N-Substituted homopiperazine barbiturates as gelatinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.06.055

Matrix metalloproteinases are implicated in a wide range of pathophysiological processes and potent selective inhibitors for these enzymes continue to be eagerly sought. 5,5-Disubstituted barbiturates hold promise as inhibitor types being stable in vivo and relatively selective for the gelatinases (MMP-2 and MMP-9). In this paper we describe the synthesis of 5-piperazine and -homopiperazine substituted barbiturates. The activity of these compounds as gelatinase inhibitors was evaluated using supernatants from 12-O-tetradecanoylphorbol-13-acetate (PMA)-stimulated HT-1080 cells as well as using recombinant human MMPs. N-Acyl homopiperazine compounds were found to be potent inhibitors of the gelatinases (range in nM) and generally more potent than the corresponding piperazine analogues. The panel of N-acyl homopiperazines was enlarged in order to exploit differences between the gelatinases at the S2′ site in order to design MMP-2- or MMP-9-selective inhibitors. Compounds in this group exhibited single digit nano-molar potency and some selectivity between the two enzymes. Representative potent compounds were effective inhibitors of cancer cell migration.

Full text of DOI:10.1016/j.bmc.2011.06.055

Bioorganic & Medicinal Chemistry 19 (2011) 4985–4999
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
N-Substituted homopiperazine barbiturates as gelatinase inhibitors
⇑
Jun Wang, Carlos Medina, Marek W. Radomski, John F. Gilmer
School of Pharmacy and Pharmaceutical Sciences, Trinity College, Dublin 2, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Matrix metalloproteinases are implicated in a wide range of pathophysiological processes and potent
selective inhibitors for these enzymes continue to be eagerly sought. 5,5-Disubstituted barbiturates hold
promise as inhibitor types being stable in vivo and relatively selective for the gelatinases (MMP-2 and
MMP-9). In this paper we describe the synthesis of 5-piperazine and -homopiperazine substituted barbi-
turates. The activity of these compounds as gelatinase inhibitors was evaluated using supernatants from
12-O-tetradecanoylphorbol-13-acetate (PMA)-stimulated HT-1080 cells as well as using recombinant
human MMPs. N-Acyl homopiperazine compounds were found to be potent inhibitors of the gelatinases
(range in nM) and generally more potent than the corresponding piperazine analogues. The panel of N-
acyl homopiperazines was enlarged in order to exploit differences between the gelatinases at the S20 site
in order to design MMP-2- or MMP-9-selective inhibitors. Compounds in this group exhibited single digit
nano-molar potency and some selectivity between the two enzymes. Representative potent compounds
were effective inhibitors of cancer cell migration.
Received 6 April 2011
Revised 16 June 2011
Accepted 20 June 2011
Available online 25 June 2011
Keywords:
MMP
Gelatinase
Barbiturate
Enzyme inhibitor
Metastasis
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
In 2001, following a random screen for anti-tumour agents,
workers from the Hoffman La-Roche company reported the surpris-
Matrix metalloproteinases (MMPs) comprise a family of zinc
and calcium-dependent endopeptidases that are involved in the
remodelling and degradation of the extracellular matrix (ECM)
during physiological processes and in pathological conditions such
as inflammation, tumour growth and metastasis.1 MMPs have been
classified into the collagenases, gelatinases and stromelysins based
on their substrate specificity. These enzymes possess a high degree
of sequence similarity (56–64%) in the catalytic domain. The active
site is a groove, shallow in places, usually presented horizontally
across the protein with three un-primed sub-sites to the left of
the co-ordinated zinc and three primed sub-sites to the right
(S3-S2-S1-Zn-S10-S20-S30).2,3 Inhibitors of the MMPs have been
intensively pursued for the past thirty years because of their
involvement in disease processes. Inhibitor design usually employs
a zinc binding group (ZBG) attached to a hydrophobic moiety
sometimes based on a collagen fragment. Zinc binding groups in-
clude thiols, phosphinic acids, carboxylic acids and perhaps most
