Imidazolopiperazines: Lead optimization of the second-generation antimalarial agents

Article abstract of DOI:10.1021/jm300041e

On the basis of the initial success of optimization of a novel series of imidazolopiperazines, a second generation of compounds involving changes in the core piperazine ring was synthesized to improve antimalarial properties. These changes were carried out to further improve the potency and metabolic stability of the compounds by leveraging the outcome of a set of in vitro metabolic identification studies. The optimized 8,8-dimethyl imidazolopiperazine analogues exhibited improved potency, in vitro metabolic stability profile and, as a result, enhanced oral exposure in vivo in mice. The optimized compounds were found to be more efficacious than the current antimalarials in a malaria mouse model. They exhibit moderate oral exposure in rat pharmacokinetic studies to achieve sufficient multiples of the oral exposure at the efficacious dose in toxicology studies.

Full text of DOI:10.1021/jm300041e

Article
pubs.acs.org/jmc
Imidazolopiperazines: Lead Optimization of the Second-Generation
Antimalarial Agents
Advait Nagle,†,# Tao Wu,†,# Kelli Kuhen,† Kerstin Gagaring,† Rachel Borboa,† Caroline Francek,†
Zhong Chen,† David Plouffe,† Xuena Lin,‡ Christopher Caldwell,‡ Jared Ek,† Suzanne Skolnik,‡
Fenghua Liu,‡ Jianling Wang,‡ Jonathan Chang,† Chun Li,† Bo Liu,† Thomas Hollenbeck,†
Tove Tuntland,† John Isbell,† Tiffany Chuan,† Philip B. Alper,† Christoph Fischli,§,∥ Reto Brun,§,∥
Suresh B. Lakshminarayana,⊥ Matthias Rottmann,§,∥ Thierry T. Diagana,⊥ Elizabeth A. 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
‡Novartis Institute for Biomedical Research, Preclinical Profiling Group, Cambridge, Massachusetts
§Swiss Tropical and Public Health Institute, Parasite Chemotherapy, CH-4002 Socinstrasse 57, CD-4002 Basel, Switzerland
∥University of Basel, CH-4003 Basel, Switzerland
⊥Novartis Institute for Tropical Diseases, 10 Biopolis Road, 05-01 Chromos, Singapore-138670
S
* Supporting Information
ABSTRACT: On the basis of the initial success of optimi-
zation of a novel series of imidazolopiperazines, a second genera-
tion of compounds involving changes in the core piperazine ring
was synthesized to improve antimalarial properties. These
changes were carried out to further improve the potency and
metabolic stability of the compounds by leveraging the out-
come of a set of in vitro metabolic identification studies. The
optimized 8,8-dimethyl imidazolopiperazine analogues exhib-
ited improved potency, in vitro metabolic stability profile and,
as a result, enhanced oral exposure in vivo in mice. The optimized compounds were found to be more efficacious than the
current antimalarials in a malaria mouse model. They exhibit moderate oral exposure in rat pharmacokinetic studies to achieve
sufficient multiples of the oral exposure at the efficacious dose in toxicology studies.
the three peripheral parts had yielded decent in vitro and in
vivo antimalarial activities as well as favorable physiochemical
and pharmacokinetic properties.
INTRODUCTION
■
After decades of neglect, the search for novel, effective anti-
malarials has been revived primarily due to emergence of drug
resistance.1 Widespread resistance has been reported not only
for 8-aminoquinolines but recent reports indicate the appear-
ance of increased parasite tolerance to artemisinin analogues,
potentially predicating clinical resistance.2 The absence of
highly efficacious malaria vaccines highlights the importance of
developing small-molecule drugs.3 With support from the
Wellcome Trust and Medicines for Malaria Venture (MMV),
the NGBS consortium (the Novartis Institute for Tropical
Diseases (NITD), the Genomics Institute of the Novartis
Research Foundation (GNF), the Biomedical Primate Research
Centre (BPRC), and the Swiss Tropical and Public Health
Institute (STPHI)) was formed and set up to discover new
antimalarials which are effective against multidrug resistant
parasite strains. This collaboration has produced promising new
leads.1a,4 Here we report our continued effort to optimize a
series of imidazolopiperazine compounds in search of a pre-
clinical candidate. The lead compound we identified was com-
pound 1.5 As described previously, an extensive SAR study on
Our efforts toward the first-generation compounds led to
candidates (like compound 1) with double-digit nanomolar
potency and moderate oral exposure in mice. However, most
potent compounds with an unsubstituted piperazine had
poor oral exposure which resulted in inferior in vivo efficacy
when administered orally, for example, compounds 28 and
31 (Scheme 1) in the previous communication (Figure 1).5 We
were intrigued by the possibility of further refining the core
structure of the molecule to further improve the properties.
Our hypothesis was further reinforced by chemical stability and
mouse in vitro metabolic stability results which led to the identi-
fication of two biologically inactive metabolites (1a and 1b)
arising from the oxidation of piperazine ring. Our plan was to
substitute three possible positions (positions 5, 6, and 8) to
Received: January 11, 2012
Published: April 23, 2012
© 2012 American Chemical Society
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Cbz-protected glycine derivative 2 and bromide 3. Adduct 4
was then refluxed with NH4OAc in toluene to give imidazole
5 with removal of water using a Dean−Stark apparatus.7
Regioselective N-alkylation with ethyl 2-bromoacetate followed
by a one-pot deprotection/cyclization sequence furnished the
lactam core, which was reduced by borane to furnish amine 8.
The quaternary β-carbon did not pose a significant amount of
steric hindrance for the HATU mediated amidation reaction
with N-Boc-glycine. Amide 9 was obtained at room temper-
ature in decent yield and further was brominated to provide
intermediate 10. Then a standard Buchwald−Hartwig amina-
tion reaction with substituted aniline followed by deprotection
finished the synthesis of compound 12 with the desired alkyl
modification at position 8. The overall sequence involved nine
steps with an overall yield of 17%.
Scheme 1. Unsubstituted Piperazines Previously Disclosed
evaluate the corresponding effects on potency as well as a
means to improve oral exposure.
Scheme 3 describes the synthesis of 6,6-dimethyl analogues.
Analogous chemistry to compound 12 was used for the first
two steps to provide imidazole 5a. The compound was
regioselectively N-alkylated with methallyl chloride to obtain
the key intermediate for the piperazine cyclization step. The
intramolecular hydroamination reaction proved to be difficult,
and after extensive screening, treating the substrate 5a with a
5:1 mixture of acetic acid and methanesulfonic acid at 210 °C
produced the cyclized core with simultaneous loss of the
CHEMISTRY
■
On the basis of the designed molecules, it was clear that
previous synthetic routes were not amenable for the desired
compounds because we could not utilize the 3 + 2 cyclization.6
Distinct syntheses were designed and executed to obtain
substitution at different positions of a piperazine ring. Scheme 2
summarizes how the 8-substituted piperazines were prepared.
The synthesis started with an alkylation reaction between
Figure 1. Central piperazine ring oxidation is a major metabolic and chemical instability site in compound 1.
a
Scheme 2. Modification on the 8 Position of Imidazolopiperazine Core
a
Reagents and conditions: (a) K2CO3, DMF, rt, 84%; (b) NH4OAc, toluene, reflux, 88% (c) ethyl 2-bromoacetate, Cs2CO3, DMF, rt, 83%; (d) Pd/
C, H2 (balloon), MeOH, rt, 91%; (e) BH3·THF, THF, reflux, 95%; (f) HATU, DIEA, CH2Cl2, rt, 70%; (g) Br2, AcOH, CH2Cl2, rt, 100%; (h)
ArNH2, Pd2(dba)3, xantphos, Cs2CO3, dioxane, 150 °C, 89% (for example 22, Ar = 4F-Ph and R1 = R2 = Me); (i) TFA, CH2Cl2, rt, 52% (for
example 22, Ar = 4F-Ph and R1 = R2 = Me).
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a
Scheme 3. Modification on the 6 Position of Imidazolopiperazine Core
a
Reagents and conditions: (a) methallyl chloride, K2CO3, KI, DMF, rt, 55%; (b) AcOH/MsOH (6:1), 210 °C, 39%; (c) N-Boc-glycine, HATU,
DIEA, DMF, rt; 57%; (d) Br2, AcOH, CH2Cl2, rt; 55%; (e) ArNH2, Pd2(dba)3, xantphos, Cs2CO3, dioxane, 150 °C; (f) TFA, CH2Cl2, rt, 55% over 2
steps (for example 25, Ar = 4F-Ph).
a
Scheme 4. Modification on the 5 Position of Imidazolopiperazine Core
a
Reagents and conditions: (a) PMBCl, KOH, DMF, 0 °C to rt, 63%; (b) MeI, NaH, DMF, rt, 86%; (c) BH3·THF, THF, reflux, 100%; (d) TFA,
70 °C, quantitative; (e) N-Boc-glycine, HATU, TEA, DMF, rt, 49%; (f) Br2, AcOH, CH2Cl2, 90%; (g) ArNH2, Pd2(dba)3, xantphos, Cs2CO3,
dioxane, 150 °C; (h) TFA, CH2Cl2, rt, 46% over 2 steps for example 26 where Ar = 4F-Ph.
carbamate protecting group. Despite the harsh reaction
condition, an acceptable yield of the product (39%) was
obtained and the regiochemistry was confirmed by 2D NMR of
the intermediate. The cyclized core was subjected to amide
coupling with N-Boc glycine to yield intermediate 13. The final
steps in the sequence are analogous to Scheme 2 to furnish
analogue 14. The sequence involved six steps from inter-
mediate 5a with an overall yield of 3.7%. The synthesis of the
5,5-dimethyl modified analogues diverged from their 8,8-
dimethyl modified counterparts at intermediate 7a. N-PMB
protection was necessary for lactam 7a before the NaH-
mediated bis-methylation at the 5-position (Scheme 4). The
lactam was then reduced using borane THF complex to afford
7b. The bicyclic intermediate was then deprotected and amine
was subjected to a HATU mediated amidation to obtain
compound 7c. The final sequence involved bromination
followed by palladium-catalyzed aniline amination and Boc-
deprotection to furnish 5,5-dimethyl modified compound 26.
The sequence involved eight steps from intermediate 7a with
an overall yield of 10.9%.
