) TBSCl, imidazole, DMF, 0 °C to rt, 73%; (f) Ac2O, py, 0 °C to rt, 71%; (g) Boc2O, DMAP, TEA, MeCN, rt, 91%; (h)
9, Pd(PPh3)4, CsF, Ag2O, DME, 85 °C, 89%; (i) DDQ, DCM, reflux, 94%; (j) HCl, DCM−MeOH, 30 °C, 93%.
Figure 3. Selected HPLC profiles of 8a−f on chiral stationary phase columns.
a
DYRK1A. CDKs, CLK3, and CK1 were not inhibited with
this enantiomer (IC50 > 10 μM). The dramatic change in the
inhibitory pattern caused by the axial chirality of 3 is quite
striking. The selectivity observed for (aS)-3a is especially
interesting from an application viewpoint.
Scheme 2. Suzuki−Miyaura Coupling of 5 with 11
Docking Studies. The results described above indicate that
CDKs and CLK3 can discriminate the chiral structures of (aS)-
3a and (aR)-3b, whereas GSK-3α/β, PIM1, and DYRK1A
cannot. To rationalize these observations, docking studies were
performed. We selected CDK2 as the kinase that can
discriminate (aS)-3a and (aR)-3b, and we selected GSK-3β as
the kinase that cannot. For the docking simulations, CDK2/
staurosporine42 and GSK-3β/staurosporine43 cocrystal struc-
ture data were employed because staurosporine and lamellarins
share common structural features of planar and fused
polyaromatic frameworks.44 Thus, the ligand in the ATP-
a
Reagents and conditions: (a) 11, Pd(PPh3)4, CsF, Ag2O, DME, 85
°C, 32%.
Table 1. (aR)-Isomer 3b strongly inhibited all protein kinases
except CK1. This profile of potent and nonselective inhibition
is similar to that of the parental 2. On the other hand, (aS)-
isomer 3a selectively inhibited GSK-3α/β, PIM1, and
C
dx.doi.org/10.1021/jm400719y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 3. Synthesis of Racemic 3
Figure 4. HPLC profile of racemic 19 on a Chiralpac IC column.
Figure 5. CD spectra of enantiomers 19a and 19b.
a
Scheme 5. Synthesis of Bromide 21b
a
Reagents and conditions: (a) 14, Cs2CO3, DMF, rt, 98%; (b) NBS,
DMF, 0 °C to rt, 93%; (c) 11, Pd(PPh3)4, CsF, Ag2O, DME, 85 °C,
80%; (d) NBS, THF, 0 °C to rt, 86%; (e) Pd(PPh3)4, K2CO3, DMA,
125 °C, 87%; (f) DDQ, DCM, reflux, 99%; (g) BCl3, DCM, −78 to 0
°C, 75%.
Scheme 4. Synthesis of Optically Active (aS)-3a and (aR)-3b
a
by HPLC Resolution of 19
a
Reagents and conditions: (a) HCl, DCM−MeOH, 30 °C, 92%; (b)
NBS, DMF, 0 °C to rt, 92%.
Figure 6. X-ray crystal structure of 21b.
a
Reagents and conditions: (a) BCl3, DCM, −78 to 0 °C, (aS)-3a 99%,
(aR)-3b 72%.
complexes were minimized using the MOE program.45 The
models receiving the highest score are depicted in Figure 7
(CDK2 complexes) and Figure 8 (GSK-3β complexes).
binding pocket of the staurosporine complexes was replaced by
docking with 2, (aS)-3a, and (aR)-3b, and the resulting
D
dx.doi.org/10.1021/jm400719y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
pentacyclic core is apparently less stable as shown by the
scoring data (GBVI/WSA dG,48 22A, −9.65 kcal/mol; 22B,
−9.78 kcal/mol; 22C, −9.19 kcal/mol). Isomer (aS)-3a may
not be able to take a normal and stable orientation of the
pentacyclic core in the ATP-binding pocket because of severe
steric repulsion between the 16-methyl group and the rigid β-
sheet of the N-terminal lobe. These analyses clearly show why
CDK2 discriminates between the axially chiral structures of 16-
methyllamellarin N and allows us to present a rational
explanation for the much lower activity of the (aS)-3a isomer
over the (aR)-3b isomer.
The inability of GSK-3β to discriminate between the chiral
structures of (aS)-3a and (aR)-3b is also clearly explained by
comparing the docking models. The favorable orientation of 2
in the ATP-binding pocket of GSK-3β is similar to the
orientation in that of CDK2. The A-ring is directed to the
solvent channel, whereas the E-ring is directed to the specificity
pocket. The B-ring carbonyl is hydrogen-bonded to the NH of
Val135. The phenolic OH at C8 is also hydrogen-bonded to the
salt bridge Asp200. Differing from the CDK2 complexes, the
pentacyclic lamellarin cores of (aR)-3b and (aS)-3a in GSK-3β
complexes 23B and 23C adopt an orientation similar to that
observed in the parental complex 23A. The scoring data
assessed for 23B and 23C are very similar to each other,
suggesting that the bulky 16-methyl group can direct upward or
downward without any energetic preferences probably because
of the larger capacity of the ATP-binding pocket of GSK-3β
compared with that of CDK2. The similar inhibitory activity of
(aS)-3a and (aR)-3b on GSK-3β can thus be explained by their
similar binding abilities to the kinase target.
Table 1. Kinase Inhibitory Activities of 2, (aR)-3b, and (aS)-
3a
IC50 (μM)
a
protein kinase
2
(aR)-3b
(aS)-3a
CDK1/cyclin B
CDK2/cyclin A
CDK5/p25
GSK-3α/β
PIM1
0.070
0.052
0.067
0.024
0.21
>10
>10
≥10
0.37
0.22
0.44
>10
>10
0.025
0.005
0.055
0.035
0.13
DYRK1A
CLK3
0.042
0.15
CK1
>10
>10
a
Data from ref 17.
In CDK2−lamellarin N complex 22A, the pentacyclic and
planar lamellarin core is bound in the cleft between the N- and
C-terminal lobes. The A ring is directed toward the solvent
channel, and the E-ring is oriented to the specificity pocket.46
The B-ring carbonyl forms a hydrogen bond with the NH of
Leu83 located in the hinge region. The phenolic OH at C8
forms a hydrogen bond with the side chain of Asp145 located
in the catalytic salt bridge. Additionally, the OH at C20 and the
OMe at C21 chelatively interact with the side chain of Lys89.
The phenolic OH at C13 of the F-ring is directed upward to
Glu12 in the P-loop. There might be a favorable hydrogen-
bonding interaction.47
The binding mode obtained for CDK2-(aR)-16-methylla-
mellarin N complex 22B is quite similar to the parental 22A.
The bulky methyl group on the F-ring is appropriately
accommodated in the hydrophobic cavity existing in the C-
terminal lobe. Alternatively, in CDK2−(aS)-16-methyllamellar-
in N complex 22C, the pentacyclic lamellarin core is rotated
180° compared to 22A or 22B. This unusual orientation of the
Topoisomerase I Inhibitory Activity. Compound 1 has
been shown to be a potent inhibitor of topoisomerase I.13,14
Therefore, we compared the topoisomerase I inhibitory
activities of 1, 2, and chiral (aS)-3a and (aR)-3b using a
Figure 7. Highest score models obtained by docking of 2, (aR)-3b, and (aS)-3a into ATP-binding pocket of CDK2.
E
dx.doi.org/10.1021/jm400719y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 8. Highest score models obtained by docking of 2, (aR)-3b, and (aS)-3a into ATP-binding pocket of GSK-3β.
DNA relaxation assay.49 The experiments were performed
using three different concentrations of lamellarins (2, 10, and
50 μM) (Figure 9). CPT was used as the reference compound
at the same concentrations. The strong bands of nicked DNA
observed at 50 μM indicate that the topoisomerase I inhibitory
activity of 1 is as potent as that of CPT. On the other hand, the
activity of 2 is somewhat less potent than that of 1. Both
enantiomers of (aS)-3a and (aR)-3b are completely inactive
even at 50 μM. This interesting result may be accounted by
considering the unfavorable steric interactions between the 16-
methyl group of 3a or 3b and the base pairs of DNA,
preventing the formation of a stable 3a (or 3b)−DNA−
topoisomerase I ternary complex.14
Cytotoxicity. Finally, we compared the cytotoxicity of 2 and
the chiral (aS)-3a and (aR)-3b on three different cancer cell
lines (HeLa, SH-SY5Y, and IMR32). The results are shown in
Table 2. The order of cyctotoxity on these cell lines is 2 > (aR)-
Table 2. Cell Survival/Proliferation Inhibitory Activities of
2, (aR)-3b, and (aS)-3a
IC50 (μM)
cell line
2
(aR)-3b
(aS)-3a
a
HeLa
0.032
0.040
0.160
0.79
0.41
2.95
4.1
b
SH-SY5Y
b
IMR32
0.019
2.0
a
b
Colony formation assay. MTS assay.
3b > (aS)-3a. The lower cytotoxicity of (aS)-3a and (aR)-3b
may be considered by the lack of their topoisomerase I
inhibitory activity. The decreased cytotoxicity of (aS)-3a
compared to (aR)-3b is likely due to the lack of CDK
inhibitory activity of (aS)-3a.
CONCLUSION
■
In this paper, we report the first successful synthesis and
separation of axially chiral lamellarins, (aS)-3a and (aR)-3b, and
also report the biological activities of the two enantiomers. The
kinase inhibitory action of these enantiomers is quite different.
Isomer (aR)-3b showed potent but nonselective inhibition of
CDK1, CDK2, CDK5, GSK-3α/β, PIM1, DYRK1A, and CLK3,
whereas (aS)-3a exhibited selective inhibition of GSK-3α/β,
Figure 9. Topoisomerase I inhibition assay of lamellarins 1, 2, (aS)-3a,
and (aR)-3b.
