Synthesis and antiproliferative activity of novel heterocyclic indole-trimethoxyphenyl conjugates

Article abstract of DOI:10.3390/ph10030062

The synthesis and biological evaluation of a series of novel heterocyclic indole derivatives is described. The consolidation of the combretastatin and bisindolylmaleimide templates towards the inclusion of a novel heterocyclic ring proffered a versatile pharmacophore with which to pursue chemical diversification. Given literature precedent, maleimide was initially investigated in this role and the bioactivity assessed by measurement of NCI-60 cell panel growth. Subsequently, a range of 5-aminopyrazoles was designed and developed to explore the specific effect of heterocycle hydrogen bonding on cell growth. The unique electronic nature of the 5-aminopyrazole moiety allowed for regiospecific monosubstitution on different sites of the ring, such as thiourea substitution at the N(1) position for derivative 45 or trifluoroacetylation on the 5-amino position for 43. Further derivatisation led to the ultimate development of bicyclic pyrazolotriazinedione 41 and pyrimidine 42 systems. The antiproliferative activities of these 3,4-diaryl-5-aminopyrazoles were assessed using the NCI-60 cell screen, disclosing the discovery of distinct selectivity profiles towards a number of cell lines, such as SNB-75 CNS cancer, UO-31 and CAKI-1 renal cancer cells. A series of DNA topological assays discounted the interaction with topoisomerase II as a putative mechanism of action.

Full text of DOI:10.3390/ph10030062

pharmaceuticals
Article
Synthesis and Antiproliferative Activity of Novel
Heterocyclic Indole-Trimethoxyphenyl Conjugates
Michael M. Cahill, Kevin D. O’Shea, Larry T. Pierce, Hannah J. Winfield, Kevin S. Eccles,
Simon E. Lawrence and Florence O. McCarthy *
School of Chemistry, Analytical and Biological Chemistry Research Facility, University College Cork,
Western Road, Cork T12 YN60, Ireland; mickcahill72@hotmail.com (M.M.C.);
kevin_oshea@umail.ucc.ie (K.D.O.); lpierce9184@yahoo.co.uk (L.T.P.); 106432930@umail.ucc.ie (H.J.W.);
kevin.s.eccles@gmail.com (K.S.E.); simon.lawrence@ucc.ie (S.E.L.)
*
Correspondence: f.mccarthy@ucc.ie; Tel.: +353-214-901-695
Academic Editor: Jean Jacques Vanden Eynde
Received: 26 May 2017; Accepted: 27 June 2017; Published: 5 July 2017
Abstract: The synthesis and biological evaluation of a series of novel heterocyclic indole derivatives
is described. The consolidation of the combretastatin and bisindolylmaleimide templates towards
the inclusion of a novel heterocyclic ring proffered a versatile pharmacophore with which to pursue
chemical diversification. Given literature precedent, maleimide was initially investigated in this role
and the bioactivity assessed by measurement of NCI-60 cell panel growth. Subsequently, a range of
5-aminopyrazoles was designed and developed to explore the specific effect of heterocycle hydrogen
bonding on cell growth. The unique electronic nature of the 5-aminopyrazole moiety allowed for
regiospecific monosubstitution on different sites of the ring, such as thiourea substitution at the N(1)
position for derivative 45 or trifluoroacetylation on the 5-amino position for 43. Further derivatisation
led to the ultimate development of bicyclic pyrazolotriazinedione 41 and pyrimidine 42 systems.
The antiproliferative activities of these 3,4-diaryl-5-aminopyrazoles were assessed using the NCI-60
cell screen, disclosing the discovery of distinct selectivity profiles towards a number of cell lines,
such as SNB-75 CNS cancer, UO-31 and CAKI-1 renal cancer cells. A series of DNA topological assays
discounted the interaction with topoisomerase II as a putative mechanism of action.
Keywords: diarylmaleimide; diaryl-aminopyrazole; 5-aminopyrazole regioselective substitution;
drug discovery; NCI anticancer screen
1
. Introduction
The 3,4,5-trimethoxyphenyl moiety is a commonly-used fragment in anticancer drug discovery
due to its provenance from natural products and their derivatives. One such example is Combretastatin
A4 (CA-4, ) which was first isolated from the African willow tree, Combretum caffrum, by Pettit and
1
co-workers (Figure 1) and is a highly potent tubulin and cell growth inhibitor. The water-soluble
prodrug of CA-4, fosbretabulin is currently in clinical trials [1–3].
One of the primary mechanisms by which CA-4
such as podophyllotoxin ( ) can mediate anticancer activity is by binding to tubulin, thus preventing
microtubule formation and arresting the cell cycle [
1 or other 3,4,5-trimethoxyphenyl derivatives
2
4
]. An additional chemotherapeutic effect of CA-4
1
is its ability to disrupt established vasculature within solid tumours, while simultaneously rendering
normal vascular networks intact [5].
Pharmaceuticals 2017, 10, 62; doi:10.3390/ph10030062
www.mdpi.com/journal/pharmaceuticals

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Figure 1. Structures of Combretastatin A4 (CA-4, 1) and podophyllotoxin 2.
Examining the structural motif of CA-4, it is established that the 3,4,5-trimethoxyphenyl ring is
an essential moiety for cytotoxicity [ ]. For example, the 3,4,5-trimethoxyphenyl ring has been found
in many other natural products with activities against tubulin, such as steganacin ( , Figure 2) [ ].
In addition, variants of the moiety are also to be found in other anticancer agents, such as the
topoisomerase II (topo II) inhibitors etoposide ( ) and azatoxin ( ), which incorporates an indole
in design [ ]. Significantly, at lower concentrations, azatoxin ( ) is found to exhibit considerable
tubulin inhibition, while at higher doses, topo II activity is primarily observed [ ]. Alterations to the
,4,5-trimethoxyphenyl functional group of CA-4 , such as their complete removal or absence, result
in loss of potency [10,11].
6
3
7
4
5
8,
9
5
9
3
1
A number of subsequent chemical elaborations have primarily been focused on modifications to
the ethene double bond and the 2-methoxyphenol subunit. For example, in many instances, the ethene
double bond of CA-4
such as a pyrazole in the case of
cyclopropylamides and bicyclic thiazolopyrimidinones [12
1
has been modified towards the inclusion of a heterocyclic functionality,
or an imidazole for (Figure 3) and, more recently, tetrazoles,
16]. In other combretastatin analogues,
6
7
–
both the ethene double bond and 2-methoxyphenol ring have been replaced with heterocycles, often
resulting in the mediation of highly promising chemotherapeutic activity [17].
Figure 2. Structures of steganacin (3), etoposide (4) and azatoxin (5).
Figure 3. Structures combretastatin derivatives 6 and 7.

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In terms of kinase activity, Peifer and co-workers described the synthesis of moguntinone
Figure 4), containing maleimide and indole moieties, which was found to be an extraordinarily
potent kinase inhibitor of VEGF-R2 (IC50 = 2.5 nM) and VEGF-R3 (IC50 = 5 nM) [18]. Modification of
the maleimide moiety of towards regioisomeric lactam derivatives produced strikingly different
biological profiles. Though pyrrol-2-one was found to potently inhibit VEGF-R2 kinase (IC50 = 31 nM),
its regioisomeric analogue 10 displayed significantly weaker activity (IC50 = 11 M), thus illustrating
8
(
8
9
µ
the importance of the orientation of the pyrrolone within the active site for the effective exploitation of
H-bonding interactions [19].
Elevated levels of angiogenic factors, such as basic fibroblast growth factor, vascular endothelial
growth factor (VEGF) and matrix metalloproteinases (MMPs), have been implicated in the promotion
of angiogenesis [20,21]. A correlation has previously been demonstrated between high expression of
VEGF, which promotes dense formation of tumour microvessels and a poor prognosis in different
types of human tumour [22,23].
Recent work on analogous derivatives by Dannhardt et al., such as maleimides 11 and 12
containing 7-azaindolyl and 6-azaindolyl functionalities, respectively, has illustrated potent inhibition
of angiogenesis and cell proliferation (Figure 4) [24,25]. In examining the molecular targets associated
with the candidates 11 and 12, significant activity was observed for both against VEGFR-2 and FLT-3
protein kinases. Furthermore, it was found that although 6-azaindolyl 12 was a potent inhibitor of
GSK-3β, 7-azaindolyl 11 is completely inactive against the same enzyme. In addition, this group has
described selective treatment of colorectal cancer using this template [24,25].
Figure 4. Maleimides 8, 11 and 12 and pyrrolones 9 and 10.
Therefore, it is evident that structural diversification of this template can lead to potent
anticancer and kinase inhibitory activity. From Structures 2–12, it is obvious that this pharmacophore
incorporating an indole, trimethoxyphenyl and a linking headgroup has direct relevance to the
disruption of cancer progression. Rationalising the enhancement of existing H-bonding interactions
or potential exploitation of new contacts, we embarked on the synthesis of derivatives of the
3
,4-diaryl-1-hydroxymaleimide scaffold 13, in order to probe the effects of oxygen insertion and indole
N-substitution. Subsequently, replacement of the maleimide/pyrrolone moieties with a closely
analogous 5-aminopyrazole ring (14 along with a regioisomeric series of general structure 15; Figure 5)
was undertaken to validate the approach and explore new chemical space [26,27].
In this work, we report the synthesis, full characterization and initial screening of effects on cancer
cell growth to investigate the anticancer activity of this novel template. We also examine the interaction
of these derivatives with topoisomerase II as a potential mechanism of action via a series of DNA
topological assays, given the inherent activity of azatoxin and etoposide.

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Figure 5. General structures of 3,4-diaryl maleimides 13 and 3,4-diaryl-5-aminopyrazoles 14 and 15.
. Results and Discussion
2
2
.1. Synthesis of the 3,4-Diaryl-1-Hydroxymaleimide Scaffold 13
The synthesis of a series of 3,4-diaryl-1-hydroxymaleimides was initially undertaken in order to
investigate the H-bonding framework required to affect cell growth. Synthesis begins from indole
Scheme 1, 16), which may be alkylated under standard conditions (19 and 22) to investigate the
(
effect of N-H capping and incorporate an element of solubility. This substitution has the possibility
to probe two effects: the influence of the indole N-H on binding and a malleable nitrile functional
group to modify solubility. Indole potassium glyoxalate salt formation is undertaken in two steps
with acylation using oxalyl chloride followed by treatment with base (18, 21 and 24). Difficulties of
translation to 7-azaindole necessitated an alternate method, with aluminium trichloride followed by
ethyl chlorooxoacetate and then base hydrolysis yielding the glyoxalate (Scheme 2, 27).
O
O
O
O
OH
-
O K+
(
COCl)2
KOH
Et O
2
N
N
EtOH
N
R
R
R
1
1
2
6: R = H
9: R = CH (86%)
17: R = H (92%)
18: R = H (60%)
3
20: R = CH (50%)
3
21: R = CH (71%)
3
2: R = (CH ) CN (77%)
23: R = (CH ) CN (86%)
24: R = (CH ) CN (33%)
2
5
2 5
2 5
Scheme 1. Synthesis of indole potassium glyoxalate salts 18, 21 and 24.
O
O
O
O
OEt
-
O K+
i. AlCl , DCM
3
K CO
2
3
N
ii. ClCOCO Et
2
N
H O MeOH
H
N
2
N
N
53%
H
N
H
70%
2
5
26
27
Scheme 2. Synthesis of 7-azaindole potassium glyoxalate salt 27.
The synthesis of the maleic anhydrides 28 31 was facilitated by a modified Perkin condensation
–
with trimethoxyphenyl acetic acid and acetic anhydride in low to moderate yield. Conversion of this
anhydride to the 1-hydroxymaleimides 32–35 proceeded smoothly and in excellent yield (Scheme 3).

