A Developability-Focused Optimization Approach Allows Identification of in Vivo Fast-Acting Antimalarials: N -[3-[(Benzimidazol-2-yl)amino]propyl]amides

Article abstract of DOI:10.1021/acs.jmedchem.5b00114

Malaria continues to be a major global health problem, being particularly devastating in the African population under the age of five. Artemisinin-based combination therapies (ACTs) are the first-line treatment recommended by the WHO to treat Plasmodium falciparum malaria, but clinical resistance against them has already been reported. As a consequence, novel chemotypes are urgently needed. Herein we report a novel, in vivo active, fast-acting antimalarial chemotype based on a benzimidazole core. This discovery is the result of a medicinal chemistry plan focused on improving the developability profile of an antichlamydial chemical class previously reported by our group. (Graph Presented).

Full text of DOI:10.1021/acs.jmedchem.5b00114

Article
pubs.acs.org/jmc
A Developability-Focused Optimization Approach Allows
Identification of in Vivo Fast-Acting Antimalarials: N‑[3-
[
(Benzimidazol-2-yl)amino]propyl]amides
†
†
‡
‡
Leena Keurulainen, Mikko Vahermo, Margarita Puente-Felipe, Elena Sandoval-Izquierdo,
Benigno Crespo-Fernan
Laura de las Heras-Duen
Pablo Castan
‡
‡
‡
‡
́
dez, Laura Guijarro-Lop
́
ez, Leticia Huertas-Valentín,
†
†
‡
‡
̃
a, Teppo O. Leino, Antti Siiskonen, Lluís Ballell-Pages, Laura M. Sanz,
‡
‡
‡
‡
̃
eda-Casado, M. Belen
́
Jimen
́
ez-Díaz, María S. Martínez-Martínez, Sara Viera,
†
‡,§
,†
́ ́
Paula Kiuru, Felix Calderon,
and Jari Yli-Kauhaluoma*
†Faculty of Pharmacy, Division of Pharmaceutical Chemistry and Technology, University of Helsinki, Viikinkaari 5 E (P.O. Box 56),
FI-00014 Helsinki, Finland
‡
Tres Cantos Medicines Development Campus, GlaxoSmithKline, Severo Ochoa 2, Tres Cantos, Madrid 28760, Spain
*
S Supporting Information
ABSTRACT: Malaria continues to be a major global health
problem, being particularly devastating in the African
population under the age of five. Artemisinin-based combina-
tion therapies (ACTs) are the first-line treatment recom-
mended by the WHO to treat Plasmodium falciparum malaria,
but clinical resistance against them has already been reported.
As a consequence, novel chemotypes are urgently needed.
Herein we report a novel, in vivo active, fast-acting antimalarial
chemotype based on a benzimidazole core. This discovery is
the result of a medicinal chemistry plan focused on improving
the developability profile of an antichlamydial chemical class previously reported by our group.
INTRODUCTION
complicated our ability to discover and develop novel
■
antimalarials (the last new chemical entity, atovaquone, was
launched more than 30 years ago), the public release of novel
antiplasmodial data sets is opening up new opportunities for
both drug discovery and target identification programs. These
sets are the result of the screening of the corporate collections
of GlaxoSmithKline (GSK; Tres Cantos Antimalarial set,
Malaria is an infectious disease caused by parasite of the genus
Plasmodium associated with nearly 600,000 deaths in 2014.
1
Five species are known to affect humans: P. falciparum, P. ovale,
P. malariae, P. vivax, and P. knowlesi. The infection is triggered
by a female Anopheles mosquito blood meal, where infective
forms of the parasite are injected into the bloodstream of a
mammalian host. In the liver, these forms will develop schizonts
and subsequently infective merozoites, which will be released
into the bloodstream, invading the red blood cells. It is at this
stage when the symptoms of the disease start to be evident.
5
6
TCAMS), Novartis, and St. Jude Children’s Research
7
Hospital. The three sets are available for download from the
ChEMBL-NTD database. Subsequent in-depth analysis of the
8
GSK set resulted in the characterization of 5 out of the 47
2
These symptoms include chill, fever, or even cause death.
chemical classes released as benzimidazole scaffolds (Figure
According to the WHO, Africa represents 90% of the deaths
reported, 78% of which are associated with children under the
age of five. Malaria is also hampering the economic develop-
ment in countries affected because of expenditures on
9−11
1
).
