Acyl derivatives of p-aminosulfonamides and dapsone as new inhibitors of the arginine methyltransferase hPRMT1

Article abstract of DOI:10.1016/j.bmc.2011.02.032

Arginine methylation is an epigenetic modification that receives increasing interest as it plays an important role in several diseases. This is especially true for hormone-dependent cancer, seeing that histone methylation by arginine methyltransferase I (PRMT1) is involved in the activation of sexual hormone receptors. Therefore, PRMT inhibitors are potential drugs and interesting tools for cell biology. A dapsone derivative called allantodapsone previously identified by our group served as a lead structure for inhibitor synthesis. Acylated derivatives of p-aminobenzenesulfonamides and the antilepra drug dapsone were identified as new inhibitors of PRMT1 by in vitro testing. The bis-chloroacetyl amide of dapsone selectively inhibited human PRMT1 in the low micromolar region and was selective for PRMT1 as compared to the arginine methyltransferase CARM1 and the lysine methyltransferase Set7/9. It showed anticancer activity on MCF7a and LNCaP cells and blocked androgen dependent transcription specifically in a reporter gene system. Likewise, a transcriptional block was also demonstrated in LNCaP cells using quantitative RT-PCR on the mRNA of androgen dependent genes.

Full text of DOI:10.1016/j.bmc.2011.02.032

Bioorganic & Medicinal Chemistry 19 (2011) 3717–3731
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Acyl derivatives of p-aminosulfonamides and dapsone as new inhibitors
of the arginine methyltransferase hPRMT1
Elisabeth-Maria Bissinger a, Ralf Heinke b, Astrid Spannhoff c, Adrien Eberlin d, Eric Metzger d,
Vincent Cura e, Pierre Hassenboehler e, Jean Cavarelli e, Roland Schüle d, Mark T. Bedford c,
Wolfgang Sippl b, Manfred Jung a,
⇑
a Institute of Pharmaceutical Sciences, Albert-Ludwigs-University of Freiburg, Germany
b Department of Pharmaceutical Chemistry, Martin-Luther University of Halle-Wittenberg, 06120 Halle/Saale, Germany
c Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithville, TX 78957, USA
d Department of Urology/Women’s Hospital and Center for Clinical Research, University of Freiburg Medical Center, Freiburg, Germany
e Département de Biologie et Génomique Structurales, Institute de génétique et de biologie moléculaire et cellulaire, 67404 Illkirch Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Arginine methylation is an epigenetic modification that receives increasing interest as it plays an impor-
tant role in several diseases. This is especially true for hormone-dependent cancer, seeing that histone
methylation by arginine methyltransferase I (PRMT1) is involved in the activation of sexual hormone
receptors. Therefore, PRMT inhibitors are potential drugs and interesting tools for cell biology. A dapsone
derivative called allantodapsone previously identified by our group served as a lead structure for inhib-
itor synthesis. Acylated derivatives of p-aminobenzenesulfonamides and the antilepra drug dapsone were
identified as new inhibitors of PRMT1 by in vitro testing. The bis-chloroacetyl amide of dapsone selec-
tively inhibited human PRMT1 in the low micromolar region and was selective for PRMT1 as compared
to the arginine methyltransferase CARM1 and the lysine methyltransferase Set7/9. It showed anticancer
activity on MCF7a and LNCaP cells and blocked androgen dependent transcription specifically in a repor-
ter gene system. Likewise, a transcriptional block was also demonstrated in LNCaP cells using quantita-
tive RT-PCR on the mRNA of androgen dependent genes.
Received 9 December 2010
Revised 15 February 2011
Accepted 19 February 2011
Available online 27 February 2011
Keywords:
Arginine methyltransferase
PRMT1
Allantodapsone
Androgen
Cancer
S-Adenosylmethionine
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Stilbamidine and allantodapsone showed indirect antiestrogen
properties in a reporter gene based assay,5 thus in this study we
Epigenetics is defined as inheritable changes in the phenotype
without changes in the genome.1 The major biochemical mecha-
nisms behind epigenetics are DNA methylation and posttransla-
tional histone modifications such as acetylation or methylation.2
Histone methylation is mediated by histone methyltransferases,
which can be divided into lysine and arginine methyltransferases.
These enzymes have repeatedly been linked to cancer formation
and progression and have therefore emerged as interesting targets
for drug discovery.3 Especially arginine methyltransferases also
target non-histone protein substrates and are therefore often
called protein methyltransferases (PRMTs).3 The subtype PRMT1
is particularly interesting as it has been linked to the activation
of estrogen and androgen4–6 receptors and therefore may represent
a new treatment option for hormone-dependent cancer.5 So far,
only a limited number of inhibitors are available for PRMT1.5,6
Examples are AMI-1,4,5,7–9 inhibitor 6e,7 stilbamidine,5 allantodap-
sone5 and C-72809488,9 (see Fig. 1).
set out to synthesize analogs of allantodapsone. Due to the poten-
tial lability of the aminal structure we wanted to prepare more sta-
ble simple acyl derivatives of allantodapsone as well as similar
derivatives of C-7280948.8 The synthesis of the modified analogs
of these lead inhibitors was based on docking studies carried out
using structural information of the PRMT1 protein. Inspection of
potential interaction points at the substrate binding pocket of
PRMT1 was used to guide the synthesis of the new analogs (details
in the Modelling section). These new compounds were tested for
PRMT1 inhibition in vitro and inhibitors in the low micromolar re-
gion were obtained. A chloroacetyl derivative showed potent anti-
proliferative activity and blocked androgen-dependent protein
expression in a reporter gene model.
2. Chemistry
2.1. Analogs of allantodapsone
Acyl analogs of allantodapsone were synthesized by adding acyl
chlorides at room temperature to commercially available dapsone
⇑
Corresponding author. Tel.: +49 761 203 4896; fax: +49 761 203 6351.
E-mail address: manfred.jung@pharmazie.uni-freiburg.de (M. Jung).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.032

3718
E.-M. Bissinger et al. / Bioorg. Med. Chem. 19 (2011) 3717–3731
O
H2N
Br
O-
NH
NH2
S
O
Na+
OH
COOH
Br
OH
Br
O
H2N
O-
HO
O
O
N
H
N
H
S
Br
Br
Na+
O
NH
O
AMI-1
Inhibitor 6e
Stilbamidine
O
S
O
O
S
O
N
H
O
O
NH2
O
N
H
N
H2N
O
N
H
Allantodapsone
C-7280948
Figure 1. Examples of known PRMT1 inhibitors.
in pyridine (see Fig. 2a). In most cases diacetylation products were
formed as well. Due to their very similar physicochemical behav-
iour, mono- and diacetylated compounds were difficult to separate
from each other by column chromatography and only a limited
amount of the respective compounds could be obtained in a pure
form. Although typical yields were below 10%, the easy accessibil-
ity of dapsone allowed collection of sufficient amounts of testing
material and did not require optimization of the synthesis.
To analyze the role of the amide function, we also synthesized a
sulfonamide and an urea analogue. Using 4-chlorobenzenesulfonyl
chloride, which was conjugated with dapsone (see Fig. 2b), we ob-
tained compound 3 as a sulfonyl analogue of 1b. To obtain the urea
derivative 4, benzyl isocyanate and dapsone were used as the start-
ing materials (see Fig. 2b).
give a set of acylated derivatives. Since the first inhibition studies
with the acylated dapsones (see below) revealed a higher potency
for derivatives containing an aromatic residue, only aromatic acyl
chlorides were used as reagents. To obtain an acylated derivative
that would again bear a p-amino function for a possible hydrogen
bond interaction with the active site, derivative 7h (see Fig. 3a)
was reduced with stannous chloride dihydrate to the aniline 7i.
Again, 4-chlorobenzenesulfonyl chloride was used to synthesize a
representative sulfonamide analogue 7j (see Fig. 3a).
For further structural variation of the lead compound 6a, we re-
duced the spacer length between the sulfonamide nitrogen and the
aromatic substituent, also introducing branched chain substitu-
ents. 4-Nitrobenzenesulfonyl chloride was treated with benzyl
amine or racemic
a-methylbenzyl amine, respectively, using the
same conditions as for the synthesis of 6a (see Fig. 3a). For further
structural variation, substituted benzyl and phenethyl amines
were also used as starting materials. The nitro group was again
reduced to the corresponding amine by the use of stannous
chloride dihydrate. Additionally, the benzene in the amine part
2.2. Acylated derivatives of C-7280948
A similar approach was used with the lead inhibitor C-7280948,
which was also treated with various acyl chlorides (see Fig. 3a) to
O
S
O
S
O
S
a
O
N
O
O
O
+
+
O
O
O
Cl
R
R
N
H
R
N
H
R
N
H
H2N
NH2
H2N
R=
Compound
2d
R=
CH3
Compound
1a
R=
Compound
1d
R=
Compound
2a
CH3
2b
CH3
1b
1c
2e
Cl
1e
Cl
2c
b
O
S
O
O
S
O
S
O
S
O
Cl
C
N
O
O
O
Cl
O
O
S
N
H
NH2
NH2
N
N
NH2
H2N
O
H
H
dapsone
4
3
Cl
Figure 2. (a) Route for the synthesis of acylated dapsone derivatives with monoacylated and diacylated dapsone derivatives, (b) synthesis of a sulfonyl and an urea derivative
of dapsone.

E.-M. Bissinger et al. / Bioorg. Med. Chem. 19 (2011) 3717–3731
3719
O
S
O
a
O
S
O
SnCl2 x 2H2O
rt
N
H
Cl
+
H2N
Δ
NMM
O2N
5a
O2N
O
O
S
O
S
O
RCOCl (7a-g)
for 7h
N
H
N
O
7i
H
SnCl2 x 2H2O, Δ
N
6a
R
H2N
7a-g
H
4-Chlorophenylsulfonyl chloride
7j
O
S
O
7a: R =
7d: R =
7e: R =
7g: R =
7j:
O
S
O
N
H
I
Cl
Cl
7b: R =
7c: R =
7h: R =
7i: R =
N
H
Cl
Cl
NO2
7f: R =
Br
NH2
b
O
S
O
R2
O
S
O
R2
O
S
O
H
R2
SnCl2 x 2 H2O,
X
X
X
5h: KOH
H2N
n
N
N
Cl
n
n
H
Δ
R1
R3
R1
R2
R1
R3
5b: R1 = NO2, R2 = H, R3 = H, X = C, n = 1
5c: R1 = NO2, R2 = CH3, R3 = H, X = C, n = 1
5d: R1 = NO2, R2 = H, R3= H, X = N, n = 1
5e: R1 = NO2, R2 = H, R3 = Cl, X = C, n = 1
5f: R1 = NO2, R2 = H, R3 = OCH3, X = C, n = 1
5g: R1 = NO2, R2 = H, R3 = Cl, X = C, n = 2
6b: R1 = NH2, R2 = H, R3 = H, X = C, n = 1
6c: R1 = NH2, R2 = CH3, R3 = H, X = C, n = 1
6d: R1 = NH2, R2 = H, R3 = H, X = N, n = 1
6e: R1 = NH2, R2 = H, R3 = Cl, X = C, n = 1
6f: R1 = NH2, R2 = H, R3= OCH3, X = C, n = 1
6g: R1 = NH2, R2 = H, R3 = Cl, X = C,n = 2
6h: R1 = NH2, R2 = H, R3 = OH, X = C, n = 2
6i: R1 = Cl, R2 = H, R3 = H, X = C, n = 2
5h: R1 = NO2, R2= H, R3 = OSO2C6H4NO2, X = C, n = 2
Figure 3. (a) Synthesis of acylated derivatives of C-7280948 (6a) and (b) derivatives with varied aliphatic side chain length.
a
b
R1
R1
O
S
O
O
S
O
O
S
O
SnCl2 x 2 H2O,
NH2
5h: KOH
Cl
N
H
N
H
Δ
R1
R2
R2
R2
5g: R1 = Cl, R2 = NO2
5h: R1 = OSO2C6H4NO2, R2 = NO2
6g: R1 = Cl, R2 = NH2
6h: R1 = OH, R2 = NH2
6i: R1 = H, R2 = Cl
O
R
O
R
O
R
SnCl2 x 2 H2O
Cl
nN
nH
N
nNH2
H
Δ
NO2
NH2
NO2
9a: R = H, n = 2
9b: R = H, n = 1
9c: R = CH3, n = 1
8a: R = H, n = 2
8b: R = H, n = 1
8c: R = CH3, n = 1
Figure 4. (a) Synthesis of derivatives of 6a with p-substituted phenethylamides, (b) synthesis of carbamide analogues of 6a with varied aliphatic side chain.

