Application of sequential Cu(I)/Pd(0)-catalysis to solution-phase parallel synthesis of combinatorial libraries of dihydroindeno[1,2-c]isoquinolines

Article abstract of DOI:10.1021/co200027c

Parallel solution-phase synthesis of combinatorial libraries of dihydroindenoisoquinolines employing a sequential Cu(I)/Pd(0)-catalyzed multicomponent coupling and annulation protocol was realized. The scope and limitations of the protocol with respect to the substitution pattern in the aryl ring of the indene core, as well as the N-substituent have been defined, revealing that the methodology is compatible with a wide-range of aliphatic linear, branched, and ester functionalized N-substituents. Unexpectedly, the formation of regioisomers featuring a 1,2,3-contiguous substitution pattern in the aromatic ring of the indene core was observed. Three distinct combinatorial libraries with a total of 111 of members were synthesized, and 80 highly substituted dihydroindenoisoquinolines structurally related to known medicinal agents including some consisting of mixtures of two regioisomers were made available for biological activity testing.

Full text of DOI:10.1021/co200027c

RESEARCH ARTICLE
pubs.acs.org/acscombsci
Application of Sequential Cu(I)/Pd(0)-Catalysis to
Solution-Phase Parallel Synthesis of Combinatorial
Libraries of Dihydroindeno[1,2-c]isoquinolines
Sarvesh Kumar, Thomas O. Painter, Benoy K. Pal, Benjamin Neuenswander, and Helena C. Malinakova*
Department of Chemistry, The University of Kansas, 1251 Wescoe Hall Drive, Lawrence, Kansas 66045, United States, and Center of
Excellence in Chemical Methodologies and Library Development, The University of Kansas, 2034 Becker Drive, Shankel Structural
Biology Center, West Campus Lawrence, Kansas 66047-3761, United States
S Supporting Information
b
ABSTRACT: Parallel solution-phase synthesis of combinatorial libraries of
dihydroindenoisoquinolines employing a sequential Cu(I)/Pd(0)-catalyzed
multicomponent coupling and annulation protocol was realized. The scope
and limitations of the protocol with respect to the substitution pattern in the
aryl ring of the indene core, as well as the N-substituent have been defined,
revealing that the methodology is compatible with a wide-range of aliphatic
linear, branched, and ester functionalized N-substituents. Unexpectedly, the
formation of regioisomers featuring a 1,2,3-contiguous substitution pattern in
the aromatic ring of the indene core was observed. Three distinct combinator-
ial libraries with a total of 111 of members were synthesized, and 80 highly
substituted dihydroindenoisoquinolines structurally related to known medic-
inal agents including some consisting of mixtures of two regioisomers were
made available for biological activity testing.
KEYWORDS: solution-phase parallel synthesis, combinatorial libraries of heterocycles, dihydroindenoisquinolines, multicompo-
nent reactions, transition metal-catalyzed reactions
’ INTRODUCTION
Solution-phase parallel synthesis serves as a powerful tool for
the preparation of large libraries of compounds needed for drug
discovery.1 To deliver libraries featuring structurally complex and
chemically diverse chemotypes, new technically challenging
synthetic protocols must be adapted to the parallel synthesis
format.2 Multicomponent reactions, and particularly those cata-
lyzed by transition metals,3 are well suited for a rapid construc-
tion of diverse libraries. However, the application of transition
metal-catalyzed reactions often utilizing sensitive organometallic
reagents or reactive intermediates to parallel synthesis still
presents a synthetic and technical challenge.2
Recently, we have reported a new methodology for the
synthesis of indenoisoquinolines IV relying on a combination
of Cu(I)-catalyzed three-component coupling followed by an
intramolecular Pd(0)-catalyzed annulation, and demonstrated its
utility by the preparation of a series of indenoisoquinolines in a
classical synthetic format (Figure 1).4 Mechanistically, the process
involves an in situ formation of an acyliminium chloride from imines
I and acyl chlorides III. The acylimminium intermediate is then
attacked by an organocuprate generated from the vinyl stannane II
and the Cu(I) catalyst yielding the amide product (Figure 1).5 In the
second step, Pd(0) catalyst initiates an intramolecular Heck reaction
followed by an electrophilic palladation of the aromatic ring, and the
entire sequence is terminated by a reductive elimination that closes
the five-membered ring4 (Figure 1).
Figure 1. Synthetic strategy for the preparation of indenoisoquinolines.
Indenoisoquinolines V came into prominence in medicinal
chemistry as prototypes of novel anticancer chemotherapeutic
agents inspired by the structures of naturally occurring topoi-
somerase I inhibitors benzophenanthridine alkaloids, for exam-
ple, fagaronine, and a natural product camptothecin believed to
function as DNA intercalators (Figure 2).6 Dihydroindenoiso-
quinolines VI have been shown to exhibit potent cytotoxicity
Received: February 10, 2011
Revised:
April 16, 2011
Published: May 01, 2011
r
2011 American Chemical Society
466
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|

ACS Combinatorial Science
RESEARCH ARTICLE
Figure 2. Naturally occurring and synthetic cytotoxic topoisomerase I
inhibitors.
Figure 4. Building blocks for the validation library and Library I.
to solution-phase parallel synthesis of medium-size combinator-
ial libraries of indenoisoquinolines IV (Figure 3). The method
was successfully adapted for the parallel synthesis format, and the
scope and limitations in the substitution patterns on the inde-
noisoquinoline cores have been surveyed. The ability to achieve a
broad variation in the N-substituent in the imine building block
was demonstrated for the first time. Our methodology delivers a
highly modular approach to substituted indenoisoquinolines and
serves as a useful alternative to the traditionally employed
routes.8 Compounds prepared by this study have been made
available for a broad array of biological screens.9
Figure 3. Libraries of indenoisoquinolines synthesized by our methodology.
serving as prodrugs of indenoisoquinolines in cancer cells.7 In the
context of those studies, the angularly substituted dihydroinde-
noisoquinoline VII was synthesized and found to be less
cytotoxic but retained a moderate topoisomerise 1 inhibitory
activity, apparently acting via a mechanism distinct from the
DNA intercalation precluded by its nonplanar structure.7a This
finding is a source of motivation for a broader survey of biological
activities of angularly substituted indenoisoquinolines. Thus, the
development of a viable solution-phase parallel synthetic meth-
odology for the preparation of combinatorial libraries of novel
indenoisoquinolines is essential to providing an efficient access
to these valuable heterocyclic structures.
