Activity of diphenyl ether benzyl amines against Human African Trypanosomiasis

Article abstract of DOI:10.1016/j.bioorg.2020.103590

Insect-borne parasite Trypanosoma brucei plagues humans and other animals, eliciting the disease Human African trypanosomiasis, also known as African sleeping sickness. This disease poses the biggest threat to the people in Sub-Saharan Africa. Given the high toxicity and difficulties with administration of currently available drugs, a novel treatment is needed. Building on known Human African trypanosomiasis structure–activity relationship (SAR), we now describe a number of functionally simple diphenyl ether analogs which give low micromolar activity (IC₅₀ = 0.16–0.96 μM) against T. b. rhodesiense. The best compound shows favorable selectivity against the L6 cell line (SI = 750) and even greater selectivity (SI = 1200) against four human cell lines. The data herein provides direction for the ongoing optimization of antitrypanosomal diphenyl ethers.

Full text of DOI:10.1016/j.bioorg.2020.103590

Journal Pre-proofs
Activity of diphenyl ether benzyl amines against Human African Trypanoso-
miasis
James P. Hagen, Grant Darner, Samuel Anderson, Katie Higgins, Derek A.
Leas, Ananya Mitra, Victoria Mashinson, Tasloach Wol, Carlos Vera-
Esquivel, Bret Belter, Monica Cal, Marcel Kaiser, Alexander Wallick, Rosalie
C. Warner, Paul H. Davis
PII:
S0045-2068(19)31421-X
DOI:
https://doi.org/10.1016/j.bioorg.2020.103590
YBIOO 103590
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
20 September 2019
29 November 2019
15 January 2020
Please cite this article as: J.P. Hagen, G. Darner, S. Anderson, K. Higgins, D.A. Leas, A. Mitra, V. Mashinson, T.
Wol, C. Vera-Esquivel, B. Belter, M. Cal, M. Kaiser, A. Wallick, R.C. Warner, P.H. Davis, Activity of diphenyl
ether benzyl amines against Human African Trypanosomiasis, Bioorganic Chemistry (2020), doi: https://doi.org/
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0.1016/j.bioorg.2020.103590
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Activity of diphenyl ether benzyl amines against Human African Trypa-
nosomiasis.
a
a
a
a
a
a
James P. Hagen, * Grant Darner, Samuel Anderson, Katie Higgins, Derek A. Leas, Ananya Mitra,
a
a
a
a
b
b
Victoria Mashinson, Tasloach Wol, Carlos Vera-Esquivel, Bret Belter, Monica Cal, Marcel Kaiser,
c
c
c
Alexander Wallick, Rosalie C. Warner , Paul H. Davis,
a
Department of Chemistry, University of Nebraska at Omaha, Omaha, Nebraska 68182-0109, United States
bSwiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland, University of Basel, CH-4003 Ba-
sel, Switzerland
cDepartment of Biology, University of Nebraska at Omaha, Omaha, Nebraska 68182
1

Abstract
Insect-borne parasite Trypanosoma brucei plagues humans and other animals, eliciting the disease Human African trypanosomiasis,
also known as African sleeping sickness. This disease poses the biggest threat to the people in Sub-Saharan Africa. Given the high
toxicity and difficulties with administration of currently available drugs, a novel treatment is needed. Building on known Human
African trypanosomiasis SAR (structure–activity relationship), we now describe a number of functionally simple diphenyl ether an-
alogs which give low micromolar activity (IC50 = 0.16–0.96 μM) against T. b. rhodesiense. The best compound shows good selec-
tivity against the L6 cell line (SI = 750) and better selectivity (SI = 1200) against four human cell lines. The data herein provides
direction for the ongoing optimization of antitrypanosomal diphenyl ethers.
2

1
. Introduction
In a recent review, Patterson and Wyllie estimate 120,000 annual fatalities from Kinetoplastida genus protazoans (Trypa-
1
nosomiasis, Chagas, Leishmania). In the case of Human African trypanosomiasis (HAT), the World Health Organization (WHO)
2
estimates approximately 20,000 annual cases with 65 million people at risk. The transmission of this disease is caused primarily
by the bite of the tsetse fly, which passes the parasitic protozoan species Trypanosoma brucei (T. b.) from its saliva to the blood-
stream of the human. In humans, HAT is caused by two trypanosomal sub-species, T. b. gambiense in Central and Western Africa
and T. b. rhodesiense in Western Africa. HAT infections are characterized by two stages. In the first stage, trypanosomes multiply
in the circulatory system causing symptoms such as fever, headaches, and joint pain. In the second stage, the parasites cross the
blood brain barrier and affect the central nervous system. This results in behavioral changes, sleep disturbances, coma, and eventu-
ally death.
Currently, five drugs are used to treat HAT (Figure 1). Problems with these drugs are myriad with some of the most seri-
ous being drug-resistance, impractical administration protocols, and high toxicity. Treatment options for T. b. rhodesiense infec-
3
tions are suramin for stage I and melarsoprol for stage II of the disease. Arsenic-based melarsoprol is highly toxic. Available
treatments for T. b. gambiense infections include pentamidine for stage I and melarsoprol and eflornithine for stage II. In recent
years, the first line treatment of stage II T. b. gambiense infections has been nifurtimox-eflornithine combination therapy (NECT).4
Trypanosomal drug resistance has been observed with the leading treatment drugs melarsoprol and eflornithine.4,5 Given these
facts, novel orally active compounds are in high demand for HAT and related neglected tropical diseases.6,7 Much work has focused
on second generation pentamidine analogs (Figure 1).8,9,10,11 Furamidine and an orally administered pro-drug pafuramidine (Figure
2
) have shown promise.12 Unfortunately, phase III clinical trials of pafuramidine were discontinued due to liver and kidney tox-
icity.13
Figure 1. Current drugs registered for treatment of HAT.
Attention has also been focused on nitroaromatic compounds that might improve upon nifurtimox (Figure 1), and indeed
fexinidazole (Figure 2) has passed late stage human trials14,15,16 and has been approved for human use by the European Medicines
Agency. Bruhn et al. have reported new pentacyclic nitrofurans active at low nanomolar levels against a nifurtimox-resistant T.
brucei brucei strain.17 Another nitroaromatic shows promise in early screens (Hwang et al.).18 Papadopoulou et al. have recently
3

reported nitroaryl N-(diarylether)-amides that are active against HAT but more active against T. cruzi and L. donovani.19 Nitroaro-
matic compounds have been associated with mutagenicity and an ideal drug candidate might best avoid this moiety. A promising
bis-amidine with an N-arylbenzamide core (Wang et al., Figure 2) showed good antitrypanosomal activity and low cytotoxicity in
early screens, but was toxic to mice.20 The N-aryl benzamide core is a feature of oxaborole21,22 which has entered clinical trials.23
Graca et al. have reported symmetrical bisnaphthalimidopropyl derivatives against T. brucei brucei with low nanomolar IC50
,
acceptable toxicity, and metabolic stability.24 Unfortunately, the pharmacologic properties of these compounds were less than satis-
factory.
Figure 2. A survey of lead compounds active against HAT.
Low micromolar IC50 against T. cruzi has been reported by Gudes-da-Silva et al. in a series of bis-amidines.25 Quinolone amides
have shown good activity in vivo (Hiltensperger et al.).26 Recently two groups have reported good anti-trypanosome activity and
low toxicity in a series of thiazole derivatives that contain N-piperidinyl urea moieties, a structural motif that represents a notable
departure from the N-arylbenzamide and nitroaryl themes. The lead reported by Patrick et al.27 is the transpositional isomer of the
compound reported by Russell et al.28 with a very similar low nanomolar IC50 against trypanosomes. Both studies reported that
oxidation of the thiazole moiety by cytochrome P450 impairs their metabolic stability and complicates the development of these
4

promising leads. Buchynskyy et al.29 have reported good activity against T. brucei in thiohydantoin derivatives. Finally, Kuettel et
al. reported 1.0 M activity for a morpholine diphenylether derivative and proposed a mechanism of action where it binds and hy-
peractivates the T. b. rhodesiense adenosine kinase.30
We have found that two diphenylether benzyl amines (3b and 4) without nitroaryl, benzamide, alkylurea or amidine func-
tionality exhibited low micromolar IC50 against T. b. rhodesiense (Table 1). We now report results from a structure-activity study
of these two lead compounds. We started by varying the length and oxidation states of the side chain (Table 1). Next, we exam-
ined a family of bis-benzylic amines (Table 2) and introduced other variations to the core structure. (Table 3 and 4). We replaced
the benzylic amine of the leads with larger moieties (Table 5 and 6). Finally, we investigated the effects of halogen substituents
(
Table 7).
2
. Chemistry and Results
Aldehydes 1a–1g were prepared by nucleophilic aromatic substitution (Scheme I). The syntheses of target compounds 3b,
3
1
4
, and 6 through 8 are shown in Scheme 2. Aldehyde 1a was converted by Wadsworth-Horner-Emmons reaction with tri-
ethylphosphonoacetate to the cyanopropenoate 2. Hydrogenation over palladium on carbon gave the propanoate amine 3a which
was then converted to the tosylate salt 3b.
2 3
Scheme 1. Synthesis of cyanobiphenyl aldehyde intermediates 1a–1g. Reagents and conditions: (a) K CO , DMA, 100–120 °C,
2
9–91%*. *See experimental section for individual reaction yields.
Hydrolysis of 3a gave the carboxylic acid ammonium salt 4. Hydrogenation of 2 over platinum on carbon gave the cyanopropano-
ate 5a and byproducts due to hydrogenolysis and amine alkylation (5b and 9a). Reduction of 5a with a superhydride system led to
alcohol ammonium salt 6. Hydrolysis of 5a gave carboxylic acid 7 which was converted into the imidazolidinium derivative 8.
5

