Synthesis and topoisomerase i inhibitory activity of a novel diazaindeno[2,1-b]phenanthrene analogue of Lamellarin D

Article abstract of DOI:10.1016/j.bmc.2011.06.056

A novel 5-oxa-6a,8-diazaindeno[2,1-b]phenanthren-7-one scaffold was designed and synthesized as an active analogue of the cytotoxic marine alkaloid Lamellarin D. The design was based on molecular modeling of the site of interaction of Lamellarin D with DNA-topoisomerase I cleavable complex, whereas the synthesis capitalized on a simple Friedel-Crafts cyclization of indole to a β-carbolinone nucleus. The product exhibited topoisomerase I poisoning activity and submicromolar cytotoxicity on human non-small cell lung cancer H460 cell line.

Full text of DOI:10.1016/j.bmc.2011.06.056

Bioorganic & Medicinal Chemistry 19 (2011) 4971–4984
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and topoisomerase I inhibitory activity of a novel
diazaindeno[2,1-b]phenanthrene analogue of Lamellarin D
Salvatore Cananzi a, Lucio Merlini a, Roberto Artali b, Giovanni Luca Beretta c, Nadia Zaffaroni c,
Sabrina Dallavalle a,
⇑
a Department of Molecular and Agrifood Sciences, Università di Milano, Via Celoria 2, 20133 Milano, Italy
b Department of Pharmaceutical Sciences ‘P. Pratesi’, Università di Milano, Via Mangiagalli 25, 20133 Milano, Italy
c Department of Experimental Oncology and Molecular Medicine, Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Venezian 1, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel 5-oxa-6a,8-diazaindeno[2,1-b]phenanthren-7-one scaffold was designed and synthesized as an
active analogue of the cytotoxic marine alkaloid Lamellarin D. The design was based on molecular mod-
eling of the site of interaction of Lamellarin D with DNA–topoisomerase I cleavable complex, whereas the
synthesis capitalized on a simple Friedel–Crafts cyclization of indole to a b-carbolinone nucleus. The
product exhibited topoisomerase I poisoning activity and submicromolar cytotoxicity on human non-
small cell lung cancer H460 cell line.
Received 20 April 2011
Revised 16 June 2011
Accepted 20 June 2011
Available online 2 July 2011
Keywords:
Lamellarin
Ó 2011 Elsevier Ltd. All rights reserved.
Synthesis
Molecular modeling
Topoisomerase I
Antitumor
H3CO
OH
20
1. Introduction
HO
14
21
Nature continues to be the most prolific source of biologically
active compounds. Indeed, more than 60% of the recently marketed
drugs have been isolated or derived from natural products.1
The marine environment has proven to be a very rich source of
potent compounds. The oceans, with their more than two million
species of plants and animals, offer a vast reservoir for new com-
pounds: more than 12,000 new chemical entities have so far been
discovered, their number being increased every year by several
hundreds of new marine compounds, a significant percentage of
which typically show antitumor activity.2
Lamellarins are a group of marine alkaloids. The first four
members of this group, Lamellarins A–D, were originally isolated
in 1985 by Faulkner and co-workers3 from six specimens of the
marine prosobranch mollusc Lamellaria sp. Since then, more
than 70 lamellarins and structurally-related pyrrole alkaloids
have been isolated from molluscs, ascidians, and sponge
species.4 Most of them possess a common 6H-[1]benzopyrano-
[4,3;4,5]pyrrolo[2,1-a]isoquinoline ring system, and differ in
the number and position of the hydroxy and methoxy groups
on the scaffold.
H3CO
13
2
1
O
H3CO
HO
9
8
3
N
O
5
6
Lamellarin D (1)
Chart 1. Structure of Lamellarin D.
Lamellarins and related pyrrole-derived alkaloids have shown
promising biological activities such as antitumor activity, reversal
of multidrug resistance (MDR), and HIV-1 integrase inhibition
activity.4 Lamellarin D (Lam-D, 1, Chart 1) is one of the most cyto-
toxic compounds in the series.4a,5
This compound exhibits potent anticancer activity at nanomo-
lar concentration. Recently, Bailly and co-workers reported that
Lam-D is a potent poison of the enzyme topoisomerase I (topI).6
Interactions between Lam-D and specific aminoacid residues of
the topI–DNA complex have been identified from molecular mod-
eling studies.6 Additionally, the proapoptotic activity of Lam-D has
been correlated with its ability to promote DNA cleavage through
stabilization of topI–DNA covalent complexes,7 similarly to what
happens with camptothecins.8
⇑
Corresponding author. Tel.: +39 2 50316818; fax: +39 2 50316801.
E-mail address: sabrina.dallavalle@unimi.it (S. Dallavalle).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.056

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S. Cananzi et al. / Bioorg. Med. Chem. 19 (2011) 4971–4984
the ligands were placed in the DNA–topI complex in almost the
R'''
R'''
R''
R''
same orientation. The comparative analysis of the best poses
obtained from the docking experiments shows how the intercala-
tion binding site of both complexes results from conformational
changes of the phosphodiester bond between the +1 (upstream)
and ꢀ1 (downstream) base pairs of the uncleaved strand. This
effectively splits the DNA duplex allowing both compounds to
intercalate within the site, stabilized mainly by the formation
of stacking interactions with both the +1(CG) and ꢀ1(AT) base
pairs.
O
NH
O
N
R'
R'
N
R
O
N
R
3
2
OH
OH
H3CO
H3CO
The binding mode of compound 2a revealed that the molecule
forms base-stacking interactions with both the ꢀ1 and +1 base
HO
HO
H3CO
H3CO
HO
4'
4
4''
1
pairs, with a strong
p–p stacking between the pyridone ring and
H3CO
H3CO
HO
Gꢀ1, and that the ternary structure is further stabilized by five
hydrogen bonds with specific amino acid residues of the protein.
According to the Lam-D binding model,6a two hydroxy groups at-
tached to the main aromatic moiety (C-3 and C-10, corresponding
to C-20 and C-8, respectively, in Lam-D) are located at hydrogen
bond distance from the Asn722 and Glu356 residues of the enzyme,
whereas the carbonyl is interacting with the Arg364 residue. This
disposition is in perfect agreement with the interaction pattern
in the lamellarin series. Interestingly, the phenol ring in the C-13
position of 2a is pointing out to the protein–DNA interface, show-
ing a so far never reported direct interaction between the methoxy
group on this ring and the protein residue Asn352. It is important to
note that the presence of this interaction for both compounds 2a
and 3a could mean that this extra ring (on C-13 in 2a and C-4 in
3a, respectively), although not essential for cytotoxicity, can be
useful to modulate the binding to the enzyme. Finally, the free in-
dole NH group did not seem to play a role: in fact, the N-methyl
substituted derivative showed the same interaction pattern and
the same free energy of binding found for 2a (data not shown).
The interactions between compound 2a and the DNA–topI com-
plex are reported in Figure 1.
O
4
6
13
NH
O
N
5
8
12
9
O
N
H
N
H
3a
2a
Chart 2. Chemical structure of compounds 2 and 3.
Several strategies for the synthesis of lamellarins and their ana-
logues have been devised by numerous research groups.9 The syn-
thesis of Lam-D derivatives with different substitution patterns on
the pentacyclic framework would allow to delineate comprehen-
sive structure–activity relationships.6b,10 Hence, the hydroxy
groups at both C-8 and C-20 position and the planar pentacyclic
core are crucial for cytotoxic activity and for topI inhibition.
Moreover, most of the Lam-D derivatives with an opened lactone
ring were found to be considerably less cytotoxic than the parent
compound except when a potential lactonization was preserved.11
All the analogues of lamellarins so far prepared have main-
tained completely or in part their pyrrolo[2,1-a]isoquinoline skele-
ton. Nevertheless, the identification of alternative molecular
scaffolds endowed with similar properties is highly desirable.
We were intrigued by the possibility of obtaining cytotoxic
compounds containing a new scaffold, that could mimic the crucial
interactions of lamellarins with the topI–DNA complex. To our
knowledge, no attempt of this kind has been reported in the
literature.
The same experiment was run also on the compound 3a. The
structure of the complex thus obtained was fully consistent with
that of the compound 2a and Lam-D. Indeed, also compound 3a
is characterized by the presence of base-stacking interactions with
both the ꢀ1 and +1 base pairs, and the ternary structure is again
stabilized by hydrogen bonds with the same residues of the pro-
tein, as shown in Figure 1(C). The two hydroxyl groups attached
to the main aromatic moiety (C-7 and C-40, corresponding to C-8
and C-20 in Lam-D) form a hydrogen bond with Asn722 and
Glu356. Differently from 2a, in compound 3a the carbonyl group
is closer to Arg364 residue. As mentioned above, the phenol ring
in the C-4 position shows a hydrogen bond between the methoxy
group and the protein residue Asn352. The two compounds are dif-
ferently oriented in the binding site because of their different tor-
Therefore, on the basis of Lam-D structure, we designed the
replacement of the pyrrolo[2,1-a]isoquinoline core with
a
b-carbolin-1-one moiety and the substitution of the labile lactone
ring with a 3,4-dihydro-2H-[1,3]oxazine (compounds 2, Chart 2).
Simplified analogues (3), that according to the modeling
studies, could satisfy these requirements as well, were also
synthesized.
sion angles s1 and s2, as shown in Figure 2.
In fact, while compound 2a adopt an almost completely planar
conformation of the main skeleton, with angles s1 and s2 of ꢀ2.3°
and 82.2°, respectively, 3a is more flexible and the interaction with
the enzyme pocket leads to a partial disruption of the planarity,
2. Results and discussion
2.1. Molecular modeling
with angles of ꢀ24.1° (
s1) and 84.0° (s2).
Molecular modeling studies were performed on compounds 2a
and 3a maintaining the crucial hydroxy and methoxy groups of
Lam-D. A preliminary simple MM2 overlay study on the isolated
molecules indicated a satisfactory superposition of the two 3D
structures 2a and 3a with that of 1. Therefore, molecular docking
studies were performed to elucidate the mode of interaction be-
tween the planned compounds and the DNA–topI complex. We
started from the crystal structure8 of human topI covalently linked
to double-stranded DNA and bound to topotecan, a camptothecin
in clinical use. In particular, in our initial model, at first topotecan
was replaced with compound 2a and then the structure of the
resulting ternary complex was minimized. As shown in Figure 1,
The modeling analysis suggests that the two compounds are
placed in a similar way at the DNA–topI interface and the obtained
molecular models confirm the possibility of the binding of the de-
signed compounds to the enzyme–DNA complex.
The promising binding provided structural support for synthe-
sizing these novel Lam-D analogues. Accordingly, we developed a
general synthetic route to compounds with a diazaindeno[2,1-
b]phenanthrene skeleton.
In order to check the feasibility of the sequence, a route for the
synthesis of the structurally simpler 3,4-diphenyl-b-carbolin-1-
one 3 was devised, that could be later extended to the preparation
of the more complex derivative 2.

