Series of Alkynyl-Substituted Thienopyrimidines as Inhibitors of Protozoan Parasite Proliferation

Article abstract of DOI:10.1021/acsmedchemlett.8b00245

Discovery of new chemotherapeutic lead agents can be accelerated by optimizing chemotypes proven to be effective in other diseases to act against parasites. One such medicinal chemistry campaign has focused on optimizing the anilinoquinazoline drug lapatinib (1) and the alkynyl thieno[3,2-d]pyrimidine hit GW837016X (NEU-391, 3) into leads for antitrypanosome drugs. We now report the structure-activity relationship studies of 3 and its analogs against Trypanosoma brucei, which causes human African trypanosomiasis (HAT). The series was also tested against Trypanosoma cruzi, Leishmania major, and Plasmodium falciparum. In each case, potent antiparasitic hits with acceptable toxicity margins over mammalian HepG2 and NIH3T3 cell lines were identified. In a mouse model of HAT, 3 extended life of treated mice by 50%, compared to untreated controls. At the cellular level, 3 inhibited mitosis and cytokinesis in T. brucei. Thus, the alkynylthieno[3,2-d]pyrimidine chemotype is an advanced hit worthy of further optimization as a potential chemotherapeutic agent for HAT.

Full text of DOI:10.1021/acsmedchemlett.8b00245

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Letter
A Series of Alkynyl Substituted Thienopyrimidines
as Inhibitors of Protozoan Parasite Proliferation
Jennifer L Woodring, Ranjan Behera, Amrita Sharma, Justin Wiedeman, Gautam Patel,
Baljinder Singh, Paul J. Guyett, Emanuele Amata, Jessey Erath, Norma Roncal, Erica Penn,
Susan E. Leed, Ana Rodriguez, Richard J. Sciotti, Kojo Mensa-Wilmot, and Michael P. Pollastri
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00245 • Publication Date (Web): 04 Sep 2018
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A Series of Alkynyl-Substituted Thienopyrimidines as Inhibitors of
Protozoan Parasite Proliferation
Jennifer L. Woodring,† Ranjan Behera,‡ Amrita Sharma,‡ Justin Wiedeman,‡ Gautam Patel,† Baljinder
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Singh,† Paul Guyett,‡ Emanuele Amata,† Jessey Erath,∥, Norma Roncal,⊥ Erica Penn,⊥ Susan E. Leed,⊥
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Ana Rodriguez,∥, Richard J. Sciotti,⊥ , Kojo Mensa-Wilmot,‡ and Michael P. Pollastri*a†
†Department of Chemistry & Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA USA. Tel:
617-373-2703; E-mail: m.pollastri@northeastern.edu
‡Department of Cellular Biology, Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA
USA
∥Department of Microbiology, New York University School of Medicine, 430 E. 29th St. New York, NY USA
⊥Experimental Therapeutics, Walter Reed Army Institute of Research, 2460 Linden Lane, Silver Spring, MD, 20910
§Anti-Infectives Screening Core, New York University School of Medicine, New York, NY 10010
ABSTRACT: Discovery of new chemotherapeutic lead agents can be accelerated by optimizing chemotypes proven to be effective
in other diseases to act against parasites. One such medicinal chemistry campaign has focused on optimizing the anilinoquinazoline
drug lapatinib (1) and the alkynyl thieno[3,2-d]pyrimidine hit GW837016X (NEU-391, 3) into leads for anti-trypanosome drugs. We
now report the structure-activity relationship studies of 3 and its analogs against Trypanosoma brucei, which causes human African
trypanosomiasis (HAT). The series was also tested against Trypanosoma cruzi, Leishmania major, and Plasmodium falciparum. In
each case, potent antiparasitic hits with acceptable toxicity margins over mammalian HepG2 and NIH3T3 cell lines were identified.
In a mouse model of HAT, 3 extended life of treated mice by 50%, compared to untreated controls. At the cellular level, 3 inhibited
mitosis and cytokinesis in T. brucei. Thus, the alkynylthieno[3,2-d]pyrimidine chemotype is an advanced hit worthy of further opti-
mization as a potential chemotherapeutic agent for HAT.