Scheme 2. Synthesis of acyclic aliphatic side chain derivatives from cyanobiphenyl aldehdye intermediates. Reagents, conditions,
and yields: (a) triethyl phosphonoacetate, NaH, THF, 0 to 62 °C, 72%; (b) H , Pd/C, EtOH, rt, 75%; (c) TsOH, Et O, rt, 77%; (d)
from 3a, 2N NaOH, EtOH, H O, rt, then conc HCl, 47%; (e) H , 60 psi, Pt/C, rt, 50% for 5a, 4% for 5b; (f) from 5a, LiBH
LiEt BH, THF, rt; 2N NaOH, then 1N HCl, 93%; (g) from 5a, 2N NaOH, EtOH, rt, then conc HCl, 77%; (h) ethylenediamine,
2
2
2
2
4
,
3
NaHS, DMA, 120 °C, then conc HCl, 82%; (i) 1N HCl/EtOAc, rt, 69%.
6

Table 1. Alkyl side chain derivatives and the resulting in-vitro activity against T. b. rhod and cytotoxicity against the L6 cell
line.
Compound
X
n
Y
Z
W
T. b. rhod.
IC50 (M) [SI]
1.4 [79]
2.0 [150]
0.96 [177]
160
L6 cytotoxicity
IC50 (M)
110
3
4
6
7
8
1
1
1
1
1
1
1
1
1
2
2
2
b
EtO
2
C
2
2
3
2
2
2
2
2
1
2
2
2
2
0
1
1
3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
CH
CH
CH
CN
2
2
2
NH
NH
NH
2
2
2
·HOTs
·HCl
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
HO
HO
HO
HO
HO
HO
2
C
>300
170
·HCl
2
2
C
C
>370
>290
20
C=NCH
CN
2
CH
2
NH·HCl
180
0
1
3a
4
5
6
7
8
9
0
3
6
71
CH
CN
CN
CN
CN
CH
CH
CN
CH
CH
CH
2
NH
2
·HCl
6.6 [18]
16
120
(EtO)
HO
CH
BnO
CH
BnO
2
PO
10
2
C
130
280
3
O
7.0
120
7.5
290
3
O
2
2
NH
2
2
·HCl
·HCl
4.1 [44]
2.9 [18]
120
180
NH
51
HO
HO
HO
HO
2
C
340
2
2
2
NH
NH
NH
2
2
2
·HCl
·HCl
·HCl
5.7 [35]
2.8 [57]
0.49 [100]
200
160
O
49
2
7
H
2
NCO
2
H
CONH
2
O
160
227
aActive against L. don. 2.3 M
As shown in Scheme 3, 10 through 13 were prepared from tyrosol and 4-fluorobenzonitrile by nucleophilic aromatic sub-
stitution to form nitrile alcohol 10. Reduction of 10 with superhydride followed by acidification gave alcohol ammonium salt 11.
Treatment of 10 with thionyl chloride followed by Arbuzov reaction with triethylphosphite produced nitrile phosphonate 13.
7

Scheme 3. Synthesis of alkyl side chain phenoxy ether derivatives 13 through 20. Reagents, conditions, and yields: (a) 4-fluoroben-
zonitrile, K CO , DMA, 120 °C, 63%; (b) LiBH , LiEt BH, THF/Et O, rt; 3N NaOH then conc HCl, 65%; (c) SOCl , reflux, 78%;
d) P(OEt) , NaI, reflux, 87%; (e) Jones reagent, acetone, 0 °C to rt; 2N NaOH then conc HCl, 28%; (f) KOH, CH I or BnCl,
DMSO, rt, 98%; (g) LiEt BH, THF, rt; 3N NaOH then EtOAc/conc HCl, 48%; (h) Jones reagent, acetone, 0 °C to rt; 2N NaOH
then conc HCl, 66%; (i) LiBH , LiEt BH, THF/Et O, rt; 3N NaOH then conc HCl, 95%.
2
3
4
3
2
2
(
3
3
3
4
3
2
Oxidation of 10 with Jones reagent gave cyano acid 14. Jones oxidation of 1a gave the cyano acid 19 while reduction of 1a with
superhydride gave amino alcohol 20. Cyano acids 14 and 19 were reduced with superhydride, however no pure product could be
isolated. Compounds 17 and 18 were made by alkylation of 10 followed by superhydride reduction. Compound 23 was made as
3
2
shown in Scheme 4. Known 21 and 4-fluorobenzaldehdye gave cyano aldehyde 22. Subsequent reduction of 22 with superhy-
dride gave alcohol ammonium salt 23.
8

Scheme 4. Synthesis of diphenyl thioether and ether derivatives 23, 26, and 27. Reagents, conditions, and yields: (a) 4-fluoroben-
zonitrile, K CO , DMA, 120 °C, 57%; (b) LiBH , LiEt BH, THF/Et O, rt; 2N NaOH then Et O/conc HCl, 37%; (c) triethyl phos-
phonoacetate, NaH, THF, 0 to 55 °C, 52%; (d) Cu(OAc) ·H O, 1,2-BDP, PMHS, t-BuOH, toluene, rt, 49%; (e) LiBH , LiEt BH,
THF, rt; 10% NaOH then CH OH/0.5 M HCl, 69%; (f) SOCl , reflux, 67%; (g) NaI, acetone, reflux, then KCN, DMF, rt; K CO
then 30% H , DMSO, 0 °C to rt, 13%.
2
3
4
3
2
2
2
2
4
3
3
2
2
3
,
2
O
2
The synthesis of 26 required a modification of the method shown in Scheme 2. Ester 24 appeared to undergo loss of
3
3
chlorine in reductions involving zinc/acetic acid or hydrogenation. A Lipshutz modification of a Stryker reagent gave clean re-
duction to 25. Superhydride reduction gave the chlorinated alcohol 26. Bis-amide 27 was made (Scheme 4) to test the need for
3
4,35
both benzylic amines. Following literature precedent
the bis-nitrile was made by conversion of 10 to the chloride, then the
iodide, then the nitrile. Basic hydrogen peroxide gave the bis-amide 27.
Compounds 28 through 41 in Table 2 were made by reductive amination followed by nitrile reduction with superhydride as shown
in Scheme 5.
3
Scheme 5. Synthesis of diphenyl ether analogs 28 through 47. Reagents, conditions, and yields: (a) amine, NaHB(OAc) , DCM or
DCE, rt*; (b) LiBH
4
, LiEt
3
BH, THF, rt*. *See experimental section for individual reaction yields.
9

Table 2. T. b. rhod. activity and L6 cell cytotoxicity of diphenyl ether derivatives 28–41.
Compound
W
X, Y
Z
T. b. rhod.
IC50 (M) [SI]
110
L6 cytotoxicity
IC50 (M)
110
2
2
3
3
8
9
0
1
EtO
2
CCH
2
NHCH
2
·HCl
H, Cl
H, Cl
H, Cl
H, Cl
CN
CN
CH
CH
HO
2
CCH
2
2
NHCH
2
2
·HCl
·HCl
110
210
HO
2
CCH
NHCH
2
2
NH
2
2
·HCl
·HCl
4.3
>250
52
HOCH
2
CH
2
NHCH
2
·HCl
NH
3.12
3
2
H, Cl
CN
140
2.9
48
·HCl
3
3
H, Cl
CH
2
NH
2
·HCl
38
·HCl
3
3
3
4
5
6
H, Cl
H, Cl
H, Cl
CN
CH
CN
5.4 [18]
12 [4]
>270
97
53
70
2
NH
2
·HCl
·HCl
·HCl
·HCl
3
3
3
7
8
9
H, Cl
Cl, H
Cl, H,
CH
CN
CH
2
NH
2
·HCl
·HCl
0.53 [152]
81
43
45
12
2
NH
2
1.1
1
0

4
4
0
1
H, H
H, H
CN
CH
7.5
110
120
·
HCl
HCl
2
NH
2
·HCl
1.4 [86]
·
Variation of the aliphatic group of the molecule is shown in Table 3 and Scheme 6. The preparation of a diphenylsulfide,
9, is also shown. Piperidine and N-methylpiperazine derivatives (42 through 47) were made by reductive amination of 1b. The
5
derivatives, 49 and 50 (Scheme 6) were prepared from Boc protected piperazine. The 4-tetrahydropyranidine compound 55 was
formed by reduction of 1b to 51 followed by treatment with concentrated HCl under phase transfer conditions to give the benzylic
chloride 52. Displacement of chlorine with triphenylphosphine produced salt 53. Wittig olefination of 53 gave 54 and the hydroly-
sis product 56. Subsequent superhydride reduction of 54 led to 55. Treatment of 3-chloro-4-fluorobenzaldehyde with 4-mercapto-
benzonitrile gave 57. Reductive amination gave 58. Superhydride reduction produced 59. Compound 60 was derived from 4-phe-
noxybenzonitrile by Friedel Crafts reaction with succinic anhydride. Esterification of 60 gave 61 which was converted to the Boc
protected amino lactone 62 by simultaneous reduction of the ketone and nitrile groups in a nickel catalyzed sodium borohydride
system. Treatment with HCl/EtOAc produced the racemic lactone salt 63.
Table 3. T. b. rhod. activity and L6 cell cytotoxicity of diphenyl ether derivatives with variable aliphatic ring variation.
Compound
V
W, X, Y
H, Cl, O
Z
T. b. rhod.
L6 cytotoxicity
IC50 (M) [SI]
IC50 (M)
4
2
CN
3.0
42
·
HCl
HCl
4
4
4
3
4
5
H, Cl, O
H, H, O
H, H, O
CH
CN
CH
2
NH
2
·HCl
0.92 [14]
8.6
13
85
76
·
·2HCl
2
NH
2
·3HCl
1.2 [63]
·2HCl
1
1

