Synthesis and Mechanism Studies of 1,3-Benzoazolyl Substituted Pyrrolo[2,3-b]pyrazine Derivatives as Nonintercalative Topoisomerase II Catalytic Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.5b01284

Novel topoisomerase II (Topo II) inhibitors have gained considerable interest for the development of anticancer agents. In this study, a series of 1,3-benzoazolyl-substituted pyrrolo[2,3-b]pyrazine derivatives were designed, synthesized, and evaluated as potential Topo II catalytic inhibitors. It was found that some of derivatives had good antiproliferative activity on seven cancer cell lines, especially on HL-60/MX2, a cancer cell line derivative from HL-60 that is resistant to Topo II poison. Topo II mediated DNA relaxation assay results showed that derivatives could significantly inhibit the activity of Topo II, and the structure-activity relationship studies indicated the importance of the alkylamino side chain and the benzoazolyl group. Further mechanism studies revealed that derivatives function as Topo II nonintercalative catalytic inhibitors and may block the ATP binding site of Topo II. Moreover, flow cytometric analysis showed that this class of compounds could induce apoptosis of HL-60 cells.

Full text of DOI:10.1021/acs.jmedchem.5b01284

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Article
Synthesis and Mechanism Studies of 1, 3-Benzoazolyl
Substitued Pyrrolo[2,3-b]pyrazine Derivatives as Non-
intercalative Topoisomerase II Catalytic Inhibitors
Peng-Hui Li, Ping Zeng, Shuo-Bin Chen, Pei-Fen Yao, Yan-Wen Mai, Jia-Heng
Tan, Tian-Miao Ou, Shi-liang Huang, Ding Li, Lian-Quan Gu, and Zhi-Shu Huang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01284 • Publication Date (Web): 09 Dec 2015
Downloaded from http://pubs.acs.org on December 14, 2015
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Synthesis and Mechanism Studies of 1, 3-Benzoazolyl Substituted
Pyrrolo[2,3-b]pyrazine Derivatives as Non-intercalative Topoisomerase II
Catalytic Inhibitors
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PengꢀHui Li, Ping Zeng, ShuoꢀBin Chen, PeiꢀFen Yao, YanꢀWen Mai, JiaꢀHeng Tan, TianꢀMiao Ou,
ShiꢀLiang Huang*, Ding Li, LianꢀQuan Gu, ZhiꢀShu Huang*
School of Pharmaceutical Sciences, Sun Yatꢀsen University, Guangzhou 510006, People’s Republic
of China
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ABSTRACT:
Novel topoisomerase II (Topo II) inhibitors have gained considerable interests for development of
anticancer agents. In this study, a series of 1,3ꢀbenzoazolylꢀsubstituted pyrrolo[2,3ꢀb]pyrazine
derivatives were designed, synthesized, and evaluated as potential Topo II catalytic inhibitors. It was
found that some of derivatives had good antiproliferative activity on seven cancer cell lines,
especially on HLꢀ60/MX2, a cancer cell line derivative from HLꢀ60 that is resistant to Topo II poison.
Topo II mediated DNA relaxation assay results showed that derivatives could significantly inhibit the
activity of Topo II, and the structureꢀactivity relationship studies indicated the importance of the
alkylamino side chain and the benzoazolyl group. Further mechanism studies revealed that
derivatives function as Topo II nonꢀintercalative catalytic inhibitor and may block the ATP binding
site of Topo II. Moreover, flow cytometric analysis showed that this class of compounds could
induce apoptosis of HLꢀ60 cells.
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KEYWORDS: Pyrrolo[2,3ꢀb]pyrazines; benzoazolyl; Topo II; nonꢀintercalative catalytic inhibitor;
antiꢀproliferation
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INTRODUCTION
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Human topoisomerase (Topo) has been recognized as an important target in anticancer drug
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discovery. Two types of topoisomerase exist in humans, namely, type I topoisomerase (Topo I)
and type II topoisomerase (Topo II). Both isomers are nuclear enzymes essential to resolve
topological problems that occur during DNA transcription, replication, and chromosome
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segregation.
Topo I, introduces singleꢀstrand breaks in DNA, whereas Topo II introduces
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doubleꢀstrand breaks and requires ATP for full activity. Given that the activity of Topo is essential
for several cellular processes, investigating the inhibitory activities of eukaryotic Topo is widely
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performed in anticancer drug development.
Topo II is the specific target of some of the most active anticancer drugs such as etoposide,
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doxorubicin, mitoxantrone, amonafide, and amsacrine. Drugs or agents targeting Topo II based on
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their mode of action are classified into two types: Topo II poison and Topo II catalytic inhibitor.
Topo II poison (e.g., doxorubicin, etoposide, amsacrine, and mitoxantrone) stabilizes the
TopoꢀDNA complex and prevents the cleaved DNA strand (s) from religation, leading to an
accumulation of undesired truncated DNA. The rest of the Topo II inhibitors, aside from Topo II
poisons, can be grouped as Topo II catalytic inhibitors. These catalytic inhibitors act by preventing
the binding of Topo II to DNA (e.g., suramin), blocking the ATPꢀbinding site of the enzyme (e.g.,
novobiocin and salvicine), or inhibiting the cleavage of DNA (e.g., merbarone and
bisdioxopiperazines). A series of Topo II poisons is currently widely used in clinical treatment of
cancer. However, some mutations and clinic studies have indicated that Topo II poison, related to the
level of enzymeꢀassociated DNA breaks versus recombinationꢀrepair pathways for the apoptosis,
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may trigger chromosomal translocations that lead to specific luckemia.
Thus, development of
Topo II catalytic inhibitors that modulate the cytotoxic effect of Topo II poison and overcome
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multidrug resistance has become important in recent years.
Several Topo II catalytic inhibitors
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are clinically used to increase the efficacy of other drugs, for example, aclarubicin as antineoplastic
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agents, (S)ꢀ(+)ꢀ1,2ꢀbis(3,5ꢀdioxopiperazinyl)propane (ICRFꢀ187)
as cardioprotectors, and
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novobiocin as modulators.
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Pyrrolo[2,3ꢀb]pyrazine derivatives are a class of biologically active compounds with
antibronchospastic effect and ability to inhibit important kinases, including p38 MAP kinase,
cyclinꢀdependent kinases, and glycogen synthase kinaseꢀ3, displaying an anticancer
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effect.
Among the most efficient pyrrolo[2,3ꢀb]pyrazine derivatives are aloisine A and B (Figure
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1). Aloisines bind to the kinase ATP–binding pocket as competitive inhibitors.
Recently, Daniel
Reker et al, reported a series of aloisine derivatives as antimalarial agents. A selfꢀorganizing map
was used to predict the potent targets of these compounds and the results indicated that the
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topꢀranking targets may be kinases and DNA Topo.
Nꢀfused imidazole is a class of heterocyclic compound represented by marketed drugs such as
zolpidem (a hypnotic drug) and zolimidine (an antiulcer drug). Several derivatives of benzoxazoles,
benzimidazoles, and related fused heterocyclic compounds have exhibited significant antimicrobial
effects and antiviral activities in vitro. More detailed investigations on benzimidazole derivatives
revealed that these compounds constitute a new class of Topo II inhibitors (Figure 1). Studies on this
class of compounds have indicated that the benzimidazole ring in the structure is critical for the
activity. The 3DꢀQSAR study showed that the phenyl group linked to benzimiazole has a special
contribution to the activity of the compound. Mechanism studies and molecular docking analysis
showed that benzimiazole derivatives function as nonꢀintercalative Topo II catalytic inhibitors and
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their catalytic inhibition mode may be through blocking the ATPꢀbinding site of enzyme.
To search for new anticancer agents that target Topo II with high potency, we introduced the
,3ꢀbenzoazole pharmacophore to the R′ position of aloisine moiety to produce a new class of
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1,3ꢀbenzoazolylꢀsubsituted pyrrolo[2,3ꢀb]pyrazine derivatives (BPPs, Figure 2). We found that the
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new derivatives were similar to adenosine (ANP). When the new scaffold was aligned with ANP in
the ATP binding site of Topo II, the aloisine moiety was aligned with pruine rine, and the
benzimidazolyl group was partly laminated with the glycosyl group of ANP (Figure 2). The new
scaffold overlap with ANP indicated that BPPs may have potential as Topo II inhibitors. Based on
this information, a series of BPPs derivatives was synthesized and bioꢀevaluated. The structure
activity relationships and activity mechanism were investigated in this study. The results revealed
that BPPs function as potential nonꢀintercalation catalytic inhibitors of Topo II. Further study in the
cellular level showed that BPPs displayed strong antiꢀproliferation activity and showed obvious
relationship between cytotoxicity and Topo II inhibition.
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RESULT AND DISCUSSION
Chemistry. The BPPs were synthesized following the general method, as shown in Schemes 1,
and 2. A twoꢀstep nucleophilic reaction was used to build the BPP scaffold. The starting materials
,6ꢀdichloropyrazineꢀ2,3ꢀdicarbonitrile and αꢀazaheteroarylacetonitriles (1a–e) were used in the first
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step as Cꢀnucleophiles to produce intermediates 2a–e. The next step was the nucleophilic substitution
of the second chlorine atom by different Nꢀnucleophiles of the primary amines, followed by the
addition of the secondary amine to the nitrile group and the formation of the pyrrolo[2,3ꢀb]pyrazine
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cyclic system.
Compounds 3a–3w, 4m–4u, 5d–5t, 6c–6u and 7c–7u were synthesized with
different side chains at the Nꢀposition of pyrrole ring. Compounds 8t and 8u were synthesized
through the route shown in Scheme 2. The 2ꢀ(cyanomethyl)benzimidazole reaction with
1,4ꢀdibromobutane provided intermediates 8-1, 8-2 and 8-3, which were synthesized according to
methods described previously. Compounds 3x were synthesized through the route shown in Scheme
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according to the reported method. The structure of the BBPs is shown on Table 1.
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BPPs as Topo II potential inhibitors. Several in vitro experiments, such as relaxation,
cleavage complex, unwinding assay, and ATP competition assay, were performed for the
bioꢀevaluation of the synthesized BPPs on their ability to inhibit Topo II and investigate their mode
of action.
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Topo IIꢀmediated DNA relaxation assay was first performed to test whether this class of
compounds target Topo II similar to our hypothesis, using etoposide as standard and pBR322 DNA
as plasmid. Compounds 3a–3w with different amine side chains were first synthesized and tested.
The results are presented in Figure 3 and Table 2. Compounds 3m, 3n, 3t, and 3u almost completely
inhibited Topo II activity at 50 ꢁM, whereas the other tested compounds did not show any inhibition.
In particular, compounds 3m, 3n, 3t, and 3u, n = 3 or 4, with respectively a dimethylamino or
diethylamino terminal, would seem to be more potent Topo II inhibitors，while introducing a shorter
(n = 2, 3c, 3d,) or longer side chain (n = 5, 3w)，which had same terminal as 3m, 3n, 3t, and 3u,
would decrease the inhibitory activity to Topo II. On the other hand, when the length of side chain is
n = 3, compound 3q, 3r, 3s, and 3v, with respectively a alkly, hydroxyl, cyclic amino, and
aromatic amino terminal groups, did not show any inhibition on Topo II at 50 ꢁM. The results
indicated that the length and terminal groups of side chain have significant impact on the compounds
in inhibiting the Topo II activity and should be maintained in future structure modification.
