Imidazolopiperazines: Hit to lead optimization of new antimalarial agents

Article abstract of DOI:10.1021/jm2003359

Starting from a hit series from a GNF compound library collection and based on a cell-based proliferation assay of Plasmodium falciparum, a novel imidazolopiperazine scaffold was optimized. SAR for this series of compounds is discussed, focusing on optimi

Full text of DOI:10.1021/jm2003359

ARTICLE
pubs.acs.org/jmc
Imidazolopiperazines: Hit to Lead Optimization of New
Antimalarial Agents
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Tao Wu, Advait Nagle, Kelli Kuhen, Kerstin Gagaring, Rachel Borboa, Caroline Francek,
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Zhong Chen, David Plouffe, Anne Goh, Suresh B. Lakshminarayana, Jeanette Wu, Hui Qing Ang,
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§
^
^
Peiting Zeng, Min Low Kang, William Tan, Maria Tan, Nicole Ye, Xuena Lin, Christopher Caldwell,
†
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^
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†
Jared Ek, Suzanne Skolnik, Fenghua Liu, Jianling Wang, Jonathan Chang, Chun Li,
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‡,||
‡,||
Thomas Hollenbeck, Tove Tuntland, John Isbell, Christoph Fischli, Reto Brun, Matthias Rottmann,
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Veronique Dartois, Thomas Keller, Thierry Diagana, Elizabeth Winzeler, Richard Glynne,
†
,†
David C. Tully, and Arnab K. Chatterjee*
†
Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121,
United States
‡
Swiss Tropical and Public Health Institute, Parasite Chemotherapy, Socinstrasse 57, Basel, Switzerland CH-4002
§
Novartis Institute for Tropical Diseases, 10 Biopolis Road, no. 05-01 Chromos, Singapore 138670
University of Basel, Petersplatz 1, Basel, Switzerland CH-4003
^
Novartis Institute for Biomedical Research, Preclinical Profiling Group, Cambridge, Massachusetts, United States
ABSTRACT: Starting from a hit series from a GNF compound
library collection and based on a cell-based proliferation assay of
Plasmodium falciparum, a novel imidazolopiperazine scaffold was
optimized. SAR for this series of compounds is discussed,
focusing on optimization of cellular potency against wild-type
and drug resistant parasites and improvement of physiochemical
and pharmacokinetic properties. The lead compounds in this
series showed good potencies in vitro and decent oral exposure
levels in vivo. In a Plasmodium berghei mouse infection model,
one lead compound lowered the parasitemia level by 99.4% after
administration of 100 mg/kg single oral dose and prolonged mice survival by an average of 17.0 days. The lead compounds were also
well-tolerated in the preliminary in vitro toxicity studies and represents an interesting lead for drug development.
’
INTRODUCTION
using a cell-based proliferation assay of Plasmodium falciparum
5
(
strain 3D7). Five-thousand of the reconfirmed hits have been
After thousands of years, malaria is still one of the major
subsequently disclosed in a public malaria database (see https://
www.ebi.ac.uk/chembldb/index.php), and we analyzed the hit
list with the following criteria: (1) good potency, where IC50
values were less than 1 μM against wild type as well as drug
resistant strain; (2) good safety index, greater than 20-fold in a
six-cell line toxicity panel; (3) easy synthesis and preferably with
multiple active hits within the scaffold.
Compounds 1aÀc consisting of imidazolopiperazine moiety
presented themselves as an attractive hit series based on these
criteria (Table 1). Their structures were distinct from the current
known antimalarials such as aminoquinolines and endo-perox-
ides. Compounds 1aÀc represented decent starting points in
terms of potency in both wild type and drug resistant strain, and
the scaffold exhibited a high selectivity index (>20-fold) against
Huh7 cell line. Moreover, one of the representative compound,
infectious diseases that affect millions of people, especially those
in underdeveloped countries. In the absence of effective anti-
malarial vaccines, low molecular weight antimalarial drugs are
important weapons against the disease. Quinine, chloroquine,
mefloquine, and artemisinin derivatives have played an important
role in fighting against malaria. However, widespread drug
resistance has made them less effective with artemisinin deriva-
tives as the only exception. Artemisinin based combination
therapies (ACT) recommended by WHO will help to delay the
emergence of clinical resistance against artemisinins, but reports
1
2
3
of increased parasite clearance times have emerged recently.
Therefore, it is important to discover antimalarials with novel
chemical entities that are effective against the multidrug resistant
parasite strains. Here we report our continued effort to develop
4
novel antimalarials from a cell-based screening strategy.
In our quest to discover novel chemotypes that inhibit
Plasmodium falciparum, we previously disclosed a more extensive
screening campaign on a library of about 2 million compounds
Received: March 22, 2011
Published: June 06, 2011
r 2011 American Chemical Society
5116
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|J. Med. Chem. 2011, 54, 5116–5130

Journal of Medicinal Chemistry
ARTICLE
Table 1. Structures of Hits from the HTS Screen
a
P. falciparum strain IC50 (nM)
cyotoxicity (μM)
compd
EBI code
R
1
3D7
W2
Huh7
1
1
1
a
b
c
GNF-Pf-5069
GNF-Pf-5179
GNF-Pf-5466
Gly
63 [460]
235 [119]
116 [30]
97 [473]
271 [122]
119 [29]
>10 [>100]
>10 [>79]
>10 [42]
(DL)-Phe
(DL)-Leu
a
Values are the mean of at least two experiments. Data in brackets are for resynthesized powders; otherwise data are from HTS DMSO stock solutions.
amidation reaction with N-Boc-glycine followed by a TFA
mediated deprotection of the amino group. When not commer-
cially available, isocyanides were prepared in a formylationÀ
8
dehydration sequence from the corresponding anilines.
To efficiently evaluate the R section and avoid repeated
3
preparation and usage of notoriously noxious isocyanides, com-
pound 6 was prepared from the same GroebkeÀBlackburn three-
component reaction of 2-aminopyrazine with tert-butyl isocya-
nide and 4-fluorobenzaldehyde. After removal of the tert-butyl
group, the obtained compound 7 was then used as a common
intermediate. A palladium mediated amination with different aryl
Figure 1. Initial hit of imidazolopiperazine series and plans to optimize
the three peripheral parts.
9
bromides provided various analogues of compound 7.
1
a, was highly soluble (>175 μM at pH 6.8) and clean on most
’
RESULTS AND DISCUSSION
commonly drug affected cytochrome P450 isoforms (IC values
50
were greater than 10 μM for CYP1A2, CYP2C19, CYP2C9,
CYP2D6, and CYP3A4). Despite the basic amine functionality
StructureÀActivity Relationship. The potency of the com-
pounds was determined against the 3D7 and W2 multidrug
resistant strains using Sybergreen assay previously described in
on R , compound 1a was a moderate hERG substrate as deter-
1
5
mined by binding assay (IC = 19 μM, 57% inhibition at 10 μM).
our HTS disclosure (Table 2). Each assay plate had mefloquine
50
However, compound 1a exhibited poor plasma oral exposure with
Cmax = 320 nM and AUC = 972 (h nM) in an oral mouse
and artemisinin as standards to ensure the reliability of the assay.
We decided to (1) test various natural and unnatural amino acids
(0À5h)
3
snapshot PK study, indicating the presence of metabolic soft spots
on R , (2) add substituents to the right side phenyl ring, and (3)
1
6
within the molecule or overall poor permeability properties.
replace the methylenedioxyl moiety with substituted phenyl
rings, heterocycles, and aliphatic rings. We began exploring the
SAR studies on the initial hit compounds 1aÀc by determining
the minimal pharmacophore needed for the antiplasmodial
activity. On the basis of the preliminary results, it was clear that
dearomatized compound 3 provided us with a decent starting
point for further SAR exploration on all the three parts of the
molecule. Installation of a glycine group gave us compound 5,
which was 4-fold more potent than the original hit compound 1a.
On the basis of this result, we focused our efforts on evaluating
various amino acids. Increasing the chain length of the amino acid
moiety led to 8, which had inferior activity. Various monosub-
stitution on the R position provided potent compounds
(compounds 9, 10, 12, and 13) which were less than 100 nM
in potency range. The testing of both of the enantiomers of
phenylalanine derivatives indicated that the stereochemistry of
the amino acid moiety was not important. Removal of chiral
center from compound 9 led us to R-methylalanine derivative 11.
The major goal of optimization was to improve potency and
remove metabolically vulnerable functionality of the compounds.
Figure 1 shows our initial objectives. In line with the three
possible metabolic soft spots of compound 1a, we decided to
study the three peripheral sections: the amino acid moiety R , the
1
phenyl moiety R , and the aniline moiety R . We hoped changes
2
3
in these parts would help us deliver a candidate with enhanced
potency as well as improved oral exposure.
’
CHEMISTRY
The synthesis of imidazolopiperazine compounds such as 5 is
outlined in Scheme 1. A GroebkeÀBlackburn three-component
reaction of 2-aminopyrazine with 4-fluorophenyl isocyanide and
7
4
-fluorobenzaldehyde efficiently furnished the skeleton. A PtO
2
mediated reduction of the pyrazine ring then afforded desired
8
core structure. Compound 5 was obtained by a HATU mediated
5
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Journal of Medicinal Chemistry
ARTICLE
a
Scheme 1. Representative Synthetic Route of Imidazolopiperazines
a
4 2 2
Reagents and conditions: (a) HClO , MeOH, room temp, 48%; (b) PtO , H (balloon), MeOH, room temp, quantitative; (c) N-Boc-glycine, HATU,
DIEA, DMF, room temp, 72%; (d) TFA, DCM, room temp, quantitative; (e) TFA, DCM, room temp, quantitative; (f) 1-bromo-3-fluorobenzene,
Pd (dba) , Xantphos, Cs CO , dioxane, 150 ꢀC, 66%.
