Investigation of the Structure-Activity Relationships of Aza-A-Ring Indenoisoquinoline Topoisomerase i Poisons

Article abstract of DOI:10.1021/acs.jmedchem.6b00003

Several indenoisoquinolines have shown promise as anticancer agents in clinical trials. Incorporation of a nitrogen atom into the indenoisoquinoline scaffold offers the possibility of favorably modulating ligand-binding site interactions, physicochemical properties, and biological activities. Four series of aza-A-ring indenoisoquinolines were synthesized in which the nitrogen atom was systematically rotated through positions 1, 2, 3, and 4. The resulting compounds were tested to establish the optimal nitrogen position for topoisomerase IB (Top1) enzyme poisoning activity and cytotoxicity to human cancer cells. The 4-aza compounds were the most likely to yield derivatives with high Top1 inhibitory activity. However, the relationship between structure and cytotoxicity was more complicated since the potency was influenced strongly by the side chains on the lactam nitrogen. The most cytotoxic azaindenoisoquinolines 45 and 46 had nitrogen in the 2- or 3-positions and a 3′-dimethylaminopropyl side chain, and they had MGM GI₅₀ values that were slightly better than the corresponding indenoisoquinoline 64.

Full text of DOI:10.1021/acs.jmedchem.6b00003

Article
pubs.acs.org/jmc
Investigation of the Structure−Activity Relationships of Aza-A-Ring
Indenoisoquinoline Topoisomerase I Poisons
†
†
†
‡
‡
Daniel E. Beck, P. V. Narasimha Reddy, Wei Lv, Monica Abdelmalak, Gabrielle S. Tender,
‡
‡
‡
‡
,†
Sophia Lopez, Keli Agama, Christophe Marchand, 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
‡
Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer
Institute, National Institutes of Health, Bethesda, Maryland 20892, United States
*
S Supporting Information
ABSTRACT: Several indenoisoquinolines have shown promise as
anticancer agents in clinical trials. Incorporation of a nitrogen atom
into the indenoisoquinoline scaffold offers the possibility of favorably
modulating ligand-binding site interactions, physicochemical proper-
ties, and biological activities. Four series of aza-A-ring indenoisoqui-
nolines were synthesized in which the nitrogen atom was
systematically rotated through positions 1, 2, 3, and 4. The resulting
compounds were tested to establish the optimal nitrogen position for
topoisomerase IB (Top1) enzyme poisoning activity and cytotoxicity
to human cancer cells. The 4-aza compounds were the most likely to yield derivatives with high Top1 inhibitory activity.
However, the relationship between structure and cytotoxicity was more complicated since the potency was influenced strongly by
the side chains on the lactam nitrogen. The most cytotoxic azaindenoisoquinolines 45 and 46 had nitrogen in the 2- or 3-
positions and a 3′-dimethylaminopropyl side chain, and they had MGM GI50 values that were slightly better than the
corresponding indenoisoquinoline 64.
INTRODUCTION
rapid diffusion from their binding site in the Top1−DNA
cleavage complex, which may necessitate longer drug infusion
times in order to maintain adequate concentrations of the
ternary cleavage complexes; (4) dose-limiting toxicities
including bone marrow suppression and severe diarrhea; (5)
susceptibility to drug resistance by several Top1 point
■
DNA relaxation catalyzed by topoisomerase IB (Top1) is
essential for replication and transcription in eukaryotic cells.
During this enzyme-mediated process, Tyr723 carries out a
nucleophilic attack on a phosphodiester of DNA, resulting in
the cleavage of a single strand of DNA and a product in which
12
1
−3
mutations; and (6) efficient removal from cancer cells by
the enzyme is covalently linked through a 3-phosphodiester.
3
drug efflux pumps that results in drug resistance.
Normally, cleavage complexes reverse rapidly and are
undetectable in cells. However, DNA damage and certain
cancer chemotherapeutic agents known as Top1 poisons can
stabilize the cleavage complexes by preventing their reversal.
As a result of the extended lifetimes of the cleavage complexes,
advancing replication forks can collide with the DNA cleavage
sites and produce DNA double-strand breaks. The DNA
damage eventually causes the cell to enter apoptosis.
Several distinct Top1 poison chemotypes have been
developed since the discovery of the natural product
camptothecin (1) and its novel mechanism of action.
These limitations have resulted in the discovery of improved
Top1 poisons. Two compounds first synthesized in our
laboratory, the indenoisoquinolines indotecan (LMP 400,
3
,4
1
3
13
2
)
and indimitecan (LMP776, 3), have been promoted to
14,15
Phase I clinical trials at the National Cancer Institute.
A
5
third, structurally related indenoisoquinoline known as MJ-III-
16,17
3
,6
65 (LMP744, 4)
has shown promising preclinical activity.
The indenoisoquinolines overcome many of the drawbacks
associated with the camptothecins, with improvements that
include: (1) greater chemical stability; (2) longer residence
7
−10
16
times in the binding site; (3) retention of activity versus
Two derivatives of camptothecin are FDA-approved drugs used
for the treatment of solid tumors, and several analogues are
16
camptothecin-resistant Top1 mutants; and (4) diminished or
11
abolished ejection by the ABCG2 and MDR-1 drug efflux
being investigated for the treatment of various cancers. The
potent anticancer activities of the members of this class are
counterbalanced by problems with physicochemical properties,
drug resistance, and patient tolerability. The shortcomings of
the camptothecins include: (1) poor water solubility; (2)
instability of the E-ring lactone at physiological pH, which
hydrolyzes to a hydroxyacid that binds to plasma proteins; (3)
3
pumps.
The structure−activity relationships of the indenoisoquino-
line Top1 poisons have not been fully investigated in the area
Received: January 3, 2016
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.6b00003
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of scaffold atom substitution. Previously, the carbon atoms at
positions 7, 8, 9, or 10 were replaced by a nitrogen atom
(
please see structures 2−4 for position numbering and ring
1
8−20
lettering).
The working hypothesis for the design of these
compounds was that the π−π stacking interactions between the
drug and the neighboring base pairs in the ternary DNA−
drug−Top1 complex would be strengthened by installation of
electronegative nitrogen atoms in the aromatic system that
would facilitate charge transfer complex formation between the
neighboring base pairs and the drug. It was found that (1)
certain aza-D-ring indenoisoquinolines have approximately 10-
fold increased aqueous solubility relative to indenoisoquinoline
analogues without compromising their Top1 and growth
19
inhibitory activities; (2) particular compounds exhibit equal
or greater activity than 1 in the Top1-mediated DNA cleavage
2
0
assay; and (3) cancer cell growth inhibition can be achieved
at nanomolar concentrations, as measured by mean graph
20
midpoint (MGM) GI50 values in the NCI-60 assay. In the
present investigation, the pharmacological effects of carbon-to-
nitrogen substitution at positions 1, 2, 3, or 4 of the
indenoisoquinoline A-ring were systematically investigated.
This necessitated the creation of viable synthetic pathways to
make sufficient quantities of the desired compounds using
practical methods. The target “azaindenoisoquinolines” com-
pounds are formally novel indenonaphthyridinediones. The 3-
21
azaindenoisoquinoline system has recently been reported,
while the 1-, 2-, and 4-azaindenoisoquinolines are novel
heterocyclic systems that have never been synthesized and
studied before.
CHEMISTRY
A second motivation for this study was to investigate the
structural requirements for potent tyrosyl DNA phosphodies-
terase 1 and 2 (TDP1 and TDP2) inhibition by indenoisoqui-
nolines. TDP1 and TDP2 are DNA repair enzymes that process
Top1- and Top2-mediated DNA lesions, respectively. TDP1
catalyzes the hydrolysis of the 3′-phosphotyrosyl-DNA linkages
that result from degradation of Top1−DNA cleavage
complexes. TDP2 catalyzes the hydrolysis of the 5′-
phosphotyrosyl-DNA linkages that result from degradation of
Top2-DNA cleavage complexes and it also displays weak
activity against 3′-phosphotyrosyl-DNA linkages. There are
currently no promising TDP2 inhibitor series. A series of
deazaflavins (e.g., 6) with low nanomolar TDP2 inhibitory
potencies was recently reported. However, the authors
remarked that the chemical series is marred by poor cellular
■
The strategy used to synthesize aza-A-ring indenoisoquinolines
centered on the preparation of key tetracyclic lactone
precursors, such as 13. An advantage of this approach is that
several azaindenoisoquinolines that differ at the lactam nitrogen
side chain can be prepared using a divergent pathway from a
single common intermediate. It was anticipated that the
location of the nitrogen atom in the azaphthalide intermediates
e.g., 8) could be controlled by regioselective reactions of
carbonyl groups attached to C-2 and C-4 as opposed to C-3 of
the pyridine ring system.
locations of the nitrogen in the final products.
(
2
9−31
This would ultimately dictate the
The lactone 13 with a nitrogen atom in the 1-position was
made by the route outlined in Scheme 1. Quinolinic acid
anhydride 7 was regioselectively reduced with NaBH in THF-
4
AcOH, and the intermediate hydroxyacid was cyclized under
the acidic reaction conditions to yield 4-azaphthalide (8). The
55% yield of 8 obtained this way was superior to the 15−25%
2
2
permeability. TDP1 and TDP2 can serve as mutual backups
yields reported for LAH reduction of 7 followed by
23
for the repair of stalled Top1−DNA cleavage complexes,
30,32
sublimation.
Compound 8 was monobrominated with
3
2
which would make dual TDP1 and TDP2 inhibition a
significant advancement. Bis(indenoisoquinoline) 5 displays
potent Top1 inhibitory activity, and its IC50 values versus
purified and whole cell extract-containing TDP1 are each
NBS to afford 9, which was then hydrolyzed to provide 4-
aza-3-hydroxyphthalide (10). Condensation of 10 with
phthalide (11) under basic conditions involving a Dieckmann
3
3,34
condensation sequence
ate 12, which was treated with refluxing Ac O to close the B-
ring, yielding the lactone 13.