In order to investigate the effect of benzimidazole ring on the activity, this moiety was changed
in following four ways: replacement of benzimidazole to benzoxazole (4m, 4s, 4t, and 4u) or
benzothiazole (5c, 5m, and 5t); Nꢀmethylation of benzimidazole (6c, 6d, 6m, 6n, 6t, and 6u);
introduction of chlorine to the phenyl ring of benzimidazole (7c, 7d, 7m, 7n, 7t, and 7u); and
removing of benzimidazole group (3x). As shown in Figures 3D, compounds 4m, 4t, 5m, 5t, 6t, and
6u showed strong activity in the inhibition of Topo II at 20 ꢁM. Comparing those compounds with
same pyrrolo[2,3ꢀb]pyrazine moiety, it was found that benzimidazole derivatives (3m and 3t) and
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benzothiazole derivatives (5m and 5t) showed an equal activity in the inhibition of Topo II, but
benzoxazole derivatives (4m and 4t) had a weaker inhibition. It was found that Nꢀmethylation of
benzimidazole led to a decrease of inhibitory activity on Topo II when compared the activity of
methylation compounds 6m, 6n, 6t, and 6u with that of nonmethylation compounds 3m, 3n, 3t, and
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3u (Figure 3C and 3D). In addition, as shown in Figure 3C, compounds 7c, 7d, 7m, 7n, 7t, and 7u
showed a weak Topo II inhibition activity at 50 ꢁM, indicating the introduction of chlorine to
benzimidazole has an unfavorable effect on the activity. Moreover, removing of benzimidazole from
the scaffold, compounds 3x did not showed any Topo II inhibition activity at 50 ꢁM (Figure 3C).
These results revealed the importance of the benzoazolyl as a functional scaffold.
In addition, compounds 8t and 8u, which moved the amine side chain from pyrrolo ring to
imidazole ring, also showed a moderate Topo II inhibition activity (Figures 3C and 3D).
Topo Iꢀmediated DNA relaxation assay was performed to test whether this class of compounds
also target Topo I. The result is presented in Figure S1. All of the tested BPPs did not exhibit Topo I
inhibitory activity at 50 ꢁM, indicating that BPPs selectively inhibited the activity of Topo II.
As compounds 3t and 3u showed the best Topo II inhibition activity, these two compounds
were selected for the further mechanism studies.
BPPs as non-intercalative Topo II catalytic inhibitor. Topo II inhibitors are classified
according to their ability to induce DNA doubleꢀstrand breaks with (Topo II poisons) or without
(Topo II catalytic inhibitors) the formation of cleaved complex, reflecting different inhibition
mechanisms. Moreover, Topo II catalytic inhibitors can antagonize Topo II poisonꢀmediated DNA
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damage. To gain insight into the mode of action, cleavage complex assay was performed to verify
whether BPPs function as Topo II poisons or catalytic inhibitors. Etoposide, a Topo II poison,
exhibited its activity by stabilizing Topo IIꢀDNA covalent complexes in vitro and in vivo, leading to
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the formation of linear DNA.
In contrast to etoposide, the linear form of the DNA is not visible
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in compounds 3t and 3u (Figure 4A). These results showed that the tested compound did not act as
classical Topo II poison even at 20 ꢁM. In addition, the linear band was reduced when the etoposide
was pretreated with compounds 3t and 3u, which revealed its antagonizing effect on Topo II poison
(Figure 4A). These observations indicate that these BPPs act as Topo II catalytic inhibitor.
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Furthermore, the ability of compound 3t to induce DNA damage in cells was examined to confirm
this mode of inhibition. A hallmark of DNA double–strand breaks termed γꢀH AX was measured by
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western blot analysis in the HLꢀ60 cell line. Figure 4B showed that etoposide induced DNA
doubleꢀstrand break, whereas compound 3t did not induce γꢀH AX formation. Moreover,
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pretreatment with 3t blocked etoposide induced γꢀH AX accumulation, suggesting that compound 3t
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antagonized Topo II poisonꢀmediated DNA break. These finding are consistent with the results
obtained from the cleave assay.
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Topo II catalytic inhibitors have two types, namely, DNA intercalators and nonꢀintercalators.
In order to examine whether compounds function as DNA intercalators or not, we first employed
Topo I mediated DNA intercalating/unwinding assay using a pBR322 DNA as a substrate, since the
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reformation of supercoiled DNA in this assay is a characterization of intercalative drugs. As shown
in Figure 4C, the selected compounds 3t and 3u cannot intercalate into plasmids by transforming
relaxed DNA into a supercoiled DNA substrate in the presence of Topo I even at high concentration
(50 ꢁM), unlike the action of ethidium bromide (EB), which is a classic intercalator of DNA. The
ability of 3t to interact with DNA was also confirmed by fluorescenceꢀbased EB displacement assay
(Figure S2). The DNA bound form of EB has a stronger fluorescence emission than free EB, so the
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displacement of EB from DNA could be monitored by a decrease in the fluorescence signal. As
expected, the addition of the DNA intercalator mAMSA (amsacrine) was accompanied by a
concomitant decrease in EB fluorescence. In contrast, the addition of increasing concentration of 3t
did not alter the fluorescence intensity of EB, indicating that 3t was incapable of intercalating into
DNA. In addition, UVꢀtitration was also performed to measure the binding affinity of 3t to calf
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thymus DNA (CTꢀDNA). The apparent binding constant (K ) of 3t to CTꢀDNA was 2.8×10 M
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(
Figure S3), indicating that BPPs showed weak interaction with CTꢀDNA. Taken together, these
finding indicates that the tested compounds are nonꢀintercalative Topo II catalytic inhibitors.
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BPPs blocking the ATP-binding site of Topo II. Topo II requires ATP to carry out its
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essential catalytic cycle. Thus, compounds blocking the ATP binding site of Topo II can affect
ATP hydrolysis and influence the progression of the enzyme through catalytic functions. The ATP
competition assay was performed to identify the potential action of BPP derivative 3t to the ATPase
domain of Topo II. The result showed that the inhibitory activity of 3t reduced with the increase in
ATP concentrations in Topo IIꢀmediated DNA relaxation. As shown in Figure 5, the concentrations
of ATP increased from 1 mM to 4 mM, the corresponding inhibitory activity IC value for Topo II
5
0
were increased from 0.9 ꢁM to 16.5 ꢁM. These results indicate that BPPs may act as ATP competitor
inhibitors by blocking the ATPꢀbinding site of the enzyme.
ATP competition assay showed that the BPPs catalytic mode of inhibition may be through
blocking the ATPase domain of the enzyme. Molecular docking analysis was carried out to identify
4
1, 42
the potential interaction of BPPs with the ATPase domain of Topo II.
Compounds 3c and 3t
were docked into the catalytic site of the ATPase domain of Topo II (PDB code: 1ZXM) by using the
Surflexꢀdock program incorporated with the SYBYL software package (Tripos, Inc. St. Louis. MO).
Both compounds can fit into the ATP binding pocket, as showed in Figure 6.
For active compound 3t (Figure 6B), the amino group (NH ) formed a special hydrogen bond
2
interaction with the carboxylic group of residues Thr147, which may provide extra stability to the
enzyme–ligand interactions. The benzimidazole group showed hydrophobic interaction with residues
Ile141 and Ser149, whereas the pyrazine group formed a hydrophobic interaction with residue
Phe142. In addition, the terminal amino group of compound 3t can fit into the cave formed by
residues Asn163, Gly164, Tyr165, Gly166, and Gln376 in the deep site of the ATPase domain. The
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non–active compound 3c (Figure 6A) formed a weak hydrogen interaction with residue Ser149;
benzimidazole also displayed hydrophobic interaction with residues Ile141 and Ser149. However,
the hydrogen bond interaction with residues Thr147 disappeared, and the terminal amino group
cannot fit into the cave formed in the deep site of the ATPase domain. These results showed that
BPPs are catalytic inhibitors that act by occupying the ATP binding pocket of the ATPase domain of
Topo II and making favorable interactions with its key residues. The molecular docking analysis also
explained the important contribution of the terminal side chain to the activity of compounds.
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In vitro cytotoxic activity. The cell proliferation inhibitory activities of the compounds are
listed in Table 2 as values of IC . All substances were evaluated for antiꢀproliferative activities
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against the human acute leukemia cell line (HLꢀ60), human cervical cancer cell line (Hela), human
breast cancer cell line (MDAꢀMBꢀ201), adenocarcinomic human alveolar basal epithelial cancer cell
line (A549), human chronic myeloid leukemia cell (K562), human lymphoma cancer cell line (Raji),
HLꢀ60/MX2 (a cancer cell line derivative from HLꢀ60 that is resistant to Topo II poison), and human
embryonic kidney 293 cells (HEKꢀ293, a normal cell line) using the MTT assay as described by
43
Mosmann with modifications.
Etoposide was tested as a reference compound. Most of compounds displayed significant
antitumor activities with low nanomolar to micromolar IC values, as shown in Table 2. It was
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found that most of the compounds showed good correlation between their Topo II inhibitory activity
and cytotoxicity toward seven cancer cell lines. The compounds 3m, 3n, 3t, 3u, 4m, 4t, 4u, 5m, 5t,
6m, 6n, 6t, 6u, 8t, and 8u, which showed strong inhibition of Topo II, displayed promising
antiꢀproliferative activity against all the tested seven cancer lines. Compounds 3t and 3u, which
displayed the most significant Topo II inhibition activity, showed the best antiꢀproliferation activity,
with an IC50 ranging between 0.43–11.24 ꢁM and 0.46–10.07 ꢁM. Meanwhile, the other tested
compounds that had no effect on Topo II activity showed low or moderate activity, which could be
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attributable to their lack of Topo II inhibition. The relationship between cytotoxicity and Topo II
inhibition was obvious. However, some compounds, such as 3p, 3s, 7m, 7n, 7t, and 7u, displayed
significant cytotoxicity against all the tested cancer cells, although they showed weak Topo II
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inhibition, suggesting these compounds may target other proteins in cancer cells.
Moreover, this
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class of compounds showed a cytotoxic selectivity for the HLꢀ60 cell line, some of the tested
compounds displayed a nanomolar level IC . But BPPs also had cytotoxicity toward HEKꢀ293,
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which indicated these series of compounds did not display a meaningful selectivity between cancer
cells and normal cells.
The HLꢀ60/MX2, a cell line derived from HLꢀ60 cells selected for resistance to mitoxantrone,
displays atypical multidrug resistance with altered Topo II catalytic activity and reduced levels of
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Topo IIα and Topo IIβ proteins. A reduced Topo II catalytic activity means that a decrease of DNA
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damage; hence, resistance to Topo II posion. Topo II catalytic inhibitor kills cancer cells depending
on the inhibition of Topo II; thus, cells with reduced Topo II are sensitive to Topo II catalytic
inhibitors. As shown in Table 2, HLꢀ60/MX2 resistant to etoposide, with resistant index (RI) of 56.