’ RESULTS AND DISCUSSION
To asses the challenges of adapting our methodology to the
solution-phase parallel library synthesis, we embarked on the
preparation of a limited validation library of six indenoisoquino-
lines 6{1ꢀ3, 1, 1ꢀ2}. The validation library featured two
elements of diversity, including three aromatic aldehydes
1{1ꢀ3} (R1) and two aroyl chlorides 5{1ꢀ2} (R3) (Figure 4).
In all cases N-benzylamine 2{1} was used to prepare the
corresponding imines 3{1ꢀ3, 1} (Scheme 1).
In the initial attempt at the validation library synthesis, the
reactions were performed in SPE tubes at room temperature
under argon atmosphere. No reaction was observed, presumably
because of lower reactivity of the vinyl tin reagent at room
Herein, we describe the application of our sequential Cu(I)/
Pd(0)-catalyzed multicomponent coupling/annulation protocol
467
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ACS Combinatorial Science
RESEARCH ARTICLE
temperature. Indeed reactions performed well at elevated tem-
peratures (45 °C, 6 h) in glass tubes. Furthermore, higher quality
of CuCl (>99% purity), improved drying of the imines building
blocks produced in bulk quantities, and the addition of molecular
sieves were employed. Thus, the protocol for the validation
library synthesis was modified. The preparation was performed in
the Miniblock XT synthesizer (24 membered) fitted with glass
vials and reflux condensers, and the reactions were run at 45 °C
for 6 h with magnetic stirring. Both the reagent addition and the
reaction were carried out under closed dry argon atmosphere(for
details see the Supporting Information).
steps and very respectable crude purities (38ꢀ78%). The final
products reached the purity standard higher than 90% measured
by HPLC (UV 214 nm) required for the samples to be acceptable
for biological testing.10 Identities of the products 6 were con-
firmed by 1H NMR analyses, and data for compounds
6{1,1,1ꢀ2} were compared to data obtained for samples prod-
uced in the classical format.4 The relative stereochemistry in
products 6 reported in this study was assigned in accordance with
the analytical data secured in our prior work.4 As anticipated,
both the C-1 and C-3 carbon in the 2-naphthyl substituted imine
underwent the terminal carbonꢀcarbon bond forming event,
providing 1:1 mixtures of isomeric indenoisoquinolines
6{3,1,1ꢀ2} as indicated by 1H NMR analyses, showing two sets
of signals for the benzylic protons in the N-Bn group (signals at
5.61 ppm, and at 4.81 ppm in the spectra for 6{3,1,1}) and for the
methine CHN protons, signals at 5.54 ppm and at 5.46 ppm in
the spectra for 6{3,1,1}). However, HPLC analyses did not
achieve separation of the peaks for the regioisomers, only giving
rise to unsymmetrical peaks in the HPLC chromatograms.
Satisfied with the results of the validation library synthesis, we
proceeded to explore the scope of the parallel synthesis protocol
with respect to the range of structural diversity that could be
tolerated in the aldehyde component. A series of imines
3{1ꢀ13,1} all derived from N-benzyl amine component 2{1}
were prepared (Figure 4). Electronically distinct substituents
(-Cl, -OMe, -Me, and ꢀNMe2) were placed at the para position
(C-4) of the aldehydes, and components with a varied placement
(meta and para) of electronically distinct substituents (-Cl,
-OMe, -Me) were included. The substituents and substitution
patterns in the aromatic rings were chosen based on the opera-
tional scheme for aromatic substitution suggested by Topliss in a
study on the methods for designing the most potent medicinal
agents in the series of compounds bearing aromatic rings.11 Since
electrophilic aromatic palladation reactions performed intermo-
lecularly were shown to strongly favor the formation of the less
sterically hindered regioisomers, failing to yield a contiguous
1,2,3-trisubstitution pattern,12 we were reasoning that steric
effects would disfavor the terminal palladation at the C-2 position
of the meta-substituted aldehydes 1{7,9,10}, and the formation
Gratifyingly, this protocol afforded all six intended validation
library members (Table 1) in good yields over the two synthetic
Scheme 1. Synthetic Scheme for the Validation Library and
Library I Synthesis
Table 1. Results of the Validation Library Synthesis
entry
compd
R1
R3
5-H
yielda (%)
purity crudeb (%)
purity finalb (%)
HRMSc
1
2
3
4
5
6
6{1,1,1}
6{1,1,2}
6{2,1,1}
6{2,1,2}
6{3,1,1}
6{3,1,2}
H
H
42
48
50
42
71
57
67
97
398.1755 (398.1756)
428.1859 (428.1861)
428.1862 (428.1861)
458.1963 (458.1967)
448.1906 (448.1912)
478.2009 (478.2018)
5-OMe
5-H
68
100
4-MeO
64
100
4-MeO
5-OMe
5-H
38
100
2-naphthyl
2-naphthyl
78(1:1)d
70 (1:1)d
92(1:1)d
92(1:1)d
5-OMe
a Isolated yield calculated for the entire two-step sequence. b UV purity determined at 214 nm. c The HRMS data for the M þ 1 molecular ion of the
compound 6 detected in the corresponding product. The calculated HRMS datafor the M þ 1 molecular ion are given in parentheses. d Ratio of the
regioisomers established by 1H NMR.
