Optimization of Physicochemical Properties for 4-Anilinoquinoline Inhibitors of Plasmodium falciparum Proliferation

Article abstract of DOI:10.1021/acsinfecdis.7b00212

We recently reported the medicinal chemistry reoptimization of a known human tyrosine kinase inhibitor, lapatinib, against a variety of parasites responsible for numerous tropical diseases, including human African trypanosomiasis (Trypanosoma brucei), Chagas disease (T. cruzi), Leishmaniasis (Leishmania spp.), and malaria (Plasmodium falciparum). Herein, we report our continuing efforts to optimize this series against P. falciparum. Through the design of a library of compounds focused on reducing the lipophilicity and molecular weight, followed by an SAR exploration, we have identified NEU-1953 (40). This compound is a potent inhibitor of P. falciparum with an improved ADME profile over the previously reported compound, NEU-961 (3).

Full text of DOI:10.1021/acsinfecdis.7b00212

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Article
Optimization of Physicochemical Properties for 4-
Anilinoquinoline Inhibitors of Plasmodium falciparum Proliferation
Naimee Mehta, Lori Ferrins, Susan E. Leed, Richard J. Sciotti, and Michael P. Pollastri
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00212 • Publication Date (Web): 04 Jan 2018
Downloaded from http://pubs.acs.org on January 5, 2018
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Optimization of Physicochemical Properties for 4ꢀ
Anilinoquinoline Inhibitors of Plasmodium
falciparum Proliferation
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a
Naimee Mehta, Lori Ferrins, Susan E. Leed, Richard J. Sciotti, and Michael P. Pollastri.*
a
Northeastern University Department of Chemistry & Chemical Biology, 360 Huntington
Avenue, Boston, MA 02115 USA. Tel: 617ꢀ373ꢀ2703; Eꢀmail: m.pollastri@northeastern.edu
b
Experimental Therapeutics, Walter Reed Army Institute for Research, 2460 Linden Lane, Silver
Spring, MD, 20910
We recently reported the medicinal chemistry reꢀoptimization of a known human tyrosine kinase
inhibitor, lapatinib, against a variety of parasites responsible for numerous tropical diseases,
including: human African trypanosomiasis (Trypanosoma brucei), Chagas disease (T. cruzi),
Leishmaniasis (Leishmania spp.) and malaria (Plasmodium falciparum). Herein, we report our
continuing efforts to optimize this series against P. falciparum. Through the design of a library
of compounds focused on reducing the lipophilicity and molecular weight, followed by an SAR
exploration, we have identified NEU-1953 (40). This compound is a potent inhibitor of P.
falciparum with an improved ADME profile over the previously reported compound, NEU-961
(3).
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Keywords: drug repurposing, tropical diseases, malaria, target class repurposing
In subꢀSaharan Africa tropical diseases remain the dominant cause of disease burden. Malaria
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is caused by protozoan parasites of the genus Plasmodium and in 2015, it was responsible for
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12 million new cases worldwide. Eradication of malaria has proven challenging due to the
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complex life cycle of Plasmodium and the emergence of drug resistance. The parasite is
transmitted to the human host through the bite of an infected Anopheles mosquito, the injected
sporozoites are taken up by the liver where they develop first into schizonts, and then into
merozoites. It is these merozoites which are responsible for infection of red blood cells. The
clinical signs of malaria arise due to this infection and consist of fever, chills, anemia and
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ultimately cerebral malaria. Many current antimalarial therapies target the asexual blood stage
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of Plasmodium, in which the parasite replicates within erythrocytes. However, in P. vivax and
P. ovale a proportion of the parasites remain dormant in the hepatocytes for months to years,
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known as hypnozoites. These parasites can then initiate a new cycle of asexual reproduction at a
later stage causing further clinical symptoms.
In an effort to expedite the drug discovery process, we employed a target class repurposing
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approach, wherein we screened known human tyrosine kinase inhibitors against a number of
related pathogenic protozoan parasites: Trypanosoma brucei (human African trypanosomiasis),
T. cruzi (Chagas disease), Leishmania major (leishmaniasis), and P. falciparum. We previously
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described the results of this screening and the subsequent discovery of NEU-617 (2) a potent
antiꢀtrypanosomal compound derived from the approved therapeutic lapatinib (1). We have since
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disclosed NEU-961 (3), a derivative of 2, which is a highly potent growth inhibitor of P.
falciparum.
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Figure 1. Progression from 1 to 2 was achieved through the implementation of a target class
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repurposing program. Following scaffold exploration we arrived at the anilinoquinoline, 3
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which showed significantly improved activity against P. falciparum.
Clear limitations of 3 include its high molecular weight (MW) and lipophilicity, which may in
part be responsible for its poor aqueous solubility. To move this chemical series towards more
drugꢀlike space and to lower the propensity for future toxicity issues, significant reductions in
size and lipophilicity of the head group (red in Figure 1) were envisioned. We now report here
our efforts to achieve improved properties while maintaining high potency by recapitulating
sterics and electronics of this head group region of 3. Simultaneously, we explored variations to
the tail portion (blue in Figure 1) to reduce the lipophilicity and maintain potency against blood
stage malaria.
Results
Since we previously observed that the structureꢀactivity relationships (SAR) in the head group
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region (red in Figure 1) of the quinazoline core tolerated relatively large changes in size, we
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hypothesized that this region could be amenable to a reduction in size and lipophilicity without a
loss of potency. We utilized an in silico design strategy to prioritize compounds for synthesis.
Using a set of commercially available heterocyclic primary amines, we enumerated a virtual
library of analogs of 3, which was subsequently filtered on the basis of cLogP <5 and MW <500
g/mol, obtaining a subꢀlibrary of 84 quinolines.
