Efficient one-pot synthesis of biologically active polysubstituted aromatic amines

Article abstract of DOI:10.1016/j.bmc.2007.08.048

An efficient and modular one-pot synthesis of polysubstituted aromatic amines by a mild reductive amination procedure is described and the biological potential of these nitrogen-centered compounds is demonstrated by growth inhibition of murine connective tissue cells and microscopy-based morphological studies.

Full text of DOI:10.1016/j.bmc.2007.08.048

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7311–7317
Efficient one-pot synthesis of biologically active
polysubstituted aromatic amines
a,
a
a
a
b
*
Dirk Menche, Fatih Arikan, Jun Li, Sven Rudolph and Florenz Sasse
a
Helmholtz-Zentrum f u¨ r Infektionsforschung GmbH, Medizinische Chemie, Inhoffenstr 7, 38124 Braunschweig, Germany
b
Helmholtz-Zentrum f u¨ r Infektionsforschung GmbH, Chemische Biologie, Inhoffenstr 7, 38124 Braunschweig, Germany
Received 14 June 2007; revised 13 August 2007; accepted 17 August 2007
Available online 29 August 2007
Abstract—An efficient and modular one-pot synthesis of polysubstituted aromatic amines by a mild reductive amination procedure
is described and the biological potential of these nitrogen-centered compounds is demonstrated by growth inhibition of murine con-
nective tissue cells and microscopy-based morphological studies.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
R
N
Polysubstituted aromatic amines present prevalent con-
stitutional chemotypes and underlying structural motifs
in registered drugs and bioactive synthons, such as
benzodiazepines (1), phenethylamines (2), and arylpi-
peridines (3, all shown in Fig. 1a), or tertiary amines
N
N
N R
N
R
R
1
2
3
1
,2
of type 4 in general (Fig. 1b), which renders the devel-
opment of efficient procedures for their synthesis an
objective of high priority from the perspective of medic-
inal and bioorganic chemistry. The reductive amination
of carbonyls is widely recognized as one of the most
important and convergent methods for the preparation
of substituted amines and numerous applications have
R3
H
R3
O
6
N
R2
NH2
+
O
R2
R1
4
R1
5
7
3
,4
H
been reported.
Surprisingly, applications of such
methods to a modular and in particular one-pot access
to tertiary aromatic amines are much less common
and established, due to increased steric hindrance in
the second amination step and/or lack of synthetic
Figure 1. Polysubstituted aromatic amines as bioactive synthons:
established motifs (a) and amines of general type 4, elaborated in this
communication (b).
2
,5
selectivity.
assembly of 4 from three building blocks, a starting
amine 5 and two different carbonyl components (6 and
Herein, we report an efficient one-pot procedure for the
modular synthesis of sterically hindered aromatic
amines of type 4 and demonstrate the biological poten-
tial of these compounds in whole cell-based assays and
microscopy-based morphological studies. The mild and
operationally simple method relies on the convergent
7
) and proceeds by selective reductive amination.
2. Results and discussion
Based on an innovative biosynthetic concept, we have
recently developed a direct reductive amination proce-
dure, which uses the Hantzsch ester (HEH, 8) for
transfer hydrogenation and proceeds in the presence of
molecular sieves and catalytic amounts of thiourea for
Keywords: Aromatic amines; Reductive amination; Diversity-oriented
synthesis; One-pot synthesis; Cytotoxicity.
*
Corresponding author. Tel.: +49 531 61819407; fax: +49 531
1819499; e-mail: dirk.menche@helmholtz-hzi.de
6
0
968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.048

7312
D. Menche et al. / Bioorg. Med. Chem. 15 (2007) 7311–7317
H
R3
H
NH2
R2
R2
O
R2
N
N
7
HEH (
thiourea]
Å MS
8)
R1
R1
5
9
R1
4
[
(
yield)
5
H
R3
+
NO
2
O
6
H H
R3
N
N
EtO C
CO Et
2
2
R2
4a
(86%)
4b
(83%)
N
MeO
MeO
N
H
O N
2
R1
HEH (
8
)
4
Scheme 1. Modular one-pot synthesis of tertiary aromatic amines.
N
N
N
OMe
4
c
4d
6
,7
MeO
imine activation. In detail, we have successfully devel-
oped protocols for the reductive alkylation of primary
(
65%)
(82%)
6
a
and secondary aromatic amines to obtain secondary
6
c
and tertiary amines, respectively. The efficiencies of
these procedures prompted us to investigate whether this
method can be further expanded also to a modular
one-pot synthesis of tertiary amines, by stepwise alkyl-
ation of a primary amine, yet without isolation of inter-
N
4
e
4f
(76%)
(
64%)
6
c
mediates, in contrast to our previous protocols. As
shown in Scheme 1, this approach would start by react-
ing amine 5 with a first carbonyl component (7) giving
initially intermediate 9, which would then be treated
in situ with a second carbonyl (6) to give the desired
tertiary product 4 in a direct fashion. Notably, the syn-
thetic design is highly convergent and modular and
should thus be amenable to combinatorial library
synthesis.
