Novel symmetrical ureas as modulators of protein arginine methyl transferases

Article abstract of DOI:10.1016/j.bmc.2013.01.017

Methylation of histone arginine residues is an epigenetic mark related to gene expression that is implicated in a variety of biological processes and can be reversed by small-molecule modulators of protein arginine methyltransferases (PRMTs). A series of symmetrical ureas, designed as analogues of the known PRMT1 inhibitor AMI-1 have been synthesized using Pd-catalyzed Ar-N amide bond formation processes or carbonylation reactions as key steps. Their inhibitory profile has been characterized. The enzymatic assays showed a weak effect on PRMT1 and PRMT5 activity for most of the compounds. The acyclic urea that exhibited the strongest effect on the inhibition of the PRMT1 activity also showed the greatest effect on the expression of some androgen receptor target genes (TMPRSS2 and FKBP5), which may be related with its enzymatic activity. Surprisingly, AMI-1 behaved as an activator of PRMT5 activity, a result not reported so far.

Full text of DOI:10.1016/j.bmc.2013.01.017

Bioorganic & Medicinal Chemistry 21 (2013) 2056–2067
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel symmetrical ureas as modulators of protein arginine methyl
transferases
⇑
⇑
Noelia Fontán, Patricia García-Domínguez, Rosana Álvarez , Ángel R. de Lera
Departamento de Química Orgánica, Facultade de Química, Universidade de Vigo, 36310 Vigo, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Methylation of histone arginine residues is an epigenetic mark related to gene expression that is impli-
cated in a variety of biological processes and can be reversed by small-molecule modulators of protein
arginine methyltransferases (PRMTs). A series of symmetrical ureas, designed as analogues of the known
PRMT1 inhibitor AMI-1 have been synthesized using Pd-catalyzed Ar–N amide bond formation processes
or carbonylation reactions as key steps. Their inhibitory profile has been characterized. The enzymatic
assays showed a weak effect on PRMT1 and PRMT5 activity for most of the compounds. The acyclic urea
that exhibited the strongest effect on the inhibition of the PRMT1 activity also showed the greatest effect
on the expression of some androgen receptor target genes (TMPRSS2 and FKBP5), which may be related
with its enzymatic activity. Surprisingly, AMI-1 behaved as an activator of PRMT5 activity, a result not
reported so far.
Received 13 December 2012
Revised 2 January 2013
Accepted 6 January 2013
Available online 22 January 2013
Keywords:
Epigenetics
Histone methylation
PRMT inhibitors
Ureas
Ó 2013 Elsevier Ltd. All rights reserved.
AMI-1
1. Introduction
marks only (PRMT7 has shown this activity on certain substrates),
and Type IV monomethylates the internal guanidinium nitrogen
Methylation of lysine and arginine residues of histones by
methyl transferases (KMTs and PRMTs, respectively) is related to
gene expression.1 Contrarily to histone acetylation, histone meth-
ylation is an epigenetic mark that does not alter the total charge
of the histone tails, although it reduces the affinity for anionic mol-
ecules such as DNA due to the increase of basicity and hydropho-
bicity of the histone tail. A large number of methyltransferases of
arginine and lysine residues has been identified,2 and most use
and has only been characterized in yeast. The activity of PRMT2,
10 and 11 has yet to be fully characterized.
Arginine methylation is implicated in a variety of biological pro-
cesses that include euchromatin maintenance, RNA processing, sig-
nal transduction, transcriptional regulation and DNA repair.1c,4b
PRMT4/CARM1 is a co-activator of androgen and estrogen recep-
tors, and is found overexpressed in hormone-dependent prostate
and breast tumors. Targeting PRMTs with small-molecule modula-
tors is potentially a promising approach to treat different diseases,
for instance cancer of the prostate and breast where this approach
would allow indirect targeting of the steroid receptors via their
associated cofactors.
the methyl group donor S-adenosyl-L-methionine (S-AdoMet or
SAM). The process can be viewed as a classical nucleophilic substi-
tution reaction at the methyl group by the partially deprotonated
terminal amino groups of histones lysine or arginine residues.3
Whereas histone lysine methylation produces increasingly methyl-
ated amines, histone arginine methylation can generate both
mono- and/or dimethylarginine derivatives, and the latter as sym-
metric or non-symmetric isomers.
At least eleven mammalian PRMT family members that share a
highly conserved catalytic domain have been identified to date.1c,4
They are classified in four groups according to their product selec-
tivity. Type I PRMTs (PRMT1, 3, 4, 6 and 8) produce non-symmetric
dimethylarginines, Type II PRMTs (PRMT5, 7 and 9) form the sym-
metric counterparts, and both types catalyze the formation of
monomethylarginine, Type III produces monomethylarginine
Inhibitors of methyl-transferase enzymes that are based on the
structure of the SAM cofactor or its product S-adenosyl-L-homocys-
teine (AdoHcy), such as AdOx, methylthioadenosine and sinefungin,
are in general unselective.5 A bisubstrate structure which incorpo-
rates an arginine end group at the position of the methyl sulfonium
ion of SAM is however more selective for PRMT inhibition and dis-
criminates between PRMT1 and CARM1 in favor of the former.6
Non-mechanism based inhibitors of arginine methylation
(AMIs; for example, AMI-1, 1 and AMI-3, 2) were discovered by
HTS of compound libraries (Fig. 1).7 AMI-1 1, which contains a
symmetrical sulfonated naphthylurea, was characterized as a
selective and cell-permeable PRMT inhibitor and was proposed to
dock to the AdoMet binding pocket, based on molecular modeling.8
Also shown in Figure 1 are selected PRMT inhibitors, including
⇑
Corresponding authors. Tel.: +34 986 812316; fax: +34 986 811940 (Á.R.L.); tel.:
+34 986 812632 (R.Á.).
E-mail address: qolera@uvigo.es (Á.R. de Lera).
allantodapsone 3,9 stilbamidine 4,9
a-methylthioglycolic amide 5
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.017

N. Fontán et al. / Bioorg. Med. Chem. 21 (2013) 2056–2067
2057
OH
OH
Me
N
O
O
O
NaO3S
N
H
N
H
SO3Na
1, AMI-1
OMe
AMI-3
2
,
NH
O
O
H2N
S
O
O
O
H
N
S
O
O
S
N
H
N
N
H
NH2
NH2
NH
O
O
N
H
O
5, RM65
3
4
, stilbamidine (NSC35605)
, allantodapsone (NSC34238)
O
O
S
N
H
NO2
H2N
7, CID 2818500
6
F3C
H
N
F3C
H
N
N
H
N
N
MeO
MeO
N
N
O
N
N
O
OMe
N
H
H
N
S
NH
H2N
Me
BocHN
9
10
O
8
O
Figure 1. Selected PRMT modulators.
(RM-65),10 benzo[b]imidazoles,10b,11 sulfonamide 6,10b nitroderiva-
tive 7,12 and pyrazoles 8, 9 and 10, which are nanomolar inhibitors
of CARM1.11,13
the symmetrical structure of AMI-1 1. We designed analogues
(Fig. 2) in which ureas are part of pyrimidin-2-ones and imidazoli-
din-2-ones heterocycles (Series I), and others with greater confor-
mational flexibility around the termini by using unsaturated and
saturated monocyclic and acyclic analogues of the naphthyl groups
(Series II). We also considered a third group of analogues in which
the carboxylic acid was replaced by a sulfonamide, which confers
greater length to these uncharged molecules (Series III).
Analogues of AMI-1 1 where the sulfonic acid and urea groups
are replaced by carboxylic acid and bis-amide functionalities,
respectively, have recently been reported (Fig. 2),14 with bis-car-
boxylic acid derivative 11 and its positional isomer as the most po-
tent and selective PRMT inhibitors.14 Diazocompounds related to
AMI-1 that preserve the naphthosulfonic acid (12) have been found
to directly target the substrate.15 Acyl derivatives of para-amino-
sulfonamides and dapsone 13 also contain a urea but in non-sym-
metrical structures.16 Recently, small-molecule enhancers of
arginine methylation with aryl ureido acetamido indole carboxyl-
ate structures which function as CARM1 activators have been
discovered.17
2. Chemistry
The synthesis of Series I includes an amidation of halobenzalde-
hydes with ureas18 and a Horner–Wadsworth–Emmons reaction to
construct the unsaturated chain of the cinnamates. For the first
step Xantphos 17 was selected as ligand of Pd(0) or Pd(II) precata-
lysts in a 1:3 Pd/L ratio (to avoid aryl exchange processes in the
phosphine),19 with Cs2CO3 as base in dioxane,20 conditions that
Based on the scaffolds discovered by Bedford et al.7 we started a
program aiming at developing inhibitors of PRMT1 and focused on
AcHN
H
N
OH
OH
OH
OH
O
O
N
N
N
O
N
O
Ph
N
H
N
H
N
H
R
Na
SO3
O S
N
H
N
H
HOOC
N
H
N
H
COOH
Na
3
13
12, NS1
11
COOH
HOOC
O
O
O
H
H
N
m, p
N
X
N
N
X
HOOC
N
H
N
H
COOH
Ar
S
N
H
N
H
S
Ar
O
O
O
O
n
Series I, n = 1, 2, X = CH, N
Series II
Series III
Figure 2. AMI-1-based PRMT inhibitors and novel structures reported in this work.

2058
N. Fontán et al. / Bioorg. Med. Chem. 21 (2013) 2056–2067
had proven successful for the preparation of both symmetrical and
non-symmetrical ureas.21 The reaction of bromobenzaldehydes
with urea led to disappointing yields (4–7% symmetrical substi-
tuted ureas), which did not improve with changes in reaction con-
ditions. Using ethyl p-bromobenzoate and a 1:3 Pd/L ratio the
major products were the monosubstituted urea in dioxane (22%
yield), and the symmetrical urea (62% yield) in DMF. This general
procedure using DMF proved useful for the direct coupling of cyclic
ureas to bromoarylaldehydes. The reaction of the bromobenzalde-
hyde isomers 14a–b and the analogue 2-pyridinecarboxaldehyde
14c with tetrahydropyrimidinin-2-(1H)-one 15 and imidazolidin-
2-one 16 provided the corresponding adducts 18a–c and 19a–c.
In general the coupling product was obtained in good to excellent
yields with minor amounts of the monosubstituted derivative in
selected cases (4% and 1% yield, respectively, in the case of 18c
and 19c). Only the reaction of pyridinecarboxaldehyde 14c with
imidazolidin-2-one 16 was inefficient (28% yield). In addition, no
reaction was obtained when p-bromobenzaldehyde was treated
with other cyclic ureas, such as 1H-benzo[d]imidazol-2(3H)-one,
with thiourea or with tetrahydropyrimidinin-2-(1H)-thione. In
the case of N,N0-dimethylurea the reaction afforded instead a
diarylamine, 4,40-(methylazanediyl)dibenzaldehyde, the result of
a rearrangement of the monosubstituted urea with release of
methylisocyanate, as described previously.22
with heterocyclic ureas as connectors 22a–c and 24a–c in variable
yields that were in general higher for the tetrahydropyrimidinin-2-
(1H)-ones 22 (Scheme 1).