Emboldened by the increase in the potency based on the
substitution on the 8-position, we decided to study the effect of
diMe group on the ethylene carbons of the piperazine ring. On
the basis of examples 25 and 26, it was abundantly clear that
the dimethyl group on the 8-position had the most significant
positive effect on potency. The substitution on the other
positions of the piperazine ring only provided modest potencies
when compared to 8-substitution, with activities in the double-
digit nanomolar range. As the 5-substituted compounds and the
6-substituted compounds were less potent, we decided to focus
our attention on the effect of the amide substituent of the
piperazine nitrogen with the 8,8-dimethyl group constant. Our
previous efforts demonstrated that dimethyl glycine had slightly
inferior potency but offered superior oral exposure. In the case
of the substituted piperazines, installation of α methylalanine
amide provided equipotent compounds (15 vs 27, 22 vs 28,
and 23 vs 29). We proceeded to further understand the overall
effect of the dimethylpiperazine on the minimal pharmaco-
phore.
After studying the effects of various substitutions on the
piperazine ring and obtaining a significant improvement in
activity, we decided to reconfirm the minimal pharmacophore
needed for antiplasmodial activity. On the basis of the earlier
results, we were able to achieve antiplasmodial activity with
EC50 in ∼160−400 nM range for the core devoid of the amino
acid substituent on the piperazine nitrogen (compound 31,
Table 2). Modification of the piperazine ring into the
corresponding lactam led to a ∼7-fold decrease in activity in
the micromolar range (32). However, the antiplasmodial
activity is regained by introduction of dimethyl substituent on
the 8-position with a 7-fold improvement (33). The most
optimal compound from this lactam subseries was a compound
with 3Cl-4F aniline substitution with an activity around 50−
100 nM (34). The dimethyl substitution on the reduced
piperazine core led to ∼10-fold improvement in potency (31 vs 35)
with compounds to ∼10 nM range. These results led us to
conclude that the amino acid substitution had minimal to no
effect on the potency of the compound with substitution on the
8-position of the imidazolopiperazine core and that the basicity
RESULTS AND DISCUSSION
■
Structure−Activity Relationship. We began exploring the
SAR on the piperazine ring by substituting at the 8-position
first, and we decided to use 4-F-aniline and 3-F-4-Cl aniline
based on our previous work at the R6 position (Table 1).5
Adding a methyl group to the 8-position led to loss of potency
by 10-fold (cmpd 17), but the compound was still equipotent
to compound 15 (Scheme 5). Moreover, there was no pro-
found effect on the potency by changing the absolute
configuration of the methyl group. Increasing the size of
substituent to isopropyl group led to dramatic loss in potency
(examples 19 and 20). A dimethyl group on the 8-postion not
only led to more favorable compound in terms of completely
blocking the benzylic carbon as indicated by our metabolite
studies but also removing the stereogenic carbon. Surprisingly,
substitution of 3Cl-4F aniline in the 8-disubstitution case
provided a less potent compound when compared to other
anilines (21 vs 22, 23, or 24).
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a
Table 1. SAR Study for Analogues with Substitution on the Piperazine Core
P. falciparum strain EC50
3D7 (nM) W2 (nM)
compd
R1
R2
R3
R4
R5
R6
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
H
H
H
H
gly
gly
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
4-FPh
3Cl,4F-Ph
3Cl,4F-Ph
3Cl,4F-Ph
3Cl,4F-Ph
3Cl,4F-Ph
3Cl,4F-Ph
4-FPh
24
4
2
3
2
4
23
4
8
2
1
3
(S)-Me
(R)-Me
(S)-i-Pr
(R)-i-Pr
Me
H
gly
29
31
24
29
H
gly
H
gly
343 59
168 15
470 130
202 38
H
gly
Me
Me
Me
Me
H
gly
27
10
8
1
3
2
1
7
5
5
1
1
3
24
6
1
1
1
1
Me
gly
Me
gly
4-MePh
3,4-diFPh
4-FPh
6
Me
gly
4
5
H
gly
82
80
20
10
2
111 17
76 15
H
H
gly
4-FPh
H
H
α-Me-Ala
α-Me-Ala
α-Me-Ala
α-Me-Ala
4-FPh
25
9
2
1
1
1
Me
Me
Me
Me
4-FPh
Me
4-MePh
3,4-diFPh
4
Me
13
7
a
Values are means of two experiments. Each assay plate has mefloquine, sulfadoxine, and artimesinin as internal standards. The EC50 values for
standard compounds match the literature values.
Scheme 5. Structures of Key Compounds
of the piperazine nitrogen could be modulated without
complete loss in antimalarial activity.
(31, 32, 34, and 35). The permeability of the compounds was
reduced once the side chain amino acid was installed, but
metabolic stability is improved (28 vs 35). There was no clear
correlation between Caco-2 and PAMPA assay. However, the %
absorbed was lower in the PAMPA assay for compounds with
poor permeability in the Caco-2 assay. Upon comparing the
calculated log P values with PAMPA as well as Caco-2 values,
we discovered no correlation with any of the permeability param-
eters. Unsurprisingly, log P of 4.5 or higher was needed for
having excellent percentage absorbed values in PAMPA assay
(see Supporting Information). Removing the basicity of the
piperazine ring by introduction of carbonyl group improved
the mouse and human microsome stability of the compound,
presumably by blocking the ethylene bridge (31 vs 32) but is
ADME and Pharmacokinetic Properties. In parallel to
the SAR studies to optimize the potency of the compounds
series, we evaluated the in vitro ADME and in vivo phar-
macokinetic properties of key analogues. In general, this series
of compounds had very good Lipinski's rule-of-five compliance
and most of the analogues were highly soluble (>175 μM at pH
6.8), with the neutral lactams being least soluble among all the
compounds. Table 3 summarizes the in vitro ADME profiles
of the important compounds. Typically, a range of values in
the permeability assays was observed. In the Caco-2 assays, the
core piperazine compounds (both lactams as well as nonamide
piperazine compounds) exhibited high apical to basal values
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Table 2. SAR Study for Analogues with Substitution on the Piperazine Core
P. falciparum strain EC50
3D7 (nM) W2 (nM)
compd
R1
R2
R3
mouse microsome hepatic extraction ratio
31
32
33
34
35
36
H
H
O
O
O
H
4-MePh
4-MePh
4-MePh
3Cl,4F-Ph
4-MePh
4-FPh
0.80
0.70
0.72
0.61
0.77
0.80
161 13
1079 285
138 10
178 12
1168 188
136 20
H
Me
Me
Me
Me
36
13
5
2
2
1
65
13
5
3
1
1
H
Table 3. In Vitro ADME of Selected Analogues
a
b
b
c
d
e
entry
PAMPA
Caco-2 A−B
Caco-2 B−A
CYP Met CL-Ms (ER)
CYP Met CL-rat (ER)
CYP Met CL-Hu (ER)
hERG IC50 (μM)
15
22
24
27
28
29
31
32
33
34
35
36
52
0.70
1.26
0.61
3.05
1.2
2.55
2.99
1.3
0.47
0.48
0.46
0.51
0.4
nt
<0.21
0.49
0.53
0.52
0.55
0.47
0.79
0.44
nt
nt
13.4
>30
5.6
21.9
>30
nt
51.8
51.5
81
0.66
0.69
0.63
0.89
0.79
0.96
0.95
nt
8.38
0.97
2.37
2.36
14.37
nt
99.5
99.5
99.5
80.9
nt
3.5
0.41
0.87
0.66
0.72
0.61
0.87
0.8
8.2
15
nt
nt
nt
97.1
99.9
99.9
3.22
15.56
nt
3.35
6.96
nt
0.68
0.93
0.72
0.88
0.92
>30
nt
0.92
nt
a
b
c
d
e
Measured as percentage absorbed. units = (cm/s ×106). Ms = mouse. Hu = human. Q-patch assay; nt = not tested.
a
Table 4. Complete in Vivo Pharmacokinetic Profile of Selected Analogues in Mice
IV PK parameters
PO PK parameters
entry
Vd (L/kg)
CL (mL/min/kg)
T1/2 (h)
AUC(0−24 h) (h·nM)
Cmax (nM)
Tmax (h)
T1/2 (h)
AUC(0−24 h) (h·nM)
F (%)
15
22
24
27
29
34
36
12.3
10.2
7.5
188.9
49.2
27.5
60.0
36.1
12.5
25.9
2.7
2.0
1072
4119
7062
3442
7607
16525
9090
235
1538
1723
1803
1642
3651
6404
2.0
1.0
3.0
1.7
1.0
1.0
1.0
4.20
4.35
5.21
8.95
14.54
2.71
2.95
965
12155
21058
17894
32409
50619
19447
15
74
5.5
75
23.7
7.5
6.2
87
18.0
2.8
107
77
2.7
1.2
0.8
53
a
PO dosing; dose (mg/kg), 20; species/strain, mouse, male balb/c; formulation, 2.5 mg/mL in PEG300/D5W, 3:1, solution; Vd = volume of
distribution; CL = clearance; Cmax = maximal concentration; Tmax = time at maximal concentration; AUC = area under the curve; F = oral
bioavailability.
not evident in rat microsomes. Introduction of the dimethyl
group on the 8-position of the piperazine ring does not im-
prove overall metabolic stability of the compound (15 vs 28 or
31 vs 35) despite the metabolite identification results presented
earlier. This may be due to the ethylene bridge in the pipera-
zine ring representing a metabolic soft spot and enabling meta-
bolic switching. In general, this scaffold exhibited poor rat
metabolic stability when compared to other species. One inter-
esting finding with the substituted piperazine analogues was
the improvement of activity against the hERG channel as deter-
mined in the automated patch clamp assay. Compared to the
unsubstituted piperazine analogue 27, we were able to decrease
inhibition of the hERG channel by at least 2−3-fold, with most
analogues (such as 22) suggesting a lower potential for QT
interval prolongation in compounds with a substituted
piperazine.
Pharmacokinetic Properties. Compounds series found to
have favorable potency and in vitro ADME properties were
tested in snapshot PK, an abbreviated PK study shown to
accurately bin compounds into low, moderate, or high oral
exposure.9 Compounds predicted to have reasonable oral
exposure would be tested in mice using both PO and IV routes
of administration (Table 4). The in vivo clearance generally
was low to moderate and significantly improved when com-
pared to the corresponding unsubstituted analogues (15, 22).