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E.-M. Bissinger et al. / Bioorg. Med. Chem. 19 (2011) 3717–3731
was replaced by a pyridine in a representative compound (6d, see
Fig. 3b).
with the GRID, fields of the binding ideas for the derivatization of
both compounds were obtained.
Furthermore, polar substituents were introduced to give rise to
the substituted phenethyl amides 6g–i (see Fig. 4a). For the synthe-
sis of carbamide derivates of 6a, phenethyl and benzyl amines
were treated with acid chlorides. Again, the aniline was synthe-
sized from the nitro precursor by reduction with stannous chloride
(see Fig. 4b).
3.1. Ligand docking
All compounds were docked in the hPRMT1 substrate binding
pocket using the GOLD 4.0 docking software (Cambridge Crystallo-
graphic Centre).11 The top-ranked docking solution obtained for
compounds 1a–e, 2a, 2b, 2e, 3, and 4 was similar to the one ob-
tained for the original compound allantodapsone, with the aro-
matic amino group interacting with Glu152 of the catalytic site
(1e, see Fig. 6). The docking of compound 2a and 2b showed that
the modification of the amino group with an alkyl group (ethyl
or n-propyl) is sterically unfavourable. Compounds 2c and 2d as
well as the anilide derivatives of C-7280948 (7a–j) could not be
docked into the substrate binding pocket due to steric clashes of
the aromatic ring attached to the anilide group and the SAM cofac-
tor. However, when docking the inhibitors into the PRMT1 struc-
ture without the bound cofactor SAM, a favourable interaction
with cofactor and substrate binding pocket could be observed.
The orientation of the most active compounds 2c and 7e is shown
exemplarily in Figures S3 and S4.
3. Molecular modelling
Since there is no crystal structure of human PRMT1 (hPRMT1)
available yet, a recently generated homology model5,8 was used
for the docking studies. The hPRMT1 model has been successfully
used before to identify the inhibitors stilbamidine, allantodapsone
and C-7280948.5,8 The substrate binding pocket was analyzed by
calculating the molecular interaction fields using the GRID software
(Molecular Discovery Inc.). The GRID molecular interaction fields10
show favourable areas of interaction calculated with the hydro-
phobic probe, the aromatic C2 probe (see Fig. 5), an amine probe
and a chlorine probe (see Fig. S1). Starting from the docking solu-
tions of allantodapsone and C-7280948 (see Fig. 5, S2), together
Interestingly, the active inhibitor 6h, in which the aromatic
amino group has been replaced by a chlorine substituent, showed
a similar binding mode as observed for C-7280948 (6a). This is in
agreement with the GRID interaction fields obtained with the chlo-
rine probe, where a favourable interaction site for chloro-substitu-
ents was detected near the catalytic site (see Fig. S1). A para
substitution of the phenyl ring of C-7280948 (6a) significantly low-
ers the activity (6i, g). The same effect is observed even with a
shorter linker (6e, f). The docking results showed that this para
substituent hinders the favourable p/p-stacking with Tyr47 as ob-
served for C-7280948. As expected, derivatives in which the sulfo-
neamide moiety is replaced by a heteroaromatic ring (6d) or the
sulfoneamide is replaced by an amide (11a–c) showed a binding
mode similar to the lead structure.
4. Biological testing
All compounds were tested for inhibition of recombinant hu-
man PRMT1 using the non-histone protein Npl3 as a substrate in
an ELISA in vitro assay. IC50 values were usually only determined
Figure 5. Docking result for C-7280948 (colored orange) at the PRMT1 substrate
binding pocket. Only relevant amino acids are displayed in green, whereas the
PRMT1 backbone is shown as grey ribbon. The favourable interaction field derived
with the aromatic C2 GRID probe is colored orange (contour level ꢁ2.0 kcal/mol).
Hydrogen bonds are shown as dashed lines.
if more than 50% inhibition was observed at 50 lM.
4.1. Analogues of allantodapsone
Among the monoacyl derivatives of dapsone, only the biphenyl
compound 1e showed good enzyme inhibition in vitro with an
IC50-value of 11.8 lM. Among the bisubstituted dapsones, aro-
matic rings in 2c and 2d led to good inhibition and the bis-chloro-
acetyl compound 2e was the strongest inhibitor with an IC50
around 1
lM, allantodapsone has been published with an IC50 of
1.7
l
M (see Table 1).5
4.2. Derivatives of C-7280948
Among the derivatives of 6a, the best inhibition was seen with
the 4-chlorobenzoyl (7d) and the 3,4-dichlorobenzoyl amide 7e.
The sulfonyl analogue 7j showed a somewhat improved activity
as compared to its carbonyl congener 7d (see Table 2).
Reduction of the spacer length in the phenethylamide part of 6a
did not lead to improved derivatives (see Table 3).
To check the binding mode of the two most potent inhibitors 2e
and 7e, a jump dilution test of reversibility was performed by incu-
bation of the compounds at the IC50 with 10ꢀ enzyme, followed by
Figure 6. Docking solution obtained for 1e. Only relevant amino acids are displayed
in green, whereas the PRMT1 backbone is shown as grey ribbon. Hydrogen bonds
are shown as dashed lines.

E.-M. Bissinger et al. / Bioorg. Med. Chem. 19 (2011) 3717–3731
3721
Table 1
Inhibitory properties of the acylated dapsone derivatives against hPRMT1
Core
R
Compound
IC50 SEM (lM) or inhibition (%) (concentration)
O
S
O
70.6 (50
23.5 (10
l
l
M)
M)
O
CH3
1a
N
H
R
H2N
1b
45.9 2.7
65.6 7.9
Cl
1c
41.2 (50
35.3 (10
l
M)
1d
lM)
1e
2a
11.8 3.9
O
S
O
O
R
nia (50
lM)
O
CH 3
N
R
N
H
H
CH3
44.1 (50
14.7 (10
l
l
M)
M)
2b
2c
14.1 1.7
2d
2e
30.4 3.8
1.5 0.2
Cl
O
S
O
O
3
4
35.1 2.9
S
N
NH2
O
H
Cl
3
O
S
O
13.6 (50
nia (10
l
M)
M)
O
l
NH2
N
N
H
H
4
a
ni—no inhibition, inhibition <5%.
dilution of enzyme to 1ꢀ in the reaction volume. The compounds
went to about 0.1ꢀ IC50. The loss of inhibition in this procedure
indicates a reversible mode of inhibition (see Fig. S5). Furthermore,
a mechanism of action study was conducted by varying the sub-
strate concentration over a large range within the in vitro assay
with fixed inhibitor concentrations for inhibitors 2e and 7e. Both
inhibitors showed greater inhibitory potential compared to normal
assay conditions at low substrate concentration whereas higher
substrate concentrations led to lower inhibitory potential (see Figs.
S6 and S7). Thus, the analogues seem to retain the substrate com-
petitive mode of inhibition of the lead compound allantodapsone.
To analyze for selectivity among arginine methyltransferases,
we tested the inhibitors 2e and 7j for inhibition of the isoform
CARM1 (=PRMT4), which has also been linked to hormone depen-
dent gene transcription.12,13 Up to very high micromolar concen-
gen receptor a by methylation which is necessary for efficient
recruitment of the receptor to its target genes.14 Again, we used
an antibody based detection of methylation on a peptide sub-
strate.14 All compounds exhibited little inhibition below 50
lM.
The phenylacetic acid derivative of dapsone 1d and the bis-chloro-
methyl compound 2e were very selective for PRMT1 (see Table 4).
4.3. Cellular characterization of the most potent inhibitors
Selected inhibitors were then characterized for their cellular
activity. The bis-chloroacetyl compound 2e was analyzed for
growth inhibition of MCF7a breast cancer and LNCaP prostate
cancer cells using an MTS assay. The GI50 values for 2e were
1.97 0.14
lM on MCF7a cells and 4.49 0.14 lM on LNCaP cells
(growth curves not shown). Inhibitor 7e showed very moderate
trations, no inhibition of CARM1 could be shown using
radioactive in vitro assay (see Fig. S8).
a
growth inhibition on MCF7a cells (52.24 5.4
analyzed on LNCaP cells.
lM) and was not
In order to test the selectivity towards lysine methyltransfer-
ases, the best inhibitors were assayed for the inhibition of human
recombinant Set7/9 as a representative example. Among others,
Set7/9 has recently been shown to play a role in stabilizing estro-
To analyze for potential indirect antihormone properties, we
used a reporter gene assay that analyzes luciferase activity in an
androgen dependent expression system. Among the most potent
inhibitors, only compound 2e showed the desired effect. In order

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E.-M. Bissinger et al. / Bioorg. Med. Chem. 19 (2011) 3717–3731
Table 2
Inhibitory properties of amide derivatives of 6a against hPRMT1
Core
R
Compound
IC50 SEM (lM) or inhibition (%) (concentration)
6a
26.7 3.5
20 (50
15 (10
l
l
M)
M)
7a
7b
10 (50
nia (10
l
l
M)
M)
42 (50
39 (10
l
l
M)
M)
7c
7d
48.9 2.4
10.2 0.5
Cl
Cl
O
S
O
N
H
O
R
7e
Cl
N
H
41 (50
23 (10
l
l
M)
M)
7f
7g
7i
Br
68.1 1.2
I
40 (50
40 (10
l
l
M)
M)
NH2
O
S
O
N
H
O
S
O
7j
5.0 0.2
N
H
Cl
a
ni—no inhibition, inhibition <5%.
to rule out unspecific effects on transcription or translation, we
also used a control reporter gene system. Here, the luciferase gene
is under control of a thymidine kinase promotor which is not
untransfected prostate cancer cells. Chloroacetamidines possess a
similar alkylating group and were shown to be inhibitors of histone
deamidation in vitro15,16 but at least in vitro our data suggests a
reversible mode of action. In the case of the sulfonamide lead inhib-
itor 6a, structural variation of the phenethyl group did not lead to
optimized derivatives. Acylated derivatives 7a were also less active,
with the exception of the 3,4-dichloro derivative 7e.
Thus, structure–activity studies led to an improved inhibitor of
PRMT1 with cellular activity. This makes 2e an interesting starting
point for the further optimization of inhibitors as useful tools in
epigenetic research, and may also serve as a basis for the develop-
ment of therapeutic agents against prostate cancer.
responsive to androgen stimulation. At 10 and 1
was observed, indicating a specific effect on androgen dependent
transcription. The reduction seen at 25 M is due to cytotoxicity,
lM no effect
l
as activity also decreased in non-stimulated cells (see Fig. 7).
We then analyzed the effects of the inhibitor 2e on androgen
dependent gene transcription in LNCap cells that are not transfected.
To this end, we quantitated the mRNA of androgen dependent target
genes after androgen stimulation with and without treatment with
2e. For all genes investigated, a statistically significant reduction in
the mRNA levels could be detected. The expression of GAPDH, which
is not androgen dependent, was not altered (see Fig. 8).
6. Experimental section
5. Conclusion
6.1. Materials and methods
Synthetic variation of sulfonamide and sulfone inhibitors of
PRMT1 led to the establishment of structure–activity relationships.
Among the monoacyl analogues of allantodapsone, the most potent
inhibitor was the biphenylylamide 1e. Among the diacyl derivatives,
especially the bis-chloroacetylated derivative 2e showed potent en-
zyme inhibition. It has also significant cytotoxic activity. While this
might at least in part be due to unspecific cytotoxicity caused by its
potentially alkylating properties, it specifically blocks androgen
dependent gene transcription in a reporter gene model and in
6.1.1. In vitro methylation assay (hPRMT1)
The assay was performed as published.4
6.1.2. In vitro methylation assay (CARM1)
A solution containing 20 lM of recombinant histone H3, 10 lg/
mL of bovine serum albumin and 250 nM of flag-CARM1 (CARM1
was expressed from the original mouse FlagCARM1 construct given
by Evans17 using the baculorvirus system) in 50 mM Tris–HCl,
0.2 M NaCl pH 8.0, 0.5 mM DTT was incubated with increasing