The BPPs showed an RI range from 0.6 to 4.4, indicating that HLꢀ60/MX2 is not crossꢀresistant to
the BPPs. This result indicates that BPPs act as Topo II catalytic inhibitors, which consistent with the
results obtained from the mechanism studies.
Apoptosis Assay by Annexin V/PI binding. Apoptosis is the process of programmed cell
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death involving a series of biochemical events. In cancer, the apoptosis cell division ratio is altered.
47
Many chemotherapy agents exert their anticancer effects by apoptosis. To investigate the effect of
BPPs on apoptosis of HLꢀ60 cells, double staining for FITCꢀlabeled annexinꢀV binding to
phosphatidylserine of membrane and propidium iodide (PI) binding for cellular DNA was carried
out, followed by flow cytometry. As shown in Figure 7, after treating HLꢀ60 cell with compounds 3t
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and 3u (0.5–2 ꢁM) for 12 h, followed by annexin VꢀFITC/PI staining, the percentages of apoptotic
cells greatly increased at a concentration of 2 ꢁM. After incubation (0.25–1 ꢁM) for 24 h,
compounds 3t and 3u induced the major population of HLꢀ60 cells into the late apoptotic stage
(90.51%, 45.05%) at 1 ꢁM concentration, indicating that compounds 3t and 3u suppressed HLꢀ60
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cell proliferation by inducing apoptosis in a dose and timeꢀdependent manner.
ꢀ
CONCLUSION
In this study, we designed and synthesized a series of BPPs as potent Topo II inhibitors,
dissected the Topo II inhibition mechanism, and antiꢀproliferation activity. Fortyꢀfive compounds
were synthesized, which showed easy preparation and good water solubility. The Topo II inhibitory
activity of these compounds was tested by relaxation and cleavage complex in vitro assays.
Unwinding assay and EB displacement assay were performed with EB as positive control. The
results revealed that 15 compounds, namely, 3m, 3n, 3t, 3u, 4m, 4t, 4u, 5m, 5t, 6m, 6n, 6t, 6u, 8t,
and 8u, acted as potent DNA nonꢀintercalating Topo II catalytic inhibitors. The ATP competition
assay and molecular docking analysis suggested that the catalytic mode of inhibition of the
compounds may be through blocking the ATPꢀbinding site of the enzyme. The Topo IIꢀDNA cleave
assay and western blot analysis revealed that these compounds can antagonize Topo II
poisonꢀmediated DNA break.
Most of the compounds showed good correlation between their Topo II inhibitory activity and
cytotoxicity toward seven cancer cell lines. Investigation of the structureꢀactivity relationship
indicated that the length of the side chain and terminal amino groups are important in enhancing both
enzyme inhibition and cytotoxicity of BPPs. Furthermore, flow cytometric analysis showed that this
class of compounds can significantly induce HLꢀ60 cells apoptosis in a dose and timeꢀdependent
manner.
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The result indicated that in addition to the 1,3ꢀbenzoazole compounds (reported Topo II
inhibitor) with aloisines (kinases inhibitor and predicted Topo II inhibitor) exhibited significant Topo
II inhibitory activity and antiꢀproliferation activity. Since Topo II is important target for cancer
chemotherapy, these findings may provide advance opportunities for the design and development
new chemotherapy agents.
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ꢀ
EXPERIMENTAL SECTION
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Chemistry. H and C NMR spectra were recorded using TMS as the internal standard in
DMSOꢀd or CDCl with a Bruker BioSpin GmbH spectrometer at 400 and 101 MHz, respectively.
6
3
1
The chemical shifts are reported in parts per million (ppm) relative to residual CHCl (δ = 7.26, H; δ
3
13
1
13
=
77.0, C) and DMSOꢀd (δ = 2.50, H; δ = 39.5, C) in the corresponding deuterium agents.
6
Highꢀresolution mass spectra (HRMS) were recorded on Shimadzu LCMSꢀITꢀTO. The melting point
Mp) were determined using an SRSꢀOptiMelt automated melting point instrument without
(
correction. The purities of synthesized compounds were confirmed by analytical HPLC performed
with a dual pump Shimadzu LCꢀ20AB system equipped with an Ultimate XBꢀC18 column and
−1
eluted with methanol/water (90%) at a flow rate of 0.7 mL min and the purities were proved to be
higher than 95%. All the reagents were commercially available. The compounds BPPs were prepared
2
8, 29
following the reported method
as mentioned in Scheme 1, and 2. All the products were identified
1
13
by H and C NMR and HRMS spectrometry.
General procedure for the synthesis of intermediates 2a, 2b, 2c, 2d, and 2e. A solution of 5,
6
ꢀdichloropyrazineꢀ2, 3ꢀdicarbonitrile (5.0 mmol) and αꢀAzaheteroarylacetonitriles (5.0 mmol) in
DMF (7mL) was stirred at 40 °C for 3 h. The mixture was left standing at 25 °C for 16 h. The
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formed precipitate was filtered off, washed with water and DMF, and dried in vacuo to give bright
yellow product.
General procedure for the synthesis of compound BPPs. A solution of 2a, 2b, 2c, 2d or 2e
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(0.5 mmol), primary amines (0.55 mmol) and TEA (1.0 mmol) in DMF (3 mL) was stirred at 60 °C
for 3 h. The mixture was concentrated in vacuo and the residue taken up in water (100 mL). The
precipitate was filtered off, taken up in MeOH (50mL), and stirred at 65 °C for 30 min. The
precipitate was filtered off and dried in vacuo.
2
-(1-Methyl-1H-benzo[d]imidazol-2-yl)acetonitrile
(1d).
A
suspension
of
2
ꢀ(cyanomethyl)benzimidazol (3.14 g, 20 mmol) and K CO (2.76 g, 20 mmol) in DMF (10 mL) was
2
3
treated with Me SO (2.52 mL, 20 mmol) and stirred at 40 °C for 5 h. K CO (2.76 g, 20 mmol) and
2
4
2
3
Me SO (2.52 mL, 20 mmol) were added. The mixture was stirred at 40 °C for 5 h, poured on brine
2
4
(100 mL), and extracted with EtOAc. The combined org. phases were kept standing at 4 °C over
night, the formed precipitated was filtered off, and the filtrate was concentrated in vacuo. The residue
1
were purified by CC (SiO ; EA:PA = 1:3) to yield 1d (0.71 mg, 18%) as a brown solid. H NMR
2
1
3
(400 MHz, CDCl ) δ: 7.79 – 7.68 (m, 1H); 7.36 – 7.29 (m, 3H), 4.09 (s, 1H), 3.87 (s, 3H). C NMR
3
(
101 MHz, CDCl ) δ: 146.2, 142.2, 136.4, 122.9, 122.3, 119.3, 116.8, 110.6, 30.3, 17.9.
3
2
-(1-(4-Bromobutyl)-1H-benzo[d]imidazol-2-yl)acetonitrile (8-1).
A
suspension of
2ꢀ(cyanomethyl)benzimidazol (1.57g, 10 mmol) and cesium carbonate (50 mmol) in Me O (50 ml)
2
was treated with 1,4ꢀdibromobutane (50 mmol), removed Me O, poured on brine (150 ml),
2
extracted with EtOAc, the org. phases was concentrated in vacuo. The residue were purified by CC
1
(
SiO ; EA:PA = 1:3) to yield 8-1 (1.51g, 50%). H NMR (400 MHz, DMSOꢀd ) : 7.93 (d, J = 8.1 Hz,
2
6
1H), 7.71 (dd, J = 6.2, 2.7 Hz, 1H), 7.59 – 7.49 (m, 2H), 4.42 (t, J = 7.4 Hz, 2H), 3.66 (t, J = 6.5 Hz,
3H), 3.52 (s, 2H), 2.00 – 1.92 (m, 2H), 1.89 – 1.69 (m, 2H).
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-((1-(4-Bromobutyl)-1H-benzo[d]imidazol-2-yl)(cyano)methyl)-6-chloropyrazine-2,3-dica
rbonitrile (8-2). A solution of 5, 6ꢀdichloropyrazineꢀ2, 3ꢀdicarbonitrile (5.0 mmol) and 8-1(5.0
5
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mmol) in DMF (7mL) was stirred at 40 °C for 3 h. The mixture was left standing at 25 °C for 16 h.
The formed precipitate was filtered off, washed with water and DMF, and dried in vacuo to give
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bright yellow product. H NMR (400 MHz, DMSOꢀd ) : 14.28 (s, 1H), 7.93 (d, J = 7.9 Hz, 1H), 7.72
6
(dd, J = 6.2, 2.3 Hz, 1H), 7.59 – 7.49 (m, 2H), 4.42 (t, J = 7.3 Hz, 2H), 3.66 (t, J = 6.5 Hz, 2H), 2.01
–
1.93 (m, 2H), 1.89 – 1.71 (m, 2H).
-Amino-7-(1-(4-bromobutyl)-1H-benzo[d]imidazol-2-yl)-5-methyl-5H-pyrrolo[2,3-b]pyra
6
zine-2,3-dicarbonitrile (8-3). A solution of 8-2 (0.5 mmol), methylamine (0.55 mmol) and TEA (1.0
mmol) in DMF (3 mL) was stirred at 60 °C for 3 h. The mixture was concentrated in vacuo and the
residue taken up in water (100 mL). The precipitate was filtered off, taken up in MeOH (50mL), and
1
stirred at 65 °C for 30 min. The precipitate was filtered off and dried in vacuo. H NMR (400 MHz,
DMSOꢀd ): 9.26 (s, 2H), 7.62 (dd, J = 8.7, 7.1 Hz, 2H), 7.24 (dd, J = 12.4, 6.3 Hz, 2H), 4.77 – 4.57
6
(m, 2H), 3.70 (s, 3H), 3.60 (t, J = 6.4 Hz, 2H), 1.94 – 1.52 (m, 4H).
6
-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-methyl-5H-pyrrolo[2,3-b]pyrazine-2,3-dicarbonit
rile (3a). Methylamine and 2a were used as reactants to give 3a.Yield: 83%.Yellow solid. Mp >300
1
°C. H NMR (400 MHz, DMSOꢀd ) δ: 11.89 (s, 1H), 9.15 (s, 2H), 7.63 (d, J = 5.6 Hz, 1H), 7.59 (dd,
6
13
J = 5.6, 3.2Hz, 1H), 7.20 – 7.14 (m, 2H), 3.68 (s, 3H). C NMR (101 MHz, DMSOꢀd ) δ: 158.5,
6
147.0, 142.8, 141.2, 139.3, 134.2, 126.2, 122.0, 118.2, 117.7, 117.0, 116.4, 112.1, 84.3, 28.0. HR
+
ESIꢀMS (M + H) m/z = 315.1092 (Cacld for C H N : 315.1101), HPLC purity: 97.8%.
1
6
10
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-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-ethyl-5H-pyrrolo[2,3-b]pyrazine-2,3-dicarbonitri
le(3b). Ethylamine and 2a were used as reactants to give 3b. Yield: 91%.Yellow solid. Mp >300
1
°
C. H NMR (400 MHz, DMSOꢀd ): 11.97 (s, 1H), 9.24 (s, 2H), 7.68 – 7.58 (m, 2H), 7.24 – 7.18 (m,
6
13
2
H), 4.30 (q, J = 7.1 Hz, 2H), 1.32 (t, J = 6.9 Hz, 3H). C NMR (101 MHz, DMSOꢀd ) δ: 157.1,
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+
HR ESIꢀMS (M + H) m/z = 327.1118 (Cacld for C H N : 327.1112), HPLC purity: 99.4%.