2
3
2
3
This compound had a decent activity of 20 nM against the
parasites but offered an enormous advantage of blocking a
potential metabolic soft spot and removing chirality in the
molecule. A number of second and tertiary amines on the amino
acid side chain had deleterious effects on the potency
Table 2. SAR Study of R Moiety
1
(
compounds not shown). The future analogues were optimized
using glycine or R-methylalanine as the side chain.
The study of the R group revealed that this portion of the
2
molecule was not amenable to broad changes (Table 3). Com-
pounds 5, 14, and 15 indicated that only small size substituents
such as fluorine or hydrogen were tolerated. A comparison of
compounds 5, 18, and 19 indicated that para and meta substitu-
tions were tolerated but not ortho substitution. 3,4-Difluoro
substitution (compound 20) did not provide additional boost
in potency, and hence, it was inferred that 4-fluoro substitution
provided the best atom economy for further SAR optimization.
Attempts to replace the phenyl ring with a cyclohexyl group led to
total loss in potency (compound not shown).
a
P. falciparum strain IC50 (nM)
compd
R
1
3D7
W2
2
3
5
8
9
>10
200
20
5520
175
23
H
Gly
β-Ala
70
75
(L)-Ala
(L)-Val
90
64
1
1
1
1
0
1
2
3
30
24
After optimizing the R and R groups, we focused our attention
1
2
R-Me-Ala
(L)-Phe
20
25
on the aniline portion of the molecule (Table 4). One general trend
110
70
121
59
was that compounds with glycine as R were usually more potent
1
(D)-Phe
than their counterparts with dimethylglycine as R . The potencies
1
mefloquine
pyrimethamine
artemisnin
12
8
for compounds 21 and 22 indicated that aliphatic rings and
heterocyclic rings were not tolerated. A number of different
substituents (compounds 23À28) at either meta or para position
gave active compounds. With the same substituents, 3,4-disubstitu-
tion was preferred over 2,4-disubsitution and 3,5-disubstitution
patterns(compounds33 and 34). The most potent compound was
29
>10000
14
12
a
Values are the mean of at least two experiments.
Lipinski’s rule-of-five compliance and most of the analogues were
highly soluble (>175 μM at pH 6.8).
3
1, which exhibited an EC of 3 nM on 3D7 and of 4 nM on W2.
50
ADMET and Pharmacokinetic Properties. Along with the
Table 5 summarizes the in vitro ADMET profiles of com-
pounds with good in vitro potency. In general, metabolic stability
of the compounds was good to moderate across three species,
with rat being the most sensitive species and thus exhibiting the
highest hepatic extraction ratio (ER). One interesting finding was
extensive SAR study to optimize the potency of this series, some
of the more potent analogues in different categories were
evaluated for their in vitro ADME and in vivo pharmacokinetic
properties. In general, this series of compounds had very good
5
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Journal of Medicinal Chemistry
ARTICLE
Table 3. SAR Study of R Moiety
Table 4. SAR Study of R Moiety
3
2
a
IC50 (nM)
compd
R
1
R
2
R
3
3D7
W2
a
P. falciparum strain IC50 (nM)
2
1
2
R-Me-Ala
R-Me-Ala
Gly
4F-Ph
4F-Ph
4F-Ph
3F-Ph
4F-Ph
4F-Ph
4F-Ph
4F-Ph
4F-Ph
4F-Ph
4F-Ph
4F-Ph
4F-Ph
4F-Ph
cyclohex-
3-pyridine
3F-Ph
9610
1413
50
5980
3320
34
compd
R
1
R
2
3D7
W2
2
14
15
16
17
18
19
20
R-Me-Ala Ph
200
660
2270
3140
10
168
437
1702
3360
30
23
24
25
26
27
28
29
30
31
Gly
Gly
Gly
Gly
4Cl-Ph
R-Me-Ala
Gly
4F-Ph
50
71
4OMe-Ph
4Me-Ph
3F-Ph
4Me-Ph
4Me-Ph
4Cl-Ph
10
13
R-Me-Ala
Gly
20
24
10
9
R-Me-Ala 2F-Ph
1390
60
1284
50
R-Me-Ala
Gly
4Cl-Ph
30
24
b
R-Me-Ala 3F,4F-Ph
3F,4F-Ph
3F,4F-Ph
3F,4Cl-Ph
3F,4Cl-Ph
2F,4F-Ph
3F,5F-Ph
30
23
a
b
Values are the mean of at least two experiments. Compound with
-methylphenyl as R3.
R-Me-Ala
Gly
44
36
4
3
4
3
2
R-Me-Ala
R-Me-Ala
R-Me-Ala
40
52
that analogues with glycine as R and analogues with dimethyl-
glycine as R behaved quite differently in terms of hERG activity.
33
34
110
220
90
1
1
244
Although the glycine analogues offered a safety advantage in
terms of low affinity to hERG channel, it was negated by low
permeability, as indicated by PAMPA and Caco-2 assays. In
general, this also led to lower bioavailability as discussed below.
While the dimethylglycines indicated some efflux due to high
BÀA values, this generally did not lead to lower bioavailability,
potentially indicating a saturation of efflux mechanism (such as
p-glycoproteins) at the doses used in the oral PK studies.
The in vivo pharmacokinetic properties were studied in non-
infected male balb/C or female CD-1 mice. The overall trend for
the oral exposure was in line with the predictions from the
permeability assays. Four pairs of head to head comparison
a
Values are the mean of at least two experiments.
chloroquine (CQ) and artesunate (AS). At 1 Â 100 mg/kg dose
level, most of the compounds could reduce the parasitemia for
more than 99% and prolong mice survival for more than 10 days.
Interestingly, for the three head-to-head comparison pairs (entry
2
5/26, entry 31/32, and entry 18/24), the glycine analogues were
less efficacious at 1 Â 100 and 1 Â 30 mg/kg dose levels despite
their better potencies. It was likely that the better oral exposure of
the dimethylglycine analogues is responsible for this outcome.
Among all the tested compounds, compounds11and30stood out
as the most efficacious compounds at 1 Â 100 and 1 Â 30 mg/kg
dose levels. They both showed parasitemia reduction activities and
survival prolongation comparable those of CQ and AS. When the
(
entry 11/5, entry 18/24, entry 25/26, and entry 31/32) of the
oral arm Cmax, AUC, and bioavailability revealed that dimethylgly-
cine analogues generally had low hepatic in vivo clearance which
resulted in higher AUCs as well as making the compounds more
orally available (Table 6). Upon comparison of the pharmacoki-
netic profiles (Figure 2) for both compounds, it was observed that
compound 32 has much higher exposure in the oral arm whereas
the intravenous kinetics were comparable for both molecules (graph
for compound 31 not shown for clarity). Some of the tested
compounds exhibited high volume of distribution (>10 L/kg)
and high plasma clearance (>90 (mL/min)/kg) in mice. On the
basis of data from in vitro mouse blood to plasma partitioning of
structural analogues with blood to plasma ratio of >1, these
compounds might be partitioned into red blood cells leading
to overestimation of blood clearance and extent of tissue distribution.
In Vivo Efficacy. The in vivo antimalarial activities of the
optimized compounds were evaluated using a Plasmodium berghei
1
 30 mg/kg and 3  30 mg/kg dose levels are compared, both
compounds 11 and 30 demonstrated improved efficacy.
In Vitro Toxicology. Because of their good in vitro potency
and in vivo efficacy results, compounds 11 and 30 were evaluated
for their toxicity profile. Both compounds did not inhibit cyto-
chromes P450 isoforms (IC values were greater than 8.9 μM for
CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4). The
observed cytotoxicity TC50 values (293T, Ba/F3, CHO, HEp2,
HeLa, Huh7) were all greater than 12 μM, which translates to a
good selectivity index (SI > 100). Compound 11 was well
tolerated in the maximum tolerable dose test in rats in vivo with
no gross effects at doses up to 500 mg/kg. Compound 30 was
negative in the mini-Ames and micronucleus tests. Theonly safety
concern for these two lead compounds were their hERG liability.
Although compounds 11 and 30 exhibited IC of 5.6 and 4.8 μM,
50
50
1
0
mouse model. In this test, groups of three P. berghei infected
mice were treated orally with compound starting 1 day after
infection. The key readouts were the percentage of parasitemia
reduction on day 3 postinfection (day 4 postinfection in the case
of a 3-day treatment schedule) compared to the untreated mice
and the prolongation of survival of the infected mice.
respectively, there was a toxicity index of 100-fold when compared
to the cellular potency against the parasites.