N
N
N
OMe
OMe
4g
87%)
4h
MeO
MeO
MeO
MeO
(
(91%)
NO
2
N
2
.1. One-pot synthesis of polysubstituted amines
4i
4j
(87%)
(
73%)
In order to evaluate the viability of such a process, we
first analyzed the selectivity of the two amination steps
involved. This revealed the first reductive alkylation to
be much faster, presumably due to steric reasons, en-
abling a highly selective monoalkylation. Upon extended
reaction times, however, also the second reductive ami-
nation may proceed under similar reaction conditions
N
n-octyl
N
4k
82%)
4l
(90%)
MeO
MeO
(
(
i.e., temperature and solvent) to give the desired prod-
Figure 2. Tertiary amines prepared with the indicated yields by one-
pot direct reductive amination (yields correspond to attachment in the
uct 4. After optimizing reaction conditions, an effective
protocol was established which involved use of 3 equiv
Hantzsch ester, 0.2 equiv of thiourea, 1.05 equiv of the
first, and 1.5 equiv of the second carbonyl component
2
order of R and then R according to general structure 4).
3
8
and proceeds at 60 °C in toluene.
2.2. Biological activity
As shown in Figure 2, this protocol was applied to the
9
This direct and modular access to substituted tertiary
aromatic amines allowed assessment of their bioactivities,
synthesis of variously substituted tertiary amines. The
desired products were obtained in good (4a, 4b, 4d, 4g,
6
a
relative to representative secondary congeners. In view
1
0
4
h, 4j–l) to useful (4c, 4e, 4f, 4i, 4m) yields without
of the cytotoxic activity of simple aromatic amines, it
appeared rewarding to likewise test these more substi-
tuted nitrogen-centered structures in whole cell-based
assays. Consequently, the inhibitory effect on the growth
of the murine connective tissue cell line L-929 was ana-
lyzed. As shown in Table 1, various representatives
showed potent cytotoxicity with IC50 values in the low
micromolar range. Interestingly, the trisubstituted struc-
further adaption of conditions to specific substrates.
Aliphatic and aromatic aldehydes are accepted as sub-
strates (e.g., 4b, 4j, 4k) and variations in the electronic
and steric properties are tolerated (e.g., 4c, 4g, 4m),
which demonstrates the usefulness of this method to
the modular synthesis of diverse as well as sterically
demanding tertiary amines.

D. Menche et al. / Bioorg. Med. Chem. 15 (2007) 7311–7317
7313
Table 1. Inhibitory effects of tertiary aromatic amines (4) in compar-
ison to secondary amines (9) on the growth of mammalian murine
connective tissue cell line
moieties appeared to increase biological activity (e.g., 4a
vs 4i, 4f vs 4 e) which might be related to variations in
membrane permeability. Among the aromatics studied,
electron donating groups (e.g., OH: 9g or OMe: 4j)
appeared to be beneficial for activity, while electron-with-
drawing groups lead to a slight decrease (e.g., 9k, 9j) in
this assay. These data demonstrate that the biological
function of such polyfunctional amines is quite versatile.
No activity below 40 lg/mL was observed for various
secondary and tertiary amines, e.g., 9a, 9d, 4b, and 4h,
which further corroborates the modular potential of these
polysubstitued amines as flexible bioactive synthons.
a
a
Compound Growth inhibition Compound Growth inhibition
L-929 IC50 L-929 IC50
,
,
lg/mL (lM)
lg/mL (lM)
4
4
4
4
4
4
4
4
4
4
4
4
9
a
b
c
d
e
f
10 (37)
>40
>40
9b
9c
9d
9e
9f
30 (141)
30 (132)
>40
11 (38)
13 (59)
5 (20)
15 (50)
>40
12 (48)
25 (103)
9 (37)
9g
9h
9i
g
h
i
>40
>40
To get further hints about the mode of action, the
amines 4f and 9g as exemplary representatives of this
compound class were checked for effects on the mor-
25 (106)
>40
9j
22 (98)
35 (190)
20 (73)
40 (184)
30 (129)
j
9k
9l
k
l
20 (66)
12 (40)
>40
9m
9n
phology of PtK potoroo cells. The cultured cells were
2
a
stained by labeling the nuclei and a marker protein of
the endoplasmatic reticulum, and inspected by fluores-
cence microscopy. As shown in Figure 3, treated cells
showed striking alterations of the inner membrane
structure of the cytoplasm. They displayed big vacu-
oles near the nucleus or a cushion-like pattern, the
first being more pronounced in the 9g treated cells.