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In addition to improving size and lipophilicity, we hoped to recapitulate shape and
electrostatic features of the Rꢀgroup (Figure 1) that likely gave rise to its high potency. Using
9ꢀ10
ROCS and EON (OpenEye Scientific Software),
a threeꢀdimensional shape and electrostatics
comparison was carried out between 3 and each member of the virtual library. Members of the
conformer library were rankꢀordered in terms of shape and electrostatics using the ET_Combo
score. Fourteen compounds were chosen for synthesis considering computational scoring,
calculated properties, and qualitative analysis of synthetic feasibility. Figure 2 shows the
comparison of the 246ꢀmembered virtual library (red) and the shaped library (blue). The average
MW (487 g/mol) and cLogP (3.2; desirable range ≤ 3) of the selected analogs were substantially
lower than those of 3 (MW 617 g/mol, cLogP 6.4).
cLogP
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Figure 2. Comparison of all the compounds with variation around the Rꢀgroup (see Figure 1) in
the virtual library (red) with the shaped library (blue).
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Synthesis of the library of substituted anilines started with Suzuki coupling of 4 with boronic
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ester 5, which provided 6 in good yield. Installation of the primary aromatic amine occurred in
reasonable yield under inert conditions with microwave irradiation (Scheme 1). In the case of
12ꢀ13
electron deficient amines it was necessary to employ BuchwaldꢀHartwig conditions
the desired product in good yield following preparative HPLC purification.
to obtain
Scheme 1. Synthesis of head group replacement analogs.
o
Reagents and conditions: (i) 5, Pd(OAc) , triethylamine, 1:1 water:ethanol, 120 C, 1h,
2
o
microwave, 71%; (ii) ArꢀNH , 4M HCl in dioxane, 2ꢀpropanol, 145 C, 30min, microwave, 19ꢀ
5%; (iii) ArꢀNH , Pd(OAc) , Xantphos, KO Bu, xylene, 160 C, 1 h, microwave, 24ꢀ45%.
2
t
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4
2 2
All synthesized compounds were tested for their antimalarial activity against three P.
falciparum strains: D6 (chloroquine (CQ) sensitive, mefloquine (MQ) resistant), W2 (CQ
resistant, MQ sensitive), and C235 (CQ, MQ and pyrimethamine resistant), and for their toxicity
against mammalian cells (HepG2 cells). The SAR for the D6 strain will be discussed herein
along with the toxicity data. The biological activity against the W2 and C235 cell lines is
summarized in the Supplementary Information, Table S1.
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Most of the compounds tested exhibited activities within threeꢀfold of that observed for D6,
against both the W2 and C235 strains. Of the compounds tested 15% (8 out of 53 total
compounds assayed; ID: 7, 11, 14, 19, 21, 23ꢀ24, 36) did fall outside of the 3ꢀfold range and
were less potent against the W2 strain than the D6 strain. Further, 2% (1 out of 53 total
compounds assayed; ID: 21) of compounds fell outside the 3ꢀfold range and were less potent
against the C235 strain than the D6 strain. This suggests a lack of sensitivity to CQ resistance for
the majority of compounds presented herein (see Supplementary Information, Figure S2-S3).
The screening results from this first round of analogs are shown in Table 1. Truncation of the
halogenated benzyl ether head group of 3 to the 3ꢀchloroꢀ4ꢀmethoxyaniline (7; EC : 0.012 µM)
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led to an analog that was approximately equipotent and demonstrated a significant improvement
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in lipophilic ligand efficiency (LLE = pEC ꢀ cLogP, desired range ≥4). However, removal of
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the 4ꢀmethoxy (8; EC : 0.20 µM) was detrimental to the potency, whilst removal of the 3ꢀchloro
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(9; EC : 0.018 µM) led to a compound which was equipotent with 3 and showed a marked
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improvement in the selectivity versus HepG2 cells (SI: 1100), highlighting the importance of the
ꢀmethoxy substituent. Substitution of the 4ꢀmethoxy by a 4ꢀtrifluoromethoxy (10) and the 3ꢀ
4
chloroꢀ4ꢀmethyl (11) was also tolerated. All the substituted aniline analogs had a lower cLogP
and improved LLE as compared to 3. The 3ꢀpyridyl (12; EC : 0.13 µM) saw a 10ꢀfold reduction
5
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in potency compared to 3, the pyridazine (13; EC : 2.1 µM) highlighted the detrimental effect of
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the endocyclic nitrogen at the 4ꢀposition. Further substitution of the 3ꢀpyridyl with a 4ꢀmethyl
(15; EC : 0.022 µM) led to a threeꢀfold improvement in potency, and a marked improvement in
5
0
selectivity (SI: > 1300), while substitution with a 4ꢀtrifluoromethyl (16; EC : 0.16 µM) had no
50
impact on potency. A trend is observed between 8 and 12, and 11 and 15 where replacement of
the 3ꢀchloro with an endocyclic nitrogen atom yielded a boost in the selectivity for the parasite
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versus HepG2 cells. Replacement with other Nꢀcontaining heterocycles such as the substituted
pyrimidine, 17 (EC : 0.050 µM) and 21 (EC : 0.058 µM) was favorable, though pyrazines (18
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– 19), 2ꢀpyridyl (20) and the triazine (23) were less favored, the latter also showed greater
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overall toxicity which was of concern.
Table 1. Analogs of the head group on the 4ꢀaminoquinoline series.
P. fal. D6 HepG2 TC50
ID
R
a
b,c,d
SI
cLogP LLE
EC (ꢁM) (ꢁM)
50
3
0.019
>4
>210
6.4
1.3
7
8
9
0.012
0.20
2.8
>5.5
20
230
4.6
4.7
3.9
3.3
2.0
3.8
>28
1100
0.018
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0.13
>30
>36
>470
>280
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2.1
>36
>33
>29
>17
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0.20
0.022
>170
>1300 3.0
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0.16
0.050
0.13
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>140
420
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>31
>28
>6
540
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>1000 3.5
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>38
2.2
3.4
4.7
3.6
24
0.11
>35
3.3
>320
660
Chloroquine
0.005
a
b
c
d
2
All r values >0.9 unless noted otherwise
n = 1 biological replicate made up of 2 technical replicates
concentration which is toxic to 50% of the cell population
tested concentration ranges were determined based on compound solubility
Systematic exploration of the tail region was targeted next and could be readily achieved
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through 26, which was synthesized from 4 and 25 as previously reported. Miyaura coupling
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followed by a Suzuki reaction led to the successful isolation of the desired products (Scheme 2).
Alternatively, BuchwaldꢀHartwig coupling allowed the installation of aromatic amines.
