Discovery of N-(2-aminoethyl)-N-benzyloxyphenyl benzamides: New potent Trypanosoma brucei inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.11.019

A phenotypic screen of a compound library for antiparasitic activity on Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT), led to the identification of N-(2-aminoethyl)-N-phenyl benzamides as a starting point for hit-to-lead m

Full text of DOI:10.1016/j.bmc.2016.11.019

Accepted Manuscript
Discovery of N-(2-aminoethyl)-N-benzyloxyphenyl benzamides: New potent
Trypanosoma brucei inhibitors
Andriy Buchynskyy, J. Robert Gillespie, Matthew A. Hulverson, Joshua
McQueen, Sharon A. Creason, Ranae M. Ranade, Nicole A. Duster, Michael H.
Gelb, Frederick S. Buckner
PII:
S0968-0896(16)31182-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.019
BMC 13386
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 August 2016
9 November 2016
11 November 2016
Please cite this article as: Buchynskyy, A., Robert Gillespie, J., Hulverson, M.A., McQueen, J., Creason, S.A.,
Ranade, R.M., Duster, N.A., Gelb, M.H., Buckner, F.S., Discovery of N-(2-aminoethyl)-N-benzyloxyphenyl
benzamides: New potent Trypanosoma brucei inhibitors, Bioorganic & Medicinal Chemistry (2016), doi: http://
dx.doi.org/10.1016/j.bmc.2016.11.019
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Discovery of N-(2-aminoethyl)-N-benzyloxyphenyl benzamides:
Trypanosoma brucei inhibitors
New potent
Andriy Buchynskyya, J. Robert Gillespieb, Matthew A. Hulversonb, Joshua McQueena, Sharon A.
Creasonb, Ranae M. Ranadeb, Nicole A. Dusterb, Michael H. Gelb*a, Frederick S. Buckner*b
aDepartment of Chemistry, University of Washington, Seattle, WA 98195 USA
bDepartment of Medicine, University of Washington, Seattle, WA 98109 USA
*Corresponding authors:
Chemistry: M.H.G.: Phone, 206-543-7142; email: gelb@chem.washington.edu
Parasitology: F.S.B.: 206-616-9214; email: fbuckner@uw.edu

Abstract: A phenotypic screen of a compound library for antiparasitic activity on Trypanosoma brucei,
the causative agent of Human African Trypanosomiasis (HAT), led to the identification of N-(2-
aminoethyl)-N-phenyl benzamides as a starting point for hit-to-lead medicinal chemistry. Eighty two
analogues were prepared, which led to the identification of a set of highly potent N-(2-aminoethyl)-N-
benzyloxyphenyl benzamides with the most potent compound 73 having an in vitro EC50 = 0.001 µM.
The compounds displayed drug-like properties when tested in a number of in vitro assays. Compound
73 was orally bioavailable and displayed good plasma and brain exposure in mice, cured 2 out of 3
mice infected with Trypanosoma brucei in acute model when dosed orally at 50 mg/kg once per day for
4 days. Given its potent antiparasitic properties and its ease of synthesis, compound 73 represents a
potential lead for the development of drug to treat Human African Trypanosomiasis.

1. Introduction:
Human African Trypanosomiasis (HAT, sleeping sickness) occurs in 36 sub-Saharan Africa countries
where biting tse-tse flies transmit the disease. The etiologic agent, Trypanosoma brucei, is a flagellated
protozoan parasite that disseminates through the body during the early hemolymphatic stage and
eventually enters the central nervous system to cause late-stage disease. Symptoms of late-stage HAT
include sleep disturbance, cognitive dysfunction, coma, and death. Unless diagnosed and treated
during the early stage, drugs must cross the blood-brain barrier to be effective. As a result, treatment
options are severely limited for late-stage disease and consist of nifurtimox-eflornithine combination
treatment (NECT) or the arsenical drug, melarsoprol 1-2. NECT is expensive and requires intravenous
administration of the eflornithine component. Melarsoprol causes severe side effects including
fatalities in 3-10% of patients. Two drugs are still being studied in clinical trials, fexinidazole3 and
SCYX-71584. New drugs that are safer and simpler to use (preferably by oral administration) are
urgently needed to address this pernicious disease.
Despite modern advances in chemistry, genomics, and high-throughput screening technology,
antiparasitic drug discovery remains an immense challenge.5-7 Debates are ongoing whether target-
8,9
based versus cell-based (“phenotypic”) screening are preferable for identifying novel drug classes.
With respect to developing new drugs for HAT, we considered the additional requirement for needing
compounds with brain permeability properties. The vast majority of small molecule (>98%) do not
cross the blood-brain barrier.10 As a result, we felt that it was advantageous to use a cell-based
screening strategy that would identify membrane permeable small molecules and provide a broad
diversity of chemical scaffolds from which brain-permeable compounds might be identified.
A high throughput screen of a library of 700,000 compounds for growth inhibitory activity against
Trypanosoma brucei was conducted as previously described.11 The 1035 confirmed and selective hits
could be grouped into 115 distinct scaffolds. We have previously described a series of substituted 2-
phenylimidazopyridines derived from this high throughput screening that was optimized by medicinal
chemistry to result in compounds showing curative activity in the murine model of acute T. brucei

infection.11 Other hits from the screening were evaluated for their potential to be further developed
based on selectivity (parasite vs. mammalian cells), chemical tractability, and compliance with Lipinski
rules. One of these hits, compound 1 (GNF-00-0394-8224-1), became the object of a hit-to-lead
medicinal chemistry project and is described herein.
2. Results and discussion
2.1. Properties of Lead Compound (1). Lead compound 1 was selected from the available hits based
on drug-like features including low MW of 363.6, clog P of 3.48, H-bond donors of 1, H-bond
acceptors of 2. Additional measurements from biological assays are shown in Table 1. It had good
activity on T. brucei cells with selectivity over mammalian cells of >30-fold. It resisted metabolism in
mouse liver microsomes with t½ >60 min. Importantly, it showed excellent permeability into brain
tissue following intraperitoneal injection in mice (Supporting information, Fig. S1), a necessary
attribute for treating late-stage trypanosomiasis. As a hit compound, the one disadvantage is fairly
potent activity on CYP3A4 enzyme with an IC50 of 0.074 µM (average of 2 independent assays). The
CYP3A4 activity was determined to be attributable to the primary amine which was also necessary for
the antiparasitic activity (discussed below). In the literature, other benzamides with activity against
T.brucei are reported but with no primary amino group and completely different SAR profile12-15
.

Table 1
Properties of the original hit compound (1) from high-throughput screen
Compound (1)
MW
363.6
cLogP
3.48
1.21
40.0
30.0
>60
T. brucei brucei EC50 (µM)a
HepG2 cells CC50 (µM)b
CRL-8150 CC50 (µM)c
Mouse liver microsome t1/2 (min)d
CYP3A4 IC50 (µM)e
0.074
aConcentration of compound required to inhibit growth by 50% (EC50) of T. brucei brucei strain BF427. b,c Concentration of
compound required to inhibit growth by 50% (CC50) of mammalian cell lines human hepatocytes (HepG2) and human
lymphoblasts (CRL-8150) respectively. dTime required by liver microsomes (mouse) to reduce the amount of compound by
e
half. Concentration of compound required to inhibit by 50% (IC50) of human cytochrome P450 (3YP3A4 isoform)
enzyme.
2.2. Synthesis of 1 and its analogues.
Scheme 1. Synthesis of N-(2-aminoethyl)-N-phenyl-benzamidesa (1-44).