In general, the benzimidazole core can be regarded as a
reasonable scaffold from a drug discovery point-of-view, as
shown by the high number of hits, leads, and even clinical
3
prevention and treatments.
12
candidates belonging to this space. Furthermore, various
The current recommended treatment by the WHO,
artemisinin-based combination therapies (ACTs), are increas-
ingly at risk of resistance development. It is now almost six
years since the first data for artemisinin resistance were
benzimidazole-derived compounds have been recently studied
13−20
as antiplasmodial agents.
Herein we report design and
synthesis of the N-[3-[(benzimidazol-2-yl)amino]propyl]-
amides, evaluation of their antimalarial activity, and further
4
reported in the Southeast Asia. This situation highlights the
urgent need to find new drugs to treat the disease.
Although chronic underinvestment and a very limited
Received: January 21, 2015
number of clinically validated modes of action have
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.5b00114
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flexible linker between the left- and right-hand sides of the
molecule could result in compounds with reduced lipophilicity
and improved solubility. Simultaneously, we tried to mitigate
the genotoxicity risk associated with potential aniline formation
by removing the aromatic moiety. A broad variety of aliphatic
linkers were prepared (Table 2). The diversity explored
includes cyclic groups (12a−c) and saturated chains, inspired
by the TCAMS antimalarial compounds. Chains with different
lengths (12d−f) and substitutions (12g−m) were explored.
The three-carbon linker (7) resulted in the most promising
substitution, showing 1 order of magnitude improvement in
potency (Pf IC50 = 0.094 μM, Tables 1 and 2) and a 3-fold
increase in solubility (CLND method, Table 1). Encouragingly,
the oral bioavailability, in vivo clearance in mice, and in vitro
permeability data associated with this compound were
promising (F = 81%, Cl = 39.7 mL/min·kg, and permeability
Figure 1. Benzimidazole representatives from the GlaxoSmithKline
antimalarial set.
=
380 nm/s; cf. pharmacokinetic data in Supporting
Information, Table 1). Interestingly, the solubility in simulated
fasting-intestine fluids (FaSSIF solubility) was found to be
above 100 μg/mL.
pharmacokinetic and antimalarial in vivo studies for the
selected compound.
Compound 7 and its analogs (Table 3) can be easily accessed
by heating the mixture of 2-chlorobenzimidazole and the
corresponding diamine alkyl derivative without solvent. The
target compounds are obtained by a standard amide coupling
reaction with EDC and the desired aromatic carboxylic acid
(Scheme 1A). SAR exploration of the molecule’s left-hand side
was undertaken by reacting the isothiocyanate 21 with various
o-diaminobenzenes in the presence of DCC (Scheme 1B).
Although compound 7 presents an in vitro selectivity index
of 386, the impact of further reduction in lipophilicity was
explored (Figure 2, Phase II). Efforts to increase polarity
through replacement of one halogen atom or, alternatively,
through the introduction of different heterocycles resulted in a
decreased potency (Table 3). However, combination of the last
two strategies afforded 8 (Figure 2). This compound presented
RESULTS AND DISCUSSION
■
The benzimidazole derivatives of TCAMS gave inspiration to
screen a set of benzimidazole-containing compounds from the
University of Helsinki (designed originally as antichlamydial
2
1
agents) against P. falciparum. Testing delivered the novel
antimalarial scaffold 6 (Figure 2). However, 6 presented a weak
antiplasmodial potency, a high degree of planarity because of
the presence of several aromatic rings, high lipophilicity, scarce
solubility, and potential to deliver aniline-like genotoxic
metabolites (Table 1).
With the aim of identifying a more developable hit, we
focused our attention first on the central unit (Figure 2, Phase
I). The hypothesis behind the selection of this first point of
synthetic intervention was based on the possibility that having a
Figure 2. Medicinal chemistry strategy. Figure shows the key analogs.