KEYWORDS: Trypanosomes, human African trypanosomiasis (HAT), leishmaniasis, Chagas, malaria, protozoan parasite inhibti-
ors, thienopyrimidines
Target class repurposing is a method for discovering new chem-
ical matter against human African trypanosomiasis (HAT),1, 2
Following our success in identifying lapatinib as a lead for anti-
HAT drug discovery15-17, we tested other lapatinib-related com-
pounds synthesized by GlaxoSmithKline18 for activity against
T. brucei. Compound GW837016X (NEU-391, Figure 2, com-
pound 3) is a covalent inhibitor of human ErbB-2 kinase.18-21
This compound inhibits proliferation of bloodstream T. brucei
in vitro (EC50 of 0.4 µM).22 Unlike compound 1, 3 features a
thieno[3,2-d]pyrimidine scaffold and an alkyne warhead at the
6-position that covalently modifies a cysteine residue of ErB-
2.21 We have previously explored bioisosteric replacement of
the alkynyl functionality,22 and we herein report a wider range
of substituted alkynes and other scaffolds (Figure 2). Besides
the thieno[3,2-d]pyrimidine chemotype, we assessed matched
isomeric thieno[2,3-d]pyrimidine and quinazoline scaffolds. Fi-
nally, we describe (i) the biological effects of 3 on T. brucei, (ii)
characterization of the effects on trypanosome cell cycle, and
(iii) efficacy of compound 3 in a mouse model of HAT.
The thienopyrimidine (4, 5) and quinazoline (6) templates
were synthesized by previously published methods.22-25 Each
template was treated with a terminal alkyne via a Sonogashira
coupling reaction, shown in Scheme 1, to achieve the desired
product. Those alkynes that were not commercially available
were prepared as outlined in Scheme S1 in the Supporting In-
formation.
a
life-threatening disease caused by the protozoan parasite Tryp-
anosoma brucei.3 By taking known human inhibitors of a well-
characterized family of targets, inhibitors can be identified and
re-optimized for antiparasitic activity. We have applied this ap-
proach against T. brucei.4-9 For example, during a screen of hu-
man tyrosine kinase inhibitors, lapatinib (Figure 1, compound
1), an FDA approved drug for breast cancer tumors,10-13 was
found to be effective against T. brucei growth with an EC50 of
1.4 µM.14 Optimization of the lapatinib scaffold led to the syn-
thesis of NEU-617 (2), which was 40-fold more potent than
lapatinib (1) and had improved selectivity over human HepG2
cells.7 Further optimization of 2 to improve the physicochemi-
cal properties led to NEU-1961 (2a) with improved LogP and
LLE.15
Figure 1. Optimization of lapatinib
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8
T.b. brucei
EC50
SEM (µM)
or % inh at
5 µMb
±
HepG2
TC50
R
Entry
Scaffolda
SIc
(µM)
7k
9k
A
C
12%
>28
>29
--
--
19%
9
7l
9l
A
C
0.76 ± 0.07
2%
>31
>41
--
10
11
12
13
14
15
16
17
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19
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21
22
23
24
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>3.0
Figure 2. Analog designs.
7m
A
15%
>24
--
Scheme 1. Synthesis of alkynyl derivativesa
7n
7o
A
A
11%
33%
>32
>28
--
--
7p
7q
A
A
19%
31%
30
--
--
>5.0
7r
7s
A
A
1.3 ± 0.03
1.2 ± 0.03
24
18
aReagents and conditions: (i) Alkyne, PdCl2(PPh3)2, CuI, TEA,
DMF, 55 oC, 12 h, 6-52%
>26
>22
Table 1. Inhibition profile of alkynes against T. brucei. Com-
pare compounds to 3, T. brucei EC50 =0.26/0.4 µM*
7t
A
0.22 ± 0.02
0.04 ± 0
9.6
44
Suramin
*EC50 =0.26 µM is obtained from our initial screening program with parasite
concentration of 2 x103 cells/mL and for direct comparison with other com-
pounds, 3 was retested in same assays that its derivatives were tested in at 5 x104
cells/mL (EC50 =0.4 µM). aAr is 3-chloro-4-((3-fluorobenzyl)oxy)phenyl. bCom-
pounds showing >75% growth inhibition at 5 µM for T. brucei, were tested for
EC50 values. T. brucei EC50 values are the result of triplicate experiments. cSI is
Selectivity index= HepG2 TC50/ Tbb EC50.