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RESEARCH ARTICLE
Table 2. Results of Library I Synthesis
entr compd
R1
R3
5-H
yielda (%) run 1f purityb (%) run 1f yielda (%) run 2 purityb (%) run 2 amt (mg)c pass/faild
HRMSe
1
2
3
4
5
6
7
8
9
6{1,1,1}
6{1,1,2}
6{1,1,3}
H
H
H
31 (42)
97 (97)
94 (100)
92
28
31
14
25
30
33
42
57
33
29
24
ꢁ
99
68
59
12
85
60
50
86
97
0
þ
398.1752 (398.1756)
428.1859 (428.1861)
434.1556 (434.1567)
428.1875 (428.1861)
458.1963 (458.1967)
464.1671 (464.1673)
448.1906 (448.1912)
478.2009 (478.2018)
484.1700 (484.1724)
432.1356 (432.1366)
462.1464 (462.1472)
468.1163 (468.1178)
412.1895 (412.1912)
442.2014 (442.2018)
448.1711 (448.1724)
448.1903 (448.1912)
478.2010 (478.2018)
484.1716 (484.1724)
432.1359 (432.1366)
5-OMe
4,5-F2
5-H
2 (48)
98
þ
16
86
þ
6{2,1,1} 4-MeO
6{2,1,2} 4-MeO
6{2,1,3} 4-MeO
6{3,1,1} 2-naphthyl
6{3,1,2} 2-naphthyl
6{3,1,3} 2-naphthyl
42 (50)
99 (100)
97 (100)
93
95
þ
5-OMe
4,5-F2
5-H
4 (42)
99
þ
30
93
þ
ꢁ (71)
ꢁ (92)
85 (92)
85
97 (1: 1)g
97 (1: 1)g
73 (1: 1)g
86
þ
5-OMe
4,5-F2
5-H
6 (57)
32
28
5
þ
ꢀ
10 6{4,1,1} 4-Cl
94
20
22
0
þ
11 6{4,1,2} 4-Cl
5-OMe
4,5-F2
5-H
70
94
þ
12 6{4,1,3} 4-Cl
7
88
ꢁ
97
ꢀ
13 6{5,1,1} 4-Me
14 6{5,1,2} 4-Me
15 6{5,1,3} 4-Me
16 6{6,1,1} 1-naphthyl
17 6{6,1,2} 1-naphthyl
18 6{6,1,3} 1-naphthyl
19 6{7,1,1} 3-Cl
35
4
98
36
38
22
7
50
32
43
15
0
þ
5-OMe
4,5-F2
5-H
94
96
þ
35
13
ꢁ
98
93
þ
95
95
EXh
EXh
EXh
ꢀ
5-OMe
4,5-F2
5-H
ꢁ
15
ꢁ
88
9
64
37g
ꢁ
0
20
12
29g
59
0
46
20 6{7,1,2} 3-Cl
5-OMe
19
61g
30
15
46g
15
þ
462.1468 (462.1472)
34
21 6{7,1,3} 3-Cl
4,5-F2
5ꢀH
5ꢀOMe
4,5-F2
5-H
ꢁ
ꢁ
10
32
26
17
34
13
0
ꢀ
ꢀ
ꢀ
ꢀ
þ
468.1167 (468.1178)
441.2177 (441.2178)
471.2280 (471.2283)
477.1980 (477.1989)
428.1859 (428.1861)
22 6{8,1,1} 4ꢀMe2N
23 6{8,1,2} 4ꢀMe2N
24 6{8,1,3} 4-Me2N
25 6{9,1,1} 3-OMe
31
14
14
29
80
86
0
80
84
0
76
72g
85
63g
0
50
27
29
26 6{9,1,2} 3-OMe
27 6{9,1,3} 3-OMe
28 6{10,1,1} 3-Me
29 6{10,1,2} 3-Me
30 6{10,1,3} 3-Me
5-OMe
4,5-F2
5-H
36
24
43
41
28
66
30
21
33
36
31
62g
31
21g
52
35
53
57
45
þ
þ
þ
þ
þ
458.1963 (458.1967)
464.1665 (464.1673)
412.1916 (412.1912)
442.2019 (442.2018)
448.1712 (448.1724)
33g
65g
28
66
66g
63g
32
67g
27g
74
31
5-OMe
4,5-F2
62g
35
63g
34
31 6{11,1,1} 3,4,5-(OMe)3 5-H
32 6{11,1,2} 3,4,5-(OMe)3 5-OMe
33 6{11,1,3} 3,4,5-(OMe)3 4,5-F2
37
41
45
24
15
27
91
39
34
9
93
92
82
69
75
87
62
66
40
0
þ
488.2066 (488.2073)
518.2188 (518.2178)
524.1886 (524.1884)
388.1539 (388.1548)
418.1649 (418.1654)
424.1354 (424.1360)
93
þ
97
þ
34 6{12,1,1} 3-furyl
35 6{12,1,2} 3-furyl
36 6{12,1,3} 3-furyl
5-H
74
19
17
16
EXh
EXh
EXh
5-OMe
4,5-F2
75
0
75
0
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Table 2. Continued
entr compd
R1
R3
5-H
yielda (%) run 1f purityb (%) run 1f yielda (%) run 2 purityb (%) run 2 amt (mg)c pass/faild
HRMSe
37 6{13,1,1} 2-indolyl
38 6{13,1,2} 2-indolyl
39 6{13,1,3} 2-indolyl
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
0
0
0
EXh
EXh
EXh
5-OMe
4,5-F2
a Isolated yield after HPLC purification calculated for the entire two-step sequence. b UV purity determined at 214 nm. c Amount of the sample that is
available in UV purity >90% (in mg). d Pass rating signified as (þ) denotes a library member that was produced in quantity of 5 mg or higher, and higher
than 90% UV purity in at least one of the two runs. Fail rating signified by (ꢀ) denotes a library member that did not fulfill these criteria both in
run 1 and run 2. e The HRMS data for the M þ 1 molecular ion of the compound 6 detected in the corresponding product. The calculated
HRMS datafor the M þ 1 molecular ion are given in parentheses. f Results from the validation library run are indicated in parentheses for comparison.
g Ratio of the regioisomeric products shown on the two lines (established from HPLC chromatograms) or in parentheses (established by 1H NMR). h
EX = excluded, building blocks were found to be outside the scope of the methodology, data was not included into the final evaluation. ꢁ = failed to yield
any product.
of single regioisomers of the corresponding indenoisoquinolines
was anticipated. The selection of aldehydes 1{12, 13} was driven
by the desire to incorporate additional heteroatoms into the
indenoisoquinolines 6. The diversity of the aroyl chloride
component was limited to three components 5{1ꢀ3} represent-
ing electronically neutral, electron rich, and electron deficient
substrates. Conceivably, electron deficient aroyl chlorides might
favor the imminium salt formation, as well as the oxidative
addition of the palladium(0) catalyst into the Csp2-Br bond in
the second step of the cascade event. Through the entire project,
ethoxycarbonyl-substituted vinyl stannane 4 was employed,
reasoning that the ester group in the indenoisoquinolines would
allow for subsequent elaboration into any number of desirable
appendages.