F
dx.doi.org/10.1021/jm400719y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
ethyl acetate = 20:1) and column chromatography over Chromatorex
NH-DM1020 (DCM−ethyl acetate = 20:1) to give 6 as a colorless
solid (78.4 mg, 80%). Physical and spectroscopic data of this
compound have been reported previously.27
3,11-Diisopropoxy-14-(3-isopropoxy-4-methoxyphenyl)-
2,12-dimethoxy-6H-[1]benzopyrano[4′,3′:4,5]pyrrolo[2,1-a]-
isoquinolin-6-one (8a). Under an argon atmosphere, a mixture of 7
(78.4 mg, 0.125 mmol) and DDQ (42.5 mg, 0.187 mmol) in toluene
(8.0 mL) was refluxed for 18 h. After cooling, the mixture was
evaporated. The residue was purified by column chromatography over
Chromatorex NH-DM1020 (hexane−ethyl acetate = 1:1) to give 8a as
a colorless solid (75.6 mg, 97%). Physical and spectroscopic data of
this compound have been reported previously.27
3,11-Dihydroxy-14-(3-hydroxy-4-methoxyphenyl)-2,12-di-
methoxy-6H-[1]benzopyrano-[4′,3′:4,5]pyrrolo[2,1-a]-
isoquinolin-6-one (Lamellarin N) (2). Under an argon atmosphere,
a heptane solution of BCl3 (1.0 M, 719 μL, 0.719 mmol) was added
dropwise to a solution of 8a (100 mg, 0.160 mmol) in DCM (5.0 mL)
at −78 °C. After being stirred for 30 min at this temperature, the
reaction mixture was allowed to warm to room temperature and stirred
for an additional 3 h. The mixture was quenched with saturated
aqueous NaHCO3 and extracted with ethyl acetate. The extract was
washed with brine, dried over Na2SO4, and evaporated. The residue
was purified by column chromatography over silica gel 60N (acetone−
methanol = 10:1) to give 2 as a pale gray powder (77.4 mg, 97%).
Physical and spectroscopic data of this compound have been reported
previously.27
PIM1, and DYRK1A. These differences are rationalized by
docking studies. Although parental 2 inhibited topoisomerase I,
(aS)-3a and (aR)-3b were inactive on this enzyme. Thus, both
(aS)-3a and (aR)-3b are the kinase-selective inhibitors. The
different activities of 2, (aS)-3a, and (aR)-3b at the molecular
level were reflected in their phenotypic cytotoxicity. The results
obtained in this study open a new avenue in the chemical and
pharmacological use of the lamellarin scaffold. Mastering the
chirality of the C1−C11 single bond allows access to a
subfamily of lamellarins. Lamellarins have been reported as
potent kinase inhibitors;17 however, they were rather broad
spectrum inhibitors in terms of selectivity, and their
pharmaceutical use was therefore limited. Starting from the
lamellarin (aS)-3a isomer, we can envisage improving the
potency of analogues toward the DYRK1A, GSK-3, and PIM1
kinases, all of which represent promising therapeutic targets for
potent drugs to treat neurodegenerative diseases like
Alzheimer’s. The selection of the lamellarin (aS)-3a isomer
removes inhibitory activities toward the cell cycle regulating
CDKs and therefore associated antiproliferative and proapop-
totic properties. This is an obvious advantage for the
development of antineurodegenerative molecules. Studies on
this new lamellarin scaffold are in progress in our laboratories.
3,11-Bis(tert-butyldimethylsilyloxy)-14-[3-(tert-butyldime-
thylsilyloxy)-4-methoxyphenyl]-2,12-dimethoxy-6H-[1]-
benzopyrano[4′,3′:4,5]pyrrolo[2,1-a]isoquinolin-6-one (8b).
To a mixture of 2 (30.0 mg, 0.0601 mmol) and TBSCl (40.7 mg,
0.270 mmol) in DMF (0.5 mL) was added imidazole (40.9 mg, 0.601
mmol) at 0 °C. After being stirred for 10 min at 0 °C, the mixture was
allowed to warm to room temperature, stirred for 15 h at room
temperature, and quenched with water. The product was extracted
with DCM, and the extract was washed with water and brine, dried
over Na2SO4, and evaporated. The residue was purified successively by
column chromatography over silica gel 60N (DCM) and column
chromatography over silica gel 60N (hexane−ethyl acetate = 10:1) to
give 8b as a pale yellow solid (36.8 mg, 73%). Recrystallization from
diethyl ether−hexane gave a colorless powder. Mp 241−242.5 °C. IR
EXPERIMENTAL SECTION
■
Synthesis: General. Melting points are uncorrected. IR spectra are
reported in terms of wavenumber of absorption (cm−1). H NMR
1
spectra were recorded at 400 MHz and are reported relative to Me4Si
1
(δ 0.0). Data for H NMR spectra are reported as follows: chemical
shift (δ ppm), multiplicity, coupling constant (Hz), and integration.
13C NMR spectra were recorded at 100 MHz and are reported relative
to Me4Si (δ 0.0). Data for 13C NMR spectra are reported in terms of
chemical shift. High resolution mass spectra were measured by the EI
or FAB method. Elemental analysis was performed for C, H, and N.
Column chromatography was conducted on silica gel 60N, 63−210
μm, or Chromatorex NH-DM1020. Solvents were dried and distilled
by standard methods if necessary.
1
(KBr): 1710, 1432, 1283, 1218, 1157 cm−1. H NMR (400 MHz,
14-Bromo-3,11-diisopropoxy-2,12-dimethoxy-8,9-dihydro-
6H-[1]benzopyrano[4′,3′:4,5]pyrrolo[2,1-a]isoquinolin-6-one
(5). A solution of NBS (151 mg, 0.848 mmol) in DMF (13 mL) was
added dropwise to a solution of 425 (382 mg, 0.824 mmol) in DMF
(10 mL) at 0 °C. The mixture was stirred for 24 h at 0 °C. The
solution was diluted with water, and the product was extracted with
DCM. The extract was washed with water and brine, dried over
Na2SO4, and evaporated. The residue was purified by column
chromatography over silica gel 60N (DCM−ethyl acetate = 20:1) to
give 4 as a pale yellow solid (398 mg, 89%). Recrystallization from
DCM−hexane gave a colorless powder. Mp 191.5−192.5 °C. IR
CDCl3): δ 0.15 (s, 3H), 0.16 (s, 3H), 0.17 (s, 3H), 0.18 (s, 9H), 0.97
(s, 9H), 0.99 (s, 9H), 1.01 (s, 9H), 3.43 (s, 3H), 3.44 (s, 3H), 3.91 (s,
3H), 6.76 (s, 1H), 6.95 (s, 1H), 6.98 (d, J = 7.3 Hz, 1H), 7.10 (d, J =
2.0 Hz, 1H), 7.12 (s, 1H), 7.13 (d, J = 8.2 Hz, 1H), 7.18 (s, 1H), 7.20
(dd, J = 2.0 and 8.2 Hz, 1H), 9.21 (d, J = 7.3 Hz, 1H). 13C NMR (100
MHz, CDCl3): δ −4.6, −4.6, −4.6, −4.6, −4.6, −4.5, 18.5, 18.5, 18.5,
25.7, 25.7, 54.8, 55.3, 56.0, 106.0, 106.1, 108.0, 109.6, 111.1, 111.4,
112.2, 113.1, 117.4, 120.1, 123.0, 124.1, 124.8, 124.9, 128.6, 129.3,
134.4, 145.8, 146.2, 146.4, 146.5, 147.8, 151.1, 151.3, 155.6. HREIMS
m/z calcd for C46H64NO8Si3 [(M + H)+]: 842.3940. Found: 842.3935.
3,11-Bis(acetoxy)-14-(3-acetoxy-4-methoxyphenyl)-2,12-di-
methoxy-6H-[1]benzopyrano[4′,3′:4,5]pyrrolo[2,1-a]-
isoquinolin-6-one (8c). To a solution of 2 (29.6 mg, 0.0593 mmol)
in pyridine (6.0 mL) was added acetic anhydride (25.2 μL, 0.267
mmol) as a neat liquid at 0 °C. The mixture was allowed to warm to
room temperature, stirred for 14.5 h at room temperature, and
quenched with water. The product was extracted with DCM, and the
extract was washed with 2 M aqueous HCl, water, and brine, dried
over Na2SO4, and evaporated. The residue was purified by column
chromatography over silica gel 60N (hexane−ethyl acetate = 3:1) to
give 8c as a pale yellow solid (26.2 mg, 71%). Recrystallization from
DCM−hexane gave a colorless powder. Mp 289−291 °C. IR (KBr):
(KBr): 1707, 1420, 1210, 1163, 1040 cm−1. H NMR (400 MHz,
1
CDCl3): δ 1.43 (d, J = 6.1 Hz, 12H), 3.04 (t, J = 6.5 Hz, 2H), 3.95 (s,
3H), 3.96 (s, 3H), 4.58 (sep, J = 6.1 Hz, 1H), 4.63 (sep, J = 6.1 Hz,
1H), 4.71−4.78 (m, 2H), 6.83 (s, 1H), 6.91 (s, 1H), 8.14 (s, 1H), 8.20
(s, 1H). 13C NMR (100 MHz, CDCl3): δ 21.8, 22.1, 28.9, 42.6, 56.3,
56.3, 71.4, 86.6, 103.3, 104.8, 109.5, 109.7, 114.1, 114.7, 119.2, 127.1,
127.3, 135.3, 146.0, 146.6, 147.7, 148.1, 148.8, 154.8. Anal. Calcd for
C27H28BrNO6: C, 59.79; H, 5.20; N, 2.58. Found: C, 59.93; H, 5.09;
N, 2.37.
3,11-Diisopropoxy-14-(3-isopropoxy-4-methoxyphenyl)-
2,12-dimethoxy-8,9-dihydro-6H-[1]benzopyrano[4′,3′:4,5]-
pyrrolo[2,1-a]isoquinolin-6-one (7). Under an argon atmosphere,
a mixture of 4 (84.6 mg, 0.156 mmol), 6 (59.3 mg, 0.282 mmol), CsF
(57.2 mg, 0.377 mmol), Ag2O (52.3 mg, 0.226 mmol), and Pd(PPh3)4
(21.7 mg, 18.8 μmol) in DME (6 mL) was heated at 85 °C for 24 h.
After cooling, the mixture was diluted with water and the product was
extracted with DCM. The extract was washed with water and brine,
dried over Na2SO4, and evaporated. The residue was purified
successively by column chromatography over silica gel 60N (DCM−
1
1770, 1709, 1187, 1149, 1035 cm−1. H NMR (400 MHz, CDCl3): δ
2.32 (s, 3H), 2.32 (s, 3H), 2.34 (s, 3H), 3.48 (s, 6H), 3.93 (s, 3H),
6.78 (s, 1H), 7.02 (dd, J = 2.0 and 7.4 Hz, 1H), 7.13 (d, J = 2.0 Hz,
1H), 7.20 (s, 1H), 7.23 (d, J = 8.3 Hz, 1H), 7.33 (d, J = 2.1 Hz, 1H),
7.37 (s, 1H), 7.44 (dd, J = 2.1 and 8.3 Hz, 1H), 9.20 (dd, J = 2.0 and
7.4 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 20.5, 20.6, 20.6, 55.4,
55.6, 56.4, 106.2, 106.4, 109.0, 111.7, 112.1, 112.8, 113.2, 115.8, 120.7,
G
dx.doi.org/10.1021/jm400719y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
123.1, 123.8, 123.8, 125.9, 127.8, 128.5, 130.1, 133.8, 139.7, 140.9,
141.0, 145.5, 147.7, 151.0, 151.8, 155.1, 168.5, 168.7, 168.9. HREIMS
m/z calcd for C34H28NO11 [(M + H)+]: 626.1662. Found: 626.1664.
