Azaindenoisoquinolines as topoisomerase i inhibitors and potential anticancer agents: A systematic study of structure-activity relationships

Article abstract of DOI:10.1021/jm201512x

A comprehensive study of a series of azaindenoisoquinoline topoisomerase I (Top1) inhibitors is reported. The synthetic pathways have been developed to prepare 7-, 8-, 9-, and 10-azaindenoisoquinolines. The present study shows that 7-azaindenoisoquinolines possess the greatest Top1 inhibitory activity and cytotoxicity. Additionally, the introduction of a methoxy group into the D-ring of 7-azaindenoisoquinolines improved their biological activities, leading to new lead molecules for further development. A series of QM calculations were performed on the model "sandwich" complexes of azaindenoisoquinolines with flanking DNA base pairs from the Drug-Top1-DNA ternary complex. The results of these calculations demonstrate how changes in two forces contributing to the π-π stacking (dispersion and charge-transfer interactions) affect the binding of the drug to the Top1-DNA cleavage complex and thus modulate the drug's Top1 inhibitory activity.

Full text of DOI:10.1021/jm201512x

Article
pubs.acs.org/jmc
Azaindenoisoquinolines as Topoisomerase I Inhibitors and Potential
Anticancer Agents: A Systematic Study of Structure−Activity
Relationships
Evgeny Kiselev,† Keli Agama,‡ 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
ABSTRACT: A comprehensive study of a series of azainde-
noisoquinoline topoisomerase I (Top1) inhibitors is reported.
The synthetic pathways have been developed to prepare 7-, 8-,
9-, and 10-azaindenoisoquinolines. The present study shows
that 7-azaindenoisoquinolines possess the greatest Top1
inhibitory activity and cytotoxicity. Additionally, the introduc-
tion of a methoxy group into the D-ring of 7-azaindenoisoqui-
nolines improved their biological activities, leading to new lead molecules for further development. A series of QM calculations
were performed on the model “sandwich” complexes of azaindenoisoquinolines with flanking DNA base pairs from the Drug−
Top1−DNA ternary complex. The results of these calculations demonstrate how changes in two forces contributing to the π−π
stacking (dispersion and charge-transfer interactions) affect the binding of the drug to the Top1−DNA cleavage complex and
thus modulate the drug’s Top1 inhibitory activity.
thus greater stability of drug−Top1−DNA ternary complexes.
These advantages of indenoisoquinolines triggered their develop-
ment as anticancer agents. The structure optimization of the lead
compound 1 resulted in the identification and promotion of two
members of the family, LMP400 (4) and LMP776 (5), into
phase I clinical study at the NCI (Figure 1).8−10
INTRODUCTION
■
After the discovery of NSC 314622’s (1, Figure 1) cytotoxic
and topoisomerase I (Top1) inhibitory properties, the indeno-
isoquinolines emerged as potential anticancer agents. Following
the demonstration of similarity between the cytotoxicity
profiles of 1 and the plant alkaloid Top1 inhibitor camptothecin
(2) by the National Cancer Institute’s (NCI’s) COMPARE
analysis, it was shown that 1 is also capable of inducing single-
strand DNA breaks in the presence of Top1 at micromolar
concentrations.1 Top1 plays its vital role in cell survival and
replication by unwinding supercoiled DNA, thus allowing its
processing. The major step in Top1-mediated DNA relaxation
is reversible single strand cleavage of DNA by Top1 that results
in the formation of a covalent Top1−DNA cleavage complex
(Top1-DNAcc).2,3 A series of elegant crystallographic studies
of Top1-DNAcc in the presence of inhibitors 2 and MJ-III-65
(3) revealed that by binding between DNA base pairs at the site
of cleavage, molecules like 2 and 3 cause separation of the ends
of the cleaved strand, preventing DNA religation and release of
active enzyme.4,5 Both camptothecins and indenoisoquinolines
have demonstrated similarity in their mode of action by
showing the ability to intercalate their polyaromatic cores
between DNA base pairs and to selectively form hydro-
gen bond interactions with Top1, thereby acting as interfacial
inhibitors (Figure 2).6
Through the synthesis and evaluation of a large number of
analogues of 1, a series of important modifications that
advantageously affect the inhibitory and cytotoxic properties
of the indenoisoquinolines have been identified. It was shown
that replacement of lactam methyl group with ω-aminoalkyl
substituents having two to four carbon atoms (e.g., 4 and 5)
positively affects the biological activities of the drug.8,11,12
Diverse substituents on the A- and D-rings of the core indeno-
isoquinoline molecules have been investigated leading to drugs
with greatly improved cytotoxic properties.11,13 The modifica-
tions of the indenone side of the indenoisoquinolines were
almost exclusively confined to electron-donating alkoxy groups,
and only a very limited number of electron withdrawing
substituents at the ninth position of indenoisoquinoline were
studied.14,15 Recently, the syntheses of 7-azaindenoisoquino-
lines 6 and 7 have been reported.16 The replacement of the
benzene D-ring with a pyridine ring has afforded improved water
solubility. The introduction of the pyridine motif into the
indenoisoquinoline system was in accordance with our hypothesis
that the increase in electron deficiency of the system would
potentially provide an increase in charge-transfer interactions.
In comparison with 2, indenoisoquinolines offer greater
chemical stability. The lactone hydrolysis of 2 at physiological
pH results in loss of its biological activity.7 The indenoisoqui-
noline Top1 inhibitors express DNA cleavage cite specificity
different from that of 2, in addition to slower reversibility and
Received: November 8, 2011
Published: February 13, 2012
© 2012 American Chemical Society
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A series of 8-, 9-, and 10-azaindenoisoquinolines have therefore
been prepared in order to complete the systematic study of
indenoisoquinolines with electron-deficient D-rings.
CHEMISTRY
■
The desired series of 8-, 9-, and 10-azaindenoisoquinolines was
prepared from the corresponding cyanomethylpyridines
(Schemes 1−3), similar to the synthesis of 7-azaindenoisoqui-
nolines from 2-cyano-3-methylpyridine.16,17 Synthesis of 8-
azaindenoisoquinolines was started with preparation of the key
starting material, 4-methylnicotinonitrile (12, Scheme 1).18
a
Scheme 1
Figure 1. Representative Top1 inhibitors.
a
Reagents and conditions: (a) KOH, methanol, reflux, 12 h (75%);
(b) POCl3, sealed tube, 150−180 °C, 8 h (67%); (c) H2, PdCl2,
CH3CO2Na, methanol, 23 °C, 14 h (93%); (d) (1) NBS, AIBN, CCl4,
reflux, 3.5 h, (2) 14, triethylamine, acetonitrile, reflux 14 h (13%); (e)
SeO2, 1,4-dioxane, reflux, 4 h (49%); (f) (1) DIAD, triphenylphos-
phine, 3-dimethylamino-1-propanol (for 17) or 4-(3-hydroxypropyl)-
morpholine (for 18), THF, 23 °C, 3 h, (2) TFA, diethyl ether,
chloroform, 23 °C (17 34%, 18 35%).
Cyanoacetamide (8) was condensed with ethyl acetoacetate (9)
in the presence of potassium hydroxide to afford dihydrox-
ypyridine 10. Conversion of 10 to dichloride 11 by heating
with excess of phosphorus oxychloride in a sealed reaction
vessel followed by hydrogenation in the presence of palladium
dichloride yielded 12. Further synthesis proceeded as pre-
viously described for 7-azaindenoisoquinolines.16 In short,
intermediate bromide 13 obtained by bromination of 12 with
N-bromosuccinimide (NBS) was reacted with 4,5-dimethox-
yhomophthalic anhydride (14)19 to produce 15 (Scheme 1).
Figure 2. Superimposition of ternary complexes of 2 (green) and
3 (blue) with Top1-DNAcc.
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a
Oxidation of 15 with selenium dioxide afforded the key
intermediate dioxoindenoisoquinoline 16. The synthesis of 8-
azaindenoisoquinoline analogues was completed with alkylation
of 16 by means of Mitsunobu reaction to prepare the
dimethylaminopropyl and morpholinopropyl analogues 17
and 18, respectively.
Scheme 3
The 3-methyl-4-cyanopyridine (23) necessary for the
preparation of 9-azaindenoisoquinolines was obtained from
3,4-lutidine (19, Scheme 2). Oxidation of 19 with selenium
a
Scheme 2
a
Reagents and conditions: (a) DMF, reflux, 3 days (31%); (b) POCl3,
reflux, 6 h (54%); (c) HCO2NH4, Pd/C, methanol, 23 °C, 12 h
(81%); (d) (1) NBS, AIBN, 1,2-dichloroethane, reflux, 9 h, (2) 14,
triethylamine, acetonitrile, reflux 2 days (31%); (e) SeO2, 1,4-dioxane,
reflux, 3 days (90%); (f) (1) DIAD, triphenylphosphine, 3-dimethy-
lamino-1-propanol (for 37) or 4-(3-hydroxypropyl)morpholine (for
38), THF, 23 °C, 12 h, (2) TFA, diethyl ether, chloroform, 23 °C (37
22%, 38 7%).
and condensation of the intermediate bromide with 14 yielded
compound 35, which contains the 10-azaindenoisoquinoline
polycyclic core. Compound 36, the product of selenium dioxide
oxidation of 35, was introduced into Mitsunobu reaction to derive
37 and 38.
It is interesting to note that reduction of 32 to 33 required
different reaction conditions than were employed to convert 11
to 12. Instead of catalytic hydrogenation of 32, reduction with
ammonium formate in the presence of palladium on carbon was
required (Scheme 3). When conditions required for the reduction
of 11 were applied to compound 32, the only isolated product was
tetrahydropyridine 39 (Scheme 4).22 In contrast, attempted
a
Reagents and conditions: (a) SeO2, Ph2O, 180 °C, 1 h (47%); (b)
(1) SOCl2, reflux, 3 h, (2) conc aq NH3, 0−5 °C (60%); (c) POCl3,
reflux, 24 h (90%); (d) (1) NBS, AIBN, CCl4, reflux, 2 h, (2) 14,
triethylamine, acetonitrile, reflux 14 h (14%); (e) SeO2, 1,4-dioxane,
reflux, 4 h (94%); (f) (1) DIAD, triphenylphosphine, 3-dimethylami-
no-1-propanol (for 27) or 4-(3-hydroxypropyl)morpholine (for 28),
THF, 23 °C, 3 h, (2) TFA, diethyl ether, chloroform, 23 °C (27 62%,
28 10%).
dioxide in hot diphenyl ether yielded acid 20.20 Treatment of
20 with thionyl chloride gave crude acyl chloride 21 that was
added in small portions to a cold concentrated ammonium
hydroxide solution in order to obtain amide 22. Dehydration of
22 in phosphorus oxychloride produced the key starting
material 23. The crude bromide 24 derived from 23 was
condensed with 14 to prepare 25. Oxidation of 25 to 26 with
selenium dioxide, followed by derivatization through Mitsuno-
bu reaction, yielded the final products 27 and 28 (Scheme 2).
The synthesis of 10-azaindenoisoquinolines began with the
preparation of 3-cyano-2-methylpyridine (33, Scheme 3). Nitrile
33 was obtained from 3-aminocrotonitrile (29) and ethyl
propiolate (30) in three steps. Condensation of 29 and 30
afforded pyridone 31, which was further converted into chloro-
pyridine 32 by treatment with phosphorus oxychloride.21
Compound 32 was reduced to 33 with ammonium formate in
the presence of palladium on charcoal. Benzylic bromination of 33
a
Scheme 4
a
Reagents and conditions: (a) H2, PdCl2, CH3CO2Na, methanol,
23 °C (84%).
reduction of compound 11 with ammonium formate in the
presence of palladium on activated carbon resulted in isolation of
monochloride 40 in high yield, but pyridine 12 was not detected
(Scheme 5). A similar reported reduction produced 40 in less than
10% yield in a mixture with 12 as a major component.23 The
conversion of 11 to 40 reported here might provide a pathway for
preparation of similarly substituted pyridines.
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a
The evaluation of the Top1 inhibitory activity and cytotoxicity
of the azaindenoisoquinolines showed that the introduction of a
methoxy group into the ninth position of 10-azaindenoisoqui-
noline resulted in an increase in Top1 inhibitory activity.14,15
But unfortunately, it did not improve cytotoxicity and 10-
azaindenoisoquinolines 37, 45, and 46 remained largely
nontoxic to cancer cell lines (Table 1). In contrast, the 7-
azaindenoisoquinoline series has demonstrated the greatest
cytotoxicity of all of the currently available azaindenoisoquino-
lines (Table 1).16 In order to investigate the effect of the 9-
methoxy substituent in the 7-azaindenoisoquinoline series,
analogues 55 and 56 were synthesized (Scheme 7).