4
4
4
5
6
7
9
0
H, Cl, O
H, Cl, O
H, Cl, O
H, Cl, O
CN
CH
CN
CH
3.4
1.9
1.5
1.8
36
33
37
40
·
2HCl
2
NH
2
·HCl
·HCl
·
2HCl
·
2HCl
2
NH
2
·2HCl
5
5
5
9
H, Cl, O
H, Cl, S
CH
2
2
NH
2
2
·HCl
·HCl
0.43 [51]
0.33 [30]
22
10
CH
NH
·HCl
6
3
H, H, O
CH
2
NH
2
·HCl
6.7[18]
120
Scheme 6. Methods for the synthesis of chemical intermediates and alicyclic ring derivatives. Reagents, conditions, and yields: (a)
-boc-piperazine, NaHB(OAc) , DCE, rt, 1N NaOH, then CH OH/0.5 M HCl and Et O, 75%; (b) MsOH, THF, rt, then 10%
1
3
3
2
1
2

NaOH; CH
EtOH, rt, then 2N NaOH, 97%; (e) conc HCl, t-Bu
-one, THF, -78 °C, 46%; (h) LiBH , LiHBEt , THF, then 1N NaOH; EtOAc/CH
zaldehyde, K CO , DMA, 115 °C, 68%; (j) morpholine, NaHB(OAc) , CH Cl , rt, 60%; (k) LiBH
EtOAc/O.5 M HCl in CH OH, 46%; (l) succinic anhydride, AlCl , 1,1,2,2-tetrachloroethane, 50 °C, 3M HCl then 2N NaOH, 46%;
m) CH COCl, EtOH, reflux, 97%; (n) Boc O, NiCl ·6 H2O, NaBH , diethylenetriamine, CH OH, 0 °C, 60%; conc HCl, EtOAc, rt,
1%.
3
OH/0.5 M HCl, 30%; (c) LiBH
4
, LiHBEt
3
, THF, rt, then 2N NaOH; CH
, rt, 93%; (f) Ph P, toluene, reflux, 18%; (g) LDA, tetrahydro-4H-pyran-
OH/O.5 M HCl, 61%; (i) 3-chloro-4-fluoroben-
, LiHBEt , THF, then 1N NaOH;
3 2 4
OH/0.5 M HCl and Et O, 34%; (d) NaBH ,
4
NHSO
4
3
4
4
3
3
2
3
3
2
2
4
3
3
3
(
3
2
2
4
3
7
A series of imidazole derivatives (Table 4) were made as shown in Scheme 7. Reduction of aldehydes 1a and 1b gave the
corresponding alcohols 51 and 64. The alcohols were treated with concentrated HCl under phase transfer conditions to produce the
benzylic chlorides 52 and 65. Treatment of 64 with concentrated HCl gave 65 cleanly; however, 51 with the same reagent gave 52,
recovered starting material, and a small quantity of 70. Treatment of 65, 52 or 70 with imidazole in toluene gave 66, 67, and 71
respectively. Superhydride reduction of 66 and 67 generated analogs 68 and 69.
Scheme 7. Synthesis of diphenyl ether imidazole derivatives 66–69, and 71. Reagents, conditions, and yields: (a) NaBH
4
, EtOH, rt,
, LiBH
3
OH, 20–70%*; (e) imidazole, toluene, 90 °C, 49%. *See experimental section
3
6–93%*; (b) conc HCl, t-Bu NHSO , rt, 93–97%*; (c) imidazole, toluene, 83 °C, then EtOAc/HCl, 79–92%*; (d) LiHB(Et)
4
4
3
4
,
THF, 20 °C, then 1N NaOH; then 0.5 M HCl in CH
for individual reaction yields.
Table 4. T. b. rhod. activity and L6 cell cytotoxicity of diphenyl ether imidazole derivatives.
Compound
T. b. rhod.
IC50 (M) [SI]
13
L6 cytotoxicity
IC50 (M)
82
6
6
1
3

6
6
6
7
7
8
9
1
12
12.5
12.4
59
4.3
2.7 [22]
5.55
15
Variations of the benzylic amine were synthesized as shown in Scheme 8. Potency and cytotoxicity results are shown in
Table 5. DIBAL-H reduction of the nitrile 36 followed by reductive amination with dimethylamine hydrochloride gave 73. Nitrile
6 was converted to imidazoline 74 upon treatment with ethylenediamine. Nucleophilic aromatic substitution of 4-fluoro-3-chloro-
benzaldehyde with 4-hydroxybenzaldehyde gave 75. Reductive amination with morpholine produced 76. The imidazole derivative
7 was made from 3-chloro-4-fluorobenzaldehyde and 4-(1H-imidazol-1-yl)-phenol via nucleophilic substitution. Reductive ami-
3
7
nation with morpholine gave compound 78. During the reductive amination of 77, aldehyde reduction to 79 occurred to a small
extent. Alcohol 79 was not significantly active against T. b. rhodesiense.
Scheme 8. Synthetic route for diphenyl ether derivatives with variation of the benzylic amine fragment. Reagents, conditions, and
yields: (a) DIBAL-H, CH
2
Cl
2
, -45 °C, 89%; (b) NaHB(OAc)
3
, DCE, rt, then 1N NaOH, then CH
3
OH/0.5 M HCl and EtOAc, 42%;
o
(c) ethylenediamine, NaHS, DMA, 120 °C, then conc HCl, 88%; (d) 4-hydroxybenzaldehyde, K
2
CO
3
, DMA, 115 C, 86%; (e)
1
4

o
NaHB(OAc)
3
, DCE, rt, 1N NaOH, then CH
3
OH/0.5 M HCl and Et
2 2 3
O, 72%; (f) 4-(1H-imidazol-1-yl)phenol, K CO , DMA, 115 C,
6
5%; (g) NaHB(OAc)
3
, DCE, rt, then 1N NaOH, then CH
3
OH/0.5 M HCl and EtOAc, 26%.
Table 5. T. b. rhod. activity and L6 cell cytotoxicity of diphenyl ether derivatives with variation of the benzylic amine portion.
Compound
T. b. rhod.
L6 cytotoxicity
IC50 (M)
13.7
4.5
IC50 (M)
123
7
7
7
7
7
3
4
6
8
9
53
11
56
18
26
43
NDa
aNot determined
A number of bulky amines (Table 6) were isolated during the course of this study. Reaction of 9a with HCl in EtOAc
gave 9b (Scheme 1). Cyano amide 80 was made from 7 via ammonia attack on the succinoyl active ester (Scheme 9). Amino dia-
mide 81 was isolated as a byproduct from hydrogenation of cyano amide 80. Amine 82 formed over a long period of time at room
temperature from neat amino ester 3a. The dicarboxylic acid ammonium salt 83 was obtained by hydrolysis of 9a, which itself was
a byproduct of hydrogenation of 2 (Schemes 2 and 9). Tertiary amine 84 was formed from 72 via reductive amination.
1
5

Scheme 9. Synthesis of bulky byproduct amines 81–84. Reagents, conditions, and yields: (a) N-hydroxysuccimide/CH
3
CN, then
, EtOH, rt, 6%; (c) formed as a byproduct of 3a after 6 months,
, DCE, rt, then CH Cl /0.5 M HCl and Et O, 38%.
EDCI, then H
2
O, then conc NH
3
(aq), rt, 78%; (b) conc NH
3
(aq), H
2
9
%; (d) 2 N NaOH, EtOH, rt, then conc HCl, 79%; (e) NaHB(OAc)
3
2
2
2
Table 6. T. b. rhod. activity and L6 cell cytotoxicity of large diphenyl ether amine dimers.
Compound
T.b.rhod.
L6 cytotoxicity
IC50 (M)
IC50 (M)
27
9
8
8
8
8
b
1
2
3
4
12
14
2.4
83
2.6
>190
38
>180
13
The halogenated derivatives of Table 7 were generated from compounds 1b, 1d, 1e, 1f, and 1g by reductive amination with mor-
pholine followed by superhydride reduction (Scheme 10).
1
6