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6-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-(2-(dimethylamino)ethyl)-5H-pyrrolo[2,3-b]pyra
zine-2,3-dicarbonitrile(3c). N, NꢀDimethylamine and 2a were used as reactants to give 3c.Yield:
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91%.Yellow solid. Mp >270 °C (decomp). H NMR (400 MHz, DMSOꢀd ) δ: 11.94 (s, 1H), 9.50 (s,
6
2H), 7.68 – 7.54 (d, J = 10.4 Hz, 2H), 7.22 – 7.14 (m, J = 41.9, 5.8, 3.0 Hz, 2H), 4.48 (s, 2H), 3.13 –
1
3
2
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.92 (m, 2H). C NMR (101 MHz, DMSOꢀd ) δ: 157.8, 146.3, 142.0, 140.7, 138.8, 133.7, 125.6,
6
+
21.5, 117.6, 117.2, 116.4, 115.8, 111.7, 83.9, 55.7, 44.0, 38.1. HR ESIꢀMS (M + H) m/z =
372.1664 (Cacld for C H N : 372.1680),HPLC purity: 95.9%.
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-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-(2-(diethylamino)ethyl)-5H-pyrrolo[2,3-b]pyrazi
ne-2,3-dicarbonitrile(3d). N, NꢀDiethylamine and 2a were used as reactants to give 3d. Yield: 88%.
1
Yellow solid. Mp >243 °C (decomp). H NMR (400 MHz, DMSOꢀd ) δ: 11.97 (s, 1H), 9.70 – 9.10
6
(br s, 2H), 7.70 – 7.58 (m, 2H), 7.21 – 7.13 (m, 2H), 4.37 (q, J = 7.0 Hz, 2H), 2.76 (d, J = 21.7 Hz,
13
2H), 0.86 (s, 6H). C NMR (101 MHz, DMSOꢀd ) δ: 157.9, 146.4, 142.2, 140.9, 138.8, 133.7,
6
+
125.6, 121.5, 117.6, 117.2, 116.4, 115.8, 111.7, 83.9, 50.4, 46.3, 25.5 11.3. HR ESIꢀMS (M + H)
m/z = 400.1983 (Cacld for C H N : 400.1993), HPLC purity: 99.8%.
2
1
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6-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-(2-(pyrrolidin-1-yl)ethyl)-5H-pyrrolo[2,3-b]pyraz
ine-2,3-dicarbonitrile (3e). 1ꢀPyrrolidineethanamine and 2a were used as reactants to give 3e.Yield:
1
8
6%. Yellow solid. Mp >300 °C. H NMR (400 MHz, DMSOꢀd ) δ: 11.97 (s, 1H), 9.32 (br s, 2H),
6
7.69 – 7.55 (m, 2H), 7.22 – 7.08 (m, 2H), 4.39 (t, J = 5.8 Hz, 2H), 2.86 (t, J = 5.7 Hz, 2H), 2.58(s,
13
4H), 1.69 (s, 4H). C NMR (101 MHz, DMSOꢀd ) δ: 158.4, 146.9, 142.7, 141.2, 139.3, 134.2,
6
+
126.2, 122.1, 118.2, 117.7, 117.0, 116.4, 112.3, 84.5, 53.9, 53.8, 41.0, 23.6. HR ESIꢀMS (M + H)
m/z = 398.1826 (Cacld for C H N : 398.1836), HPLC purity: 97.6%.
2
1
19
9
6
-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-(2-(4-methylpiperazin-1-yl)ethyl)-5H-pyrrolo[2,3
-b]pyrazine-2,3-dicarbonitrile (3f). 1ꢀMethylꢀ4ꢀ(2ꢀaminoethyl)piperazine and 2a were used as
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reactants to give 3f. Yield: 77%. Yellow solid. Mp >300 °C. H NMR (400 MHz, DMSOꢀd ) δ: 11.98
6
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
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4
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5
5
5
5
5
5
5
5
6
(
s, 1H), 9.45 (s, 2H), 7.70 – 7.56 (m, 2H), 7.23 – 7.12 (m, 2H), 4.38 (t, J = 5.6 Hz, 2H), 2.71 (t, J =
1
3
5
1
4
.6 Hz, 2H), 2.35 – 2.20 (br s, 4H), 2.14 (s, 3H). C NMR (101 MHz, DMSOꢀd ) δ: 157.9, 146.5,
6
42.3, 140.9, 138.9, 133.8, 125.7, 121.6, 117.7, 117.3, 116.5, 115.9, 111.8, 83.9, 55.3, 54.4, 52.4,
0
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8
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3
4
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7
8
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5
6
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8
9
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2
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4
5
6
7
8
9
0
+
5.4. HR ESIꢀMS (M + H) m/z = 427.2083 (Cacld for C H N : 427.2102), HPLC purity: 99.8%.
2
2
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6-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-(2-morpholinoethyl)-5H-pyrrolo[2,3-b]pyrazine-
2
,3-dicarbonitrile (3g). Nꢀethylꢀ2ꢀmorpholinoethanamine and 2a were used as reactants to give
1
3
g.Yield: 77%. Yellow solid. Mp >300 °C. H NMR (400 MHz, DMSOꢀd ) δ: 11.96 (s, 1H), 9.40 (s,
6
2H), 7.76 – 7.49 (m, 2H), 7.18 (dd, J = 5.7, 3.2 Hz, 2H), 4.37 (d, J = 5.3 Hz, 2H), 3.51 (s, 4H), 2.70
13
(t, J = 5.3 Hz, 2H), 2.59 (s, 4). C NMR (101 MHz, DMSOꢀd ) δ: 158.2, 147.0, 142.8, 141.4, 139.3,
6
1
34.3, 126.3, 122.0, 118.2, 117.7, 117.0, 116.4, 112.3, 84.4, 66.5, 56.3, 53.6, 39.0. HR ESIꢀMS (M +
+
H) m/z = 414.1773 (Cacld for C H N O: 414.1785), HPLC purity: 97.1%.
2
1
19
9
6
-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-phenethyl-5H-pyrrolo[2,3-b]pyrazine-2,3-dicarb
onitrile(3h). Phenylethylamine and 2a were used as reactants to give 3h.Yield: 89%. Yellow solid;
1
Mp >300 °C. H NMR (400 MHz, DMSOꢀd ) δ: 11.96 (s, 1H), 9.25 (s, 2H), 7.70 – 7.58 (m, 2H), 7.28
6
13
–
7.15 (m, 7H), 4.52 (t, J = 7.4 Hz, 2H), 3.05 (t, J = 7.3 Hz, 2H). C NMR (101 MHz, DMSOꢀd ) δ:
6
157.6, 146.9, 142.7, 140.9, 139.0, 137.8, 134.2, 129.5, 128.7, 127.1, 126.1, 122.1, 122.0, 118.2,
+
1
17.7, 116.9, 116.3, 112.3, 84.3, 42.4, 34.0. HR ESIꢀMS (M + H) m/z = 405.1548 (Cacld for
C H N : 405.1571), HPLC purity: 98.3%.
23
16
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6-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-(4-methoxyphenethyl)-5H-pyrrolo[2,3-b]pyrazin
e-2,3-dicarbonitrile (3i). 2ꢀ(4ꢀMethoxyꢀphenyl) ethylamine and 2a were used as reactants to give 3i.
1
Yield: 87%.Yellow solid; Mp >196 °C (decomp). H NMR (400 MHz, DMSOꢀd ) δ: 11.98 (s, 1H),
6
9
.25 (s, 2H), 7.21 – 7.14 (m, 2H), 7.11 (d, J = 8.6 Hz, 2H), 6.79 (d, J = 8.6 Hz, 2H), 4.47 (t, J = 7.3
13
Hz, 2H), 3.69 (s, 3H), 2.98 (t, J = 7.3 Hz, 2H). C NMR (101 MHz, DMSOꢀd ) δ: 158.5, 157.6,
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2
2
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3
3
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46.8, 140.8, 139.1, 130.4, 129.7, 126.1, 122.0, 118.2, 116.8, 116.3, 114.2, 84.2, 49.0, 42.5, 33.2.
+
HR ESIꢀMS (M + H) m/z = 435.1657 (Cacld for C H N O: 435.1676), HPLC purity: 97.5%.
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5
-(2-(1H-indol-2-yl)ethyl)-6-amino-7-(1H-benzo[d]imidazol-2-yl)-5H-pyrrolo[2,3-b]pyrazin
e-2,3-dicarbonitrile (3j). 3ꢀ(2ꢀAminoethyl)indole and 2a were used as reactants to give 3j.Yield:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
9
1%.Yellow solid. Mp >300 °C. H NMR (400 MHz, DMSOꢀd ) δ: 11.97 (s, 1H), 10.85 (s, 1H), 9.27
6
(s, 2H), 7.71 – 7.58 (m, 2H), 7.53 (d, J = 7.8 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H), 7.24 – 7.11 (m, 3H),
1
3
7.05 (t, J = 7.2 Hz, 1H), 6.94 (t, J = 7.2 Hz, 1H), 4.62 – 4.51 (m, 2H), 3.17 (t, J = 7.3 Hz, 2H). C
NMR (101 MHz, DMSOꢀd ) δ: 157.8, 147.0, 142.8, 141.0, 139.3, 136.5, 134.3, 127.5, 126.0, 123.7,
6
122.1, 122.0, 121.5, 118.8, 118.4, 118.3, 117.7, 116.9, 116.4, 112.3, 111.8, 110.4, 84.4, 41.92, 24.1.
+
HR ESIꢀMS (M + H) m/z = 444.1658 (Cacld for C H N : 444.1680), HPLC purity: 99.9%.
25
17
9
6
-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-propyl-5H-pyrrolo[2,3-b]pyrazine-2,3-dicarbonit
rile (3k). 1ꢀPropylamine and 2a were used as reactants to give 3k.Yield: 83%.Yellow solid. Mp
1
>
300 °C. H NMR (400 MHz, DMSOꢀd ) δ: 11.97 (s, 1H), 9.22 (s, 2H), 7.68 – 7.58 (m, 2H), 7.21 –
6
13
7.15 (m, 2H), 4.21 (t, J = 7.4 Hz, 2H), 2.03 – 1.46 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H). C NMR (101
MHz, DMSOꢀd ) δ: 157.8, 147.0, 142.8, 141.0, 139.3, 134.3, 126.0, 123.7, 121.8, 118.4, 117.7,
6
+
1
16.9, 116.4, 112.3, 84.4, 41.92, 24.1, 15.8. HR ESIꢀMS (M + H) m/z = 341.1278 (Cacld for
C H N : 341.1269, HPLC purity: 97.6%.
18
14
8
6
-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-(2-hydroxyethyl)-5H-pyrrolo[2,3-b]pyrazine-2,3-
dicarbonitrile (3l). 2ꢀEthylaminoethanol and 2a were used as reactants to give 3l.Yield:
1
90%.Yellow solid. Mp >300 °C. H NMR (400 MHz, DMSOꢀd ) δ: 12.00 (s, 1H), 9.19 (s, 2H), 7.83
6
–
7.50 (m, 2H), 7.32 – 7.05 (m, 2H), 5.04 (s, 1H), 4.36 (t, J = 5.3 Hz, 2H), 3.78 (t, J = 5.9 Hz, 2H).