T.b. brucei
EC50
±
HepG2
TC50
(µM)
R
Entry
Scaffolda
SEM (µM)
or % inh at
5 µMb
16%
48%
27%
2.5 ± 0.04
1.0 ± 0.06
1.0 ± 0.07
27%
SIc
All compounds in series 7, 8 and 9 had different alkyne
groups, and they were first tested against T. brucei cultures at a
single concentration (5 or 10 µM); those inhibiting proliferation
>75% were advanced to EC50 determination. Several com-
pounds demonstrated sub-micromolar potencies. Of the new an-
alogs, 7t, 7j, and 7l were the most potent with EC50 values of
0.23 µM (selectivity index, SI = 43.6), 0.28 µM (SI = 17.5), and
0.76 µM (SI = 40.8), respectively. Compound 7t (EC50 = 0.22
µM) is the enantiomer of the reference compound 3 (EC50 = 0.4
µM)and is slightly better in terms of potency. . Addition of the
Boc-protecting group to the pyrrolidine ring in 7s resulted in a
5-fold loss of potency against T. brucei. This is likely due to an
increase in steric encumbrance of the Boc group or reduction of
cellular permeability and not due to the loss of the hydrogen
bond donor, given the activity of both 7j and 7l. Furthermore,
small aliphatic cyclic rings found in 7t, 7j, and 7l were more
potent than both pyridine 7r (EC50 = 1.3 µM) and larger cyclic
aliphatic systems such as morpholine 7a (16% inh/5 µM), pi-
perazine 7b (EC50 = 2.5 µM) or thiomorpholine 7k (12% inh/5
µM). This may suggest either unfavorable steric interactions
within the putative binding site of the target(s), or unfavorable
effects on cellular permeability. Conversely, some steric bulk is
needed in this region as evidenced by a loss in activity exhibited
7a
8a
9a
7b
8b
9b
7c
8c
A
B
C
A
B
C
A
B
>29
>29
>30
11
15
8.2
26
--
--
--
4.3
15
8.2
--
53%
>28
--
9c
C
23%
>28.0
--
7d
8d
7e
A
B
A
18%
0%
>27
21.6
29.3
--
--
--
31%
8e
7f
8f
B
A
B
1%
1%
0%
>32
26.5
17
--
--
--
7g
8g
A
B
52%
>32
>32
--
2.5 ± 0.04
>13
7h
8h
A
B
16%
>29
7.7
--
1.9 ± 0
4.1
by smaller aliphatic substituted compounds such as 7i (EC50
=
7i
8i
7j
8j
A
B
A
B
3.4 ± 0.05
18%
0.28 ± 0.07
>33
9.5
4.9
>9.7
--
18
3.4 µM) and 7g (52% inh/5 µM) when compared with 3 (EC50
= 0.26 µM). The methylated piperazine tail 8b (EC50 = 1.0 µM)
was slightly more potent than the unsubstituted piperazine 8h
>2.5
>30
--
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Table 2. Inhibition profile of alkynes against T. cruzi amastigotes, L. major amastigotes and P. falciparum D6
1
2
3
4
5
6
7
8
T. cruzi amastigotes
NIH3T3
TC50 (µM)
L. major amastigotes
P. falciparum D6
HepG2
TC50 (µM)
Entry
SIb
SId
SIe
EC50 ± SEM (µM)a
EC50 in µM (r2)c
EC50 in µM (r2)c
7a
8a
9a
7b
8b
9b
7c
8c
9c
7d
8d
7e
8e
7f
8f
7g
8g
7h
8h
7i
8i
7j
8j
7k
9k
7l
9l
7m
7n
7o
7p
7q
3.4 ± 3.4
nt
4.7
nt
33
4.8
6.7
7.2
52
1.4
--
41
2.3
1.9
2.9
5.4
--
>6.3
--
1.3
--
>1.7
--
1.3
>6.0
16
7.6 (0.92)
6.5 (0.94)
5.5 (0.85)
>15
8.1 (0.94)
10 (0.81)
4.6 (0.85)
9.0 (0.87)
>15
3.8
4.5
>5.5
--
1.9
0.8
5.9
3.1
--
3.0 (0.98)
3.0 (0.90)
5.9 (0.83)
0.64 (0.99)