Scheme 10. Synthesis of halogenated cyanobiphenyl ether morpholines from cyanobiphenyl aldehdye intermediates followed by
reductive amination to give benzyl amine products. Reagents, conditions, and yields: (a) morpholine, NaHB(OAc) , DCE, rt, 51–
6%*; (b) LiHB(Et) , LiBH , THF, rt , 41–81%*. *See experimental section for individual reaction yields.
3
9
3
4
Table 7. T. b. rhod. activity and L6 cell cytotoxicity of halogenated morpholine derivatives and alcohol derivative 95.
Compound
T. b. rhod.
L6 cytotoxicity
IC50 (M) [SI]
IC50 (M)
3
8
7
6
0.53 [150]
0.71 [180]
81
124
8
9
8
0
1.24 [64]
0.88 [22]
80
19
9
9
2
5
0.16 [750]
11.0
120
NDa
aNot determined
In light of the improved activity seen with difluorination in the morpholinyl series, 95, the difluoro variant of the mono-chlorinated
hydroxpropyl 26, was prepared using the same sequence used to make 26 (Scheme 11).
Scheme 11. Three step synthesis of difluoro benzyl amine 95 from 1f. Reagents, conditions, and yields: (a) triethyl phosphonoace-
tate, NaH, THF, 60 °C, 37%; (b) Cu(OAc)
2 2 4 3
·H O, 1,2-BDP, PMHS, t-BuOH, toluene, rt, 59%; (c) LiBH , LiEt BH, THF, rt, then
1
0% NaOH, then CH OH/ 0.5 M HCl, 20%.
3
1
7

An additional group of difluoro compounds is reported in Table 8. These compounds were made according to Scheme 12.
The geminal dimethylamine 96 was prepared by reaction of 91 with an organocerium reagent followed by hydrolysis. The boronate
ester 97 was prepared from 1f and a known phenolic boronate by nucleophilic aromatic substitution. Reductive amination of 97 with
morpholine gave 98. This boronate ester was cleaved by brief reaction with NaIO
zoate and 1f gave 100 by nucleophilic aromatic substitution. Reductive amination of 100 with morpholine gave morpholine salt 101.
Reduction of 101 with LiAlH and subsequent basification generated alcohol 102.
4
to form boronic acid 99. Methyl 4-hydroxyben-
4
Scheme 12. Synthetic route for difluoro diphenyl ether morpholine derivatives 96, 99, and 102. Reagents, conditions, and yields: (a)
o
o
o
CeCl
3
, MeLi, THF, -78 C, then rt 3 days, then 0.5 M HCl/MeOH; 62 %;(b) 1f, K
, DCM, rt, 2 h, then 0.5 M HCl/MeOH, 72%; (d) NaIO , 1 M HCl (aq), THF, rt, 11 min, then 0.5 M HCl,
, DMF, 80 C, 11 h, then 93 C, 3.5 h, 80%; (f) morpholine, NaHB(OAc) , DCM, rt, 1 h, then 0.5 M HCl/MeOH,
, THF, reflux, 1.5 h, 100%.
2 3
CO , DMF, 80 C 11 h, then 93 C, 6 h; 70% (c)
morpholine, NaHB(OAc)
3
4
o
o
7
6
5%; (e) 1f, K
2
CO
3
3
8%; (g) LiAlH
4
Table 8. T. b. rhod. activity and L6 cell cytotoxicity of difluoro diphenyl ether morpholine derivatives 96, 99, and 102.
Compound
T. b. rhod.
L6 cytotoxicity
IC50 (M)
5.6
IC50 (M)
110
9
9
1
6
9
22
22
02
127
160
3
. Discussion
A set of alkyl side chain derivatives (Table 1) revealed activity for various benzylic ammonium salts. The three-carbon
alcoholic side chain in 6 gave maximum activity in this series. The corresponding ester 3b and carboxylic acid 4 were somewhat
less active. The dihydroimidazoline 8 was much less active than 4, a fact of some interest since amidines are a feature of pentami-
dine and have appeared in much published work as reviewed above. Analogues 20 and 23 with one carbon alcohol-terminated side
1
8

chains, retain some activity with the sulfide 23 somewhat more active than 20. The two-carbon alcohol-terminated 11 is less active
than either 20 or 23. The methyl ether 17, made from 11, shows somewhat improved activity and is less toxic than 11. Ether 17
shows better selectivity than 20 or 23. The benzyl ether, 18, is also more active than 11 but is more toxic. The most cytotoxic mate-
rial in this series was the cyanophosphonate 13. Chlorinated 26 gave improved activity compared to 6 albeit with increased tox-
icity. The bis-amide 27 was not active and indicated that a benzylic amine is needed on the right side of the molecule for activity.
A series of diamino compounds is shown in Table 2. Once again, the benzylic ammonium salts show better activity than
their nitrile precursors with one exception. The nitrile 34 with a 4-hydroxypiperidine terminus is unusual in showing more activity
than the free amine 35. Of the benzylic ammonium salts the best activity, 0.53M, occurs with the morpholine derivative 37 which
represents an improvement compared to the 0.96M activity of 6 in Table 1. However, 37 shows less selectivity than 6. The iso-
meric morpholine 39 and the non-chlorinated derivative 41 are also active but less so than 37. Interestingly, the cyano precursors to
these amines, 38 and 40, show better activity than is typical in this series with the exception of 34.
Structural modifications to the aliphatic ring of the molecule are shown in Table 3. The 4-tetrahydropyranilidiene group
5
5 gave improved activity but increased toxicity significantly compared to the acyclic analogue 6. The piperidine, pyrazine, and N-
methylpyrazine variations (42 through 50) did not give more active compounds and were more toxic than 37. The sulfur analogue
of 34 reported here (59) is more active but shows the greatest toxicity in the series. Lactone 63 showed modest activity against T.b.
rhodesiense.
The imidazole analogues of Table 4 did not show improvement in activity. The best of these, 69, with 2.7 M activity (SI
=
22) has a chloro substituent ortho to the aryl ether and is a benzylic amine. Table 5 shows compounds in which the benzyl amine
is replaced by an amidine or tertiary amines. An increase in size of the amine gives uniformly reduced activity. Table 6 shows a
collection of larger amines. Two of these, 81 and 83, are not significantly active, but 82 shows modest 2.4M activity.
Since the presence of chlorine gave improved activity in both the acyclic alcohol 26 and the morpholinyl derivative 37
compared to the halogen free parent compounds, and since saturated cyclic moieties seemed to improve activity, we prepared an-
other series of halogenated compounds. The activities of these compounds are compared to 37 in Table 7. When fluorine was sub-
stituted for chlorine (86) the selectivity increased. However, bromine substitution for chlorine (88) gave reduced selectivity.
Fluoro substitution on the opposite ring (90) was deleterious to selectivity. Difluoro substitution (92) gave significant improvement
in activity with less toxicity (SI = 750 against the L6 cell line).
Unfortunately, the activity of the difluoro variant 95 (11 M) was considerably reduced compared to that of our original
3
-hydroxypropyl lead 6 (0.49 M). This loss of activity in 95 is surprising if one presumes some binding interaction around the
fluorine atoms. Perhaps the better activity that the fluorine substituents impart to 92 might be due simply to decreased basicity at
the morpholine nitrogen.
1
9

We made difluoronated morpholino compounds with and without the benzyl amine (Table 8). Dimethylation at the ben-
zylic position drops activity significantly (96). Replacing the benzylic amine entirely with a boronic acid group (99) or with the
corresponding alcohol (102) dropped the activity even more.
The most active compounds were submitted to toxicity tests against four different human cells (Table 9). The best com-
pound shows an SI of 750 against rat myoblast L6 and an SI >1000 against these human cell lines. Our more active compounds
against T. b. rhodesiense are compared to other protozoans in Table 10 and to Toxoplasma gondii. All compounds in Table 10 are
less active against T. cruzi, L. donovani and P. falciparum than against T. b. rhodesiense. Against T. gondii, 92 also showed the
best activity but negligible activity against L. donovani amastigotes. This may indicate possible stage specificity which bears fur-
ther investigation.
Table 9. Effect of lead compounds tested for toxicity against four human cell lines and corresponding SI values.
T. b. rhod.
IC50 (M)
HFF IC50
(M) [SI]
U-2 OS IC50
(M) [SI]
HC-04 IC50 (M) HEK293 IC50
[SI]
(M) [SI]
Compound
6
0.96
0.49
0.53
1.1
NR [>420]
NR [>420]
>200 [410]
320 [600]
NR [>420]
2
3
6
7
>200 [410]
400 [750]
>200 [410]
300 [570]
150 [300]
3
9
400 [360]
NR [> 360]
NR [>290]
184.7 [440]
149.0 [450]
NR [>150]
>200 [1200]
NR [> 360]
NR [>290]
178.0 [424]
128.0 [390]
NR [>150]
>200 [1200]
4
5
5
6
9
1
5
9
8
2
1.4
NR [>290]
>200 [480]
184.8 [560]
NR [>150]
>200 [1200]
0.42
0.33
2.7
150 [360]
148 [55]
0.16
160 [1000]
NR= Not Reached at Concentrations up to 400µM
HFF: human foreskin fibroblasts
HC-04: human liver cell line
U-2 OS: human bone marrow cell line
HEK293 (Human Embryonic Kidney Cell Line)
Table 10. Activity of lead compounds compared with a panel of other parasitic protozoans.
T. b. rhod.
IC50 (M)
T. cruzi
IC50 (M)
L. don. ax am
IC50 (M)
P. falc NF54
IC50 (M)
L6
IC50 (M)
RH-dTom
IC50 (M)
Compound
6
0.96
0.49
0.53
71
40
72
184
201
151
11
7.6
16
170
49
NR
2
3
6
7
29.2
81
48.12
2
0