E.-M. Bissinger et al. / Bioorg. Med. Chem. 19 (2011) 3717–3731
3723
Table 3
Inhibitory properties of further analogues of 6a against hPRMT1
Table 4
Selectivity of the best inhibitors tested against Set7/9
Core
Compound IC50 SEM (
l
M) or
Compound
IC50 SEM (
inhibition (%)
l
M) or
Compound
IC50 SEM (
inhibition (%)
(concentration)
lM) or
inhibition (%)
(concentration)
1d
1e
2d
2e
nia (50
66.4 0.7
l
M)
7e
7j
6d
11a
37 (50
58 (50
14 (50
20 (50
lM)
lM)
lM)
lM)
O
S
O
31 (50
lM)
N
H
6a
26.7 3.5
nia (50
lM)
a
H2N
H2N
H2N
ni—no inhibition, inhibition <5%.
O
S
O
28 (50
22 (10
l
l
M)
M)
and stopped by mixing to 4 lL of SDS PAGE loading buffer. The
N
H
6b
6c
6d
6e
6f
reaction product was analyzed on a 12% polyacrylamide denatur-
ing gel. After electrophoresis, the gel was stained with Coomassie
Brilliant Blue, dried on a Whatman 3MM paper sheet and exposed
for 24 h to 5 days against a GP2025 imaging plate (Kodak). The
amount of radioactivity incorporated specifically by histone H3
was quantified by phosphorimager analysis (Typhoon, Molecular
Dynamic).
O
S
O
40 (50
28 (10
l
l
M)
M)
N
H
O
S
O
N
N
H
70.7 4.4
6.1.3. In vitro time-resolved fluorescence immunosorbent
methyltransferase assay (Set7/9)
The heterogeneous assay is performed in streptavidine-coated
96-well plates (Nunc). After each incubation step, six washing
steps are necessary to remove the non bound fraction (Tecan
H2N
H2N
H2N
O
S
O
nia (50
lM)
N
H
Cl
Columbus Plate washer; 300 lL/ step in an overflow modus, using
O
S
O
100 mM Tris, containing 0.1% Tween 20; pH 7.5). In the first incu-
bation step, the biotinylated histone peptide (aa 1–21 of human
histone H3, Upstate, 20 pmol/ well in the same buffer as mentioned
above) is bound to the cavities. In the second step, the bound sub-
strate is turned over in an enzymatic reaction. Therefore, preincu-
bation of enzyme (Set7/9, BPS biosciences, human, recombinant,
N-terminal GST tag, 5
NaCl, 1 mM EDTA, 1 mM DTT, pH 8.5), 5
(DMSO) and 10 L of 15 mM Tris-buffer takes place for 10 min at
30 °C. Controls are treated in the same manner, using buffer in-
stead of the inhibitor solution. Then, each mixture is transferred
to the plate. Each well is filled with 15 mM Tris-buffer to obtain
an incubation volume of 100
reaction, 5 L of an 800 M SAM solution is added to each well. The
amount of the turnover is detected by a primary rabbit antibody
(anti-methyl H3K4, Upstate, 100 of 1:1000 dilution in
19 (50
12 (10
l
l
M)
M)
N
H
OMe
Cl
O
S
O
6g
6h
nia (50
lM)
lL/well, diluted in 50 mM Tris, 100 mM
N
H
l
L of inhibitor solution
H2N
l
OH
O
S
O
37 (50
30 (10
l
l
M)
M)
N
H
lL in the end. To start the enzymatic
H2N
Cl
l
l
O
S
O
lL
a
6i
nia (50
lM)
N
H
100 mM Tris containing 0.1% Tween 20 and 0.5 % BSA, protease-
free; pH 7.5) followed by an europium-labeled secondary antibody
directed against rabbit IgG (Perkin–Elmer, 5 pmol/well, using the
same buffer as for the dilution of the first antibody). The europium
O
N
H
9a
49.7 1.7
label is cleaved off by adding 100 lL of enhancement solution
(Perkin–Elmer) to all wells. After shaking the plate for 10 min at
room temperature, the TRF measurement (340/615 nm, BMG
Polarstar) is performed for the quantitation.
H2N
H2N
H2N
O
M)b
M)b
N
H
9b
9c
47 (50
34 (50
l
l
6.2. Luciferase assay
O
The day before transfection, 2 ꢀ 105 LNCaP cells were cultured
in RPMI1640 supplemented with 10% Fetal Calf Serum (FCS). LNCaP
cells were washed one time with PBS and cultured in phenol-
red-free RPMI1640 supplemented with 10% double-stripped FCS
(dsFCS) just before transfection with Effecten (Qiagen). 500 ng of
MMTV-Luc or TK-Luc reporter plasmid18 were used for each well.
Six hours later, cells were treated with indicated inhibitors at indi-
cated concentrations. When cells were not treated by inhibitor,
DMSO was added as vehicle. Just after adding inhibitors or vehicle,
cells were treated over night with or without 10ꢁ10 M R1881 as
indicated. Luciferase activity was assayed as described.19 All exper-
iments were repeated three times in duplicate. Bars represent
mean + SD (n = 6).
N
H
a
ni—no inhibition, inhibition <5%.
Data obtained as published.5
b
concentrations of inhibitor (0.01–2 mM) for 30 min at room tem-
perature. A 5 min centrifugation step at 15,000g was performed
to eliminate any precipitated protein. The reaction was initiated
in a volume of 10
lL by addition of 10 lM
14C-labeled SAM at
50 mCi mmolꢁ1 (GE Healthcare), performed for 15 min at 25 °C

3724
E.-M. Bissinger et al. / Bioorg. Med. Chem. 19 (2011) 3717–3731
Figure 7. Transcriptional regulation by PRMT1 inhibitors, investigated in a reporter gene system. The MMTV-Luc reporter is androgen driven, the TK-Luc promotor not.
R1881 is a synthetic androgen that is used for receptor activation. Differences for androgen dependent cells are only significant for 2e (all concentrations) and 7e (25 lM, p
<0.01).
6.3. Effects on androgen receptor target genes
6.5. Purity
LNCaP cells were washed one time with PBS and have been
starved during 24 h in phenol-red-free RPMI1640 supplemented
with 0.5% double stripped FCS (dsFCS). Cells were then treated or
not with inhibitor as indicated. When cells were not treated by
inhibitor, DMSO was added as vehicle. After (10 min) adding inhib-
itors or vehicle, cells were treated over night with or without
10ꢁ9 M R1881 as indicated.20 DNaseI-treated RNA isolated using
Trizol (Invitrogen) was used for reverse transcription. Quantitative
PCR was performed in a LightCycler 480 (Roche). Product forma-
tion was detected by incorporation of SYBR green I using ROX as
a passive reference (ABgene). For qRT-PCR, the following primers
were used: TMPRSS221 50-TCACACCAGCCATGATCTGT-30 and 50-
CTGTCACCCTGGCAAGAATC-30; ELK4 50-CTGTTGCTCCCCTAAGTC
CA-30 and 50-CCAGCCCAGACAGAGTGAAT-30; IGF1-R21 50-CTGTAT
If purity of the final compounds was determined by HPLC anal-
yses, the following protocols were used:
System 1:
Column: Phenomenex LunaÒ
5
l
Phenyl–Hexyl 250 ꢀ 4.6 mm;
mobile phase: acetonitrile/water (70/30 v/v); flow: 0.5 mL/min;
detection UV 254 nm; samples: 0.1 mg/mL; injection volume:
20 ll; manual integration after blind subtraction from same series.
System 2:
Column: Phenomenex SynergiÒ
4.60 mm; mobile phase: A: water + 0.05% TFA, B: acetonitrile +
0.05% TFA; flow: 1 mL/min; detection UV 210 nm; samples:
4
l
m Max-RP 80 Å, 150 ꢀ
1 mg/mL; injection volume: 5 ll; gradient elution:
Time (min)
Eluent A (%)
Eluent B (%)
GCCTCTGTGA
ACC-30
and
50-TAGACCATCCCAAACGAC-30;
0.0
4.0
29.0
31.0
31.5
40.0
90.0
90.0
0.0
0.0
90.0
90.0
10.0
10.0
100.0
100.0
10.0
10.0
NKX3.122 50-AGAACGACCAGCTGA GCAC-3’ and 50-AAGACCC-
CAAGTGCCTTTCT-30; CXCR423 50-CTGTGAGCAGAGGGTCCAG-30
and 50-ATGAATGTCCACCTCGCTTT-30; MAK24 50-TGGACTTGCAAGA
GAATTAAGGT-30 and 50-CTTCAGGGGCACGATACC-30; MAF25 50-
AGCGGCTTCCGAGAAAAC-30 and 50-GCGAGTGGGCTCAGTTATG-30;
N4A1
50-ACAGCTTGCTTGTCGATGTC-30
and
50-GGTTCTGC
AGCTCCTCCAC-30; PER126 50-AGGTACCTGGAGAGCTGCAA-3’ and
50-GATCTTTCTTGGTCCCCACA-30; GREB125 50-AAGCTGAGCAGCA
CAGACAA-30 and 50-GGCTTCTCTTCTCCGAGGTAG-30; FKBP5 50-TTT
TTGAGATTGAGCTCCTTGA-30 and 50-TTGTGTTCACCTTTGCCAAC-30;
GAPDH27 50-GAGTCCACTGGCGTCTTCAC-30 and 50-GTTCACAC
Manual integration after blind subtraction from same series.
Compounds were synthesized according to general procedures
as described in the following.
CCATGACGAACA-30. Bars represent mean + SD (n = 6). P-value:
ns = non significant; ⁄ = <0.05;
<0.001 (Supplementary data).
⁄⁄⁄
6.6. General procedure 1: synthesis of amides with acyl
chlorides and dapsone28
6.4. Inhibitors
Dapsone (3 mmol) is dissolved in pyridine (5 mL/mmol). The
acyl chloride (1 mmol) is dissolved in dioxane (5 mL/mmol) and
added dropwise to the reaction mixture which is then stirred over
night at room temperature.
To purify the crude product, approximately 50 mL of
diethylether are added to the solution. The formed precipitate
is separated and dissolved in DMF. The solution is poured into
200 mL of 2 M hydrochloric acid and the resulting precipitate
is filtered off and washed thoroughly with water. The product
is dried in vacuo and can be further purified by flash column
chromatography.
Standard chemicals were purchased from Acros, Aldrich, Sigma
or Fluka. NMR spectra were obtained on a Bruker Avance DRX
400 MHz spectrometer or a Varian 100 MHz, respectively. Chemi-
cal shifts are reported in ppm (d) relative to tetramethylsilane.
EI- and CI-mass spectra were measured with a TSQ700 mass
spectrometer (Thermoelectron). ESI- and PCI-mass spectra were
recorded with a LCQ-Advantage mass spectrometer. Merck Silica
Gel 60 was used for flash chromatography with cyclohexane/ethyl
acetate mixtures as eluent so that the Rf value of the desired prod-
uct was about 0.3.

