Development of Potent PfCLK3 Inhibitors Based on TCMDC-135051 as a New Class of Antimalarials

Article abstract of DOI:10.1021/acs.jmedchem.0c00451

The protein kinase PfCLK3 plays a critical role in the regulation of malarial parasite RNA splicing and is essential for the survival of blood stage Plasmodium falciparum. We recently validated PfCLK3 as a drug target in malaria that offers prophylactic,

Full text of DOI:10.1021/acs.jmedchem.0c00451

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Article
Development of potent PfCLK3 inhibitors based
on TCMDC-135051 as a new class of antimalarials
Amit Mahindra, Omar Janha, Kopano Mapesa, Ana Sanchez Azqueta, Mahmood M Alam,
Alfred Amambua-Ngwa, Davies C Nwakanma, Andrew B Tobin, and Andrew G. Jamieson
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00451 • Publication Date (Web): 28 Jul 2020
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Journal of Medicinal Chemistry
Development of potent PfCLK3 inhibitors based on TCMDC-135051 as
a new class of antimalarials
Amit Mahindra,†# Omar Janha,‡# Kopano Mapesa,† Ana Sanchez-Azqueta,‡ Mahmood M Alam,§ Alfred
Amambua-Ngwa,$ Davis C. Nwakanma,$ Andrew B. Tobin‡* and Andrew G. Jamieson†*.
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†School of Chemistry, University of Glasgow, Joseph Black Building, University Avenue, Glasgow G12 8QQ, U.K.
‡Centre for Translational Pharmacology, Institute of Molecular Cell and Systems Biology, Davidson Building, University of
Glasgow, Glasgow G12 8QQ, U.K.
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Wellcome Centre for Integrative Parasitology and Centre for Translational Pharmacology, Institute of Infection Immunity and
Inflammation, University of Glasgow, Glasgow G12 8TA, U.K.
$Medical Research Council Unit The Gambia at LSHTM, Banjul, The Gambia.
Abstract
The protein kinase PfCLK3 plays a critical role in the regulation of malarial parasite RNA splicing and is
essential for the survival of blood stage Plasmodium falciparum. We recently validated PfCLK3 as a drug
target in malaria that offers prophylactic, transmission blocking and curative potential. Herein we describe
the synthesis of our initial hit TCMDC-135051 (1) and efforts to establish a structure−activity relationship
(SAR) with a 7-azaindole-based series. A total of 14 analogues were assessed in a TR-FRET assay against the
full length recombinant protein kinase PfCLK3 and 11 analogues were further assessed in asexual 3D7
(chloroquine sensitive) strains of P. falciparum parasites. SAR relating to rings A and B was established.
These data together with analysis of activity against parasites collected from patients in the field suggest
that TCMDC-135051 (1) is a promising lead compound for the development of new antimalarials with a
novel mechanism of action targeting PfCLK3.
Introduction
In the global fight against malaria the distribution of over 0.5 billion insecticide impregnated bed nets since
2015, together with artemisinin-based combination therapies have contributed to 20 million fewer cases of
malaria being reported in 2017 compared to 2010.1 Despite this success, the rate of reduction in malaria
has stalled with no significant reduction seen over the last 3 years.1 This, together with evidence of the
emergence of resistance to both artemisinin2,3 and partner drugs including piperaquine and mefloquine4,5
means that there is a danger that the progress made in the reduction of malaria will be reversed unless
new medicines with novel mechanisms of action are discovered. To address this, we have been testing the
notion that targeting malaria parasite protein kinases, known to be essential for the survival of the
parasite, might offer a novel strategy for the development of next generation anti-malarials.6,7
In 2011 we determined that 36 of the 65 eukaryotic protein kinases are essential for the survival of the
blood stage of the most virulent species of human malaria, Plasmodium falciparum.8 Among these protein
kinases was PfCLK3 (PF3D7_1114700), one of four members of the cyclin-dependent like protein kinase
family (PfCLK1-4). The plasmodium CLK-family are closely related to the mammalian CLK family and the
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serine-arginine-rich protein kinase (SRPK) family9 both of which are crucial mediators of multiple
phosphorylation events on splicing factors necessary for the correct assembly and catalytic activity of
spliceosomes.10 Our in vitro studies showed that PfCLK3 can phosphorylate parasite SR proteins11
indicating that PfCLK3, together with other members of the PfCLK family12, plays a role in the processing of
parasite RNA.9 We reasoned therefore that inhibitors to PfCLK3 might have parasiticidal activity at any
stage of the parasite life cycle where RNA splicing played an essential role.
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Screening of ~25,000 compounds including all 13,533 compounds of the Tres Cantos Antimalarial Set
(TCAMS) resulted in the discovery of TCMDC-135051 (1, Figure 1), a compound with nanomolar (nM)
activity against PfCLK3 in vitro kinase assays and submicromolar (µM) parasiticidal activity in asexual blood
stage P. falciparum parasites (Figure 1).13 Subsequent studies revealed that TCMDC-135051 rapidly killed P.
falciparum at the trophozoite to schizont stages as well as preventing the development of stage V
gametocytes and inhibiting the development of liver stage parasites. TCMDC-135051 also showed
equivalent in vitro kinase activity at CLK3 from P. falciparum, P. berghei (mouse malaria), P. vivax (human
malaria), P. knowlesi (monkey and human malaria).13 Our recent studies have therefore validated PfCLK3 as
a target with the potential to deliver a curative treatment, be transmission blocking and act as a
prophylactic target. 13
SAR4
CO2H
SAR3
SAR1
Ring B
N
.CF3CO2H
Ring A
N
H
N
MeO
SAR2
PfCLK3 (IC50) = 40 nM (pIC50 = 7.4)
3D7 (EC50) = 180 nM (pEC50 = 6.7)
ClogP = 3.3
Aq. Solubility = 8.0 mg/L
Figure 1. Structure and biological profile of hit PfCLK3 inhibitor TCMDC-135051, 1
Key Structural features of 1 include a core 7-azaindole scaffold with aromatic rings in the 2- and 4-positions
(ring-A and –B respectively). These aromatic rings are further substituted with various functional groups,
including a tertiary amine (ring A) and carboxylic acid (ring B) resulting in a zwitterionic compound at
physiological pH.
7-Azaindoles are a widely studied pharmacophore incorporated in several therapeutic agents.14 Kinases are
the predominate target with the 7-azaindole moiety generally interacting at the ATP binding site within the
kinase hinge region.15,16 Interaction occurs between the kinase hinge region peptide backbone and the
azaindole via two H-bonds. The first involving the pyridine nitrogen lone pair and peptide backbone NH,
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Journal of Medicinal Chemistry
and the second between the pyrrole NH and peptide backbone carbonyl. The resulting H-bond acceptor
and donor bidentate 7-azaindole interaction with the hinge region of the kinase can occur in the more
common “normal” orientation or the “flipped” orientation with the 2-position of the 7-azaindole projected
out of the hinge region into the solvent exposed space.17,18
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A number of small molecule kinase 7-azaindole inhibitors have progressed to different stages of clinical
trials.19 Potential drug candidate GSK1070916 is being developed as an Aurora kinase (Ser/Thr protein
kinases family) inhibitor and has reached human clinical trials (Figure 2A).20 The core scaffold is a 7-
azaindole with aromatic substituents in the 2- and 4-positions. A X-ray crystal structure of the
molecule:aurora kinase complex revealed a flipped hinge region binding mechanism, with 2-aryl projecting
out of the hinge region into solvent and 4-aryl bound within the ribose pocket.20
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Substructure analysis using Scifinder revealed several 7-azaindole analogues described as antimalarials. In
1972, Verbiscar et al. reported the first example of a 7-azaindole with antimalarial activity.21 1-p-
Chlorobenzyl-7-azaindole-3--piperidylmethanol displayed antimalarial activity in mice against
Plasmodium berghei (Figure 2B). More recently, Picard et al. reported 7-azaindole compounds that were
identified from an in silico structure-based drug screen (10,13,483 compounds, Chembridge).22 Compound
IND31119 (Figure 2C) binds to the recombinant N-terminal domain of PfHsp90 and showed selectivity over
human Hsp90 and PfHsp90 mutant.22
In addition to antimalarial activity, the 7-azaindole scaffold has been identified as having a wide variety of
biological applications including antitumor activity and act as HIV-1 inhibitor in infected patients.23,24 The 7-
azaindole motif can be regarded as a privileged scaffold in medicinal chemistry as it is found in three
clinical candidates (Vemurafenib, PLX3397, and AZD5363) (Figure 2D), which suggests its usefulness for
developing novel pharmaceuticals.25-27
Figure 2. A) Aurora kinase inhibitor GSK1070916, B) 1-p-Chlorobenzyl-7-azaindole-3--piperidylmethanol has in-vivo activity
against Plasmodium berghei, C) PfHsp90 inhibitor IND31119, D) Drug candidates incorporating a core 7-azaindole scaffold,
Vemurafenib, PLX3397, and AZD5363.
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To investigate the structure-activity relationship of hit compound 1, a series of analogues were prepared
through varying different substituents on ring-A and ring-B (Figure 1). The N-diethyl amine group (ring A)
was initially replaced with different amine substituents to investigate lipophilicity and solubility (Figure 1,
SAR1). Next, we replaced the methoxy moiety (ring A) with hydroxyl or hydrogen to modify polarity and to
explore the role of the methoxy group on activity (Figure 1, SAR2). The isopropyl substituent (ring B) was
replaced with other small hydrophobic substituents to probe non-coplanar conformations that could
potentially lower the energy of crystal packing and consequently improve aqueous solubility and logP
(Figure 1, SAR3). Finally, we exchanged the carboxylic acid group (ring B) with other substituents to
investigate its role in binding, potentially improve metabolic stability and to explore the effect of increased
lipophilic character (Figure 1, SAR4).
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TCMDC-135051 is a promising hit compound for a medicinal chemistry programme to develop as a
preclinical lead that meets many of the criteria set by the Medicines for Malaria Venture (capable of
rapidly clearing the parasite, has multi-stage potency, killing multiple parasite species with action as a
transmission blocker).28,29 Here we describe the synthetic route to TCMDC-135051 and determine a
structure-activity-relationship that will be key for lead development.
Chemistry
To investigate the effect of the N-diethyl group of ring-A on antimalarial activity, analogues of 4-(2-(5-
((diethylamino)methyl)-2-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-isopropylbenzoic acid (TCMDC-
135051) 1, 8a-c were prepared as shown in scheme 1. Protection of 4-bromo-7-azaindole 2 was achieved
using p-toluenesulfonyl chloride under basic conditions to provide N-tosyl-7-azaindole 3. Regioselective
iodination of 3 using lithium diisopropylamide (LDA) and iodine in THF at -78 °C provided 2-iodoazaindole
4, which was subsequently subjected to Suzuki coupling with (5-formyl-2-methoxyphenyl)boronic acid to
give 2-aryl substituted azaindole 5. Reductive amination of the aldehyde functionality of 5 was performed
with various amines in the presence of sodium triacetoxyborohydride to give amines 6a-d in excellent
yields. Next, the N-tosyl protecting group was removed under basic conditions at reflux for 18 h to yield 7a-
d. Finally, the desired analogues 1 and 8a-c were obtained by Suzuki cross-coupling with 2-isopropyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (see SI for boronate ester synthesis).
Compound 9 was prepared as described in Scheme 1. The carboxylic acid of 1 was converted to its
corresponding ethyl ester by refluxing in thionyl chloride and ethanol for 18h.
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Journal of Medicinal Chemistry
Scheme 1. Synthesis of TCMDC-135051 1 and analogues 8a-c & 9a
1
2
3
4
5
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7
Br
Br
Br
Br
N
CHO
(i)
(ii)
(iii)
I
N
N
N
N
N
N
N
H
Ts
(90%)
Ts
4
(88%)
OMe
(70%)
Ts
2
3
5
8
9
R2
(iv)
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Br
N
R1
Br
N
R1
R1
(vi)
(v)
N
N
N
N
H
H
MeO
MeO
OMe
Ts
1
2
1
1
1
7a
7b
7c
7d
6a
6b
6c
6d
: R = NEt2, R = CO2H (68%)
: R = NEt2 (89%)
: R = NEt2 (88%)
1
2
1
1
8a
8b
8c
: R = NMe2, R = CO2H (37%)
: R = NMe2 (92%)
: R = NMe2 (70%)
1
2
1
1
: R = N-pyrrolidinyl, R = CO2H (49%)
: R = N-pyrrolidinyl (77%)
: R = N-pyrrolidinyl (99%)
1
2
1
1
: R = N-morpholinyl, R = CO2H (60%)
: R = N-morpholinyl (80%)
: R = N-morpholinyl (80%)
(vii)
1
2
9
: R = NEt2, R = CO2Et (98%)
aReagents and conditions: (i) TsCl, NaH, THF, 0 °C, 2 h; (ii) LDA, I2, THF, -78 °C, 3 h; (iii) (5-formyl-2-methoxyphenyl)boronic acid,
Pd(PPh3)4, Na2CO3, 1,4-Dioxane, 110 °C, 12 h; (iv) amine, NaBH(AcO)3, 1,4-Dioxane, 20 °C, 12 h; (v) CH3OH, K2CO3, 55 °C, 18 h; (vi)
2-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid, Pd(dppf)Cl2.CH2Cl2, Na2CO3, 1,4-dioxane, 110 °C, 0.5 h,
MW; (vii) SOCl2, CH3CH2OH, Reflux, 18 h.