aReagents and conditions: (a) N-Boc-2-aminoacetaldehyde, NaCNBH3, chloroform, rt ; (b) R2COCl ,
DIPEA, dichloromethane, 40C to rt; (c) 4M HCl in dioxane, rt.
The N-(2-aminoethyl)-N-phenyl benzamide derivatives (2-44) and compound 1 were synthesized
starting from corresponding commercial anilines 84 by reductive alkylation with N-Boc-2-
aminoacetaldehyde (Scheme 1). Benzoylation of amine 85 and subsequent removal of the Boc
protecting group with hydrochloric acid in dioxane gave final compounds purified by flash
chromatography or HPLC.
Scheme 2 shows the synthetic route to make N-(2-aminoethyl)-N-benzyloxyphenyl benzamides
analogues. The compounds were synthesized in four steps starting with alkylation of 4-nitrophenol (87)
with alkyl or benzyl bromides (86) (Scheme 2, condition (a)). A second step - reduction of the aromatic
nitro group was performed in two different conditions depending on presence of halogens in the
aromatic ring (Scheme 2, condition (b)). The reduction of molecule containing aromatic halogens
(chlorine) was performed with activated zinc/copper pair in aqueous ammonium chloride to prevent
dehalogenation observed with use of palladium catalyst. The third step - an N-alkylation of anilines 88
by reductive amination with N-Boc-2-aminoacetaldehyde (Scheme 2, condition (c)). Final step is
benzoylation of amine 89 and subsequent removal of the Boc protecting group under room temperature
with hydrochloric acid in dioxane that yield final compounds 64-83 as hydrochloric salt (Scheme 2,
conditions (d,e)). All final compounds were purified by HPLC.
Scheme 2. Synthesis of N-(2-aminoethyl)-N-benzyloxyphenyl-benzamidesa (64-83).

aReagents and conditions: (a) Acetone, 600C, K2CO3;( b) Pt/C, H2, EtOAc, rt or Zn/Cu, aq. NH4Cl /
diethylether, rt; (c) N-Boc-2-aminoacetaldehyde, Na(OAc)3BH, chloroform, rt; (d) R2COCl , DIPEA,
dichloromethane, 40C to rt; (e) 4M HCl in dioxane, rt.
2.3. Structure−Activity Studies of Analogues of (1).
2.3.1. Substitutions at the aniline ring (R1)
The biochemical target of action for compound 1 is not known, thus an unbiased approach was taken to
investigate different substitutions at varying parts of the molecule. First, changes to the para-fluoro
substituent (R1 position) were investigated (Table 2). Replacement with chlorine (2) or bromine (3) led
to a 4-fold enhancement of potency. Trifluoromethyl (4) methyl (5) and phenyl (6) derivatives retained
potency, while isopropyl (7) was less active in comparison to 1. Replacement of fluorine (1) by
methoxy (8), nitro (9) or amino (10) group eliminated activity on T. brucei cells.
Table 2
SAR optimization of site R1 of N-(2-aminoethyl)-N-phenyl-2.4-dichlorobenzamides*
Compound
R1
EC50 (µM) a
CC50 (µM) b
4-F
1.21
0.31
30.0c, 40.0d
12.5 c, 14.2d
1
2
4-Cl
4-Br
4-CF3
4-CH3
4-Ph
0.33
1.05
1.52
1.83
11.5c,11.0d
13.2 e
3
4
5
6
24.9c

4-CH(CH3)2
4-OCH3
3.89
>6
7
56.1e
8
4-NO2
>10
>10
3.05
0.51
1.68
2.72
4.52
0.17
0.28
3.60
2.13
9
4-NH2
11.0 c
10
11
12
13
14
15
16
17
18
19
3-Cl
2,4-di-Cl
3,4-di-Cl
3-Cl-4F
4-Cl-3-OCH3
4-(4-Cl-Ph-O)
4-(3-Cl-Ph-O)
4-(2-Cl-Ph-O)
3-(4-Cl-Ph-O)
*All compounds prepared as HCl salts
b
aConcentration of compound required to inhibit growth by 50% (EC50) of T. brucei brucei strain BF427. Concentration of
c
compound required to inhibit growth by 50% (CC50) of mammalian cell lines. Human lymphoblasts (CRL-8155). dHuman
hepatocytes (HepG2). eRat myoblasts (L6)
To investigate the influence of substitution position in aromatic ring on activity, the meta-chloro
substituted analogue 11 was prepared and showed 10-fold reduction in potency over corresponding
para-chloro (2) derivative, indicating the importance of para-position (R1) for activity. From 2,4-di-
chloro (12), 3,4-dichloro (13), 4-fluoro-3-chloro (14) and 4-chloro-3-methoxy (15) substituted
analogues, only 2,4-dichloro (12) derivative has more than 2-fold improved activity with respect to 1
and just small reduction of potency with regards to 2.
Substitution at para-position with the bigger and more lipophilic 4-chlorophenoxy (16) or 3-
chlorophenoxy (17) group led to enhanced antiparasitic activity by 7-fold and 4-fold respectively,
while 2-chlorophenoxy (18) derivative resulted in reduced potency. Moving 4-chlorophenoxy
substituent to the meta-position (19) drastically reduced the potency indicating importance of 4-

substitution positon for antiparasitic activity with no obvious size limitation of substituents in this
preferred substitution position.
2.3.2 Substitutions at the benzoyl position (R2)
The influence of varying the benzoyl part of the molecule (R2) on antiparasitic activity was assessed
while keeping the 4-chloro substituent unchanged (Table 3). First, the unsubstituted benzoyl derivative
20 is less active than all other compound in this series. Among isomers of mono-substituted benzoyl
derivatives the most active are the 2-substituted benzoyl analogues. The 2-chlorobenzoyl derivative 23
is 3 fold more active than 3-chloro (22) and almost 8-fold more active than corresponding 4-
chlorobenzoyl derivative 21. 2-Methyl derivative 24 is 14-fold more active than 4-methyl derivative 25
and 2-methoxy analogue 26 is at least 4-times more potent than corresponding 4-methoxy (27). The 2-
chloro (23) and 2-methyl (24) analogues are equipotent while 2-methoxy derivative 26 is at least 5 time
less active compere to 23, 24. In the case of mono-fluoro isomers 28, 29, 30 there is no difference in
activity indicating that substituents larger than F are needed to influence potency favorably. Among the
dichloro-isomers (2, 31-34) 2,4-dichloro (2) and 2,3-dichloro (31) are the most active and equally
potent with 2-chloro derivative 23.
Table 3
SAR optimization of benzoyl site R2 of N-(2-aminoethyl)-N-4-Cl-phenyl-benzamides*
Compound
R2
EC50 (µM) a
CC50 (µM) b
Ph
13.00
3.12
20
21
4-Cl-Ph
3-Cl-Ph
2-Cl-Ph
1.20
0.44
22
23
>50c, >50d

2-CH3
4-CH3
0.52
7.00
2.51
>10
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
2-OCH3
4-OCH3
4-F-Ph
4.01
4.03
3.90
0.31
0.60
0.78
1.08
2.90
1.20
0.23
0.97
4.82
1.24
0.54
0.33
0.18
0.59
3-F-Ph
2-F-Ph
104.1e
2,3-di-Cl
2,5-di-Cl
2,6-di-Cl
3,5-di-Cl
4-Cl-3-NO2
2-Cl-5-NO2
2,4,6-tri-Cl
2,4-di-CH3
2,4-di-OCH3
2,4-di-F
>50c,>50d
8.5c, 21.5d
2,3,4,5,6-penta-F
2-CF3
2-CF3-4-F
2-CF3-3-Pyr
42.3c, 37.7d
*All compounds prepared as HCl salts
aConcentration of compound required to inhibit growth by 50% (EC50) of T. brucei brucei strain BF427. Pentamidine
isethionate was used as control with EC50 = 0.0021±0.00001 µM bConcentration of compound required to inhibit growth by
c
d
e
50% (CC50) of mammalian cell lines. Human lymphoblasts (CRL-8155). Human hepatocytes (HepG2). Rat myoblasts
(L6)
4-Chloro-3-nitro analogue 35 retains the potency of mono-chloro derivative 21, while 2-chloro-5-nitro
compound 36 is 3 times less potent than corresponding 2-chloro analogue 23. 2,4,6-tri-Chlorobenzoyl
derivative 37 is the most active from all chlorobenzoyl derivatives. No increase in activity was
observed comparing the 2,4-dimethyl (38) and 2,4 –dimethoxy (39) derivatives with corresponding
mono-ortho-substituted analogues. Interestingly pentafluoro derivative 41 showed 26-fold

improvement in activity compared to 20 and the 2,4-difluorobenzoyl compound 40 was at least 3-fold
more active than 2-floro (30) or 4-fluoro (28) derivatives. Making a bigger ortho-benzoyl substituent
like 2-trifluoromethyl derivative 42 improves the potency and the most active analogue in this SAR
series was 2-trifluoromethyl-4-flurobenzoyl derivative 43. In order to make the compounds more water
soluble the 2- trifluoromethyl-pyridinoyl compound 44 was made and it showed a high potency against
T.brucei parasites (EC50 = 0.59 µM). Selected compounds were tested for growth inhibition activity on
mammalian cells and were observed to have low toxicity (Table 3).
2.3.3. Substitutions at the ethylamino position (R3)
To investigate the SAR of ethylamino group (R3), we synthesized compounds derivatives of 2 (Table
4). Removing amino group at position R3 (45) as well as acylation (46) and dimethylation (47) of
amino group eliminated anti-T. brucei activity. The IC50 of compound 45 on CYP3A4 was 11.9 µM
(>100-fold weaker than compound 2 with CYP3A4 EC50 = 0.070 µM) indicating that the CYP450
inhibitory activity was due to the free amino group.
Table 4
SAR optimization of ethylamino site R3 of N-4-Cl-phenyl-2,4-benzoylmides
Compound
R3
EC50a (µM)
CH2CH3
>10
>10
45
46
CH2CH2NHC(O)CH3
CH2CH2N(CH3)2
>10
47