E.-M. Bissinger et al. / Bioorg. Med. Chem. 19 (2011) 3717–3731
3725
Figure 8. Block of androgen dependent gene transcription by PRMT1 inhibitor 2e. mRNA levels of androgen dependent target genes were measured by qRT-PCR. R1881 is a
synthetic androgen that is used for receptor activation.
6.7. General procedure 2: synthesis of urea derivatives with
isocyanates29
tate is filtered off. The crude product is then purified by reversed
phase column chromatography.
The amine (1 mmol) is dissolved in dioxane (20 mL/mmol) and
the isocyanate (1 mmol), also dissolved in dioxane, is added drop-
wise. The reaction mixture is heated under reflux for 3 h, control-
ling the end of the reaction by thin layer chromatography. Water
is added to the cooled reaction mixture and the resulting precipi-
6.8. General procedure 3: reaction of amines with 4-
nitrobenzenesulfonylchloride30
The amine is dissolved in dichloromethane, cooled to 0 °C and
N-methylmorpholine (0.24 mL/mmol amine) is added. An

3726
E.-M. Bissinger et al. / Bioorg. Med. Chem. 19 (2011) 3717–3731
equimolar amount of sulfonylchloride, also dissolved in dichloro-
methane, is added dropwise to the solution. After stirring over
night at room temperature, the solution is washed three times
with 2 M hydrochloric acid, once with a saturated solution of NaH-
CO3 and once with brine. The organic layer is dried over MgSO4. To
obtain the crude product, the solvent is removed in vacuo. The
crude product may be purified by flash column chromatography.
137.24 (Car), 133.51 (Car), 130.19 (CHar), 129.64 (CHar), 128.96
(CHar), 127.99 (CHar), 126.41 (Car), 120.62 (CHar), 113.40 (CHar);
CIMS (direct mode): calcd/found (m/z): 386.0/386.0 [M]+, 404.0/
404.1 [M+NH4]+; Purity (HPLC): 99.3% (System 2).
6.14. N-[4-(4-Aminophenyl)sulfonylphenyl]benzamide, 1c
General procedure: 1, white crystals; Yield: 4%; 1H NMR
(DMSO-d6): d = 10.59 (s, 1H, NH), 8.00–7.92 (m, 4H, Har), 7.85–
7.79 (m, 2H, Har), 7.65–7.58 (m, 1H, Har), 7.57–7.50 (m, 4H, Har),
6.65–6.58 (m, 2H, Har), 6.14 (s, 2H, NH2); 13C NMR (DMSO-d6):
d = 166.44 (C@O), 153.86 (C–NH2), 143.33 (C–NH), 137.89 (C–
SO2), 134.80 (Car), 132.39 (Car), 129.64 (CHar), 128.88 (CHar),
128.22 (CHar), 127.99 (CHar), 126.41 (CHar), 120.52 (CHar), 113.37
(CHar); EIMS (direct mode): calcd/found (m/z): 352.1/352.1 [M]+,
353.1/353.2 [M+H]+; Purity (HPLC): 95.4% (System 2).
6.9. General procedure 4: reduction of aromatic nitro
compounds30
The aromatic nitro compounds are dissolved in ethyl acetate
and SnCl2 ꢀ 2H2O (1.125 g/mmol) is added. The reaction mixture
is heated under reflux for at least 2 h. The reaction is monitored
by thin layer chromatography. When the reaction is completed,
the batch is cooled. A solution of NaHCO3 is then added until
pH 7–8 is reached. The organic layer is separated and the aque-
ous residue is washed three times with ethyl acetate. The com-
bined organic layers are washed with brine and dried over
MgSO4. The solvent is removed in vacuo to obtain the crude
product.
6.15. N-[4-(4-Aminophenyl)sulfonylphenyl]-2-phenyl-
acetamide, 1d
General procedure: 1, beige crystals; Yield: 1%; 1H NMR
(DMSO-d6): d = 10.54 (s, 1H, NH), 7.80–7.71 (m, 4H, Har), 7.53–
7.47 (m, 2H, Har), 7.34–7.29 (m, 4H, Har), 7.28–7.21 (m, 1H, Har),
6.63–6.56 (m, 2H, Har), 6.13 (s, 2H, NH2), 3.67 (s, 2H, CH2); 13C
NMR (DMSO-d6): d = 170.20 (C@O), 153.82 (C–NH2), 143.23 (C–
NH), 137.50 (C–SO2), 135.93 (Car), 129.58 (CHar), 129.54 (CHar),
128.75 (CHar), 128.17 (CHar), 127.05 (CHar), 126.42 (Car), 119.41
(CHar), 113.35 (CHar), 43.69 (CH2); ESIMS (positive mode): calcd/
found (m/z): 367.1/367.3 [M+H]+; Purity (HPLC): 95.5% (System 2).
6.10. General procedure 5: synthesis of amides with acyl
chlorides and aromatic amines
The corresponding acyl chlorides is dissolved in dioxane and
added to a solution of the appropriate aromatic amine in hot
toluene. The reaction mixture is heated under reflux for 2 h. When
the reaction is over, the solvent is removed in vacuo and the
crude product is purified by recrystallisation or flash column
chromatography.
6.16. N-[4-(4-Aminophenyl)sulfonylphenyl]-4-phenyl-
benzamide, 1e
6.11. General procedure 6: hydrolysis of esters under basic
conditions
General procedure: 1, white crystals; Yield: 6%; 1H NMR
(DMSO-d6): d = 10.63 (s, 1H, NH), 8.11–7.94 (m, 4H, Har), 7.89–
7.78 (m, 4H, Har), 7.79–7.72 (m, 2H, Har), 7.58–7.47 (m, 4H, Har),
7.46–7.40 (m, 1H, Har), 6.66–6.59 (m, 2H, Har), 6.15 (s, 2H, NH2);
13C NMR (DMSO-d6): d = 166.04 (C@O), 153.86 (C–NH2), 143.91
(C–NH), 143.36 (Car), 139.41 (Car), 137.88 (Car), 133.51 (Car),
129.64 (CHar), 129.51 (Car), 128.96 (CHar), 128.67 (CHar), 128.01
(CHar), 127.63 (CHar), 127.07 (CHar), 126.42 (CHar), 120.55 (CHar),
113.39 (CHar); EIMS (direct mode): calcd/found (m/z): 429.1/
429.1 [M]+; Purity (HPLC): 99.2% (System 2).
The ester is dissolved in ethanol and refluxed with a 10%
solution of KOH (40 mL/mmol) for 30 min. After the end of the
reaction, monitored by thin layer chromatography, the reaction
mixture is cooled to room temperature. The excess solvent is
evaporated. The crude product is dissolved in ethyl acetate and
is washed three times with water, once with a saturated solution
of NaHCO3 and once with brine. The organic layer is dried over
Na2SO4 and the solvent is evaporated. The obtained crude prod-
uct can be further purified by recrystallisation or flash column
chromatography.
6.17. N-[4-[4-(Propanoylamino)phenyl]sulfonylphenyl]-
propanamide, 2a
6.12. N-[4-(4-Aminophenyl)sulfonylphenyl]propanamide, 1a
General procedure: 1, yellowish crystals; Yield: 5%; 1H NMR
(DMSO-d6): d = 10.26 (s, 2H, NH), 7.86–7.74 (m, 8H, Har), 2.35 (q,
4H, 3J = 7.57 Hz, CH2), 1.07 (t, 6H, 3J = 7.57 Hz, CH3); 13C NMR
(DMSO-d6): d = 173.17 (C@O), 144.10 (C–NH), 135.42 (C–SO2),
128.77 (Car, C2/6), 119.35 (Car, C3/5), 30.01 (CH2), 9.76 (CH3); CIMS
(direct mode): calcd/found (m/z): 360.1/360.1 [M]+, 378.1/378.1
[M+NH4]+; Purity (HPLC): >99.5% (System 1).
General procedure: 1, yellowish crystals; Yield: 2%; 1H NMR
(DMSO-d6): d = 10.2 (s, 1H, NH), 7.74 (s, 4H, Har 2/3/5/6), 7.53–
7.47 (m, 2H, Har 20/60), 6.64–6.58 (m, 2H, Har 30/50), 6.09 (s, 2H,
NH2), 2.35 (q, 2H, 3J = 7.52 Hz, CH2), 1.11 (t, 3H, 3J = 7.49 Hz,
CH3); 13C NMR (DMSO-d6): d = 173.07 (C@O), 153.83 (C–NH2),
143.45 (C–NH), 132.26 (C–SO2), 129.56 (CHar, C20/60), 128.13 (CHar,
C2/6), 126.63 (C0–SO2), 119.26 (CHar, C3/5), 113.41 (CHar, C30/50),
30.02 (CH2), 9.81 (CH3); CIMS (direct mode): calcd/found (m/z):
322.1/322.1 [M+NH4]+; Purity (HPLC): 95.6% (System 1).
6.18. N-[4-[4-(Butanoylamino)phenyl]sulfonylphenyl]-
butanamide, 2b
6.13. N-[4-(4-Aminophenyl)sulfonylphenyl]-4-chloro-
benzamide, 1b
General procedure: 1, white crystals; Yield: 34%;1H NMR
(DMSO-d6): d = 10.30 (s, 2H, NH), 7.86–7.76 (m, 8H, Har), 2.31 (t,
4H, 3J = 7.38 Hz,
a-CH2), 1.65–1.54 (m, 4H, b-CH2), 0.90 (t, 6H,
General procedure: 1, yellow crystals; Yield: 4%; 1H NMR
(DMSO-d6): d = 10.62 (s, 1H, NH), 8.01–7.92 (m, 4H, Har), 7.85–
7.80 (m, 2H, Har), 7.65–7.59 (m, 2H, Har), 7.56–7.50 (m, 2H, Har),
6.65–6.58 (m, 2H, Har), 6.12 (s, 2H, NH2); 13C NMR (DMSO-d6):
d = 165.32 (C@O), 153.87 (C–NH2), 143.13 (C–NH), 138.11 (C–Cl),
3J = 7.38 Hz, CH3); 13C NMR (DMSO-d6): d = 173.35 (C@O), 144.03
(C–NH), 135.44 (C–SO2), 128.79 (Car, C2/6), 119.36 (Car, C3/5),
38.76 (a-CH2), 18.77 (b-CH2), 13.95 (CH3); CIMS (direct mode):
calcd/found (m/z): 388.2/388.2 [M]+, 406.2/406.2 [M+NH4]+; Purity
(HPLC): 97.8% (System 1).