For Series II, which features alkenyl or alkylureas as substitu-
ents of benzoic acids, we selected instead the carbonylation of
the corresponding amines (Scheme 2). The reaction of
commercial methyl 6-aminohexanoate with carbonyldiimidazole
2624 or with Co2(CO)8 as carbonyl sources under microwave irra-
diation25 led to the substituted urea in 65% and 44%, respec-
tively. However, saponification of the ester was unsuccessful
using a variety of bases (10% aqueous NaOH, Ba(OH)2, LiOH,
KOSiMe3, Me3SnOH). Alternatively, the carbonylation of 6-
aminohexanol 25 with carbonyldiimidazole 26 (30%) followed
by Jones oxidation (72%) of 27 provided the desired urea 28
(Scheme 2).
The synthesis of the ureas attached to benzoic acids via an alkyl
chain involved the carbonylation of the amines obtained by reduc-
tion of the nitriles (Scheme 2). Commercial 3-cyanobenzoate 29a
and homologue 29b (prepared by displacement of methyl 3-bro-
momethylbenzoate with NaCN in DMF in 85% yield) were hydroge-
nated in the presence of 10% Pd/C and HCl (83% and 63%,
respectively) and the hydrochlorides 30 neutralized with Et3N.
Using triphosgene 32 as carbonyl source, the ureas 33a and 33b
were acquired in 89% and 86% yield, respectively, and then were
saponified by treatment with 10% LiOH in THF to afford carboxylic
acids 34a (82%) and 34b (61%).
Chain extension was based on the HWE reaction23 of the corre-
sponding benzaldehydes with the anion of ethyl 2-(diethoxyphos-
phonyl)acetate 20 generated with n-BuLi in THF/DMPU (for 19c,
HNa in DMF was used due to solubility problems). Yields were
good to excellent in all cases with the exception of the reaction
of 19b. The condensation took place with complete stereoselectiv-
ity and only the E unsaturated diesters 21a–c and 23a–c were ob-
tained, as judged from the analysis of the vinyl signals on the 1H
NMR spectra. Final saponification using aqueous NaOH in EtOH
at 80 °C (25 °C for 22b) afforded the desired symmetrical acrylates
The carbonylation method was also successful for the prepa-
ration of 38 (Scheme 2). In this case, the steps were reversed,
with a HWE to cinnamate 35 preceding the formation of the ani-
line 36 upon treatment of 35 with sodium azide in a DMSO/H2O
mixture using a CuI/
-proline catalytic system at 110 °C.26 Addi-
L
tion of triphosgene 32 to the aniline provided diester 37 and fi-
nal hydrolysis led to dicarboxylic acid 38 in the yields shown in
Scheme 2.
HO2C
CO2H
CO2H
O
O
HO2C
X
X
N
N
N
N
O
22a, X = CH
22c, X = N
c
22b
c
HN
NH
a
ROOC
COOR
O
O
b
15
CHO
OHC
m, p
X
N
N
X
X
N
N
X
m, p
OEt
OEt
EtO2C
P
O
OHC
20
X
Br
21a-c, X = CH, N; R = Et
18a, X = CH, 3-CHO
18b
, X = CH, 4-CHO
O
14
18c, X = N, 6-CHO
HN
NH
a
16
ROOC
COOR
m, p
O
O
b
OHC
CHO
X
N
N
X
X
N
N
X
m, p
OEt
OEt
EtO2C
P
O
23a-c
20
, X = CH, N; R = Et
c
19a
19b
, X = CH, 3-CHO
PPh2
PPh2
, X = CH, 4-CHO
c
O
19c, X = N, 6-CHO
HO2C
CO2H
O
N
O
N
N
17, Xantphos
HO2C
X
N
X
CO2H
24b
24a, X = CH
24c
, X = N
Scheme 1. Synthesis of analogues of Series I. Reagents and conditions: (a) 15 or 16, Pd2dba3ꢀCHCl3, Xantphos 17, Cs2CO3, DMF, 100 °C (18a, 52%; 18b, 81%; 18c, 71%; 19a, 91%;
19b, 72%; 19c, 28%); (b) n-BuLi, DMPU, THF, 0 to ꢁ78 °C, 2 h (21a, 90%; 21b, 81%; 21c, 90%; 23a, 81%; 23b, 12%; 23c, 60%); (c) 10% aq NaOH, EtOH, 80 °C, 17 h (22a, 83%; 22b,
80%; 22c, 36%; 24a, 48%; 24b, 30%; 24c, 86%).

N. Fontán et al. / Bioorg. Med. Chem. 21 (2013) 2056–2067
2059
O
Im
Im
a
26
O
HO
NH2
R
N
H
N
H
R
25
27, R = CH2OH
b
28
, R = CO2H
O
e
36
Cl3CO
OCCl3
Cl
NH3
O
c
d
CN
n
MeO2C
MeO2C
MeO2C
NH2
R
N
H
N
H
R
n
n
n
n
29, n = 1, 2
31
30
33
34
, R = CO2Me, n = 0, 1
, R = CO2H, n = 0, 1
a
: n = 0
f
b: n = 1
O
e
32
OCCl3
Cl3CO
g
O
h
14a
OEt
OEt
R
N
H
N
H
R
EtO2C
Br
EtO2C
NH2
EtO2C
P
O
35
36
20
37
, R = CO2Me
f
38, R = CO2H
Scheme 2. Synthesis of analogues of Series II. Reagents and conditions: (a) Et3N, THF, 25 °C, 120 h, 30%; (b) CrO3, H2SO4, acetone/H2O, 25 °C, 2 h, 72%; (c) H2, Pd/C 10%, HCl,
25 °C, 17 h (30a, 83%; 30b, 63%); (d) Et3N, CH2Cl2, 30 min; (e) (i) Et3N, C6H6, 25 °C, 12 h, (ii) 31 or 36, acetone, 25 °C, 2 h (33a, 89%; 33b, 86%; 37, 71%); (f) 10% aq LiOH, THF,
80 °C, 24 h (34a, 82%; 34b, 61%; 38, 86%); (g) n-BuLi, DMPU, THF, from ꢁ78 to 25 °C, 6 h, 96%; (h) NaN3, CuI, L-proline, NaOH, DMSO, EtOH, 110 °C, 20 h, 56%.
Unfortunately, the formation of thioureas with Lawesson’s re-
agent27 was unsuccessful for models 33a and 34a, which were fully
recovered after 17 h stirring at 70 °C.
both showed very potent inhibition of enzymatic activity. Other
compounds like 22a, 24a and 22c also inhibited PRMT1 activity
but less potently than those mentioned before. The remaining
compounds exhibited significantly weaker inhibition. The IC50 val-
Lastly, longer analogues of AMI-1, Series III, represented by sul-
fonamides 45, were prepared from 7-amino-2-naphthalenesul-
phonic acid 39.28 The sequence (Scheme 3) comprises acetylation
of the aniline (Ac2O, pyridine, 84%), formation of the sodium sul-
phonate (MeONa, MeOH, 57%), activation as sulphonyl chloride
(POCl3, DMA, 44%) and addition of the corresponding anilines 41
in the presence of Hünig’s base. No reaction took place with halo-
genated anilines (2,5-dibromoaniline, 4-chloro-3-trifluoromethyl-
aniline and 4-ciano-3-trifluoromethylaniline). Reaction of 42 with
5 M NaOH and work-up afforded the ammonium salts, which were
treated with Et3N and used in the next reaction. Their treatment
with triphosgene 32 according to the above method provided
45a and 45b in low yields (23% and 7%), which might be due to sol-
ubility problems and difficulties in the purification process.
ues of the two most potent compounds, 28 and 24a are 6.1
25.2 M, respectively (see SI).
lM and
l
A similar assay performed to measure the PRMT5 enzymatic
activity assay revealed that AMI-1 1 is an activator of PRMT5,
whereas 24c and 34b proved to be the most potent inhibitors
(Fig. 3B).
3.2. Cellular characterization of the AMI-1 analogues
3.2.1. Effect on cell viability and proliferation
After completing the enzymatic PRMT assays, the effect of the
compounds on the proliferation and viability of VCaP and LAPC-4
prostate cancer cell lines was tested. None of these compounds
showed an effect on proliferation, except compounds 45a and
45b, which had inhibitory effects on the LAPC-4 cell line. The
3. Results and discussion
IC50 values for 45a and 45b were 1.5 and 8.6 lM, respectively.
The effect in the case of 45b may be due to toxic (non-apoptotic)
effects related to the high concentration used, since a dose-depen-
dent effect on the proliferation was not observed.
3.1. Activity on PRMT enzymes
We first tested the activity of the synthesized compounds on re-
combinant PRMTs using AMI-1 1 as standard for enzyme inhibi-
tion. The assays measured the modulation of human
recombinant PRMT1 (expressed in E. coli) and PRMT5 (expressed
in Sf9 cells) enzymes using histone H4 peptide as substrate pre-
coated in a commercially available enzymatic assay kit.29 PRMT1
and PRMT5 were chosen as representative members of the type I
and type II enzymes and catalyze the non-symmetric and symmet-
ric dimethylation of arginines, respectively. AMI-3 2 (IC50 of
3.2.2. Effect on the expression of androgen receptor target
genes
As mentioned before, several PRMTs have been described as
cofactors/co-activators of the androgen and estrogen class of nu-
clear receptors, making them putative targets for the treatment
of hormone-dependent cancers.7,16,30 In order to determine the ef-
fects of the analogues on the expression of androgen-dependent
genes in VCaP cells, we quantified the mRNA levels of selected
genes after androgen stimulation in the absence or in the presence
of the compounds (not shown). Among all the modulators tested,
only 28 showed a significant effect. We therefore analyzed in detail
the effects of this compound on a number of androgen-controlled
genes in the VCaP cell line. Using different concentrations we could
16.3
der to have a broader comparison with other PRMT inhibitors.