3
4
5
5
9
9
1
5
9
2
1.1
1.4
62
129
14
>247
>269
66
12
8.3
8.7
1.2
21
45
120
22
NR
NR
0.42
0.33
0.16
31.5
21.0
12.1
5.2
56
10
44
>246
120
RH-dTom: Toxoplasma gondii strain RH
4
. Conclusion
We have shown herein an extensive SAR on a series of diphenylethers active against T.b. rhodesiense with our best lead
compound being the difluorinated double salt 92. It shows an SI of 750 against rat myoblast L6 and > 1000 against four human
cell lines. The monochloro derivative 26 shows poorer selectivity (100) compared to our simple lead 6 (177) against the L6 line.
Both show comparable selectivity (~410) against these human cell lines. In contrast, against the L6 line, chlorine substituted 37
shows better selectivity (152) than the unsubstituted compound 41 (86). The greater selectivity of 37 vs. 41 is also seen against
human cell lines (~ 600 vs ~ 300 respectively). Selectivity data using the L6 cell line is available for the HAT drugs melarsoprol
(5800), pentamidine isethionate (15,000), and suramin (2,100). Our lead 92 falls short by this metric; however, the low toxicity
against human cells lines is promising and will guide ongoing identification of more effective antitrypanosomal diphenyl ethers.
5
. Experimental
5
.1. General.
1
13
Melting points are uncorrected. IR were obtained on a Perkin-Elmer Spectrum 100 FT-IR in ATR mode. H and C NMR spectra
were recorded on a Bruker AVIII-HD 400 MHz spectrometer. All chemical shifts are reported in parts per million and are relative
1 6
to TMS (0.0 ppm, 77.0 ppm) for proton and carbon spectra respectively in chloroform-d and to DMSO- d for proton (2.50 ppm)
and carbon NMR (39.52 ppm). GC-MS analysis was conducted on an Agilent 7890A-GC with 5975C VL MSD. Target compounds
show 95% or greater purity by combustion analyses or by NMR. Combustion analyses were done by M-H-W Laboratories. High res-
olution mass spectra were obtained from the Midwest Center for Mass Spectrometry. Preparative chromatography was done using the
3
6
flash method or by Biotage preparative chromatography. Unless otherwise noted, reagents were purchased from Acros Organics and
used as obtained.
5
.2. Protozoan panel
2
1

In vitro antiprotozoal activity was measured using theSTIB 900strainof T.bruceirhodesiense (trypomastigotes stage), the
MHOM-ET-67/L82 strain of L. donovani (axenic grown amastigotes), the Tulahuen C4 strain of T. cruzi (amastigotes stage), and
the NF54 strain of Plasmodium falciparum (IEF stage).
5
.2.1 Activity against Trypanosoma brucei rhodesiense STIB900. This stock was isolated in 1982 from a human patient in Tanzania
37
and after several mouse passages cloned and adapted to axenic culture conditions. Minimum Essential Medium (50 µl) supple-
mented with 25 mM HEPES, 1g/l additional glucose, 1% MEM non-essential amino acids (100x), 0.2 mM 2-mercaptoethanol,
1
mM Na-pyruvate and 15% heat inactivated horse serum was added to each well of a 96-well microtiter plate. Serial drug dilutions
of eleven 3-fold dilution steps covering a range from 100 to 0.002 μg/ml were prepared. Then 4x103 bloodstream forms of T. b.
rhodesiense STIB 900 in 50 µl was added to each well and the plate incubated at 37 °C under a 5% CO atmosphere for 70 h. 10 µl
Alamar Blue (resazurin, 12.5 mg in 100 ml double-distilled water) was then added to each well and incubation continued for a fur-
2
38
ther 2–4 h. Then the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation,
Sunnyvale, CA, USA) using an excitation wave length of 536 nm and an emission wave length of 588 nm. Data were analyzed with
the graphic programme Softmax Pro (Molecular Devices Cooperation, Sunnyvale, CA, USA), which calculated IC50 values by lin-
39
ear regression and 4-parameter logistic regression from the sigmoidal dose inhibition curves. Melarsoprol (Arsobal Sanofi-
Aventis, received from WHO) is used as control.
5
.2.2 Activity against T. cruzi. Rat skeletal myoblasts (L-6 cells) were seeded in 96-well microtiter plates at 2000 cells/well in
00 μL RPMI 1640 medium with 10% FBS and 2 mM L-glutamine. After 24 h the medium was removed and replaced by 100 μl
1
4
0
per well containing 5000 trypomastigote forms of T. cruzi Tulahuen strain C2C4 containing the β-galactosidase (Lac Z) gene. Af-
ter 48 h the medium was removed from the wells and replaced by 100 μl fresh medium with or without a serial drug dilution of
eleven 3-fold dilution steps covering a range from 100 to 0.002 μg/ml. After 96 h of incubation, the plates were inspected under an
inverted microscope to assure growth of the controls and sterility. Then the substrate CPRG/Nonidet (50 μl) was added to all wells.
A color reaction developed within 2–6 h and could be read photometrically at 540 nm. Data were analyzed with the graphic pro-
gramme Softmax Pro (Molecular Devices), which calculated IC50 values by linear regression (Huber 1993) and 4-parameter logistic
regression from the sigmoidal dose inhibition curves. Benznidazole is used as control (IC50 0.5±0.2 g/ml).
5
.2.3 Activity against L. donovani axenic amastigotes. Amastigotes of L. donovani strain MHOM/ET/67/L82 are grown in axenic
culture at 37 °C in SM medium (Cunnigham et al. 1977) at pH 5.4 supplemented with 10% heat-inactivated fetal bovine serum un-
5
der an atmosphere of 5% CO
2
in air. 100 μl of culture medium with 10 amastigotes from axenic culture with or without a serial
2
2

drug dilution are seeded in 96-well microtiter plates. Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100
to 0.002 μg/ml are prepared. After 70 h of incubation the plates are inspected under an inverted microscope to assure growth of the
controls and sterile conditions. 10 μl of Alamar Blue (12.5 mg resazurin dissolved in 100 ml distilled water) are then added to each
well and the plates incubated for another 2 h. Then the plates are read with a Spectramax Gemini XS microplate fluorometer (Mo-
lecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wave length of 536 nm and an emission wave length of
5
88 nm. Data are analyzed using the software Softmax Pro (Molecular Devices Cooperation, Sunnyvale, CA, USA). Decrease of
fluorescence (= inhibition) is expressed as percentage of the fluorescence of control cultures and plotted against the drug concentra-
tions. From the sigmoidal inhibition curves the IC50 values are calculated.
5
.2.4 Activity against P. falciparum. In vitro activity against erythrocytic stages of P. falciparum was determined using a 3H-hypo-
4
1,42
43
xanthine incorporation assay , using the drug sensitive NF54 strain and the standard drugs chloroquine (Sigma C6628) and ar-
tesunate (Sigma A3731). Compounds were dissolved in DMSO at 10 mg/ml and further diluted in medium before added to parasite
3
cultures incubated in RPMI 1640 medium without hypoxanthine, supplemented with HEPES (5.94 g/l), NaHCO (2.1 g/l), neomy-
cin (100 U/ml), AlbumaxR (5 g/l) and washed human red cells A+ at 2.5% haematocrit (0.3% parasitaemia). Serial drug dilutions
of eleven 3-fold dilution steps covering a range from 100 to 0.002 μg/ml were prepared. The 96-well plates were incubated in a
2 2 2
humidified atmosphere at 37 °C; 4% CO , 3% O , 93% N . After 48 h 50 μl of 3H-hypoxanthine (=0.5 μCi) was added to each well
of the plate. The plates were incubated for a further 24 h under the same conditions. The plates were then harvested with a Be-
taplate™ cell harvester (Wallac, Zurich, Switzerland), and the red blood cells transferred onto a glass fibre filter then washed with
distilled water. The dried filters were inserted into a plastic foil with 10 ml of scintillation fluid, and counted in a Betaplate™ liquid
scintillation counter (Wallac, Zurich, Switzerland). IC50 values were calculated from sigmoidal inhibition curves by linear regres-
sion (Huber 1993) using Microsoft Excel. Chloroquine and artemisinin are used as control.
5
.3. Cytotoxicity Assay
4
4
5
.3.1 In vitro cytotoxicity with L-6 cells. Assays were performed in 96-well microtiter plates, each well containing 100 l of RPMI
1
640 medium supplemented with 1% L-glutamine (200mM) and 10% fetal bovine serum, and 4000 L-6 cells (a primary cell line
45,46
derived from rat skeletal myoblasts).
Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002
μg/ml were prepared. After 70 hours of incubation the plates were inspected under an inverted microscope to assure growth of the
controls and sterile conditions. 10l of Alamar Blue was then added to each well and the plates incubated for another 2 hours. Then
the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA)
2
3