The protocol developed for the preparation of the validation
library was applied to the synthesis of Library I (39 members)
without modification. The crude reaction mixtures obtained in
the final step were analyzed by HPLC to establish the crude
purity (UV 214 nm), and were subsequently submitted to
preparative HPLC with mass-directed fractionation to produce
the final samples, the purity of which was established by HPLC
(UV 214 nm) (run 1, Table 2). To evaluate reproducibility of the
protocol, the preparation of the Library I was repeated under
identical conditions for the synthesis (run 2, Table 2). A minor
difference in the purification conditions involved the use of
neutral mobile phase for the run one (1) analysis and purification,
and an alkaline pH 9.8 mobile phase for analysis and purification
of the run two (2). The yields, purities (HPLC, UV 214 nm) for
both the runs one (1) and two (2) as well as final amount (mg) of
material available in purity higher than 90% are given in Table 2.
Furthermore, compound identities were confirmed by 1H NMR
analyses that were performed on 21 out of the 39 members 6.
Selected library members (6 members) were fully characterized
(see the Supporting Information).
C-6 carbons of the aromatic ring of the carbaldehydes in
significant quantities (1:2ꢀ1:3).13 Thus, an unexpected example
of an intramolecular electrophilic palladation providing the
contiguous 1,2,3-trisubstitution pattern was uncovered.14,15
-
When integrations for both the partially resolved peaks of the
regioisomeric indenoisoquinolines from the HPLC chromato-
grams were entered into the results in Table 2, the combined
purities were found to be significantly higher than 90% for
products 6{9ꢀ10,1,1ꢀ3}, and 88% and 91% for products
6{7,1,1} and 6{7,1,2}, respectively, bearing an electron-with-
drawing Cl substituent. As expected (vide infra) indenoisoqui-
nolines 6{3,1,1ꢀ3} derived from 2-naphthyl carbaldehyde were
produced as 1:1 mixture of regioisomers that were not separated
by HPLC.
Aldehydes bearing a hydrogen or an electron-donating sub-
stituent in the para position (15 entries) all afforded the
corresponding indenoisoquinolines in good yields (20ꢀ40%
over the two step sequence) and good purities (>90%), regard-
less of the choice of the acyl chloride component. Indenoisoqui-
nolines 6{8, 1, 1ꢀ3} derived from 4-Me2N-substituted
carbaldehyde 1{8} proved to be an intriguing exception, being
isolated in good yields but only 80ꢀ86% purity by HPLC (UV
214 nm). The samples of indenoisoquinolines 6{8,1,1ꢀ3} were
repurified by flash column chromatography. However, the purity
by HPLC (UV 214 nm) had not improved. It is possible that the
presence of the amine group and the choice of pH of the elution
phase for HPLC leads to a retention of an impurity, the nature of
which could not be elucidated from 1H and 13C NMR analyses.
As anticipated based on our prior work,5 electron deficient
aldehydes 1{4} and 1{7} afforded the indenoisoquinoline pro-
ducts 6{4, 1, 1ꢀ3} and 6{7, 1, 1ꢀ3} in diminished yields lower
than 30% (mostly 7ꢀ15%) and somewhat lower purities
(88ꢀ94%).
The preparation of Library I allowed us to survey the scope of
the aldehyde building block for the novel indenoisoquinoline
synthesis in the parallel format, identifying three aldehydes (1-
naphthyl, 3-furyl, and 2-indolyl, a total of 9 entries from 39, 23%)
as unsuitable for the protocol. An unexpected formation of
regioisomers via electrophilic aromatic palladation from meta-
substituted aromatic carbaldehydes was observed. We confirmed
that electron deficient carbaldehydes gave rise to lower yields,
still providing useful product quantities. After subtracting the
nine (9) entries that employed aldehydes outside the scope of
this protocol and combining both the regioisomers of the
indenoisoquinolines to signify a “combined final purity”, the
success rate within the remaining 30-membered suite of com-
pounds, defined as obtaining more than 5 mg of the product with
Data in Table 2 indicate that imines derived from 1-naphthyl-,
3-furyl-, and 2-indolyl carbaldehydes 1{6}, 1{12} and1{13} did
not prove to be competent substrates for this methodology,
giving heterocycles 6 in low yields, and purities, or failing to
deliver the products at all. Intrigued by the unexpectedly low
purities initially reported for indenoisoquinolines 6 derived from
the meta-substituted imines 3{7,1}, 3{9,1} and3{10,1} (3-Cl,
3-OMe and 3-Me), we carefully examined the HPLC chromato-
1
grams, MS data, and H NMR analyses of the corresponding
indenoisoquinolines 6. In fact, the analytical data revealed that
purified samples of indenoisoquinolines 6 formed from imines
3{7,1}, 3{9,1}, and 3{10,1} possessed both the regioisomeric
indenoisoquinolines arising from substitution at either C-2 or
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Scheme 2. Synthesis of Library II
opportunity to significantly modify the physical, chemical, and
biological properties of the resulting heterocycles.16 Thus we
embarked on parallel synthesis of Library II (24 members).
Previously, N-benzyl amine 2{1} was employed exclusively in all
our syntheses of indenoisoquinolines. For the construction of
Library II, six amines 2{2ꢀ7} including aliphatic amines with
straight chains, branching in R-positions or an ester (ꢀCOOMe)
group at either R- or in β-positions were combined with
aldehydes 1{2} and 1{4} bearing an electron donating (OMe)
or electron withdrawing (Cl) substituents in the para positions to
afford imines substrates 3{2,2ꢀ7} and 3{4,2ꢀ7} (Figure 5).
Aroyl chlorides 5{1ꢀ2} were chosen to complete the building
block set for Library II (Figure 5).
The protocol for the synthesis and purification used in the
preparation of Library I was applied to the preparation of Library
II without modifications. The synthetic sequence is depicted in
Scheme 2.