The results show (Fig. 3A) that at 100 M AMI-1 1 was the most
potent inhibitor of PRMT1 enzymatic activity (an IC50 value of
8.8 M was previously measured for AMI-1). Compound 28 proved
to be the second most potent compound, followed by AMI-3 2, and
l
M for human PRMT1)7 was also included in this study in or-
l
l

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N. Fontán et al. / Bioorg. Med. Chem. 21 (2013) 2056–2067
a
b
c
O
O
Cl
H
N
H
N
HO3S
R =
NH2
ClO2S
N
H
R
S
N
H
R
S
NH3
O2
O2
42
43
39
40
41
(
RNH2
)
d
O
O
O
e
H
H
H
Ph
N
N
O
b
N
R
S
N
H
N
S
R
R
S
NH2
a
O2
H
O2
O2
45
44
Scheme 3. Synthesis of analogues of Series III. Reagents and conditions: (a) (i) Ac2O, pyridine, 25 °C, 8 h, 84%, (ii) MeONa, MeOH, 25 °C, 12 h, 57%; (iii) POCl3, DMA, 25 °C, 24 h,
44%; (b) R-NH2 41, Hünig base, THF/CHCl3, 25 °C, 12 h (42a, 44%; 42b, 53%); (c) 5 M NaOH, MeOH, 60 °C, 120 h (43a, 88%; 43b, 91%). (d) Et3N, CH2Cl2, 30 min; (e) (i)
triphosgene 32, Et3N, C6D6, 25 °C, 12 h, (ii) 44, acetone 2 h (45a, 23%; 45b, 7%).
Figure 3. In vitro methylation assays to evaluate the effect of compounds on PRMT activity. (A) PRMT1 assay in the presence of AMI-1 1, AMI-3 2 and compounds of Series I, II
and III. (B) PRMT5 assay in the presence of the same compounds. The data represent the average value of independent duplicates; error bars represent standard deviation (SD)
of biological duplicates. See Section 5 for a detailed experimental procedure.

N. Fontán et al. / Bioorg. Med. Chem. 21 (2013) 2056–2067
2061
Figure 4. Transcriptional regulation of androgen-dependent genes by compound 28. The first bar of the graphs represents the expression without androgen stimulation. The
second bar shows the expression after treatment with R1881, a synthetic non-aromatizable androgen used for AR activation. The other bars correspond to the expression
levels in presence of androgen and of increasing concentrations of compound 28. The data represent the average value of independent triplicates.
demonstrate the specific, dose-dependent inhibitory effects of 28
on the expression of several genes that are stimulated by andro-
gens such as TMPRSS2 and FKBP5. This was however not seen for
all androgen-regulated genes since the levels of KLK2 remained
unaltered. The expression of PRMT1, which is not androgen depen-
dent, was not affected either. AR levels were reduced by androgen
treatment of VCaP cells, as reported before,31 but did not vary after
application of 28 (Fig. 4).
was noticed with 28, 22a, 22c and 24a, whereas 24c and 34b were
the most active inhibitors of PRMT5. In this regard, it is not
straightforward to establish a correlation between the structure
of these analogues and their activity. In addition, in this assay,
AMI-1 behaved as an activator of PRMT5 activity, a result not re-
ported so far. Acyclic urea 28 showed the greatest effect on the
expression of some androgen receptor target genes, which may
correlate with its PRMT1 enzymatic inhibitory activity. The ab-
sence of effects of 22a, 22c and 24a may be due to less efficient cell
penetration. Since the potency of the compounds is low, we can
conclude that these symmetrical ureas inspired by AMI-1 are not
apparent inhibitors of PRMT1 and 5. These scaffolds should be ru-
led out as starting points to develop more potent PRMT inhibitors.
4. Conclusions
Three series of analogues of the known PRMT1 inhibitor AMI-1
have been synthesized using as key steps Pd-catalyzed Ar–N amide
bond formation processes for cyclic ureas or carbonylation reac-
tions for amines/anilines for the parent and substituted ureas. All
compounds share the symmetrical structure, composed of a central
urea, which can be part of pyrimidin-2-one or imidazolidin-2-one
heterocycles, and terminal moieties with carboxylic acid or sulph-
onamide functionalities. Differences are found in the length of the
chain linking the central urea to the termini, in the relative position
of these two functional groups as aryl ring substituents or in the
presence of partially or totally saturated chains, which confers in
the latter case greater conformational flexibility to the compounds.
The biological evaluation of the three series using in vitro enzy-
matic assays showed a low effect on the inhibition of PRMT1 and
PRMT5 enzymes for most of the compounds. The strongest effect
on the symmetric (PRMT1 assay) arginine dimethylation reactions
5. Experimental section
5.1. Biochemistry methods
5.1.1. In vitro methylation assay
The assay was performed with PRMT Direct Activity Assay kits
for PRMT1 and PRMT5 purchased from BPS Bioscience (San Diego,
CA, USA).29
PRMT1 and PRMT5 assay kits: PRMT kits are designed to measure
PRMT1 or PRMT5 activity using purified PRMT1 or PRMT5 or
extracts cells containing PRMT1 or PRMT5. These kits include a
96-well plate precoated with histone H4 peptide substrate, the pri-
mary antibody against methylated arginine-3 residue of histone

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N. Fontán et al. / Bioorg. Med. Chem. 21 (2013) 2056–2067
H4, the secondary HRP-labeled antibody, S-adenosylmethionine,
methyltransferase assay buffer and purified human recombinant
PRMT1 (expressed in E. coli) or PRMT5 (expressed in HEK293 cells)
enzyme for 100 enzyme reactions. In addition to PRMT enzymes
provided with both kits, PRMT1 and PRMT5 expressed in Sf9 cells
were also purchased to compare the results obtained with both en-
zymes and select the one with the highest activity. The highly spe-
cific primary antibody recognizes methylated R3 residue of histone
H4 (asymmetric methylation in case of the antibody for PRMT1 and
symmetric methylation for PRMT5). Detection of methyltransfer-
ase activity is done in three steps. First, S-adenosylmethionine is
incubated with a sample containing assay buffer and methyltrans-
ferase enzyme for one hour. Next, a primary antibody is added fol-
lowed by an incubation time of one hour. Finally, the plate is
treated with a secondary HRP-labeled antibody, and the HRP sub-
strate to produce chemiluminiscence that can be measured using a
chemiluminiscence reader.
with a solution of phosphomolibdic acid. Flash column chromatog-
raphy was carried out using Merck Kieselgel 60 (230–400 mesh)
under pressure. Infrared spectra were obtained on a JASCO FTIR
4200 spectrophotometer, from a thin film deposited onto a NaCl
glass. 1H NMR spectra were recorded in CDCl3, CD3OD, DMSO-d6
and (CD3)2CO at ambient temperature on a Bruker AMX-400 spec-
trometer at 400 MHz with residual protic solvent as the internal
reference (CDCl3, dH = 7.26 ppm; (CD3)2CO, dH = 2.05 ppm; CD3OD,
dH = 3.31; DMSO-d6, dH = 2.50); chemical shifts (d) are given in
parts per million (ppm), and coupling constants (J) are given in
Hertz (Hz). The proton spectra are reported as follows: d (multiplic-
ity, coupling constant J, number of protons, assignment). 13C NMR
spectra were recorded in CDCl3 CD3OD, DMSO-d6 and (CD3)2CO at
ambient temperature on the same spectrometer at 100 MHz, with
the central peak of CDCl3 (dC = 77.0 ppm) CD3OD (dC = 49.0 ppm),
DMSO-d6 (dC = 39.4 ppm) or (CD3)2CO (dC = 30.8 ppm) as the internal
reference. DEPT135 sequence was used to aid in the assignment of
signals in the 13C NMR spectra. Melting points were determined on
a Stuart SMP10 apparatus. Elemental analyses were determined on
a Carlo Erba EA 1108 analyzer. Mass Spectrometry. Experiments
were performed on an APEX III FT-ICR MS (Bruker Daltonics, Billerica,
MA), equipped with a 7T actively shielded magnet. Ions were gener-
ated using an Apollo API electrospray ionization (ESI) source, with a
voltage between 1800 and 2200 V (to optimize ionisation efficiency)
applied to the needle, and a counter voltage of 450 V applied to the
capillary. Samples were prepared by adding a spray solution of
70:29.9:0.1 (v/v/v) CH3OH/water/formic acid to a solution of the
sample at a v/v ratio of 1 to 5% to give the best signal-to-noise ratio.
Data acquisition and data processing were performed using the
XMASS software, version 6.1.2 (Bruker Daltonics). FAB Experiments
were performed on a VG AutoSpec instrument, using 3-nitroben-
zylalcohol or glycerol as matrices.
25 mM stock solutions of the compounds in DMSO were pre-
pared. PRMTs assays were performed in the presence of the ana-
logues at 100
composed of 20
assay buffer provided with the assay kit (1 ꢂ TBS, pH 8.0, contain-
ing 0.05% Tween20), 2.5 L of S-adenosylmethionine (400
stock solution) and 15 L of the solution of the compounds in
water. IC50 values were calculated by measuring the enzymatic
activity after treatment with 1.56, 6.25, 25 and 100 M solutions
lM (solutions in water) plus a reaction mixture
l
L of a solution of PRMT enzyme, 12.5 L of TBST
l
l
lM
l
l
of the compounds. The programme BioDataFit1.02 was used to fit
the data to a sigmoidal function (logEC50), which provided the de-
sired values (see Supplementary data).
5.1.2. Cell viability assay
The LAPC-4 and VCaP prostate cancer cell lines were obtained
from ATCC (Manassas, VA, USA). LAPC-4 cells were grown in
RPMI1640 without phenol red supplemented with 10% cFCS and
The purity of the compounds was established in most cases by
elemental analysis and, for those that did not gave suitable crys-
tals, by HPLC, and found to be greater than 95%.
2 mM L-glutamine at a concentration of 4000 cells/well in a 96-
well microtiter plate. One day later, the cells were treated with
1 nM R1881 and different compound concentrations. VCaP cells
were grown in DMEM with phenol red supplemented with 10%
cFCS at a concentration of 16,000 cells/well in a 96-well microtiter
plate. One day later, the cells were treated with 0.1 nM R1881 and
different compound concentrations. For both cell lines, the cell
number was determined seven days later by Alamar Blue staining
(Invitrogen, Life Technologies, Darmstadt, Germany) in a Victor3
luminometer (PerkinElmer, Rodgau, Germany).
5.2.2. (1,3)-Bis-(1-formyl-phen-3-yl)-tetrahydropyrimidin-
2(1H)-one (18a)
5.2.2.1. General procedure for the palladium-catalyzed amida-
tion of arylbromides with ureas.
A solution of 3-bromobenz-
aldehyde 14a (0.2 g, 0.125 mL, 1.08 mmol), tetrahydropyrimidin-
2(1H)-one 15 (0.07 g, 0.7 mmol), Pd2dba3.CHCl3 (0.0056 g,
0.0054 mmol), Xantphos (0.0094 g, 0.016 mmol) and dried Cs2CO3
(0.49 g, 1.51 mmol) in DMF (6 mL) was stirred at 100 °C for 4 h.