The synthesis of lactone 21 began with isonicotinic acid (14,
Scheme 2). After the formation of N-phenyl amide 15,
lithiation and functionalization with DMF provided lactam
generated indanedione intermedi-
2
4,25
2
approximately 1 μM,
and a number of monomeric
indenoisoquinolines have also displayed TDP1 inhibitory
activity, suggesting that the azaindenoisoquinolines should
also be investigated for TDP1 and TDP2 inhibitory
2
6−28
activity.
16. Reduction of lactam 16 with NaBH in MeOH delivered
4
B
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a
a
Scheme 1
Scheme 3
a
a
Reagents and conditions: (a) i. NaBH , PhMe, DMF, −20 to 35 °C, 2
Reagents and conditions: (a) i. NaBH , THF, AcOH, 15 °C, 4 h; ii.
4
4
h; ii. 5 M HCl, reflux, 0.5 h; (b) NBS, AIBN, CCl , reflux, 2 h; (c)
H O, reflux, 2 h; (d) i. NaOMe, MeOH, EtOAc, reflux, 24 h; ii. Ac O,
reflux, 6 h.
Ac O, AcOH, 100 °C, 3 h; (b) NBS, AIBN, CCl , reflux, 2 h; (c) H O,
reflux, 2 h; (d) i. NaOMe, MeOH, EtOAc, reflux, 15 h, then HCl; ii.
Ac O, reflux, 6 h.
4
2
4
2
2
2
2
a
a
Scheme 4
Scheme 2
a
Reagents and conditions: (a) MeOH, reflux, 2 h; (b) CDI, THF,
room temp., 1 h, then NaBH , MeOH, room temp., 2 h; (c) NBS,
4
AIBN, CCl , reflux, 24 h; (d) H O, reflux, 2 h; (e) i. NaOMe, MeOH,
4
2
EtOAc, reflux, 15 h, then HCl; ii. Ac O, reflux, 6 h.
2
a
Reagents and conditions: (a) i. SOCl , reflux, 2 h; ii. PhNH , K CO ,
2
2
2
3
THF, room temp., 24 h; (b) i. n-BuLi, THF, −78 °C (0.5 h) to 0 °C
(
0.5 h); ii. DMF, −78 °C (1 h) to 0 °C (1 h); (c) NaBH , MeOH,
CDI, followed by selective reduction of the mixed anhydride
and cyclization yielded 7-azaphthalide 28. Oxidation with NBS
afforded 29, followed by hydrolysis of the bromide to yield
4
room temp., 5 h; (d) 15% HCl, 60 °C, 2 d; (e) NBS, AIBN, CCl ,
4
CH Cl , reflux, 2 d; (f) H O, reflux, 2 h; (g) i. NaOMe, MeOH,
2
2
2
32
EtOAc, reflux, 15 h, then HCl; ii. Ac O, reflux, 6 h.
2
30. Condensation of 30 with phthalide (11) as before gave
the desired lactone 31. The regioselectivities of the key
carbonyl reactions observed in Schemes 1, 3, and 4 (7 → 8, 22
→ 23, and 7 → 27) are the result of the greater reactivity of
carbonyl substituents at C-2 and C-4 vs C-3 on the pyridine
ring, which is a consequence of the greater electronegativity of
1
7, and lactonization in aqueous HCl yielded azaphthalide 18.
From this point, oxidation with NBS to afford 19, hydrolysis to
yield 20, and condensation of 20 with 11 followed by
cyclization of the resulting intermediate in refluxing Ac O
2
29−31
produced the final product 21.
the nitrogen atom vs a carbon atom.
The synthesis of lactone 26 was accomplished according to
our previously published procedure (Scheme 3).
Lactones 13, 21, 26, and 31 were condensed with primary
21
amines 32−35 in CHCl , with or without MeOH as a
3
The final lactone 31 was produced starting from anhydride 7
Scheme 4). Heating the latter to reflux in MeOH yielded half-
cosolvent, to yield aza-A-ring indenoisoquinolines 36−39, 40−
43, 44−47, and 48−51 (Scheme 5). The syntheses of
(
3
0
21
ester 27. Activation of the carboxylic acid present in 27 with
compounds 38, 42, and 46 were previously published.
C
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Journal of Medicinal Chemistry
Article
a
Scheme 5
a
Reagents and conditions: (a) CHCl , reflux, 15 h; (b) CHCl , MeOH, reflux or room temperature.
3
3
Lastly, lactones 13, 21, 26, and 31 were each condensed with
N-Boc-1,3-diaminopropane (52), and the resulting intermedi-
ates 53−56 were deprotected with 5 N HCl in MeOH and
phoresis, the cleaved DNA strands can be visualized as cleavage
bands on the gel. The intensity and amount of cleavage bands
observed are used to assign a semiquantitative score to a test
agent’s Top1 inhibitory activity. A representative gel is shown
in Figure 1. The caption in Figure 1 provides further detail on
the scoring system used in this assay, and the Experimental
Section gives a description of the experimental protocol.
Top1 scores were generally moderate (++) for 1-, 2-, and 3-
azaindenoisoquinolines, with only five exceptions out of these
CHCl to yield aza-A-ring indenoisoquinoline dihydrochloride
3
21
salts 57−60 (Scheme 6). The selection of a three-carbon
polymethylene linker between the lactam nitrogen and the side
chain nitrogen in the final products is consistent with prior
studies indicating that the optimal length is 2−4 atoms in
36
35
comparable systems.
1
5 compounds (Table 1). The 4-azaindenoisoquinolines
BIOLOGICAL RESULTS AND DISCUSSION
■
performed best in the Top1 assay, and these five compounds
The Top1-mediated DNA cleavage assay was used to measure
Top1 inhibitory activity. This assay measures the ability of the
(
(
37, 42, 48, 49, and 59) have a greater average Top1 score
2.4) than any of the other azaindenoisoquinolines series
3
2
compound to stabilize DNA cleavage in a 3′-[ P]-labeled DNA
substrate via Top1 poisoning. Putative Top1 poisons are tested
at 0.1, 1, 10, and 100 μM concentrations, alongside positive
controls camptothecin (1) and 4 at 1 μM concentration. In the
absence of a Top1 poison, Top1 can execute its DNA
relaxation mechanism, and Top1−DNA cleavage complexes are
not trapped. However, in the presence of a Top1 poison,
Top1−DNA cleavage complexes are stabilized through
intercalation of the Top1 poisons between the base pairs at
the cleavage site, and following denaturation and gel electro-
prepared in this study. The worst side chain for Top1 activity
appears to be 2′-S-propanediol: three of the four compounds
with this moiety were the worst in their respective series. The
2
′-S-propanediol side chain is the most polar of the five side
chains, both by computed polar surface area and effect on
37
ClogP (calculated in ChemBioDraw ). This would be to the
detriment of the hydrophobic effect that helps to drive the
binding of an indenoisoquinoline to the Top1−DNA cleavage
complex.
D
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Article
a
Scheme 6
are produced when topoisomerase II (Top2) is trapped on the
DNA in a cleavage complex. Correspondingly, the TDP2 gel-
based assay measures the ability of a drug to inhibit the cleavage
of a 5′-phosphotyrosyl-DNA bond by TDP2 (Y19, Figure 3).
Upon catalytic cleavage of the phosphotyrosyl linkage, a 5′-
phosphate DNA product is generated (p19, Figure 3). Both a
schematic representation of the assay and a representative gel
are displayed in Figure 3.
TDP1 and TDP2 inhibitory activity was observed only for
analogues with sterically undemanding 3′-dimethylaminopropyl
or 3′-aminopropyl side chains. This observation is consistent
21,26
with our previous findings.
The new indenoisoquinolines were evaluated for cancer cell
growth inhibitory activities in The National Cancer Institute’s
human cytotoxicity assay, which measures the ability of test
agents to inhibit the growth of approximately 60 different
cancer cell lines. In the initial single-concentration assay, cells
are treated with a 10 μM concentration of test agent. The
growth of treated cells versus untreated cells is then compared.
If a test agent induces a sufficiently low mean growth percent, it
is promoted to five-concentration testing to determine a mean
graph midpoint (MGM) GI50 value. In five-concentration
testing, cells are treated with test agent concentrations ranging
from 10− to 10 M. The concentration required to achieve
8
−4
5
0% growth inhibition in a particular cell line is calculated. In
situations in which this value is greater than 100 μM or less
than 0.01 μM, it is recorded as 100 and 0.01 μM, respectively.
The values for each of the tested cell lines are averaged to
obtain a mean graph midpoint (MGM) GI value. MGM GI
50
50
values are often, but not always, determined from two separate
38
rounds.
The mean growth percentages observed after 48 h incubation
at 10 μM concentration are listed in Table 1. On the basis of
this one-concentration testing, the most active compounds are
4
5 (−8.83%), 46 (−9.25%), 47 (5.45%), and 60 (12.2%).
These four of the compounds met the NCI criteria for testing
in the five-concentration cytotoxicity assay. Compounds 45−47
contain the 3′-dimethylaminopropyl side chain, and compound
a
Reagents and conditions: (a) CHCl , reflux, 24 h; (b) 5 N HCl in
3
60 contains the aminopropyl side chain. Compounds 46, 47,
MeOH, CHCl , 6 h.
3
and 60 displayed single-digit micromolar GI50 against most of
the cell lines and compound 45 had submicromolar activity
TDP1 is involved in the repair of DNA damage that results
from the action of Top1 poisons, which induce the formation
of DNA−protein adducts that include 3′-phosphotyrosyl
linkages. The TDP1 gel-based assay assesses the ability of a
compound to inhibit the enzyme-induced cleavage of the
phosphotyrosyl-DNA bond at the 3′end of the DNA (N14Y,
Figure 2). Cleavage of the phosphodiester bond between the
(
Table 2).