The synthetic route outlined in Scheme 1 was modified to prepare primary amine 12. The first step of this
synthetic route involved coupling 2-iodo-azaindole 4 with the appropriate boronate ester under Suzuki
conditions to yield nitrile 10 in 87% yield (Scheme 2). Tosyl deprotection was then achieved using K2CO3,
followed by a Suzuki coupling at the 4-bromo-azaindole scaffold with the appropriate pinacol boronate
ester. The crude material was reduced in situ using cobalt (II) chloride hexahydrate and sodium
borohydride to provide the corresponding amine 12 (Scheme 2).
Scheme 2. Synthesis of 4-(2-(5-(aminomethyl)-2-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-isopropylbenzoic
acid 12.a
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Br
Br
N
Br
N
CN
CN
1
2
3
4
5
6
(i)
(ii)
I
N
N
N
N
H
OMe
Ts
10
MeO
Ts
11
(59%)
(87%)
4
7
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9
(iii), (iv)
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CO2H
NH2
N
H
N
MeO
12
(60%)
aReagents and conditions: (i) 4-methoxy-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile, Pd(PPh3)4, Na2CO3, 1,4-
Dioxane, 110 °C, 18 h; (ii) CH3OH, K2CO3, 55 °C, 18 h; (iii) 2-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid,
Pd(dppf)Cl2.CH2Cl2, Na2CO3, 1,4-dioxane, 110 °C, 0.5 h, MW; (iv) CoCl2·6H2O, NaBH4, CH3OH.
Similarly, analogue 15 was synthesised via Suzuki coupling of 4 with 2-methoxyphenyl boronic acid,
followed by tosyl deprotection and Suzuki coupling (Scheme 3).
To investigate the precise role of the methoxy group of ring-A on activity, we prepared analogues 19 and
23 in which the OMe is replaced by a hydroxy group (19) and OMe is removed (23) (Scheme 3). The
synthetic route toward 19 starts with Suzuki coupling of 4 with methoxymethyl ether (MOM) protected
boronate ester (see SI for boronate ester synthesis) to yield 16. Reductive amination of 16 followed by
removal of the tosyl group gave 18. Finally, 18 was coupled with boronate ester under Suzuki conditions
and the MOM protecting group was removed under acidic conditions to provide the desired analogue 19 in
excellent yield. Analogue 23 was synthesized in 4 steps using the same methods.
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Journal of Medicinal Chemistry
To investigate whether the substitution pattern on ring A is important for antimalarial activity, we
produced analogue 27 in which the methoxy group is moved from the para to ortho position relative to the
methylene N-diethyl functionality (Scheme 3).
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9
Scheme 3. Synthesis of 2-isopropyl-4-(2-(2-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)benzoic acid 15, 4-(2-(5-
((diethylamino)methyl)-2-hydroxyphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-isopropylbenzoic acid 19, 4-(2-(3-
((diethylamino)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-isopropylbenzoic
acid
23
and
[4-(2-(3-
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((diethylamino)methyl)-2-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-isopropylbenzoic acid] 27.a
CO2H
Br
N
Br
N
(i)
(ii)
(iii)
N
N
H
OMe
MeO
Ts
N
N
H
MeO
13
14
(80%)
(32%)
15
(84%)
4
(iv)
Br
N
R1
N
Br
N
R1
R2
CHO
Br
N
R1
N
(v)
(ii)
N
N
N
H
R2
R2
Ts
Ts
1
2
1
2
1
2
16
: R = H, R = OMOM (77%)
17
18
22
26
: R = H, R = OMOM (83%)
: R = H, R = OMOM (84%)
1
2
1
2
1
2
20
24
: R = H, R = H (76%)
21
25
: R = H, R = H (83%)
: R = H, R = H (89%)
1
2
1
2
1
2
: R = OMe, R = H (72%)
: R = OMe, R = H (81%)
: R = OMe, R = H (85%)
(iii)
(vi)b
CO2H
R1
N
N
H
N
R2
1
2
19
23
27
: R = H, R = OH (76%)
1
2
b
: R = H, R = H (70%)
1
2
: R = OMe, R = H (74%)
aReagents and conditions: (i) (2-methoxyphenyl)boronic acid, Pd(PPh3)4, Na2CO3, 1,4-Dioxane, 110 °C, 12 h; (ii) CH3OH, K2CO3, 55
°C, 18 h; (iii) 2-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid, Pd(dppf)Cl2 CH2Cl2, Na2CO3, 1,4-dioxane,
.
110 °C, 0.5 h, MW; (iv) 4-(methoxymethoxy)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde or 3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde, Pd(PPh3)4, Na2CO3, 1,4-Dioxane, 110 °C, 18 h, (v) Et2NH, NaBH(AcO)3, 1,4-
dioxane, rt, 18 h; (vi) HCl, MeCN/H2O 3:1.
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To investigate the effect of the iso-propyl substituent (ring-B) on efficacy, we prepared analogues 28-29, as
shown in scheme 4. 4-Bromo-7-azaindole 7a was coupled with various boronate esters under Suzuki
conditions to yield the desired 4-aryl-7-azaindole analogues 28-29.
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2
3
4
5
6
7
8
9
To investigate the role of the carboxylic acid substituent of ring B on antimalarial activity, several
analogues 30-32 were synthesised as depicted in scheme 4. Synthesis of analogues 30-32 was achieved by
Suzuki coupling 7a with various boronate esters (R2 = 1H-tetrazole or H) to yield target compounds 30-31 in
good yields.
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An isomer in which the isopropyl and carboxylic acid were switched, 32 was synthesized to investigate the
role of these functional groups on antimalarial activity (Scheme 4).
Scheme 4. Synthesis of 4-aryl-7-azaindole analogues,28-32.a
R2
R1
Br
N
NEt2
(i)
N
H
N
N
N
H
MeO
MeO
7a
1
2
28:
29:
R = CH3, R = CO2H (75%)
1
2
R = H, R = CO2H (85%)
1
30
31
32
: R = CH(CH3)2, R2 = 1H-tetrazole (62%)
1
: R = CH(CH3)2, R2 = H (82%)
1
: R = CO2H, R2 = CH(CH3)2 (78%)
aReagents and conditions: (i) boronate esters, Pd(dppf)Cl2.CH2Cl2, Na2CO3, 1-4-dioxane, 110 °C, 0.5 h, MW.
Result and discussion
In this work, we have maintained the core 7-azaindole molecular scaffold of TCMDC-135051 and focused
on the structure-activity relationships (SARs) of the substitutes on ring-A and –B to assess the requirement
for this functionality. All synthesised analogues were assessed in a TR-FRET PfCLK3 in vitro kinase assay
against the full recombinant protein kinase (Table 1 and Table 2). Analogues which gave low nanomolar
activity were then further assessed in live parasite viability (parasiticidal) assays using laboratory strain 3D7
(chloroquine sensitive) P. falciparum (Table 1 and Table 2). The synthesized analogues were evaluated for
LogD7.4 (Distribution co-efficient), metabolic stability, and the results are shown in Table 1 and 2.
SAR1 corresponding to analogues 8a-c, 12 and 15 were designed to examine the effect of the N-diethyl
group of ring-A on antimalarial activity. In analogue 8a, the N-diethyl group was replaced with an N-
dimethyl group to investigate the effect of alkyl group size and molecular lipophilicity (logD7.4 = 0.85). In
vitro kinase activity of 8a the half maximal inhibitory activity (IC50) IC50 = 29 nM (pIC50 = 7.5 ± 0.224)
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remains the same in recombinant PfCLK3 kinase whereas a 2-fold decrease in the half maximal parasite
growth inhibition effect (EC50) EC50 = 457 nM (pEC50 = 6.3 ± 0.129) was observed (Table 1). To further
investigate the steric requirements of this functionality and polarity, pyrrolidine (8b) and morpholino
groups (8c) were introduced. Analogue 8b shows comparable in vitro kinase activity, IC50 = 38 nM (pIC50 =
7.4 ± 0.113) and 2-fold decrease in parasite growth inhibition 8b EC50 = 382 nM (pEC50 = 6.4 ± 0.081) was
observed. Whereas the more polar 8c shows a slight improvement in vitro kinase activity of IC50 = 9 nM
(pIC50 = 8.0 ± 0.191), yet a 7-fold decrease in parasite growth inhibition was observed 8c, EC50 = 1339 nM
(pEC50 = 5.9 ± 0.118) (Table 1). To further investigate the polarity of this moiety we replaced the N-diethyl
functionality with a more polar primary amine (logD7.4 = 0.61). Analogue 12 incorporating a primary amine
gave an IC50 = 76 nM (pIC50 = 7.1 ± 0.142), thus was 2-fold less potent in vitro and showed a dramatic loss
of efficacy EC50 = 2801 nM (pEC50 = 5.6 ± 0.104) in parasites, indicating the need to decrease the polarity of
the amine group for optimal parasite growth inhibition (Table 1). Analogue 15, with the alkyl amine group
removed (logD7.4 = 2.45) was less potent in vitro, IC50 = 79.0 nM (pIC50 = 7.1 ± 0.132) against recombinant
PfCLK3 protein and in parasites, EC50 = 1456 nM (pEC50 = 5.8 ± 0.152). There was a 7-fold drop in the
parasite growth inhibition, suggesting that the alkyl amine on ring A is important for antimalarial activity
(Table 1).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
COOH
R3
R2
R1
N
N
H
Table 1. Physicochemical properties and activity data of TCMDC-135051 ring A analogues.
c
Analogue
R1
R2
R3
PfCLK3
3D7
logD7.4
Clint (mL/min/g
liver)d
1.12
IC50 (nM)a
pIC50
EC50 (nM)b
180
pEC50
1
NEt2
NMe2
N-pyrrolidinyl
N-morpholinyl
NH2
OMe
OMe
OMe
OMe
OMe
OMe
OH
H
H
H
H
H
H
H
H
OMe
40
29
38
9
76
79
22
25
17
7.4 (±0.221)
7.5 (±0.224)
7.4 (±0.113)
8.0 (±0.191)
7.1 (±0.142)
7.1 (±0.132)
7.7 (±0.115)
7.6 (±0.089)
7.7 (±0.116)
6.7 (±0.126)
6.3 (±0.129)
6.4 (±0.081)
5.9 (±0.118)
5.6 (±0.104)
5.8 (±0.152)
5.5 (±0.133)
6.5 (±0.114)
5.6 (±0.109)
0.93
0.85
2.43
1.20
0.61
2.45
0.59
0.80
0.74
8a
8b
8c
12
15
19
23
27
457
382
2.53
1.94
1.60
2.92
2.54
1.94
0.85
1.65
1339
2801
1456
3529
309
H
NEt2
NEt2
NEt2
H
H
3167
aIC50 (the concentration of an inhibitor where the response is reduced by half); bEC50 (the concentration of a drug that gives half-
maximal response). IC50 and EC50 values are means ± standard error of the mean (S.E.M) of three independent experiments run
d
in triplicates (n=3); cLogD7.4 (Distribution co-efficient) was estimated using HPLC chromatography; In vitro intrinsic clearance in mouse liver
microsomes.
To explore the importance of the methoxy group (OMe) on ring-A, two analogues, hydroxyl 19 and 23, with
methoxy removed, were prepared. We first replaced the OMe group with a hydroxyl to investigate the role
of polarity on activity (logD7.4 = 0.59). Next, we replaced the OMe group with hydrogen (23) to investigate
the importance of this functionality for activity. For both compounds 19 and 23 in vitro kinase potency was
comparable IC50 = 22 nM (pIC50 = 7.7 ± 0.115) and IC50 = 25 nM (pIC50 = 7.6 ± 0.089) respectively (Table 1).
When tested in parasites 19 shows significant loss of activity EC50 = 3529 nM (pEC50 = 5.5 ± 0.133).
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Interestingly, replacing the OMe group with a hydrogen, 23 also shows 1.5-fold decrease in potency, EC50 =
309 nM (pEC50 = 6.5 ± 0.114) against parasites. These data demonstrated that the OMe group on ring A is
required for in vitro kinase and parasite growth inhibition. Overall, analogues (8a-c, 12, 15, 19 and 23)
showed good metabolic stability in mouse liver microsomes (Table 1).