commonly, hydroxamates. Attempts to introduce selectivity have
focussed almost exclusively on the primed side of the catalytic
groove and especially the S10 channel.3,4
ing finding that certain 5,5-disubstituted pyrimidine-2,4,6,-triones
(barbiturates) are inhibitors of the MMPs with good gelatinase
selectivity.3,5 MMP inhibitory barbiturates are without the sedative
effects of the classical agents, but exhibit some of their pharmaco-
kinetic strengths; some other Zn binding groups impart poor in vivo
stability (e.g., hydroxamates).6 The structural basis for barbiturate
interactions with the MMPs was revealed in several X-ray studies.7
The ionised barbiturate ring chelates the zinc atom in a tridentate
mode directing the 5-substituents into the S10 and S20 substrate
binding pockets. The class exhibit broadly similar SAR to non-barbi-
turate MMP inhibitor families. The 5-aromatic substituents (biphe-
nyl or phenyloxyphenyl) bind in the deep hydrophobic S10
substrate pocket. The second 5-substitutent is directed in this
arrangement into the S20 pocket towards solvent. Several elegant
studies have demonstrate that C5- piperazine or piperidine
substitution is favourable for high potency and moreover that the
heterocycle imparts flexibility in the design of a range of barbitu-
rate-based pharmacological, clinical and biochemical tools.8–10
Our laboratory has research programs investigating the role of
MMPs in inflammatory bowel disease, cancer-platelet interactions
and heart failure. We have an ongoing need for inhibitor tools with
selectivity for MMP-2 or MMP-9 (gelatinases A and B). In designing
such molecules we decided to adopt the barbiturate template
mainly because of its pharmacokinetic characteristics.
Abbreviations: MMP, matrix metalloproteinase; ECM, extracellular matrix; ZBG,
zinc binding group; PMA, 12-O-tetradecanoylphorbol-13-acetate; TGF-b, trans-
forming growth factor beta; HGF, hepatocyte growth factor; FCS, fetal calf serum;
APMA, p-aminophenylmercuric acetate.
We are currently interested in probing for interactions at the
mouth of the S20 pocket that might impart gelatinase type or
sub-type selectivity. We were in this regard attracted to the
⇑
Corresponding author. Tel.: +353 1 896 2795; fax: +353 1 896 2793.
E-mail address: gilmerjf@tcd.ie (J.F. Gilmer).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.055

4986
J. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4985–4999
initial problems with the incorporation of the phenoxyphenyl
O
Vary at X which binds close to
Ile/Met exchange site at P2'
PhO
group. This remains an occasionally problematic transformation
in organic chemistry. A number of different methods were tried
such as microwave assisted reactions or the Ullmann coupling.
These approaches either gave low yield or presented problems
with purification. Eventually it was found that a copper-mediated
coupling with phenyl boronic acid in the presence of copper ace-
tate and pyridine worked well producing methyl 2-(4-phenoxy-
phenyl) acetate (2) in good yield.11
N
X
Optimal for
S1 binding
(CH2)n
N
O
H
O
H
n=2 shows improved potency
N
N
Zn binding group
O
The key intermediate 5 was either converted directly to the
N-acyl or N-alkyl piperazines or homopiperazine barbiturate deriv-
atives 8a–j and 9a–d or reacted with piperazine or homopiperazine
to produce the intermediates 10 and 11, which then yielded 12 and
13 using the appropriate acid chlorides or carboxylic acids (Scheme
Figure 1. Strategy for exploring the P20 site on the gelatinases.
piperazine model as these provide the possibility for distal substi-
tution through simple N-acylation or alkylation. However, it
seemed to us from inspection of X-ray models of the barbiturate
docking into collagenases that the S20 pocket might be better oc-
cluded with a seven membered ring (Fig. 1). We were also inter-
ested in differences between the gelatinases at this site that
might endow interacting compounds with some sub-type selectiv-
ity. This paper describes synthesis and evaluation of acyl 5-homo-
piperazine barbiturates, their potential to exhibit gelatinase
subtype selectivity and their effects on cancer cell migration.
1).
A wider panel of N-acyl homopiperazines was obtained
(Scheme 2) by acylation of 11 at low temperature with the appro-
priate acid chloride or by diimide coupling.