The regular (nonaza) indenoisoquinoline analogues 61−65
39,40
were previously studied
and their relevant biological data
are shown in Table 3. Their average Top1 score is +++, and 63
was scored at ++++. Although the non-aza compounds are
superior in terms of Top1 activity, their MGM GI50 values are
not substantially greater than those of the tested azaindenoi-
soquinolines. In fact, the 3′-dimethylaminopropyl non-aza
compound 64 (MGM 1.86 μM) is slightly less cytotoxic than
the three identically substituted azaindenoisoquinolines 45 (2-
aza, MGM 0.437 μM), 46 (3-aza, MGM 0.977 μM), and 47 (4-
aza, MGM 1.26 μM). Most of the compounds in Table 3 show
MGM GI values that are greater than 1 μM, and only one that
3
′-phosphate and tyrosine generates a 3′-phosphate DNA
product (N14P, Figure 2). Compounds are tested in a
concentration-dependent manner, and inhibition of TDP1 is
represented by disappearance of the gel band corresponding to
the N14P product (Figure 2). A schematic representation of
the assay and a representative gel are shown in Figure 2, and
5
0
the IC values for the inhibition of TDP1 are listed in Table 1.
50
is submicromolar. Although many of these compounds are
more potent versus Top1 than their azaindenoisoquinolines
counterparts, this did not always translate to more potent
antiproliferative activities.
The docking model for 43 bound to the Top1−DNA
cleavage complex indicates that the ligand adopts nearly the
same binding orientation as 66 in its cocrystal structure with
41
TDP2, as opposed to TDP1, cleaves the phosphotyrosyl
linkage at the 5′-end of DNA. The latter DNA−protein adducts
Top1 and DNA (Figure 4). The nitrogen in the 4-position is
oriented toward the major groove and faces toward the solvent.
E
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Journal of Medicinal Chemistry
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Figure 1. Representative Top1-mediated DNA cleavage assays. Two independent experiments are shown for the indicated compounds at the
indicated concentrations. (A) Concentration−response for 49 and 51. (B) Comparison of four selected compounds 48−51 (the gel was cropped to
only show the selected concentrations). The numbers and arrows at the left indicate cleavage site positions (see Experimental Section). Gel-based
assays include positive controls (i.e., 1 and 6). The combined intensities of the bands observed at different drug concentrations in the DNA cleavage
electrophoresis gels are used to estimate the abilities of the topoisomerase I poisons to stabilize the cleavage complexes through inhibition of the
religation reaction at several different DNA cleavage sites. The “+”-based scoring system is based on the activity of 1 μM camptothecin (1): 0, no
activity; +, between 20 and 50% activity; and ++, between 50 and 75% activity at the most effective drug concentration. Gel-based assays were usually
run in two independent experiments.
Table 1. Top1 Poisoning and TDP1 and TDP2 Inhibitory Activities of Aza-A-Ring Indenoisoquinolines
a
b
c
d
compd
Top1
TDP1
TDP2
mean growth %
side chain
e
1
++++
++
++
++
+
N.A.
N.A.
>111
>111
>111
>111
>111
>111
>111
37
N.A.
87.9
83.6
44.4
100
1-aza
2-aza
3-aza
4-aza
36
>111
>111
90
morpholinopropyl
imidazolylpropyl
dimethylaminopropyl.
dihydroxypropyl
aminopropyl
4
4
4
5
0
4
8
7
>111
e
++
+++
++
++
+
43, 63 (n = 2)
N.T.
37
>111
N.T.
67.8
−8.83
85.5
N.T.
N.T.
47.5
−9.25
N.T.
N.T.
42.9
49.6
5.45
61.7
12.2
morpholinopropyl
imidazolylpropyl
dimethylaminopropyl
dihydroxypropyl
aminopropyl
41
45
49
58
>111
63
>111
>111
++
++
+
19, 30 (n = 2)
∼111
>111
>111
80
38
>111
morpholinopropyl
imidazolylpropyl
dimethylaminopropyl
dihydroxypropyl
aminopropyl
42
46
50
59
>111
++
++
0/+
+++
+++
+++
+
63
>111
>111
∼111
>111
>111
>111
>111
>111
30, 48 (n = 2)
>111
39
morpholinopropyl
imidazolylpropyl
dimethylaminopropyl
dihydroxypropyl
aminopropyl
43
47
51
60
>111
∼111
>111
++
60, > 111 (n = 2)
a
Compound-induced DNA cleavage due to Top1 poisoning, with scores given according to the following system based on the activity of 1 μM 1: 0,
b
no activity; +, between 20 and 50% activity; ++, between 50 and 75% activity; +++, between 75 and 95% activity; ++++, equal activity. IC values
for the inhibition of TDP1 (μM). IC50 values for the inhibition of TDP2 (μM). The mean-graph midpoint of the percent growth of 60 human
cancer cell lines treated with 10 μM drug concentration for 48 h relative to no-drug control and relative to the time zero number of cells. Not
available. Not tested because the compound was not accepted for evaluation by the National Cancer Institute, Developmental Therapeutics
5
0
c
d
e
f
Program.
F
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Journal of Medicinal Chemistry
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Figure 2. TDP1 inhibition assay gel. The concentrations of positive controls 5 and 61 and test compounds were 1.4, 4.1, 12.3, 37, and 111 μM (left
to right). The 5′-labeled N14Y TDP1 DNA substrate corresponds to a 3′-phosphotyrosyl 14-mer oligonucleotide, and the 5′-labeled N14P DNA
product corresponds to a 3′-phosphate 14-mer oligonucleotide (see Experimental Section). Gel-based assays are usually run in two independent
experiments.
Figure 3. TDP2 inhibition assay gel. The concentrations of positive control 6 were 0.006, 0.017, 0.05, 0.15, and 0.46 μM, and the concentrations of
6
1 and the test compounds were 0.46, 1.4, 4.1, 12.3, 37, and 111 μM (left to right). The 3′-labeled Y19 TDP2 DNA substrate corresponds to a 5′-
phosphotyrosyl 19-mer oligonucleotide and the 3′-labeled p19 TDP2 DNA product corresponds to a 5′-phosphate 19-mer oligonucleotide (see
Experimental Section).
Table 2. Antiproliferative Activities of Aza A-Ring Indenoisoquinolines
cytotoxicity (GI , μM)
50
lung, HOP-
62
colon, HCT-
116
CNS, SF-
539
melanoma,
UACC-62
ovarian,
OVCAR-3
renal,
SN12C
prostate, DU-
145
breast,
MCF7
a
compd
MGM
1
0.01
0.03
0.01
0.01
0.309
1.23
1.29
1.35
0.22
1.10
2.00
2.40
2.29
0.02
0.01
0.01
0.0405
0.437
0.977
1.26
4
4
4
6
5
6
7
0
0.398
0.871
1.26
0.245
0.380
0.437
0.537
0.380
0.708
0.851
1.38
0.288
0.955
1.12
0.490
0.977
1.91
0.245
0.331
0.363
0.417
1.45
1.12
1.91
1.51
a
Mean graph midpoint GI50 from the NCI-60 five-concentration assay.
The clustering of hydrophilic functionality in this position may
contribute to the 4-azaindenoisoquinolines’ greater average
Top1 scores compared to the other regioisomers. The
clustering theoretically decreases the extent of desolvation
that must occur for these ligands to bind the target or pass
through the cell membrane interior. No specific interactions
between the side chain imidazole and the surrounding DNA
and enzyme were observed, and the side chain projects into the
solvent-filled major groove of the DNA. The ligand’s aromatic
ring system is calculated to form van der Waals interactions
with the flanking DNA base pairs, and the ligand’s C-ring
carbonyl oxygen is calculated to engage Arg364 in a hydrogen
bonding interaction.
this approach proved to be of no value in correlating calculated
binding energies with Top1 inhibitory potencies (data not
shown).
CONCLUSION
Practical synthetic pathways were successfully pioneered to the
-, 2-, 3-, and 4-azaindenoisoquinolines, and the resulting series
■
1
of compounds were tested as Top1 poisons, TDP1 and TDP2
inhibitors, and as cytotoxic agents in human cancer cell
cultures. These compounds allowed the determination of the
most favorable location for the nitrogen atom in the A-ring.
The average Top1 scores were greatest for the 4-azaindeno-
isoquinoline series. The aza-A-ring indenoisoquinolines were
generally inactive against TDP1 and TDP2, although sterically
undemanding aminopropyl side chains were found to impart
some degree of activity. Comparisons against regular (non-aza)
indenoisoquinolines show that Top1 inhibition scores are
diminished by nitrogen atom incorporation, although MGM
GI50 values for the most cytotoxic compounds were slightly
better for the azaindenoisoquinolines in most cases. The
An attempt was also made to correlate the calculated DNA
binding energies with the Top1 inhibitory activities using an
MP2 quantum mechanics model that had previously been
successful in predicting indenoisoquinoline and camptothecin
DNA binding site orientations and DNA binding site
4
2−44
selectivities,
as well as the Top1 inhibitory activities of
45
camptothecin isomers and camptothecin-lactam. However,
G
DOI: 10.1021/acs.jmedchem.6b00003
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Journal of Medicinal Chemistry
Article
Table 3. Top1 Poisoning and Antiproliferative Activities of
Indenoisoquinolines 61−65
cultures. The anticancer activities were highly dependent on the
side chain attached to the lactam nitrogen, with the 3′-
dimethylaminopropyl substituent being the most favorable. A
molecular modeling study showed that a nitrogen in the 4-
position is clustered with other polar functionality at the edge
of the indenoisoquinoline that faces the solvent-filled major
groove. These molecules might therefore require less desolva-
tion upon cleavage site binding.
a
compd
Top1
MGM GI50 (μM)
61
62
63
64
65
+++
++
0.32 ± 0.23
15.1
++++
+++
+++
1.86
1.86
8.71
a
Mean graph midpoint GI50 from the NCI-60 five-concentration assay.
EXPERIMENTAL SECTION
■
Reactions were monitored by silica gel analytical thin-layer
chromatography, and 254 nm UV light was used for visualization.