1
2
3
4
5
6
7
8
9
Next, we turned our attention to study the SAR of various functional groups on ring-B. First, we examined
the role of binding through Van der Waals interactions in vitro and lipophilicity in parasites, imparted by
the ring-B isopropyl group, on antimalarial activity. In analogue 28 (logD7.4 = 0.66) the isopropyl group was
replaced with methyl, whereas in analogue 29 (logD7.4 = 0.65) the isopropyl was removed and replaced
with hydrogen. Analogues 28 and 29 had a modest increase on in vitro kinase activity IC50 = 24 nM (pIC50 =
7.6 ± 0.10) and IC50 = 34 nM (pIC50 = 7.5 ± 0.089) respectively. However, a dramatic loss of potency was
observed for both compounds 28 and 29 with EC50 = 1185 nM (pEC50 = 5.6 ± 0.097) and EC50 = 3272 nM
(pEC50 = 5.5 ± 0.124) respectively, representing a 6-fold and 16-fold decrease in parasite growth inhibition
(Table 2). Therefore, this group is not required for binding in vitro, however the effect of lipophilicity
appears to be very important for parasite growth inhibition. Analogues 28 and 29, were then evaluated for
intrinsic clearance in mouse microsome. 28 (Clint = 1.33 mL/min/g liver) showed a similar clearance value to
that of 1 (TCMDC-051) in mouse liver microsomes whereas 29 had a higher clearance under the same
conditions (Clint = 11.01 mL/min/g liver) indicating the importance of isopropyl group.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
At this point we decided to increase the lipophilicity and replace the isopropyl group to the larger tert-
butyl group. However due to a restriction of the chemistry of boronate esters (instability of tert-butyl anion
required for SNAr reaction) we could not synthesize the proposed molecule. From these data, the alkyl
substituent on ring-B appears not to be particularly important in the molecular recognition event with
PfCLK3, however is key for overall molecular lipophilicity that contributes to parasite growth inhibition.
To investigate the requirements (i.e. ionic and H-bonding) of the carboxylic acid (ring B) for antimalarial
activity we employed a series of structural changes. The presence of a carboxylic acid functionality in a
drug molecule has several potential drawbacks including limited permeability across biological
membranes, metabolic instability, and potential idiosyncratic toxicities. A common isostere of a carboxylic
acid, that overcomes many of these physicochemical limitations, is a tetrazole. We therefore designed
tetrazole analogue 30. This change was orchestrated to increase the lipophilicity of the molecule, retain H-
bonding capability and investigate the role of potential ionic interactions with the enzyme. The tetrazole
analogue 30 shows improved in vitro kinase potency with an IC50 = 19 nM (pIC50 = 7.7 ± 0.089), with
comparable parasite growth inhibition, EC50 = 270 nM (pEC50 = 6.6 ± 0.158) (Table 2). The potency of
analogue 30 was tested against resistant mutant PfCLK3 (PfCLK3_G449P) 3D7 parasites.13 Against the
mutant parasites (PfCLK3_G449P) parasite growth inhibition of the analogue 30, EC50 = 3494 nM (pEC50 =
5.5 ± 0.09) was 2-fold lower compared to 1 EC50 = 1806 nM (pEC50 = 5.7 ± 0.07).
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Analogue 31 where the carboxylic acid group was replaced with a hydrogen shows significant loss of in
vitro kinase activity IC50 = 1300 nM (pIC50 = 6.0 ± 0.091). Analogue 31 has high lipophilicity (logD7.4 = 4.45)
and in vitro high intrinsic clearance (Clint = 9.54 mL/min/g liver) demonstrating the importance of the
carboxylic acid group (Table 2).
1
2
3
4
5
6
7
R2
8
R1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NEt2
N
N
H
MeO
Table 2. Physicochemical properties and activity data of TCMDC-135051 ring B analogues.
c
Analogue
R1
R2
PfCLK3
3D7
logD7.4
Clint (mL/min/g
liver)d
IC50 (nM)a
pIC50
IC50 (nM)b
1185
pIC50
28
29
30
31
9
CH3
H
CH(CH3)2
CH(CH3)2
CH(CH3)2
CO2H
CO2H
CO2H
1H-tetrazole
H
CO2CH2CH3
CH(CH3)2
24
34
19
7.6 (±0.10)
7.5 (±0.089)
7.7 (±0.089)
6.0 (±0.091)
6.4 (±0.087)
4.9 (±0.093)
5.6 (±0.097)
5.5 (±0.124)
6.6 (±0.158)
NDe
0.66
0.65
0.93
4.45
2.45
NDe
1.33
3272
270
NDe
NDe
NDe
11.01
2.32
9.54
16.06
NDe
1300
390
1385
NDe
NDe
32
aIC50 (the concentration of an inhibitor where the response is reduced by half); bEC50 (the concentration of a drug that gives half-
maximal response). IC50 and EC50 values are means ± standard error of the mean (S.E.M) of three independent experiments run
in triplicates (n=3); cLogD7.4 (Distribution co-efficient) was estimated using HPLC chromatography; In vitro intrinsic clearance in mouse liver
d
microsomes; eND, not determined
Analogue 9 has improved lipophilicity (logD7.4 = 2.45) yet the in vitro kinase potency was low IC50 = 390 nM
(pIC50 = 6.4 ± 0.087), indicating the presence of a functional group capable of donating a hydrogen bond is
important for activity (Table 2). This analogue was not tested in parasites because of the low potency
against recombinant PfCLK3 kinase.
The final part of our SAR assessment was dedicated to exploring the possibility of orientation of key
substituents. Varying the position of the OMe group from para to the ortho-position 27 resulted in 2-fold
increase in vitro kinase activity IC50 = 17 nM (pIC50 = 7.7 ± 0.116). Whereas when 27 was tested in parasites
the activity was lowered by 15-fold EC50 = 3167 nM (pEC50 = 5.6 ± 0.109) (Table 1).
We next investigated the position of the isopropyl and carboxylic acid substituents of ring B 32 on
antimalarial activity. The change of orientation of the substituent is detrimental for the in vitro kinase
activity IC50 = 1385nM (pIC50 = 4.9 ± 0.093) and therefore was not tested in parasites. These data suggested
that the positioning of the isopropyl and carboxylic acid group was essential for binding to its cellular
target.
No X-ray crystal structure has been reported for PfCLK3 and so a homology model of PfCLK3 was created
using SWISS-MODEL to provide evidence of our proposed binding mechanism (i.e. hinge binder), which we
hypothesise based on other literature examples of 7-azaindole scaffolds binding at the hinge region of the
kinase domain. The structure of human PRPF4B kinase domain was selected as the template as the closest
homologue (PDB 6CNH)(sequence identity 53.2%)).30 Model accuracy was determined to be reasonable
using SWISS-MODEL with QMEAN (Qualitative Model Energy Analysis) score = -2.26 and GMQE (Global
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Model Quality Estimation) = 0.77. Overlay with Human Jnk1alpha kinase with 4-phenyl-7-azaindole IKK2
inhibitor bound (PDB 4AWI) facilitated identification of the proposed binding pocket.31 Based on this model
we propose that the 7-aza-indole scaffold interacts with the hinge region in the flipped conformation, H-
bonding to the peptide backbone of the hinge region. The benzoic acid on ring B appears to occupy the
ribose pocket and interacts with Lys-394. In the model, the ring-B iso-propyl group occupies a hydrophobic
back pocket. Ring A projects into the solvent exposed space. The diethyl-amine and methoxy substituents
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
are solvent exposed and may contribute to orientating the 7-azaindole in the flipped binding conformation.
A
B
Figure 3. Putative binding mode of TCMDC-135051 1 in a PfCLK3 homology model.
We previously demonstrated that TCMDC-135051 showed selective inhibition of PfCLK3 when compared
against the closely related human kinases PRPF4B and CLK2, as well as the closest parasite kinase, PfCLK1,
and other parasite kinases (PfPKG, PfCDPK1).13 To further assess kinase selectivity, and the potential for off
target toxicity, TCMDC-135051 was screened against 140 human kinases at 1 µM concentration. Only 9
kinases were found to have less than 20% activity at this concentration (See Supporting Information).
TCMDC-135051 inhibits the growth of clinical field isolates
An important property for next generation anti-malarials is effectiveness against parasites that are
resistant to currently used anti-malarials. To determine if the parent molecule TCMDC-135051 1 might
offer such activity we tested the efficacy of this compound against parasites that were collected from
malaria patients in The Gambia. These parasite strains were sequenced and those that contained genetic
markers of resistance were selected (Table 3). Parasites were then tested for resistance to pyrimethamine,
a frontline antimalarial where resistance has been documented in parasites originating from The Gambia.32
This analysis identified nine parasite isolates from patients with varying degrees of resistance to
pyrimethamine (Figure 4A, B) and which contained mutations in one or more of three genes; PfCRT (P.
falciparum chloroquine resistant transporter gene), PfMDR1 (P. falciparum multidrug resistant gene 1), and
PfDHFR (P. falciparum dihydrofolate reductase gene). The mutations identified in these genes (Table 3)
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Journal of Medicinal Chemistry
have previously been associated with resistance to commonly used anti-malarials including chloroquine
and pyrimethamine.33 An EC50 concentration of pyrimethamine was then determined using the laboratory
strain parasite 3D7 (EC50 = 18 nM). Using this concentration in parasite reduction rate (PRR) assays all
isolates showed a reduction in parasitaemia by at least 70% except for three isolates (GB0020, GB002 and
GB0026), which showed resistance to pyrimethamine treatment. These three isolates uniquely contained
triple mutations in the PfDHFR gene and a single mutation in PfCRT (Table 3). For these isolates, resistance
was evident after 24 hours of treatment with pyrimethamine where parasitaemia was only reduced by 45,
64, and 36% respectively (Figure 4A). Resistance to pyrimethamine of these three isolates was still evident
after 72 hours of drug treatment (Figure 4A) and in parasites exposed to 10X the EC50 (180 nM) of
pyrimethamine (Figure 4B).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In contrast, all isolates treated with the EC50 of TCMDC-135051, as determine in 3D7 parasites (EC50 = 180
nM), demonstrated total susceptibility including isolates GB0020, GB002 and GB0026 (Figure 4C). This was
true both at the EC50 concentration and 10xEC50 for TCMDC-135051 (Figure 4D). Hence in the field isolates
tested, TCDMDC-135051 showed equivalent activity against parasites that carried genetic markers of
resistance and those that showed actual resistance to a front-line anti-malarial. This indicates the
possibility that the parent molecule, and potentially analogues of this molecule, would be active against
naturally circulating malaria parasites harbouring mutations that promoted resistance to current anti-
malaria drugs. The PRR assay was setup using clinical isolates previously cryopreserved in liquid nitrogen
(LN2). These were thawed and put in culture for at least one cycle (~48hour ± 2). The life cycle stage of the
parasites was monitored using a blood film and greater than 95 % of the parasites are expected egress and
form new invading rings. These new rings, mainly 0-3 hours old post invasion, were used as time point zero
hours (t=0) for the assay. The parasites are then cultured with drugs for 24, 48 and 72 hours. For the 24
hours treatment, the drug was removed by washing twice with wash media and for longer treatments (48
and 72 hour time points) drug was replenished with fresh drug every 24 hours. After the treatment period,
parasites were grown in fresh media for an additional 48 hours in freshly stained erythrocytes to allowed
invasion from any viable parasites.
Table 3: Genotypes of field isolated parasite strains: Whole genome Sanger sequencing identified mutations in
three parasite genes (Cholorquine resistant transporter (CRT), multi-drug resistant gene (PfMDR1) and dihydrofolate
reductase (PfDHFR) associated with drug resistance. Shown are the amino acid changes associated with each of
these three genes and whether the parasite strain contained the mutation.
PfCRT
PfMDR1
PfDHFR
Sample ID
GB0006
GB0004
GB0026
GB002
GB0021
GB0048
K76T
N86Y
X
N
N
N
Y184F
N1042D
N51I
C59R
S108N
I164L
X
K
K
T
K
X
X
Y
X
X
F
F
N
N
N
N
N
N
X
N
N
I
X
I
X
R
R
C
X
C
N
N
N
N
N
N
L
L
L
L
L
L
N
N
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GB0071
GB0087
GB0020
X
T
T
N
N
Y
Y
Y
Y
N
N
N
N
I
I
C
C
C
N
N
N
L
L
L
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4: Parasite Reduction Rate of clinical isolates comparing pyrimethamine and TCMDC-135051 at EC50 and 10 times the EC50
of each drug. This represent the average of triplicate
Conclusions
In summary, we report the synthesis of hit PfCLK3 inhibitor TCMDC-135051 1 (PfCLK3 IC50 = 40 nM, 3D7
EC50 = 180 nM) and a series of related 7-azaindole-based analogues. Of the 14 analogues, 11 had low
nanomolar activity and were further assessed in a live parasite viability assays using the 3D7 (chloroquine
sensitive) strain of P. falciparum. Tetrazole analogue 30 was identified with improved in vitro kinase
activity (PfCLK3, IC50 = 19 nM) and comparable activity in parasites 3D7 (EC50 = 271 nM). SAR was
established for both ring A, highlighting the importance of H-bonding functionality in the 4-aryl position
and for the alkyl amino group on ring B. Together these data provide a good starting point for the hit to
lead development of novel PfCLK3 inhibitors based on TCMDC-135051 (1).
Experimental Section
General information
Chemicals and solvents were purchased from standard suppliers and used without additional purification.