The key starting material required for the preparation of this
series, 2-cyano-5-methoxy-3-methylpyridine (51), was prepared
in four steps from commercially available 2-amino-3-picoline
(47, Scheme 7). Bromination of 47 with NBS in acetonitrile in
the presence of ammonium acetate yielded 2-amino-5-bromo-
3-methylpyridine (48).24 The amino group of 48 was replaced
with bromide to provide 49, followed by further displacement
with cyanide to yield pyridine 50. For this transformation
sodium nitrite was added to the solution of aminopyridine 48
and bromine in concentrated hydrobromic acid at −15 °C.25−27
Treatment of 49 with 1 equiv of copper(I) cyanide in dry DMF
led to the selective substitution of only the bromide in the
second position of the pyridine, yielding 50.28 The direct
conversion of aminopyridine 48 to nitrile 50 via Sandmeyer-
type reaction is prevented by the instability of the intermediate
2-pyridyldiazonium salt that is apparently capable of decom-
posing at relatively low temperatures prior to its transformation
into nitrile 50 in the presence of copper(I) cyanide.
Nucleophilic substitution of the second bromide with
methoxide provided 51.
Scheme 5
a
Reagents and conditions: (a) HCO2NH4, Pd/C, methanol, 23 °C,
3 days (87%).
It was found in earlier SAR studies of indenoisoquinolines
that the introduction of alkoxy groups in the ninth position
generally benefits the Top1 inhibitory activities and cytotoxic
properties of the target compounds. Having the chloropyridine
32 in hand provided the opportunity to investigate the effect of
the methoxy group on the activity of 10-azaindenoisoquino-
lines.
Luckily, the arrangement of substituents in the pyridine 32 is
such that the displacement of chlorine with a methoxy group
would produce 41, which could be converted to the 9-methoxy
derivatives of 10-azaindenoisoquinolines (Scheme 6). 3-Cyano-6-
methoxy-2-methylpyridine (41) was subjected to radical
bromination with NBS followed by condensation with 14
(Scheme 6). The isolated azaindenoisoquinoline 43 was oxidized
a
Scheme 6
A series of transformations starting from 51 to the targets 55
and 56 were performed in a way that is similar to that described
for other azaindenoisoquinolines (Scheme 7). Bromination of
51 in the presence of radical initiator AIBN, followed by
condensation with 14, produced 53. Oxidation of 53 to 54 and
Mitsunobu reaction of 54 with 3-dimethylamino-1-propanol
and 4-(3-hydroxypropyl)morpholine led to analogues 55 and
56, respectively.
In order to further explore the potential of the 7-aza-9-
methoxyindenoisoquinoline series as anticancer agents, com-
pounds 63−66 were prepared (Scheme 8). These analogues
contain a differently substituted indenoisoquinoline A-ring
compared to compounds 55 and 56. Pyridine 51 was utilized in
the preparation of 63−66 (Scheme 8). Bromination of 51
followed by condensation with homophthalic and 5-nitro-
homophthalic anhydrides29 (57 and 58) yielded azaindenoiso-
quinolines 59 and 60, respectively. Compounds 59 and 60
underwent oxidation to 61 and 62, respectively, followed by
Mitsunobu transformation to yield 63−66.
BIOLOGICAL RESULTS AND DISCUSSION
■
a
Reagents and conditions: (a) NaOCH3, methanol, reflux, 1.5 h
All of the target compounds were tested for induction of DNA
damage in Top1-mediated DNA cleavage assays.30 For this
purpose, a 32P 3′-end-labeled 117-bp DNA fragment was
incubated with human recombinant Top1 and increasing
concentrations of the test compounds. The DNA fragments
were separated on a denaturing gel (Figure 3). The Top1
inhibitory activity was assigned based on the visual inspection
of the number and intensities of the DNA cleavage bands and
expressed in semiquantitative fashion relative to the Top1
inhibitory activities of compounds 1 and 2: 0, no detectable
(87%); (b) (1) NBS, AIBN, 1,2-dichloroethane, reflux, 3.5 h, (2) 14,
triethylamine, acetonitrile, reflux 14 h (19%); (c) SeO2, 1,4-dioxane,
reflux, 24 h (89%); (d) DIAD, triphenylphosphine, 3-dimethylamino-
1-propanol (for 45) or 4-(3-hydroxypropyl)morpholine (for 46),
THF, 23 °C, 3 days, (2) TFA, diethyl ether, chloroform, 23 °C (45
30%, 46 31%).
with selenium dioxide, and the product 44 was reacted with 3-
dimethylamino-1-propanol and 4-(3-hydroxypropyl)morpholine
under Mitsunobu conditions to prepare 45 and 46, respectively.
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Table 1. Top1 Inhibitory and Antiproliferative Activity of Azaindenoisoquinolines
c
cytotoxicity (GI50, μM)
Top1
lung,
colon,
CNS,
melanoma,
UACC-62
ovarian,
renal,
prostate,
DU-145
breast,
MCF7
a
b
compd
cleavage
MGM
8.5
HOP-62
HCT-116
SF-539
OVCAR-3
SN12C
1
++
2.8
11.5
0.030
1.6
1.7
0.56
0.010
13
22
26
4.8
1.9
2
++++
+++
++
0.040
4.5
0.010
3.4
0.010
4.1
0.22
3.6
0.020
3.2
0.010
1.7
0.013
0.44
0.052
6
7
0.30
0.30
0.22
0.29
0.10
0.37
0.52
0.31
d
d
d
d
d
d
d
d
d
17
18
27
28
37
38
45
46
55
56
63
64
65
66
++
ND
ND
ND
ND
ND
ND
ND
ND
ND
++
16
6.5
6.5
5.0
2.5
40
48
24
7.4
12
16
24
4.1
3.9
1.9
+
6.5
9.5
0.62
0.39
12
>100
85
8.5
5.9
2.9
7.6
++
>100
d
d
d
d
d
d
d
d
d
++
ND
ND
ND
ND
ND
ND
ND
ND
ND
e
e
e
e
e
e
e
e
e
e
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
d
d
d
d
d
d
d
d
d
+++
+++
+++
++
ND
ND
ND
ND
ND
ND
ND
ND
ND
d
d
d
d
d
d
d
d
d
ND
ND
ND
ND
ND
ND
ND
ND
ND
1.8
0.92
0.24
0.30
4.1
1.5
1.1
3.9
2.9
3.6
0.88
0.34
0.30
3.6
0.13
0.10
0.21
0.60
0.024
0.020
0.48
0.40
3.0
0.33
0.34
2.5
0.27
0.57
3.5
0.22
0.54
1.7
0.31
0.94
6.3
0.34
0.26
2.6
+++
++
+++
++++
0.11
0.085
0.054
0.051
0.074
0.050
0.078
0.035
0.052
0.040
0.14
0.11
0.057
0.043
0.051
0.040
a
The relative Top1 inhibitory potencies of the compounds are presented as follows: 0: no detectable activity; +: weak activity; ++: similar activity as
b
compound 1; +++ and ++++: greater activity than compound 1; ++++: similar activity as 1 μM 2. Mean graph midpoint (MGM) for growth
inhibition of all human cancer cell lines successfully tested. The cytotoxicity GI50 values listed are the concentrations corresponding to 50% growth
inhibition and are the result of single determinations. GI50 values were not determined because the low activities revealed in the initial single-
concentration testing at 10 μM did not warrant the multiple-concentration testing required for determination of GI50 values. Not tested.
c
d
e
a
activity; +, weak activity; ++, similar activity to compound 1;
+++, greater activity than 1; ++++, equipotent to 2 (Table 1).
The antiproliferative activity of each compound was
determined in the National Cancer Institute (NCI) screen.31,32
Cells of approximately 60 different human cancer cell lines
were incubated for 48 h with five 10-fold dilutions of the test
compounds starting from 100 μM and then treated with
sulforhodamine B dye. The ratios of recorded optical densities
relative to those of the control were plotted as a function of the
common logarithm of the tested compound concentrations.
The interpolation between the points located above and below
the 50% percentage growth provided 50% growth inhibition
(GI50) values. The GI50 and the mean graph midpoint (MGM)
values of the prepared indenoisoquinolines in selected cell lines
are presented in Table 1. The MGM is based on a calculation of
the average GI50 for all of the cell lines tested in which GI50
values above and below the tested range (10−4−10−8 M) are
taken as the maximum (10−4 M) and minimum (10−8 M) drug
concentrations used in the screening test. The Top1 inhibitory
and cytotoxicity (MGM and GI50) data for lead compound
1,1,33 compound 2, and previously reported 7-azaindenoisoqui-
nolines 6 and 716 are included in Table 1 for comparison
purposes.
Scheme 7
The results of the biological evaluations presented in Table 1
clearly show that 7-azaindenoisoquinolines 6 and 7 are more
active than their corresponding isomers, 8-, 9-, and 10-azainde-
noisoquinolines, 17, 18, 27, 28, and 37. The Top1 inhibitory
activities of 6 and 7 are at the +++/++ level, and their
cytotoxicities are in the low micromolar concentration range.
Top1 inhibition is observed at only the ++/+ level for 8-, 9-, and
10-azaindenoisoquinolines. The MGM of compounds 18, 27,
and 28 increased 2−50 times relative to the MGM of 6 and 7.
The 8-azaindenoisoquinoline 17 and 10-azaindenoisoquinoline
37 were not cytotoxic enough for GI50 and MGM values to be
a
Reagents and conditions: (a) NBS, CH3CO2NH4, acetonitrile, 0−
23 °C, 25 min (65%); (b) Br2, NaNO2, aq HBr, −15 °C, then 23 °C,
3 h (94%); (c) CuCN, DMF, reflux, 2 h (74%); (d) NaOCH3,
methanol, reflux, 12 h (79%); (e) (1) NBS, AIBN, 1,2-dichloroethane,
reflux, 24 h, (2) 14, triethylamine, acetonitrile, reflux 24 h (21%); (f)
SeO2, 1,4-dioxane, reflux, 24 h (92%); (g) (1) DIAD, triphenylphos-
phine, 3-dimethylamino-1-propanol (for 55) or 4-(3-hydroxypropyl)-
morpholine (for 56), THF, 23 °C, 2 days, (2) HCl, methanol, chloro-
form, 23 °C (55 52%, 56 59%).
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a
7-aza-9-methoxyindenoisoquinoline 55, a nearly 3-fold im-
provement in cytotoxicity was observed relative to 7-
azaindenoisoquinoline 6 (Table 1).
Scheme 8
Having achieved the desired effect, 7-aza-9-methoxyindenoi-
soquinolines 63−66 were prepared that bear different sub-
stituents on the A-ring (Scheme 8). An improvement in cyto-
toxicity was observed for the dimethylaminopropyl analogues
63 and 65, which had MGM values of 0.40 and 0.11 μM. No
loss of Top1 inhibitory activity was observed for 63 and 65 in
comparison with the closely related compounds 6 and 55. In
the case of compound 66, there was a surprising improvement
in Top1 inhibitory activity, up to ++++, making it equipotent
with 2. The MGM value obtained for compound 66 was
85 nM, making it the most active compound to date in the
entire azaindenoisoquinoline series.
The cleavage band intensities displayed in Figure 3 indicate
that the azaindenoisoquinolines offer different DNA cleavage
site specificity in comparison to 2. Most notably, the bands at
positions 44, 62, and 106 become more prominent whereas
bands at positions 32 and 97 are weaker than in the case of 2.
The cleavage site specificity of the azaindenoisoquinoline series
is consistent with that previously observed for 1, 4, and 5.1,10 By
trapping Top1-DNA cleavage complexes having different DNA
cleavage sites than the camptothecins, the indenoisoquinolines
and azaindenoisoquinolines target the genome differently than
the camptothecins. This suggests that the cancer treatment
profiles of the indenoisoquinolines might be different from the
camptothecins.
In an attempt to understand the effect of introduction of
nitrogen into various positions of the indenoisoquinoline
D-ring on the π−π stacking interactions, a series of hypothetical
azaindenoisoquinoline structures were modeled and subjected
to quantum mechanical single point energy calculations. It was
shown earlier that π−π stacking interactions of indenoisoquino-
lines with the flanking base pairs are responsible for their
orientation in the ternary complex and their DNA cleavage site
specificities. The results of these calculations indicate that π−π
stacking interactions are the major complex stabilizing
force.34−36
a
Reagents and conditions: (a) (1) NBS, AIBN, 1,2-dichloroethane,
reflux, 24 h, (2) homophthalic anhydride (57, for 59) or 5-
nitrohomophthalic anhydride (58, for 60), triethylamine, acetonitrile,
reflux 24 h (59 46%, 60 26%); (b) SeO2, 1,4-dioxane, reflux, 24 h (61
76%, 62 86%); (c) DIAD, triphenylphosphine, 3-dimethylamino-1-
propanol (for 63 and 65) or 4-(3-hydroxypropyl)morpholine (for 64
and 66), THF, 23 °C, 2 days (63 38%, 64 40%, 65 61%, 66 47%).