All yields refer to isolated compounds. Unless otherwise stated,
chemicals and solvents were of reagent grade and used as obtained
from commercial sources without further purification. Melting points
1
were determined using capillary tubes and are uncorrected. H Nuclear
magnetic resonance spectroscopy was performed using a 300 MHz
spectrometer. Infrared spectra were obtained using an FTIR
spectrometer. Mass spectral analyses were performed at the Purdue
University Campus-Wide Mass Spectrometry Center. HPLC analyses
were performed on a Waters 1525 binary HPLC pump/Waters 2487
dual λ absorbance detector system, using a 5 μm C18 reversed phase
column and UV detection at 254 nm. HPLC purities of all tested
compounds were estimated from the major peak areas, which were
≥
95% of the combined total peak areas. Compounds 23−26, 38, 42,
21
and 46 were prepared according to previously reported procedures.
4
-Azaphthalide (8). NaBH (1.14 g, 33.5 mmol) was added to a
4
solution of quinolinic acid anhydride (7, 5.0 g, 33.5 mmol) in THF
35 mL) at 15 °C under argon. Acetic acid (4 g, 67 mmol) was added
(
dropwise, and the resulting mixture was stirred at 15 °C for 4 h. The
solvent was removed in vacuo. The residue was dissolved in acetic acid
(13.5 mL) and acetic anhydride (13.5 mL), and the resulting solution
was stirred for 3 h at 100 °C. The mixture was concentrated in vacuo,
incorporation of nitrogen in the 2-, 3-, or 4-position led to
moderate improvement in cytotoxicity in human cancer cell
Figure 4. Energy-minimized hypothetical binding pose of 43 (green) within the X-ray crystal structure of a stalled Top1−DNA cleavage complex
4
1
cocrystallized with an indenoisoquinoline (66, PDB ID 1SC7). The major groove of the DNA is always oriented toward the viewer. Heavy atom
distance (in Å) appears next to the dashed line. The stereoviews are programmed for wall-eyed (relaxed) viewing.
H
DOI: 10.1021/acs.jmedchem.6b00003
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Journal of Medicinal Chemistry
Article
and the residue was dissolved in a solution of H O (35 mL) + NaCl
300 MHz) δ 8.97 (s, 1 H), 8.86 (d, J = 4.9 Hz, 1 H), 7.77 (m, 3 H),
7.48 (t, J = 6.1 Hz, 2 H), 7.28 (t, J = 7.5 Hz, 1 H), 7.10 (br s, 1 H),
6.69 (s, 1 H).
2
(
6.7 g). The water phase was extracted with CHCl (3 × 40 mL), and
3
the combined organic layers were concentrated. Recrystallization from
i-PrOH yielded compound 8 (2.5 g, 55%) as a light yellow solid: mp
3-(Hydroxymethyl)-N-phenylisonicotinamide (17). Com-
pound 16 (3.0 g, 13 mmol) was dissolved in MeOH (65 mL),
−1 1
1
23−125 °C. IR (KBr) 1778, 1567, 1423, 1355, 1000, 743 cm ; H
NMR (300 MHz, CDCl ) δ 8.87 (dd, J = 1.5, 4.9 Hz, 1 H), 8.22 (dd, J
NaBH (0.902 g, 26.4 mmol) was added, and the mixture was stirred
3
4
=
1.5, 7.7 Hz, 1 H), 7.50 (dd, J = 5.0, 7.8 Hz, 1 H), 5.33 (s, 2 H);
at room temperature for 5 h. MeOH was evaporated under vacuum,
and water (28 mL) was added to the residue. This mixture was
extracted with EtOAc (2 × 100 mL). The combined organic layer was
dried over Na SO , concentrated, and purified by silica gel flash
+
ESIMS m/z (rel intensity) 136 (MH , 100).
-Aza-3-bromophthalide (9). Compound 8 (1.0 g, 7.40 mmol)
4
was heated at reflux with NBS (1.44 g, 8.1 mmol) and AIBN (20 mg)
2
4
in dry CCl (40 mL) for 2 h. The reaction mixture was cooled to room
column chromatography (70:30 EtOAc/hexanes) to afford compound
17 (2.5 g, 82%) as a light yellow syrup. IR (KBr) 3451, 1675, 666
4
temperature, the succinate salts were filtered off, and the filtrate was
concentrated and purified by silica gel flash column chromatography
−
1 1
cm ; H NMR (CDC1 , 300 MHz) δ 9.77 (s, 1 H), 8.60 (d, J = 5.1
3
(
75:25 EtOAc/hexanes) to afford product 9 (1.4 g 87%) as a colorless
Hz, 1 H), 8.46 (s, 1 H), 7.67 (m, 3 H), 7.38 (t, J = 7.5 Hz, 2 H), 7.17
(t, J = 6.6 Hz, 1 H), 4.72 (s, 2 H).
−1 1
syrup. IR (film) 1782, 1594, 1428, 986, 667 cm ; H NMR (CDCl ,
3
300 MHz) δ 8.99 (dd, J = 1.4, 4.9 Hz, 1 H), 8.26 (dd, J = 1.4, 7.8 Hz, 1
5-Azaphthalide (18). Compound 17 (2.5 g, 11 mmol) was added
H), 7.59 (dd, J = 4.8, 7.8 Hz, 1 H), 7.38 (s, 1 H); CIMS m/z (rel
to hydrochloric acid (15%, 22 mL), and the reaction mixture was
+
intensity) 214 (MH , 100).
heated at 60 °C for 2 d. The mixture was adjusted to pH 5−6
4-Aza-3-hydroxyphthalide (10). Compound 9 (1.4 g, 6.5 mmol)
(
NaHCO ). The solution was extracted with CHCl (3 × 75 mL). The
3
3
was heated at reflux in H O (40 mL) for 2 h before the solvent was
2
combined organic layer was dried over Na SO , concentrated, and
2 4
evaporated to afford compound 10 (0.9 g, 91%) as a light yellow solid:
mp 209−211 °C. IR (KBr) 3145, 1784, 1721, 1619, 1214, 1076, 765
purified by silica gel flash column chromatography (60:40 EtOAc/
hexanes) to afford compound 18 (1.0 g, 68%) as a light yellow solid:
−1 1
cm ; H NMR (CDC1 , 300 MHz) δ 8.94 (dd, J = 1.5, 4.9 Hz, 1 H),
−1 1
3
mp 101−103 °C. IR (KBr) 1778, 1589, 1423, 1003, 741 cm ; H
8
1
.29 (dd, J = 1.4, 7.8 Hz, 1 H), 7.69 (dd, J = 4.9, 7.7 Hz, 1 H), 6.61 (s,
H); ESIMS m/z (rel intensity) 152 (MH , 100).
General Procedure A. The appropriate aza-3-hydroxyphthalides
NMR (CDC1 , 300 MHz) δ 8.94 (s, 1 H), 8.85 (d, J = 4.9 Hz, 1 H),
3
+
7
(
.79 (d, J = 4.9 Hz, 1 H), 5.43 (s, 2 H); ESIMS m/z (rel intensity) 136
+
MH , 100).
(
0.5 g, 3 mmol) and phthalide (11, 0.41 g, 3.1 mmol) were diluted in
5-Aza-3-bromophthalide (19). Compound 18 (1.0 g, 7.4 mmol)
EtOAc (15 mL). Sodium metal (0.35 g, 15 mmol) was dissolved in
MeOH (30 mL), and the solution was added to reaction mixture. The
solution was heated at reflux for 24 h, cooled to room temperature,
acidified with 37% HCl (3−4 mL), and concentrated. The obtained
was combined with NBS (1.4 g, 8.1 mmol) and AIBN (20 mg) in dry
CH Cl −CCl (10:50 mL), and the mixture was heated to reflux for
2
2
4
4
8 h. The reaction mixture was cooled to room temperature, solids
were filtered off, and the filtrate was concentrated and purified by silica
gel flash column chromatography (50:50 EtOAc/hexanes) to afford 19
solid was diluted with Ac O (20 mL), and the mixture was heated at
2
reflux for 6 h. The solution was concentrated, diluted with CHCl (100
3
(
0.750 g, 51%) as a light brown syrup. IR (film) 1784, 1587, 1422, 667
mL), and washed with sat. NaHCO (3 × 50 mL). The organic layer
−1 1
3
cm ; H NMR (CDCl , 300 MHz) δ 9.05 (s, 1 H), 8.94 (d, J = 5.0
3
was washed with sat. NaCl (75 mL), dried over Na SO , concentrated,
2
4
Hz, 1 H), 7.82 (d, J = 5.1 Hz, 1 H), 7.48 (s, 1 H); CIMS m/z (rel
and purified by silica gel column chromatography (9:1 CHCl3/
hexanes) to yield product 13, 21, or 31.
+
intensity) 214 (MH , 100).
5-Aza-3-hydroxyphthalide (20). Compound 19 (0.75 g, 3.5
1-Azaindeno[1,2-c]isochromene-5,11-dione (13). Following
mmol) was heated to reflux in H O (25 mL) for 2 h before the solvent
2
general procedure A, 13 (0.125 g, 15%) was obtained as an orange−
was evaporated to afford compound 20 (0.500 g, 95%) as a light
red solid: mp 167−168 °C. IR (KBr) 1754, 1712, 1492, 998, 695
−1 1
−1
1
yellow thick syrup: IR (film) 3215, 1726, 1622, 1078, 756 cm ; H
cm ; H NMR (CDC1 , 300 MHz) δ 8.94 (d, J = 4.5 Hz, 1 H), 8.24
3
NMR (CDC1 , 300 MHz) δ 9.01 (s, 1 H) 8.78 (d, J = 3.7 Hz, 1 H),
3
(
(
2
d, J = 7.0 Hz, 2 H), 7.53 (m, 4 H); ESIMS m/z (rel intensity) 250
+
+
8.22 (d, J = 7.7 Hz, 1 H), 6.67 (s, 1 H); ESIMS m/z (rel intensity) 152
MH , 100); HRESIMS calcd for C H NO 250.0504 (MH ), found
15
8
3
+
(
MH , 100).
50.0501.
