Synthesis and biological evaluation of indenoisoquinolines that inhibit both tyrosyl-DNA phosphodiesterase i (Tdp1) and topoisomerase i (Top1)

Article abstract of DOI:10.1021/jm3014458

Tyrosyl-DNA phosphodiesterase I (Tdp1) plays a key role in the repair of damaged DNA resulting from the topoisomerase I (Top1) inhibitor camptothecin and a variety of other DNA-damaging anticancer agents. This report documents the design, synthesis, and evaluation of new indenoisoquinolines that are dual inhibitors of both Tdp1 and Top1. Enzyme inhibitory data and cytotoxicity data from human cancer cell cultures were used to establish structure-activity relationships. The potencies of the indenoisoquinolines against Tdp1 ranged from 5 μM to 111 μM, which places the more active compounds among the most potent known inhibitors of this target. The cytotoxicity mean graph midpoints ranged from 0.02 to 2.34 μM. Dual Tdp1-Top1 inhibitors are of interest because the Top1 and Tdp1 inhibitory activities could theoretically work synergistically to create more effective anticancer agents.

Full text of DOI:10.1021/jm3014458

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of Indenoisoquinolines That
Inhibit Both Tyrosyl-DNA Phosphodiesterase I (Tdp1) and
Topoisomerase I (Top1)
Martin Conda-Sheridan,†,§ P. V. Narasimha Reddy,†,§ Andrew Morrell,† Brooklyn T. Cobb,†
Christophe Marchand,‡ Keli Agama,‡ Adel Chergui,‡ Amelie Renaud,‡ Andrew G. Stephen,∥
́
Lakshman K. Bindu,∥ Yves Pommier,‡ and Mark Cushman*,†
†Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, and the Purdue Center for Cancer
Research, Purdue University, West Lafayette, Indiana 47907, United States
‡Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892-4255,
United States
∥Protein Chemistry Laboratory, Advanced Technology Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 21702,
United States
ABSTRACT: Tyrosyl-DNA phosphodiesterase I (Tdp1) plays a key role in the repair of damaged DNA
resulting from the topoisomerase I (Top1) inhibitor camptothecin and a variety of other DNA-damaging
anticancer agents. This report documents the design, synthesis, and evaluation of new indenoisoquinolines
that are dual inhibitors of both Tdp1 and Top1. Enzyme inhibitory data and cytotoxicity data from human
cancer cell cultures were used to establish structure−activity relationships. The potencies of the
indenoisoquinolines against Tdp1 ranged from 5 μM to 111 μM, which places the more active compounds
among the most potent known inhibitors of this target. The cytotoxicity mean graph midpoints ranged
from 0.02 to 2.34 μM. Dual Tdp1−Top1 inhibitors are of interest because the Top1 and Tdp1 inhibitory activities could
theoretically work synergistically to create more effective anticancer agents.
One approach to chemotherapy is based on forming lesions
in tumor DNA. Thus, stalled Top1−DNA complexes can also
arise when Top1 poisons, such as camptothecins (CPTs) or
indenoisoquinolines, are used for cancer therapy.7−9 The
function of the CPTs is to stabilize the DNA−Top1 complex
and inhibit the religation process. As a consequence, DNA
replication forks collide with drug-stabilized complexes causing
double-stranded DNA breaks that ultimately result in tumor
cell death. It is believed that Tdp1 may be responsible for the
drug resistance of some cancers by virtue of repairing DNA
lesions caused by CPTs.5,10 This hypothesis is further
supported by the fact that hypersensitivity to CPTs is observed
when Tdp1 is inactivated in DNA repair- or checkpoint-
deficient yeast.11
Given the relationship between Top1 and Tdp1, inhibitors of
Tdp1 could potentiate the effects of Top1 poisons.2,5 Although
the association between these enzymes makes Tdp1 an
attractive target for cancer treatment, there are few known
inhibitors in the literature.5,12−15 Moreover, these compounds
show weak inhibitory activity with IC50’s usually in the
micromolar range. A recent publication reported that
indenoisoquinolines bearing three or four methylene units on
the amine-substituted side chain are good Tdp1 inhibitors.16
The binding interactions between Tdp1 and indenoisoquino-
line 116 (Figure 2) have previously been characterized using
INTRODUCTION
■
The phospholipases are a heterogeneous family of enzymes that
catalyze the hydrolysis of phosphodiester bonds.1 One member
of this family, tyrosyl-DNA phosphodiesterase I (Tdp1),
catalyzes the hydrolysis of 3′-phosphotyrosyl linkers.2 This
enzyme is unique because its catalytic site possesses two
histidine and two lysine residues but lacks the aspartate residue
that is found in the other members of the family.3 When DNA
topoisomerase I (Top1) nicks double-stranded DNA, a
covalent Top1−DNA complex is made.4 The formed 3′-
adducts must be removed by Tdp1 to repair damaged DNA in
stalled Top1−DNA complexes in which the normal DNA
religation reaction has not occurred. The enzyme mechanism is
believed to occur in two sequential steps.5,6 The first step
consists of the nucleophilic attack by His263 on the
phosphorus atom linked to the oxygen of the Top1 catalytic
residue Tyr723 at the 3′ end of DNA (Figure 1). The function
of the Lys265 and Lys495 residues found in the catalytic site is
to coordinate the oxygen atoms of the phosphate group, which
enables the covalent linkage of His263 to the 3′-phosphate end
of the DNA. In the second step, this intermediate is hydrolyzed
by a His493-activated water molecule. The overall reaction
frees the tyrosine residue and affords a DNA strand that has a
3′-phosphate end. The process of DNA restoration is then
finished by DNA polymerases and DNA ligases. The role of
Tdp1 is to hydrolyze phosphotyrosyl-DNA linkages in
denatured or proteolytically degraded Top1−DNA complexes.7
Received: October 4, 2012
© XXXX American Chemical Society
A
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Figure 1. Mechanism of Tdp1.5
compounds such as 2, did not provide any advantage when
present on the side chain attached to the lactam of
indenoisoquinolines. This paper expands the previously
reported SAR studies regarding Tdp1 inhibitors and introduces
novel indenoisoquinolines that are active against this target.
Additionally, compounds with and without dual activity against
both Tdp1 and Top1 enzymes are presented.
CHEMISTRY
■
Figure 2. Tdp1 inhibitors.
It is known that the Tdp1 active site possesses two lysine (265
and 495) and two histidine (263 and 493) residues. Some Tdp1
inhibitors have functional groups (e.g., carbonyls) that can
hydrogen bond with these residues, and a lipophilic core that
interacts with the hydrophobic region (Ala520, Phe259,
surface plasmon resonance (SPR) and fluorescence resonance
energy transfer (FRET). During the previous studies, it was
concluded that the sulfonate substituent, key for the activity of
B
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Gly260, Tyr261, etc.) of the active site.5 Indenoisoquinolines
with an ester moiety might therefore be inhibitors of the
enzyme. The partially negatively charged carbonyl oxygen may
interact with some of the charged polar residues in the binding
pocket while the indenoisoquinoline aromatic system might sit
in the hydrophobic portion of the cavity. To build on the
previous work,16 an ester moiety was therefore added to 3-
amino-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione
(3)17 as shown in Scheme 1. Briefly, the amino group of 3 was
a
Scheme 2
a
Scheme 1
a
Reagents and conditions: (a) CHCl3, Et3N; (b) i. TFA, CHCl3; ii.
NH3, MeOH.
a
Reagents and conditions: (a) THF, Et3N; (b) NaOH, MeOH, DMF
or NaOH, EtOH.
displacement with azide to provide 50. Subsequent reduction of
50 under Staudinger conditions gave indenoisoquinoline 51.
Precursor 49 was treated with imidazole or morpholine to
afford compounds 52 and 53 (Scheme 6). Compounds 54−63
(Figure 3),18,20,24 from our existing library of compounds
prepared to investigate Top1 inhibition, were also evaluated to
identify key features for Tdp1 inhibitory activity.
reacted with methyl oxalyl chloride (4) to provide compound 9
that was hydrolyzed, under basic conditions, to yield 14.
Compound 9 was inactive, but 14 showed modest Tdp1
inhibitory activity with an IC50 of 61.7 μM. Therefore,
compounds 5−8 with several methylene units between the
acyl chloride and the esters functionalities were reacted with 3
to afford compounds 10−13. The ester 10 was converted to the
acid derivative 15.
Additionally, new indenoisoquinolines bearing nitrile and
iodo substituents on the A ring were synthesized. There are not
many substitutions reported in the literature on this side of the
indenoisoquinoline system; thus, the current study provided a
prime opportunity to expand the substitution pattern on the A
ring. Moreover, given the fact that the methoxy or
methylenedioxy substituents have been shown to improve
Top1 activity, these substituents were added to some of the
new compounds.26 Isochromenone 68 (Scheme 7) was
prepared by condensing 6-cyano-3-hydroxyphthalide (66),
obtained from 3-cyanophthalide (64), with phthalide (67).27
The product was reacted with various substituted aminopropyl
compounds to yield indenoisoquinolines 72−74 (Scheme 7).
Compound 66 was also condensed with 5,6-methylenediox-
yphthalide28 (75, Scheme 8) to provide the methylenedioxy-
substituted compound 76, which was reacted N′,N′-dimethy-
laminopropylamine (71) to yield indenoisoquinoline 77.
Compound 85 (Scheme 9) was prepared according to previous
procedures from 5-nitrohomophthalic acid (47) and Schiff base
78.20,22−24 The nitro group of compound 80 was reduced to
aniline 81 by catalytic hydrogenation. The amine functionality
of compound 81 was replaced by an iodine atom using
Sandmeyer chemistry to provide indenoisoquinoline 82, which
was converted to the amino analogue 84 by azide displacement
Compound 1618 was reacted with acyl chlorides 4−8 to
produce indenoisoquinolines 17−21, which were converted to
the amines 22−26 by deprotection under acidic conditions
(Scheme 2). The acids 32−36 were prepared from
intermediates 17−21 (Scheme 3) by hydrolysis under basic
conditions followed by acid treatment. Also, precursors 3 and
16 were reacted with ethyl glyoxylate using the Borch reduction
to give esters 38 and 39 (Scheme 4) that were further
hydrolyzed to acids 41 and 43. Compound 40 was prepared by
deprotection of 39 (Scheme 4).
A set of 3-nitroindenoisoquinolines was prepared because a
high-priority goal was to synthesize dual Tdp1−Top1
inhibitors, and it is well established that a 3-nitro substituent
increases potency vs Top1.19−21 Compound 51 (Scheme 5)
was prepared using the Schiff base−homophthalic anhydride
condensation approach.20,22−24 Briefly, Schiff base 46,25
obtained by the condensation of m-methoxybenzaldehyde
(44) and 3-bromopropylamine hydrobromide (45), was
reacted with 5-nitrohomophthalic anhydride (47) to provide
acid 48. This compound was subjected to Friedel−Crafts
conditions to afford indenoisoquinoline 49, followed by halide
C
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a
a
Scheme 3
Scheme 5
a
Reagents and conditions: (a) Et3N, CHCl3; (b) CHCl3; (c) i. SOCl2,
PhH, ii. AlCl3, PhNO2; (d) NaN3, DMSO, 90 °C; (e) i. P(OEt)3, PhH,
ii. HCl, MeOH.
a
Scheme 6
a
Reagents and conditions: (a) NaOH, THF, MeOH; (b) HCl, ethyl
ether, CHCl3.
a
Scheme 4
a
Reagents and conditions: (a) (i) Nal, dioxane, DMF, (ii) imidazole or
morpholine, K2CO3.
compound 86 (Scheme 10) was prepared following published
procedures.20 Reduction of the nitro group of analogue 86 was
attempted with several conditions, including Raney nickel as
previously performed.24 Ultimately, it was discovered that using
comparable amounts (by weight) of 5% Pd−C and
indenoisoquinoline 86 while hydrogenating at 1 atm in THF
provided the most consistent and reproducible yield of
intermediate 87. Treatment of compound 87 with sodium
azide in DMSO at 100 °C provided analogue 88, which was
subsequently reduced with triethyl phosphite to provide
compound 89, isolated as the dihydrochloride salt (Scheme
10).
a
Reagents and conditions: (a) i. HOAc, MeOH, ii. NaCNBH3; (b)
BIOLOGICAL RESULTS AND DISCUSSION
The IC50 of the oxalic acid derivative 14 vs Tdp1 was 61
■
KOH (for 41) or NaOH (for 42), MeOH, THF, H2O; (c) 2 M HCl,
ether, CHCl3
7
μM as shown in the titration curve (Figure 4). Surface plasmon
resonance studies indicated that the compound binds the
protein in a 1:1 ratio (Figure 5). The association and
dissociation rates were very fast but within the limit of
detection of the instrument. The experiment also indicated that
the compound did not bind to the single-stranded DNA
substrate. Based on previous results16 and the activity of
compound 14, computational studies using GOLD and Sybyl
and Staudinger reduction. Reaction with formaldehyde under
Borch reduction conditions afforded the dimethylamino
analogue 85.
Finally, to expand the diversity of the set, the effect of
combining an aniline group at the 3 position with a methoxy
group at the 9 position was investigated. To accomplish this,
D
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a
Scheme 7
a
Reagents and conditions: (a) 3-Cl-perBzOH, NBS, hν, CCl4; (b)
H2O, reflux; (c) (i) NaOMe, MeOH, EtOAc, (ii) HCl, (iii) pTsOH,
PhH; (d) THF, Et3N, reflux; (e) CHCl3, Et3N, reflux.
a
Scheme 8
Figure 3. Previously reported indenoisoquinolines prepared to study
Top1 inhibitory activity.
were performed.29,30 Docking 14 in the catalytic site of Tdp1
suggested interactions between the oxygen atoms of 14 and the
Tdp1 residues Thr261, His263, Thr281, Asn283, and Ser400
(Figure 6). Thus, a series of compounds containing
homologous side chains and ester moieties at the 3 position
were prepared to probe the left side of the binding pocket as
portrayed in Figure 6.
An analysis of the biological data for the rest of the
indenoisoquinolines allowed the determination of some of the
important features needed for Tdp1 inhibitory activity. The
aminopropyl side chain is clearly important for enzyme
inhibition as observed in 22, +(+); 23, ++(+); 40, ++(+); 43
+++; 51, ++; 54, +++; 55, +++; 57, ++; 60, +(+); 62, ++(+);
74, ++; 84, +++; 85, +(+); and 89, +++; (note: the “+ system”
is a semiquantitative scale expressing IC50 values at a given
range as explained in Table 1, and the IC50 values of the more
active Tdp1 inhibitors are reported in Table 2). The
indenoisoquinoline 40 was chosen as a representative example
of an N-(3-aminopropyl) compound for GOLD docking and
molecular mechanics energy minimization studies, which
resulted in the hypothetical binding mode displayed in Figure
7. The structure suggests the existence of bonding interactions
between the charged ammonium cation of the ligand and the
Val401 backbone carbonyl, the Pro461 backbone carbonyl, and
the Thr466 side chain oxygen with distances of 2.7 Å, 2.6 Å,
and 3.0 Å, respectively. These bonding interactions would help
to explain the Tdp1 inhibitory activity of the 3-aminopropyl-
substituted compounds 40 and 43 vs the inactivity of their N-
a
Reagents and conditions: (a) i. NaOMe, MeOH, EtOAc, ii. HCl,
PhH, pTsOH; (b) 71, CHCl3, Et3N.
methylated and Boc-protected counterparts 38, 41, and 42.
Additionally, Thr261 with its side-chain hydroxyl was within H-
bonding distance, 2.7 Å, to the carbonyl oxygen of the ligand
40.
In general, compounds in which the aminopropyl side chain
was Boc-protected were either inactive or had very low Tdp1
inhibitory activity (Table 1). The weakly active Boc-protected
compounds were 29 and 31. One N-methylated compound 14
showed weak activity, +, but all of the other analogues
containing an N-methyl side chain (9−13, 15, 38, and 41) were
inactive. Molecules 15, 30, and 32−36 with an acidic side chain
at C-3 on the A ring were inactive but compounds 14 (+) and
43 (+++) were the exceptions. The ester-substituted
indenoisoquinolines containing an aminopropyl side chain
were all active, but the activity decreased when more methylene
units were added to the ester side chain, going from +(+) and
E
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a
Scheme 9
Figure 4. Titration curve of compound 14 against Tdp1.
a
Reagents and conditions: (a) CHCl3; (b) (i)SOCl2, PhH, (ii) AlCl3,
PhNO2; (c) H2, Pd−C, THF, MeOH, EtOAc; (d) (i) NaNO2, HCl,
H2O, dioxane, (ii) Cul, Kl, H2O; (e) NaN3, DMSO, 90 °C; (f) (i)
P(OEt)3, PhH (ii) HCl, MeOH, (iii) K2CO3; (g) (i) CH2(O), MeOH,
HOAc, (ii) NaCNBH3.
a
Scheme 10
Figure 5. Surface plasmon resonance for compound 14 with Tdp1.
The binding of compound 14 to Tdp1 was examined using SPR
spectroscopy. Different concentrations of 14 (50, 25, 12.5, and 6.25
μM) were injected over immobilized Tdp1. The kinetics were fit to a
1:1 binding model yielding the following parameters, ka 7.7e4 1/Ms, kd
0.24 1/s, and KD 31 μM.
a
Reagents and conditions: (a) Pd−C, H2 (1 atm), THF; (b) NaN3,
DMSO, 100 °C; (c) (i) P(OEt)3, PhH, reflux, (ii) HCl, MeOH, reflux.
++(+) for 22 and 23 to + for 24−26 (Table 1). The ethyl ester
40, which has a short ester side chain, was active.
Given the apparent importance of the aminopropyl side
chain for Tdp1 inhibition, 10 compounds 54−63 from our
existing Top1 inhibitor library were selected for testing to
further explore this feature (Figure 3).16,18,20,24 These
compounds were all active as long as the 3-aminopropyl side
chain and the 11-keto functional group were both present.