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Scheme 3. Route to 3,4-diaryl-1-hydroxymaleimides 32–35.
2
.2. Antiproliferative Activity of 3,4-Diaryl-1-Hydroxymaleimide Derivatives
Assessment of antiproliferative activity was undertaken using the NCI-60 cell screen, where
compounds are tested at a single dose (10 M) against the panel of 60 cancer cells (see Section 2.6
µ
for details). It is evident from the mean growth percentages (Table 1) that there is little inhibition of
growth, and on inspection of the individual cell lines, UO-31 appears to be the most susceptible with
some limited effect on HOP-92. Substitution of the indole nitrogen or replacement of the indole with
7
-azaindole disappointingly led to little difference in activity. Overall, there is minimal effect on cell
growth, which led to a revision of our strategy to incorporate greater functionality in the headgroup as
seen in Templates 14 and 15.
2
.3. Synthetic Routes towards 3,4-Diaryl-5-Aminopyrazoles of General Structure 15
Synthesis of novel 3,4-diaryl-5-aminopyrazoles 15 to probe the influence of headgroup on
cytotoxicity proved relatively straightforward. Initially,
β-ketonitrile 39 was fashioned via reaction of
acetonitrile 37 with 7-azaindolyl-3-acyl chloride (38, Scheme 4).
Scheme 4. Synthesis of β-ketonitrile 39.
Reaction of
β-ketonitrile 39 with hydrazine hydrate, employing hydrochloric acid as catalyst,
in absolute ethanol at reflux temperature led to aminopyrazole 40 (Scheme 5).
Scheme 5. Formation of 5-aminopyrazole 40 from β-ketonitrile 39.

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The versatile nature of 5-aminopyrazole 40 easily lends itself towards further chemical elaboration
of the headgroup space. Our initial plan set out to probe the chemical space by use of hydrophilic and
lipophilic components and monosubstitution in tandem with ring formation. Following treatment with
bidentate electrophiles N-chlorocarbonyl isocyanate and hexafluoroacetylacetone, bicyclic systems 41
and 42 were formed, respectively (Scheme 6).
Structural confirmation of the barbiturate moiety of triazinedione 41 is afforded by the two broad
1
singlets arising from the NH protons at 11.59 and 11.74 ppm in the H NMR spectrum. A second
cyclic headgroup was furnished by condensation of aminopyrazole 40 with hexafluoroacetylacetone in
an acidic medium gave highly conjugated pyrazolo[1,5-a]pyrimidine 42.
An intriguing pattern of regioselectivity was observed upon reaction of 5-aminopyrazole 40
with a number of monodentate electrophiles. Reaction with trifluoroacetic anhydride afforded
trifluoroacetamide 43 (Scheme 6). The presence of two broad singlets at 11.37 and 13.30 ppm in
1
the H NMR spectrum confirm substitution on the exocyclic amino functionality rather than at the
N(1) nitrogen.
Scheme 6. Reaction of aminopyrazole 40 with bidentate and monodentate electrophiles.
Acetylated Derivative 44 was generated following treatment of aminopyrazole 40 with acetic
anhydride in acetonitrile at reflux. In contrast to trifluoroacetamide 43, the site of substitution
for 44 was confirmed to be the N(1) position of the pyrazole, due to the presence of a 2H broad
1
singlet at 5.56 ppm in the H NMR spectrum corresponding to the exocyclic amino functionality.
Reaction of aminopyrazole 40 with methyl isothiocyanate led to the formation of thiourea 45
(Scheme 6). Spectroscopic analysis again provided for structural confirmation of substitution at
the N(1) pyrazolic position.
2
.4. Synthetic Routes to 3,4-Diaryl-5-Aminopyrazoles of General Structure 14 [22]
The synthesis of a series of 3,4-diaryl-5-aminopyrazoles 14 was initiated from
β-ketonitriles
3
6, which has previously been described (Scheme 7) [26]. Cyclocondensation of Intermediate 36
with hydrazine hydrate under reflux conditions allowed for the synthesis of a highly versatile
-aminopyrazole core. Subsequent reaction with a range of mono- and bi-dentate electrophiles resulted
in the formation of both monosubstituted and bicyclic systems of general Structure 14 (Derivatives
6–50, X = N; 51–55, X = CH; Table 1).
5
4

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Scheme 7. Route to aminopyrazoles 14 via β-ketonitrile 36.
2
.5. X-ray Crystal Structure Analysis of Substituted 3,4-Diaryl-5-Aminopyrazole Derivatives
Precedence for the difference in regioselectivity observed for the substitution of aminopyrazoles
such as 40 exists in the literature [28]. For example, in the development of a series of novel
protein kinase inhibitors, Nie et al. described the substitution of substituted 5-aminopyrazoles
with ethoxycarbonyl isothiocyanate, with the regioselectivity of the reaction dependant on both
the conditions employed and the nature of the ring substituent at the C(4) position [29].
Therefore, in order to confirm the existence of both monosubstitution and the bicyclic templates,
X-ray crystallographic studies were undertaken on a select panel of aminopyrazoles. As can be seen in
Figure 6, acetyl aminopyrazole 54 and thiourea 55 demonstrate selective monosubstitution at the N(1)
position of the pyrazole ring.
In contrast, pyrazolo[1,5-a]pyrimidine 53 is consistent with the structure proposed, confirming
the reactivity of both the exocyclic amino functionality and the nitrogen at the N(1) position of the
parent aminopyrazole.
Figure 6. Crystal structures of the bicyclic pyrazolo[1,5-a]pyrimidine 53 and monosubstituted
aminopyrazoles 54 and 55.
Comparing three analogous series of derivatized aminopyrazoles, it is evident that the pattern of
regioselectivity with regard to reaction with monodentate electrophiles is not entirely dependent on
the position or nature of the aryl substituents. In the case of the reaction with methyl isothiocyanate
and acetic anhydride, substitution occurred at the N(1) position of the aminopyrazole ring in each case,
i.e., the kinetic product of the reaction was more favourable than the thermodynamic product.

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However, when treated under similar conditions with trifluoroacetic anhydride, the most reactive
of the three monodentate electrophiles used, substitution occurred on the exocyclic amine of the
aminopyrazole (such as with derivative 43), i.e., the thermodynamic product was the more favourable
of the two. In this scenario, it is possible that the kinetic product of the reaction was more unstable
than the analogous thiourea and acetyl derivatives, due to the greater electron withdrawing ability
of the trifluoroacetyl moiety. Hence, substitution on the amino group may result in the more stable
and favoured product.
2
.6. Antiproliferative Activity of 3,4-Diaryl-5-Aminopyrazole Derivatives
Assessment of antiproliferative activity elicited by the three 5-aminopyrazole series was
undertaken using the NCI-60 cell screen, where compounds are tested at a single dose (10 µM) against
the panel of 60 cells. Using this platform, distinctive patterns corresponding to differential sensitivities
of cell lines to particular compounds can be correlated to the mechanism of cell growth inhibition,
and so, it is an important probe in the investigation of mechanistic action in cancer chemotherapy [30].
Examining the first series of 5-aminopyrazoles (40–45), parent 40 displays appreciable selectivity
for HOP-92 lung, SNB-75 CNS and UO-31 and CAKI-1 renal cancer cell lines, all of which display
inhibition of growth greater than 50% after 48 h of incubation (Table 1). Extension of aminopyrazole 40
towards bicyclic systems 41 and 42 subsequently resulted in a decrease in overall growth inhibition.
In contrast, however, it was found that monosubstitution in the case of acetyl Derivative 44 and
thiourea 45 led to both an overall increase in potency against the NCI-60 panel and also an increase in
selectivity for UO-31, CAKI-1 renal and SNB-75 CNS cancer cells. For example, an average growth
of 32.2% was recorded for thiourea 45 across these three cell lines, compared with a mean growth of
8
1.1% against the remainder of the panel.
Aminopyrazole 46 and thiourea 50 from the second series (Derivatives 46–50) also display
considerable selectivity for the SNB-75, UO-31 and CAKI-1 cell lines, though acetylated derivative
is, somewhat surprisingly, relatively inactive by comparison (Table 1). Although conversion of
4
9
aminopyrazole 46 towards bicyclic Analogues 47 and 48 resulted in an increase in mean growth in
both cases, greater selectivity was achieved towards the HOP-92 cell line. This was particularly evident
for pyrimidine 48, which completely arrests growth in this tumour cell line and even exhibits a degree
of cytotoxicity ( 3.1% growth after 48 h). Utilising the COMPARE algorithm, a high correlation was
−
obtained between the growth inhibition data for three different sets of regioisomers (where one is
an identical result): aminopyrazoles 40 and 46 (0.791), pyrimidines 42 and 48 (0.632) and thioureas 45
and 50 (0.805). Given the close agreement found, there exists the likelihood of a common mechanism
of action, which provides the scope for further SAR study.
The final series of 3,4-diaryl-5-aminopyrazoles (51–55), containing an indolyl functionality rather
than a 7-azaindolyl subunit, displayed the most potent overall antiproliferative activity across
the derivatives tested. In keeping with the trend displayed in the previous two series, parent
aminopyrazole 51 and monosubstituted Derivatives 54 and 55 are more potent than their bicyclic
Analogues 52 and 53. In particular, thiourea 55 almost completely arrests growth in HOP-92 lung
cancer cells (0.3% growth after 48 h), whilst also exhibiting excellent selectivity for SNB-75 CNS and
UO-31 and CAKI-1 renal cancer cells. Once more, this pattern of selectivity compares favourably to
that seen for analogous thioureas 45 and 50. Of great interest also is the fact that aminopyrazole 51
acetylated Derivative 54 and thiourea 55 exhibit a highly similar level of growth inhibition against the
renal cancer cell lines UO-31 and CAKI-1 (Table 1).
,
2
.7. Topoisomerase II Decatenation Assay
Consequent to screening the four series for antiproliferative activity, interaction with
topoisomerase II of a subset was investigated as a potential mechanism of action. Topo II helps
to modulate DNA processes via transient double-strand breaks in the DNA helix [31 32]. With higher
expression of topo II in proliferating cells than inactive cells, it is a clinical target for the induction
,