This resulted in higher maximum concentration as well as
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improvement in AUC values in the oral arms as well. Lactam
compound 34 exhibited highest oral AUC than all other ana-
logues, and it also had lower intrinsic clearance with moderate
volume of distribution. To test whether red cell partitioning
was occurring to explain the high volume of distribution for the
basic compounds, we carried out an in vitro experiment in
the second animal species for oral exposure evaluation as it may
serve as the in vivo preclinical toxicity species. Taking into
account the nonoptimal metabolic stability data for rats for this
scaffold, we decided to study the oral exposure of the com-
pound in rat for a period of 5 h in the oral arm only to get an
estimation of the exposure (Table 6).9 The results show that
̈
which compound 22 was incubated in naive rat whole blood as
well as plasma for 30 min. After centrifuging the blood and
quantifying the amount of compound in the red blood cells, we
determined that the concentration of the compound was 3.47
times higher in whole blood than in plasma alone, indicating
that the compound is either adhering to the RBC’s or getting
distributed inside the cells.
In Vivo Efficacy. Because we had examples of compounds
with different modifications with acceptable bioavailability, we
decided to carry on the efficacy experiment in mice. The in vivo anti-
malarial activities of the optimized compounds were evaluated using a
Plasmodium berghei mouse survival model.8 In this test, groups of
three P. berghei infected mice were treated orally with compound
one day after infection. The key readouts were the percentage of
parasitemia reduction on day 3 postinfection compared to the
untreated mice and the prolongation of survival of the infected mice.
Table 5 summarizes the in vivo efficacy test results of com-
pound in comparison with standard antimalarials chloroquine
Table 6. Rat Snapshot PK Results for Interesting
Compounds
a
PK parameters
AUC(0−5 h)
(h·nM)
Cmax
(nM)
AUC(0−5 h)/dose
[(min·μg/mL)/(mg/kg)]
entry
22
24
29
34
36
487
367
NA
126
96.66
BLQ
504.44
202
1.2
0.95
2097
423
5.07
0.9
a
PO dosing; dose (mg/kg), 20; species/strain, rat, Wistar;
formulation, 2.5 mg/mL in PEG300/D5W, 3:1, solution; BLQ,
below level of quantification; NA = could not be determined.
the 8-substituted piperazine compounds had inferior exposure.
α-Methylalanine subclass of compounds like 29 did not exhibit
any in vivo exposure and were not further pursued. In line with
the better rat metabolic stability (ER = 0.68), nonbasic lactam
compound 34 demonstrated the best rat exposure despite lower
mouse efficacy.
Table 5. In Vivo Efficacy of Compounds on Plasmodium
berghei Mouse Survival Model Following Different Dose
a
Levels in Comparison with Known Antimalarials
1 × 30 mg/kg
activity survival
1 × 100 mg/kg
3 × 30 mg/kg
activity survival
As the oral exposure of compounds 22 was low in rats but
demonstrated superior mouse efficacy, we decided to dose
compound 22 at higher doses to make sure that sufficient
multiples of exposure of the mouse efficacious dose could
be achieved (Table 7). We observe that when the dose was
increased to 30 mg/kg from 10 mg/kg, there was over-
proportional 7-fold increase in AUC as well as 2-fold
increase in bioavailability. Increasing the dose further to
100 mg/kg dose with suspension formulation led to 55-fold
increase in AUC with a 3-fold increase in oral bioavail-
ability. The AUC at the mouse efficacious dose (2 mg/kg)
was 780 h·nM, therefore a ∼45× exposure multiple is achi-
eved at 100 mg/kg in the rat. The multiple of exposure
provides a sufficient window to use the rat as a rodent
toxicology species. It is unclear at this time if this over-
proportional exposure at higher doses is due to saturating
metabolism or an efflux transporter, but given the partial
efflux observed in Caco-2, this may explain this multiple
dose exposure results.
In Vitro Toxicology. Compound 22 was subjected to a
series of assays to evaluate the toxicity profile. This drug
candidate was inactive on a panel of human cytochromes
P450 (IC50s are greater than 6 μM for CYP1A2, CYP2C19,
CYP2C9, CYP2D6, and CYP3A4), and the observed
cytotoxicity CC50 were all greater than 12 μM on a panel
of mammalian cell lines (293T, Ba/F3, CHO, HEp2, HeLa,
Huh7), which translates to an adequate selectivity index
(SI > 100). Compound 20 was found negative in the mini-
Ames and micronucleus tests, which indicated a low mutagenic
potential. Finally, considering the efficacious plasma Cmax in the
mouse at 2 mg/kg is low (∼75 nM), the risk of cardiotoxicity is
considered to be minimal where the hERG channel inhibitory
activity IC50 = 13.4 μM, providing a greater than 170-fold
window.
activity
survival
(day)
entry
(%) (day)
(%)
(%) (day)
22
24
29
34
36
99.5
99.0
99.0
47.0
90.0
99.5
95.6
16.3
15.0
15.3
6.3
99.4
99.1
99.0
97.0
99.3
99.6
98.0
14.0
16.7
16.7
6.7
99.8
99.7
99.7
66.0
99.7
99.8
99.0
17.7
17.0
18.0
7.0
7.0
9.7
12.0
13.6
12.2
b
CQ
9.0
12.5
7.0
b
AS
5.8
a
Activity = average parasitemia reduction; survival = average lifespan
after infection (6−7 days for untreated control mice); compounds
were formulated in 75% PEG300/25% D5W (5% dextrose in water);
b
CQ, chloroquine; AS, artesunate. 10% ethanol, 30% PEG400, 6%
vitamin ETPGS.
(CQ) and artesunate (AS). It was observed that compounds
22, 24, and 29 exhibited >99% parasitemia reduction at a low
dose of 30 mg/kg with a modest survival of 15 days or higher.
Increasing the dose to 100 mg/kg or dosing the compound 3 ×
30 mg/kg did not lead to increase in survival. Currently, we are
investigating the possibility of recrudescence of parasites or
development of resistance for the compound in vivo to better
explain the lack of curative activity in this model. We decided to
determine the ED99 representative examples for the substituted
and nonsubstituted piperazine cores and found the substituted
piperazine core was ∼10-fold more efficacious (compound 22
ED99 = 2.2 mg/kg) than the unsubstituted core (compound 1
ED99 = 26.5 mg/kg). Compounds 34 and 36 clearly proved
inferior both in terms of parasitemia reduction as well as
survival in mice, indicating the need for the amino acid side
chain in providing better mouse efficacy.
On the basis of the efficacy results, we decided to study the
oral exposure of several compounds in rats. Rat was chosen as
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Table 7. In Vivo Rat Dose Escalation PK Tests for Compound 22
IV PK parameters
PO PK parameters
Cmax (nM) Tmax (h) T1/2 (h) AUC(all) (h·nM) F (%)
entry Vd (L/kg) CL (mL/min/kg) T1/2 (h) AUC (h·nM)
dose mg/kg
22
13.7
67.5
4.6
1831
10
30
91
580
1.5
4.3
8.0
4.7
6.3
8.4
974
7337
34885
20
40
57
100
34885
dilution). On the basis of the measured parasitemia levels, each para-
site strain was diluted to yield 0.5% parasitemia, and 50% hematocrit
blood (uninfected erythrocytes) was added to a concentration of
4.17%. This diluted culture was 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 was 0.3% and final
hematocrit was 2.5% in a total volume of 50 μL. Assay plates were
incubated at 37 °C for 72 h in a gas chamber filled with low oxygen
blood gas. After the 72 h incubation (equivalent to 1−2 cycles during
the blood stage), a prepared mixture of the lysis buffer (5 mM EDTA,
1.6% Triton X-100, 20 mM Tris-HCl, 0.16% saponin) in water and
SYBR Green detection (0.1%) reagent was dispensed at 10 μL per
well using the MicroFlo. Cultures were incubated for an additional
24 h at 25 °C before measuring fluorescence intensity using the Envi-
sion plate reader (Perkin-Elmer) with a 505 dichroic mirror. Excitation
and emission filters are 485 and 530 nm, respectively. Data were
normalized based on maximum fluorescence signal values for DMSO
treated wells (no inhibition by compound) and the minimum fluo-
rescence signal values for wells containing the highest concentration of
inhibitor control compounds, for example, pyrimethamine at a final
concentration of 10 μM. Data were analyzed on a plate-by-plate basis
and compared to reference compounds that were always included on
every plate, typically artemisinin, mefloquine, and pyrimethamine.
EC50 values were obtained using a custom curve fitting model, and
a standard logistic regression model was applied for curve fitting.
The quality of the assay run was 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. Additionally, all compounds were
typically assayed at a minimum in duplicate (independent assay
plates) and EC50 values ideally must not vary by more than 2-fold
between plates.
Metabolic Stability. The metabolic stability of drug candidates
was determined in human, mouse, and rat liver microsomes using the
compound depletion approach, quantified by LC/MS/MS. The assay
measured the rate and extent of metabolism of chemical compounds
by measuring the disappearance of the parent compound. The assay
determined the compound’s in vitro half-life (T1/2) and hepatic
extraction ratios (ER) and predicted metabolic clearance in human,
rat, and mouse species.11
PAMPA Assay. Permeation experiments were carried out in 96-
well microtiter filter plates. The compound concentration was deter-
mined by UV absorption measured with a SpectraMax190 microplate
spectrophotometer (Molecular Devices Corporation, Sunnyvale, CA,
USA) at absorption wavelengths between 260 and 290 nm.12
Caco-2 Assay. The Caco-2 assay was carried out in a 96-well
format, and compound concentrations in each chamber were mea-
sured by LC/MS. The assay offers apparent permeability data of test
compounds from the apical to the basolateral chambers [Papp (A−B)]
and from the basolateral to the apical chambers [Papp (B−A)]. The
results can be used to predict the in vivo oral absorption (using
Papp (A−B)) and the transport mechanism of the test compounds
across GI membrane (ratio of Papp (B−A):Papp (A−B) and log P
lipophilicity data).13
SUMMARY
■
Imidazolopiperazine compounds had been proven to be
effective against multidrug resistant parasite strains with mini-
mum toxicity to host-cell lines. However, early lead compound
such as compound 15 and 27 still suffer from moderate
potencies, relatively poor rat PK, and potent hERG inhibitory
activity. Directed by the identification of in vitro metabolites
formed by compound 1, the piperazine core of 15 was sys-
tematically modified by a gem-dimethyl functionality at all three
possible places. The 8,8-dimethyl analogues demonstrated
excellent potency as well as improved oral exposure in the
mouse. This also allowed us to use the glycine amide that re-
duced our hERG risk when compared to 27. The first genera-
tion of compounds exhibited efficacy that was comparable to
the known antimalarials in the P. berghei mouse model, how-
ever, compound 22 was more efficacious than chloroquine and
artesunate especially when parasitemia reduction is compared
in terms of ED99 values. Although compound 22 demonstrated
poor rat PK at low doses, with higher doses it was able to
achieve the necessary exposure multiples calculated based
on the plasma concentration found to be efficacious in the
mouse model. Compound 22 is thus currently under pre-
clinical safety assessment in rats as a potential candidate for
human trials.