3,11-Bis(tert-butoxycarbonyloxy)-14-[3-(tert-butoxycarbo-
nyloxy)-4-methoxyphenyl]-2,12-dimethoxy-6H-[1]-
benzopyrano[4′,3′:4,5]pyrrolo[2,1-a]isoquinolin-6-one (8d).
To a mixture of 2 (30.0 mg, 0.0601 mmol), DMAP (6.0 mg, 0.0491
mmol), and TEA (37.6 μL, 0.270 mmol) in MeCN (3.0 mL) was
added Boc2O (59.0 mg, 0.270 mmol) as a neat liquid. After being
stirred for 17 h, the mixture was evaporated. The residue was purified
by column chromatography over silica gel 60N (hexane−ethyl acetate
= 1:1) to give 8d as a pale yellow solid (43.6 mg, 91%).
Recrystallization from DCM−hexane gave a colorless powder. Mp
(0.8 mL). After being stirred for 14 h at 30 °C, the mixture was diluted
with water and evaporated. The product was extracted with DCM, and
the extract was washed with water and brine, dried over Na2SO4, and
evaporated. The residue was purified by column chromatography over
silica gel 60N (hexane−ethyl acetate = 1:1) to give 8f as a colorless
solid (70.8 mg, 93%). Recrystallization from DCM−diethyl ether gave
a colorless powder. Mp 241−242 °C (sealed capillary). IR (KBr):
1701, 1429, 1265, 1038 cm−1. 1H NMR (400 MHz, CDCl3): δ 1.40 (d,
J = 6.1 Hz, 6H), 1.43 (d, J = 6.1 Hz, 6H), 3.46 (s, 3H), 3.48 (s, 3H),
4.00 (s, 3H), 4.57 (sep, J = 6.1 Hz, 1H), 4.69 (sep, J = 6.1 Hz, 1H),
5.91 (br s, 1H), 6.78 (s, 1H), 6.95 (s, 1H), 7.00 (d, J = 7.4 Hz, 1H),
7.08 (s, 1H), 7.10 (s, 1H), 7.11 (s, 1H), 7.19 (s, 1H), 7.21 (s, 1H),
9.20 (d, J = 7.4 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 21.8, 21.8,
21.9, 21.9, 55.1, 55.5, 56.4, 71.2, 71.5, 98.4, 103.4, 105.7, 105.8, 107.8,
110.0, 110.4, 110.9, 111.4, 112.3, 117.9, 119.0, 123.2, 123.5, 124.7,
129.0, 129.4, 134.4, 146.5, 146.5, 146.6, 146.6, 147.8, 148.4, 150.1,
155.6. HRFABMS m/z calcd for C34H33NO8 (M+): 583.2206. Found:
583.2206.
3,11-Diisopropoxy-2,12-dimethoxy-14-[4-methoxy-5-(me-
thoxymethoxy)-2-methylphenyl]-8,9-dihydro-6H-[1]-
benzopyrano[4′,3′:4,5]pyrrolo[2,1-a]isoquinolin-6-one (12).
According to the procedure described for the preparation of 7,
compound 5 (30.0 mg, 55.3 μmol), compound 11 (18.8 mg, 83.0
μmol), CsF (16.8 mg, 0.111 mmol), Ag2O (15.4 mg, 66.4 μmol), and
Pd(PPh3)4 (6.4 mg, 5.5 μmol) were reacted in DME (2.0 mL). After
chromatographic purification over silica gel 60N (DCM−ethyl acetate
= 20:1), 12 was obtained as a colorless solid (11.4 mg, 32%).
Recrystallization from ethyl acetate gave a colorless powder. Mp 187−
188 °C. IR (KBr): 1709, 1417, 1270, 1216, 1161 cm−1. 1H NMR (400
MHz, CDCl3): δ 1.38 (d, J = 6.1 Hz, 6H), 1.39 (d, J = 6.1 Hz, 6H),
2.07 (s, 3H), 3.11 (t, J = 6.8 Hz, 2H), 3.35 (s, 3H), 3.43 (s, 3H), 3.43
(s, 3H), 3.94 (s, 3H), 4.54 (sep, J = 6.1 Hz, 1H), 4.56 (sep, J = 6.1 Hz,
1H), 4.81 (t, J = 6.8 Hz, 2H), 5.15 (d, J = 6.7 Hz, 1H), 5.17 (d, J = 6.7
Hz, 1H), 6.53 (s, 1H), 6.67 (s, 1H), 6.77 (s, 1H), 6.92 (s, 1H), 6.97 (s,
1H), 7.21 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 19.9, 21.8, 21.9,
22.1, 22.1, 28.6, 42.5, 55.1, 55.5, 56.1, 56.3, 71.3, 71.4, 95.8, 103.3,
104.4, 108.4, 110.6, 113.4, 113.9, 114.0, 114.6, 119.8, 120.4, 126.1,
126.9, 128.3, 132.9, 135.8, 145.1, 145.9, 146.7, 147.0, 147.3, 148.8,
149.9, 155.7. Anal. Calcd for C37H41NO9: C, 69.04; H, 6.42; N, 2.18.
Found: C, 69.08; H, 6.42; N, 2.13.
1
197.5−198.5 °C. IR (KBr): 1760, 1710, 1255, 1141 cm−1. H NMR
(400 MHz, CDCl3): δ 1.55 (s, 9H), 1.55 (s, 18H), 3.50 (s, 6H), 3.96
(s, 3H), 6.79 (s, 1H), 7.05 (d, J = 7.4 Hz, 1H), 7.21 (s, 1H), 7.23 (d, J
= 8.3 Hz, 1H), 7.23 (s, 1H), 7.41 (dd, J = 2.1 and 8.3 Hz, 1H), 7.44 (d,
J = 2.1 Hz, 1H), 7.47 (s, 1H), 9.23 (d, J = 7.4 Hz, 1H). 13C NMR (100
MHz, CDCl3): δ 27.6, 55.5, 55.8, 56.5, 83.7, 83.9, 84.0, 106.3, 106.5,
109.0, 111.7, 111.9, 112.8, 113.5, 115.7, 120.3, 123.2, 123.7, 123.8,
125.6, 127.8, 128.5, 130.1, 133.8, 140.1, 141.1, 141.4, 145.4, 147.9,
151.0, 151.1, 151.1, 151.9, 155.1. HREIMS m/z calcd for C43H46NO14
[(M + H)+]: 800.2918. Found: 800.2938.
3,11-Diisopropoxy-2,12-dimethoxy-14-[4-methoxy-3-
(methoxymethoxy)phenyl]-8,9-dihydro-6H-[1]benzopyrano-
[4′,3′:4,5]pyrrolo[2,1-a]isoquinolin-6-one (10). According to the
procedure described for the preparation of 7, compound 5 (30.0 mg,
55.3 μmol), compound 9 (24.4 mg, 83.0 μmol), CsF (16.8 mg, 0.111
mmol), Ag2O (15.4 mg, 66.4 μmol), and Pd(PPh3)4 (6.4 mg, 5.5
μmol) were reacted in DME (2.0 mL). After chromatographic
purification over silica gel 60N (DCM−ethyl acetate = 20:1), 10 was
obtained as a colorless solid (30.9 mg, 89%). Recrystallization from
DCM−hexane gave a colorless powder. Mp 220.5−221.5 °C. IR
1
(KBr): 1708, 1485, 1417, 1267, 1176 cm−1. H NMR (400 MHz,
CDCl3): δ 1.37 (d, J = 6.1 Hz, 6H), 1.38 (d, J = 6.1 Hz, 6H), 3.09 (t, J
= 6.8 Hz, 2H), 3.35 (s, 3H), 3.45 (s, 6H), 3.95 (s, 3H), 4.53 (sep, J =
6.1 Hz, 1H), 4.56 (sep, J = 6.1 Hz, 1H), 4.69−4.78 (m, 1H), 4.79−
4.88 (m, 1H), 5.21 (d, J = 6.8 Hz, 1H), 5.23 (d, J = 6.8 Hz, 1H), 6.65
(s, 1H), 6.69 (s, 1H), 6.77 (s, 1H), 6.91 (s, 1H), 7.09 (d, J = 8.3 Hz,
1H), 7.16 (dd, J = 2.0 and 8.3 Hz, 1H), 7.33 (d, J = 2.0 Hz, 1H). 13C
NMR (100 MHz, CDCl3): δ 21.8, 22.1, 28.7, 42.4, 55.1, 55.5, 56.2,
56.3, 71.3, 71.4, 95.6, 103.5, 105.0, 109.2, 110.4, 112.5, 113.7, 114.5,
114.7, 119.5, 120.2, 125.4, 126.4, 128.2, 128.3, 136.1, 146.0, 146.5,
147.0, 147.1, 147.3, 148.6, 149.7, 155.7. Anal. Calcd for C36H39NO9:
C, 68.67; H, 6.24; N, 2.22. Found: C, 68.41; H, 6.45; N, 2.14.
3,11-Diisopropoxy-2,12-dimethoxy-14-[4-methoxy-3-
(methoxymethoxy)phenyl]-6H-[1]benzopyrano[4′,3′:4,5]-
pyrrolo[2,1-a]isoquinolin-6-one (8e). Under an argon atmosphere,
a mixture of of 10 (104 mg, 0.165 mmol) and DDQ (56.2 mg, 0.248
mmol) in DCM (7.0 mL) was refluxed for 24 h. After cooling, the
mixture was evaporated. The residue was purified by column
chromatography over Chromatorex NH-DM1020 (hexane−ethyl
acetate = 1:1) to give 8e as a colorless solid (97.5 mg, 94%).