CH2CH2N(CH2)4O
CH2CH2OH
>10
>10
2.90
1.05
3.56
>10
>10
>10
>10
4.47
1.65
>10
>10
>10
4.11
>10
48
49
CH2CH2CH2NH2
CH2CH(CH3)NH2
CH(CH3)CH2NH2
CH2-2-furane
CH2-2-thiophene
CH2-2-imidazole
CH2-5-imidazole
3-pirrolidine
50*
51*
52*
53
54
55*
56*
57*
58*
59
CH2CH2NHCH3
CH2CH2C(O)NH2
CH2CN
60
3-piperidine
61*
62*
63*
4-piperidine
3-pyridine
*Compounds prepared as HCl salts
aConcentration of compound required to inhibit growth by 50% (EC50) of T. brucei brucei strain BF427. Pentamidine
isethionate was used as control with EC50 = 0.0021±0.00001 µM.
The morpholino analogue 48 was also inactive in the T.brucei EC50 assay. Changing NH2 group to OH
led to an inactive alcohol 49. Elongation of the alkyl chain to propylamine (50) resulted in 10-fold loss
of activity with almost the same gain in activity for propyl chain isomers (51, 52). Methylene-
heterocycle derivatives 53-56 were made to keep the three C-C bond distance between heteroatoms the
same as in compound 2, however, all of these compounds 53-56 were inactive. 3-Pyrrolidine derivative
57 showed low activity with EC50 of 4.5 µM. Monomethylation of the amino group (58) decreased
activity 5-fold compared to 2. The carbonyl amide (59) and CN (60) derivatives were inactive. Among
piperidine isomers, the 4-piperidine derivative 62 was at ~2.5 fold more active than 3-piperidine (61)
but 10-fold less active than compound 2. The 3-pyridine analogue 63 was inactive. Based on this SAR
the unprotected, unsubstituted ethylamino group is indispensable for antiparasitic activity.

2.3.4. Substitutions at the aniline ring (R1) – round 2
After the first round of optimization leading to analogues 15 and 42 with EC50 (T.b.b.) = 0.17 µM and
0.18 µM respectively, we performed a second round of optimization on site R1 (Table 5).
Table 5
N-(2-aminoethyl)-N-benzyloxyphenyl-benzamides*
Compound
R1
R2
EC50 (µM) a
CC50 (µM) b
4-chlorophenyl
2,4-dichlorophenyl
2-trifluoromethyl-4-fluorophenyl
2-trifluoromethyphenyl
2-chloro-3-pyridyl
0.031
0.007
0.007
0.030
0.003
0.005
0.002
0.003
0.002
0.001
0.001
0.001
0.020
0.005
0.010
0.015
0.003
0.043
2.00c, 2.00d
3.67c, 7.53 d
3.08 c, 5.72 d
11.46 c, 15.22 d
8.01c, 10.22 d
3.10 c, 3.19 d
2.83 c, 3.66 d
2.22 c, 3.99 d
4.03 c, 10.53 d
2.03 c, 3.83 d
3.03c
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
“
“
“
“
2-trifluoromethyl-3-pyridyl
2,4-dichlorophenyl
4-isopropylphenyl
“
2-trifluoromethyl-4-fluorophenyl
2-trifluoromethyphenyl
2-chloro-3-pyridyl
“
“
“
2-trifluoromethyl-3-pyridyl
2-nitrophenyl
“
“
2-bromophenyl
1.91c
3,5-dimethylphenyl
2,4-dichlorophenyl
2.45c, 4.10d
2.24c, 4.53d
2.94c, 3.94d
5.12c, 8.38d
4.74c, 6.46d
2.38c, 3.78d
“
2-trifluoromethyl-4-fluorophenyl
2-trifluoromethyphenyl
2-chloro-3-pyridyl
“
“
“
2-trifluoromethyl-3-pyridyl
2,4-dichlorophenyl
3-chlorophenyl

4-tert-buthylphenyl
4-ethylphenyl
2,4-dichlorophenyl
2,4-dichlorophenyl
0.005
0.015
2.12c, 3.83d
2.59c, 5.60d
82
83
*All compounds prepared as HCl salts
aConcentration of compound required to inhibit growth by 50% (EC50) of T. brucei brucei strain BF427. Pentamidine
isethionate was used as control with EC50 = 0.0021±0.00001 µM bConcentration of compound required to inhibit growth by
50% (CC50) of mammalian cell lines. cHuman lymphoblasts (CRL-8155). dHuman hepatocytes (HepG2).
It was found that 4-chlorobenzyloxy derivatives substantially increased activity with analogue 64
having an EC50 of 0.031 µM. Switching to 2-trifluoromethyl-4-fluorobenzoyl and 2-trifluoromethyl
derivatives 65, 66 further improve potency to single digit nanomolar EC50 values. From nicotinoyl
derivatives the 2-trifluoromethylnicotinoyl (68) was 10-fold more active than 2-chloronicotinoyl (67)
even though both are very active with EC50 of 0.003 µM and 0.03 µM, respectively. Changing the
position of the chlorine substituent in the chlorobenzoxy part to 3-chlorobenzoxy (81) resulted in
comparable potency to 4-chlorobenzoxy analogue 64. Introduction of an isopropyl group at position 4
further improved potency. The most active compounds 69-75, containing a 4-isopropylbenzyloxy
moiety, had EC50 values in the low nanomolar range. Interestingly, the 2-nitorobenzoyl (74) and 2-
bromobenzoyl (75) compounds were as active as 2-trifluoromethylnicotinoyl derivative 73, each with
EC50 values of 0.001 µM. The 2-trifluoromethyl-4-fluorobenzoyl (70) and 2-trifluoromethylbenzoyl
(71) derivatives had EC50 values of 0.002 and 0.003 µM, respectively. 3,5-Dimethyl analogues 78-80
showed slight reduction in potency compared to corresponding 4-isopropyl derivatives but still
retained low nanomolar antiparasitic activity with EC50 of 0.003 µM for the most active 2-
trifluoromethylnicotinoyl analogue 80. Switching to 4-terbutyl (82) or 4-ethylbenzyloxy (83)
derivatives retained the potency. The second-generation SAR effort around aniline ring R1 shows that
oxybenzyl moiety with the lipophilic substituent in aromatic ring (chloro, methyl, iso-propyl, tert-
butyl) at 4 and/or 3 position(s) significantly improves in-vitro antiparasitic activity of lead compounds.
3. Biological studies
3.1. Pharmacological testing