S. Cananzi et al. / Bioorg. Med. Chem. 19 (2011) 4971–4984
4973
Figure 1. Theoretical model of 2a and 3a bound to the covalent topoisomerase I–DNA (PDB code: 1K4T). (A) Solid ribbon (protein) and ladder (DNA) representation of
topoisomerase I–DNA. The transparent rectangle correspond to the position of the binding site used for the docking runs. (B) Best pose for compound 2a (in stick).
Topoisomerase I residues forming hydrogen bond interactions with 2a are in stick, hydrogen bond interactions showed as green dot lines. (C) Best pose for compound 3a (in
stick). Topoisomerase I residues forming hydrogen bond interactions with 3a are in stick, hydrogen bond interactions shown as green dot lines.
Figure 2. Graphical representation (in stick) of 2a (left) and 3a (right).
s
1 and
s
2 refer to the respective torsion angles, as discussed in the text. Atoms are coloured following
the CPK notation.
2.2. Chemistry
substituted 2-amino-1,2-diphenyl-ethanone 6 (Scheme 1). This
strategy was based on an efficient procedure recently developed
The synthesis was envisioned to proceed via a key intramolecular
Friedel–Crafts cyclization of indole-2-carboxylic acid b-oxoamide
(4), on its turn obtained by coupling 2-indole carboxylic acid 5 and
by us for the preparation of 3- or 4-substituted b-carbolinones.12
The 2-amino-1,2-diphenyl-ethanone fragment 15 was obtained
starting from vanillic acid (7), following the sequence depicted in

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S. Cananzi et al. / Bioorg. Med. Chem. 19 (2011) 4971–4984
migration reaction, following a procedure described in the litera-
R'''
ture on different substrates.13 The yield was optimized using three
equivalents of LDA in the presence of DMPU (3%) at ꢀ78 °C. Inter-
estingly, other strong bases, such as NaH, KH, KHMDS and s-BuLi
with sparteine, did not give any acyl migrated product. Treatment
with TFA in DCM at room temperature afforded compound 15 as a
trifluoroacetate salt.
R''
NH
R'
O
N
With the protected aminoketone in hand, attention turned to the
indole fragment. We planned to synthesize differently substituted
rings to verify whether a change in the position of the hydroxy
groups could influence the cytotoxic activity. Compounds 17 were
obtained by reacting aldehydes 16 with ethyl azidoacetate in the
presence of sodium ethoxide at ꢀ15 °C in ethanol, followed by cycli-
zation in xylene at reflux. N-alkylation and basic hydrolysis of the
ester group gave the indolecarboxylic acids 18, that were coupled
3
R
R'''
R''
O
NH
with the previously obtained a-aminoketone 15 in the presence of
R'
HOBt and WSC in THF (Scheme 3). Subsequently, b-oxoamides 19
were treated with TFA to perform the crucial Friedel–Crafts cycliza-
tion step by which the b-carbolin-1-one derivatives 20 were ob-
tained. The cleavage of the protecting groups was carried out with
AlCl3 in anisole under heating. The treatment of compound 20a with
AlCl3 in DCM produced the N-benzyl protected compound 21a.
The synthetic strategy reported in Schemes 2 and 3 was applied
to the synthesis of compound 2. To obtain the desired compound it
was necessary to introduce an additional OH group on the benzyl-
amine fragment, for the construction of the oxazine ring. Moreover,
the new phenolic moiety had to be protected with a differently
cleavable protecting group, in order to remove it in a different step
with respect to the isopropyl ethers, that were to be maintained
throughout the whole process.
O
N
R
4
R''
R'''
OH
O
R'
+
N
R
O
6
5
NH2
Scheme 1. Retrosynthetic approach to compounds 3.
Due to the sensitivity of the free indole ring to acidic reagents, it
appeared more appropriate to use indole synthon with a stable
protecting group, such as methyl. This choice was supported by
the modeling studies that indicated no specific interaction due to
the NH indole moiety. The synthesis of compound 27 (Scheme 4)
was performed starting from commercially available 2,4,5-tri-
methoxybenzaldehyde (23). Selective ortho/para methylether
cleavage by AlCl3 in DCM provided compound 24 in good yield.
The treatment with 2-bromopropane and sodium bicarbonate in
DMF allowed a selective protection of the p-hydroxy group, due
Scheme 2. The phenol group was protected as isopropyl ether 8,
that could be selectively cleaved at the end of the synthetic route
providing the desired hydroxy–methoxy alternation. Protection
of vanillin 9 as isopropylether 10, followed by conversion into
the corresponding oxime and reduction with Raney-Ni alloy, gave
the benzylamine derivative 11, that was coupled with 8 using
HOBt (1-hydroxybenzotriazole) and WSC [N-(3-dimethylamino-
propyl)-N0-ethylcarbodiimide] in THF. After protection of the
amide nitrogen with a BOC group, compound 13 was treated with
an excess of LDA to obtain the
a-aminoketone 14 via a N–C
COOH
COOH
a, b
OCH3
i-PrO
OCH3
OCH3
OCH3
g
OH
f
OiPr
8
7
OPr-i
HN
CHO
OiPr
CHO
CH2NH2
12
c
d, e
O
OCH3
10
OCH3
OCH3
OH
OiPr
9
11
OCH3
H3CO
OPr-i
i-PrO
H3CO
H3CO
OPr-i
i-PrO
H3CO
i-PrO
OCH3
OPr-i
h
i
N
O
+
Boc
O
NH3
NHBoc
CF3COO-
13
15
14
O
Scheme 2. Reagents and conditions: (a) iPrBr, K2CO3, DMF, reflux, 4 h, 98%; (b) KOH, ethanol, H2O, reflux, 4 h, 86%; (c) iPrBr, Na2CO3, DMF, reflux, 4 h, 91%; (d) NH2OHꢁHCl, Py,
ethanol, rt, 90 min, 71%; (e) Ni/Al alloy, NaOH 2 N, ethanol, rt, 90 min, 98%; (f) HOBt, WSC, THF, rt, 24 h, 96%; (g) di-tert-butyldicarbonate, DMAP, acetonitrile, rt, 16 h, 95%; (h)
LDA, DMPU, THF, ꢀ78 °C, 8 h, 55%; (i) CF3COOH, DCM, rt, 2 h, 98%.

S. Cananzi et al. / Bioorg. Med. Chem. 19 (2011) 4971–4984
4975
R1
R2
CHO
R1
R2
R1
R2
e
a, b
c, d
COOEt
17 a-b
COOH
18 a-c
N
H
N
R3
16 a-b
OPr-i
H3CO
OPr-i
i-PrO
OPr-i
OCH3
H3CO
R1
R2
OCH3
f
O
N
R1
R2
NH
O
NH
O
a R1 = OCH3 R2 = OBn R3 = Bn
b R1 = OBn R2 = OCH3 R3 = Bn
c R1 = H R2 = H R3 = CH3
N
R3
19 a-c
20 a-c
R3
g
g
OH
OCH3
HO
H3CO
H3CO
HO
OH
HO
OCH3
H3CO
R1
h
a R1 = OCH3 R2 = OH R3 = H
b R1 = OH R2 = OCH3 R3 = H
c R1 = H R2 = H R3 = CH3
NH
O
NH
O
21 a
3a-c
R2
N
N
Bn
R3
Scheme 3. Reagents and conditions: (a) ethyl azidoacetate, EtONa, EtOH, ꢀ15 °C, 3 h, 73–88%; (b) xylene, reflux, 12 h, 32–76%; (c) for 17a–b: BnBr, NaH, DMF, 0 °C–rt, 2 h,
67–97%; (d) LiOH, H2O, THF, MeOH, rt, 4 h, 64–76%; (e) HOBt (1-hydroxybenzotriazole), WSC, 15, TEA, THF, rt, 14 h, 56–58%; (f) TFA, acetonitrile, reflux, 15 min, 58–71%; (g)
AlCl3, DCM, 0 °C–rt, 24 h, 30–48%; (h) for 3a: AlCl3, anisole, 100 °C, 5 h, 20%.
CHO
OPr-i
CHO
OPr-i
CHO
CHO
OH
OH
OMEM
OCH3
OH
a
b
c
d,e
H3CO
OCH3
H3CO
H3CO
H3CO
24
26
25
OCH3
23
CH2NH2
i-PrO
OMEM
i-PrO
H3CO
OMEM
OCH3
OMEM
g
f
OPr-i
OPr-i
H3CO
H3CO
N
O
HN
29
Boc
28
27
OPr-i
O
H3CO
OPr-i
i-PrO
H3CO
OPr-i
i-PrO
H3CO
i
h
H3CO
+OMEM
NH3
CF3COO-
OMEM
O
O
NH
31
30
Boc
Scheme 4. Reagents and conditions: (a) AlCl3, DCM, N2, rt, 19 h, 84%; (b) iPrBr, NaHCO3, DMF, 70 °C, 4 h, 34%; (c) MEMCl, NaH, THF, rt, 12 h, 94%; (d) NH2OHꢁHCl, Py, EtOH,
0 °C, 90 min, 89%; (e) Al/Ni alloy, EtOH, rt, 90 min, 97%; (f) 8, HOBt, WSC, THF, rt, 24 h, 95%; (g) di-tert-butyl dicarbonate, DMAP [4-(dimethylamino)pyridine], acetonitrile, rt,
16 h, 67%; (h) LDA (lithium diisopropylamide), DMPU [1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone], THF, ꢀ78 °C, 6 h, 29%; (i) CF3COOH, DCM, 0 °C, 2 h.
to the different reactivity of the two phenolic hydroxyls, one of
them H-bonded to the ortho carbonyl. Finally, compound 25 was
protected with a MEM (2-methoxyethoxymethyl) group by react-
ing it with MEM chloride in the presence of NaH. The aldehyde
26 was then converted into the corresponding oxime, then reduced
using Ni/Al alloy and NaOH in ethanol to give benzylamine 27 in
good yield.
Synthesis of the N-BOC a-aminoketone was achieved following
the same procedure described in Scheme 2. In this case the N-BOC
removal resulted troublesome, due to the presence of the MEM
protecting group, which is cleavable under the same acidic condi-
tions. For this reason, alternative procedures were investigated,
such as thermolytic treatment in solid phase under nitrogen, silica
gel promoted cleavage, and cerium ammonium nitrate treatment

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S. Cananzi et al. / Bioorg. Med. Chem. 19 (2011) 4971–4984
in acetonitrile. All these efforts failed to produce a selective
removal. Finally, a kinetic cleavage of the two protecting groups
in acidic conditions was attempted. The reaction was carried out
in DCM and TFA under nitrogen at 0 °C (monitoring the course
the same cytotoxic activity, confirming that the introduction of a
methyl group on the indole nitrogen was not deleterious for activ-
ity. Collectively, the modest activity shown by this series of com-
pounds may be presumably due to lack of planarity, as already
observed for other analogues of Lam-D.11
Remarkably, compound 2b, possessing a planar 3,4-dihydro-
2H-[1,3]oxazine core, showed a significant increase in cytotoxicity
compared to the previously discussed derivatives. This is consis-
tent with the docking experiments. Moreover, comparing 2b with
the unsubstituted analogue 38, it can be seen that deletion of the
OH groups has a dramatic effect on the activity, giving a 30-fold
drop in cytotoxicity. This can probably be attributed to the capacity
of 2b to form hydrogen bonds with the active site, as described for
Lam-D6a and predicted by molecular modeling.
by 1H NMR analysis) and afforded
acetate salt.
a-aminoketone 31 as a trifluoro-
The coupling with indolecarboxylic acid 32 was performed fol-
lowing the same procedure described in Scheme 3 and gave amide
33 (Scheme 5). Attempts to obtain the key b-carbolin-1-one scaf-
fold following the intramolecular Friedel–Crafts cyclization failed,
due to the instability of MEM group in acidic conditions: in the
presence of TFA it released formaldehyde that immediately at-
tacked the extremely reactive position 3 of the indole moiety. To
avoid this side reaction, other conditions were investigated. Inter-
estingly, it was found that treatment of 33 with two equivalents of
trimethylsilyl iodide (TMSI) afforded the desired compound 34.
Ring-closure to give the tetracyclic compound 35 was achieved
using diiodomethane in DCM under basic conditions. Finally, re-
moval of protecting isopropyl and benzyl ether groups with AlCl3
in DCM at room temperature gave the final compound 2b in good
yield.
2.3.2. Topoisomerase I-dependent DNA cleavage assay
Topoisomerase I-mediated DNA cleavage experiments were
performed to investigate the ability of the new compounds to stim-
ulate DNA damage. Lam-D and SN-38 (10-hydroxy-7-ethylcampto-
thecin) were used as reference compounds.
The results of topoisomerase I-mediated DNA damage are pre-
sented in Figure 3. Marked topI poisoning was detected for
SN-38, used as a positive control. Similarly, DNA damage was ob-
served in the presence of Lam-D, indicating that this natural prod-
uct also stabilizes DNA–topI covalent complex. Virtually no
cleavage was detected in the presence of 20a, 21a, 3a, this result
being consistent with the very low cytotoxicity showed by these
compounds. In contrast, compound 2b was found to stimulate
DNA cleavage by topI, although less efficiently than SN-38 and
Lam-D. Interestingly, the new molecule showed the same cleavage
profile of the controls, therefore validating the conclusion that 2b
can be identified as a new topI poison.
To investigate the role of the oxygenated groups on the aro-
matic rings, the unsubstituted analogue 38 was synthesized fol-
lowing a similar sequence (Scheme 6).
2.3. Biological activity
2.3.1. Cytotoxicity studies
The antiproliferative effect of the new molecules was deter-
mined at 1 h exposure on human non-small cell lung cancer
H460 cell line (Table 1). Topotecan, a camptothecin derivative in
clinical use, and Lam-D were used as reference drugs. The antipro-
liferative effect of the most active compounds was also evaluated
at 72 h exposure.
All the tested derivatives, except for 2b, exhibited a moderate
cytotoxicity, much lower than Lamellarin D. In a general overview,
compounds possessing the more flexible skeleton of 3 were less po-
tent in reducing cell growth. It can be seen that the cytotoxic po-
tency varies considerably depending on the presence of free OH
substituents on the ring system. Comparing compounds 20a–
21a–3a we can notice that 20a, with bulky substituents on all the
aromatic rings, proved to be almost totally inactive, whereas pro-
gressive removal of protecting groups (21a, 3a) corresponded to
an increase in cytotoxic activity. Compounds 3a and 3c showed
Overall, our results are consistent with previous findings on
lamellarin derivatives, such as the critical importance of the pla-
narity of the molecule skeleton.11 In addition, the presence of
OH–hydrogen bond donors positionally equivalent to hydroxy
groups at C-8 and C-20 in Lam-D led to a strong gain in
cytotoxicity.
3. Conclusions
In conclusion, on the basis of molecular modeling studies, we
identified
a
new molecular scaffold that could mimic the
OPr-i
H3CO
OPr-i
i-PrO
H3CO
H3CO
H3CO
BnO
OCH3
+
OPr-i
b
COOH
CH3
a
N
+OMEM
NH3
CF3COO-
O
O
N
32
33
31
OMEM
H3CO
BnO
NH
O
CH3
H3CO
i-PrO
H3CO
H3CO
BnO
OPr-i
H3CO
OPr-i
i-PrO
H3CO
H3CO
BnO
H3CO
OH
O
HO
H3CO
d
c
OH
O
H3CO
HO
NH
O
N
O
N
O
N
CH3
N
CH3
N
34
35
2b
CH3
Scheme 5. Reagents and conditions: (a) HOBt, WSC, TEA, THF, rt, 26 h, 55%; (b) NaI, TMSCl, acetonitrile, ꢀ20 °C, 30 min, 31%; (c) CH2I2, DCM, reflux, 1 h, 12%; (d) AlCl3, DCM,
reflux, 6 h, 83%.