The model for these calculations was derived from the X-ray
crystal structure of the 3−Top1−DNA ternary complex (PDB
entry 1SC7).5 The deoxyribose rings of the flanking base pairs
and the lactam side chain of 3 were replaced with methyl
groups because they are considered to be nonparticipating
groups in π−π stacking interactions.35 The geometry
optimizations and frequency calculations at the HF/6-31G**
level were performed on the indenoisoquinoline molecule, as
well as A−T and G−C base pairs, using the Gaussian 09
software package.37 The original complex was then replaced
with the geometry optimized parts (Figure 4 and Figure 5,
model A). The CH groups in positions 7−10 of the D-ring of
the inhibitor were replaced with nitrogen to produce 7-, 8-, 9-,
and 10-azaindenoisoquinoline models (Figure 5, models B−F).
Geometry optimizations were performed on these molecules
before fitting them into their corresponding complexes with the
DNA base pairs.
After the complexes were assembled, MP2/6-31G* single
point energy calculations were performed. The basis set
superposition error (BSSE) for each complex was calculated
using the counterpoise correction method within the Gaussian
09 package, specifying two fragments: the first being the two
flanking DNA base pairs and the second being the intercalating
indenoisoquinoline inhibitor. The π−π staking interaction
determined in the NCI testing concentration range. These
results indicate that the introduction of a nitrogen atom in posi-
tions other than 7 of the indenoisoquinoline polycyclic system
results in compounds with diminished activity.
The synthetic protocol developed for the preparation of the
10-azaindenoisoquinolines 37 and 38 was modified, allowing
for an addition of the methoxy group to position 9, which
resulted in the preparation of compounds 45 and 46 (Schemes
3 and 6). It was previously noted that introduction of a
methoxy group into position 9 of the indenoisoquinolines
positively affects the inhibitory and cytotoxic activities of the
target compounds.14,15 10-Aza-9-methoxyindenoisoquinolines
45 and 46 (Scheme 6) were ranked +++ in the Top1-mediated
DNA cleavage assay, showing an improvement in comparison
to the corresponding analogue 37 that lacks a 9-methoxy group
(Top1: ++, Table 1). Unfortunately, the 10-azaindenoisoquino-
lines were largely noncytotoxic in the tested cancer cell lines.
The 7-aza-9-methoxyindenoisoquinoline series was prepared
(Scheme 7) in view of the positive Top1 inhibition results
observed on the introduction of a 9-methoxy group in the 10-
aza series. In the case of 7-azaindenoisoquinolines, there was no
apparent increase in the Top1 inhibitory activity of compounds
55 and 56 over 6 and 7, respectively. However, in the case of
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Figure 3. Top1-mediated DNA cleavage induced by azaindenoisoquinolines. Lanes 1, 29: DNA alone. Lanes 2, 30: Top1 alone. Lanes 3, 31: Top1 +
2 (1 μM). Lane 4: Top1 + 1 (100 μM). Lanes 5−8: Top1 + 6 at 0.1, 1, 10, 100 μM. Lanes 9−12: Top1 + 17 at 0.1, 1, 10, 100 μM. Lanes 13−16:
Top1 + 27 at 0.1, 1, 10, 100 μM. Lanes 17−20: Top1 + 37 at 0.1, 1, 10, 100 μM. Lanes 21−24: Top1 + 45 at 0.1, 1, 10, 100 μM. Lanes 25−28:
Top1 + 55 at 0.1, 1, 10, 100 μM. Lanes 32−35: Top1 + 65 at 0.1, 1, 10, 100 μM. Lanes 36−39: Top1 + 66 at 0.1, 1, 10, 100 μM. Numbers on right
and arrows show the cleavage site positions.
Figure 4. Model of the complex of the inhibitor with DNA base pairs.
energy was calculated as Eint = Ecomplex − Eligand − Ebp + BSSE,
where Ecomplex, Eligand, and Ebp are the corresponding MP2/6-
31G* calculated energies of complex, ligand, and DNA base
pairs at their normal distance in the absence of intercalator
(Table 2). The polarizable continuum model (PCM) was used
at the MP2/6-31G* level of theory to estimate the effect of
solvation on stabilization of the complex (Eint,aq, Table 2). The
dispersion energy Ecorr was calculated as Ecorr = Eint(MP2) −
Eint(HF). The energy associated with charge transfer was
obtained as result of natural bond orbital (NBO) analysis in
Gaussian 09.38 The NBO analysis was performed at the HF/6-
31G** level of theory and presented as ECT in Table 2. The
Figure 5. Modeled indenoisoquinoline−DNA complexes.
calculated transfer of 0.01e can be equated to 1 kcal/mol of
complex stabilization energy.39
stabilization resulting from π−π stacking. Analysis of the ECT
column on the other hand suggests that as expected the charge-
transfer stabilization energy is greater by 1.08 kcal/mol for
7-azindenoisoquinoline represented by model B, in comparison
to indenoisoquinoline in model A. Table 2 indicates that the
dispersion interaction is decreased significantly for models C−E
but not B (relative to A). This decrease in dispersion energy
contributes to the decrease of Top1 inhibitory activity and
Although Ecorr and ECT could not be summed directly to
obtain the whole or at least a part of Eint, they could be used to
evaluate the effect of various modifications on particular forces
responsible for stabilization of drug−Top1−DNA ternary com-
plexes. For example, comparison of Ecorr for “classical” indenoi-
soquinoline (model A) and 7-azaindenoisoquinoline (model B)
shows that introduction of nitrogen into the seventh position
did not decrease the dispersion energy contribution to the
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Top1-DNAcc might explain the increase of Top1 inhibitory
and antiproliferative potency of 66 in comparison to other
azaindenoisoquinolines such as 7 and 56.
Table 2. MP2 Calculated Energies for Models A−F
a
b
c
Eint
Eint,aq
Ecorr
ECT
model
Eint (au)
(kcal/mol) (kcal/mol) (kcal/mol) (kcal/mol)
In conclusion, a series of 7-, 8-, 9-, and 10-azaindenoisoqui-
nolines have been prepared. The established synthetic protocols
allow the preparation of these compounds from easily affordable
starting materials. Azaindenoisoquinolines have been evaluated
for their ability to stabilize the Top1-DNAcc and thus inhibit
Top1. The ab initio calculations show that the values of the com-
ponents of the π−π stacking interaction energy, i.e., dispersion
forces (Ecorr) and charge-transfer interactions (ECT), significantly
depend on the position of the nitrogen atom. The 7-azainden-
oisoquinoline system allows for an increase of charge transfer
interaction without compromising the level of dispersion interac-
tions. Previous studies have also revealed that incorporation of
nitrogen into position 7 of indenoisoquinolines allows for improve-
ment of water solubility without compromising the Top1 inhibitory
activities and cytotoxicities of the resulting 7-azaindenoisoquino-
lines.16 Further development of the 7-azaindenoisoquinolines
presented in this study led to the discovery of 7-aza-9-methoxy-3-
nitroindenoisoquinoline 66, which demonstrated high activity in the
Top1-mediated DNA cleavage assay, ++++, and in cytotoxicity
screening at the NCI, with an MGM of 85 nM. Further amino-
propyl chain modifications and optimization8,13 could be done in
order to further improve the Top1 inhibitory and cytotoxic
performance of compounds such as 65 and 66.
A
B
C
D
E
F
−0.01108
−0.01273
−0.01452
−0.01663
−0.01459
−0.01305
−6.95
−7.99
−9.11
−10.43
−9.16
−8.19
−15.17
−15.57
−16.20
−17.01
−18.59
−17.03
−26.03
−25.79
−15.11
−14.73
−14.27
−40.04
−11.50
−12.58
−13.57
−11.99
−10.05
−10.91
a
Eint is derived from MP2/6-31G* single point energy calculations.
Ecorr = Eint(MP2/6‑31G*) − Eint(HF/6‑31G*). The magnitude of the charge
transfer as estimated by NBO analysis at the HF/6-31G** level.
b
c
cytotoxicity. Even in the case of 8-azaindenosioquinolines
(model C), despite the increase of the absolute magnitude of
ECT to almost 13.6 kcal/mol, the decrease in dispersion
interactions was significant enough to decrease Top1 inhibitory
activity. Comparison of the experimental results of Top1-mediated
DNA cleavage for compounds 37 and 45 and calculated ECT and
Ecorr values for models E and F demonstrates the importance of
the methoxy group in position 9 of the indenoisoquinolines
(Tables 1 and 2). This methoxy substitution drastically increases
dispersion (Ecorr) interaction energy while maintaining charge-
transfer (ECT) interactions. The increase in the dispersion inter-
action of 9-methoxy analogues could be attributed to the electron
donating properties of the methoxy group and increase in electron
delocalization in comparison to the analogues lacking this group.
The increase of the Top1 inhibitory activity that corresponds to
the addition of a methoxy group is observed in the case of 10-aza-
and 10-aza-9-methoxyindenoisoquinolines, 37 and 45, respectively.
These findings were extrapolated to 7-azaindenoisoquinolines. The
resulting 7-aza-9-methoxyindenoisoquinolines 55, 56, and 63−66
demonstrated improvement in their biological properties relative to
those lacking the methoxy group in position 9.
EXPERIMENTAL SECTION
■
General. Melting points were determined using capillary tubes with
a Mel-Temp apparatus and are uncorrected. The nuclear magnetic
resonance spectra (1H and 13C NMR) were recorded using ARX300
300 MHz and DRX500 500 MHz Bruker NMR spectrometers. IR
spectra were recorded using a Perkin-Elmer 1600 series FTIR spectro-
meter. Purities of all tested compounds were ≥95%, as established by
combustion and/or estimated HPLC analysis. Combustion micro-
analyses were performed at the Purdue University Microanalysis
Laboratory or Galbraith Laboratories Inc., and the reported values
were within 0.4% of the calculated values. HPLC analyses were
performed on a Waters 1525 binary HPLC pump/Waters 2487 dual λ
absorbance detector system. For purities estimated by HPLC, the
major peak accounted for ≥95% of the combined total peak area when
monitored by a UV detector at 254 nm. Analytical thin-layer
chromatography was carried out on Baker-flex silica gel IB2-F plates,
and compounds were visualized with UV light at 254 nm. Silica gel
flash chromatography was performed using 230−400 mesh silica gel.
3-Cyano-2,6-dihydroxy-4-methylpyridine (10).18 Cyanoaceta-
mide (8, 34 g, 0.40 mol) and ethyl acetoacetate (9, 52 g, 0.40 mol)
were dissolved in methanol (250 mL) at room temperature. A solution
of potassium hydroxide (28 g, 0.42 mol) in methanol (200 mL) was
slowly added, and the resulting mixture was heated to reflux for 12 h.
The reaction mixture was cooled to room temperature, and the white
amorphous precipitate was filtered and washed with methanol (2 × 50
mL). The solid product was redissolved in hot water. The solution was
carefully acidified, and the off-white precipitate was allowed to form.
The precipitate was filtered and washed with water and methanol to
yield 10 (46 g, 75%): mp >300 °C (dec) [lit.18 mp 315−320 °C
(dec)]. 1H NMR (300 MHz, DMSO-d6) δ 5.58 (s, 1 H), 2.22 (s, 3 H).
3-Cyano-2,6-dichloro-4-methylpyridine (11).18 3-Cyano-2,6-
dihydroxy-4-methylpyridine (10, 10 g, 0.07 mol) and phosphorus
oxychloride (25 mL, 0.27 mol) were sealed in a heavy-walled tube, and
the mixture was heated to 150−180 °C in an oil bath for 8 h. The
resulting mixture was allowed to cool to room temperature and carefully
quenched by pouring it into ice (200 g). The light brown precipitate was
filtered, washed with water, and dried to yield 11 (8.3 g, 67%): mp
114−118 °C (lit.18 mp 109−110 °C). 1H NMR (300 MHz, DMSO-d6)
δ 6.77 (s, 1 H), 1.49 (s, 3 H).
It was previously hypothesized that a nitro group in the third
position of the indenoisoquinoline system is capable of forming
a direct hydrogen bond with Asn722.14 Overlaying the structure
of 66 with 3 in its ternary complex (PDB entry 1SC7)5 revealed
that this interaction would be possible in addition to the
contact with Arg364 formed by the carbonyl oxygen of the
indenone moiety (Figure 6). Addition of an extra hydro-
gen bond to the network of contacts of the drug with the
Figure 6. Hypothetical binding mode of 66 in complex with Top1-
DNAcc. Hydrogen bonds (dashed lines) are represented as distances
between corresponding heavy atoms.