Recrystallization from DCM−hexane gave a colorless powder. Mp
200−201 °C. IR (KBr): 1700, 1432, 1423, 1264 cm−1. 1H NMR (400
MHz, CDCl3): δ 1.40 (d, J = 6.1 Hz, 6H), 1.43 (d, J = 6.1 Hz, 6H),
3.46 (s, 3H), 3.46 (s, 3H), 3.47 (s, 3H), 3.99 (s, 3H), 4.57 (sep, J = 6.1
Hz, 1H), 4.70 (sep, J = 6.1 Hz, 1H), 5.23 (d, J = 6.8 Hz, 1H), 5.25 (d, J
= 6.8 Hz, 1H), 6.72 (s, 1H), 6.96 (s, 1H), 7.01 (d, J = 7.4 Hz, 1H),
7.09 (s, 1H), 7.15 (s, 1H), 7.17 (d, J = 8.2 Hz, 1H), 7.27 (dd, J = 2.0
and 8.2 Hz, 1H), 7.41 (d, J = 2.0 Hz, 1H), 9.21 (d, J = 7.4 Hz, 1H).
13C NMR (100 MHz, CDCl3): δ 21.8, 21.8, 21.9, 21.9, 55.1, 55.5, 56.3,
2-(3-Isopropoxy-4-methoxyphenyl)ethyl p-Toluenesulfo-
nate (14). Under an argon atmosphere, p-toluenesulfonyl chloride
(1.09 g, 5.71 mmol) in DCM (30 mL) was added dropwise to a
solution of 2-(4-isopropoxy-3-methoxyphenyl)ethanol25 (1.00 g, 4.76
mmol), DMAP (58.1 mg, 0.476 mmol), and triethylamine (729 μL,
5.23 mmol) in DCM (30 mL) at 0 °C. The mixture was allowed to
warm to room temperature and stirred for 13 h at room temperature
and quenched with water. The product was extracted with DCM, and
the extract was washed water and brine, dried over Na2SO4, and
evaporated. The residue was purified by column chromatography over
silica gel 60N (hexane−ethyl acetate = 3:1) to give 14 as a colorless oil
1
(1.47 g, 85%). IR (KBr): 1515, 1357, 1262, 1177, 1139 cm−1. H
NMR (400 MHz, CDCl3): δ 1.33 (d, J = 6.1 Hz, 6H), 2.43 (s, 3H),
2.87 (t, J = 7.1 Hz, 2H), 3.82 (s, 3H), 4.17 (t, J = 7.1 Hz, 2H), 4.45
(sep, J = 6.1 Hz, 1H), 6.65 (dd, J = 2.1 and 8.7 Hz, 1H), 6.66 (d, J =
2.1 Hz, 1H), 6.76 (d, J = 8.7 Hz, 1H), 7.28 (d, J = 8.2 Hz, 2H), 7.69
(d, J = 8.2 Hz, 2H). 13C NMR (100 MHz, CDCl3): δ 21.6, 22.1, 34.9,
56.0, 70.9, 71.4, 112.0, 116.7, 121.5, 127.8, 128.6, 129.7, 133.0, 144.7,
147.2, 149.4. Anal. Calcd for C19H24O5S: C, 62.61; H, 6.64. Found: C,
62.63; H, 6.80.
7-Isopropoxy-3-[2-(3-isopropoxy-4-methoxyphenyl)ethyl]-
8-methoxy[1]benzopyrano[3,4-b]pyrrol-4(3H)-one (15). Under
an argon atmosphere, a mixture of 1325 (614 mg, 2.25 mmol), 14
(1.29 g, 3.37 mmol), and Cs2CO3 (2.19 g, 6.72 mmol) in DMF (20
mL) was stirred for 15 h at room temperature. The mixture was
quenched with saturated aqueous NH4Cl and diluted with water. The
product was extracted with DCM, and the extract was washed with
water and brine, dried over Na2SO4, and evaporated. The residue was
purified by column chromatography over silica gel 60N (DCM−ethyl
acetate = 20:1) to give 15 as a colorless solid (1.03 g, 98%). Physical
56.3, 71.2, 71.4, 95.7, 103.4, 105.6, 105.7, 107.8, 110.0, 110.4, 110.7,
112.3, 112.6, 119.0, 120.0, 123.2, 124.7, 125.9, 128.5, 129.5, 134.5,
146.5, 146.6, 147.3, 147.9, 148.5, 150.0, 150.2, 155.6. Anal. Calcd for
C36H37NO9: C, 68.89; H, 5.94; N, 2.23. Found: C, 68.75; H, 5.93; N,
2.22.
14-(3-Hydroxy-4-methoxyphenyl)-3,11-diisopropoxy-2,12-
dimethoxy-6H-[1]benzopyrano[4′,3′:4,5]pyrrolo[2,1-a]-
isoquinolin-6-one (8f). To a mixture of 8e (82.2 mg, 0.131 mmol),
DCM (10 mL), and methanol (5.0 mL) was added concentrated HCl
H
dx.doi.org/10.1021/jm400719y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
and spectroscopic data of this compound have been reported
previously.25
Na2SO4, and evaporated. The residue was purified by column
chromatography over silica gel 60N (toluene−ethyl acetate = 5:1)
to give 12 as a colorless solid (1.54 g, 87%).
1 - B r o m o - 7 - i s o p r o p o x y - 3 - [ 2 - ( 3 - i s o p r o p o x y - 4 -
methoxyphenyl)ethyl]-8-methoxy[1]benzopyrano[3,4-b]-
pyrrol-4(3H)-one (16). According to the previously reported
procedure,25 compound 16 was synthesized. A solution of NBS
(0.772 g, 4.34 mmol) in DMF (30 mL) was added dropwise to a
solution of 15 (2.00 g, 4.29 mmol) in DMF (90 mL) at 0 °C. The
mixture was stirred for 1 h at 0 °C and for 14 h at room temperature.
The solution was diluted with water, and the product was extracted
with DCM. The extract was washed with water and brine, dried over
Na2SO4, and evaporated. The residue was purified by column
chromatography over silica gel 60N (hexane−ethyl acetate = 3:1) to
give 16 as a colorless solid (2.18 g, 93%). Physical and spectroscopic
data of this compound have been reported previously.25
7-Isopropoxy-3-[2-(3-isopropoxy-4-methoxyphenyl)ethyl]-
8-methoxy-1-[4-methoxy-5-(methoxymethoxy)-2-
methylphenyl][1]benzopyrano[3,4-b]pyrrol-4(3H)-one (17). Ac-
cording to the procedure described for the preparation of 7,
compound 16 (2.18 g, 4.00 mmol) was reacted with arylboronic
acid 11 (1.81 g, 7.99 mmol). After chromatographic purification over
silica gel 60N (hexane−ethyl acetate = 2:1), 17 was obtained as a
colorless solid (2.07 g, 80%). Recrystallization from DCM−hexane
gave a colorless powder. Mp 81−82 °C. IR (KBr): 1712, 1512, 1258,
1232, 1154 cm−1. 1H NMR (400 MHz, CDCl3): δ 1.29 (d, J = 6.1 Hz,
3H), 1.29 (d, J = 6.1 Hz, 3H), 1.39 (d, J = 6.1 Hz, 3H), 1.40 (d, J = 6.1
Hz, 3H), 2.03 (s, 3H), 3.11 (dt, J = 3.1 and 6.8 Hz, 2H), 3.46 (s, 3H),
3.48 (s, 3H), 3.80 (s, 3H), 3.91 (s, 3H), 4.44 (sep, J = 6.1 Hz, 1H),
4.55 (sep, J = 6.1 Hz, 1H), 4.55−4.63 (m, 1H), 4.67−4.75 (m, 1H),
5.16 (d, J = 6.6 Hz, 1H), 5.19 (d, J = 6.6 Hz, 1H), 6.62 (s, 1H), 6.63
(dd, J = 2.0 and 8.2 Hz, 1H), 6.69 (s, 1H), 6.71 (d, J = 2.0 Hz, 1H),
6.75 (d, J = 8.2 Hz, 1H), 6.83 (s, 1H), 6.93 (s, 1H), 6.99 (s, 1H). 13C
NMR (100 MHz, CDCl3): δ 20.0, 21.8, 21.9, 22.0, 22.1, 37.6, 50.9,
55.7, 56.0, 56.1, 56.2, 71.4, 71.4, 95.8, 103.4, 104.5, 110.8, 112.1, 113.4,
114.6, 116.6, 117.3, 119.6, 121.5, 125.6, 128.1, 130.5, 131.8, 132.1,
144.2, 146.0, 146.8, 147.2, 147.3, 149.2, 149.6, 155.6. Anal. Calcd for
C37H43NO9: C, 68.82; H, 6.71; N, 2.17. Found: C, 68.68; H, 6.83; N,
1.99.
3,11-Diisopropoxy-2,12-dimethoxy-14-[4-methoxy-5-(me-
thoxymethoxy)-2-methylphenyl]-6H-[1]benzopyrano-
[4′,3′:4,5]pyrrolo[2,1-a]isoquinolin-6-one (19). According to the
procedure described for the preparation of compound 8e, 12 (204 mg,
0.317 mmol) and DDQ (108 mg, 0.476 mmol) were reacted for 24 h.
After chromatographic purification over Chromatorex NH-DM1020
(hexane−ethyl acetate = 2:1), 19 was obtained as a colorless solid
(201 mg, 99%). Recrystallization from DCM−diethyl ether gave a
colorless powder. Mp 195−196 °C. IR (KBr): 1702, 1423, 1267, 1209,
1179 cm−1. 1H NMR (400 MHz, CDCl3): δ 1.41 (d, J = 6.1 Hz, 6H),
1.44 (d, J = 6.1 Hz, 6H), 2.08 (s, 3H), 3.43 (s, 3H), 3.45 (s, 3H), 3.46
(s, 3H), 3.98 (s, 3H), 4.58 (sep, J = 6.1 Hz, 1H), 4.71 (sep, J = 6.1 Hz,
1H), 5.17 (d, J = 6.7 Hz, 1H), 5.19 (d, J = 6.7 Hz, 1H), 6.66 (s, 1H),
6.98 (s, 1H), 7.04 (d, J = 7.4 Hz, 1H), 7.05 (s, 1H), 7.10 (s, 1H), 7.11
(s, 1H), 7.29 (s, 1H), 9.23 (d, J = 7.4 Hz, 1H). 13C NMR (100 MHz,
CDCl3): δ 19.9, 21.8, 21.9, 21.9, 21.9, 55.2, 55.5, 56.2, 56.3, 71.1, 71.4,
95.9, 103.3, 105.0, 108.1, 109.6, 110.2, 110.3, 112.3, 114.0, 119.2,
120.3, 123.3, 124.7, 127.1, 129.3, 133.6, 134.2, 145.2, 146.6, 146.7,
147.9, 148.5, 150.1, 150.4, 155.6. Anal. Calcd for C37H39NO9: C,
69.25; H, 6.13; N, 2.18. Found: C, 68.95; H, 6.44; N, 1.94.