’
SUMMARY
A series of imidazolopiperazines was selected as one of the
good hits from a high throughput screen of two million library
compounds against a cell based parasite proliferation assay. After
Table 7 summarized the in vivo efficacy test results of
selected compounds compared with the standard antimalarials
5
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Journal of Medicinal Chemistry
ARTICLE
a
Table 5. In Vitro ADME of Selected Analogues
hepatic microsome metabolic stability (extraction ratio)
PAMPA
Caco-2 AÀB
Caco-2 BÀA
hERG (Q-patch)
6
6
compd
(% absorbed)
(cm/s  10 )
(cm/s  10 )
mouse
rat
nt
human
IC50 (μM)
5
52
0.70
3.05
0.56
1.07
1.63
0.96
0.44
1.46
0.99
0.14
0.75
0.84
2.55
8.38
2.32
2.6
0.47
0.51
0.31
0.46
0.17
0.19
0.17
0.17
0.21
0.17
0.57
0.76
<0.21
0.52
nt
nt
1
1
2
2
2
2
2
2
3
3
3
1
8
4
5
6
7
8
9
0
1
2
81
0.63
nt
5.6
>30
5.7
11.1
1.5
nt
low
92
nt
nt
52
3.58
1.66
1.25
1.86
2.99
1.03
0.96
0.87
nt
nt
96
0.87
0.63
0.43
0.38
0.12
0.6
0.54
0.55
0.54
0.21
0.39
0.37
0.48
52
89
2.3
nt
low
86
4.8
21
low
94
0.67
5.6
a
nt = not tested.
a
Table 6. In Vivo Pharmacokinetic Profile of Selected Analogues after Oral Dose at 30 mg/kg and iv Dose at 5 mg/kg in Mice
intravenous PK parameters
oral PK parameters
compd
V
d
(L/kg)
CL (mL/min/kg)
T
1/2 (h)
AUC(inf) (nM h)
Cmax (nM)
T
max (h)
T
1/2 (h)
AUC(inf) (nM h)
F (%)
3
3
5
12.3
23.7
21.6
12.8
39.7
41.1
40.5
14.0
1.5
188.9
60.0
192.3
134.2
222.2
42.0
65.2
27.7
10.3
5.8
2.7
6.2
1.8
2.3
3.6
13.7
8.7
7.7
3.8
7.8
1072
3442
1232
1319
976
234
1803
417
2.0
4.2
9.0
965
17894
1330
15
87
18
47
40
57
158
76
5
1
1
2
2
2
2
3
3
3
1
8
4
5
6
8
0
1
2
1.67
2.0
1.0
778
0.50
2.0
2.3
3719
343
4.8
2343
5253
2985
7003
20582
25464
1007
749
2.0
15.5
37.6
7.7
17964
28301
31758
6175
1.00
1.67
2.0
1940
790
3.6
1.7
3319
2.0
9.1
36668
24
a
Intravenous dosing was at 5 mg/kg. Compounds 11 and 30 were dosed at 20 mg/kg. PK values were normalized to 30 mg/kg for comparison. For
compounds 11 and 30: Species/strain, Balb/c mice. Formulation: 2.5 mg/mL in PEG300/D5W, 3:1, solution. All of the other compounds were dosed at
0 mg/kg po; strain, CD-1 female mice. Solution formulation with citrate buffer 50 mM (pH 4.5) for iv route and citrate buffer 100 mM (pH 3.5) for po route for
3
compounds 5, 18, 24, 25, 26, and 28. Solution formulation for compounds 31 and 32: Ethanol/PEG400/10% vitamin ETPGS (10:30:60) was used.
physicochemical and pharmacokinetic properties as well as safety
profile. In the rodent P. berghei model, compounds 11 and 30
were as efficacious as the known antimalarials at 1 Â 100 and 1 Â
3
0 mg/kg dose levels.
The mechanism of actions for the imidazolopiperazine series is
not clear. The tested compounds were active against 15 Plasmo-
dium falciparum parasite strains with no potency shift, which
proved their effectiveness against the existing multidrug resistant
parasite stains. In a time when artemisinin based therapy is the last
defenseline formultidrug resistant malariaparasite strains, a novel
effective chemical entity with distinct structure and mechanism of
action will help to offer a new layer of protection. The continued
optimization of this series will be reported in due course.
Figure 2. Graphical representation of po and iv kinetics for compounds
1 and 32.
’
EXPERIMENTAL SECTION
3
Biology. Maintenance of Plasmodium falciparum Cul-
tures. Plasmodium falciparum cultures are grown in O RBCs using
culturing medium: RPMI 1640 medium (no phenol red) with
+
extensive optimization, the most potent compounds achieved
single digit nanomolar range. The lead compounds showed good
5
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Journal of Medicinal Chemistry
ARTICLE
Table 7. In Vivo Efficacy of Selected Analogues on P. berghei
Rodent Malaria Model after Different Dose Levels
fluorescence intensity using the Envision plate reader (Perkin-Elmer)
with a 505 dichroic mirror. Excitation and emission filters are 485 and
a
530 nm, respectively. Data are normalized based on maximum fluores-
1
 30 mg/kg
parasitemia
reduction survival reduction survival reduction survival
1 Â 100 mg/kg
3 Â 30 mg/kg
cence signals for DMSO treated wells (no inhibition by compound) and
the minimum fluorescence signals for wells containing the highest
concentration of inhibitor control compounds, for pyrimethamine, at
a final concentration of 10 μM. Data are analyzed on a plate-by-plate
basis and compared to reference compounds that are always included on
every plate, typically artemisinin, mefloquine, and pyrimethamine. EC50
values are obtained using a custom curve fitting model, and a standard
logistic regression model is applied for curve fitting. The quality of the
assay run is assessed by the performance of the reference compounds
where the EC50 must be within 3-fold of the standard reference values
for the assay plate to pass requisite data quality control needs. Addi-
tionally, all compounds are typically assayed at a minimum in duplicate
(independent assay plates) and EC50 values ideally must not vary by
more than 2-fold between plates.
parasitemia
parasitemia
compd
(%)
(day)
(%)
(day)
(%)
(day)
11
18
24
25
26
28
30
31
32
99.3
86
7.7
6.0
7.0
6.3
7.3
6.0
7.7
6.7
7.0
8.7
7.2
99.6
99.7
99.7
99.6
99.6
99.5
99.4
99.6
99.8
>99.9
97
13.3
10.0
12.7
10.3
13
99.9
14.0
83
83
94
98
13.0
17.0
9.0
b
99.4
77
99.8
17.7
Metabolic Stability. The metabolic stability of drug candidates is
determined in human, mouse, and rat liver microsomes using the
compound depletion approach, quantified by LC/MS/MS. The assay
measures the rate and extent of metabolism of chemical compounds by
measuring the disappearance of the parent compound. The assay deter-
mines thecompound’s in vitrohalf-life (T1/2) andhepaticextraction ratios
93
11.0
12
CQ
AS
99.7
89
98.6
98
18.8
7.2
6.7
a
Activity = average parasitemia reduction. Survival = average lifespan
after infection; 6À7 days for untreated mice; 7% Tween 80/3% ethanol
b
12
formulation. 75% PEG300/25%D5W formulation.
(ER) and predicts metabolicclearanceinhuman, rat, and mouse species.
Pampa Assay. Permeation experiments were carried out in 96-well
L-glutamine and containing 0.05 mg/mL gentamicin, 0.014 mg/mL
hypoxanthine, 38.4 mM Hepes, 0.20% sodium bicarbonate, 0.20%
glucose, 3.4 nM NaOH, 5% human serum, and 1.25% albumax. By use
of traditional P. falciparum protocols published previously, 25 mL
microtiter filter plates. The compound concentration was determined by
UV absorption measured with a SpectraMax190 microplate spectro-
photometer (Molecular Devices Corporation, Sunnyvale, CA, U.S.) at
1
3
absorption wavelengths between 260 and 290 nm.
2
cultures are maintained in 75 cm flasks (Fisher catalog no. 10-126-41)
Caco-2 Assay. The Caco-2 assay was carried out in a 96-well format,
and compound concentrations in each chamber are measured by
LC/MS. The assay offers apparent permeability data of test compounds
from the apical to the basolateral chambers [Papp(AÀB)] and from the
basolateral to the apical chambers [Papp(BÀA)]. The results can be used
to predict the in vivo oral absorption (using Papp(AÀB)) and the
transport mechanism of the test compounds across GI membrane
1
1
at 5% hematocrit and parasitemia ranging between 1% and 10%.
Once parasitemia reaches 8À10%, cultures are diluted to 1% or higher
depending on needs. Maintenance requires daily medium changes and
fresh blood every 2 weeks. Cultures are gassed for approximately 30 s
to 1 min using a bloodÀgas mixture to maintain a gas composition of
9
3
6% nitrogen, 3% carbon dioxide, and 1% oxygen and incubated at
7 ꢀC. On day 1 of the assay, percent parasitemia is determined by
1
4
(ratio Papp(BÀA)/Papp(AÀB) and log P lipophilicity data).
obtaining a 1 μL blood smear. The smear is fixed onto the slide by
placing in methanol for 30 s and staining in 10% Geimsa stain, and the
percent of parasitized erythrocytes vs uninfected erythrocytes is
determined by microscopy with a light microscope.
hERG (Q-Patch) Assay. The assay utilizes electrophysiology
measurements on the electric current passing through hERG channel
that is heterologously expressed in a stable CHO cell line. Channels are
open by a hERG-specific voltage protocol, and the compound effects are
directly characterized by their block of the hERG current. The assay is
performed on an automated platform, Q-Patch. All compounds are
tested by four-point dose response curves, and IC50 values (between 0.2
Antimalarial Proliferation Inhibition Assay: 384-Well Plate
Format. P. falciparum strains to be used for screening are prepared with
screening medium (culturing medium without human serum but
supplemented with 0.5% Albumax II) and fresh erythrocytes. An amount
of 20 μL of screening medium is dispensed via the MicroFlo (BioTek)
into 384-well, black clear-bottom assay plates (μClear GNF custom
plates by Griener Bio-One). An amount of 50 nL of compounds is
transferred using a PlateMate Plus (Matrix) or the GNF Systems
PinTool into the assay plates containing screening medium to a final
maximum concentration of 10 μM in a 12-point dose response (1/2 log
serial dilution). On the basis of the measured parasitemia levels, each
parasite strain is diluted to yield 0.5% parasitemia, and 50% hematocrit
blood (uninfected erythrocytes) is added to a concentration of 4.17%.
This diluted culture is used in dispensing the remaining 60% of the assay
volume (30 μL) into the prewarmed assay plates via the MicroFlo liquid
dispenser. The final parasitemia is 0.3% and the final hematocrit is 2.5%
in a total volume of 50 μL. Assay plates are incubated at 37 ꢀC for 72 h in
a gas chamber filled with low oxygen blood gas. After the 72 h of
incubation (equivalent to one to two cycles during the blood stage) a
prepared mixture of the lysis buffer (5 mM EDTA, 1.6% Triton X-100,
1
5
and 30 μM) are measured.
In Vivo Pharmacokinetics Studies in Mice. NITD’s Animal
Care and Use Committee (IACUC), registered with Agri-Food and
Veterinary Authority (AVA), Government of Singapore, approved all
animal experimental protocols. Mice were allowed for acclimatization
before initiation of pharmacokinetic (PK) experiments. Feed and water
were given ad libitum.