0.26 (0.94)
0.16 (0.98)
3.8 (0.91)
>15
>15
>15
4.8 (0.93)
>15
5.7 (0.95)
>15
>9.7
>9.7
>5.1
17
58
51
7.1
--
--
--
4.6
--
>29
>29
>30
11
15
8.2
0.8 ± 0.06
2.1 ± 0.18
3.5 ± 0.11
2.5 ± 0.46
9.6 ± 1.43
>50
8.0 ± 0.30
>50
1.9 ± 0.08
>50
30 ± 7.9
>50
1.7 ± 0.11
8.2 ± 0.27
2.6 ± 0.07
>50
0.61 ± 0.16
30.7 ± 8.7
1.5 ± 0.18
1.8 ± 0.17
nte
5.0 ± 0.53
2.5 ± 0.21
>50
2.0 ± 0.16
13 ± 3.5
>50
>50
19 ± 1.6
>50
12 ± 0.45
>50
3.7 ± 0.23
0.79 ± 0.01
27
>50
>50
>50
2.5
>50
>50
>50
2.2
>50
41
>50
3.9
>50
1.7
6.4
nte
>50
>50
>100
>50
47
>50
>50
47
>28
>28
>27
22
29
>32
27
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
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
>15
11 (0.92)
>15
--
2.0
--
>15
--
>5.6
--
21
6.0 (0.85)
1.4 (0.92)
2.5 (0.91)
5.5 (0.89)
4.0 (0.86)
9.1 (0.88)
9.4 (0.77)
7.3 (0.93)
4.1 (0.77)
4.3 (0.95)
5.9 (0.81)
5.4 (0.93)
>15
4.5
12
13
>5.8
7.3
0.85
3.5
1.3
1.2
7.0
4.8
>5.4
--
0.80 (0.99)
>15
17
--
>32
>32
>29
7.7
>33
9.5
1.5 (0.98)
1.9 (0.97)
0.38 (0.98)
5.9 (0.89)
3.02 (0.97)
1.3 (0.96)
0.14 (0.99)
>15
1.1 (0.96)
3.6 (0.99)
>15
1.5 (0.99)
>15
5.7 (0.98)
1.1 (0.97)
>3.6
2.0 (0.90)
1.7 (0.99)
0.94 (0.97)
>21
>15
20
>5.6
3.2
3.8
>214
--
>26
>8.6
--
>19
--
4.9
27
--
12
>15
10
--
6.4
>1.6
1.1
3.6
--
>10
>20
--
>25
3.6
--
--
2.5
--
4.9
>30
>28
>29
>31
>3.0
>24
>32
>28
30
>5.0
24
>26
9.6
>2
6.9 (0.83)
>15
>15
>15
--
3.5
--
--
--
>50
27
>100
10
>3
--
7r
7s
7t
2.3
--
2.7
--
0.38 (0.94)
1.6 (0.97)
4.2 (0.94)
63
16
2.3
Benznidazole
Amphotericin B
Chloroquine
--
0.035 (0.90)
--
--
--
--
--
--
--
--
0.0089 (0.97)
aT. cruzi EC50 values are the result of triplicate experiments. bSelectivity index= NIH3T3TC50/ Tc EC50. nt: not tested. cCompounds screened against L. major
amastigotes or P. falciparum (D6 strain) either in duplicate or quadruplicate. dSelectivity index= HepG2 TC50/ Lmj EC50. eSelectivity index= HepG2 TC50/ Pfal EC50
.
(EC50 = 1.9 µM), though the analogous morpholine 8a showed
little activity (48% inh/5 µM). The quinazoline series (9) gen-
erally displayed only modest potencies.
Against Leishmania major amastigotes, 15 compounds
were active in the micromolar range (< 10 µM) but only one
compound had sub-micromolar activity (7r, EC50 value of 0.38
µM, SI=62.1, Table 2). The P. falciparum screen yielded 9 an-
alogs with sub-micromolar activity. The most potent com-
pounds were 8j (EC50 = 0.14 µM, Table 2) and 9b (EC50 = 0.16
µM, Table S2, Supporting Information). Several alkynes exhib-
ited sub-micromolar activity irrespective of the central scaffold,
they included 7b, 8b, 9b, 8f, 8h, 8j, 7t (Table 2) and 9b (Table
S2, Supporting Information). These compounds were also
screened against the drug-resistant W2 and C235 strains of P.
falciparum (Table S2, Supporting Information); all compounds
were within a 3-fold potency window compared to the drug-
sensitive D6 strain, indicating no cross-resistance with chloro-
quine, mefloquine or pyrimethamine.