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3-Cyano-4-methylpyridine (12).18 Palladium dichloride (50 mg,
0.3 mmol) was added to a degassed solution of 11 (5.0 g, 27 mmol)
and sodium acetate (4.5 g, 55 mmol) in methanol (100 mL). The
resulting mixture was stirred under hydrogen (1 atm) for 14 h at room
temperature. The precipitate was filtered and washed with methanol
(3 × 20 mL). The combined filtrates were evaporated under reduced
pressure, and chloroform (50 mL) was added to the residue. The
chloroform solution was filtered through a thin pad of silica gel,
washing with additional portions of chloroform. The filtrate was
THF (10 mL). Diisopropyl azodicarboxylate (0.13 mL, 0.65 mmol) was
added to the THF solution, and the resulting mixture was stirred at room
temperature for 3 h. The reaction mixture was then evaporated to
dryness under reduced pressure. The residue was purified by flash
column chromatography (silica gel), eluting with 10% methanol in
chloroform, followed by preparative TLC (silica gel), eluting with 5%
methanol in chloroform, to provide an orange solid. The solid was
redissolved in chloroform (5 mL), and trifluoroacetic acid (2 M in
diethyl ether, 1 mL) was added. The precipitate was collected by
filtration and washed with ether (2 × 2 mL) to yield the product in the
form of its trifluoroacetate salt (62 mg, 35%): mp 213−214 °C. IR (KBr)
1778, 1753, 1679, 1614 cm−1. 1H NMR (300 MHz, DMSO-d6) δ 10.15
(s, 1 H), 8.83−8.58 (m, 2 H), 7.69 (s, 1 H), 7.44 (d, J = 4.5 Hz, 1 H),
7.31 (s, 1 H), 4.69 (t, J = 5.8 Hz, 2 H), 4.02 (d, J = 12.3 Hz, 2 H), 3.92
(s, 3 H), 3.90 (s, 3 H), 3.67 (t, J = 11.5 Hz, 2 H), 3.54 (d, J = 12.1 Hz,
2 H), 3.40 (s, 2 H), 3.16 (s, 2 H), 2.32 (s, 2 H). Positive ion ESIMS m/z
(rel intensity): 436 (MH+, 100). HRMS-ESI m/z: MH+ calcd for
C24H25N3O5, 436.1872; found, 436.1769. HPLC purity: 97.05% [C-18
reverse phase, MeOH (1% CF3COOH)/H2O, 90:10]; 97.36% [C-18
reverse phase, MeOH (1% CF3COOH)/H2O, 70:30].
1
evaporated to dryness to provide 12 (2.9 g, 93%) as a yellow oil. H
NMR (300 MHz, DMSO-d6) δ 8.77 (s, 1 H), 8.63 (s, J = 6.0 Hz, 1 H),
7.3 (d, J = 6.0 Hz, 1 H), 2.56 (s, 3 H). 3-Cyano-4-methylpyridine (12)
was used further without additional purification.
8-Aza-5,6-dihydro-2,3-dimethoxy-5-oxo-11H-indeno[1,2-c]-
isoquinoline (15). 3-Cyano-4-methylpyridine (12, 1.5 g, 13 mmol),
NBS (3.3 g, 19 mmol), and AIBN (100 mg, 0.6 mmol) were diluted
with carbon tetrachloride (60 mL), and the mixture was heated at
reflux for 3.5 h. The reaction mixture was concentrated to one-half its
original volume, filtered, and the filtrate was evaporated to dryness
under reduced pressure. The residue was diluted with acetonitrile
(60 mL). 4,5-Dimethoxyhomophthalic anhydride (14, 5.6 g, 25 mmol)
was added, followed by triethylamine (3.5 mL, 25 mmol), and the
solution was heated at reflux for 14 h. The solution was allowed to
cool to room temperature and the precipitate was filtered and washed
with acetonitrile (2 × 15 mL) to provide a gray solid (500 mg, 13%):
mp 270−272 °C. IR (KBr) 1633, 1611, 1593 cm−1; 1H NMR
(300 MHz, CDCl3) δ 12.31 (s, 1 H), 9.08 (s, 1 H), 8.48 (d, J = 6.0 Hz,
1 H), 7.61 (m, 2 H), 7.13 (s, 1 H) 3.93 (s, 5 H), 3.86 (s, 3 H). Positive
ESIMS m/z (rel intensity): 295 (MH+, 100).
3-Methyl-4-nicotinic Acid (20).20 A solution of 3,4-lutidine (19,
30 g, 0.28 mol) in diphenyl ether (150 mL) was heated to 150−
170 °C, and selenium dioxide (62 g, 0.56 mol) was carefully added to
the hot solution in small portions in the course of 1 h. The resulting
mixture was heated to 180 °C for 1 h. The reaction mixture was
filtered while hot, and the collected precipitate was washed with
boiling water (3 × 300 mL). The combined filtrates were
extracted with chloroform (3 × 300 mL). The aqueous phase
was evaporated to dryness and the remaining product was
recrystallized from ethanol (450 mL) to obtain pure acid 20
8-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno-
[1,2-c]isoquinoline (16). 8-Aza-5,6-dihydro-2,3-dimethoxy-5-oxo-
11H-indeno[1,2-c]isoquinoline (15, 250 mg, 0.85 mmol) and SeO2
(190 mg, 1.7 mmol) were diluted with 1,4-dioxane (20 mL), and the
mixture was heated at reflux for 4 h. The reaction mixture was filtered
while hot, and the precipitate was washed with hot dioxane (3 ×
10 mL). The combined filtrates were evaporated to dryness under reduced
pressure. The solid residue was purified by flash column chromatography
(silica gel), eluting with 5% methanol in chloroform, to obtain 16 (130 mg,
(18 g, 47%): mp 220−222 °C (lit.40 mp 232 °C). H NMR (300
1
MHz, DMSO-d6) δ 8.59 (s, 1 H), 8.04 (s, 1 H), 8.47 (d, J = 4.8,
1 H), 7.69 (d, J = 4.8, 1 H), 2.48 (s, 3 H).
3-Methyl-4-nicotinamide (22). A solution of 3-methyl-4-
nicotinic acid (20, 5.0 g, 37 mmol) in thionyl chloride (20 mL, 0.28
mol) was heated at reflux for 3 h. The thionyl chloride was evaporated.
The solid acid chloride 21 was added in small portions to a
concentrated ammonium hydroxide solution (300 mL) while cooling
the reaction mixture to 0−5 °C. The reaction mixture was saturated
with potassium carbonate, and the solution was extracted with
chloroform (2 × 150 mL) and ethyl acetate (2 × 150 mL). The
aqueous phase was evaporated to dryness, and the resulting solid was
extracted with hot ethyl acetate (3 × 150 mL). The combined extracts
were evaporated to dryness to yield crude 22 (3.0 g, 60%): mp 140−
142 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.47 (s, 1 H), 8.44 (d, J =
4.9 Hz, 1 H), 7.94 (s, 1 H), 7.65 (s, 1 H), 7.29 (d, J = 4.9 Hz, 1 H),
2.32 (s, 3 H).
−1
49%): mp 300−302 °C. IR (KBr) 1708, 1648, 1611, 1579 cm . 1H NMR
(300 MHz, DMSO-d6) δ 12.96 (s, 1 H), 8.56 (d, J = 6.2 and 1.4 Hz, 1 H),
7.86−7.81 (m, 2 H), 7.59 (s, 1 H), 7.40 (t, J = 6.8 Hz, 1 H). Positive
ESIMS m/z (rel intensity): 309 (MH+, 100).
8-Aza-5,6-dihydro-6-(3-dimethylaminopropyl)-2,3-dime-
thoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline Trifluoroace-
tate (17). 8-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno[1,2-c]-
isoquinoline (16, 92 mg, 0.30 mmol), 3-dimethylamino-1-propanol
(0.1 mL, 0.9 mmol), and PPh3 (240 mg, 0.92 mmol) were diluted with
THF (15 mL). Diisopropyl azodicarboxylate (0.18 mL, 0.92 mmol) was
added to the THF solution, and the resulting mixture was stirred at
room temperature for 3 h. The reaction mixture was then evaporated to
dryness under reduced pressure. The residue was purified by flash
column chromatography (silica gel), eluting with 10% methanol in
chloroform, followed by preparative TLC (silica gel), eluting with 5%
methanol in chloroform, to provide an orange solid. The solid was
redissolved in chloroform (5 mL), and trifluoroacetic acid (2 M in
diethyl ether, 1 mL) was added. The precipitate was collected by
filtration and washed with ether (2 × 2 mL) to yield the product in the
form of its trifluoroacetate salt (53 mg, 34%): mp 230−232 °C (dec). IR
3-Methyl-4-cyanopyridine (23). Phosphorus oxychloride (100
mL, 1.1 mol) was slowly added to the crude amide 22 (15 g, 0.11 mol)
while cooling the mixture in an ice bath. The resulting solution was
heated at reflux for 24 h. The reaction mixture was cooled to room
temperature, and the excess phosphorus oxychloride was removed
under reduced pressure. Crushed ice (150 g) was slowly added to the
oily residue, and the solution was neutralized with saturated
ammonium hydroxide. The crude product was extracted with
chloroform (3 × 100 mL). The combined extracts were filtered
through a layer of silica gel, washing with extra portions of chloroform.
The filtrates were evaporated to dryness to yield 23 as colorless
crystals (12 g, 90%): mp 45−47 °C (lit.41 mp 50 °C). H NMR (300
1
1
(KBr) 1690, 1612 cm−1. H NMR (300 MHz, CD3OD) δ 8.68−8.60
MHz, CDCl3) δ 8.66 (s, 1 H), 8.59 (d, J = 5.0 Hz, 1 H), 7.45 (d, J =
5.0 Hz, 1 H), 2.54 (s, 3 H).
(m, 2 H), 7.73 (d, J = 5.5 Hz, 1 H), 7.69 (s, 1 H), 7.26 (s, 1 H), 4.57 (t,
J = 6.1 Hz, 2 H), 3.76 (s, 3 H), 3.70 (s, 3 H), 3.24−3.08 (m, 2 H), 2.69
(s, 6 H), 2.24−2.06 (m, 2 H). Positive ion ESIMS m/z (rel intensity):
394 (MH+, 100). HRMS-ESI m/z: MH+ calcd for C22H23N3O4,
394.1767; found, 394.1769. HPLC purity: 97.44% [C-18 reverse phase,
MeOH (1% CF3COOH)/H2O, 80:20].
9-Aza-5,6-dihydro-2,3-dimethoxy-5-oxo-11H-indeno[1,2-c]-
isoquinoline (25). 3-Methylisonicotinonitrile (23, 590 mg, 5.0
mmol), NBS (1.2 g, 7.0 mmol), and AIBN (50 mg, 0.3 mmol) were
diluted with carbon tetrachloride (20 mL), and the mixture was heated
at reflux for 2 h. The reaction mixture was concentrated to one-half its
original volume, filtered, and the filtrate was evaporated to dryness
under reduced pressure. The residue was diluted with acetonitrile
(25 mL), and 14 (2.2 g, 10 mmol) was added, followed by triethyla-
mine (5 mL, 36 mmol), and the solution was heated at reflux for 14 h.
8-Aza-5,6-dihydro-6-(3-(4-morpholino)propyl)-2,3-dime-
thoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline Trifluoroace-
tate (18). 8-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno-
[1,2-c]isoquinoline (16, 100 mg, 0.32 mmol), 4-(3-hydroxypropyl)morpholine
(94.3 mg, 0.65 mmol), and PPh3 (170 mg, 0.65 mmol) were diluted with
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The solution was allowed to cool to room temperature, and the
precipitate was filtered and washed with acetonitrile (50 mL) to
provide a light-brown solid (200 mg, 14%): mp 306−308 °C. IR
(KBr) 1639, 1613, 1592 cm−1. 1H NMR (300 MHz, DMSO-d6)
δ 12.32 (s, 1 H), 8.73 (s, 1 H), 8.54 (d, J = 6.0 Hz, 1 H), 7.87 (d, J =
6.0 Hz, 1 H), 7.65 (s, 1 H), 7.23 (s, 1 H), 3.96 (s, 3 H), 3.88 (s, 3 H),
3.37 (s, 2 H). Positive ESIMS m/z (rel intensity): 295 (MH+, 100).
9-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno-
[1,2-c]isoquinoline (26). 9-Aza-5,6-dihydro-2,3-dimethoxy-5-oxo-
11H-indeno[1,2-c]isoquinoline (25, 100 mg, 0.34 mmol) and SeO2
(75 mg, 0.68 mmol) were diluted with 1,4-dioxane (10 mL), and the
mixture was heated at reflux for 4 h. The reaction mixture was filtered
while hot, and the precipitate was washed with hot dioxane (3 ×
10 mL). The combined filtrates were evaporated to dryness under
reduced pressure. The solid residue was purified by flash column
chromatography (silica gel), eluting with 5% methanol in chloroform,
to obtain 26 (98 mg, 94%): mp 312−314 °C. IR (KBr) 1710, 1638,
1608 cm−1. 1H NMR (300 MHz, DMSO-d6) δ 12.96 (s, 1 H), 8.56 (d,
J = 6.2 and 1.4 Hz, 1 H), 7.86−7.81 (m, 2 H), 7.59 (s, 1 H), 7.40 (t,
J = 6.8 Hz, 1 H). Positive ESIMS m/z (rel intensity): 309 (MH+, 100).
Negative ion ESIMS m/z (rel intensity): 307 [(M − H+)−, 100].