E.-M. Bissinger et al. / Bioorg. Med. Chem. 19 (2011) 3717–3731
3727
6.19. N-[4-(4-Benzamidophenyl)sulfonylphenyl]benzamide, 2c
(Car), 137.09 (C–SO2), 128.89 (CHar), 128.66 (CHar), 128.18 (CHar),
127.05 (CHar), 124.32 (CHar), 44.33 (CH2), 35.81 (NH–CH2); CIMS
(direct mode): calcd/found (m/z): 324.1/324.2 [M+NH4]+.
General procedure: 1, white crystals; Yield: 8%; 1H NMR (DMSO-
d6): d = 10.65 (s, 2H, NH), 8.06–8.00 (m, 4H, Har), 7.98–7.90 (m, 8H,
Har), 7.65–7.59 (m, 2H, Har), 7.58–7.51 (m, 4H, Har); 13C NMR
(DMSO-d6): d = 166.54 (C@O), 144.11 (C–NH), 136.06 (C–SO2),
134.74 (Car), 132.46 (Car), 128.90 (CHar), 128.71 (CHar), 128.26 (CHar),
120.64 (CHar); CIMS (direct mode): calcd/found (m/z): 474.1/474.1
[M+NH4]+, 456.1/456.1 [M]+; Purity (HPLC): 95.9% (System 2).
6.25. N-Benzyl-4-nitro-benzenesulfonamide, 5b
General procedure: 3, shining yellow crystals; Yield: 89%; 1H
NMR (DMSO-d6): d = 8.56 (s, 1H, NH), 8.39–8.33 (m, 2H, Har, H2/
6), 8.04–7.97 (m, 2H, Har, H3/5), 7.29–7.15 (m, 5H, Har), 4.07 (s,
1H, CH2); 13C NMR (DMSO-d6): d = 149.82 (C–NO2), 146.84 (Car),
137.55 (C–SO2), 128.68 (CHar), 128.84 (CHar), 128.08 (CHar),
127.68 (CHar), 124.90 (CHar), 46.58 (CH2); CIMS (direct mode):
calcd/found (m/z): 310.1/310.1 [M+NH4]+.
6.20. 2-Phenyl-N-[4-[4-[(2-phenylacetyl)amino]phenyl]-
sulfonylphenyl]acetamide, 2d
General procedure: 1, white crystals; Yield: 1%; 1H NMR
(DMSO-d6): d = 10.60 (s, 2H, NH), 7.87–7.75 (m, 8H, Har), 7.33–
7.21 (m, 10H, Har), 3.67 (s, 4H, CH2); 13C NMR (DMSO-d6):
d = 170.29 (C@O), 143.94 (C–NH), 135.86 (C–SO2), 135.70 (CHar),
129.53 (CHar), 128.83 (CHar), 128.74 (CHar), 127.05 (CHar), 119.55
(CHar), 43.71 (CH2); EIMS (direct mode): calcd/found (m/z): 484.1
/484.1 [M]+; Purity (HPLC): 95.4% (System 2).
6.26. 4-Nitro-N-(1-phenylethyl)benzenesulfonamide, 5c
General procedure: 3, yellow crystals; Yield: 65%; 1H NMR
(DMSO-d6): d = 8.63 (d, 1H, 3J = 8.08 Hz, NH), 8.24–8.17 (m, 2H,
Har, H3/5), 7.86–7.79 (m, 2H, Har, H2/6), 7.16–7.05 (m, 5H, Har),
4.50–4.38 (pq, 1H, CH), 1.27 (d, 3H, 3J = 6.98 Hz, CH3); 13C NMR
(DMSO-d6): d = 149.46 (C–NO2), 147.45 (Car), 143.02 (C–SO2),
128.50 (CHar), 128.32 (CHar), 127.27 (CHar), 126.51 (CHar), 124.53
(CHar), 53.67 (CH), 23.94 (CH3); APCIMS (direct negative mode):
calcd/found (m/z): 305.1/305.1 [MꢁH]ꢁ, 306.1/306.1 [M]ꢁ.
6.21. 2-Chloro-N-[4-[4-[(2-chloroacetyl)amino]phenyl]-
sulfonylphenyl]acetamide, 2e
General procedure: 1, yellowish crystals; Yield: 6%; 1H NMR
(DMSO-d6): d = 10.73 (s, 2H, O@C–NH), 7.93–7.87 (m, 4H, Har),
7.82–7.77 (m, 4H, Har), 4.30 (s, 4H, CH2); 13C NMR (DMSO-d6):
d = 165.82 (C@O), 143.30 (Car–NH), 136.17 (C–SO2), 129.00 (CHar),
119.88 (CHar), 43.95 (CH2); EIMS (direct mode): calcd/found (m/
z): 400.0/400.1 [35Cl35ClM]+, 401.0/401.1 [35Cl35ClM+H]+, 402.0/
402.1 [37Cl35ClM]+ 404.0/404.1 [37Cl37ClM]+; Purity (HPLC): 97.9%
(System 2).
6.27. 4-Nitro-N-(2-pyridylmethyl)benzenesulfonamide, 5d
General procedure: 3, yellow crystals; Yield: 45%; 1H NMR
(DMSO-d6): d = 8.70 (s, 1H, NH), 8.42–8.19 (m, 3H, Pyr-H6, Har,
H3/5), 8.04–7.97 (m, 2H, Har, H2/6), 7.71 (dt, 1H, 3J = 7.74 Hz,
4J = 1.89 Hz, Pyr-H5), 7.33 (d, 1H, 3J = 7.74 Hz, Pyr-H4), 7.25–7.18
(m, 1H, Pyr-H3), 4.18 (s, 1H, CH2); 13C NMR (DMSO-d6):
d = 156.98 (Pyr-C2), 149.82 (C–SO2), 149.22 (C–NO2), 146.76 (Pyr-
C6), 137.16 (Pyr-C4), 128.52 (CHar), 124.82 (CHar), 122.90 (Pyr-
C3), 122.20 (Pyr-C5), 48.33 (CH2); CIMS (direct mode): calcd/found
(m/z): 294.0/294.0 [M+H]+.
6.22. N-[4-(4-Aminophenyl)sulfonylphenyl]-4-chloro-
benzenesulfonamide, 3
General procedure: 1, yellowish crystals; Yield: 24%; 1H NMR
(DMSO-d6): d = 11.01 (s, 1H, NH), 7.84–7.78 (m, 2H, Har), 7.73–
7.68 (m, 2H, Har), 7.37–7.62 (m, 2H, Har), 7.49–7.43 (m, 2H, Har),
7.26–7.19 (m, 2H, Har), 6.61–6.55 (m, 2H, Har), 6.15 (s, 2H, NH2);
13C NMR (DMSO-d6): d = 153.93 (C–NH2), 141.91 (C–NH), 138.64
(C–SO2–NH), 138.54 (C–SO2), 138.23 (C–SO2), 130.12 (Car), 129.66
(Car), 128.96 (Car), 128.62 (Car), 125.97 (Car–Cl), 119.28 (Car, C30/
50), 113.38 (Car, C3/5); EIMS (direct mode): calcd/found (m/z):
422.0/422.0 [M]+; Purity (HPLC): 96.5% (System 2).
6.28. N-[(4-Chlorophenyl)methyl]-4-nitro-benzenesulfonamide,
5e
General procedure: 3, yellow crystals; Yield: 71%; 1H NMR
(DMSO-d6): d = 8.62 (br s, 1H, SO2–NH), 8.40–8.32 (m, 2H, Har3/5),
8.05–7.96 (m, 2H, Har, H2/6), 7.37–7.27 (m, 2H, Har), 7.26–7.20 (m,
2H, CHar), 4.08 (s, 2H, CH2); 13C NMR (DMSO-d6): d = 149.81 (C–
NO2), 146.76 (C–Cl), 136.65 (C–SO2), 132.34 (Car), 129.93 (CHar),
128.61(CHar), 128.50(CHar), 124.87(CHar), 45.83 (CH2); CIMS(direct
mode): calcd/found (m/z): 140.0/140.1 [Cl–C6H4–CH2–NH]+, 344.0/
344.1 [M+NH4]+, 346.0/346.1 [37ClM+NH4]+.
6.23. 1-[4-(4-Aminophenyl)sulfonylphenyl]-3-benzyl-urea, 4
General procedure: 1, white crystals; Yield: 79%; 1H NMR
(DMSO-d6): d = 9.06 (s, 1H, Ar-NH), 7.71–7.65 (m, 2H, Har), 7.5–
7.53 (m, 2H, Har), 7.52–7.46 (m, 2H, Har), 7.37–7.19 (m, 5H, Har),
6.79 (t, 1H, 3J = 5.94 Hz, CH2–NH), 6.63–6.56 (m, 2H, Har), 6.09 (s,
2H, NH2), 4.30 (d, 2H, 3J = 2.93 Hz, CH2); 13C NMR (DMSO-d6):
d = 155.15 (C@O), 153.65 (C–NH2), 144.80 (Car) 140.39 (Car–NH),
135.13 (C–SO2), 129.43 (CHar), 128.74 (CHar), 128.21 (CHar),
127.53 (CHar), 127.21 (CHar), 126.98 (Car), 117.69 (CHar), 113.33
(CHar), 43.15 (CH2); ESIMS (direct positive mode): calcd/found
(m/z): 382.1/382.2 [M+H]+; Purity (HPLC): 97.7% (System 2).
6.29. N-[(4-Methoxyphenyl)methyl]-4-nitro-benzene-
sulfonamide, 5f
General procedure: 3, yellow crystals; Yield: 95%; 1H NMR
(DMSO-d6): d = 8.48 (t, 1H, 3J = 6.13 Hz, SO2–NH), 8.38–8.31 (m,
2H, Har, H3/5), 8.00–7.94 (m, 2H, Har, H2/6), 7.13–7.06 (m, 2H,
Har), 6.82–6.75 (m, 2H, Har), 4.00 (t, 1H, 3J = 5.00 Hz, CH2), 3.69 (s,
3H, CH3); 13C NMR (DMSO-d6): d = 158.94 (C–NO2), 149.74 (Car),
146.95 (C–SO2), 129.52 (CHar), 129.34 (Car), 128.47 (CHar), 124.82
(CHar), 114.03 (CHar), 55.46 (CH3), 46.15 (CH2); ESIMS (direct neg-
ative mode): calcd/found (m/z): 321.1/321.4 [MꢁH]ꢁ.
6.24. 4-Nitro-N-phenethyl-benzenesulfonamide, 5a
General procedure: 3, yellowish crystals; Yield: 81%; 1H NMR
(CDCl3): d = 8.35–8.29 (m, 2H, Har), 7.99–7.93 (m, 2H, Har), 7.32–
7.22 (m, 3H, Har), 7.12–7.06 (m, 2H, Har), 4.66 (t, 1H, 3J = 5.97 Hz,
NH), 3.33 (dt, 2H, 3J = 6.68, 6.31 Hz, NH-CH2), 2.82 (t, 2H,
3J = 6.84 Hz, CH2); 13C NMR (CDCl3): d = 149.96 (C–NO2), 145.79
6.30. N-[2-(4-Chlorophenyl)ethyl]-4-nitro-benzenesulfonamide,
5g
General procedure: 3, white crystals; Yield: 76%; 1H NMR
(DMSO-d6): d = 8.40–8.31 (m, 2H, Har, H3/5), 8.09 (br s, 1H, NH),