HepG2 cells were employed to screen for overt toxicity,
and to calculate the selectivity index of the anti-parasitic hits.
Three compounds had selectivity index of 20 or more against T.
brucei over HepG2 cells. The selectivity ranged from 43x (7t)
to 4.3x (7b). In case of selectivity of compounds against P. fal-
ciparum D6 over HepG2 cells, there were eight compounds
with selectivity of 20 or more; for P. falciparum 8j had the best
selectivity index of more than 200x. We also tested compounds
against NIH 3T3 cells, which are the host cell line utilized for
We have had previous success in “parasite hopping”,17
wherein compounds prepared as potential T. brucei inhibitors
are tested against other protozoan parasites. Thus, this library
was screened against intracellular amastigotes of T. cruzi (re-
sponsible for Chagas disease) and of Leishmania major (leish-
maniasis). Based on our previous observation that thienopyrim-
idines have activity against Plasmodium falciparum,22 the li-
brary was also tested against different strains of Plasmodium
falciparum (malaria). From these assays, compounds 8h (EC50
= 0.61 µM) and 9a (EC50 = 0.81 µM) (Table 2) showed sub-
micromolar activity against T. cruzi. There does appear to be a
preference for the quinazoline core over either thienopyrimi-
dine regioisomer. This is apparent when comparing compounds
9a and 7a, where there is a ~4-fold improvement in potency for
the quinazoline core. A similar trend is observed for 7k (EC50
=
5.0 µM) versus 9k (EC50 = 2.5 µM) and 7l (EC50 = >50 µM)
versus 9l (EC50 = 2.0 µM). Interestingly, there appears to be a
preference for the morpholine (9a; EC50 = 0.81 µM) and piper-
azine (8h; EC50 = 0.61 µM) over the thiomorpholine (9k; EC50
= 2.5 µM), indicating a sensitivity to electronegativity differ-
ences at this position.
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culturing the intracellular amastigotes of T. cruzi; This data is
V ehicle
C o m p o u n d
3
10 1 0
10 9
10 8
10 7
10 6
10 5
p < 0 .0 0 1
1
2
3
4
5
6
7
8
summarized in Table 2. Three compounds had selectivity index
of 20 or more against T. cruzi over NIH 3T3 host cells; Com-
pound 9a had the best selectivity index of 41.
The dose response curves of selected compounds against
T. brucei, P. falciparum and L. major are provided in Figure S2,
S3 and S4 respectively in Supporting Information. Protocols for
in vitro proliferation inhibition assays of T. brucei, T. cruzi, L.
major, P. falciparum, and host cells are also provided in the
Supporting Information. These methods have been previously
described.8, 26-28
In the end, compounds 3, 7j and 7t were the most promis-
ing anti-T. brucei agents (Table 1). Noting that compound 3 was
previously dosed in mice orally at 100 mg/kg,21 we evaluated
its pharmacokinetics at 130 mg/kg for possible anti-HAT effi-
cacy studies in mice: It’s in vitro ADME properties were also
evaluated. These data, along with an optimized synthetic route
for scaling up compound 3, are described in the Supporting In-
formation.
N S
U N D
Ve h .
3
Ve h .
3
3
3
3
3
3
3
D P I
11
13
3
5
9
12
6
7
(B)
Figure 3. Evaluation of compound 3 efficacy in mouse model of acute
HAT. (A) Mouse (n = 4) survival curve post-infection for control and
compound 3 treated mice. (B) Parasitemia during compound 3 treat-
ment. Mice (four per group) were infected with 105 T. brucei Lister427
(day 0). After 24 h, compound 3 (130 mg/kg) was administered by oral
gavage once daily for 10 days. Control mice received vehicle (10%
NMP and 90% PEG). Parasitemia was determined every 24 h. Different
shapes in graph represent individual mice; DPI=days post-infection.
(Statistical significance of the difference in mean parasitemia was de-
termined with Student’s T-test)
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Compound 3 was dosed at 130 mg/kg orally (formulated in
10% NMP and 90% PEG) to female Swiss Webster mice, blood
samples were collected at 0.5, 1, 2, 4, 8, and 24 h and analyzed
by LC−MS/MS. The results are summarized in Figure S1 and
presented in Tables S4 and S5 in the Supporting Information.