using an excitation wave length of 536 nm and an emission wave length of 588 nm. The IC50 values were calculated by linear re-
gression (Huber 1993) and 4-parameter logistic regression from the sigmoidal dose inhibition curves using SoftmaxPro software
(Molecular Devices Cooperation, Sunnyvale, CA, USA). Podophyllotoxin (Sigma P4405) is used as control.
5
.3.2 Cytotoxicity IC50 Assay on Human Cell Lines: Compounds were screened for toxicity against host cell lines grown in 96 well
plates, and assessed for viability with Alamar blue as previously reported (Pubmed ID 29081387). Cultured host cells were human
foreskin fibroblasts (HFF), U-2 OS osteosarcoma cells, and HC-04 hepatocytes.
5
.4. Toxoplasma IC50
Toxoplasma gondii strain RH IC50's were assessed with a fluorescent plate reader following infection of foreskin fibroblast cells in
9
6 well plates, as previously described (Pubmed ID 29081387).
5
.5. General Procedures for nitrile reductions
5
.5.1. Method A. Nitrile ammonium salt reduction. To the nitrile salt (0.676 mmol) in anhydrous THF (2 mL) under nitrogen with
stirring was added a 4 M THF solution of LiBH
4 3
(0.21 mL, 0.831 mmol), and then after 5 min a 1.7 M THF solution of LiHBEt
(0.89 mL, 1.51 mmol). After 2 days at rt 2 M NaOH (6 mL) was added and the solution was then stirred for 45 minutes. The solu-
tion was then extracted with EtOAc (3 x 25 mL). The organic phase was washed with brine, dried over MgSO
the solvent removed by rotary evaporation.
4
, filtered, and then
5
.5.2. Method B. Hydroxy nitrile reduction. To a solution of the hydroxy nitrile(1.43 mmol) in anhydrous ether (2.5 mL, Fischer)
and anhydrous THF (0.5 mL, Aldrich) under a nitrogen atmosphere was added 4 M LiBH4 in THF (0.726 mL, 2.90 mmol) fol-
lowed by 1 M lithium LiHBEt in THF (1.72 mL, 1.72 mmol, Alfa Aesar). The resulting mixture was stirred at rt for 24 hours be-
fore a second addition of 1 M LiHBEt in THF (1.15 mL, 1.15 mmol) was added. The reaction was allowed to stir for another 24
hours at rt. Then the mixture was diluted with ether (15 mL), NaOH (3M, 2.5 mL) was added and the mixture then was stirred for
0 minutes. More NaOH (2.5 mL) was added and the two layers were separated. The organic layer was then washed with distilled
water (2 x 2.5 mL) followed by brine (2 x 2.5 mL). The organic layer was dried with MgSO and filtered. The filtrate was acidified
3
3
3
4
with 12 M HCl. This solution was concentrated by rotary evaporation until a white precipitate formed which was collected by filtra-
tion.
5
.5.3. Method C. Ester nitrile reduction. To the ester nitrile (1.00 mmol) under nitrogen was added anhydrous THF (4 mL), a 4 M
solution of LiBH in THF (0.55 mL, 2.20 mmol) and a 1.7 M solution of LiHBEt in THF (0.3 mL, 0.500 mmol). After 4.5 h of
stirring at rt additional LiHBEt (1.2 mL, 2 mmol) was added. After an additional 19.5 h of stirring 2 M NaOH (6 mL) was added.
4
3
3
2
4

After 1 h of stirring the mixture was diluted with water (14 mL), extracted with DCM (3 x 20 mL), and then dried over Mg
The solution was filtered and then the solvent removed by rotary evaporation to give the crude amine.
2 4
SO .
5
.5.4. Method D. for hydroxy nitrile ammonium salt substrates Method B was followed with one additional mole equivalent of
LiBH
.5.5. Method E. for ester nitrile ammonium salt substrates Method C was followed with one additional mole equivalent of LiBH
General Procedures for Reductive Aminations
4
.
5
4
.
5
.5.6. Method F. for neutral amine substrates. The direct amination procedure (Method l) of Abdel-Magid et al. was followed using
4
7
either DCM or DCE as solvent.
5
.5.7. Method G. for hydrochloride substrates. Method F was followed with one equivalent of triethylamine added before addition
of the reductant.
5
5
.6 Procedures and Spectral Data
.6.1. 4-(4-Formylphenoxy)benzonitrile 1a.
-
1
1
A literature procedure was followed (Li et al.) IR (neat, cm ): 3100, 2854, 2747, 1693, 1584, 1493, 1109, 831, 854. H NMR (400
) δ 7.28 (AA’, 2H), 7.30 (AA’, 2H), 7.93 (BB , 2H), 8.00 (BB’, 2H), 9.99 (s, 1H); 13C NMR (100 MHz, CHCl
’
-d
)
MHz, DMSO-d
6
3
1
r
δ 107.5, 118.3, 119.3, 119.5, 132.0, 132.6, 134.3, 159.4, 160.4, 190.5; GS-MS t , = 10.50 m/z = 121.0.
5
.6.2. 4-(2-Chloro-4-formylphenoxy)benzonitrile 1b.
4
8
A variation of a literature procedure was employed.
A mixture of 4-hydroxybenzonitrile (5.00 g, 0.042 mol), 3-chloro-4-fluoro-
CO (5.805 g, 0.042 mol) were heated to 110 °C in DMA (30 mL) for 10 h.
benzaldehyde (6.433 g, 0.0406 mol), and anhydrous K
2
3
The reaction mixture was poured into 10% NaOH (6 mL) containing 25 g of ice. The residual material was transferred with water
giving a solution volume of 75 mL. A solid formed while the mixture was stirred. Suction filtration produced a dull yellow crude
solid (9.369 g, 91%). mp 83.0–85.0 °C (lit 85.1–87.3 °C). The spectra matched the literature and the compound was used without
further purification.
5
.6.3. 4-(3-Chloro-4-formylphenoxy)benzonitrile 1c.
4
9
The compound is reported in a patent. A mixture of 4-hydroxybenzonitrile (5.00 g, 0.042 mol), 2-chloro-4-fluorobenzaldehyde
6.347 g, 0.040 mol), and anhydrous K CO (5.805 g, 0.042 mol) was heated in DMA (30 mL) in an oil bath (133–155 °C) for 2.5
(
2
3
h. When cool, the reaction was transferred to a 400 mL beaker with 2.5 M NaOH (70 mL). To this mixture was added water (50
mL) and ice (50 g). After stirring the light tan solid was collected by suction filtration. After drying in a vacuum oven a brown-
orange solid (10.1g, 98%) was obtained. Purification of a 2 g sample by Kugelrohr distillation (190-243 °C, 0.05 mm Hg) gave
mostly white solid with some yellow contamination (1.82 g, 89%) and a black residue (0.174 g). Recrystallization from DCM or
2
5

-
1
EtOH did not significantly improve the purity. mp (from EtOH) 105.5–107.1 °C. IR (neat, cm ) 3098, 3064, 3047, 2893, 2233,
1
1
7
681, 1607, 1587, 1502, 1481, 1225, 921, 850, 821, 790. H NMR (400 MHz, CHCl3-
d
1
) δ 7.02 (ddd, J = 8.6, 3.4, 0.75 Hz, 1H),
.03 (d, J = 2.3 Hz, 1H), 7.15 (AA’, 2H), 7.72 (BB’, 2H), 7.97 (d, J = 8.6 Hz, 1H), 10.4 (d, J = 0.77 Hz, 1H); 13C NMR (100 MHz,
) δ 108.2, 117.6, 118.1, 120.1, 128.7, 131.4, 134.2, 134.7, 158.7, 160.8, 188.2 The product was used without
(400 MHz, CHCl3-
d
1
further purification.
5
.6.4. 4-(2-Fluoro-4-formylphenoxy)benzonitrile 1d.
2 3
A mixture of 3,4-difluorobenzaldehyde (5.68 g, 0.040 mol), 4-hydroxybenzonitrile (5.00 g, 0.042 mol), and K CO (5.805
g, 0.042 mol) in DMA (31 mL) under nitrogen was heated with stirring at 115 °C for 5 h. The mixture was poured onto ice (25 g)
and NaOH (10%, 6 mL). With stirring a solid appeared which was isolated and washed with water (7.245 g). The crude material
-
1
was recrystallized from ethanol/water (6.38 g, 66%). mp 91–92 °C. IR (neat, cm ) 3069, 2842, 2738, 2228, 1696, 1614, 1595, 1497,
274, 1226, 1168, 969, 949, 863, 836, 781, 747. 1H NMR (400 MHz, CHCl
BB’, 2H), 7.72 (ddd, J = 8.2, 1.9, 1.0 Hz, 1H), 7.76 (dd, J = 10.2, 1.9 Hz, 1H), 9.97 (d, J = 1.9 Hz, 1H). 13C NMR (100 MHz, CHCl
-
3
1
3 1
-d ): δ 7.03 (AA’, 2H), 7.26 (t, J = 7.8 Hz, 1H), 7.68
(
d
1
): δ 107.7, 117.4, 117.6, 118.0, 118.3, 122.2, 127.3, 127.4. 134.3, 134.4, 147.4, 147.5, 153.1, 155.6, 159.7, 189.4. Anal. Calcd for
C14H FNO2: C, 69.71; H, 3.34; N, 5.81. Found: C, 69.90; H, 3.65; N 5.65.
.6.5. 4-(2-Bromo-4-formylphenoxy)benzonitrile 1e.
Under nitrogen with stirring 3-bromo-4-fluorobenzaldehyde (8.12 g, 0.040 mol), 4-hydroxybenzonitrile (5.00 g 0.042 mol),
CO (5.805 g, 0.042 mol), in DMA (31 mL) were heated for 10 h. The mixture was poured into ice (25 g) and 10% NaOH (6
8
5
and K
2
3
mL). EtOAc was added. The organic phase was extracted with water (3 x 30 mL) and then dried over MgSO4. After rotary evapo-
ration a yellow oil (11.4 g) was obtained. The compound was recrystallized from EtOH/water to give the product (9.78 g, 81%). mp
9.5–80.0 °C. IR (neat, cm ) 3065, 3095, 2855, 2232, 1683, 1587, 1563, 1498, 1265, 150, 1165, 1038, 906, 896, 862, 811. 1H
-
1
7
NMR (400 MHz, CHCl
3
-d
.9 Hz, 1H), 9.96 (s, 1H). 13C NMR (100 MHz, CHCl
-d ): δ 107.8, 116.1, 118.3, 118.7, 121.0, 130.4, 134.2, 134.4, 135.6, 156.9,
3 1
1
): δ 7.07 (AA’, 2H), 7.14 (d, J = 8.3 Hz, 1H), 7.69 (BB’, 2H), 7.86 (dd, J = 8.3, 1.9 Hz, 1H), 8.21 (d, J =
1
1
5
3
8
59.3, 189.2. Anal. Calcd for C14H BrNO2: C, 55.66; H, 2.67; Br, 26.45; N 4.64. Found: C, 55.52; H, 2.80; Br, 26.55; N, 4.42.
.6.6. 4-(2,6-Difluoro-4-formylphenoxy)benzonitrile 1f.
2 3
,4,5-trifluorobenzaldehye (5.395 g, 0.0337 mol, Oakwood Chemical), 4-cyanophenol (4.009 g, 0.0337 mol) and anhydrous K CO
(4.644 g, 0.0337 mol) were dissolved in DMF (26 mL) and then heated to 80 °C (oil bath temperature) with stirring under nitrogen.
After 1.5 hours of stirring, the reaction mixture was cooled and then poured onto 16 g of ice containing NaOH (2.5 M, 1 mL). The
precipitate was collected by suction filtration and washed with water. The solid dissolved in EtOAc (100 mL), extracted with water
(3 x 60 mL), and then the organic phase was dried with MgSO
4
. Filtration and removal of the solvent by rotary evaporation gave a
white slightly tacky product (5.344 g, 61%). Longer reaction times or higher temperatures reduced the yield. An analytical sample
2
6