Compound 4f seems to be less effective in vacuoliza-
tion, rather leading to cell enlargement. The effect of
H
N
H
N
MeO
MeO
MeO
MeO
9
a
9b
H
N
H
N
9g is very similar to the corallidicyals, sesquiterpene
hydroquinones from the Caribbean Sponge Aka coral-
9
c
9d
1
2
liphagum, which suggests that a similar mode of ac-
tion might be involved. Possibly, redox processes
might be associated. The notion that the tertiary
amine 4f is less effective in vacuolization as compared
to 9g, might be related to the fact that the central
nitrogen is sterically much more hindered and thus
less accessible to oxidation–reduction processes. Signif-
icantly, tertiary amines are more potent in the cell cul-
ture assays in comparison to primary or secondary
amines, which might suggest that also other effects
may be involved for these more encumbered nitro-
gen-containing structures.
H
N
H
N
n-octyl
OMe
MeO
MeO
9
e
9f
NO2
NO2
H
N
H
N
HO
9
g
9h
H
N
H
N
HO C
2
9
i
9j
3. Conclusions
O
OMe
H
N
H
N
In conclusion, we have reported polysubstituted aro-
matic amines as promising and modular bioactive
synthons. The synthesis of these compounds was
enabled in a versatile, one-pot fashion by reductive
amination, which enables a rapid entry into diverse
tertiary aromatic amines. These compounds exhibited
pronounced inhibitory effects on the growth of the
murine connective tissue cell line L-929 in the low
micromolar range. Two exemplary representatives
were shown to cause alterations of the inner mem-
brane structure of the cytoplasm by microscopy-based
studies. Tertiary amines were more active than the
corresponding secondary or primary congeners in
the cell culture assays, while being less effective in
vacuolization of the membrane, which warrants fur-
ther research into this class of compounds a poten-
tially rewarding task. It is expected, that our
procedure will find further applications in medicinal
chemistry and chemical biology.
O N
2
N
HO
9
k
9l
S
O
H
N
H
N
MeO
MeO
9
m
9n
a
The synthesis of the secondary amines (9) by application of our
6
methodology has been previously reported in preliminary form.
Full details are given in the Supplementary material.
a,b
tures were 2–10 times more potent than the respective
secondary amines (as shown e.g., by comparing 4a/4l with
9
b, 4g with 9a, or 4i with 9a) and also more potent as
1
1
compared to simple primary aromatic amines. In com-
parison to aliphatic residues, additional aromatic

7314
D. Menche et al. / Bioorg. Med. Chem. 15 (2007) 7311–7317
(TLC aluminum sheets silica gel Si 60 F254 (Merck), sol-
vent: mixtures of ethylacetate/petroleum ether, detec-
tion: UV absorption at 254 nm, dark blue spots on
staining with cerium(IV)sulfate-phosphomolybdic acid
in sulfuric acid followed by charring. Reagents were
obtained from commercial suppliers and used without
further purification. Dry toluene was obtained from
Fluka.
4.1. General procedure for the synthesis of tertiary
aromatic amines
A solution of the amine (5, 1.00 mmol) and the first car-
bonyl component (7, 1.05 mmol) in toluene (5 mL) was
treated with the Hantzsch ester (8, 950 mg, 3 mmol),
˚
thiourea (15.2 mg, 0.200 mmol), and MS 5 A (2.0 g)
and the mixture was stirred under nitrogen at 60 °C
for 24 h, after which the second carbonyl component
(
6, 1.50 mmol) was added and the reaction mixture
was stirred at 60 °C until complete conversion (24–
2 h). After filtration over Celite, the solvent was evap-
7
orated and the residue purified by flash chromatography
on silica gel using mixtures of petroleum ether and ethyl
acetate as eluents to give the product tertiary amine (4)
in analytically pure form.