3,11-Dihydroxy-14-(5-hydroxy-4-methoxy-2-methylphen-
yl)-2,12-dimethoxy-6H-[1]benzopyrano[4′,3′:4,5]pyrrolo[2,1-
a]isoquinolin-6-one (3). According to the procedure described for
the preparation of 8a, compound 19 (30.0 mg, 46.8 μmol) was reacted
with BCl3 (1.0 M, 421 μL, 0.421 mmol). After chromatographic
purification over silica gel 60N (ethyl acetate), 3 was obtained as a pale
gray powder (17.9 mg, 75%). Mp 292−294 °C (sealed capillary). IR
1
(KBr): 3377, 1679, 1433, 1275, 1148 cm−1. H NMR (400 MHz,
DMSO-d6): δ 1.99 (s, 3H), 3.40 (s, 3H), 3.41 (s, 3H), 3.88 (s, 3H),
6.67 (s, 1H), 6.89 (s, 1H), 6.90 (s, 1H), 7.10 (s, 1H), 7.18 (s, 1H),
7.22 (s, 1H), 7.24 (d, J = 7.4 Hz, 1H), 9.02 (d, J = 7.4 Hz, 1H), 9.14
(br s, 1H), 9.92 (br s, 2H). 13C NMR (100 MHz, DMSO-d6): δ 19.0,
54.5, 55.0, 56.0, 103.6, 104.5, 105.0, 106.6, 108.3, 109.1, 111.4, 112.3,
114.8, 117.6, 118.1, 122.1, 124.5, 125.9, 128.5, 128.6, 133.6, 144.7,
145.5, 146.2, 147.8, 147.9, 148.3, 148.7, 154.3. HRFABMS m/z calcd
for C29H23NO8 (M+): 513.1424. Found: 513.1423.
Resolution of 19. The resolution was performed using HPLC with
a Daicel semipreparative CHIRALPAK IC column (10 mm i.d. × 250
mm). The eluent was composed of n-hexane−DCM−ethanol
(60:40:1, v/v/v). Flow rate was set at 2.0 mL/min, and detection
wavelength was fixed at 305 nm. Racemate 19 (33.3 mg) was treated
to give (aS)-19a (15.6 mg) and (aR)-19b (14.9 mg).
3-[2-(2-Bromo-5-isopropoxy-4-methoxyphenyl)ethyl]-7-iso-
propoxy-8-methoxy-1-[4-methoxy-5-(methoxymethoxy)-2-
methylphenyl][1]benzopyrano[3,4-b]pyrrol-4(3H)-one (18). A
solution of NBS (0.577 g, 3.21 mmol) in THF (100 mL) was added
dropwise to a solution of 17 (2.07 g, 3.21 mmol) in THF (100 mL) at
0 °C. The mixture was stirred for 15 h at 0 °C and for 24 h at room
temperature. The solution was diluted with water, and the product was
extracted with DCM. The extract was washed with water and brine,
dried over Na2SO4, and evaporated. The residue was purified by
column chromatography over silica gel 60N (hexane−ethyl acetate =
3:1) to give 18 as a colorless solid (2.00 g, 86%). Recrystallization
from DCM−hexane gave a colorless powder. Mp 82.5−83.5 °C. IR
(aS)-3,11-Diisopropoxy-2,12-dimethoxy-14-[4-methoxy-5-(methoxyme-
thoxy)-2-methylphenyl]-6H-[1]benzopyrano[4′,3′:4,5]pyrrolo[2,1-a]-
20
isoquinolin-6-one [(aS)-19a]. >99% ee. Mp 106−107.5 °C. [α]D
−7.15 (c 0.695, CHCl3).
(aR)-3,11-Diisopropoxy-2,12-dimethoxy-14-[4-methoxy-5-(methoxy-
methoxy)-2-methylphenyl]-6H-[1]benzopyrano[4′,3′:4,5]pyrrolo[2,1-a]-
1
(KBr): 1702, 1498, 1252, 1217, 1160 cm−1. H NMR (400 MHz,
20
CDCl3): δ 1.16 (d, J = 6.1 Hz, 3H), 1.19 (d, J = 6.1 Hz, 3H), 1.39 (d, J
= 6.1 Hz, 3H), 1.40 (d, J = 6.1 Hz, 3H), 2.01 (s, 3H), 3.18−3.34 (m,
2H), 3.46 (s, 3H), 3.48 (s, 3H), 3.80 (s, 3H), 3.91 (s, 3H), 4.28 (sep, J
= 6.1 Hz, 1H), 4.55 (sep, J = 6.1 Hz, 1H), 4.63−4.76 (m, 2H), 5.17 (d,
J = 6.7 Hz, 1H), 5.19 (d, J = 6.7 Hz, 1H), 6.58 (s, 1H), 6.60 (s, 1H),
6.72 (s, 1H), 6.83 (s, 1H), 6.93 (s, 1H), 6.96 (s, 1H), 6.98 (s, 1H). 13C
NMR (100 MHz, CDCl3): δ 19.9, 21.8, 21.8, 21.8, 21.9, 37.6, 48.6,
55.7, 56.1, 56.1, 56.2, 71.4, 95.8, 103.4, 104.5, 110.8, 113.4, 114.3,
114.8, 115.9, 117.5, 117.6, 119.6, 125.5, 128.2, 129.1, 131.9, 132.1,
144.2, 146.0, 146.8, 146.8, 147.2, 149.6, 149.7, 155.6. Anal. Calcd for
C37H42BrNO9: C, 61.33; H, 5.84; N, 1.93. Found: C, 61.05; H, 5.86;
N, 1.84.
3,11-Diisopropoxy-2,12-dimethoxy-14-[4-methoxy-5-(me-
thoxymethoxy)-2-methylphenyl]-8,9-dihydro-6H-[1]-
benzopyrano[4′,3′:4,5]pyrrolo[2,1-a]isoquinolin-6-one (12).
Under an argon atmosphere, a mixture of 18 (2.00 g, 2.76 mmol),
K2CO3 (0.838 g, 6.06 mmol), and Pd(PPh3)4 (0.159 g, 0.140 mmol)
in DMA (54 mL) was heated at 125 °C for 20 h. After cooling to room
temperature, the mixture was diluted with water and extracted with
DCM. The extract was washed with water and brine, dried over
isoquinolin-6-one [(aR)-19b]. >99% ee. Mp 105.5−107.5 °C. [α]D
+5.04 (c 0.745, CHCl3).
(aS)-3,11-Dihydroxy-14-(5-hydroxy-4-methoxy-2-methyl-
phenyl)-2,12-dimethoxy-6H-[1]benzopyrano[4′,3′:4,5]pyrrolo-
[2,1-a]isoquinolin-6-one [(aS)-3a]. According to the procedure
described for the preparation of 8a, compound 19a (30.0 mg, 46.8
μmol) was reacted with BCl3 (1.0 M, 421 μL, 0.421 mmol). After
chromatographic purification over silica gel 60N (ethyl acetate), (aS)-
3a was obtained as a pale gray powder (23.7 mg, 99%). The
enantiomeric excess of this compound was determined to be >99% ee
by HPLC analysis (Daicel CHIRALCEL OD-H, hexane−2-propanol =
3:7) after conversion to the corresponding triacetate. Mp > 300 °C
20
(sealed capillary). [α]D +3.6 (c 0.46, DMSO).
(aR)-3,11-Dihydroxy-14-(5-hydroxy-4-methoxy-2-methyl-
phenyl)-2,12-dimethoxy-6H-[1]benzopyrano[4′,3′:4,5]pyrrolo-
[2,1-a]isoquinolin-6-one [(aR)-3b]. According to the procedure
described for the preparation of 8a, compound 19b (30.0 mg, 46.8
μmol) was reacted with BCl3 (1.0 M, 421 μL, 0.421 mmol). After
chromatographic purification over silica gel 60N (ethyl acetate), (aR)-
3b was obtained as a pale gray powder (88.0 mg, 72%). The
I
dx.doi.org/10.1021/jm400719y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
enantiomeric excess of this compound was determined to be >99% ee
by HPLC analysis (Daicel Chiralcel OD-H, hexane−2-propanol = 3:7)
after conversion to the corresponding triacetate. Mp > 300 °C (sealed
be obtained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
Biological Assays: General. The purity of all tested compounds
[1, 2, (aS)-3a, and (aR)-3b] was determined to be >95% using HPLC
with a Nacalai Cosmosil 5C18-AR-II column (4.6 mm i.d. × 250 mm).
The eluent was composed of MeOH−H2O (70:30, v/v). Flow rate
was set at 0.5 mL/min, and detection wavelength was fixed at 254 nm.
CPT was purchased from TopoGEN, and its purity (>95%) was
determined by HPLC as described above.
Protein Kinase Inhibition Assays. Buffers. Homogenization
buffer consisted of 60 mM β-glycerophosphate, 15 mM p-nitrophenyl
phosphate, 25 mM Mops (pH 7.2), 15 mM EGTA, 15 mM MgCl2, 1
mM DTT, 1 mM sodium vanadate, 1 mM NaF, 1 mM phenyl-
phosphate, 10 μg of leupeptin/mL, 10 μg of aprotinin/mL, 10 μg of
soybean trypsin inhibitor/mL, and 100 μM benzamidine. Buffer A
consisted of 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 25 mM Tris-
HCl, pH 7.5, and 50 μg of heparin/mL.
Kinase Preparations and Assays. Kinase activities for each enzyme
were assayed in buffer A, with their corresponding substrates, in the
presence of 15 μM ATP in a final volume of 30 μL. After a 30 min
incubation at 30 °C, the reaction was stopped by harvesting, using a
FilterMate harvester (Packard), onto P81 phosphocellulose papers
(GE Healthcare), which were washed in 1% phosphoric acid. Then 20
μL of scintillation fluid was added and the radioactivity measured in a
Packard counter. Blank values were subtracted and activities calculated
as picomoles of phosphate incorporated during the 30 min incubation.
The activities were expressed in % of the maximal activity, i.e., in the
absence of inhibitors. Controls were performed with appropriate
dilutions of DMSO.
CDK1/cyclin B was extracted in homogenization buffer from M
phase starfish (Marthasterias glacialis) oocytes and purified by affinity
chromatography on p9CKShs1-Sepharose beads, from which it was
eluted by free p9CKShs1 as previously described.52 The kinase activity
was assayed in buffer C, with 1 mg of histone H1/mL, in the presence
of 15 μM [γ-33P]ATP (3000 Ci/mmol, 10 mCi/mL) in a final volume
of 30 μL.