N-Phenylisonicotinamide (15). A solution of isonicotinic acid
14, 5.0 g, 40.6 mmol) in thionyl chloride (40 mL) was heated at
2-Azaindeno[1,2-c]isochromene-5,11-dione (21). Following
general procedure A, 21 (0.400 g, 48%) was obtained as an orange−
(
red solid: mp 208−209 °C. IR (KBr) 1755, 1702, 1490, 996, 691
reflux for 2 h. After completion of the reaction, thionyl chloride was
−
1 1
cm ; H NMR (CDC1 , 300 MHz) δ 9.69 (s, 1 H), 8.80 (d, J = 5.1
3
removed under reduced pressure. THF (50 mL), K CO (16.8 g,
2
3
Hz, 1 H), 8.04 (d, J = 5.1 Hz, 1 H), 7.63 (dd, J = 1.0, 7.4 Hz, 1 H),
1
21.9 mmol), and aniline (3.78 g, 40.6 mmol) were added to the
residue and the reaction mixture was stirred at room temperature for
4 h. The reaction mixture was diluted with water (100 mL) and
+
7
.49 (m, 3 H); ESIMS m/z (rel intensity) 250 (MH , 100); HRESIMS
+
calcd for C H NO 250.0504 (MH ), found 250.0503.
15
8
3
2
2-(Methoxycarbonyl)nicotinic Acid (27). Compound 7 (5.0 g,
extracted with EtOAc (2 × 100 mL). The combined organic layer was
concentrated, and the residue was recrystallized from EtOAc/hexanes
3
4 mmol) was dissolved in MeOH (25 mL). The mixture was heated
to reflux for 2 h, and the solvent was then removed. The resulting
white solid was dissolved in EtOAc (25 mL) at reflux and filtered to
remove insoluble byproducts. The filtrate was concentrated in vacuo
(
60:40) to afford the product 15 (8.0 g, 99%) as a light yellow solid:
−1 1
mp 166−168 °C. IR (KBr) 1344, 1653, 1465, 665 cm ; H NMR
(
DMSO-d , 300 MHz) δ 10.49 (s, 1 H), 8.78 (m, 2 H), 7.86 (m, 2 H),
6
and recrystallized from EtOAc to provide 27 (4.5 g, 71%) as a white
7
.78 (m, 2 H), 7.39 (t, J = 8.0 Hz, 2 H), 7.15 (t, J = 6.5 Hz, 1 H).
-Hydroxy-2-phenyl-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-
-one (16). A 2.5 M n-BuLi solution in hexanes (17.8 mL, 44.4
1
solid: mp 157−159 °C. H NMR (300 MHz, CDCl ) δ 8.84 (dd, J =
3
3
1
1.7, 5.0 Hz, 1 H), 8.34 (dd, J = 1.6, 7.9 Hz, 1 H), 7.57 (dd, J = 5.0, 8.1
Hz, 1 H), 4.00 (s, 3 H).
mmol) was added to a solution of 15 (4.0 g, 20 mmol) in dry THF
(
120 mL) at −78 °C. The solution was held at −78 °C for 0.5 h and
7-Azaphthalide (28). A solution of compound 27 (4.5 g, 25
mmol) in THF (100 mL) was treated with CDI (5.35 g, 33.1 mmol) at
then allowed to rise to 0 °C and kept at 0 °C for 6 min. The mixture
was cooled to −78 °C and DMF (2.94 mL, 40.4 mmol) was added.
After 1 h at −78 °C, the reaction mixture was warmed to 0 °C and
0 °C. After 1 h, NaBH (1.40 g, 41.2 mmol) was added in portions.
4
The mixture was stirred for 2 h and then quenched carefully with
MeOH. EtOAc (50 mL) was added. The organic layer was washed
kept at 0 °C for 1 h. H O (40 mL) was added, the organic layer was
2
separated, and the water layer was extracted with CHCl (2 × 40 mL).
with water (75 mL) and brine (25 mL), dried over anhydrous Na SO ,
2 4
3
The combined organic layer was dried over Na SO , concentrated, and
and concentrated. The residue was purified by flash chromatography
(80:20 EtOAc/hexanes) to give 28 (1.5 g, 45%) as a white solid: mp
2
4
purified by silica gel flash column chromatography (60:40 EtOAc/
−1 1
hexanes) to afford compound 16 (3.0 g, 67%) as an off-white solid: mp
142−144 °C. IR (KBr) 1785, 1577, 1005, 754 cm ; H NMR (300
−1 1
2
09−211 °C. IR (KBr) 3356, 1721, 776 cm ; H NMR (DMSO-d ,
MHz, CDCl ) δ 8.87 (d, J = 4.6 Hz, 1 H), 7.95 (d, J = 8.4 Hz, 1 H),
6
3
I
DOI: 10.1021/acs.jmedchem.6b00003
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Journal of Medicinal Chemistry
Article
7
.59 (dd, J = 4.7, 7.3 Hz, 1 H), 5.38 (s, 2 H); ESIMS m/z (rel
layer was dried over Na SO , filtered, and concentrated to provide a
crude solid. The solid was purified by silica gel flash column
2
4
+
intensity) 136 (MH , 100).
-Aza-3-bromophthalide (29). Compound 28 (1.0 g, 7.4 mmol)
7
chromatography (3% MeOH in CHCl ) to provide the product 40,
3
was heated to reflux with NBS (2.10 g, 12.1 mmol) and AIBN (30 mg)
41, or 43.
in dry CCl (60 mL) for 24 h. The reaction mixture was cooled to
room temperature, solids were filtered off, and the filtrate was
concentrated and purified by silica gel flash column chromatography
1-Aza-5,6-dihydro-6-(3-(1H-imidazol-1-yl)propyl)-5,11-
dioxo-indeno[1,2-c]isoquinoline (40). Following general proce-
dure C, 40 (0.052 g, 82%) was obtained as an orange−red solid: mp
4
−1 1
(
60:40 hexanes/EtOAc) to afford product 29 (0.550 g 42%) as a white
242−243 °C. IR (KBr) 1702, 1667, 1495, 761, 665 cm ; H NMR
−1 1
solid: mp 112−113 °C. IR (KBr) 1782, 1590, 982, 665 cm ; H NMR
(
(CDCl , 300 MHz) δ 9.09 (dd, J = 1.9, 4.5 Hz, 1 H), 8.58 (dd, J = 1.9,
3
CDC1 , 300 MHz) δ 8.96 (d, J = 3.5 Hz, 1 H), 8.03 (dd, J = 1.3, 8.0
8.2 Hz, 1 H), 7.71 (d, J = 7.0 Hz, 1 H), 7.64 (s, 1 H), 7.43 (m, 3 H),
7.19 (s, 1 H), 7.07 (s, 1 H), 6.75 (d, J = 7.5 Hz, 1 H), 4.58 (t, J = 7.4
3
Hz, 1 H), 7.69 (dd, J = 4.8 Hz, 8.0 Hz, 1 H), 7.43 (s, 1 H); CIMS m/z
+
(
rel intensity) 214 (MH , 100).
-Aza-3-hydroxyphthalide (30). Compound 29 (0.75 g, 3.5
Hz, 2 H), 4.27 (t, J = 6.2 Hz, 2 H), 2.43 (m, 2 H); ESIMS (m/z rel
+
7
intensity) 357 (MH , 100); HRESIMS calcd for C21
H
17
N
4
O
2
+
mmol) was heated at reflux in H O (25 mL) for 2 h before the solvent
357.1352 (MH ), found 357.1359; HPLC purity, 98.36% (1% TFA
2
was evaporated to afford compound 30 (0.530 g, 99%) as a light
yellow thick syrup. IR (film) 3165, 1729, 1622, 1075, 758 cm ; H
in MeOH/H O, 90:10).
2
−1 1
2-Aza-5,6-dihydro-6-(3-(1H-imidazol-1-yl)propyl)-5,11-
dioxo-indeno[1,2-c]isoquinoline (41). Following general proce-
dure C, 41 (0.063 g, 87%) was obtained as an orange−red solid: mp
NMR (CDC1 , 300 MHz) δ 8.89 (d, J = 3.7 Hz, 1 H), 8.19 (d, J = 7.7
3
Hz, 1 H), 7.77 (m, 1 H), 6.71 (s, 1 H); ESIMS m/z (rel intensity) 152
+
−1 1
(
MH , 100).
274−275 °C. IR (KBr) 1708, 1689, 768, 656 cm ; H NMR (CDCl
3
4-Azaindeno[1,2-c]isochromene-5,11-dione (31). Following
+ CD OD, 300 MHz) δ 9.86 (s, 1 H), 8.56 (d, J = 5.5 Hz, 1 H), 8.02
3
general procedure A, 31 (0.350 g, 42%) was obtained as an orange−
(d, J = 5.5 Hz, 1 H), 7.69 (s, 1 H), 7.57 (dd, J = 1.2, 6.8 Hz, 1 H), 7.36
(m, 2 H), 7.06 (d, J = 4.2 Hz, 1 H), 6.81 (d, J = 7.3 Hz, 1 H), 4.49 (t, J
= 7.5 Hz, 2 H), 4.21 (t, J = 6.5 Hz, 2 H), 2.33 (m, 2 H); ESIMS (m/z
red solid: mp 262−263 °C. IR (KBr) 1756, 1710, 1367, 998, 692
−1 1
cm ; H NMR (CDC1 , 300 MHz) δ 8.86 (dd, J = 1.5, 4.5 Hz, 1 H),
3
+
8
.70 (dd, J = 1.5, 8.2 Hz, 1 H), 7.71 (m, 1 H), 7.62 (d, J = 6.8 Hz, 1
rel intensity) 357 (MH , 100); HRESIMS calcd for C21
H
17
N
O
4
2
+
+
H), 7.52 (m, 3 H); CIMS (m/z rel intensity) 250 (MH , 100);
HRESIMS calcd for C H NO 250.0504 (MH ), found 250.0506.
357.1352 (MH ), found 357.1354; HPLC purity, 96.57% (1% TFA in
+
MeOH/H O, 90:10).