S. Cananzi et al. / Bioorg. Med. Chem. 19 (2011) 4971–4984
4977
OH
H2N
HOH2C
N
OCH3
OCH3
O
a
b-e
NH
O
N
O
+
COOH
N
N
CH3
38
37
36
18c
CH3
CH3
Scheme 6. Reagents and conditions: (a) HOBt, WSC, TEA, THF, rt, 2 h, 97%; (b) IBX (2-iodoxybenzoic acid), DMSO, acetonitrile, rt, 3 h; (c) TFA, acetonitrile, reflux, 15 min, 64%;
(d) BBr3, DCM, rt, overnight, 38%; e) CH2I2, DCM, reflux, 1 h, 28%.
rings. The synthesized compounds were tested to evaluate their
antiproliferative effect on human non small cell lung cancer H460
cell line and the topI-mediated DNA cleavage. Collectively, the com-
Table 1
Antiproliferative activity of Lamellarin D analogues
IC50 (l
M)a
pounds resulted less active than Lam-D. However, the obtained data
provided information about the importance of a planar structure
and of hydrogen bond donors at appropriate positions on the aro-
matic ring. In fact, compound 2b, possessing a planar 3,4-dihydro-
2H-[1,3]oxazine core, and phenolic OHs positionally equivalent to
hydroxy groups at C-8 and C-20 in Lam-D, showed remarkable cyto-
toxicity and topI–DNA cleavage ability.
Therefore, we have reached the proof of concept that the scaf-
fold of lamellarins can be modified to obtain new cytotoxic and
topI-active compounds, provided that the crucial peripheral inter-
actions are maintained.
1 h
72 h
Topotecan
Lam-D
20a
20c
21a
3a
3b
3c
2b
38
1.75 0.22
0.066 0.004
>150
60.8 2.6
118.8 5.6
60.8 5.3
60.8 4.2
60.8 7.1
4.02 0.95
0.016 0.005
0.003 0.0006
–
–
–
–
–
–
0.35 0.030
–
117.5
3
a
Drug concentration required for 50% reduction of cell growth as compared with
untreated controls after 1 and 72 h exposure to the drug.
4. Experimental section
4.1. General
interactions of lamellarins with the topI–DNA complex. We de-
signed the replacement of the pyrrolo[2,1-a]isoquinoline core with
a b-carbolin-1-one moiety, while maintaining the crucial hydroxy
and methoxy groups at the appropriate positions on the aromatic
All reagents and solvents were reagent grade or were purified
by standard methods before use. Melting points were determined
Figure 3. Topoisomerase I-mediated DNA cleavage by SN38, Lamellarin D, and analogues. Samples were reacted with 1, 10, and 50 lM (100 lM for 2b) drug at 37 °C for
30 min. Reaction was then stopped by adding 0.5% SDS, 0.3 mg/mL of proteinase K, and incubating for 45 min at 42 °C before loading on a denaturing 8% polyacrylamide gel. C,
control DNA; T, reaction without drug; M, purine markers.

4978
S. Cananzi et al. / Bioorg. Med. Chem. 19 (2011) 4971–4984
in open capillaries. NMR spectra were recorded with a Bruker AMX
300 spectrometer. Mass spectra were recorded with a Fourier
transform ion cyclotron resonance (FT-ICR) mass spectrometer
4.4. (4-Isopropoxy-3-methoxyphenyl)methanamine (11)
A solution of 10 (4.0 g, 20.6 mmol) in 95% ethanol (44 mL)
was treated with a solution of hydroxylamine hydrochloride
(1.82 g, 26.2 mmol) in dry pyridine (8.13 mL) under stirring
at room temperature for 90 min, then at 0 °C for 30 min to facil-
itate 4-isopropoxy-3-methoxybenzaldoxime precipitation. The
white solid was filtered, dried (3.20 g, 74%) and used without
further purification; mp 130 °C. 1H NMR (CDCl3) d: 8.09 (1H,
s), 7.23 (1H, d, J = 1.9 Hz), 7.03 (1H, dd, J = 1.9 and 8.2 Hz),
6.89 (1H, d, J = 8.2 Hz), 4.60 (1H, m), 3.89 (3H, s,), 1.40 (6H, d,
J = 6.1 Hz).
A solution of the oxime (3.15 g, 15.0 mmol) in 95% ethanol
(38.5 mL) was treated with an equal volume of 2 M NaOH followed
by Al/Ni alloy (4 g) under stirring at room temperature for 90 min.
The Al/Ni alloy was removed by filtration and washed with fresh
ethanol. Filtrate and washings were combined, acidified with
0.8 M HCl (pH 2) and extracted with DCM (3 ꢂ 15 mL). The aque-
ous phase was treated with solid KOH up to pH 14 and extracted
with diethyl ether (3 ꢂ 10 mL). The extracts, after drying over
anhydrous Na2SO4 and removal of the solvent, provided 11
(2.88 g, 98%) as a brown oil. 1H NMR (CDCl3) d: 6.90 (1H, dd,
J = 2.1 and 8.2 Hz), 6.80 (1H, d, J = 8.2 Hz), 6.75 (1H, d, J = 2.1 Hz),
4.56 (1H, m), 3.89 (3H, s), 3.75 (2H, s), 1.40 (6H, d, J = 6.10 Hz).
Anal. Calcd for C11H17NO2: C, 67.66; H, 8.78; N, 7.17. Found: C,
67.62; H, 8.75; N, 7.12.
APEX II
& Xmass software (Bruker Daltonics)-4.7T Magnet
(Magnex). The Elemental Analyses were recorded with a CARLO
ERBA EA 1108 instrument. Solvents were routinely distilled prior
to use; anhydrous tetrahydrofuran (THF) and ether (Et2O) were ob-
tained by distillation from sodium–benzophenone ketyl; dry
dichloromethane was obtained by distillation from phosphorus
pentoxide. All reactions requiring anhydrous conditions were per-
formed under a positive nitrogen flow, and all glassware were oven
dried and/or flame dried. Isolation and purification of the com-
pounds were performed by flash column chromatography on silica
gel 60 (230–400 mesh). Analytical thin-layer chromatography
(TLC) was conducted on TLC plates (silica gel 60 F254, aluminum
foil) visualized by UV light and spraying with phosphomolybdic
acid and p-anisaldehyde.
4.2. 4-Isopropoxy-3-methoxybenzoic acid (8)
Vanillic acid 7 (12 g, 71.4 mmol) was dissolved in DMF (70 mL)
under nitrogen atmosphere and K2CO3 (24.53 g, 0.177 mol) was
added. The suspension was stirred for 15 min at room tempera-
ture and subsequently 2-bromopropane (35.11 g, 285.4 mmol,
26 mL) was added dropwise. After refluxing for 4 h, the suspen-
sion was filtered and the solvent was evaporated under reduced
pressure. The crude product was dissolved in ethyl acetate
(80 mL) and the solution was washed with NaHCO3 (3 ꢂ 20 mL)
and brine (3 ꢂ 20 mL). The organic layer was dried over Na2SO4,
filtered and evaporated under reduced pressure giving 4-isopro-
poxy-3-methoxybenzoic acid isopropyl ester as a yellow solid
(17.65 g, 98%). 1H NMR (CDCl3) d: 7.62 (1H, dd, J = 1.7 and
8.4 Hz), 7.53 (1H, d, J = 1.7 Hz), 6.87 (1H, d, J = 8.4 Hz), 5.21
(1H, m), 4.62 (1H, m), 3.89 (3H, s), 1.38 (6H, d, J = 6.1 Hz), 1.34
(6H, d, J = 6.1 Hz).
4.5. N-(4-Isopropoxy-3-methoxybenzyl)-4-isopropoxy-3-meth
oxybenzamide (12)
To a suspension of HOBt (1.89 g, 14.0 mmol) and WSC (2.68 g,
14.0 mmol) in THF (110 mL) were added under nitrogen atmo-
sphere 8 (1.46 g, 6.9 mmol) and 11 (2.70 g, 13.8 mmol). The reac-
tion mixture was stirred at room temperature for 24 h and the
solvent was evaporated under reduced pressure. The crude product
was dissolved in ethyl acetate, the solution washed with NaHCO3,
HCl 2 M and brine, dried over Na2SO4 and evaporated under re-
duced pressure to afford 12 as a yellow oil (2.58 g, 96%). 1H NMR
(CDCl3) d: 7.47 (1H, s), 7.28 (1H, s), 6.81–6.93 (4H, m), 6.37 (1H,
br s), 4.45–4.70 (4H, m), 3.91 (3H, s), 3.85 (3H, s), 1.40 (6H, d,
J = 6.2 Hz), 1.37 (6H, d, J = 6.3 Hz). 13C NMR (DMSO-d6) d: 166.2,
150.5, 149.8, 149.6, 146.1, 133.4, 127.2, 120.9, 119.9, 116.5,
114.2, 112.5, 111.7, 71.0, 70.6, 56.0, 42.9, 22.5, 22.4. Anal. Calcd
for C22H29NO5: C, 68.20; H, 7.54; N, 3.61. Found: C, 68.24; H,
7.51; N, 3.70.
The ester (17.60 g, 69.7 mmol) was dissolved in 90% ethanol
(100 mL) and KOH (5.89 g, 104.9 mmol) was added. The solution
was refluxed for 4 h. The solvent was evaporated under reduced
pressure and the crude product was dissolved in water adding
HCl solution until complete precipitation occurred (pH 3). The sus-
pension was extracted with ethyl acetate, the organic layer was
dried over Na2CO3, filtered and evaporated under reduced pressure
to give compound 8 (12.70 g, 86%) as a white solid; mp 120 °C. 1
H
NMR (CDCl3) d: 7.73 (1H, dd, J = 2.1 and 8.9 Hz), 7.59 (1H, d,
J = 2.1 Hz), 6.91 (1H, d, J = 8.9 Hz), 4.66 (1H, m), 3.91 (3H, s), 1.41
(6H, d, J = 6.10 Hz). Anal. Calcd for C11H14O4: C, 62.85; H, 6.71.
Found: C, 62.88; H, 6.69.
4.6. (4-Isopropoxy-3-methoxybenzoyl)-(4-isopropoxy-3-meth
oxybenzyl)-carbamic acid tert-butyl ester (13)
4.3. 4-Isopropoxy-3-methoxybenzaldehyde (10)
Compound 12 (2.20 g, 5.6 mmol) was dissolved in acetonitrile
(23 mL) under nitrogen flow and DMAP (0.073 g, 0.6 mmol) and
di-tert-butyldicarbonate (2.62 g, 12.0 mmol) were added. After
stirring the solution at room temperature for 16 h, the solvent
was evaporated under reduced pressure and the crude product
was dissolved in DCM. The solution was washed with saturated
aqueous NaHCO3 and the organic layer was dried over Na2SO4, fil-
tered and evaporated under reduced pressure. The crude product
was purified by flash chromatography (hexane:ethyl acetate, 6:4)
affording 13 (2.60 g, 95%) as a yellow oil. 1H NMR (CDCl3) d: 7.60
(1H, dd, J = 1.7 and 8.8 Hz), 7.53 (1H, d, J = 1.7 Hz), 6.77–6.94 (4H,
m), 4.63 (1H, m), 4.48 (1H, m), 3.87 (3H, s), 3.83 (3H, s), 3.62
(1H, d, J = 9.9 Hz), 1.45 (9H, s), 1.35 (12H, d, J = 6.5 Hz). Anal. Calcd
for C27H37NO7: C, 66.51; H, 7.65; N, 2.87. Found: C, 66.41; H, 7.61;
N, 2.80.
Vanillin 9 (3.95 g, 25.9 mmol) was dissolved in DMF (20 mL)
under nitrogen atmosphere and K2CO3 (4.13 g, 29.9 mmol) was
added. The suspension was stirred for 15 min at room temperature
and subsequently 2-bromopropane (6.40 g, 52.0 mmol, 4.88 mL)
was added dropwise. After refluxing for 4 h, the suspension was fil-
tered and the solvent was evaporated under reduced pressure. The
crude product was dissolved in ethyl acetate (20 mL) and the solu-
tion was washed with brine (3 ꢂ 15 mL). The organic layer was
dried over Na2SO4, filtered and evaporated affording compound
10 as a yellow solid (4.07 g, 81%); mp 114–116 °C. 1H NMR (CDCl3)
d: 9.84 (1H, s), 7.36–7.45 (2H, m), 6.97 (1H, d, J = 8.2 Hz), 4.70 (1H,
m), 3.92 (3H, s), 1.43 (6H, d, J = 5.9 Hz). Anal. Calcd for C11H14O3: C,
68.02; H, 7.27. Found: C, 68.09; H, 7.22.