B
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Table 1. Antimalarial Efficacy against P. falciparum, hERG Inhibition, Cytotoxicity, Solubility, and Selectivity Indices of the Key
Compounds
Pf IC50 3aD7A
(μM)
Pf IC50 W2
(μM)
Pf IC50 V1/S
(μM)
hERG inhibition
HepG2 Tox
CLND solubility
50
b
c
d
e
compd
(μM)
(μM)
(μM)
SI
6
7
8
9
1
1
1.52
>14.79
1.17
26.3
64
17.3
386
0.094
0.25
0.410
1.029
36.31
>100
>100
>100
25.7
239
349
322
393
29
22.39
>50.1
>50.1
>50.1
>400
>43.5
>111
171
2.30
0
1
0.90
0.15
chloroquine
0.036
0.068
>1
>1
pyrimethamine
>20
>20
a
b
c
d
Average of duplicates. Pf, P. falciparum. 50% inhibitory concentration. 50% cytotoxic concentration. Chemiluminescent nitrogen detection,
e
kinetic solubility from DMSO stock solution in phosphate buffered saline (pH 7.4). Selectivity index (HepG2 tox /Pf IC ).
50
50
Table 2. Structures and Antimalarial Activity (P. falciparum 3D7A IC (Pf IC , μM)) of the Central-Part-Modified Derivatives
5
0
50
7
and 12a−m
a competitive potency (Pf IC = 0.25 μM, Table 1) combined
Compound 7 was able to reduce the parasitemia under the limit
of detection with just two doses, showing a parasite clearance
rate consistent with fast-acting antimalarials like dihydroarte-
misinin and piperaquine (Figure 4A). The microscopic
observations suggest that 7 induces rapid in vivo killing of P.
falciparum erythrocyte stages (pyknotic parasites emerging 2
days after the initiation of treatment, Figure 4B). Regarding its
in vitro profiling, the compound was tested against P.
falciparum laboratory-adapted strains resistant to chloroquine
and pyrimethamine (W2 and V1/S), resulting in a decrease in
potency (4- and 11-fold, respectively). Given the importance of
these findings, further studies will be required to understand
the molecular events underpinning this cross-resistance and
mechanism of action of these derivatives. Compound 7 was
5
0
with improved solubility and no signs of cytotoxicity (Tox50
00 μM), showing that cytotoxicity is not intrinsically related to
the chemical class. Preliminary hERG profiling of compounds
−11 also suggested differences between the different
>
1
6
structures. Classical strategies to improve solubility by the
addition of basic amino groups (9, Figure 2) and reduction of
aromaticity (10, Figure 2) resulted in compounds with inferior
overall profiles when comparing to 8. However, single-digit
micromolar antiplasmodial activities were measured, warranting
further interest in the compound series.
Next, we moved to the left-hand side of the molecule. The
benzimidazole positions liable to oxidation were successfully
blocked without loss of potency (11, Figure 2 and Table 1), but
neither polar substitutents nor aza derivatives showed a
competitive potency (Table 4). Figure 3 summarizes the key
SAR findings during Phase I and II of the optimization process.
At this point, compound 7 was selected for further
parasitological profiling in vitro and validation of the series in
vivo to classify the type of antimalarial efficacy. The compound
23
tested also against gametocyte stage of P. falciparum, but it
did not show activity (>10 μM).
CONCLUSIONS
■
We have identified a new antimalarial chemotype, N-[3-
[(benzimidazol-2-yl)amino]propyl]amides, with promising in
vivo pharmacokinetics and efficacy, a fast-acting mode of action
(comparable to artemisinins), and amenability for optimization
from a medicinal chemistry perspective. The remaining
challenges for the lead optimization phase will include
22
was evaluated in a 4 day test using a humanized mouse model.
Mice were infected at day 0 with the human parasite (P.
falciparum Pf 3D7
0087/N9
), and 3 days after infection, the
compound was dosed once a day for four consecutive days.
C
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Journal of Medicinal Chemistry
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Table 3. Structures and Antimalarial Activity (P. falciparum 3D7A IC50 (Pf IC , μM)) of the Right-Hand Side-Modified
50
Derivatives 8−10 and 13a−ar
a
Scheme 1. Synthetic Routes for Compounds (A) 7−10 and 13a−ar and (B) 11/14e and 14g
a
1
Reagents and conditions: (a) 100 °C, 36 h, 66%. (b) R COOH, EDC, HOBt, Et N, DMF, rt, 1.5−17 h, 3−67%. (c) (1) Et N, CH Cl , 1.5 h, 84%;
3
3
2
2
(
2) TFA, CH Cl , rt, 4 h, 68% over two steps. (d) TCDI, THF, rt, 3 h, quant. (e) DCC, Et N, MeCN, 85 °C, 24 h, 18−46%.