1
3
C NMR (101 MHz, DMSOꢀd ) δ: 158.4, 146.9, 142.7, 141.4, 139.3, 134.2, 126.2, 122.0, 121.9,
6
+
1
18.2, 117.7, 116.9, 116.4, 112.3, 84.4, 58.8, 44.0. HR ESIꢀMS (M + H) m/z = 345.1185 (Cacld for
C H N O: 345.1207), HPLC purity: 99.6%.
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-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-(3-(dimethylamino)propyl)-5H-pyrrolo[2,
3-b]
pyrazine-2,3-dicarbonitrile (3m). N, NꢀDimethylꢀ1,3ꢀpropanediamine and 2a were used as
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
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4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
reactants to give 3m.Yield: 90%. Yellow solid. Mp >218 °C (decomp). H NMR (400 MHz,
DMSOꢀd ) δ: 11.96 (s, 1H), 9.19 (br s, 2H), 7.70 – 7.55 (m, 2H), 7.22 – 7.12 (m, 2H), 4.25 (t, J = 6.7
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
Hz, 2H), 2.29 (t, J = 6.6 Hz, 2H), 2.16 (s, 6H), 1.96 – 1.84 (m, 2H). C NMR (101 MHz, DMSOꢀd )
6
δ: 157.9, 146.4, 142.2, 140.6, 138.9, 133.7, 125.7, 121.5, 117.8, 117.2, 116.5, 115.9, 111.8, 83.9,
+
5
5.1, 44.7, 38.7, 25.7. HR ESIꢀMS (M + H) m/z = 386.1827 (Cacld for C H N : 386.1836), HPLC
20
19
9
purity: 98.5%.
6-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-(3-(diethylamino)propyl)-5H-pyrrolo[2,3-b]pyraz
ine-2,3-dicarbonitrile (3n). N, NꢀDiethylꢀ1,3ꢀpropanediamine and 2a were used as reactants to give
1
3
1
1
n.Yield: 92%. Yellow solid. Mp >232 °C (decomp). H NMR (400 MHz, DMSOꢀd ) δ: 11.94 (s,
6
H), 9.20 (br s, 2H), 7.72 – 7.58 (m, 2H), 7.22 – 7.10 (m, 2H), 4.26 (t, J = 7.0 Hz, 2H), 2.45 (m, 6H),
13
.94 – 1.80 (m, 2H), 0.92 (t, J = 7.1 Hz, 6H). C NMR (101 MHz, DMSOꢀd ) δ: 157.7, 146.5,
6
142.1, 140.5, 138.9, 133.5, 125.7, 121.5, 117.8, 117.4, 116.5, 115.9, 111.8, 83.9, 49.2, 45.6, 25.4,
+
1
1.2. HR ESIꢀMS (M + H) m/z = 414.2138（Cacld for C H N : 414.2149), HPLC purity: 99.5%.
2
2
23
9
6-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-(3-morpholinopropyl)-5H-pyrrolo[2,3-b]pyrazine
-
2,3-dicarbonitrile (3o). Nꢀ(3ꢀAminopropyl)pyrrolidine and 2a were used as reactants to give
1
3
o.Yield: 88%. Yellow solid. Mp >196 °C (decomp). H NMR (400 MHz, DMSOꢀd ) δ: 11.98 (s,
6
1H), 9.23 (s, 2H), 7.70 – 7.59 (m, 2H), 7.23 – 7.14 (m, 2H), 4.33 (t, J = 6.4 Hz, 2H), 3.39 (s, 4H),
13
2
.35 (t, J = 6.3 Hz, 2H), 2.23 (t, J =6.8Hz, 4H), 2.00 – 1.85 (m, 2H). C NMR (101 MHz,
DMSOꢀd ) δ: 158.2, 147.0, 142.8, 141.4, 139.5, 134.3, 126.0, 122.2, 122.0, 118.2, 117.7, 117.0,
6
+
116.5, 112.3, 84.5, 68.9, 54.7, 52.9, 23.9. HR ESIꢀMS (M + H) m/z = 428.1929 (Cacld for
C H N O: 428.1942), HPLC purity: 97.8%.
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-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-(3-(4-methylpiperazin-1-yl)propyl)-5H-pyrrolo[2
,
3-b]pyrazine-2,3-dicarbonitrile (3p). 3ꢀ(4ꢀMethylpeperazinꢀ1ꢀyl)propylamine and 2a were used as
1
reactants to give 3p.Yield: 75%. Yellow solid. Mp >272 °C (decomp). H NMR (400 MHz,
DMSOꢀd ) δ: 11.98 (s, 1H), 9.23 (s, 2H), 7.72 – 7.61 (m, 2H), 7.19 –7.13 (m, 2H), 4.31 (t, J = 6.2
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
Hz, 2H), 2.33 (t, J = 6.1 Hz, 2H), 2.23 (s, 4H), 2.12 (s, 4H), 2.03 (s, 3H), 1.98 – 1.88 (m, 2H). C
NMR (101 MHz, DMSOꢀd ) δ: 158.3, 147.1, 142.8, 141.4, 139.6, 134.3, 126.1, 122.1, 122.0, 118.2,
6
+
1
17.7, 117.0, 116.5, 112.3, 84.6, 54.8, 52.8, 46.1, 24.6. HR ESIꢀMS (M + H) m/z = 441.2231 (Cacld
for C H N : 441.2258), HPLC purity: 98.4%.
2
3
24 10
6-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-butyl-5H-pyrrolo[2,3-b]pyrazine-2,3-dicarbonitri
le (3q). Butylamine and 2a were used as reactants to give 3q. Yield: 88%.Yellow solid. Mp >300 °C.
1
H NMR (400 MHz, DMSOꢀd ) δ: 11.99 (s, 1H), 9.23 (s, 2H), 7.68 – 7.58 (m, 2H), 7.20 – 7.15 (m,
6
2
H), 4.26 (t, J = 7.3 Hz, 2H), 1.77 – 1.65 (m, 2H), 1.40 – 1.30 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H). 13C
NMR (101 MHz, DMSOꢀd ) δ: 158.3, 146.9, 142.7, 141.4, 139.3, 134.2, 126.2, 122.0, 121.9, 118.2,
6
+
117.7, 117.0, 116.4, 112.3, 84.4, 43.9, 30.5, 19.7, 14.0. HR ESIꢀMS (M + H) m/z = 355.1428 (Cacld
for C H N : 355.1425), HPLC purity: 99.2%.
1
9
16
8
6
-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-(3-hydroxypropyl)-5H-pyrrolo[2,3-b]pyrazine-2,
3-dicarbonitrile (3r). Propanolamine and 2a were used as reactants to give 3r. Yield: 83%.Yellow
1
solid. Mp >300 °C. H NMR (400 MHz, DMSOꢀd ) δ: 11.99 (s, 1H), 9.20 (s, 2H), 7.83 – 7.52 (m,
6
2
H), 7.36 – 7.03 (m, 2H), 4.68 (s, 1H), 4.32 (t, J = 7.2 Hz, 2H), 3.51 (t, J = 6.1 Hz, 2H), 1.95 – 1.87
1
3
(m, 2H). C NMR (101 MHz, DMSOꢀd ) δ: 157.5, 146.6, 142.2, 140.5, 138.9, 133.4, 125.5, 121.6,
6
+
1
21.5, 117.8, 117.2, 116.6, 115.9, 83.8, 58.0, 38.2, 31.0. HR ESIꢀMS (M + H) m/z = 357.1199
(
Cacld for C H N O: 357.1218), HPLC purity: 97.9%.
18 14 8
6
-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-(3-(piperidin-1-yl)propyl)-5H-pyrrolo[2,3-b]pyra
zine-2,3-dicarbonitrile (3s). 1ꢀPiperidinepropanamine and 2a were used as reactants to give 3s.
1
Yield: 79%. Yellow solid. Mp >300 °C. H NMR (400 MHz, DMSOꢀd ) δ: 11.96 (s, 1H), 9.26 (br s,
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2H), 7.68 – 7.58 (m, 2H), 7.22 – 7.14 (m, 2H), 4.29 (t, J = 6.3 Hz, 2H), 2.30 (t, J = 6.1 Hz, 2H), 2.20
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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3
3
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3
3
4
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4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
13
(
s, 4H), 2.02 – 1.80 (m, 2H), 1.38ꢀ1.22 (m, 6H). C NMR (101 MHz, DMSOꢀd ) δ: 158.0, 146.5,
6
1
42.3, 140.9, 139.0, 133.8, 125.5, 121.5, 117.7, 117.2, 116.5, 115.9, 111.8, 84.0, 54.9, 53.6, 39.2,
+
2
5.2, 24.3, 23.9. HR ESIꢀMS (M + H) m/z = 426.2128 (Cacld for C H N : 426.2149, HPLC
2
3
23
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
purity: 99.6%.
6-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-(4-(dimethylamino)butyl)-5H-pyrrolo[2,3-b]pyra
zine-2,3-dicarbonitrile (3t). N, NꢀDimethylbutylamine and 2a were used as reactants to give 3t.
1
Yield: 87%.Yellow solid. Mp >202 °C (decomp). H NMR (400 MHz, DMSOꢀd ) δ: 11.96 (s, 1H),
6
9.19 (br s, 2H), 7.71– 7.63 (m, 2H), 7.24 – 7.14 (m, 2H), 4.25 (t, J = 7.3 Hz, 2H), 2.29 (t, J = 7.2 Hz,
13
2
H), 2.14 (s, 6H), 1.80 – 1.70 (m, 2H), 1.52 – 1.44 (m, 2H). C NMR (101 MHz, DMSOꢀd ) δ:
6
1
57.8, 146.9, 142.7, 141.0, 139.3, 134.2, 126.3, 122.0, 118.3, 117.7, 117.0, 116.4, 112.3, 84.3, 58.7,
+
4
5.4, 41.0, 26.3, 24.1. HR ESIꢀMS (M + H) m/z = 398.1866 (Cacld for C H N : 398.1847), HPLC
21
21
9
purity: 97.1%.
6-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-(4-(diethylamino)butyl)-5H-pyrrolo[2,3-b]pyrazi
ne-2,3-dicarbonitrile (3u). N, NꢀDiethylbutylamine and 2a were used as reactants to give 3u. Yield:
1
8
7%.Yellow solid. Mp>214 °C (decomp). H NMR (400 MHz, DMSOꢀd ) δ: 11.98 (s, 1H), 9.66 (br
6
s, 2H), 7.68 – 7.58 (m, 2H), 7.23 – 7.13 (m, 2H), 4.32 – 4.26 (m, 2H), 2.48 – 2.34 (m, 6H), 1.79 –
13
1
.69 (m, 2H), 1.50 – 1.40 (m, 2H), 0.93 (t, J = 6.1 Hz, 6H). C NMR (101 MHz, DMSOꢀd ) δ:
6
1
57.8, 146.9, 140.9, 139.2, 126.2, 122.0, 118.3, 117.7, 117.0, 116.4, 112.4, 100.0, 84.3, 52.2, 46.9,
+
41.0, 26.5, 24.1, 12.4. HR ESIꢀMS (M + H) m/z = 427.1275 (Cacld for C H N ): 426.2160),
2
3
25
9
HPLC purity: 99.3%.