4
.1.1. Synthesis of N-Benzyl-N-isobutyl-4-methoxy-ani-
line (4a). According to the general procedure, para-anisidine
was treated with benzaldehyde and iso-butyraldehyde to
give after purification by flash chromatography (petroleum
ether/ethyl acetate, 15:1) amine 4a as a yellow precipitate
1
(
(
262 mg, 860 lmol, 86%). Mp 35–37 °C; H NMR
300 MHz, CDCl ) d = 0.96 (d, J = 6.7 Hz, 6H), 2.11 (tsept,
3
J = 7.1, J = 6.7 Hz, 1H), 3.14 (d, J = 7.1 Hz, 2H), 3.74 (s,
3H), 4.52 (s, 2H), 6.67 (d, J = 9.0 Hz, 2H), 6.79
1
3
(
d, J = 9.0 Hz, 2H), 7.14–7.37 (m, 5H); C NMR
(
75 MHz, CDCl ) d = 20.59, 27.4, 55.8, 56.4, 60.4, 114.7,
3
126.6, 126.9, 128.4, 139.3, 143.7, 151.4; HRMS (ESI): m/z:
calcd for C H NO [M+H] : 270.1858. Found: 270.1859.
+
18
24
4
.1.2. Synthesis of N-isobutyl-4-methoxy-N-(4-nitroben-
zyl)aniline (4b). According to the general procedure,
para-anisidine was treated with 4-nitro-benzaldehyde
and iso-butyraldehyde to give after purification by flash
chromatography (petroleum ether/ethyl acetate, 15:1)
1
amine 4b as a yellow oil (260 mg, 830 lmol, 83%). H
NMR (300 MHz, CDCl ) d = 0.95 (d, J = 6.6 Hz, 6H),
3
2
2
.11 (tsept, J = 7.1,J = 6.6 Hz, 1H), 3.12 (d, J = 7.1 Hz,
H), 3.72 (s, 3H), 6.63 (d, J = 9.1 Hz, 2H), 6.77
(
(
d, J = 9.1 Hz, 2H), 7.37 (d, J = 9.1 Hz, 2H), 8.13
d, J = 9.1 Hz, 2H);
1
3
C NMR (75 MHz, CDCl3)
Figure 3. Changes in the morphology of cultivated PtK
2
potoroo cells
d = 20.6, 27.4, 55.7, 56.4, 61.2, 114.8, 115.4, 123.7,
27.7, 142.9, 147.0, 147.5, 152.1; HRMS (ESI): m/z:
calcd for C H N O [M] : 314.1630. Found: 314.1634.
upon treatment with the aromatic amines 9 g (top), and 4f (middle) in
comparison to control cells (bottom). Cells were incubated with 50 lg/mL
for 18 h and stained for nuclei (blue) and ER structure (green).
1
+
1
8
22
2
3
4
.1.3. Synthesis of N-(2-Methoxybenzyl)-4-methoxy-N-
4
. Experimental
(4-nitrobenzyl)-aniline (4c). According to the general
procedure, para-anisidine was treated with 2-methoxy-
benzaldehyde and 4-nitro-benzaldehyde to give after
purification by flash chromatography (petroleum ether/
NMR spectra were recorded in CDCl on a Bruker AM-
3
00 spectrometer. EI and DCI mass spectra (reactant
3
gas ammonia) were obtained on a Finnigan MAT 95
spectrometer, high resolution data were acquired using
peak matching (M/DM = 10,000). Analytical TLC
ethyl acetate, 15:1) amine 4c as a yellow oil (275 mg,
650 lmol, 65%).
d = 3.71 (s, 3H), 3.81 (s, 3H), 4.56 (s, 2H), 4.64
1
H
NMR (300 MHz, CDCl3):

D. Menche et al. / Bioorg. Med. Chem. 15 (2007) 7311–7317
7315
(
2
7
s, 2H), 6.61 (d, J = 8.9 Hz, 2H), 6.75 (d, J = 8.9 Hz,
4.1.8. Synthesis of N-2-Methoxybenzyl)-N-benzyl-4-meth-
oxy-aniline (4h). According to the general procedure, para-
anisidine was treated with 2-methoxybenzaldehyde and
benzaldehyde to give after purification by flash chromatog-
raphy (petroleum ether/ethyl acetate, 15:1) amine 4h as a
H), 6.9 (d, J = 7.9 Hz, 2H), 7.14 (d, J = 7.2 Hz, 2H),
.41 (d, J = 8.9Hz, 2H), 8.15 (d, J = 8.9 Hz, 2H); HRMS
+
(
Found: 424.1509.
ESI): m/z: calcd for C H N O [M+H] : 424.1509.