9-Aza-5,6-dihydro-6-(3-dimethylaminopropyl)-2,3-dime-
thoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline Trifluoroacetate
(27). 9-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno[1,2-c]-
isoquinoline (26, 92 mg, 0.3 mmol), 3-dimethylamino-1-pro-
panol (0.1 mL, 0.9 mmol), and PPh3 (240 mg, 0.92 mmol) were
diluted with THF (15 mL). Diisopropyl azodicarboxylate (0.18 mL,
0.92 mmol) was added to the THF solution, and the resulting mixture
was stirred at room temperature for 3 h. The reaction mixture was then
evaporated to dryness under reduced pressure. The residue was purified
by flash column chromatography (silica gel), eluting with 10% methanol
in chloroform, to provide a dark-orange solid. The solid was redissolved
in chloroform (10 mL), and trifluoroacetic acid (2 M in diethyl ether,
1 mL) was added. The precipitate was collected by filtration and washed
with ether (2 × 2 mL) to yield the product in the form of its
trifluoroacetate salt (94 mg, 62%): mp 220 °C (dec). IR (KBr) 1772,
2-Methyl-6-oxo-1,6-dihydropyridine-3-carbonitrile (31).21 3-
Aminocrotonitrile (29, 2.9 g, 36 mmol) and ethyl propiolate (30, 3.0
mL, 36 mmol) were dissolved in dry DMF (17 mL). The reaction
mixture was stirred for 1 h at room temperature, and the mixture was
heated at reflux for 3 days. The precipitate formed after cooling to
room temperature was collected, washed with methanol (5 mL), ether
(10 mL), and dried to yield 31 (1.5 g, 31%): mp >300 °C. H NMR
(300 MHz, DMSO-d6) δ 12.43 (s, 1 H), 7.58 (d, J = 9.6 Hz, 1 H), 6.23
(d, J = 9.6 Hz, 1 H), 2.37 (s, 3 H).
1
6-Chloro-2-methylnicotinonitrile (32).21 A mixture of 31 (1.5 g,
11 mmol) and phosphorus oxychloride (9 mL, 0.1 mol) was heated at
reflux for 6 h. The reaction mixture was cooled to room temperature,
and the excess phosphorus oxychloride was removed under reduced
pressure. Ice cold water (50 mL) was added to the residue. The brown
precipitate was collected and washed with ice cold water (3 × 25 mL),
ether (2 × 20 mL), and dried to provide 32 as a light-brown solid
(0.9 g, 54%): mp 104−105 °C (lit.21 mp 106−108 °C). 1H NMR (300
MHz, CDCl3) δ 7.83 (d, J = 8.1 Hz, 1 H), 7.29 (d, J = 8.1 Hz, 1 H),
2.77 (s, 3 H).
2-Methylnicotinonitrile (33). 6-Chloro-2-methylnicotinonitrile
(32, 10 g, 66 mmol) and ammonium formate (41 g, 0.65 mol) were
dissolved in methanol (250 mL), and palladium (5% on activated
carbon, 3.5 g, 2.5 mol %) was added. The mixture was stirred at room
temperature for 12 h, filtered through Celite, and washed with
methanol (3 × 50 mL). The combined filtrates were evaporated, and
the yellow oily residue was subjected to flash column chromatography
on silica gel, eluting with chloroform to provide 33 as an off-white
solid (6.3 g, 81%): mp 55 °C (lit.42 mp 56−58 °C). H NMR (300
1
MHz, CDCl3) δ 7.80 (dd, J = 4.9, 1.6 Hz, 1 H), 7.02 (dd, J = 7.8, 1.7
Hz, 1 H), 6.37 (dd, J = 7.8, 5.0 Hz, 1 H), 1.88 (s, 3 H). Positive ion
ESIMS m/z (rel intensity): 119 (MH+, 100).
10-Aza-5,6-dihydro-2,3-dimethoxy-5-oxo-11H-indeno[1,2-c]-
isoquinoline (35). 2-Methylnicotinonitrile (33, 3.3 g, 34 mmol),
NBS (5.5 g, 33 mmol), and AIBN (600 mg, 4 mmol) were diluted
with 1,2-dichloroethane (60 mL), and the mixture was heated at reflux
for 9 h. The reaction mixture was concentrated to the half its original
volume, filtered, and the filtrate was evaporated to dryness under
reduced pressure. The residue was redissolved in acetonitrile (70 mL).
14 (11 g, 48 mmol) was added, followed by triethylamine (7 mL, 50
mmol), and the solution was heated at reflux for 2 days. The hot
solution was filtered, and the precipitate was washed with boiling
acetonitrile (2 × 25 mL) to provide a gray solid (2.8 g, 31%): mp
270−272 °C. IR (KBr) 1635, 1610, 1528, 1503 cm−1. 1H NMR
(300 MHz, DMSO-d6) δ 12.26 (s, 1 H), 8.40 (d, J = 4.9 Hz, 1 H), 8.19
(d, J = 7.7 Hz, 1 H), 7.63 (s, 1 H), 7.34 (dd, J = 7.6, 5.1 Hz, 1 H),
7.19 (s, 1 H), 3.95 (s, 3 H), 3.91 (s, 2 H), 3.86 (s, 3 H). Positive
ESIMS m/z (rel intensity): 295 (M+, 100).
10-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno-
[1,2-c]isoquinoline (36). 10-Aza-5,6-dihydro-2,3-dimethoxy-5-oxo-
11H-indeno[1,2-c]isoquinoline (35, 1.5 g, 5.1 mmol) and SeO2
(1.13 g, 10.2 mmol) were diluted with 1,4-dioxane (50 mL), and
the mixture was heated at reflux for 3 days. The reaction mixture was
filtered while hot, and the precipitate was extracted in a Soxhlet
extractor with a chloroform−methanol mixture (4:1). The extracts
were evaporated to dryness to get 36 (1.4 g, 90%): mp >300 °C. IR
(KBr) 1708, 1659, 1600, 1574 cm−1. 1H NMR (300 MHz, DMSO-d6)
δ 8.49 (d, J = 4.9 Hz, 1 H), 8.07 (d, J = 7.6 Hz, 1 H), 7.83 (s, 1 H),
7.56 (s, 1 H), 7.42 (dd, J = 7.4, 5.2 Hz, 1 H), 3.93 (s, 3 H), 3.87 (s,
3 H). Negative ion ESIMS m/z (rel intensity): 307 [(M − H+)−, 100].
10-Aza-5,6-dihydro-6-(3-dimethylaminopropyl)-2,3-dime-
thoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline Trifluoroacetate
(37). 10-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno-
[1,2-c]isoquinoline (36, 92 mg, 0.3 mmol), 3-dimethylamino-1-pro-
panol (0.1 mL, 0.9 mmol), and PPh3 (240 mg, 0.92 mmol) were
diluted with THF (15 mL). Diisopropyl azodicarboxylate (0.18 mL,
0.92 mmol) was added to the THF solution, and the resulting mixture
was stirred at room temperature for 12 h. The reaction mixture was
then evaporated to dryness under reduced pressure. The residue was
purified by flash column chromatography (silica gel), eluting with 10%
methanol in chloroform, followed by preparative TLC (silica gel),
1
1688, 1633, 1612 cm−1. H NMR (300 MHz, CD3OD) δ 8.61 (d, J =
5.2 Hz, 1 H), 8.44 (s, 1 H), 7.54 (d, J = 7.0 Hz, 2 H), 7.16 (s, 1 H), 4.62
(t, J = 6.0 Hz, 2 H), 3.84 (s, 3 H), 3.81 (s, 3 H), 3.40−3.29 (m, 2 H),
2.89 (s, 6 H), 2.38−2.24 (m, 2 H). Positive ion ESIMS m/z (rel
intensity): 394 (MH+, 100). HRMS-ESI m/z: MH+ calcd for
C22H23N3O4, 394.1767; found, 394.1770. HPLC purity: 96.18% [C-18
reverse phase, MeOH (1% CF3COOH)/H2O, 70:30]; 97.23% [C-18
reverse phase, MeOH (1% CF3COOH)/H2O, 80:20].
9-Aza-5,6-dihydro-6-(3-(4-morpholino)propyl)-2,3-dime-
thoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline Trifluoroacetate
(28). 9-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno[1,2-c]-
isoquinoline (26, 85 mg, 0.28 mmol), 4-(3-hydroxypropyl)morpholine
(120 mg, 0.84 mmol), and PPh3 (230 mg, 0.84 mmol) were diluted with
THF (10 mL). Diisopropyl azodicarboxylate (0.17 mL, 0.84 mmol) was
added to the THF solution, and the resulting mixture was stirred at
room temperature for 3 h. The reaction mixture was then evaporated to
dryness under reduced pressure. The residue was purified by flash
column chromatography (silica gel), eluting with 10% methanol in
chloroform, to provide a dark-orange solid. The solid was redissolved in
chloroform (2 mL), and trifluoroacetic acid (2 M in diethyl ether, 1 mL)
was added. The precipitate was collected by filtration and washed with
ether (2 × 2 mL) to yield the product in the form of its trifluoroacetate
salt (16 mg, 10%): mp 222−224 °C (dec). IR (KBr) 1772, 1712, 1677,
1
1635, 1612 cm−1. H NMR (300 MHz, CD3OD) δ 8.73 (s, 1 H), 8.62
(s, 1 H), 8.55 (d, J = 8.3 Hz, 1 H), 8.24 (d, J = 8.0 Hz, 1 H), 7.80 (t, J =
7.6 Hz, 2 H), 7.59 (t, J = 7.7 Hz, 1 H), 4.75 (d, J = 6.4 Hz, 2 H), 3.99
(d, J = 11.5 Hz, 2 H), 3.68 (t, J = 12.7 Hz, 2 H), 3.50 (d, J = 12.2 Hz,
2 H), 3.44−3.35 (m, 2 H), 3.13−2.98 (m, 2 H), 2.36 (td, J = 11.7,
5.7 Hz, 2 H). Positive ion ESIMS m/z (rel intensity): 436 (MH+, 100).
HRMS-ESI m/z: MH+ calcd for C20H19N3O2, 436.1872; found,
436.1870. HPLC purity: 98.14% [C-18 reverse phase, MeOH (1%
CF3COOH)/H2O, 70:30]; 96.84% [C-18 reverse phase, MeOH (1%
CF3COOH), 100].
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eluting with 5% methanol in chloroform, to provide an orange solid.
The solid was redissolved in chloroform (5 mL), and trifluoroacetic
acid (2 M in diethyl ether, 1 mL) was added. The precipitate was
collected by filtration and washed with ether (2 × 2 mL) to yield the
product in the form of its trifluoroacetate salt (34 mg, 22%): mp 212−
214 °C (dec). 1H NMR (300 MHz, DMSO-d6) δ 10.60 (s, 1 H), 8.50
(dd, J = 5.1, 1.3 Hz, 1 H), 7.83 (dd, J = 7.5, 1.3 Hz, 1 H), 7.73 (s, 1 H),
7.41 (dd, J = 7.5, 5.1 Hz, 1 H), 7.32 (s, 1 H), 4.67 (t, J = 6.1 Hz, 2 H),
3.93 (s, 3 H), 3.90 (s, 3 H), 3.34−3.25 (m, 2 H), 2.81 (d, J = 4.7 Hz, 6
H), 2.38−2.28 (m, 2 H). Positive ion ESIMS m/z (rel intensity): 394
(MH+, 100). HRMS-ESI m/z: MH+ calcd for C22H23N3O4, 394.1767;
found, 394.1769. HPLC purity: 98.32% [C-18 reverse phase, MeOH
(1% CF3COOH)/H2O, 80:20].
was removed by filtration, and the filtrate was concentrated to dryness.
The crude solid was redissolved in chloroform, and the resulting solution
was filtered through a layer of silica gel, washing with extra portions of
chloroform. The combined filtrates were evaporated to dryness to yield
41 (8.3 g, 87%): mp 81−82 °C (lit.21 mp 80−80.5 °C). H NMR (300
1
MHz, CDCl3) δ 7.66 (d, J = 8.5 Hz, 1H), 6.59 (d, J = 8.5 Hz, 1H), 3.94
(s, 3H), 2.62 (s, 3H).
10-Aza-5,6-dihydro-2,3,9-trimethoxy-5-oxo-11H-indeno-
[1,2-c]isoquinoline (43). 6-Methoxy-2-methylnicotinonitrile (41, 2.2 g,
15 mmol), NBS (2.9 g, 16 mmol), and AIBN (100 mg, 0.6 mmol)
were diluted with 1,2-dichloroethane (50 mL), and the mixture was
heated at reflux for 3.5 h. The reaction mixture was concentrated to
one-half its original volume, filtered, and the filtrate was evaporated to
dryness under reduced pressure. The residue was diluted with acetoni-
trile (60 mL), and 14 (5.3 g, 24 mmol) was added, followed by
triethylamine (3.5 mL, 25 mmol), and the solution was heated at reflux
for 14 h. The solution was allowed to cool to room temperature and
the obtained precipitate was filtered and washed with acetonitrile (2 ×
15 mL) to provide an off-white solid (0.9 g, 19%): mp 284−286 °C.