Replacement of the primary amine with bromide, azide,
morpholine, or imidazole rendered the compounds very weak
or inactive, as exemplified by 49, 50, 52, and 53 (Table 1). The
primary alcohols 58 and 63 were also inactive, indicating that a
primary ammonium ion may be a critical feature that is
important for activity as opposed to hydrogen bonding
Figure 6. Hypothetical binding model of 14 in the binding pocket of
Tdp1.
capabilities per se. Compounds 59 and 62, with hydrogen
bond acceptors at the 3 position of the A ring, are also Tdp1
F
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Table 1. Tdp1 and Top1 Inhibitory Activity
a
b
compd
Tdp1
Top1
compd
Tdp1
Top1
1
++
+++
NT
0
40
41
42
43
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
72
73
74
77
84
85
89
++(+)
0
++(+)
0
2
+++
9
0
0
0
10
11
12
13
14
15
18
19
20
22
23
24
25
26
29
30
31
32
33
34
35
36
38
0
0
+++
0
0
0
(+)
+
+++
+
0
0
0
0
++
(+)
0
++
+
++
+(+)
0
+++
++
0
0
+++
+++
0
++++
++++
++
Figure 7. Hypothetical binding model of 40 in the binding pocket of
Tdp1.
0
0
0
0
+(+)
0
++
0
0
interact with either the Ser400 or Thr261 side-chain hydroxyl
groups as seen in Figures 6 and 7. In the case of 14, the lactam
carbonyl binds Thr261, whereas in 40, the ketone binds, so the
ring systems are “flipped” relative to each other. This suggests
that the amino group of the ligand 40 and other N-(3-
aminopropyl)indenoisoquinolines can play a major role in
orienting the ligand in the binding site of the enzyme.
++(+)
++(+)
(+)
0
+
+
+
+
+
0
+
0
0
0
0
0
0
+
+(+)
++(+)
0
+(+)
0
(+)
(+)
0
++(+)
0
++(+)
++
(+)
+
0
++
Compound 61, which contains a hydroxyl group instead of
the ketone, was not active, suggesting that a hydrogen bond-
accepting carbonyl oxygen is important for activity. The
presence of a 9-methoxy group on the D ring of the
indenoisoquinolines seems to have a positive effect on the
Tdp1 inhibitory activity, as observed for 54 (+++) vs 60 +(+)
or 89 (+++) vs 62 ++(+). Also, the position of the methoxy
group at C7, C8, or C9 affects the IC50’s as seen with 51 (15
μM), 54 (11 μM), and 55 (5.8 μM). Figure 8 shows a
0
++
+
++
0
+++
++(+)
+(+)
+++
++(+)
+
0
+++
+(+)
+++
+
0
a
Tdp1 IC50 was determined in duplicate using a semiquantitative
scale: 0, IC50 > 111 μM; +, IC50 between 37 and 111 μM; ++, IC50
between 12 and 37 μM; +++, IC50 between 1 and 12 μM, ++++, IC50
< 1 μM. Active compounds were further evaluated for a more accurate
b
value. Compound-induced DNA cleavage due to Top1 inhibition is
graded by the following semiquantitative scale relative to 1 μM
camptothecin (90) or MJ-III-65 (91): 0, no detectable activity; +,
weak activity; ++, similar activity to compound 91; +++, greater
activity than 91; ++++, equipotent to 90. The (+) ranking indicates
the activity lies between two given values. NT: not tested.
a
Table 2. IC50 Values of Tdp1-Active Compounds
compd
Tdp1 (μM)
61
45 10
compd
Tdp1 (μM)
11
5.8 0.8
14
22
23
40
43
51
7
54
55
57
62
84
89
1
18
11
8
5
18
12
1
4
5.0 1.4
15
5.2 0.1
6.7 0.8
3
a
The IC50 values of Tdp1-active compounds are expressed as mean
SD from at least three independent experiments.
inhibitors. It seems that the Tdp1 inhibitory activity of the
compounds decreases as the electron-withdrawing strength of
the substituent at the 3 position increases, as seen with the
IC50’s of compounds 60 > 57 > 62, which are >37, 18, and 12
μM, respectively. Compounds 72−74 support the trend that a
nitrogen that is more basic than morpholine or imidazole on
the aminopropyl side chain is necessary for Tdp1 inhibitory
activity. The primary amine 84, +++, was more active than the
tertiary amine 85, +(+), suggesting a steric limitation to binding
that may also be operating in the 72−74 series.
Figure 8. (A) Representative gel showing Tdp1 inhibition for
compounds 54 and 55. (B) Titration curves for determination of
Tdp1 IC50 values for compounds 54 and 55.
representative gel electrophoresis assay and titration curves for
determination of Tdp1 IC50 values for compounds 54 and 55.
The 9-methoxy substituent seems to play a bigger role in Tdp1
inhibitory activity than the substituent at the 3 position. Within
the 9-methoxy indenoisoquinolines, the electron-withdrawing
3-nitro group decreases the activity slightly when compared
with its 3-iodo or 3-amino counterparts. The IC50’s of the active
Molecular modeling indicates that the carbonyl of the five-
membered C ring or the amide-carbonyl of the B ring may
G
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compounds 54 (3-NO2), 84 (3-I), and 89 (3-NH2) are 11 1,
5.2 0.1, and 6.7 0.8 (Table 2), respectively. Compound 77,
which contains an 8,9-methylenedioxy group, was inactive vs
Tdp1.
Top1−DNA cleavage complexes by the indenoisoquinolines
are similar to each other, but the pattern is different from
camptothecin, indicating that the indenoisoquinolines target
the genome differently from camptothecin. However, the
indenoisoquinolines 52, 77, and 85 differ from each other in
their abilities to suppress DNA cleavage at high drug
concentrations. As is evident from the gel, the indenoisoquino-
lines 85 and 77, having terminal dimethylamino substituents on
the side chain, suppress DNA cleavage at a high concentration
of 100 μM, but compound 52, having an imidazole substituent
at the end of the chain, does not. DNA unwinding studies on
similar 7-azaindenoisoquinolines have shown that compounds
with an N-(3-dimethylaminopropyl) substituent intercalate into
free DNA at high drug concentrations, making the DNA a
poorer substrate for Top1, but compounds with an N-(3-
imidazolylpropyl) substituent do not intercalate into free DNA
so DNA cleavage is not suppressed at high drug concen-
tration.35 Although slight suppression is evident at high
concentrations of the imidazole analogue 52, it does not
resemble the complete suppression observed with the
dimethylamino analogues 77 and 85.
The Top1 inhibitory activities of some of the active
compounds have previously been analyzed and published.16
Regarding the new compounds, 49−53 were less active
compared with 54 and 55. For example, compounds 52 and
53 present Top1 inhibitory activities of +++ and ++,
respectively, compared to ++++ observed for both 54 and
55. The 3-cyano-substituted compounds 72−74 showed Top1
inhibitory activities of ++, ++, and +++, respectively. The 3-
cyano compound 77 had good Top1 activity of ++(+).
The iodo-substituted compounds 84 and 85 were also active
as Top1 inhibitors, although not as active as the previously
published analogues such as 54 having a 3-nitro substituent.18
However, the 3-iodo substituent could be easily exchanged,
thus expanding the alternatives for substitution. When the nitro
group of compound 54 was replaced with an amine to obtain
89, the Top1 inhibitory activity dropped from ++++ to ++(+).
The addition of a methoxy group at the 9 position of
compound 60 increased Top1 inhibitory activity as seen with
54 (++++) vs 60 [++(+)]. However, this trend is not observed
with the aniline analogues as seen in 62 with an activity of +
+(+) vs 89 with an activity of ++(+).
All of the compounds were tested for induction of Top1−
DNA cleavage complexes that are stabilized by inhibition of the
DNA religation reaction due to intercalation of the drugs
between DNA base pairs (Figure 9).31 Camptothecin (90) and
To investigate their potential as anticancer agents, a set of
indenoisoquinolines was examined for antiproliferative activity
against the human cancer cell lines in the National Cancer
Institute screen, in which the activity of each compound was
evaluated with approximately 55 different cancer cell lines of
diverse tumor origins.36,37 The GI50 values obtained with
selected cell lines, along with the mean graph midpoint
(MGM) values, are summarized in Table 3. The MGM is based
on a calculation of the average GI50 for all of the cell lines tested
in which GI50 values below and above the test range (10−8 to
10−4 M) are taken as the minimum (10−8 M) and maximum
(10−4 M) drug concentrations used in the screening test. For
comparison purposes, the activities of previously reported
compounds in Figure 3 are included on the Table 3. Many of
the new compounds display significant potencies against
various cell lines with GI50’s in the low micromolar or
submicromolar range. All of the compounds in Table 3 except
22, 25, 43, and 63 display some degree of inhibitory activity
against both Tdp1 and Top1. The most promising new
compounds are 52 and 89, with mean graph midpoint (MGM)
GI50 values of 0.02 μM and 0.04 μM, respectively. Overall, the
cytotoxicities do not correlate very well with the potencies vs
the isolated enzymes. For example, compounds 23, 40, and 62
all have the same ++(+) potencies vs both enzymes, but their
cytotoxicity MGM values range from 1.86 to 0.16 μM. Another
example would be 54 (MGM 0.02 μM) vs 55 (MGM 1.41
μM), both of which have +++ potency vs Tdp1 and ++++
potency vs Top1. Compounds 52 (MGM 0.02 μM) and 54
(MGM 0.02 μM) provide another way of stating the case,
because they both have the same MGM value but on the basis
of the Tdp1 and Top1 inhibitory potencies, 54 would be
expected to be more cytotoxic than 52. These differences in
cytotoxicities may reflect different uptake, distribution,
metabolism, and excretion profiles in the cellular systems as
well as off-target effects.
Figure 9. Top1-mediated DNA cleavage induced by 85, 77, and 52.
Lane 1: DNA alone; lane 2: Top1 alone; lane 3: 1, 1 μM; lane 4: 5, 1
μM; lane 5−16: 85, 77, and 52 at 0.1, 1, 10, and 100 μM, respectively,
from left to right. Numbers and arrows on the left indicate arbitrary
cleavage site positions. The activity of the compounds to produce
Top1-mediated DNAcleavage was expressed semiquantitatively as
follows: +, weak activity; ++ and +++, moderate activity; ++++, similar
activity as 1 μM camptothecin (90).
the previously synthesized lead compound 9132−34 were
included for comparison. The cleavage complexes were
monitored using a 32P 3′-end labeled, 117-bp DNA fragment
that was reacted with recombinant human Top1 in the
presence of increasing concentrations of the indenoisoquino-
lines while separation of the DNA fragments was carried out on
a denaturing gel. The sequence preferences for trapping the
H
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Table 3. Cytotoxicities of Selected Compounds
a
cytotoxicity (GI50 in μM)
lung HOP- colon HCT-
CNS SF- melanoma UACC-
ovarian
OVCAR-3
renal
SN12C
prostate DU-
145
breast MDA-MB-
435
b
compd
62
116
539
62
MGM (μM)
c
22
23
25
26
40
43
51
52
54
55
60
62
63
74
84
89
0.65
1.56
0.79
2.32
0.93
0.65
0.14
<0.01
<0.01
1.15
−
0.27
0.39
0.26
0.14
<0.01
0.57
0.54
6.90
4.03
1.47
5.65
8.91
1.79
0.61
<0.01
<0.01
1.45
0.14
0.34
0.39
1.12
0.39
0.03
1.22
1.69
1.22
2.43
2.25
0.72
0.14
<0.01
<0.01
2.34
0.03
0.30
0.31
0.19
0.14
0.10
4.01
3.22
1.47
3.52
3.12
1.6
2.53
1.46
1.11
1.57
0.54
0.77
1.68
1.51
0.18
<0.01
−
1.07
0.01
0.12
0.43
0.24
0.05
<0.01
3.36
2.33
2.05
4.3
1.86 0.38
1.86
1.09
2.34
1.69
0.38
1.02
0.51
1.82
0.44
1.75
3.05
1.14
0.58
0.03
3.31
1.62
0.12
0.23
1.97
0.62
0.96
0.12
0.33
0.49
0.87 0.014
0.17 0.017
0.02 0.0006
0.02 0.0008
1.41 0.43
0.15 0.10
0.16
0.06
0.54
0.03
2.82
2.29
0.08
0.13
0.87
1.72
1.32
0.02
0.15
<0.01
<0.01
0.72
<0.01
<0.01
7.08
<0.01
0.18
<0.01
0.23
0.19
0.47
0.89 0.22
0.30 0.014
0.25 0.004
0.04
0.05
0.19
0.07
0.05
<0.01
<0.01
a
The cytotoxicity GI50 values are the concentrations corresponding to 50% growth inhibition. The compounds were tested at concentrations ranging
b
c
up to 10 μM. Mean graph midpoint for growth inhibition of all human cancer cell lines successfully tested. For MGM GI50 values in which a
standard error appears, the GI50 values for individual cell lines are the average of two determinations; values without standard error are from one
determination.
clear rationale for dual inhibition. However, the poor
correlation of cytotoxicity with activities against the two
isolated enzymes observed here does not allow a
convincing argument to be made that inhibition of
Tdp1 increases the cytotoxicity resulting from Top1
inhibition.
CONCLUSIONS
The key points of this study are as follows:
■
(1) A 3-aminopropyl side chain on the lactam nitrogen is
important for Tdp1 inhibitory activity, because com-
pounds that lack this structural feature are either inactive
or have very low activity.
(8) The indenoisoquinolines 54 and 89 both have significant
dual enzyme inhibitory activities and are cytotoxic
enough in human cancer cell cultures to warrant further
preclinical development.
(2) The 11-keto group appears to be important for Tdp1
inhibitory activity because its reduction in 57 to an
alcohol 61 abolished the activity.
(3) Consistent with previous results,35 indenoisoquinolines
77 and 85 with acyclic 3-aminopropyl side chains
suppress Top1-mediated DNA cleavage at high drug
concentrations, but the 3-imidazolylpropyl compound 52
does not.
EXPERIMENTAL SECTION
■
General. NMR spectra were obtained at 300 or 500 (1H) and 75 or
125 (13C) MHz using Bruker ARX300 or Bruker DX-2 500 [QNP
probe or multinuclear broadband observe (BBO) probe, respectively]
spectrometers. Column chromatography was performed with 230−400
mesh silica gel. The melting points were determined using capillary
tubes with a Mel-Temp apparatus and are uncorrected. IR spectra were
obtained using a Perkin-Elmer 1600 series FTIR spectrometer on salt
plates or as KBr pellets. ESI-MS analyses were recorded in a
FinniganMAT LCQ Classic mass spectrometer. APCI-MS analyses
were performed in an Agilent 6320 ion trap mass spectrometer. EI/CI-
MS analyses were obtained in a Hewlett-Packard Engine mass
spectrometer. All mass spectral analyses were performed at the
Campus-Wide Mass Spectrometry Center of Purdue University.
Microanalyses were performed at Midwest Microlab. HPLC analyses
were carried out on a Waters 1525 binary HPLC pump/Waters 2487
dual λ absorbance detector system using a 5 μM C18 reverse phase
column. All reported yields refer to pure isolated compounds.
Chemicals and solvents were of reagent grade and used as obtained
from commercial sources without further purification. The purities of
all of the tested compounds were >95% as estimated by HPLC or
determined by elemental analysis. For HPLC, the peak area of the
major product was ≥95% of the combined total peak areas when
monitored by a UV detector at 254 nm.
(4) Installation of a 9-methoxy substituent into 60 (MGM
0.15 μM) results in an increase in cytotoxicity (see 54,
MGM 0.02 μM), while an 8-methoxy has little effect (see
51, MGM 0.17 μM) and a 7-methoxy is detrimental (see
55, MGM 1.41 μM). These trends are not reflected in
their activities vs the isolated enzymes (compare 54 and
55, Table 1).
(5) With the exception of 14 and 43, installation of C-3 side
chains of various lengths ending in a carboxylate
generally resulted in compounds that are inactive vs
Tdp1 and have low activity or no activity vs Top1 (see
32−36, Table 1). However, the corresponding esters
22−26 retained low to moderate activity vs Tdp1 and
had mixed effects on Top1 compared with the
corresponding acids (Table 1).
(6) Within the similar series of compounds 54 (3-NO2), 89
(3-NH2), and 84 (3-I), the substituent at C-3 has no
effect on Tdp1 inhibitory activity, but it does have an
effect on Top1 inhibitory activity, with the potency
ranking being NO2 > NH2 > I. This is reflected in the
relative cytotoxicities of these compounds.