The analogues with the highest potency against T. brucei cultures were subjected to additional
pharmacological profiling (Table 6). Eight out of 10 compounds that were tested showed half-life time
>60 min in human liver microsome incubations. This may be related to strong potency against CYP450
enzymes as indicated by inhibition of CYP3A4 in the range of 0.003-0.179 µM. Pharmacokinetics in
mice were assessed by oral gavage dosing at 50 mg/kg and sampling plasma at intervals out to 8 hours.
The maximum plasma concentration (Cmax) was generally in the range of 1-2 μM, with the exceptions
of 65 and 76 having Cmax values of 4.4 and 5.9 µM, respectively.
Table 6
Antiparasitic activity, pharmacological data and efficacy of selected compounds.
Liver
Brain/
AUC±SEM
Acute
cure rate
(mice)g
CYP3A4
Cmax
EC50(μM) a
(min*μM)f
IC50(μM)b
(μM)e
Comp.
microsome Plasma
T1/2(min) c
ratiod
T.brucei
T.rhod
35
(mouse),
>60
(human)
>60
0.42 ±
0.06
0.53 ±
0.23
205.05 ±
91.48
0.031
0.007
0.005
0.179
0.010
4/5
2/3
64
(mouse),
>60
0.38 ±
0.05
4.39 ±
0.56
1560.83 ±
364.67
65
(human)
>60
(human)
>60
(human)
52
(human)
>60
(human),
>60
(mouse)
>60
(human)
>60
(human)
>60
0.88 ±
0.35
0.32±
0.07
0.22 ±
0.10
1.67 ±
0.41
1.49 ±
0.36
1.78 ±
0.31
616.97 ±
146.07
1260.70 ±
128.72
665.63 ±
204.69
0.007
0.005
0.002
1/3
2/3
1/3
66
69
70
0.019
0.002
0.015
0.16±
0.09
1.89 ±
0.28
735.07 ±
72.62
0.002
2/3
72
0.23 ±
0.19
0.18 ±
0.09
0.96 ±
0.24
0.06 ±
0.01
1.00 ±
0.77
5.90 ±
1.31
1.01 ±
0.41
2.07 ±
1.52
334.63 ±
236.47
2032.63 ±
689.16
390.43 ±
155.95
653.33 ±
432.23
0.001
0.020
0.010
0.015
0.144
0.003
2/3h
1/3
1/2
2/3
73
76
78
79
(human)
39
(human)
aConcentration of compound required to inhibit growth by 50% (EC50) of T. brucei brucei strain BF427 and T.brucei
rhodesiense strain STIB900 . Pentamidine isethionate was used as control with EC50 = 0.0021±0.00001 µM bConcentration
c
of compound required to inhibit by 50% (IC50) of human cytochrome P450 (3YP3A4 isoform) enzyme. Time required by
liver microsomes (mouse, human) to reduce the amount of compound by half. d Ratio of compound concentration in brain to

e
concentration in plasma 1h after intraperitoneal injection of 5 mg/kg (values are the mean of three mice, n=3). Maximum
f
concentration of compound in blood by oral admission (values are the mean of three mice, n=3). Area under the curve of
concentration of compound in blood over the time (values are the mean of three mice, n=3). G Ratio of number of cured mice
h
over total number of mice in each compound experiment after 60 days post infection. The parasitemia relapse (in one
mice) was observed 31 days after treatment.
These two compounds also had the highest area under the curve (AUC0-8 hr) measurements (Table 6).
Penetration into brain tissue was assessed by comparing brain to plasma concentrations of compounds
at 60 minutes following a single intraperitoneal injection at 5 mg/kg. Compounds 64, 65, 66, 69, 78
shows high CNS exposure with the brain-to-plasma concentration ratios >0.3.
3.2. Efficacy studies in mice
Given the favorable pharmacological properties and antiparasitic activity, ten compounds were selected
for efficacy testing in the mouse model of acute HAT infection using the STIB900 strain of T. brucei
rhodesiense. Mice were treated for 4 days beginning 48 hours post infection at 50 mg/kg once per day
by oral gavage. Parasitemia was followed out to 60 days. Compounds 64, 65, 69, 72, 73, 79 had cure
rates of 67-80% while compounds 66, 70, 76, 78 showed partial cure rates of 33% (Table 6). The
highest parasitemia rebound time (parasitemia free period) was observed for compound 73 which was
31 days post infection (see Supporting information, Fig. S3).
Selected compounds were tested in a murine model of late-stage HAT. Mice were injected with the
TREU667 strain that was allowed to establish infection for 21 days. Dosing with high concentration
and long duration was chosen as a proof of concept to see if the full cure could be achieved. Mice were
treated twice a day at 50 mg/kg by oral gavage from day 21 to 34 (14 days). Diminazene aceturate
single dose of 10 mg/kg on day 21 was used as one of the controls. Diminazene does not cross the
blood-brain barrier. It causes temporary clearance of parasitemia which later relapses, most likely from
parasites leaving the brain and returning to the bloodstream. Compounds 76 and 65 showed partial
suppression during treatment whereas compounds 72 and 73 led to complete suppression during the

treatment phase, however, all mice relapsed with parasites in the blood. The longest parasite free
(blood) time (12 days post-treatment/46 days post infection) was observed for treatment with
compound 65 (Table 7).
Table 7
Late-stage efficacy model in mice. The data indicate the number of mice free of parasitemia / number
of mice in the group.
Day post-
infection
Day post-
treatment
0
21-34
34 35 36 37 38 39 40 41-42 43-45
46-49
50-51 52-180
16-17 18-180
0
1
2
3
4
5
6
7-8
--
9-11
--
12-15
--
infected DOSING 0/4 --
--
--
--
--
--
--
--
--
Vehicle
Diminazene
infected DOSING 3/3 3/3 3/3 3/3 3/3 3/3 3/3
3/3
3/3
1/3
0/3
day 21a
65b
72b
73b
76b
infected DOSING 4/4 4/4 4/4 2/4 1/4 1/4 1/4
infected DOSING 5/5 5/5 5/5 4/5 3/5 2/5 1/5
infected DOSING 5/5 5/5 5/5 4/5 4/5 3/5 0/5
infected DOSING 4/4 3/4 3/4 3/4 3/4 2/4 1/4
1/4
0/5
--
1/4
--
0/4
--
--
--
--
--
--
--
--
--
--
--
0/4
--
--
SCYX-
7158c
infected DOSING 5/5 5/5 5/5 5/5 5/5 5/5 5/5
5/5
5/5
5/5
5/5
5/5
aSingle intraperitoneal dose of diminazene aceturate at 10 mg/kg in water on day 21 (clear parasites from blood and
b
peripheries but not from the brain). Mice were injected with the T.brucei brucei (strain TREU667) that was allowed to
c
establish infection for 21 days. Mice were treated at 50 mg/kg twice a day by oral gavage from day 21 to 34 (14 days).
Control oxaborole compound, SCYX-7158 , developed by Anacor Pharmaceuticals
This indicated that the compounds did not fully suppress parasites in the periphery or were unable to
clear parasites from the central nervous system and they re-established infection in the blood. The
control compound, SCYX-7158, cured all mice at the same dose (50 mg/kg PO twice per day).
3.3. Washout experiments

Although N-(2-aminoethyl)-N-benzyloxyphenyl benzamide compounds demonstrated very low EC50
values on cultured T. brucei and reasonably good pharmacokinetic profiles in mice, they were only
successful at giving partial cures in the acute infection model. Therefore, washout experiments were
performed to evaluate the time and concentrations of compounds required to kill T. brucei cultures.
The compounds in this series required relatively long exposures at high concentrations compared to the
clinical drug pentamidine (Table 8).
Table 8
Washout experiments with T. bruceia
Compound
24H
48H
72H
96H
4X
4X
1X
1X
Pentamidine
64
32X
16X
16X
16X
16X
16X
32X
8X
16X
8X
32X
4X
69
72
79
66
32X
64X
64X
16X
128X
128X
64X
64X
aThe values indicate the concentration of compound relative to the 48-hr EC50 and the duration of exposure that resulted in
no outgrowth of cells after another 10 days of observation in media without compound.
For example, parasites incubated in vitro with compound 66 for 96 hours required a concentration of
64-times the EC50 to completely kill the culture. This contrasted with pentamidine which killed all the
parasites at 4-times the EC50 after only 24 hours exposure. Thus, it appears that parasites are able to
rebound after exposures to compounds well above the EC50 values.
4. Conclusions
Eighty two analogues of compound 1 were synthesized to optimize anti-trypanosomal activity and
pharmacological properties. Several compounds with EC50 values as low as 0.001-0.002 µM were
identified. They demonstrated reasonably good oral bioavailability and plasma exposures, and had