Simultaneously, we explored modifications to the tail region with the aim of reducing the
lipophilicity of the series as compared to 3 (cLogP 6.4) (Table 2).
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Scheme 2. Synthesis of linker replacement analogs.
o
Reagents and conditions: (i) 25, 2ꢀpropanol, 80 C, 12 h, 90% (ii) B pin , Pd(PPh ) , KOAc,
2
2
3 4
o
o
dioxane, 120 C, 2.5 h, microwave, 91%; (iii) ArꢀBpin or ArꢀBr, Pd(PPh ) , dioxane, 120 C, 1h,
microwave, 33ꢀ67%; (iv) ArꢀNH , Pd(OAc) , XantPhos, Cs CO , dioxane, 160 C 1h,
3
4
o
2
2
2
3
microwave, 32ꢀ57%.
To determine whether there was a favored substitution pattern around the phenyl linker we
investigated the meta (28) substituted analog and found it to be equipotent with 3. Replacement
of the phenyl sulfonamide with the pyrimidine (29) led to another equipotent compound (EC50:
0
.026 µM) that maintained excellent selectivity. The ꢀNH linked analogs 32-35 all exhibited a
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reduction in potency and decrease in the selectivity index, most markedly for 35 (SI: 19). The
tertꢀbutyl carbamate (36) similarly exhibited an almost threeꢀfold drop in potency (EC : 0.050
5
0
µM) and a decrease in the SI (56). The 1,2,5ꢀthiadiazoleꢀlinked N-methylpiperazine (30) led to a
sixꢀfold reduction in potency. In an effort to reduce the overall cLogP, the pyrazole (31) was
tested and found to be equipotent to 3 with a SI of 100.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Phenyl linker replacement analogs.
O
Cl
F
HN
N
R
HepG2
P. fal. D6
ID
R
a
TC50
SI
cLogP
LLE
1.3
EC (ꢁM)
b,c,d
50
(ꢁM)
3
0.019
0.010
0.026
>4
>210 6.4
2
2
8
9
4.2
3.0
420
115
6.4
6.2
1.6
2.2
3
0
0.10
5.7
57
6.8
0.15
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
1
0.021
2.1
4.8
100
150
5.8
7.3
1.9
32
0.033
0.031
0.025
0.20
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
3.1
3.0
100
120
6.0
6.6
1.5
34
35
0.97
0.24
4.6
19
6.6
6.8
ꢀ0.020
0.51
3
6
0.050
2.8
3.3
56
Chloroquine
0.0050
660
a
b
c
d
2
All r values >0.9
n = 1 biological replicate made up of 2 technical replicates
concentration which is toxic to 50% of the cell population
tested concentration ranges were determined based on compound solubility
A series of crossꢀover analogs was designed based upon the biological data and the ADME
profile that was obtained for the head group and linker/tail replacement analogs shown in Tables
1
and 2. The head groups that were selected for further analysis included the 2ꢀaminopyrazine
from compound 14), 3ꢀchloroꢀ4ꢀmethoxyaniline (7), and the 5ꢀaminobenzotriazole (22). The tail
groups that were selected included the 2ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)pyrimidine (from compound
(
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2
3
4
5
6
7
8
9
2
9), pyrazole (31), 4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)aniline (32), 4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)pyrimidinꢀ
ꢀamine (34), and 2ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)pyrimidinꢀ4ꢀamine (35). The synthesis of these
2
analogs utilized the protocols summarized in Scheme 3 and consisted of either a Buchwaldꢀ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hartwig or Suzuki coupling to obtain the desired product in reasonable to good yields.
Scheme 3. Synthesis of crossꢀover analogs, combining favorable head and tail/linker groups.
o
Reagents and conditions: (i) ArꢀBpin, Pd(OAc) , Pd(PPh ) , dioxane, 120 C, 1h, microwave,
2
3 4
o
2
1ꢀ63%; (ii) ArꢀNH , Pd(OAc) , Xantphos, Cs CO , dioxane, 160 C, 1h, microwave, 19ꢀ66%.
2 2 2 3
Clear trends in lipophilicity were observed across matched pairs with different headgroups
(Table 3). Compounds with the 3ꢀchloroꢀ4ꢀmethoxyaniline head group (B; 41, 44, 47, and 49)
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3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
typically had an increased cLogP, decreased LLE, and decreased selectivity as compared to their
matched pairs with other headgroups. While the incorporation of the 2ꢀaminopyrazine head
group (A) resulted in compounds with the highest LLE of the series (40, LLE = 5.5 and 46, LLE
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
=
4.8). The pyrimidine linker analogs (40ꢀ42) generally demonstrated an improvement in cLogP
with no significant loss in potency. Replacement with the pyrazole (43-44) was generally
unfavorable for activity though did lead to an improvement in the cLogP and LLE. In an attempt
to improve the aqueous solubility of the series, analogs with an amine linking the tail to the
quinoline core were synthesized (45-49) as it was thought that this would break up the planarity
of the molecule. The aminoꢀpyrimidine linked analogs (46–49) were largely equipotent and
showed a boost in the selectivity when compared to 40-42 with the direct linkage.
Table 3. Biological assay data for crossꢀover series against P. falciparum and HepG2 cells.
HepG2
P. fal. D6
1
2
ID
R
R
a
TC50
SI
cLogP LLE
EC (ꢁM)
b,c,d
50
(ꢁM)
4
4
4
4
4
0
1
2
3
4
A
B
C
A
B
0.026
0.035
0.093
0.94
>4
>3
21
4.5
23
>150 2.1
5.5
3.1
3.8
4.4
2.6
>86
230
4.8
92
4.4
3.1
1.6
3.9
0.25
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5
6
7
8
9
4
5
A
0.13
17
540
3.1
3.8
46
47
A
B
A
B
0.054
0.099
0.18
22
410
77
2.4
4.7
2.5
4.7
4.8
2.2
4.3
2.3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7.6
21
4
4
8
9
120
60
0.078
0.0050
4.7
3.3
Chloroquine
660
a
b
c
d
2
All r values >0.9
n = 1 biological replicate made up of 2 technical replicates
concentration which is toxic to 50% of the cell population
tested concentration ranges were determined based on compound solubility
Analysis of the ADME data for the crossꢀover analogs is summarized in Table 4 (for ADME
data on all other compounds see Supplementary Information, Table S3). Incorporation of the ꢀ
NH linker did, in some cases, lead to an increase in the aqueous solubility, and to an
improvement in the overall ADME profile. For example, 45 exhibited the most significant
improvement in aqueous solubility (810 µM; desired range > 10 µM) and had a lower plasma
protein binding (88%; desired range < 95%) though this was also associated with a loss in
potency (EC : 0.13 µM). While the cLogP and LLE were improved, the pyrazole replacement
5
0
analogs (43-44) exhibited poor solubility, and 43 demonstrated high microsomal clearance.