Compounds 5, 18, 24, 25, 26, 28, 31, and 32 were formulated at
3 mg/mL for a dose of 30 mg/kg given orally (po) and at 1 mg/mL for a
dose of 5 mg/kg given intravenously (iv). Citrate buffer 100 mM, pH 3.5,
was used to formulate compounds 5, 18, 24, 25, 26, and 28 prior to po
administration, while citrate buffer 50 mM, pH 4.5, was used for iv
administration. Both were clear solutions. For compounds 31 and 32,
the solution formulation for both po and iv dosing was ethanol/
PEG400/10% vitamin ETPGS (10:30:60). Blood samples were col-
lected at 0.08, 0.25, 0.5, 1, 2, 4, 8, 16, and 24 h following po dosing.
Sampling was identical following iv dosing except that the first time point
was 0.02 h. Groups of three mice were used for each time point. Blood
was centrifuged at 13 000 rpm for 7 min at 4 ꢀC and plasma harvested
and stored at À20 ꢀC until analysis.
2
(
0 mM Tris-HCl, 0.16% saponin) in water and SYBR Green detection
0.1%) reagent is dispensed at 10 μL per well using the MicroFlo.
Cultures are incubated for an additional 24 h at 25 ꢀC before measuring
5
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Journal of Medicinal Chemistry
ARTICLE
PK studies for compounds 11 and 30 were conducted at GNF. In-life
studieswerecarriedout under protocolsapprovedbythe AnimalCareand
Use Committee (IACUC) of GNF. Compounds 11 and 30 were
formulated at 2.5 mg/mL in 75% PEG300 and 25% D5W (5% dextrose
in distilled water) solution and were filtered using a 0.45 μm syringe filter.
The filtered solutions were dosed intravenously via the lateral tail vein
at 5 mg/kg with a dosing volume of 2 mL/kg to male Balb/c mice (n =
regression analysis using an in-house fitting program developed at
GNF. The oral bioavailability (F) was calculated as the ratio between
the area under the curve (AUC) following oral administration and the
AUC following intravenous administration corrected for dose (F =
[(AUC )(dose )]/[(AUC )(dose )].
po
iv
iv
po
In Vivo Antimalarial Activity. All in vivo efficacy studies were
approved by the veterinary authorities of the Canton Basel-Stadt. In vivo
antimalarial activity was usually assessed for groups of three female7
NMRI mice (20À22 g) intravenously infected on day zero with 2 Â 10
erythrocytes parasitized with P. berghei GFP ANKA malaria strain
(PbGFPCON donation from AP Waters and CJ Janse, Leiden Uni-
3
(
3
per group). The filtered solutions were also administered orally at 20 mg/kg
with a dosing volume of 8 mL/kg) to another groups of mice (n =
per group). Six blood samples of 50 μL each were collected serially
from each animal via retro-orbital sinus up to 24 h after dosing. Blood
samples were collected into heparin microtainer and centrifuged, and the
plasma was separated and frozen until analysis. Plasma samples were
analyzed by LC/MS/MS.
Extraction and LC/MS Analysis. Plasma samples were extracted
with acetonitrile/methanol/acetic acid (90:9.8:0.2) for analytes and
internal standard (analogue from the same series) using a 8 to 1
extractant to plasma ratio. Analyte quantitation was performed by
LC/MS/MS. Liquid chromatography was performed using an Agilent
1
0a,16
versity, The Netherlands).
Untreated control mice die typically
between day 6 and day 7 after infection. Experimental compounds were
formulated in 7% Tween 80/3% ethanol or 75% PEG300/25% D5W as
indicated. Compounds were administered orally in a volume of 10 mL/kg
either as a single dose (24 h post infection) or as three consecutive daily
doses (24, 48, and 72 h after infection). With the single dose regimen
parasitemia was determined (72 h after infection). For the triple dose
regimen (96 h after infection), standard flow cytometry techniques were
1
6
1
100 HPLC system (Santa Clara, CA), with the Agilent Zorbax XDB
phenyl (3.5 μ, 4.6 mm  75 mm) column at an oven temperature of
5 ꢀC, coupled with a QTRAP4000 triple quadruple mass spectrometer
Applied Biosystems, Foster City, CA). Instrument control and data
used. Activity was calculated as the difference between the mean percent
parasitemia for the control and treated groups expressed as a percentage of
the control group. The survival time in days was also recorded up to 30
days after infection. A compound was considered curative if the animal
survived to day 30 after infection with no detectable parasites.
4
(
acquisition were performed using the Applied Biosystems software
Analyst, version 1.4.2. The mobile phases used were (A) waterÀacetic
acid (99.8:0.2, v/v) and (B) acetonitrileÀacetic acid (99.8:0.2, v/v),
using a gradient, with flow rate of 1.0 mL/min, and run time of 5 min.
Compound detection on the mass spectrometer was performed in
electrospray positive ionization mode and using multiple reaction
monitoring (MRM) for specificity (transitions: compound 5 384.2/
Chemistry. Unless otherwise noted, materials were obtained from
commercial suppliers and were used without purification. Removal of
solvent under reduced pressure refers to distillation using B €u chi rotary
evaporator attached to a vacuum pump (∼3 mmHg). Products obtained
as solids or high boiling oils were dried under vacuum (∼1 mmHg).
Purification of compounds by high pressure liquid chromatography
was achieved using a Waters autopurification system consisting of a 2767
autosampler/fraction collector, a 2525 binary gradient module, a 2487
UV detector, and a ZQ mass spectrometer. Compounds were purified
using flow rate of 30 mL/min with a 50 mm  20 mm i.d. Ultra 120 5 μm
C18Q column (Peeke Scientific, Novato, CA). A 7.5 min linear gradient
from 10% solvent A (acetonitrile with 0.035% trifluoroacetic acid) in
solvent B (water with 0.05% trifluoroacetic acid) to 90% A was used,
1
01.2, compound 18 384.3/101.2, compound 24 412.3/327.2, com-
pound 25 380.4/118.2, compound 26 408.5/280.3, compound 28
28.2/343.2, compound 31 418.3/101.4, compound 32 446.3/361.2,)
4
together with their optimized MS parameters. The lower limit of
quantification ranged between 0.5 and 23 ng/mL for all the compounds
studied.
For PK studies of compounds 11 and 30 in mice, plasma samples
1
followed by a 2.5 min hold at 90% A. H NMR spectra were recorded on
(20 μL) were extracted with a solution of acetonitrile/methanol (3:1)
Bruker XWIN-NMR (400 or 600 MHz) spectrometer. Proton reso-
containing internal standard. The samples were vortexed, then centri-
fuged with an Eppendorf centrifuge 5810R (Eppendorf, Hamburg,
Germany) at a setting of 4000 rpm for 10 min at room temperature.
The supernatant was transferred to a clean 96-well analysis plate for LC/
MS/MS analysis. An aliquot (10 μL) was injected onto a Phenomenex
C18 guard column (4 mm  2 mm) followed by a Zorbax SB-C8
analytical column (2.1 mm  50 mm, 5 μm, Agilent Technologies Inc.,
Palo Alto, CA, U.S.). Separation was carried out using a gradient elution
method with a mobile phase consisting of 0.05% formic acid in water
nances are reported in parts per million (ppm) downfield from tetra-
1
methylsilane (TMS). H NMR data are reported as multiplicity
(s, singlet; d, doublet; t, triplet; q, quartet; quint, quintuplet; sept
septuplet; dd, doublet of doublets; dt, doublet of triplets; bs, broad
singlet), number of protons, and coupling constant in hertz. For
spectra obtained in CDCl
7.27, 2.50, and 3.31 ppm, respectively) were used as the reference.
Analytical thin-layer chromatography (TLC) was performed on
3 6 3
, DMSO-d , CD OD, the residual protons
(
commercial silica plates (Merck 60-F 254, 0.25 mm thickness). Com-
pounds were visualized by UV light (254 nm). Flash chromatography was
performed either by CombiFlash (separation system Sg. 100c, ISCO) or
using silica gel (Merck Kieselgel 60, 230À400 mesh). Elemental analyses
were carried out by Midwest Microlabs LLC, Indianapolis, IN.
Purity of Compounds. AllcompoundswereanalyzedwithaWaters
ZQ 2000 LC/MS system, which used an Agilent binary pump and
photodiode array detector (PDA) along witha Sedere 75evaporative light
scattering detector (ELSD). Samples were injected from 96-well or 384-
well plates with a Leap Technologies HTS Pal autosampler. The mobile
(
solvent A) and 0.05% formic acid in acetonitrile (solvent B). The flow
rate was 600 μL/min. The HPLC system, consisting of Agilent 1100
series binary pump (Agilent Technologies Inc.), Agilent 1100 series
microvacuum degasser (Agilent Technologies Inc.), HTC PAL CTC
analytics autosampler (LEAP Technologies, Carborro, NC), and VICI
two-position actuator (Valco Int., Huston, Texas, U.S.), was interfaced
to a MDS SCIEX API-4000 triple quadruple mass spectrometer (MDS
Inc., Toronto, Canada). Mass spectral analyses were carried out using
electrospray source in the positive ion mode and using multiple reaction
monitoring (MRM) for quantification. MRM transitions of 412.2/327.2
and 430.1/304.0 were used for compounds 11 and 30, respectively.