S. Cananzi et al. / Bioorg. Med. Chem. 19 (2011) 4971–4984
4979
4.7. tert-Butyl 1,2-bis(4-isopropoxy-3-methoxyphenyl)-2-oxo
ethylcarbamate (14)
45 min, then benzyl bromide (3.7 mmol, 0.43 mL) was added and
the mixture was reacted for 2 h. When the reaction was completed
the solvent was removed under vacuo. The crude product was dis-
solved in ethyl acetate and washed with brine. The organic layer
was dried over Na2SO4, filtered and evaporated under reduced
pressure to afford ethyl 1-benzyl-6-(benzyloxy)-5-methoxy-1H-in-
dole-2-carboxylate as a white solid (0.730 g, 67%); mp 190–191 °C.
1H NMR (CDCl3) d: 7.11–7.47 (9H, m), 7.07 (1H, s), 6.91–7.00 (2H,
m), 6.74 (1H, s), 5.69 (2H, s), 5.11 (2H, s), 4.29 (2H, q, J = 7.1 Hz),
3.91 (3H, s), 1.33 (3H, t, J = 7.1 Hz).
The ester (0.725 g, 1.7 mmol) was dissolved in 3:2:1 THF:MeOH:-
H2O (6 mL), treated with LiOHꢁH2O (0.292 g, 6.9 mmol), and the mix-
ture was stirred for 4 h at 25 °C. 1 N aqueous HCl was added and the
solution was extracted with ethyl acetate (3 ꢂ 10 mL). The com-
bined organic extracts were washed with brine, dried over Na2SO4,
and concentrated in vacuo to afford 18a (0.500 g, 76%); mp 200 °C.
1H NMR (CDCl3) d: 7.13–7.43 (9H, m), 7.07 (1H, s), 6.91–6.99 (2H,
m), 6.72 (1H, s), 5.68 (2H, s), 5.12 (2H, s), 3.91 (3H, s). 13C NMR
(CDCl3) d: 165.9, 149.6, 147.1, 138.0, 136.7, 135.5, 129.2, 128.6,
128.2, 127.8, 127.5, 126.8, 126.1, 124.8, 119.5, 95.7, 70.8, 55.5,
53.9. Anal. Calcd for C24H21NO4: C, 74.40; H, 5.46; N, 3.62. Found:
C, 74.29; H, 5.41; N, 3.69.
LDA was prepared by mixing diisopropylamine (1.52 g,
15.0 mmol, 2.10 mL) and DMPU (1.05 mL) with n-BuLi (1.59 M in
hexane, 9.4 mL, 15.0 mmol) in THF (14 mL) at ꢀ78 °C under nitro-
gen atmosphere. A solution of 13 (2.40 g, 4.9 mmol) in freshly dis-
tilled THF (43 mL) was added to the LDA solution at ꢀ78 °C. The
mixture was stirred for 8 h, then it was quenched with aqueous
NH4Cl, diluted with ethyl acetate, washed with saturated aqueous
NH4Cl and brine, and dried over Na2SO4. Solvent evaporation fol-
lowed by flash chromatography (hexane:ethyl acetate, 7:3) gave
product 14 (1.21 g, 55%) as a white solid; mp 120–122 °C. 1H
NMR (CDCl3) d: 7.60 (1H, dd, J = 1.7 and 8.8 Hz), 7.53 (1H, d,
J = 1.7 Hz), 6.77–6.94 (4H, m), 4.63 (1H, m), 4.48 (1H, m), 3.87
(3H, s), 3.83 (3H, s), 3.62 (1H, d, J = 9.9 Hz), 1.45 (9H, s), 1.35
(12H, d, J = 6.5 Hz). Anal. Calcd for C27H37NO7: C, 66.51; H, 7.65;
N, 2.87. Found: C, 66.47; H, 7.62; N, 2.91.
4.8. 2-Amino-1,2-bis(4-isopropoxy-3-methoxyphenyl)ethanone
trifluoroacetate (15)
Compound 14 (1.21 g, 2.3 mmol) was dissolved in dry DCM
(4 mL) under nitrogen flow and TFA (2 mL) was added at 0 °C.
The solution was stirred at rt for 2 h. The solvent was evaporated
under vacuo and the crude product was used for the next step
without further purification.
4.11. N-(1,2-Bis(4-isopropoxy-3-methoxyphenyl)-2-oxoethyl)-
1-benzyl-6-(benzyloxy)-5-methoxy-1H-indole-2-carboxamide
(19a)
To a suspension of HOBt (0.739 g, 5.4 mmol) and WSC (1.04 g,
5.4 mol) in THF (44 mL) under nitrogen atmosphere 18a (2.09 g,
5.4 mmol) was added. The reaction mixture was stirred at room
temperature for 12 h until the activated ester was formed, then
compound 15 dissolved in THF (10 mL) and TEA (5.4 mmol,
0.75 mL) were added. After stirring the reaction mixture at room
temperature for 14 h, the solvent was evaporated under reduced
pressure. The crude product was dissolved in ethyl acetate, the
solution washed with aqueous NaHCO3, HCl 2 M and brine, dried
over Na2SO4 and evaporated under reduced pressure to afford
19a as a yellow solid (2.24 g, 55%); mp 140–141 °C. 1H NMR
(CDCl3) d: 7.50–7.68 (3H, m), 7.21–7.43 (5H, m), 7.09–7.18 (3H,
m), 7.06 (1H, s), 6.88–7.00 (5H, m), 6.76–6.87 (2H, m), 6.73 (1H,
s), 6.59 (1H, d, J = 7.8 Hz), 5.72 (1H, AB, J = 16.1 Hz), 5.59 (1H, AB,
J = 16.1 Hz), 5.10 (2H, s), 4.62 (1H, m), 4.49 (1H, m), 3.91 (3H, s),
3.86 (3H, s), 3.79 (3H, s), 1.38 (6H, d, J = 6.1 Hz), 1.32 (6H, d,
J = 6.1 Hz). 13C NMR (CDCl3) d: 194.8, 162.0, 151.9, 150.2, 149.6,
148.1, 147.0, 146.5, 139.3, 137.5, 133.7, 129.7, 129.6, 128.8,
128.6, 128.4, 127.3, 119.3, 113.5, 107.3, 103.5, 96.3, 70.1, 58.0,
56.9, 56.3, 54.7, 22.5, 22.4. Anal. Calcd for C46H48N2O8: C, 73.00;
H, 6.39; N, 3.70. Found: C, 73.08; H, 6.31; N, 3.77.
4.9. Ethyl 6-(benzyloxy)-5-methoxy-1H-indole-2-carboxylate
(17a)
Na (1.13 g, 49.2 mmol) was dissolved in anhydrous EtOH
(45 mL), treated with 4-benzyloxy-3-methoxybenzaldehyde (3 g,
12.4 mmol), and the mixture was cooled to ꢀ15 °C. Ethyl azidoac-
etate (ethanol solution 25%, 49.2 mmol, 25 mL) was added drop-
wise to the reaction mixture. The mixture was stirred for 3 h at
ꢀ15 °C, then warmed to room temperature and treated with satu-
rated aqueous NH4Cl. The beige solid that formed was collected by
filtration and washed with water to afford (Z)-ethyl 2-azido-3-(4-
(benzyloxy)-3-methoxyphenyl)acrylate (2.95 g, 73%); mp 102–
103 °C. 1H NMR (CDCl3) d: 7.53 (1H, d, J = 1.9 Hz), 7.26–7.50 (6H,
m), 6.85–6.93 (2H, m), 5.22 (2H, s), 4.38 (2H, q, J = 7.2 Hz), 3.95
(3H, s), 1.41 (3H, t, J = 7.2 Hz).
The above compound (2.90 g, 8.1 mmol) was dissolved in xy-
lenes (20 mL) and the solution was warmed at 130 °C for 12 h.
The solution was cooled to room temperature and hexane was
added (5 mL). The beige solid was collected by filtration. The fil-
trated was concentrated in vacuo and purified by flash chromatog-
raphy (hexane:ethyl acetate, 8:2). The precipitate was combined
with the product isolated by chromatography to afford 17a
(0.847 g, 32%); mp 218–220 °C. 1H NMR (CDCl3) d: 8.63 (1H, br
s), 7.23–7.50 (5H, m), 7.01–7.12 (2H, m), 6.83 (1H, s), 5.20 (2H,
s), 4.35 (2H, q, J = 7.1 Hz), 3.92 (3H, s), 1.38 (3H, t, J = 7.1 Hz). 13C
NMR (CDCl3) d: 161.5, 148.9, 146.6, 136.7, 131.6, 128.4, 127.7,
126.9, 126.0, 120.8, 108.4, 103.0, 96.3, 71.0, 60.5, 56.2, 14.2. Anal.
Calcd for C19H19NO4: C, 70.14; H, 5.89; N, 4.31. Found: C, 70.22;
H, 5.79; N, 4.35.
4.12. 9-Benzyl-7-(benzyloxy)-3,4-bis(4-isopropoxy-3-methoxy
phenyl)-6-methoxy-2H-pyrido[3,4-b]indol-1(9H)-one (20a)
To a solution of 19a (1.10 g, 1.4 mmol) in acetonitrile (8 mL)
was added TFA (0.16 mL) and the reaction mixture was refluxed
for 15 min. The precipitate formed was filtered and washed with
acetonitrile to afford 20a as a white solid (0.764 g, 71%); mp
238 °C. 1H NMR (DMSO-d6) d: 7.05–7.51 (12H, m), 6.63–7.03 (7H,
m), 6.04 (2H, m), 5.16 (2H, s), 4.36–4.60 (2H, m), 3.60 (3H, s),
3.57 (3H, s), 3.37 (3H, s), 1.25 (6H, d, J = 5.5 Hz), 1.21 (6H, d,
J = 5.6 Hz). 13C NMR (DMSO-d6) d: 156.1, 150.5, 146.7, 146.3,
139.2, 137.3, 134.7, 130.3, 129.3, 128.7, 128.3, 128.0, 127.7,
127.4, 126.0, 124.5, 114.7, 114.2, 79.5, 70.6, 70.1, 56.7, 54.2, 22.6,
22.3. Anal. Calcd for C46H46N2O7: C, 74.78; H, 6.28; N, 3.79. Found:
C, 74.81; H, 6.25; N, 3.74. HRMS (ESI positive) Calcd for
4.10. 1-Benzyl-6-(benzyloxy)-5-methoxy-1H-indole-2-carbox
ylic acid (18a)
Indole 17a (0.840 g, 2.6 mmol) was dissolved in dry DMF (8 mL)
under nitrogen. The solution was cooled at 0 °C and NaH (mineral
oil susp. 60%, 0.146 g, 3.7 mmol) was added slowly. The reaction
mixture was warmed to room temperature and stirred for
C
46H46N2O7Na: 761.31972. Found: 761.31644 [M+Na]+.