Despite the reduced selectivity observed with the 3ꢀchloroꢀ4ꢀmethoxy aniline head group (B), it
tended to impart favorable metabolic stability to the analogs (44, 47, 49). Finally, 40 was potent
against P. falciparum (EC : 0.026 µM), exhibited an improvement in aqueous solubility (44
50
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
µM), and a lower plasma protein binding (87%) compared with 3, though microsomal stability
was low in both humans and rats. Similarly, 7 was potent against P. falciparum (EC : 0.012
5
0
µM), exhibited an improvement in aqueous solubility (100 µM) though plasma protein binding
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(99%) and microsomal stability were still outside the desirable range (human: 47 µL/min/mg;
6
rat: ≤ 27 µL/min/10 ). After considering the potency and ADME profile of all the analogs, 7 and
4
0 were identified as our most promising candidates to date.
Table 4. ADME data for crossꢀover analogs.
Polar
surface
area (Ų) pH 7.4 (µM)
Aqueous
Solubility
Human
Microsome CLint CLint
(µL/min/mg)
Liver Rat Hepatocyte
Human
PPB (%)
ID
LogD7.4
6
(µL/min/10 )
3
75
83
66
99
79
63
69
95
78
95
78
0.3
44
> 99
87
98
91
nd
97
88
nd
99
nd
nd
140
180
170
120
130
8
20
nd
4
4
4
0
1
2
130
45
3.3
4.3
3.3
2.6
3.5
2.2
2.8
3.3
2.9
nd
< 1
< 1
1.0
2.0
810
26
51
43
44
45
46
47
48
49
5.0
5.0
10
nd
nd
9.3
7.2
nd
570
2.0
nd
nd
nd
45
2.3
nd = not determined.
We note that overall, the aqueous solubility of this series does not correlate well with either
cLogP or LogD (see Supporting Information, Figure S1). Despite this, we have made progress
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2
3
4
5
6
7
8
9
towards our goal of identifying potent compounds with improved aqueous solubility as
exemplified by the fact that 21% (11 out of 53) of the compounds presented herein have aqueous
solubility > 10 µM. Further efforts to fully understand the relationship between these properties
is underway and will be presented in due course.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Given the ability to successfully modulate the ADME profile of the series seemed to reside
with the tail region we explored additional heterocyclic and amine linked analogs (Table 5). No
significant change in potency was observed upon capping the free ꢀNH of the pyrazole with a
methyl group (50-51), and insertion of an amine linker (56; EC : 0.40 µM) led to an equipotent
5
0
compound. Replacement with the furan (52) led to a reduction in potency. A series of amine
linked heterocycles was also trialed, including variously substituted pyrazoles (55-59),
isothiazoles (60), thiazoles (61-62), isoxazoles (63) and triazines (64-65), in the hopes of
recapitulating the boost in aqueous solubility observed with the crossꢀover analogs. In general,
these compounds exhibited a loss of potency, with exception to 60, and 62-63 which showed
improved, potent activity and excellent selectivity. However, the ADME profile of these
compounds (see Supplementary Information, Table S3) showed that they had poor aqueous
solubility suggesting that this strategy could not be generally applied to the series to address the
poor ADME profile.
Table 5. Biological assay data for further tail group replacement analogs against P. falciparum.
1
2
HepG2
TC50
ID
R
R
P. fal.
SI
cLogP LLE
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
b,c,d
EC50
(ꢁM)
a
(ꢁM)
5
5
0
1
A
B
0.50
0.16
40
80
1.8
4
4.5
2.8
>44
>280
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
5
2
3
A
0.30
>53
>180 2.4
>50 2.6
>420 4.9
4.1
A
B
1.0
>50
>42
3.4
2.1
54
55
0.10
A
1.2
>39
>33
1.9
4.0
5
5
6
7
A
A
A
0.40
0.60
0.86
29
73
1.6
1.7
2.0
4.8
4.5
4.1
35
58
58
>42
>49
5
6
6
9
0
1
A
A
A
6.8
>40
>36
7.1
>5.9
1.8
3.4
5.1
3.7
0.031
0.63
>1200 2.4
11
2.5
62
63
A
A
0.053
0.093
>40
>48
>760 3.2
>520 1.8
4.1
5.3
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2
3
4
5
6
7
8
9
6
4
5
A
A
0.18
11
61
0.95
1.2
5.8
5.8
6
0.11
19
170
660
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Chloroquine
0.0050
3.3
a
b
c
d
2
All r values >0.9
n = 1 biological replicate made up of 2 technical replicates
concentration which is toxic to 50% of the cell population
tested concentration ranges were determined based on compound solubility
In addition to demonstrating activity against bloodꢀstage Plasmodium, it is useful to determine
compound activity during the liver stage of the infection particularly for P. vivax and P. ovale
malaria. It is during this stage that sporozoites develop into merozoites, which then invade and
3
multiply in red blood cells; compounds that target both the liver and blood stage of the infection
1
5
are therefore more effective in preventing relapsing malaria and the symptomatic phase.
However, it is not critical to demonstrate activity in the hepatic stage for progression of
promising compounds; most drugs that target bloodstream Plasmodium have little activity in the
1
6
17ꢀ18
hepatic stage and only primaquine is currently approved to eliminate hypnozoites.
Given
this, a representative subset of our anilinoquinoline compounds were assessed in a P. berghei
liver stage model (see Supplementary Information, Table S2). In general, the compounds
exhibited low micromolar inhibition in the assay despite demonstrating nanomolar activity in the
P. falciparum bloodstream form assays. It is important to acknowledge the differential activity
that has been previously observed between human and murine models which could be due to
differences in the humanized mouse model of P. falciparum and the normal mouse model for P.