6
2
2
22
3
slightly yellow solid (303 mg, 910 lmol, 91%). Mp 53–
1
4
.1.4. Synthesis of N,N-dibenzyl-4-methylaniline (4d). In
55 °C. H NMR (300 MHz, CDCl ) d = 3.72 (s, 3H), 3.83
3
analogy to the general procedure, 4-methylaniline was
directly treated with an excess of benzaldehyde
(s, 3H), 4.60 (s, 2H), 4.62 (s, 2H), 6.65 (d, J = 9.1 Hz, 2H)
6.76 (d, J = 9.1 Hz, 2H), 6.90 (t, J = 8.1 Hz, 2H), 7.15–
1
3
(
2.55 mmol) to give after purification by flash chroma-
tography (petroleum ether/ethyl acetate, 15:1) amine
d (235 mg, 820 lmol, 82%) as a white solid. Mp 63–64 °C;
7.37 (m, 7H); C NMR (75 MHz, CDCl ) d = 50.3, 55.1,
3
55.8, 110.0, 113.6, 114.8, 120.4, 126.7, 127.4, 127.7, 128.5,
139.4, 143.8, 151.4, 157.3; HRMS (ESI): m/z: calcd for
4
1
+
C H NO [M+H] : 334.1807. Found: 334.1807.
H NMR (300 MHz, CDCl ): d = 2.22 (s, 3H), 4.61
3
22 24
2
(
2
s, 4H), 6.66 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 8.7 Hz,
C
1
3
H), 7.23 (m, 6H), 7.31 (m, 4H);
NMR:
4.1.9. Synthesis of N,N-diisobutyl-4-methoxy-aniline (4i).
In analogy to the general procedure, para-anisidine was
directly treated with an excess of iso-butyraldehyde
(2.55 mmol) to give after purification by flash chroma-
tography (petroleum ether/ethyl acetate, 20:1) amine 4i
d = (75 MHz, CDCl ): 20.2, 54.5 (C-2), 112.9 (C-2),
3
1
26.1, 126.8 (C-6), 128.6 (C-4), 129.8 (C-2), 139.0
C-2), 147.2; HRMS (ESI): m/z: calcd for C H N
(
2
1
21
+
M] : 287.1674. Found: 287.1674.
[
1
as a colorless oil (171 mg, 730 lmol, 73%). H NMR
4.1.5. Synthesis of N,N-diisobutyl-4-methylanilin (4e). In
analogy to the general procedure, 4-methylaniline was
directly treated with an excess of iso-butyraldehyde
(300 MHz, CDCl ): d = 0.91 (d, J = 6.6 Hz, 12H), 2.00
3
(tsept, J = 7.2 Hz, J = 6.8 Hz, 2H), 3.04 (d, J = 7.2 Hz,
4H), 3.76 (s, 3H), 6.67 (m, 2H), 6.82 (m, 2H);
1
3
C
(
tography (petroleum ether/ethyl acetate, 15:1) amine
2.55 mmol) to give after purification by flash chroma-
NMR d = (75 MHz, CDCl ): 20.5, 26.5, 55.8, 61.4,
3
114.7, 115.0, 143.7, 151.1; HRMS (ESI): m/z: calcd for
C H NO [M+H] : 236.2014. Found: 236.2013.
+
4
e as a yellow solid (140 mg, 640 lmol, 64%). Mp
1
5
26
1
5
J = 6.6 Hz, 12H), 2.05 (tsept, J = 6.9 Hz, J = 6.6 Hz,
8–60 °C; H NMR (300 MHz, CDCl ): d = 0.88 (d,
3
4.1.10. Synthesis of N-benzyl-4-methoxy-N-(4-nitroben-
zyl)aniline (4j). According to the general procedure,
para-anisidine was treated with 4-nitro-benzaldehyde
and benzaldehyde to give after purification by flash
chromatography (petroleum ether/ethyl acetate, 15:1)
2
H), 2.23 (s, 3H), 3.09 (d, J = 7.2 Hz, 4H), 6.58
C
1
3
(
d, J = 8.8 Hz, 2H), 7.00 (d, J = 8.3 Hz, 2H);
NMR d (75 MHz, CDCl ): 20.17, 20.47, 26.43, 60.09,
3
112.90, 124.37, 129.59, 146.38; HRMS (ESI): m/z: calcd
for C H N [M+H] : 220.2065. Found: 220.2065.
+
amine 4j as a red solid (263 mg, 870 lmol, 87%). Mp
1
5
26
1
9
3–95 °C; H NMR (300 MHz, CDCl ) d = 3.74 (s,
3
4.1.6. Synthesis of N-Benzyl-N-isobutyl-4-methyl-aniline
3H), 4.56 (s, 2H), 4.60 (s, 2H), 6.71 (d, J = 9.1 Hz,
2H), 6.79 (d, J = 9.1 Hz, 2H), 7.23–7.37 (m, 5H), 7.42
(d, J = 9.1 Hz, 2H), 8.16 (d, J = 8.7 Hz, 2H);
(4f). According to the general procedure, 4-methylaniline
was treated with benzaldehyde and iso-butyraldehyde to
1
3
C
give after purification by flash chromatography (petroleum
ether/ethyl acetate, 20:1) amine 4f as a colorless oil
NMR (75 MHz, CDCl ) d = 54.9, 55.7, 56.1, 114.9,
3
115.4, 123.8, 127.2, 127.7, 128.6, 138.3, 143.2, 147.1,
152.6; HRMS (ESI): m/z: calcd for C H NO
1
(
192 mg, 760 lmol, 76%). H NMR (300 MHz, CDCl )
3
21 22
+
[M+H] : 304.1701. Found: 304.1701.