3728
E.-M. Bissinger et al. / Bioorg. Med. Chem. 19 (2011) 3717–3731
8.01–7.91 (m, 2H, Har, H2/6), 7.30–7.22 (m, 2H, Har), 7.20–7.13 (m,
2H, Har), 3.09 (t, 2H, 3J = 7.14 Hz, NH–CH2), 2.69 (t, 2H, 3J = 7.14 Hz,
CH2); 13C NMR (DMSO-d6): d = 149.82 (C–NO2), 146.48 (Car),
137.96 (C–SO2), 131.45 (Car), 131.09 (CHar), 128.59 (CHar), 128.40
(CHar), 124.96 (CHar), 44.28 (NH–CH2), 34.82 (CH2); CIMS (direct
mode): calcd/found (m/z): 358.0/358.1 [M+NH4]+, 360.0/360.1
130.29 (Car), 129.32 (CHar), 128.90 (CHar), 126.06 (C–SO2), 114.01
(CHar), 113.09 (CHar), 55.49 (CH3), 46.05 (CH2); CIMS (direct mode):
calcd/found (m/z): 121.1/121.1 [H3CO–C6H4–CH2]+, 136.1/136.1
[H3CO–C6H4–CH2–NH]+, 293.1/293.2 [M+H]+; Purity (HPLC):
98.3% (System 2).
[
37ClM+NH4]+.
6.36. 4-Amino-N-[2-(4-
chlorophenyl)ethyl]benzenesulfonamide, 6g
6.31. 4-Amino-N-phenethyl-benzenesulfonamide, 6a
General procedure: 4, yellow crystals; Yield: 89%; 1H NMR
(DMSO-d6): d = 7.42–7.36 (m, 2H, Har, H2/6), 7.34–7.29 (m, 2H,
Har), 7.21–7.14 (m, 3H, SO2–NH, Har), 6.62–6.57 (m, 2H, Har), 5.93
(s, 2H, NH2), 2.90–2.81 (m, 2H, NH–CH2), 2.64 (t, 2H, 3J = 7.17 Hz,
CH2); 13C NMR (DMSO-d6): d = 152.86 (C–NH2), 138.46 (Car),
131.25 (Car), 130.93 (CHar), 128.84 (CHar), 128.59 (CHar), 125.96
(C–SO2), 113.15 (CHar), 44.18 (NH–CH2), 34.87 (CH2); CIMS (direct
mode): calcd/found (m/z): 311.1/311.1 [35ClM+H]+, 313.1/313.1
General procedure: 4, yellow crystals; Yield: 89%; 1H NMR
(CDCl3): d = 7.63–7.56 (m, 2H, Har, H2/6), 7.34–7.19 (m, 4H, Har,
H20/30/50/60), 7.14–7.05 (m, 1H, Har, H40), 6.72–6.63 (m, 2H, Har,
H3/5), 4.26 (t, 1H, 3J = 6.03 Hz, SO2–NH), 4.13 (s, 2H, NH2), 3.25–
3.17 (m, 2H, NH–CH2), 2.78 (t, 2H, 3J = 6.89 Hz, CH2); 13C NMR
(CDCl3): d = 150.42 (C–NH2), 137.75 (Car), 129.20 (CHar), 128.72
(CHar), 127.98 (C–SO2), 126.73 (Car), 114.05 (CHar), 44.06 (CH2),
35.69 (NH–CH2); CIMS (direct mode): calcd/found (m/z): 277.1/
277.1 [M+H]+. Purity (HPLC): 97.3% (System 2).
[
37ClM+H]+; Purity (HPLC): 98.3% (System 2).
6.37. 4-Amino-N-[2-(4-
6.32. 4-Amino-N-benzyl-benzenesulfonamide, 6b
hydroxyphenyl)ethyl]benzenesulfonamide, 6h
General procedure: 4, yellow crystals; Yield: 98%; 1H NMR
(DMSO-d6): d = 7.63 (t, 1H, 3J = 6.37 Hz, NH), 7.48–7.42 (m, 2H,
Har, H2/6), 7.33–7.19 (m, 5H, Har), 6.64–6.58 (m, 2H, Har, H3/5),
5.93 (s, 2H, NH2), 3.87 (d, 2H, 3J = 6.23 Hz, CH2); 13C NMR
(DMSO-d6): d = 152.89 (C–NH2), 138.49 (Car), 128.88 (CHar),
128.59 (CHar), 127.97 (CHar), 127.43 (CHar), 125.98 (C–SO2),
113.08 (CHar), 46.51 (CH2); CIMS (direct mode): calcd/found (m/
z): 263.1/263.1 [M+H]+, 280.1/280.1 [M+NH4]+; Purity Elemental
Anal. Calcd C, 59.52; H, 5.38; N, 10.68; S, 12.22. Found: C, 59.53;
H, 5.48; N, 10.44; S, 11.92.
General procedure: 6, brown crystals; Yield: 84%; 1H NMR
(DMSO-d6): d = 9.18 (s, 1H, OH), 7.50–7.37 (m, 2H, Har, H20/60),
3
7.11 (t, 1H, J = 5.85 Hz, SO2–NH), 6.99–6.87 (m, 2H, Har, H30/50),
6.73–6.56 (m, 4H, Har), 5.87 (s, 2H, NH2), 2.89–2.75 (m, 2H, NH–
CH2), 2.60–2.47 (m, 2H, CH2); 13C NMR (DMSO-d6): d = 156.11
(C–OH), 152.83 (C–SO2), 129.87 (CHar), 129.43 (Car), 128.88 (CHar),
125.98 (C–NH2), 115.56 (CHar), 113.16 (CHar), 44.95 (NH–CH2),
35.86 (CH2); CIMS (direct mode): calcd/found (m/z): 293.1/293.1
[M+H]+; Purity (HPLC): 96.8% (System 2).
6.38. 4-Chloro-N-phenethyl-benzenesulfonamide, 6i
6.33. 4-Amino-N-(1-phenylethyl)benzenesulfonamide, 6c
General procedure: 3, white crystals; Yield: 60%; 1H NMR
(DMSO-d6): d = 7.84 (s, 1H, NH), 7.80–7.74 (m, 2H, Har), 7.68–
7.62 (m, 2H, Har), 7.29–7.22 (m, 2H, Har), 7.21–7.12 (m, 3H, Har),
2.98 (t, 2H, 3J = 7.29 Hz, NH–CH2) ,2.68 (t, 2H, 3J = 7.37 Hz, CH2);
13C NMR (DMSO-d6): d = 139.68 (Car), 138.99 (Car), 137.61 (Car),
129.76 (CHar), 129.08 (CHar), 128.85 (CHar), 128.74 (CHar), 126.67
(CHar), 44.42 (NH–CH2), 35.63 (CH2); CIMS (direct mode): calcd/
found (m/z): 296.0/296.1 [35ClM+H]+, 298.0/298.1 [37ClM+H]+; Pur-
ity (HPLC): 99.1% (System 2).
General procedure: 4, colorless crystals; Yield: 1%; 1H NMR
(DMSO-d6): d = 7.68 (d, 1H, 8.03 Hz, NH), 7.39–7.32 (m, 2H, Har,
H 2/6), 7.27–7.13 (m, 5H, Har), 6.56–6.48 (m, 2H, Har, H3/5), 5.86
(s, 2H, NH2), 4.26–4.14 (m, 1H, CH), 2.09 (s, 3H, CH3); 13C NMR
(DMSO-d6): d = 152.63 (C–NH2), 144.58 (Car), 128.74 (CHar),
128.46 (CHar), 127.01 (CHar), 126.47 (CHar), 112.93 (CHar), 52.86
(CH), 23.83 (CH3); CIMS (direct mode): calcd/found (m/z): 277.1/
277.2 [M+H]+, 294.1/294.2 [M+NH4]+; Purity (HPLC): 99.8% (Sys-
tem 2).
6.39. N-[4-(Phenethylsulfamoyl)phenyl]benzamide, 7a
6.34. 4-Amino-N-[(4-
chlorophenyl)methyl]benzenesulfonamide, 6e
General procedure: 5, white crystals; Yield: 92%; 1H NMR
(DMSO-d6): d = 10.58 (s, 1H, CONH), 8.02–7.92 (m, 4H, Har), 7.81–
7.73 (m, 2H, Har), 7.65–7.50 (m, 4H, Har), 7.31–7-23 (m, 2H, Har),
7.22–7.12 (m, 3H, Har), 3.02–2.91 (m, 2H, CH2–NH), 2.69 (t, 2H,
3J = 7.62 Hz, CH2); 13C NMR (DMSO-d6): d = 166.47 (C@O), 143.12
(Car–NH), 139.15 (Car), 135.06 (C–SO2), 134.91 (Car), 132.37 (Car),
129.08 (CHar), 128.89 (CHar), 128.75 (CHar), 128.22 (CHar), 127.95
(CHar), 126.65 (Car), 120.39 (CHar), 44.53 (NH–CH2), 35.66 (CH2);
CIMS (direct mode): calcd/found (m/z): 381.1/381.2 [M+H]+,
389.1/398.3 [M+NH4]+; Purity (HPLC): 99.5% (System 2).
General procedure: 4, yellow crystals; Yield: 70%; 1H NMR
(DMSO-d6): d = 7.69 (t, 1H, 3J = 4.42 Hz, SO2–NH), 7.46–7.70 (m,
2H, Har), 7.37–7.32 (m, 2H, Har), 7.30–7.23 (m, 2H, Har, H2/6),
6.63–6.57 (m, 2H, Har, H3/5), 5.94 (s, 2H, NH2), 3.87 (d, 2H,
3J = 6.42 Hz, CH2); 13C NMR (DMSO-d6): d = 152.95 (C–NH2),
137.67 (C–Cl), 131.96 (Car), 129.79 (CHar), 128.88 (CHar), 128.52
(CHar), 125.86 (C–SO2), 113.09 (CHar), 45.73 (CH2); CIMS (direct
mode): calcd/found (m/z): 297.0/297.1 [35ClM+H]+, 299.0/299.1
[
37ClM+H]+; Purity (HPLC): 98.6% (System 2).
6.40. N-[4-(Phenethylsulfamoyl)phenyl]-2-phenyl-acetamide,
6.35. 4-Amino-N-[(4-
7b
methoxyphenyl)methyl]benzenesulfonamide, 6f
General procedure: 5, yellow needles; Yield: 70%; 1H NMR
(DMSO-d6): d = 10.53 (s, 1H, Ar-NH), 7.82–7.75 (m, 2H, CHar),
7.74–7.68 (m, 2H, Har), 7.65 (t, 1H, 3J = 5.76 Hz, SO2–NH), 7.37–
7.30 (m, 4H, Har), 7.29–7.22 (m, 3H, Har), 7.21–7.12 (m, 3H, Har),
3.69 (s, 2H, CH2–CO), 2.98–2.89 (m, 2H, CH2–NH), 2.67 (t, 2H,
3J = 7,58 Hz, Phe-CH2); 13C NMR (DMSO-d6): d = 170.17 (C@O),
General procedure: 4, light yellow crystals; Yield: 84%; 1H NMR
(DMSO-d6): d = 7.54 (s, 1H, 3J = 6.45 Hz, SO2-NH), 7.46–7.40 (m, 2H,
Har), 7.17–7.11 (m, 2H, Har), 6.87–6.81 (m, 2H, Har), 6.63–6.57 (m,
2H, Har), 6.93 (s, 2H, NH2), 3.79 (d, 2H, 3J = 6.21 Hz, CH2), 3.72 (s,
3H, CH3); 13C NMR (DMSO-d6): d = 158.78 (C–NH2), 152.87 (Car),