1
6
berghei.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The exquisite human kinase selectivity of lapatinib (1) is attributed to the large, lipophilic head
group. Compound 1 has previously demonstrated >300ꢀfold selectivity versus numerous human
1
9
kinases involved in cellular proliferation. Given the structural modifications to the anilino head
group presented herein, as well as modifications to other regions, we sought to assess the human
kinase activity profile of 40 in a panel of human kinases (see Supplementary Information,
Figure S4). At a test concentration of 5 µM, 40 showed significant inhibition of seven kinases
largely belonging to the serine/threonine kinase family (Aurora A, MNK2 and MAP4K4) and the
tyrosine kinase family (EGFR, Abl and KDR), which is unsurprising given the activity of 1. In
addition, 40 showed moderate to weak inhibition of 10 kinases, and insignificant inhibition of an
additional 28 kinases.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Finally, 7 and 40 had the best combination of potency and aqueous solubility amongst all the
members in the library and were tested in a modified Thompson in vivo infection model against
P. berghei. This measures the parasite clearance and survivability of mice following
administration of the compound on days 3 to 5 postꢀinfection. Mice alive on day 31 with no
parasitemia are considered cured. The mice in the study were given a single intraperitoneal dose
of 160 mg/kg/day × 3 days and in both cases there was a reduction in parasitemia, but only mice
treated with 7 experienced suppression of malaria and a life extension of 5 days compared to the
control group. Hence, both 7 and 40 were suppressive and not curative of the P. falciparum
infection. The poor in vivo efficacy is most likely due to the rapid clearance of 7 and 40. Despite
this, a positive impact was observed on the life span of infected mice, highlighting the promise of
this series as antiꢀmalarials.
Conclusion
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5
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7
8
9
We have previously observed that compounds bearing the large lipophilic head group of
lapatinib have problematic physicochemical properties which are likely a result of the high MW
and lipophilicity of the compounds in general. Given this, we designed a library of analogs
which would mimic the key features of the head group while focusing on reducing the size and
lipophilicity. These designs were further informed by shape and electrostatics similarity
comparison to a known active, 3. We also explored numerous tail region replacements and
incorporated structural features that led to an improvement in the aqueous solubility of the series.
Combining these features led us to the identification of 7 and 40, potent inhibitors of
bloodstream P. falciparum, with improved aqueous solubility over 3. Progression of these
compounds into an in vivo model demonstrated suppression of parasitemia, though neither were
curative; it is likely that the metabolic instability of these analogs contributed to the outcome.
Current efforts are centered upon further improving the overall ADME profile of this series with
a focus on the microsomal stability, as well as further improvements to the aqueous solubility
and plasma protein binding with an eye to identifying a lead compound for P. falciparum.
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Experimental
General Chemistry Experimental
All starting materials were commercially procured and were used without further purification,
unless specified. Reaction solvents were purified by passage through alumina columns on a
purification system manufactured by Innovative Technology (Newburyport, MA). NMR spectra
1
were obtained on Varian NMR systems, operating at 400 MHz or 500 MHz for H acquisitions
13
and at 126 MHz for C acquisitions. LCMS analysis was performed using a Waters Alliance
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reverse phase HPLC (columns Waters SunFire C18 4.6x50mm, 3.5ꢁm or Waters SunFire C8
4
.6x50mm, 3.5 ꢁm), with singleꢀwavelength UV–visible detector and LCT Premier timeꢀofꢀ
flight mass spectrometer (electrospray ionization) or Waters Micromass ZQ detector
0
1
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0
(
electrospray ionization). All final compounds were purified by preparative reverse phase HPLC
columns Waters Symmetry RP8 30 × 50 mm, 5 ꢁm column or OBD RP18 30 × 50 mm, 5 ꢁm),
(
with a singleꢀwavelength UV–visible detector and Waters Micromass ZQ (electrospray
ionization). For further details regarding preparative HPLC and LCMS protocols, see Supporting
Information. All final compounds have purities greater than 95% based upon LC/MS.
General library condition A:
In a dry 2mL microwave vial equipped with stir bar were combined 6 (0.018 g, 0.045 mmol),
0
.067 mmol of amine and 4M hydrogen chloride in dioxane (0.017 mL, 0.067 mmol). The sealed
vial was evacuated and backfilled three times with nitrogen followed by addition of 2ꢀpropanol
(1 mL). The resultant mixture was stirred under nitrogen for 5 min. The vial was irradiated at
°
1
45 C for between 30 and 60 min; the reaction progress was monitored by LCꢀMS. The reaction
was cooled to rt and filtered. The precipitate was sequentially washed with 2ꢀpropanol (5 mL)
and DCM (5 mL) to afford final product. Further purification, if required, was carried out via
preparative HPLC using a gradient 10ꢀ25% acetonitrile in water containing 0.1% formic acid to
give the desired product.
General library condition B:
In a dry 2mL microwave vial equipped with stir bar was combined 6 (0.045 mmol),
heterocyclic amine (0.067 mmol), palladium(II) acetate (2.5 mol%, 1.831 µmol), XantPhos (5
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mol%, 3.57 µmol), and potassium tertꢀbutoxide (0.089 mmol). The sealed vial was evacuated
and backfilled three times with nitrogen followed by addition of xylene (1 mL). The contents
was stirred under nitrogen for 5 min. The vial was irradiated at 160 °C for between 60 and 90
min; the reaction was monitored by LCꢀMS. The crude material was concentrated under reduced
pressure. The solid was reꢀdissolved in ethyl acetate and filtered through a celite plug, which was
washed sequentially with ethyl acetate (5mL) and methanol (5 mL) and concentrated. The crude
material was solubilized in 1 mL DMSO and filtered through 17 mm cellulose syringe filter
1
1
1
1
1
1
1
1
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1
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5
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0
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9
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6
7
8
9
0
(0.45 µm). The crude product was purified via preparative HPLC using a gradient of 10ꢀ25%
acetonitrile in water containing 0.1% formic acid to afford the desired product. HRMS data was
collected using an Agilent Technologies 6520 AccurateꢀMass QꢀTOF MS system equipped with
an IonSense DART SVPꢀ100 ionization source.