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of cytotoxic effects in many cancer types, and inhibitors include the podophyllotoxin (
2
) derivative,
etoposide 4.
Candidates 45–50 were screened for their ability to inhibit topoisomerase II-mediated circular
kinetoplast DNA (kDNA) decatenation. The standard topo II inhibitor, ellipticine, was used as
a reference compound in this study. From the assay, it was apparent that none of the derivatives
displayed any activity against topo II (see the Supporting Information). It is possible that the
lack of a core planar region in these derivatives prevents intercalation with DNA from occurring,
thus eliminating the possibility of topo II inhibition as a function of their antiproliferative activity.
Table 1. NCI-60 single dose results for 3,4-diaryl-5-aminopyrazoles.
Selected Cells (% Growth)
Regioisomer
Correlation
No.
Substituents
NSC No.
Mean (%)
Renal Cells Mean (%)
HOP-92SNB-75 CAKI-1 UO-31
X
R
3
3
3
3
2
3
4
5
CH
CH
CH
N
H
CH3
(CH2)5CN
H
776698
776695
776696
776697
95.8
95.4
96.8
98.4
89.6
-
69.1
83.4
91.0
77.9
96.3
94.0
103.3
100.4
106.4
101.9
71.5
66.4
77.3
86.1
94.9
93.4
94.9
97.3
R1
R2
40
41
42
44
45
NH2
H
763892
763893
763894
763896
763895
79.8
101.5
95.4
76.6
78.6
47.9
87.7
-
-
-
34.4
67.2
73.2
8.5
48.9
89.8
82.3
39.7
34.1
49.6
94.2
71.2
33.1
35.9
76.4
100.7
92.6
68.7
71.1
0.791
0.366
0.632
0.475
0.805
-NHCONHCO-
-NC(CF3)CHC(CF3)-
NH2
NH2
COCH3
CSNHCH3
26.5
R3
R4
46
47
48
49
50
NH2
H
754616
754617
754618
763899
763898
75.1
104.3
94.0
104.2
66.5
30.9
53.5
-3.1
86.4
-
38.1
99.7
67.2
101.9
39.7
50.0
118.4
-
81.0
18.2
51.7
94.8
73.7
92.8
25.3
70.8
101.4
92.5
97.6
61.5
0.791
0.366
0.632
0.475
0.805
-NHCONHCO-
-NC(CF3)CHC(CF3)-
NH2
NH2
COCH3
CSNHCH3
R5
R6
51
52
53
54
55
NH2
H
763905
763910
763909
763906
763907
70.6
75.1
92.9
69.4
59.3
-
-
4.8
18.8
52.9
55.2
18.1
16.3
24.6
43.9
61.4
23.7
17.9
65.0
80.3
87.5
60.6
53.2
-NHCONHCO-
-NC(CF3)CHC(CF3)-
NH2
NH2
47.2
56.3
40.3
27.8
48.6
-
0.3
COCH3
CSNHCH3
3
. Materials and Methods
3
.1. Topoisomerase II Decatenation Assay
The decatenation assay kit was obtained from Inspiralis (Norwich, U.K.) and comprised of the
following: (i) 10 assay buffer containing 50 mM Tris-HCl (pH 7.5), 125 mM NaCl, 10 mM MgCl , 5 mM
DTT and 100 g/mL albumin; (ii) dilution buffer containing 50 mM Tris.HCl (pH 7.5), 100 mM NaCl,
mM DTT, 0.5 mM EDTA, 50% (v/v) glycerol, 50 g/mL albumin; (iii) ATP 30 mM; (iv) kDNA
100 ng/ L); (v) 10 U/ L human topo II in dilution buffer; (vi) 5 stop buffer containing 2.5% SDS,
5% Ficoll-400, 0.05% bromophenol blue, 0.05% xylene cyanol and 25 mM EDTA. Tris-acetate-EDTA
buffer) and agarose were obtained from Sigma Life Sciences (Dublin, Ireland).
The topo II decatenation assay protocol involved initial incubation of each inhibitor candidate
×
2
µ
1
(
1
µ
µ
µ
×
buffer (supplied as 10
×
(
100
µ
M) along with a stock solution containing water, ATP, assay buffer, kDNA obtained from the
◦
mitochondrial DNA of Crithidia fasciculate, and topo II, at 37 C for 1 h. Following the addition of
stop buffer, agarose DNA gel electrophoresis was run at 50 V for 2 h using a Consort EV243 power
pack, to determine the relative amounts of decatenated DNA bands obtained in each compound lane.

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Positive (water), as well as negative controls (ellipticine) were incorporated in order to validate the
results of each run [33]. The resulting gels were viewed under UV light using a DNR Bio-Imaging
System and photographed using GelCapture software.
3
.2. NCI-60 Anticancer Screening
The experimental methodology involves initial growth of the tumour cell lines in RPMI 1640
medium containing 5% foetal bovine serum and 2 mM L-glutamine. For a typical screening experiment,
cells are inoculated into 96-well microtiter plates in 100 L of medium at plating densities ranging from
000–40,000 cells/well, depending on the doubling time of individual cell lines. After cell inoculation,
µ
5
◦
the microtiter plates are incubated at 37 C, 5% CO , 95% air and 100% relative humidity for 24 h prior
2
to the addition of candidate compounds.
After 24 h, two plates of each cell line are fixed in situ with trichloroacetic acid (TCA), to represent
a measurement of the cell population for each cell line at the time of drug addition (time zero (Tz)).
Candidate compounds are dissolved in DMSO at 400-fold the desired final maximum test concentration
and stored frozen prior to use. The single dose screen is carried out at a concentration of 10 µM.
◦
Following drug addition, the plates are incubated for an additional 48 h at 37 C, 5% CO , 95% air
2
and 100% relative humidity. For adherent cells, the assay is terminated by the addition of cold TCA.
Cells are fixed in situ by the gentle addition of 50
µ
L of cold 50% (w/v) TCA and incubated for 60 min
◦
at 4 C. Sulforhodamine B (SRB) solution (100
µL) at 0.4% (w/v) in 1% acetic acid is added to each well,
and plates are incubated for 10 min at room temperature. Absorbance is read on an automated plate
reader at a wavelength of 515 nm, and using the absorbance measurements of time zero (Tz), control
growth (C) and test growth in the presence of the drug at a concentration of 10
growth is calculated [34].
µM, the percentage of
3
.3. Chemical Data
The data for Compounds 17–21 are available in the Supplementary Information.
6
-(1H-Indol-1-yl)hexanenitrile (22). To a solution of indole (2.51 g, 21.4 mmol) in dry DMF (60 mL)
◦
at 0 C was added sodium hydride (1.31 g, 32.75 mmol) in a portion-wise manner. The resultant
mixture was allowed to stir at room temperature for 30 min after which time 6-bromohexanitrile
(
4.25 mL, 1.328 g/mL, 32 mmol) was added with care. The reaction mixture was then allowed to warm
to room temperature and stirred overnight. The reaction mixture was subsequently and carefully
poured into ice-cold water, and this resulting mixture was extracted with ethyl acetate (6 50 mL).
Combined organic layers were then washed with water (5 50 mL) and brine (3 50 mL) before
×
×
×
being dried over anhydrous magnesium sulphate and concentrated under reduced pressure to yield
a brown oil, which was subject to flash column chromatography (65:35, hexane/ethyl acetate) to
yield a viscous yellow oil, which was used without further purification (3.49 g, 16.4 mmol, 77%):
−
1
νmax/cm (NaCl) 3053, 2937, 2866, 2244, 1611;
δ (300 MHz, CDCl ) 1.43 (m, 2H, CH (CH ) CN),
H 3 2 2 2
1
.61 (m, 2H, CH CH CN), 1.84 (m, 2H, CH (CH ) CN), 2.25 (t, 2H, J = 7.1 Hz, CH -CN), 4.11 (t, 2H,
2 2 2 2 3 2
J = 6.9 Hz, N-CH ), 6.48 (dd, 1H, J = 3.2, 0.86 Hz, C-H ), 7.05 (d, 1H, J = 3.1 Hz, C-H ) 7.09 (overlapping
2
3
2
ddd, 1H, J = 0.9, 7.1, 7.9 Hz, C-H ), 7.19 (m, 1H, J = 1.1, 7.1 Hz, C-H ), 7.30 (dd, 1H, J = 8.3, 0.8 Hz,
5
6
C-H ), 7.62 (dt, 1H, J = 7.9, 0.9 Hz, C-H );
δ
C
(75 MHz, CDCl ) 17.1 (CH , CH ), 25.1 (CH , CH ),
7
4
3
2
2
2
2
2
6.2 (CH , CH ), 29.5 (CH , CH ), 46.0 (CH , NCH ), 101.3 (CH, aromatic CH), 109.3 (CH, aromatic
2 2 2 2 2 2
CH), 119.4 (C, CN), 119.5 (CH, aromatic CH), 121.1 (CH, aromatic CH), 121.5 (CH, aromatic CH),
+
1
27.7 (CH, aromatic CH), 128.7 (C, aromatic C), 135.9 (C, aromatic C); m/z (ES+) 213.4 [M + H] (100%);
HRMS (ES+): exact mass calculated for C H N 213.1392. Found 213.1386.
14
17
2
2
-(1-(5-Cyanopentyl)-1H-indol-3-yl)-2-oxoacetic acid (23). To a solution of 6-(1H-indol-1-yl)hexanenitrile 6
◦
(
3.49 g, 16.4 mmol) in diethyl ether (100 mL) at 0 C was added oxalyl chloride (2.20 mL, 1.48 g/mL,
24.64 mmol) dropwise over a 20 min. The resultant deep orange slurry was allowed to stir at room
temperature for 30 min. After this time saturated, aqueous sodium bicarbonate solution was added

Pharmaceuticals 2017, 10, 62
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very carefully (15 mL). The resulting biphasic mixture was left to stir overnight. Following this,
the reaction mixture was vacuum filtered, and the product cake was washed with ice-cold diethyl
ether (10 mL) and ice-cold water (5 mL) before being allowed to dry to yield the oxoacetic acid as
◦
−1
a pink solid (4.03 g, 14.2 mmol, 86%), m.p. 133–135 C: νmax/cm (KBr) 3428, 2952, 2241, 1752, 1628,
520, 1148; (400 MHz, DMSO-d ) 1.38 (m, 2H, CH (CH ) CN), 1.60 (m, 2H, CH CH CN), 1.83
1
δ
H
6
2
2 2
2
2
(
m, 2H, CH (CH ) CN), 2.48 (t, 2H, J = 2.4 Hz, CH -CN), 4.34 (t, 2H, J = 7.2 Hz, N-CH ), 7.33 (m,
2 2 3 2 2
2
H, C-H , C-H ), 7.68 (d, 1H, J = 8.1 Hz, C-H ), 8.22 (d, 1H, J = 8.0 Hz, C-H ), 8.52 (s, 1H, C-H ); δ
5 6 7 4 2 C
(
100 MHz, DMSO-d ) 16.0 (CH , CH ), 24.3 (CH , CH ), 25.2 (CH , CH ), 28.6 (CH , CH ), 46.2 (CH ,
6 2 2 2 2 2 2 2 2 2
NCH ), 111.3 (CH, aromatic CH), 111.4 (C, aromatic C), 120.6 (C, CN), 121.4 (CH, aromatic CH), 123.0
2
(
CH, aromatic CH), 123.7 (CH, aromatic CH), 126.2 (C, aromatic C), 136.6 (C, aromatic C), 140.3 (CH,
+
aromatic CH), 165.2 (C, aromatic C=O), 180.4 (C, C=O); m/z (ES+) 285.3 [M + H] (100%); HRMS (ES+):
exact mass calculated for C H N O : 285.1239. Found 285.1236.
16
17
2
3
Potassium 2-(1-(5-Cyanopentyl)-1H-indol-3-yl)-2-oxoacetate (24). To a solution of 2-(1-(5-cyanopentyl)-
H-indol-3-yl)-2-oxoacetic acid 7 (4.03 g, 14.17 mmol) in ethanol (200 mL) was added potassium
1
hydroxide (0.83 g, 14.8 mmol). The reaction mixture was allowed to stir for 4 h before being isolated
by vacuum filtration. The resulting product cake was allowed to dry to yield a beige solid (1.48 g,
◦
−1
4
.6 mmol, 33 %), m.p. 167–170 C: νmax/cm (KBr) 3406, 2944, 2245, 1884, 1765, 1632 (broad), 1523,
1
149; (400 MHz, DMSO-d ) 1.35 (m, 2H, CH (CH ) CN), 1.58 (m, 2H, CH CH CN), 1.79 (m, 2H,
δ
H
6
2
2 2
2
2
CH (CH ) CN), 2.47 (t, 2H, J = 7.12 Hz, CH -CN), 4.25 (t, 2H, J = 7.26, N-CH ), 7.19 (m, 2H, C-H ,
2
2 3
2
2
5
C-H ), 7.55 (d, 1H, J = 8.09 Hz, C-H ), 8.18 (s, 1H, C-H ), 8.20 (d, 1H, J = 7.58 Hz, C-H ); δ (100 MHz,
C
6
7
2
4
DMSO-d ) 16.0 (CH , CH ), 24.4 (CH , CH ), 25.3 (CH , CH ), 28.8 (CH , CH ), 45.7 (NCH ), 110.5
6
2
2
2
2
2
2
2
2
2
(
CH, aromatic CH), 113.1 (C, CN), 120.6 (C, aromatic C), 121.4 (CH, aromatic CH), 121.5 (CH, aromatic
CH), 122.3 (CH, aromatic CH), 126.5 (C, aromatic C), 136.2 (C, aromatic C), 138.3 (CH, aromatic CH),
−
1
69.6 (C, C=O), 192.9 (C, C=O); m/z (ES
−
) 283.4 [M
−
H] (100%); HRMS (ES+): exact mass calculated
for C H N O : 285.1239. Found 285.1232.
16
17
2
3
Ethyl 2-oxo-2-(1H-pyrrolo[2,3-b]pyridin-3-yl)acetate (26). 7-Azaindole (5.00 g, 42 mmol) was added to
a suspension of aluminium trichloride (30.00 g, 225 mmol) in DCM (500 mL), and the resulting mixture
was allowed to stir for 30 min at room temperature. After this time, ethyl chlorooxoacetate (24 mL,
2
15 mmol) was added dropwise over 20 min with the resulting mixture left to stir vigorously at room
temperature overnight. The reaction mixture was then poured over ice cold water, and the layers were
separated. The aqueous layer was washed with DCM (4 50 mL). Combined organic layers were
then washed very carefully with ice-cold, saturated aqueous sodium bicarbonate solution (3 20 mL)
and brine (1 100 mL) before being dried over anhydrous magnesium sulphate and concentrated
×
×
×
under reduced pressure to yield a brown oil. Purification by flash column chromatography (60:40,
hexane/ethyl acetate) afforded the oxoacetate as a fluffy, light yellow solid (4.83 g, 22.1 mmol, 53%),
m.p. 157–158 C; νmax/cm (KBr) 3418, 1872, 1723, 1611, 1174;
◦
−1
δ (400 MHz, DMSO-d ) 1.35 (t, 3H,
H 6
J = 7.1 Hz, OCH CH ), 4.38 (q, 2H, J = 14.2, 7.1 Hz, OCH CH ), 7.34 (dd, 1H, J = 7.9, 4.7 Hz, C-H ),
2
3
2
3
5
8
2
.40 (dd, 1H, J = 4.7, 1.6 Hz, C-H ), 8.47 (dd, 1H, J = 7.9, 1.6 Hz, C-H ), 8.57 (s, 1H, C-H ); m/z (ES+)
6 4 2
19.3 [M + H] 100%.
+
Potassium 2-oxo-2-(1H-pyrrolo[2,3-b]pyridin-3-yl)acetate (27). To a suspension of ethyl 2-oxo-2-(1H-
pyrrolo[2,3-b]pyridin-3-yl)acetate 9 (4.83 g, 22.1 mmol) in a 1:1 solution of water/MeOH was added
potassium carbonate (5.67 g, 41.03 mmol). The resulting mixture was allowed to stir for 5 h before
the product was isolated by vacuum filtration and the cake dried overnight to yield the glyoxylate
◦
−1
salt as a brilliant white powder (3.53 g, 15.5 mmol, 70%), m.p. > 250 C; νmax/cm (KBr) 3244, 3061,
530, 1980, 1860, 1780, 1634, 1455; (400 MHz, DMSO-d ) 7.18 (q, 1H, J = 7.9, 4.5 Hz, C-H ), 8.13 (s,
2
δ
H
6
5
1
H, C-H ), 8.27 (dd, 1H, J = 4.7, 1.6 Hz, C-H ), 8.42 (dd, 1H, J = 7.8, 1.6 Hz, C-H ); m/z (ES ) 189.3
−
2
6
4
−
[M−H] 100%.