The purities of the final products were established by HPLC
(UV 214 nm), and the identities of products 6 were confirmed by
recording 1H NMR spectra for 6 out of the total of 24 of library
members (Table 3). Selected library members (5 members) were
fully characterized (see the Supporting Information). Data in
Table 3 confirm that a broad diversification in the N-substituent
of indenoisoquinolines 6 can be realized. Indenoisoquinolines 6
were obtained in yields 29ꢀ45% over the two synthetic steps with
three exceptions, predictably involving the electron deficient para
chloro-substituted aldehyde 1{4}. Imines 3{2,6ꢀ7} and 3{4,6ꢀ7}
featuring the N-glycine and its homologue as the N-substituents
proved to be the most challenging substrates, giving lower yields
and purities of the corresponding indenoisoquinolines. Never-
theless, five (5) out of eight (8) total entries with these N-
substituents afforded acceptable quantities and purities of the
indenoisoquinolines 6 with the glycine or homoglycine N-substit-
uents (entries 9ꢀ12 and 21ꢀ24 Table 3). Twenty library members
were obtained in quantities higher than 5 mg (10ꢀ38 mg) and
purities higher than 90% (HPLC, UV 214 nm) corresponding to
83% success rate for Library II preparation. A single diastereomer of
each product was obtained.The relative stereochemistry at the C-5/
C-6 ring juncture in the heterocycles 6{2,2ꢀ7,1ꢀ2} and
6{4,2ꢀ7,1ꢀ2} was assigned in analogy to the structures of
indenoisoquinolines 6 bearing the N-benzyl substituent, and an
NOE study on product 6{2,3,1} was also attempted.17
Figure 5. Building blocks for Library II.
HPLC (UV 214 nm) purity higher than 90% was 77%.10 The
seven (7) members considered as failed (from the 30-compound
suite) include six compounds possessing relatively high purities
(84ꢀ88% HPLC, UV 214 nm) and including the three 4-Me2N-
substituted products 6{8,1,1ꢀ3} (84ꢀ86% HPLC purity) for
which the contaminant could not be detected by H and 13C
1
NMR spectroscopy.
Having defined the scope of the methodology with respect to
the aldehyde building block 1, we turned our attention to
exploring for the first time the diversification of the amine
building block 2. Variations in the N-substituent present an
471
dx.doi.org/10.1021/co200027c |ACS Comb. Sci. 2011, 13, 466–477

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RESEARCH ARTICLE
Table 3. Results from Library II Synthesis
entry
compd
R1
R2
R3
yielda (%)
purityb (%)
amt (mg)c
pass/faild
HRMSe
1
6{4,2,1}
6{2,2,1}
6{4,3,1}
6{2,3,1}
6{4,4,1}
6{2,4,1}
6{4,5,1}
6{2,5,1}
6{4,6,1}
6{2,6,1}
6{4,7,1}
6{2,7,1}
6{4,2,2}
6{2,2,2}
6{4,3,2}
6{2,3,2}
6{4,4,2}
6{2,4,2}
6{4,5,2}
6{2,5,2}
6{4,6,2}
6{2,6,2}
6{4,7,2}
6{2,7,2}
4-Cl
Me
Me
H
37
33
17
45
20
31
20
39
10
17
20
41
41
29
37
29
ꢁ
94
100
100
97
100
97
100
93
98
98
76
95
98
100
96
94
ꢁ
22
20
12
31
13
20
14
28
7
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
ꢀ
þ
þ
þ
þ
þ
ꢀ
þ
þ
þ
ꢀ
þ
ꢀ
þ
356.1049 (356.1053)
352.1577 (352.1548)
412.1666 (412.1679)
408.2171 (408.2174)
384.1375 (384.1366)
380.1885 (380.1861)
424.1671 (424.1679)
420.2175 (420.2174)
414.1122 (414.1108)
410.1606 (410.1603)
428.1270 (428.1264)
424.1781 (424.1760)
386.1188 (386.1159)
382.1664 (382.1654)
442.1798 (442.1785)
438.2293 (438.2280)
2
4-OMe
4-Cl
H
3
n-C5H11
H
4
4-OMe
4-Cl
n-C5H11
H
5
i-C3H7
H
6
4-OMe
4-Cl
i-C3H7
H
7
C6H11 cyclohexyl
C6H11 cyclohexyl
CH2COOMe
CH2COOMe
(CH2)2COOMe
(CH2)2COOMe
Me
H
8
4-OMe
4-Cl
H
9
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
4-OMe
4-Cl
H
12
14
30
27
19
28
21
ꢁ
25
21
24
4
H
4-OMe
4-Cl
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
4-OMe
4-Cl
Me
n-C5H11
4-OMe
4-Cl
n-C5H11
i-C3H7
4-OMe
4-Cl
i-C3H7
35
27
31
6
99
96
92
17
98
84
94
410.1978 (410.1967)
454.1773 (454.1785)
450.2276 (450.2280)
444.1211 (444.1213)
440.1694 (440.1709)
458.1363 (458.1370)
454.1848 (454.1865)
C6H11 cyclohexyl
C6H11 cyclohexyl
CH2COOMe
CH2COOMe
(CH2)2COOMe
(CH2)2COOMe
4-OMe
4-Cl
4-OMe
4-Cl
16
20
45
12
15
38
4-OMe
a Isolated yield after HPLC purification calculated for the entire two-step sequence. b UV purity determined at 214 nm. c Amount of the sample that is
available in UV purity >90% (in mg). d Pass rating signified as (þ) denotes a library member that was produced in quantity of 5 mg or higher, and higher
than 90% UV purity. Fail rating signified by (ꢀ) denotes a library member that did not fulfill these criteria. e The HRMS data for the M þ 1 molecular ion
of the compound 6 detected in the corresponding product.The calculated HRMS datafor the M þ 1 molecular ion are given in parentheses.ꢁ = failed to
yield any product.