2
2
3
D
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Table 4. Structures and Antimalarial Activity (P. falciparum 3D7A IC50 (Pf IC , μM)) of the Left-Hand Side-Modified
50
Derivatives 10 and 14a−g
Figure 3. Structure−activity relationships around derivative 7 as antimalarial activity against P. falciparum (3D7A), structure, and substituent order
are based on Pf IC . See detailed antimalarial activities of derivatives in Tables 1−4.
50
mitigation of the observed cardiotoxicity risk (on the basis of
values of hERG IonWorks) and understanding of the origin of
the cross-resistance observed with W2 and V1/S clones.
Na
SO , and evaporated. Purification by preparative HPLC (MeCN/
2 4
H O, NH HCO ) gave compound 7 as off-white powder (0.17 g,
2
4
3
1
6
2
2
7%). H NMR (400 MHz, CD OD) δ ppm 1.94 (quin, J = 6.7 Hz,
H), 3.46 (t, J = 6.8 Hz, 2H), 3.50 (t, J = 6.8 Hz, 2H), 6.94−6.98 (m,
H), 7.17−7.19 (m, 2H), 7.63 (t, J = 1.8 Hz, 1H), 7.81 (d, J = 2.0 Hz,
3
EXPERIMENTAL SECTION
13
■
2H). C NMR (100 MHz, CD
3
OD) δ ppm 30.7, 38.7, 41.3, 121.4,
+
1
27.3, 32.3, 136.6, 139.1, 157.1, 167.4. MS: m/z 363 [M + H] . Purity
Chemistry. LC-MS analyses for purity were performed using a
diode-array detector and an ESI ion source. Signal separation was
carried out by use of Acquity UPLC BEH C18 column (1.7 μm, 3.0
mm × 50 mm) or Luna C18 (5 μm, 4.6 mm × 50 mm), eluent: 25
was determined as >95% by HPLC (λ 285 nm), R : 1.16 min (Acquity
UPLC BEH C18 1.7 μm, 3 mm × 50 mm, 25 mM NH OAc/MeCN,
pH 6.6).
t
4
mM NH OAc + 10% MeCN at pH 6.6/MeCN (gradient run 100:0 →
N-[3-[(1H-Benz[d]imidazol-2-yl)amino]propyl]-2-
trifluoromethyl)isonicotinamide (8). Compound 17 (49 mg, 0.26
4
(
1
0:90 for Acquity and 100:0 → 0:100 for Luna), and flow = 0.8 mL/
mmol), 2-(trifluoromethyl)isonicotinic acid (54 mg, 0.28 mmol), EDC
min. Purity of the all tested compounds was 95% or higher.
(56 mg, 0.29 mmol), and HOBt (39 mg, 0.29 mmol) were dissolved in
N-(1H-Benz[d]imidazol-2-yl)propane-1,3-diamine (17). 1,2-Dia-
minopropane 16 (8.30 mL, 0.100 mol) and 2-chloro-1H-benzimida-
zole 15 (1.53 g, 10.0 mmol) were heated in sealed tube at 100 °C for
dry DMF (3 mL). Et N (0.040 mL, 0.29 mmol) was added, and the
resulting mixture was stirred at room temperature overnight under N2.