was prepared by Kugelrohr distillation (175–185°C at 0.02 mm Hg) followed by recrystallization with EtOAc/Hexane. mp 129.8–
-
1
1
1
30.2 °C. IR (cm ) 3059, 2888, 2228, 1694, 1594, 1504, 1446, 1240, 1203, 984, 878, 863, 839. H NMR (400 MHz, DMSO-d
6
): δ
):
7
.05 (AA’, 2H), 7.61 (BB’ from fluorine coupling, 2H), 7.66 (BB’, 2H), 9.95 (t, J = 1.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d
6
7 2 2
δ 107.5, 113.7, 115.9, 116.3, 118.3, 133.9, 135.3, 157.4, 159.8, 188.3. Anal. Calcd for C14H F NO : C, 64.87; H, 2.72; N, 5.40.
Found: C, 64.53; H, 2.99; N, 5.09.
5
.6.7. 4-(2-Chloro-4-formylphenoxy)-3-fluorobenzonitrile 1g.
-Chloro-4-fluorobenzaldehyde (2.0 g, 0.012 mol, Oakwood Chemical), 4-cyanophenol (1.73 g, 0.0126 mol, Oakwood
Chemical) and anhydrous K CO (1.65 g, 0.012 mol) were mixed in DMA (9.0 mL) and heated to 100 °C (oil bath temperature).
3
2
3
After 12 hours of stirring, the reaction mixture was cooled producing a dark red-brown viscous oil. NaOH (2 M, 4 mL) and 25 g ice
were added at rt. The reaction mixture was refrigerated for over 24 hours. A dark red-brown solid precipitated. This was then filtered
and purified via Kugelrohr distillation (73.1–76.9 °C at 0.04 mm Hg) followed by recrystallization from diethyl ether/hexane to give
-
1
a white powder (1.004 g, 29%). mp 76.6–78.2 °C. IR (cm ) 3090, 3059, 2852, 2745, 2237, 1690, 1508, 1236, 1205, 963, 844, 736,
1
6
8
1
46. H NMR (400 MHz, DMSO-d
6
): δ 7.07 (m, 2H), 7.48 (dt, J = 1.6, 8.8 Hz, 1H), 7.55 (dd, J = 1.6, 9.6 Hz, 1H), 7.79 (dd, J = 2,
): δ 108.9, 117.0, 119.3, 121.2, 126.3, 129.59,
29.63, 129.77, 132.3, 133.8, 146.97, 151.7, 154.2, 155.8, 189.3. Anal. Calcd for C14H7ClFNO2: C, 61.00; H, 2.56; N, 5.08. Found:
.4 Hz, 1H), 7.92 (d, J = 9.3 Hz, 1H), 9.96 (s, 1H). 13C NMR (100 MHz, DMSO-d
6
C, 61.18; H, 2.75; N, 4.92.
.6.8. (E)-Ethyl 3-[4-(4-cyanophenoxy)phenyl]propenoate 2.
5
To a 250 mL 3-neck round bottom flask, fitted with a condenser, mechanical stirrer, a Newman funnel, and a nitrogen inlet, in an
ice bath, was added sodium hydride (1.276 g, 53.17 mmol) and then hexane (20 mL). After agitation the hexane was removed.
THF (23 mL) was then added, followed by dropwise addition of triethylphosphonoacetate (distilled, 4.82 g, 21.5 mmol). Then 4-
(
4-formylphenoxy)benzonitrile (4.80 g, 21.5 mmol) was added in one portion. The flask was stirred for 30 min and then warmed at
0 °C for 10 minutes with an oil bath. The reaction mixture was poured over ice and then the tan solid isolated by vacuum filtration.
Kugelrohr distillation gave colorless solid (4.282 g, 68%). In another run the crude brown product (6.91 g) was recrystallized from
6
-
1
ethanol (95%, 20 mL) to give a beige solid (4.568 g, 72%). mp 87.0–89.6 °C. IR (neat, cm ) 3064, 2983, 2228, 1707, 1629, 1590,
1
1
6
6
508, 1499, 1474, 1245, 1171, 1110, 1007, 884, 876, 855, 834, 823. H NMR (400 MHz, DMSO-d
6
) δ 1.27 (t, 3H), 4.20 (q, 2H),
) δ 14.2,
= 9.00 m/z = 293.1. HRMS-
.62 (d, 1H), 7.16 (AA , 2H), 7.18 (AA , 2H) 7.68 (d, 1H), 7.83 (BB , 2H), 7.88 (BB , 2H). 13C NMR (100 MHz, DMSO-d
’
’
’
’
6
0.0, 105.8, 117.9, 118.6, 118.8, 120.1, 130.6, 130.8, 134.8, 143.4, 156.4, 160.3, 166.2. GC-MS t
r
+
+
TOF MS EI (m/z): [M ] calcd for C18
H15NO
3
293.1052; found 293.1045. Anal. Calcd for C18
3
H15NO : C, 73.71; H, 5.15; N, 4.78.
Found: C, 73.80, H, 5.13; N, 4.64.
5
.6.9. 4-(4-(2-Carboxyethyl)phenoxy)phenyl)methanaminium chloride 4.
2
7