CDK2/cyclin A (human, recombinant, expressed in insect cells,
from A. Echalier) was assayed as described for CDK1/cyclin B.
CDK5/p25 was reconstituted by mixing amounts of recombinant
mammalian CDK5 and p25 expressed in E. coli as GST (glutathione-S-
transferase) fusion proteins and purified by affinity chromatography on
glutathione-agarose (vectors kindly provided by Dr. L. H. Tsai) (p25 is
a truncated version of p35, the 35 kDa CDK5 activator). Its activity
was assayed with histone H1 in buffer A as described for CDK1/cyclin
B.
GSK-3α/β was purified from porcine brain by affinity chromatog-
raphy on immobilized axin.53 It was assayed, following a 1/100 dilution
in 1 mg BSA/mL of 10 mM DTT, with 5 μL of 4 μM GS-1 peptide
substrate (GS-1, YRRAAVPPSPSLSRHSSPHQpSEDEEE where pS
stands for phosphorylated serine), in buffer A, in the presence of 15
μM [γ-33P]ATP (3000 Ci/mmol, 10 mCi/mL) in a final volume of 30
μL.
PIM1 was expressed as a GST-fusion protein in E. coli and purified
by affinity chromatography on glutathione-agarose. Its kinase activity
was assayed for 30 min with histone H1 in buffer C as described for
CDK1/cyclin B.
DYRK1A (human, recombinant, expressed in E. coli as a GST fusion
protein) was purified by affinity chromatography on glutathione-
agarose and assayed as described for CDK1/cyclin B.
20
capillary). [α]D −4.0 (c 0.46, DMSO).
(aR)-14-(5-Hydroxy-4-methoxy-2-methylphenyl)-3,11-diiso-
propoxy-2,12-dimethoxy-6H-[1]benzopyrano[4′,3′:4,5]-
pyrrolo[2,1-a]isoquinolin-6-one [(aR)-20b]. According to the
procedure described for the preparation of 8f, compound (aR)-19b
(20.2 mg, 31.5 μmol) was treated with concentrated HCl (0.4 mL) in
a mixture of DCM (5.0 mL) and methanol (2.5 mL) for 8 h to give
(aR)-20b as a colorless solid (17.3 mg, 92%). The enantiomeric excess
of this compound was determined to be >99% ee by HPLC analysis
(Daicel CHIRALPAK AD-H, hexane−2-propanol = 8:2). Recrystalli-
zation from DCM−hexane gave a colorless powder. Mp 217−219.5 °C
1
(sealed capillary). IR (KBr): 3428, 1701, 1423, 1268, 1207 cm−1. H
NMR (400 MHz, CDCl3): δ 1.41 (d, J = 6.1 Hz, 6H), 1.43 (d, J = 6.1
Hz, 6H), 2.05 (s, 3H), 3.46 (s, 3H), 3.47 (s, 3H), 3.98 (s, 3H), 4.58
(sep, J = 6.1 Hz, 1H), 4.70 (sep, J = 6.1 Hz, 1H), 5.71 (br s, 1H), 6.70
(s, 1H), 6.97 (s, 1H), 7.00 (s, 1H), 7.02 (d, J = 7.3 Hz, 1H), 7.09 (s,
1H), 7.10 (s, 1H), 7.13 (s, 1H), 9.22 (d, J = 7.3 Hz, 1H). 13C NMR
(100 MHz, CDCl3): δ 19.8, 21.8, 21.9, 21.9, 21.9, 55.1, 55.5, 56.3,
71.2, 71.5, 103.3, 105.1, 105.2, 108.1, 109.7, 110.2, 110.3, 112.3, 112.7,
117.9, 119.2, 123.3, 124.6, 127.5, 129.2, 130.8, 134.1, 144.5, 146.6,
20
146.7, 146.7, 147.9, 148.5, 150.4, 155.7. [α]D −9.90 (c 0.675,
CHCl3). HRFABMS m/z calcd for C35H36NO8 [(M + H)+]: 598.2341,
found 598.2439.
(aS)-14-(2-Bromo-3-hydroxy-4-methoxy-6-methylphenyl)-
3,11-diisopropoxy-2,12-dimethoxy-6H-[1]benzopyrano-
[4′,3′:4,5]pyrrolo[2,1-a]isoquinolin-6-one [(aS)-21b]. A solution
of (aR)-20b (35.3 mg, 59.1 μmol) in DMF (7.0 mL) was added
dropwise to a solution of NBS (10.6 mg, 59.7 μmol) in DMF (3.0 mL)
at 0 °C. The mixture was stirred for 1 h at 0 °C and for 9 h at room
temperature. The solution was diluted with water, and the product was
extracted with ethyl acetate. The extract was washed with water and
brine, dried over Na2SO4, and evaporated. The residue was purified by
column chromatography over silica gel 60N (hexane−ethyl acetate =
1:1) to give (aS)-21b as a pale brown solid (36.9 mg, 92%). The
enantiomeric excess of this compound was determined to be >99% ee
by HPLC analysis (Daicel CHIRALPAK AD-H, hexane−2-propanol =
9:1). Recrystallization from DCM−diethyl ether gave pale brown
granules. Mp 262.5−264.5 °C. IR (KBr): 3410, 1710, 1427, 1269,
1208 cm−1. 1H NMR (400 MHz, CDCl3): δ 1.41 (d, J = 6.1 Hz, 6H),
1.44 (d, J = 6.1 Hz, 6H), 2.05 (s, 3H), 3.45 (s, 3H), 3.47 (s, 3H), 4.02
(s, 3H), 4.59 (sep, J = 6.1 Hz, 1H), 4.71 (sep, J = 6.1 Hz, 1H), 6.03 (br
s, 1H), 6.56 (s, 1H), 6.98 (s, 1H), 6.99 (s, 1H), 6.99 (s, 1H), 7.07 (d, J
= 7.4 Hz, 1H), 7.12 (s, 1H), 9.25 (d, J = 7.4 Hz, 1H). 13C NMR (100
MHz, CDCl3): δ 21.0, 21.9, 21.9, 55.2, 55.6, 56.7, 71.2, 71.5, 103.3,
104.3, 104.6, 108.5, 108.5, 109.9, 110.2, 111.9, 112.5, 113.7, 119.0,
123.4, 124.8, 128.2, 129.2, 132.2, 134.0, 142.1, 146.7, 146.9, 147.1,
148.2, 148.7, 150.7, 155.6. [α]D20 +20.6 (c 0.700, CHCl3). HRFABMS
m/z calcd for C35H35BrNO8 [(M + H)+]: 676.1546. Found: 676.1561.
X-ray Crystallographic Analysis of (aS)-21b. Results are as
follows: compound formula C38H39BrNO8, Mw = 717.63, monoclinic,
P21, a = 10.905(3) Å, b = 18.705(5) Å, c = 16.375(4) Å, β = 96.40(2)°,
V = 3319(2) Å3, Z = 4, Dcalc = 1.436 g/cm3, monochromatized
radiation λ(Cu Kα) = 1.541 87 Å, μ = 21.532 mm−1, F(000) =
1492.00, T = 113 K. Data were collected on a Rigaku RAXIS RAPID
imaging plate to a θ limit of 136.5° which yielded 41 261 reflections.
There are 11 457 unique reflections with 8028 observed at the 2σ level.
The structure was solved by direct methods (SIR92)50 and refined
using full-matrix least-squares on F2 (SHELXL-97).51 The final model
was refined using 849 parameters and all 11 457 data. All non-
hydrogen atoms were refined with isotropic thermal displacements.
The final agreement statistics are as follows: R = 0.0610 (based on
8028 reflections with I > 2σ(I)), wR = 0.1512, S = 0.946. The
maximum peak height in a final difference Fourier map is 1.19 e Å3,
and this peak is without chemical significance. The absolute
configuration was determined based on the Flack parameter,
−0.008(18), refined using 5230 Friedel pairs. CCDC 886003 contains
the supplementary crystallographic data for this paper. These data can
CLK3 (mouse, recombinant, and expressed in E. coli as GST fusion
proteins) was assayed in buffer A (+0.15 mg BSA/mL) with RS
peptide (GRSRSRSRSRSR) (1 μg/assay).
CK1δ/ε was purified from porcine brain by affinity chromatography
on an immobilized axin fragment.54 It was assayed as described for
CDK1 but using CKS (RRKHAAIGpSAYSITA), a CK1-specific
peptide substrate.
Topoisomerase I Inhibition Assay. Topoisomerase I inhibition was
assessed by measuring relaxation of supercoiled DNA pBR322
according to the reported method49 with a slight modification. In
brief, a mixture of 250 ng of plasmid pBR322 (Takara Bio, Inc.), 5 U
J
dx.doi.org/10.1021/jm400719y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
of calf thymus topoisomerase I (Takara Bio, Inc.), 1 μL of a DMSO
solution of the inhibitor, and 0.01% bovine serum albumin in
topoisomerase I relaxation buffer (Takara Bio, Inc.) comprising 35
mM Tris-HCl (pH 8.0), 72 mM KCl, 5 mM MgCl2, 5 mM
dithiothreitol, and 5 mM spermidine in final volume of 20 μL was
incubated at 37 °C for 30 min. The reaction was terminated by adding
2 μL of 10% SDS solution followed by digestion with 2.2 μL of
proteinase K solution (TopoGEN, Inc.) at 0.5 mg/mL at 37 °C for 30
min. Then 2.4 μL of 10× gel loading dye followed by 20 μL of 24:1
chloroform/isoamyl alcohol (CIA) was added. The mixture was
vortexed and centrifuged at 10 000 rpm at 4 °C for 1 min. The blue
aqueous phase was applied to 1% agarose gel and electrophoresed for
2 h at 50 V/cm in Tris−borate−EDTA buffer containing 89 mM Tris,
89 mM H3BO4, and 2 mM Na EDTA. The gel was stained in 0.5 μg/
mL solution of ethidium bromide.