E.-M. Bissinger et al. / Bioorg. Med. Chem. 19 (2011) 3717–3731
3729
143.02 (Car–NH), 139.15 (Car), 136.01 (Car), 134.76 (Car), 129.56
(CHar), 129.05 (CHar), 128.75 (CHar), 128.72 (CHar), 128.09 (CHar),
127.05 (CHar), 126.62 (CHar), 119.28 (CHar), 44.47 (CH2), 43.74
(CH2–NH), 35.66 (Phe-CH2); EIMS (direct positive mode): calcd/
found (m/z): 395.1/395.2 [M+H]+, 412.1/412.3 [M+NH4]+; Purity
(HPLC): 98.6% (System 2).
(NH–CH2), 35.66 (CH2); CIMS (direct mode): calcd/found (m/z):
459.0/458.9
79BrM+H]+, 461.0/461.0 81BrM]+; Purity (HPLC):
98.4% (System 2).
[
[
6.45. 4-Iodo-N-[4-(phenethylsulfamoyl)phenyl]benzamide, 7g
General procedure: 5, white crystals; Yield: 32%; 1H NMR
(DMSO-d6): d = 10.66 (s, 1H, CONH), 8.04–7.88 (m, 4H, Har, A/A0,
C/C0), 7.81–7.73 (m, 4H, Har, B/B0), 7.64 (t, 1H, 3J = 5.91 Hz, SO2–
NH), 7.32–7-23 (m, 2H, Har), 7.22–7.10 (m, 3H, Har), 3.02–2.89
(m, 2H, CH2–NH), 2.68 (t, 2H, 3J = 7.52 Hz, CH2); 13C NMR (DMSO-
d6): d = 165.75 (C@O), 142.93 (Car–NH), 139.15 (C–SO2), 137.76
(CHar), 135.20 (Car), 134.22 (Car), 130.17 (CHar), 129.08 (CHar),
128.75 (CHar), 127.95 (CHar), 126.65 (CHar), 120.47 (CHar), 100.27
(C–I), 44.53 (NH–CH2), 35.65 (CH2); CIMS (direct mode): calcd/
found (m/z): 507.0/506.9 [M+H]+, 524.0/524.0 [M+NH4]+; Purity
(HPLC): 99.0% (System 2).
6.41. N-[4-(Phenethylsulfamoyl)phenyl]-4-phenyl-benzamide,
7c
General procedure: 5, white crystals; Yield: 40%; 1H NMR
(DMSO-d6): d = 10.62 (s, 1H, CONH), 8.12–8.04 (m, 2H, Har), 8.03–
7.95 (m, 2H, Har), 7.90–7.84 (m, 2H, Har), 7.82–7.73 (m, 4H, Har),
7.60 (t, 1H, SO2–NH, 3J = 5.82 Hz), 7.55–7.49 (m, 2H, Har), 7.47–
7.41 (m, 1H, Har), 7.31–7.24 (m, 2H, Har), 7.23–7.13 (m, 3H, Har),
3.02–2.93 (m, 2H, CH2–NH), 2.70 (t, 2H, 3J = 7.65 Hz, CH2); 13C
NMR (DMSO-d6): d = 166.11 (C@O), 143.92 (Car–NH), 143.18 (Car)
139.45 (Car), 139.17 (C–SO2), 135.08 (Car), 133.63 (Car), 129.52
(CHar), 129.09 (CHar), 128.98 (CHar), 128.75 (CHar), 128.67 (Car),
127.98 (CHar), 127.37 (CHar), 127.08 (CHar), 126.66 (Car), 120.44
(CHar), 44.56 (CH2–NH), 35.69 (CH2); CIMS (direct mode): calcd/
found (m/z): 457.2/457.3 [M+H]+, 474.2/474.3 [M+NH4]+; Purity
(HPLC): >99.5% (System 2).
6.46. 4-Nitro-N-[4-(phenethylsulfamoyl)phenyl]benzamide, 7h
General procedure: 5, yellowish crystals; Yield: 5%; 1H NMR
(DMSO-d6): d = 10.90 (s, 1H, CONH), 8.42–8.34 (m, 2H, Har), 8.23–
8.15 (m, 2H, Har), 8.01–7.93 (m, 2H, Har), 7.83–7.75 (m, 2H, Har),
7.66 (t, 1H, 3J = 5.80 Hz, SO2–NH), 7.30–7.23 (m, 2H, Har), 7.22–
7.10 (m, 3H, Har), 3.01–2.90 (m, 2H, CH2–NH), 2.68 (t, 2H,
3J = 7.49 Hz, CH2); 13C NMR (DMSO-d6): d = 164.85 (C@O), 149.77
(Car–NO2), 142.60 (Car), 140.54 (Car), 139.14 (C–SO2), 135.63 (Car),
129.81 (CHar), 129.08 (CHar), 128.75 (CHar), 128.03 (CHar), 126.66
(CHar), 124.04 (CHar), 120.62 (CHar), 44.51 (CH2–NH), 35.66 (CH2);
APCIMS (direct positive mode): calcd/found (m/z): 426.1/ 425.9
[M+H]+.
6.42. 4-Chloro-N-[4-(phenethylsulfamoyl)phenyl]benzamide,
7d
General procedure: 5, off-white crystals; Yield: 10%; 1H NMR
(DMSO-d6): d = 10.64 (s, 1H, CONH), 8.05–7.92 (m, 4H, Har), 7.83–
7.74 (m, 2H, Har), 7.67–7.55 (m, 3H, Har, SO2–NH), 7.31–7.23 (m,
2H, Har), 7.22–7.11 (m, 3H, Har), 3.03–2.92 (m, 2H, CH2–NH), 2.70
(t, 2H, 3J = 7.59 Hz, CH2); 13C NMR (DMSO-d6): d = 165.51 (C@O),
142.86 (Car–NH), 139.08 (C–SO2), 137.27 (Car), 135.20 (Car),
133.50 (Car), 130.16 (CHar), 129.06 (CHar), 128.99 (CHar), 128.77
(CHar), 127.96 (CHar), 126.68 (Car), 120.57 (CHar), 44.51 (NH–CH2),
35.596 (CH2); CIMS (direct mode): calcd/found (m/z): 415.0/415.0
6.47. 4-Amino-N-[4-(phenethylsulfamoyl)phenyl]benzamide, 7i
General procedure: 4, yellowish crystals; Yield: 97%; 1H NMR
(DMSO-d6): d = 10.09 (s, 1H, CONH), 8.00–7.89 (m, 2H, Har), 7.76–
7.68 (m, 4H, Har), 7.57 (t, 1H, 3J = 5.75 Hz, SO2-NH), 7.30–7.23 (m,
2H, Har), 7.22–7.12 (m, 3H, Har), 6.64–6.57 (m, 2H, Har), 5.85 (s,
2H, NH2), 3.00–2.90 (m, 2H, CH2-NH), 2.68 (t, 2H, 3J = 7.67 Hz,
CH2); 13C NMR (DMSO-d6): d = 152.95 (C@O), 143.72 (Car), 139.12
(C–SO2), 134.11 (Car), 130.04 (CHar), 129.06 (CHar), 128.78 (CHar),
127.84 (CHar), 126.74 (Car), 126.69 (Car), 120.75 (CHar), 120.13
(CHar), 113.06 (CHar), 44.52 (CH2–NH), 35.56 (CH2); CIMS (direct
mode): calcd/found (m/z): 396.1/396.1 [M+H]+; Purity (HPLC):
99.2% (System 2).
[
35ClM+H]+, 417.0/417.1 [37ClM+H]+; Purity (HPLC): 97.5% (System
2).
6.43. 3,4-Dichloro-N-[4-
(phenethylsulfamoyl)phenyl]benzamide, 7e
General procedure: 5, yellowish crystals; Yield: 78%; 1H NMR
(DMSO-d6): d = 10.68 (s, 1H, CONH), 8.23 (d, 1H, 4J = 2.03 Hz, Har,
H20), 8.00–7.91 (m, 3H, Har, A/A0, H50), 7.84 (d, 2H, 3J = 8.42 Hz,
Har, H60), 7.60 (1H, t, 5.88 Hz, SO2–NH), 7.31–7.23 (m, 2H, Har, B/
B0), 7.22–7.13 (m, 3H, Har), 3.03–2.95 (m, 2H, CH2–NH), (t, 2H,
3J = 7.40 Hz, CH2); 13C NMR (DMSO-d6): d = 164.11 (C@O), 142.66
(Car–NH), 139.14 (C–SO2), 135.49 (Car), 135.17 (Car), 135.16 (Car),
131.79 (Car), 131.27 (Car), 130.16 (Car), 129.07 (CHar), 128.75 (CHar),
128.62 (Car), 127.99 (CHar), 126.67 (Car), 120.58 (CHar), 44.53 (NH–
CH2), 35.65 (CHar2); CIMS (direct mode): calcd/found (m/z): 449.0/
448.9 [35Cl35ClM+H]+, 466.0/466.1 [35Cl35ClM+NH4]+ 468.0/468.1
6.48. 4-Chloro-N-[4-(phenethylsulfamoyl)phenyl]benzene-
sulfonamide, 7j
Generalprocedure:5, whitecrystals;Yield82%;1HNMR(DMSO-
d6): d = 10.93 (s, 1H, SO2-NH-AR), 7.88–7.79 (m, 2H, Har), 7.71–7.64
(m, 2H, Har), 7.67–7.79 (m, 2H, Har), 7.56 (t, 1H, 3J = 5.70 Hz, SO2-
NH), 7.33–7.14 (m, 5H, Har), 7.12–7.04 (m, 2H, Har), 3.00–2.84 (m,
2H, CH2-NH), 2.62 (t, 2H, 3J = 7.60 Hz, CH2); 13C NMR (DMSO-d6):
d = 141.49 (C–NH2), 139.05 (C–SO2), 138.66 (Car), 138.39 (Car),
135.73 (Car), 130.04 (CHar), 129.01 (CHar), 128.71 (CHar), 128.57
(CHar), 126.63 (CHar), 119.38 (CHar), 44.44 (CH2–NH), 35.58 (CH2);
CIMS (direct mode): calcd/found (m/z): 541.0/541.0 [35ClM+H]+,
453.0/453.0 [37ClM+H]+, 468.0/468.0 [35ClM+NH4]+, 470.0/470.0
[
35Cl37ClClM+H]+, 470.1 [37Cl37ClM+NH4]+; Purity (HPLC): 98.9%
(System 2).
6.44. 4-Bromo-N-[4-(phenethylsulfamoyl)phenyl]benzamide, 7f
General procedure: 5, off-white crystals; Yield: 98%; 1H NMR
(DMSO-d6): d = 10.65 (s, 1H, CONH), 8.02–7.86 (m, 4H, Har), 7.81–
7.72 (m, 4H, Har), 7.63 (t, 1H, SO2–NH, 3J = 5.96 Hz), 7.31–7-23
(m, 2H, Har), 7.22–7.10 (m, 3H, Har), 3.02–2.89 (m, 2H, CH2-NH),
2.68 (t, 2H, 3J = 7.37 Hz, CH2); 13C NMR (DMSO-d6): d = 165.49
(C@O), 142.89 (Car–NH), 139.15 (C–SO2), 135.26 (Car), 133.94
(Car), 131.93 (CHar), 130.37 (CHar), 129.08 (CHar), 128.75 (CHar),
127.97 (CHar), 126.66 (CHar), 126.19 (Car), 120.48 (CHar), 44.53
[
37ClM+NH4]+; Purity (HPLC): 97.8% (System 2).
6.49. [4-[2-[(4-Aminophenyl)sulfonylamino]ethyl]phenyl] 4-
aminobenzenesulfonate, 8
General procedure: 4, yellowish crystals; Yield: 45%; 1H NMR
(DMSO-d6): d = 7.48–7.35 (m, 4H, Har), 7.18–7.07 (m, 3H, Har,