-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-(3-(aniline)propyl)-5H-pyrrolo[2,3-b]pyrazine-2,
6
3
-dicarbonitrile (3v). Nꢀphenylpropaneꢀ1,3ꢀdiamine and 2a were used as reactants to give 3v. Yield:
1
9
1%. Mp >212 °C (decomp). H NMR (400 MHz, DMSOꢀd ) δ: 11.99 (s, 1H), 9.24 (s, 2H), 7.73 –
6
7.55 (m, 2H), 7.19ꢀ7.15 (m, 2H), 7.05 (t, J = 7.8 Hz, 2H), 6.58ꢀ6.52 (m, 3H), 5.55 (s, 1H), 4.37 (t, J
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Journal of Medicinal Chemistry
Page 22 of 51
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5
5
5
5
6
1
3
=
6.8 Hz, 2H), 3.09 (t, J = 6.2 Hz, 2H), 2.12 – 1.95 (m, 2H). C NMR (101 MHz, DMSOꢀd ) δ:
6
1
2
57.8, 149.1, 146.9, 140.9, 139.2, 129.3, 126.2, 122.0, 118.3, 116.9, 116.5, 116.3, 112.5, 84.3, 40.7,
+
8.1. HR ESIꢀMS (M + H) m/z = 414.2162 (Cacld for C H N ): 414.2149), HPLC purity: 97.0%.
22 23
9
6
-Amino-7-(1H-benzo[d]imidazol-2-yl)-5-(5-(dimethylamino)pentyl)-6,7-dihydro-5H-pyrro
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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4
5
6
7
8
9
0
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6
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8
9
0
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5
6
7
8
9
0
lo[2,3-b]pyrazine-2,3-dicarbonitrile (3w). N, Nꢀdimethylpentaneꢀ1,5ꢀdiamine and 2a were used as
1
reactants to give 3w. Yield: 88%. Mp > 232°C (decomp). H NMR (400 MHz, DMSOꢀd ) δ: 11.94 (s,
6
1
H), 9.26 (br s, 2H), 7.66ꢀ7.58 (m, 2H), 7.21ꢀ7.13 (m, 2H), 4.22 (t, J = 6.8 Hz, 2H), 2.18 (t, J = 7.0
13
Hz, 2H), 2.10 (s, 6H), 1.81 – 1.67 (m, 2H), 1.49ꢀ1.41 (m, 2H), 1.38 – 1.25 (m, 2H). C NMR (101
MHz, DMSOꢀd ) δ: 157.8, 146.9, 141.0, 139.2, 126.3, 122.1, 118.3, 117.1, 116.4, 84.3, 59.3, 45.6,
6
+
4
1.1, 28.3, 27.0, 24.1. HR ESIꢀMS (M + H) m/z = 434.1847 (Cacld for C H N ): 434.1836),
2
4
19
9
HPLC purity: 96.9%.
6
-Amino-5-(4-(dimethylamino)butyl)-5H-pyrrolo[2,3-b]pyrazine-2,3,7-tricarbonitrile (3x).
48
The 3x were synthesized following the reported method. A solution of 2f (0.5 mmol) and N,
Nꢀdimethylbutyldiamine (1.0 mmol) in DMF (3 ml) was stirred at 60 °C for 5 h. It was then cooled,
acidified with acetic acid (0.5 ml), the precipitate filtered off, and recrystallized from dioxane. Yield:
1
7
0%. Yellow solid. Mp > 208°C (decomp). H NMR (400 MHz, DMSOꢀd ) δ: 9.26 (br s, 2H), 4.22
6
(t, J = 6.8 Hz, 2H), 2.66 (t, J = 6.0 Hz, 2H), 2.44 (s, 6H), 2.03 – 1.95 (m, 2H), 1.82 – 1.75 (m, 2H).
13
C NMR (101 MHz, DMSOꢀd ) δ: 151.7, 141.0, 139.4, 132.6, 126.3, 118.5, 117.6, 115.8, 88.5,
6
+
6
1.3, 50.0, 47.0, 28.6, 25.5. HR ESIꢀMS (M + H) m/z = 308.3476 (Cacld for C H N : 308.3411),
1
5
16
8
HPLC purity: 96.2%.
6
-Amino-7-(benzo[d]oxazol-2-yl)-5-(3-(dimethylamino)propyl)-5H-pyrrolo[2,3-b]pyrazine-
2
4
7
,3-dicarbonitrile (4m). N, NꢀDimethylꢀ1,3ꢀpropanediamine and 2b were used as reactants to give
1
m.Yield: 38%.Yellow solid. Mp >300 °C. H NMR (400 MHz, CDCl ) δ: 9.24 (br s, 2H), 7.65 –
3
.55 (m, 2H), 7.30 – 7.20 (m, 2H), 4.22 (t, J = 6.7 Hz, 2H), 2.35 – 2.25(m, 8H), 2.10 – 2.00 (m, 2H).
1
3
C NMR (101 MHz, DMSOꢀd ) δ: 157.8, 146.5, 142.2, 140.6, 138.6, 133.7, 125.7, 121.5, 117.7,
6
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Page 23 of 51
Journal of Medicinal Chemistry
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+
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17.2, 116.5, 116.0, 111.7, 83.8, 55.0, 44.7, 38.7, 25.7. HR ESIꢀMS (M + H) m/z = 387.1648 (Cacld
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
for C H N O: 387.1676), HPLC purity: 99.6%.
2
0
18
8
6-Amino-7-(benzo[d]oxazol-2-yl)-5-(3-(piperidin-1-yl)propyl)-5H-pyrrolo[2,3-b]pyrazine-2
,3-dicarbonitrile (4s). 1ꢀPiperidinepropanamine and 2b were used as reactants to give 4s. Yield:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
4
0%.Yellow solid. Mp >300 °C. H NMR (400 MHz, DMSOꢀd ) δ: 9.29 (br s, 2H), 7.81 (d, J = 6.8
6
Hz, 1H), 7.70 (d, J =6.8 Hz, 1H), 7.41 – 7.30 (m, 2H), 4.29 (t, J = 6.3 Hz, 2H), 2.31 (d, J = 5.9 Hz,
1
3
2H), 2.26 – 2.18 (m, 4H), 1.978 – 1.87(m, 2H), 1.36 – 1.25 (m, 6H). C NMR (101 MHz,
DMSOꢀd ) δ: 159.5, 158.7, 149.5, 141.7, 141.2, 126.5, 125.1, 124.6, 119.5, 118.7, 116.6, 116.3,
6
+
111.0, 82.2, 55.2, 54.1, 25.7, 24.6, 24.4. HR ESIꢀMS (M + H) m/z = 427.1987 (Cacld for
C H N O: 427.1989), HPLC purity: 98.1%.
23
22
8
6
-Amino-7-(benzo[d]oxazol-2-yl)-5-(4-(dimethylamino)butyl)-5H-pyrrolo[2,3-b]pyrazine-2
,3-dicarbonitrile (4t). N, NꢀDimethylbutylamine and 2b were used as reactants to give 4t.Yield:
1
4
5%.Yellow solid. Mp >300 °C. H NMR (400 MHz, CDCl ) δ: 9.40 (br s, 2H), 7.71 (dd, J = 6.8,
3
2.8, 1H), 7.67(dd, J =6.0, 2.0Hz, 1H ), 7.40 – 7.32(m, 2H), 4.35 – 4.18 (m, 2H), 2.66 (t, J = 6.0 Hz,
1
3
2
1
H), 2.44 (s, 6H), 2.03 – 1.95 (m, 2H), 1.82 – 1.75 (m, 2H). C NMR (101 MHz, DMSOꢀd ) δ:
6
59.3, 156.7, 151.7, 141.0, 139.4, 132.6, 126.3, 125.8, 124.0, 122.0, 121.0, 118.5, 116.3, 115.8, 88.5,
+
57.5, 44.0, 40.3, 25.5, 22.7. HR ESIꢀMS (M + H) m/z = 401.1816 (Cacld for C H N O: 401.1833),
2
1
20
8
HPLC purity: 95.3%.
-Amino-7-(benzo[d]oxazol-2-yl)-5-(4-(diethylamino)butyl)-5H-pyrrolo[2,3-b]pyrazine-2,3
6
-dicarbonitrile (4u). N, NꢀDiethylbutylamine and 2b were used as reactants to give 4u.Yield:
1
4
1
3%.Yellow solid. Mp >300 °C. H NMR (400 MHz, DMSOꢀd ) δ: 9.37 (br s, 2H),7.85 – 7.76 (m,
6
H), 7.70 (d, J = 6.9 Hz, 1H), 7.42 – 7.30 (m, 2H), 4.26 (t, J = 7.2 Hz, 2H), 2.59 (s, 6H), 1.84 – 1.66
1
3
(m, 2H), 1.57 – 1.45 (m, 2H), 1.00 (t, J = 7.0 Hz, 6H). C NMR (101 MHz, DMSOꢀd ) δ: 159.9,
6
157.5, 148.8, 140.8, 140.6, 139.3, 126.2, 124.7, 124.1, 119.0, 118.2, 116.1, 115.8, 110.5, 81.5, 51.3,
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Journal of Medicinal Chemistry
Page 24 of 51
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2
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2
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5
5
5
5
5
5
5
5
6
+
4
6.2, 40.6, 25.6, 22.7, 10.8. HR ESIꢀMS (M + H) m/z = 429.2123 (Cacld for C H N O: 429.2146),
2
3
24
8
HPLC purity: 98.6%.
6-Amino-7-(benzo[d]thiazol-2-yl)-5-(2-(dimethylamino)ethyl)-5H-pyrrolo[2,3-b]pyrazine-2
,
3-dicarbonitrile (5c). N, NꢀDimethylethylamine and 2c were used as reactants to give 5c.Yield:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
9
1%.Yellow solid. Mp >300 °C. H NMR (400 MHz, DMSOꢀd ) δ: 9.31 (br s, 2H), 8.08 (d, J = 7.9
6
Hz, 1H), 7.98 (d, J = 7.9 Hz, 1H), 7.50 (td, J = 8.0, 1.2 Hz, 1H), 7.36 (td, J = 8.0, 1.2 Hz, 1H), 4.35
1
3
(
t, J = 5.6 Hz, 2H), 2.74 – 2.66 (m, 2H), 2.27 (s, 6H). C NMR (101 MHz, DMSOꢀd ) δ: 159.8,
6
1
58.0, 152.2, 141.8, 140.0, 133.3, 126.8, 124.4, 122.4, 121.4, 118.9, 116.7, 116.2, 89.1, 57.0, 45.4.
+
HR ESIꢀMS (M + H) m/z = 387.1156 (Cacld for C H N S): 387.1146, HPLC purity: 95.4%.