All glassware was dried with a flame under flushing argon gas or stored in the oven and let cool under an
inert atmosphere prior to use. Anhydrous solvents (THF, DCM and Et2O) were obtained by passage through
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Journal of Medicinal Chemistry
solvent filtration systems (Pure Solv) and solvents were transferred by syringe. PET ether refers to
petroleum (bp. 40-60 °C, reagent grade, Fisher Scientific). All reactions carried out under inert or dry
atmosphere were carried out under a blanket of nitrogen. Thin-layer chromatography (TLC) was performed
using aluminium plates precoated with silica gel (0.25 mm, 60 A° pore-size) impregnated with a fluorescent
indicator (254 nm). Visualization on TLC was achieved by the use of UV light (254 nm). Flash column
chromatography was undertaken on silica gel (400-630 mesh). Proton nuclear magnetic resonance spectra
(1H NMR) were recorded on AVANCE III 400 Bruker (400 MHz). Proton chemical shifts are expressed in
parts per million (ppm, δ scale) and are referenced to residual protium in the NMR solvent (CDCl3, δ 7.26;
CD3OD, δ 3.31 and DMSO-d6, δ 2.50). The following abbreviations were used to describe peak patterns
when appropriate: br = broad, s = singlet, d = doublet, t = triplet, q = quadruplet, sept = septet, m =
multiplet. Coupling constants, J, were reported in Hertz unit (Hz). Carbon 13 nuclear magnetic resonance
spectroscopy (13C NMR) was recorded on AVANCE III 400 Bruker (101 MHz) and was fully decoupled by
broad band decoupling. Chemical shifts were reported in ppm referenced to the centre line of a triplet at
77.0, 49.0, 39.5 ppm of CDCl3, CD3OD and DMSO-d6. Low-resolution mass spectrometry (LRMS) was
performed on a Thermo Scientific LCQ Fleet quadrupole mass spectrometer using electrospray ionisation in
positive mode (ESI+), employing a 150 mm x 4 mm C18 column (Dr. Maisch Reprosil Gold). High-resolution
mass spectrometry (HRMS) was performed on a Bruker microTOF-Q II (ESI+). Preparative HPLC was carried
out on a Dionex HPLC system equipped with Dionex P680 pumps and a Dionex UVD170U UV-vis detector
(monitoring at 214 nm and 280 nm), using a Phenomenex, Gemini, C18, 5 µm, 250 x 21.2 mm column.
Gradients were performed using solvents consisting of A (H2O + 0.1% TFA) and B (CH3CN + 0.1% TFA) and
fractions were lyophilised on a Christ Alpha 2-4 LO plus freeze dryer. Final molecules were analysed on a
Shimadzu reverse-phase HPLC (RP-HPLC) system equipped with Shimadzu LC-20AT pumps, a SIL-20A
autosampler and a SPD-20A UV-vis detector (monitoring at 214 nm) using a Phenomenex, Aeris, 5 µm,
peptide XB-C18, 150 x 4.6 mm column at a flow rate of 1 mL/min. RP-HPLC gradients were run using a
solvent system consisting of solution A (5% CH3CN in H2O + 0.1% TFA) and B (5% H2O in CH3CN + 0.1% TFA).
A gradient from 0% to 100% solution B over 20 min 0%-100% solution B. Purity of all final compounds is >
95% as determined by RP-HPLC.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Experimental procedures and characterisation data:
4-Bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (3)
To a solution of sodium hydride (1.83 g, 76.1 mmol, 3 equiv.) and tetrabutylammonium bromide (0.25 g,
0.76 mmol, 0.03 equiv.) in dichloromethane (80 mL) at 0 °C was added 4-bromo-1H-pyrrolo[2,3-b]pyridine,
2 (5 g, 25.4 mmol, 1 equiv.), the mixture was then left to stir at 0 °C for 15 mins. Toluene sulphonylchloride
(5.81 g, 30.5 mmol, 1.2 equiv.) in dichloromethane (20 mL) was slowly added over 5 mins. The mixture was
then left to warm up to room temperature and stirred for 1 hour. The reaction was quenched by addition
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of water and extracted with dichloromethane. The organic layer was washed with brine and dried over
magnesium sulphate. The residue was then purified by flash column chromatography (10% ethyl acetate-
PET Ether) to give 3 as a colourless solid (8.83 g, 99%); 1H NMR (400 MHz, CDCl3) δ: 8.22 (d, J = 5.2 Hz, 1H),
8.06 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 4.0 Hz, 1H), 7.35 (d, J = 5.3 Hz, 1H), 7.28 (d, J = 8.0 Hz, 2H), 6.64 (d, J =
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4.0 Hz, 1H), 2.37 (s, 3H); C NMR (101 MHz, CDCl3) δ: 146.8, 145.5, 145.0, 135.1, 129.7, 128.2, 127.0,
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125.7, 124.4, 122.1, 104.9, 21.7; HRMS m/z calcd for C14H11BrN2NaO2S [M+Na]+ 372.9617 found 372.9608
(Δ = 2.3 ppm).
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4-Bromo-2-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (4)
n-Butyllithium (2.5 M; 6.3 mL, 15.6 mmol, 1.1 equiv.) was added dropwise to diisopropylamine (2.4 mL,
17.2 mmol, 1.2 equiv.) in diethyl ether (30 mL) at -78 °C over a period of 5 mins. The resulting solution was
stirred at -78 °C for 60 mins and then slowly added via cannula to a solution of 4-bromo-1-tosyl-1H-
pyrrolo[2,3-b]pyridine, 3 (5.0 g, 14.2 mmol, 1 equiv.) and tetramethylethylenediamine (2.3 mL, 15.7 mmol,
1.1 equiv.) in diethyl ether (170 mL) over a period of 10 mins at -78 °C. The resulting solution was then
stirred at -78 °C for 90 mins. Iodine (5.4 g, 21.4 mmol, 1.5 equiv.) was added in one portion, and the
reaction mixture was stirred at -78 °C for 60 mins. The reaction was quenched with saturated ammonium
chloride solution and the organic layer was washed with aqueous sodium thiosulphate and brine before
drying over magnesium sulphate. The residue was then purified by column chromatography (20% ethyl
acetate-PET ether) to give 4 as a colourless solid (5.59 g, 85 %; 1H NMR (400 MHz, CDCl3) δ: 8.11 (d, J = 5.2
Hz, 1H), 8.01 (d, J = 8.5 Hz, 2H), 7.23 (d, J = 5.2 Hz, 1H), 7.22-7.19 (m, 2H), 6.96 (s, 1H), 2.30 (s, 3H); 13C NMR
(101 MHz, CDCl3) δ: 149.1, 145.7, 144.7, 135.4, 129.8, 128.3, 125.3, 123.6, 122.4, 119.4, 21.7; HRMS m/z
calcd for C14H10BrIN2NaO2S [M+Na]+ 498.8583 found 498.8602 (Δ = -3.8 ppm).
Method A: General Method of Suzuki Cross-Coupling
To a solution of desired aryl bromide (1.0 equiv.) and tetrakis(triphenylphosphine)palladium(0) (0.05
equiv.) in 1,4-dioxane was added boronic acid/ester (1.1 equiv.) under a nitrogen atmosphere. Aqueous
sodium carbonate (2 M, 7.0 equiv.) was then added and the reaction mixture left to stir at 110 °C for 18
hrs. Solvent was removed under vacuum and the crude was dissolved in ethyl acetate and poured into
water and extracted with ethyl acetate. The organic layer was washed with brine before drying over
magnesium sulphate and purified by flash column chromatography as indicated.
Method B: Reductive Amination of Aldehydes
To a solution of aryl aldehyde (1.0 equiv.) in 1,4-dioxane was added amine (1.5 equiv.) and the solution
was allowed to stir for 2 mins before the addition of sodium triacetoxyborohydride (2.5 equiv.). The
reaction mixture was stirred at room temperature for 18 h before quenching with ammonium hydroxide.
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Journal of Medicinal Chemistry
The reaction mixture was extracted with ethyl acetate and washed with brine. The organic layer was dried
over magnesium sulphate and the residue purified by flash column chromatography as indicated.
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Method C: Deprotection of azaindole
To a solution of protected 7-azaindole (1 equiv.) in methanol was added potassium carbonate (3.5 equiv.)
and refluxed for 18 h. Poured the reaction into a mixture of EtOAc (10 mL) and H2O in a separatory funnel.
Solvent was then removed under vacuum and the residue was then purified by flash column
chromatography as indicated.
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Method D: Suzuki Cross-Coupling with boronate ester
To a 10 mL microwave vial containing the required bromo-7-azaindole (1 equiv.) in 1,4- dioxane was added
boronic acid/ester (1.1 equiv.), Pd(dppf)Cl2·DCM complex (0.05 equiv.) under a nitrogen atmosphere. The
solution was purged with nitrogen for 5 mins and the reaction microwaved at 110 °C for 0.5 h. The reaction
was allowed to cool to room temperature and the mixture was filtered through celite eluting with
methanol. The filtrate was evaporated and the resulting residue was purified by preparative HPLC: 10-95%
acetonitrile in water + 0.1% TFA to give the desired products.
Method E: Synthesis of boronate ester
Boronate esters required for Suzuki coupling were prepared according to procedure reported in
literature.35 To a solution of aryl bromide (1 equiv.), bis(pinacolato)diboron (1.5 equiv.) and potassium
acetate (3 equiv.) in 1,4-dioxane (20 ml), PdCI2(dppf).CH2Cl2 complex (0.1 equiv.) were added under
nitrogen and stirred at 100 °C for 3 hour. The reaction mixture was quenched with saturated NaHCO3 and
extracted with ethyl acetate. The organic phase was dried (Na2SO4), filtered and concentrated to dryness.
The crude product was purified by chromatography (20% ethyl acetate-PET Ether) to give the desired
boronate esters.
3-[4-Bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl]-4-methoxybenzaldehyde (5)
Prepared according to method A. Purification by flash chromatography (50% ethyl acetate-PET ether)
1
afforded 5 as a yellow oil (1.72 g, 70%); H NMR (400 MHz, CDCl3) δ: 9.90 (s, 1H), 8.15 (d, J = 5.2 Hz, 1H),
7.95 (dd, J = 8.5, 2.1 Hz, 1H), 7.84 (d, J = 2.1 Hz, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 5.3 Hz, 1H), 7.13 (d,
J = 8.2 Hz, 2H), 7.04 (d, J = 8.5 Hz, 1H), 6.52 (s, 1H), 3.85 (s, 3H), 2.27 (s, 3H); 13C NMR (101 MHz, CDCl3) δ:
190.4, 163.5, 148.7, 145.1, 144.8, 137.6, 135.8, 134.4, 131.4, 129.4, 129.4, 128.1, 125.1, 123.3, 123.1,
122.3, 110.6, 107.9, 56.1, 21.6; HRMS m/z calcd for C22H17BrN2NaO4S [M+Na]+ 506.9985 found 506.9977 (Δ
= 1.5 ppm).
N-(3-(4-bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-4-methoxybenzyl)-N-ethylethanamine (6a)
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Prepared according to method B. Purification by flash chromatography (5% methanol-dichloromethane)
afforded 6a as a yellow oil (920 mg, 72%); 1H NMR (400 MHz, CD3OD) δ: 8.15 (d, J = 5.3 Hz, 1H), 7.76 (d, J =
8.4 Hz, 2H), 7.65 (dd, J = 8.5, 2.4 Hz, 1H), 7.62 (d, J = 2.3 Hz, 1H), 7.47 (d, J = 5.3 Hz, 1H), 7.31 (d, J = 8.0 Hz,
2H), 7.21 (d, J = 8.5 Hz, 1H), 6.65 (s, 1H), 4.45-4.35 (m, 2H), 3.81 (s, 3H), 3.31-3.23 (m, 4H), 2.36 (s, 3H), 1.38
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(t, J = 7.3 Hz, 7H); C NMR (101 MHz, CD3OD) δ: 159.3, 148.4, 145.7, 144.3, 138.2, 135.5, 133.7, 133.6,
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129.1, 127.6, 124.7, 123.3, 122.3, 122.3, 120.8, 111.1, 107.6, 55.2, 54.9, 20.1; HRMS m/z calcd for
C26H28BrN3O3S [M+H]+ 541.1035 found 542.1093 (Δ = 2.7 ppm).
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1-({3-[4-Bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl]-4-methoxyphenyl}methyl)-N,N-dimethylamine (6b)
Prepared according to method B. Purification by flash chromatography (5% methanol-dichloromethane)
afforded 6b as a yellow oil (225 mg, 70%); 1H NMR (400 MHz, CD3OD) δ: 8.17 (d, J = 5.3 Hz, 1H), 7.78 (d, J =
8.4 Hz, 2H), 7.65 (dd, J = 8.5, 2.3 Hz, 1H), 7.61 (d, J = 2.3 Hz, 1H), 7.50 (d, J = 5.3 Hz, 1H), 7.33 (d, J = 8.0 Hz,
2H), 7.21 (d, J = 8.5 Hz, 1H), 6.66 (s, 1H), 4.27 (d, J = 14.4 Hz, 2H), 3.83 (s, 3H), 2.84 (s, 7H), 2.38 (s, 3H); 13C
NMR (101 MHz, CD3OD) δ: 156.2, 147.9, 142.1, 137.0, 131.0, 129.6, 129.5, 124.1, 122.6, 119.3, 118.8,
111.5, 98.8, 62.6, 55.0, 43.6; HRMS m/z calcd for C24H25BrN3O3S [M+H]+ 514.0795 found 514.0790 (Δ = 0.9
ppm).