General library condition C
In a dry 0.5ꢀ2 mL microwave vial were combined 26 (0.11 mmol), heterocyclic amine (0.13
mmol), palladium (II) acetate (10 mol%, 10.9 µmol), XantPhos (0.019 µmol), and cesium
carbonate (0.218 mmol). The sealed vial was evacuated and backfilled three times with nitrogen
followed by addition of dioxane (volume: 2 mL). The mixture was stirred under nitrogen for 5
min. The vial was irradiated at 140°C for 60ꢀ90 min; the reaction was monitored by LCꢀMS.
Once the reaction was complete based on LCꢀMS the reaction mixture was concentrated under
reduced pressure. The residue was partitioned between EtOAc and 2M NaOH. The aqueous layer
was extracted twice with EtOAc (25 mL), the combined organic layers were washed with brine,
dried over sodium sulfate and concentrated in vacuo. The crude material was solubilized in 1 mL
DMSO and filtered through a 17 mm cellulose syringe filter (0.45 µm). The crude product was
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purified via preparative HPLC using a gradient 10ꢀ25% acetonitrile in water containing 0.1%
formic acid to give the desired product.
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9
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8
9
0
General conditions D
In a 2mL microwave vial equipped with stir bar were appropriate bromide partner (1 eq), Arꢀ
Bpin (1.2 eq), palladium (II) acetate (10 mol%), triethylamine (3 eq). 1:1 mixture of water and
EtOH (1 mL) was added to the mixture and the sealed tube was irradiated at 120 °C for between
6
0 and 90 min. The reaction progress was monitored by LCꢀMS. Once the reaction was complete
the crude material was filtered through a celite plug and washed with ethanol (10 mL). The
filtrate was concentrated under vacuum. The crude material was solubilized in 1 mL DMSO and
filtered through 17 mm cellulose syringe filter (0.45 µm). The crude material was purified via
preparative HPLC using a 0ꢀ50% acetonitrile/water gradient containing 0.1% formic acid to give
the desired product.
General library condition E
In a 2 mL microwave vial equipped with stir bar were appropriate bromide partner (50 mg, 1
eq), ArꢀNH (1.2 eq), palladium (II) acetate (10 mol %), XantPhos (11.1 mg, 0.019 mmol)
2
cesium carbonate (71.2 mg, 0.21 mmol). The sealed vial was evacuated and backfilled three
times with nitrogen followed by addition of anhydrous dioxane (2 mL). The contents were stirred
under nitrogen for 5 min. The sealed vial was irradiated at 140 °C for 60ꢀ90 min. The reaction
progress was monitored by LCꢀMS. Once the reaction was complete the crude was filtered
through a celite plug and washed with dioxane (10 mL). The filtrate was concentrated under
vacuum. The crude material was solubilized in 1 mL DMSO and filtered through 17 mm
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cellulose syringe filter (0.45 µm). The crude material was purified via preparative HPLC using a
0
ꢀ50% acetonitrile/water gradient containing 0.1% formic acid to give the desired product.
1
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4
-Chloro-7-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)quinoline (6)
In a dry 20 mL microwave vial equipped with a stir bar was combined 4 (2.07 mmol), 5 (2.69
mmol), palladium(II) acetate (1 mol%, 0.027 mmol), triethylamine (6.21 mmol) and 1:1 mixture
of water and ethanol (8 mL). The sealed vial was irradiated at 120 °C for 1 h. The crude material
was filtered under vacuum and the precipitate was transferred to a conical flask. The precipitate
was diluted with water (50 mL), and extracted with DCM (3 x 50 mL). The combined organic
layers were washed with aq. NaOH (1M, 2 x 50 mL), water (50 mL), and brine (50 mL), dried
over sodium sulfate and concentrated to afford 6 (584 mg, 1.45 mmol) as a pale white solid in
1
7
0% yield. H NMR (DMSOꢀd , 500 MHz) δ 8.93 (d, J=4.9 Hz, 1 H), 8.49 (d, J=2.0 Hz, 1 H),
6
8.35 (d, J=8.8 Hz, 1 H), 8.16 ꢀ 8.21 (m, 3 H), 7.87 ꢀ 7.90 (m, 2 H), 7.83 (d, J=4.4 Hz, 1 H), 2.96
+
(
br. s., 4 H), 2.36 ꢀ 2.43 (m, 4 H), 2.16 ppm (br. s., 3 H). ESIꢀMS: m/z = 402.1 (M + H) .
N-(3-Chloro-4-methoxyphenyl)-7-{4-[(4-methylpiperazin-1-yl)sulfonyl]phenyl}quinolin-
4
-amine, formate salt (7) Synthesized using general library condition A. Yield 42%
1
H NMR (DMSOꢀd , 500 MHz) δ 8.46 ꢀ 8.50 (m, 2 H), 8.22 (d, J=2.0 Hz, 1 H), 8.20 (s, 1 H),
6
8
.14 (d, J=8.3 Hz, 2 H), 7.94 (dd, J=8.8, 2.0 Hz, 1 H), 7.85 (d, J=8.8 Hz, 2 H), 7.43 (d, J=2.4 Hz,
1 H), 7.34 (dd, J=8.8, 2.4 Hz, 1 H), 7.22 (d, J=8.8 Hz, 1 H), 6.75 (d, J=5.4 Hz, 1 H), 3.87 (s, 3
+
H), 2.94 (br. s., 4 H), 2.37 (br. s., 4 H), 2.13 ppm (s, 3 H). ESIꢀMS: m/z = 523.1 (M + H) .