Cytotoxicity Assay. Colony Formation Assay. The cytotoxicity of
test compounds was determined by the inhibitory activity of colony
formation of HeLa cells as described previously.11 In brief, 100−150
cells per well in a 24-well plate were cultured with varying
concentrations of each test compound in the growth medium
(MEM supplemented with streptomycin (100 μg/mL), penicillin
(100 U/mL), and 10% fetal calf serum) for 72 h at 37 °C under 5%
CO2/95% air. The number of colonies formed was counted after
staining with 1% methylene blue in 50% methanol. Clusters of 40 or
more cells were considered as colonies. All assays were performed in
duplicate with at least four different concentrations of the inhibitor,
and the IC50 values were calculated by least-squares fit to a logarithm-
probit analysis. The IC50 values (95% confidence limits) of 2, (aS)-3a,
and (aR)-3b thus obtained were 0.032 (0.028−0.037), 2.95 (1.57−
9.02), and 0.16 (0.140.18) μM, respectively.
force field refinement (side chain, free) and rescored by GBVI/WSA
dG scoring function (the maximum number of poses, 100).
The obtained poses were evaluated using the GBVI/WSA dG
scoring function, and the best scoring poses are shown in Figures 7
and 8.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis details of 11, analytical data, and H NMR and 13C
1
NMR spectra of all compounds synthesized in this work. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +81-95-819-2681. E-mail: iwao@nagasaki-u.ac.jp.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by a Grant-in-Aid for
Scientific Research (B) (Grant No. 20310135) from the Japan
Society for the Promotion of Science (JSPS). This research was
supported by grants (L.M.) from the “Fonds Unique
Interminister
France-Alzheimer (Finister
“Canceropole Grand-Ouest”, the “Association France-Alz-
heimer Finistere”, the “Ligue Nationale contre le Cancer
(Comite Grand-Ouest)”, the Fondation Jer me Lejeune. This
́
iel” (FUI) PHARMASEA project, the “Association
̀
e)”, “CRITT-Sante Bretagne”, the
́
́
MTS Assay. SH-SY5Y or IMR32 human neuroblastoma cells were
grown in DMEM supplemented with 2 mM L-glutamine (Invitrogen,
Cergy Pontoise, France), plus antibiotics (penicillin−streptmycin) and
10% volume of fetal calf serum (FCS) (Invirogen). Cell viability was
determined by means of the MTS [3-(4,5-dimethylthiazol-2-yl)-5-
(carbomethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium] reduction
method. All the assays were carried out in triplicate, and the values
did not differ by more than 10%. The IC50 values shown in Table 2
were estimated graphically from the dose−response curves performed
with at least six concentrations of the inhibitor.
̀
́
́ ̂
o
research was partly supported by an FP7-KBBE-2012 grant
(BlueGenics) to L.M. The HPLC analyses shown in Figure 3
were performed at the laboratories of Daicel Corporation. We
thank Daicel Corporation for these experiments.
ABBREVIATIONS USED
■
Docking Simulation. Docking studies were performed using MOE
2011.10.45 Crystal structures of CDK2−staurosporine complex (PDB
code 1AQ1)42 and GSK-3β-staurosporine complex (PDB code
1Q3D)43 were obtained from the Protein Data Bank. The protein
structures for the docking were prepared by the following sequence:
(i) A kinase−staurosporine complex was loaded. (ii) To the complex
were added hydrogen atoms and electric charge by Protonate 3D
(default settings). (iii) The hydrogen atoms were optimized by
MMFF94x force field (the heavy atoms were fixed during the
optimization). (iv) The dummy atoms were disposed in the docking
site by Site Finder (default settings). On the other hand, the
conformers of 2, (aS)-3a, and (aR)-3b were obtained by conforma-
tional search using the LowModeMD search method with default
parameters except for the following: the hydrogens check box was
selected in order to include both hydrogen and heavy atoms in the
rmsd calculation for duplication detection, and the value of energy
window was set to 10 kcal/mol. Finally, the ligands were docked into
the binding site of the kinases by using the Dock docking program
according to the following sequence: (i) Initial poses were obtained
using the Triangle Matcher placement (timeout, 3000 s; no. of return
poses, 10 000), London dG rescoring 1 (the maximum number of
poses, 500), GridMin refinement (default settings), and GBVI/WSA
dG48 rescoring 2 (the maximum number of poses, 100). (ii) The poses
obtained in (i) were refined by force field refinement (default settings)
and rescored by GBVI/WSA dG scoring function (the maximum
number of poses, 100). (iii) The poses obtained in (ii) were refined by
force field refinement (side chain, tether, the tether value was set to
10) and rescored by GBVI/WSA dG scoring function (the maximum
number of poses, 100). (iv) The poses obtained in (iii) were refined by
CDK, cyclin-dependent kinase; GSK-3, glycogen synthase
kinase 3; PIM-1, proto-oncogene serine/threonine-protein
kinase Pim 1; DYRK1A, dual specificity, tyrosine phosphor-
ylation regulated kinase 1A; CLK3, cdc2-like kinase 3; CK1,
casein kinase 1; MDR, multidrug resistant; SAR, structure−
activity relationship; TEA, triethylamine; DMA, dimethylaceta-
mide; CPT, camptothecin; Topo I, topoisomerase I
REFERENCES
■
́
(1) Cironi, P.; Albericio, F.; Alvarez, M. Lamellarins: Isolation,
Activity and Synthesis. Prog. Heterocycl. Chem. 2004, 16, 1−26.
(2) Fan, H.; Peng, J.; Hamann, M. T.; Hu, J.-F. Lamellarins and
Related Pyrrole-Derived Alkaloids from Marine Organisms. Chem. Rev.
2008, 108, 264−287.
(3) Fukuda, T.; Ishibashi, F.; Iwao, M. Synthesis and Biological
Activity of Lamellarin Alkaloids: An Overview. Heterocycles 2011, 83,
491−529.
(4) Bailly, C. Lamellarins, from A to Z: A Family of Anticancer
Marine Pyrrole Alkaloids. Curr. Med. Chem.: Anti-Cancer Agents 2004,
4, 363−378.
(5) Kluza, J.; Marchetti, P.; Bailly, C. Lamellarin Alkaloids: Structure
and Pharmacological Properties. In Modern Alkaloids: Structure,
Isolation, Synthesis and Biology; Fattorusso, E., Taglialatela-Scafati, O.,
Eds.; Wiley-VCH: Weinheim, Germany, 2008; pp 171−187.
́
(6) Quesada, A. R.; Gravalos, M. D. G.; Puentes, J. L. F. Polyaromatic
Alkaloids from Marine Invertebrates as Cytotoxic Compounds and
Inhibitors of Multidrug Resistance Caused by P-Glycoprotein. Br. J.
Cancer 1996, 74, 677−682.
K
dx.doi.org/10.1021/jm400719y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(7) Reddy, M. V. R.; Rao, M. R.; Rhodes, D.; Hansen, M. S. T.;
Rubins, K.; Bushman, F. D.; Venkateswarlu, Y.; Faulkner, D. J.
Lamellarin α 20-Sulfate, an Inhibitor of HIV-1 Integrase Active against
HIV-1 Virus in Cell Culture. J. Med. Chem. 1999, 42, 1901−1907.
(8) Ridley, C. P.; Reddy, M. V. R.; Rocha, G.; Bushman, F. D.;
Faulkner, D. J. Total Synthesis and Evaluation of Lamellarin α 20-
Sulfate Analogues. Bioorg. Med. Chem. 2002, 10, 3285−3290.
(9) Recently, we have indicated that the anti-HIV-1 activity of
lamellarin sulfates is caused by inhibition of the virus entry step rather
than the integration step. See the following: Kamiyama, H.; Kubo, Y.;
Sato, H.; Yamamoto, N.; Fukuda, T.; Ishibashi, F.; Iwao, M. Synthesis,
Structure−Activity Relationships, and Mechanism of Action of Anti-
HIV-1 Lamellarin α 20-Sulfate Analogues. Bioorg. Med. Chem. 2011,
19, 7541−7550.
(26) Qiu, W.; Chen, S.; Sun, X.; Liu, Y.; Zhu, D. Suzuki Coupling
Reaction of 1,6,7,12-Tetrabromoperylene Bisimide. Org. Lett. 2006, 8,
867−870.
(27) Fujikawa, N.; Ohta, T.; Yamaguchi, T.; Fukuda, T.; Ishibahsi, F.;
Iwao, M. Total Synthesis of Lamellarins D, L, and N. Tetrahedron
2006, 62, 594−604.
(28) Wolf, C. Dynamic Stereochemistry of Chiral Compounds Principles
and Applications; The Royal Society of Chemistry: Cambridge, U.K.,
2008; pp 136−179.
(29) The rotational energy barriers around the C1−C11 single bond
of O-protected lamellarins N were assumed to be around 83−87 kJ/
1
mol by means of variable-temperature H and 13C NMR techniques.
Fukuda, , T.; Itoyama, , R.; Minagawa, , T.; Iwao, , M. Rotational
Energy Barrier around the C1−C11 Single Bond in Lamellarins: A
Study by Variable-Temperature NMR.. Heterocycles [Online early
access].DOI: DOI: 10.3987/COM-13-S(S)69,. Published Online:
Aug 26, 2013,.
(10) Ishibashi, F.; Miyazaki, Y.; Iwao, M. Total Syntheses of
Lamellarin D and H. The First Synthesis of Lamellarin-Class Marine
Alkaloids. Tetrahedron 1997, 53, 5951−5962.
(11) Ishibashi, F.; Tanabe, S.; Oda, T.; Iwao, M. Synthesis and
Structure−Activity Relationship Study of Lamellarin Derivatives. J.
Nat. Prod. 2002, 65, 500−504.
(30) Alberico, D.; Scott, M. E.; Lautens, M. Aryl−Aryl Bond
Formation by Transition-Metal-Catalyzed Direct Arylation. Chem. Rev.
2007, 107, 174−238.
(12) Chittchang, M.; Batsomboon, P.; Ruchirawat, S.; Ploypradith, P.
Cytotoxicities and Structure−Activity Relationships of Natural and
Unnatural Lamellarins toward Cancer Cell Lines. ChemMedChem
2009, 4, 457−465.
(31) Knockaert, M.; Greengard, P.; Meijer, L. Pharmacological
Inhibitors of Cyclin-Dependent Kinases. Trends Pharmacol. Sci. 2002,
23, 417−425.
(32) Meijer, L.; Flajolet, M.; Greengard, P. Pharmacological
Inhibitors of Glycogen Synthase Kinase 3. Trends Pharmacol. Sci.
2004, 25, 471−480.
́
(13) Facompre, M.; Tardy, C.; Bal-Mahieu, C.; Colson, P.; Perez, C.;
Manzanares, I.; Cuevas, C.; Bailly, C. Lamellarin D: A Novel Potent
Inhibitor of Topoisomerase I. Cancer Res. 2003, 63, 7392−7399.