15
8
3
2
General Procedure B. 3-Aminopropylmorpholine (32, 0.043 g,
.301 mmol) was added to a solution of the corresponding lactone
4-Aza-5,6-dihydro-6-(3-(1H-imidazol-1-yl)propyl)-5,11-
dioxo-indeno[1,2-c]isoquinoline (43). Following general proce-
dure C, 43 (0.061 g, 86%) was obtained as an orange−red solid: mp
0
(
0.050 g, 0.2 mmol) in CHCl (30 mL). The solution was allowed to
3
−1 1
2
51−252 °C. IR (KBr) 1712, 1687, 778, 666 cm ; H NMR (CDCl ,
stir at reflux temperature for 15 h, diluted with CHCl (45 mL), and
3
3
3
00 MHz) δ 9.00 (dd, J = 1.6, 8.3 Hz, 1 H), 8.84 (dd, J = 1.7, 4.4 Hz, 1
washed with H O (3 × 25 mL) and sat. NaCl (25 mL). The organic
2
H), 7.64 (s, 1 H), 7.62 (m, 2 H), 7.58 (m, 2 H), 7.18 (s, 1 H), 7.07 (s,
H), 6.66 (d, J = 7.3 Hz, 1 H), 4.60 (t, J = 7.5 Hz, 2 H), 4.28 (t, J =
layer was dried over sodium sulfate, filtered, and concentrated to
provide a crude solid. The solid was purified by silica gel flash column
1
+
chromatography (1% MeOH in CHCl ) to provide the product 36,
6.2 Hz, 2 H), 2.28 (m, 2 H); ESIMS (m/z rel intensity) 357 (MH ,
100); HRESIMS calcd for C21
3
+
3
7, or 39.
-Aza-5,6-dihydro-6-(3-morpholinopropyl)-5,11-dioxo-
indeno[1,2-c]isoquinoline (36). Following general procedure B, 36
H
17
N
4
O
2
357.1352 (MH ), found
1
357.1358; HPLC purity, 99.80% (1% TFA in MeOH/H
2
O, 90:10).
General Procedure D. N,N-Dimethylaminopropylamine (34,
0.030 g, 0.301 mmol) was added to a solution of corresponding
(
(
0.057 g, 85%) was obtained as an orange solid: mp 223−224 °C. IR
−1 1
KBr) 1702, 1658, 1494, 763 cm ; H NMR (CDCl , 300 MHz) δ
lactone (0.050 g, 0.2 mmol) in CHCl (30 mL). The solution was
3
3
9
(
.09 (dd, J = 1.7, 4.5 Hz, 1 H), 8.59 (dd, J = 1.7 Hz, 8.0 Hz, 1 H), 7.82
m, 1 H), 7.73 (m, 1 H), 7.47 (m, 2 H), 7.39 (m, 1 H), 4.66 (t, J = 7.8
Hz, 2 H), 3.72 (m, 4 H), 2.59 (t, J = 6.3 Hz, 2 H), 2.49 (m, 4 H), 2.11
allowed to stir at reflux temperature for 15 h, diluted with CHCl (60
3
mL), and washed with H O (3 × 30 mL) and brine (30 mL). The
2
organic layer was dried over Na SO , filtered, and concentrated to
2
4
+
(
m, 2 H); CIMS (m/z rel intensity) 376 (MH , 100); HRESIMS calcd
provide a crude solid. The solid was purified by silica gel flash column
+
for C H N O 376.1662 (MH ), found 376.1667; HPLC purity,
chromatography (4% MeOH in CHCl ) to afford the product 44, 45,
22
22
3
3
3
9
9.39% (1% TFA in MeOH/H O, 90:10).
2
or 47.
2
-Aza-5,6-dihydro-6-(3-morpholinopropyl)-5,11-dioxo-
indeno[1,2-c]isoquinoline (37). Following general procedure B, 37
0.066 g, 90%) was obtained as an orange−red solid: mp 218−219 °C.
1-Aza-5,6-dihydro-6-(3-(dimethylamino)propyl)-5,11-dioxo-
indeno[1,2-c]isoquinoline (44). Following general procedure D, 44
(0.055 g, 84%) was obtained as an orange−red solid: mp 187−188 °C.
(
−1
1
−1 1
IR (KBr) 1723, 1678, 789 cm ; H NMR (CDCl , 300 MHz) δ 10.0
IR (KBr) 1704, 1659, 1554, 764 cm ; H NMR (CDCl , 300 MHz) δ
3
3
(
=
=
s, 1 H), 8.68 (d, J = 5.4 Hz, 1 H), 8.06 (d, J = 5.8 Hz, 1 H), 7.78 (d, J
7.8 Hz, 1 H), 7.66 (dd, J = 1.3, 6.2 Hz, 1 H), 7.46 (m, 2 H), 4.63 (t, J
9.09 (dd, J = 1.8, 4.6 Hz, 1 H), 8.61 (dd, J = 1.8, 8.0 Hz, 1 H), 7.88 (m,
1 H), 7.85 (m, 1 H), 7.49 (m, 2 H), 7.46 (m, 1 H), 4.64 (m, 2 H), 2.52
(t, J = 6.5 Hz, 2 H), 2.29 (s, 6 H), 2.06 (m, 2 H); CIMS (m/z rel
7.8 Hz, 2 H), 3.72 (t, J = 4.2 Hz, 4 H), 2.60 (t, J = 5.9 Hz, 2 H), 2.50
+
+
(
m, 4 H), 2.08 (m, 2 H); CIMS (m/z rel intensity) 376 (MH , 100);
intensity) 334 (MH , 100); HRESIMS calcd for C H N O
2
0
20
3
2
+
+
HRESIMS calcd for C H N O 376.1662 (MH ), found 376.1663;
334.1556 (MH ), found 334.1562; HPLC purity, 98.32% (1% TFA
22
22
3
3
HPLC purity, 95.54% (1% TFA in MeOH/H O, 90:10).
in MeOH/H O, 90:10).
2
2
4
-Aza-5,6-dihydro-6-(3-morpholinopropyl)-5,11-dioxo-
indeno[1,2-c]isoquinoline (39). Following general procedure B, 39
0.067 g, 90%) was obtained as an orange−red solid: mp 177−178 °C.
2-Aza-5,6-dihydro-6-(3-(dimethylamino)propyl)-5,11-dioxo-
indeno[1,2-c]isoquinoline (45). Following general procedure D, 45
(0.059 g, 86%) was obtained as an orange−red solid: mp 177−178 °C.
(
−1
1
−1 1
IR (KBr) 1707, 1685, 1494, 765 cm ; H NMR (CDCl , 300 MHz) δ
IR (KBr) 1709, 1667, 1492, 768 cm ; H NMR (CDCl , 300 MHz) δ
3
3
9
.03 (dd, J = 1.6, 8.3 Hz, 1 H), 8.83 (dd, J = 1.6, 4.3 Hz, 1 H), 7.81 (d,
10.0 (s, 1 H), 8.68 (d, J = 5.2 Hz, 1 H), 8.07 (d, J = 5.3 Hz, 1 H), 7.81
(d, J = 7.2 Hz, 1 H), 7.66 (d, J = 7.0 Hz, 1 H), 7.47 (m, 2 H), 4.61 (m,
2 H), 2.54 (t, J = 6.5 Hz, 2 H), 2.06 (m, 2 H); CIMS (m/z rel
J = 6.5 Hz, 1 H), 7.64 (m, 2 H), 7.59 (m, 2 H), 4.70 (t, J = 7.9 Hz, 2
H), 3.69 (t, J = 4.6 Hz, 4 h), 2.59 (t, J = 6.2 Hz, 2 H), 2.48 (m, 4 H),
+
+
2.09 (m, 2 H); CIMS (m/z rel intensity) 376 (MH , 100); HRESIMS
intensity) 334 (MH , 100); HRESIMS calcd for C H N O
334.1556 (MH ), found 334.1560; HPLC purity, 98.21% (1% TFA
2
0
20
3
2
+
+
calcd for C H N O 376.1662 (MH ), found 376.1664; HPLC
purity, 99.49% (1% TFA in MeOH/H O, 90:10).
22
22
3
3
2
in MeOH/H O, 90:10).
2
General Procedure C. 3-Aminopropylimidazole (33, 0.037 g,
4-Aza-5,6-dihydro-6-(3-(dimethylamino)propyl)-5,11-dioxo-
indeno[1,2-c]isoquinoline (47). Following general procedure D, 47
(0.056 g, 84%) was obtained as an orange−red solid: mp 183−184 °C.
0.301 mmol) was added to a solution of the corresponding lactone
(
0.050 g, 0.2 mmol) in CHCl (30 mL). The solution was allowed to
3
−1 1
stir at reflux temperature for 15 h, diluted with CHCl (50 mL), and
IR (KBr) 1712, 1678, 1489, 767 cm ; H NMR (CDCl , 300 MHz) δ
3
3
washed with H O (3 × 20 mL) and sat. NaCl (20 mL). The organic
9.03 (dd, J = 1.5 Hz, 8.2 Hz, 1 H), 8.82 (dd, J = 2.7, 4.2 Hz, 1 H), 7.86
2
J
DOI: 10.1021/acs.jmedchem.6b00003
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(
(
d, J = 7.1 Hz, 1 H), 7.62 (m, 2 H), 7.58 (m, 2 H), 4.67 (m, 2 H), 2.53
reaction, CHCl was removed under reduced pressure. The crude
product was purified by silica gel flash column chromatography (95:5
3
t, J = 6.5 Hz, 2 H), 2.28 (s, 6 H), 2.07 (m, 2 H); CIMS (m/z rel
+
intensity) 334 (MH , 100); HRESIMS calcd for C H N O
3
in MeOH/H O, 90:10).
CHCl /MeOH) to afford Boc-protected compounds 53−56.