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N-(3-Chlorophenyl)-7-{4-[(4-methylpiperazin-1-yl)sulfonyl]phenyl}quinolin-4-amine, bis-
formate salt (8) Synthesized using general library condition A. Yield 68%
1
H NMR (DMSOꢀd , 500 MHz) δ 9.17 (br. s., 1 H), 8.57 (d, J=5.4 Hz, 1 H), 8.48 (d, J=8.8 Hz,
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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6
7
8
9
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9
0
1
H), 8.26 (d, J=1.5 Hz, 1 H), 8.22 (s, 2 H), 8.15 (d, J=8.3 Hz, 2 H), 7.98 (dd, J=8.8, 1.5 Hz, 1
H), 7.85 (d, J=8.3 Hz, 2 H), 7.39 ꢀ 7.44 (m, 2 H), 7.36 (d, J=7.8 Hz, 1 H), 7.15 (d, J=8.3 Hz, 1
H), 7.08 (d, J=5.4 Hz, 1 H), 2.94 (br. s., 4 H), 2.37 (br. s., 4 H), 2.13 ppm (s, 3 H). ESIꢀMS: m/z
+
=
493.1 (M + H) .
N-(4-Methoxyphenyl)-7-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)quinolin-4-amine,
bis-formate salt (9) Synthesized using general library condition A. Yield 56%
1
H NMR (500 MHz, DMSOꢀd ) δ 8.54 (d, J=8.8 Hz, 1 H), 8.43 (d, J=5.9 Hz, 1 H), 8.21 (d,
6
J=2.0 Hz, 1 H), 8.13 ꢀ 8.15 (m, 2 H), 8.13 (s, 2 H), 7.94 (dd, J=9.0, 1.7 Hz, 1 H), 7.85 (d, J=8.3
Hz, 2 H), 7.30 (d, J=8.8 Hz, 2 H), 7.03 (d, J=8.8 Hz, 2 H), 6.65 (d, J=5.4 Hz, 1 H), 3.78 (s, 3 H),
+
2
.94 (br. s., 4 H), 2.38 (br. s., 4 H), 2.14 ppm (s, 3 H). ESIꢀMS: m/z = 489.2 (M + H) .
7-(4-((4-Methylpiperazin-1-yl)sulfonyl)phenyl)-N-(4-(trifluoromethoxy)phenyl)quinolin-
4
-amine, bis-formate salt (10) Synthesized using general library condition A. Yield 56%
1
H NMR (500 MHz, DMSOꢀd ) δ 8.54 (d, J=5.4 Hz, 1 H), 8.50 (d, J=8.8 Hz, 1 H), 8.26 (d,
6
J=2.0 Hz, 1 H), 8.14 (s, 2 H), 8.12 ꢀ 8.18 (m, 2 H), 7.97 (dd, J=8.8, 2.0 Hz, 1 H), 7.85 (d, J=8.3
Hz, 2 H), 7.45 ꢀ 7.51 (m, 2 H), 7.38 ꢀ 7.43 (m, 2 H), 7.02 (d, J=4.9 Hz, 1 H), 2.94 (br. s., 4 H),
+
2
.37 (br. s., 4 H), 2.13 ppm (s, 3 H). ESIꢀMS: m/z = 543.2 (M + H) .
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N-(3-Chloro-4-methylphenyl)-7-{4-[(4-methylpiperazin-1-yl)sulfonyl]phenyl}quinolin-4-
amine, formate salt (11) Synthesized using general library condition A. Yield 56%
1
H NMR (500 MHz, DMSOꢀd ) δ 8.53 (d, J=5.4 Hz, 1 H), 8.48 (d, J=8.8 Hz, 1 H), 8.24 (d,
6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
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2
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8
9
0
J=1.5 Hz, 1 H), 8.13 ꢀ 8.15 (m, 2 H), 8.15 (s, 1 H), 7.96 (dd, J=9.0, 1.7 Hz, 1 H), 7.85 (d, J=8.3
Hz, 2 H), 7.40 (d, J=2.0 Hz, 1 H), 7.38 (d, J=8.3 Hz, 1 H), 7.28 (dd, J=8.3, 2.0 Hz, 1 H), 6.96 (d,
J=5.4 Hz, 1 H), 2.94 (br. s., 4 H), 2.90 ꢀ 3.00 (m, 4 H), 2.37 (br. s., 4 H), 2.33 (s, 3 H), 2.13 ppm
+
(
s, 3 H). ESIꢀMS: m/z = 507.1 (M + H) .
7-{4-[(4-Methylpiperazin-1-yl)sulfonyl]phenyl}-N-(pyridin-3-yl)quinolin-4-amine
(12)
Synthesized using general library condition A. Yield 19%
1
H NMR (500 MHz, DMSOꢀd ) δ 9.21 (br. s., 1 H), 8.65 (br. s., 1 H), 8.51 ꢀ 8.59 (m, 2 H),
6
8.40 (s, 1 H), 8.36 (d, J=3.9 Hz, 1 H), 8.28 (s, 1 H), 8.16 (d, J=8.8 Hz, 2 H), 8.00 (d, J=7.8 Hz, 1
H), 7.80 ꢀ 7.89 (m, 3 H), 7.46 (dd, J=7.8, 4.9 Hz, 1 H), 6.99 (d, J=4.9 Hz, 1 H), 2.96 (br. s., 4 H),
+
2
.36 ꢀ 2.41 (m, 4 H), 2.15 ppm (s, 3 H). ESIꢀMS: m/z = 460.1 (M + H) .
7
-{4-[(4-Methylpiperazin-1-yl)sulfonyl]phenyl}-N-(pyridazin-4-yl)quinolin-4-amine (13)
Synthesized using general library condition A. Yield 37%
1
H NMR (500 MHz, METHANOLꢀd ) δ 9.19 (d, J=4.4 Hz, 1 H), 9.10 (d, J=7.8 Hz, 1 H), 8.76
4
(
d, J=2.9 Hz, 1 H), 8.56 (d, J=1.0 Hz, 1 H), 8.11 ꢀ 8.18 (m, 3 H), 8.06 (d, J=8.8 Hz, 1 H), 8.00
(
d, J=8.3 Hz, 2 H), 7.85 (d, J=4.4 Hz, 1 H), 7.36 (dd, J=7.3, 2.9 Hz, 1 H), 2.98 ꢀ 3.21 (m, 4 H),
+
2
.67 ppm (br. s, 3 H). ESIꢀMS: m/z = 461.1 (M + H) . *Four of the piperazine protons are
coincidental with the methanol.