(14) Marco, E.; Laine, W.; Tardy, C.; Lansiaux, A.; Iwao, M.;
Ishibashi, F.; Bailly, C.; Gago, F. Molecular Determinants of
Topoisomerase I Poisoning by Lamellarins: Comparison with
Camptothecin and Structure−Activity Relationships. J. Med. Chem.
2005, 48, 3796−3807.
(33) Bachmann, M.; Moroy, T. The Serine/Threonine Kinase Pim-1.
̈
̈
Int. J. Biochem. Cell Biol. 2005, 37, 726−730.
(34) Wang, J.; Anderson, P. D.; Luo, W.; Gius, D.; Roh, M.;
Abdulkadir, S. A. Pim1 Kinase Is Required To Maintain Tumor-
igenicity in MYC-Expressing Prostate Cancer Cells. Oncogene 2012,
31, 1794−1803.
(15) Kluza, J.; Gallego, M.-A.; Loyens, A.; Beauvillain, J.-C.; Sousa-
Faro, J.-M. F.; Cuevas, C.; Marchetti, P.; Bailly, C. Cancer Cell
Mitochondria Are Direct Proapoptotic Targets for the Marine
Antitumor Drug Lamellarin D. Cancer Res. 2006, 66, 3177−3187.
(16) Ballot, C.; Kluza, J.; Lancel, S.; Martoriati, A.; Hassoun, S. M.;
Mortier, L.; Vienne, J.-C.; Briand, G.; Formstecher, P.; Bailly, C.;
Neviere, R.; Marchetti, P. Inhibition of Mitochondrial Respiration
̀
Mediates Apoptosis Induced by the Anti-Tumoral Alkaloid Lamellarin
D. Apoptosis 2010, 15, 769−781.
(17) Baunbæk, D.; Trinkler, N.; Ferandin, Y.; Lozach, O.;
Ploypradith, P.; Ruchirawat, S.; Ishibashi, F.; Iwao, M.; Meijer, L.
Anticancer Alkaloid Lamellarins Inhibit Protein Kinases. Marine Drugs
2008, 6, 514−527.
(35) Tejedor, F. J.; Hammerle, B. MNB/DYRK1A as a Multiple
̈
Regulator of Neuronal Development. FEBS J. 2011, 278, 223−235.
(36) Wegiel, J.; Gong, C.-X.; Hwang, Y.-W. The Role of DYRK1A in
Neurodegenerative Diseases. FEBS J. 2011, 278, 236−245.
(37) Becker, W.; Sippl, W. Activation, Regulation, and Inhibition of
DYRK1A. FEBS J. 2011, 278, 246−256.
(38) Tahtouh, T.; Elkins, J. M.; Filippakopoulos, P.; Soundararajan,
M.; Burgy, G.; Durieu, E.; Cochet, C.; Schmid, R. S.; Lo, D. C.;
Delhommel, F.; Oberholzer, A. E.; Pearl, L. H.; Carreaux, F.; Bazureau,
J.-P.; Knapp, S.; Meijer, L. Selectivity, Cocrystal Structures, and
Neuroprotective Properties of Leucettines, a Family of Protein Kinase
Inhibitors Derived from the Marine Sponge Alkaloid Leucettamine B.
J. Med. Chem. 2012, 55, 9312−9330.
(39) Duncan, P. I.; Stojdl, D. F.; Marius, R. M.; Scheit, K. H.; Bell, J.
C. The Clk2 and Clk3 Dual-Specificity Protein Kinases Regulate the
Intranuclear Distribution of SR Proteins and Influence Pre-mRNA
Splicing. Exp. Cell Res. 1998, 241, 300−308.
(18) Anderson, R. J.; Faulkner, D. J.; Cun-heng, H.; Van Duyne, G.
D.; Clardy, J. Metabolites of the Marine Prosobranch Mollusk
Lamellaria sp. J. Am. Chem. Soc. 1985, 107, 5492−5495.
(19) Urban, S.; Capon, R. J. Lamellarin-S: A New Aromatic
Metabolite from an Australian Tunicate, Didemnum sp. Aust. J.
Chem. 1996, 49, 711−713.
(40) Muraki, M.; Ohkawara, B.; Hosoya, T.; Onogi, H.; Koizumi, J.;
Koizumi, T.; Sumi, K.; Yomoda, J.; Murray, M. V.; Kimura, H.;
Furuichi, K.; Shibuya, H.; Krainer, A. R.; Suzuki, M.; Hagiwara, M.
Manipulation of Alternative Splicing by a Newly Developed Inhibitor
of Clks. J. Biol. Chem. 2004, 279, 24246−24254.
(20) Crossley, R. J. Chirality and the Biological Activity of Drugs; CRC
Press: Boca Raton, FL, 1995.
(21) Clayden, J.; Moran, W. J.; Edwards, P. J.; LaPlante, S. R. The
Challenge of Atropisomerism in Drug Discovery. Angew. Chem., Int.
Ed. 2009, 48, 6398−6401.
(41) Knippschild, U.; Wolff, S.; Giamas, G.; Brockschmidt, C.;
Wittau, M.; Wurl, P. U.; Eismann, T.; Stoter, M. The Role of the
̈
̈
(22) LaPlante, S. R.; Edwards, P. J.; Fader, L. D.; Jakalian, A.; Hucke,
O. Revealing Atropisomer Axial Chirality in Drug Discovery.
ChemMedChem 2011, 6, 505−513.
Casein Kinase 1 (CK1) Family in Different Signaling Pathways Linked
to Cancer Development. Onkologie 2005, 28, 508−514.
(42) Lawrie, A. M.; Noble, M. E. M.; Tunnah, P.; Brown, N. R.;
Johnson, L. N.; Endicott, J. A. Protein Kinase Inhibition by
Staurosporine Revealed in Details of the Molecular Interaction with
CDK2. Nat. Struct. Biol. 1997, 4, 796−801.
(23) Takahashi, H.; Wakamatsu, S.; Tabata, H.; Oshitari, T.; Harada,
A.; Inoue, K.; Natsugari, H. Atropisomerism Observed in Indometacin
Derivatives. Org. Lett. 2011, 13, 760−763.
(24) Tabata, H.; Nakagomi, J.; Morizono, D.; Oshitari, T.; Takahashi,
H.; Natsugari, H. Atropisomerism in the Vaptan Class of Vasopressin
Receptor Ligands: The Active Conformation Recognized by the
Receptor. Angew. Chem., Int. Ed. 2011, 50, 3075−3079.
(25) Ohta, T.; Fukuda, T.; Ishibashi, F.; Iwao, M. Design and
Synthesis of Lamellarin D Analogues Targeting Topoisomerase I. J.
Org. Chem. 2009, 74, 8143−8153.
(43) Bertrand, J. A.; Thieffine, S.; Vulpetti, A.; Cristiani, C.; Valsasina,
B.; Knapp, S.; Kalisz, H. M.; Flocco, M. Structural Characterization of
the GSK-3β Active Site Using Selective and Non-Selective ATP-
Mimetic Inhibitors. J. Mol. Biol. 2003, 333, 393−407.
(44) It is noteworthy that staurosporine derivatives and lamellarins
were isolated from the similar prosobranch mollusks of the Coriocella
family. See the following: Cantrell, C. L.; Groweiss, A.; Gustafson, K.
L
dx.doi.org/10.1021/jm400719y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
R.; Boyd, M. R. A New Staurosporine Analog from the Prosobranch
Mollusk Coriocella nigra. Nat. Prod. Lett. 1999, 14, 39−46.
(45) Molecular Operating Environment (MOE), version 2011.10;
Chemical Computing Group Inc.: Montreal, Quebec, Canada, 2011;
http://www. chemcomp.com.
(46) Ghose, A. K.; Herbertz, T.; Pippin, D. A.; Salvino, J. M.;
Mallamo, J. P. Knowledge Based Prediction of Ligand Binding Modes
and Rational Inhibitor Design for Kinase Drug Discovery. J. Med.
Chem. 2008, 51, 5149−5171.
́
(47) Echalier, A.; Bettayeb, K.; Ferandin, Y.; Lozach, O.; Clement,
M.; Valette, A.; Liger, F.; Marquet, B.; Morris, J. C.; Endicott, J. A.;
Joseph, B.; Meijer, L. Meriolins (3-(Pyrimidin-4-yl)-7-azaindoles):
Synthesis, Kinase Inhibitory Activity, Cellular Effects, and Structure of
a CDK2/Cyclin A/Meriolin Complex. J. Med. Chem. 2008, 51, 737−
751.
(48) Corbeil, C. R.; Williams, C. I.; Labute, P. Variability in Docking
Success Rates Due to Dataset Preparation. J. Comput.-Aided Mol. Des.
2012, 26, 775−786.
(49) Liu, L. F.; Miller, K. G. Eukaryotic DNA Topoisomerases: Two
Forms of Type I DNA Topoisomerases from HeLa Cell Nuclei. Proc.
Natl. Acad. Sci. U.S.A. 1981, 78, 3487−3491.
(50) Altomare, A.; Cascarano, G.; Giacovazzo, C.; Guagliardi, A.;
Burla, M. C.; Polidori, G.; Camalli, M. SIR 92A Program for
Automatic Solution of Crystal Structures by Direct Methods. J. Appl.
Crystallogr. 1994, 27, 435.
(51) Sheldrick, G. M. A Short History of SHELX. Acta Crystallogr.,
Sect. A: Found. Crystallogr. 2008, 64, 112−122.
(52) Meijer, L.; Borgne, A.; Mulner, O.; Chong, J. P. J.; Blow, J. J.;
Inagaki, N.; Inagaki, M.; Delcros, J.-G.; Moulinoux, J.-P. Biochemical
and Cellular Effects of Roscovitine, a Potent and Selective Inhibitor of
the Cyclin-Dependent Kinases cdc2, cdk2 and cdk5. Eur. J. Biochem.
1997, 243, 527−536.
(53) Primot, A.; Baratte, B.; Gompel, M.; Borgne, A.; Liabeuf, S.;
Romette, J.-L.; Jho, E.; Costantini, F.; Meijer, L. Purification of GSK-3
by Affinity Chromatography on Immobilized Axin. Protein Expression
Purif. 2000, 20, 394−404.
(54) Reinhardt, J.; Ferandin, Y.; Meijer, L. Purification of CK1 by
Affinity Chromatography on Immobilised Axin. Protein Expression
Purif. 2007, 54, 101−109.
M
dx.doi.org/10.1021/jm400719y | J. Med. Chem. XXXX, XXX, XXX−XXX