2.2. Enzyme inhibition and modelling
The novel barbiturates were screened by zymography for inhib-
itory effects on MMP-2 and MMP-9 using the HT1080 fibrosarcoma
cell line following stimulation with 12-O-tetradecanoylphorbol-
13-acetate (PMA). Residual gelatinolytic activity in the presence
of the test compounds was measured by densitometry. Represen-
2. Results and discussion
2.1. Chemistry
tative results of% inhibition of MMP-2 and MMP-9 at 0.5
shown in Table 1.
lM are
The zymography results indicated that compound 9d, a 5-homo-
piperazine caused notably good inhibition of the gelatinases,
The compounds were prepared based on a standard approach to
5-substituted barbiturates outlined in Scheme 1. We had some
O
OH
O
O
O
O
vi
iv
i
ii
iii
NH
N
O
n= 1, 2
O
O
Br
O
O
O
O
O
O
O
HN NH
HN NH
O
O
O
HN NH
O
O
O
10 n=1
11 n=2
O
3
1
4
5
2
vii
v
BOC
N
O
BOC
N
O
R2
vii
viii
ix
R1
N
N
N
O
n=1, 2
N
O
O
N
n= 1, 2
N
H
O
HN NH
HN NH
O
O
12 n=1
13 n=2
8 n=1
9 n=2
7
6
R1 =
R2=
O
O
O
O
CH3
O
CH3
HO
8e
8j
8a
8b
8d
8f
8g
8h
8i
9a
9b
9c
9d
8c
O
O
O
O
O
O
O
S
S
N
N
CN
CN
Cl
Cl
12a
12b 12c 13a
13b 13c
13d
Scheme 1. Preparation of phenoxyphenyl substituted piperazine- and homopiperazine-based barbiturate derivatives. Reagents and conditions: (i) C6H5B(OH)2, copper(II)
acetate, pyridine, DCM, rt, 24 h, 32%; (ii) (CH3O)2CO, NaH, dry THF, 105 °C, 5 h, 86%; (iii) Na, urea, EtOH, 100 °C, 7 h, 78%; (iv) Br2, HBr, H2O, 0 °C, 5 h, 82%; (v) substituted
piperazines or homopiperazines, MeOH, rt, 24 h, 37–63%; (vi) piperazine or homopiperazine, MeOH, rt, 24 h 54–58%; (vii) acid chlorides, THF, ꢀ70 °C, 5 h or carboxylic acids,
DCC, DMAP, dry THF, rt, 24 h, 31–42%; (vii) C3H5CH2Br, MeOH, rt, 12 h, 64%; (viii) CF3COOH, DCM, 0 °C, overnight; (ix) MeOH, compounds 5, rt, 24 h, 41%.

J. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4985–4999
4987
O
O
R
N
NH
i
N
O
N
O
O
O
HN NH
HN NH
O
O
14-46
11
CH3
CH3
O
O
O
O
O
O
R =
S
CF3
O
18
O
O
14
O
15
17
16
H3C
O
O
O
CH3
O
Cl
O
CH3
N
O
22
N
S
NH
O
Cl
O
F
23
21
20
19
CH3
O
O
O
O
O
S
N
N
CH3
O
CF3
Cl
N
O
28
27
26
CH3
25
24
Cl
O
O
O
O
O
Br
Cl
CH3
32
33
30
31
29
O
O
Cl
O
S
N
O
N
O
O
Cl
N
O
S CH3
N
CH3
F
H3C
38
35
36
37
34
O
O
O
O
H
NH
N
O
CH3
O
S
N
S
O
S
O
O
O
N
H3C
CH3
N
CH3
O
40
41
42
39
O
S
O
O
O
O
CH3
NH
O
HN
O
CH3
N
O
S
S
HN
S
O
O
NH
CH3
CH3
O
CH3
O
F
46
45
44
43
Scheme 2. Preparation of 5-N-acyl homopiperazine barbiturates. Reagents and conditions: (i) acid chlorides, THF, ꢀ70 °C, 5 h, or DCC, DMAP, carboxylic acids, THF, rt, 12 h,
26–52%.
supporting the idea that the larger ring might impart improved
affinity/potency. To further investigate this, a series of directly anal-
ogous piperazine and homo piperazine compounds 8a–j, 9a–c, 12a–
c, and 13a–d were assessed. Inhibitory potency of these modified
compounds was measured using human recombinant MMP-2 and
MMP-9 and a fluorogenic assay.12 The results are shown in Table 2.