good penetration into brain tissue. However, in murine efficacy models of HAT infection, the
compounds showed only partial cures or suppression. In vitro washout studies suggested that the
compounds completely eliminate parasites only with long exposure at concentrations many fold above
EC50 values and this probably is responsible for the suboptimal results in the efficacy experiments.
5. Experimental section
5.1. Chemistry. All starting materials were purchased from various chemical vendors and used without
further purification unless noted. Thin-layer chromatography was performed on Merck Silica Gel 60
F254 pre-coated plates. Column chromatography was conducted under medium pressure on silica
(Cleanert Silica (40-60 µm)) from Agela Technologies. 1H NMR spectra were recorded on a Bruker
AV-300 or AV-500 spectrometers. Chemical shifts were referenced with respect to the residual
solvents signals. Electrospray (ESI) mass spectra were obtained on Bruker Esquire Ion Trap Mass
Spectrometer. All target compounds were purified by Varian semi-preparative HPLC (Varian PrepStar,
model 218, column YMC ODS-A, 100x20 mm, 5µm, flow: 10 mL/min, UV detector at : 218 nm and
254 nm) with mobile phase 1 (water: methanol, gradient 50% to 80% methanol over 15 min) or
mobile phase 2 (water (0.01% HCl): methanol, gradient 15% to 50% methanol over 15 min). All final
compounds are judged to be > 95% pure by HPLC (UV at 254 nm and 218 nm).
5.1.1 . General synthesis of compounds 2-19
Compounds 2-19 were synthesized by reductive alkylation of commercially available anilines with N-
Boc-2-aminoacetaldehyde and benzoylation with 2,4-dichlorobenzoyl chloride with subsequent
removal of Boc-protecting group with 4N HCl in dioxane. Compound precipitated from hexane/
dioxane reaction mixture as HCl salt.
5.1.1.1. N-(2-aminoethyl)-2,4-dichloro-N-(4-chlorophenyl)benzamide (2)
was prepared from 4-chloroaniline, N-Boc-2-aminoacetaldehyde and 2,4-dichlorobenzoyl chloride as
HCl salt (65 mg, 84%) ;1H NMR (300 MHz, CDCl3): δ 7.23 (1H, bs), 7.18 (2H, d, J=9Hz), 7.08 (2H,

d, J=9Hz), 7.07 (2H, bs), 3.96 (2H, t, J=9Hz), 2.94 (2H, t, J=9Hz); ESI MS m/z 343.4 (M+H)+, 326.2
(M-NH3+H)+.
5.1.1.2. N-(2-aminoethyl)-N-(4-bromophenyl)-2,4-dichlorobenzamide hydrochloride (3)
was prepared from 4-bromoaniline, N-Boc-2-aminoacetaldehyde and 2,4-dichlorobenzoyl chloride as
HCl salt (100 mg, 80%); 1H NMR (300 MHz, CDCl3): δ 8.55 (3H, bs), 7.90 (1H, d, J=9Hz), 7.26 (2H,
d, J=9Hz), 7.19 (2H, d, J=9Hz), 7.12 (1H, d, J=3Hz), 7.04 (1H, dd, J=9Hz, J=3Hz), 4.27 (2H, bs), 3.34
(2H, bs); ESI MS m/z 387.5 (M+H)+, 370.7 (M-NH3+H)+.
5.1.1.3. N-(2-aminoethyl)-2,4-dichloro-N-[4-(trifluoromethyl)phenyl]benzamide hydrochloride (4)
was prepared from 4-trifluoroaniline, N-Boc-2-aminoacetaldehyde and 2,4-dichlorobenzoyl chloride
1
as HCl salt (38 mg, 50%); H NMR (300 MHz, CDCl3): δ 8.68 (3H, bs), 7.98 (1H, d, J=9Hz), 7.55-
7.35 (4H, bs), 7.12 (1H, s), 7.06 (1H, d, J=8Hz), 7.12 (1H, d, J=3Hz), 4.31 (2H, bs), 3.34 (2H, bs); ESI
MS m/z 377.3 (M+H)+, 360.7 (M-NH3+H)+.
5.1.1.4. N-(2-aminoethyl)-2,4-dichloro-N-(4-methylphenyl)benzamide hydrochloride (5)
was prepared from p-toluidine, N-Boc-2-aminoacetaldehyde and 2,4-dichlorobenzoyl chloride as HCl
1
salt (65 mg, 90%); H NMR (300 MHz, CDCl3): δ 8.55 (3H, bs), 7.87 (1H, bs), 7.14 (2H, bs), 7.07
(1H, s), 7.00 (1H, s), 6.92 (2H, d, J=6Hz), 4.25 (2H, bs), 3.32 (2H, bs), 2.21 (3H, s); ESI MS m/z 323.7
(M+H)+, 306.8 (M-NH3+H)+.
5.1.1.5. N-(2-aminoethyl)-2,4-dichloro-N-(4-phenylphenyl)benzamide (6)
was prepared from 4-phenylaniline, N-Boc-2-aminoacetaldehyde and 2,4-dichlorobenzoyl chloride as
HCl salt (17 mg, 45%); 1 H NMR (300 MHz, CDCl3): δ 8.71 (3H, bs), 8.03 (1H, d, J=9Hz), 7.50-7.30
(9H, bs), 7.10 (2H, bs), 4.37 (2H, bs), 3.43 (2H, bs); ESI MS m/z 385.3 (M+H)+, 368.6 (M-NH3+H)+.
5.1.1.6. N-(2-aminoethyl)-2,4-dichloro-N-[4-(propan-2-yl)phenyl]benzamide hydrochloride (7)

was prepared from 4-isopropylaniline, N-Boc-2-aminoacetaldehyde and 2,4-dichlorobenzoyl chloride
as HCl salt (75 mg, 83%); 1H NMR (300 MHz, d6-DMSO): δ 8.15 (3H, bs), 7.62 (1H, d, J=9Hz), 7.49
(1H, d, J=2Hz), 7.33 (1H, dd, J=9Hz, J=2Hz), 7.26 (2H, d, J=9Hz), 7.15 (2H, d, J=9Hz), 4.04 (2H, bs),
2.99 (2H, bs), 2.80 (1H, m), 1.11 (3H, s), 1.08 (3H, s); ESI MS m/z 351.3 (M+H)+, 334.2 (M-NH3+H)+.
5.1.1.7. N-(2-aminoethyl)-2,4-dichloro-N-(4-methoxyphenyl)benzamide hydrochloride (8)
was prepared from p-anisidine, N-Boc-2-aminoacetaldehyde and 2,4-dichlorobenzoyl chloride as HCl
1
salt (60 mg, 80%); H NMR (300 MHz, CDCl3): δ 8.56 (3H, bs), 7.90 (1H, d, J=9Hz), 7.22 (2H, d,
J=9Hz), 7.09 (1H, s), 7.02 (1H, d, J=9Hz), 6.63 (2H, d, J=9Hz), 4.25 (2H, bs), 3.70 (3H, s), 3.33 (2H,
bs); ESI MS m/z 339.6 (M+H)+, 322.3 (M-NH3+H)+.
5.1.1.8. N-(2-aminoethyl)-2,4-dichloro-N-(4-nitrophenyl)benzamide hydrochloride (9)
was prepared from 4-nitroaniline, N-Boc-2-aminoacetaldehyde and 2,4-dichlorobenzoyl chloride as
1
HCl salt (20 mg, 77%); H NMR (300 MHz, d6-DMSO): δ 8.25 (3H, bs), 8.16 (2H, d, J=9Hz), 7.81
(1H, d, J=9Hz), 7.67 (2H, d, J=9Hz), 7.51 (1H, bs), 7.43 (1H, d, J=9Hz), 4.16 (2H, bs), 2.99 (2H, bs);
ESI MS m/z 354.8 (M+H)+, 337.2 (M-NH3+H)+.
5.1.1.9 N-(2-aminoethyl)-N-(4-aminophenyl)-2,4-dichlorobenzamide (10)
1
was prepared from 9 by reduction with SnCl2 in ethyl acetate (10 mg, 77%); H NMR (300 MHz,
CDCl3): δ 7.21 (1H, d, J=2Hz), 7.02 (2H, d, J=2Hz), 6.88 (2H, d, J=9Hz), 6.45 (2H, d, J=9Hz), 3.90
(2H, t, J=9Hz), 3,67 (2H, bs), 2.91 (2H, t, J=9Hz); ESI MS m/z 324.5 (M+H)+, 307.4 (M-NH3+H)+.
5.1.1.10. N-(2-aminoethyl)-2,4-dichloro-N-(3-chlorophenyl)benzamide hydrochloride (11)
was prepared from 3-chloroaniline, N-Boc-2-aminoacetaldehyde and 2,4-dichlorobenzoyl chloride as
1
HCl salt (50 mg, 74%); H NMR (300 MHz, d6-DMSO): δ 8.20 (3H, bs), 7.74 (1H, d, J=9Hz), 7.58
(1H, s), 7.51 (1H, d, J=2Hz), 7.45-7.25 (4H, m), 4.10 (2H, bs), 3.00 (2H, m); ESI MS m/z 343.3
(M+H)+, 326.9 (M-NH3+H)+.