d = 0.96 (d, J = 6.6 Hz, 6H), 2.14 (tsept, J = 7.1, 6.6 Hz,
1H), 2.23 (s, 3H), 3.20 (d, J=7.1 Hz, 2H), 4.57 (s, 2H), 6.62
(
d, J = 8.6 Hz, 2H), 6.99 (d, J = 8.6 Hz, 2H), 7.18 - 7.32
4.1.11. Synthesis of N-isobutyl-4-methoxy-N-nonylaniline
(4k). According to the general procedure, para-anisidine
was treated with nonanal and iso-butyraldehyde to give
after purification by flash chromatography (petroleum
ether/ethyl acetate, 15:1) amine 4k as a slightly yellow
13
(m, 19H); C NMR (75 MHz, CDCl ) d = 20.1, 129.6,
3
1
[
39.2, 146.8; HRMS (ESI): m/z: calcd for C H N
18
24
+
M+H] : 254.1909. Found: 254.1908.
1
4
.1.7. Synthesis of N-(2-Methoxybenzyl)-N-isobutyl-4-
oil (250 mg, 820 lmol, 82%). H NMR (300 MHz,
methoxy-aniline (4g). According to the general procedure,
para-anisidine was treated with 2-methoxybenzaldehyde
and iso-butyraldehyde to give after purification by flash
chromatography (petroleum ether/ethyl acetate, 15:1)
CDCl ): d = 0.89 (m, 3H), 0.92 (d, J = 6.6 Hz, 6H),
3
1.28 (br s, 12H), 1.53 (m, 2H), 1.97 (tsept, J = 7.4 Hz,
J = 6.6 Hz, 1H), 2.96 (d, J = 7.2 Hz, 2H), 3.21
(t, J = 7.5 Hz, 2H), 3.75 (s, 3H), 6.64 (d, J = 9.2 Hz,
1
3
amine 4g as a slightly yellow oil (260 mg, 870 lmol,
2H), 6.81 (d, J = 9.2 Hz, 2H); C NMR (75 MHz,
CDCl ): d = 14.1, 20.5 (C-2), 22.7, 26.8, 27.1; 27.3;
1
H
87%).
NMR (300 MHz, CDCl3) d = 0.96 (d,
3
J = 6.6 Hz, 6H), 2.11 (tsept, J = 7.1, 6.6 Hz, 1H), 3.17 (d,
J = 7.1 Hz, 2H), 3.73 (s, 3H), 3.87 (s, 3H), 4.51 (s, 2H),
29.3, 29.6, 29.7, 31.9, 53.0, 55.9, 60.2, 114.5 (C-2),
114.8 (C-2), 143.7, 151.0; HRMS (ESI): m/z: calcd for
+
C H NO [M+H] : 306.2797. Found: 306.2791.
6.60 (d, J = 9.1 Hz, 2H), 6.77 (d, J = 9.1 Hz, 2H), 6.83 (t,
J = 7.3 Hz, 1H) 6.88 (d, J = 8.1 Hz, 1H), 7.02 (d,
J = 7.1 Hz, 1H), 7.19 (t, J = 8.3 Hz, 1H); C NMR
2
0
36
13
4.1.12. Synthesis of N,N-dibenzyl-4-methoxy-aniline (4l).
In analogy to the general procedure, para-anisidine was
directly treated with an excess of benzaldehyde
(2.55 mmol) to give after purification by flash chroma-
tography (petroleum ether/ethyl acetate, 15:1) amine 4l
(
75 MHz, CDCl ) d = 20.6, 27.6, 50.8, 55.2, 55.8, 60.3,
3
109.9, 113.5, 114.8, 120.3, 126.7, 127.4, 127.5, 143.7,
1
[
50.8, 157.3; HRMS (ESI): m/z: calcd for C H NO
2
19
26
+
M+H] : 300.1964. Found: 300.1962.

7316
D. Menche et al. / Bioorg. Med. Chem. 15 (2007) 7311–7317
as a colorless solid (273 mg, 900 lmol, 90%). Mp 85–
H., M u¨ ller, G. (Eds.), Chemogenomics in Drug Discov-
ery—A Medicinal Chemistry Perspective. In Methods and
Principles in Medicinal Chemistry; Mannhold, R., Kub-
inyi, H., Folkers, G., Eds.; Wiley-VCH: Weinheim, Vol.