The reaction mixture was cooled down to room temperature and fil-
tered through a pad of CeliteÒ. The solvent was evaporated to afford,
after purification by column chromatography (silica gel, 97.5:2.5
CH2Cl2/MeOH), 0.087 g (52%) of a solid identified as (1,3)-bis-(1-for-
myl-phen-3-yl)-tetrahydropyrimidin-2(1H)-one 18a. 1H NMR
(400.13 MHz, CDCl3): d 9.99 (s, 2H, CHO), 7.87 (t, J = 1.8 Hz, 2H,
ArH), 7.71–7.66 (m, 4H, ArH), 7.51 (t, J = 7.8 Hz, 2H, ArH), 3.91 (t,
J = 5.9 Hz, 4H, 2 ꢂ CH2), 2.35 (quint., J = 5.9 Hz, 2H, CH2) ppm. 13C
NMR (100.62 MHz, CDCl3): d 191.9 (d, 2ꢂ), 154.2 (s), 144.5 (s, 2ꢂ),
137.2 (s, 2ꢂ), 132.0 (d, 2ꢂ), 129.4 (d, 2ꢂ), 127.2 (d, 2ꢂ), 126.1 (d,
2ꢂ), 49.0 (t, 2ꢂ), 23.0 (t) ppm. MS (EI): m/z (%) 309 ([M+1]+, 7),
308 ([M]+, 100), 307 (19), 133 (45), 132 (35), 105 (15), 77 (14). HMRS
5.1.3. Effect on the expression of androgen receptor target
genes
RNA was prepared from VCaP cells using the RNeasy extraction
kit (Qiagen, Hilden, Germany) and reverse-transcribed with the
SuperScript Reverse Transcriptase kit (Invitrogen). Gene expres-
sion was measured using specific fluorogenic probes and measured
on a Fast Real-Time PCR system (Applied Biosystems, Life Technol-
ogies, Darmstadt, Germany). Human cyclophilin A levels were
determined as internal control for normalization.
5.2. Chemistry methods
(EI): Calcd for C18H16N2O3, 308.1161; found, 308.1164. IR (NaCl):
m
5.2.1. General
2839 (w, C–H), 1694 (s, C@O), 1650 (s, C@O), 1590 (m), 1482 (s),
1426 (s), 1307 (s), 1203 (s) cmꢁ1. UV (MeOH): kmax 242 nm. Mp:
146–148 °C (EtOAc/hexane).
Solvents were dried according to published methods and dis-
tilled before use. HPLC grade solvents were used for HPLC purifica-
tion. All other reagents were commercial compounds of the highest
purity available. All reactions were carried out under argon atmo-
sphere, and those not involving aqueous reagents were carried out
in oven-dried glassware. Analytical thin layer chromatography
(TLC) was performed on aluminium plates with Merck Kieselgel
60F254 and visualized by UV irradiation (254 nm) or by staining
5.2.3. (E,E)-(1,3)-Bis-[3-(1-ethoxycarbonyl-ethen-2-yl)-phen-1-
yl]-tetrahydropyrimidin-2(1H)-one (21a)
5.2.3.1. General procedure for the Horner–Wadsworth–
Emmons reaction.
A cooled (0 °C) solution of ethyl 2-(dieth-
oxyphosphoryl)acetate 20 (0.07 g, 0.06 mL, 0.31 mmol) in THF

N. Fontán et al. / Bioorg. Med. Chem. 21 (2013) 2056–2067
2063
(0.75 mL) was treated with n-BuLi (0.18 mL, 1.61 M en hexane,
0.29 mmol) and DMPU (1 mL) and the mixture was stirred for
30 min. The reaction was cooled down to ꢁ78 °C, a solution of
(1,3)-bis-(1-formyl-phen-3-yl)-tetrahydropyrimidin-2(1H)-one
18a (0.04 g, 0.13 mmol) in THF (0.75 mL) was added and the result-
ing mixture was stirred for 1.5 h at ꢁ78 °C and then was allowed to
warm up to 25 °C for 30 min. H2O was added and the mixture was
extracted with Et2O (3ꢂ). The combined organic layers were
washed with water (3ꢂ), brine (3ꢂ) and dried (Na2SO4) and the
solvent was evaporated. The residue was purified by column chro-
matography (silica gel, 97.5:2.5 CH2Cl2/MeOH) to afford 0.052 g
(90%) of a solid identified as (E,E)-(1,3)-bis-[3-(1-ethoxycarbonyl-
ethen-2-yl)-phen-1-yl]-tetrahydropyrimidin-2(1H)-one 21a. 1H
NMR (400.13 MHz, CDCl3): d 7.65 (d, J = 16.0 Hz, 2H), 7.53 (s, 2H,
ArH), 7.37–7.34 (m, 6H, ArH), 6.42 (d, J = 16.0 Hz, 2H), 4.25 (q,
J = 7.1 Hz, 4H, CO2CH2CH3), 3.85 (t, J = 5.8 Hz, 4H, 2 ꢂ CH2), 2.31
(quint., J = 5.8 Hz, 2H, CH2), 1.32 (t, J = 7.1 Hz, 6H, CO2CH2CH3)
ppm. 13C NMR (100.62 MHz, CDCl3): d 167.0 (s, 2ꢂ), 154.3 (s),
144.4 (s, 2ꢂ), 144.2 (d, 2ꢂ), 135.3 (s, 2ꢂ), 129.3 (d, 2ꢂ), 127.6 (d,
2ꢂ), 125.4 (d, 2ꢂ), 125.3 (d, 2ꢂ), 118.9 (d, 2ꢂ), 60.6 (t, 2ꢂ), 49.2
(t, 2ꢂ), 23.2 (t), 14.4 (q, 2ꢂ) ppm. MS (EI): m/z (%) 449 ([M+1]+,
13), 448 ([M]+, 89), 419 (69), 403 (15), 374 (14), 373 (100), 184
(13), 172 (12), 158 (61), 130 (25), 102 (22). HMRS (EI): Calcd for
(m, 4H, 2 ꢂ CH2), 1.54–1.46 (m, 4H, 2 ꢂ CH2), 1.42–1.33 (m, 4H,
2 ꢂ CH2) ppm. 13C NMR (100.62 MHz, CD3OD): d 177.6 (s, 2ꢂ),
161.3 (s), 40.8 (t, 2ꢂ), 34.9 (t, 2ꢂ), 31.0 (t, 2ꢂ), 27.5 (t, 2ꢂ),
25.8 (t, 2ꢂ) ppm. HMRS (ESI+): Calcd for C13H24N2NaO5
([M+Na]+), 311.1572; found, 311.1577. IR (NaCl):
m 3330 (br, O–
H/N–H), 2926 (m, C–H), 2859 (m, C–H), 1694 (s, C@O), 1610
(m, C@O), 1570 (s), 1261 (s), 1203 (m) cmꢁ1
.
Mp: 136–
139 °C (CH2Cl2/MeOH). This compound has been previously
described.32
5.2.6. Methyl 3-(cyanomethyl)benzoate (29b)
A
suspension of methyl-3-(bromomethyl)benzoate (0.5 g,
2.18 mmol) and sodium cyanide (0.16 g, 3.27 mmol) in DMF
(0.93 mL) and H2O (0.04 mL) was stirred at 75 °C for 5 h. The reac-
tion was quenched with water and extracted with EtOAc. The com-
bined organic layers were washed with H2O (3ꢂ), dried (Na2SO4)
and the solvents were evaporated. The residue was purified by col-
umn chromatography (silica gel, from 80:20 hexane/EtOAc to
70:30 hexane/EtOAc) to afford 0.324 g (85%) of a colourless oil
identified as methyl 3-(cyanomethyl)benzoate 29b. 1H NMR
(400.13 MHz, CDCl3): d 8.02–8.00 (m, 2H, ArH), 7.56–7.53 (m, 1H,
ArH), 7.50–7.46 (m, 1H, ArH), 3.93 (s, 3H, CH3), 3.81 (s, 2H, CH2)
ppm. 13C NMR (100.62 MHz, CDCl3): d 166.3 (s), 132.3 (d), 131.0
(s), 130.5 (s), 129.3 (d), 129.2 (d), 129.1 (d), 117.5 (s), 52.3 (q),
23.4 (t) ppm. HMRS (ESI+): Calcd for C10H10NO2 ([M+H]+),
C
26H28N2O5 ([M]+), 448.1998; found, 448.1995. IR (NaCl):
m 2980
(w, C–H), 1709 (s, C@O), 1645 (s, C@O), 1585 (w, C@C), 1482
(m), 1426 (m), 1303 (s), 1176 (s) cmꢁ1. UV (MeOH): kmax 261 nm.
Mp: 136–139 °C (CH2Cl2/MeOH).
176.0706; found, 176.0703. IR (NaCl):
m 3003 (w, C–H), 2954 (w,
C–H), 2252 (w, C„N), 1722 (s, C@O), 1439 (m), 1287 (m) cmꢁ1
UV (MeOH): kmax 283, 229 nm.
.
5.2.4. (E,E)-3,30-[(2-Oxodihydropyrimidine-1,3-diyl)-bis-(1,3-
phenylene)]-diacrylic acid (22a)
5.2.4.1. General procedure for the hydrolysis of esters with 10%
5.2.7. (3-Methoxycarbonyl-phen-1-yl)-methanammonium
chloride (30a)
NaOH.