2
0
20
3
2
3
+
34.1556 (MH ), found 334.1559; HPLC purity, 99.62% (1% TFA
1-Aza-6-(N-Boc-3-aminopropyl)-5,6-dihydro-5,11-dioxo-
indeno[1,2-c]isoquinoline (53). Following general procedure E, 53
(0.075 g, 92%) was obtained as an orange solid: mp 202−203 °C. IR
2
(
2′S)-1-Aza-5,6-dihydro-6-(2′,3′-dihydroxypropyl)-5,11-
−1 1
dioxo-indeno[1,2-c]isoquinoline (48). Lactone 13 (54 mg, 0.22
mmol) was suspended with stirring in CHCl (10 mL) and MeOH
(
mL) was added to the suspension, and it was stirred with heating to
reflux for 17.5 h. The mixture was cooled to room temperature and
concentrated in vacuo. H O (5 mL) was added, and the suspension
was filtered to collect the solid. Compound 48 (27 mg, 39%) was
obtained as a red−orange solid: mp 245−250 °C (dec). H NMR (300
MHz, DMSO-d ) δ 9.00 (dd, J = 4.5, 1.9 Hz, 1 H), 8.50 (dd, J = 8.1,
1
=
(KBr) 1711, 1698, 1676, 665 cm ; H NMR (CDCl , 300 MHz) δ
3
3
9.13 (dd, J = 1.7, 4.5 Hz, 1 H), 8.62 (dd, J = 1.7, 8.1 Hz, 1 H), 7.75 (d,
J = 8.1 Hz, 1 H), 7.63 (d, J = 8.0 Hz, 1 H), 7.50 (m, 2 H), 7.41 (m, 1
H), 4.66 (t, J = 7.0 Hz, 2 H), 3.29 (m, 2 H), 2.13 (m, 2 H), 1.45 (s, 9
2.5 mL). Amine 35 (32 mg, 0.35 mmol) dissolved in MeOH (0.5
+
H); CIMS (m/z rel intensity) 406 (MH , 100).
2
2-Aza-6-(N-Boc-3-aminopropyl)-5,6-dihydro-5,11-dioxo-
indeno[1,2-c]isoquinoline (54). Following general procedure E, 54
(0.072 g, 90%) was obtained as an orange−red solid: mp 184−185 °C.
1
−1 1
6
IR (KBr) 1715, 1685, 1675, 666 cm ; H NMR (CDCl , 300 MHz) δ
3
.9 Hz, 1 H), 8.14 (d, J = 7.3 Hz, 1 H), 7.67−7.42 (m, 4 H), 5.15 (d, J
5.0 Hz, 1 H), 5.00 (t, J = 5.6 Hz, 1 H), 4.58 (d, J = 6.7 Hz, 2 H),
.10−3.90 (m, 1 H), 3.60 (t, J = 5.4 Hz, 2 H); ESIMS m/z (rel
1
(
=
0.0 (s, 1 H), 8.70 (d, J = 5.4 Hz, 1 H), 8.10 (d, J = 5.4 Hz, 1 H), 7.69
d, J = 6.5 Hz, 1 H), 7.59 (d, J = 6.8 Hz, 1 H), 7.49 (m, 2 H), 4.63 (t, J
7.0 Hz, 2 H), 3.29 (m, 2 H), 2.12 (m, 2 H), 1.45 (s, 9 H); CIMS
4
+
intensity) 345 (MNa , 100); HRESIMS m/z calcd for C H N O Na
3
1
+
1
8
14
2
4
(m/z rel intensity) 406 (MH , 100).
-Aza-6-(N-Boc-3-aminopropyl)-5,6-dihydro-5,11-dioxo-
indeno[1,2-c]isoquinoline (55). Following general procedure E, 55
0.070 g, 86%) was obtained as an orange−red solid: mp 176−177 °C.
+
45.0852 (MNa ), found 345.0861; HPLC purity, 100% (MeOH,
3
00).
(
2′S)-2-Aza-5,6-dihydro-6-(2′,3′-dihydroxypropyl)-5,11-
(
dioxo-indeno[1,2-c]isoquinoline (49). Lactone 21 (50 mg, 0.20
mmol) was suspended with stirring in CHCl (10 mL) and MeOH
(
mL) was added to the suspension, and it was stirred with heating to
reflux for 23 h. The mixture was cooled to room temperature and
concentrated in vacuo. H O (1 mL) was added, and the suspension
was filtered to provide a solid. Compound 49 (27 mg, 42%) was
−1 1
IR (KBr) 1708, 1694, 1671, 665 cm ; H NMR (CDCl , 300 MHz) δ
3
3
9
.50 (s, 1 H), 8.75 (d, J = 5.5 Hz, 1 H), 8.40 (d, J = 5.6 Hz, 1 H), 7.89
m, 1 H), 7.68 (m, 1 H), 7.50 (m, 2 H), 4.61 (m, 2 H), 2.53 (t, J = 6.4
Hz, 2 H), 2.10 (m, 2 H), 1.46 (s, 9 H); CIMS (m/z rel intensity) 406
2.5 mL). Amine 35 (26 mg, 0.29 mmol) dissolved in MeOH (0.5
(
+
(
MH , 100).
-Aza-6-(N-Boc-3-aminopropyl)-5,6-dihydro-5,11-dioxo-
indeno[1,2-c]isoquinoline (56). Following general procedure E, 56
2
4
1
obtained as a red−orange solid: mp 229−232 °C. H NMR (300
MHz, DMSO-d ) δ 9.82 (d, J = 1.0 Hz, 1 H), 8.66 (d, J = 5.3 Hz, 1 H),
(
(
0.077 g, 95%) was obtained as an orange solid: mp 197−198 °C. IR
−1 1
6
KBr) 1721, 1659, 1634, 656 cm ; H NMR (CDCl , 300 MHz) δ
3
8
(
4
.07 (d, J = 7.4 Hz, 1 H), 8.01 (dd, J = 5.4, 1.0 Hz, 1 H), 7.61−7.46
m, 3 H), 5.14 (d, J = 5.0 Hz, 1 H), 5.01 (t, J = 5.6 Hz, 1 H), 4.60−
.46 (m, 2 H), 4.05−3.92 (m, 1 H), 3.65−3.53 (m, 2 H); CIMS m/z
9
.06 (d, J = 8.3 Hz, 1 H), 8.86 (dd, J = 1.5 Hz, 4.3 Hz, 1 H), 7.66 (m, 3
H), 7.58 (m, 2 H), 4.69 (t, J = 6.6 Hz, 2 H), 3.26 (m, 2 H), 2.12 (m, 2
+
H), 1.43 (s, 9 H); CIMS (m/z rel intensity) 406 (MH , 100).
+
(
rel intensity) 323 (MH , 100); HRESIMS m/z calcd for C H N O
18 15 2 4
General Procedure F. Appropriate Boc-protected azaindenoiso-
+
3
23.1032 (MH ), found 323.1045; HPLC purity, 100% (MeOH, 100).
quinolines 53−56 (0.065 g, 0.160 mmol) in CHCl (20 mL) were
3
(
2′S)-3-Aza-5,6-dihydro-6-(2′,3′-dihydroxypropyl)-5,11-
treated with 5 N HCl in MeOH (4 mL). The reaction mixture was
stirred at room temperature for 6 h. After completion of the reaction,
the solvents were removed under reduced pressure, and the crude
dioxo-indeno[1,2-c]isoquinoline (50). Lactone 26 (11 mg, 0.044
mmol) was suspended with stirring in CHCl (1 mL), and a solution
of the amine 35 (7 mg, 0.08 mmol) in MeOH (1 mL) was added. The
mixture was stirred at room temperature for 1 h and concentrated in
vacuo. The residue was purified by silica gel column chromatography,
eluting with 95:5 CHCl /MeOH, to yield 50 (6 mg, 42%) as a
yellow−orange solid: mp 237−238 °C. H NMR (300 MHz, DMSO-
d ) δ 9.32 (s, 1 H), 8.79 (d, J = 5.5 Hz, 1 H), 8.32 (dd, J = 5.5, 0.9 Hz,
3
product was washed with 10% MeOH in CHCl (20 mL) and filtered
3
to afford the dihydrochloride salts.
6
-(3-Aminopropyl)-1-aza-5,6-dihydro-5,11-dioxo-indeno-
3
1
[1,2-c]isoquinoline Dihydrochloride (57). Following general
procedure F, 57 (0.053 g, 88%) was obtained as an orange−red
6
−1 1
solid: mp 226−228 °C. IR (KBr) 3370, 1696, 1676, 763, 666 cm ; H
1
H), 8.17 (d, J = 7.3 Hz, 1 H), 7.68−7.48 (m, 3 H), 5.17 (d, J = 5.0
NMR (DMSO-d , 300 MHz) δ 9.01 (d, J = 4.3 Hz, 1 H), 8.57 (d, J =
6
Hz, 1 H), 5.02 (t, J = 5.6 Hz, 1 H), 4.65−4.46 (m, 2 H), 4.07−3.93
8
.0 Hz, 1 H), 8.04 (br s, 3 H), 7.91 (d, J = 7.7 Hz, 1 H), 7.64 (m, 4 H),
(
(
m, 1 H), 3.60 (t, J = 5.3 Hz, 2 H); ESIMS m/z (rel intensity) 323
MH , 100); HRESIMS m/z calcd for C H N O 323.1032 (MH ),
+
+
4.60 (t, J = 7.0 Hz, 2 H), 2.99 (m, 2 H), 2.16 (m, 2 H); ESIMS (m/z
18
15
2
4
+
rel intensity) 306 (MH , 100); HRESIMS calcd for C H N O
3
found 323.1024; HPLC purity, 100% (MeOH, 100).
18 16
3
2
+
06.1243 (MH ), found 306.1247; HPLC purity, 96.16% (1% TFA in
(
2′S)-4-Aza-5,6-dihydro-6-(2′,3′-dihydroxypropyl)-5,11-
dioxo-indeno[1,2-c]isoquinoline (51). Lactone 31 (46 mg, 0.18
mmol) was suspended with stirring in CHCl (10 mL) and MeOH
(
MeOH/H O, 90:10).
2
6-(3-Aminopropyl)-2-aza-5,6-dihydro-5,11-dioxo-indeno-
1,2-c]isoquinoline Dihydrochloride (58). Following general
3
[
2.5 mL). A solution of the amine 35 (21 mg, 0.23 mmol) in MeOH
procedure F, 58 (0.052 g, 86%) was obtained as an orange−red
(
0.5 mL) was added to the suspension, and the mixture was stirred at
−1 1
solid: mp 272−274 °C. IR (KBr) 3376, 1698, 1685, 765, 665 cm ; H
room temperature for 2 h and with heating to reflux for 23 h. The
mixture was cooled to room temperature and concentrated in vacuo.