5.1.1.11. N-(2-aminoethyl)-2,4-dichloro-N-(2,4-dichlorophenyl)benzamide (12)
was prepared from 2.4-dichloroaniline, N-Boc-2-aminoacetaldehyde and 2,4-dichlorobenzoyl chloride
as HCl salt (75 mg, 98%); 1 H NMR (300 MHz, d6-DMSO): δ 8.16 (3H, bs), 7.72 (1H, d, J=2Hz), 7.69
(1H, d, J=9Hz), 7.57 (1H, d, J=2Hz), 7.52 (1H, d, J=9Hz), 7.43 (1H,dd, J=9Hz, J=2Hz), 7.34 (1H, dd,
J=9Hz, J=2Hz), 4.59 (2H,bs), 3.05 (2H, bs) ); ESI MS m/z 377.3 (M+H)+, 360.7 (M-NH3+H)+.
5.1.1.12. N-(2-aminoethyl)-2,4-dichloro-N-(3,4-dichlorophenyl)benzamide hydrochloride (13)
was prepared from 2.4-dichloroaniline, N-Boc-2-aminoacetaldehyde and 2,4-dichlorobenzoyl chloride
as HCl salt (78 mg, 57%); 1 H NMR (300 MHz, d6-DMSO): δ 8.24 (3H, bs), 7.95-7.75 (2H, bs), 7.65-
7.35 (4H, bs), 4.11 (2H,bs), 3.00 (2H, bs). ESI MS m/z 377.5 (M+H)+, 360.4 (M-NH3+H)+.
5.1.1.13.N-(2-aminoethyl)-2,4-dichloro-N-(3-chloro-4-fluorophenyl)benzamide hydrochloride (14)
was prepared from 3-chloro-4-fluoro-aniline, N-Boc-2-aminoacetaldehyde and 2,4-dichlorobenzoyl
1
chloride as HCl salt (74 mg, 54%); H NMR (300 MHz, d6-DMSO): δ 8.27 (3H, bs), 7.82 (2H, d,
J=9Hz), 7.55-7.30 (4H, bs), 4.09 (2H,bs), 3.01 (2H, bs); ESI MS m/z 361.3 (M+H)+, 344.4 (M-
NH3+H)+.
5.1.1.14. N-(2-aminoethyl)-2,4-dichloro-N-(4-chloro-3-methoxyphenyl)benzamide hydrochloride
(15)
was prepared from 4-chloro-3-methoxy-aniline, N-Boc-2-aminoacetaldehyde and 2,4-dichlorobenzoyl
1
chloride as HCl salt (27 mg, 63%); H NMR (300 MHz, d6-DMSO): δ 8.20 (3H, bs), 7.79 (1H, d,
J=9Hz), 7.50 (1H, d, J=2Hz), 7.42-7.26 (3H, bm), 6.96 (1H, dd, J=9Hz, J=2Hz), 4.10 (2H, bs), 3.76
(3H, s), 3.01 (2H, bs); ESI MS m/z 373.2 (M+H)+, 356.4 (M-NH3+H)+.
5.1.1.15. N-(2-aminoethyl)-2,4-dichloro-N-[4-(4-chlorophenoxy)phenyl]benzamide hydrochloride
(16)

was prepared from 4-(4-chlorophenoxyl)-aniline, N-Boc-2-aminoacetaldehyde and 2,4-dichlorobenzoyl
chloride as HCl salt (35 mg, 79%); 1 H NMR (300 MHz, d6-DMSO): δ 8.17 (3H, bs), 7.66 (1H, d, J=9
Hz), 7.51 (1H, d, J=3Hz), 7.43-7.36 (5H, bm), 6.92 (2H, d, J=9Hz), 6.91 (2H, d, J=9Hz), 4.07 (2H, bs),
3.01 (2H, bs). ESI MS m/z 435.2 (M+H)+, 418.4 (M-NH3+H)+.
5.1.1.16. N-(2-aminoethyl)-2,4-dichloro-N-[4-(3-chlorophenoxy)phenyl]benzamide hydrochloride
(17)
was prepared from 4-(3-chlorophenoxy)-aniline, N-Boc-2-aminoacetaldehyde and 2,4-dichlorobenzoyl
chloride. Purified by HPLC as HCl salt (100 mg, 75%); 1 H NMR (500 MHz, d6-DMSO): δ 8.11 (3H,
bs), 7.63 (1H, d, J=9Hz), 7.51 (1H, d, J=2Hz), 7.45-7.35(4H, bm), 7.20 (1H, d, J=9Hz), 6.98 (2H, d,
J=9Hz), 6.90-6.80 (2H, bm), 4.08 (2H, bs), 3.03 (2H, t, J=8Hz); ESI MS m/z: 435.4 (M+H)+, 418.5 (M-
NH3+H)+.
5.1.1.17. N-(2-aminoethyl)-2,4-dichloro-N-[4-(2-chlorophenoxy)phenyl]benzamide hydrochloride
(18)
was prepared from 4-(2-chlorophenoxy)aniline, N-Boc-2-aminoacetaldehyde and 2,4-dichlorobenzoyl
1
chloride. Purified by HPLC as HCl salt (52 mg, 51%); H NMR (300 MHz, d6-DMSO): δ 8.14 (3H,
bs), 7.63 (1H, d,J=9Hz), 7.58 (1H, dd, J=9 Hz, J=2Hz), 7.51 (1H, d, J=2 Hz), 7.40-7.30(4H, bm), 7.24
(1H, dd, J=9Hz, J=2Hz), 6.96 (1H, dd, J=9Hz, J=2 Hz), 6.83 (2H, d, J=9Hz), 4.06 (2H, bs), 3.02 (2H,
bs); ESI MS m/z: 435.5 (M+H)+, 418.2 (M-NH3+H)+.
5.1.1.18. N-(2-aminoethyl)-2,4-dichloro-N-[3-(4-chlorophenoxy)phenyl]benzamide hydrochloride
(19)
was prepared from 3-(4-chlorophenoxy)aniline, N-Boc-2-aminoacetaldehyde and 2,4-dichlorobenzoyl
chloride. Purified by HPLC as HCl salt (18 mg, 90%); 1 H NMR (300 MHz, d6-DMSO): δ 8.19 (3H,
bs), 7.63 (1H, d,J=9Hz), 7.56 (1H, d, J=2Hz), 7.45-7.30 (4H, bm), 7.23 (1H, dd, J=9Hz, J=2Hz), 7.08

(1H, bs), 6.94 (1H, dd, J=9Hz, J=2 Hz), 6.73 (2H, d, J=9Hz), 4.09 (2H, bs), 3.00 (2H, bs); ESI MS m/z:
435.4 (M+H)+, 418.7 (M-NH3+H)+.
5.1.2. General synthesis of compounds 20-44
Compounds
20-44
were
synthesized
by
benzoylation
of
tert-butyl
N-{2-[(4-
chlorophenyl)amino]ethyl}carbamate (obtained by reductive amination of N-Boc-2-aminoacetaldehyde
with 4-chloroaniline) with corresponding benzoyl chlorides and subsequent removal of Boc-protecting
group by HCl. All compounds precipitated from Boc-removal reaction mixture as HCl salt.
5.1.2.1 N-(2-aminoethyl)-N-(4-chlorophenyl)benzamide hydrochloride (20)
was prepared by reaction of amine with benzoyl chloride and subsequent removal of Boc-protecting
group by HCl (16 mg, 53%); 1 H NMR (300 MHz, CDCl3): δ 8.65 (3H, bs), 7.35 (2H, bs), 7.20-6.95
(7H, bs), 4.33 (2H, bs), 3.34 (2H, bs); ESI MS m/z: 275.2 (M+H)+, 258.7 (M-NH3+H)+.
5.1.2.2.N-(2-aminoethyl)-4-chloro-N-(4-chlorophenyl)benzamide hydrochloride (21)
was prepared by reaction of amine with 4-chlorobenzoyl chloride and subsequent removal of Boc-
protecting group by HCl (35 mg, 92%); 1 H NMR (300 MHz, d6-DMSO): δ 8.16 (3H, bs), 7.37 (4H, s),
7.32 (4H, s), 4.07 (2H, t, J = 6Hz), 2.95 (2H, m); ESI MS m/z: 309.1 (M+H)+, 292.3 (M-NH3+H)+.
5.1.2.3. N-(2-aminoethyl)-3-chloro-N-(4-chlorophenyl)benzamide hydrochloride (22)
was prepared by reaction of amine with 3-chlorobenzoyl chloride and subsequent removal of Boc-
protecting group by HCl (35 mg, 91%); 1 H NMR (300 MHz, d6-DMSO): δ 8.17 (3H, bs), 7.45-7.30
(6H, m), 7.27-7.18 (2H, m), 4.06 (2H, t, J = 6Hz), 2.95 (2H, m); ESI MS m/z: 309.3 (M+H)+, 292.5
(M-NH3+H)+.
5.1.2.4. N-(2-aminoethyl)-2-chloro-N-(4-chlorophenyl)benzamide hydrochloride (23)
was prepared by reaction of amine with 2-chlorobenzoyl chloride and subsequent removal of Boc-
protecting group by HCl (30 mg, 79%); 1 H NMR (300 MHz, d6-DMSO): δ 8.14 (3H, bs), 7.60 (1H,