To a solution of (E,E)-(1,3)-bis-[3-(ethoxycarbonyle-
A
suspensión of methyl 3-cyanobenzoate 29a (1.0 g,
then-1-yl)-phenyl]-tetrahydropyrimidin-2(1H)-one 21a (0.04 g,
0.09 mmol) in EtOH (2.7 mL) was added a 10% aqueous solution
of NaOH (0.09 mL, 0.89 mmol) and the mixture was stirred at
80 °C for 17 h. The reaction was quenched with a 10% aqueous
solution of HCl and the solid was filtered off to afford, 0.029 g
(83%) of a solid identified of (E,E)-3,30-[(2-oxodihydropyrimidin-
1,3-diyl)-bis-(1,3-phenylene)]-diacrylic acid 22a. 1H NMR
(400.13 MHz, DMSO-d6): d 12.39 (br s, 2H, CO2H), 7.68 (s, 2H,
ArH), 7.58 (d, J = 16.0 Hz, 2H), 7.47 (d, J = 6.9 Hz, 2H, ArH), 7.41–
7.35 (m, 4H, ArH, 2 ꢂ CH2), 6.54 (d, J = 16.0 Hz, 2H, CH2), 3.81 (t,
J = 5.7 Hz, 4H, 2 ꢂ CH2), 2.21 (quint., J = 5.7 Hz, 2H, CH2) ppm. 13C
NMR (100.62 MHz, DMSO-d6): d 167.5 (s, 2ꢂ), 153.4 (s), 144.7 (s,
2ꢂ), 143.6 (d, 2ꢂ), 134.6 (s, 2ꢂ), 128.8 (d, 2ꢂ), 127.8 (d, 2ꢂ),
125.1 (d, 2ꢂ), 124.9 (d, 2ꢂ), 119.4 (d, 2ꢂ), 48.7 (t, 2ꢂ), 22.5 (t)
ppm. MS (FAB+): m/z (%) 393 ([M+H]+, 100), 392 ([M]+, 21), 157
(11), 154 (30). HMRS (FAB+): Calcd for C22H21N2O5 ([M+H]+),
6.20 mmol), 10% Pd/C (0.48 g) and concd HCl. (0.66 mL, 6.20 mmol)
in MeOH (60 mL) was stirred overnight under a hydrogen atmo-
sphere. The mixture was filtered through a pad of CeliteÒ, the sol-
vent was evaporated and the solid was crystallized (EtOH/Et2O) to
afford 1.04 g (83%) of a solid identified as (3-methoxycarbonyl)-
phen-1-yl)-methanamonium chloride 30a. 1H NMR (400.13 MHz,
CD3OD) d 8.14 (s, 1H, ArH), 8.06 (d, J = 7.8 Hz, 1H, ArH), 7.69 (d,
J = 7.3 Hz, 1H, ArH), 7.57 (t, J = 7.7 Hz, 1H, ArH), 4.18 (s, 2H, CH2),
3.91 (s, 3H, CH3) ppm. 13C NMR (100.62 MHz, CD3OD): d 167.8
(s), 135.1 (s), 134.8 (d), 132.2 (s), 131.1 (d, 2ꢂ), 130.5 (d), 52.8
(q), 43.9 (t) ppm. HMRS (ESI+): Calcd for C9H12NO2 ([MꢁCl]+),
166.0863, found 166.0858. IR (neat):
m 3200–2600 (br, N–H),
3158 (w, N–H), 2959 (w, C–H), 2807 (m, C–H), 1689 (s, C@O),
1607 (w), 1473 (w), 1450 (w), 1289 (s), 1213 (s) cmꢁ1. UV (MeOH):
kmax 282, 228 nm. Mp: 165–166 °C (EtOH/Et2O).
393.1450; found, 393.1450. IR (neat):
m
3500–2500 (br, O–H),
5.2.8. 1-[3-(Methoxycarbonyl)-phen-1-yl]-ethan-1-ammonium
chloride (30b)
1678 (s, C@O), 1642 (s, C@O), 1485 (s), 1424 (s), 1307 (s), 1201
(s) cmꢁ1. UV (MeOH): kmax 259 nm. Mp: 292–295 °C (EtOH). Purity
Following the general procedure for reduction of nitriles, the
reaction of methyl 3-(cyanomethyl)benzoate 29b (0.22 g,
1.23 mmol), 10% Pd/C (0.095 g) and concd HCl (0.039 mL,
1.227 mmol) in MeOH (12.0 mL) afforded 0.166 g (63%) of a solid
identified as 2-[3-(methoxycarbonyl)-phen-1-yl)-ethan-1-amoni-
um chloride 30b. 1H NMR (400.13 MHz, CD3OD) d 7.96 (s, 1H,
ArH), 7.94–7.91 (m, 1H, ArH), 7.57 (d, J = 7.7, 1H, ArH), 7.48 (t,
J = 7.7, 1H, ArH), 3.90 (s, 3H), 3.24–3.20 (m, 2H), 3.06 (t, J = 7.8,
2H) ppm. 13C NMR (100.62 MHz, CD3OD): d 168.2 (s), 138.6 (s),
134.7 (d), 132.0 (s), 130.8 (d), 130.2 (d), 129.40 (d), 52.7 (q), 41.7
(t), 34.2 (t) ppm. HMRS (ESI+): Calcd for C10H14NO2 ([MꢁCl]+),
trace: RPHPLC-ESI (SunfireÒ C18
95:5 to 0:100 H2O/CH3CN, 25 min, 1 mL/min, tR = 14.4 min; 98%
5
l
m, 250 ꢂ 46 mm, gradient from
purity).
5.2.5. 6,60-Ureylen-di-hexanoic acid (28)
To a cooled (0 °C) solution of 1,3-bis-(6-hydroxyhex-1-yl)-urea
27 (0.05 g, 0.12 mmol) in acetone (0.65 mL) was added dropwise
the Jones reagent (0.07 g CrO3, 0.059 mL H2SO4, 0.69 mL H2O)
and the mixture was stirred for 2 h at 25 °C. The reaction mixture
was quenched with water and the mixture was extracted with
EtOAc (5ꢂ). The combined organic layers were washed with brine
(3ꢂ), dried (Na2SO4) and the solvent was evaporated to
afford 0.04 g (72%) of a white solid identified as 6,60-ureylen-di-
hexanoic acid 28. 1H NMR (400.13 MHz, CD3OD): d 3.11 (t,
J = 6.9 Hz, 4H, 2 ꢂ CH2), 2.30 (t, J = 7.4 Hz, 4H, 2 ꢂ CH2), 1.67–1.60
180.1019, found 180.1027. IR (neat):
m 3200–2800 (br, N–H),
2971 (m, C–H), 2893 (m, C–H), 2798 (w, C–H), 2732 (w, C–H),
1719 (s, C@O), 1595 (w), 1479 (m), 1437 (m), 1279 (s), 1213
(s) cmꢁ1. UV (MeOH): kmax 285, 224 nm. Mp: 150–151 °C (EtOH/
Et2O).

2064
N. Fontán et al. / Bioorg. Med. Chem. 21 (2013) 2056–2067
5.2.9. N,N0-bis-(3-Methoxycarbonyl-phen-1-yl-methyl)urea (33a)
([M+H]+), 329.1132, found, 329.1135. IR (neat):
m 3309 (w, N–H),
5.2.9.1. General procedure for the carbonylation reaction using
2877 (m, C–H), 1678 (m, C@O), 1571 (m), 1421 (m), 1286 (s)
triphosgene.
A solution of methyl 3-(aminomethyl)benzoate
cmꢁ1. UV (MeOH): kmax 283 nm. Mp: 260–261 °C (MeOH/Et2O).
31a (previously obtained from (3-methoxycarbonyl-phen-1-yl)-
methanammonium chloride 30a by treatment of Et3N) (0.23 g,
1.53 mmol) and Et3N (0.48 mL, 0.35 g, 3.48 mmol) in benzene
Purity trace: RPHPLC-ESI (SunfireÒ C18
ent from 95:5 to 0:100 H2O/CH3CN, 25 min, 1 mL/min,
5
l
m, 250 ꢂ 46 mm, gradi-
tR = 12.3 min; 100% purity).
(22.2 mL) was added
a solution of triphosgene 32 (0.23 g,
1.53 mmol) in benzene (22.2 mL). The mixture was stirred over-
night at 25 °C and then the solvent was evaporated. The residue
was dissolved in acetone (9.8 mL), another portion of 3-(amino-
methyl)benzoate 31a (0.23 g, 1.53 mmol) was added , and the mix-
ture was stirred for 2 h at 25 °C. The residue was purified by
crystallization (EtOH/Et2O) to afford 0.242 g (89%) of a solid iden-
tified as N,N0-bis-(3-methoxycarbonyl-phen-1-yl-methyl)urea
33a. 1H NMR (400.13 MHz, DMSO-d6) d 7.87 (s, 2H, NH), 7.82 (d,
J = 7.6 Hz, 2H, ArH), 7.53 (d, J = 7.7 Hz, 2H, ArH), 7.46 (t,
J = 7.6 Hz, 2H, ArH), 6.64 (t, J = 6.1 Hz, 2H, 2 ꢂ NH), 4.29 (d,
J = 6.1 Hz, 4H, 2 ꢂ CH2), 3.84 (s, 6H, 2 ꢂ CH3) ppm. 13C NMR
(100.62 MHz, DMSO-d6): d 166.3 (s, 2ꢂ), 158.0 (s), 141.8 (s, 2ꢂ),
131.9 (d, 2ꢂ), 129.6 (s, 2ꢂ), 128.6 (d, 2ꢂ), 127.5 (d, 2ꢂ), 127.4 (d,
2ꢂ), 52.1 (q, 2ꢂ), 42.6 (t, 2ꢂ) ppm. HMRS (ESI+): Calcd for
5.2.12. 3,30-(Ureylen-di-N,N0-ethan-2-yl)-dibenzoic acid (34b)
Following the general procedure for hydrolysis of esters with
LiOH, the reaction of N,N0-bis-2-[3-(methoxycarbonyl)-phen-1-
yl]-ethan-1-ylurea (0.07 g, 0.18 mmol), a 10% aqueous solution of
LiOH (0.04 mL, 1.82 mmol) in THF (5.6 mL) at 80 °C for 24 h affor-
ded, after purification by crystallization (EtOH/Et2O), 0.4 g (62%) of
a
solid identified as 3,30-(ureylen-di-N,N0-ethan-2-yl)dibenzoic
acid 34b. 1H NMR (400.13 MHz, DMSO-d6) d 7.78–7.76 (m, 4H,
ArH), 7.44–7.39 (m, 4H, ArH), 3.22 (t, J = 7.1 Hz, 4H, 2 ꢂ CH2),
2.72 (t, J = 7.1 Hz, 4H, 2 ꢂ CH2) ppm. 13C NMR (100.62 MHz,
DMSO-d6) d 167.4 (s, 2ꢂ), 158.0 (s), 140.2 (s, 2ꢂ), 133.3 (d, 2ꢂ),
130.8 (s, 2ꢂ), 129.6 (d, 2ꢂ), 128.6 (d, 2ꢂ), 127.1 (d, 2ꢂ), 40.8 (t,
2ꢂ), 35.9 (t, 2ꢂ) ppm. HMRS (ESI+): Calcd for C19H21N2O5
([M+H]+), 357.1445, found, 357.1429. IR (neat):
m 3360 (w, N–H),
C
19H21N2O5 ([M+H]+), 357.1445, found 357.1445. IR (neat):
m
3164 (w, N–H), 2943 (w, C–H), 1683 (s, C@O), 1626 (m, C@O),
1584 (m), 1419 (m) cmꢁ1. UV (MeOH): kmax 318, 284, 226 nm.
Mp: 213–215 °C (EtOH/Et2O). Purity trace: RPHPLC-ESI (SunfireÒ
3328 (w, N–H), 3009 (w, C–H), 2952 (w, C–H), 1714 (s, C@O),
1617 (s, C@O), 1564 (m), 1499 (m) cmꢁ1. UV (MeOH): kmax
287 nm. Mp: 180–182 °C (EtOH/Et2O).
C18
5
l
m, 250 ꢂ 46 mm, gradient from 95:5 to 0:100 H2O/CH3CN,
25 min, 1 mL/min, tR = 13.0 min; 94% purity).