EXPERIMENTAL SECTION
Experimental Protocols (Biology). Maintenance of Plasmo-
dium falciparum Cultures. Plasmodium falciparum cultures were
grown in O+ RBCs using culturing media: RPMI 1640 media
■
(no phenol red) with 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. Using traditional
P. falciparum protocols published previously, 25 mL cultures
were maintained in 75 cm2 flasks (Fisher cat. no. 10-126-41) at
5% hematocrit and parasitemia ranging between 1% and 10%.10
Once parasitemia reached 8−10%, cultures are diluted down to
1% or higher depending on needs. Maintenance required daily
media changes and fresh blood every two weeks. Cultures
were gassed for approximately 30 s to 1 min using a blood gas
mixture to maintain a gas composition of 96% nitrogen, 3%
carbon dioxide, and 1% oxygen and incubated at 37 °C. On day
1 of the assay, percent parasitemia was determined by obtain-
ing a 1 μL blood smear. The smear was fixed onto the slide
by placing in methanol for 30 s, stained in 10% Geimsa stain,
and the percent of parasitized erythrocytes vs uninfected
erythrocytes was determined by microscopy with a light
microscope.
Antiplasmodial Proliferation Inhibition Assay: 384-Well Plate
Format. P. falciparum strains to be used for screening were prepared
with screening media (culturing media without human serum but
supplemented with 0.5% Albumax II) and fresh erythrocytes. Twenty
μL of screening media were dispensed via the MicroFlo (BioTek) into
384-well, black clear-bottom assay plates (μClear GNF custom plates
by Griener Bio-One). Then 50 nL of compounds were transferred
using a PlateMate Plus (Matrix) or the GNF Systems PinTool into the
assay plates containing screening media to a final maximum
concentration of 10 μM in a 12 point dose−response (1/2 log serial
hERG (Q-Patch) Assay. The assay utilizes electrophysiology
measurement on the electric current passing through hERG channel
that is heterologously expressed in a stable CHO cell line. Channels
were open by a hERG-specific voltage protocol, and the compound
effects were directly characterized by their block of the hERG current.
The assay was performed on an automated platform, Q-Patch. All
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compounds were tested by 4-point dose−response curves, and IC50
values (between 0.2 and 30 μM) were measured.14
2 × 107 erythrocytes parasitized with P. berghei GFP ANKA malaria
strain (PbGFPCON donation from A. P. Waters and C. J. Janse,
Leiden University).8a,15 Untreated control mice die typically between
day six and day seven post infection. Experimental compounds were
formulated in 75% PEG300/25% D5W as indicated. Chloroquine and
artesunate were formulated in 10% ethanol, 30% PEG400, and 6%
vitamin E TPGS. 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 post infection). With the
single dose regimen, parasitemia was determined (72 h post infection)
and for the triple dose regimen (96 h post infection) using standard
flow cytometry techniques.15 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.
In Vivo PK. In Vivo Pharmacokinetics Studies in Mice. In-life
studies were carried out under protocols approved by the
Animal Care and Use Committee (IACUC) of GNF. All
compounds were formulated at a concentration of 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 = 3 per group). The filtered solutions were also
administered orally at 20 mg/kg (with a dosing volume of
8 mL/kg) to another group of mice (n = 3 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
plasma separated and frozen until analysis. Plasma samples
were analyzed by LC-MS/MS.
In Vivo Pharmacokinetics Studies in Rats (Snapshot and Full PK).
Compounds were formulated at a concentration of 2.5 mg/mL in 75%
PEG300 and 25% D5W solution. In rat snapshot PK, two rats (Wistar
rats) were administrated orally at 10 mg/kg dose and four blood
samples (100 μL) were taken at 0.5, 1, 3, and 5 h post dosing. Before
the sample analysis, the plasma samples were pooled based on the
sampling time. In the rat full PK studies, the formulations were filtered
before the administration to rats. Three rats were dosed intravenously
(3 mg/kg), and three rats were administrated orally (10 mg/kg). Six
blood samples (100 μL per sample) were collected serially from each
rat within 24 h post dosing.
Extraction and LCMS Analysis. Plasma samples (20 μL) were
extracted with solution of acetonitrile:methanol (3:1) containing
internal standard. The samples were vortexed and then centrifuged
with an Eppendorf centrifuge 5810R (Eppendorf, Hamburg,
Germany) at a setting of 4000 rpm for 5 min at 4 °C. 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 × 30 mm, 3.5 μm, Agilent Technologies
Inc., Palo Alto, CA, USA). Separation was carried out using a gradient
elution method with a mobile phase consisting of 0.05% formic acid in
water (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 micro vacuum degasser (Agilent Technologies Inc.), HTC
PAL CTC analytics autosampler (LEAP Technologies, Carborro,
NC), and VICI two-position actuator (Valco Int., Houston, TX, USA)
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 transition of compound 15 383.9/101.2, compound 22 412.2/
312.1, compound 24 430.1/330.1, compound 27 412.2/327.2,
compound 29 436.1/308.1, compound 34 430.0/346.1, and
compound 36 355.0/272.3 was used with its optimized MS
parameters, respectively.
Pharmacokinetic Analysis. The mean value from the three
animals at each time point was plotted against time to give plasma
concentration time profile. Pharmacokinetic parameters were
determined using Watson LIMS, version 7.2 (Thermo Electron
Corporation, PA, USA), by noncompartmental analysis. Pharmacoki-
netic parameters for compounds were calculated by noncompartmen-
tal 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 =
AUCpo × doseiv/AUCiv × dosepo).
Experimental Protocols (Chemistry). Materials and Meth-
ods. Unless otherwise noted, materials were obtained from
commercial suppliers and were used without purification.
Removal of solvent under reduced pressure refers to distilla-
tion using Buchi 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 com-
pounds by high pressure liquid chromatography was achieved
using a Waters 2487 series with Ultra 120 5 μm C18Q column
with a linear gradient from 10% solvent A (acetonitrile with
0.035% trifluoroacetic acid) in solvent B (water with 0.05%
trifluoroacetic acid) to 90% A in 7.5 min, followed by 2.5 min
elution with 90% A.
1H NMR spectra were recorded on Bruker XWIN-NMR (400 or
600 MHz). Proton resonances are reported in parts per million (ppm)
1
downfield from tetramethylsilane (TMS). H NMR data are reported
as multiplicity (s singlet, d doublet, t triplet, q quartet, quint quinted,
sept septed, dd doublet of doublets, dt doublet of triplet, bs broad
singlet), number of protons, and coupling constant in hertz. For
spectra obtained in CDCl3, DMSO-d6, and CD3OD, the residual
protons (7.27, 2.50, and 3.31 ppm, respectively) were used as the
reference.
Analytical thin-layer chromatography (TLC) was performed on
commercial silica plates (Merck 60-F 254, 0.25 mm thickness);
compounds 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, 230−
400 mesh). The purity and quantitative analysis were determined with
a Waters ZQ 2000 LC/MS system, which employed an Acquity UPLC
system (binary pump, column compartment, autosampler, and diode
array UV detector). The eluent was split between the mass
spectrometer and an Antek chemiluminescent nitrogen detector
(CLND). The mobile phases used were (A) 95% H2O/5% MeOH/
IPA (75/25, v/v) + 0.05% formic acid and (B) MeOH/IPA (75/25,
v/v) + 0.035% formic acid. A gradient HPLC method with flow of
0.4 mL/min started at 2% B, with a hold of 1.0 min before a linear
increase to 95% B at 3.50 min, with a 30 s hold. From 4.0−4.25 min,
the mobile phases were returned to starting conditions. Total run time
was 5.0 min. The column used was a Thermo Syncronis C18 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
and desolvation temperatures were 130 and 400 °C, respectively, with
600 L/h of nitrogen desolvation flow. All the final compounds
reported in this communication had the purity of at least 95%.
Elemental analyses were carried out by Midwest microlabs LLC,
Indianapolis, IN, USA.
Synthesis of 1a. Compound 1a was prepared from 1 by the
following method: to a stirred solution of 1 (12 mg, 0.03 mmol) was
added MnO2 (52 mg, 0.60 mmol). The reaction mixture was stirred at
room temperature (rt) for 2 h. LC/MS test showed that 1 was almost
consumed and the desired product ([M + 1] = 428) was detected as
one of the major peaks. Solid was filtered off, and solvent was
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
female NMRI mice (20−22 g) intravenously infected on day zero with
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removed. The residue was subjected to a mass-triggered HPLC
purification to give 1a as well as 1b.
To a solution of compound 6 (70 g, 159.45 mmol, 1.00 equiv) in
methanol (800 mL) was added palladium on carbon (10 g). The
resulting solution was degassed and backfilled with hydrogen. The
solution was stirred for 3 days at 25 °C. The solids were filtered out
1H NMR for 1a (400 MHz, CD3OD) δ 7.59−7.63 (m, 2H), 7.09 (t,
J = 6.4 Hz, 2H), 6.95−7.02 (m, 1H), 6.46−6.52 (m, 1H), 6.34−6.38
(m, 1H), 5.92 (s, 1H), 4.33 (dd, J = 4.4, 14 Hz, 1H), 3.96 (dd, J = 4.8,
13.2 Hz, 1H), 3.70−3.78 (m, 1H), 3.46−3.54 (m, 1H), 1.73 (s, 3H),
1
1.36 (s, 3H). m/z = 428.1 (M + 1). H NMR for 1b (400 MHz,
CD3OD) δ 7.75−7.78 (m, 2H), 6.95−7.01 (m, 3H), 6.44−6.50 (m,
1H), 6.32−6.36 (m, 1H), 4.19 (dd, J = 1.6, 6.8 Hz, 2H), 4.00 (dd, J =
1.6, 6.8 Hz, 2H), 1.47 (s, 6H). m/z = 426.1 (M + 1).