19
16
8
6
-Amino-7-(benzo[d]thiazol-2-yl)-5-(3-(dimethylamino)propyl)-5H-pyrrolo[2,3-b]pyrazine
-
2,3-dicarbonitrile (5m). N, NꢀDimethylꢀ1,3ꢀpropanediamine and 2c were used as reactants to give
1
5
m. Yield: 93%.Yellow solid. Mp >300 °C. H NMR (400 MHz, DMSOꢀd ) δ: 9.24 (br s, 2H), 8.11
6
(
d, J = 7.4 Hz, 1H), 7.98 (d, J = 7.4 Hz, 1H), 7.52 (dt, J = 8.0, 1.2 Hz, 1H), 7.37 (dt, J = 8.0, 1.2 Hz,
1
3
1H), 4.34 (t, J = 6.7 Hz, 2H), 2.92 – 2.80 (m, 2H), 2.54 (s, 6H), 2.12 – 2.02 (m, 2H). C NMR (101
MHz, DMSOꢀd ) δ: 159.4, 157.0, 151.8, 141.2, 139.7, 132.6, 126.4, 125.8, 124.0, 122.0, 121.0,
6
+
1
18.5, 116.4, 115.6, 88.6, 54.3, 43.2, 38.4, 24.2. HR ESIꢀMS (M + H) m/z = 403.1440 (Cacld for
C H N S: 403.1448), HPLC purity: 96.8%.
20
18
8
6
-Amino-7-(benzo[d]thiazol-2-yl)-5-(4-(dimethylamino)butyl)-5H-pyrrolo[2,3-b]pyrazine-2
,
3-dicarbonitrile (5t). N, NꢀDimethylbutylamine and 2c were used as reactants to give 5t.Yield:
1
87%.Yellow solid. Mp >300 °C. H NMR (400 MHz, DMSOꢀd ) δ: 9.14 (br s, 2H), 8.12 (d, J = 7.4
6
Hz, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.51 (td, J =8.0, 1.2 Hz, 1H), 7.37 (td, J = 8.0, 1.2 Hz, 1H), 4.28 (t,
13
J = 7.1 Hz, 2H), 2.60 – 2.54 (m, 2H), 2.33 (s, 6H), 1.80 – 1.70 (m, 2H), 1.58 – 1.50 (m, 2H). C
NMR (101 MHz, DMSOꢀd ) δ: 159.5, 156.8, 151.6, 141.0, 139.5, 126.3, 125.8, 125.3, 124.0, 122.0,
6
+
1
21.0, 118.5, 117.9, 116.3, 115.8, 88.5, 57.5, 44.0, 40.3, 25.5, 22.8. HR ESIꢀMS (M + H) m/z =
417.1598 (Cacld for C H N S: 417.1604), HPLC purity: 99.2%.
2
1
20
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Page 25 of 51
Journal of Medicinal Chemistry
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-Amino-5-(2-(dimethylamino)ethyl)-7-(1-methyl-1H-benzo[d]imidazol-2-yl)-5H-pyrrolo[2
,
3-b]pyrazine-2,3-dicarbonitrile (6c). N, NꢀDimethylamine and 2d were used as reactants to give
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
6
2
3
c. Yield: 83%. Yellow solid. Mp >258 °C (decomp). H NMR (400 MHz, DMSOꢀd ) δ: 9.29 (br s,
6
H), 7.64 (d, J = 6.9 Hz, 1H), 7.58 (d, J = 7.2 Hz, 1H), 7.33 – 7.20 (m, 2H), 4.38 (s, 2H), 4.01 (s,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
H), 2.69 (s, 2H), 2.26 (s, 6H). C NMR (101 MHz, DMSOꢀd ) δ: 159.3, 148.2, 142.9, 141.1, 139.6,
6
136.3, 125.9, 122.4, 122.2, 118.5, 117.7, 116.9, 116.4, 110.5, 84.4, 57.2, 45.5, 32.8. HR ESIꢀMS (M
+
+
H) m/z = 386.1821 (Cacld for C H N : 386.1836), HPLC purity: 97.6%.
2
0
19
9
6
-Amino-5-(2-(diethylamino)ethyl)-7-(1-methyl-1H-benzo[d]imidazol-2-yl)-5H-pyrrolo[2,3
-b]pyrazine-2,3-dicarbonitrile (6d). N, NꢀDiethylamine and 2d were used as reactants to give 6d.
1
Yield: 89%. Yellow solid. Mp >239 °C (decomp). H NMR (400 MHz, DMSOꢀd ) δ: 9.23 (br s, 2H),
6
7
3
1
.67 (d, J = 7.3 Hz, 1H), 7.60 (d, J = 7.4 Hz, 1H), 7.33 – 7.21 (m, 2H), 4.75 (s, 2H), 4.00 (s, 3H),
1
3
.47 (s, 2H), 3.40 (s, 4H), 1.25 (s, 6H). C NMR (101 MHz, DMSOꢀd ) δ: 158.6, 147.8, 142.8,
6
41.7, 140.8, 136.2, 126.2, 122.5, 122.3, 118.6, 117.6, 116.9, 116.3, 110.6, 84.8, 47.1, 32.7, 9.0. HR
+
ESIꢀMS (M + H) m/z = 414.2133 (Cacld for C H N : 414.2149), HPLC purity: 96.9%.
2
2
23
9
6
-Amino-5-(3-(dimethylamino)propyl)-7-(1-methyl-1H-benzo[d]imidazol-2-yl)-5H-pyrrolo
[
2,3-b]pyrazine-2,3-dicarbonitrile (6m). N, NꢀDimethylꢀ1,3ꢀpropanediamine and 2d were used as
1
reactants to give 6m. Mp >204 °C (decomp).Yield: 91%. Yellow solid. H NMR (400 MHz,
DMSOꢀd ) δ: 9.28 (br s, 2H), 7.65 (dd, J = 6.8, 1.5 Hz, 1H), 7.59 (dd, J = 6.6, 1.4 Hz, 1H), 7.29 –
6
7
.16 (m, 2H), 4.38 (t, J = 5.9 Hz, 2H), 4.01 (s, 3H), 2.68 (t, J = 5.7 Hz, 2H), 2.25 (s, 6H), 2.24 (s,
1
3
2H). C NMR (101 MHz, DMSOꢀd ) δ: 159.3, 148.2, 143.0, 141.0, 139.8, 136.3, 125.9, 122.4,
6
+
1
4
22.2, 118.6, 117.8, 117.0, 116.4, 110.6, 84.4, 55.5, 45.2, 32.7, 26.2. HR ESIꢀMS (M + H) m/z =
00.2003 (Cacld for C H N : 400.1993), HPLC purity: 98.0%.
21 21
9
6
-Amino-5-(3-(diethylamino)propyl)-7-(1-methyl-1H-benzo[d]imidazol-2-yl)-5H-pyrrolo[2,
3
-b]pyrazine-2,3-dicarbonitrile (6n). N, NꢀDiethylꢀ1,3ꢀpropanediamine and 2d were used as
1
reactants to give 6n. Yield: 76%. Yellow solid. Mp >192 °C (decomp). H NMR (400 MHz,
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Page 26 of 51
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
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4
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5
5
5
5
5
5
5
5
5
5
6
DMSOꢀd ) δ: 9.24 (br s, 2H), 7.66 (d, J = 7.2 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.30 – 7.23 (m, 2H),
6
4
6
1
.28 (t, J = 6.9 Hz, 2H), 3.99 (s, 3H), 2.46 – 2.38 (m, 6H), 1.95 – 1.82 (m, 2H), 0.94 (t, J = 7.0 Hz,
1
3
H). C NMR (101 MHz, DMSOꢀd ) δ: 159.0, 148.1, 143.0, 140.9, 139.8, 136.3, 125.9, 122.6,
6
+
22.4, 118.6, 117.8, 117.0, 116.4, 110.5, 84.4, 49.5, 46.1, 32.7, 25.8, 11.5. HR ESIꢀMS (M + H) m/z
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
=
428.2306 (Cacld for C H N : 428.2306), HPLC purity: 96.5%.
23 25 9
6-Amino-5-(4-(dimethylamino)butyl)-7-(1-methyl-1H-benzo[d]imidazol-2-yl)-5H-pyrrolo[2
,3-b]pyrazine-2,3-dicarbonitrile (6t). N, NꢀDimethylbutylamine and 2d were used as reactants to
1
give 6t. Yield: 92%. Yellow solid. Mp >228 °C (decomp). H NMR (400 MHz, DMSOꢀd ) δ: 9.03
6
(br s, 2H), 7.66 (dd, J = 6.8, 1.4 Hz, 1H), 7.59 (d, J = 7.2 Hz,1H), 7.32 – 7.13 (m, 2H), 4.26 (t, J =
7
2
1
.2 Hz, 2H), 3.99 (s, 3H), 2.27 (t, J = 7.1 Hz, 2H), 2.13 (s, 6H), 1.76 – 1.68 (m, 2H), 1.53 – 1.45 (m,
1
3
H). C NMR (101 MHz, DMSOꢀd ) δ: 158.6, 148.1, 142.9, 140.7, 139.6, 136.3, 125.9, 122.4,
6
22.2, 118.5, 117.8, 117.0, 116.4, 110.5, 84.3, 58.8, 45.5, 41.0, 32.7, 26.4, 24.2. HR ESIꢀMS (M +
+
H) m/z = 414.2141 (Cacld for C H N : 414.2149), HPLC purity: 97.1%.
2
2
23
9
6-Amino-5-(4-(diethylamino)butyl)-7-(1-methyl-1H-benzo[d]imidazol-2-yl)-5H-pyrrolo[2,3
-b]pyrazine-2,3-dicarbonitrile (6u). N, NꢀDiethylbutylamine and 2d were used as reactants to give
1
6
u. Yield: 88%. Yellow solid. Mp >239 °C (decomp). H NMR (400 MHz, DMSOꢀd ) δ: 8.97 (br s,
6
2H), 7.67 (dd, J = 6.9, 1.5 Hz, 1H), 7.60 (dd, J = 6.8, 1.2 Hz, 1H), 7.32 – 7.22 (m, 2H), 4.28 (t, J =
7.3 Hz, 2H), 4.01 (s, 3H), 2.49 – 2.43 (m, 6H), 1.80 – 1.66 (m, 2H), 1.51 – 1.43 (m, 2H), 0.95 (t, J =
1
3
6
.7 Hz, 6H). C NMR (101 MHz, DMSOꢀd ) δ: 158.6, 148.1, 142.9, 140.7, 139.6, 136.3, 125.9,
6
122.4, 122.2, 118.6, 117.8, 116.9, 116.4, 110.5, 84.3, 52.1, 46.7, 41.1, 32.7, 26.5, 24.0, 12.1. HR
+
ESIꢀMS (M + H) m/z = 442.2451 (Cacld for C H N : 442.2462), HPLC purity: 99.8%.