The drug concentration observed in plasma was above EC50
value for up to 4h. Given the good in vitro potency and accepta-
ble plasma exposure in mice, we performed an efficacy study
of 3 in a mouse model of acute phase human African trypano-
somiasis.
Mice infected with T. brucei (Lister427) were dosed orally
with 130 mg/kg once daily for 10 days. The control group was
treated with vehicle (10% NMP and 90% PEG). Animals were
monitored on alternate days for parasitemia. In untreated (con-
trol) mice, parasitemia reached 108/mL of plasma by day 5
(mean parasitemia = 9.30 × 108/mL) (Figure 3b) and the mice
were euthanized. The mean survival of compound 3-treated
mice was 7.5 days compared to 5 days for untreated mice (Fig-
ure 3a). The difference in mouse survival times (2.5 days) was
not statistically significant (determined from chi-square analy-
sis of Kaplan-Meier survival data (p = 0.127)). Mice treated
with compound 3 did not show signs of toxicity. By day 5, the
treated group had two animals with 3-fold decrease in para-
sitemia and two animals with 400-fold decrease in parasitemia.
One of the latter mice had parasitemia of 4 ×104/mL on day 9,
representing a projected 108-fold decrease of trypanosomes in
blood compared to the untreated mice (Figure 3b). Drug dosing
was stopped on day 10 after which trypanosome proliferation
resumed, reaching 8 ×108/mL on day 13 all the mice were eu-
thanized (Figure 3b).
Figure 4. Compound 3 inhibits mitosis and cytokinesis. Trypanosomes
(5x105/mL) were treated with compound 3 (9.5 µM) or DMSO (0.1%)
in HMI-9 medium for 3 hours. Cells were incubated with mCLING,
and the kinetoplast and nuclear DNA was counterstained with DAPI.
The number of kinetoplasts and nuclei per trypanosome (n= >120) was
quantitated. The mean percentage of trypanosomes with indicated num-
ber of nuclei (N) or kinetoplasts (K) is shown. Trypanosomes with two
kinetoplasts and one or two nuclei are binned together for clarity of the
3 effect. Error bars represent standard deviation among three biological
replicates. The difference in distribution of kinetoplasts and nuclei be-
tween control and compound 3-treated trypanosomes was evaluated
with Pearson χ2 analysis (p = 5.85 x 10-7). Inset: Representative images
of mCLING and DAPI-stained trypanosomes after treatment with
DMSO. Scale bar, 2 µm. K= kinetoplast, N= nucleus.
Recently, we have reported that lapatinib (1) inhibited up-
take of transferrin, a growth factor29 in bloodstream trypano-
somes. Based upon the structure similarity of 3 to 1, we hypoth-
esized that these two compounds might inhibit proliferation via
the same mechanism. Thus, the possible effect of 3 on transfer-
rin (Tf-647) uptake was tested (Biological assay protocols in
Supporting Information). We found that compound 3 had no ef-
fect on transferrin uptake (data not shown), indicating that 3 in-
hibits T. brucei proliferation through a different mechanism
than lapatinib.
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C o m p o u n d 3
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0
Stages in trypanosome division can be classified by enu-
meration of several single organelles (e.g., kinetoplast (a nucle-
oid containing mitochondrial DNA termed kinetoplast DNA
30, 31
(kDNA), nucleus, flagellum).
Synthesis of mitochondrial
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and nuclear DNA occurs in S-phase in cells containing one nu-
cleus and one kinetoplast (1K1N) (Figure 4).32 Division of ki-
netoplasts is completed in G232 yielding 2K1N trypanosomes
(Figure 4) in which nucleus division occurs, producing 2K2N
S u rv iv a l P e rio d (D P I)
(A)
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We are grateful to AstraZeneca for performing the in vitro ADME
experiments tabulated in Table S3 in the Supporting Information
cells (Figure 4). Cytokinesis takes place in 2K2N trypanosomes
yielding new progeny with 1K1N genotype that can re-enter S-
phase.33
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REFERENCES
In “mode of action” studies to learn how compound 3 af-
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log phase T. brucei (5x105 cells/mL) were treated with either 3
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or DMSO for 3 hours, fixed, stained with mCLING
and
DAPI, and examined with a fluorescence microscope. The num-
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62.1), 8b (P. falciparum EC50=0.26 µM, SI = 57.7), 8j (P. fal-
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ASSOCIATED CONTENT
Supporting Information
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Patel, G.; Karver, C. E.; Behera, R.; Guyett, P. J.;
Sullenberger, C.; Edwards, P.; Roncal, N. E.; Mensa-Wilmot,
K.; Pollastri, M. P. Kinase scaffold repurposing for neglected
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derived lead compound for trypanosomiasis. J. Med. Chem.