Synthesis of 10 (R1R2Me). To a solution of 2-bromo-1-(4-
fluorophenyl)ethanone (46.5 g, 214.29 mmol) in DMF (400 mL) was
and washed with MeOH. The filtrate was concentrated under reduced
pressure. This resulted in 7 (38 g, 145.56 mmol, 91%) as a white solid.
LC-MS: (ES, m/z) [M + H]+ calcd for C14H14FN3O, 260; found, 260.
1H NMR (300 Hz, DMSO-d6) δ 7.79−7.74 (m, 2H), 7.13−7.07 (m,
3H), 6.35 (s, 1H), 4.73 (s, 2H), 1.79 (s, 6H).
To a stirred solution of compound 7 (5 g, 19.2 mmol, 1 equiv) was
dissolved in 50 mL of THF, 1 M borane/THF complex (57 mL, 57
mmol, 3 equiv) was added slowly and reaction was refluxed overnight.
LCMS indicated that the reaction was complete. THF was removed
under reduced pressure. The reaction was quenched with MeOH. The
crude product of 8 (4.5 g, 18.3 mmol, 95%) was used in the next step.
LC-MS: (ES, m/z) [M + H]+ calcd for C14H17FN3 246, found 246. 1H
NMR (DMSO, 300 Hz) δ 7.75−7.70 (m, 2H), 7.4 (s, 1H), 7.14 (t, J =
added 2-(benzyloxycarbonyl)-2-methylpropanoic acid (55.8 g, 236.4
mmol) and potassium carbonate (35.4 g, 256.5 mmol). The resulting
solution was stirred for 4 h at rt. The resulting solution was diluted
with 1000 mL of water. The resulting solution was extracted with ethyl
acetate (2 × 800 mL), and the combined organic layer was washed
with water (2 × 800 mL) and then brine (1 × 800 mL). The resulting
organic phase was concentrated under vacuum. This resulted in 67 g
(84%) of compound 4 as a white solid. m/z = 374 (M + 1).
9.0 Hz, 2H), 3.9 (t, J = 5.4 Hz, 2H), 2.51 (t, J = 5.4 Hz, 2H), 1.41(s,
6H); NH proton not observed.
To a solution of compound 4 (70 g, 187.7 mmol) in toluene (700 mL)
was added NH4OAc (144.5 g, 1.88 mol). The resulting solution was
heated to reflux for 3 h in an oil bath. The resulting mixture was
concentrated under vacuum. The residue was dissolved in 800 mL of water.
The resulting solution was extracted with ethyl acetate (2 × 500 mL), and
the combined organic layer was washed with water (2 × 800 mL) and brine
(1 × 800 mL). The resulting mixture was concentrated under vacuum.
This resulted in 58 g (88%) of compound 5 as a white solid. 1H NMR
To a stirred solution of compound 8 (2.9 g, 11.82 mmol, 1.1 equiv)
and 2-(tert-butoxycarbonylamino)acetic acid (2.27 g, 13 mmol, 1.1
equiv) in 15 mL of dichloromethane were added DIEA (2.47 mL,
14.18 mmol, 1.2 equiv) and HATU (5.39 g, 14.18 mmol, 1.2 equiv).
The reaction mixture was stirred at rt for 8 h. HPLC/MS analysis
showed that desired product compound 9 was the major product. The
reaction was further diluted with dichloromethane (70 mL). The
organic layer was washed with washed with water (1 × 30 mL),
followed by saturated NaHCO3 (1 × 30 mL) and finally with brine
(1 × 30 mL). The organic layer was then dried over anhydrous Na2SO4
and concentrated in vacuo. The resulting oil was purified using column
chromatography with hexanes/ethyl acetate (0−100% linear gradient)
used as eluant. The desired product compound 9 was obtained as oil
(3.3 g, 8.27 mmol, 70%). 1H NMR (300 Hz, DMSO-d6,) δ 7.77−7.72
(m, 2H), 7.54 (s, 1H), 7.20−7.14 (m, 2H), 6.84−6.80 (m, 1H), 4.07
(s, 2H), 3.90 (d, J = 3.0 Hz, 2H), 3.70 (s, 2H), 1.80 (s, 6H), 1.40 (s,
(300 Hz, DMSO-d6) δ 11.91 (s, NH), 7.80−7.75 (m, 2H), 7.54−7.12
(m, 8H), 4.97 (m, 2H), 1.6 (s, 6H).
To a solution of compound 5 (58 g, 164.3 mmol) in DMF (400 mL)
was added cesium carbonate (134 g, 411.0 mmol). This was followed
by the addition of ethyl 2-bromoacetate (33 g, 197.6 mmol) dropwise
with stirring at rt in 30 min. The resulting solution was stirred for 2 h
at rt. The resulting solution was diluted with 1000 mL of water/ice.
The resulting solution was extracted with ethyl acetate (2 × 700 mL),
and the combined organic layer was washed with water (2 × 800 mL)
and brine (1 × 800 mL). The resulting mixture was dried concentrated
under vacuum. This resulted in 60 g (83%) of compound 6 as a yellow
solid. 1H NMR (300 Hz, DMSO-d6) δ 7.76−7.72 (m, 2H), 7.32−7.28
(m, 5H), 7.08−7.03 (m, 3H), 5.08 (m, 2H), 4.87 (m, 2H), 4.23 (q, J =
5.4 Hz, 2H), 2.0 (s, 6H), 1.28 (t, J = 5.4 Hz, 3H).
9H). LC-MS: (ES, m/z) [M + H]+ calcd for C21H28FN4O3, 403;
found, 403.
To a stirred solution of compound 9 (3.02 g, 7.51 mmol) in
dichloromethane (30 mL) was added Br2 (0.43 mL, 8.26 mmol) in
acetic acid (3 mL). The reaction mixture was stirred at rt for 30 min.
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HPLC/MS test showed that desired product (II) was the only peak.
Solvent was removed via rotavap at a temperature no higher than
20 °C. After neutralization, white solid (3.60 g) was obtained. The
product was confirmed by 400 MHz proton NMR to be the title
compound 10. The product was used in the next step without further
purification and exhibited quantitative mass recovery. LC-MS: (ES,
m/z) [M + H]+ calcd for C21H27BrFN4O3, 482; found, 482. 1H NMR
(MeOH-d4, 400 Hz) δ 7.84−7.81 (m, 2H), 7.14 (t, J = 8.8 Hz, 2H),
4.09−4.01 (m, 4H), 3.81 (t, J = 4.8 Hz, 2H), 1.89 (s, 6H), 1.46
(s, 9H).
Synthesis of 20. (R)-2-Amino-1-(3-(4-chloro-3-fluorophenylami-
no)-2-(4-fluorophenyl)-8-isopropyl-5,6-dihydroimidazo[1,2-a]pyrazin-
7(8H)-yl)ethanone:
Compound 20 was prepared from compound 10 (R1 = isopropyl,
R2 = H) by a Pd2(dba)3 mediated amination reaction with 4-chloro-3-
fluoroaniline followed by a TFA mediated deprotection in a protocol
1
similar to compound 22. H NMR (MeOH-d4, 400 MHz) δ 7.69−
7.67 (m, 2H), 7.27−7.20 (m, 3H), 6.68−6.65 (dd, J = 2.5 Hz, J =
11.0 Hz, 1H), 6.60−6.55 (dd, J = 2.25 Hz, J = 8.7 Hz, 1H), 5.78 (d,
J = 7.7 Hz, 1H), 4.42−4.02 (m, 4H), 3.94−3.87 (m, 2H), 2.55
(m,1H), 1.24(d, J = 6.8 Hz, 3H), 1.05 (d, J = 6.8 Hz, 3H). m/z =
460.2 (M + 1).
Synthesis of 17. (S)-2-Amino-1-(3-(4-chloro-3-fluorophenylami-
no)-2-(4-fluorophenyl)-8-methyl-5,6-dihydroimidazo[1,2-a]pyrazin-
7(8H)-yl)ethanone:
Compound 17 was prepared from compound 10 (R1 = Me, R2 = H)
by a Pd2(dba)3 mediated amination reaction with 4-chloro-3-
fluoroaniline followed by a TFA mediated deprotection in a protocol
similar to compound 22. 1H NMR (MeOH-d4, 400 MHz) δ 7.61−7.58
(m, 2H), 7.16−7.12 (m, 1H), 7.07−7.02 (m, 2H), 6.47−6.42 (m, 2H),
5.74 (m, 1H), 4.07−3.68 (m, 6H), 1.56 (d, J = 6.8 Hz, 3H). m/z =
432.0 (M + 1).
Synthesis of 21. 2-Amino-1-(3-(4-chloro-3-fluorophenylamino)-2-
(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-
7(8H)-yl)ethanone:
Compound 21 was prepared from compound 10 (R1 = Me, R2 =
Me) by a Pd2(dba)3 mediated amination reaction with 4-chloro-3-
fluoroaniline followed by a TFA mediated deprotection in a protocol
similar to compound 22.
1H NMR (MeOH-d4, 400 MHz) δ 7.59−7.55 (m, 2H), 7.20−7.13
(m, 3H), 6.60 (dd, J = 11.2, 10.8 Hz), 1H), 6.53 (dd, J = 2.4, 0.8 Hz,
1H), 4.0 (m, 4H), 3.78 (m, 2H), 2.00 (s, 6H). m/z = 446.0 (M + 1).
Elemental analysis (compound + 2.0 HCl + 2.0 H2O): % C, 47.62; %
H, 5.09; %N, 12.62; (calc). %C = 47.54/47.38; %N = 12.44/12.46; %
H = 4.62/4.57 (experimental). LC/MS major mass 446.2.
Synthesis of 22 (Analogue of Compound 12 in Scheme 2 where
R1R2Me). 2-Amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-
8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)ethanone:
Synthesis of 18. (R)-2-Amino-1-(3-(4-chloro-3-fluorophenylami-
no)-2-(4-fluorophenyl)-8-methyl-5,6-dihydroimidazo[1,2-a]pyrazin-
7(8H)-yl)ethanone:
Compound 18 was prepared from compound 10 (R1 = Me, R2 = H)
by a Pd2(dba)3 mediated amination reaction with 4-chloro-3-
fluoroaniline followed by a TFA mediated deprotection in a protocol
similar to compound 22. m/z = 432.2 (M + 1).