Pharmaceuticals 2017, 10, 62
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3
2
3
-(1-Acetyl-1H-indol-3-yl)-4-(3,4,5-trimethoxyphenyl)furan-2,5-dione (28). Potassium 2-(1H-indol-3-yl)-
-oxoacetate 2 (703 mg, 3.09 mmol) was added to a round-bottomed flask along with
,4,5-trimethoxyphenylacetic acid (700 mg, 3.09 mmol). Acetic anhydride was added (25 mL) and the
◦
reaction mixture was heated for 24 h at 80 C. Following this, the acetic anhydride was removed under
reduced pressure, and to the crude residue was added saturated aqueous sodium bicarbonate solution.
This mixture was extracted with ethyl acetate (5
saturated aqueous sodium bicarbonate solution (4
before being dried over anhydrous magnesium sulphate and concentrated under reduced pressure
to yield a brown residue. Purification by flash chromatography (65:35, hexane/ethyl acetate) yielded
a bright yellow solid (216 mg, 0.5 mmol, 16%), m.p. 153–155 C; νmax/cm (KBr) 3417, 2942, 1824,
×
50 mL). The ethyl acetate was then washed with
50 mL), water (4 50 mL) and brine (2 100 mL)
×
×
×
◦
−1
1
3
763, 1718, 1676, 1477;
δ
(400 MHz, CDCl ) 2.77 (s, 3H, acyl CH ), 3.50 (s, 6H, 2
×
m-OCH ), 3.89 (s,
3
H
3
3
H, p-OCH ), 6.50 (d, 1H, J = 8.1 Hz, C-H ), 6.84 (s, 2H, Ar-CH
0
and Ar-CH6 ), 7.03 (overlapping ddd,
0
3
7
2
1
8
6
H, J = 8.1, 7.6, 0.9 Hz, C-H ), 7.34 (overlapping ddd, 1H, J = 8.4, 7.3, 1.0 Hz, C-H ), 8.23 (s, 1H, C-H ),
5
6
2
.49 (d, 1H, J = 8.4 Hz, C-H4);
δ
(100 MHz, CDCl ) 24.0 (CH , Acyl-CH ), 56.0 (2
×
CH , 2
×
m-OCH3),
C
3
3
3
3
1.0 (CH , p-OCH ), 108.2 (2
×
CH, 2
×
aromatic CH), 110.2 (C, aromatic C), 116.9 (CH, aromatic CH),
3
3
1
22.3 (CH, aromatic CH), 122.7 (C, aromatic C), 124.0 (CH, aromatic CH), 125.4 (C, aromatic C), 126.3
(CH, aromatic CH), 130.0 (C, aromatic C), 130.5 (CH, aromatic CH), 134.5 (C, aromatic C), 135.9 (C,
aromatic C), 141.0 (C, aromatic C), 153.0 (2
C=O); m/z (ES+) 422.2 [M + H] (20%); HRMS (ES+): exact mass calculated for C H NO 422.1240.
×
C, 2
×
aromatic C), 165.0 (C, C=O), 165.3 (C, C=O), 168.6 (C,
+
23
20
7
Found 422.1227.
3-(1-Methyl-1H-indol-3-yl)-4-(3,4,5-trimethoxyphenyl)furan-2,5-dione (29). Potassium 2-(1-methyl-1H-
indol-3-yl)-2-oxoacetate 5 (1.43 g, 5.93 mmol) and 3,4,5-trimethoxyphenylacetic acid (1.31 g, 5.8 mmol)
were placed in a round-bottomed flask. Acetic anhydride (55 mL) was added, and the resulting
◦
mixture was heated for 24 h at 80 C. Following this, the excess acetic anhydride was removed under
reduced pressure, and to the solid residue was added saturated aqueous sodium bicarbonate solution.
The crude organic material was extracted with ethyl acetate (4
washings were then washed with saturated aqueous sodium bicarbonate solution (3
100 mL) and brine (2 100 mL) before being dried over anhydrous magnesium sulphate and
concentrated under reduced pressure to yield a sticky red solid. Purification by flash chromatography
×
50 mL). The combined ethyl acetate
×
100 mL), water
(3
×
×
(
1
70:30, hexane/ethyl acetate) yielded the desired maleic anhydride as a red solid (293 mg, 0.75 mmol,
◦ −1
3 %), m.p. 175–177 C; νmax/cm (KBr) 2940, 1987, 1846, 1814, 1748, 1601, 1247, 1124; δ (400 MHz,
H
CDCl ) 3.51 (s, 6H, 2
×
m-OCH ), 3.89 (s, 3H, p-OCH ), 3.92 (s, 3H, N-CH ), 6.42 (d, 1H, J = 8.2 Hz,
3
3
3
3
C-H ), 6.81 (s, 2H, Ar-CH
0
and Ar-CH6 ), 6.90 (overlapping ddd, 1H, J = 8.1, 7.2, 0.9 Hz, C-H ), 7.21
0
7
2
5
(
overlapping ddd, 1H, J = 8.0, 7.1, 0.9 Hz, C-H ), 7.35 (d, 1H, J = 8.3 Hz, C-H ), 8.05 (d, 1H, J = 1.3
6
4
Hz, C-H2);
δ
(75 MHz, CDCl ) 33.7 (CH , N-CH ), 55.9 (2
×
CH , 2
×
m-OCH ), 61.0 (CH , p-OCH ),
C
3
3
3
3
3
3
3
1
04.2 (C, aromatic C), 107.7 (2
×
CH, 2
×
aromatic CH), 110.1 (CH, aromatic CH), 121.3 (CH, aromatic
CH), 123.2 (CH, aromatic CH), 123.3 (CH, aromatic CH), 124.1 (C, aromatic C), 124.2 (C, aromatic C),
1
28.1 (C, aromatic C), 132.5 (C, aromatic C), 136.0 (CH, aromatic CH), 137.5 (C, aromatic C), 139.7 (C,
aromatic C), 152.8 (2 C, 2
aromatic C), 165.9 (C, C=O), 166.4 (C, C=O); m/z (ES+) 394.4 [M + H]+
40%); HRMS (ES+): exact mass calculated for C H NO 394.1291. Found 394.1283.
×
×
(
22
20
6
6
-(3-(2,5-Dioxo-4-(3,4,5-trimethoxyphenyl)-2,5-dihydrofuran-3-yl)-1H-indol-1-yl)hexanenitrile
(511 mg, 1.58 mmol) and
,4,5-trimethoxyphenylacetic acid (365 mg, 1.62 mmol) were added to a round-bottomed flask.
(30).
Potassium 2-(1-(5-cyanopentyl)-1H-indol-3-yl)-2-oxoacetate
3
Acetic anhydride (15 mL) was added, and the mixture was heated at 80 C for 24 h. Following this,
the excess acetic anhydride was removed under reduced pressure. Saturated aqueous sodium
bicarbonate solution was added to the residue to remove any residual acetic anhydride. Crude organic
8
◦
material was then extracted with ethyl acetate (4
then washed with saturated aqueous sodium bicarbonate solution (3
brine (3 50 mL) before being dried over anhydrous magnesium sulphate and concentrated under
×
50 mL). The combined ethyl acetate extracts were
×
50 mL), water (3 50 mL) and
×
×