1-({3-[4-Bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl]-4 methoxyphenyl}methyl)pyrrolidine (6c)
Prepared according to method B. Purification by flash chromatography (5% methanol-dichloromethane)
afforded 6c as a yellow oil (330 mg, 97%); 1H NMR (400 MHz, CD3OD) δ: 8.13 (d, J = 5.2 Hz, 1H), 7.77 (d, J =
8.4 Hz, 2H), 7.65-7.67 (m, 2H), 7.42 (d, J = 5.3 Hz, 1H), 7.28 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 9.1 Hz, 1H), 6.63
13
(s, 1H), 4.44-4.29 (m, 1H), 3.78 (s, 3H), 2.32 (s, 3H), 2.10-2.06 (p, 4H), 1.95 (s, 4H); C NMR (101 MHz,
CD3OD) δ: 176.8, 159.1, 148.4, 145.6, 144.3, 138.6, 135.6, 129.2, 127.6, 124.6, 123.3, 122.8, 122.3, 122.1,
110.9, 107.5, 56.9, 54.9, 52.8, 22.5, 21.5, 20.2; HRMS m/z calcd for C26H27BrN3O3S [M+H]+ 540.0951 found
540.0934 (Δ; = 3.2 ppm).
4-({3-[4-Bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl]-4-methoxyphenyl}methyl)morpholine (6d)
Prepared according to method B. Purification by flash chromatography (5% methanol-dichloromethane)
1
afforded 6d as a yellow oil (270 mg, 80%); H NMR (400 MHz, CD3OD) δ: 8.03 (m, 3H), 7.71 (d, J = 7.9 Hz,
1H), 7.52 (d, J = 8.4 Hz, 1H), 7.31 (d, J = 4.8 Hz, 1H), 7.23-7.18 (m, 3H), 6.96 (s, 1H), 4.36 (s, 2H), 4.04 (s, 3H),
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3.34 (t, J = 6.2 Hz, 4H), 2.08 (t, J = 6.2 Hz, 4H), 1.99 (s, 3H); C NMR (101 MHz, CD3OD) δ: 157.5, 147.9,
142.5, 140.4, 136.2, 131.8, 130.4, 128.5, 125.6, 124.3, 123.6, 122.4, 120.1, 118.9, 112.2, 99.4, 62.9, 57.2,
55.2, 53.2, 22.5; HRMS m/z calcd for C26H27BrN3O4S [M+H]+ 556.0900 found 556.0902 (Δ = -0.2 ppm).
N-(3-(4-bromo-1H-pyrrolo[2,3-b]pyridin-2-yl)-4-methoxybenzyl)-N-ethylethanamine (7a)
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Journal of Medicinal Chemistry
Prepared according to method C. Purification by flash chromatography (10% methanol-dichloromethane)
afforded 7a as a colourless oil (560 mg, 79%); 1H NMR (400 MHz, CD3OD) δ: 8.00 (d, J = 5.3 Hz, 1H), 7.83 (d,
J = 2.3 Hz, 1H), 7.36 (dd, J = 8.5, 2.2 Hz, 1H), 7.29 (d, J = 5.3 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 6.91 (s, 1H),
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4.00 (s, 3H), 3.75 (s, 2H), 3.31 (p, J = 1.6 Hz, 1H), 2.71 (q, J = 7.2 Hz, 4H), 1.15 (t, J = 7.2 Hz, 6H); C NMR
(101 MHz, CD3OD) δ: 156.3, 147.9, 142.1, 136.9, 131.2, 129.7, 124.11, 122.5, 119.4, 118.8, 111.6, 98.8,
56.1, 54.9, 9.4; HRMS m/z calcd for C19H22BrN3O [M+H]+ 387.0946 found 388.1008 (Δ = 2.7 ppm).
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1-[(3-{4-Bromo-1H-pyrrolo[2,3-b]pyridin-2-yl}-4-methoxyphenyl)methyl]dimethylamine (7b)
Prepared according to method C. Purification by flash chromatography (10% methanol-dichloromethane)
afforded 7b as a colourless oil (280 mg, 92%); 1H NMR (400 MHz, CD3OD) δ: 7.91 (d, J = 5.3 Hz, 1H), 7.70 (d,
J = 2.2 Hz, 1H), 7.23 (dd, J = 8.4, 2.2 Hz, 1H), 7.20 (d, J = 5.3 Hz, 1H), 7.04 (d, J = 8.5 Hz, 1H), 6.82 (s, 1H),
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3.91 (s, 3H), 3.44 (s, 2H), 2.21 (s, 6H); C NMR (101 MHz, CD3OD) δ: 156.3, 147.9, 142.1, 137.0, 131.0,
129.7, 129.5, 124.1, 122.6, 119.3, 118.8, 111.5, 98.8, 62.6, 54.9, 43.6; HRMS m/z calcd for C17H19BrN3OS
[M+H]+ 360.0706 found 360.0701 (Δ = 1.4 ppm).
1-[(3-{4-Bromo-1H-pyrrolo[2,3-b]pyridin-2-yl}-4-methoxyphenyl)methyl]pyrrolidine (7c)
Prepared according to method C. Purification by flash chromatography (10% methanol-dichloromethane)
afforded 7c as a yellow oil (165 mg, 77%); 1H NMR (400 MHz, CD3OD) δ: 7.88 (d, J = 5.2 Hz, 1H), 7.84 (d, J =
2.3 Hz, 1H), 7.36 (dd, J = 8.5, 2.3 Hz, 1H), 7.16 (dd, J = 8.5, 3.4 Hz, 1H), 7.04 (d, J = 8.6 Hz, 1H), 6.81 (s, 1H),
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4.06 (s, 2H), 3.89 (s, 3H), 3.04 (t, J = 6.8 Hz, 4H), 1.91 (p, J = 3.2 Hz, 4H); C NMR (101 MHz, CD3OD) δ:
157.0, 148.0, 142.4, 136.5, 131.3, 130.0, 126.4, 124.2, 122.4, 119.9, 118.9, 112.0, 99.1, 58.1, 55.1, 53.4,
22.6; HRMS m/z calcd for C19H21BrN3O [M+H]+ 386.0900 found 386.0903 (Δ = 0.8 ppm).
4-[(3-{4-Bromo-1H-pyrrolo[2,3-b]pyridin-2-yl}-4-methoxyphenyl)methyl]morpholine (7d)
Prepared according to method C. Purification by flash chromatography (10% methanol-dichloromethane)
afforded 7d as a yellow oil (269 mg, 80%); 1H NMR (400 MHz, DMSO-d6) δ: 12.18 (s, 1H), 8.08 (d, J = 5.1 Hz,
1H), 7.80 (d, J = 2.0 Hz, 1H), 7.65-7.60 (m, 2H), 7.58-7.55 (m, 1H), 7.33 (d, J = 5.1 Hz, 1H), 7.32 (d, J = 2.0 Hz,
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1H), 7.15 (d, J = 8.5 Hz, 1H), 6.89 (d, J = 2.2 Hz, 1H), 3.94 (s, 3H), 3.58 (t, 4H), 3.47 (s, 2H), 2.38 (t, 4H); C
NMR (101 MHz, DMSO-d6) δ: 156.2, 149.1, 143.5, 136.8, 132.5, 130.9, 129.7, 129.2, 123.4, 122.1, 119.4,
119.1, 112.4, 100.2, 66.7, 62.3, 56.2, 53.6; HRMS m/z calcd for C19H21BrN3O2 [M+H]+ 402.0812 found
402.0810 (Δ = 0.5 ppm).
4-(2-(5-((diethylamino)methyl)-2-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-isopropylbenzoic
acid, 1
Prepared according to method D. Purification by preparative HPLC 10-95% MeCN/H2O to give 1 in a (85
mg, 68%); 1H NMR (400 MHz, CD3OD) δ: 8.39 (d, J = 5.7 Hz, 1H), 8.01 (d, J = 2.2 Hz, 1H), 7.97 (d, J = 8.0 Hz,
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1H), 7.91 (d, J = 1.8 Hz, 1H), 7.74 (dd, J = 8.0, 1.8 Hz, 1H), 7.58 (dd, J = 8.6, 2.3 Hz, 1H), 7.52 (d, J = 5.8 Hz,
1H), 7.32 (d, J = 8.6 Hz, 1H), 7.31 (s, 1H), 4.37 (s, 2H), 4.07 (s, 3H), 3.94 (p, J = 6.9 Hz, 1H), 3.25 (dq, J = 14.2,
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7.1 Hz, 4H), 1.39-1.34 (m, 12H); C NMR (101 MHz, CD3OD) δ: 169.8, 157.9, 150.1, 145.6, 143.9, 140.0,
137.8, 136.8, 132.9, 131.8, 131.0, 130.3, 126.4, 125.6, 122.1, 121.8, 119.6, 114.9, 112.5, 100.3, 55.3, 55.2,
29.4, 23.0, 7.6; HRMS m/z calcd for C29H33N3O3 [M+H]+ 471.2522 found 472.2576 (Δ = 2.1 ppm).
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4-(2-{5-[(Dimethylamino)methyl]-2-methoxyphenyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-(propan-2-
yl)benzoic acid (8a)
Prepared according to method D. Purification by preparative HPLC 10-95% MeCN/H2O to give 8a in a (11
mg, 29%); 1H NMR (400 MHz, CD3OD) δ: 8.27 (d, J = 5.4 Hz, 1H), 7.89 (d, J = 2.3 Hz, 1H), 7.87 (d, J = 8.1 Hz,
1H), 7.79 (d, J = 1.7 Hz, 1H), 7.63 (dd, J = 8.0, 1.8 Hz, 1H), 7.44 (dd, J = 8.6, 2.3 Hz, 2H), 7.32 (d, J = 5.5 Hz,
1H), 7.23 (d, J = 8.6 Hz, 1H), 7.13 (s, 1H), 4.23 (s, 2H), 3.97 (s, 3H), 3.89-3.81 (m, 1H), 2.79 (s, 6H), 1.27 (d, J =
6.9 Hz, 6H); 13C NMR (101 MHz, CD3OD) δ: 169.9, 158.1, 150.2, 146.7, 142.6, 139.6, 138.4, 135.1, 133.2,
132.2, 131.1, 130.4, 126.5, 125.7, 122.3, 120.4, 119.2, 115.0, 112.5, 100.5, 60.1, 55.3, 41.4, 29.5, 23.0;
HRMS m/z calcd for C27H29N3O3 [M+H]+ 470.2438 found 470.2433 (Δ = 1.1 ppm).
4-(2-{2-Methoxy-5-[(pyrrolidin-1-yl)methyl]phenyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-(propan-2-yl)benzoic
acid (8b)
Prepared according to method D. Purification by preparative HPLC 10-95% MeCN/H2O to give 8b in a (27
1
mg, 37%); H NMR (400 MHz, CD3OD) δ: 8.27 (br s, 1H), 7.90 (s, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.79 (s, 1H),
7.62 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 8.6 Hz, 1H), 7.36 (s, 1H), 7.21-7.15 (m, 2H), 4.29 (s, 2H), 3.96 (s, 3H),
3.85-3.81 (m, 1H), 3.42 (br. s., 2H), 3.14 (br s, 2H), 2.08 (br. s., 2H), 1.93 (br. s., 2H), 1.26 (d, J = 6.8 Hz, 6H);
13C NMR (101 MHz, CD3OD) δ: 169.9, 157.9, 150.1, 144.5, 140.5, 137.4, 132.1, 131.5, 130.4, 130.3, 126.3,
125.5, 123.4, 119.9, 112.4, 100.0, 57.2, 55.2, 53.3, 29.4, 23.0, 22.4; HRMS m/z calcd for C29H32N3O3 [M+H]+
470.2438 found 470.2433 (Δ = 1.1 ppm).
4-(2-{2-Methoxy-5-[(morpholin-4-yl)methyl]phenyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-(propan-2-
yl)benzoic acid (8c)
Prepared according to method D. Purification by preparative HPLC 10-95% MeCN/H2O to give 8c in a (34
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mg, 60%); H NMR (400 MHz, DMSO-d6) δ: 8.43 (d, J = 5.8 Hz, 1H), 8.04 (d, J = 2.3 Hz, 1H), 8.00 (d, J = 8.0
Hz, 1H), 7.93 (d, J = 1.8 Hz, 1H), 7.78 (dd, J = 8.0, 1.8 Hz, 1H), 7.61 (dd, J = 8.6, 2.2 Hz, 1H), 7.56 (d, J = 5.7
Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.32 (s, 1H), 4.41 (s, 2H), 4.10 (s, 3H), 4.07 (s, 2H), 3.98-3.93 (m, 1H), 3.76
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(s, 2H), 3.44 (s, 2H), 3.26 (s, 2H), 1.39 (d, J = 6.9 Hz, 6H); C NMR (101 MHz DMSO-d6) δ: 170.3, 169.7,
149.6, 147.1, 136.0, 133.0, 133.0, 132.2, 132.1, 131.6, 131.3, 130.6, 128.3, 126.5, 125.9, 121.7, 120.6,
112.9, 100.5, 63.8, 59.4, 56.4, 51.2, 49.1, 29.4, 24.4, 24.3; HRMS m/z calcd for C29H32N3O4 [M+H]+ 486.2387
found 486.2384 (Δ = 0.8 ppm).