In a glass vial, Cs2CO3, 4F-aniline (0.462 g, 4.1 mmol, 2.0
equiv), Pd2(dba)3 (0.095 g, 0.104 mmol, 0.05 equiv), xantphos
(0.120 g, 0.208 mmol, 0.1 equiv), and dioxane were stirred for 5
min at rt. Compound 10 (1 g, 2.08 mmol, 1.0 equiv) was added to
the reaction mixture, after which the reaction mixture was
degassed for 15 min and then stirred at 120 °C under N2 for 8
h. HPLC/MS test showed that the starting material compound 10
was consumed and desired product was formed predominantly
along with some 9. The reaction was filtered to remove solids. The
reaction mixture was concentrated and then purified by normal
phase column chromatography (silica gel 80 g) using a gradient of
100−0% to 0−100% hexane:EtOAc. The desired product eluted at
60:40 EtOAc:hexanes. The organic layer was concentrated at
reduced pressure to yield the Boc derivative (950 mg, 89%) yield.
Synthesis of 19. (S)-2-Amino-1-(3-(4-chloro-3-fluorophenylami-
no)-2-(4-fluorophenyl)-8-isopropyl-5,6-dihydroimidazo[1,2-a]pyrazin-
7(8H)-yl)ethanone:
Compound 19 was prepared from compound 10 (R1 = isopropyl,
R2 = H) by a Pd2(dba)3 mediated amination reaction with 4-
chloro-3-fluoroaniline followed by a TFA mediated deprotection
1
in a protocol similar to compound 22. H NMR (MeOH-d4, 400
MHz) δ7.61−7.58 (m, 2H), 7.26−7.20 (m, 3H), 6.45−6.38 (m,
2H), 5.5 (d, J = 7.9 Hz, 1H), 4.11−3.75 (m, 6H), 2.36−2.31 (m,
1H), 1.15 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.7 Hz, 3H). m/z =
460.2 (M + 1).
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Table 8. Regiochemistry of the diMe Groups Assigned Using 2-D NMR Techniques
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Table 8. continued
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Table 8. continued
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Table 8. continued
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Table 8. continued
LC-MS: (ES, m/z) [M + H]+ calcd for C27H32F2N5O3, 512;
found, 512.
was dissolved in dichloromethane and carefully neutralized by satd
NaHCO3. The organic layer was successively washed with brine fol-
lowed by water. The organic layer was concentrated to yield 22 (450
mg, 52%). LC-MS: (ES, m/z) [M + H]+ calcd for C22H23F2N5O,
The Boc compound was treated with 20% TFA in CH2Cl2 (50 mL)
and was added to the mixture. After the completion of this reaction
(monitored by LCMS), the resulting mixture was concentrated under
reduced pressure. The resulting residue was purified by reverse phase
HPLC to yield product as a TFA salt. The acetonitrile−water layer
was concentrated in vacuo to remove all the solvents. The residue
1
412.2; found, 412.1. H NMR (MeOH-d4, 400 Hz) δ 7.61−7.57 (m,
2H), 6.94 (t, J = 8.8 Hz, 2H), 6.81 (t, J = 8.8 Hz, 2H), 6.47 (m, 2H),
3.72 (m, 2H), 3.58 (m, 2H), 3.42 (m, 2H), 1.85 (s, 6H). Elemental
analysis compound 22 with 0.65 equiv H2O: C, 62.44; N, 16.55; H,
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5.79 (calculated). C = 62.54/62.44; N = 16.35/16.29; H = 5.52/5.61
(experimental).
sodium sulfate, and concentrated under vacuum. The residue was
applied onto a silica gel column with petroleum ether/EtOAc
The regiochemistry of the diMe groups was unambiguously
assigned using 2-D NMR techniques (Table 8).
(5:1) to give 0.7 g (55%) of compound 5a-1 as a light-yellow solid.
m/z = 380 (M + 1).
Synthesis of 23. 2-Amino-1-(2-(4-fluorophenyl)-8,8-dimethyl-3-
(p-tolylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
ethanone:
Compound 23 was prepared from compound 10 (R1 = Me, R2 =
Me) by a Pd2(dba)3 mediated amination reaction with 4-
methylaniline followed by a TFA mediated deprotection in a
protocol similar to compound 22. 1H NMR (MeOH-d4, 400 MHz)
δ 7.79−7.69 (m, 2H), 7.21 (t, J = 8.4 Hz, 2H), 7.04 (d, J = 8.0 Hz,
2H), 6.76 (d, J = 8.2 Hz, 2H), 4.11 (s, 2H), 3.88 (m, 4H), 2.22 (s,
3H), 2.12 (s, 6H). LC/MS major mass: 408.1 (M + H).
Into a 30 mL sealed tube, was placed compound 5a-1 (2.0 g,
5.28 mmol, 1.00 equiv), acetic acid (12 mL), and methanesul-
fonic acid (2 mL). The reaction mixture was stirred for 12 h at
260 °C (the temperature of the sand bath). The reaction mixture
was cooled to rt. The mixture was poured into 20 mL of water.
The aqueous layer was washed with ethyl acetate (3 × 10 mL).
Aqueous sodium hydroxide (1N) was added to adjust pH to 8.
The aqueous layer was extracted with ethyl acetate (3 × 10 mL).
The combined organic layers were washed with brine (3 ×
10 mL), dried over sodium sulfate, and concentrated under
vacuum. The solid was collected by filtration and washed with
5 mL of n-hexane to give 500 mg (39%) of compound 5a-2 as a
1
white solid. H NMR (400 MHz, CDCl3) δ 7.70−7.65 (m, 2H),
Synthesis of 24. 2-Amino-1-(3-((3,4-difluorophenyl)amino)-2-(4-
fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-
yl)ethanone:
Compound 24 was prepared from compound 10 (R1= Me, R2 =
Me) by a Pd2(dba)3 mediated amination reaction with 3,4-
difluoroaniline followed by a TFA mediated deprotection in a
protocol similar to compound 22. 1H NMR (MeOH-d4, 400 MHz)
δ 7.73−7.69 (m, 2H), 7.21 (t, J = 8.8 Hz, 2H), 7.06−7.03 (m, 1H),
6.76−6.65 (m, 1H), 6.53−6.51 (m, 1H), 4.08−4.06 (m, 4H),
3.86−3.83 (m, 2H), 2.07 (s, 6H). Elemental analysis (compound
+2.9 HCl): %C, 49.37; %H, 4.69; %N, 13.09; (calc). %C = 49.43/
49.06; %N = 12.89/12.77; %H = 4.7/4.81 (experimental). m/z =
430.2 (M + 1).
7.04−6.9 (m, 2H), 6.96 (s, 1H), 4.12 (s, 2H), 3.69 (s, 2H), 1.23
(s, 6H).
To a stirred solution of compound 5a-2 (280 mg, 1.14 mmol,
1.0 equiv) in DMF (10 mL) was added 2-(tert-butoxycarbonyl)-
acetic acid (600 mg, 3.43 mmol, 3.0 equiv), HATU (1.3 g,
3.42 mmol, 3.0 equiv), and DIEA (880 mg, 6.82 mmol, 6.0 equiv)
at rt. The reaction mixture was stirred overnight at rt. The reac-
tion mixture was diluted with ethyl acetate (100 mL). The organic
layer was washed with brine (3 × 10 mL), dried over sodium
sulfate, and concentrated under vacuum. The residue was applied
onto a silica gel column with CH2Cl2/MeOH (10:1) to give
1
280 mg (57%) of compound 13 as a brown solid. H NMR (400
MHz, CDCl3) δ 7.72−7.68 (m, 2H), 7.23 (s, 1H), 7.13−7.07 (m,
2H), 5.46 (s, NH), 4.65 (s, 2H), 4.05−4.04 (m, 2H), 3.97 (s, 2H),
1.55−1.39 (m, 15H).
The structure of 13 was verified by HMQC, COSY, HMBC, and
ROESY (Table 9).
Synthesis of 13. tert-Butyl 2-(3-bromo-2-(4-fluorophenyl)-6,6-
dimethyl-5,6-dihydroimidazo [1,2-a]pyrazin-7(8H)-yl)-2-oxoethylcar-
bamate.
Synthesis of 25 (Analogue of Compound 14 in Scheme 3).
2-Amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-6,6-
dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
ethanone:
13 was prepared from compound 5a by the following way:
To a stirred solution of compound 5a (1.1 g, 3.38 mmol, 1.00
equiv) in DMF (30 mL) was added 3-chloro-2-methylprop-1-ene
(500 mg, 5.49 mmol, 1.50 equiv), potassium carbonate (560 mg,
4.06 mmol, 1.10 equiv), and potassium iodide (1.12 g, 6.75 mmol,
2.00 equiv) at rt. The reaction mixture was stirred for 48 h at 40
°C. The reaction mixture was diluted with ethyl acetate (100 mL).
The mixture was washed with brine (3 × 10 mL), dried over
To a stirred solution of compound 13 (386 g, 0.96 mmol) in 6
mL of dichloromethane was added Br2 (55 μL, 1.06 mmol) in
acetic acid (2 mL). The reaction mixture was stirred at rt for 30
min. Solvent was removed via rotavap at a temperature no higher
than 20 °C. After neutralization, the residue was subjected to flash
chromatography (40 g, 0−100% ethyl acetate in hexane, 50 min,
dry loading) purification to give 256 mg (55%) of the title
compound 13a as a colorless oil. 1H NMR (400 MHz, CDCl3)
δ 7.87−7.83 (m, 2H), 7.03 (t, J = 8.8 Hz, 2H), 5.46 (s, 1H),
4.5 (s, 2H), 3.97 (d, J = 4.2 Hz, 2H), 3.81 (s, 2H), 1.45 (s, 6H),
1.39 (s, 9H).