10-Aza-5,6-dihydro-6-[3-(4-morpholino)propyl]-2,3-dime-
thoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline Trifluoroacetate
(38). 10-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno-
[1,2-c]isoquinoline (36, 100 mg, 0.32 mmol), 4-(3-hydroxypropyl)-
morpholine (94 mg, 0.65 mmol), and PPh3 (170 mg, 0.65 mmol) were
diluted with THF (10 mL). Diisopropyl azodicarboxylate (130 mg,
0.65 mmol) was added to the THF solution, and the resulting mixture
was stirred at room temperature for 12 h. The reaction mixture was
then evaporated to dryness under reduced pressure. The residue was
purified by flash column chromatography (silica gel), eluting with 10%
methanol in chloroform, followed by preparative TLC (silica gel),
eluting with 5% methanol in chloroform, to provide an orange solid.
The solid was redissolved in chloroform (5 mL), and trifluoroacetic
acid (2 M in diethyl ether, 1 mL) was added. The precipitate was col-
lected by filtration and washed with ether (2 × 2 mL) to yield the
product in the form of its trifluoroacetate salt (12 mg, 7%): mp 208−
210 °C. 1H NMR (300 MHz, DMSO-d6) δ 11.05 (s, 1 H), 8.51 (d, J =
4.0 Hz, 1 H), 7.88 (d, J = 7.4 Hz, 1 H), 7.81 (s, 1 H), 7.44 (dd, J = 7.4,
5.1 Hz, 1 H), 7.40 (s, 1 H), 4.71 (t, J = 5.8 Hz, 2 H), 3.96 (t, J = 10.8
Hz, 8 H), 3.81 (dd, J = 20.1, 8.5 Hz, 2 H), 3.50 (d, J = 12.1 Hz, 2 H),
3.11 (dd, J = 21.2, 9.3 Hz, 4 H), 2.41−2.33 (m, 2 H). Positive ion
ESIMS m/z (rel intensity): 436 (MH+, 100). HRMS-ESI m/z: MH+
calcd for C24H25N3O5, 436.1872; found, 436.1769. HPLC purity:
98.28% [C-18 reverse phase, MeOH (1% CF3COOH)/H2O, 90:10];
97.14% [C-18 reverse phase, MeOH(1% CF3COOH)/H2O, 70:30].
2-Methyl-1,4,5,6-tetrahydropyridine-3-carbonitrile (39). A
solution of 6-chloro-2-methylnicotinonitrile (32, 6.0 g, 39 mmol)
and potassium acetate (7.8 g, 80 mmol) in methanol (80 mL) was
degassed. Palladium dichloride (350 mg, 2.0 mmol) was added to the
solution, and the reaction vessel was filled with hydrogen (1 atm.).
The mixture was stirred at room temperature until no more hydrogen
was consumed. The solvent was removed under reduced pressure and
the oily residue was subjected to flash column chromatography on silica
gel, eluting with chloroform, to provide 39 as light-brown solid (4.0 g,
1
IR (KBr) 1648, 1614 cm−1. H NMR (300 MHz, DMSO-d6) δ 12.20
(s, 1 H), 8.14 (d, J = 8.4 Hz, 1 H), 7.61 (s, 1 H), 7.10 (s, 1 H), 6.81
(d, J = 8.5 Hz, 1 H), 3.94 (s, 3 H), 3.91 (s, 3 H), 3.86 (s, 3H), 3.85 (s, 2 H).
EIMS m/z: 324 (M+). CIMS m/z (rel intensity): 325 (MH+, 100).
10-Aza-5,6-dihydro-2,3,9-trimethoxy-5,11-dioxo-11H-
indeno[1,2-c]isoquinoline (44). 10-Aza-5,6-dihydro-2,3,9-trime-
thoxy-5-oxo-11H-indeno[1,2-c]isoquinoline (43, 0.7 g, 2.2 mmol)
and SeO2 (0.48 mg, 4.3 mmol) were diluted with 1,4-dioxane (50 mL),
and the mixture was heated at reflux for 24 h. The reaction mixture
was filtered while hot, and the precipitate was washed with hot dioxane
(3 × 100 mL). The combined filtrates were evaporated to dryness
under reduced pressure to obtain 44 (0.65 g, 89%): mp >350 °C. IR
(KBr) 1713, 1645, 1624, 1612, 1592 cm−1. NMR data have not been
obtained because of poor solubility of the sample. Negative ion ESIMS
m/z (rel intensity): 337 [(M − H+)−, 100].
10-Aza-5,6-dihydro-6-(3-dimethylaminopropyl)-2,3,9-trime-
thoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline Trifluoroace-
tate (45). 10-Aza-5,6-dihydro-2,3,9-trimethoxy-5,11-dioxo-11H-
indeno[1,2-c]isoquinoline (44, 110 mg, 0.32 mmol) was added to a
stirred solution of PPh3 (170 mg, 0.65 mmol) and diisopropyl
azodicarboxylate (0.13 mL, 0.65 mmol) in tetrahydrofuran (10 mL).
The mixture was stirred at room temperature until the solid material
completely disappeared to form a dark-red solution. 3-Dimethylamino-
1-propanol (67 mg, 0.65 mmol) was added dropwise to the resulting
solution over the course of 30 min, and the reaction mixture was
stirred at room temperature for 3 days. The resulting mixture was
evaporated to dryness under reduced pressure. The residue was
purified by flash column chromatography (silica gel), eluting with 3%
methanol in chloroform, to provide a red solid. The solid was
redissolved in chloroform (10 mL), and trifluoroacetic acid (2 M in
diethyl ether, 1 mL) was added. The precipitate was collected by
filtration and washed with ether (2 × 2 mL) to yield the product in the
form of its trifluoroacetate salt (52 mg, 30%): mp 250−252 °C (dec).
IR (KBr) 1692, 1621, 1605 1562 cm−1. 1H NMR (500 MHz, CD3OD)
δ 7.28 (d, J = 8.1 Hz, 1 H), 7.16 (s, 1 H), 6.95 (s, 1 H), 6.54 (d, J = 8.1
Hz, 1 H), 4.57 (t, J = 5.8 Hz, 2 H), 3.94 (s, 3 H), 3.83 (s, 3 H), 3.82 (s,
3 H), 3.58−3.48 (m, 2 H), 3.07 (s, 6 H), 2.46−2.32 (m, 2 H). Positive
ion ESIMS m/z (rel intensity): 424 (MH+, 100). Anal. Calcd for
C25H26F3N3O7: C, 55.87; H, 4.88; N, 7.82. Found: C, 55.45; H, 4.62;
N, 7.75.
1
84%): mp 49−52 °C. H NMR (300 MHz, CDCl3) δ 4.43 (s, 1 H),
3.18 (td, J = 6.2, 3.0 Hz, 2 H), 2.20 (t, J = 6.3 Hz, 2 H), 1.97 (s, 3 H),
1.80−1.64 (m, 2 H). 13C NMR (75 MHz, CDCl3) δ 153.22, 123.85,
71.19, 41.22, 23.27, 20.75, 19.85.
3-Cyano-2-chloro-4-methylpyridine (40). 3-Cyano-2,6-di-
chloro-4-methylpyridine (11, 35.1 g, 0.19 mol) and ammonium
formate (235 g, 3.73 mol) were added to a suspension of palladium
(5% on activated carbon, 4.0 g, 0.8 mol %) in methanol (600 mL), and
the mixture was stirred at room temperature for 3 days. The mixture
was filtered through a Celite bed. The filtrate was evaporated to
dryness, and water (100 mL) and chloroform (100 mL) were added.
The organic layer was separated, and the aqueous layer was extracted
with chloroform (3 × 50 mL). The combined extracts were washed
with water (50 mL), brine (100 mL), dried with sodium sulfate, and
filtered through a thin pad of silica gel, washing with chloroform. The
combined filtrates were evaporated to yield a light-brown solid (25.3 g,
87%): mp 104−106 °C (lit.23 mp 105−108 °C). 1H NMR (300 MHz,
CDCl3) δ 8.39 (d, J = 5.1 Hz, 1 H), 7.21 (d, J = 5.1 Hz, 1 H), 2.57
(s, 3 H).
10-Aza-5,6-dihydro-6-(3-(4-morpholino)propyl)-2,3,9-trime-
thoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline Trifluoroace-
tate (46). 10-Aza-5,6-dihydro-2,3,9-trimethoxy-5,11-dioxo-11H-
indeno[1,2-c]isoquinoline (44, 110 mg, 0.32 mmol) was added to a
stirred solution of PPh3 (170 mg, 0.65 mmol) and diisopropyl
azodicarboxylate (0.13 mL, 0.65 mmol) in tetrahydrofuran (10 mL).
The mixture was stirred at room temperature until the solid material
completely disappeared to form a dark-red solution. 4-(3-
Hydroxypropyl)morpholine (94 mg, 0.65 mmol) was added dropwise
to the resulting solution over the course of 30 min, and the reaction
mixture was stirred at room temperature for 3 days. The resulting
6-Methoxy-2-methylnicotinonitrile (41). Sodium methoxide
(20 g, 0.4 mol) was added to a solution of 6-chloro-2-methylnicotinoni-
trile (32, 10 g, 66 mmol) in methanol (150 mL), and the mixture was
heated at reflux for 1.5 h and cooled to room temperature. The precipitate
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1
mixture was evaporated to dryness under reduced pressure. The
residue was purified by flash column chromatography (silica gel),
eluting with 3% methanol in chloroform, to provide a red solid. The
solid was redissolved in chloroform (10 mL), and hydrochloric acid (2
M in methanol, 1 mL) was added. The precipitate was collected by
filtration and washed with ether (2 × 2 mL) to yield the product in the
form of its trifluoroacetate salt (61 mg, 31%): mp 237−238 °C (dec).
IR (KBr) 1709, 1618, 1605, 1561 cm−1. 1H NMR (300 MHz, DMSO-
d6) δ 10.81 (s, 1 H), 7.79 (d, J = 8.2 Hz, 1 H), 7.70 (s, 1 H), 7.33
(s, 1 H), 6.86 (d, J = 8.2 Hz, 1 H), 4.68 (s, 2 H), 4.00 (s, 2 H), 3.94
(s, 3 H), 3.92 (s, 3 H), 3.90 (s, 3 H), 3.78 (t, J = 11.8 Hz, 2 H), 3.62−
3.40 (m, 4 H), 3.18−3.04 (m, 2 H), 2.35 (s, 2 H). Positive ion ESIMS
m/z (rel intensity): 466 (MH+, 100). HRMS-ESI m/z: MH+ calcd for
C25H27N3O6, 466.1978; found, 466.1980. HPLC purity: 96.75% [C-18
reverse phase, MeOH (1% CF3COOH)/H2O, 70:30]; 95.27% [C-18
reverse phase, MeOH (1% CF3COOH)].
196/198 (M+). CIMS 197/199 (MH+). The H NMR spectrum is
consistent with previously published data.45
5-Methoxy-3-methylpicolinonitrile (51). 5-Bromo-3-methylpi-
colinonitrile (50, 35 g, 0.18 mol) was added to a solution of sodium
methoxide (18 g, 0.54 mol) in methanol (200 mL), and the mixture
was heated at reflux for 12 h. The solution was cooled to room
temperature and concentrated to one-third of its volume. The
concentrated solution was diluted with water (150 mL), and the
products were extracted with chloroform (3 × 50 mL). The combined
extracts were washed with water (2 × 50 mL), brine (50 mL), dried
with sodium sulfate, and filtered through a pad of silica gel, washing
with chloroform, to produce 51 as an off-white solid (21 g, 79%): mp
1
80−81 °C. IR (film) 2225, 1645, 1589 cm−1. H NMR (300 MHz,
CDCl3) δ 8.06 (d, J = 2.6 Hz, 1 H), 7.03 (d, J = 2.4 Hz, 1 H), 3.83
(s, 3 H), 2.43 (s, 3 H). 13C NMR (75 MHz, CDCl3) δ 157.65, 139.99,
137.68, 125.05, 120.52, 116.76, 55.84, 18.71. EIMS m/z (rel intensity):
148 (M+, 100). CIMS m/z (rel intensity): 149 (MH+, 100).
5-Bromo-3-methylpyridin-2-amine (48).24 N-Bromosuccini-
mide (170 g, 0.95 mol) was added to a solution of 47 (99 g, 0.92
mol) and ammonium acetate (7 g, 10 mol %) in acetonitrile (500 mL).
The temperature of the reaction mixture during addition was controlled
with an ice bath. After the full amount of NBS was added, the ice bath
was removed and the reaction mixture was stirred at room temperature
for 25 min and acetonitrile was removed under reduced pressure. A
mixture of ethyl acetate (1 L) and water (1 L) was added to the solid
residue. The resulting mixture was stirred, and the organic layer was
separated. The water layer was extracted with ethyl acetate (3 × 500
mL). The combined extracts were washed with water (300 mL),
saturated sodium bicarbonate solution (500 L), dried with sodium
sulfate, and evaporated to dryness to give a dark brown solid. The crude
product was redissolved in chloroform (300 mL), and the solution was
filtered through a thin pad of silica gel, eluting with chloroform. The
combined filtrates were evaporated under reduced pressure to yield 48
as a light-brown solid (113 g, 65%): mp 89−90 °C (lit.43 mp 91−93 °C).