The mixture was poured into H O and extracted (3×) with CH Cl /2-
propanol (1:1), washed (2×) with saturated NH Cl, dried over
anhydrous Na
with preparative HPLC (H
8 as a white solid (17 mg, 17%). H NMR (400 MHz, CD
3
1.98 (quin, J = 6.69 Hz, 2H), 3.47 (t, J = 6.8 Hz, 2H), 3.55 (t, J = 6.7
Hz, 2H) 6.96 (m, 2H), 7.17 (m, 2H), 8.01 (d, J = 4.8 Hz, 1H), 8.19 (s,
3
9
2 h and then evaporated to dryness. Purification by flash-column
2
2
2
chromatography (amino column, CH Cl /MeOH, twice) and
4
2
2
recrystallization from MeCN gave compound 17 as off-white crystals
2
SO
4
, and evaporated. The crude product was purified
O, NH HCO /MeCN) to give compound
OD) δ ppm
1
(
1.30 g, 66%). H NMR (400 MHz, CD OD) δ ppm 1.74 (quin, J =
2
4
3
3
1
7
.0 Hz, 2H), 2.69 (t, J = 7.1 Hz, 2H), 3.35 (t, J = 6.8 Hz, 2H), 6.86−
6
.90 (m, 2H), 7.08−7.11 (m, 2H).
N-[3-[(1H-Benz[d]imidazol-2-yl)amino]propyl]-3,5-dichloroben-
+
zamide (7). Compound 17 (0.13 g, 0.70 mmol), EDC (0.15 g, 0.77
1H), 8.85 (d, J = 5.1 Hz, 1H). MS: m/z 364 [M + H] . Purity was
mmol), HOBt (0.10 g, 0.77 mmol), Et N (0.11 mL, 0.77 mmol), and
determined as >95% by HPLC (λ 212 nm), R : 1.01 min (Acquity
3
t
3
,5-dichlorobenzoic acid (0.14 g, 0.74 mmol) in DMF (6 mL) were
UPLC BEH C18 1.7 μm, 3 mm × 50 mm, 25 mM NH OAc/MeCN,
4
stirred at room temperature for 1.5 h. The reaction mixture was
pH 6.6).
partitioned between EtOAc and saturated NaHCO , and the organic
N-[3-[(1H-Benz[d]imidazol-2-yl)amino]propyl]-4-(pyrrolidin-1-
ylmethyl)benzamide (9). Following the procedure for compound 7
3
phase was washed (2×) with H O and brine, dried over anhydrous
2
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and TFA (2 mL) in CH Cl (3 mL) was stirred at room temperature
2
2
for 4 h, evaporated, and purified by SCX-2 column to give compound
1
2
0 as a yellowish solid (0.68 g, 68%). H NMR (400 MHz, CD OD) δ
3
ppm 1.76 (quin, J = 6.9 Hz, 2H), 2.70 (t, J = 6.9 Hz, 2H), 3.44 (t, J =
.8 Hz, 2H), 7.63 (t, J = 1.8 Hz, 1H), 7.78 (d, J = 2.0 Hz, 2H).
,5-Dichloro-N-(3-isothiocyanatopropyl)benzamide (21). To a
6
3
suspension of 20 (0.68 g, 2.73 mmol) in dry THF (130 mL) was
added 1,1′-thiocarbonyldiimidazole (0.54 g, 3.00 mmol). The resulting
mixture was stirred at room temperature for 3 h and washed with
brine. The organic phase was dried over anhydrous Na SO and
2
4
evaporated. The resulting oily crude product (1.03 g) was carried to
the next step without purification.
Methyl 2-[[3-(3,5-Dichlorobenzamido)propyl]amino]-1H-benz-
d]imidazole-5-carboxylate (11). Compound 21 (0.48 g, 1.65
[
mmol), methyl 3,4-diaminobenzoate (0.25 g, 1.5 mmol), DCC (0.37
g, 1.8 mmol), and Et N (0.21 mL, 1.5 mmol) in MeCN were heated at
3
8
5 °C for 24 h and evaporated to dryness. Purification by flash-column
chromatography (amino column, MeOH/CH Cl ) gave compound 11
2
2
1
(0.29 g, 46%). H NMR (400 MHz, DMSO-d ) δ ppm 1.82 (quin, J =
6
6
.7 Hz, 2H), 3.36−3.40 (m, 4H), 3.80 (s, 3H), 7.01 (br. s, 1H), 7.16
(
d, J = 8.3 Hz, 1H), 7.58 (d, J = 8.1 Hz, 1H), 7.71 (m, 1H), 7.82 (t, J =
.9 Hz, 1H), 7.88 (d, J = 1.8 Hz, 2H), 8.8 (m, 1H), 10.90 (m, 1H). C
1
3
1
NMR (100 MHz, DMSO-d ) δ 29.0, 36.8, 39.5, 51.4, 125.8, 130.4,
6
+
1
34.1, 137.6, 163.3, 166.9. MS: m/z 421 [M + H] . Purity was
determined as >95% by HPLC (λ 208 nm), R : 1.12 min (Acquity
t
UPLC BEH C18 1.7 μm, 3 mm × 50 mm, 25 mM NH OAc/MeCN,
4
pH 6.6).