The barbiturate homopiperazine compounds exhibited greater
potency than the corresponding piperazine-substituted com-
pounds apart from compounds 13d (IC50 (MMP-2) = 110 nM,
(MMP-9) = 104 nM) with homopiperazine ring, being somewhat
less potent than 12c (IC50 (MMP-2) = 45 nM, (MMP-9) = 54 nM)
with piperazine ring (Table 2). The MMP-2 and MMP-9 inhibition
by cyclopropylcarbonyl substituted barbiturate derivatives slightly
increased when the piperazine ring 8d (IC50 (MMP-2) = 27 nM,
(MMP-9) = 15 nM) was replaced by the homopiperazine ring 13a
(IC50 (MMP-2) = 13 nM, (MMP-9) = 13 nM).
Benzyl homopiperazine 9b (IC50 (MMP-9) = 22 nM) showed
fourfold higher potency towards MMP-9 than its piperazine

4988
J. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4985–4999
Table 1
Representative gelatinase inhibition by 8a–i and 9d determined by zymographically separating gelatinases from PMA-stimulated human fibrosarcoma HT1080 cells and
incubating in the presence of test compounds at 0.5 M (n = 3)
l
Compd
MMP-9 inhibition (%)
MMP-2 inhibition (%)
Compd
MMP-9 inhibition (%)
25
55 11
53 12
MMP-2 inhibition (%)
30 11
8a
8b
8c
8d
9d
64
62
37
5
5
8
74
71
5
5
8e
8f
8g
8h
8i
7
64
52 11
63
54 11
7
55 10
76 12
69 13
28
42
49
6
8
7
7
77
7
Table 2
IC50 values comparing piperazine and homopiperazine substituted compounds
Piperazines
Homopiperazines
Compd
Compd
MMP-2 IC50 (nM)
MMP-9 IC50 (nM)
MMP-2 IC50 (nM)
MMP-9 IC50 (nM)
8c
8d
8g
8i
8j
12a
12b
12c
5.2 (3.5–7.7)
27 (21–34)
131 (114–151)
14 (14–16)
16 (11–23)
43 (31–62)
43 (34–56)
45 (34–61)
10 (8–13)
9a
13a
9b
9c
9d
13b
13c
13d
1.1 (0.7–1.5)
13 (11–16)
26 (19–35)
1.5 (1.1–2.1)
1.9 (1.6–2.3)
19 (13–27)
28 (20 – 41)
110 (97–125)
1.1 (0.8–1.5)
13 (8–19)
15 (12–20)
86 (71–104)
16 (13–20)
9.4 (7.8–11)
73 (58–91)
45 (36–56)
54 (45–65)
22 (16–30)
2.9 (2.1–3.9)
7.5 (6.2–9.1)
16 (12–20)
9.8 (7.7–13)
104 (89–123)
analogue 8g (IC50 (MMP-9 = 86). The acetyl substituted homopip-
erazine inhibitor 9c (IC50 (MMP-2) = 1.50 nM, (MMP-9) =
2.90 nM) had a ninefold increased potency on MMP-2 and fivefold
in MMP-9 compared with piperazine-based compound 8i (IC50
(MMP-2) = 14 nM, (MMP-9) = 16 nM). The changing of piperazine
substitution in the cyclopropylmethyl compound 8c (IC50 (MMP-
2) = 5.20 nM, (MMP-9) = 10 nM), the most potent piperazine-based
compound for MMP-2 in the library, to homopiperazine ring 9a
(IC50 (MMP-2) = 1.08 nM, (MMP-9) = 1.15 nM) improved the po-
tency fivefold in MMP-2 and 10-fold in MMP-9. Molecular model-
ling methods were used to investigate initial biochemical findings.