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Journal of Medicinal Chemistry
Ethyl
4-(2-(5-((diethylamino)methyl)-2-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-
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isopropylbenzoate (9)
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9
Purification by preparative HPLC 10-95% MeCN/H2O to give 9 in a (36 mg, 98%); 1H NMR (400 MHz, CD3OD)
δ: 8.39 (d, J = 5.6 Hz, 1H), 8.01 (d, J = 2.2 Hz, 1H), 7.92 (d, J = 8.2 Hz, 2H), 7.77-7.72 (m, 1H), 7.57 (dd, J = 8.6,
2.2 Hz, 1H), 7.49 (d, J = 5.7 Hz, 1H), 7.33 (d, J = 8.6 Hz, 1H), 7.28 (s, 1H), 4.42 (q, J = 7.1 Hz, 2H), 4.37 (s, 2H),
4.07 (s, 3H), 3.82 (p, J = 6.9 Hz, 1H), 3.25 (p, J = 7.4 Hz, 4H), 1.50-1.29 (m, 16H); 13C NMR (101 MHz, CD3OD)
δ: 168.1, 157.9, 149.9, 140.7, 138.8, 137.2, 132.6, 131.1, 130.9, 130.1, 126.4, 125.6, 122.0, 120.1, 114.9,
112.5, 99.9, 60.9, 55.3, 29.6, 22.9, 13.2, 7.7; HRMS m/z calcd for C31H37N3O3 [M+H]+ 499.2835 found
500.2913 (Δ = 4.1 ppm)
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3-[4-Bromo-1-(4-methylbenzenesulfonyl)-1H-pyrrolo[2,3-b]pyridin-2-yl]-4-methoxybenzonitrile (10)
Prepared according to method B. Purification by flash chromatography (50% ethyl acetate-PET ether)
afforded 10 as a yellow oil (586 mg, 87%; 1H NMR (400 MHz, DMSO-d6) δ: 8.23 (d, J = 5.2 Hz, 1H), 8.03-7.99
(m, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 5.2 Hz, 1H), 7.41-7.37 (m, 2H), 7.34-7.29 (m, 2H), 6.81 (s, 1H),
3.84 (s, 3H), 2.34 (s, 3H); 13C NMR (101 MHz, DMSO- d6) δ: 161.8, 148.3, 145.9, 145.7, 143.5, 137.1, 136.4,
135.5, 134.8, 130.2, 128.2, 125.0, 123.0, 119.3, 112.4, 109.5, 108.3, 103.1, 56.6, 21.6; HRMS m/z calcd for
C22H16BrN3NaO3S [M+Na]+ 503.9988 found 503.9973 (Δ = 3.0 ppm).
3-{4-Bromo-1H-pyrrolo[2,3-b]pyridin-2-yl}-4-methoxybenzonitrile (11)
Prepared according to method C. Purification by flash chromatography (10% methanol-dichloromethane)
afforded 11 as a yellow oil (327 mg, 59%); 1H NMR (400 MHz, DMSO-d6) δ: 12.24 (s, 1H), 8.29 (d, J = 2.1 Hz,
1H), 8.06 (d, J = 5.1 Hz, 1H), 7.81 (dd, J = 8.7, 2.1 Hz, 1H), 7.33 – 7.29 (m, 2H), 6.96 (s, 1H), 3.98 (s, 3H); 13C
NMR (101 MHz, DMSO-d6) δ: 207.0, 160.3, 149.1, 144.4, 134.3, 132.5, 124.1, 121.1, 119.4, 119.3, 113.7,
103.8, 101.8, 56.9, 31.2; HRMS m/z calcd for C15H11BrN3O [M+H]+ 328.0080 found 328.0076 (Δ = 1.1 ppm).
4-{2-[5-(aminomethyl)-2-methoxyphenyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}-2-(propan-2-yl)benzoic acid (12)
Prepared according to method D. The filtrate residue was dissolved in methanol (10 mL), CoCl2·6H2O (3
equiv) was then added before cooling to 0 °C. NaBH4 (10 equiv) was slowly added to the reaction and the
mixture was allowed to stir at room temperature for a further 1 hour before filtration through celite and
purification by preparative HPLC 10-95% MeCN/H2O to give 13 (26 mg, 60%); 1H NMR (400 MHz, DMSO-d6)
δ: 12.01 (s, 1H), 8.30 (d, J = 5.0 Hz, 1H), 8.17 (br. s., 2H), 7.94 (d, J = 2.3 Hz, 1H), 7.82- 7.80 (m, 2H), 7.62
(dd, J = 8.0, 1.8 Hz, 1H), 7.41 (dd, J = 8.6, 2.2 Hz, 1H), 7.24 (d, J = 5.0 Hz, 1H), 7.18 (d, J = 8.6 Hz, 1H), 7.09 (s,
13
1H), 3.98 (q, J = 5.6 Hz, 2H), 3.89 (s, 3H), 3.83- 3.76 (m, 1H), 1.25 (d, J = 6.9 Hz, 6H).; C NMR (101 MHz,
DMSO-d6) δ: 169.7, 157.0, 149.6, 149.2, 142.9, 141.4, 140.4, 136.6, 131.4, 130.6, 130.6, 129.7, 126.7,
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126.6, 125.9, 120.1, 119.0, 115.2, 112.7, 100.0, 56.3, 42.4, 29.4, 24.3; HRMS m/z calcd for C25H26N3O3 [M +
H]+ 416.1969 found 416.1968 (Δ = 0.2 ppm).
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4-Bromo-2-(2-methoxyphenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (13)
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Prepared according to method A. Purification by flash chromatography (50% ethyl acetate-PET ether)
afforded 14 as a colourless oil (960 mg, 32%); 1H NMR (400 MHz, CDCl3) δ: 8.14 (d, J = 5.3 Hz, 1H), 7.78 (d, J
= 8.4 Hz, 2H), 7.41 (td, J = 8.2, 1.7 Hz, 1H), 7.29-7.24 (m, 2H), 7.12 (d, J = 8.1 Hz, 2H), 6.99 (t, J = 7.5 Hz, 1H),
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6.91 (d, J = 8.2 Hz, 1H), 6.45 (s, 1H), 3.73 (s, 3H), 2.27 (s, 3H); C NMR (101 MHz, CDCl3) δ: 158.3, 148.7,
144.8, 144.4, 139.4, 136.1, 131.1, 130.9, 129.2, 128.2, 124.8, 123.5, 122.1, 121.8, 120.0, 110.4, 107.3, 55.5,
21.6; HRMS m/z calcd for C21H17BrN2NaO3S [M+Na]+ 479.0035 found 479.0035 (Δ = 0.0 ppm).
4-Bromo-2-(2-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (14)
Prepared according to method C. Purification by flash chromatography (10% methanol-dichloromethane)
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afforded 15 as a colourless oil (300 mg 80%); H NMR (400 MHz, CDCl3) δ: 10.38 (s, 1H), 8.09 (d, J = 5.2 Hz,
1H), 7.92 (dd, J = 7.8, 1.6 Hz, 1H), 7.41-7.34 (m, 1H), 7.30-7.25 (m, 2H), 7.14-7.07 (m, 2H), 6.89 (s, 1H), 4.04
13
(s, 3H); C NMR (101 MHz, CDCl3) δ: 156.1, 148.1, 143.0, 137.0, 129.9, 128.4, 124.3, 122.2, 121.6, 119.4,
119.3, 112.0, 97.8, 55.9; HRMS m/z calcd for C14H12BrN2O [M+H]+ 303.0128 found 303.0122 (Δ = 1.8 ppm).
4-[2-(2-Methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl]-2-(propan-2-yl)benzoic acid (15)
Prepared according to method D. Purification by preparative HPLC 10-95% MeCN/H2O to give 16 in a (30
1
mg, 59%); H NMR (400 MHz, DMSO-d6) δ: 12.05 (s, 1H), 8.32 (d, J = 5.0 Hz, 1H), 7.92 (dd, J = 7.8, 1.7 Hz,
1H), 7.90 (d, J = 1.8 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.68 (dd, J = 8.1, 1.8 Hz, 1H), 7.38 (ddd, J = 8.7, 7.4, 1.7
Hz, 1H), 7.27 (d, J = 5.0 Hz, 1H), 7.20-7.18 (m, 2H), 7.08 (td, J = 7.6, 1.0 Hz, 1H), 3.92 (s, 3H), 3.90-3.83 (m,
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1H), 1.32 (d, J = 6.9 Hz, 6H); C NMR (101 MHz, DMSO-d6) δ: 169.7, 157.1, 149.9, 149.6, 141.7, 136.5,
131.1, 130.7, 129.9, 126.5, 125.9, 121.2, 100.3, 45.4, 29.4; HRMS m/z calcd for C24H23N2O3 [M+H]+
387.17034 found 387.1710 (Δ = -1.7 ppm).
3-(4-Bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-4-(methoxymethoxy)benzaldehyde (16)
Prepared according to method A. Purification by flash chromatography (50% ethyl acetate-PET ether)
afforded 17 as a colourless oil (850 mg, 77%); 1H NMR (400 MHz, CDCl3) δ: 9.92 (s, 1H), 8.16 (d, J = 5.3 Hz,
1H), 7.92 (dd, J = 8.5, 2.1 Hz, 1H), 7.84 (d, J = 2.1 Hz, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.5 Hz, 1H),
7.29 (d, J = 5.3 Hz, 1H), 7.12 (d, J = 8.1 Hz, 3H), 6.53 (s, 1H), 5.26-5.22 (m, 2H), 3.46 (s, 3H), 2.27 (s, 3H); 13C
NMR (101 MHz, CDCl3) δ: 190.6, 161.7, 148.6, 145.2, 144.9, 137.7, 135.5, 134.4, 131.0, 130.0, 129.4, 128.0,
125.1, 123.6, 122.3, 113.9, 108.0, 95.1, 56.9, 21.6; HRMS m/z calcd for C23H19BrN2NaO5S [M+Na]+ 537.0090
found 537.0098 (Δ = -1.7 ppm).
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Journal of Medicinal Chemistry
({3-[4-bromo-1-(4-methylbenzenesulfonyl)-1H-pyrrolo[2,3-b]pyridin-2-yl]-4-
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(methoxymethoxy)phenyl}methyl)diethylamine (17)
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Prepared according to method B. Purification by flash chromatography (5% methanol-dichloromethane)
afforded 18 as a colourless oil (672 mg, 87%); 1H NMR (400 MHz, CD3OD) δ: 8.16 (d, J = 5.3 Hz, 1H), 7.77 (d,
J = 8.4 Hz, 2H), 7.59 (d, J = 7.9 Hz, 2H), 7.50 (d, J = 5.3 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.31 (d, J = 8.2 Hz,
2H), 6.68 (s, 1H), 5.18 (br. s., 1H), 5.28 (br. s., 1H), 4.20 (d, J = 5.4 Hz, 2H), 3.43 (s, 3H), 3.10 (q, J = 7.1 Hz,
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4H), 2.37 (s, 3H), 1.33 (t, J = 7.2 Hz, 6H); C NMR (101 MHz, CD3OD) δ: 156.7, 148.4, 145.7, 144.3, 138.7,
135.5, 133.1, 132.9, 129.2, 127.6, 124.7, 123.3, 122.8, 122.4, 114.1, 107.5, 94.7, 55.4, 55.4, 46.2, 20.1, 8.4;
HRMS m/z calcd for C27H31BrN3O4S [M+H]+ 572.1213 found 572.1208 (Δ = 0.9 ppm).
[(3-{4-Bromo-1H-pyrrolo[2,3-b]pyridin-2-yl}-4-(methoxymethoxy)phenyl)methyl]diethylamine (18)
Prepared according to method C. Purification by flash chromatography (10% methanol-dichloromethane)
afforded 19 as a yellow oil (420 mg, 69%); 1H NMR (400 MHz, CD3OD) δ: 8.03 (d, J = 5.2 Hz, 1H), 7.84 (d, J =
2.1 Hz, 1H), 7.34 (dd, J = 8.5, 2.1 Hz, 1H), 7.31-7.28 (m, 2H), 6.96 (s, 1H), 5.39 (s, 2H), 4.88 (s, 3H), 3.73 (s,
2H), 3.50 (s, 3H), 2.70 (q, J = 7.2 Hz, 4H), 1.15 (t, J = 7.2 Hz, 6H); 13C NMR (101 MHz, CD3OD) δ: 153.9, 148.0,
142.3, 136.7, 130.9, 130.5, 129.7, 124.2, 122.6, 120.4, 118.9, 115.2, 99.3, 94.7, 56.1, 55.4, 46.1, 9.5; HRMS
m/z calcd for C21H25BrN2O2 [M+H]+ 418.1113 found 418.1112 (Δ = -0.2 ppm).