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Table 9. Structure of 13 Verified by HMQC, COSY, HMBC, and ROESY
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Table 9. continued
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Table 9. continued
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Table 9. continued
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Table 9. continued
To a solution of compound 7a (231 mg, 1.0 mmol) in DMF (10
mL) were added KOH (168 mg, 3.0 mmol) and PMBCl (405 μL,
3.0 mmol) at 0 °C. The reaction mixture was stirred at the same
temperature for 2 h and at rt for 2 additional hours. The reaction
mixture was purified by reverse phase HPLC. The HPLC fraction
were evaporated under reduced pressure and neutralized by satd
NaHCO3. The compound was extracted by ethyl acetate and dried
over sodium sulfate. The organic layer was concentrated under
reduced pressure to give compound 7a-1 (221 mg, 63%) as a white
Example 25 was obtained from 13a by a Pd2(dba)3 mediated
amination reaction with 4-fluoroaniline followed by a TFA
mediated deprotection by analogy to compound 22 (55% yield
1
solid. H NMR (400 MHz, CDCl3) δ 7.67−7.63 (m, 2H), 7.28−
7.25 (m, 2H), 7.07−7.02 (m, 2H), 6.89−6.86 (m, 2H), 4.74 (s,
2H), 4.70 (m, 2H), 4.53 (s, 2H), 3.79 (s, 3H).
1
over 2 steps). H NMR (400 MHz, DMSO-d6): δ 8.06 (m, 2H),
7.98 (m, 1H), 7.80 (m, 2H), 7.16 (m, 2H), 6.98 (m, 2H), 6.60
(m, 2H), 4.64 (m, 2H), 4.02 (m, 2H), 3.80 (m, 2H), 1.38 (s, 6H).
m/z = 412.1 (M + 1).
To a solution of compound 7a-1 (253 mg, 0.72 mmol) in DMF
(15 mL) were added 60% sodium hydride (87 mg, 0.085 mmol)
and methyl iodide (0.45 mL, 7.2 mmol) at rt. The reaction mixture
was stirred at rt for 2 h. The reaction mixture was quenched with
methanol and directly subjected to mass-triggered HPLC
purification to give 7a-2 as a white solid after evaporation of
Synthesis of 7b. 2-(4-Fluorophenyl)-7-(4-methoxybenzyl)-5,5-
dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine:
Compound 7b was prepared from compound 7a (R1 = R2 = H) by
the following way:
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Table 10. Structure of 7a-2 Verified by 2-D NMR Techniques
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Table 10. continued
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Table 10. continued
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Table 10. continued
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Table 10. continued
concentrated under reduced pressure to yield the desired
compound 7a-2 (234 mg, 86%). The structure of 7a-2 was verified
by 2-D NMR techniques (Table 10).
Synthesis of Compound 7b. To a solution of compound 7a-2 (170
mg, 0.45 mmol) in THF (9 mL) was added 1.0 N BH3·THF (2.70 mL,
2.70 mmol) at rt. The reaction mixture was stirred at reflux for 2 h.
acetonitrile−water in vacuo. The TFA salt was subsequently
neutralized by saturated sodium bicarbonate solution and
extracted with dichloromethane to get the compound as a free
base. The organic layer was dried over sodium sulfate and
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resulting solution was stirred overnight at rt. The residue was dissolved
in ethyl acetate (200 mL). The resulting mixture was washed with
brine (3 × 100 mL), dried over anhydrous sodium sulfate, and
concentrated under vacuum. The residue was applied onto a silica
gel column with petroleum ether/ethyl acetate (4:1). This resulted
in 600 mg (49%) of 7c as a pale-yellow solid. 1H NMR (300 MHz,
CDCl3): δ 7.75 (m, 2H), 7.22 (s, 1H), 7.13−7.07 (m, 2H), 4.99−
4.87 (m, 2H), 4.12 (s, 2H), 3.91 3.65 (m, 2H), 1.57 (m, 6H), 1.43
(s, 9H).
To a stirred solution of compound 7b-2 (583 g, 1.45 mmol) in
dichloromethane (6 mL) was added Br2 (82 μL, 1.60 mmol) in
acetic acid (2 mL). The reaction mixture was stirred at rt for 30
min. Solvent was removed via rotavap at a temperature no higher
than 20 °C. After neutralization, the residue was subjected to flash
chromatography (40 g, 0−100% ethyl acetate in hexane, 50 min,
dry loading) purification to give 628 mg (90%) of the title
1
compound 7c as a yellow solid. H NMR (400 MHz, CDCl3) δ
781−7.78 (m, 2H), 7.07 (t, J = 8.6 Hz, 2H), 5.28 (s, NH), 4.84−
4.7 (m, 2H), 4.07−3.58 (m, 4H), 1.49−1.44 (m, 6H), 1.28
(s, 9H).
Pd/C was added (gas generated). The reaction mixture was stirred for
1 h. Solid was filtered off, and solvent was removed. The structure of
crude compound 7b was verified by proton NMR. The product was
assumed to be of 100% yield and used in the next step without further
purification. 1H NMR (400 MHz, CD3OD) δ 8.1 (s, 1H), 7.74−
7.70 (m, 2H), 7.34 (d, J = 8.6 Hz, 2H), 7.27 (m, 2H), 6.94 (d, J =
8.6 Hz, 2H), 3.91 (s, 2H), 3.80 (s, 3H), 3.79 (s, 2H), 2.87 (s, 2H),
1.61 (s, 6H).
7-(4-Methoxybenzyl)-2-(4-fluorophenyl)-5,5-dimethyl-5,6,7,8-
tetrahydroimidazo[1,2-a]pyrazine (1.07 g, 2.93 mmol, 1.00 equiv)
(7b) was added to trifluoroacetic acid (15 mL). The resulting solution
was stirred for 2 h at 70 °C in an oil bath. The resulting mixture was
concentrated under vacuum. The residue was dissolved in 200 mL of
dichloromethane. The pH value of the solution was adjusted to 8 with
Synthesis of 26. 2-Amino-1-(2-(4-fluorophenyl)-3-(4-fluoropheny-
lamino)-5,5-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-
ethanone:
Compound 26 was prepared from compound 7c by a Pd2(dba)3
mediated amination reaction with 4-fluoroaniline followed by a TFA
mediated deprotection in 46% yield over 2 steps. 1H NMR (400 MHz,
CD3OD): δ 7.57 (m, 2H), 7.02 (m, 2H), 6.77 (m, 2H), 6.52 (m, 2H),
4.97−4.91 (m, 2H), 4.09−3.74 (m, 4H), 1.56 (s, 3H), 1.51 (s, 3H).
m/z = 412.2 (M + 1).
Synthesis of 27. 2-Amino-1-(2-(4-fluorophenyl)-3-((4-
fluorophenyl)amino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-2-
methylpropan-1-one. The synthesis has been reported in the previous
communication.5
aqueous sodium bicarbonate. 7b-1 was extracted from the aqueous
layer using 3 × 100 mL of ethyl acetate. The mixture was dried over
sodium sulfate and concentrated under vacuum. This resulted in 7b-1
(718 mg, quantitative) as a yellow solid. m/z = 246 (M + H).
To a solution of 7b-1 (750 mg, 3.06 mmol, 1.00 equiv) in N,N-
dimethylformamide (30 mL) were added 2-(tert-butoxycarbonyl)acetic
acid (850 mg, 4.86 mmol, 1.50 equiv), HATU (1.3 g, 3.42 mmol, 1.10
equiv), and triethylamine (650 mg, 6.44 mmol, 2.00 equiv). The
Synthesis of 28. 2-Amino-1-(2-(4-fluorophenyl)-3-(4-fluoropheny-
lamino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-2-
methylpropan-1-one:
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7.75−7.71 (m, 2H), 7.09−7.02 (m, 3H), 4.84(s, NH), 4.09−4.0 (m,
4H), 1.98 (s, 6H), 1.55 (s, 6H), 1.45 (s, 9H).
To a stirred solution of compound 8-4 (32 mg, 0.074 mmol) in
dichloromethane (3 mL) was added Br2 (4.2 μL, 0.082 mmol) in
acetic acid (1 mL). The reaction mixture was stirred at rt for 30 min.
Solvent was removed via rotavap at a temperature no higher than
20 °C. After neutralization, the residue was subjected to flash chro-
matography (4 g, 0−60% ethyl acetate in hexane, 16 min) purification
to give the compound 8-5 as colorless oil, was collected by filtration,
and washed with n-hexane (5 mL) to give compound 8-4 as a white
Compound 28 was prepared from compound 8 (R1 = R2 = Me) in
the following fashion using a slightly modified route to the one
described in Scheme 2.
To a stirred solution of compound 8 (1.48 g, 6.04 mmol) and NEt3
(6.0 g, 59.4 mmol) in dichloromethane (20 mL) was added 2-bromo-
2-methylpropanoyl bromide (14 g, 60.9 mmol) dropwise at rt. After
being stirred for 3 h at rt, the reaction was then quenched by the
addition of water (30 mL). The resulting solution was extracted with
1
solid. H NMR (400 MHz, CDCl3) δ 7.91−7.88 (m, 2H), 7.10−7.04
(m, 2H), 5.11(s, NH), 4.01 (m, 2H), 3.96 (s, 2H), 1.94 (s, 6H), 1.51
(s, 6H), 1.41 (s, 9H).
Compound 28 was prepared from compound 8-5 by a Pd2(dba)3
mediated amination reaction with 4-fluoroaniline followed by a TFA
1
mediated deprotection. H NMR (400 MHz, MeOH-d4) δ 7.66 (dd,
J = 9.2, 5.2 Hz, 2H), 7.11 (t, J = 8.8 Hz, 2H), 6.81 (t, J = 8.8 Hz, 2H),
6.72−6.68 (m, 2H), 4.04−3.99 (m, 4H), 2.01 (s, 6H), 1.66 (s, 6H).
m/z = 440.1 (M + 1).
Synthesis of 29. 2-Amino-1-(2-(4-fluorophenyl)-8,8-dimethyl-3-
(p-tolylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-2-methyl-
propan-1-one:
Compound 29 was prepared from 35 by the following way:
To a stirred solution of 35 (21 mg, 0.06 mmol) and Et3N (83 μL,
0.60 mmol) in dry dichloromethane (6 mL) was added 2-bromo-2-
methylpropanoyl bromide (71 μL, 0.60 mmol). The reaction mixture
was stirred at rt for 5 h. The reaction mixture was concentrated and
subjected to mass-triggered LC/MS purification directly. The obtained
solution was concentrated to give 22 mg (73%) of compound 35-1
3 × 30 mL of ethyl acetate. The combined organic layers were washed
with brine, dried over anhydrous sodium sulfate, and concentrated
under vacuum. This resulted in crude product as a dark solid, which
was washed with EtOAc:petroleum ether (1:10) to remove the impurities
to produce compound 8-1 as a gray solid. m/z 396 (M + H).