Pharmaceuticals 2017, 10, 62
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reduced pressure to yield a red residue. Purification by flash chromatography (50:50, hexane/ethyl
◦
acetate) yielded the maleic anhydride as a red solid (333 mg, 0.7 mmol, 44%), m.p. 134–136 C;
νmax/cm (KBr) 3056, 2247, 1815, 1748, 1464;
−
1
δ (400 MHz, CDCl ) 1.56 (m, 2H, CH (CH ) CN),
H 3 2 2 2
1.73 (m, 2H, CH CH CN), 1.99 (m, 2H, CH (CH ) CN), 2.37 (t, 2H, J = 7 Hz, CH CN), 3.52 (s, 6H,
2 2 2 2 3 2
2
×
m-OCH ), 3.89 (s, 3H, p-OCH ), 4.26 ppm (t, 2H, J = 7.1 Hz, N-CH ), 6.46 (d, 1H, J = 8.12 Hz,
3
3
2
C-H ), 6.81 (s, 2H, Ar-CH
0
and Ar-CH6 ), 6.92 (dt, 1H, J = 8.0, 0.9 Hz, C-H ), 7.23 (dt, 1H, J = 8.0, 0.9
0
7
2
5
Hz, C-H ), 7.37 (d, 1H, J = 8.3 Hz, C-H ), 8.07 (d, 1H, J = 1.46 Hz, C-H );
δ (100 MHz, CDCl ) 17.1
C 3
6
4
2
(
2
CH , CH ), 24.9 (CH , CH ), 26.0 (CH , CH ), 29.2 (CH , CH ), 46.8 (CH , N-CH ), 55.9 (2
×
CH3,
aromatic CH), 110.1 (CH,
aromatic CH), 124.0 (C,
aromatic C), 124.3 (C, aromatic C), 128.4 (C, aromatic C), 132.4 (C, aromatic C), 134.7 (CH, aromatic
2
2
2
2
2
2
2
2
2
2
×
m-OCH ), 61.0 (CH , p-OCH ), 104.5 (C, aromatic C), 107.8 (2
×
CH, 2
CH, 2
×
3
3
3
aromatic CH), 119.2 (C, CN), 121.4 (CH, aromatic CH), 123.4 (2
×
×
CH), 136.7 (C, aromatic C), 139.8 (C, aromatic C), 152.9 (2
C=O); m/z (ES+) 475.2 [M + H] (100%); HRMS (ES+): exact mass calculated for C H N O 475.1869.
×
C, 2
×
aromatic C), 165.8 (C, C=O), 166.4 (C,
+
27
27
2
6
Found 475.1863
3-(1-Acetyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-(3,4,5-trimethoxyphenyl)furan-2,5-dione (31). Potassium 2-
oxo-2-(1H-pyrrolo[2,3-b]pyridin-3-yl)acetate 10 (410 mg, 1.8 mmol) and 3,4,5-trimethoxyphenylacetic
acid (407 mg, 1.8 mmol) were added to a round-bottomed flask. Acetic anhydride (20 mL) was
◦
added and the mixture was heated for 24 h at 80 C. Following this, the excess acetic anhydride was
removed under reduced pressure. Saturated aqueous sodium bicarbonate solution was added to the
residue in order to remove any excess acetic anhydride. Crude organic material was then extracted
with ethyl acetate (4
aqueous sodium bicarbonate solution (3
being dried over anhydrous magnesium sulphate and concentrated under reduced pressure to yield
a dull golden residue. Purification by flash chromatography (65:35, hexane/ethyl acetate) yielded the
maleic anhydride as a light orange solid (208 mg, 0.49 mmol, 27%), m.p. 103–105 C; νmax/cm (KBr)
×
50 mL). The combined ethyl acetate extracts were then washed with saturated
50 mL), water (3 50 mL) and brine (3 50 mL) before
×
×
×
◦
−1
3
3
311, 1824, 1757, 1625, 1580;
δ
(400 MHz, CDCl ) 3.12 (s, 3H, acyl CH ), 3.58 (s, 6H, 2
×
m-OCH3),
H
3
3
.90 (s, 3H, p-OCH ), 6.81 (s, 2H, Ar-CH2
0
and Ar-CH6
0
), 6.88 (dd, 1H, J = 8.05, 1.38 Hz, C-H ), 7.00
3
4
(
m, 1H, J = 8.0, 4.7 Hz, C-H ), 8.38 (dd, 1H, J = 4.7, 1.6 Hz, C-H ), 8.76 (s, 1H, C-H ); δ (100 MHz,
5 6 2 C
CDCl ) 26.1 (CH , Acyl CH ), 56.1 (2
×
CH , 2
×
m-OCH ), 61.1 (CH , p-OCH ), 107.0 (C, aromatic C),
3 3 3
3
3
3
3
107.9 (2
×
CH, 2
×
aromatic CH), 118.9 (CH, aromatic CH), 119.3 (C, aromatic C), 122.7 (C, aromatic C),
1
30.4 (C, aromatic C), 130.7 (CH, aromatic CH), 131.0 (CH, aromatic CH), 135.0 (C, aromatic C), 141.0
(
C, aromatic C), 145.2 (CH, aromatic CH), 147.6 (C, aromatic C), 153.2 (2
×
C, 2
×
aromatic C), 164.5
+
(C, C=O), 164.8 (C, C=O), 168.4 (C, C=O); m/z (ES+) 423.2 [M + H] (40%); HRMS (ES+): exact mass
calculated for C H N O 423.1192. Found 423.1189.
22
19
2
7
Synthesis of aryl hydroxymaleimides: General Procedure (i): To a solution of the maleic anhydride
(
(
1 eq.) in acetonitrile (or DMF) was added hydroxylamine hydrochloride (5 eq.) and triethylamine
5 eq.). The mixture was heated for 24 h at 80 C. Following this, 1 M aqueous HCl was
◦
added to quench the reaction. Organic components were extracted with ethyl acetate, and the
combined extracts were washed with saturated aqueous sodium bicarbonate solution, water, brine,
dried over anhydrous magnesium sulphate and concentrated under reduced pressure to yield the
corresponding hydroxymaleimide.
1-Hydroxy-3-(1H-indol-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-dione (32). Applying General
Procedure (i) starting from maleic anhydride 11 (125 mg, 0.33 mmol), hydroxylamine HCl
(
115 mg, 1.65 mmol) and triethylamine (0.23 mL, 0.726 g/mL, 1.65 mmol) in acetonitrile (15 mL),
◦
hydroxymaleimide 15 was formed as a deep red solid (107.3 mg, 0.27 mmol, 82%), m.p. 123–125 C;
νmax/cm (KBr) 3262, 2943, 1767, 1711 (broad), 1601, 1562, 1124;
−
1
δ (400 MHz, DMSO-d ) 3.40 (s, 6H,
H 6
2
6
×
m-OCH ), 3.69 (s, 3H, p-OCH ), 6.37 (d, 1H, J = 8.05 Hz, C-H ), 6.75 (s, 2H, Ar-CH and Ar-CH6 ),
0
0
3 3 7
2
.79 (t, 1H, J = 7.7 Hz, C-H ), 7.11 (t, 1H, J = 7.6 Hz, C-H ), 7.48 (d, 1H, J = 8.17 Hz, C-H ), 8.05 (s,
5
6
4
1
H, C-H ), 10.54 (brs, 1H, N-H), 11.98 (brs, 1H, N-OH);
δ
C
(100 MHz, DMSO-d ) 55.5 (2
×
CH , 2
×
2
6
3

Pharmaceuticals 2017, 10, 62
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m-OCH ), 60.1 (CH , p-OCH ), 104.1 (C, aromatic C), 107.6 (2
aromatic CH), 119.8 (CH, aromatic CH), 121.6 (CH, aromatic CH), 122.2 (CH, aromatic CH), 123.6 (C,
aromatic C), 124.6 (C, aromatic C), 125.2 (C, aromatic C), 128.8 (C, aromatic C), 131.6 (CH, aromatic
×
CH, 2
×
aromatic CH), 112.2 (CH,
3
3
3
CH), 136.5 (C, aromatic C), 138.2 (C, aromatic C), 152.3 (2
C=O); m/z (ES+) 395.6 [M + H] (10%); HRMS (ES+): exact mass calculated for C H N O 395.1243.
×
C, 2
×
aromatic C), 167.6 (C, C=O), 167.9 (C,
+
21
19
2
6
Found 395.1226.
-Hydroxy-3-(1-methyl-1H-indol-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-dione (33). Applying General
Procedure (i) starting from maleic anhydride 12 (257 mg, 0.65 mmol), hydroxylamine HCl
248 mg, 3.25 mmol) and triethylamine (0.45 mL, 0.726 g/mL, 3.25 mmol) in dry DMF (30 mL),
1
(
◦
−1
hydroxymaleimide 16 was formed as a red solid (226 mg, 0.55 mmol, 85%), m.p. > 250 C; νmax/cm
KBr) 3262, 3125, 2942, 1769, 1715 (broad), 1597, 1467, 1125; (400 MHz, DMSO-d ) 3.40 (s, 6H,
(
δ
H
6
2
×
m-OCH ), 3.68 (s, 3H, p-OCH ), 3.93 (s, 3H, N-CH ), 6.32 (d, 1H, J = 7.98 Hz, C-H ), 6.73 (s, 2H,
3
3
3
7
Ar-CH2
0
and Ar-CH6 ), 6.82 (overlapping ddd, 1H, J = 8.1, 7.7, 1.0 Hz, C-H ), 7.18 (overlapping ddd,
0
5
1
H, J = 8.2, 7.5, 0.9 Hz, C-H ), 7.53 (d, 1H, J = 8.25 Hz, C-H ), 8.11 (s, 1H, C-H ), 10.56 (brs, 1H, N-OH);
6
4
2
δ
(100 MHz, DMSO-d ) 33.1 (CH , N-CH ), 55.5 (2
×
CH , 2
×
m-OCH ), 59.7 (CH , p-OCH ), 103.2 (C,
C
6
3
3
3
3
3
3
aromatic C), 107.7 (2
×
CH, 2
×
aromatic CH), 110.6 (CH, aromatic CH), 120.1 (CH, aromatic CH), 121.8
(CH, aromatic CH), 122.3 (CH, aromatic CH), 124.0 (C, aromatic C), 124.3 (C, aromatic C), 125.2 (C,
aromatic C), 128.3 (C, aromatic C), 135.2 (CH, aromatic CH), 137.0 (C, aromatic C), 138.2 (C, aromatic
+
C), 152.3 (2
×
C, 2
×
aromatic C), 167.6 (C, C=O), 167.8 (C, C=O); m/z (ES+) 409.2 [M + H] (30%);
HRMS (ES+): exact mass calculated for C H N O 409.1400. Found 409.1394.
22
21
2
6
6-(3-(1-Hydroxy-2,5-dioxo-4-(3,4,5-trimethoxyphenyl)-2,5-dihydro-1H-pyrrol-3-yl)-1H-indol-1-yl)hexanenitrile (34).
Applying general procedure (i) starting from maleic anhydride 13 (257 mg, 0.54 mmol), hydroxylamine
HCl (188 mg, 2.7 mmol) and triethylamine (0.38 mL, 0.726 g/mL, 2.7 mmol) in dry DMF (30 mL),
◦
hydroxymaleimide 17 was formed as a light red solid (219 mg, 0.45 mmol, 83%), m.p. 182–184 C;
νmax/cm (KBr) 3467, 3213, 3000, 2942, 2260, 1720 (broad), 1626, 1580;
−
1
δ (400 MHz, DMSO-d ) 1.34
H 6
(
m, 2H, CH (CH ) CN), 1.58 (m, 2H, CH CH CN), 1.83 (m, 2H, CH (CH ) CN), 2.46 (t, 2H, J = 7.03
2 2 2 2 2 2 2 3
Hz, CH -CN), 3.34 (s, 6H, 2
×
m-OCH ), 3.68 (s, 3H, p-OCH ), 4.34 (t, 2H, J = 6.81 , N-CH ), 6.40 (d,
3 3 2
2
1
H, J = 8.10 Hz, CH ), 6.75 (s, 2H, Ar-CH and Ar-CH6 ), 6.82 (t, 1H, J = 7.74 Hz, C-H ), 7.17 (t, 1H,
0
0
7
2
5
J = 7.62 Hz, C-H ), 7.59 (d, 1H, J = 8.21 Hz, C-H ), 8.09 (s, 1H, C-H ), 10.54 (brs, 1H, N-OH);
δ (100
C
6
4
2
MHz, DMSO-d ) 16.0 (CH , CH ), 24.2 (CH , CH ), 25.1 (CH , CH ), 28.7 (CH , CH ), 45.6 (CH ,
6
2
2
2
2
2
2
2
2
2
N-CH ), 55.4 (2
×
CH , 2
×
m-OCH ), 60.1 (CH , p-OCH ), 103.4 (C, aromatic C), 107.5 (2
×
CH, 2
×
2
3
3
3
3
aromatic CH), 110.7 (CH, aromatic CH), 120.0 (CH, aromatic CH), 120.5 (C, CN), 121.8 (CH, aromatic
CH), 122.3 (CH, aromatic CH), 124.2 (C, aromatic C), 124.7 (C, aromatic C), 125.1 (C, aromatic C), 128.3
(
C, aromatic C), 134.1 (CH, aromatic CH), 136.3 (C, aromatic C), 138.2 (C, aromatic C), 152.3 (2
×
C, 2
×
+
aromatic C), 167.5 (C, C=O), 167.8 (C, C=O); m/z (ES+) 490.2 [M + H] (70%); HRMS (ES+): exact mass
calculated for C H N O 490.1978. Found 490.1992.
27
28
3
6
1-Hydroxy-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-dione (35). Applying General
Procedure (i) starting from maleic anhydride 14 (147 mg, 0.38 mmol), hydroxylamine HCl (136 mg,
2
3
3
mmol) and triethylamine (0.27 mL, 0.726 g/mL, 1.9 mmol) in acetonitrile (15 mL), hydroxymaleimide
◦
−1
5
was formed as an orange solid (106 mg, 0.27 mmol, 71%), m.p. > 250 C; νmax/cm (KBr) 3157,
098, 2937, 1712 (broad), 1616, 1500; (400 MHz, DMSO-d ) 3.45 (s, 6H, 2 m-OCH ), 3.70 (s, 3H,
δ
H
×
6
3
p-OCH ), 6.68 (dd, 1H, J = 8.05, 1.54 Hz, C-H ), 6.74 (s, 2H, Ar-CH and Ar-CH6 ), 6.88 (m, 1H, J =
0 0
2
3
4
8
.01, 4.69 Hz, C-H ), 8.14 (s, 1H, C-H ), 8.23 (dd, 1H, J = 4.69, 1.53 Hz, C-H ), 10.61 (brs, 1H, N-OH),
5
2
6
1
2.50 (brs, 1H, N-H);
δ
(100 MHz, DMSO-d ) 55.6 (2
×
CH , 2
×
m-OCH ), 60.2 (CH , p-OCH ), 102.9
C
6
3
3
3
3
(
C, aromatic C), 107.6 (2
×
CH, 2
×
aromatic CH), 116.1 (CH, aromatic CH), 116.2 (C, aromatic C), 124.9
(C, aromatic C), 126.1 (C, aromatic C), 128.3 (C, aromatic C), 129.5 (CH, aromatic CH), 131.7 (CH,
aromatic CH), 138.5 (C, aromatic C), 143.7 (CH, aromatic CH), 148.8 (C, aromatic C), 152.5 (2
aromatic C), 167.3 (C, C=O), 167.5 (C, C=O); m/z (ES+) 396.2 [M + H] (50%); HRMS (ES+): exact mass
×
C, 2
×
+
calculated for C H N O 396.1196. Found 396.1181.
20
18
3
6