Pharmacokinetic Analysis. The mean value from the three
animals at each time point was plotted against time to give plasma
concentrationÀtime profile. Pharmacokinetic parameters were deter-
mined using Watson LIMS, version 7.2 (Thermo Electron Corporation,
PA, U.S.), by noncompartmental analysis. Pharmacokinetic parameters
for compounds 11 and 30 were calculated by noncompartmental
2
phases used were (A) H O + 0.05% TFA and (B) ACN + 0.035% TFA. A
gradient HPLC method with flow of 1.0 mL/min started at 5% B, with a
hold of 0.1 min before a linear increase to 95% B at 2.70 min. At 2.71 min
the B mobile phase was increased to 100% and held until 2.98 min. Total
run time was 3.0 min. The column used was a Waters Atlantis dC18 2.1
mm  30 mm, 3 μm. The mass spectrometer was operated in positive
mode, with a spray voltage of 3.2 kV and cone voltage of 30 V. The source
5
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Journal of Medicinal Chemistry
ARTICLE
and desolvation temperatures were 130 and 400 ꢀC, respectively, with
at room temperature overnight. The solid was filtered off, and solvent
6
00 L/h of nitrogen desolvation flow. The progress of reactions and the
was removed. The 400 MHz proton NMR of the crude product proved it
1
purity of products were measured using 254 and 220 nm wavelengths and
electrospray ionization (ESI) positive mode. Mass spectra were obtained
in ESI positive mode. All the reported compounds were found to be>95%
when analyzed at the 254 nm wavelength.
to be 1-3. H NMR (MeOH-d
4
, 400 MHz) δ 7.72À7.4 (m, 2H),
7.38À7.33 (m, 3H), 6.66 (d,, J = 8.29 Hz, 1H), 6.33 (d, J = 2.32 Hz, 1H),
6.14 (dd, J = 8.3, 2.3 Hz, 1H), 5.86 (s, 2H), 4.57 (s, 2H), 4.08 (t, J = 5.7
Hz, 2H), 3.68 (t, J = 5.7 Hz, 2H).
Compound 1a: 2-Amino-1-(3-(benzo[d][1,3]dioxol-5-
ylamino)-2-phenyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
ethanone. The structure of 1a is as follows:
Synthesis of Compound 1a: 2-Amino-1-(3-(benzo[d][1,3]
dioxol-5-ylamino)-2-phenyl-5,6-dihydroimidazo[1,2-a]pyrazin-
7(8H)-yl)ethanone. To a stirred solution of N-BOC-glycine (142
mg, 0.81 mmol) in 2 mL of DMF were added HATU (308 mg, 0.81
mmol) and DIEA (0.28 mL, 1.62 mmol). After the mixture was
stirred for 10 min, compound 1-3 (180 mg, 0.54 mmol) was added.
The reaction mixture was stirred at room temperature for 3 h.
Solvent was removed, and the crude product was subjected to MS-
triggered HPLC purification. The collected MeCN/water solution
was concentrated until no more MeCN was left. The remaining
aqueous solution was neutralized with NaHCO and extracted with
3
DCM. The organic solution was dried and concentrated. The residue
was dissolved in 1:1 MeCN/water solvent and dried on a lyophilizer
to give 192 mg of powdery amide coupled product.
Synthesis of 1-2: N-(Benzo[d][1,3]dioxol-5-yl)-2-phenyl-
,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine
5
A solution of the above obtained amide (265 mg, 0.54 mmol) in
10 mL of 4:1 DCM and TFA was stirred at room temperature for 2 h.
Solvent was removed, and the crude product was subjected to MS-
triggered HPLC separation. The collected MeCN/water solution was
concentrated until no more MeCN was left. The remaining aqueous
solution was neutralized with NaHCO and extracted with DCM. The
3
organic solution was dried and concentrated. The oil was then dissolved
in 1:1 MeCN/water solvent and dried on a lyophilizer to give a powdery
1
product in quantitative yield. H NMR (400 MHz, MeOH-d
4
) δ 7.75 (m,
2H), 7.19(m, 2H), 7.18(m, 1H), 6.61(m, 1H), 6.22(m, 1H), 6.03(m, 1H),
5
.82 (m, 2H), 4.75 (m, 2H), 3.78À4.05 (m, 6H). M/Z = 392.2 (M + 1).
Compound 1b: 2-Amino-1-(3-(benzo[d][1,3]dioxol-5-
ylamino)-2-phenyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
3-phenylpropan-1-one
To a stirred solution of isocyanide (158 mg, 1.66 mmol) in 10 mL of
MeOH were added benzaldehyde (0.25 mL, 2.49 mmol) and 2-amino-
pyrazine (281 mg, 1.91 mmol), followed by 1.0 N HClO
4
in MeOH
(0.17 mL, 0.17 mmol). The reaction mixture was stirred at room
Compound 1b was synthesized from compound 1-3 by a HATU
mediated coupling with 2-(tert-butoxycarbonylamino)-3-phenylpropa-
noic acid followed by TFA mediated deprotection. H NMR (400 MHz,
temperature for 3 h. The reaction mixture was directly taken to mass-
triggered HPLC purification. The collected MeCN/water solution was
1
concentrated and dried on a lyophilizer to give 296 mg of compound 1-2
MeOH-d
4
) δ 7.67 (m, 2H), 7.37À7.20 (m, 8H), 6.65 (m, 1H), 6.25 (m,
1
as a powdery product. H NMR (CDCl
3
, 400 MHz) δ 9.23 (d, J =
1H), 6.07 (td, J = 2.29 Hz, J = 8.14 Hz, 1H), 5.85 (m, 2H), 4.79 (dd,
J =5.86 Hz, J = 9.79 Hz, 1H), 4.59À4.19 (m, 2H), 3.57À3.83 (m, 3H),
3.16À2.93 (m, 3H). M/Z = 482.3 (M + 1).
1
.2 Hz, 1H), 8.03 (d, J = 4.8 Hz, 1H), 7.91À7.94 (m, 3H), 7.26À7.44
(m, 3H), 6.68 (d, J = 8.4 Hz, 1H), 6.32 (br, 1H), 6.25 (d, J = 2.4 Hz, 1H),
6.00 (dd, J = 8.4, 2.4 Hz, 1H), 5.93 (s, 2H).
Compound 1c: 2-Amino-1-(3-(benzo[d][1,3]dioxol-5-
ylamino)-2-phenyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
4-methylpentan-1-one
Synthesis of 1-3: N-(Benzo[d][1,3]dioxol-5-yl)-2-phenyl-
,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine
5
To a stirred solution of compound 1-2 (181 mg, 0.54 mmol) in 10 mL of
MeOH was added Pd/C (58 mg, 0.054 mmol). The reaction mixture
was evacuated and back-filled with H . The reaction mixture was stirred
2
Compound 1c was synthesized from compound 1-3 by a HATU mediated
coupling with 2-((tert-butoxycarbonyl)amino)-4-methylpentanoic acid
5
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Journal of Medicinal Chemistry
ARTICLE
1
followed by TFA mediated deprotection. H NMR (400 MHz, MeOH-d
δ 7.78 (m, 2H), 7.42 (m, 3H), 6.66 (m, 1H), 6.36 (m, 1H), 6.13 (m, 1H),
.85 (m, 2H), 5.24 (m, 1H), 4.58 (m, 1H), 4.05 (m, 4H), 3.35 (s, 2H), 1.76
4
)
(compound + 3HCl): % C, 48.75; % H, 4.50; % N, 14.21 (calculated).
% C = 48.86/48.58; % N = 14.11/13.95; % H = 4.69/4.49 (experimental).
Compound 6: N-(tert-butyl)-2-(4-fluorophenyl)imidazo
[1,2-a]pyrazin-3-amine
5
(
m, 2H), 1.03 (m, 6H). M/Z = 448.2 (M + 1).
Compound 2: N,2-Bis(4-fluorophenyl)imidazo[1,2-a]pyrazin-
-amine
3
To a stirred solution of 2-aminopyrazine (571 mg, 6.0 mmol) in 10 mL of
MeOH were added 4-fluorobenzaldehyde (0.97 mL, 9.0 mmol), 2-isocyano-
2
4
-methylpropane (0.78 mL, 6.9 mmol), and 1.0 N HClO in MeOH
Compound 2 was synthesized by the following way: To a
(0.60 mL, 0.60 mmol). The reaction mixture was stirred at room temperature
stirred solution of 2-aminopyrazine (683 mg, 7.18 mmol) in 50 mL
of MeOH were added 4-fluorobenzaldehyde (1.16 mL, 10.8 mmol)
and 1-fluoro-4-isocyanobenzene (1.0 g, 8.25 mmol), followed by 1.0 N
for 3 h. Solvent was removed and the residue was subjected to flash chromato-
1
graphy purification to give compound 6as white solid. HNMR(CDCl , 400
3
MHz) δ 8.97 (d, J = 1.6 Hz, 1H), 8.10 (dd, J = 1.6, 4.4 Hz, 1H), 7.90À7.94
(m, 2H), 7.86 (d, J = 4.66 Hz, 1H), 7.13 (t, J = 8.8 Hz, 2H), 1.04 (s, 9H).
Compound 7: 2-(4-fluorophenyl)imidazo[1,2-a]pyrazin-3-
amine
4
HClO in MeOH (0.72 mL, 0.72 mmol). The reaction mixture was
stirred at room temperature overnight. The reaction mixture was
concentrated, and the residue was subjected to flash chromato-
graphy purification. The collected organic solution was concentrated
1
to give the compound 2 (1.1 g, 48%) as a yellow oil. H NMR
(
(
400 MHz, MeOH-d
t, J = 8.84 Hz, 2H), 6.82 (t, J = 8.77 Hz, 2H),6.45 (m, 2H). M/Z =
4
) δ 8.96 (s, 1H), 7.95 (m, 3H), 7.82 (s, 1H), 7.07
3
23.3 (M + 1).
Compound 3: N,2-Bis(4-fluorophenyl)-5,6,7,8-tetrahy-
droimidazo[1,2-a]pyrazin-3-amine
A solution of compound 6 (284 mg, 1.0 mmol) in 10 mL of 4:1 DCM and
TFA was stirred at room temperature for 2 h. Solvent was removed, and the
1
product was directly used in the next step after neutralization. H NMR (300
Hz, CDCl ) δ8.82 (s, 1H), 8.28 (d, J= 4.8 Hz, 1H), 8.05À8.09 (m, 2H), 7.76
3
(
d, J = 4.5 Hz, 1H), 7.27À7.33 (m, 2H), 5.79 (s, 2H). M/Z = 229 (M + 1).