Methyl 2-[(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno-
[1,2-c]isoquinolin-3-yl)amino]-2-oxoacetate (9). 3-Amino-6-
methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione10 (3, 100 mg,
0.36 mmol) was dissolved in tetrahydrofuran (10 mL) and the reaction
mixture cooled to 0 °C. Methyl 2-chloro-2-oxoacetate (4, 0.1 mL, 1.09
(7) There is increasing interest in obtaining drugs that act on
more than one target, and in the present case there is a
I
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mmol) was added dropwise to the indenoisoquinoline solution, and
the reaction mixture was stirred for 30 min. Triethylamine (0.1 mL)
was slowly added, and the reaction mixture was stirred for another 30
min, keeping the temperature at 0 °C. The reaction mixture was
diluted with water (30 mL), and chloroform (20 mL) was added. The
organic phase was separated and washed with water (30 mL) and brine
(30 mL). The organic phase was concentrated and the compound
purified by silica gel column chromatography, eluting with chloro-
form−methanol, 50:1. The product was obtained as a brick-red solid
(53 mg, 39%): mp 287−289 °C. IR (film) 3336, 1642, 1514, 1480,
Methyl 5-[(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno-
[1,2-c]isoquinolin-3-yl)amino]-5-oxopentanoate (12). The com-
pound was eluted with chloroform−methanol, 30:1. The product was
obtained as a dark red solid (225 mg, 96%): mp 184−186 °C. IR
(film) 3335, 3119, 2945, 1730, 1689, 1650, 1580, 1525, 1431, 1316,
1274, 1194, 901, 844, 757, 718 cm−1; 1H NMR (500 MHz, DMSO-d6)
δ 10.35 (s, 1 H), 8.49 (d, J = 1.9 Hz, 1 H), 8.35 (d, J = 8.7 Hz, 1 H),
7.85 (dd, J = 8.7 Hz, J = 2.1 Hz, 1 H), 7.76 (d, J = 7.5 Hz, 1 H), 7.45−
7.32 (m, 3 H), 3.88 (s, 3 H), 3.56 (s, 3 H), 2.46 (m, 4 H), 1.81 (m, 2
H); 13C NMR (125 MHz, DMSO-d6) δ 190.3, 173.4, 171.2, 162.5,
155.6, 138.3, 137.7, 134.5, 133.8, 131.2, 127.3, 125.8, 124.2, 123.7,
123.5, 122.7, 116.9, 107.0, 51.6, 35.6, 32.9, 32.4, 20.6.; ESIMS m/z (rel
intensity) 405 (MH+, 100); HRESIMS calcd for C23H20N2O5
405.1450 (MH+), found 405.1446 (MH+); HPLC purity: 96.0%
(MeOH−H2O, 80:20).
1
1406, 1358, 1333, 1231, 1045, 815, 759 cm−1; H NMR (300 MHz,
CDCl3) δ 11.08 (s, 1 H), 8.67 (d, J = 1.9 Hz, 1 H), 8.40 (d, J = 8.7 Hz,
1 H), 8.06 (dd, J = 8.8 Hz, J = 2.1 Hz, 1 H), 7.82 (d, J = 7.2 Hz, 1 H),
7.50−7.43 (m, 3 H), 3.93 (s, 3 H), 3.87 (s, 3 H); ESIMS m/z (rel
intensity) 363 (MH+, 100); HRESIMS calcd for C20H14N2O5
363.0910 (MH+), found 363.0907 (MH+); HPLC purity: 98.3%
(MeOH−H2O, 90:10), 95.2% (MeOH−H2O, 85:15).
Methyl 6-[(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno-
[1,2-c]isoquinolin-3-yl)amino]-6-oxohexanoate (13). The com-
pound was eluted with chloroform−methanol, 30:1. The product was
obtained as a red solid (221 mg, 91.2%): mp 177−179 °C. IR (film)
3341, 3071, 2950, 2868, 1732, 1692, 1659, 1573, 1522, 1512, 1434,
Methyl 3-[(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno-
[1,2-c]isoquinolin-3-yl)amino]-3-oxopropanoate (10). 3-Amino-
6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (3, 150 mg,
0.54 mmol) was dissolved in tetrahydrofuran (20 mL) and the solution
was cooled to 0 °C. Methyl malonyl chloride (5, 0.1 mL, 0.93 mmol)
was added and the reaction mixture was stirred for 5 min at 0 °C.
Triethylamine (0.2 mL) was added and the reaction mixture was
stirred for 2 h at 0 °C. The solvent was removed under vacuum and
the residue was purified by silica gel column chromatography, eluting
with chloroform−methanol, 95:5, and then chloroform-ethyl acetate-
methanol, 45:45:10. The fractions were combined, the solvent was
removed under vacuum and the solid was washed with hexane−
dichloromethane, 1:1 (25 mL). The product was obtained as a
reddish-brown solid (60 mg, 29%): mp 230 °C (decomp). IR (film)
3309, 3071, 2952, 1744, 1692, 1661, 1574, 1531, 1317, 1276, 1054,
1
1316, 1276, 1196, 1055, 902, 842, 762, 750 cm−1; H NMR (500
MHz, DMSO-d6) δ 10.20 (s, 1 H), 8.49 (d, J = 2.0 Hz, 1 H), 8.36 (d, J
= 8.8 Hz, 1 H), 7.88 (dd, J = 8.7 Hz, J = 2.0 Hz, 1 H), 7.79 (d, J = 7.5
Hz, 1 H) 7.51−7.36 (m, 3 H), 3.91 (s, 3 H), 3.55 (s, 3 H), 2.31 (m, 4
H), 1.56 (m, 4 H); 13C NMR (125 MHz, DMSO-d6) δ 190.3, 173.6,
171.6, 162.5, 155.8, 138.5, 137.7, 134.6, 133.9, 131.2, 127.3, 125.9,
124.2, 123.7, 123.5, 122.8, 117.0, 107.1, 51.6, 36.4, 33.4, 32.5, 24.8,
24.4; ESIMS (rel intensity) m/z 419 (MH+, 100); HRESIMS calcd for
C24H22N2O5 419.1607 (MH+), found 419.1613 (MH+); HPLC purity:
97.0% (MeOH−H2O, 85:15), 96.9% (MeOH−H2O, 90:10).
(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]-
isoquinolin-3-yl)carbamic Acid (14). 3-Amino-6-methyl-5H-
indeno[1,2-c]isoquinoline-5,11(6H)-dione (3, 201 mg, 0.72 mmol)
was dissolved in tetrahydrofuran (30 mL) and cooled to 0 °C. Methyl
oxalyl chloride (4, 0.1 mL, 1.16 mmol) and triethylamine (0.2 mL)
were added dropwise and the reaction mixture was stirred for 2 h at 0
°C. The reaction mixture was diluted with water (100 mL) and
extracted with chloroform (4 × 50 mL). The solvent was removed
under vacuum and the compound passed through a short silica gel
column chromatography, eluting with chloroform−methanol, 9:1. The
impure solid, compound 9, was dissolved in a solution of sodium
hydroxide (0.1 g, 2.5 mmol) in ethanol (50 mL) and water (1 mL) and
the reaction mixture was stirred overnight at room temperature. The
solvent was removed under vacuum and the residue purified by silica
gel column chromatography, eluting with chloroform−methanol-acetic
acid, 90:9:1. Compound 14 was obtained as a reddish-brown solid
(103 mg, 44.7%, after two steps): mp 360 °C (decomp). IR (film)
3325, 2928, 1692, 1657, 1600, 1580, 1533, 1435, 1319, 1197, 903, 701
cm −1; 1H NMR (300 MHz, DMSO-d6) δ 10.9 (s, 1 H), 8.70 (s, 1 H),
8.35 (d, J = 8.2 Hz, 1 H), 8.02 (s, 1 H), 7.90 (br s, 1 H), 7.45 (m, 3
H), 3.89 (s, 3 H); 13C NMR (125 MHz, DMSO-d6) δ 190.1, 162.4,
162.2, 157.9, 156.3, 137.5, 136.9, 134.6, 134.0, 131.4, 128.4, 126.9,
124.4, 123.5, 123.4, 122.8, 118.5, 106.9; negative ion ESIMS m/z (rel
intensity) 347 [(M − H+)−]; negative ion HRESIMS calcd for
C19H12N2O5 347.0668 [(M − H+)−], found 347.0664 [(M − H+)−];
HPLC purity: 95.2% (MeOH−H2O, 85:15), 95.7% (MeOH−H2O,
75:25).
3-[(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]-
isoquinolin-3-yl)amino]-3-oxopropanoic Acid (15). Methyl 3-
[(6-methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-
yl)amino]-3-oxopropanoate (10, 158 mg, 0.42 mmol) was dissolved in
methanol (20 mL) and dimethylformamide (2 mL). A solution of
sodium hydroxide (103 mg) in water (2 mL) was added dropwise and
the reaction mixture was stirred at room temperature for 24 h.
Concentrated hydrochloric acid (1 mL) was added and the solution
diluted with chloroform (30 mL). A dark-red precipitated was formed
between the two phases. The solid was filtered and the organic and
water layers discarded. The solid was washed with water (10 mL) and
ether (2 × 10 mL). The solid was dried and compound 15 was
obtained as a reddish-brown solid (93 mg, 61%): mp 262 °C
1
1017, 901 cm −1; H NMR (300 MHz, DMSO-d6) δ 10.46 (s, 1 H),
8.42 (d, J = 1.8 Hz, 1 H), 8.31 (d, J = 8.8 Hz, 1 H), 7.79 (dd, J = 8.8
Hz, J = 2.0 Hz, 1 H), 7.72 (d, J = 7.4 Hz, 1 H) 7.45−7.37 (m, 3 H),
3.85 (s, 3 H), 3.66 (s, 3 H), 3.49 (s, 2 H); 13C NMR (125 MHz,
DMSO-d6) δ 190.3, 168.4, 164.6, 162.5, 156.0, 137.9, 137.7, 134.6,
133.9, 131.3, 127.8, 125.8, 124.3, 123.7, 122.8, 117.2, 107.0, 52.4, 43.8,
32.5; ESIMS m/z (rel intensity) 775 (2MNa+, 100), 752 (2MH+, 37),
377 (MH+, 10); HRESIMS calcd for C21H16N2O5 377.1137 (MH+),
found 377.1140 (MH+); HPLC purity: 95.0% (75:25), 95.0%
(MeOH−H2O, 70:30).
General Procedure for the Synthesis of Indenoisoquinolines
11−13. 3-Amino-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-
dione (3, 160 mg, 0.58 mmol) was dissolved in tetrahydrofuran (15
mL) and the reaction mixture cooled to 0 °C. The desired acyl
chloride (6−8, 100−125 μL, 0.81 mmol) was added dropwise to the
indenoisoquinoline solution, and the reaction mixture was stirred for
10 min. Triethylamine (0.4 mL) was slowly added, and the reaction
mixture was stirred for 1 h, keeping the temperature at 0 °C.
Chloroform (40 mL) was added to the reaction mixture, and the
organic solution was washed with water (3 × 40 mL) and brine (1 ×
50 mL). The organic phase was concentrated, and the compounds
were purified by silica gel column chromatography.
Methyl 4-[(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno-
[1,2-c]isoquinolin-3-yl)amino]-4-oxobutanoate (11). The com-
pound was eluted with chloroform−methanol, 20:1. The product was
obtained as an orange solid (101 mg, 44%): mp 250−252 °C. IR
(film) 3339, 3314, 2951, 1735, 1720, 1688, 1658, 1643, 1573, 1518,
1432, 1315, 1195, 1157, 1055, 891, 845, 763, 700 cm−1; 1H NMR (500
MHz, DMSO-d6) δ 10.37 (s, 1 H), 8.49 (d, J = 1.9 Hz, 1 H), 8.35 (d, J
= 8.7 Hz, 1 H), 7.87 (dd, J = 8.7 Hz, J = 2.1 Hz, 1 H), 7.78 (d, J = 7.5
Hz, 1 H) 7.49−7.37 (m, 3 H), 3.89 (s, 3 H), 3.57 (s, 3 H), 2.61 (m, 4
H); 13C NMR (125 MHz, DMSO-d6) δ 190.9, 173.8, 171.0, 163.1,
156.3, 139.0, 138.3, 135.1, 134.5, 131.8, 127.9, 126.3, 124.8, 124.3,
124.1, 123.3, 117.4, 107.6, 52.3, 33.0, 31.8, 29.3; ESIMS m/z (rel
intensity) 413 (MNa+, 100); HRESIMS calcd for C22H18N2O5
413.1113 (MNa+), found 413.1119 (MNa+); HPLC purity: 98.7%
(MeOH−H2O, 85:15), 98.8% (MeOH−H2O, 95:5).
J
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Journal of Medicinal Chemistry
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(decomp). IR (KBr) 3447, 3323, 1731, 1695, 1573, 1529, 1433, 1317,
1.45 (s, 9 H); 13C NMR (CDCl3 + DMSO-d6, 125 MHz) δ 190.0,
173.0, 170.0, 162.9, 155.8, 153.6, 137.9, 136.7, 134.5, 133.0, 130.3,
127.4, 126.0, 123.7, 123.4, 122.6, 122.0, 117.2, 108.3, 78.7, 51.4, 41.6,
36.9, 31.1, 29.6, 28.6, 28.1; ESIMS m/z (relative intensity) 556
(MNa+, 12); HRESIMS calcd for C29H31N3O7Na 556.2060, found
556.2069; HPLC purity: 95.0% (MeOH−H2O, 85:15).
1
1195, 1054, 899, 763 cm −1; H NMR (300 MHz, DMSO-d6) δ 10.5
(s, 1 H), 8.45 (s, 1 H), 8.31 (d, J = 8.6 Hz, 1 H), 7.80 (d, J = 7.0 Hz, 1
H), 7.46−7.38 (m, 3 H), 3.86 (s, 3 H), 3.39 (s, 2 H); 13C NMR (125
MHz, DMSO-d6) δ 190.1, 169.5, 165.1, 162.4, 155.7, 138.1, 137.5,
134.5, 133.8, 131.2, 127.6, 125.7, 124.1, 123.6, 122.7, 117.0, 116.9,
106.9, 44.4, 32.4; MALDIMS m/z (rel intensity) 363 (MH+, 100);
negative ion ESIMS (m/z, relative intensity) 361 [(M − H+)−], 100);
negative ion HRESIMS calcd for C20H14N2O5 361.0824 [(M − H+)−],
found 361.0828 [(M − H+)−]; HPLC purity: 99.4% (MeOH), 98.6%
(MeOH−H2O, 95:5).
Methyl 5-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-
dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-
5-oxopentanoate (20). Compound 16 (0.150 g, 0.357 mmol) was
dissolved in chloroform (100 mL), triethylamine (0.108 g, 1.07 mmol)
was added followed by methyl 5-chloro-5-oxopentanoate (7, 0.088 g,
0.536 mmol) at room temperature, and the reaction mixture was
stirred for 2 h. The reaction mixture was washed with water (2 × 25
mL), extracted with chloroform (2 × 60 mL), and dried over
anhydrous sodium sulfate. The solvent was removed under vacuum
and the residue purified by silica gel column chromatography, eluting
with chloroform−methanol, 9.6:0.4, to afford the product 20 (0.10 g,
50%) as a red solid: mp 118−120 °C. IR (KBr) 3329, 1696, 1679,
Methyl 2-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-
dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-
2-oxoacetate (17). Compound 1618 (0.3 g, 0.715 mmol) was
dissolved in chloroform (100 mL). Triethylamine (0.261 g, 2.14
mmol) was added at room temperature followed by methyl oxaloyl
chloride (4, 0.113 g, 0.930 mmol) and the reaction mixture was stirred
for 2 h. The reaction mixture was washed with water (2 × 20 mL) and
dried over anhydrous sodium sulfate. The solvent was removed under
vacuum and the residue purified by silica gel column chromatography,
eluting with chloroform−methanol, 9.4:0.6, to furnish the product 17
(0.230 g, 65%) as an orange solid: mp 157−159 °C. IR (KBr) 3339,
1
1663, 1596, 1528, 1169, 760 cm−1; H NMR (CDCl3, 300 MHz) δ
8.52 (d, J = 5.3 Hz, 1 H), 8.21 (br s, 1 H), 8.01 (br s, 1 H), 7.48 (d, J =
4.7 Hz, 1 H), 7.40 (m, 2 H), 7.26 (m, 1 H), 5.48 (t, J = 4.5 Hz, 1 H),
4.51 (t, J = 4.5 Hz, 2 H), 3.69 (s, 3 H), 3.20 (m, 2 H), 2.49 (m, 4 H),
2.10 (m, 4 H), 1.45 (s, 9 H); 13C NMR (CDCl3, 125 MHz) δ 190.2,
173.8, 170.8, 163.2, 156.1, 154.0, 137.1, 136.9, 134.7, 133.3, 130.7,
128.3, 126.6, 124.3, 123.7, 123.1, 122.3, 117.9, 108.6, 79.3, 51.7, 41.9,
37.3, 36.1, 32.9, 30.0, 28.4, 20.6; ESIMS m/z (relative intensity) 570
(MNa+, 100); HRESIMS calcd for C30H33N3O7Na 570.2216 (MNa+),
found 570.2209 (MNa+); HPLC purity: 96.4% (MeOH−H2O, 85:15).
Methyl 6-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-
dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-
6-oxohexanoate (21). Compound 16 (0.150 g, 0.357 mmol) was
dissolved in chloroform (100 mL), triethylamine (0.090 g, 0.892
mmol) was added followed by methyl 6-chloro-6-oxohexanoate (8,
0.095 g, 0.536 mmol) at room temperature, and the reaction mixture
was stirred for 2 h. The reaction mixture washed with water (2 × 30
mL), extracted with chloroform (2 × 75 mL), and dried over
anhydrous sodium sulfate. The solvent was removed under vacuum
and the residue purified by silica gel column chromatography, eluting
with chloroform−methanol, 9.7:0.3, to yield the product 21 (0.116 g,
55%) as a red solid: mp 143−145 °C. IR (KBr) 3332, 2974, 1696,
1
2976, 1702, 1662, 1580, 1570, 1534, 1514, 1163, 759, 665 cm−1; H
NMR (CDCl3, 300 MHz) δ 9.02 (s, 1 H), 8.73 (d, J = 8.7 Hz, 1 H),
8.32 (s, 1 H), 8.10 (dd, J = 1.8, J = 5.6 Hz, 1 H), 7.68 (m, 1 H), 7.48
(m, 1 H), 7.30 (m, 2 H), 5.30 (m, 1 H), 4.58 (t, J = 4.8 Hz, 2 H), 3.99
(s, 3 H), 3.26 (m, 2 H), 2.10 (m, 2 H), 1.45 (s, 9 H); 13C NMR
(CDCl3 + CD3OD, 75 MHz) δ 163.2, 160.6, 156.5, 154.6, 136.5,
135.6, 134.5, 133.4, 130.9, 129.2, 126.4, 124.3, 123.5, 123.1, 122.5,
118.3, 108.4, 79.3, 53.6, 42.2, 37.2, 29.5, 28.0; ESIMS m/z (relative
intensity) 528 (MNa+, 62); HRESIMS calcd for C27H27N3O7Na
528.1747 (MNa+), found 528.1740 (MNa+).