2013, 56, 3820-3832.
The Supporting Information is available free of charge on the ACS
Publications website. Details of synthetic chemistry, biological as-
say protocols and other data (annotated with NEU registry num-
bers) are contained in the Supporting Information, as are ADME
and PK properties of selected analogs.
8.
Patel, G.; Roncal, N. E.; Lee, P. J.; Leed, S. E.; Erath,
J.; Rodriguez, A.; Sciotti, R. J.; Pollastri, M. P. Repurposing
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AUTHOR INFORMATION
Corresponding Author
9.
Devine, W.; Woodring, J. L.; Swaminathan, U.;
Amata, E.; Patel, G.; Erath, J.; Roncal, N. E.; Lee, P. J.; Leed,
S. E.; Rodriguez, A.; Mensa-Wilmot, K.; Sciotti, R. J.; Pollastri,
M. P. Protozoan Parasite Growth Inhibitors Discovered by
Cross-Screening Yield Potent Scaffolds for Lead Discovery. J.
Med. Chem. 2015, 58, 5522-5537.
*E-mail: m.pollastri@northeastern.edu. Tel: 617-373-2703
ORCID ID:
Michael P. Pollastri: 0000-0001-9943-7197
Baljinder Singh: 0000-0003-2828-5768
10.
Rusnak, D. W.; Lackey, K.; Affleck, K.; Wood, E. R.;
Author Contributions
Alligood, K. J.; Rhodes, N.; Keith, B. R.; Murray, D. M.;
Knight, W. B.; Mullin, R. J.; Gilmer, T. M. The effects of the
novel, reversible epidermal growth factor receptor/ErbB-2
tyrosine kinase inhibitor, GW2016, on the growth of human
normal and tumor-derived cell lines in vitro and in vivo. Mol.
Cancer Ther. 2001, 1, 85-94.
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manu-
script.
Funding Sources
Funding from the National Institutes of Health (R01AI124046,
R56AI099476 to M.P. and K.M-W.) is gratefully acknowledged.
11.
Xia, W.; Mullin, R. J.; Keith, B. R.; Liu, L. H.; Ma,
H.; Rusnak, D. W.; Owens, G.; Alligood, K. J.; Spector, N. L.
Anti-tumor activity of GW572016: a dual tyrosine kinase
inhibitor blocks EGF activation of EGFR/erbB2 and
downstream Erk1/2 and AKT pathways. Oncogene 2002, 21,
6255-6263.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
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A Series of Alkynyl Substituted Thienopyrimidines as Inhibitors of
Trypanosoma brucei Proliferation
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Jennifer L. Woodring,† Ranjan Behera,‡ Amrita Sharma,‡ Justin Wiedeman,‡ Gautam Patel,†
,§
Baljinder Singh,† Paul Guyett,‡ Emanuele Amata,† Jessey Erath,∥ Norma Roncal,⊥ Erica
,§
Penn,⊥ Susan E. Leed,⊥ Ana Rodriguez,∥ Richard J. Sciotti,⊥ , Kojo Mensa-Wilmot,‡ and
Michael P. Pollastri*a†
†Department of Chemistry & Chemical Biology, Northeastern University, 360 Huntington Avenue,
Boston, MA USA. Tel: 617-373-2703; E-mail: m.pollastri@northeastern.edu
‡Department of Cellular Biology, Center for Tropical and Emerging Global Diseases, University of
Georgia, Athens, GA USA
∥Department of Microbiology, New York University School of Medicine, 430 E. 29th St. New York, NY
USA
⊥Experimental Therapeutics, Walter Reed Army Institute of Research, 2460 Linden Lane, Silver
Spring, MD, 20910
§Anti-Infectives Screening Core, New York University School of Medicine, New York, NY 10010
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