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S. Cananzi et al. / Bioorg. Med. Chem. 19 (2011) 4971–4984
4.13. 9-Benzyl-7-hydroxy-3,4-bis(4-hydroxy-3-methoxyphenyl)
4.16. 1-Benzyl-5-(benzyloxy)-6-methoxy-1H-indole-2-carbox
-6-methoxy-2H-pyrido[3,4-b]indol-1(9H)-one (21a)
ylic acid (18b)
Anhydrous AlCl3 (0.360 g, 2.7 mmol) was suspended in dry DCM
(5 mL) under nitrogen and compound 20a (0.700 g, 0.9 mmol) was
added. After stirring the reaction mixture at room temperature for
24 h, saturated aqueous NH4Cl was added and the mixture was ex-
tracted with ethyl acetate. The organic layer was washed with
brine, dried over Na2SO4 and evaporated under reduced pressure.
The obtained solid was crystallized from ethanol to give product
21a (0.056 g, 11%); mp 297 °C. 1H NMR (DMSO-d6) d: 11.25 (1H,
s), 9.30 (1H, s), 9.07 (1H, s), 9.02 (1H, s), 7.15–7.35 (5H, m), 6.56–
6.93 (7H, m), 6.13 (2H, s), 6.02 (2H, m), 3.64 (3H, s), 3.60 (3H, s),
3.39 (3H, s). 13C NMR (DMSO-d6) d: 156.1, 148.4, 148.0, 147.0,
146.6, 146.2, 144.2, 139.4, 136.5, 134.8, 129.5, 128.4, 128.1,
127.0, 126.5, 126.1, 124.4, 115.3, 114.3, 114.1, 57.0, 55.3, 53.9.
Anal. Calcd for C33H28N2O7: C, 70.20; H, 5.00; N, 4.96. Found: C,
70.14; H, 5.09; N, 4.99. HRMS (ESI positive) Calcd for C33H28N2O7-
Na: 587.17887. Found: 587.17698 [M+Na]+.
Indole 17b (0.740 g, 2.3 mmol) was dissolved in dry DMF (7 mL)
under nitrogen. The solution was cooled at 0 °C and NaH (oil min-
eral susp. 60%, 0.170 g, 4.2 mol) was slowly added. The reaction
mixture was warmed to room temperature and stirred for
45 min, then benzyl bromide (4.2 mmol, 0.5 mL) was added and
the mixture was stirred for 2 h. The solvent was removed under va-
cuo, the crude product was dissolved in ethyl acetate and washed
with brine. The organic layer was dried over Na2SO4, filtered and
evaporated under reduced pressure to afford ethyl 1-benzyl-5-
(benzyloxy)-6-methoxy-1H-indole-2-carboxylate (0.900 g, 97%)
as a white solid; mp 90–91 °C. 1H NMR (CDCl3) d: 7.20–7.48 (9H,
m), 7.12 (1H, s), 7.02 (2H, m), 6.79 (1H, m), 6.79 (1H, s), 5.72
(2H, s), 5.18 (2H, s), 4.32 (2H, q, J = 7.1 Hz), 3.95 (3H, s), 1.36 (3H,
t, J = 7.1 Hz).
The protected indole (0.890 g, 2.1 mmol) was dissolved in a
solution 3:2:1 THF:MeOH:H2O (5 mL), then treated with LiOHꢁH2O
(0.359 g, 8.5 mmol). The mixture was stirred for 4 h at 25 °C. 1 N
aqueous HCl was added and the solution was extracted with ethyl
acetate (3 ꢂ 10 mL). The combined organic extracts were washed
with brine, dried over Na2SO4, and concentrated in vacuo to afford
18b (0.600 g, 73%); mp 130 °C. 1H NMR (CDCl3) d: 7.17–7.48 (9H,
m), 7.10 (1H, s), 6.94–7.06 (2H, m), 6.75 (1H, s), 5.71 (2H, s), 5.15
(2H, s), 3.94 (3H, s). 13C NMR (CDCl3) d: 165.8, 149.7, 147.2,
138.0, 136.5, 135.9, 129.5, 129.0, 128.3, 128.0, 127.9, 127.4,
119.5, 78.2, 57.0, 53.9. Anal. Calcd for C24H21NO4: C, 74.40; H,
5.46; N, 3.62. Found: C, 74.45; H, 5.41; N, 3.61.
4.14. 7-Hydroxy-3,4-bis(4-hydroxy-3-methoxyphenyl)-6-metho
xy-2H-pyrido[3,4-b]indol-1(9H)-one (3a)
Anhydrous AlCl3 (0.188 g, 1.4 mmol) was suspended in anisole
(10 mL) under nitrogen and compound 21a (0.05 g, 0.1 mmol)
was added. After refluxing the reaction mixture at 100 °C for 5 h,
MeOH was added and the solvent was evaporated under reduced
pressure. The crude product was purified by flash chromatography
(DCM–MeOH, 9:1) affording product 22a as a white solid (9.5 mg,
20%); mp 312–315 °C. 1H NMR (DMSO-d6) d: 11.55 (1H, s), 11.13
(1H, s), 9.25 (1H, s), 9.05 (1H, s), 8.99 (1H, s), 6.58–6.91 (7H, m),
6.22 (1H, s), 3.62 (3H, s), 3.59 (3H, s), 3.42 (3H, s). 13C NMR
(DMSO-d6) d: 156.0, 147.8, 146.9, 143.2, 143.9, 141.2, 139.5,
139.2, 134.8, 128.7, 126.7, 125.1, 123.1, 122.9, 122.6, 115.4, 114.9,
114.6, 113.9, 112.6, 109.9, 109.3, 55.6, 55.3, 55.2 34.1. Anal. Calcd
for C26H22N2O7: C, 65.82; H, 4.67; N, 5.90. Found: C, 65.86; H,
4.17. N-(1,2-Bis(4-isopropoxy-3-methoxyphenyl)-2-oxoethyl)-
1-benzyl-5-(benzyloxy)-6-methoxy-1H-indole-2-carboxamide
(19b)
To a suspension of HOBt (0.335 g, 2.5 mmol) and WSC (0.474 g,
2.5 mmol) in THF (20 mL) under nitrogen atmosphere compound
18b (0.481 g, 1.2 mmol) was added. The reaction mixture was stir-
red at room temperature for 12 h until the activated ester was
formed. Then compound 15 dissolved in THF (5 mL) and TEA
(2.8 mmol, 0.346 mL) were added. After stirring the reaction mix-
ture at room temperature for 14 h, the solvent was evaporated un-
der reduced pressure. The crude product was dissolved in ethyl
acetate, the solution washed with aqueous NaHCO3, HCl 2 M and
brine, dried over Na2SO4, and evaporated under reduced pressure
to afford 19b as a yellow solid (0.520 g, 56%); mp 127 °C. 1H
NMR (CDCl3) d: 7.53–7.69 (3H, m), 7.23–7.44 (4H, m), 7.11–7.18
(3H, m), 7.07 (1H, s), 6.92–7.01 (5H, m), 6.77–6.87 (2H, m), 6.74
(1H, s), 6.61 (1H, d, J = 7.4 Hz), 5.74 (1H, AB, J = 16.2 Hz), 5.61
(1H, AB, J = 16.2 Hz), 5.12 (2H, s), 4.65 (1H, m), 4.49 (1H, m), 3.93
(3H, s), 3.88 (3H, s), 3.81 (3H, s), 1.40 (6H, d, J = 6.10 Hz), 1.34
(6H, d, J = 6.10 Hz). 13C NMR (DMSO-d6) d: 194.8, 162.0, 151.9,
150.2, 149.6, 148.1, 147.1, 146.5, 139.3, 137.6, 133.7, 129.8,
129.6, 128.9, 128.8, 128.4, 127.9, 127.4, 123.8, 121.9, 119.4,
115.5, 113.6, 113.5, 112.0, 107.2, 103.7, 96.4, 70.7, 58.3, 56.5,
56.0, 22.5, 22.3. Anal. Calcd for C46H48N2O8: C, 73.00; H, 6.39; N,
3.70. Found: C, 73.04; H, 6.35; N, 3.71.
4.62; N, 5.98. HRMS (ESI positive) Calcd for
C26H22N2O7Na:
497.13192. Found: 497.13346 [M+Na]+.
4.15. Ethyl 5-(benzyloxy)-6-methoxy-1H-indole-2-carboxylate
(17b)
Na (0.377 g, 16.4 mmol) was dissolved in anhydrous EtOH
(15 mL), treated with 3-benzyloxy-4-methoxybenzaldehyde
(1.00 g, 4.1 mol), and the mixture was cooled to ꢀ15 °C. Ethyl azi-
doacetate (ethanol solution 25%, 16.0 mol, 1.044 mL) was added
dropwise to the reaction mixture. The mixture was stirred for 3 h
at ꢀ15 °C, then warmed to room temperature and treated with sat-
urated aqueous NH4Cl. The beige solid that formed was collected by
filtration and washed with water to afford (Z)-ethyl-2-azido-3-(3-
(benzyloxy)-4-methoxyphenyl)acrylate (1.20 g, 88%); mp 94–
95 °C. 1H NMR (CDCl3) d: 7.25–7.50 (7H, m), 6.85–6.95 (2H, m),
5.21 (2H, s), 4.38 (2H, q, J = 7.1 Hz), 3.95 (3H, s), 1.39 (3H, t,
J = 7.1 Hz).
The azido derivative (1.15 g, 3.1 mol) was dissolved in xylenes
(5 mL) and the solution was warmed at 130 °C for 12 h. The solu-
tion was cooled to room temperature and hexane was added
(5 mL). The beige solid was collected by filtration. The filtrated
was concentrated in vacuo and purified by flash chromatography
(hexane:ethyl acetate, 8:2). The precipitate was combined with
the product isolated by chromatography to afford 17b (0.800 g,
76%); mp 177–178 °C. 1H NMR (CDCl3) d: 8.69 (1H, s), 7.30–7.58
(6H, m), 7.12 (1H, m), 6.88 (1H, s), 5.25 (2H, s), 4.40 (2H, s), 4.40
(2H, q, J = 7.02 Hz), 3.95 (3H, s), 1.40 (3H, t, J = 7.07 Hz). Anal. Calcd
for C19H19NO4: C, 70.14; H, 5.89; N, 4.31. Found: C, 70.21; H, 5.85;
N, 4.27.
4.18. 9-Benzyl-6-(benzyloxy)-3,4-bis(4-isopropoxy-3-methoxy
phenyl)-7-methoxy-2H-pyrido[3,4-b]indol-1(9H)-one (20b)
To a solution of 19b (0.500 g, 0.66 mmol) in acetonitrile (5 mL)
was added TFA (0.9 mmol, 0.07 mL)and thereaction mixture was re-
fluxed for 15 min. The precipitate was filtered and washed with ace-
tonitrile to afford 20b as a white solid (0.340 g, 70%); mp 230 °C. 1H
NMR (CDCl3) d: 7.11–7.51 (11H, m), 6.65–7.04 (7H, m), 6.43 (1H, s),
5.96 (2H, m), 5.17 (2H, s), 4.56 (1H, m), 4.44 (1H, m), 3.70 (3H, s), 3.57