Methyl 3-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-
dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-
3-oxopropanoate (18). Compound 16 (0.100 g, 0.234 mmol) was
dissolved in chloroform (75 mL), triethylamine (0.060 g, 0.591 mmol)
was added followed by methyl 3-chloro-3-oxopropionate (5, 0.055 g,
0.290 mmol) at room temperature, and the reaction mixture was
stirred for 2 h. The reaction mixture was washed with water (2 × 20
mL), extracted with chloroform (2 × 75 mL), and dried over
anhydrous sodium sulfate. The solvent was removed under vacuum
and the residue purified by silica gel column chromatography, eluting
with chloroform−methanol, 9.3:0.7, to furnish the product 18 (0.075
g, 65%) as a red solid: mp 144−145 °C. IR (KBr) 3325, 2976, 1744,
1
1662, 1580, 1568, 1527, 1169, 760, 666 cm−1; H NMR (CDCl3, 300
MHz) δ 8.46 (d, J = 8.7 Hz, 1 H), 8.28 (br s, 1 H), 8.03 (br s, 1 H),
7.93 (d, J = 5.2 Hz, 1 H), 7.52 (d, J = 5.2 Hz, 1 H), 7.31 (m, 2 H), 7.28
(d, J = 6.3 Hz, 1 H), 5.43 (t, J = 5.4 Hz, 1 H), 4.48 (t, J = 6.6 Hz, 2 H),
3.67 (s, 3 H), 3.18 (m, 2 H), 2.38 (m, 4 H), 2.05 (m, 2 H), 1.76 (m, 4
H), 1.45 (s, 9 H); 13C NMR (CDCl3, 125 MHz) δ 190.3, 174.1, 171.1,
163.1, 156.1, 154.1, 137.2, 136.9, 134.8, 133.3, 130.7, 128.3, 126.7,
124.3, 123.7, 123.1, 122.3, 118.0, 108.6, 79.3, 51.6, 41.9, 37.3, 37.0,
33.6, 30.0, 28.4, 24.8, 24.2; ESIMS m/z (relative intensity) 584
(MNa+, 17); HRESIMS calcd for C31H35N3O7Na 584.2373, found
584.2370.
Methyl 2-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-
indeno[1,2-c]isoquinolin-3-yl)amino]-2-oxoacetate (22). Com-
pound 17 (0.070 g, 0.130 mmol) was treated with trifluoroacetic acid
(0.5 mL) in chloroform (5 mL) for 2 h at room temperature. The
solvent was removed on a rotary evaporator, and the residue was then
basified with 2 N NH3 in methanol to get the free amine, which was
purified by silica gel column chromatography, eluting with chloro-
form−methanol, 8.7:1.3, to yield the product 22 (0.035 g, 65%) as a
brown solid: mp 185−186 °C. IR (KBr) 3014, 1692, 1581, 1570, 1533,
1307, 1198, 722, 455 cm−1; 1H NMR (CD3OD, 300 MHz) δ 8.55 (s, 1
H), 8.23 (d, J = 3.2 Hz, 1 H), 7.71 (d, J = 3.5 Hz, 1 H), 7.54 (m, 1 H),
7.36 (m, 3 H), 4.52 (m, 2 H), 3.91 (s, 3 H), 3.05 (m, 2 H), 2.28 (m, 2
H); 13C NMR (CD3OD, 125 MHz) δ 191.6, 169.9, 166.8, 164.9,
155.4, 138.8, 137.9, 135.7, 134.9, 132.2, 129.6, 127.2, 125.0, 124.8,
124.1, 123.9, 118.6, 109.6, 52.9, 42.5, 38.2, 28.6; ESIMS m/z (relative
intensity) 420 (MH+, 100); HRESIMS calcd for C23H21N3O5
420.1559 (MH+), found 420.1554 (MH+); HPLC purity: 98.2% (1%
TFA in MeOH).
1
1696, 1664, 1572, 1532, 1512, 1433, 1168, 761, 665 cm−1; H NMR
(CDCl3, 300 MHz) δ 9.47 (s, 1 H), 8.65 (d, J = 8.8 Hz, 1 H), 8.35 (s,
1 H), 8.06 (m, 1 H), 7.59 (m, 1 H), 7.50 (m, 1 H), 7.41 (m, 2 H), 5.40
(br s, 1 H), 4.60 (t, J = 6.6 Hz, 2 H), 3.82 (s, 3 H), 3.53 (s, 2 H), 3.50
(m, 2 H), 2.08 (m, 2 H), 1.45 (s, 9 H); 13C NMR (CDCl3, 300 MHz)
δ 190.0, 169.8, 163.3, 163.0, 156.1, 154.1, 136.7, 136.5, 134.6, 133.2,
130.6, 128.5, 126.5, 124.2, 123.6, 123.0, 122.3, 118.2, 108.3, 79.2, 52.6,
42.0, 41.7, 37.3, 29.9, 28.4; ESIMS m/z (relative intensity) 542
(MNa+, 100); HRESIMS calcd for C28H29N3O7Na 542.1903 (MNa+),
found 542.1906 (MNa+); HPLC purity: 95.7% (MeOH−H2O, 85:15).
Methyl 4-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-
dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-
4-oxobutanoate (19). Compound 16 (0.150 g, 0.357 mmol) was
dissolved in chloroform (100 mL), triethylamine (0.090 g, 0.892
mmol) was added followed by methyl 4-chloro-4-oxobutanoate (6,
0.082 g, 0.536 mmol) at room temperature, and the reaction mixture
was stirred for 2 h. The reaction mixture was washed with water (2 ×
20 mL), extracted with chloroform (2 × 75 mL), and dried over
anhydrous sodium sulfate. The solvent was removed under vacuum
and the residue purified by silica gel column chromatography, eluting
with chloroform−methanol, 9.5:0.5, to furnish the product 19 (0.114
g, 60%) as an orange solid: mp 176−178 °C. IR (KBr) 3307, 2917,
1728, 1703, 1718, 1695, 1575, 1165, 666 cm−1; 1H NMR (CDCl3, 300
MHz) δ 8.68 (d, J = 5.3 Hz, 1 H), 8.21 (br s, 1 H), 8.02 (m, 2 H), 7.68
(d, J = 4.8 Hz, 1 H), 7.32 (m, 4 H), 5.41 (br s, 1 H), 4.57 (t, J = 3.2
Hz, 2 H), 3.72 (s, 3 H), 3.20 (m, 2 H), 2.79 (m, 4 H), 2.08 (m, 2 H),
K
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Journal of Medicinal Chemistry
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Methyl 3-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-
indeno[1,2-c]isoquinolin-3-yl)amino]-3-oxopropanoate (23).
Compound 18 (0.080 g, 0.145 mmol) was treated with TFA (0.5
mL) in chloroform (5 mL) for 2 h at room temperature. The solvent
was removed on a rotary evaporator and the residue was then basified
with 2 N NH3 in methanol to get the free amine, which was purified by
silica gel column chromatography, eluting with chloroform−methanol,
8.8:1.2, to yield the product 23 (0.040 g, 62%) as a brown solid: mp
225−227 °C. IR (KBr) 3067, 1739, 1673, 1574, 1535, 1511, 1202,
1134, 722 cm−1; 1H NMR (CD3OD, 300 MHz) 8.52 (d, J = 2.1 Hz, 1
H), 8.29 (d, J = 6.8 Hz, 1 H), 7.43 (m, 5 H), 4.50 (t, J = 2.1 Hz, 2 H),
3.78 (s, 3 H), 3.10 (m, 2 H), 2.22 (m, 2 H); 13C NMR (CD3OD, 125
MHz) δ 191.6, 169.9, 166.8, 164.9, 155.4, 138.8, 137.9, 135.7, 134.9,
132.2, 129.6, 127.2, 125.0, 124.8, 124.1, 123.9, 118.6, 109.6, 52.9, 42.5,
38.2, 28.6; ESIMS m/z (relative intensity) 420 (MH+, 100);
HRESIMS calcd for C23H21N3O5 420.1559 (MH+), found 420.1554
(MH+); HPLC purity: 100% (1% TFA in MeOH−H2O, 50:50).
Methyl 4-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-
indeno[1,2-c]isoquinolin-3-yl)amino]-4-oxobutanoate (24).
Compound 19 (0.060 g, 0.112 mmol) was treated with trifluoroacetic
acid (0.5 mL) in chloroform (5 mL) for 2 h at room temperature. The
solvent was removed on a rotary evaporator and the residue was then
basified with 2 N NH3 in methanol to get the free amine, which was
purified by silica gel column chromatography, eluting with chloro-
form−methanol, 8.8:1.2, to yield the product 24 (0.028 g, 60%) as a
brown solid: mp 272−274 °C. IR (KBr) 2952, 1735, 1690, 1656, 1572,
130.9, 127.0, 125.6, 123.5, 123.4, 123.3, 122.5, 116.5, 107.2, 51.2, 42.1,
38.2, 36.0, 33.0, 30.9, 24.4, 24.0; ESIMS (m/z, relative intensity) 462
(MH+, 100); HRESIMS calcd for C26H27N3O5 462.2023 (MH+),
found 462.2031 (MH+); HPLC purity: 98.5% (1% TFA in MeOH−
H2O, 70:30).
General Procedure for Synthesis of Indenoisoquinolines 27-
31. The esters 17−21 (0.1 g) were dissolved in methanol (10 mL)
and tetrahydrofuran (10 mL). An aqueous NaOH solution (4 N, 5
mL) was added at room temperature, and the reaction mixture was
stirred at room temperature for 6 h. The solvent was removed on a
rotary evaporator and the residue washed with 1 N HCl (30 mL),
extracted with chloroform (2 × 50 mL), dried over anhydrous sodium
sulfate, concentrated, and purified by silica gel column chromatog-
raphy, eluting with chloroform−methanol 9.6:0.4 to 9:1, to afford
acids 27−31 in 55−75% yields as orange solids.
2-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-
6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-2-oxo-
acetic Acid (27). mp 267−268 °C. IR (KBr) 3295, 1758, 1698, 1646,
1
1605, 1580, 1535, 1350, 1161, 847, 665 cm−1; H NMR (CD3OD +
DMSO-d6, 300 MHz) δ 8.87 (s, 1 H), 8.63 (d, J = 8.7 Hz, 1 H), 8.14
(d, J = 9.3 Hz, 1 H), 7.80 (d, J = 7.2 Hz, 1 H), 7.64 (m, 2 H), 7.55 (m,
1 H), 4.60 (t, J = 7.2 Hz, 2 H), 3.21 (m, 2 H), 2.07 (m, 2 H), 1.50 (s, 9
H).
3-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-
6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-3-oxo-
propanoic Acid (28). mp 211−212 °C. IR (KBr) 3310, 1768, 1710,
1976, 1522, 1385, 1355, 847, 665 cm−1; 1H NMR (CD3OD, 300
MHz) δ 8.56 (d, J = 2.2 Hz, 1 H), 8.30 (d, J = 5.6 Hz, 1 H), 7.51 (m, 1
H), 7.48 (m, 1 H), 7.44 (m, 1 H), 7.39 (m, 1 H), 7.30 (m, 1 H), 4.52
(t, J = 4.5 Hz, 2 H), 3.52 (s, 2 H), 3.15 (t, J = 5.8 Hz, 2 H), 2.18 (m, 2
H), 1.43 (s, 9 H).
1
1532, 1510, 1160, 765, 455 cm−1; H NMR (CD3OD, 300 MHz) δ
8.28 (s, 1 H), 7.92 (m, 1 H), 7.31 (m, 3 H), 7.20 (s, 2 H), 4.30 (t, J =
4.8 Hz, 2 H), 3.71 (s, 3 H), 2.83 (m, 2 H), 2.68 (m, 4 H), 2.00 (m, 2
H); 13C NMR (CD3OD, 125 MHz) δ 191.8, 174.9, 172.6, 164.4,
155.6, 139.2, 138.2, 135.9, 134.8, 131.9, 129.3, 127.1, 125.0, 124.9,
124.0, 123.8, 122.9, 118.5, 114.8, 109.5, 57.6, 57.4, 57.2, 43.6, 36.3,
32.2, 30.3, 29.7; ESIMS m/z (relative intensity) 434 (MH+, 100);
HRESIMS calcd for C24H23N3O5 434.1852 (MH+), found 434.1835
(MH+); HPLC purity: 98.0% (1% TFA in MeOH).
4-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-
6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-4-oxo-
butanoic Acid (29). mp 155−157 °C. IR (KBr) 3315, 1763, 1698,
1
1678, 1543, 1345, 1165, 667 cm−1; H NMR (CD3OD, 300 MHz) δ
8.44 (m, 2 H), 7.82 (dd, J = 2.1, J = 5.5 Hz, 1 H), 7.68 (d, J = 5.5 Hz, 1
H), 7.51 (m, 2 H), 7.34 (m, 1 H), 4.45 (m, 2 H), 3.12 (m, 2 H), 2.14
(s, 4 H), 1.96 (m, 2 H), 1.44 (s, 9 H); 13C NMR (CDCl3 + DMSO-d6,
75 MHz) δ 188.4, 172.3, 168.8, 160.8, 154.3, 152.3, 136.8, 135.3,
133.0, 131.8, 129.0, 125.7, 124.0, 122.1, 121.9, 121.2, 120.9, 115.2,
106.2, 40.9, 35.9, 29.7, 29.1, 28.1, 27.3, 26.7; ESIMS (m/z, relative
intensity) 542 (MNa+, 100), 420 (loss of Boc, 16); HRESIMS calcd
for C28H29N3O7Na 542.1903 (MNa+), found 542.1912 (MNa+).
5-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-
6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-5-oxo-
pentanoic Acid (30). mp 198−200 °C. IR (KBr) 3307, 1696, 1690,
1663, 1569, 1528, 1401, 1366, 1250, 1167, 759 cm−1; 1H NMR
(CD3OD, 300 MHz) δ 8.52 (d, J = 5.3 Hz, 1 H), 8.21 (br s, 1 H), 8.01
(br s, 1 H), 7.48 (d, J = 4.7 Hz, 1 H), 7.40 (m, 2 H), 7.26 (m, 1 H),
4.51 (t, J = 4.5 Hz, 2 H), 3.20 (m, 2 H), 2.49 (m, 4 H), 2.10 (m, 4 H),
1.45 (s, 9 H); 13C NMR (CD3OD + DMSO-d6, 75 Hz) δ 191.5, 176.3,
173.2, 164.1, 158.0, 155.6, 139.3, 138.2, 135.9, 134.8, 131.9, 129.0,
127.0, 125.0, 124.8, 124.2, 123.8, 118.4, 109.1, 79.8, 79.7, 36.9, 34.2,
30.9, 29.0, 21.9; ESIMS (m/z, relative intensity) 556 (MNa+, 100);
HRESIMS calcd for C29H31N3O7 556.2059 (MNa+), found 556.2063
(MNa+).
Methyl 5-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-
indeno[1,2-c]isoquinolin-3-yl)amino]-5-oxopentanoate (25).
Compound 20 (0.150 g, 0.274 mmol) was treated with trifluoroacetic
acid (1.0 mL) in chloroform (10 mL) for 2 h at room temperature.
The solvent was removed on a rotary evaporator, and the residue was
then basified with 2 N NH3 in methanol to get the free amine, which
was purified by silica gel column chromatography, eluting with
chloroform−methanol, 9.0:1.0, to afford the product 25 (0.092 g,
75%) as a brown solid: mp 215−217 °C. IR (KBr) 3075, 1729, 1687,
1673, 1572, 1532, 1433, 1202, 760, 722, 455 cm−1; 1H NMR (DMSO-
d6, 300 MHz) δ 10.2 (s, 1 H), 8.60 (d, J = 1.8 Hz, 1 H), 8.47 (d, J =
8.7 Hz, 1 H), 7.89 (dd, J = 1.8, J = 8.7 Hz, 1 H), 7.74 (m, 3 H), 7.54
(m, 2 H), 7.47 (m, 1 H), 4.53 (t, J = 3.2 Hz, 2 H), 3.59 (s, 3 H), 2.96
(m, 2 H), 2.38 (m, 4 H), 2.13 (m, 2 H), 1.87 (m, 2 H); 13C NMR
(DMSO-d6, 75 MHz) δ 190.1, 173.0, 171.0, 162.6, 154.4, 138.3, 136.6,
134.2, 134.0, 131.0, 127.1, 125.8, 123.5, 123.4, 122.6, 116.6, 107.4,
79.1, 51.3, 41.2, 38.6, 36.6, 35.2, 32.6, 27.3, 20.2; ESIMS m/z (relative
intensity) 448 (MH+, 100); HRESIMS calcd for C25H25N3O5
448.1867 (MH+), found 448.1877 (MH+); HPLC purity: 100% (1%
TFA in MeOH−H2O, 70:30).
Methyl 6-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-
indeno[1,2-c]isoquinolin-3-yl)amino]-6-oxohexanoate (26).
Compound 21 (0.030 g, 0.053 mmol) was treated with trifluoroacetic
acid (0.25 mL) in chloroform (3 mL) for 2 h at room temperature.