4-[2-(2-Methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl]-2-(propan-2-yl)benzoic acid (19)
Prepared according to method D. The filter residue was then stirred in a mixture of acetonitrile/HCl (3:1)
for 30 mins before filtering through celite and purification by preparative HPLC 10-95% MeCN/H2O to give
21 in a (72 mg, 70%); 1H NMR (400 MHz, CD3OD) δ: 8.30 (d, J = 5.3 Hz, 1H), 7.92 (d, J = 2.2 Hz, 1H), 7.89 (d, J
= 8.1 Hz, 1H), 7.84 (d, J = 1.6 Hz, 1H), 7.68 (dd, J = 8.1, 1.7 Hz, 1H), 7.46 (d, J = 5.7 Hz, 1H), 7.3-7.31 (m, 2H),
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7.04 (d, J = 8.4 Hz, 1H), 4.24 (s, 2H), 3.85 (sept, J = 6.9 Hz, 1H), 3.19-3.12 (m, 4H), 1.30-1.27 (m, 12H); C
NMR (101 MHz, CD3OD) δ: 169.8, 156.5, 150.2, 139.7, 138.9, 135.4, 132.7, 132.1, 130.8, 130.4, 126.6,
125.7, 121.0, 117.4, 117.1, 115.0, 99.7, 55.4, 46.4, 29.5, 23.0; HRMS m/z calcd for C28H32N3O3 [M + H]+
458.2438 found 458.2430 (Δ = 1.8 ppm).
3-(4-bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)benzaldehyde (20)
Prepared according to method A. Purification by flash chromatography (50% ethyl acetate-PET ether)
afforded 22 as a colourless oil (500 mg, 76%); 1H NMR (400 MHz, CDCl3) δ: 10.04 (s, 1H), 8.23 (d, J = 5.2 Hz,
1H), 7.96 (t, J = 1.8 Hz, 1H), 7.93 (d, J = 7.7 Hz, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.59 (t, J
= 7.7 Hz, 1H), 7.33 (d, J = 5.3 Hz, 1H), 7.14 (d, J = 8.3 Hz, 2H), 6.56 (s, 1H), 2.29 (s, 3H); 13C NMR (101 MHz,
CDCl3) δ: 191.8, 149.5, 145.5, 145.2, 141.0, 136.0, 135.9, 135.2, 133.3, 130.6, 130.3, 129.6, 128.5, 127.9,
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Journal of Medicinal Chemistry
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125.3, 123.6, 122.9, 109.1, 21.7; HRMS m/z calcd for C21H15BrN2NaO3S [M+Na]+ 476.9879 found 476.9884
(Δ = -1.0 ppm).
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({3-[4-Bromo-1-(4-methylbenzenesulfonyl)-1H-pyrrolo[2,3-b]pyridin-2-yl]-4-
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(methoxymethoxy)phenyl}methyl)diethylamine (21)
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Prepared according to method B. Purification by flash chromatography (5% methanol-dichloromethane)
afforded 23 as a colourless oil (154 mg, 83%); 1H NMR (400 MHz, CD3OD) δ: 8.22 (d, J = 5.3 Hz, 1H), 7.87 (d,
J = 1.9 Hz, 1H), 7.75-7.70 (m, 2H), 7.68 (d, J = 7.4 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 5.3 Hz, 1H),
7.29 (d, J = 8.4 Hz, 2H), 6.79 (s, 1H), 4.50 (s, 2H), 3.35-3.30 (m, 4H), 2.35 (s, 3H), 1.42 (t, J = 7.3 Hz, 6H); 13C
NMR (101 MHz, CD3OD) δ: 156.7, 148.4, 145.7, 144.3, 138.7, 135.5, 133.1, 132.9, 129.2, 127.6, 124.7,
123.3, 122.8, 122.4, 114.1, 107.5, 94.7, 55.4, 55.4, 46.2, 20.1, 8.4; HRMS m/z calcd for C25H27BrN3O2S
[M+H]+ 512.1002 found 512.1013 (Δ = -2.3 ppm).
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[(3-{4-Bromo-1H-pyrrolo[2,3-b]pyridin-2-yl}phenyl)methyl]diethylamine (22)
Prepared according to method C. Purification by flash chromatography (10% methanol-dichloromethane)
afforded 24 as a colourless oil (95 mg, 89%); 1H NMR (400 MHz, CD3OD) δ: 7.92 (d, J = 5.3 Hz, 1H), 7.78 (s,
1H), 7.71 (d, J = 7.8 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H), 7.20 (d, J = 5.3 Hz, 1H), 6.76 (s,
1H), 3.70 (s, 2H), 2.62 (q, J = 7.2 Hz, 4H), 1.06 (t, J = 7.1 Hz, 6H); 13C NMR (101 MHz, CD3OD) δ: 148.9, 142.4,
139.7, 131.5, 129.7, 128.9, 126.8, 124.7, 124.2, 123.2, 118.9, 96.9, 56.8, 46.3, 9.6; HRMS m/z calcd for
C18H21BrN3 [M+H]+ 358.0913 found 358.0907 (Δ = 1.7 ppm).
4-(2-{3-[(Diethylamino)methyl]phenyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-(propan-2-yl)benzoic acid (23)
Prepared according to method D. Purification by preparative HPLC 10-95% MeCN/H2O to give 25 in a (30
mg, 70%); 1H NMR (400 MHz, CD3OD) δ: 8.25 (s, 1H), 7.94 (s, 1H), 7.91 (d, J = 6.9 Hz, 1H), 7.85 (d, J = 8.0 Hz,
1H), 7.73 (s, 1H), 7.62 (s, 2H), 7.53 (s, 2H), 7.45 (d, J = 7.7 Hz, 1H), 7.28 (d, J = 5.2 Hz, 1H), 7.04 (s, 1H), 4.32
13
(s, 2H), 3.83 (sept, J = 13.9, 6.9 Hz, 1H), 3.16 (br. s., 4H), 1.27 (q, J = 7.2 Hz, 12H); C NMR (101 MHz,
CD3OD) δ: 170.0, 150.1, 132.3, 130.8, 130.6, 130.3, 129.9, 128.1, 127.0, 126.2, 125.5, 115.1, 111.9, 97.8,
55.7, 46.7, 29.4, 23.0, 7.6; HRMS m/z calcd for C28H32N3O2 [M + H]+ 442.2489 found 442.2476 (Δ = 3.0 ppm).
3-(4-bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-2-methoxybenzaldehyde (24)
Prepared according to method A. Purification by flash chromatography (50% ethyl acetate-PET ether)
afforded 26 as a colourless oil (990 mg, 72%); 1H NMR (400 MHz, CDCl3) δ: 10.49 (s, 1H), 8.28 (d, J = 5.3 Hz,
1H), 8.07-7.90 (m, 3H), 7.59 (dd, J = 7.4, 1.8 Hz, 1H), 7.41 (d, J = 5.3 Hz, 1H), 7.37-7.30 (m, 1H), 7.25 (d, J =
6.7 Hz, 2H), 6.66 (s, 1H), 3.75 (s, 3H), 2.38 (s, 3H); 13C NMR (101 MHz, CDCl3) δ: 189.6, 162.0, 148.7, 145.4,
145.0, 137.1, 137.1, 135.6, 130.2, 129.4, 129.0, 128.6, 127.3, 125.2, 123.7, 123.1, 122.5, 108.1, 63.6, 21.7;
HRMS m/z calcd for C22H17BrN2O4SNa [M+Na]+ 507.9990 found 508.0068 (Δ = 0.2 ppm).
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Journal of Medicinal Chemistry
N-(3-(4-bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-2-methoxybenzyl)-N-ethylethanamine (25)
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Prepared according to method B. Purification by flash chromatography (50% ethyl acetate-PET ether)
afforded 27 as a yellow oil (765 mg, 81%; 1H NMR (400 MHz, CD3OD) δ: 8.18 (d, J = 5.3 Hz, 1H), 7.73 (d, J =
8.4 Hz, 2H), 7.69 (dd, J = 7.7, 1.7 Hz, 1H), 7.63 (dd, J = 7.6, 1.7 Hz, 1H), 7.51 (d, J = 5.3 Hz, 1H), 7.41 (t, J = 7.7
Hz, 1H), 7.33-7.27 (m, 2H), 6.85 (s, 1H), 4.56 (d, J = 13.3 Hz, 1H), 4.28 (d, J = 13.3 Hz, 1H), 3.58 (s, 3H), 2.35
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(s, 3H), 1.39 (t, J = 7.3 Hz, 6H); C NMR (101 MHz, CD3OD) δ: 178.1, 158.6, 148.6, 145.9, 144.8, 138.4,
135.2, 133.7, 132.8, 129.2, 127.8, 126.4, 124.9, 124.2, 124.1, 123.2, 122.6, 108.0, 60.3, 50.2, 22.4, 20.2, 8.1;
HRMS m/z calcd for C26H28BrN3O3S [M+H]+ 541.1035 found 542.1095 (Δ = 3.6 ppm).
N-(3-(4-bromo-1H-pyrrolo[2,3-b]pyridin-2-yl)-2-methoxybenzyl)-N-ethylethanamine (26)
Prepared according to method C. Purification by flash chromatography (5% methanol-dichloromethane)
afforded 28 as a yellow oil (630 mg, 85%); 1H NMR (400 MHz, CD3OD) δ: 8.12 (d, J = 5.4 Hz, 1H), 7.92 (dd, J
= 7.8, 1.7 Hz, 1H), 7.59 (dd, J = 7.6, 1.7 Hz, 1H), 7.46-7.37 (m, 2H), 7.03 (s, 1H), 4.43 (s, 2H), 3.69 (s, 3H),
3.32-3.28 (m, 4H), 1.43 (t, J = 7.3 Hz, 6H); 13C NMR (101 MHz, CD3OD) δ: 157.0, 147.6, 142.1, 135.7, 132.8,
131.9, 125.6, 125.4, 125.3, 124.5, 123.2, 119.2, 100.5, 60.6, 50.9, 7.7; HRMS m/z calcd for C19H22BrN3O
[M+H]+ 387.0946 found 388.1008 (Δ = 2.7 ppm).
4-(2-(3-((diethylamino)methyl)-2-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-isopropylbenzoic acid
(27)
Prepared according to method D. Purification by preparative HPLC 10-95% MeCN/H2O to give 29 in a (30
mg, 74%); 1H NMR (400 MHz, CD3OD) δ: 8.41 (s, 1H), 7.98-7.92 (m, 2H), 7.89 (d, J = 1.7 Hz, 1H), 7.73 (dd, J =
8.1, 1.7 Hz, 1H), 7.58 (dd, J = 7.6, 1.6 Hz, 1H), 7.47-7.43 (m, 1H), 7.41 (d, J = 7.7 Hz, 1H), 7.23 (s, 1H), 4.43 (s,
2H), 3.94 (p, J = 6.8 Hz, 1H), 3.72 (s, 3H), 3.31-3.25 (m, 5H), 1.42 (t, J = 7.3 Hz, 6H), 1.36 (d, J = 6.9 Hz, 6H);
13C NMR (101 MHz, CD3OD) δ: 169.9, 157.1, 150.1, 140.6, 136.3, 132.8, 131.9, 131.4, 130.3, 126.3, 125.5,
125.4, 124.6, 100.5, 60.7, 50.8, 29.4, 23.0, 7.7; HRMS m/z calcd for C29H33N3O3 [M+H]+ 471.2522 found
472.2579 (Δ = 3.4 ppm).
4-(2-(5-((diethylamino)methyl)-2-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-methylbenzoic acid
(28)
Prepared according to method D. Purification by preparative HPLC 10-95% MeCN/H2O to give 30 in a (50
mg, 75%); 1H NMR (101 MHz, CD3OD) δ: 8.27 (d, J = 5.6 Hz, 1H), 8.04 (d, J = 8.7 Hz, 1H), 7.94 (d, J = 2.3 Hz,
1H), 7.70 – 7.61 (m, 2H), 7.46 (dd, J = 8.6, 2.3 Hz, 1H), 7.36 (d, J = 5.6 Hz, 1H), 7.23 (d, J = 8.6 Hz, 1H), 7.16
(s, 1H), 4.27 (s, 2H), 3.99 (s, 3H), 3.15 (p, J = 7.2 Hz, 4H), 2.63 (s, 3H), 1.27 (t, J = 7.3 Hz, 6H); 13C NMR (400
MHz, CD3OD) δ: 168.9, 157.8, 140.9, 140.3, 136.5, 133.0, 131.5, 131.3, 131.2, 125.9, 122.2, 119.6, 115.1,
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112.5, 99.7, 55.3, 20.6, 7.6; HRMS m/z calcd for C27H29N3O3 [M+H]+ 443.2209 found 444.2214 (Δ = 1.4
1
2
3
ppm).