To a solution of compound 8-1 (2.0 g, 5.08 mmol, 1.00 equiv) in
DMF (10 mL) was NaN3 (1.0 g, 15.38 mmol, 3.00 equiv) at rt. The
1
as yellow oil after neutralization. H NMR (400 MHz, MeOH-d4) δ
7.71−7.66 (m, 2H), 7.23−7.19 (m, 2H), 7.02 (d, J = 8.4 Hz, 2H), 6.66
(d, J = 8.4 Hz, 2H), 4.31 (m, 2H), 4.09 (m, 2H), 2.21 (s, 3H), 2.06 (s,
6H), 2.00 (s, 6H).
To a solution of compound 35-1 (22 mg, 0.044 mmol) in DMF
(3 mL) was added NaN3 (8.6 mg, 0.132 mmol) at rt. The reaction
mixture was stirred at rt for 2 h. The reaction mixture was directly
subjected to mass-triggered HPLC purification directly. The obtained
MeCN/aqueous solution was combined and concentrated to give
1
15 mg (75%) of compound 35-2 as yellow oil after neutralization. H
reaction mixture was stirred overnight at rt. The resulting solution was
diluted with 300 mL of ethyl acetate. The resulting mixture was
washed with 3 × 20 mL of brine, dried over anhydrous sodium sulfate,
NMR (400 MHz, MeOH-d4) δ 7.70 −7.66 (m, 2H), 7.23−7.19 (m,
2H), 7.04 (d, J = 8.3 Hz, 2H), 6.66 (d, J = 8.4 Hz, 2H), 4.26 (m, 2H),
4.07−4.05 (m, 2H), 2.24 (s, 3H), 2.03 (s, 6H), 1.58 (s, 6H).
To a solution of compound 35-2 (15 mg, 0.033 mmol) in MeOH
(3 mL) was added 10% Pd/C (4 mg, 0.003 mmol) at rt. Air was
removed, and H2 was filled. The reaction mixture was stirred at rt for
2 h. Solid was filtered off, and solvent was removed. The reaction mix-
ture was directly subjected to mass-triggered HPLC purification to give
15 mg (100%) of the title compound yellow oil. 1H NMR (MeOH-d4,
400 Hz) δ 7.78−7.75 (m, 2H), 7.06−6.97 (m, 4H), 6.56 (d, J = 8.4
Hz, 2H), 5.16 (s, NH), 4.33 (t, J = 4.8 Hz, 2H), 3.74 (t, J = 4.8 Hz,
2H), 2.29 (s, 3H), 1.95 (s, 6H), 1.45 (s, 6H). m/z = 436.2 (M + 1).
Synthesis of 30. 2-Amino-1-(3-(3,4-difluorophenylamino)-2-
(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-
7(8H)-yl)-2-methylpropan-1-one:
and concentrated under vacuum. The residue was applied onto a silica
gel column with petroleum ether/EtOAc (5:1) to give compound 8-2
as a white solid. m/z 357 (M + H).
Compound 30 was prepared from compound 8-5 by a Pd2(dba)3
mediated amination reaction with 3,4-difluoroaniline followed by a
TFA mediated deprotection.1H NMR (400 MHz, MeOH-d4) δ 7.63−
7.60 (m, 2H), 7.14 (t, J = 8.4 Hz, 2H), 7.01 (dd, J = 18.8, 8.8 Hz, 1H),
6.68−6.63 (m, 1H), 6.5 (m, 1H), 4.03 (m, 2H), 4.01 (m, 2H), 2.01 (s,
6H), 1.66 (s, 6H). m/z = 458.1 (M + 1).
To a stirred solution of compound 8-2 (1.2 g, 3.37 mmol, 1.00
equiv) in methanol (20 mL) was added Pd/C (80 mg, 0.75 mmol,
0.20 equiv) at rt. The reaction mixture was evacuated and backfilled
with H2. The reaction mixture was stirred overnight at rt. The solids
were filtered out. The resulting mixture was concentrated under
vacuum. The solid was washed with petroleum ether. This resulted in
compound 8-3 as a white solid. m/z 331 (M + H).
Synthesis of 31. 2-(4-Fluorophenyl)-N-(p-tolyl)-5,6,7,8-
tetrahydroimidazo[1,2-a]pyrazin-3-amine:
To a stirred solution of compound 8-3 (910 mg, 2.76 mmol, 1.00
equiv) in THF (50 mL) was added BOC anhydride (3.2 g, 14.68
mmol, 5.00 equiv), followed by aqueous NaOH (1N, 6 mL, 2.0 equiv)
at rt. The resulting solution was stirred for 24 h at 40 °C. The resulting
solution was concentrated under vacuum. The mixture was diluted
with EtOAc (60 mL). The organic layer was washed with 3 × 10 mL
of brine, dried over Na2SO4, and concentrated under vacuum. The
solid was collected by filtration and washed with n-hexane (5 mL) to
The details of the synthesis involving an Ugi reaction between 4-
fluorobenzaledehyde, 2-aminopyrazine, and 1-isocyano-4-methylben-
zene has been reported in the previous communication.5 1H NMR
(400 MHz, MeOH-d4) δ 7.73 (d, J = 6.8 Hz, 2H), 7.00−6.99 (m, 4H),
6.49 (d, J = 8.0 Hz, 2H), 4.01 (s, 2H), 3.72 (m, 2H), 3.15 (m, 2H),
2.19 (s, 3H). m/z = 324.2 (M + 1).
Synthesis of 32. 2-(4-Fluorophenyl)-3-(p-tolylamino)-7,8-
dihydroimidazo[1,2-a]pyrazin-6(5H)-one:
1
give compound 8-4 as a white solid. H NMR (300 MHz, CDCl3) δ
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dx.doi.org/10.1021/jm300041e | J. Med. Chem. 2012, 55, 4244−4273

Journal of Medicinal Chemistry
Article
Compound 32 was synthesized from compound 7 (R1 = R2 = H)
Pd2(dba)3 mediated coupling reaction with p-toluidine. 1H NMR (400
MHz, DMSO-d6) δ 8.55 (s, 1H), 7.77−7.75 (m, 2H), 7.26−7.14 (m,
4H), 7.01−6.95 (m, 2H), 6.53 (m, 1H), 4.53 (m, 2H), 4.22 (m, 2H),
2.17 (s, 3H). m/z = 337.2 (M + 1).
Tropical Diseases, the Genomics Institute of the Novartis
Research Foundation, and the Swiss Tropical Institute
REFERENCES
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1
mediated amination reaction with p-toluidine. H NMR (MeOH-d4,
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8.4 Hz, 2H), 6.52 (d, J = 8.4 Hz, 2H), 4.42 (s, 2H), 2.12 (s, 3H), 1.71
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Compound 34 was prepared from compound 7b by a Pd2(dba)3
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1
mediated amination reaction with 4-chloro-3-fluoroaniline. H NMR
(MeOH-d4, 400 Hz) δ 7.74−7.70 (m, 2H), 7.21 (t, J = 8.4 Hz, 1H),
7.08 (m, 2H), 6.49−6.40 (m, 2H), 4.43 (s, 2H), 1.72 (s, 6H). m/z =
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Synthesis of 35. 2-(4-Fluorophenyl)-8,8-dimethyl-N-p-tolyl-5,6,7,8-
tetrahydroimidazo[1,2-a]pyrazin-3-amine:
Compound 35 was prepared from compound 35-1 (shown below
and prepared by analogy to compound 9 and using Cbz-glycine) by a
Pd2(dba)3 mediated amination reaction with p-toluidine followed by a
1
6N HCl mediated hydrolysis. H NMR (MeOH-d4, 400 Hz) δ 7.78−
7.75 (m, 2H), 7.06−6.97 (m, 4H), 6.56 (d, J = 8.4 Hz, 2H), 5.16 (s,
NH), 4.33 (t, J = 4.8 Hz, 2H), 3.74 (t, J = 4.8 Hz, 2H), 2.29 (s, 3H),
1.95 (s, 6H). m/z = 436.2 (M + 1).
Synthesis of 36. N,2-Bis(4-fluorophenyl)-8,8-dimethyl-5,6,7,8-
tetrahydroimidazo[1,2-a]pyrazin-3-amine:
Compound 36 was prepared from compound 35-1 by a Pd2(dba)3
mediated amination reaction with 4-fluoroaniline followed by a 6N
1
HCl mediated hydrolysis. H NMR (400 MHz, MeOH-d4) δ 7.62−
7.58 (m, 2H), 7.12−7.08 (m, 2H), 6.68−6.63 (m, 2H), 6.67 (m, 2H),
4.16 (m, 2H), 3.78 (m, 2H), 1.92 (s, 6H). m/z = 355.1 (M + 1).
ASSOCIATED CONTENT
■
S
* Supporting Information
In vitro ADME of selected analogues; PAMPA vs Log P; Caco-
2 (A−B) vs Log P; Caco-2 A−B/B−A ratio vs Log P; Caco-2
(B−A) vs Log P. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
(5) Wu, T. N.; Nagle, A.; Kuhen, K.; Gagaring, K.; Borboa, R.;
Francek, C.; Chen, Z.; Plouffe, D.; Goh, A.; Lakshminarayana, S. B.;
Wu, J.; Ang, H. Q.; Zeng, P.; Kang, M. L.; Tan, W.; Tan, M.; Ye, N.;
Lin, X.; Caldwell, C.; Ek, J.; Skolnik, S.; Liu, F.; Wang, J.; Chang, J.; Li,
C.; Hollenbeck, T.; Tuntland, T.; Isbell, J.; Fischli, C.; Brun, R.;
Rottmann, M.; Dartois, V.; Keller, T.; Diagana, T.; Winzeler, E.;
Glynne, R.; Tully, D. C.; Chatterjee, A. K. Imidazolopiperazines: Hit to
Lead Optimization of New Antimalarial Agents . J. Med. Chem. 2011,
54, 5116−5130.
Corresponding Author
*Phone: 858-332-4751. E-mail: akc@gnf.org.
Author Contributions
#Equal contribution to this work
Notes
The authors declare no competing financial interest.
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isonitriles: efficient parallel synthesis of a diverse and druglike library
of imidazo- and tetrahydroimidazo[1,2-d] heterocycles. J. Comb. Chem.
2007, 9 (6), 1177−1187.
ACKNOWLEDGMENTS
■
This work was supported by a translational research grant
(WT078285) from the Wellcome Trust and a grant from the
Medicines for Malaria Venture to the Novartis Institute for
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