1
8
4
7 °C; H NMR (300 MHz, CDCl ): d = 3.72 (s, 3H),
3
.55 (s, 4H), 6.69 (d, J = 9.0 Hz, 2H), 6.76
C NMR
1
3
(
d, J = 9.2 Hz, 2H), 7.28 (m, 10H);
2
. Recent examples of bioactive tertiary amines, which are
2, 2004.
(
75 MHz, CDCl ): d = 55.2 (C-2), 55.8, 114.6 (C-2),
3
2
1
1
14.8 (C-2), 126.9 (C-2), 127.0 (C-4), 128.6 (C-4),
39.1 (C-2), 143.9, 151.9; HRMS (ESI): m/z: calcd for
structurally closely related to type 4, are described in:
(
a) Dinsmore, C. J.; Williams, T. M.; O’Neill, T. J.; Liu,
+
C H NO [M] : 303.1623. Found: 303.1626.
21
2
1
D.; Rands, E.; Culberson, J. C.; Lobell, R. B.; Koblan,
K. S.; Kohl, N. E.; Gibbs, J. B.; Oliff, A. I.; Graham, S.
L.; Hartman, G. D. Bioorg. Med. Chem. Lett. 1999, 9,
3301; (b) Ding, C. Z.; Hunt, J. T.; Ricca, C.; Manne, V.
Bioorg. Med. Chem. Lett. 2000, 10, 273; (c) Masubuchi,
K.; Taniguchi, M.; Umeda, I.; Hattori, K.; Suda, H.;
Kohchi, Y.; Isshiki, Y.; Sakai, T.; Kohchi, M.; Shirai,
M.; Okabe, H.; Sudoh, M.; Yamazaki, T.; Shimma, N.
Bioorg. Med. Chem. Lett. 2000, 10, 1459; (d) Massa, M.
A.; Spangler, D. P.; Durley, R. C.; Hickory, B. S.;
Connolly, D. T.; Witherbee, B. J.; Smith, M. E.;
Sikorski, J. A. Bioorg. Med. Chem. Lett. 2001, 11,
1625; (e) Perez, M.; Maraval, C.; Dumond, S.; Lamothe,
M.; Schambel, P.; Eti e´ vant, C.; Hill, B. Bioorg. Med.
Chem. Lett. 2003, 11, 1455.
4.2. Biological activity: cell culture and growth inhibition
assay
L-929 mouse cell line was from the German collection of
Microorganisms and Cell Cultures (DSMZ) and culti-
vated in DME medium (GIBCO BRL) plus 10% new-
born calf serum at 37 °C and 10% CO in a moist
2
atmosphere. Growth inhibition was measured on
microtiterplates. Aliquots of 120 lL of the suspended
À1
cells (50,000 mL ) were given to 60 lL of a serial dilu-
tion of the inhibitor. After 5 days, metabolic activity per
1
3
well was determined using the MTT assay.
3
4
5
. For leading references, see: (a) Martens, J. In Methods of
Organic Chemistry (Houben-Weyl); Helmchen, G., Ed.;
Thieme: New York, 1995; Vol. E21d, p 4199; (b)
Kobayashi, S.; Ishitani, H. Chem. Rev. 1999, 99,
4
.3. Biological activity: cell staining
PtK cells (ATCC CCL-56) grown on glass coverslips
2
1
069; (c) Overman, L. E.; Baxter, E. W.; Reitz, A. B.
were fixed with cold (À20 °C) MeOH/acetone (1:1) for
Organic Reactions; Wiley: New York, 2002; Vol. 59,
p. 1–53.
1
0 min, incubated with a primary antibody against
GRP-94 (1:1000; Affinity Bioreagents) and then with a
secondary Alexa Fluor 488 goat anti-rat IgG antibody
(1 lg/mL; Molecular Probes), and mounted in ProLong
Antifade Gold (Molecular Probes), which included
. For more recent developments, i.e., asymmetric vari-
ants, see Refs. 7c,d,f as well as: (a) Tararov, V. I.;
B o¨ rner, A. Synlett 2005, 203; (b) Nugent, T. C.;
Ghosh, A. K.; Wakchaure, V. N.; Mohanty, R. R.
Adv. Synth. Catal. 2006, 348, 1289; (c) Menche, D.;
Arikan, F.; Li, J.; Rudolph, S. Org. Lett. 2007, 9,
DAPI to stain the nuclei.
2
67.