After evaluating the results from the Library I and Library II
syntheses, building blocks for the preparation of Library III were
selected, aiming to assess the performance of the parallel
synthetic methodology in systems diversifying both the amine
and the aldehyde components. Building blocks that performed
successfully in syntheses of Libraries I and II were chosen. Thus,
electron neutral, electron rich, and electron deficient as well as
one meta-substituted aldehyde 1{1,2,4,5,10 and 11} (Figure 4)
were combined with four (4) amines 2{2,4,5, and 7} (Figure 5)
to deliver the corresponding imines 3 (Figure 6). Utilizing the
imines 3 shown in Figure 6 and two (2) aroyl chlorides 5{1ꢀ2} a
48-membered Library III of indenoisoquinoliens 6 was synthe-
sized proceeding according to the synthetic sequence outlined in
Scheme 3 and employing the previously established method for
the synthesis and purification (Table 4).
seven (7) indenoisoquionolines were fully characterized (see the
Supporting Information). Representative structures of indenoi-
soquinolines 6 prepared in Library III are shown in Figure 7.
In agreement with our prior observations, the inspection of
HPLC and 1H NMR data for indenoisoquinolines arising from
the meta-Me substituted aldehyde 1{10} indicated the formation
of significant quantities (2:3 to 1:2 ratios) of both the regioi-
somers featuring two distinct substitution patterns in the aro-
matic ring of the indene substructure (Figure 7).18
When the final purities of these library members were adjusted
for considering the “combined” content of both the regioisomers,
37 out of a total of 48 samples were obtained in purities higher
than 90% (HPLC, UV 214 nm) and sufficient quantities. Out of
the 11 members that failed to fulfill these criteria, 6 library
members were obtained in good quantities and purities 81ꢀ89%.
These results translate into a success rate of 77% for compounds
with HPLC purities >90%, and a success rate 89% for
1
To confirm the identities of the library members, H NMR
was recorded on 15 out of the total of 48 library members, and
472
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ACS Combinatorial Science
RESEARCH ARTICLE
Figure 6. Building blocks for Library III.
Scheme 3. Synthesis of Library III
N-homoglycine substituent and the electron deficient aldehyde
were among the members that failed the purity criteria giving
products with only 72% and 84% purity by HPLC (UV 214 nm).
’ CONCLUSION
In conclusion, a new and efficient method for solution phase
parallel synthesis of combinatorial libraries of dihydroindeno-
[1,2-c]isoquinolines relying on a sequence of Cu(I)-catalyzed
three-component coupling and Pd(0)-catalyzed intramolecular
annulation4 was developed. The scope and limitations of the
protocol with respect to the substitution pattern in the aryl ring
of the indene core, as well as the N-substituent have been defined,
revealing that the methodology is compatible with a wide-range
of aliphatic linear, branched, and ester functionalized N-substit-
uents. Unexpectedly, the formation of regioisomers possessing a
1,2,3-contiguous substitution pattern in the aromatic ring of the
indene core via an intramolecular electrophilic palladation was
observed. Since preparative HPLC purification methods that
have not been specifically optimized for the separation of the
regioisomers already achieved a partial separation, we conclude
that preparation of pure individual regioisomers is feasible, and
work toward this goal is ongoing. To date, three distinct
combinatorial libraries were synthesized and delivered 80 com-
pounds with novel highly substituted indenoisoquinoline struc-
tures (some consisting of mixtures of regioisomers) that are
compounds with HPLC purities (>80%). As anticipated, lower
yields (10ꢀ15%) in comparison to the yields of other members
(20ꢀ30%) were achieved with the electron deficient para-chloro
substituted aldehyde, and 4 out of the 11 library members that
failed the 90% purity criteria employed the aldehyde 1{4}.
Furthermore, the two library members utilizing the challenging
473
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Table 4. Results from Library III Synthesis
entry
compd
R1
R2
R3
yielda (%)
purityb (%)
amt (mg)c
pass/faild
HRMSe
1
2
3
4
5
6
7
8
9
6{1,2,1}
6{1,2,2}
6{2,2,1}
6{2,2,2}
6{4,2,1}
6{4,2,2}
6{5,2,1}
6{5,2,2}
6{10,2,1}
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
ꢁ
ꢁ
0
ꢀ
þ
þ
ꢀ
þ
ꢀ
þ
þ
þ
ꢀ
OMe
H
32
27
ꢁ
100
100
13
99
87
100
99
58f
42
56f
43
95
97
94
98
99
96
100
89
99
78
38f
58
61f
33
95
96
89
94
81
86
96
98
97
91
95g
91g
89
97
96
94
97
ꢁ
19
16
1
352.1553 (3521548)
352.1550 (352.1548)
382.1650 (382.1654)
356.1058 (356.1053)
386.1170 (386.1159)
336.1605 (336.1599)
366.1724 (366.1705)
336.1607 (336.1599)
4-OMe
4-OMe
4-Cl
OMe
H
26
31
27
31
40
16
21
16
19
17
4-Cl
OMe
H
4-Me
4-Me
3-Me
OMe
H
10
6{10,2,2}
3-Me
Me
OMe
30
19
þ
366.1711 (366.1705)
11
12
13
14
15
16
17
18
19
20
21
6{11,2,1}
6{11,2,2}
6{1,4,1}
6{1,4,2}
6{2,4,1}
6{2,4,2}
6{4,4,1}
6{4,4,2}
6{5,4,1}
6{5,4,2}
6{10,4,1}
3,4,5-(OMe)3
3,4,5-(OMe)3
H
Me
Me
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
H
27
33
16
22
16
23
10
15
14
27
11
19
25
9
þ
þ
þ
þ
þ