H O (1 mL) was added, and the suspension was filtered to yield a
NMR (DMSO-d , 300 MHz) δ 9.78 (s, 1 H), 8.69 (d, J = 5.4 Hz, 1
6
H), 8.08 (d, J = 5.3 Hz, 1 H), 7.86 (d, J = 7.8 Hz, 1 H), 7.64 (m, 3 H),
4.93 (br s, 3 H), 4.56 (t, J = 7.1 Hz, 2 H), 2.99 (m, 2 H), 2.15 (m, 2
2
solid. Compound 51 (27 mg, 45%) was obtained as a yellow−orange
1
+
solid: mp 247−255 °C. H NMR (300 MHz, DMSO-d ) δ 8.90 (dd, J
H); ESIMS (m/z rel intensity) 306 (MH , 100); HRESIMS calcd for
6
+
=
1
5
8.3, 1.7 Hz, 1 H), 8.78 (dd, J = 4.3, 1.7 Hz, 1 H), 8.09 (d, J = 7.5 Hz,
H), 7.77 (dd, J = 8.3, 4.3 Hz, 1 H), 7.62−7.41 (m, 3 H), 5.14 (d, J =
.0 Hz, 1 H), 5.00 (t, J = 5.6 Hz, 1 H), 4.65−4.43 (m, 2 H), 4.09−3.94
C
18
H
16
N
3
O
2
306.1243 (MH ), found 306.1249; HPLC purity, 99.62%
(1% TFA in MeOH/H O, 90:10).
2
6-(3-Aminopropyl)-3-aza-5,6-dihydro-5,11-dioxo-indeno-
[1,2-c]isoquinoline Dihydrochloride (59). Following general
procedure F, 59 (0.054 g, 89%) was obtained as an orange−red
(
(
m, 1 H), 3.60 (t, J = 5.3 Hz, 2 H); ESIMS m/z (rel intensity) 323
MH , 100); HRESIMS m/z calcd for C H N O 323.1032 (MH ),
+
+
18
15
2
4
−1 1
found 323.1038; HPLC purity, 100% (MeOH, 100).
solid: mp 253−254 °C. IR (KBr) 3410, 1702, 1695, 784, 655 cm ; H
General Procedure E. A solution of N-Boc-1,3-diaminopropane
NMR (DMSO-d , 300 MHz) δ 9.88 (s, 1 H), 8.78 (d, J = 5.6 Hz, 1
6
(
52, 0.068 g, 0.4 mmol) in CHCl3 (10 mL) was added to the
H), 8.18 (d, J = 5.5 Hz, 1 H), 7.94 (d, J = 8.0 Hz, 1 H), 7.74 (m, 3 H),
5.03 (br s, 3 H), 4.68 (t, J = 7.5 Hz, 2 H), 3.05 (m, 2 H), 2.10 (m, 2
H); ESIMS (m/z rel intensity) 306 (MH , 100); HRESIMS calcd for
appropriate lactone (0.050 g, 0.2 mmol) in CHCl (25 mL). The
reaction mixture was heated at reflux for 24 h. After completion of the
3
+
K
DOI: 10.1021/acs.jmedchem.6b00003
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
+
C H N O 306.1243 (MH ), found 306.1244; HPLC purity, 96.81%
18
16
3
2
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b00003.
■
(
1% TFA in MeOH/H O,90:10).
2
*
S
6
-(3-Aminopropyl)-4-aza-5,6-dihydro-5,11-dioxo-indeno-
1,2-c]isoquinoline Dihydrochloride (60). Following general
procedure F, 60 (0.050 g, 83%) was obtained as an orange solid:
mp 284−286 °C. IR (KBr) 3395, 1710, 1698, 765, 666 cm ; H
NMR (D O, 300 MHz) δ 8.31 (s, 1 H), 8.13 (br s, 1 H), 7.28 (m, 3
H), 7.10 (br s, 1 H), 6.87 (br s, 1 H), 4.20 (t, J = 6.8 Hz, 2 H), 3.13 (t,
J = 6.8 Hz, 2 H), 2.12 (m, 2 H); ESIMS (m/z rel intensity) 306 (MH ,
00); HRESIMS calcd for C H N O 306.1243 (MH ), found
06.1245; HPLC purity, 98.75% (1% TFA in MeOH/H O, 80:20).
Topoisomerase I-Mediated DNA Cleavage Reactions. A 3′-
P]-labeled 117-bp DNA oligonucleotide was prepared as previously
described. The oligonucleotide contains previously identified Top1
cleavage sites in 161-bp pBluescript SK(−) phagemid DNA.
Approximately 2 nM radiolabeled DNA substrate was incubated
with recombinant Top1 in 20 μL of reaction buffer [10 mM Tris-HCl
[
−1
1
2
SMILES molecular formula strings (CSV)
PDB file for the structure displayed in Figure 4 (PDB)
+
+
1
3
18
16
3
2
2
AUTHOR INFORMATION
■
*
[32
Corresponding Author
Phone: 765-494-1465. Fax: 765-494-6970. E-mail: cushman@
purdue.edu.
Notes
The authors declare the following competing financial
interest(s): M.C. is on the Board of Directors and is an
investor in Linus Oncology, Inc., which has licensed
indenoisoquinoline intellectual property owned by Purdue
University. Neither Linus Oncology, Inc., nor any other
commercial company sponsored or provided other direct
financial support to the author or his laboratory for the research
reported in this article.
(
pH 7.5), 50 mM KCl, 5 mM MgCl , 0.1 mM EDTA, and 15 μg/mL
2
BSA] at 25 °C for 20 min in the presence of various concentrations of
test 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 mM sodium hydroxide, 1 mM
sodium EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue).
Aliquots of each reaction mixture were subjected to 20% denaturing
PAGE. Gels were dried and visualized by using a phosphoimager and
ImageQuant software (Molecular Dynamics). Cleavage sites are
numbered to reflect actual sites on the 117 bp oligonucleotide.
Recombinant TDP1 Assay. A 5′-[ P]-labeled single-stranded
DNA oligonucleotide containing a 3′-phosphotyrosine (N14Y) was
incubated at 1 nM with 10 pM recombinant TDP1 in the absence or
presence of inhibitor for 15 min at room temperature in the LMP1
assay buffer containing 50 mM Tris HCl, pH 7.5, 80 mM KCl, 2 mM
3
6
ACKNOWLEDGMENTS
■
32
This work was made possible by the National Institutes of
Health (NIH) through support with Research Grants
U01CA089566 and P30CACA023168. This research was also
supported in part by the Intramural Research Program of the
NIH, National Cancer Institute, Center for Cancer Research. In
vitro cytotoxicity testing was performed by the Developmental
Therapeutics Program at the National Cancer Institute, under
contract NO1-CO-56000.
4
6
2
5
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 with multiple loadings at 12 min intervals.
Gels were dried and exposed to a PhosphorImager screen (GE
Healthcare). Gel images were scanned using a Typhoon 8600 (GE
Healthcare), and densitometry analyses were performed using
ImageQuant software (GE Healthcare).
ABBREVIATIONS USED
■
CPT, camptothecin; Top1, topoisomerase IB; TDP1, tyrosyl-
DNA phosphodiesterase 1; TDP2, tyrosyl-DNA phosphodies-
terase 2; CDI, carbonyl diimidazole
Recombinant TDP2 Assay. TDP2 reactions were carried out as
described previously with the following modifications. The 19-mer
47
REFERENCES
single-stranded oligonucleotide DNA substrate containing a 5′-
■
32
phosphotyrosine (Y19, α P-cordycepin-3′-labeled) was incubated at
(1) Stewart, L.; Redinbo, M. R.; Qiu, X.; Hol, W. G. J.; Champoux, J.
J. A Model for the Mechanism of Human Topoisomerase I. Science
998, 279, 1534−1541.
2) Redinbo, M. R.; Stewart, L.; Kuhn, P.; Champoux, J. J.; Hol, W.
G. J. Crystal Structures of Human Topoisomerase I in Covalent and
Noncovalent Complexes with DNA. Science 1998, 279, 1504−1513.
3) Pommier, Y. Topoisomerase I Inhibitors: Camptothecins and
1
nM with 25 pM recombinant human TDP2 in the absence or
1
(
presence of inhibitor for 15 min at room temperature in the LMP2
assay buffer containing 50 mM Tris-HCl, pH 7.5, 80 mM KCl, 5 mM
MgCl , 0.1 mM EDTA, 1 mM DTT, 40 μg/mL BSA, and 0.01%
Tween 20. Reactions were terminated and treated similarly to
recombinant TDP1 reactions (see above).
Modeling Studies. The 1SC7 X-ray crystal structure file was
prepared for molecular modeling as previously described. Selected
indenoisoquinolines were constructed and optimized using the Tripos
force field with default parameters in SYBYL. Ligands were docked
into the prepared crystal structure using GOLD 3.2. The centroid of
the binding site was defined by the crystallized ligand. Ten GOLD
algorithm runs were executed per ligand, and default parameters were
used. The top ten docking poses per ligand were inspected visually
following the docking runs. Energy minimizations were performed for
selected ligands in SYBYL. Ligand SYBYL atom types were inspected
and corrected as necessary prior to minimization. Minimization was
executed by allowing only the ligand to move while freezing the
surrounding crystal structure, which was defined as a static set. The
details of the minimization were set as follows: Powell method;
2
(
Beyond. Nat. Rev. Cancer 2006, 6, 789−802.
4
8
(4) Staker, B. L.; Hjerrild, K.; Feese, M. D.; Behnke, C. A.; Burgin, A.
B.; Stewart, L. The Mechanism of Topoisomerase I Poisoning by a
Camptothecin Analogue. Proc. Natl. Acad. Sci. U. S. A. 2002, 99,
15387−15392.
49
50
(
5) Hsiang, Y. H.; Lihou, M. G.; Liu, L. F. Arrest of Replication Forks
by Drug-Stabilized Topoisomerase I-DNA Cleavable Complexes as a
Mechanism of Cell Killing by Camptothecin. Cancer Res. 1989, 49,
5
(
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