S. Cananzi et al. / Bioorg. Med. Chem. 19 (2011) 4971–4984
4981
(3H, s), 3.55 (3H, s), 1.40 (6H, d, J = 5.7 Hz), 1.35 (6H, d, J = 5.7 Hz). 13
C
55.5, 55.2, 34.2, 21.5, 21.4. Anal. Calcd for C32H34N2O5: C, 72.98;
H, 6.51; N, 5.32. Found: C, 73.09; H, 6.56; N, 5.42. HRMS (ESI posi-
NMR (CDCl3) d: 155.6, 150.0, 148.9, 148.8, 146.5, 145.0, 144.7, 137.7,
136.2, 135.5, 132.9, 129.4, 128.1, 128.0, 127.4, 127.0, 126.8, 126.6,
126.0, 124.0, 120.9, 115.4, 114.7, 114.6, 114.3, 114.0, 112.9, 103.7,
95.0, 71.1, 70.6, 55.5, 55.2, 55.1, 21.6, 21.4. Anal. Calcd for
tive) Calcd for
C32H34N2O5Na: 549.23599. Found: 549.23754
[M+Na]+.
C
46H46N2O7: C, 74.78; H, 6.28; N, 3.79. Found: C, 74.87; H, 6.32; N,
4.22. 3,4-Bis(4-hydroxy-3-methoxyphenyl)-9-methyl-2H-
pyrido[3,4-b]indol-1(9H)-one (3c)
3.70. HRMS (ESI positive) Calcd for C46H46N2O7Na: 761.31972.
Found: 761.32011 [M+Na]+.
Anhydrous AlCl3 (0.197 g, 1.5 mmol) was suspended in dry DCM
(5 mL) under nitrogen and compound 20c (0.300 g, 0.6 mmol) was
added. After stirring the reaction mixture at room temperature for
6 h, saturated aqueous NH4Cl was added and the mixture was ex-
tracted with ethyl acetate. The organic layer was washed with
brine, dried over Na2SO4 and evaporated under reduced pressure.
The crude solid was crystallized from ethanol to give product 3c
(0.08 g, 30%), together with unreacted starting material; mp
315 °C. 1H NMR (DMSO-d6) d: 11.40 (1H, br s), 11.32 (1H, br s),
9.05 (1H, br s), 6.53–6.99 (10H, m), 4.28 (3H, s), 3.60 (3H, s), 3.33
(3H, s). 13C NMR (DMSO-d6) d: 156.0, 147.4, 147.1, 146.03, 145.6,
142.4, 134.6, 128.0, 127.8, 125.5, 123.8, 123.5, 122.6, 122.2,
119.7, 119.3, 115.5, 115.5, 114.7, 114.3, 113.9, 110.7, 107.3, 55.7,
55.2, 34.0. Anal. Calcd for C26H22N2O5: C, 70.58; H, 5.01; N, 6.33.
Found: C, 70.64; H, 5.09; N, 6.22. HRMS (ESI positive) Calcd for
4.19. 6-Hydroxy-3,4-bis(4-hydroxy-3-methoxyphenyl)-7-meth
oxy-2H-pyrido[3,4-b]indol-1(9H)-one (3b)
Anhydrous AlCl3 (0.094 g, 0.70 mmol) was suspended in dry
DCM (4 mL) under nitrogen and compound 20b (0.100 g,
0.13 mmol) was added. After stirring the reaction mixture at room
temperature for 24 h, saturated aqueous NH4Cl was added and the
mixture was extracted with ethyl acetate. The organic layer was
washed with brine, dried over Na2SO4 and evaporated under re-
duced pressure. The crude solid was crystallized from ethanol to
give product 3b (0.030 g, 48%); mp > 350 °C. 1H NMR (CDCl3) d:
11.53 (1H, s), 11.15 (1H, s), 9.25 (1H, s), 9.08 (1H, s), 9.00 (1H, s),
6.60–6.95 (7H, m), 6.22 (1H, s), 3.65 (3H, s), 3.60 (3H, s), 2.45
(3H, s). 13C NMR (DMSO-d6) d: 156.4, 147.4, 147.3, 143.2, 142.4,
140.6, 139.7, 139.2, 134.2, 127.9, 127.7, 125.3, 123.8, 123.5,
122.6, 115.5, 114.8, 114.1, 113.9, 110.7, 109.8, 108.3, 55.6, 55.2,
55.1 34.1. Anal. Calcd for C26H22N2O7: C, 65.82; H, 4.67; N, 5.90.
Found: C, 65.79; H, 4.60; N, 5.82. HRMS (ESI positive) Calcd for
C
26H22N2O5Na: 465.14209. Found: 465.14334 [M+Na]+.
4.23. 2,4-Dihydroxy-5-methoxy-benzaldehyde (24)
C
26H22N2O7Na: 497.13192. Found: 497.13256 [M+Na]+.
To a stirred suspension of AlCl3 (68 g, 509.9 mmol) in dry DCM
(450 mL), a solution of 2,4,5-trimethoxybenzaldehyde 23 (25 g,
127.4 mmol) in dry DCM (125 mL) was added dropwise. After stir-
ring for 4 h at room temperature, another portion of AlCl3 (68 g,
509.9 mmol) was added. The suspension was further stirred for
19 h and the reaction mixture was poured into 1 kg of ice to which
45 mL of concentrated hydrochloric acid were added. The organic
layer was separated and the aqueous phase was extracted twice
with DCM (200 mL). The combined organic layers were filtered
over silica gel, dried over Na2SO4, evaporated and the residue crys-
tallized from ethyl acetate to give compound 24 (17.95 g, 84%); mp
150 °C. 1H NMR (CDCl3) d: 11.95 (1H, s), 9.71 (1H, s), 7.29 (1H, s),
6.90 (1H, s), 6.55 (1H, s), 3.92 (3H, s). Anal. Calcd for C8H8O4: C,
57.14; H, 4.80. Found: C, 57.17; H, 4.85.
4.20. N-(1,2-Bis(4-isopropoxy-3-methoxyphenyl)-2-oxoethyl)-
1-methyl-1H-indole-2-carboxamide (19c)
To a suspension of HOBt (0.988 g, 7.3 mmol) and WSC (1.39 g,
7.3 mmol) in THF (60 mL) under nitrogen atmosphere commer-
cially available 18c (0.540 g, 3.1 mmol) was added. The reaction
mixture was stirred at room temperature for 12 h until the acti-
vated ester was formed, then compound 15 dissolved in THF
(10 mL) and TEA (7.3 mmol, 1 mL) were added. After stirring the
reaction mixture at room temperature for 14 h, the solvent was
evaporated under reduced pressure. The crude product was dis-
solved in ethyl acetate, the solution washed with aqueous NaHCO3,
HCl 2 M and brine, dried over Na2SO4 and evaporated under re-
duced pressure to afford 19c as a white solid (0.978 g, 58%); mp
111 °C. 1H NMR (DMSO-d6) d: 8.98 (1H, d, J = 7.07 Hz), 7.76 (1H,
d, J = 8.2 Hz), 7.40–7.67 (3H, m), 7.23–7.33 (2H, m), 7.19 (1H, s),
6.80–7.15 (4H, m), 6.64 (1H, d, J = 7.07 Hz), 4.70 (1H, m), 4.50
(1H, m), 3.96 (3H, s), 3.79 (3H, s), 3.75 (3H, s), 1.27 (6H, d,
J = 6.3 Hz), 1.22 (6H, d, J = 6.3 Hz). 13C NMR (DMSO-d6) d: 194.7,
162.0, 151.9, 150.2, 149.6, 147.1, 139.0, 131.9, 129.2, 127.8,
126.1, 124.1, 115.4, 113.5, 113.3, 70.4, 70.1, 62.9, 56.5, 54.4, 22.7,
22.4. Anal. Calcd for C32H36N2O6: C, 70.57; H, 6.66; N, 5.14. Found:
C, 70.65; H, 6.58; N, 5.22.
4.24. 2-Hydroxy-4-isopropoxy-5-methoxy-benzaldehyde (25)
2,4-Dihydroxy-5-methoxybenzaldehyde 24 (35.18 g, 209.2
mmol) was dissolved in dry DMF (345 mL). NaHCO3 (26.29 g,
313.0 mmol) and 2-bromopropane (38.5 g, 313.0 mol) were added
under nitrogen flow. The reaction mixture was heated at 70 °C
and stirred. After 4 h, the suspension was filtered and the organic
layer was evaporated. Ethyl acetate was added to the crude product
and the suspension was filtered. The organic phase was dried over
Na2SO4, filtered and evaporated under reduced pressure. The crude
product was purified by flash chromatography (hexane:ethyl ace-
tate, 1:1) affording a yellow oil (15 g, 34%). 1H NMR (CDCl3) d:
11.36 (1H, s), 9.66 (1H, s), 6.89 (1H, s), 6.44 (1H, s), 4.63 (1H, s),
3.85 (3H, s), 1.42 (6H, d, J = 5.9 Hz). 13C NMR (CDCl3) d: 194.0,
159.4, 156.2, 143.7, 114.2, 112.7, 101.6, 72.8, 56.8, 22.2, 21.9. Anal.
Calcd for C11H14O4: C, 62.85; H, 6.71. Found: C, 62.77; H, 6.78.
4.21. 3,4-Bis(4-isopropoxy-3-methoxyphenyl)-9-methyl-2H-
pyrido[3,4-b]indol-1(9H)-one (20c)
To a solution of 19c (0.970 g, 1.8 mmol) in acetonitrile (10 mL)
was added TFA (0.2 mL) and the reaction mixture was refluxed
for 15 min. The precipitate formed was filtered and washed with
acetonitrile to afford 20c as a white solid (0.556 g, 58%); mp
287 °C. 1H NMR (DMSO-d6) d: 9.10 (1H, br s), 6.50–6.85 (10H, m),
4.62 (1H, m), 4.55 (1H, m), 4.28 (3H, s), 3.60 (3H, s), 3.33 (3H, s),
1.40 (6H, d, J = 6.2 Hz), 1.35 (6H, d, J = 6.2 Hz). 1H NMR (DMSO-
d6) d: 155.6, 148.9, 148.8, 146.5, 146.0, 144.7, 137.7, 136.2, 135.5,
132.3, 129.4, 128.2, 128.0, 127.4, 127.0, 126.8, 123.1, 120.8,
115.4, 114.7, 114.6, 114.3, 114.0, 112.9, 103.7, 95.0, 71.1, 70.6,
4.25. 4-Isopropoxy-5-methoxy-2-(2-methoxy-ethoxymethoxy)-
benzaldehyde (26)
To a suspension of K2CO3 (29.16 g, 211.0 mmol) in dry DMF
(150 mL) was added compound 25 (14.80 g, 70.4 mmol). After stir-
ring for 15 min, MEMCl (26.63 g, 213.8 mmol) was added slowly
and the reaction mixture was stirred for 12 h. The solvent was