þ
þ
ꢀ
þ
ꢀ
þ
412.1763 (412.1760)
442.1859 (442.1865)
350.1754 (350.1756)
380.1859 (380.1861)
380.1867 (380.1861)
410.1967 (410.1967)
384.1349 (384.1366)
414.1475 (414.1472)
364.1920 (364.1912)
394.2017 (394.2018)
364.1914 (364.1912)
OMe
H
H
OMe
H
14
10
16
7
4-OMe
4-OMe
4-Cl
OMe
H
4-Cl
OMe
H
11
8
4-Me
4-Me
OMe
H
18
7
3-Me
22
6{10,4,2}
3-Me
i-Pr
OMe
26
17
þ
394.2023 (394.2018)
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
6{11,4,1}
6{11,4,2}
6{1,5,1}
6{1,5,2}
6{2,5,1}
6{2,5,2}
6{4,5,1}
6{4,5,2}
6{5,5,1}
6{5,5,2}
6{10,5,1}
6{10,5,2}
6{11,5,1}
6{11,5,2}
6{1,7,1}
6{1,7,2}
6{2,7,1}
6{2,7,2}
3,4,5-(OMe)3
3,4,5-(OMe)3
H
i-Pr
i-Pr
H
23
21
23
24
25
23
15
7
17
17
15
17
18
18
11
5
þ
þ
þ
þ
ꢀ
ꢀ
þ
þ
þ
þ
þ
þ
ꢀ
þ
þ
þ
þ
ꢀ
440.2079 (440.2073)
470.2180 (470.2178)
390.2076 (390.2069)
420.2174 (420.2174)
420.2183 (420.2174)
450.2276 (450.2280)
424.1680 (424.1679)
454.1777 (454.1785)
404.2233 (404.2225)
434.2333 (434.2331)
404.2238 (404.2225)
434.2337 (434.2331)
480.2370 (480.2386)
510.2476 (510.2491)
394.1669 (394.1654)
424.1760 (424.1760)
424.1776 (424.1760)
ꢀ
OMe
H
cyclohexyl
H
cyclohexyl
OMe
H
4-OMe
4-OMe
4-Cl
cyclohexyl
cyclohexyl
OMe
H
cyclohexyl
4-Cl
cyclohexyl
OMe
H
4-Me
cyclohexyl
17
28
21
26
14
29
14
20
22
ꢁ
12
20
15
19
12
25
9
4-Me
cyclohexyl
OMe
H
3-Me
cyclohexyl
3-Me
cyclohexyl
OMe
H
3,4,5-(OMe)3
3,4,5-(OMe)3
H
cyclohexyl
cyclohexyl
OMe
H
(CH2)2 COOMe
(CH2)2 COOMe
(CH2)2 COOMe
(CH2)2 COOMe
H
OMe
H
15
16
0
4-OMe
4-OMe
OMe
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RESEARCH ARTICLE
Table 4. Continued
entry
compd
R1
R2
R3
yielda (%)
purityb (%)
amt (mg)c
pass/faild
HRMSe
41
42
43
44
45
6{4,7,1}
6{4,7,2}
6{5,7,1}
6{5,7,2}
6{10,7,1}
4-Cl
(CH2)2 COOMe
(CH2)2 COOMe
(CH2)2 COOMe
(CH2)2 COOMe
(CH2)2 COOMe
H
13
21
28
26
25
72
84
93
98
53f
37
58
37f
96
95
10
17
20
19
18
ꢀ
ꢀ
þ
þ
þ
428.1279 (428.1264)
458.1358 (458.1370)
408.1812 (408.1810)
438.1903 (438.1916)
408.1820 (408.1810)
4-Cl
OMe
H
4-Me
4-Me
3-Me
OMe
H
46
6{10,7,2}
3-Me
(CH2)2 COOMe
OMe
22
16
þ
438.1907 (438.1916)
47
48
6{11,7,1}
6{11,7,2}
3,4,5-(OMe)3
3,4,5-(OMe)3
(CH2)2 COOMe
(CH2)2 COOMe
H
28
32
23
28
þ
þ
484.1965 (484.1971)
514.2053 (514.2077)
OMe
a Isolated yield after HPLC purification calculated for the entire two-step sequence. b UV purity determined at 214 nm. c Amount of the sample that is
available in UV purity >90% (in mg). d Pass rating signified as (þ) denotes a library member that was produced in quantity of 5 mg or higher, and higher
than 90% UV purity. Fail rating signified by (ꢀ) denotes a library member that did not fulfill these criteria. e The HRMS data for the M þ 1 molecular ion
of the compound 6 detected in the corresponding product.The calculated HRMS datafor the M þ 1 molecular ion are given in parentheses. f Ratio of the
regioisomeric products established from the HPLC chromatogram is shown on the two lines. g The peaks for the regioisomers were not resolved by
HPLC, the presence of regioisomers was detected by 1H NMR. ꢁ = failed to yield any product.
Figure 7. Examples of indenoisoquinolines prepared in Library III.
currently being evaluated in high-throughput screens for a wide
range of biological activities.9
diluted with ethyl acetate (1.0 mL) and saturated aqueous KF
solution (1.0 mL) and stirred further at room temperature for 16 h.
The reaction mixtures were filtered into reaction vials (17 ꢁ 100
mm) through PrepSep silica gel tubes (SPE) under air pressure, and
each tube was rinsed with ethyl acetate (2 ꢁ 3 mL) and the rinsed
portions were eluted through SPE tubes. The eluents were
evaporated using a GeneVac EZ-2 evaporator.
Anhydrous sodium acetate (13.9 mg, 0.17 mmol, 1.0 equiv)
and palladium acetate (1.9 mg, 0.0085 mmol, 0.05 equiv) were
added by solid dispensers to reaction vials (17 ꢁ 100 mm)
containing the crude products from the first step, the reaction
vials were flushed with dry argon for 5 min at room temperature,
and anhydrous DMF (1.8 mL) was added via a syringe. Reaction
mixtures were heated under argon atmosphere at 120 °C for 24 h
on IKA plates with the thermocouple inserted into the metal
block. The reaction mixtures were cooled to room temperature
and diluted with ethyl acetate (2.0 mL) and filtered into barcoded
CCT tubes through PrepSep silica gel columns (SPE) under air
pressure. The reaction tubes were washed with ethyl acetate
’ EXPERIMENTAL PROCEDURES
General Procedure for the Parallel Synthesis of the Libraries.
24-Member MiniBlock XT synthesizer vials (17 ꢁ 100 mm) were
charged with activated 3 Å MS (2.0 g) and CuCl (g99.0% purity,
3.4 mg, 0.034 mmol, 0.2 equiv) utilizing a solid dispenser. Reaction
vials were flushed with dry argon for 5 min. The stock solutions
of imines 3 (0.17 mmol, 1.5 mL, 1.0 equiv) and aroyl chlorides
5 (0.22 mmol, 1.5 mL, 1.3 equiv) in acetonitrile were injected via a
syringe into the reaction vials. The reaction mixtures were stirred at
room temperature for 5 min under dry argon atmosphere followed
by the addition of the stock solution of the vinylstannane 4
(0.34 mmol, 1.5 mL, 2.0 equiv) in dichloromethane. The reaction
vials were stirred at 45 °C for 6 h under argon atmosphere on IKA
stirring plates with a thermocouple inserted into the metal block.