5.2.10. N,N0-bis-2-[3-(Methoxycarbonyl)-phen-1-yl]-ethan-1-
ylurea (33b)
5.2.13. (E)-Ethyl 3-bromophenylacrylate (35)
Following the general procedure for the carbonylation reaction
with triphosgene, the reaction of methyl 3-(2-aminoethyl)benzo-
ate 31b (previously obtained from 2-[3-(methoxycarbonyl)-phen-
1-yl]-ethan-1-ammonium chloride 30b by treatment with Et3N)
(0.1 g, 0.61 mmol), Et3N (0.19 mL, 0.14 g, 1.38 mmol) and triphos-
gene 32 (0.09 g, 0.31 mmol) in benzene (15.7 mL) at 25 °C
overnight, and the reaction of the residue with another portion
of 3-(2-aminoethyl)benzoate 31b (0.1 g, 0.61 mmol) in acetone
(3.9 mL) afforded, after purification by column chromatography
(silica gel, from 95:5 CH2Cl2/MeOH to 90:10 CH2Cl2/MeOH),
0.10 g (86%) of a solid identified as N,N0-bis-2-[3-(methoxycar-
bonyl)-phen-1-yl]-ethan-1-ylurea 33b. 1H NMR (400.13 MHz,
CDCl3) d 7.89–7.86 (m, 4H), 7.39–7.34 (m, 4H), 4.24 (t, J = 5.5 Hz,
2H, NH), 3.90 (s, 6H, 2 ꢂ CH3), 3.44 (dd, J = 6.8, 5.5 Hz, 4H,
2 ꢂ CH2), 2.85 (t, J = 6.8 Hz, 4H, 2 ꢂ CH2) ppm. 13C NMR
(100.62 MHz, CDCl3): d 167.1 (s, 2ꢂ), 158.5 (s), 139.7 (s, 2ꢂ),
133.5 (d, 2ꢂ), 130.1 (s, 2ꢂ), 129.8 (d, 2ꢂ), 128.5 (d, 2ꢂ), 127.6 (d,
2ꢂ), 52.0 (q, 2ꢂ), 41.3 (t, 2ꢂ), 36.3 (t, 2ꢂ) ppm. HMRS (ESI+): Calcd
Following the general procedure for the Horner–Wadsworth–
Emmons reaction, the reaction of 3-bromobenzaldehyde 14a
(1.0 g, 0.63 mL, 5.40 mmol), ethyl 2-(diethoxyphosphoryl)acetate
20 (2.18 g, 1.95 mL, 9.73 mmol), n-BuLi (7.07 mL, 1.30 M in hexane,
9.19 mmol), DMPU (1.87 g, 1.76 mL, 14.59 mmol) in THF (9.1 mL)
afforded, after purification by column chromatography (silica gel,
95:5 hexane/EtOAc), 1.322 g (96%) of a solid identified as (E)-ethyl
3-bromophenylacrylate 35. 1H NMR (400.13 MHz, CDCl3): d 7.66 (t,
J = 1.7 Hz, 1H, ArH), 7.59 (d, J = 16.0 Hz, 1H, 1H), 7.49 (dt, J = 7.9,
1.0 Hz, 1H, ArH), 7.42 (d, J = 7.9 Hz, 1H, ArH), 7.24 (t, J = 7.9 Hz,
1H, ArH), 6.42 (d, J = 16.0 Hz, 1H), 4.26 (q, J = 7.1 Hz, 2H,
CO2CH2CH3), 1.33 (t, J = 7.1 Hz, 3H, CO2CH2CH3) ppm. 13C NMR
(100.62 MHz, CDCl3): d 166.6 (s), 142.9 (d), 136.7 (s), 133.1 (d),
130.8 (d), 130.5 (d), 126.8 (d), 123.1 (s), 119.9 (d), 60.8 (t), 14.4
(q) ppm. MS (EI): m/z (%) 256 ([M]+ 81[Br], 28), 254 ([M]+ 79[Br],
30), 228 (26), 211 (82), 209 (86), 183 (26), 102 (100). HMRS (EI):
Calcd for C11H11O281Br ([M]+ 81[Br]), 255.9922; found, 255.9929,
and Calcd for
253.9950. IR (NaCl): m 2981 (w, C–H), 1712 (s, C@O), 1639 (m),
C
11H11O279Br ([M]+ 79[Br]), 253.9942; found,
for C21H25N2O5 ([M+H]+), 385.1758, found 385.1769. IR (neat):
m
3309 (m, N–H), 2956(w, C–H), 2873 (w, C–H), 1720 (s, C@O),
1583 (s), 1434 (m), 1282(s), 1200 (s) cmꢁ1. UV (MeOH): kmax
283 nm. Mp: 182–184 °C (EtOH/Et2O).
1561 (w), 1473 (w), 1309 (s), 1177 (s) cmꢁ1. UV (MeOH): kmax
273, 223 nm. Mp: 34–36 °C (hexane/EtOAc).
5.2.14. (E)-Ethyl 3-aminophenylacrylate (36)
To a solution of (E)-ethyl 3-bromophenylacrylate 35 (0.55 g,
2.16 mmol) in DMSO/EtOH (31:16 mL) were added NaN3 (1.81 g,
5.2.11. 3,30-(Ureylen-di-N,N0-methyl)-dibenzoic acid (34a)
5.2.11.1. General procedure for hydrolysis of esters using
LiOH.
A 10% aqueous solution of LiOH (0.67 mL, 2.81 mmol)
27.81 mmol),
L-proline (0.22 g, 1.94 mmol), NaOH (0.08 g,
was added to N,N0-bis-(3-methoxycarbonyl-phen-1-yl-methyl)-
urea 33a (0.1 g, 0.28 mmol) in THF (8.6 mL) and the mixture was
stirred at 80 °C for 22 h. The solvent was evaporated and the resi-
due was purified by crystallization (MeOH/Et2O) to afford 0.076 g
(82%) of a solid identified as 3,30-(ureylen-di-N,N0-methyl)-diben-
zoic acid 34a. 1H NMR (400.13 MHz, DMSO-d6) d 12.95 (s, 2H,
OH), 7.86 (s, 2H, ArH), 7.80 (d, J = 7.6 Hz, 2H, ArH), 7.50 (d,
J = 7.6 Hz, 2H, ArH), 7.43 (t, J = 7.6 Hz, 2H, ArH), 6.66 (t, J = 6.1 Hz,
2H, NH), 4.29 (d, J = 6.1 Hz, 4H, 2 ꢂ CH2) ppm. 13C NMR
(100.62 MHz, DMSO-d6) d 167.4 (s, 2ꢂ), 158.1 (s), 141.6 (s, 2ꢂ),
131.5 (d, 2ꢂ), 130.7 (s, 2ꢂ), 128.5 (d, 2ꢂ), 127.8 (d, 2ꢂ), 127.5 (d,
2ꢂ), 42.7 (t, 2ꢂ) ppm. HMRS (ESI+): Calcd for C17H17N2O5
2.09 mmol) and CuI (0.37 g, 1.94 mmol) and the mixture was stir-
red at 110 °C for 20 h. The reaction was cooled down to 25 °C and a
1:1 mixture of EtOAc/H2O was added, and the undissolved solid
was filtered off. The layers were separated, the organic layer was
dried (Na2SO4), and the solvent was evaporated. The residue was
purified by chromatography (silica gel, from CH2Cl2 to 95:5
CH2Cl2/MeOH) to afford 0.233 g (56%) of an oil identified as (E)-
ethyl 3-aminophenylacrylate 36. 1H NMR (400.13 MHz, CD3OD):
d 7.56 (d, J = 16.0 Hz, 1H), 7.11 (t, J = 7.8 Hz, 1H, ArH), 6.92 (s, 1H,
ArH), 6.87 (d, J = 7.6 Hz, 1H, ArH), 6.75 (dd, J = 7.9, 1.6 Hz, 1H,
ArH), 6.40 (d, J = 16.0 Hz, 1H), 4.22 (q, J = 7.1 Hz, 2H, CO2CH2CH3),
1.31 (t, J = 7.1 Hz, 3H, CO2CH2CH3) ppm. 13C NMR (100.62 MHz,

N. Fontán et al. / Bioorg. Med. Chem. 21 (2013) 2056–2067
2065
CD3OD): d 168.8 (s), 149.5 (s), 146.8 (d), 136.4 (s), 130.6 (d), 119.2
(d), 118.6 (d), 118.2 (d), 115.2 (d), 61.6 (t), 14.6 (q) ppm. MS (EI):
m/z (%) 192 ([M+1]+, 12), 191 ([M]+, 100), 162 (22), 147 (30), 146
(85), 119 (51), 118 (55), 117 (43), 91 (37). HMRS (EI): Cacld for
5.2.17.2. General procedure for the synthesis of chlorosulpho-
nates. To a solution of 7-acetamidonaphthalene-2-sulphonic
acid (0.426 g, 1.613 mmol) in MeOH (4 mL) was added MeONa
(0.09 g, 1.61 mmol) and the mixture was stirred at 25 °C overnight.
The solid was filtered off and dried to afford 0.264 (57%) of a solid
identified as sodium 7-acetamidonaphthalene-2-sulphonate,
which was used in the next step without further purification.
To a solution of sodium 7-acetamidonaphthalene-2-sulphonate
(0.27 g, 0.93 mmol) was added phosphorus oxychloride (4.94 g,
32,185 mmol, 3 mL), the resulting suspension was cooled down
to 0 °C and N,N-dimethylacetamide (0.12 g, 1,40 mmol, 0.13 mL)
was added dropwise. The mixture was warmed up to 25 °C and
stirred for 24 h. Then the suspension was poured into ice-cold
H2O (2 mL) and the mixture was stored for 3 days in the fridge.
Then more H2O was added and the precipitate was filtered off
and washed with H2O to obtain 0.115 (44%) of a white solid iden-
tified as 7-acetamidonaphthalene-2-sulphonyl chloride 40. 1H
NMR (400.13 MHz, DMSO-d6) d 10.25 (s, 1H, N–H), 8.23 (s, 1H,
N–H), 8.00 (s, 1H, ArH), 7.83 (d, J = 8.8 Hz, 1H, ArH), 7.78 (d,
J = 8.5 Hz, 1H, ArH), 7.69 (dd, J = 8.8, 1.8 Hz, 1H, ArH), 7.60 (dd,
J = 8.5, 1.4 Hz, 1H, ArH), 2.10 (s, 3H, CH3) ppm. 13C NMR
(100.62 MHz, DMSO-d6) d 168.8 (s), 145.5 (s), 137.6 (s), 132.6 (s),
129.5 (s), 128.1 (d), 127.4 (d), 123.7 (d), 122.4 (d), 120.7 (d),
115.6 (d), 24.2 (q) ppm. HMRS (ESI+): Calcd for C12H11ClNO3S
C11H13NO2, 191.0946; found, 191.0947. IR (NaCl): m 3456 (w, N–
H), 3367 (w, N–H), 2924 (m, C–H), 2853 (w, C–H), 1703 (s, C@O),
1633 (s), 1602 (m), 1305 (m), 1176 (s) cmꢁ1. UV (MeOH): kmax
283, 251 nm.