Pharmaceuticals 2017, 10, 62
15 of 20
3-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-3-oxo-2-(3,4,5-trimethoxyphenyl)propanenitrile (39). A solution
of Lithium diisopropylamide (LDA) (1.8 M, 9.8 mL, 17.7 mmol) in THF (10 mL) was then cooled
◦
to
(
−
78 C under a nitrogen atmosphere. A second solution of 3,4,5-trimethoxyphenyl acetonitrile
37, 1.36 g, 6.5 mmol) in THF (5 mL) was then added in a dropwise manner, with the resulting mixture
stirred for 1 h at
38, 1.40 g, 7.2 mmol) in THF (25 mL) was slowly added to the reaction mixture over a 20-min period.
Once the addition was complete, the reaction mixture was warmed to 35 C and allowed to stir at this
temperature, still under an inert atmosphere, for 12 h. The solvent was evaporated under reduced
pressure, and the resultant residue was dissolved in ethyl acetate (40 mL). The organic phase was
washed with a saturated aqueous sodium bicarbonate solution (50 mL), water (50 mL) and then brine
◦
−
78 C. A suspension of 1-methyl-1H-pyrrolo[2,3-b]pyridine-3-carbonyl chloride
(
◦
(50 mL), before being dried over magnesium sulphate. The solvent was removed under reduced
pressure, and the crude residue was subjected to flash column chromatography (hexane/ethyl acetate,
◦ −1
0:70) to yield oxopropanenitrile 39 as a yellow solid (1.09 g, 46%): m.p. 137–140 C; vmax/cm (KBr)
3
2
933, 2243, 1728, 1643, 1595, 1450, 1130;
δ
H
(400 MHz, CDCl ) 3.83 [3H, s, p-OCH ], 3.87 [6H, s, 2
×
3
3
m-OCH3], 3.94 [3H, s, NCH ], 5.23 [1H, s, CH
α
], 6.70 [2H, s, C-H ], 7.28 [1H, q, J 7.9, 4.8, C-H ],
0 0
3
2 6
5
7
3
.97 [1H, s, C-H ], 8.43 [1H, dd, J 4.8, 1.6, C-H ], 8.60 [1H, dd, J 7.9, 1.6, C-H ]; δ (75 MHz, CDCl3)
2 6 4 C
2.3 (CH , NCH ), 47.8 (CH, CHCN), 56.4 (2CH , 2
×
m-OCH ), 60.9 (CH , p-OCH ), 105.1 (2CH, 2
×
3
3
3
3
3
3
aromatic CH), 111.4 (C, aromatic C), 117.3 (C, aromatic C), 119.3 (C, C
26.5 (C, aromatic C), 131.2 (CH, aromatic CH), 136.0 (CH, aromatic CH), 138.5 (C, aromatic C), 145.4
CH, aromatic CH), 148.2 (C, aromatic C), 154.0 (2C, 2 aromatic C), 182.6 (C, C=O); m/z (ES+) 364.1
M−H] (100%); HRMS (ES+): exact mass calculated for C H N O 366.1454. Found 366.1472.
≡
N), 119.4 (CH, aromatic CH),
1
(
[
×
−
20
20
3
4
5-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrazol-3-amine (40). To a solution of
3
-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-3-oxo-2-(3,4,5-trimethoxyphenyl)propanenitrile (39, 1.02 g,
2.8 mmol) in absolute alcohol (30 mL) was added 12 M aqueous HCl (1 mL) in a dropwise manner.
Hydrazine hydrate (50% aqueous solution, 0.87 mL, 13.9 mmol) was then added, and the resulting
mixture was heated to reflux for 26 h. Once cooled to room temperature, the solvent was evaporated
under reduced pressure. The residue was dissolved in ethyl acetate (50 mL), then washed successively
with saturated aqueous sodium bicarbonate solution (40 mL), water (40 mL) and then brine (40 mL),
before being dried over magnesium sulphate. The solvent was removed under reduced pressure, and
the crude brown residue was subjected to flash column chromatography (ethyl acetate/methanol,
◦
−1
9
0:10) to yield pure aminopyrazole 40 as a light brown solid (0.66 g, 62%): m.p. 135–137 C; vmax/cm
(300 MHz, DMSO-d ) 3.53 [6H, s, 2 m-OCH ], 3.64
(
KBr) 3199, 2931, 1602, 1518, 1470, 1351, 1115;
δ
×
H
6
3
[3H, s, p-OCH ], 3.82 [3H, s, NCH ], 4.68 [2H, bs, NH ], 6.53 [2H, s, C-H
0 0
], 6.98 [1H, q, J 7.9, 4.6,
3
3
2
2 6
C-H ], 7.55 [2H, m, C-H ], 8.24 [1H, dd, J 4.6, 1.3, C-H ], 11.73 [1H, bs, NH]; δ (75 MHz, DMSO-d )
5
2,4
6
C
6
3
2
0.8 (C, NCH ), 55.5 (2C, 2
×
m-OCH ), 60.0 (C, p-OCH ), 94.9 (C, broad aromatic C), 106.1 (2CH,
3 3
3
×
aromatic CH), 115.5 (CH, aromatic CH), 117.9 (C, aromatic C), 125.4 (C, broad aromatic C), 128.2
(CH, aromatic CH), 128.8 (CH, aromatic CH), 129.4 (C, aromatic C), 135.4 (C, aromatic C), 141.9 (C,
broad aromatic C), 142.7 (CH, aromatic CH), 147.2 (C, aromatic C), 151.8 (C, aromatic C), 152.7 (2C, 2
×
+
aromatic C); m/z (ES+) 380.2 [M + H] (100%); HRMS (ES+): exact mass calculated for C H N O
20
22
5
3
3
80.1723. Found 380.1724.
-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-8-(3,4,5-trimethoxyphenyl)pyrazolo[1,5-a][1,3,5]triazine-2,4(1H,3H)-
dione (41). 5-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrazol-3-amine
7
(
40, 100 mg, 0.26 mmol) was dissolved in DCM (5 mL) containing three drops of triethylamine, and the
◦
resulting solution was cooled to 0 C in an ice bath. This solution was then treated, while stirring,
with N-chlorocarbonyl isocyanate (0.03 mL, 0.29 mmol). The reaction mixture was subsequently
allowed to warm to room temperature and was stirred for 15 h. After careful addition of water (3 mL),
a solid precipitated, which was filtered off and then washed with water (4
×
10 mL), followed by ether
◦
(
2
×
10 mL). The precipitate was desiccated at 50 C overnight to give the desired pyrazolotriazinedione
◦ −1
41 as a white solid (51 mg, 45%): m.p. 284–285 C; vmax/cm (KBr) 3215, 3067, 1761, 1713, 1647,