Biology. Pf IC50 Determination. Parasite growth inhibition assays
and IC50 determination were carried out following standard methods
3
24
Figure 4. (A) Efficacy of 7 in the P. falciparum (Pf 3D70087/N9) mouse
using the H-hypoxanthine incorporation assay. Briefly, this assay
relies on the parasite incorporation of labeled hypoxanthine that is
proportional to P. falciparum growth. A culture of P. falciparum 3D7
parasitized red blood cells (RBC; 0.5% parasitemia, 2% hematocrit) in
RPMI-1640, albumax 5%, and 5 μM hypoxanthine is exposed to serial
model. Data shows individual parasitemia for two mice treated with
compound 7 at 100 mg/kg (white dots and squares) or vehicle (black
dots). (B) Giemsa-stained peripheral blood smears from mice treated
with vehicle (left) and derivative 7 (right) after 48 h starting the
treatment.
drug dilutions. Plates are incubated 24 h at 37 °C, 5% CO , 5% O ,
2
2
3
9
0% N . After 24 h of incubation, H-hypoxanthine is added, and
2
plates are incubated for additional 24 h period. After that, parasites are
harvested on a glass-fiber filter using a TOMTEC Cell harvester 96.
Filters are dried and melted on scintillator sheets to determine the
and using the corresponding carboxylic acid, compound 9 was
obtained as a white powder (60 mg, 40%). H NMR (400 MHz,
1
3
incorporation of H-hypoxanthine. Radioactivity is measured using a
CD OD) δ ppm 1.82 (m, 4H), 1.95 (quin, J = 6.7 Hz, 2H), 2.56 (m,
3
microbeta counter. Data are normalized using the incorporation of the
positive control (parasitized RBCs without drug). IC50s are determined
using Grafit 7 program.
4
H), 3.47 (t, J = 6.8 Hz, 2H), 3.51 (t, J = 6.7 Hz, 2H), 3.70 (s, 2 H),
6
.94−6.97 (m, 2H), 7.17−7.19 (m, 2H), 7.45 (d, J = 7.8 Hz, 2H), 7.82
+
(
d, J = 8.1 Hz, 2H). MS: m/z 378 [M + H] . Purity was determined as
hERG Inhibition Determination and Cell Cytotoxicity Assays. Use
>
95% by HPLC (λ 212 nm), R : 0.98 min (Acquity UPLC BEH C18
t
of hERG channel by IonWorks electrophysiology is described in
1
.7 μm, 3 mm × 50 mm, 25 mM NH OAc/MeCN, pH 6.6).
4
2
5−27
28
literature.
Cell cytotoxicity assays are described in literature.
tert-Butyl 4-[[3-[(1H-Benz[d]imidazol-2-yl)amino]propyl]-
carbamoyl]piperidine-1-carboxylate (10). Following the procedure
for compound 7, using the corresponding carboxylic acid and
In Vivo Studies. Authors declare that all animal studies were
ethically reviewed and approved by the DDW Ethical Committee on
Animal Research, performed at the DDW Laboratory Animal Science
facilities accredited by AAALAC and carried out in accordance with
European Directive 2010/63/EU and the GSK Policy on the Care,
Welfare, and Treatment of Animals.
Pharmacokinetic Studies in Mice. For pharmacokinetic studies in
mice, animals were dosed by either intravenous (IV) or oral (PO)
route. For IV administration, the dosing volume was 10 mL/kg for a
total dose of 1 mg/kg, and for PO administration, the dosing volume
was 10 mL/kg for a total dose of 10 mg/kg. Dosing solution for
intravenous administration was prepared in 20% encapsine and 5%
DMSO in saline solution (C = 0.1 mg/mL), and for PO
administration, a suspension in 1% methylcellulose (w:v) was
formulated (C = 1 mg/mL). Following IV dosing, blood samples
were collected at 5, 15, and 30 min and 1, 2, 4, 6, 8, and 24 h postdose.