m), 7.40-7.32 (4H, m), 7.28-7.23 (3H, m), 4.07 (2H, bt), 3.00 (2H, bs); ESI MS m/z 309.1 (M+H)+,
292.2 (M-NH3+H)+.
5.2.1.5. N-(2-aminoethyl)-N-(4-chlorophenyl)-2-methylbenzamide hydrochloride (24)
was prepared by reaction of amine with o-toluoyl chloride and subsequent removal of Boc-protecting
group by HCl (19 mg, 51%); 1 H NMR (300 MHz, d6-DMSO): δ 8.16 (3H, bs), 7.32-7.01 (8H, m),
4.07 (2H, bs), 2.98 (2H, bs), 2.24 (3H,s); ESI MS m/z 289.0 (M+H)+.
5.2.1.6. N-(2-aminoethyl)-N-(4-chlorophenyl)-4-methylbenzamide hydrochloride (25)
was prepared by reaction of amine with p-toluoyl chloride and subsequent removal of Boc-protecting
group by HCl (27 mg, 93%);1 H NMR (300 MHz, d6-DMSO): δ 8.10 (3H, bs), 7.39-7.30 (4H, m), 7.19
(2H, d, J=9Hz), 7.04 (2H, d, J=9Hz), 4.06 (2H, t, J = 9Hz), 2.96 (2H, m), 2.23 (3H, s); ESI MS m/z
289.1 (M+H)+, 272.2 (M-NH3+H)+
5.2.1.7. N-(2-aminoethyl)-N-(4-chlorophenyl)-2-methoxybenzamide hydrochloride (26)
was prepared by reaction of amine with 2-methoxybenzoyl chloride and subsequent removal of Boc-
protecting group by HCl (29 mg, 87%); 1 H NMR (500 MHz, d6-DMSO): δ 8.11 (3H, bs), 7.38 (1H, d,
J=9Hz), 7.30-7.15 (5H, m), 6.85 (1H, m), 6.75 (1H, d, J=9Hz), 4.04 (2H, bs), 3.56 (3H, s), 2.94 (2H,
bs); ESI MS m/z 305.0 (M+H)+, 288.0 (M-NH3+H)+.
5.2.1.8. N-(2-aminoethyl)-N-(4-chlorophenyl)-4-methoxybenzamide hydrochloride (27)
was prepared by reaction of amine with 4-methoxybenzoyl chloride and subsequent removal of Boc-
protecting group by HCl (25 mg, 68%); 1 H NMR (300 MHz, d6-DMSO): δ 8.14 (3H, bs), 7.39-7.30
(4H, m), 7.24 (2H, d, J=9Hz), 6.77 (2H, d, J=9Hz), 4.05 (2H, t, J = 9Hz), 3.70 (3H, s), 2.94 (2H, m);
ESI MS m/z 305.2 (M+H)+, 288.1 (M-NH3+H)+.
5.2.1.9. N-(2-aminoethyl)-N-(4-chlorophenyl)-4-fluorobenzamide hydrochloride (28)

was prepared by reaction of amine with 4-fluorobenzoyl chloride and subsequent removal of Boc-
protecting group by HCl (30 mg, 83%); 1 H NMR (300 MHz, d6-DMSO): δ 8.13 (3H, bs), 7.40-7.34
(6H, m), 7.09 (2H, t, J= 9Hz), 4.07 (2H, t, J = 6Hz), 2.95 (2H, m); ESI MS m/z 293.4 (M+H)+, 276.7
(M-NH3+H)+.
5.2.1.10. N-(2-aminoethyl)-N-(4-chlorophenyl)-3-fluorobenzamide hydrochloride (29)
was prepared by reaction of amine with 3-fluorobenzoyl chloride and subsequent removal of Boc-
protecting group by HCl (33 mg, 92%); 1 H NMR (300 MHz, d6-DMSO): δ 8.13 (3H, bs), 7.38 (4H, s),
7.30-7.08 (4H, m), 4.06 (2H, t, J = 6Hz), 2.95 (2H, m); ESI MS m/z 293.2 (M+H)+, 276.2 (M-
NH3+H)+.
5.2.1.11. N-(2-aminoethyl)-N-(4-chlorophenyl)-2-fluorobenzamide hydrochloride (30)
was prepared by reaction of amine with 2-fluorobenzoyl chloride and subsequent removal of Boc-
protecting group by HCl (31 mg, 86%); 1 H NMR (300 MHz, d6-DMSO): δ 8.10 (3H, bs), 7.53 (1H, t,
J=9Hz), 7.34 (5H, bs), 7.13 (1H, t, J=6Hz), 7.02 (1H, t, J= 6 Hz), 4.08 (2H, t, J= 6Hz), 2.96 (2H, bs);
ESI MS m/z 293.1 (M+H)+, 276.0 (M-NH3+H)+
5.2.1.12. N-(2-aminoethyl)-2,3-dichloro-N-(4-chlorophenyl)benzamide hydrochloride (31)
was prepared by reaction of amine with 2,3-dichlorobenzoyl chloride and subsequent removal of Boc-
protecting group by HCl (43 mg, 77%); 1 H NMR (300 MHz, d6-DMSO): δ 8.22 (3H, bs), 7.65 (1H, d,
J=9Hz), 7.50 (1H, d, J=9Hz), 7.45-7.35 (4H, m), 7.27 (1H, t, J=9Hz), 4.07 (2H, bs), 3.00 (2H, bs);
ESI MS m/z 343.4 (M+H)+, 326.5 (M-NH3+H)+.
5.1.2.13. N-(2-aminoethyl)-2,5-dichloro-N-(4-chlorophenyl)benzamide hydrochloride (32)
was prepared by reaction of amine with 2,5-dichlorobenzoyl chloride and subsequent removal of Boc-
protecting group by HCl (48 mg, 85%); 1 H NMR (300 MHz, d6-DMSO): δ 8.21 (3H, bs), 7.90 (1H, d,
J=2Hz), 7.47-7.29 (5H, m), 4.08 (2H, bs), 3.00 (2H, bs); ESI MS m/z 343.2 (M+H)+, 326.3 (M-
NH3+H)+.