2
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3
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5
5
6
7
-{4-[(4-Methylpiperazin-1-yl)sulfonyl]phenyl}-N-(pyrazin-2-yl)quinolin-4-amine,
formate salt (14) Synthesized using general library condition B. Yield 29%
1
H NMR (500 MHz, DMSOꢀd ) δ 9.88 (br. s., 1 H), 8.78 (d, J=4.9 Hz, 1 H), 8.75 (s, 1 H), 8.65
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
d, J=8.8 Hz, 1 H), 8.39 (d, J=4.9 Hz, 1 H), 8.34 (br. s., 2 H), 8.22 (s, 1 H), 8.19 (s, 1 H), 8.18 (d,
J=5.9 Hz, 2 H), 8.06 (dd, J=8.8, 2.0 Hz, 1 H), 7.87 (d, J=8.8 Hz, 2 H), 2.96 (br. s., 4 H), 2.39 (br.
+
s., 4 H), 2.15 ppm (s, 3 H). ESIꢀMS: m/z = 461.1 (M + H) .
7
-(4-((4-Methylpiperazin-1-yl)sulfonyl)phenyl)-N-(6-methylpyridin-3-yl)quinolin-4-
amine, formate salt (15) Synthesized using general library condition B. Yield 56%
1
H NMR (500 MHz, DMSOꢀd ) δ 8.55 (d, J=8.8 Hz, 1 H), 8.48 ꢀ 8.53 (m, 2 H), 8.25 (br. s., 1
6
H), 8.15 (d, J=7.8 Hz, 2 H), 8.12 (s, 1 H), 8.00 (d, J=8.8 Hz, 1 H), 7.86 (d, J=8.3 Hz, 2 H), 7.72
(dd, J=8.3, 2.9 Hz, 1 H), 7.33 (d, J=8.3 Hz, 1 H), 6.82 (d, J=5.4 Hz, 1 H), 2.97 (br. s., 4 H), 2.19
+
ppm (s, 3 H). ESIꢀMS: m/z = 474.2 (M + H) . *Four of the piperazine protons and the methyl are
coincident with the DMSO.
7
-(4-((4-Methylpiperazin-1-yl)sulfonyl)phenyl)-N-(6-(trifluoromethyl)pyridin-3-
yl)quinolin-4-amine, formate salt (16) Synthesized using general library condition B. Yield
3
8
4%
1
H NMR (500 MHz, DMSOꢀd ) δ 9.56 (br. s., 1 H), 8.78 (br. s., 1 H), 8.68 (d, J=3.9 Hz, 1 H),
6
.48 (d, J=8.8 Hz, 1 H), 8.32 (br. s., 1 H), 8.16 (d, J=8.3 Hz, 2 H), 8.15 (br. s., 1 H), 8.04 (d,
J=8.3 Hz, 1 H), 7.96 (br. s., 1 H), 7.83 ꢀ 7.89 (m, 3 H), 7.33 (d, J=3.9 Hz, 1 H), 2.94 (br. s., 4 H),
+
2.37 (br. s., 4 H), 2.13 ppm (s, 3 H). ESIꢀMS: m/z = 528.2 (M + H) .
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N-(2-Methoxypyrimidin-5-yl)-7-{4-[(4-methylpiperazin-1-yl)sulfonyl]phenyl}quinolin-4-
amine, formate salt (17) Synthesized using general library condition A. Yield 45%
1
H NMR (500 MHz, DMSOꢀd ) δ 8.68 (s, 2 H), 8.49 (d, J=9.3 Hz, 2 H), 8.45 (br. s, 1 H), 8.25
6
1
1
1
1
1
1
1
1
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0
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(
s, 1 H), 8.15 (d, J=8.3 Hz, 2 H), 8.13 (s, 1 H), 7.99 (d, J=8.8 Hz, 1 H), 7.85 (d, J=8.3 Hz, 2 H),
6
.66 (d, J=5.4 Hz, 1 H), 3.95 (s, 3 H), 2.94 (br. s., 4 H), 2.37 (br. s., 4 H), 2.13 ppm (s, 3 H). ESIꢀ
+
MS: m/z = 491.1 (M + H) .
N-(5-Methoxypyrazin-2-yl)-7-{4-[(4-methylpiperazin-1-yl)sulfonyl]phenyl}quinolin-4-
amine, formate salt (18) Synthesized using general library condition A. Yield 32%
1
H NMR (500 MHz, DMSOꢀd ) δ 9.62 (br. s., 1 H), 8.65 (d, J=5.4 Hz, 1 H), 8.62 (d, J=8.8 Hz,
6
1
H), 8.34 (d, J=1.0 Hz, 1 H), 8.29 (d, J=1.5 Hz, 1 H), 8.18 (s, 2 H), 8.17 (s, 1 H), 8.15 (d, J=1.5
Hz, 1 H), 8.02 (dd, J=8.8, 2.0 Hz, 1 H), 7.93 (d, J=5.4 Hz, 1 H), 7.87 (d, J=8.3 Hz, 2 H), 3.92 (s,
+
3
H), 2.96 (br. s., 4 H), 2.39 (br. s., 4 H), 2.15 ppm (s, 3 H). ESIꢀMS: m/z = 491.1 (M + H) .
5
-[(7-{4-[(4-Methylpiperazin-1-yl)sulfonyl]phenyl}quinolin-4-yl)amino]pyrazine-2-
carbonitrile, formate salt (19) Synthesized using general library condition B. Yield 23%
1
H NMR (DMSOꢀd , 500 MHz) δ 8.88 (d, J=4.9 Hz, 1 H), 8.82 (s, 1 H), 8.73 (s, 1 H), 8.59 (d,
6
J=8.8 Hz, 1 H), 8.39 (s, 1 H), 8.29 (d, J=5.4 Hz, 1 H), 8.25 (s, 1 H), 8.19 (d, J=8.3 Hz, 2 H), 8.10
(d, J=8.8 Hz, 1 H), 7.88 (d, J=8.3 Hz, 2 H), 2.96 (br. s., 4 H), 2.39 (br. s., 4 H), 2.15 ppm (s, 3
+
H). ESIꢀMS: m/z = 486.1 (M + H) .
N-(5-Chloropyridin-2-yl)-7-{4-[(4-methylpiperazin-1-yl)sulfonyl]phenyl}quinolin-4-
amine (20) Synthesized using general library condition B. Yield 32%
2
9
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