The solvent was removed on a rotary evaporator, and the residue was
then basified with 2 N NH3 in methanol to get the free amine, which
was purified by silica gel column chromatography, eluting with
chloroform−methanol, 9.2:0.8, to yield the product 26 (0.015 g, 55%)
as a red solid: mp 153−155 °C. IR (KBr) 2947, 1739, 1705, 1693,
1661, 1570, 1530, 1431, 1197, 760, 665 cm−1; 1H NMR (CD3OD, 300
MHz) δ 8.51 (d, J = 2.4 Hz, 1 H), 8.34 (d, J = 9.0 Hz, 1 H), 7.70 (dd, J
= 2.4, J = 9.0 Hz, 1 H), 7.60 (t, J = 9.0 Hz, 1 H), 7.51 (m, 2 H), 7.31
(m, 1 H), 4.55 (t, J = 4.5 Hz, 2 H), 3.67 (s, 3 H), 3.13 (m, 2 H), 2.39
(m, 4 H), 2.26 (m, 2 H), 1.70 (m, 2 H); 13C NMR (DMSO-d6, 125
MHz) δ 190.0, 173.3, 171.3, 162.3, 154.4, 138.3, 136.6, 134.2, 133.9,
6-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-
6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-6-oxo-
hexanoic Acid (31). mp 206−208 °C. IR (KBr) 3312, 1702, 1695,
1
1670, 1573, 1534, 1376, 1250, 760, 665 cm−1; H NMR (CDCl3 +
CD3OD, 300 MHz) δ 8.55 (d, J = 6.2 Hz, 1 H), 8.18 (s, 1 H), 8.01 (m,
1 H), 7.50 (d, J = 3.5 Hz, 1 H), 7.40 (d, J = 3.5 Hz, 1 H), 7.32 (m, 1
H), 7.28 (d, J = 3.5 Hz, 1 H), 4.50 (m, 2 H), 3.12 (m, 2 H), 2.29 (m, 4
H), 1.96 (m, 2 H), 1.65 (m, 4 H), 1.37 (s, 9 H); 13C NMR (DMSO-d6,
75 Hz) δ 190.0, 174.5, 171.5, 162.2, 155.8, 154.5, 138.3, 136.7, 134.3,
133.9, 131.0, 127.1, 125.7, 123.5, 123.3, 123.2, 122.7, 116.6, 107.3,
77.8, 42.5, 37.5, 36.2, 33.5, 29.6, 28.3, 24.6, 24.9; ESIMS (m/z, relative
intensity) 570 (MNa+, 23), 448 (loss of Boc, 100); HRESIMS calcd
for C30H33N3O7Na 570.2216 (MNa+), found 570.2207 (MNa+).
General Procedure for Synthesis of Indenoisoquinolines 32-
36. Acids 27−31 (0.050 g) were treated with HCl in diethyl ether (2
L
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Journal of Medicinal Chemistry
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M, 2 mL) at room temperature for 5 h. The solvent was removed on a
rotary evaporator to yield the solid hydrochloride salts, which were
washed with 5% MeOH in chloroform to remove impurities and afford
the pure products 32−36 in quantitative yields.
2 H), 3.96 (d, J = 6.3 Hz, 2 H), 3.87 (s, 3 H), 1.12 (t, J = 7.1 Hz, 3 H);
13C NMR (125 MHz, CDCl3) δ 190.7, 170.6, 163.0, 152.2, 146.1,
138.3, 134.8, 132.8, 130.0, 125.0, 124.7, 123.7, 122.9, 121.9, 121.2,
109.0, 107.0, 61.4, 45.3, 32.2, 14.1; ESIMS m/z (rel intensity) 363
(MH+, 100); HRESIMS calcd for C21H18N2O4 363.1345 (MH+),
found 363.1349 (MH+); HPLC purity: 99.42% (MeOH−H2O−TFA,
95:5:1), 95.33% (MeOH−TFA, 100:1).
2-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno-
[1,2-c]isoquinolin-3-yl)amino]-2-oxoacetic Acid Hydrochloride
(32). mp 251−252 °C. IR (KBr) 2955, 1742, 1733, 1641, 1578, 1535,
1
1431, 1195, 665 cm−1; H NMR (DMSO-d6, 300 MHz) δ 11.04 (s, 1
Ethyl 2-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-
dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl]amino)-
acetate (39). Compound 16 (0.200 g, 0.477 mmol) was dissolved in
acetic acid (20 mL) and methanol (1 mL). Ethyl glyoxalate (37, 0.058
g, 0.577 mmol) was added to the reaction mixture, which was stirred at
room temperature for 1.5 h. Sodium cyanoborohydride (0.071 g, 1.19
mmol) was added, and stirring was continued for another 0.5 h. The
solvents were removed on a rotary evaporator. The residue was
washed with sodium bicarbonate (2 × 15 mL) and extracted with
CHCl3 (2 × 60 mL), the combined organic layers were dried over
anhydrous sodium bicarbonate, and the mixture was concentrated to
get a crude product. The crude product was precipitated from
hexane−chloroform (7 + 3 mL) to afford a pure violet solid 39 (0.215
g, 90%): mp 203−204 °C. IR (KBr) 2977, 1739, 1698, 1650, 1619,
H), 8.75 (dd, J = 1.7, J = 8.9 Hz, 1 H), 8.49 (dd, J = 4.0, J = 8.8 Hz),
8.11 (m, 2 H), 7.69 (m, 1 H), 7.54 (m, 3 H), 4.54 (m, 2 H), 3.01 (m, 2
H), 2.07 (m, 2 H); ESIMS (m/z, relative intensity) 392 (MH+, 100);
HRESIMS calcd for C21H17N3O5 392.1246 (MH+), found 392.1249
(MH+); HPLC purity: 96.6% (1% TFA in MeOH).
3-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno-
[1,2-c]isoquinolin-3-yl)amino]-3-oxopropanoic Acid Hydro-
chloride (33). mp 205−207 °C. IR (KBr) 2917, 2356, 1670, 1582,
1565, 1538, 1191, 665 cm−1; 1H NMR (DMSO-d6, 300 MHz) δ 10.65
(d, J = 8.9 Hz, 1 H), 8.48 (s, 1 H), 8.40 (d, J = 4.5 Hz, 1 H), 7.88 (m, 4
H), 7.71 (d, J = 4.5 Hz, 1 H), 7.56 (m, 2 H), 7.41 (m, 1 H), 4.54 (m, 2
H), 3.64 (s, 1 H), 3.52 (s, 1 H), 3.00 (m, 2 H), 2.09 (m, 2 H); ESIMS
(m/z, relative intensity) 406 (MH+, 100); HRESIMS calcd for
C22H19N3O5 406.1403 (MH+), found 406.1400 (MH+); HPLC purity:
95.2% (1% TFA in MeOH).
1
1579, 1523, 1390, 1365, 1198, 1171, 1020, 758, 455 cm−1; H NMR
(300 MHz, CDCl3) δ 8.51 (d, J = 8.7 Hz, 1 H), 7.53 (d, J = 6.4 Hz, 1
H), 7.39 (m, 4 H), 7.09 (dd, J = 2.6, J = 8.7 Hz, 1 H), 5.44 (m, 1 H),
4.66 (m, 1 H), 4.56 (t, J = 4.5 Hz, 2 H), 4.30 (q, J = 7.1 Hz, 2 H), 4.01
(d, J = 5.1 Hz, 2 H), 3.23 (m, 2 H), 2.06 (m, 2 H), 1.45 (s, 9 H), 1.31
(t, J = 7.1 Hz, 3 H); 13C NMR (CDCl3, 125 MHz) δ 190.9, 170.6,
163.6, 156.1, 151.4, 146.3, 137.6, 134.9, 133.3, 130.1, 125.0, 124.9,
123.9, 123.1, 122.3, 121.7, 109.6, 107.2, 79.2, 61.6, 45.3, 41.7, 28.4,
14.2; ESIMS m/z (rel intensity) 528 (MNa+, 100); HRESIMS calcd
for C28H31N3O6Na 528.2111 (MNa+), found 528.2108 (MNa+).
Ethyl 2-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-
indeno[1,2-c]isoquinolin-3-yl)amino]acetate Hydrochloride
(40). Ester 39 (0.060 g, 0.011 mmol) was treated with 2 M HCl in
diethyl ether (2 mL) at room temperature for 2 h. The solvent was
removed on a rotary evaporator to yield the solid hydrochloride salt,
which was washed with 5% MeOH in chloroform to remove impurities
and afford the pure solid product 40 in quantitative yield: mp 253−
254 °C. IR (KBr) 3356, 2972, 1711, 1689, 1634, 1598, 1544, 1354,
4-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno-
[1,2-c]isoquinolin-3-yl)amino]-4-oxobutanoic Acid Hydro-
chloride (34). mp 167−169 °C. IR (KBr) 3080, 1671, 1571, 1533,
1508, 1197, 734, 665 cm−1; 1H NMR (CD3OD, 300 MHz) δ 8.68 (d, J
= 2.3 Hz, 1 H), 8.55 (d, J = 5.2 Hz, 1 H), 7.78 (dd, J = 2.3, J = 5.4 Hz,
1 H), 7.64 (m, 1 H), 7.52 (m, 2 H), 7.37 (m, 1 H), 4.62 (m, 2 H), 3.06
(m, 2 H), 2.30 (m, 2 H); ESIMS (m/z, relative intensity) 420 (MH+,
100); HRESIMS calcd for C23H21N3O5 420.1559 (MH+), found
420.1565 (MH+); HPLC purity: 96.71% (1% TFA in MeOH).
5-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno-
[1,2-c]isoquinolin-3-yl)amino]-5-oxopentanoic Acid Hydro-
chloride (35). mp 242−244 °C. IR (KBr) 3583, 2348, 1761, 1723,
1
1695, 1510, 1497, 906, 673 cm−1; H NMR (DMSO-d6, 300 MHz) δ
10.33 (s, 1 H), 8.63 (d, J = 2.1 Hz, 1 H), 8.49 (d, J = 8.7 Hz, 1 H), 7.89
(dd, J = 2.1, J = 8.7 Hz, 1 H), 7.84 (br s, 2 H), 7.76 (d, J = 7.2 Hz, 1
H), 7.54 (m, 2 H), 7.40 (m, 1 H), 4.53 (m, 2 H), 3.04 (m, 2 H), 2.40
(m, 2 H), 2.04 (m, 4 H), 2.13 (m, 2 H), 1.85 (m, 2 H); ESIMS (m/z,
relative intensity) 434 (MH+, 100); HRESIMS calcd for C24H23N3O5
434.1716, found 434.1710; HPLC purity: 95.3% (1% TFA in MeOH).
6-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno-
[1,2-c]isoquinolin-3-yl)amino]-6-oxohexanoic Acid Hydro-
chloride (36). mp 203−205 °C. IR (KBr) 3583, 2952, 1730, 1719,
1
1129, 665 cm−1; H NMR (DMSO-d6, 300 MHz) δ 8.31 (d, J = 8.7
Hz, 1 H), 8.04 (br s, 3 H), 7.64 (m, 1 H), 7.47 (m, 2 H), 7.40 (m, 1
H), 7.21 (dd, J = 2.2, J = 8.7 Hz, 1 H), 7.14 (d, J = 2.2 Hz, 1 H), 4.49
(m, 2 H), 4.13 (q, J = 7.1 Hz, 2 H), 4.00 (s, 2 H), 2.92 (m, 2 H), 2.09
(m, 2 H), 1.20 (t, J = 5.9 Hz, 3 H); 13C NMR (DMSO-d6, 300 MHz)
δ 190.7, 190.2, 170.8, 162.8, 160.6, 156.0, 155.5, 137.3, 136.7, 134.3,
130.7, 124.2, 123.1, 118.8, 87.0, 60.4, 56.3, 36.8, 27.6, 18.8, 14.3;
ESIMS (m/z, relative intensity) 406 (MH+, 100); HRESIMS calcd for
C23H23N3O4 406.1767 (MH+), found 406.1772 (MH+); HPLC purity:
95.66% (1% TFA in MeOH).
1
1646, 1570, 1528, 1431, 1195, 759, 667 cm−1; H NMR (DMSO-d6,
300 MHz) δ 10.30 (d, J = 6.6 Hz, 1 H), 8.85 (d, J = 9.9 Hz, 1 H), 8.51
(m, 1 H), 7.91 (m, 1 H), 7.76 (m, 3 H), 7.51 (m, 3 H), 4.50 (m, 2 H),
3.32 (m, 1 H), 3.22 (m, 1 H), 2.39 (m, 2 H), 2.27 (m, 2 H), 2.11 (m, 1
H), 1.91 (m, 1 H), 1.58 (m, 4 H); ESIMS (m/z, relative intensity) 448
(MH+, 100); HRESIMS calcd for C25H26N3O5 448.1872 (MH+),
found 448.1868 (MH+); HPLC purity: 96.5% (1% TFA in MeOH).
Ethyl 2-[(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-
c]isoquinolin-3-yl)amino]acetate (38). 3-Amino-6-methyl-5H-
indeno[1,2-c]isoquinoline-5,11(6H)-dione (3, 400 mg, 1.45 mmol)
and ethyl glyoxylate (37, 0.3 mL, 50% solution in toluene) were
dissolved in glacial acetic acid (25 mL). The reaction mixture was
stirred overnight at room temperature. Sodium cyanoborohydride
(1.00 g) was added in portions. Once the production of bubbles
stopped, the reaction mixture was heated at reflux for 30 min. Water
(150 mL) was added and the compound extracted with chloroform (3
× 50 mL). The organic layers were combined and washed with water
(2 × 150 mL), aqueous sodium bicarbonate (100 mL), and brine (100
mL). The solvent was removed under vacuum and the compound
purified by silica gel column chromatography, eluting with chloro-
form−methanol, 50:1. The product was obtained as a dark solid (351
mg, 67%): mp 207 °C (decomp). IR (film) 3380, 2929, 1735, 1694,
1654, 1560, 1434, 1212, 1017 cm−1; 1H NMR (500 MHz, DMSO-d6)
δ 8.21 (d, J = 8.7 Hz, 1 H), 7.69 (d, J = 7.5 Hz, 1 H), 7.43−7.38 (m, 2
H), 7.31 (t, J = 7.4 Hz, 1 H), 7.13 (dd, J = 8.8 Hz, J = 2.5 Hz, 1 H),
7.10 (d, J = 2.5 Hz, 1 H), 6.67 (t, J = 6.4 Hz, 1 H), 4.09 (q, J = 7.1 Hz,
2-[(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]-
isoquinolin-3-yl)amino]acetic Acid (41). Ethyl 2-((6-methyl-5,11-
dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino)acetate
(38, 213 mg, 0.59 mmol) was dissolved in methanol (5 mL) and
tetrahydrofuran (5 mL). A solution of potassium hydroxide (112 mg,
2.00 mmol) in water (5 mL) was added to the reaction mixture, which
was stirred at room temperature for 10 h. Concentrated hydrochloric
acid (1 mL) was added to the reaction mixture and the solvent
removed under vacuum. Water (20 mL) was added, and the mixture
was sonicated and filtered. The solid was washed with water (20 mL).
The residue was then dissolved in a mixture of dimethylformamide
(0.5 mL) and methanol (9.5 mL), heated, and allowed to reach room
temperature. Ethyl ether (25 mL) and hexane (10 mL) were added,
and the reaction mixture was placed inside the refrigerator overnight.
The solvent was filtered off and the residue dried. The product was
obtained as a black solid (59 mg, 30.0%): mp 205 °C (decomp). IR
(KBr) 3361, 3032, 1725, 1698, 1650, 1619, 1578, 1527, 1432, 1391,
1
1318, 1217, 1197, 1054, 1016, 901, 839, 758 cm−1; H NMR (300
MHz, CDCl3) δ 12.63 (br s, 1 H), 8.21 (d, J = 8.7 Hz, 1 H), 7.68 (d, J
= 7.5 Hz, 1 H), 7.43−7.38 (m, 2 H), 7.30 (t, J = 7.4 Hz, 1 H), 7.14
(dd, J = 8.8 Hz, J = 2.5 Hz, 1 H), 7.09 (d, J = 2.5 Hz, 1 H), 6.56 (br s,
M
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1 H), 3.87 (s, 3 H), 3.86 (s, 2 H); 13C NMR (125 MHz, CDCl3) δ
190.7, 172.5, 162.4, 152.7, 147.9, 138.3, 134.6, 133.8, 130.5, 124.8,
123.8, 123.4, 122.6, 122.1, 121.8, 107.9, 106.5, 44.7, 32.3; negative ion
ESIMS m/z (rel intensity) 333 ([M − H+]−, 100); HRESIMS calcd
for C19H14N2O4Na 357.0851 (MNa+), found 357.0857 (MNa+). Anal.
Calcd for C19H14N2O4: C, 68.26; H, 4.22; N, 8.38. Found: C, 68.03;
H, 4.16; N, 8.35.
2-((6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-
6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino)acetic
Acid (42). The ester 39 (0.1 g, 0.02 mmol) was dissolved in MeOH−
THF (10 +10 mL), and aq NaOH (4 N, 5 mL) was added at room
temperature. The reaction mixture was stirred at room temperature for
4 h. The solvent was removed on a rotary evaporator and the residue
washed with 1 N HCl (30 mL) and extracted with CHCl3 (2 × 50
mL). The extract was dried over Na2SO4 and concentrated to afford
pure 42 (0.075 g, 80%), which was obtained as a brown solid: mp
190−192 °C. IR (KBr) 3350, 2973, 1692, 1645, 1619, 1578, 1520,
1364, 1164, 666 cm−1; 1H NMR (DMSO-d6, 300 MHz) δ 8.30 (d, J =
8.7 Hz, 1 H), 7.59 (m, 1 H), 7.45 (m, 2 H), 7.39 (m, 1 H), 7.21 (dd, J
= 2.4, J = 8.7 Hz, 1 H), 7.12 (d, J = 2.4 Hz, 1 H), 7.07 (m, 1 H), 4.40
(m, 2 H), 3.90 (s, 2 H), 3.09 (m, 2 H), 1.86 (m, 2 H), 1.33 (s, 9 H);
ESIMS (m/z, relative intensity) 500 (MNa+, 100); HRESIMS calcd for
C26H27N3O6Na 500.1798 (MNa+), found 500.1806 (MNa+); HPLC
purity: 95.5% (MeOH−H2O, 75:25).
and the liquid was decanted again. The solid was purified by silica gel
column chromatography, eluting with chloroform−methanol, 20:1, to
provide a red-orange solid (1.02 g, 55%): mp 282−284 °C (decomp).