Compound 8: 3-Amino-1-(2-(4-fluorophenyl)-3-(4-fluoro-
phenylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
propan-1-one
Compound 3 was synthesized by the following way: To a
stirred solution of compound 2 (761 mg, 2.36 mmol) in 10 mL of
MeOH was added Pd/C (258 mg, 0.24 mmol). The reaction mixture
2
was evacuated and back-filled with H . The reaction mixture was stirred
at room temperature overnight. The solid was filtered off, and solvent
was removed. The product was subjected to mass-triggered HPLC
purification. The obtained MeCN/water solution was combined and
concentrated to give the final product as a yellow oil after neutralization
1
(
(
3
quantitative yield). H NMR (300 MHz, DMSO-d ) δ 7.81À7.72
6
Compound 8 was synthesized from compound 3 by a HATU mediated
m, 2H), 7.11 (m, 2H), 6.97 (m, 2H), 6.56À6.52 (m, 2H), 3.86 (s, 2H),
coupling with 3-(tert-butoxycarbonylamino)propanoic acid followed by
.55 (m, 2H), 3.02 (m, 2H). M/Z = 327.2 (M + 1).
1
TFA mediated deprotection. H NMR (CD OD, 400 MHz) δ 7.61À7.65
3
Compound 5: 2-Amino-1-(2-(4-fluorophenyl)-3-(4-fluoro-
phenylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
ethanone
(
m, 2H), 6.87À6.92 (m, 2H), 6.75À6.80 (m, 2H), 6.45À6.49 (m, 2H),
4
2
.71 (d, J = 4.0 Hz, 2H), 3.82À3.90 (m, 2H), 3.62À3.74 (m, 2H),
.81À2.84 (m, 2H), 2.51À2.57 (m, 2H). M/Z = 398.4 (M + 1).
Compound 9: (S)-2-Amino-1-(2-(4-fluorophenyl)-3-(4-fluoro-
phenylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
propan-1-one
Compound 5 was synthesized from compound 3 by a HATU mediated
coupling with N-BOC-glycine followed by TFA mediated deprotection
1
(
72% isolated yield) using a protocol for synthesis of compound 1a. H
NMR (400 MHz, MeOH-d
2
4
4
) δ 7.59À7.56 (m, 2H), 7.12 (t, J = 8.8 Hz,
H), 6.86 (t, J = 8.8 Hz, 2H), 6.68À6.65 (m, 2H), 5.04À5.00 (m, 2H),
.08À3.92 (m, 6H). M/Z = 384.2 (M + 1). Elemental analysis
Compound 9 was synthesized from compound 3 by a HATU mediated
coupling with (S)-2-(tert-butoxycarbonylamino)propanoic acid followed by
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ARTICLE
1
TFA mediated deprotection. H NMR (CD
(
3
3
OD, 400 MHz) δ 7.63
Compound 13: (R)-2-Amino-1-(2-(4-fluorophenyl)-3-(4-
fluorophenylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-
yl)-3-phenylpropan-1-one
m, 2H), 6.95 (m, 2H), 6.82 (m, 2H), 6.50 (m, 2H), 4.41 (m, 1H),
.92À3.74 (m, 6H), 1.42 (d, J = 6.95 Hz, 3H). M/Z = 398.2 (M + 1).
Compound 10: (S)-2-Amino-1-(2-(4-fluorophenyl)-3-(4-
fluorophenylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-
yl)-3-methylbutan-1-one
Compound 13 was synthesized from compound 3 by a HATU
mediated coupling with (R)-2-(tert-butoxycarbonylamino)-3-phenyl-
1
propanoic acid followed by TFA mediated deprotection. H NMR
Compound 10 was prepared from compound 3 by a HATU mediated
(
(
(
(
400 MHz, MeOH-d ) δ 7.65 (m, 1H), 7.18 (m, 8H), 6.96
4
coupling reaction with (S)-2-(tert-butoxycarbonylamino)-3-methylbutanoic
m, 2H), 6.82 (m, 1H), 6.49 (m, 1H), 4.65À4.8 (m, 2H), 4.00À4.49
m, 2H), 3.38À3.57 (m, 3H), 2.73À3.00 (m, 2H). M/Z = 474.4
M + 1).
1
acid followed by a TFA mediated deprotection. H NMR (DMSO-d , 400
6
MHz) δ 8.15 (bs, 1H), 8.02 (s, 1H), 7.77 (m, 2H), 7.19 (m, 2H), 7.00
(
4
2
m, 2H), 6.61 (m, 2H), 5.05À4.87 (m, 1H), 4.69À4.41 (m, 1H),
Compound 14: 2-Amino-1-(3-((4-fluorophenyl)amino)-2-
phenyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-2-methyl-
propan-1-one
.23À4.11 (m, 1H), 3.98À3.77 (m, 1H), 3.00 (m, 1H), 2.51 (s, 2H),
.1 (m, 1H), 0.95 (dd, J = 22, 6.72 Hz, 6H). M/Z = 426 (M + 1)
Compound 11: 2-Amino-1-(2-(4-fluorophenyl)-3-(4-fluo-
rophenylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
2
-methylpropan-1-one
Compound 14 was synthesized following the same sequence as
compound 1 involving an Ugi reaction, reduction using palladium
on carbon followed by HATU mediated coupling with N-BOC-
Compound 11 was synthesized from compound 3 by a HATU
1
dimethylglycine and TFA mediated deprotection. H NMR
mediated coupling with 2-(tert-butoxycarbonylamino)-2-methylpropa-
1
(
DMSO-d , 400 MHz) δ 8.25 (bs, 1H), 8.01 (s, 1H), 7.73 (m, 2H),
noic acid followed by TFA mediated deprotection. H NMR (MeOH-d
00 Hz) δ7.70À7.67 (m, 2H), 7.22 (t, J = 8.8 Hz, 2H), 6.97(t, J = 8.8
Hz,2H), 6.8À6.77 (m, 2H), 5.16 (s, 2H), 4.24 (m, 2H), 4.07 (m, 2H),
.76 (s, 6H). M/Z = 412.2 (M + 1). Elemental analysis of HCl salt
4
,
6
7
4
.35 (m, 2H), 7.25 (m, 1H), 7.00 (t, J = 8.04 Hz, 2H), 6.62 (m, 2H),
.40 (m, 2H), 4.08 (m, 2H), 3.76 (m, 2H), 1.60 (s, 6H). M/Z = 394.4
4
(M + 1).
1
(
(
(
compound + 2HCl + 0.50 H O): % C, 53.56; % H, 5.31; % N, 14.19
Compound 15: 2-Amino-1-(2-(4-chlorophenyl)-3-((4-fluoro-
phenyl)amino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
ethanone
2
calculated). % C = 53.98/53.69; % N = 14.07/13.97; % H = 5.16/4.98
experimental).
Compound 12: (S)-2-Amino-1-(2-(4-fluorophenyl)-3-(4-
fluorophenylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-
yl)-3-phenylpropan-1-one
Compound 15 was synthesized following the same sequence as
compound 1 involving an Ugi reaction, reduction using palladium
Compound 12 was synthesized from compound 3 by a HATU
mediated coupling with (S)-2-(tert-butoxycarbonylamino)-3-phenyl-
propanoic acid followed by TFA mediated deprotection. H NMR
on carbon followed by HATU mediated coupling with N-BOC-
1
1
glycine and TFA mediated deprotection. H NMR (400 MHz,
MeOH-d ) δ 7.68 (m, 2H), 7.38 (m, 2H), 6.94 (m, 2H), 6.69 (m, 2H),
4
(
(
CDCl
3
, 400 Hz) δ 9.06 (bs), 7.94 (bs), 7.3À6.93 (m, 11H), 6.55
5
.05À5.00 (m, 2H), 4.12 (s, 2H), 3.83À3.99 (m, 4H). M/Z = 400.2
m, 2H), 5.5À4.62 (m, 2H), 4.28 (m, 1H), 3.68À2.60 (m, 6H).
M/Z = 474.4 (M + 1).
(M + 1).
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Compound 16: 2-Amino-1-(3-((4-fluorophenyl)amino)-
-(4-methoxyphenyl)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-
Compound 19 was synthesized following the same sequence as
compound 1 involving an Ugi reaction, reduction using palladium on
2
yl)ethanone
carbon followed by HATU mediated coupling with N-BOC-dimethyl-
1
glycine and TFA mediated deprotection. H NMR (MeOH-d
4
, 400 Hz)
δ 7.41 (m, 1H), 7.28 (m, 1H), 7.08 (m, 2H), 6.79 (m, 2H), 6.56 (m,
H), 4.93(m, 2H), 4.00 (m, 2H), 3.86 (t, J = 5.17 Hz, 2H), 1.64 (s, 6H).
2
M/Z = 412.2 (M + 1).
Compound 20: 2-Amino-1-(2-(3,4-difluorophenyl)-3-(p-
tolylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-2-
methylpropan-1-one
Compound 16 was synthesized following the same sequence as
compound 1 involving an Ugi reaction, reduction using palladium on
carbon followed by HATU mediated coupling with N-Boc-glycine and
1
TFA mediated deprotection. H NMR (400 MHz, MeOH-d
4
) δ 7.58 (d,
J = 8.79 Hz, 2H), 6.98 (m, 4H), 6.75 (m, 2H), 5.07À5.00 (m, 2H), 4.14
(s, 2H), 4.02 (m, 4H), 3.18 (s, 3H). M/Z = 396.2 (M + 1).