The reaction mixtures were cooled to room temperature and
475
dx.doi.org/10.1021/co200027c |ACS Comb. Sci. 2011, 13, 466–477

ACS Combinatorial Science
RESEARCH ARTICLE
(2 ꢁ 2 mL) and filtered through SPE tubes as well. The solvents
were removed on GeneVac HT4 evaporator. Crude products
were subjected to HPLC (UV 214 nm) analysis followed by
preparative HPLC purification with mass directed fractionation.
annulation protocol for the synthesis of indenoisoquinolines. Org. Lett.
2009, 11, 815.
(5) Black, D. A.; Arndtsen, B. A. Copper-Catalyzed Cross-Coupling
of Imines, Acid Chlorides, and Organostannanes: A Multicomponent
Synthesis of R-Substituted Amides. J. Org. Chem. 2005, 70, 5133.
(6) (a) Tillequin, F. Rutaceous alkaloids as models for the design of
novel antitumor drugs. Phytochem. Rev. 2007, 6, 65. (b) Antony, S.;
Agama, K. K.; Miao, Z.-H.; Takagi, K.; Wright, M. H.; Robles, A. I.;
Varticovski, L.; Nagarajan, M.; Morrell, A.; Cushman, M.; Pommier, Y.
Novel Indenoisoquinolines NSC 725776 and NSC 724998 Produce
Persistent Topoisomerase I Cleavage Complexes and Overcome Multi-
drug Resistance. Cancer. Res. 2007, 67, 10397. (c) Nagarajan, M.;
Morrell, A.; Fort, B. C.; Meckley, M. R.; Antony, S.; Kohlhagen, G.;
Pommier, Y.; Cushman, M. Synthesis and Anticancer Activity of
Simplified Indenoisoquinoline Topoisomerase I Inhibitors Lacking
Substituents on the Aromatic Rings. J. Med. Chem. 2004, 47, 5651.
(d) Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M.
Synthesis of nitrated indenoisoquinolines as topoisomerase I inhibitors.
Bioorg. Med. Chem. Lett. 2004, 14, 3659.
(7) (a) Xiao, X.; Miao, Z.-H.; Antony, S.; Pommier, Y.; Cushman, M.
Dihydroindenoisoquinolines function as prodrugs of indenoisoquino-
lines. Bioorg. Med. Chem. Lett. 2005, 15, 2795. (b) Jayaraman, M.; Fox,
B. M.; Hollingshead, M.; Kohlhagen, G.; Pommier, Y.; Cushman, M.
Synthesis of New Dihydroindeno[1,2-c]isoquinoline and Indenoisoqui-
nolinium Chloride Topoisomerase I Inhibitors Having High in Vivo
Anticancer Activity in the Hollow Fiber Animal Model. J. Med. Chem.
2002, 45, 242.
’ ASSOCIATED CONTENT
S
Supporting Information.
General experimental pro-
b
cedures, full characterization data for 18 compounds and copies
of 1H and 13C NMR spectra and HPLC traces for fully
characterized compounds (18) and additional selected products.
This material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 1-785-864-4743. Fax: 1-785-864-5396. E-mail: hmalina@
ku.edu.
Funding Sources
This work was supported by the National Institutes of Health
Kansas University Chemical Methodologies and Library Devel-
opment Center of Excellence (NIH0063950 P50).
(8) (a) Mahmoud, M. R.; EL-Shahawi, M. M.; El-Bordany, E. A. A.;
Abu, E.-A.; Fatma, S. M. Synthesis of Novel Indeno[1,2-c]isoquinoline
Derivatives. Synth. Commun. 2010, 40, 666. (b) Van, H. T. M.; Le, Q. M.;
Lee, K. Y.; Lee, E.-S.; Kwon, Y.; Kim, T. S.; Le, T. N.; Lee, S.-H.; Cho,
W.-J. Convenient synthesis of indeno[1,2-c]isoquinolines as constrained
forms of 3-arylisoquinolines and docking study of a topoisomerase I
inhibitor into DNA-topoisomerase I complex. Bioorg. Med. Chem. Lett.
2007, 17, 5763. (c) Morrell, A.; Placzek, M.; Parmley, S.; Grella, B.;
Antony, S.; Pommier, Y.; Cushman, M. Optimization of the Indenone
Ring of Indenoisoquinoline Topoisomerase I Inhibitors. J. Med. Chem.
2007, 50, 4388.
(9) Evaluation of the biological activity of the submitted compounds
in high-throughput screens is currently underway, and results will
become available via PubChem (http://www.ncbi.nlm.nih.gov/
pccompound).
(10) The KU-CMLD is required, under the terms of its grant, to
adhere to these standards (HPLC assay >90% (UV 214 nm), 5 mg
minimum mass) for submission of compounds to the Molecular
Libraries and Small Molecule Repository (MLSMR). KU biological
collaborators obtain the identical compounds directly from CMLD.
(11) Topliss, J. G. Utilization of operational schemes for analog
synthesis in drug design. J. Med. Chem. 1972, 15, 1006.
’ ACKNOWLEDGMENT
We thank our colleague Dr. Conrad Santini for his assistance
with the organization of the library synthesis.
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Hz, 0.74 H) corresponding to the isomer with 1,2,4-substituted
aromatic ring.1H NMR data for compound 6{10,1,1} featured signals
at 7.01 ppm (t, J = 7.5 Hz, 0.45 H) indicating the presence of the
regioisomer with a contiguously substituted aromatic ring, and at 6.70
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ACS Combinatorial Science
RESEARCH ARTICLE
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(17) The NOE data recorded for the indenoisoquinoline 6{2,3,1}
were consistent with the proposed structure, but were inconclusive in
terms of assigning the relative stereochemistry. Efforts to obtain single
crystals of several indenoisoquinolines 6 with diverse N-groups are in
progress, but so far did not afford satisfactory results.
(18) 1H NMR spectra recorded for compound 6{10,5,2} (no
resolution of regioisomers was indicated by HPLC chromatogram)
indicated the presence of two regioisomers in approximately 1:1
ratio, revealed by the signals at 6.94 ppm (s, 0.5 H); 5.33 (s, 0.5 H),
5.32 (s, 0.5 H).
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