5.1.2.14. N-(2-aminoethyl)-2,6-dichloro-N-(4-chlorophenyl)benzamide hydrochloride (33)
was prepared by reaction of amine with 2,6-dichlorobenzoyl chloride and subsequent removal of Boc-
protecting group by HCl (45 mg, 80%); 1 H NMR (300 MHz, d6-DMSO): δ 8.18 (3H, bs), 7.40-7.25
(7H, m), 4.06 (2H, t, J=9Hz), 3.04 (2H, bs); ESI MS m/z 343.3 (M+H)+, 326.5 (M-NH3+H)+.
5.1.2.15. N-(2-aminoethyl)-3,5-dichloro-N-(4-chlorophenyl)benzamide hydrochloride (34)
was prepared by reaction of amine with 3,5-dichlorobenzoyl chloride and subsequent removal of Boc-
protecting group by HCl (48 mg, 86%); 1 H NMR (300 MHz, d6-DMSO): δ 8.15 (3H, bs), 7.55 (1H, s),
7.43 (4H, s), 7.39 (2H, s), 4.05 (2H, t, J=9Hz), 2.96 (2H, bs); ESI MS m/z 343.6 (M+H)+, 326.8 (M-
NH3+H)+.
5.1.2.16. N-(2-aminoethyl)-4-chloro-N-(4-chlorophenyl)-3-nitrobenzamide hydrochloride (35)
was prepared by reaction of amine with 4-chloro-3-nitrobenzoyl chloride and subsequent removal of
Boc-protecting group by HCl (26 mg, 62%); 1 H NMR (300 MHz, d6-DMSO): δ 8.23 (3H, bs), 8.10
(1H, bs), 7.68 (1H, d, J=9Hz), 7.56 (1H, d, J=9Hz) 7.48-7.40 (4H, m) , 4.09 (2H, t, J=9Hz), 2.96 (2H,
bs); ESI MS m/z 354.4 (M+H)+, 337.5 (M-NH3+H)+.
5.1.2.17. N-(2-aminoethyl)-2-chloro-N-(4-chlorophenyl)-5-nitrobenzamide hydrochloride (36)
was prepared by reaction of amine with 2-chloro-5-nitrobenzoyl chloride and subsequent removal of
Boc-protecting group by HCl (27 mg, 63%); 1 H NMR (300 MHz, d6-DMSO): δ 8.71 (1H, d, J=2Hz),
8.17 (3H, bs), 8.09 (1H, dd, J=9Hz, J=2 Hz), 7.59 (1H, d, J=9Hz), 7.47 (2H, d, J=9Hz) 7.37 (2H, d,
J=9Hz) , 4.13 (2H, bs), 3.00 (2H, bs); ESI MS m/z 354.3 (M+H)+, 337.2 (M-NH3+H)+.
5.1.2.18. N-(2-aminoethyl)-2,4,6-trichloro-N-(4-chlorophenyl)benzamide hydrochloride (37)
was prepared by reaction of amine with 2,4,6-trichlorobenzoyl chloride and subsequent removal of
Boc-protecting group by HCl (35 mg, 76%); 1 H NMR (300 MHz, CDCl3): δ 7.30-7.10 (6H, m), 4.09
(4H, bs), 3.14 (2H, bs); ESI MS m/z 377.4 (M+H)+, 360.6 (M-NH3+H)+.

5.1.2.19. N-(2-aminoethyl)-N-(4-chlorophenyl)-2,4-dimethylbenzamide hydrochloride (38)
was prepared by reaction of amine with 2,4-dimethylbenzoyl chloride and subsequent removal of Boc-
protecting group by HCl (14 mg, 38%); 1 H NMR (300 MHz, d6-DMSO): δ 8.09 (3H, bs), 7.39-7.32
(4H, m), 7.20 (2H, d, J=9Hz), 7.03 (2H, d, J=9Hz), 4.05 (2H, t, J = 9Hz), 2.97 (2H, m), 2.25 (3H, s),
2.23 (3H, s); ESI MS m/z 303.5 (M+H)+, 286.7 (M-NH3+H)+.
5.1.2.20. N-(2-aminoethyl)-N-(4-chlorophenyl)-2,4-dimethoxybenzamide hydrochloride (39)
was prepared by reaction of amine with 2,4-dimethoxylbenzoyl chloride and subsequent removal of
Boc-protecting group by HCl (26 mg, 65%); 1 H NMR (500 MHz, d6-DMSO): δ 8.13 (3H, bs), 7.32-
7.29 (3H, m), 7.23 (2H, d, J=9 Hz), 6.45 (1H, d, J=2 Hz), 6.28 (1H, bs), 4.02 (2H, bs), 3.69 (3H, s),
3.52 (3H, s), 2.91 (2H, bs); ESI MS m/z 335.6 (M+H)+, 318.7 (M-NH3+H)+.
5.1.2.21. N-(2-aminoethyl)-N-(4-chlorophenyl)-2,4-difluorobenzamide hydrochloride (40)
was prepared by reaction of amine with 2,4-difluorobenzoyl chloride and subsequent removal of Boc-
protecting group by HCl (32 mg, 94%); 1 H NMR (500 MHz, d6-DMSO): δ 8.22 (3H, bs), 7.67 (1H,
m), 7.42-7.37 (4H, bs), 7.08 (2H, m), 4.09 (2H, bs), 2.95 (2H, bs); ESI MS m/z 311.0 (M+H)+, 294.2
(M-NH3+H)+
5.1.2.22. N-(2-aminoethyl)-N-(4-chlorophenyl)-2,3,4,5,6-pentafluorobenzamide hydrochloride (41)
was prepared by reaction of amine with 2,3,4,5,6-pentafluobenzoyl chloride and subsequent removal of
Boc-protecting group by HCl (15 mg, 38%); 1 H NMR (300 MHz, d6-DMSO): δ 8.21 (3H, bs), 7.46
(2H, d, J=9Hz), 7.37 (2H, d, J=9Hz), 4.11 (2H, t, J=9Hz), 3.00 (2H, bs); ESI MS m/z 365.2 (M+H)+,
348.4 (M-NH3+H)+
5.1.2.23. N-(2-aminoethyl)-N-(4-chlorophenyl)-2-(trifluoromethyl)benzamide hydrochloride (42)
was prepared by reaction of amine with 2-trifluoromethylbenzoyl chloride and subsequent removal of
Boc-protecting group by HCl. Product was purified by HPLC (76 mg, 92%); 1 H NMR (300 MHz,

CDCl3): δ 8.55 (3H, bs), 7.85 (1H, m), 7.43 (1H, m), 7.28 (2H, d, J = 4Hz), 7.18 (2H, d, J=9Hz), 7.04
(2H, d, J = 9Hz), 4.21 (2H, bs), 3.34 (2H, bs); ESI MS m/z 343.0 (M+H)+, 326.1 (M-NH3+H)+
5.1.2.24. N-(2-aminoethyl)-N-(4-chlorophenyl)-4-fluoro-2-(trifluoromethyl)benzamide
hydrochloride (43)
was prepared by reaction of amine with 2-trifluoromethyl-4-fluorobenzoyl chloride and subsequent
removal of Boc-protecting group by HCl. Product was purified by HPLC (76 mg, 87%); 1 H NMR
(300 MHz, CDCl3): δ 8.53 (3H, bs), 7.96 (1H, m), 7.20 (2H, d, J = 9Hz), 7.15 (1H, d, J=2Hz), 7.10
(2H, d, J = 9Hz), 6.98 (1H, dt, J=9Hz, J=2Hz), 4.13 (2H, bs), 3.34 (2H, bs); ESI MS m/z 361.2
(M+H)+, 344.5 (M-NH3+H)+
5.1.2.25. N-(2-aminoethyl)-N-(4-chlorophenyl)-2-(trifluoromethyl)pyridine-3-carboxamide
hydrochloride (44)
was prepared by reaction of amine with 2-trifluoromethyl-4-fluorobenzoyl chloride and subsequent
removal of Boc-protecting group by HCl. Product was purified by HPLC (44 mg, 89%); 1 H NMR
(500 MHz, d6-DMSO): δ 8.63 (1H, d, J= 5 Hz), 8.29 (1H, d, J= 10 Hz), 8.19 (3H, bs), 7.63 (1H, m),
7.42-7.35 (4H, m), 4.08 (2H, bs), 3.02 (2H, bs); ESI MS m/z 344.3 (M+H)+, 327.7 (M-NH3+H)+.
5.1.3. General synthesis of compounds 45-63
Compounds 45-63 were synthesized by reductive alkylation of 4-chloroaniline with corresponding
aldehydes (or alkylation with alkyl bromides) with following benzoylation with 2,4-dichlorobenzoyl
chloride and subsequent removal of Boc-protecting group .
5.1.3.1. 2,4-dichloro-N-ethyl-N-(4-fluorophenyl)benzamide (45)
was prepared by reaction of N-ethyl-N-4-fluorophenyl amine with 2,4-dichlorobenzoyl chloride and
subsequent removal of Boc-protecting group by HCl. Product was purified by flash chromatography on
silica gel with dichloromethane as mobile phase (63 mg, 27%); 1 H NMR (300 MHz, CDCl3): δ 7.22