Compound 17: 2-Amino-1-(3-((4-fluorophenyl)amino)-2-(p-
tolyl)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)ethanone
Compound 20 was synthesized following the same sequence as
compound 1 involving an Ugi reaction, reduction using palladium on
carbon followed by HATU mediated coupling with N-BOC-dimethyl-
1
glycine and TFA mediated deprotection. H NMR (MeOH-d
4
, 400 Hz)
δ 7.53À7.48 (m, 1H), 7.44À7.41 (m, 1H), 7.17 (m, 1H), 6.9 (m, 2H),
6
1
.48 (m, 2H), 4.88 (m, 2H), 4.05 (m, 2H), 3.81 (m, 2H), 2.12 (s, 3H),
.62 (s, 6H). M/Z = 426.2(M + 1).
Compound 21: 2-Amino-1-(3-(cyclohexylamino)-2-(4-
fluorophenyl)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-2-
methylpropan-1-one
Compound 17 was synthesized following the same sequence as
compound 1 involving an Ugi reaction, reduction using palladium on
carbon followed by HATU mediated coupling with N-Boc-glycine and
1
TFA mediated deprotection. H NMR (400 MHz, MeOH-d
4
) δ 7.53 (d,
J = 8.12 Hz, 2H), 7.17 (m, 2H), 6.94 (m, 2H), 6.72 (m, 2H), 5.07À5.00
(m, 2H), 4.13 (s, 2H), 4.02 (m, 4H), 2.35 (s, 3H). M/Z = 380.4 (M + 1).
Compound 18: 2-Amino-1-(3-((4-fluorophenyl)amino)-2-
3-fluorophenyl)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
(
ethanone
Compound 21 was synthesized following the same sequence as
compound 1 involving an Ugi reaction, reduction using palladium on
carbon followed by HATU mediated coupling with N-BOC-dimethyl-
1
glycine and TFA mediated deprotection. H NMR (MeOH-d
4
, 400 Hz)
δ 7.66 (m, 2H), 7.18 (t, J = 8.74 Hz, 2H), 4.95 (m, 2H), 4.13 (m, 2H),
4
4
.11 (m, 2H), 2.74 (m, 1H), 1.73 (m, 2H), 1.68 (s, 6H), 1.37À1.60 (m,
H), 1.05À1.15 (m, 4H). M/Z = 400.3 (M + 1).
Compound 18 was synthesized following the same sequence as
compound 1 involving an Ugi reaction, reduction using palladium on
Compound 22: 2-Amino-1-(2-(4-fluorophenyl)-3-(pyridin-
-ylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-2-
3
methylpropan-1-one
carbon followed by HATU mediated coupling with N-BOC-glycine and
1
TFA mediated deprotection. H NMR (MeOH-d
4
, 400 Hz) δ
7
.55À7.53 (m, 1H), 7.47À7.45 (m, 2H), 7.15 (m, 1H), 6.96À6.94
(m, 2H), 6.84À6.81 (m, 2H), 5.20 (m, 2H), 4.25À4.20 (m, 6H), 3.35
(s, NH). M/Z = 384.2 (M + 1).
Compound 19: 2-Amino-1-(2-(2-fluorophenyl)-3-((4-fluoro-
phenyl)amino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
-methylpropan-1-one
2
Compound 22 was synthesized following the same sequence as
compound 1 involving an Ugi reaction, reduction using palladium
on carbon followed by HATU mediated coupling with N-BOC-
1
dimethylglycine and TFA mediated deprotection. H NMR (400
MHz, MeOH-d
4
) δ 8.15 (m, 1H), 7.7 (m, 3H), 7.57 (m, 2H), 7.1 (t,
J = 8.74 Hz, 2H); 4.93 (m, 2H); 4.18 (s, 2H), 3.97 (m, 2H); 1.68 (s,
6
H). M/Z = 395.4 (M + 1).
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Compound 23: 2-Amino-1-(2-(4-fluorophenyl)-3-(3-fluoro-
phenylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
ethanone. The structure of 23 is as follows:
δ 7.50À7.48 (m, 2H), 7.44À7.40 (m, 1H), 7.18 (m, 1H), 6.98 (t, J = 8.8
Hz, 2H), 6.82À6.79 (m, 2H), 5.18 (m, 2H), 4.25 (t, J = 4.8 Hz, 2H), 4.07
(t, J = 4.8 Hz, 2H), 1.76 (s, 6H). M/Z = 412.0 (M + 1). Elemental
analysis of HCl salt (compound + 2HCl + 1.3H
.48; % N, 13.79 (calculated). % C = 51.95/52.02; % N = 13.37/13.5;
H = 5.26/5.32 (experimental).
Compound 25: 2-Amino-1-(2-(4-fluorophenyl)-3-(p-tolyl-
2
O): % C, 52.04; % H,
5
%
amino)-5,6-dihydroimidazo[1,2-a]pyrazine-7(8H)-yl)-
ethanone
Synthesis of 23-3: N-(3-Fluorophenyl)-2-(4-fluorophenyl)-
,6,7,8-tetrahydroimidazo[1,2-a] pyrazin-3-amine
5
Compound 25 was synthesized following the same sequence as
compound 1 involving an Ugi reaction, reduction using palladium on
To a solution of compound 7 (69 mg, 0.30 mmol) in 6 mL of dioxane
were added 1-bromo-3-fluorobenzene (66 μL, 0.60 mmol), Pd (dba)
8 mg, 0.009 mmol), Xantphos (11 mg, 0.018 mmol), and Cs CO (196
mg, 0.60 mmol) at room temperature. The reaction mixture was
degassed and stirred at 120 ꢀC under N for 5 h. The reaction mixture
was cooled to room temperature, and solid was filtered off. The resulting
filtrate was concentrated. The residue was subjected to mass-triggered
HPLC purification to give a yellow oil (64 mg, 66%) after neutralization.
To a stirred solution of the obtained adduct (64 mg, 0.20 mmol) in 5 mL
of MeOH was added Pd/C (21 mg, 0.02 mmol). The reaction mixture
carbon followed by HATU mediated coupling with N-BOC-glycine and
2
3
1
TFA mediated deprotection. H NMR (400 MHz, MeOH-d
4
) δ
(
2
3
7
6
.74À7.71 (m, 2H), 7.21À7.17 (m, 2H), 7.03 (t, J = 8.0 Hz, 2H),
.67 (d, J = 8.4 Hz, 2H), 5.17À5.15 (m, 2H), 4.22À3.98 (m, 6H), 2.22
2
(
(
(
(
s, 3H). M/Z = 380.4 (M + 1). Elemental analysis of HCl salt
compound + 3HCl + 0.1H O): % C, 51.41; % H, 5.18; % N, 14.27
calculated). % C = 51.75/51.52; % N = 14.34/14.29; % H = 5.17/5.15
experimental).
Compound 26: 2-Amino-1-(2-(4-fluorophenyl)-3-(p-tolyl-
2
amino)-5,6-dihydroimidazo[1,2-a]pyrazine-7(8H)-yl)-2-methyl-
propan-1-one
was evacuated and back-filled with H . The reaction mixture was stirred
2
at room temperature overnight. The solid was filtered off, and solvent
was removed. The residue was subjected to mass-triggered HPLC
purification to give a yellow solid (42 mg, 66%).
Synthesis of 23: 2-Amino-1-(2-(4-fluorophenyl)-3-(3-fluoro-
phenylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
ethanone
Compound 26 was synthesized following the same sequence as
compound 1 involving an Ugi reaction, reduction using palladium on
carbon followed by HATU mediated coupling with N-BOC-dimethyl-
1
glycine and TFA mediated deprotection. H-NMR (MeOH-d
4
, 400
Compound 23 was synthesized from compound 23-3 by a HATU
MHz) δ 7.72 (m, 2H), 7.18 (t, J = 8.0 Hz, 2H), 7.02 (m, 2H), 6.66 (d, J =
8.35 Hz, 2H), 5.19 (s, 2H), 4.28 (m, 2H), 4.07 (m, 2H), 2.21 (s, 3H),
1.77 (s, 6H). M/Z = 408.2 (M + 1). Elemental analysis of HCl salt
mediated coupling with N-Boc-glycine followed by TFA mediated
1
deprotection. H NMR (DMSO-d
6
, 400 MHz) δ 8.24 (bs, 1H), 8.12
(
(
s, 2H), 7.76 (m, 2H), 7.19 (m, 3H), 6.48 (m, 2H), 6.36 (m, 1H), 4.80
m, 2H), 4.04À3.69 (m, 6H). M/Z = 384.4 (M + 1).
2
(compound + 3HCl + 0.1H O): % C, 53.26; % H, 5.67; % N, 13.5
(calculated). % C = 53.28/53.18; % N = 13.39/13.32; % H = 5.92/5.81
(experimental).
Compound 27: 2-Amino-1-(3-(4-chlorophenylamino)-
2-(4-fluorophenyl)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-
yl)ethanone
Compound 24: 2-Amino-1-(2-(3-fluorophenyl)-3-(4-fluoro-
phenylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-2-
methylpropan-1-one
Compound 24 was synthesized from compound 23-3 by a HATU
mediated coupling with 3-(tert-butoxycarbonylamino)propanoic acid
Compound 27 was synthesized from compound 7 using palladium
catalyzed amination, reduction using palladium on carbon followed by
1
followed by TFA mediated deprotection. H NMR (MeOH-d , 400 Hz)
4
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ARTICLE
HATU mediated coupling with N-BOC-glycine and TFA mediated
1.36 (s, 6H). M/Z = 430.2 (M + 1). Elemental analysis of HCl salt
1
deprotection. H NMR (MeOH-d , 400 Hz) δ 7.59À7.56 (m, 2H),
(compound + 2HCl + 0.40H O): % C, 51.85; % H, 4.91; % N, 13.74
4
2
7
.15À7.09 (m, 4H), 6.68À6.65 (m, 2H), 5.05 (m, 2H), 4.04À3.87
(calculated). % C = 51.73/51.85; % N = 13.5/13.51; % H = 4.87/4.94
(experimental).