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E.-M. Bissinger et al. / Bioorg. Med. Chem. 19 (2011) 3717–3731
NH), 6.93–6.84 (m, 2H, Har), 6.69–6.56 (m, 4, Har), 6.28 (s, 2H, NH2),
5.84 (s, 2H, NH2), 2.92–2.81 (m, 2H, NH-CH2), 2.65 (t, 2H,
3J = 7.19 Hz, CH2); 13C NMR (DMSO-d6): d = 154.91 (Car), 152.88
(C–SO2–O), 148.17 (Car), 138.25 (C–SO2), 130.75 (CHar), 130.31
(CHar), 128.90 (CHar), 125.65 (C–NH2), 122.43 (CHar), 118.37 (C–
NH2), 113.18 (CHar), 113.12 (CHar), 44.16 (NH–CH2), 34.79 (CH2);
CIMS (direct mode): calcd/found (m/z): 465.1/465.1 [M+NH4]+.
6.55. 4-Amino-N-(1-phenylethyl)benzamide, 9c
General procedure: 4, yellow crystals; Yield: 98%; 1H NMR
(DMSO-d6): d = 8.31 (d, 1H, 3J = 8.12 Hz, NH), 7.66–7.59 (m, 2H,
Har, A/A0), 7.39–7.34 (m, 2H, Har), 7.33–7.26 (m, 2H, Har), 7.23–
7.17 (m, 1H, Har), 6.57–6.51 (m, 2H, Har, B/B0), 5.61 (s, 2H, NH2),
5.13 (m, 1H, CH), 2.52–2.49 (m, 3H, CH3); 13C NMR (DMSO-d6):
d = 165.90 (C@O), 152.01 (C–NH2), 145.89 (CHar), 129.31 (CHar),
128,53 (CHar), 126.80 (Car), 126.47 (CHar), 121.59 (Car-CO), 112.87
(CHar), 48.43 (CH), 22.80 (CH3); EIMS (direct mode): calcd/found
(m/z): 120.0/120.0 [Phe–CH–(CH3)–NH]+, [H2N–Phe–CO]+; Purity
(HPLC): 97.5% (System 2).
6.50. 4-Nitro-N-phenethyl-benzamide, 8a
General procedure: 3, yellow crystals; Yield: 43%; 1H NMR
(DMSO-d6): d = 8.91 (t, 1H, 3J = 5.41 Hz, NH), 8.34–8.28 (m, 2H,
Har, A/A0), 8.07–8.01 (m, 2H, Har, B/B0), 7.34–7.16 (m. 5H, Har),
3.57–3.47 (m, 2H, CH2-NH), 2.87 (t, 2H, 3J = 7.35 Hz, CH2); 13C
NMR (DMSO-d6): d = 164.93 (C@O), 149.37 (C–NO2), 140.61 (C–
CO), 139.76 (CHar), 129.08 (CHar), 129.02 (CHar), 128.78 (CHar),
126.57 (Car), 123.95 (CHar), 41.47 (NH–CH2), 35.30 (Phe-CH2); CIMS
(direct mode): calcd/found (m/z): 270.1/270.1 [M+H]+.
6.56. Molecular modeling
All calculations were performed on a Pentium IV 2.6 GHz based
Linux cluster (16 CPUs). The compounds were transformed into
three-dimensional molecular structures using the MOE modeling
package (Chemical Computing Group). All compounds were gener-
ated in the protonation state that can be assumed under physiolog-
ical condition.
6.51. N-Benzyl-4-nitro-benzamide, 8b
General procedure: 3, yellow crystals; Yield: 79%; 1H NMR
(DMSO-d6): d = 9.39 (t, 1H, 3J = 5.69 Hz, NH), 8.36–8.30 (m, 2H,
Har, A/A0), 8.15–8.10 (m, 2H, Har, B/B0), 7.36–7.31 (m, 4H, Har),
7.29–7.23 (m, 1H, Har), 4.52 (d, 2H, 3J = 5.92 Hz, CH2); 13C NMR
(DMSO-d6): d = 165.03 (C@O), 149.47 (C–NO2), 140.37 (C–CO),
139.55 (CHar), 129.20 (CHar), 128.77 (CHar), 127.72 (CHar), 127.32
(Car), 124.00 (CHar), 43.27 (CH2); CIMS (direct mode): calcd/found
(m/z): 274.1/274.1 [M+NH4]+.
6.57. Protein modeling
A homology model for human PRMT1 was generated using Clu-
stalW sequence alignment of mouse PRMT1 and PRMT3 and the
COMPOSER module within Sybyl 7.1. The model generation and
validation is described in detail in the previous publications.5,8
6.58. GRID calculations
6.52. 4-Nitro-N-(1-phenylethyl)benzamide, 8c
Interaction possibilities were analyzed using the GRID program
(Molecular Discovery Inc.). GRID is an approach to predict noncova-
lent interactions between a molecule of known three-dimensional
structure (i.e., PRMT1) and a small group as a probe (representing
chemical features of a ligand).10 The calculations were performed
using version 22 of the GRID program and the hPRMT1 homology
model.
General procedure: 3, light yellow needles; Yield: 59%; 1H NMR
(DMSO-d6): d = 9.18 (d, 1H, 3J = 7.56 Hz, NH), 8.35–8.29 (m, 2H, Har,
A/A0), 8.15–8.08 (m, 2H, Har, B/B0), 7.43–7.38 (m, 2H, Har), 7.37–7.30
(m, 2H, Har), 7.27–7.20 (m, 1H, Har), 5.18 (m, 1H, CH), 1.5 (d, 3H,
3J = 7.09 Hz, CH3); 13C NMR (DMSO-d6): d = 164.35 (C@O), 149.40
(C–NO2), 144.87 (CHar), 140.58 (C–CO), 129.33 (CHar), 128.72
(CHar), 127.16 (Car), 126.47 (CHar), 123.88 (CHar), 49.25 (CH),
22.56 (CH3); EIMS (direct mode): calcd/found (m/z): 270.1/270.2
[M]+, 271.1/271.2 [M+H]+.
6.59. Ligand docking
The docking studies were performed using GOLD (version 4.0,
Cambridge Crystallographic Data Centre) with default parameters
together with the GoldScore fitness function.11 GoldScore was cho-
sen because it has outperformed other scoring functions in our pre-
vious studies. Docking was carried out to obtain a population of
possible conformations and orientations for the inhibitors at the
putative active site. All torsion angles in each compound were al-
lowed to rotate freely. For each ligand, docking runs were per-
formed with a maximum allowed number of 10 poses.
6.53. 4-Amino-N-phenethyl-benzamide, 9a
General procedure: 4, light yellow crystals; Yield: 84%; 1H NMR
(DMSO-d6): d = 8.09 (t, 1H, 3J = 5.51 Hz, NH), 7.58–7.52 (m, 2H, Har,
A/A0), 7.33–7.26 (m, 2H, Har), 7.25–7.16 (m, 3H, Har), 6.56–6.50 (m,
2H, Har, B/B0), 5.58 (s, 2H, NH2), 3.46–3.38 (m, 2H, CH2-NH), 2.81 (t,
2H, 3J = 7.56 Hz, CH2); 13C NMR (DMSO-d6): d = 166.59 (C@O),
151.93 (Car-NH), 140.18 (CHar), 129.04 (CHar), 128.72 (CHar),
126.42 (Car), 121.75 (Car-CO), 112.93 (CHar), 41.14 (NH–CH2),
35.84 (CH2); CIMS (direct mode): calcd/found (m/z): 241.1/241.2
[M+H]+; Purity (HPLC): 98.7% (System 2).
Acknowledgment
R.S. and M.J. thank the Deutsche Krebshilfe for funding.
6.54. 4-Amino-N-benzyl-benzamide, 9b
General procedure: 4, yellow crystals; Yield: 91%; 1H NMR
(DMSO-d6): d = 8.57 (t, 1H, 3J = 5.59 Hz, NH), 7.65–7.59 (m, 2H,
Har, A/A0), 7.34–7.26 (m, 4H, Har), 7.25–7.19 (m, 1H, Har), 6.58–
6.51 (m, 2H, Har, B/B0), 5.62 (s, 2H, NH2), 4.43 (d, 2H, 3J = 6.03 Hz,
CH2); 13C NMR (DMSO-d6): d = 166.62 (C@O), 152.08 (C–NH2),
140.76 (CHar), 129.19 (CHar), 128.59 (CHar), 127.55 (Car), 126.95
(CHar), 121.41 (C–CO), 112.95 (CHar), 42.76 (CH2); CIMS (direct
mode): calcd/found (m/z): 226.1/226.2 [M]+, 227.1/227.2 [M+H]+;
Purity (HPLC): 96.5% (System 2).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.02.032.
References and notes
1. Holliday, R. Epigenetics 2006, 1, 76.
2. Jenuwein, T.; Allis, C. D. Science 2001, 293, 1074.
3. Bissinger, E. M.; Heinke, R.; Sippl, W.; Jung, M. Med. Chem. Commun. 2010, 1, 114.

E.-M. Bissinger et al. / Bioorg. Med. Chem. 19 (2011) 3717–3731
3731
4. Cheng, D.; Yadav, N.; King, R. W.; Swanson, M. S.; Weinstein, E. J.; Bedford, M. T.
J. Biol. Chem. 2004, 279, 23892.
18. Metzger, E.; Muller, J. M.; Ferrari, S.; Buettner, R.; Schule, R. EMBO J. 2003, 22,
270.
5. Spannhoff, A.; Heinke, R.; Bauer, I.; Trojer, P.; Metzger, E.; Gust, R.; Schule, R.;
Brosch, G.; Sippl, W.; Jung, M. J. Med. Chem. 2007, 50, 2319.
6. Bissinger, E. M.; Jung, M. Chem. Biol. 2010, 17, 677.
7. Ragno, R.; Simeoni, S.; Castellano, S.; Vicidomini, C.; Mai, A.; Caroli, A.;
Tramontano, A.; Bonaccini, C.; Trojer, P.; Bauer, I.; Brosch, G.; Sbardella, G. J.
Med. Chem. 2007, 50, 1241.
19. Müller, J. M.; Isele, U.; Metzger, E.; Rempel, A.; Moser, M.; Pscherer, A.; Breyer,
T.; Holubarsch, C.; Buettner, R.; Schüle, R. EMBO J. 2000, 19, 359.
20. Ngan, S.; Stronach, E. A.; Photiou, A.; Waxman, J.; Ali, S.; Buluwela, L. Oncogene
2009, 28, 2051.
21. Pandini, G.; Mineo, R.; Frasca, F.; Roberts, C. T., Jr.; Marcelli, M.; Vigneri, R.;
Belfiore, A. Cancer Res. 2005, 65, 1849.
8. Heinke, R.; Spannhoff, A.; Meier, R.; Trojer, P.; Bauer, I.; Jung, M.; Sippl, W.
ChemMedChem 2009, 4, 69.
9. Spannhoff, A.; Hauser, A. T.; Heinke, R.; Sippl, W.; Jung, M. ChemMedChem 2009,
4, 1568.
10. Goodford, P. J. J. Med. Chem. 1985, 28, 849.
11. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. J. Mol. Biol. 1997, 267,
727.
22. Prescott, J. L.; Blok, L.; Tindall, D. J. Prostate 1998, 35, 71.
23. Frigo, D. E.; Sherk, A. B.; Wittmann, B. M.; Norris, J. D.; Wang, Q.; Joseph, J. D.;
Toner, A. P.; Brown, M.; McDonnell, D. P. Mol. Endocrinol. 2009, 23, 1385.
24. Ma, A. H.; Xia, L.; Desai, S. J.; Boucher, D. L.; Guan, Y.; Shih, H. M.; Shi, X. B.;
deVere White, R. W.; Chen, H. W.; Tepper, C. G.; Kung, H. J. Cancer Res. 2006, 66,
8439.
25. Rae, J. M.; Johnson, M. D.; Cordero, K. E.; Scheys, J. O.; Larios, J. M.; Gottardis, M.
M.; Pienta, K. J.; Lippman, M. E. Prostate 2006, 66, 886.
26. Cao, Q.; Gery, S.; Dashti, A.; Yin, D.; Zhou, Y.; Gu, J.; Koeffler, H. P. Cancer Res.
2009, 69, 7619.
27. Metzger, E.; Wissmann, M.; Yin, N.; Muller, J. M.; Schneider, R.; Peters, A. H.;
Gunther, T.; Buettner, R.; Schule, R. Nature 2005, 437, 436.
28. Turpin, J. A.; Song, Y.; Inman, J. K.; Huang, M.; Wallqvist, A.; Maynard, A.;
Covell, D. G.; Rice, W. G.; Appella, E. J. Med. Chem. 1999, 42, 67.
29. Robison, M. M.; Robison, B. L. J. Am. Chem. Soc. 1955, 77, 6554.
30. Kettler, K.; Sakowski, J.; Wiesner, J.; Ortmann, R.; Jomaa, H.; Schlitzer, M.
Pharmazie 2005, 60, 323.
12. Chen, D.; Ma, H.; Hong, H.; Koh, S. S.; Huang, S. M.; Schurter, B. T.; Aswad, D.
W.; Stallcup, M. R. Science 1999, 284, 2174.
13. Chen, D.; Huang, S. M.; Stallcup, M. R. J. Biol. Chem. 2000, 275, 40810.
14. Spannhoff, A.; Machmur, R.; Heinke, R.; Trojer, P.; Bauer, I.; Brosch, G.; Schule,
R.; Hanefeld, W.; Sippl, W.; Jung, M. Bioorg. Med. Chem. Lett. 2007, 17, 4150.
15. Stone, E. M.; Schaller, T. H.; Bianchi, H.; Person, M. D.; Fast, W. Biochemistry
2005, 44, 13744.
16. Luo, Y.; Arita, K.; Bhatia, M.; Knuckley, B.; Lee, Y. H.; Stallcup, M. R.; Sato, M.;
Thompson, P. R. Biochemistry 2006, 45, 11727.
17. Xu, W.; Chen, H.; Du, K.; Asahara, H.; Tini, M.; Emerson, B. M.; Montminy, M.;
Evans, R. M. Science 2001, 294, 2507.