Pharmaceuticals 2017, 10, 62
16 of 20
1
586, 1411, 1127;
δ
(300MHz, DMSO-d ) 3.72 [6H, s, 2
×
m-OCH ], 3.74 [3H, s, p-OCH ], 3.78 [3H, s,
H
6
3
3
NCH ], 6.64 [2H, s, C-H
0
0
], 7.18 [1H, q, J 7.8, 4.9, C-H ], 7.31 [1H, s, C-H ], 8.32-8.34 [2H, m, C-H4,6],
3
2 ,6
5 2
1
1.59 [1H, bs, C=C-NHCO], 11.74 [1H, bs, CONHCO]; δ (75 MHz, DMSO-d ) 31.0 (CH , NCH ),
C 6 3 3
5
5.8 (2CH , 2
×
m-OCH ), 60.1 (CH , p-OCH ), 102.0 (C, aromatic C), 104.8 (C, aromatic C), 107.9
3 3 3
3
(2CH, 2
×
aromatic CH), 116.5 (CH, aromatic CH), 118.0 (C, aromatic C), 124.9 (C, aromatic C), 129.5
(CH, aromatic CH), 130.0 (CH, aromatic CH), 137.2 (C, aromatic C), 138.1 (C, aromatic C), 143.3 (CH,
aromatic CH), 144.1 (C, aromatic C), 147.2 (C, aromatic C), 148.7 (C, C=O), 149.7 (C, C=O), 153.0 (2C,
−
2
4
×
aromatic C); m/z (ES+) 447.2 [M
49.1573. Found 449.1587.
−
H] (100%); HRMS (ES+): exact mass calculated for C H N O
22
21
6
5
2-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-5,7-bis(trifluoromethyl)-3-(3,4,5-trimethoxyphenyl)pyrazolo[1,5-a]
pyrimidine (42). To a stirred solution of 5-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-(3,4,5-trimethoxyphenyl)-
1
H-pyrazol-3-amine (40, 60 mg, 0.16 mmol) in acetic acid (5 mL), was added hexafluoroacetylacetone
◦
(0.44 mL, 3.2 mmol). The mixture was then heated to 80 C for 22 h. Once cooled to room temperature,
the excess solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate
20 mL), then washed with water (20 mL) and brine (20 mL), before being dried over magnesium
(
sulphate. The solvent was removed under reduced pressure, and the crude residue was subjected to
flash column chromatography (hexane/ethyl acetate, 60:40) to yield 42 as an orange solid (69 mg,
◦ −1
8%): m.p. 176–178 C; vmax/cm (KBr) 2923, 2841, 1586, 1560, 1509, 1279, 1131; δ (300MHz, CDCl3)
H
7
3
8
3
.71 [6H, s, 2
.0, 4.8, C-H ], 7.33 [1H, s, C-H ], 7.44 [1H, s, CHCCF ], 8.33-8.38 [2H, m, C-H4,6]; δ (75 MHz, CDCl3)
C
×
m-OCH ], 3.81 [3H, s, p-OCH ], 3.88 [3H, s, NCH ], 6.82 [2H, s, C-H ], 7.11 [1H, q, J
0 0
3 3 3
2 6
5
2
3
0.6 (CH , NCH ), 55.1 (2CH , 2
×
m-OCH ), 60.0 (CH , p-OCH ), 100.4 (CH, aromatic CH), 104.3 (C,
3 3 3
3
3
3
aromatic C), 106.2 (2CH, 2
×
aromatic CH), 110.0 (C, aromatic C), 116.2 (CH, aromatic CH), 112.8-123.7
(
C, q, JF-C 274.9, CF ), 113.8-124.8 (C, q, J 274.6, CF ), 117.8 (C, aromatic C), 124.5 (C, aromatic C),
3
F-C
3
1
29.1 (CH, aromatic CH), 129.7 (CH, aromatic CH), 132.8-134.4 (C, q, JF-C 39.0, aromatic C), 137.0 (C,
aromatic C), 143.0 (CH, aromatic CH), 143.6-145.1 (C, q, JF-C 38.2, aromatic C), 145.3 (C, aromatic C),
47.0 (C, aromatic C), 151.8 (C, aromatic C), 152.6 (2C, 2
aromatic C); m/z (ES+) 552.2 [M + H]+
100%); HRMS (ES+): exact mass calculated for C H N O F 552.1470. Found 552.1493.
1
(
×
25
20
5
3 6
2
,2,2-Trifluoro-N-(5-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrazol-3-yl)
acetamide (43). To a solution of 5-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-(3,4,5-trimethoxyphenyl)-
H-pyrazol-3-amine (40, 60 mg, 0.16 mmol) in acetonitrile (10 mL) was added trifluoroacetic anhydride
0.03 mL, 0.19 mmol). The resulting mixture was heated to reflux for 28 h. Once cooled to room
1
(
temperature, the solvent was evaporated under reduced pressure, and the crude brown residue was
subjected to flash column chromatography (hexane/ethyl acetate, 30:70), yielding trifluoroacetamide
◦
−1
4
3
as an-off white solid (52 mg, 68%): m.p. 129–131 C; vmax/cm (KBr) 3215, 3067, 1713, 1603, 1586,
1
6
411; (300 MHz, DMSO-d ) 3.49 [6H, s, 2 m-OCH ], 3.66 [3H, s, p-OCH ], 3.91 [3H, s, NCH ],
δ
×
H
6
3
3
3
.53 [2H, s, C-H2
0
0
], 7.03 [1H, q, J 7.9, 4.6, C-H ], 7.32 [1H, d, J 6.5, C-H ], 7.86 [1H, s, C-H ], 8.31
,6
5
4
2
[
1H, d, J 3.5, C-H ], 11.37 [1H, bs, NHCOCF ], 13.30 [1H, bs, C=N-NH-C];
δ
C
(75 MHz, DMSO-d6)
6
3
3
2
1.0 (CH , NCH ), 55.4 (2CH , 2
×
m-OCH ), 60.0 (CH , p-OCH ), 102.0 (C, aromatic C), 105.8 (2CH,
3 3 3
3
3
3
×
aromatic CH), 110.2-121.6 (C, q, J 288.0, CF ), 113.9 (C, aromatic C), 115.9 (CH, aromatic CH),
F-C 3
1
(
17.3 (C, aromatic C), 127.1 (C, aromatic C), 128.2 (CH, aromatic CH), 129.4 (CH, aromatic CH), 136.2
C, aromatic C), 143.1 (CH, aromatic CH), 147.2 (C, aromatic C), 152.7 (2C, 2 aromatic C), 152.9 (C,
H] (100%); HRMS (ES+): exact
×
−
aromatic C), 155.8-157.2 (C, q, JF-C 36.2, C=O); m/z (ES+) 474.3 [M
−
mass calculated for C H N O F 476.1546. Found 476.1532.
22
21
5
4 3
1
(
3
-(5-Amino-3-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrazol-1-yl)ethanone
44). To a solution of 5-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrazol-
-amine (40, 60 mg, 0.16 mmol) in acetonitrile (10 mL) was added acetic anhydride (0.016 mL, 0.17
mmol). The resulting mixture was then heated to reflux for 20 h. Once cooled to room temperature,
the solvent was removed under reduced pressure. The crude residue was purified by flash column
chromatography (hexane/ethyl acetate, 50:50) to yield acetylated product 44 as an off-white solid

Pharmaceuticals 2017, 10, 62
17 of 20
◦ −1
48 mg, 71%): m.p. 171–172 C; vmax/cm (KBr) 3454, 3358, 2927, 1704, 1624, 1509, 1404, 1124; δ
H
(
(
300 MHz, CDCl ) 2.80 [3H, s, NCOCH ], 3.79 [3H, s, p-OCH ], 3.79 [6H, s, 2
×
m-OCH ], 3.92 [3H, s,
3
3
3
3
NCH ], 5.56 [2H, bs, NH ], 6.59 [2H, s, C-H
0
0
], 7.05 [1H, s, C-H ], 7.14 [1H, q, J 7.9, 4.7, C-H ], 8.36
3
2
2 ,6
2 5
[
1H, dd, J 4.7, 1.5, C-H ], 8.47 [1H, dd, J 7.9, 1.6, C-H ]; δ (75 MHz, CDCl ) 23.3 (CH , NCOCH ), 31.4
6 4 C 3 3 3
(
CH , NCH ), 56.2 (CH , 2
×
m-OCH ), 61.0 (CH , p-OCH ), 101.9 (C, aromatic C), 105.9 (C, aromatic
3 3 3
3
3
3
C), 107.1 (CH, 2
1
1
×
aromatic CH), 116.6 (CH, aromatic CH), 118.8 (C, aromatic C), 127.4 (C, aromatic C),
29.3 (CH, aromatic CH), 130.7 (CH, aromatic CH), 137.5 (C, aromatic C), 143.7 (CH, aromatic CH),
47.0 (C, aromatic C), 147.9 (C, aromatic C), 148.5 (C, aromatic C), 153.9 (C, 2
×
aromatic C), 173.7 (C,
+
C=O); m/z (ES+) 422.3 [M + H] (100%); HRMS (ES+): exact mass calculated for C H N O 422.1828.
22
24
5
4
Found 422.1813.
-Amino-N-methyl-3-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrazole-1-
carbothioamide (45). To a solution of 5-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-(3,4,5-trimethoxyphenyl)-
H-pyrazol-3-amine (40, 60 mg, 0.16 mmol) in acetonitrile (10 mL) was added methyl isothiocyanate
14 mg, 0.19 mmol). The mixture was heated to reflux for 22 h, before then being cooled to room
5
1
(
temperature. The solvent was evaporated under reduced pressure, and the crude residue was
subjected to flash column chromatography (hexane/ethyl acetate, 50:50), yielding pure thiourea 45 as
◦
−1
a white solid (55 mg, 76%): m.p. 162–164 C; vmax/cm (KBr) 3378, 2931, 1601, 1523, 1406, 1362, 1129;
(300 MHz, CDCl ) 3.33 [3H, d, J 5.0, NHCH ], 3.78 [6H, s, 2 m-OCH ], 3.80 [3H, s, p-OCH ], 3.92
δ
×
H
3
3
3
3
[
3H, s, NCH ], 6.47 [2H, bs, NH ], 6.59 [2H, s, C-H
0
0
], 7.08 [1H, s, C-H ], 7.12 [1H, q, J 7.9, 4.7, C-H ],
3
2
2 ,6
2
5
8
.27 [1H, dd, J 7.9, 1.6, C-H ], 8.36 [1H, dd, J 4.7, 1.5, C-H ], 9.24 [1H, bd, J 4.7, NHCH ]; δ (75 MHz,
C
4
6
3
CDCl ) 30.9 (CH , NHCH ), 31.4 (CH , NCH ), 56.2 (2CH , 2
×
m-OCH ), 61.0 (CH , p-OCH ), 102.5
3 3 3
3
3
3
3
3
3
(C, aromatic C), 105.4 (C, aromatic C), 107.2 (2CH, 2
×
aromatic CH), 116.5 (CH, aromatic CH), 118.6
(C, aromatic C), 127.6 (C, aromatic C), 129.7 (CH, aromatic CH), 130.2 (CH, aromatic CH), 137.5 (C,
aromatic C), 143.7 (CH, aromatic CH), 145.7 (C, aromatic C), 147.8 (C, aromatic C), 148.1 (C, aromatic
+
C), 153.9 (2C, 2
mass calculated for C H N O S 453.1709. Found 453.1718.
×
aromatic C), 176.8 (C, C=S); m/z (ES+) 453.2 [M + H] (100%); HRMS (ES+): exact
22
25
6
3
4
. Conclusions
We report the synthesis and evaluation of a novel template for the investigation of the inhibition
of cancer cell growth. The synthetic route allows for late stage differentiation to explore the effect of
substitution on the headgroup between indole and trimethoxyphenyl subgroups. While disappointing
in terms of hydroxymaleimide raw potency, the results of the cell growth inhibition NCI-60 cell
screen illustrate the possibility of a similar mode of bioactivity across the four series of 3,4-diaryl
heterocyclic derivatives. Appreciable selectivity was recorded for the three parent aminopyrazoles 40
,
4
4
6
1
and 51. Upon extension of the 5-aminopyrazole core towards bicyclic systems such as triazinedione
, the overall mean antiproliferative potency was diminished. However, it was highly notable
that conversion of aminopyrazole 46 to pyrimidine 48 resulted in an extraordinary increase in both
selectivity and potency against HOP-92 lung cancer cells.
In contrast to their bicyclic analogues, monosubstitution of the 5-aminopyrazole core with thiourea
and acetyl functionalities resulted in an increase in overall mean growth inhibition in each case,
except for acetylated derivative 49. Also of particular note is the substituent directed progression
seen from 33–45, 50 and finally 55, the most potent compound of the entire series. Taking the core
hydroxymaleimide 33 and converting to a 5-aminopyrazole thiourea 55 yields significant growth
inhibition, and the difference in 45 and 50 confirms which orientation is most potent. It is evident that
conversion of the headgroup to novel substituted heterocycles can imbue potency and some selectivity
in cancer cell lines. This finding is of particular relevance to Compounds 9 and 10 seen earlier, albeit
that the substituent orientation that delivers potency against VEGF-R2 kinase is reversed.
Despite the overall mean growth inhibition evident in all three series of aminopyrazole derivatives,
the lack of cytotoxicity points to a cytostatic (or cell cycle) mechanism of action. Taking this into account,
further SAR study using other monosubstituents is imperative towards the future realization of this