Following oral dosing, blood samples were collected at 15, 30, and 45
min and 1, 2, 4, 6, 8, and 24 h postdose.
extraction with EtOAc, compound 10 was obtained as a colorless
1
sticky solid (60 mg, 36%). H NMR (400 MHz, CD OD) δ ppm 1.46
3
(
6
s, 9H), 1.57 (qd, J = 12.4, 4.0 Hz, 2H), 1.74 (m, 2H), 1.83 (quin, J =
.8 Hz, 2H), 2.36 (m, 1H), 2.78 (br. s, 2H), 3.29 (t, 2H, partially
hidden under CD OD signal), 3.39 (t, J = 6.8 Hz, 2H), 4.10 (m, 2H),
3
+
6
.96 (m, 2H), 7.18 (m, 2H). MS: m/z 402 [M + H] . Purity was
determined as >95% by HPLC (λ 212 nm), R : 1.03 min (Acquity
t
UPLC BEH C18 1.7 μm, 3 mm × 50 mm, 25 mM NH OAc/MeCN,
4
pH 6.6).
N-(3-Aminopropyl)-3,5-dichlorobenzamide (20). tert-Butyl (3-
aminopropyl)carbamate 19 (0.34 mL, 2.00 mmol), 2,5-dichloroben-
zoyl chloride 18 (0.28 mL, 2.00 mmol), and Et N (0.28 mL, 2.00
3
mmol) in CH Cl (10 mL) were stirred at room temperature for 0.5 h.
2
2
The reaction mixture was partitioned between saturated NaHCO and
3
EtOAc. The organic phase was washed with brine, dried over
anhydrous Na SO , and evaporated. The crude tert-butyl [3-(3,5-
2
4
dichlorobenzamido)propyl]carbamate was obtained as a white solid
For whole blood samples analysis, 25 μL of fresh blood was mixed
with 25 μL of saponine solution (0.1% in water) and immediately
stored frozen at −80 °C until analysis.
Diluted blood samples were processed under standard liquid−liquid
extraction procedures using acetonitrile and analyzed by LC-MS/MS
in positive ion mode with electrospray. Samples were assayed for
and was carried to the next step without further purification (0.58 g,
1
8
4%). H NMR (400 MHz, CD OD) δ ppm 1.36 (s, 9H), 1.68 (quin,
3
J = 6.6 Hz, 2H), 3.05 (q, J = 6.4 Hz, 2H), 3.33 (t, J = 6.8 Hz, 2H), 7.56
t, J = 1.9 Hz, 1H), 7.72 (d, J = 1.8 Hz, 2H). A solution of tert-butyl
3-(3,5-dichlorobenzamido)propyl]carbamate (1.22 g, 3.51 mmol)
(
[
F
DOI: 10.1021/acs.jmedchem.5b00114
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
parent compound using a Sciex API 4000 Triple Quadrupole Mass
Spectrometer (Sciex, Division of MDS Inc., Toronto, Canada), against
a series of matrix-matched calibration curve standards, using multiple
reaction monitoring (MRM) at the specific transitions for the
compound. Noncompartmental analysis was performed using Phoenix,
version 6.3 (Phoenix WinNonlin, ©1998−2012, Certara L.P.), and the
main pharmacokinetic parameters were estimated. Additional statistical
analysis of the data was performed with GraphPad Prism, version 5.01
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ASSOCIATED CONTENT
■
*
S
Supporting Information
(
Additional spectroscopic data for 7 and synthetic method-
ologies for compounds 6, 12a−m, 13a−ar, and 14a−g as well
as additional biological data. The Supporting Information is
available free of charge on the ACS Publications website at
DOI: 10.1021/acs.jmedchem.5b00114.
́
AUTHOR INFORMATION
■
Corresponding Author
(
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ac.uk/chemblntd.
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*
E-mail: jari.yli-kauhaluoma@helsinki.fi. Phone: +358-
94159170.
2
(
́
́ ́
, J. M.; Fernandez,
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ABBREVIATIONS
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