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S. Cananzi et al. / Bioorg. Med. Chem. 19 (2011) 4971–4984
removed under reduced pressure and the crude product was dis-
solved in ethyl acetate, washed with brine, dried over Na2SO4, fil-
tered and evaporated in vacuo, affording a yellow oil (19.70 g,
94%). 1H NMR (CDCl3) d: 10.29 (1H, s), 7.29 (1H, s), 6.82 (1H, s),
5.31 (2H, s), 4.65 (1H, m), 3.80–3.90 (5H, m), 3.53–3.60 (2H, m),
3.36 (3H, s), 1.40 (6H, d, J = 6.1 Hz). 13C NMR (CDCl3) d: 188.2,
157.2, 155.1, 146.1, 118.8, 110.0, 102.8, 95.1, 72.9, 70.8, 59.1,
57.2, 57.1, 22.1, 22.0. Anal. Calcd for C15H22O6: C, 60.39; H, 7.43.
Found: C, 60.48; H, 7.46.
After stirring the solution at rt for 16 h, the solvent was evaporated
under reduced pressure and the crude product was dissolved in
DCM. The solution was washed with saturated aqueous NaHCO3
and the organic layer was dried over Na2SO4, filtered and evapo-
rated. The crude product was purified by flash chromatography
(hexane:ethyl acetate, 6:4) affording 29 (13.99 g, 67%) as a yellow
oil. 1H NMR (CDCl3) d: 7.05–7.17 (2H, m), 6.88 (1H, s), 6.78–6.85
(2H, m), 5.10 (2H, s), 4.89 (2H, s), 4.57 (1H, m), 4.47 (1H, m),
3.81 (3H, s), 3.76 (3H, s), 3.70–3.74 (2H, m), 3.46–3.52 (2H, m),
3.33 (3H, s), 1.35 (6H, d, J = 6.1 Hz), 1.31 (6H, d, J = 6.1 Hz), 1.15
(9H, s). 13C NMR (CDCl3) d: 172.7, 153.6, 150.2, 149.5, 149.1,
146.9, 145.1, 129.6, 121.5, 119.1, 113.2, 111.7, 104.7, 94.4, 82.2,
71.4, 71.1, 67.2, 58.7, 56.5, 55.8, 44.0, 27.3, 22.5, 21.8, 21.7. Anal.
Calcd for C31H45NO10: C, 62.93; H, 7.67; N, 2.37. Found: C, 62.88;
H, 7.60; N, 2.46.
4.26. 4-Isopropoxy-5-methoxy-2-(2-methoxy-ethoxymethoxy)-
benzylamine (27)
A solution of 26 (17.70 g, 59.3 mmol) in 95% ethanol (400 mL)
was treated with a solution of hydroxylamine hydrochloride
(5.73 g, 82.4 mmol) in dry pyridine (40 mL) under stirring at room
temperature for 90 min and at 0 °C for 30 min to facilitate the
oxime precipitation. The white solid 4-isopropoxy-5-methoxy-2-
(2-methoxy-ethoxymethoxy)-benzaldehyde oxime was filtered,
dried, and used (18.48 g, 89%) without further purification. 1H
NMR (CDCl3) d: 8.94 (1H, br s), 8.40 (1H, s), 7.19 (1H, s), 6.80
(1H, s), 5.20 (2H, s), 4.55 (1H, m), 3.76–3.85 (5H, m), 3.50–3.58
(2H, m), 3.35 (3H, s), 1.35 (6H, d, J = 6.1 Hz). 13C NMR (CDCl3) d:
150.4, 149.2, 147.0, 118.0, 114.0, 105.2, 94.6, 71.7, 71.2, 61.0,
59.7, 58.9, 57.3, 53.5, 22.1.
A solution of the oxime (18.40 g, 58.7 mmol) in 95% ethanol
(154 mL) was treated with an equal volume of 2 M NaOH followed
by Al/Ni alloy (15.97 g) under stirring at room temperature for
90 min. The Al/Ni alloy was removed by filtration and washed with
fresh ethanol. Filtrate and washings were combined, acidified with
0.8 M HCl (pH 2) and extracted with DCM (3 ꢂ 50 mL). The aque-
ous phase was treated with solid KOH up to pH 14 and extracted
with diethyl ether (3 ꢂ 30 mL). The extracts after drying over
anhydrous Na2SO4 and removal of the solvent afforded 27 (17 g,
97%) as a brown oil. 1H NMR (CDCl3) d: 6.80 (1H, s), 6.77 (1H, s),
5.20 (2H, s), 4.46 (1H, m), 3.71–3.83 (7H, m), 3.50–3.58 (2H, m),
3.38 (3H, s), 1.31 (6H, d, J = 6.1 Hz). Anal. Calcd for C15H25NO5: C,
60.18; H, 8.42; N, 4.68. Found: C, 60.09; H, 8.51; N, 4.63.
4.29. 1-[4-Isopropoxy-5-methoxy-2-(2-methoxy-etxy-
methoxy)-phenyl]-2-(4-isopropoxy-3-methoxy-phenyl)-2-oxo-
ethyl]-carbamic acid tert-butyl ester (30)
LDA was prepared by mixing diisopropylamine (7.29 g,
72.0 mmol, 10.1 mL) and DMPU (10.9 mL) with n-BuLi (2.7 M in
heptane, 26.67 mL, 72.0 mmol) in THF (70 mL) at ꢀ78 °C under
nitrogen atmosphere.
A solution of the amide 29 (13.90 g,
23.5 mmol) in freshly distilled THF (209 mL) was added to the
LDA solution at ꢀ78 °C and the mixture was stirred for 6 h. The
mixture was quenched with aqueous NH4Cl, diluted with ethyl
acetate, washed with saturated aqueous NH4Cl and brine, and
dried over Na2SO4. Concentration in vacuo followed by flash chro-
matography (hexane:ethyl acetate, 7:3) gave product 30 (4.11 g,
29%) as a yellow oil. 1H NMR (CDCl3) d: 7.69 (1H, d, J = 8.2 Hz),
7.57 (1H, s), 6.68–6.85 (3H, m), 6.49 (1H, d, J = 7.8 Hz), 5.85 (1H,
d, J = 7.8 Hz), 5.27 (2H, m), 4.62 (1H, m), 4.49 (1H, m), 3.74–3.95
(2H, m), 3.86 (3H, s), 3.77 (3H, s), 3.55–3.65 (2H, m), 3.38 (3H, s),
1.44 (9H, s), 1.28–1.40 (12H, m). Anal. Calcd for C31H45NO10: C,
62.93; H, 7.67; N, 2.37. Found: C, 63.03; H, 7.54; N, 2.43.
4.30. 1-[4-Isopropoxy-5-methoxy-2-(2-methoxy-ethoxymeth
oxy)-phenyl]-2-(4-isopropoxy-3-ethoxy-phenyl)-2-oxo-ethyl-
ammonium trifluoro-acetate (31)
4.27. 4-Isopropoxy-N-[4-isopropoxy-5-methoxy-2-(2-methoxy-
ethoxymethoxy)-benzyl]-3-methoxy-benzamide (28)
Compound 30 (1.50 g, 2.53 mmol) was dissolved in dry DCM
(30 mL) under nitrogen flow and TFA (3 mL) was added at 0 °C.
The solution was stirred at rt for 2 h. Solvent was evaporated under
vacuo and the crude product (1.24 g) was used for the next step
without further purification.
To a suspension of HOBt (6.16 g, 45.6 mmol) and WSC (8.79 g,
45.6 mmol) in THF (250 mL) under nitrogen atmosphere, 8 (7.9 g,
37.6 mmol) and 27 (16.90 g, 56.4 mmol) were added. The reaction
mixture was stirred at room temperature for 24 h and the solvent
was evaporated under reduced pressure. The crude product was
dissolved in ethyl acetate, the solution washed with NaHCO3, HCl
2 M and brine, dried over Na2SO4 and evaporated under reduced
pressure to afford 28 as a yellow oil (17.70 g, 95%). 1H NMR (CDCl3)
4.31. 6-Benzyloxy-5-methoxy-1-methyl-1H-indole-2-carboxylic
acid [1-[4-isopropoxy-5-methoxy-2-(2-methoxyethoxymethoxy
)-phenyl]-2-(4-isopropoxy-3-methoxyphenyl)-2-oxo-ethyl]-
amide (33)
d: 7.42 (1H, s), 7.21 (1H, dd, J = 1.7 and 8.4 Hz), 6.75–6.93 (3H, m),
6.65 (1H, t, J = 5.7 Hz), 5.24 (2H, s), 4.40–4.65 (4H, m), 3.88 (3H, s),
3.79–3.85 (2H, m), 3.80 (3H, s), 3.50–3.56 (2H, m), 3.30 (3H, s), 1.36
(6H, d, J = 6.5 Hz), 1.34 (6H, d, J = 6.5 Hz). 13C NMR (CDCl3) d: 166.6,
149.8, 149.4, 147.2, 145.5, 127.2, 119.7, 119.0, 114.1, 113.6, 111.2,
105.3, 94.8, 71.6, 71.4, 71.1, 67.9, 58.8, 56.4, 55.9, 39.2, 21.8, 21.7.
Anal. Calcd for C26H37NO8: C, 63.53; H, 7.59; N, 2.85. Found: C,
63.57; H, 7.54; N, 2.78.
To a suspension of HOBt (0.459 g, 3.4 mmol) and WSC (0.650 g,
3.4 mmol) in THF (30 mL) was added under nitrogen atmosphere
32 (0.519 g, 1.67 mmol).14 The reaction mixture was stirred at
room temperature for 12 h until the activated ester was formed.
Then compound 31 (1.21 g, 1.85 mmol) dissolved in THF (10 mL)
and TEA (3.4 mmol, 0.47 mL) were added. After stirring the reac-
tion mixture at room temperature for 14 h, the solvent was evap-
orated under reduced pressure. The crude product was dissolved
in ethyl acetate, the solution washed with aqueous NaHCO3, HCl
2 M and brine, dried over Na2SO4 and evaporated under reduced
pressure to afford 33 as a yellow solid (0.720 g, 55%); mp 134 °C.
1H NMR (DMSO-d6) d: 8.79 (1H, d, J = 7.5 Hz), 7.67 (1H, d,
J = 8.6 Hz), 7.46–7.58 (3H, m), 7.32–7.46, 7.03–7.25 (4H, m),
6.83–6.98 (3H, m), 5.30 (2H, m), 5.30 (2H, m), 5.15 (2H, s), 4.69
4.28. (4-Isopropoxy-3-methoxy-benzoyl)-[4-isopropoxy-5-met
hoxy-2-(2-methoxy-ethoxymethoxy)-benzyl]-carbamic acid
tert-butyl ester (29)
Compound 28 (17.65 g, 35.9 mmol) was dissolved in acetoni-
trile (150 mL) under nitrogen flow and DMAP (2.99 g, 24.5 mmol)
and di-tert-butyldicarbonate (15.71 g, 72.0 mmol) were added.