5.2.15. N,N0-bis-[3-(Ethoxycarbonyl-ethen-2-yl)-phen-1-yl]urea
(37)
Following the general procedure for the carbonylation reaction
using triphosgene, the reaction of (E)-ethyl 3-aminophenylacrylate
36 (0.17 g, 0.89 mmol), Et3N (0.20 g, 0.28 mL, 2.0 mmol) and tri-
phosgene (0.13 g, 0.44 mmol) in benzene (23.4 mL) at 25 °C over-
night, and then the reaction of the residue with another portion
of (E)-ethyl 3-aminophenylacrylate 36 (0.17 g, 0.89 mmol) in ace-
tone (5.7 mL) at 25 °C for 2 h afforded, after purification by chro-
matography (silica gel, from 80:20 hexane/EtOAc to 50:50
hexane/EtOAc) and crystallization (EtOH/Et2O), 0.129 g (71%) of a
solid identified as N,N0-bis-[3-(ethoxycarbonyl-ethen-2-yl)-phen-
1-yl]urea 37. 1H NMR (400.13 MHz, DMSO-d6): d 8.84 (s, 2H,
NH), 7.76 (s, 2H, ArH), 7.62 (d, J = 16.0 Hz, 2H), 7.50 (d, J = 6.7 Hz,
2H, ArH), 7.37–7.32 (m, 4H, ArH), 6.54 (d, J = 16.0 Hz, 2H), 4.20
(q, J = 7.1 Hz, 4H, CO2CH2CH3), 1.27 (t, J = 7.1 Hz, 6H, CO2CH2CH3)
ppm. 13C NMR (100.62 MHz, DMSO-d6) d 166.1 (s, 2ꢂ), 152.6 (s),
144.4 (d, 2ꢂ), 140.1 (s, 2ꢂ), 134.5 (s, 2ꢂ), 129.4 (d, 2ꢂ), 121.8 (d,
2ꢂ), 120.5 (d, 2ꢂ), 118.2 (d, 2ꢂ), 118.0 (d, 2ꢂ), 60.1 (t, 2ꢂ), 14.2
(q, 2ꢂ) ppm. HMRS (ESI+): Calcd for C23H25N2O5 ([M+H]+),
([M+H]+), 284.0143, found, 284.0140. IR (neat):
m 3257 (m, N–H),
3081 (w, C–H), 1659 (s, C@O), 1556 (s, C@O), 1366 (s), 1167 (s)
cmꢁ1. UV (MeOH): kmax 258 nm. Mp: 144–146 (MeOH/H2O).
5.2.18. N-[7-(N0-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-
sulphamoyl)-naphthalen-2-yl]-acetamide (42a)
409.1758; found, 409.1743. IR (neat):
m 3291 (m, N–H), 2990 (w,
C–H), 1714 (s, C@O), 1628 (w, C@O), 1559 (s), 1477 (m), 1314
(s), 1175 (s) cmꢁ1. UV (MeOH): kmax 282 nm. Mp: 182–184 °C
(EtOH/Et2O).
5.2.18.1. General procedure to the formation of sulphona-
mides.
To the solution of 2,3-dihydrobenzo[b][1,4]dioxin-6-
amine 41a (0.27 g, 1.77 mmol), N,N-diisopropylamine (0.3 g,
0.5 mL, 2.97 mmol) in CHCl3 (2 mL) was added 7-acetamidonaph-
thalene-2-sulphonyl chloride 40 (0.2 g, 0.71 mmol) in a 1:1 THF/
CHCl3 mixture (4 mL). The reaction mixture was stirred overnight
at 25 °C, then the solvent was evaporated and the residue was puri-
fied by crystallization (H2O, adjusting to pH 3 with a 37% saturated
aqueous solution of HCl) to afford 0.151 g (54%) of a solid identified
5.2.16. (E,E)-3,30-(Ureylendi-N,N0-phen-3-yl)-diacrylic acid (38)
Following the general procedure for hydrolysis of esters, the reac-
tion of N,N0-bis-[3-(ethoxycarbonyl-ethen-2-yl)-phen-1-yl]urea 37
(0.06 g, 0.15 mmol), a 10% aqueous solution of LiOH (0.04 mL,
1.5 mmol) in THF (4.6 mL) at 80 °C for 22 h afforded, after purifica-
tion by crystallization (EtOH/Et2O), 0.045 g (86%) of a solid identified
as (E,E)-3,30-(ureylendi-N,N0-phen-3-yl)-diacrylic acid 38. 1H NMR
(400.13 MHz, DMSO-d6) d 12.45 (s, 2H, CO2H), 8.85 (s, 2H), 7.72 (s,
2H, ArH), 7.56 (d, J = 15.9 Hz, 2H), 7.50 (d, J = 6.4 Hz, 2H, ArH),
7.36–7.33 (m, 4H, ArH), 6.45 (d, J = 15.9 Hz, 2H) ppm. 13C NMR
(100.62 MHz, DMSO-d6) d 167.4 (s, 2ꢂ), 152.6 (s), 144.0 (d, 2ꢂ),
140.1 (s, 2ꢂ), 134.7 (s, 2ꢂ), 129.4 (d, 2ꢂ), 121.8 (d, 2ꢂ), 120.3 (d,
2ꢂ), 119.2 (d, 2ꢂ), 117.7 (d, 2ꢂ) ppm. HMRS (ESI+): Calcd for
as
N-[7-(N0-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-sulphamoyl)-
naphthalen-2-yl]-acetamide 42a. 1H NMR (400.13 MHz, DMSO-
d6) d 10.30 (s, 1H, N–H), 10.05 (s, 1H, N–H), 8.36 (s, 1H, ArH),
8.18 (s, 1H, ArH), 7.99 (d, J = 8.7 Hz, 1H, ArH), 7.94 (d, J = 8.9 Hz,
1H, ArH), 7.74 (dd, J = 8.9, 1.8 Hz, 1H, ArH), 7.61 (dd, J = 8.6,
1.6 Hz, 1H, ArH), 6.67 (d, J = 8.6 Hz, 1H, ArH), 6.59 (d, J = 2.4 Hz,
1H, ArH), 6.54 (dd, J = 8.6, 2.5 Hz, 1H, ArH), 4.11 (s, 4H, 2 ꢂ CH2),
2.11 (s, 3H, CH3) ppm. 13C NMR (100.62 MHz, DMSO-d6) d 169.0
(s), 143.2 (s), 140.6 (s), 138.5 (s), 137.0 (s), 132.2 (s), 130.9 (s),
130.8 (s), 129.1 (d), 128.6 (d), 127.0 (d), 122.6 (d), 120.7 (d),
117.3 (d), 115.7 (d), 114.3 (d), 110.2 (d), 64.1 (t), 63.8 (t), 24.2 (q)
ppm. HMRS (ESI+): Calcd for C20H19N2O5S ([M+H]+), 399.1009,
C
19H17N2O5 ([M+H]+), 353.1132; found, 353.1130. IR (neat):
m 3306
(w, N–H), 2957 (w, C–H), 2820 (w, C–H), 1684 (s, C@O), 1635 (s,
C@O), 1588 (m), 1555 (m) 1438 (w) cmꢁ1. UV (MeOH): kmax
258 nm. Mp: >300 °C (EtOH/Et2O, dec.). Purity trace: RPHPLC-ESI
(SunfireÒ C18
5
l
m, 250 ꢂ 46 mm, gradient from 95:5 to 0:100
found, 399.1021. IR (neat): m 3263 (m, N–H), 3080 (w, C–H),
H2O/CH3CN, 25 min, 1 mL/min, tR = 14.4 min; 100% purity).
1666 (m, C@O), 1587 (m), 1558 (m), 1505 (m), 1317 (s) cmꢁ1. UV
(MeOH): kmax 254 nm. Mp: >300 °C (H2O, dec.).
5.2.17. 7-Acetamidonaphthalene-2-sulphonyl chloride (40)
5.2.17.1. General procedure for the protection of ani-
5.2.19. N-[7-(N0-4-Phenoxyphen-1-yl-sulphamoyl)-naphthalen-
2-yl]-acetamide (42b)
lines.
To a cooled (0 °C) solution of sodium 7-aminonaphtha-
Following the general procedure to the formation of sulphona-
mides, the reaction of 7-acetamidonaphthalene-2-sulphonyl
chloride 40 (0.1 g, 0.35 mmol) in a 1:1 THF/CHCl3 mixture (2 mL),
4-phenoxyaniline 41b (0.09 g, 0.76 mL) and N,N-diisopropylamine
(0.13 g, 0.26 mL, 1.5 mmol) in CHCl3 (1 mL) afforded 0.067 g (44%)
of a white solid identified as N-[7-(N0-4-phenoxyphen-1-yl-sulpha-
moyl)-naphthalen-2-yl]-acetamide 42b. 1H NMR (400.13 MHz,
DMSO-d6) d 10.28 (s, 1H, N–H), 10.21 (s, 1H, N–H), 8.37 (s, 1H,
ArH), 8.18 (s, 1H, ArH), 8.00 (d, J = 8.7 Hz, 1H, ArH), 7.94 (d,
lene-2-sulphonate 39 (2.0 g, 8.16 mmol) in pyridine (10 mL) was
added dropwise acetic anhydride (10 mL) and the reaction was al-
lowed to warm to 25 °C and stirred for 8 h. The suspension was
poured into a mixture of Et2O (35 mL) and THF (10 mL). The result-
ing solid was filtered off and washed with H2O to afford 1.81 g
(84%) of a white solid identified as 7-acetamidonaphthalene-2-sul-
phonic acid, which was used in the next reaction without further
purification.