IR (film) 1699, 1667, 1612, 1555, 1499, 1332, 1159, 1078, 840, 785,
688, 662 cm−1; 1H NMR (CDCl3, 300 MHz) δ 8.85 (d, J = 2.5 Hz, 1
H), 8.70 (d, J = 8.9 Hz, 1 H), 8.55 (d, J = 8.9 Hz, 1 H), 7.60 (d, J = 8.5
Hz, 1 H), 7.38 (s, 1 H), 7.06 (d, J = 8.3 Hz, 1 H), 4.60 (t, J = 7.3 Hz, 2
H), 3.91 (s, 3 H), 3.75 (t, J = 6.4 Hz, 2 H), 2.36 (t, J = 7.0 Hz, 2 H);
EIMS m/z (rel intensity) 444 (M+, 100), 442 (M+, 100), 363 [(M −
Br)+, 69]; HRESIMS calcd for C20H15BrN2O5 442.0164 (MH+), found
442.0158 (MH+). Anal. Calcd for C20H15BrN2O5·0.8 H2O: C, 52.49;
H, 3.66; N, 6.12; Br, 17.46. Found: C, 52.20; H, 3.54; N, 5.96; Br,
17.46.
6-(3-Azidopropyl)-5,6-dihydro-8-methoxy-3-nitro-5,11-
dioxo-11H-indeno[1,2-c]isoquinoline (50). Sodium azide (0.59 g,
9.07 mmol) and 6-(3-bromopropyl)-5,6-dihydro-8-methoxy-3-nitro-
5,11-dioxo-11H-indeno[1,2-c]isoquinoline (49, 261 mg, 0.59 mmol)
were diluted with DMSO (30 mL), and the mixture was heated at 90
°C for 12 h. The reaction mixture was diluted with chloroform (100
mL), washed with water (100 mL) and sat aq NaCl (30 mL), and
dried over sodium sulfate. The solution was concentrated to provide a
crude solid that was purified by silica gel column chromatography,
eluting with chloroform−methanol, 50:1, to afford an orange solid (92
mg, 40%): mp 310 °C (decomp). IR (KBr) 3060, 3015, 2979, 2090,
1691, 1668, 1614, 1560, 1504, 1371, 1344, 1258, 1232, 1082, 1022,
911, 876, 843, 775 cm−1; 1H NMR (CDCl3, 300 MHz) δ 8.88 (d, J =
2.5 Hz, 1 H), 8. 67 (d, J = 8.9 Hz, 1 H), 8.50 (dd, J = 8.9 Hz, J = 2.5
Hz, 1 H), 7.59 (d, J = 8.4 Hz, 1 H), 7.38 (d, J = 2.0 Hz, 1 H), 7.04 (dd,
J = 8.1 Hz, J = 1.9 Hz, 1 H), 4.57 (t, J = 7.2 Hz, 2 H), 3.91 (s, 3 H),
3.59 (t, J = 6.4 Hz, 2 H), 2.07 (t, J = 7.0 Hz, 2 H); 13C NMR (125
MHz, DMSO-d6) δ 188.6, 164.6, 162.3, 157.7, 145.7, 138.5, 136.7,
127.9, 127.3, 125.2, 124.5, 124.3, 123.2, 114.7, 113.7, 108.0, 56.6, 49.0,
42.8, 28.3; ESIMS m/z (rel intensity) 405 (MH+, 41), 322 [(M −
C3H7N3)+, 100]; HRESIMS m/z calcd for C20H15N5O5 405.1073
(MH+), found, 405.1069 (MH+); HPLC purity: 95.0% (MeOH),
95.2% (MeOH−H2O, 85:15).
6-(3-Aminopropyl)-5,6-dihydro-8-methoxy-3-nitro-5,11-
dioxo-11H-indeno[1,2-c]isoquinoline Hydrochloride (51).
Triethyl phosphite (1.0 mL) was added to a solution of 6-(3-
azidopropyl)-5,6-dihydro-8-methoxy-3-nitro-5,11-dioxo-11H-indeno-
[1,2-c]isoquinoline (50, 0.287 g, 0.710 mmol) in benzene (50 mL),
and the reaction mixture was heated at reflux for 16 h. The reaction
mixture was allowed to cool to room temperature, HCl in methanol (3
M, 10 mL) was added, and the reaction mixture was heated at reflux
for 4 h. The reaction mixture was allowed to cool to room temperature
and filtered to provide a red solid (0.180 g, 61%): mp 288−290 °C. IR
(KBr) 3060, 3015, 2979, 2090, 1691, 1668, 1614, 1560, 1504, 1371,
1344, 1258, 1232, 1082, 1022, 911, 876, 843, 775 cm−1; 1H NMR (300
MHz, CDCl3) δ 8.84 (d, J = 2.5 Hz, 1 H), 8.69 (d, J = 8.9 Hz, 1 H),
8.53 (dd, J = 8.9 Hz, J = 2.5 Hz, 1 H), 7.87 (br s, 3 H), 7.60 (d, J = 8.4
Hz, 1 H), 7.30 (d, J = 2.0 Hz, 1 H), 7.05 (dd, J = 8.1 Hz, J = 1.9 Hz, 1
H), 4.54 (t, J = 7.2 Hz, 2 H), 3.91 (s, 3 H), 2.94 (t, J = 6.4 Hz, 2 H),
2.10 (t, J = 7.0 Hz, 2 H); 13C NMR (125 MHz, DMSO-d6) δ 188.6,
164.6, 162.6, 157.6, 145.8, 138.3, 136.7, 128.0, 127.3, 125.3, 124.6,
124.2, 123.2, 114.6, 113.8, 108.2, 56.6, 42.3, 37.2, 27.3; ESIMS m/z
(rel intensity) 380 (MH+, 72), 363 ([(M − NH3]+, 100); HRESIMS
m/z calcd for C20H17N3O5 380.1246 (MH+), found, 380.1243 (MH+);
HPLC purity: 97.2% (MeOH−H2O, 90:10).
6-(3-(1H-Imidazol-1-yl)propyl)-8-methoxy-3-nitro-5H-
indeno[1,2-c]isoquinoline-5,11(6H)-dione (52). 6-(3-Bromoprop-
yl)-8-methoxy-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione
(49, 120 mg, 0.27 mmol) was dissolved in dimethylformamide (2 mL)
and dioxane (10 mL). Sodium iodide (208 mg, 1.40 mmol) was added,
and the reaction mixture was heated at 60 °C for 6 h. Imidazole (202
mg, 297 mmol) and potassium carbonate (225 mg, 1.63 mmol) were
added, and the reaction mixture was stirred at 90 °C for 12 h. Water
(15 mL) was added and a precipitated formed. The solid was filtered
and kept. The remaining solution was diluted with water (100 mL)
and the aqueous phase extracted with chloroform (3 × 30 mL). The
organic extracts were combined, washed with water (3 × 100 mL), and
2-((6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno-
[1,2-c]isoquinolin-3-yl)amino)acetic Acid Hydrochloride (43).
Acid 42 (0.070 g, 0.014 mmol) was treated with HCl in diethyl ether
(2 M, 2 mL) at room temperature for 1 h. The solvent was removed
on a rotary evaporator to provide the solid hydrochloride salt, which
was washed with 10% MeOH in chloroform to remove impurities and
afford the pure product 43 in quantitative yield: mp 263−264 °C. IR
(KBr) 3376, 2982, 1723, 1695, 1634, 1598, 1566, 1354, 1188, 667
1
cm−1; H NMR (DMSO-d6, 300 MHz) δ 8.34 (d, J = 7.8 Hz, 1 H),
8.28 (s, 1 H), 8.12 (br s, 3 H), 7.64 (m, 1 H), 7.49 (m, 2 H), 7.39 (m,
1 H), 7.19 (dd, J = 2.2, J = 8.7 Hz, 1 H), 7.12 (d, J = 2.2 Hz, 1 H), 4.50
(t, J = 6.3 Hz, 2 H), 3.90 (s, 2 H), 2.90 (m, 2 H), 2.10 (m, 2 H); 13C
NMR (DMSO-d6, 125 MHz) δ 190.2, 163.1, 162.3, 136.7, 134.1,
130.7, 125.1, 124.2, 124.1, 123.2, 122.6, 107.8, 79.2, 41.2, 36.5, 27.2;
ESIMS (m/z, relative intensity) 378 (MH+, 100); HRESIMS calcd for
C21H19N3O4 378.1454 (MH+), found 378.1458 (MH+); HPLC purity:
95.3% (1% TFA in MeOH−H2O, 50:50).
cis-2-(3-Bromopropyl)-3-(3-methoxyphenyl)-7-nitro-1-oxo-
1,2,3,4-tetrahydroisoquinoline-4-carboxylic Acid (48). The
Schiff base 46 (0,618 g, 2.41 mmol) was diluted in chloroform (50
mL) at 0 °C, and 4-nitrohomophthalic anhydride (0.5 g, 2.41 mmol)
was added. The red mixture was stirred at 0 °C for 1 h and then at
room temperature for 3 h. The creamy orange mixture was filtered,
and the residue was washed with CHCl3 to provide the product as a
pale yellow solid (0.91 g, 82%): mp 122−123 °C. IR (film) 3419,
1
3010, 1714, 1651, 1530, 1489, 1346, 1266, 1163, 758 cm−1; H NMR
(300 MHz, DMSO-d6) δ 8.60 (d, J = 2.4 Hz, 1 H), 8.25 (dd, J = 2.4, J
= 8.3 Hz, 1 H), 7.47 (d, J = 8.3 Hz, 1 H), 7.16 (m, 1 H), 6.78 (m, 1
H), 6.64 (s, 1 H), 6.59 (d, J = 7.7 Hz, 1 H), 5.10 (d, J = 2.4 Hz, 1 H),
4.08 (m, 1 H), 3.65 (s, 3 H), 3.33 (m, 2 H), 2.92 (m, 2 H), 2.10 (m, 2
H).
6-(3-Bromopropyl)-5,6-dihydro-8-methoxy-3-nitro-5,11-
dioxo-11H-indeno[1,2-c]isoquinoline (49). Thionyl chloride (10
mL) was slowly added to a solution of cis-4-carboxy-N-(3-
chloropropyl)-3,4-dihydro-3-(4-methoxyphenyl)-7-nitro-1(2H)-
isoquinolone (48, 1.94 g, 4.18 mmol) in benzene (60 mL). The
reaction mixture was heated at reflux for 30 min, allowed to cool to
room temperature, and concentrated. The residue was diluted with
nitrobenzene (80 mL) and chilled in an ice bath, and aluminum
chloride (3.00 g, 22.5 mmol) was added. The reaction mixture was
removed from the bath and heated at 100 °C for 1 h. Water (200 mL)
was added, and the solution was extracted with CHCl3 (3 × 70 mL).
The combined organic layers were washed with sodium bicarbonate (3
× 75 mL) and brine (75 mL) and dried over sodium sulfate. The
solution was concentrated, hexanes (250 mL) were added, and the
liquid was decanted. The solid was washed with hexanes (100 mL),
N
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dried over sodium sulfate. The filtered solid from the previous step was
combined with the organic extracts, the solvent was removed in vacuo,
and the compound was purified by silica gel column chromatography,
eluting with chloroform−methanol, 50:1. The compound was
obtained as a dark yellow solid (47 mg, 40%): mp 265−276 °C. IR
(film) 1693, 1673, 1614, 1559, 1336, 1234, 1075, 866, 842, 774 cm−1;
1H NMR (CDCl3, 300 MHz) δ 9.19 (d, J = 2.1 Hz, 1 H), 8.89 (d, J =
temperature and then heated at reflux for 6 h. Concentrated
hydrochloric acid (15 mL) was added and the solvent removed
under vacuum. Benzene (100 mL) and p-toluenesulfonic acid (50 mg)
were added, and the reaction mixture was connected to a Dean−Stark
trap and heated at reflux for 16 h. Chloroform (150 mL) was added,
and the reaction mixture was heated at reflux and immediately filtered.
The solvent was removed under vacuum and the solid recrystallized
from chloroform to remove unreacted phthalide. The compound was
purified by silica gel column chromatography, eluting with chloro-
form−methanol, 50:1. The product was obtained as a yellow solid
(1.03 g, 46.8%): mp 225−227 °C. IR (KBr) 3118, 3082, 2232, 1760,
8.9 Hz, 1 H), 8.48 (dd, J = 8.9 Hz, J = 2.2 Hz, 1 H), 7.68−7.60 (m, 2
H), 7.13 (br s, 1 H), 7.06 (br s, 1 H), 6.86−6.83 (m, 2 H), 4.55 (t, J =
7.1 Hz, 2 H), 4.23 (t, J = 6.5 Hz, 2 H), 3.93 (s, 3 H), 2.41 (p, J = 7.1
Hz, 2 H); 13C NMR (CDCl3, 125 MHz) δ 188.6, 164.3, 162.4, 157.6,
145.4, 137.9, 136.5, 136.1, 128.2, 126.8, 125.4, 124.4, 124.2, 122.9,
114.2, 113.7, 107.9, 56.5, 46.5, 41.7, 29.7; ESIMS m/z (rel intensity)
431 (MH+, 100); HRESIMS calcd for C23H18N4O5 431.1355 (MH+),
found 431.1352 (MH+); HPLC purity: 96.8% (MeOH−H2O, 90:10).
8-Methoxy-6-(3-morpholinopropyl)-3-nitro-5H-indeno[1,2-
c]isoquinoline-5,11(6H)-dione (53). 6-(3-Bromopropyl)-8-me-
thoxy-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (49, 117
mg, 0.26 mmol) was dissolved in dimethylformamide (2 mL) and
dioxane (10 mL). Sodium iodide (213 mg, 1.42 mmol) was added, and
the reaction mixture was heated at 60 °C for 6 h. Morpholine (0.2 mL,
2.30 mmol) and potassium carbonate (260 mg, 1.88 mmol) were
added, and the reaction mixture was stirred at 90 °C for 12 h. Water
(200 mL) was added and the compound extracted with chloroform (3
× 35 mL). The organic extracts were combined, washed with water (3
× 100 mL), and dried over sodium sulfate. The solvent was removed
in vacuo and the compound purified by silica gel column
chromatography, eluting with chloroform−methanol, 50:1. The
product was obtained as an orange solid (30 mg, 25%): mp 276−
278 °C. IR (film) 3020, 1676, 1614, 1562, 1337, 1215, 1066, 846, 757,
1
1747, 1715, 1503, 1385, 1309, 1018, 877, 777, 763, 665 cm −1; H
NMR (300 MHz, DMSO-d6) δ 8.59 (s, 1 H), 8.47 (d, J = 8.2 Hz, 1
H), 7.99 (dd, J = 8.2 Hz, J = 1.6 Hz,1 H), 7.64 (d, J = 7.9 Hz, 1 H),
7.55−7.50 (m, 3 H); 13C NMR (125 MHz, DMSO-d6) δ 188.8, 172.6,
158.8, 137.9, 135.8, 135.4, 135.1, 134.0, 132.7, 124.1, 123.6, 120.6,
119.2, 117.3, 11.7, 106.5; CIMS m/z (rel intensity) 274 (MH+, 100).
3-Cyano-5,11-dihydro-6-[3-(1H-imidazolyl)propyl]-5,11-
dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinoline (72). 3-
Cyano-5,11-dihydro-5,11-dioxoindeno[1,2-c]isochromene (68, 200
mg, 0.73 mmol) was dissolved in tetrahydrofuran (10 mL). 3-(1H-
Imidazol-1-yl)propyl-1-amine (69, 335 mg, 2.67 mmol) was dissolved
in chloroform (5 mL) and the solution added to the reaction mixture.
The reaction mixture was stirred for 3 h at room temperature and then
heated at reflux for 1 h. The solution turned red and, upon reflux, an
orange precipitate formed. The solvent was removed under vacuum
and the solid purified by silica gel column chromatography, eluting
with chloroform−methanol, 30:1. The desired compound was
obtained as a yellow solid (103 mg, 37%): mp 256−258 °C. IR
(KBr) 3103, 2952, 2225, 1694, 1671, 1611, 1534, 1509, 1433, 1192,
1
666 cm−1; H NMR (CDCl3, 300 MHz) δ 9.18 (d, J = 2.2 Hz, 1 H),
1
850, 764, 665 cm −1; H NMR (300 MHz, DMSO-d6) δ 8.80 (d, J =
8.90 (d, J = 8.9 Hz, 1 H), 8.47 (dd, J = 9.0 Hz, J = 2.3 Hz, 1 H), 7.50−
7.48 (m, 2 H), 7.10 (dd, J = 8.9 Hz, J = 2.1 Hz, 1 H), 4.63 (t, J = 7.6
Hz, 2 H), 4.03 (s, 3 H), 3.72 (m, 4 H), 2.59 (t, J = 6.2 Hz, 2 H), 2.50
(s, 4 H) 2.06 (m, 2 H); 13C NMR (CDCl3, 125 MHz) δ 188.0, 162.5,
157.5, 156.9, 145.3, 137.8, 136.8, 136.7, 128.2, 124.2, 123.1, 118.6,
118.5, 117.9, 106.9, 63.5, 56.4, 53.5, 51.3, 42.0, 23.5; ESIMS m/z (rel
intensity) 450 (MH+, 62); HRESIMS calcd for C24H13N3O6 450.1665
(MH+), found 450.1660 (MH+); HPLC purity: 97.7% (MeOH−H2O,
85:15), 97.8% (MeOH−H2O, 90:10).