S. Cananzi et al. / Bioorg. Med. Chem. 19 (2011) 4971–4984
4983
(1H, s), 4.52 (1H, s), 3.90 (3H, s), 3.78 (6H, s), 3.67 (3H, s), 3.17 (3H,
s), 1.20–1.29 (12H, m). Anal. Calcd for C44H52N2O11: C, 67.33; H,
6.68; N, 3.57; Found: C, 67.24; H, 6.53; N, 3.66.
C, 65.19; H, 4.64; N, 5.49. HRMS (ESI positive) Calcd for C28H24N2O8-
Na: 539.14249. Found: 539.14201 [M+Na]+.
4.35. 1-Methyl-1H-indole-2-carboxylic acid [2-hydroxy-1-(2-
4.32. 7-Benzyloxy-3-(2-hydroxy-4-isopropoxy-5-methoxy-phen
yl)-4-(4-isopropoxy-3-methoxy-phenyl)-6-methoxy-9-methyl-
2,9-dihydro-b-carbolin-1-one (34)
methoxyphenyl)ethyl]amide (37)
To a suspension of HOBt (3.24 g, 23.9 mmol) and WSC (4.60 g,
23.9 mmol) in THF (40 mL) under nitrogen atmosphere, 36 (1.90 g,
11.4 mmol) and 18c (1.29 g, 7.4 mmol) were added. The reaction
mixture was stirred at room temperature for 24 h and the solvent
was evaporated under reduced pressure. The crude product was dis-
solved in ethyl acetate, the solution washed with NaHCO3, HCl 2 M
and brine, dried over Na2SO4 and evaporated under reduced pres-
sure to afford 37 as a white solid (2.33 g, 97%); mp 93–95 °C. 1H
NMR (CDCl3) d: 7.64 (1H, d, J = 8.3 Hz), 7.21–7.43 (5H, m), 7.15
(1H, m), 6.29–7.03 (2H, m), 6.90 (1H, s), 5.51 (1H, m), 4.05 (3H, s),
3.93–4.03 (2H, m), 3.93 (3H, s). 13C NMR (DMSO-d6) d: 162.1,
156.9, 138.9, 132.9, 129.4, 128.6, 127.6, 126.2, 124.0, 122.0, 120.8,
120.7, 111.3, 111.0, 105.1, 63.8, 56.0, 50.9. Anal. Calcd for
TMSCl (0.67 mmol, 0.08 mL) and NaI (0.100 g, 0.67 mmol) were
added to a solution of 33 (0.450 g, 0.67 mmol) in acetonitrile
(40 mL) at ꢀ20 °C under nitrogen flow. After stirring at this tem-
perature for 15 min, an additional equivalent of both NaI and
TMSCl was added and the mixture stirred at ꢀ20 °C until no start-
ing material remained (TLC analysis). The yellow-orange mixture
was quenched with methanol and the solvent removed in vacuo.
The residue was extracted with ethyl acetate and the organic layer
washed with saturated sodium thiosulfate and brine. The crude
product was purified by flash chromatography (hexane:ethyl ace-
tate, 7:3) affording 34 (0.140 g, 31%) as a white solid; mp 220 °C.
1H NMR (DMSO-d6) d: 11.00 (1H, s), 9.09 (1H, s), 7.19–7.56 (7H,
m), 6.67–7.00 (3H,m), 6.54 (1H, s), 6.41 (1H, s), 6.27 (1H, s), 5.19
(2H, s), 4.50 (1H, s), 4.38 (1H, s), 4.24 (3H, s), 3.58 (3H, s), 3.43
(3H, s), 3.39 (3H, s), 1.22 (12H, m). Anal. Calcd for C40H42N2O8: C,
70.78; H, 6.24; N, 4.13. Found: C, 70.74; H, 6.29; N, 4.20. HRMS
C19H20N2O3: C, 70.35; H, 6.21; N, 8.64. Found: C, 70.30; H, 6.27; N,
8.60.
4.36. 8-Methyl-8H-5-oxa-6a,8-diazaindeno[2,1-b]phenanthren-
7-one (38)
(ESI positive) Calcd for
C40H43N2O8: 679.30139. Found:
679.30333 [M+H]+. Calcd for C40H42N2O8Na: 701.28334. Found:
701.28543 [M+Na]+.
The above alcohol (0.907 g, 2.8 mmol) was added to a solution
of IBX (1.55 g, 5.6 mmol) in dry DMSO (10 mL) under nitrogen
atmosphere. The reaction mixture was stirred at room temperature
for 3 h, treated with ethyl acetate, washed with NaHCO3 and brine.
The organic layer was dried over Na2SO4, and evaporated under re-
duced pressure to afford N-(formyl(2-methoxyphenyl)ethyl)-1-
methyl-1H-indole-2-carboxamide as a yellow oil (0.900 g), used
for the next step without further purification.
To a solution of the above aldehyde (0.900 g, 2.8 mmol) in aceto-
nitrile (10 mL) was added TFA (1 mL) and the reaction mixture was
refluxed for 15 min. The precipitate formed was filtered and washed
with acetonitrile to afford 3-(2-methoxyphenyl)-9-methyl-2H-pyr-
ido[3,4-b]indol-1(9H)-one as a white solid (0.540 g, 64.3%); mp
250 °C. 1H NMR (DMSO) d: 7.88–8.12 (6H, m), 7.63–7.86 (4H, m),
3.90 (3H, s), 3.31 (3H, s), 2.52 (3H, s).
To an ice-cooled suspension of the above compound (0.540 g,
1.8 mmol) in dry dichloromethane (13 mL) 1 M BBr3 in dichloro-
methane (1.33 g, 5.3 mmol, 5.3 mL) was added dropwise under
nitrogen atmosphere. The reaction mixture was allowed to warm
to room temperature, stirred overnight, quenched with aqueous
HCl and extracted with dichloromethane. The extract was washed
with brine, dried over Na2SO4, concentrated under reduced pressure
and purified by flash chromatography (dichloromethane:methanol,
9.5:0.5) to afford 3-(2-hydroxyphenyl)-9-methyl-2H-pyrido[3,4-
b]indol-1(9H)-one (0.200 g, 38%) as a yellow solid; mp 270 °C. 1H
NMR (DMSO-d6) d: 7.88–8.12 (6H, m), 7.63–7.86 (4H, m), 3.31
(3H, s), 2.52 (3H, s).
4.33. 10-Benzyloxy-3-isopropoxy-13-(4-isopropoxy-3-methoxy-
phenyl)-2,11-dimethoxy-8-methyl-8H-5-oxa-6a,8-diazaindeno
[2,1-b]phenanthren-7-one (35)
A suspension of 34 (0.132 g, 0.2 mmol) and K2CO3 (0.072 g,
52.1 mmol) in DMF (6 mL) was stirred for 30 min at room temper-
ature, then treated with diiodomethane (21.96 g, 82.0 mmol). After
being heated at 100 °C for 1 h, the solvent was evaporated under
reduced pressure and the crude product was dissolved in dichloro-
methane, washed with brine, dried over Na2SO4 and purified by
flash chromatography (petroleum ether–ethyl acetate, 1:1) to af-
ford 35 as a white solid (15 mg, 12%). 1H NMR (DMSO-d6) d:
7.17–7.58 (5H, m), 6.74–7.17 (4H, m), 6.46–6.72 (2H, m), 5.82–
6.14 (3H, m), 5.23 (2H, s), 4.42–4.72 (2H, m), 4.24 (3H, s), 3.78
(3H, s), 3.52 (3H, s), 3.26 (3H, s), 1.07–1.48 (12H, m). Anal. Calcd
for C41H42N2O8: C, 71.29; H, 6.13; N, 4.06. Found: C, 71.38; H,
6.04; N, 4.18. HRMS (ESI positive) Calcd for
C41H42N2O8Na:
713.28334. Found: 713.28421 [M+Na]+.
4.34. 3,10-Dihydroxy-13-(4-hydroxy-3-methoxy-phenyl)-2,11-
dimethoxy-8-methyl-8H-5-oxa-6a,8-diazaindeno[2,1-b]phenan
thren-7-one (2b)
Anhydrous AlCl3 (15.5 mg, 0.116 mmol) was suspended in dry
DCM (5 mL) under nitrogen and compound 35 (10 mg, 0.014 mmol)
was added. After refluxing the reaction mixture for 6 h, saturated
aqueous NH4Cl was added and the mixture was extracted with ethyl
acetate. The organic layer was washed with brine, dried over Na2SO4
and evaporated under reduced pressure. The crude product was
purified by preparative TLC to give 2b (6 mg, 83%); mp 290 °C. 1H
NMR (DMSO-d6) d: 7.03 (1H, d, J = 8.1 Hz), 6.94 (1H, d, J = 1.9 Hz),
6.88 (1H, s), 6.76 (1H, dd, J = 8.1, 1.9 Hz), 6.63 (1H, s), 6.45 (1H, s),
5.87 (1H, AB, J = 9.6 Hz), 5.80 (1H, s), 5.75 (1H, AB, J = 9.6 Hz), 4.11
(3H, s), 3.69 (3H, s), 3.16 (3H, s). 13C NMR (DMSO-d6) d: 153.3,
149.9, 149.5, 149.3, 148.9, 147.2, 144.4, 143.5, 137.6, 128.7, 126.9,
125.9, 123.3, 117.0, 113.5, 112.6, 104.2, 103.9, 71.8, 56.5, 55.6,
55.1. Anal. Calcd for C28H24N2O8: C, 65.11; H, 4.68; N, 5.42. Found:
A suspension of the above compound (0.04 g, 0.14 mmol) and
K2CO3 (0.05 g, 0.34 mmol) in DMF (4 mL) was stirred for 30 min at
room temperature, then treated with iodomethane (0.150 g,
0.56 mmol). After being refluxed for 1 h, the solvent was evaporated
and the crude product was dissolved in dichloromethane, washed
with brine, dried over Na2SO4 and purified by flash chromatography
(dichloromethane:methanol, 9.5:0.5) to afford 38 as a white solid
(12 mg, 28%); mp 280 °C. 1H NMR (DMSO-d6) d: 8.02–8.19 (2H,
m), 7.81 (1H, s), 7.65 (1H, d, J = 8.4 Hz), 7.52 (1H, m), 7.38 (1H, m),
7.18–7.32 (2H, m), 7.13 (1H, d, J = 8.1 Hz), 5.92 (2H, s), 4.23 (3H,
s). 13C NMR (DMSO-d6) d: 153.5, 152.7, 140.8, 130.2, 129.9, 127.1,
125.5, 124.1, 124.0, 123.7, 121.6, 121.2, 120.3, 119.7, 117.2, 110.8,
96.4, 70.8, 31.2. Anal. Calcd for C19H14N2O2: C, 75.48; H, 4.67; N,
9.27. Found: C, 75.42; H, 4.71; N, 9.32. HRMS (ESI positive) Calcd

4984
S. Cananzi et al. / Bioorg. Med. Chem. 19 (2011) 4971–4984
for C19H14N2O2Na: 325.09474. Found: 325.09499 [M+Na]+. Calcd for
38H28N4O4Na: 627.20027. Found: 627.20145 [2M+Na]+.
length topoisomerase I)20 for 30 min at 37 °C. Reactions were
stopped by 0.5% SDS and 0.3 mg/mL of proteinase K for 45 min at
42 °C. After precipitation DNA was resuspended in denaturing buf-
fer (80% formamide, 10 mM NaOH, 0.01 M EDTA, and 1 mg/mL
dyes) before loading on a denaturing 8% polyacrylamide gel in
TBE buffer.
C
4.37. Molecular modeling
The three-dimensional structure of the two examined ligands
were built and energy minimized within Ghemical.14 All calcula-
tions were performed on a 3.0 GHz Quad-Xeon 64-bit workstation
running under MacOSX Tiger. The 2.10 Å resolution crystal struc-
ture of human topoisomerase I covalently linked to double-
stranded DNA and bound to topotecan8 (Protein Data Bank entry
1k4t) was used as the reference structure to model the topoisomer-
ase I–DNA complex with 2a and 3. After the removal of topotecan
and water molecules, hydrogens and partial charges (using the dic-
tionary of partial charges for nucleic acid and protein atoms of the
AMBER force-field) were added to the complex. Partial charges
were also assigned to the ligands atoms. The two derivatives were
docked at the putative binding site using Autodock Vina15 with a
radius of 7 Å centered on the topotecan molecule, with a grid res-
olution of <0.36 Å. The resulting orientations were clustered, con-
sidering a root-mean-square deviation (rmsd) tolerance of 2.0 Å,
into families, and the lowest docking-energy conformations were
then equilibrated for 1.2 ns by unrestrained MD. The simulations
were performed at constant temperature and pressure (NPT
ensemble) in a periodic cubic box of pre-equilibrated TIP3P water
molecules. The water bond distances and angle were forced using
the SETTLE algorithm16 while the bond lengths within the protein
were constrained with the LINCS algorithm.17 The coupling time
was set to 1.0 ps, and the isothermal compressibility was set to
4.6 ꢂ 10ꢀ5 barꢀ1. The protein, ligand, and solvent were indepen-
dently coupled at a temperature of 298 K, coupling time 0.1 ps,
and the pressure was held at 1 bar, coupling time 0.2 ps, using a
Berendsen thermostat to maintain temperature and pressure
unvarying. The time step used was 1.0 fs. Snapshots of the recep-
tor–substrate system were saved every 0.2 ps. Hydrogen bonds
and contacts were automatically identified using a contact module
of CCP418,19 while the other interactions were identified visually.
Acknowledgments
Financial support was provided by the University of Milano
(PRIN funds). We are grateful to Prof. Mercedes Álvarez for provid-
ing a sample of Lamellarin D and to Dr. Franco Zunino for helpful
discussions.
References and notes
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ghemical.
4.38. Cytotoxic activity
The human non-small cell lung cancer carcinoma cell line NCI-
H460 (ATCC, HTB-177) was used. Cells were cultured in RPMI-1640
containing 10% foetal calf serum. Cytotoxicity was assessed by a
growth inhibition assay after 1 and 72 h drug exposure. Cells in
the logarithmic phase of growth were harvested and seeded in
duplicates into six-well plates. Twenty-four hours after seeding,
cells were exposed to the drug for 1 and 72 h. Cells were counted
72 h after drug exposure for 1 h of treatment and at the end of drug
exposure in the case of 72 h of treatment. Cells were counted with
a Coulter counter. IC50 is defined as the inhibitory drug concentra-
tion causing a 50% decrease of cell growth compared to the un-
treated control.
16. http://www.vina.scripps.edu/.
4.39. Topoisomerase I-dependent DNA cleavage assay
17. Miyamoto, S.; Kollman, P. A. J. Comput. Chem. 1992, 13, 952.
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50, 760.
A gel purified 751-bp BamHI–EcoRV fragment of SV40 DNA was
used for the cleavage assay. DNA fragment was uniquely 30-end
labeled. Cleavage reactions (10,000 cpm/sample) were performed
in 20 mL of 10 mM Tris–HCl (pH 7.6), 150 mM KCl, 5 mM MgCl2,
0.1 mM dithiotreitol, and the human recombinant enzyme (full
20. Beretta, G. L.; Binaschi, M.; Zagni, E.; Capuani, L.; Capranico, G. Cancer Res. 1999,
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