1H NMR (300 MHz, CDCl3) δ 7.97 (d, J = 2.3 Hz, 1 H), 7.36
(d, J = 2.3 Hz, 1 H), 4.50 (s, 2 H), 2.09 (s, 3 H). 13C NMR (75 MHz,
CDCl3) δ 156.10, 146.29, 140.03, 118.79, 108.58, 77.72, 77.30, 76.88,
17.27.
7-Aza-5,6-dihydro-2,3,9-trimethoxy-5-oxo-11H-indeno[1,2-
c]isoquinoline (53). 5-Methoxy-3-methylpicolinonitrile (51, 5.0 g, 34
mmol), NBS (6.6 g, 37 mmol), and AIBN (500 mg, 3 mmol) were
diluted with 1,2-dichloroethane (50 mL), and the mixture was heated
at reflux for 24 h. The reaction mixture was concentrated to one-half
its original volume, filtered, and the filtrate was evaporated to dryness
under reduced pressure. The residue was redissolved in acetonitrile
(100 mL). 4,5-Dimethoxyhomophthalic anhydride (14, 11.2 g, 50
mmol) was added, followed by triethylamine (8 mL, 58 mmol), and
the solution was heated at reflux for 24 h. The hot solution was
filtered, and the precipitate was washed with boiling acetonitrile (2 ×
25 mL) to provide a gray solid (2.4 g, 21%): mp >260 °C. IR (KBr)
1635, 1608 cm−1. H NMR (300 MHz, DMSO-d6) δ 11.94 (s, 1 H),
8.22 (d, J = 2.6 Hz, 1 H), 7.63 (s, 2 H), 7.17 (s, 1 H), 3.95 (s, 3 H),
3.89 (s, 3 H), 3.87 (s, 3 H), 3.85 (s, 2 H). Positive ion ESIMS m/z (rel
intensity): 265 (MH+, 100).
1
7-Aza-5,6-dihydro-2,3,9-trimethoxy-5,11-dioxo-11H-
indeno[1,2-c]isoquinoline (54). 10-Aza-5,6-dihydro-2,3,9-trime-
thoxy-5-oxo-11H-indeno[1,2-c]isoquinoline (53, 2.08 g, 6.4 mmol)
and SeO2 (1.42 g, 12.8 mmol) were diluted with 1,4-dioxane (100
mL), and the mixture was heated at reflux for 24 h. The reaction
mixture was filtered while hot, and the precipitate was washed with hot
dioxane (2 × 500 mL). The combined filtrates were evaporated to
dryness under reduced pressure to afford 54 (2.0 g, 92%): mp
>300 °C. IR (KBr) 1705, 1662, 1615, 1602, 1563 cm−1. NMR data have
not been obtained because of poor solubility of the sample. Negative ion
ESIMS m/z (rel intensity): 339 [(M − H+)−, 100]. The product was
introduced into the next step without additional purification.
7-Aza-5,6-dihydro-6-(3-dimethylaminopropyl)-2,3,9-trime-
thoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline Hydrochlor-
ide (55). 7-Aza-5,6-dihydro-2,3,9-trimethoxy-5,11-dioxo-11H-in-
deno[1,2-c]isoquinoline (54, 338 mg, 1 mmol) was added to a stirred
solution of PPh3 (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate
(390 mg, 1.9 mmol) in tetrahydrofuran (10 mL). The mixture was
stirred for 4 h at room temperature. 3-Dimethylamino-1-propanol (200 mg,
1.9 mmol) was added dropwise to the resulting solution over the course of
30 min, and the reaction mixture was stirred at room temperature for 12 h.
PPh3 (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate (390 mg, 1.9
mmol) were added to the reaction mixture. The mixture was stirred for 6 h,
and 3-dimethylamino-1-propanol (200 mg, 1.9 mmol) was added, forming
a dark red solution. The solution was stirred at room temperature for 24 h
and evaporated to dryness under reduced pressure. The residue was
subjected to flash column chromatography (silica gel), eluting with a
gradient of 1−5% methanol in chloroform, to provide a red solid. The solid
was redissolved in chloroform (10 mL), and hydrochloric acid (1 M in
methanol, 1 mL) was added. The precipitate was collected by filtration and
washed with ether (2 × 2 mL) to yield the product in the form of its
hydrochloride salt (238 mg, 52%): mp 245 °C (dec). IR (KBr) 3445, 1699,
2,5-Dibromo-3-methylpyridine (49).27 5-Bromo-3-methylpyri-
din-2-amine (48, 69 g, 0.37 mol) was suspended in hydrobromic acid
(200 mL, 48% in water), and the mixture was cooled to −15 °C.
Bromine (95 g, 0.59 mol) was added dropwise to the mixture followed
by addition of sodium nitrite (69 g, 1 mol) in water (100 mL).
Temperature of the reaction mixture was kept below −15 °C during
addition. After addition, the cooling bath was removed and the
reaction mixture was stirred for 3 h. The reaction mixture was cooled
to −15 °C and quenched with potassium hydroxide (112 g, 2 mol) in
water (500 mL). The cooling bath was removed, and the mixture was
stirred for 1.5 h. The products were extracted with ethyl acetate (3 ×
300 mL). The combined extracts were washed with water (2 × 200 mL),
saturated aqueous sodium bicarbonate (200 mL), dried with sodium
sulfate, and evaporated to dryness. The oily residue was redissolved in
chloroform (100 mL), and the solution was filtered through a pad of silica
gel, washing with chloroform. The combined filtrates were evaporated to
provide 49 as light-yellow solid (87 g, 94%): mp 38−40 °C (lit.44 mp
1
41−42 °C). H NMR (300 MHz, CDCl3) δ 8.22 (d, J = 2.4 Hz, 1 H),
7.61 (d, J = 2.4 Hz, 1 H), 2.33 (s, 3 H). 13C NMR (75 MHz, CDCl3) δ
148.29, 143.05, 141.15, 137.15, 119.68, 22.06.
5-Bromo-3-methylpicolinonitrile (50). Copper(I) cyanide (21 g,
0.24 mol) was added to a solution of 2,5-dibromo-3-methylpyridine
(49, 60 g, 0.24 mol) in dry DMF (200 mL), and the mixture was
heated at reflux for 2 h. After the mixture was cooled to room
temperature, water (1500 mL) was added to the mixture and the pro-
ducts were extracted with ethyl acetate (3 × 300 mL). The combined
extracts were washed with water (3 × 300 mL), brine (300 mL), dried
with sodium sulfate, and evaporated to dryness. The brown oily
residue was subjected to flash column chromatography (silica gel),
eluting with chloroform, to yield a white solid (35 g, 74%): mp 86−
88 °C. 1H NMR (300 MHz, CDCl3) δ 8.55 (d, J = 1.8 Hz, 1 H), 7.84
(d, J = 1.5 Hz, 1 H), 2.53 (s, 3 H). 13C NMR (75 MHz, CDCl3)
δ 149.75, 140.68, 139.95, 132.25, 124.61, 115.90, 18.59. EIMS m/z
−1
1
1651, 1611 cm . H NMR (300 MHz, DMSO-d6) δ 9.90 (s, 1 H), 8.21
(s, 1 H), 7.81 (s, 1 H), 7.49 (s, 1 H), 7.43 (s, 1 H), 4.78 (s, 2 H), 3.94
(s, 3 H), 3.91 (s, 3 H), 3.86 (s, 3 H), 3.15 (s, 2 H), 2.76 (s, 3 H), 2.74
(s, 3 H), 2.14 (s, 2 H). Positive ion ESIMS m/z (rel intensity): 424
(MH+, 100). HRMS-ESI m/z: MH+ calcd for C23H25N3O5, 424.1822;
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found, 424.1869. HPLC purity: 98.61% [C-18 reverse phase, MeOH];
97.99% [C-18 reverse phase, MeOH/H2O, 85:15].
1689, 1618, 1574 cm−1. NMR and MS data have not been obtained
because of poor solubility of the sample. The product was introduced
into the next step without additional purification.
7-Aza-5,6-dihydro-6-[3-(4-morpholino)propyl]-2,3,9-trime-
thoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline Hydrochlor-
ide (56). 7-Aza-5,6-dihydro-2,3,9-trimethoxy-5,11-dioxo-11H-indeno-
[1,2-c]isoquinoline (54, 338 mg, 1 mmol) was added to a stirred
solution of PPh3 (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate
(390 mg, 1.9 mmol) in tetrahydrofuran (10 mL). The mixture was
stirred for 4 h at room temperature. 4-(3-Hydroxypropyl)morpholine
(280 mg, 1.9 mmol) was added dropwise to the resulting solution over
the course of 30 min, and the reaction mixture was stirred at room tem-
perature for 12 h. PPh3 (510 mg, 1.9 mmol) and diisopropyl azodicar-
boxylate (390 mg, 1.9 mmol) were added to the reaction mixture. The
mixture was stirred for 6 h, and 4-(3-hydroxypropyl)morpholine (280
mg, 1.9 mmol) was added, forming a dark red solution. The solution
was stirred at room temperature for 24 h and evaporated to dryness
under reduced pressure. The residue was subjected to flash column
chromatography (silica gel), eluting with a gradient of 1−5% methanol
in chloroform, to provide a red solid. The solid was redissolved in
chloroform (10 mL), and hydrochloric acid (1 M in methanol, 1 mL)
was added. The precipitate was collected by filtration and washed with
chloroform (20 mL) and diethyl ether (10 mL) to yield the product in
the form of its hydrochloride salt (276 mg, 59%): mp 260−261 °C
(dec). 1H NMR (300 MHz, DMSO-d6) δ 8.18 (d, J = 2.6 Hz, 1 H), 7.76
(s, 1 H), 7.45 (s, 1 H), 7.38 (d, J = 2.6 Hz, 1 H), 4.76 (s, 2 H), 3.93
(s, 3 H), 3.90 (s, 3 H), 3.84 (s, 3 H), 3.75 (t, J = 11.6 Hz, 4 H), 3.20 (s,
2 H), 3.06 (s, 4 H), 2.20 (s, 2 H). Positive ion ESIMS m/z (rel intensity):
466 (MH+, 100). HRMS-ESI m/z: MH+ calcd for C23H25N3O5, 466.1978;
found, 466.1974. HPLC purity: 95.45% [C-18 reverse phase, MeOH];
96.67% [C-18 reverse phase, MeOH/H2O, 85:15].
7-Aza-5,6-dihydro-3-nitro-9-methoxy-5,11-dioxo-11H-
indeno[1,2-c]isoquinoline (62). 7-Aza-5,6-dihydro-3-nitro-9-me-
thoxy-5-oxo-11H-indeno[1,2-c]isoquinoline (60, 2.2 g, 7 mmol) and
SeO2 (1.6 g, 14 mmol) were diluted with 1,4-dioxane (100 mL), and
the mixture was heated at reflux for 24 h. The reaction mixture was
filtered while hot, and the precipitate was washed with hot dioxane
(2 × 500 mL). The combined filtrates were evaporated to dryness
under reduced pressure to yield 62 (1.94 g, 86%): mp >300 °C. IR
(KBr) 1693, 1618, 1571 cm−1. NMR data have not been obtained
because ofpoor solubility of the sample. Negative ion ESIMS m/z (rel
intensity): 322 [(M − H+)−, 100]. The product was introduced into
the next step without additional purification.
7-Aza-5,6-dihydro-6-(3-dimethylaminopropyl)-9-methoxy-
5,11-dioxo-11H-indeno[1,2-c]isoquinoline (63). 7-Aza-5,6-dihy-
dro-9-methoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (61, 278 mg,
1 mmol) was added to a stirred solution of PPh3 (510 mg, 1.9 mmol)
and diisopropyl azodicarboxylate (390 mg, 1.9 mmol) in tetrahydrofuran
(20 mL). The mixture was stirred at room temperature for 4 h.
3-Dimethylamino-1-propanol (200 mg, 1.9 mmol) was added dropwise
to the resulting solution over the course of 15 min, and the reaction
mixture was stirred at room temperature for 12 h. PPh3 (510 mg, 1.9
mmol) and diisopropyl azodicarboxylate (390 mg, 1.9 mmol) were
added to the reaction mixture. The mixture was stirred for 6 h, and 3-
dimethylamino-1-propanol (200 mg, 1.9 mmol) was added, forming an
orange solution. The solution was stirred at room temperature for 24 h
and evaporated to dryness under reduced pressure. The residue was
subjected to flash column chromatography (silica gel), eluting with a
gradient of 1% to 5% methanol in chloroform, to provide an orange
solid (138 mg, 38%): mp 190−192 °C. 1H NMR (300 MHz, CDCl3) δ
8.35 (d, J = 8.1 Hz, 1 H), 8.16 (d, J = 8.1 Hz, 1 H), 7.93 (d, J = 2.7 Hz,
1 H), 7.59−7.49 (m, 1 H), 7.35−7.27 (m, 1 H), 7.09 (d, J = 2.7 Hz,
1 H), 4.82−4.69 (m, 2 H), 3.81 (s, 3 H), 2.40 (t, J = 7.1 Hz, 2 H), 2.18
(s, 6 H), 1.89 (dt, J = 14.6, 7.5 Hz, 2 H). Positive ion ESIMS m/z (rel
intensity): 364 (MH+, 100). HRMS-ESI m/z: MH+ calcd for
C21H21N3O3, 364.1661; found, 364.1663. HPLC purity: 98.39% [C-18
reverse phase, MeOH]; 98.46% [C-18 reverse phase, MeOH/H2O,
85:15].