2
4
27
9
6
-Amino-7-(6-chloro-1-methyl-1H-benzo[d]imidazol-2-yl)-5-(2-(dimethylamino)ethyl)-5H-
pyrrolo[2,3-b]pyrazine-2,3-dicarbonitrile (7c). N, NꢀDimethylamine and 2e were used as reactants
1
to give 7c. Yield: 81%. Yellow solid. Mp >300 °C. H NMR (400 MHz, DMSOꢀd ) δ: 12.16 (s, 1H),
6
9.42 (br s, 2H), 7.67ꢀ7.61 (m, 2H), 7.20 (dd, J = 8.4, 1.9 Hz, 1H), 4.38 (t, J = 5.7 Hz, 2H), 2.68 (t, J
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Page 27 of 51
Journal of Medicinal Chemistry
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2
3
4
1
3
=
5.4 Hz, 2H), 2.24 (s, 6H). C NMR (101 MHz, DMSOꢀd ) δ: 158.4, 141.2, 139.5, 126.2, 126.0,
6
+
1
22.1, 118.5, 116.9, 116.4, 83.9, 55.5, 45.2, 26.2. HR ESIꢀMS (M + H) m/z = 406.1280 (Cacld for
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
C H N Cl: 406.1290), HPLC purity: 99.2%.
19 16
9
6
-Amino-7-(6-chloro-1-methyl-1H-benzo[d]imidazol-2-yl)-5-(2-(diethylamino)ethyl)-5H-py
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
rrolo[2,3-b]pyrazine-2,3-dicarbonitrile (7d). N, NꢀDiethylamine and 2e were used as reactants to
1
give 7d. Yield: 80%. Yellow solid. Mp >300 °C. H NMR (400 MHz, DMSOꢀd ) δ: 12.16 (s, 1H),
6
9.42 (br s, 2H), 7.68 – 7.60 (m, 2H), 7.20 (dd, J = 8.4, 1.9 Hz, 1H), 4.38 (t, J = 5.7 Hz, 2H), 2.76 (t, J
1
3
=
5.4 Hz, 2H), 2.52 – 2.44 (m, 4H), 0.84 (t, J = 7.2 Hz, 6H). C NMR (101 MHz, DMSOꢀd ) δ:
6
158.2, 148.2, 141.1, 139.4, 126.4, 126.2, 122.1, 118.5, 116.9, 116.4, 83.9, 49.6, 46.1, 25.8, 11.6. HR
+
ESIꢀMS (M + H) m/z = 432.1443 (Cacld for C H N Cl: 432.1457), HPLC purity: 97.1%.
2
1
20
9
6
-Amino-7-(6-chloro-1-methyl-1H-benzo[d]imidazol-2-yl)-5-(3-(dimethylamino)propyl)-5
H-pyrrolo[2,3-b]pyrazine-2,3-dicarbonitrile (7m). N, NꢀDimethylꢀ1,3ꢀpropanediamine and 2e
1
were used as reactants to give 7m. Yield: 95%. Yellow solid. Mp >300 °C. H NMR (400 MHz,
DMSOꢀd ) δ: 12.10 (s, 1H), 9.06 (s, 2H), 7.60 (s, 2H), 7.18 (d, J = 7.1 Hz, 1H), 4.23 (s, 2H), 2.30 (s,
6
1
3
2
1
H), 2.17 (s, 6H), 1.89 (s, 2H). C NMR (101 MHz, DMSOꢀd ) δ: 158.4, 141.2, 139.5, 126.3, 126.1,
6
+
22.1, 118.5, 117.0, 116.4, 84.0, 55.5, 45.2, 39.2, 26.2. HR ESIꢀMS (M + H) m/z = 420.1438 (Cacld
for C H N Cl: 420.1446), HPLC purity: 97. 6%.
2
0
18
9
6
-Amino-7-(6-chloro-1-methyl-1H-benzo[d]imidazol-2-yl)-5-(3-(diethylamino)propyl)-5H-
pyrrolo[2,3-b]pyrazine-2,3-dicarbonitrile (7n). N, NꢀDiethylꢀ1,3ꢀpropanediamine and 2e were
1
used as reactants to give 7n. Yield: 73%. Yellow solid. Mp >300 °C. H NMR (400 MHz, DMSOꢀd )
6
δ: 12.06 (s, 1H), 9.05 (BR s, 2H), 7.61 (m, 2H), 7.18 (dd, J = 8.5, 2.0 Hz, 1H), 4.25 (t, J = 6.9 Hz,
1
3
2H), 2.49 – 2.39 (m, 6H), 1.98 – 1.74 (m, 2H), 0.92 (t, J = 7.1 Hz, 6H). C NMR (101 MHz,
DMSOꢀd ) δ: 158.2, 141.2, 139.5, 126.2, 126.0, 122.1, 118.5, 116.9, 116.4, 84.0, 49.6, 46.1, 25.8,
6
+
1
1.6. HR ESIꢀMS (M + H) m/z = 448.1744 (Cacld for C H N Cl: 448.1759), HPLC purity: 98.9%.
2
2
22
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Journal of Medicinal Chemistry
Page 28 of 51
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2
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6
7
8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
-Amino-7-(6-chloro-1-methyl-1H-benzo[d]imidazol-2-yl)-5-(4-(dimethylamino)butyl)-5H-
pyrrolo[2,3-b]pyrazine-2,3-dicarbonitrile (7t). N, NꢀDimethylbutylamine and 2e were used as
1
reactants to give 7t. Yield: 90%. Yellow solid. Mp >300 °C. H NMR (400 MHz, DMSOꢀd ) δ: 12.08
6
(
s, 1H), 9.13 (s, 2H), 7.68ꢀ7.62 (m, 2H), 7.19 (dd, J = 8.5, 1.9 Hz, 1H), 4.24 (t, J = 7.1 Hz, 2H), 2.27
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
(
t, J = 7.0 Hz, 2H), 2.13 (s, 6H), 1.77 – 1.69 (m, 2H), 1.50 – 1.44 (m, 2H). C NMR (101 MHz,
DMSOꢀd ) δ: 157.7, 140.9, 139.2, 126.2, 126.0, 122.1, 118.5, 116.9, 116.3, 83.8, 58.6, 45.4, 40.8,
6
+
2
9
6.2, 24.1. HR ESIꢀMS (M + H) m/z = 434.1589 (Cacld for C H N Cl: 434.1603), HPLC purity:
2
1
20
9
8.0%.
6-Amino-7-(6-chloro-1-methyl-1H-benzo[d]imidazol-2-yl)-5-(4-(diethylamino)butyl)-5H-p
yrrolo[2,3-b]pyrazine-2,3-dicarbonitrile (7u). N, NꢀDiethylbutylamine and 2e were used as
1
reactants to give 7u. Yield: 88%. Yellow solid. Mp >300 °C. H NMR (400 MHz, DMSOꢀd ) δ:
6
1
2.03 (s, 1H), 9.20 (br s, 2H), 7.70 – 7.56 (m, 2H), 7.19 (dd, J = 8.5, 1.9 Hz, 1H), 4.24 (t, J = 7.1 Hz,
13
2
H), 2.45 – 2.37 (m, 6H), 1.77 – 1.69 (m, 2H), 1.50 – 1.39 (m, 2H), 0.93 (t, J = 7.1 Hz, 6H). C
NMR (101 MHz, DMSOꢀd ) δ: 157.8, 141.0, 139.2, 126.2, 126.1, 122.1, 118.4, 116.9, 116.3, 83.8,
6
+
5
2.0, 46.6, 40.9, 26.4, 23.8, 11.9. HR ESIꢀMS (M + H) m/z = 462.1899 (Cacld for C H N Cl:
2
3
24
9
4
62.1916), HPLC purity: 99.0%.
6-Amino-7-(1-(4-(dimethylamino)butyl)-1H-benzo[d]imidazol-2-yl)-5-methyl-5H-pyrrolo[2
,
3-b]pyrazine-2,3-dicarbonitrile (8t). Dimethylamine and 8-3 were used as reactants to give 8t.
1
Yield: 91%. Mp >190 °C (decomp). H NMR (400 MHz, DMSOꢀd ) δ: 9.01 (br s, 2H), 7.66 – 7.61
6
(m, 2H), 7.30 – 7.19 (m, 2H), 4.72 – 4.61 (m, 2H), 3.71 (s, 3H), 2.12 (t, J = 7.2 Hz, 2H), 2.02 (s,
13
6
H), 1.79 – 1.68 (m, 2H), 1.42 – 1.35 (m, 2H). C NMR (101 MHz, DMSOꢀd ) δ: 159.8, 147.8,
6
1
42.8, 140.9, 139.1, 135.4, 125.6, 122.4, 122.2, 118.5, 117.6, 116.9, 116.3, 110.7, 84.5, 58.9, 45.5,
+
4
5.0, 28.2, 24.5. HR ESIꢀMS (M + H) m/z = 414.2137 (Cacld for C H N : 414.2149), HPLC
2
2
23
9
purity: 95.7%.
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Journal of Medicinal Chemistry
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-Amino-7-(1-(4-(diethylamino)butyl)-1H-benzo[d]imidazol-2-yl)-5-methyl-5H-pyrrolo[2,3
-
b]pyrazine-2,3-dicarbonitrile (8u). Diethylamine and 8-3 were used as reactants to give 8u. Yield:
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
8
7
1
6%. Mp >212 °C (decomp). H NMR (400 MHz, DMSOꢀd ) δ: 9.14 (br s, 2H), 7.74 – 7.56 (m, 2H),
6
.34 – 7.16 (m, 2H), 4.81 – 4.56 (m, 2H), 4.35 (s, 3H), 3.60 (t, J = 5.6 Hz, 2H), 3.44 (s, 4H), 1.88 –
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
.70(m, 4H), 1.06 (t, J = 6.7 Hz, 6H). C NMR (101 MHz, DMSOꢀd ) δ: 159.8, 147.8, 142.7, 140.8,
6
139.0, 135.3, 125.6, 122.4, 122.3, 118.5, 117.7, 116.9, 116.3, 110.6, 84.3, 56.5, 45.3, 44.4, 29.6,
+
2
8.2, 27.8, 19.0. HR ESIꢀMS (M + H) m/z = 442.2454 (Cacld for C H N : 442.2462), HPLC
24
27
9
purity: 95.4%.
Biological Assay. The testing of new chemical entities was performed using a commercially
available. Purified Topo II purchased from TopoGEN, Inc. pBR322 DNA purchased from Takara.
Topo I purchased from Takara. A stock solution of epotoside of concentration, 10 mM in DMSO,
was prepared and stored at ꢀ20 °C. Synthesized compounds were also stored at ꢀ20 °C. The assay
protocols followed were same as mentioned in the supplier manual, except the concentration of the
reagents which were varied according to the requirement.
Topo II-mediated DNA relaxation assay. We used the Topo II assay kit from TopoGEN to
determine the effects of drugs on DNA relaxation catalyzed by Topo II. Relaxation assays were
carried out according to the manufacturer’s instructions with minor modifications. The assay was
performed in a final volume of 20 ꢁL in Topo II reaction buffer (1 × Topo II buffer = 50 mM
−
1
TrisꢀHCl, pH 8.0, 150 mM NaCl, 10 mM MgCl , 2 mM ATP, 0.5 mM DTT, and 30 ꢁg.ml BSA)
2
with 0.2 ꢁg pBR322 DNA. Compounds were included in the reactions at a constant solvent volume.
Reactions were initiated by addition of 1 U human Topo II, and incubated for 30 min at 37 °C.
Reaction was terminated with 5 × stop buffer (5 ꢁL per 20 ꢁL reaction volume). Stop buffer
contained 5% sarcosyl, 0.02% bromophenol blue and 25% glycerol. Reaction products were
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