Pharmaceuticals 2017, 10, 62
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class of compounds as chemotherapeutic agents. Similarly, further investigative work will aim to
identify the molecular targets associated with the cancer cell lines against which considerable selectivity
was observed, namely HOP-92 lung cancer, SNB-75 CNS and UO-31, CAKI-1 renal cancer cell lines.
As a result of the topological assays, it is clear topo II inhibition is not a relevant mechanism of action,
and to further this, screening of protein kinase and tubulin binding are proposed for the active novel
3
,4-diaryl-5-aminopyrazole derivatives.
Supplementary Materials: The supplementary material incorporating NMR spectra and NCI data are available
online at www.mdpi.com/1424-8247/10/3/62/s1.
Acknowledgments: The authors would like to acknowledge the Irish Research Council for Science, Engineering
and Technology for funding this research, the National Cancer Institute (NCI) screening program for 60-cell line
testing and Ken Devine for performing agarose gel electrophoresis.
Author Contributions: Michael M. Cahill, Kevin D. O’Shea and Larry T. Pierce performed the research in relation
to the synthesis and characterization on novel compounds. Hannah J. Winfield performed topoisomerase assays.
Kevin S. Eccles and Simon E. Lawrence are responsible for the crystal structure assignment. Florence O. McCarthy
designed the research and drafted the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
.
Pettit, G.R.; Singh, S.B.; Hamel, E.; Lin, C.M.; Alberts, D.S.; Garcia-Kendall, D. Isolation and structure of
the strong cell growth and tubulin inhibitor combretastatin A-4. Experientia 1989, 45, 209–211. [CrossRef]
[PubMed]
2
.
.
Woods, J.A.; Hadfield, J.A.; Pettit, G.R.; Fox, B.W.; McGown, A.T. The interaction with tubulin of a series of
stilbenes based on combretastatin A-4. Br. J. Cancer 1995, 71, 705–711. [CrossRef] [PubMed]
Siemann, D.W.; Chaplin, D.J.; Walicke, P.A. A review and update of the current status of the
vasculature-disabling agent combretastatin-A4 phosphate (CA4P). Expert Opin. Investig. Drug 2009, 18,
3
1
89–197. [CrossRef] [PubMed]
4.
5.
6.
7.
Tron, G.C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani, A.A. Medicinal chemistry of combretastatin
A4: Present and future directions. J. Med. Chem. 2006, 49, 3033–3044. [CrossRef] [PubMed]
Griggs, J.; Metcalfe, J.C.; Hesketh, R. Targeting tumour vasculature: The development of combretastatin A4.
Lancet Oncol. 2001, 2, 82–87. [CrossRef]
Jordan, A.; Hadfield, J.A.; Lawrence, N.J.; McGown, A.T. Tubulin as a target for anticancer drugs: Agents
which interact with the mitotic spindle. Med. Res. Rev. 1998, 18, 259–296. [CrossRef]
Zavala, F.; Guenard, D.; Robin, J-P.; Brown, E. Structure-antitubulin activity relationships in steganacin
congeners and analogs. Inhibition of tubulin polymerization in vitro by ( )-isodeoxypodophyllotoxin.
±
J. Med. Chem. 1980, 23, 546–549. [CrossRef] [PubMed]
8
.
.
Baldwin, E.L.; Osheroff, N. Etoposide, topoisomerase II and cancer. Curr. Med. Chem. Anti Cancer Agents
005, 5, 363–372. [CrossRef]
Solary, E.; Leteurtre, F.; Paull, K.D.; Scudiero, D.; Hamel, E.; Pommier, Y. Dual inhibition of topoisomerase II
2
9
and tubulin polymerization by azatoxin, a novel cytotoxic agent. Biochem. Pharmacol. 1993, 45, 2449–2456.
[CrossRef]
1
1
1
0. Cushman, M.; Nagarathnam, D.; Gopal, D.; He, H.M.; Lin, C.M.; Hamel, E. Synthesis and evaluation of
analogs of (Z)-1-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethene as potential cytotoxic and antimitotic
agents. J. Med. Chem. 1992, 35, 2293–2306. [CrossRef]
1. Gaukroger, K.; Hadfield, J.A.; Lawrence., N.J.; Nolan, S.; McGown, A.T. Structural requirements for the
interaction of combretastatins with tubulin: How important is the trimethoxy unit? Org. Biomol. Chem. 2003
, 3033–3037. [CrossRef] [PubMed]
,
1
2. LeBlanc, R.; Dickson, J.; Brown, T.; Stewart, M.; Pati, H.N.; VanDerveer, D.; Arman, H.; Harris, J.;
Pennington, W.; Holt, H.L., Jr.; et al. Synthesis and cytotoxicity of epoxide and pyrazole analogs of the
combretastatins. Bioorg. Med. Chem. 2005, 13, 6025–6034. [CrossRef] [PubMed]

Pharmaceuticals 2017, 10, 62
19 of 20
1
1
1
3. Schobert, R.; Biersack, B.; Dietrich, A.; Effenberger, K.; Knauer, S.; Mueller, T. 4-(3-Halo/amino-4,
-dimethoxyphenyl)-5-aryloxazoles and-N-methylimidazoles that are cytotoxic against combretastatin.
A resistant tumor cells and vascular disrupting in a cisplatin resistant germ cell tumor model. J. Med. Chem.
010, 53, 6595–6602. [CrossRef] [PubMed]
4. Romagnoli, R.; Baraldi, P.G.; Salvador, M.K.; Preti, D.; Tabrizi, M.A.; Brancale, A.; Fu, X-H.; Li, J.; Zhang, S-Z.;
Hamel, E.; et al. Synthesis and evaluation of 1, 5-disubstituted tetrazoles as rigid analogues of combretastatin
A-4 with potent antiproliferative and antitumor activity. J. Med. Chem. 2012, 55, 475–488. [CrossRef]
5
2
[PubMed]
5. Chen, H.; Li, Y.; Sheng, C.; Lv, Z.; Dong, G.; Wang, T.; Liu, J.; Zhang, M.; Li, L.; Zhang, T.; et al. Design
and synthesis of cyclopropylamide analogues of combretastatin-A4 as novel microtubule-stabilizing agents.
J. Med. Chem. 2013, 56, 685–699. [CrossRef] [PubMed]
1
6. Becan, L.; Wagner, E. Synthesis and anticancer evaluation of novel 3, 5-diaryl-thiazolo [4,5-d] pyrimidin-2-one
derivatives. Med. Chem. Res. 2013, 22, 2376–2384. [CrossRef] [PubMed]
1
7. Lee, L.; Robb, L.M.; Lee, M.; Davis, R.; Mackay, H.; Chavda, S.; Babu, B.; O’Brien, E.L.;
Risinger, A.L.; Mooberry, S.L.; et al.
Design, synthesis, and biological evaluations of
,5-diaryl-2,3-dihydro-1,3,4-oxadiazoline analogs of combretastatin-A4. J. Med. Chem. 2010, 53,
25–334. [CrossRef] [PubMed]
2
3
1
1
8. Peifer, C.; Krasowski, A.; Hämmerle, N.; Kohlbacher, O.; Dannhardt, G.; Totzke, F.; Schächtele, C.; Laufer, S.
Profile and Molecular Modeling of 3-(Indole-3-yl)-4-(3, 4, 5-trimethoxyphenyl)-1 H-pyrrole-2,5dione (1) as
a Highly Selective VEGF-R2/3 Inhibitor. J. Med. Chem. 2006, 49, 7549–7553. [CrossRef] [PubMed]
9. Peifer, C.; Selig, R.; Kinkel, K.; Ott, D.; Totzke, F.; Schächtele, C.; Heidenreich, R.; Röcken, M.;
Schollmeyer, D.; Laufer, S. Design, synthesis, and biological evaluation of novel 3-aryl-4-(1 H-indole-3yl)-1,
5-dihydro-2H-pyrrole-2-ones as vascular endothelial growth factor receptor (VEGF-R) inhibitors.
J. Med. Chem. 2008, 51, 3814–3824. [CrossRef] [PubMed]
2
0. Hanahan, D.; Folkman, J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.
Cell 1996, 86, 353–364. [CrossRef]
2
2
1. Risau, W. Mechanisms of angiogenesis. Nature 1997, 386, 671–674. [CrossRef] [PubMed]
2. Inoue, K.; Ozeki, Y.; Suganuma, T.; Sugiura, Y.; Tanaka, S. Vascular endothelial growth factor expression in
primary esophageal squamous cell carcinoma. Cancer 1997, 79, 206–213. [CrossRef]
2
3. Volm, M.; Koomägi, R.; Mattern, J. Prognostic value of vascular endothelial growth factor and its receptor
Flt-1 in squamous cell lung cancer. Int. J. Cancer 1997, 74, 64–68. [CrossRef]
2
4. Ganser, C.; Lauermann, E.; Maderer, A.; Stauder, T.; Kramb, J-P.; Plutizki, S.; Kindler, T.; Moehler, M.;
Dannhardt, G. Novel 3-azaindolyl-4-arylmaleimides exhibiting potent antiangiogenic efficacy, protein kinase
inhibition, and antiproliferative activity. J. Med. Chem. 2012, 55, 9531–9540. [CrossRef] [PubMed]
5. Maderer, A.; Plutiziki, S.; Kramb, J.P.; Gopfert, K.; Linning, M.; Khillimberger, K.; Ganser, C.; Lauermann, E.;
Dannhardt, G.; Galle, P.R.; et al. Moguntinones—New selective inhibitors for the treatment of human
colorectal cancer. Mol. Cancer Ther. 2014, 13, 1399–1409. [CrossRef] [PubMed]
2
2
2
6. Pierce, L.T.; Cahill, M.M.; McCarthy, F.O. Synthesis of novel 3,4-diaryl-5-aminopyrazoles as potential kinase
inhibitors. Tetrahedron 2011, 67, 4601–4611. [CrossRef]
7. Pierce, L.T.; Cahill, M.M.; Winfield, H.J.; McCarthy, F.O. Synthesis and identification of novel indolo [2,3-a]
pyrimido [5,4-c] carbazoles as a new class of anti-cancer agents. Eur. J. Med. Chem. 2012, 56, 292–300.
[CrossRef] [PubMed]
2
2
8. Anwar, H.F.; Elnagdi, M.H. Recent developments in aminopyrazole chemistry. ARKIVOC 2009, (i), 198–250.
9. Nie, Z.; Perretta, C.; Erickson, P.; Margosiak, S.; Almassy, R.; Lu, J.; Averill, A.; Yagger, K.M.; Chu, S.
Structure-based design, synthesis, and study of pyrazolo [1,5-a][1,3,5] triazine derivatives as potent inhibitors
of protein kinase CK2. Bioorg. Med. Chem. Lett. 2007, 17, 4191–4195. [CrossRef] [PubMed]
3
0. Shoemaker, R.H. The NCI60 human tumour cell line anticancer drug screen. Nat. Rev. Cancer 2006, 6, 813–823.
[CrossRef] [PubMed]
3
1. Berger, J.M.; Gamblin, S.J.; Harrison, S.C.; Wang, J.C. Structure and mechanism of DNA topoisomerase II.
Nature 1996, 379, 225–232. [CrossRef] [PubMed]

Pharmaceuticals 2017, 10, 62
20 of 20
3
3
3
2. Burden, D.A.; Osheroff, N. Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the
enzyme. BBA Gene Struct. Expr. 1998, 1400, 139–154. [CrossRef]
3. Miller, C.M.; O’Sullivan, E.C.; Devine, K.J.; McCarthy, F.O. Synthesis and biological evaluation of novel
isoellipticine derivatives and salts. Org. Biomol. Chem. 2012, 10, 7912–7921. [CrossRef] [PubMed]
4. Developmental Therapeutics Program (DTP) NCI/NIH. Available online: http://dtp.cancer.gov
(accessed on 4 July 2017).
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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