7-Aza-5,6-dihydro-6-(3-(4-morpholino)propyl)-9-methoxy-
5,11-dioxo-11H-indeno[1,2-c]isoquinoline (64). 7-Aza-5,6-dihy-
dro-9-methoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (61, 278 mg,
1 mmol) was added to a stirred solution of PPh3 (510 mg, 1.9 mmol)
and diisopropyl azodicarboxylate (390 mg, 1.9 mmol) in tetrahydrofuran
(20 mL). The mixture was stirred at room temperature for 4 h. 4-(3-
Hydroxypropyl)morpholine (280 mg, 1.9 mmol) was added dropwise to
the resulting solution over the course of 15 min, and the reaction
mixture was stirred at room temperature for 12 h. PPh3 (510 mg, 1.9
mmol) and diisopropyl azodicarboxylate (390 mg, 1.9 mmol) were
added to the reaction mixture. The mixture was stirred for 6 h, and 4-(3-
hydroxypropyl)morpholine (280 mg, 1.9 mmol) was added, forming an
orange solution. The solution was stirred at room temperature for 24 h
and evaporated to dryness under reduced pressure. The residue was
subjected to flash column chromatography (silica gel), eluting with a
gradient of 1−5% methanol in chloroform, to provide an orange solid
(163 mg, 40%): mp 218−224 °C (dec). 1H NMR (300 MHz, DMSO-
d6) δ 8.45 (d, J = 8.2 Hz, 1 H), 8.25 (d, J = 2.5 Hz, 1 H), 8.20 (d, J = 7.8
Hz, 1 H), 7.82 (t, J = 7.7 Hz, 1 H), 7.61−7.45 (m, 2 H), 4.82 (s, 2 H),
3.92 (d, J = 12.6 Hz, 5 H), 3.74 (t, J = 11.9 Hz, 2 H), 3.39 (d, J = 12.2
Hz, 2 H), 3.21 (s, 2 H), 3.03 (d, J = 11.8 Hz, 2 H), 2.22 (s, 2 H).
Positive ion ESIMS m/z (rel intensity): 406 (MH+, 100). HRMS-ESI
m/z: MH+ calcd for C21H21N3O3, 406.1767; found, 406.1773. HPLC
purity: 97.30% [C-18 reverse phase, MeOH]; 98.60% [C-18 reverse
phase, MeOH/H2O, 85:15].
7-Aza-5,6-dihydro-9-methoxy-5-oxo-11H-indeno[1,2-c]-
isoquinoline (59). 5-Methoxy-3-methylpicolinonitrile (51, 5.0 g, 34
mmol), NBS (6.6 g, 37 mmol), and AIBN (500 mg, 3 mmol) were
diluted with 1,2-dichloroethane (50 mL), and the mixture was heated
at reflux for 24 h. The reaction mixture was concentrated to one-half
its original volume, filtered, and the filtrate was evaporated to dryness
under reduced pressure. The residue was redissolved in acetonitrile
(100 mL). Homophthalic anhydride (57, 9 g, 55 mmol) was added,
followed by triethylamine (8 mL, 58 mmol), and the solution was
heated at reflux for 24 h. The hot solution was filtered, and the
precipitate was washed with boiling acetonitrile (2 × 30 mL) to
provide a gray solid (4.1 g, 46%): mp 232−233 °C. IR (KBr) 1666,
1
1621, 1607 cm−1. H NMR (300 MHz, DMSO-d6) δ 12.11 (s, 1 H),
8.29−8.19 (m, 2 H), 7.75 (d, J = 4.1 Hz, 2 H), 7.68 (s, 1 H), 7.48 (dd,
J = 8.1, 4.3 Hz, 1 H), 3.89 (s, 5 H). Positive ion ESIMS m/z (rel
intensity): 310 (MH+, 100).
7-Aza-5,6-dihydro-3-nitro-9-methoxy-5-oxo-11H-indeno-
[1,2-c]isoquinoline (60). 5-Methoxy-3-methylpicolinonitrile (51,
5.0 g, 34 mmol), NBS (6.6 g, 37 mmol), and AIBN (500 mg,
3 mmol) were diluted with 1,2-dichloroethane (50 mL), and the mixture
was heated at reflux for 24 h. The reaction mixture was concentrated to
one-half its original volume, filtered, and the filtrate was evaporated to
dryness under reduced pressure. The residue was redissolved in
acetonitrile (100 mL). 5-Nitrohomophthalic anhydride (58, 11 g, 53
mmol) was added, followed by triethylamine (8 mL, 58 mmol), and the
solution was heated at reflux for 24 h. The hot solution was filtered, and
the precipitate was washed with boiling acetonitrile (2 × 30 mL) to
provide a gray solid (2.7 g, 26%): mp >260 °C. IR (KBr) 1690, 1616,
1559 cm−1. 1H NMR (300 MHz, DMSO-d6) δ 8.89 (d, J = 2.5 Hz, 1 H),
8.47 (dd, J = 8.8, 2.5 Hz, 1 H), 8.30 (d, J = 2.6 Hz, 1 H), 7.91 (d, J = 8.8
Hz, 1 H), 7.72 (d, J = 2.5 Hz, 1 H), 3.94 (s, 2 H), 3.91 (s, 3 H). Positive
ion ESIMS m/z (rel intensity): 310 (MH+, 100).
7-Aza-5,6-dihydro-9-methoxy-5,11-dioxo-11H-indeno[1,2-c]
isoquinoline (61). 7-Aza-5,6-dihydro-9-methoxy-5-oxo-11H--
indeno[1,2-c]isoquinoline (59, 2.64 g, 10 mmol) and SeO2 (2.22 g,
20 mmol) were diluted with 1,4-dioxane (120 mL), and the mixture
was heated at reflux for 24 h. The reaction mixture was filtered while
hot, and the precipitate was washed with hot dioxane (3 × 300 mL).
The combined filtrates were evaporated to dryness under reduced
pressure to afford 61 (2.10 g, 76%): mp >350 °C. IR (KBr) 1717,
7-Aza-5,6-dihydro-6-(3-dimethylaminopropyl)-9-methoxy-
3-nitro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (65). 7-Aza-
5,6-dihydro-9-methoxy-3-nitro-5,11-dioxo-11H-indeno[1,2-c]-
isoquinoline (62, 323 mg, 1 mmol) was added to a stirred solution of
PPh3 (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate (390 mg,
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1.9 mmol) in tetrahydrofuran (20 mL). The mixture was stirred at
room temperature for 4 h. 3-Dimethylamino-1-propanol (200 mg, 1.9
mmol) was added dropwise to the resulting solution over the course of
15 min, and the reaction mixture was stirred at room temperature for
12 h. PPh3 (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate (390
mg, 1.9 mmol) were added to the reaction mixture. The mixture was
stirred for 6 h, and 3-dimethylamino-1-propanol (200 mg, 1.9 mmol)
was added, forming an orange solution. The solution was stirred at
room temperature for 24 h and evaporated to dryness under reduced
pressure. The residue was subjected to flash column chromatography
(silica gel), eluting with a gradient of 1−5% methanol in chloroform,
to provide a red solid (250 mg, 61%): mp 224−226 °C. 1H NMR (300
MHz, CDCl3) δ 9.17 (d, J = 2.2 Hz, 1 H), 8.71 (d, J = 8.8 Hz, 1 H),
8.45 (dd, J = 8.9, 2.4 Hz, 1 H), 8.21 (d, J = 2.8 Hz, 1 H), 7.41 (d, J =
2.8 Hz, 1 H), 5.12−4.88 (m, 2 H), 3.98 (d, J = 4.0 Hz, 3 H), 2.48 (t,
J = 7.0 Hz, 2 H), 2.22 (d, J = 3.9 Hz, 6 H), 1.99 (dt, J = 14.3, 7.1 Hz,
2 H). Positive ion ESIMS m/z (rel intensity): 409 (MH+, 100).
HRMS-ESI m/z: MH+ calcd for C21H20N4O5, 409.1512; found,
409.1510. HPLC purity: 100% [C-18 reverse phase, MeOH]; 99.03%
[C-18 reverse phase, MeOH/H2O, 85:15].
7-Aza-5,6-dihydro-6-(3-(4-morpholino)propyl)-9-methoxy-
3-nitro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (66). 7-Aza-
5,6-dihydro-9-methoxy-3-nitro-5,11-dioxo-11H-indeno[1,2-c]-
isoquinoline (62, 323 mg, 1 mmol) was added to a stirred solution of
PPh3 (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate (390 mg,
1.9 mmol) in tetrahydrofuran (20 mL). The mixture was stirred at room
temperature for 4 h. 4-(3-Hydroxypropyl)morpholine (280 mg, 1.9
mmol) was added dropwise to the resulting solution over the course of
15 min, and the reaction mixture was stirred at room temperature for 12
h. PPh3 (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate (390 mg,
1.9 mmol) were added to the reaction mixture. The mixture was stirred
for 6 h, and 4-(3-hydroxypropyl)morpholine (280 mg, 1.9 mmol) was
added, forming an orange solution. The solution was stirred at room
temperature for 24 h and evaporated to dryness under reduced pressure.
The residue was subjected to flash column chromatography (silica gel),
eluting with a gradient of 1−5% methanol in chloroform, to provide a
red solid (212 mg, 47%): mp 243−245 °C (dec). 1H NMR (300 MHz,
DMSO-d6) δ 8.81 (s, 1 H), 8.55 (s, 2 H), 8.32 (d, J = 2.5 Hz, 1 H), 7.59
(d, J = 2.5 Hz, 1 H), 4.84 (s, 2 H), 3.96 (s, 3 H), 3.79 (s, 4 H), 3.21 (m,
6 H), 2.22 (s, 2 H). Positive ion ESIMS m/z (rel intensity): 451 (MH+,
100). HPLC purity: 95.42% [C-18 reverse phase, MeOH]; 95.93%
[C-18 reverse phase, MeOH/H2O, 85:15].
Topoisomerase I Mediated DNA Cleavage Reactions. Human
recombinant Top1 was purified from baculovirus as previously described.46
DNA cleavage reactions were prepared as previously reported with the
exception of the DNA substrate.30 Briefly, a 117-bp DNA oligonucleotide
(Integrated DNA Technologies) encompassing the previously identified
Top1 cleavage sites in the 161-bp fragment from pBluescript SK(−)
phagemid DNA was employed. This 117-bp oligonucleotide contains a
single 5′-cytosine overhang, which was 3′-end-labeled by fill-in reaction with
[α-32P]dGTP in React 2 buffer (50 mM Tris-HCl, pH 8.0, 100 mM
MgCl2, 50 mM NaCl) with 0.5 unit of DNA polymerase I (Klenow
fragment, New England BioLabs). Unincorporated [32P]dGTP was
removed using mini Quick Spin DNA columns (Roche, Indianapolis,
IN), and the eluate containing the 3′-end-labeled DNA substrate was
collected. Approximately 2 nM radiolabeled DNA substrate was incubated
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 mM sodium hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol, and
0.1% bromphenol 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). For
simplicity, cleavage sites were numbered as previously described in the 161-
bp fragment.46
models A−F were optimized at the HF/6-31G** level of theory. The
single point energy calculations were performed at the MP2/6-31G*
and HF/6-31G* levels of theory. The NBO analysis was performed at
the HF/6-31G** level of theory.
AUTHOR INFORMATION
Corresponding Author
*Phone: 765-494-1465. Fax: 765-494-6790. E-mail: cushman@
purdue.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was made possible by the National Institutes of
Health (NIH) through support of this work with Research
Grant UO1 CA89566, a Purdue Research Foundation Research
Grant, and the Center for Cancer Research, Intramural
Program of the National Cancer Institute. We thank the
Rosen Center for Advanced Computing (RCAC), Purdue
University, IN, for providing computing facilities.
ABBREVIATIONS USED
■
AIBN, azobisisobutyronitrile; DIAD, diisopropyl azodicarbox-
ylate; DMF, N,N-dimethylformamide; DMSO-d6, dimethyl-d6
sulfoxide; NBS, N-bromosuccinimide; TFA, trifluoroacetic acid;
THF, tetrahydrofuran; Top1, topoisomerase type I; Top1-
DNAcc, topoisomerase type I−DNA cleavage complex
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■
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