To ethyl 3-[4-(4-aminomethylphenoxy)phenyl]propanoate (0.252 g, 0.842 mmol) in 95% ethanol (3.5 mL) was added 2 M NaOH
(1.1 mL). After 4 h of stirring at rt water (3.5 mL) and then 12 M HCl was added until the pH = 1. The solid formed was collected
o
by suction filtration and then recrystallized from methanol/acetonitrile (0.123 g, 47%). mp 238–240 C dec with gas evolution. IR
-
1
1
(
neat, cm ) 2972, 2876, 2583, 1701, 1602, 1579, 1505, 1445, 1259, 1223, 1200, 1173, 940, 878, 826. H NMR (400 MHz, DMSO-
’
d
7
1
2
5
6
) δ 2.54 (t, J = 7.8 Hz, 2H), 2.82 (t, J = 7.6 Hz, 2H), 3.98 (q, J = 5.8 Hz, 2H), 6.93 (AA , 2H), 7.01 (AA’, 2H) 7.26 (BB’, 2H),
.50 (BB , 2H), 8.41 (bs, 3H), 12.12 (bs, 1H). 13C (100 MHz, DMSO-d
’
) δ 29.6, 35.3, 41.6, 118.2, 118.8, 128.7, 129.8, 130.9,
6
+
+
23Na 294.1106; found
36.4, 154.4, 157.2, 173.7. HRMS-TOF MS EI in sodium acetate (m/z): [M + Na] calcd for C16
H18ClNO
3
94.1114.
.6.10. Ethyl 3-[4-(4-methylphenoxy)phenyl]propanoate 5b. Ethyl 3-[4-(4-aminomethylphenoxy)phenyl]propanoate 3a.
To 5% Pd on carbon with 55% water (42 mg) was added conjugated ester 2 (0.412 g, 1.40 mmol) in 95% ethanol (35 mL). Parr
hydrogenation (61 psi) at rt for 14.5 h was followed by filtration through celite and solvent removal giving a mixture of products
(
0.408 g). The mixture was subjected to flash chromatography with 30% EtOAc/hexane eluent giving the hydrogenolysis product
b (0.053 g, 13%). Elution with 100% EtOAc (100 mL) gave no significant amount of material. Subsequent elution with methanol
75 mL) gave the primary amine 3a (0.313 g, 75%).
5
(
-
1
1
Hydrogenolysis product 5b: oil. IR (neat, cm ) 3036, 2924, 2855, 1736, 1604, 1500, 1238, 1168, 874, 846, 817. H NMR (400
MHz, DMSO-d
6
) δ 1.16 (t, J = 7.1 Hz, 3H), 2.28 (s, 3H), 2.59 (t, J = 7.6 Hz, 2H), 2.82 (t, J = 7.6 Hz, 2H), 4.035 (q, J = 7.1 Hz,
’
’
2
1
2
3
3
H), 6.37 (AA , 4H), 7.18 (BB , 4H), 13C NMR (100 MHz, DMSO-d
6
) δ 14.1, 20.2, 29.5, 35.2, 59.8, 118.1, 118.6, 129.7, 130.3,
+
+
32.4, 135.2, 154.4, 155.4, 172.1. GC-MS t
84.1412; found 284.1408.
r
20 3
= 5.80 min, m/z = 284.1. HRMS-TOF MS EI (m/z): [M ] calcd for C18H O
-
1
1
a: oil. IR (neat, cm ) 3350, 2982, 1729, 1601, 1499, 1232, 1168, 873, 827. H NMR (400 MHz, DMSO-d
6
) δ 1.15 (t, J = 7.1 Hz,
H), 2.60 (t, J = 7.6 Hz, 2H), 2.59 (t, 2H), 2.82 (t, J = 7.6 Hz, 2H), 3.69 (s, 2H), 4.04 (q, J = 7.1 Hz, 2H), 6.89 (AA’, 2H), 6.92
AA’, 2H) 7.22 (BB’, 2H), 7.31 (BB , 2H); 13C (100 MHz, DMSO-d
’
) δ 14.1, 29.5, 35.2, 45.0, 59.8, 118.2, 118.3, 128.5, 129.7,
(
6
+
+
1
2
5
35.3, 139.4, 155.0, 155.3, 172.1. GC-MS t
99.1521; found. 299.1522.
r 3
= 5.00 min, m/z = 299.1. HRMS-TOF MS EI (m/z): [M ] calcd for C18H21NO
.6.11. Ethyl 3-[4-(4-aminomethylphenoxy)phenyl] propanoate tosylate 3b.
To 3a (0.095 g, 0.32 mmol in ether was added tosic acid (0.060 g, 0.32 mmol) in ether. The white solid formed was collected by
-
1
vacuum filtration and dried (0.116 g, 77%). mp 163.5–165.5 °C. IR (neat, cm ) 3066, 2976, 1727, 1716, 1604, 1505, 1483, 1245,
1
1
164, 1123, 1106, 1034, 1010, 878, 831, 813, 683. H NMR (400 MHz, DMSO-d
6
) δ 1.16 (t, J = 7.2 Hz, 3H), 2.29 (s, 3H), 2.62 (t,
J = 7.6 Hz, 2H), 4.01 (q, J = 6.0 Hz, 2H) 4.05 (q, J = 7.2 Hz, 2H), 6.93, (AA’, 2H), 7.02 (BB’, 2H), 7.12 (AA’, 2H), 7.27 (BB’,
H), 7.46 (AA’, 2H) 7.48 (BB’, 2H), 8.08 (s, 3H). 13C NMR (100 MHz, DMSO-d
2
6
) δ 14.6, 21.2, 30.0, 35.6, 42.2, 60.3, 118.7,
2
8

1
19.3, 126.0, 128.5, 129.1, 130.4, 131.3, 136.5, 138.0, 146.2, 155.0, 157.8, 172.6. Anal. Calcd for C25
N, 2.97; S, 6.80. Found: C, 63.76; H, 6.12; N, 2.74; S, 6.63.
.6.12. Ethyl 3-(4-(4-cyanophenoxy)phenyl)propanoate 5a and Diethyl 3,3'-((((azanediylbis(methylene))bis(4,1-phe-
6
H29NO S: C, 63.68; H, 6.20;
5
nylene))bis(oxy))bis(4,1-phenylene)) dipropionate 9a.
Ester 2 (0.779 g, 2.66 mmol) in EtOH (25 mL) was hydrogenated (Parr) in the presence of 5% platinum on carbon catalyst (0.048
g) at 60 psi for 21 h. Filtration through celite and rotary evaporation of solvent gave a mixture of products (0.671 g). The crude
products were separated by flash chromatography using 10% EtOAc/hexane and then 20% EtOAc/hexane giving the hydrogenoly-
sis product 5b (0.028 g, 4%) and then cyanoester 5a (0.393 g, 50%). Subsequent elution with 100% EtOAc give the secondary
amine 9a (0.184 g, 12%).
-1
5
d
2
1
a: oil. IR (neat, cm ) 3063, 2984, 2937, 2227, 1730, 1597, 1497, 1245, 1201, 1168, 874, 852, 837. 1H NMR (400 MHz, DMSO-
’
6
) δ 1.16 (t, J = 7.1 Hz, 3H), 2.63 (t, J = 7.6 Hz, 2H), 2.87 (t, J = 7.6 Hz, 2H), 4.05 (q, J = 7.1 Hz, 2H), 7.04 (AA , 2H), 7.07 (AA’,
H), 7.32 (BB , 2H), 7.82 (BB , 2H). 13C (100 MHz, DMSO-d
’
’
) δ 14.1, 29.6, 35.0, 59.8, 104.8, 117.7, 118.7, 120.2, 130.2, 134.6,
6
+
+
37.5, 152.6, 161.3, 172.1. GC-MS t
r
3
= 5.80 min, m/z = 295.1. HRMS-TOF MS EI (m/z): [M ] calcd for C18H17NO 295.1208;
found 295.1235.
-
1
1
9
a: oil. IR (neat, cm ) 2982, 1731, 1602, 1499, 1232, 1167, 1099, 1043, 1015, 874, 827. H NMR (400 MHz, DMSO-d
6
) δ 1.14 (t,
’
J = 7.1 Hz, 3H), 2.58 (t, J = 7.6 Hz, 2H), 2.81 (t, J = 7.5 Hz, 2H), 3.63 (s, 2H), 4.03 (q, J = 7.2 Hz, 2H), 6.88 (AA , 2H), 6.91 (AA’,
’
2
1
5
H), 7.21 (BB , 4H), 7.32 (BB’, 2H). 13C NMR (100 MHz, DMSO-d
6
) δ 14.2, 29.6, 35.3, 51.7, 60.0, 118.4, 118.5, 129.6, 129.9,
582.2856; found 582.2833.
+
+
35.6, 135.9, 155.3, 155.6, 172.4. HRMS-TOF MS EI (m/z): [M ] calcd for C36
H40NO
6
.6.13. Bis(4-(4-(3-ethoxy-3-oxopropyl)phenoxy)benzyl)ammonium chloride 9b.
To 7 (0.184 g, 0.316 mmol) was added 1 M HCl/EtOAc (0.6 mL, 0.6 mmol). Vacuum filtration gave the salt 9b (0.135 g, 69%).
mp 211–212 °C. IR (neat, cm ) 3063, 3032, 2993, 2909, 2788, 2734, 2601, 1728, 1606, 1503, 1238, 1167, 874, 857, 829, 812. 1H
-
1
NMR (400 MHz, DMSO-d
6
) δ 1.15 (t, J = 7.1 Hz, 6H), 2.61 (t, J = 7.5 Hz, 4H), 2.84 (t, J = 7.6 Hz, 4H), 4.04 (q, J = 7.1 Hz, 4H),
4
1
6
5
.11 (s, 4H), 6.94 (AA’, 2H), 7.00 (AA’, 2H), 7.26 (BB’, 2H), 7.52 (BB’, 2H), 9.47 (s, 2H). 13C NMR (100 MHz, DMSO-d
6
) δ
4.1, 19.6, 35.1, 49.2, 59.8, 118.1, 119.0, 126.5, 132.0, 136.2, 154.3, 157.6, 172.2. Anal. Calcd for C36
.52; Cl, 5.73; N, 2.27. Found: C, 70.00; H, 6.32; Cl, 5.55; N, 2.28.
H40ClNO : C, 69.92; H,
6
.6.14. (4-(4-(3-Hydroxypropyl)phenoxy)phenyl)methanaminium chloride 6.
By Method C 5a (0.295 g, 1.00 mmol) was converted to 6 (0.333 g). The impure oil was dissolved in EtOAc (5 mL) and 1 M HCl
-
1
in EtOAc (2 mL) was added dropwise. Filtration gave 6 (0.272 g, 93 %). mp 177–179 °C dec. IR (neat, cm ) 3250, 2921, 2758,
1
2
d
7
706, 2623, 1641, 1615, 1415, 1599, 1501, 1455, 1388, 1248, 1200, 1170, 1056, 1032, 877, 849, 837. H NMR (400 MHz, DMSO-
6
) δ 1.71 (m, 2H), 2.60 (t, J = 7.7 Hz), 3.42 (t, J = 6.5 Hz, 2H), 4.00 (m, 2H), 6.93 (AA’, 2H), 7.02 (AA’, 2H), 7.24 (BB’, 2H),
.48 (BB’, 2H), 8.30 (s, 3H). 13C NMR (100 MHz, DMSO-d
) δ 30.9, 34.3, 41.6, 60.0, 118.1, 118.9, 128.6, 129.8, 130.9, 137.7,
6
2
9