2066
N. Fontán et al. / Bioorg. Med. Chem. 21 (2013) 2056–2067
J = 8.9 Hz, 1H, ArH), 7.74 (dd, J = 8.9, 2.0 Hz, 1H, ArH), 7.63 (dd,
J = 8.6, 1.8 Hz, 1H, ArH), 7.4–7.3 (m, 2H, ArH), 7.2–7.0 (m, 3H,
ArH), 6.9–6.8 (m, 4H, ArH), 2.11 (s, 3H, CH3) ppm. 13C NMR
(100.62 MHz, DMSO-d6) d 168.9 (s), 156.9 (s), 153.1 (s), 138.4 (s),
136.9 (s), 133.2 (s), 132.2 (s), 130.8 (s), 130.0 (d, 2ꢂ), 129.1 (d),
128.6 (d), 127.1 (d), 123.2 (d), 122.8 (d, 2ꢂ), 122.6 (d), 120.6 (d),
119.6 (d, 2ꢂ), 118.1 (d, 2ꢂ), 115.7 (d), 24.1 (q) ppm. HMRS
(ESI+): Calcd for C24H21N2O4S ([M+H]+), 433.1217, found,
(d, J = 9.0 Hz, 2H, ArH), 7.76 (dd, J = 8.8, 1.5 Hz, 2H, ArH), 7.61 (dd,
J = 8.7, 1.3 Hz, 2H, ArH), 6.68 (d, J = 8.7 Hz, 2H, ArH), 6.61 (d,
J = 2.4 Hz, 2H, ArH), 6.56 (dd, J = 8.6, 2.4 Hz, 2H, ArH), 4.12 (s, 8H,
4 ꢂ CH2) ppm. 13C NMR (100.62 MHz, DMSO-d6) d 152.6 (s), 143.2
(s, 2ꢂ), 140.5 (s, 2ꢂ), 138.7 (s, 2ꢂ), 137.1 (s, 2ꢂ), 132.4 (s, 2ꢂ),
130.9 (s, 2ꢂ), 130.2 (s, 2ꢂ), 129.02 (d, 2ꢂ), 128.7 (d, 2ꢂ), 126.7 (d,
2ꢂ), 122.4 (d, 2ꢂ), 120.2 (s, 2ꢂ), 117.2 (d, 2ꢂ), 114.5 (d, 2ꢂ),
114.3 (d, 2ꢂ), 110.1 (d, 2ꢂ), 64.0 (t, 4ꢂ), 63.8 (t, 4ꢂ) ppm. HMRS
(ESI+): Calcd for C37H30N4NaO9S2 ([M+Na]+), 761.1346, found,
433.1204. IR (neat):
m 3243 (m, N–H), 3100 (w, C–H), 1660 (m,
C@O), 1585 (m), 1558 (m), 1497 (m), 1328 (m), 1154 (s) cmꢁ1
UV (MeOH): kmax 247 nm. Mp: >300 (H2O, dec.).
.
761.1354. IR (neat): m 3410 (w, N–H), 3365 (w, N–H), 3254 (w, N–
H), 2929 (w, N–H), 2878 (w, C–H), 1718 (w, C@O), 1541 (m), 1503
(m), 1147 (s) cmꢁ1. UV (MeOH): kmax 263, 240 nm. Mp: 258–
260 °C (CH2Cl2/MeOH).
5.2.20. 7-[N-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-
sulphamoyl]-naphthalen-2-ammonium chloride (43a)
5.2.20.1. General procedure for deprotection of amines.
To a
5.2.23. N,N0-bis-[7-(N-(4-Phenoxyphenyl)-sulphamoyl)-
solution of N-[7-(N0-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-sulpha-
moyl]-naphthalen-2-yl)acetamide 42a (0.08 g, 0.19 mmol) in MeOH
(1.2 mL) was added a 5 M aqueous solution of NaOH (0.38 g, 0.38 mL,
9.41 mmol) and the resulting solution was heated at 60 °C for 120 h.
The mixture was cooled down and then acidified with a 6 M aqueous
solution of HCl until pH 1. The resulting solid was filtered
off and then the solvent was evaporated to afford, 0.067 g (91%) of
a solid identified as 7-[N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-
sulphamoyl]-naphthalen-2-ammonium chloride 43a. 1H NMR
(400.13 MHz, DMSO-d6) d 10.06 (s, 1H, N–H), 8.04 (s, 1H), 7.87 (d,
J = 8.6 Hz, 1H, ArH), 7.79 (d, J = 8.8 Hz, 1H, ArH), 7.45 (d, J = 8.6 Hz,
1H, ArH), 7.24 (d, J = 8.2 Hz, 2H, ArH), 6.66 (d, J = 8.6 Hz, 1H, ArH),
6.60 (d, J = 2.4 Hz, 1H, ArH), 6.55 (dd, J = 8.6, 2.5 Hz, 1H, ArH), 4.11
(s, 4H, 2 ꢂ CH2) ppm. HMRS (ESI+): Calcd for C18H17N2O4S ([MꢁCl]+),
naphthalene-2-yl]-urea (45b)
Following the general procedure for the carbonylation with tri-
phosgene, the reaction of 7-[amino-N-(4-phenoxyphenyl)]-naph-
thalene-2-sulphonamide 44b (previously obtained from corresponding
ammoniun chloride 43b by treatment with Et3N) (0.09 g,
0.22 mmol), Et3N (0.07 mL, 0.05 g, 0.5 mmol) and triphosgene 32
(0.03 g, 0.11 mmol) in benzene (5.7 mL) and then the reaction of
the residue with another portion of 7-[amino-N-(4-phenoxyphe-
nyl)]-naphthalene-2-sulphonamide 44b (0.09 g, 0.22 mmol) in
acetone (1.7 mL) afforded, after purification by column chromatog-
raphy (silica gel, from 97.5:2.5 CH2Cl2/MeOH to 95:5 CH2Cl2/
MeOH), 0.021 g (23%) of a solid identified as N,N0-bis-[7-(N-(4-
phenoxyphenyl)-sulfamoyl)-naphthalene-2-yl]-urea 45b, which
still contained small amounts of triethylammonium chloride. 1H
NMR (400.13 MHz, DMSO-d6) d 10.22 (s, 2H, 2 ꢂ NH), 10.01 (s,
2H, 2 ꢂ NH), 8.15 (d, J = 11.0 Hz, 4H, ArH), 7.98 (d, J = 8.6 Hz, 2H,
ArH), 7.93 (d, J = 8.9 Hz, 2H, ArH), 7.69 (dd, J = 8.9, 2.0 Hz, 2H,
ArH), 7.61 (dd, J = 8.6, 1.6 Hz, 2H, ArH), 7.31 (t, J = 7.9 Hz, 4H,
ArH), 7.14–7.03 (m, 6H, ArH), 6.89–6.85 (m, 8H, ArH) ppm. 13C
NMR (100.62 MHz, DMSO-d6) d 156.9 (s), 153.6 (s, 2ꢂ), 153.1 (s,
2ꢂ), 138.5 (s, 2ꢂ), 136.9 (s, 2ꢂ), 133.1 (s, 2ꢂ), 132.2 (s, 2ꢂ),
130.4 (s, 2ꢂ), 129.9 (d, 4ꢂ), 129.0 (d, 2ꢂ), 128.6 (d, 2ꢂ), 126.8 (d,
2ꢂ), 123.2 (d, 2ꢂ), 122.8 (d, 4ꢂ), 122.1 (d, 2ꢂ), 120.3 (d, 2ꢂ),
357.0904, found, 357.0911. IR (neat):
m 3400–2600 (br, N–H), 2869
(w, C–H), 1625 (w), 1595 (w), 1502 (s) cmꢁ1. UV (MeOH): kmax
248 nm. Mp: >300 °C (H2O, dec.).
5.2.21. 7-[N-(4-Phenoxyphen-1-yl)-sulphamoyl]-naphthalen-2-
ammonium chloride (43b)
Following the general procedure for the deprotection of amines,
the reaction of N-[7-(N0-(4-phenoxyphenyl)-sulphamoyl)-naph-
thalen-2-yl]-acetamide (0.07 g, 0.15 mmol) and a 5 M aqueous
solution of NaOH (0.43 g, 0.42 mL, 10.79 mmol) in MeOH (1 mL)
at 60 °C for 120 h afforded 0.058 g (88%) of a solid identified as
7-[N-(4-phenoxyphen-1-yl)-sulphamoyl]-naphthalen-2-ammonium
chloride 43b. 1H NMR (400.13 MHz, DMSO-d6) d 10.29 (s, 1H, N–H),
8.11 (s, 1H, ArH), 7.93 (d, J = 8.6 Hz, 1H, ArH), 7.86 (d, J = 8.7 Hz, 1H,
ArH), 7.53 (d, J = 8.5 Hz, 1H, ArH), 7.38 (s, 1H, ArH), 7.32 (t,
J = 7.7 Hz, 3H, ArH), 7.11–7.05 (m, 3H, ArH), 6.86 (d, J = 8.8 Hz,
4H, ArH) ppm. HMRS (ESI+): Calcd for C22H19N2O3S ([MꢁCl]+),
119.6 (d, 4ꢂ), 118.1 (d, 4ꢂ), 114.3 (d, 2ꢂ) ppm. IR (neat):
m 3248
(w, N–H), 3063 (w, C–H), 2978 (w, C–H), 1707 (m, C@O), 1590
(m), 1542 (m), 1496 (s), 1215 (s), 1153 (s) cmꢁ1. UV (MeOH): kmax
246 nm.
Acknowledgements
This work was supported by the European Union LSHC-CT-
2005-518417 ‘Epitron’, MINECO (SAF2010-17935 FEDER, FPU Fel-
lowships to N.F. and P. G.-D.) and Xunta de Galicia (Consolidación,
INBIOMED-FEDER ‘Unha maneira de facer Galicia’). The authors
wish to thank Dr. Hortensia Faus and Dr. Bernard Haendler (Global
Drug Discovery, Bayer Pharma AG) for invaluable help with the
biochemistry experiments.
391.1111, found, 391.1105. IR (neat):
m 3200–2600 (br, N–H),
2853 (w, C–H), 1590 (m), 1498 (s) cmꢁ1. UV (MeOH): kmax
246 nm. Mp: >300 °C (H2O, dec.).
5.2.22. N,N0-bis-[7-(N-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-
sulphamoyl)-naphthalene-2-yl]-urea (45a)
Following the general procedure for the carbonylation with
triphosgene, the reaction of 7-amino-N-(2,3-dihydrobenzo[b][1,4]
dioxin-6-yl)-naphthalene-2-sulfonamide 44a (previously obtained
from the corresponding ammoniun chloride 43a by treatment with
Et3N) (0.07 g, 0.19 mmol), Et3N (0.06 mL, 0.04 g, 0.42 mmol) and
triphosgene 32 (0.03 g, 0.09 mmol) in benzene (4.9 mL) and then
reaction of the residue with another portion of 7-amino-N-(2,3-
dihydrobenzo[b][1,4]dioxin-6-yl)naphthalene-2-sulphonamide
(0.07 g, 0.19 mmol) in acetone (1.4 mL) afforded, after purification
by column chromatography (silica gel, 90:10 CH2Cl2/MeOH),
4.9 mg (7%) of a solid identified as N,N0-bis-[7-(N-(2,3-dihydro-
benzo[b][1,4]dioxin-6-yl)-sulphamoyl)-naphthalene-2-yl]-urea 45a.
1H NMR (400.13 MHz, DMSO-d6) d 10.05 (s, 2H, 2 ꢂ NH), 9.22 (s,
2H, 2 ꢂ NH), 8.21 (s, 4H, ArH), 8.00 (d, J = 8.8 Hz, 2H, ArH), 7.96
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.017.
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