1-Bromo-6-cyanophthalide (65). 6-Cyanophthalide (64, 2.73 g,
17.2 mmol) was dissolved in carbon tetrachloride (120 mL). 3-
Chloroperbenzoic acid (100 mg) and N-bromosuccinimide (3.20 g,
17.9 mmol) were added. Light was applied with a 250 W lamp, and the
reaction mixture was heated at reflux for 24 h. The solvent was
removed and the residue purified by silica gel column chromatography,
eluting with ethyl acetate−hexane, 1:7. The product was obtained as a
white solid (2.32 g, 56.7%): mp 132−134 °C. IR (film) 3090, 3064,
3032, 2992, 2235, 1788, 1738, 1428, 1295, 1227, 1119, 990, 717, 664
cm−1; NMR (300 MHz, CDCl3) δ 8.23 (s, 1 H), 8.05 (dd, J = 1.1 Hz, J
= 8.1 Hz, 1 H), 7.79 (d, J = 7.9 Hz, 1 H), 7.44 (s, 1 H); 13C NMR (75
MHz, CDCl3) δ 165.1, 152.2, 138.3, 130.0, 125.0, 121.5, 116.8, 115.3,
73.4; CIMS m/z (rel intensity) 238 (MH+, 13), 160 [(MH − Br)+,
100].
7-Cyano-3-hydroxyphthalide (66). 1-Bromo-6-cyanophthalide
(65, 1.99 g, 8.36 mmol) was dissolved in hot water (50 mL). The
reaction mixture was heated at reflux for 3 h. Ethanol (20 mL) was
added, and the solution was concentrated to half its volume and heated
at reflux for 5 min. Once the solution reached room temperature, the
reaction mixture was placed in the refrigerator. The desired compound
was recrystallized as off-white solid (1.23 g, 84.1%): mp 138−140 °C.
1H NMR (300 MHz, DMSO-d6) δ 8.42 (br s, 1 H), 8.37 (d, J = 1.8
Hz, 1 H), 8.24 (dd, J = 7.8 Hz, J = 1.7 Hz, 1 H), 7.90 (d, J = 7.9 Hz, 1
H), 6.75 (s, 1 H); ESIMS m/z (rel intensity) 174 (MH+, 100).
3-Cyano-5,11-dihydro-5,11-dioxoindeno[1,2-c]isochromene
(68). 7-Cyano-3-hydroxyphthalide (66, 1.41 g, 8.06 mmol) and
phthalide (67, 1.08 g, 8.06 mmol) were dissolved in ethyl acetate (15
mL). Sodium (0.80 g, 34.8 mmol) was added to methanol (30 mL)
under nitrogen at 0 °C and the resulting solution added to the reaction
mixture. The reaction mixture was stirred for 30 min at room
8.6 Hz, 1 H), 8.63 (d, J = 1.4 Hz, 1 H), 7.88 (dd, J = 8.4 Hz, J = 1.6
Hz, 1 H), 7.67−7.64 (m, 2 H), 7.43 (t, J = 7.6 Hz, 1 H), 7.35 (dt, J =
7.5 Hz, J = 1.2 Hz, 1 H), 7.21 (s, 1 H), 7.07 (s, 1 H), 6.66 (d, J = 7.5
Hz, 1 H), 4.54 (t, J = 7.8 Hz, 2 H), 4.26 (t, J = 6.2 Hz, 2 H), 2.37 (p, J
= 8.2 Hz, J = 6.6 Hz, 2 H); ESIMS m/z (rel intensity) 381 (MH+,
100); HRESIMS calcd for C23H16N4O2 381.1352 (MH+), found
381.1345 (MH+); HPLC purity: 95.0% (MeOH−H2O, 85:15), 95.4%
(MeOH−H2O, 95:5).
3-Cyano-6,11-dihydro-5,11-dioxo-6-(3-morpholinylpropyl)-
5H-indeno[1,2-c]isoquinoline (73). 3-Cyano-5,11-dihydro-5,11-
dioxoindeno[1,2-c]isochromene (68, 130 mg, 0.47 mmol) was
dissolved in chloroform (50 mL). 3-Morpholinopropyl-1-amine (70,
152 mg, 1.04 mmol) and molecular sieves were added, and the
reaction mixture was stirred at room temperature for 8 h. The reaction
mixture was then heated at reflux for 1 h. The molecular sieves were
filtered off, the solvent removed under vacuum, and the residue
purified by silica gel column chromatography, eluting with chloro-
form−methanol, 100:1. The desired compound was obtained as an
orange solid (113 mg, 60.0%): mp 259−261 °C. IR (film) 3083, 2950,
2849, 2223, 1698, 1673, 1611, 1509, 1435, 1275, 1185, 1116, 998, 959,
896, 851, 766, 665 cm−1; 1H NMR (CDCl3, 500 MHz) δ 8.80 (d, J =
8.6 Hz, 1 H), 8.62 (d, J = 1.7 Hz, 1 H), 7.88−7.84 (m, 2H), 7.69 (m, 1
H), 7.51−7.48 (m, 2 H), 4.64 (t, J = 7.8 Hz, 2 H), 3.70 (t, J = 4.4 Hz, 4
H), 2.58 (t, J = 6.2 Hz, 1 H), 2.49 (br s, 4 H), 2.05 (m, 2 H); 13C
NMR (125 MHz, CDCl3) δ 189.5, 162.0, 157.8, 136.3, 135.3, 135.0,
133.4, 131.9, 124.4, 123.6, 123.4, 123.0, 110.0, 107.2, 66.9, 56.0, 53.9,
43.9, 25.7; ESIMS m/z (rel intensity) 400 (MH+, 100); HRESIMS
calcd for C24H21N3O3 400.1661 (MH+), found, 400.1665 (MH+).
Anal. Calcd for C24H21N3O3·0.5 H2O: C, 70.57; H, 5.43; N, 10.29.
Found: C, 70.71; H, 5.22; N, 10.01.
3-Cyano-6,11-dihydro-5,11-dioxo-6-[3-(dimethylamino-
propyl]-5H-indeno[1,2-c]isoquinoline (74). 3-Cyano-5,11-dihy-
dro-5,11-dioxoindeno[1,2-c]isochromene (68, 135 mg, 0.49 mmol)
was dissolved in tetrahydrofuran (10 mL). N′,N′-Dimethylpropane-
1,3-diamine (71, 158 mg, 1.51 mmol) was dissolved in chloroform (5
mL). This solution was added to the first solution, and the reaction
mixture was stirred for 3 h at room temperature. The mixture was
heated at reflux for 1 h. The solvent was removed under vacuum and
the solid purified by silica gel column chromatography, eluting with
O
dx.doi.org/10.1021/jm3014458 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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chloroform−methanol, 50:1. The compound was obtained as a yellow
solid (96 mg, 55%): mp 210−212 °C. IR (film) 2817, 2762, 2223,
was extracted with CHCl3 (3 × 70 mL). The combined organic layer
was washed with a solution of NaHCO3 (4.0 g) in water (75 mL). The
solution was concentrated, and hexanes were added until the
precipitation of a red solid was observed. The solid was filtered,
washed with hexanes (100 mL), and purified by flash silica gel column
chromatography, eluting with chloroform−methanol, 12:1. The
product was obtained as an orange solid (0.79 g, 11.2%): mp 282−
284 °C. IR (film) 3097, 1809, 1745, 1685, 1604, 1510, 1337, 1231,
1127, 1082, 851 cm−1; 1H NMR (300 MHz, CDCl3) δ 9.17 (d, J = 2.3
Hz, 1 H), 8.81 (d, J = 8.9 Hz, 1 H), 8.47 (dd, J = 9.0 Hz, J = 2.3 Hz, 1
H), 7.81 (d, J = 8.5 Hz, 1 H), 7.28 (d, J = 2.5 Hz, 1 H), 6.94 (dd, J =
8.5 Hz, J = 2.5 Hz, 1 H), 4.51 (t, J = 6.0 Hz, 2 H), 3.84 (s, 3 H), 3.75
(dd, J = 7.5 Hz, J = 6.6 Hz, 2 H), 2.30 (br p, J = 8.5 Hz, 2 H); EIMS
m/z (rel intensity) 442 (M+, 100); HREIMS calcd for C20H15N2O5Br
442.0164 (M+), found, 442.0158 (M+).
1
1698, 1673, 1611, 1611, 1435, 1261, 1191, 1040, 897, 764 cm−1; H
NMR (500 MHz, DMSO-d6) δ 8.78 (d, J = 8.5 Hz, 1 H), 8.62 (s, 1
H), 7.87−7.84 (m, 2 H), 7.67 (d, J = 6.7 Hz, 1 H), 7.50−7.46 (m, 2
H), 4.59 (t, J = 5.9 Hz, 2 H), 2.53 (t, J = 6.5 Hz, 2 H), 2.32 (s, 6 H),
2.04 (br p, J = 8.5 Hz, 2 H); 13C NMR (125 MHz, DMSO-d6) δ 189.6,
162.0, 157.9, 136.3, 135.3, 135.1, 135.0, 133.6, 133.5, 131.8, 124.4,
123.7, 123.5, 123.0, 118.2, 109.9, 107.3, 56.6, 45.6, 43.8, 29.9; ESIMS
m/z (rel intensity) 402 (MH+, 100); HRESIMS calcd for C23H19N3O4
402.1454 (MH+), found 402.1452 (MH+); HPLC purity: 97.4%
(MeOH−H2O, 95:5), 97.4% (MeOH).
3-Cyano-5,11-dihydro-8,9-methylenedioxy-5,11-dioxo[1,2-
c]isochromene (76). 7-Cyano-3-hydroxyphthalide (66, 1.55 g, 8.85
mmol) and 5,6-methylenedioxyphthalide (75, 1.58 g, 8.06 mmol) were
dissolved in ethyl acetate (20 mL). Sodium (0.85 g, 34.8 mmol) was
dissolved in methanol (35 mL) at 0 °C and the resulting solution
added to the reaction mixture. The reaction mixture was stirred for 30
min at room temperature and then heated at reflux for 15 h.
Concentrated hydrochloric acid (20 mL) was added and the solvent
removed under vacuum. Benzene (100 mL) and p-toluenesulfonic acid
(50 mg) were added, and the reaction mixture was connected to a
Dean−Stark trap and heated at reflux for 16 h. Chloroform (100 mL)
was added, and the reaction mixture heated at reflux and immediately
filtered. The solvent was removed under vacuum and the solid
recrystallized from chloroform−methanol, 25:1, to remove unreacted
phthalide. The compound was purified by silica gel column
chromatography, eluting with chloroform−methanol, 50:1. The
product was obtained as a yellow solid (133 mg, 10%): mp >350
°C. Also, 0.81 g of the starting material 75 was recovered. IR (film)
3-Amino-6-(3-bromopropyl)-9-methoxy-5H-indeno[1,2-c]-
isoquinoline-5,11(6H)-dione (81). 6-(3-Bromopropyl)-9-methoxy-
3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (80, 1.00 g,
2.26 mmol) was dissolved in tetrahydrofuran (60 mL), methanol
(20 mL), and ethyl acetate (30 mL) and transferred to a Parr shaker
flask. The reaction vessel was purged with argon for 10 min, and then
palladium−charcoal (10%, 20 mg) was added. The reaction mixture
was shaken for 24 h under an atmosphere of hydrogen (60 psi). The
solid was filtered off, the solvent was removed under vacuum, and the
compound was purified by silica gel column chromatography, eluting
with ethyl acetate. The product was obtained as a brown solid (0.55 g,
59%): mp 330 °C (decomp). IR (film) 3367, 1696, 1655, 1506, 1480,
1
1431, 1293, 1229, 1016, 834, 787, 665 cm−1; H NMR (300 MHz,
DMSO-d6) δ 8.35 (d, J = 8.6 Hz, 1 H), 7.67−7.62 (m, 2 H), 7.06 (d, J
= 2.5 Hz, 1 H), 7.0.1−6.94 (m, 2 H), 4.51 (t, J = 6.0 Hz, 2 H), 3.84 (s,
3 H), 3.75 (dd, J = 7.5 Hz, J = 6.6 Hz, 2 H), 2.30 (br p, J = 8.5 Hz, 2
H); EIMS m/z (rel intensity) 413 (M+, 46), 333 ([M − HBr)]+, 100);
HRESIMS m/z calcd for C20H17N2O3Br 413.0501 (MH+), found,
413.0505 (MH+).
1
2233, 1692, 1417, 1313, 1152, 1029, 925, 861, 785, 665 cm−1; H
NMR (CDCl3−MeOH-d4, 300 MHz) δ 8.44 (s, 1 H), 8.29 (d, J = 8.1
Hz, 1 H), 7.88 (dd, J = 8.1 Hz, J = 1.5 Hz, 1 H), 7.05 (s, 1 H), 6.94 (s,
1 H), 6.09 (s, 2 H); EIMS m/z (rel intensity) 317 (M+, 100);
HREIMS calcd for C18H7NO5 317.0324 (M+), found 317.0327 (M+).
3-Cyano-5,11-dihydro-6-[3-(dimethylamino)propyl]-5,11-
dioxoindeno[1,2-c]isquinoline (77). 3-Cyano-5,11-dihydro-8,9-
methylenedioxy-5,11-dioxo[1,2-c]isochromene (76, 91 mg, 0.28
mmol) was dissolved in tetrahydrofuran (25 mL). N1,N1-Dimethyl-
propane-1,3-diamine (71, 156 mg, 1.56 mmol) and molecular sieves
were added, and the reaction mixture was stirred at room temperature
for 8 h. The reaction mixture was then heated at reflux for 1 h. The
molecular sieves were filtered off, the solvent was removed in vacuo,
and the residue was purified by recrystallization from chloroform−
hexane. The desired compound was obtained as a purple solid (89 mg,
79%): mp 280−282 °C. IR (film) 2968, 2909, 2825, 2271, 1694, 1672,
6-(3-Bromopropyl)-3-iodo-9-methoxy-5H-indeno[1,2-c]-
isoquinoline-5,11(6H)-dione (82). 3-Amino-6-(3-bromopropyl)-9-
methoxy-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (81, 420 mg,
1.01 mmol) was dissolved in dioxane (10 mL). Concentrated
hydrochloric acid (0.65 mL, 3.3 mmol) was added, and the reaction
mixture was stirred for 10 min and then cooled to 0 °C. A solution of
sodium nitrite (114 mg, 1.65 mmol) in water (5 mL) was slowly added
with stirring while the temperature was −20 °C. The reaction mixture
was stirred for 30 min and then slowly added to a solution of
copper(I) iodide (200 mg) and potassium iodide (300 mg) in water
(15 mL). The reaction mixture was stirred at room temperature for 8 h
and then heated at reflux for 1 h. The reaction mixture was diluted
with water (200 mL) and extracted with chloroform (3 × 100 mL).
The combined organic extracts were washed with an aqueous solution
of sodium thiosulfate (2 × 200 mL), aqueous sodium bicarbonate (2 ×
200 mL), water (100 mL), and brine (100 mL). The organic solution
was dried over sodium sulfate and the solvent removed under vacuum.
The residue was purified by silica gel column chromatography, eluting
with chloroform−methanol, 30:1. Compound 82 was obtained as a
dark red solid (402 mg, 76%): mp 207−209 °C. IR (film) 2922, 1765,
1655, 1599, 1532, 1478, 1455, 1430, 1379, 1295, 1225, 1047, 969, 812,
788, 691, 666, 592 cm−1; 1H NMR (CDCl3, 500 MHz) δ 8.60 (d, J =
1.5 Hz, 1 H), 8.32 (d, J = 8.6 Hz, 1 H), 7.93 (dd, J = 8.5 Hz, J = 1.5
Hz, 1 H), 7.64 (d, J = 8.4 Hz, 1 H), 7.15 (d, J = 2.3 Hz, 1 H), 6.82 (dd,
J = 8.3 Hz, J = 2.4 Hz, 1 H), 4.58 (t, J = 7.2 Hz, 2 H), 3.89 (s, 3 H),
3.64 (t, J = 6.2 Hz, 2 H), 2.44 (t, J = 7.2 Hz, 2 H); 13C NMR (125
MHz, CDCl3) δ 189.3, 162.5, 162.0, 156.6, 142.4, 137.6, 137.0, 131.4,
127.6, 124.7, 124.1, 123.9, 115.8, 111.1, 107.6, 91.2, 55.8, 44.2, 31.1,
1
1607, 1577, 1529, 1432, 1309, 1282, 1185, 1033, 852, 793 cm −1; H
NMR (500 MHz, CDCl3−MeOH-d4) δ 8.62 (d, J = 8.5 Hz, 1 H), 8.51
(s, 1 H), 7.86 (d, J = 8.5 Hz, 1 H), 7.44 (s 1 H), 7.08 (s, 1 H), 6.08 (s,
1 H), 4.45 (t, J = 6.9 Hz, 2 H), 2.46 (br s, 2 H), 2.25 (s, 6 H), 1.94 (m,
2 H); 13C NMR (125 MHz, CDCl3) δ 188.7, 162.2, 157.6, 152.0,
150.0, 135.2, 133.6, 131.3, 130.7, 123.8, 122.1, 118.1, 109.1, 106.6,
106.3, 105.4, 102.9, 56.4, 45.4, 43.5, 33.0, 26.8; ESIMS m/z (rel
intensity) 401 (M+, 100); HPLC purity: 100% (MeOH−H2O, 90:10),
96.7% (MeOH−H2O, 85:15).
6-(3-Bromopropyl)-9-methoxy-3-nitro-5H-indeno[1,2-c]-
isoquinoline-5,11(6H)-dione (80). 5-Nitrohomophthalic anhydride
(47, 5.02 g, 24.2 mmol) was added to solution of 3-bromo-N-(4-
methoxybenzylidene)propyl-1-amine (78, 6.21 g, 24.2 mmol) in
chloroform (115 mL), and the reaction mixture was stirred at room
temperature for 1.5 h and then placed inside the freezer overnight. The
precipitate was filtered, washed with chloroform (50 mL), and dried to
provide intermediate 79 (5.13 g), which was used without further
purification. Thionyl chloride (26 mL) was added to a suspension of
79 in benzene (200 mL), and the reaction mixture was heated at reflux
until complete dissolution. The solvent was removed under vacuum
and the residue diluted with nitrobenzene (225 mL) and chilled in an
ice bath. Aluminum chloride (4.50 g, 33.8 mmol) was added, and the
reaction mixture was heated at 100 °C for 2 h. Cool water (200 mL)
was added, the organic phase was separated, and the aqueous phase
30.2; ESIMS m/z (rel intensity) 523 (MH+, 60), 403 [(MH+
−
C3H6Br)+, 100]; HRESIMS m/z calcd for C20H15NO3IBr 523.9358
(MH+), found, 523.9362 (MH+).