. (a) Notably, all tertiary aromatic amines described in
Ref. 2 have been obtained in a non one-pot fashion and
the yields where reported for this step-wise process were
only moderate; Nonaromatic tertiary amines are more
readily available by reductive amination, see for exam-
ple: (b) Mizuta, T.; Sakaguchi, S.; Ishii, Y. J. Org.
Chem. 2005, 70, 2195.
Acknowledgments
We thank the Fonds der Chemischen Industrie (‘Liebig-
Stipendium’ to D.M., fellowship to F.A.) and the Deut-
sche Forschungsgemeinschaft (Grant: ME 2765/2-1, fel-
lowships to J.L. and S.R.) for generous funding.
Furthermore, this work was supported by the HZI.
Technical support by Tatjana Arnold, Stefanie B o¨ hm,
Wiebke Zander, and Bettina Hinkelmann (all HZI) is
gratefully acknowledged.
6
. (a) Menche, D.; Hassfeld, J.; Li, J.; Menche, G.; Ritter, A.;
Rudolph, S. Org. Lett. 2006, 8, 741; (b) Menche, D.;
Arikan, F. Synlett 2006, 6, 841; (c) Menche, D.; B o¨ hm, S.;
Li, J.; Rudolph, S.; Zander, W. Tetrahedron Lett. 2007, 48,
3
65.
7
. Hantzsch-ester mediated reductive aminations in the
presence of lewis or Brønsted acids for the synthesis of
secondary amines in a non-direct (requiring intermedi-
ate formation of the imines, Refs. a–d) or a direct
fashion (e,f) have been described by: (a) Steevens, J. B.;
Pandit, U. K. Tetrahedron 1983, 39, 1395; (b) Fujii,
M.; Aida, T.; Yoshihara, M.; Ohno, A. Bull. Chem.
Soc. Jpn. 1989, 62, 3845; (c) Rueping, M.; Sugiono, E.;
Azap, C.; Theissmann, T.; Bolte, M. Org. Lett. 2005, 7,
Supplementary data
Experimental details and spectroscopic data for second-
ary amines (9). Supplementary data associated with this
article can be found, in the online version, at
doi:10.1016/j.bmc.2007.08.048.
3
781; (d) Hoffmann, S.; Seayad, A. M.; List, B. Angew.
Chem., Int. Ed. 2005, 44, 7424; (e) Itoh, T.; Nagata,
K.; Miyazaki, M.; Ishikawa, H.; Kurihara, A.; Ohsawa,
A. Tetrahedron 2004, 60, 6649; (f) Storer, R. I.;
Carrera, D. E.; Ni, Y.; MacMillan, D. W. C. J. Am.
Chem. Soc. 2006, 128, 84.
References and notes
1
. For leading references, see: (a) Evans, B. E.; Rittle, K. E.;
Bock, M. G.; DiPrado, R. M.; Freidinger, R. M.; Whitter,
W. L.; Lundell, G. F.; Veber, D. F.; Anderson, P. S.;
Chang, R. S. L.; Lotti, V. J.; Cerino, D. J.; Chen, T. B.;
Kling, P. J.; Kunkel, K. A.; Springer, J. P.; Hirshfield, J.
J. Med. Chem. 1988, 31, 2235; (b) Ajay, W.; Walters, P.;
Murcko, M. A. J. Med. Chem. 1998, 41, 3314; (c) Kubinyi,
8. For good conversion also in the second step, extended
reaction times are beneficial (48 h as compared to 24 h in
the first alkylation).
9. Examples were selected to allow for a direct comparison to
6
c
a non one-pot protocol previously reported, demon-

D. Menche et al. / Bioorg. Med. Chem. 15 (2007) 7311–7317
7317
strating the yields were superior when following the
procedure reported herein. In detail, all tertiary amines
except for compound 4c, had been previously prepared,
Jaeschke, H.; Neumann, H.-G. Chem. Biol. Interact.
1995, 98, 85.
11. Data for representative primary aromatic amines in the
same assay were: para-anisidine: 12 lg/mL (98 lM), para-
toluidine: >40 lg/mL.
12. Grube, A.; Assmann, M.; Lichte, E.; Sasse, F.; Pawlik, J.
R.; Kock, M. J. Nat. Prod. 2007, 70, 504.
13. Mosman, T. J. Immunol. Methods 1983, 65, 55.
6
c
following a non-direct strategy.
1
0. The cytotoxicity of simple aromatic amines is
suggested to be initiated by nitrogen oxidation to the
N-oxides: (a) Rjosk, H.-K.; Neumann, H.-G. Zschr.
Krebsforsch 1971, 75, 209; (b) Hillesheim, W.;