(m, 6H). M/Z = 400.1 (M + 1).
Compound 28: 2-Amino-1-(3-(4-chlorophenylamino)-
-(4-fluorophenyl)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-
Compound 31: 2-Amino-1-(3-(4-chloro-3-fluorophenyl-
amino)-2-(4-fluorophenyl)-5,6-dihydroimidazo[1,2-a]pyrazin-
7(8H)-yl)ethanone
2
yl)-2-methylpropan-1-one
Compound 31 was synthesized from compound 7 using palladium
catalyzed amination reduction using palladium on carbon followed by
Compound 28 was synthesized from compound 7 using palladium
catalyzed amination, reduction using palladium on carbon followed by
HATU coupling using N-Boc-glycine followed by TFA mediated
1
deprotection. H NMR (400 MHz, MeOH-d
4
) δ 7.7 (m, 2H),
HATU mediated coupling with N-BOC-dimethylglycine and TFA
1
7.44À7.40 (m, 3H), 6.71 (m, 1H), 6.64 (m, 1H), 5.16 (s, 2H),
.18À4.01 (m, 6H). M/Z = 418.0 (M + 1). Elemental analysis of HCl
salt (compound + 2HCl + 2H O): % C, 45.6; % H, 4.59; % N, 13.29
calculated). % C = 47.46/47.57; % N = 13.17/13.18; % H = 4.04/4.05
mediated deprotection. H NMR (MeOH-d
4
, 400 Hz) δ 7.70
4
(
(
m, 2H), 7.21 (m, 4H), 6.8 (m, 2H), 5.19 (m, 2H), 4.26 (m, 2H), 4.0
m, 2H), 1.77 (s, 6H). M/Z = 428.1 (M + 1). Elemental analysis of HCl
2
(
(
salt (compound + 3HCl + H O): % C, 47.58; % H, 5.08, % N, 12.61
2
experimental).
Compound 32: 2-Amino-1-(3-(4-chloro-3-fluorophenyl-
(
(
calculated). % C = 47.46/47.57; % N = 12.48/12.5; % H = 4.72/4.81
experimental).
Compound 29: 3-Amino-1-(2-(4-fluorophenyl)-3-(4-fluo-
rophenylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
propan-1-one
amino)-2-(4-fluorophenyl)-5,6-dihydro imidazo[1,2-a]pyrazin-
(8H)-yl)-2-methylpropan-1-one
7
Compound 32 was synthesized from compound 7 using palladium
catalyzed amination, reduction using palladium on carbon followed by
HATU mediated coupling with N-BOC-dimethylglycine and TFA
Compound 29 was synthesized from compound 7 using palladium
catalyzed amination, reduction using palladium on carbon followed by
1
mediated deprotection. H NMR (MeOH-d
(
4
, 400 Hz) δ 7.59À7.56
HATU mediated coupling with N-BOC-glycine and TFA mediated
m, 2H), 7.2À7.11 (m, 3H), 6.6À6.57 (m, 2H), 5.04 (s, 2H), 4.14 (m,
2H), 3.97 (m, 2H), 1.69 (s, 6H). M/Z = 446.2 (M + 1). Elemental
analysis (compound + 3HCl + 0.2H O): % C,47.28; % H, 4.58; % N,
2.53 (calculated). % C = 47.07/47.19; % N = 12.33/12.34; % H = 4.86/
.82 (experimental).
Compound 33: 2-Amino-1-(3-(2,4-difluorophenylamino)-
1
deprotection. H NMR (MeOH-d , 400 Hz) δ 7.64À7.59 (m, 2H),
4
6
.96À6.89 (m, 3H), 6.38À6.34 (m, 1H), 6.27À6.23 (m, 1H),
.74À4.63 (m, 2H), 3.94À3.41 (m, 6H). M/Z = 402.1 (M + 1).
Compound 30: 2-Amino-1-(3-(3,4-difluorophenylamino)-
2
4
1
4
2-(4-fluorophenyl)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-
yl)-2-methylpropan-1-one
2-(4-fluorophenyl)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
2-methylpropan-1-one
Compound 30 was synthesized following the same sequence as
compound 1 involving an Ugi reaction, reduction using palladium on
carbon followed by HATU mediated coupling with N-BOC-dimethyl-
Compound 33 was synthesized from compound 7 using palladium
catalyzed amination, reduction using palladium on carbon followed
1
glycine and TFA mediated deprotection. H NMR (400 MHz, MeOH-
by HATU mediated coupling with N-BOC-dimethylglycine and TFA
1
d
6
4
) δ 7.64À7.70 (m, 2H), 6.95À6.91 (m, 3H), 6.33À6.39 (m, 1H),
mediated deprotection. H NMR (DMSO-d
6
, 400 MHz) δ 8.24 (bs,
.27À6.22 (m, 1H), 4.97 (s, 2H), 4.17 (m, 2H), 3.73 (t, J = 2.8 Hz, 2H),
1H), 7.83 (bs, 1H), 7.75 (m, 2H), 7.25 (m, 1H), 7.17 (m, 2H), 6.81
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Journal of Medicinal Chemistry
ARTICLE
(
(
m, 1H), 6.4 (m, 1H), 4.86 (m, 2H), 4.06 (m, 2H), 3.75 (m, 2H), 1.6
m, 6H). M/Z = 430.2 (M + 1).
Compound 34: 2-Amino-1-(3-(3,5-difluorophenylamino)-
-(4-fluorophenyl)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
-methylpropan-1-one
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2000À2010; World Health Organization: Geneva, Switzerland, 2010;
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(1), No. e1000402. (c) Sadanand, S. Malaria: an evaluation of the
current state of research on pathogenesis and antimalarial drugs. Yale J.
Biol. Med. 2010, 83 (4), 185–191. (d) Wu, T.; Nagle, A. S.; Chatterjee,
A. K. Road towards new antimalarials: overview of the strategies and
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2
2
(3) Noedl, H.; Chanthap, L.; Se, Y.; Socheat, D.; Peou, S.; Schaecher,
K.; Srivichai, S.; Teja-Isavadharm, P.; Smith, B.; Jongsakul, K.; Surasri, S.;
Fukuda, M. Artemisinin resistance in Cambodia? Trop. Med. Int. Health
2
007, 12, 69.
4) (a) Deng, X. M.; Nagle, A.; Wu, T.; Sakata, T.; Henson, K.; Chen,
(
Z.; Kuhen, K.; Plouffe, D.; Winzeler, E.; Adrian, F.; Tuntland, T.; Chang,
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Z.; Henson, K.; Chang, J.; Plouffe, D.; Tuntland, T.; Kuhen, K.;
Winzeler, E.; Gray, N.; Chatterjee, A. K. Cell-Based Optimization of
Novel Benzamides as Antimalarials. Abstracts of Papers, 236th National
Meeting of the American Chemical Society, Philadelphia, PA, Aug
Compound 34 was synthesized from compound 7 using palladium
catalyzed amination, reduction using palladium on carbon followed by
HATU mediated coupling with N-BOC-dimethylglycine and TFA
1
mediated deprotection. H NMR (MeOH-d
4
, 400 Hz) δ 7.6 (m, 2H),
7
2
.08 (t, J = 8.76 Hz, 2H), 6.25 (m, 3H), 4.98 (s, 2H), 4.11 (t, J = 5.28 Hz,
H), 3.9 (m, 2H), 1.66 (s, 6H). M/Z = 430.2 (M + 1).
’
AUTHOR INFORMATION
17À21, 2008; American Chemical Society: Washington, DC, 2008;
Corresponding Author
Phone: +1 858-332-4751. E-mail: akc@gnf.org.
MEDI 339, p 236. (c) Wu, T.; Nagle, A.; Sakata, T.; Henson, K.; Borboa,
R.; Chen, Z.; Kuhen, K.; Plouffe, D.; Winzeler, E.; Adrian, F.; Tuntland,
T.; Chang, J.; Simerson, S.; Howard, S.; Ek, J.; Isbell, J.; Deng, X.; Gray,
N. S.; Tully, D. C.; Chatterjee, A. K. Cell-based optimization of novel
benzamides as potential antimalarial leads. Bioorg. Med. Chem. Lett.
*
Author Contributions
These authors made equal contribution to this work.
#
2
009, 19 (24), 6970–6974.
5) Plouffe, D.; Brinker, A.; McNamara, C.; Henson, K.; Kato, N.;
(
’
ACKNOWLEDGMENT
Kuhen, K.; Nagle, A.; Adrian, F.; Matzen, J. T.; Anderson, P.; Nam, T. G.;
Gray, N. S.; Chatterjee, A.; Janes, J.; Yan, S. F.; Trager, R.; Caldwell, J. S.;
Schultz, P. G.; Zhou, Y.; Winzeler, E. A. In silico activity profiling reveals
the mechanism of action of antimalarials discovered in a high-through-
put screen. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (26), 9059–9064.
This work was supported by a joint grant to Genomics
Institute of the Novartis Research Foundation, the Biomedical
Primate Research Center, the Swiss Tropical and Public Health
Institute, and the Novartis Institute for the Tropical Diseases
from the U.K. Wellcome Trust and Medicines for Malaria
Venture.
(6) Liu, B.; Chang, J.; Gordon, W. P.; Isbell, J.; Zhou, Y.; Tuntland,
T. Snapshot PK: a rapid rodent in vivo preclinical screening approach.
Drug Discovery Today 2008, 13 (7À8), 360–367.
(7) (a) Varma, R. S.; Kumar, D. Microwave-accelerated three-
’
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ACT, artemisinin based combination therapy;WHO, World
Health Organization; MMV, Medicines for Malaria Venture;
GNF, Genomics Institute of the Novartis Research Foundation;
Cmax, the maximum plasma concentration; IC , half-maximal
50
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effective concentration; AUC, area under the concentrationÀ
time curve;hERG, human ether-a-go-go-related gene; PEG,
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