6-(3-Azidopropyl)-3-iodo-9-methoxy-5H-indeno[1,2-c]-
isoquinoline-5,11(6H)-dione (83). Sodium azide (0.59 g, 9.07
mmol) and 6-(3-bromopropyl)-3-iodo-9-methoxy-5H-indeno[1,2-c]-
isoquinoline-5,11(6H)-dione (82, 261 mg, 0.59 mmol) were diluted
with DMSO (30 mL), and the mixture was heated at 90 °C for 12 h.
P
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Journal of Medicinal Chemistry
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The reaction mixture was diluted with CHCl3 (100 mL), washed with
water (100 mL) and sat aq NaCl (30 mL), and dried over sodium
sulfate. The solution was concentrated to provide a crude solid that
was purified by silica gel column chromatography, eluting with
chloroform−methanol, 50:1, to afford an orange solid (0.16 g, 83%):
mp 267−269 °C (decomp). IR (KBr) 2090, 1688, 1662, 1598, 1532,
eluting with chloroform to provide a purple solid (0.080 g, 39%): mp
232−235 °C (decomp). IR (KBr) 3357, 1644, 1544, 1510, 1272, 1052
cm−1; 1H NMR (DMSO-d6) δ 8.33 (d, J = 8.6 Hz, 1 H), 7.42 (dd, J =
8.5 Hz, J = 6.8 Hz, 1 H), 7.32 (m, 2 H), 7.14 (dd, J = 6.7 Hz, J = 1.0
Hz, 1 H), 7.09 (dd, J = 8.7 Hz, J = 2.4 Hz, 1 H), 5.74 (s, 2 H), 4.68 (t,
J = 7.1 Hz, 2 H), 3.97 (s, 3 H), 3.73 (t, J = 6.6 Hz, 2 H), 2.21 (pent, J
= 7.1 Hz, 2 H); ESIMS m/z (rel intensity) 369/371 (MH+, 100/32).
Anal. Calcd for C20H17ClN2O3: C, 65.13; H, 4.65; N, 7.60. Found: C,
64.85; H, 4.74; N, 7.42.
3-Amino-6-(3-azidopropyl)-5,6-dihydro-9-methoxy-5,11-
dioxo-11H-indeno[1,2-c]isoquinoline (88). Sodium azide (0.063
g, 0.968 mmol) and 3-amino-6-(3-chloropropyl)-5,6-dihydro-9-me-
thoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (87, 0.119 g, 0.323
mmol) were diluted with DMSO (25 mL), and the mixture was heated
at 100 °C for 16 h. The reaction mixture was diluted with chloroform
(50 mL), washed with water (3 × 25 mL) and brine (25 mL), and
dried over sodium sulfate. The solution was concentrated to provide a
crude solid that was purified by silica gel column chromatography,
eluting with a gradient of chloroform to 1% MeOH−chloroform, to
provide a solid that was washed with diethyl ether to afford a purple
solid (0.102 g, 84%): mp 220−223 °C. IR (KBr) 3434, 3349, 2096,
1651, 1509, 1271 cm−1; 1H NMR (DMSO-d6) δ 8.34 (d, J = 8.7 Hz, 1
H), 7.42 (dd, J = 8.5 Hz, J = 6.7 Hz, 1 H), 7.33 (m, 2 H), 7.15 (dd, J =
6.7 Hz, J = 1.1 Hz, 1 H), 7.09 (dd, J = 8.7 Hz, J = 2.5 Hz, 1 H), 5.74 (s,
2 H), 4.65 (m, 2 H), 3.97 (s, 3 H), 3.46 (t, J = 6.7 Hz, 2 H), 1.99 (m, 2
H); ESIMS m/z (rel intensity) 376 (MH+, 55). Anal. Calcd for
C20H17N5O3: C, 63.99; H, 4.56; N, 18.66. Found: C, 63.78; H, 4.38;
N, 18.30.
1
1478, 1424, 1298, 1164, 1014, 819, 791, 694 cm−1; H NMR (300
MHz, CDCl3) δ 8.61 (d, J = 1.8 Hz, 1 H), 8.34 (d, J = 8.6 Hz, 1 H),
7.92 (dd, J = 8.6 Hz, J = 1.8 Hz, 1 H), 7.59 (d, J = 8.4 Hz, 1 H), 7.17
(d, J = 2.5 Hz, 1 H), 6.85 (dd, J = 8.3 Hz, J = 2.8 Hz, 1 H), 4.53 (t, J =
7.2 Hz, 2 H), 3.89 (s, 3 H), 3.63 (t, J = 6.1 Hz, 2 H), 2.11 (t, J = 7.2
Hz, 2 H); 13C NMR (125 MHz, CDCl3) δ 188.9, 162.5, 161.8, 156.5,
142.2, 137.7, 136.9, 131.3, 127.6, 124.6, 123.9, 123.8, 115.8, 110.9,
107.4, 91.0, 55.7, 49.2, 42.7, 28.3; ESIMS m/z (rel intensity) 509
(MNa+, 100); HRESIMS m/z calcd for C20H15N4O3I 487.0267
(MH+), found, 487.0271 (MH+).
6-(3-Aminopropyl)-5,6-dihydro-9-methoxy-3-iodo-5,11-
dioxo-11H-indeno[1,2-c]isoquinoline (84). Triethyl phosphite
(0.2 mL) was added to a solution of 6-(3-azidopropyl)-3-iodo-9-
methoxy-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (83, 0.186 g,
0.459 mmol) in benzene (30 mL), and the reaction mixture was
heated at reflux for 16 h. The reaction mixture was allowed to cool to
room temperature, HCl in methanol (prepared from 0.5 mL of acetyl
chloride in 9.5 mL of methanol) was added, and the reaction mixture
was heated at reflux for 4 h. The reaction mixture was allowed to cool
to room temperature and filtered to provide a red solid (0.159 g,
83%): mp 267−269 °C (decomp). IR (KBr) 3399, 2937, 1692, 1658,
1
1600, 1532, 1478, 1429, 1298, 1228, 909, 823, 733 cm−1; H NMR
3-Amino-6-(3-aminopropyl)-5,6-dihydro-9-methoxy-5,11-
dioxo-11H-indeno[1,2-c]isoquinoline Dihydrochloride (89).
Triethyl phosphite (0.07 mL) was added to a solution of 3-amino-6-
(3-azidopropyl)-5,6-dihydro-9-methoxy-5,11-dioxo-11H-indeno[1,2-
c]isoquinoline (88, 0.061 g, 0.163 mmol) in benzene (20 mL), and the
reaction mixture was heated at reflux for 16 h. The reaction mixture
was allowed to cool to room temperature, 3 M HCl in methanol (10
mL) was added, and the reaction mixture was heated at reflux for 3 h.
The reaction mixture was allowed to cool to room temperature and
concentrated, and the precipitate was washed with chloroform (50
mL) and filtered to provide an orange solid (0.054 g, 78%): mp 246−
248 °C (decomp). IR (KBr) 3445, 2929, 1651, 1544, 1505, 1479, 1266
(300 MHz, CDCl3) δ 8.65 (d, J = 1.5 Hz, 1 H), 8.37 (d, J = 8.5 Hz, 1
H), 7.94 (dd, J = 8.6 Hz, J = 1.6 Hz, 1 H), 7.64 (d, J = 8.4 Hz, 1 H),
7.21 (d, J = 2.4 Hz, 1 H), 6.84 (dd, J = 8.3 Hz, J = 2.4 Hz, 1 H), 4.58
(t, J = 7.2 Hz, 2 H), 3.89 (s, 3 H), 2.89 (t, J = 6.3 Hz, 2 H), 2.00 (t, J =
7.3 Hz, 2 H); ESIMS m/z (rel intensity) 461 (MH+, 100); HRESIMS
m/z calcd for C20H17N2O3I 461.0362 (MH+), found, 461.0371
(MH+); HPLC purity: 98.9% (MeOH−H2O, 90:10), 96.7%
(MeOH−H2O, 80:20).
3-Iodo-9-methoxy-6-(3-(dimethylamino)propyl)-5H-indeno-
[1,2-c]isoquinoline-5,11(6H)-dione (85). 3-Amino-6-(3-amino-
propyl)-9-methoxy-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione
(84, 111 mg, 1.93 mmol) was dissolved in methanol (10 mL) and
acetic acid (5 mL). Aqueous formaldehyde (37%, 0.15 mL) was added,
and the reaction mixture was stirred at room temperature for 2 h. The
reaction mixture was cooled to 0 °C and sodium cyanoborohydride
(300 mg) was slowly added. The reaction mixture was stirred for 1 h at
room temperature. Water (30 mL) was added and the reaction mixture
extracted with chloroform (2 × 25 mL). The organic extracts were
combined and washed with brine (30 mL). The solvent was removed
in vacuo and the compound purified by silica gel column
chromatography, eluting with chloroform. The product was obtained
as a red solid (65 mg, 55%) mp >350 °C. IR (film) 3054, 2987, 1698,
1
cm−1; H NMR (DMSO-d6) δ 8.51 (d, J = 8.7 Hz, 1 H), 8.03 (bs, 2
H), 7.77 (d, J = 2.2 Hz, 1 H), 7.49 (m, 2 H), 7.36 (d, J = 7.6 Hz, 1 H),
7.19 (dd, J = 6.8 Hz, J = 0.8 Hz, 1 H), 4.61 (t, J = 7.0 Hz, 2 H), 4.01 (s,
3 H), 2.86 (m, 2 H), 2.09 (m, 2 H); ESIMS m/z (rel intensity) 350
(MH+, 90). Anal. Calcd for C20H21Cl2N3O3·1.0 H2O: C, 54.55; H,
5.26; N, 9.54. Found: C, 54.87; H, 5.10; N, 9.21.
Molecular Modeling. The Tdp1 crystal structure (PDB: 1RFF)
was prepared by removing one of the monomers along with all
crystallized waters, the polydeoxyribonucleotide 5′-D-
(*AP*GP*TP*T)-3′, the Top1-derived peptide residues 720−727
(mutation L724Y), and all metal ions. The Lys265, Lys495, and
His493 residues were protonated. Missing hydrogens were added as
needed. GOLD docking was performed using the centroid x = 8.0128,
y = 46.5888, z = −1.5534. The hydrogen bond length was set to 4 Å
while the van der Waals parameter was set to 10 Å. The top ligand
binding-pose (highest GOLD score) was selected and merged with the
prepared protein. The ligand was surrounded by a sphere with a 6 Å
radius and minimized by the conjugate gradient method using the
MMFF94s force field and MMFF94 charges with Sybyl software. The
calculation was terminated when the gradient reached a value of 0.05
kcal/(mol·Å).
1
1649, 1531, 1477, 1430, 1301, 1265, 740 cm−1; H NMR (500 MHz,
CDCl3) δ 8.65 (d, J = 1.8 Hz, 1 H), 8.38 (d, J = 8.6 Hz, 1 H), 7.85 (dd,
J = 2.0, J = 8.6 Hz, 1 H), 7.67 (d, J = 8.3 Hz, 1 H), 7.21 (d, J = 2.5 Hz,
1 H), 6.83 (dd, J = 2.5, J = 8.4 Hz, 1 H), 4.54 (t, J = 8.1 Hz, 2 H), 3.89
(s, 3 H), 2.50 (d, J = 6.3 Hz, 2 H), 2.02 (m, 2 H); 13C NMR (125
MHz, CDCl3−MeOH-d4) δ 189.5, 162.6, 162.2, 142.4, 137.7, 136.9,
131.6, 127.6, 124.6, 124.3, 123.8, 115.9, 111.2, 107.7, 91.0, 56.2, 55.7,
44.7, 43.0, 26.4; ESIMS m/z (rel intensity) 489 (MH+, 100);
HRESIMS m/z calcd for C22H21N2O3I 489.0675 (MH+), found,
489.0670 (MH+); HPLC purity: 97.1% (MeOH−H2O, 95:5); 97.5%
(MeOH−H2O, 85:15).
Topoisomerase I-Mediated DNA Cleavage Reactions. Top1
reactions were performed as recently described.16 Briefly, a 3′-[32P]-
end-labeled 117-bp DNA oligonucleotide (Integrated DNA Tech-
nologies) was incubated at 2 nM with recombinant Top1 in 20 μL of
reaction buffer [10 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM
MgCl2, 0.1 mM EDTA, and 15 μg/mL BSA] at 25 °C for 20 min in
the presence of various concentrations of compounds. The reactions
were terminated by adding SDS (0.5% final concentration) followed
by the addition of two volumes of loading dye (80% formamide, 10
3-Amino-6-(3-chloropropyl)-5,6-dihydro-9-methoxy-5,11-
dioxo-11H-indeno[1,2-c]isoquinoline (87). 6-(3-Chloropropyl)-
5,6-dihydro-9-methoxy-3-nitro-5,11-dioxo-11H-indeno[1,2-c]-
isoquinoline (86)20 (0.220 g, 0.552 mmol) and 5% Pd/C (0.200 g)
were diluted with THF (125 mL). The solution was degassed and
stirred at room temperature under a hydrogen atmosphere for 1 h.
The solution was filtered, the filterpad was washed with chloroform
(125 mL), and the filtrate was concentrated to provide a crude purple
solid. The solid was purified by silica gel flash column chromatography,
Q
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Journal of Medicinal Chemistry
Article
mM sodium hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol, and
0.1% bromphenol blue). Reactions were subjected to 20% denaturing
PAGE. Gels were dried and visualized by using a Typhoon 8600 and
ImageQuant software (Molecular Dynamics).
mention of trade names, commercial products, or organizations
imply endorsement by the U.S. Government.
ABBREVIATIONS USED
Gel-Based Assay Measuring the Inhibition of Recombinant
Tdp1. Tdp1 reactions were performed as recently described.16 Briefly,
a 5′-[32P]-labeled single-stranded DNA oligonucleotide containing a
3′-phosphotyrosine (N14Y) incubated with 5 pM recombinant Tdp1
in the absence or presence of inhibitor for 15 min at room temperature
in a buffer containing 50 mM Tris HCl, pH 7.5, 80 mM KCl, 2 mM
EDTA, 1 mM DTT, 40 μg/mL BSA, and 0.01% Tween-20. Reactions
were terminated by the addition of 1 volume of gel loading buffer
[99.5% (v/v) formamide, 5 mM EDTA, 0.01% (w/v) xylene cyanol,
and 0.01% (w/v) bromophenol blue]. Samples were subjected to a
16% denaturing PAGE, and dried gels were exposed to a
PhosphorImager screen (GE Healthcare). Gel images were scanned
using a Typhoon 8600 (GE Healthcare), and densitometric analyses
were performed using the ImageQuant software (GE Healthcare).
Surface Plasmon Resonance Analysis. Binding experiments
were performed as recently described.16 Briefly, Tdp1 was amine-
coupled to a CM5 sensor chip (GE Healthcare, Piscataway NJ). To
protect the amine groups with the active site from modification, 1 mM
Tdp1 was incubated with 2 mM of a 14-base oligonucleotide
containing a phosphate group at the 3′ end (GATCTAAAAGACTT)
in 10 mM sodium acetate pH 4.5 for 20 min. The CM5 chip surface
was activated for 7 min with 0.1 M NHS and 0.4 M EDC at a flow rate
of 20 mL/min, and the Tdp1−oligonucleotide mixture was injected
until approximately 4000 RU’s were attached. Activated amine groups
were quenched with an injection of 1 M solution of ethanolamine pH
8.0 for 7 min. Any bound oligonucleotide was removed by washing the
surface with 1 M NaCl. A reference surface was prepared in the same
manner without coupling of Tdp1. Compound 70 was diluted into
running buffer [10 mM HEPES, 150 mM NaCl, 0.01% Tween 20 (v/
v), 5% DMSO (v/v) pH 7.5] and injected over all flow cells at 30 mL/
min at 25 °C. Following compound injections, the surface was
regenerated with a 30 s injection of 1 M NaCl, a 30 s injection of 50%
DMSO (v/v), and a 30 s running buffer injection. Each cycle of
compound injection was followed by a buffer cycle for referencing
purposes. A DMSO calibration curve was included to correct for
refractive index mismatches between the running buffer and
compound dilution series.
■
APCIMS, atmospheric pressure chemical ionization mass
spectrometry; BBO, multinuclear broadband observe; EIMS,
electron impact mass spectrometry; ESIMS, electrospray
ionization mass spectrometry; QNP, quattro nucleus probe;
Tdp1, Tyrosyl-DNA phosphodiesterase I; Top1, topoisomerase
1
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 765-494-1465; fax: 765-494-6790; e-mail: cushman@
purdue.edu.
Author Contributions
§These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was made possible by the National Institutes of
Health (NIH) through support from Research Grant UO1
CA89566. This work was also supported by the NIH, National
Cancer Institute R25CA128770 (D. Teegarden) Cancer
Prevention Internship Program (M.C.-S.) administered by the
Oncological Sciences Center and the Discovery Learning
Research Center at Purdue University. This research was also
supported in part by the Intramural Research Program of the
NIH, National Cancer Institute, Center for Cancer Research.
This project has been funded in part with federal funds from
the National Cancer Institute, National Institutes of Health,
under contract no. HHSN261200800001E. The content of this
publication does not necessarily reflect the views or policies of
the Department of Health and Human Services, nor does the
R
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