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5
4-(2-(5-((diethylamino)methyl)-2-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)benzoic acid (29)
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8
9
Prepared according to method D. Purification by preparative HPLC 10-95% MeCN/H2O to give 31 in a (100
mg, 85%); 1H NMR (400 MHz, CD3OD) δ: 8.28 (d, J = 5.5 Hz, 1H), 8.16 (d, J = 8.5 Hz, 2H), 7.95 (d, J = 2.3 Hz,
1H), 7.87 (d, J = 8.6 Hz, 2H), 7.46 (dd, J = 8.6, 2.3 Hz, 1H), 7.38 (d, J = 5.6 Hz, 1H), 7.23 (d, J = 8.6 Hz, 1H),
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7.17 (s, 1H), 4.27 (s, 2H), 3.99 (s, 3H), 3.15 (p, J = 7.3 Hz, 4H), 1.27 (t, J = 7.3 Hz, 6H); C NMR (101 MHz,
CD3OD) δ: 168.0, 157.6, 148.6, 143.1, 142.4, 140.9, 136.3, 132.0, 130.9, 130.6, 129.9, 129.3, 128.3, 122.1,
120.8, 114.9, 112.4, 98.6, 55.5, 55.2, 7.6; HRMS m/z calcd for C26H27N3O3 [M+H]+ 429.2052 found 430.2056
(Δ = 1.1 ppm).
N-ethyl-N-(3-(4-(3-isopropyl-4-(1H-tetrazol-5-yl)phenyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-4-
methoxybenzyl)ethanamine (30)
Prepared according to method D. Purification by preparative HPLC 10-95% MeCN/H2O to give 32 in a (27
mg, 62%); 1H NMR (400 MHz, CD3OD) δ: 8.39 (d, J = 5.8 Hz, 1H), 8.01 (d, J = 2.3 Hz, 1H), 7.97 (d, J = 8.0 Hz,
1H), 7.91 (d, J = 1.8 Hz, 1H), 7.74 (dd, J = 8.0, 1.9 Hz, 1H), 7.58 (dd, J = 8.6, 2.3 Hz, 1H), 7.52 (d, J = 5.8 Hz,
1H), 7.32 (d, J = 8.7 Hz, 1H), 7.31 (s, 1H), 4.37 (s, 2H), 4.07 (s, 3H), 3.94 (p, J = 6.9 Hz, 1H), 3.25 (dd, J = 12.2,
13
7.0 Hz, 4H), 1.42-1.33 (m, 12H); C NMR (101 MHz, CD3OD) δ: 169.8, 157.9, 150.1, 145.6, 143.9, 140.0,
137.8, 136.8, 132.9, 131.8, 131.0, 130.3, 126.4, 125.6, 122.1, 121.8, 119.6, 114.9, 112.5, 100.3, 55.3, 55.2,
29.4, 23.0, 7.6; HRMS m/z calcd for C29H33N7O [M+H]+ 495.2747 found 496.2829 (Δ = 0.6 ppm).
N-ethyl-N-(3-(4-(3-isopropylphenyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-4-methoxybenzyl)ethanamine (31)
Prepared according to method D. Purification by preparative HPLC 10-95% MeCN/H2O to give 33 in a (67
mg, 82%); 1H NMR (101 MHz, CD3OD) δ: 8.37 (d, J = 5.9 Hz, 1H), 8.03 (d, J = 2.2 Hz, 1H), 7.75 -7.68 (m, 2H),
7.61-7.54 (m, 2H), 7.50 (dd, J = 16.9, 6.8 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 4.37 (s, 2H), 4.08 (s, 3H), 3.25 (p, J
13
= 7.5 Hz, 4H), 3.08 (p, J = 6.9 Hz, 1H), 1.43-1.31 (m, 12H); C NMR (400 MHz, CD3OD) δ: 157.9, 149.9,
137.2, 132.8, 131.1, 128.9, 127.7, 126.5, 126.1, 122.1, 119.8, 114.9, 112.5, 100.3, 55.3, 34.1, 23.1, 7.6;
HRMS m/z calcd for C28H33N3O [M+H]+ 427.2624 found 428.2624 (Δ = 3.8 ppm).
5-(2-(5-((diethylamino)methyl)-2-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-isopropylbenzoic acid
(32)
Prepared according to method D. Purification by preparative HPLC 10-95% MeCN/H2O to give 34 in a (55
mg, 78%); 1H NMR (400 MHz, CD3OD) δ: 8.41 (d, J = 5.7 Hz, 1H), 8.26 (d, J = 2.0 Hz, 1H), 7.99 (ddd, J = 9.3,
8.0, 1.9 Hz, 2H), 7.73 (d, J = 8.2 Hz, 1H), 7.60 (dd, J = 7.6, 1.6 Hz, 1H), 7.53 (d, J = 5.7 Hz, 1H), 7.42 (t, J = 7.7
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Hz, 1H), 7.36 (s, 1H), 4.43 (s, 2H), 3.92 (p, J = 6.9 Hz, 1H), 3.73 (s, 3H), 3.31-3.25 (m, 4H), 1.42 (t, J = 7.3 Hz,
6H), 1.34 (d, J = 6.9 Hz, 6H); 13C NMR (101 MHz, CD3OD) δ: 169.8, 157.0, 151.0, 145.4, 144.8, 137.8, 136.9,
134.4, 133.1, 131.9, 131.6, 131.3, 129.8, 127.2, 125.4, 124.9, 124.7, 121.6, 114.9, 100.8, 60.7, 50.9, 29.4,
22.8, 7.7; HRMS m/z calcd for C29H33N3O3 [M+H]+ 471.2522 found 472.2579 (Δ = 3.4 ppm).
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Assay
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P. falciparum culture and synchronisation
P. falciparum cultures were maintained in RPMI-1640 media (Invitrogen) supplemented with 0.2% sodium
bicarbonate, 0.5% Albumax II, 2.0 mM L-glutamine (Sigma) and 10 mg/L gentamycin. For continuous
culture, the parasites were kept at 4% haematocrit in human erythrocytes from 0+ blood donors and
between 0.5 - 3% parasitaemia maintained in an incubator at 37 °C, 5% carbon dioxide (CO2), 5% oxygen
(O2) and 90% nitrogen (N2). To obtain highly synchronous ring stage parasites for the drug assays, cultures
were double synchronised using Percoll and Sorbitol synchronisation as previously described.35-36 First,
highly segmented schizonts were enriched by centrifugation on a 70% Percoll (GE Healthcare) cushion
gradient. The Schizont pellet was collected and washed before fresh erythrocytes were added to a final
haematocrit of 4%. The schizonts were incubated for about 1-2 hours shaking continuously to allow egress
and re-invasion of new erythrocytes. Residual schizonts were then removed by treating the pellet with
sorbitol to generate highly synchronous 1-2 hours old ring-stage parasites.
Determining the IC50 of compound inhibitors and drugs-ex vivo
To determine the IC50 of the molecules in parasites (P. falciparum 3D7) ex vivo, the molecules were diluted
1 in 3 from a starting concentration of 100 μM for 12 dilution points. Fifty microliters of freshly diluted
drugs, at twice the required final concentrations were aliquoted into black 96-well plates. To the drug
plates, 50 μl of parasites prepared at 8% haematocrit at a parasitaemia (0.3 - 0.5%) were added and mixed
by pipetting up and down several times giving a final culture volume of 100 μl at the required drug
concentration (top concentration of 100 μM) and 4% haematocrit. To the ‘no drug’ control, growth media
was added and uninfected erythrocytes were included on the plate as blank. The outer wells were filled
with media to reduce evaporation from the experimental wells and the plates incubated for 50 hours (± 2
hours) to allow the parasites sufficient time to re-invade before they are collected and frozen. To quantify
growth inhibition, the plates were thawed at room temperature for at least 1 hour and 100 μl of lysis
buffer (20 mM Tris-HCl; 5 mM EDTA; 0.004% saponin and triton X-100) in PBS containing Sybr Green I (1μl
in 5 ml) was added to each well and mixed by pipetting up and down several times and incubated for 1
hour in the dark shaking. Using a Fluroskan/ClarioStar plate reader at excitation of 485 nm and emission of
538 nm, plate absorbances were acquired. The data was normalised against the controls and graphs were
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generated using Graph Pad Prism 8 to determine the IC50 values using the non-linear regression log
(inhibitor) versus response (three parameter) curve.
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Time Resolve Florescence Energy Transfer (TR-FRET) to determine the IC50 of the inhibitors with full-
length PfCLK3 recombinant protein
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9
The TR-FRET assays, a high-throughput inhibition assay, as described previously13 was used to determine
the potency of the small molecules generated against full-length PfCLK3 recombinant protein in a kinase
buffer (containing 50 mM HEPES, 10 mM MgCl2, 2 mM DTT, 0.01% Tween 20, and 1 mM EGTA), ULight-
labeled peptide substrate (MBP peptide (sequence: CFFKNIVTPRTPPPSQGK). First, in a 10L reaction
volume, 5l of twice the required enzyme concentration (50 nM) and 2.5l of 4 times the required
substrate concentration mix containing cold ATP, and the serially diluted drugs were mixed in a black 384-
plate well plate and incubated at 37 °C for 1 hour. The reaction was stopped after incubation by adding the
stopping/detection solution (containing 30 mM EDTA in 1X Lance detection buffer and 3 nM Europium-
labeled anti-phospho specific antibody) and incubated for another hour at RT before phosphorylation
signals were measured using the ClarioStar.
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For each test compound, percent inhibition (response) which was calculated using the formula:
(퐾푖푛푎푠푒 푎푐푡푖푣푦 –푏푙푎푛푘)
Percentage inhibition (response) =
∗ 100
[
]
(푀푎푥푖푚푢푚 푘푖푛푎푠푒 푎푐푡푖푣푖푡푦 ― 푏푙푎푛푘)
was plotted against log molar concentration of compound to calculate the IC50 (potency) of each inhibitor
molecule and plotted using GraphPad prism software. All experiments were done in triplicates and the
data presented is the S.E.M of three independent experiments run in triplicates.
PfCLK3 phosphorylation of substrate results in the Europium-labeled anti-phospho specific antibody
recognizing the phosphorylated site on the substrate. The Europium donor fluorophore is excited at 320
nm or 340 nm and energy is transferred to the ULight acceptor dye on the substrate, which finally results
in the emission of light at 665 nm. The level of ULight peptide phosphorylation correlates with the intensity
of the emission. For normalization, a no kinase and a no inhibitor reaction wells were included and all
experiments conducted in triplicates. Drug dilutions, protein concentrations and incubation times were the
same for easy comparison of results.
Microsomal stability
Compounds were incubated at 37 °C at a concentration of 1 µM with CD1 mouse liver microsomes
(GIBCO™, Thermo Fisher Scientific) in a suspension of 50 mM potassium phosphate buffer (pH 7.4) with a
final protein concentration of 0.5 mg/mL. The reaction was started by the addition of excess NADPH and
then quenched at several time points starting from time zero then at 3, 6, 9, 15 and 30 min addition of
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Journal of Medicinal Chemistry
acetonitrile to an aliquot of the sample. Internal standard was added to each sample before centrifugation
to remove any precipitates before monitoring loss of parent compound by HPLC analysis using Shimadzu
LC-20A (Shimadzu, UK). Prism (Graphpad, USA) was used to fit an exponential decay for substrate
depletion and subsequently rate constant (k) from the peak area of the parent compound to internal
standard at each time point. Rate of intrinsic clearance (CLint) was then calculated according to the
methods of Obach using the equation:37
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CLint (mL/min/g liver) = k x v x microsomal protein yield
Where V is the incubation volume (volume/mg protein) and microsomal protein yield is assumed to be
52.5 mg protein/ g liver. With verapamil used as a positive control.
Distribution coefficient (LogD7.4)
Distribution coefficient (LogD7.4) was estimated by correlation of the compounds chromatographic
retention properties to those of 10 standard compounds with known distribution coefficients ranging from
-0.5 to 5.5 at pH 7.4. A fast gradient HPLC methodology was used based on the method developed by Valkó
et al.38
Kinase Screen Method
Each enzyme is assayed in its linear range with 0.3 µM substrate in 50 mM Tris pH 7. 5, 0.1 mM EGTA, 0.01
mM DTT, relevant Mg/ATP (5, 20 or 50 µM) for 30 min at room temp. Assays are stopped by the addition
of 3% orthophosphoric acid and harvester onto p81 filter paper using the Perkin elmer unifilter harvester.
Once dried, they are read on a Perkin elmer Topcount NXT scintillation counter for 30 sec/well.39
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXX.
1
13
Experimental procedures and characterization data for all compounds, copies of H, C, NMR spectra for
all compounds, LogD7.4 (Distribution co-efficient), metabolic stability and analytical HPLC traces for final
compounds. P. falciparum culture, synchronisation and TR-FRET assay data. Molecular formula strings (CSV)
file
AUTHOR INFORMATION
Corresponding Authors:
Andrew G. Jamieson: School of Chemistry, University of Glasgow, Joseph Black Building, University Avenue,
Glasgow G12 8QQ, U.K.; orcid.org/ 0000-0003-1726-7353; E mail: andrew.jamieson.2@glasgow.ac.uk
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