Piperazine and homopiperazine compounds were docked into both
MMP-2 and MMP-9 using Autodock 4. The docking experiments
placed the C5-phenoxyphenyl group deep into the S10 pocket as ex-
pected, with the piperazine/homopiperazine directed into the S20
site and towards solvent. Docking of several of the piperazine/
homopiperazine pairs suggested that the homopiperazine com-
pounds are more potent because of increased occlusion of the S2
with better surface contacts with both MMP2 and MMP9.
A larger panel of compounds was constructed in order to ex-
plore the potential of homopiperazines to exhibit gelatinase sub-
type selectivity (Scheme 2). We deliberately included fragments
for conjugation with 11 that could potentially donate a H-bond
to the sulfur atom of Met422 of MMP-9 in the expectation that this
might impart selectivity over MMP-2, which is substituted with an
Ile at the corresponding position. Initial modelling studies indi-
cated that compounds 38–46 would position functionality close
to Met422 to make favourable contacts for binding.
Table 3
IC50 values of C5 homopiperazine-based barbiturate derivatives estimated from
sigmoids obtained at five concentration levels in duplicate
Compd
MMP-2 (nM)
MMP-9 (nM)
MMP-9 selectivity
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
14 (12–17)
36 (28–47)
13 (11–16)
29 (24–35)
269 (220–330)
60 (51–71)
50 (42–59)
329 (286–378)
86 (64–115)
264 (218–320)
2.55 (1.96–3.32)
308 (251–377)
24 (20–29)
1.08
1.24
4.42
1.17
3.10
1.14
0.33
0.72
1.13
0.70
1.04
1.13
1.09
1.13
0.75
0.55
1.05
3.78
2.00
1.82
0.77
3.24
0.96
0.24
0.23
8.92
0.99
1.44
0.75
0.69
0.52
0.56
0.41
1190 (974–1453)
70 (61–81)
155 (121–198)
376 (294–480)
28 (22–36)
191 (156–233)
2.88 (2.35–3.52)
215 (181–255)
25 (20–30)
34 (28–43)
51 (44–59)
30 (25–37)
47 (39–57)
120 (96–149)
198 (172–228)
44 (37–51)
282 (232–343)
87 (77–97)
62 (50–76)
173 (127–236)
77 (69–86)
81 (68–97)
24 (19–30)
74 (66–84)
93 (76–113)
214 (169–271)
698 (595–818)
978 (800–1196)
15 (13–18)
106 (83–135)
264 (213–327)
80 (69–93)
268 (220–327)
23 (20–26)
31 (22–43)
95 (72–125)
100 (82–124)
25 (21–30)
25 (19–32)
311 (232–417)
411 (267–635)
24 (21–28)
706 (524–953)
681 (525–901)
20 (15–25)
83 (39–101)
54 (38–75)
142 (118–170)
184 (153–221)
Results for the homopiperazine panel are presented in Table 3.
Compound 16 had the highest IC50 value on MMP-2, exhibiting >4-
fold selectivity for MMP-9 over MMP-2 (IC50: MMP-2 = 1190 nM,
MMP-9 = 269 nM). Compound 31 (IC50: MMP-2 = 87 nM, MMP-
9 = 23 nM) was >11-fold more potent than 16 in inhibition of
gelatinases and had nearly fourfold selectivity for MMP-9 over
57 (48–68)
28 (22–35)
80 (64–99)
76 (65–90)
MMP-2. Compounds 18 (IC50: (MMP-2) = 155 nM, MMP-9 =
50 nM) and 35 (IC50: MMP-2 = 81 nM, MMP-9 = 25 nM) also exhib-
ited MMP-9 selectivity. Compound 38 (IC50: MMP-2 = 93 nM,
MMP-9 = 411 nM) was the weakest MMP-9 inhibitor, but was
the most MMP-2 selective inhibitor. Other compounds such as 20
(IC50: MMP-2 = 28 nM, MMP-9 = 86 nM) and 37 (IC50: MMP-2 =
74 nM, MMP-9 = 311 nM) also showed moderate selectivity for
MMP-2 over MMP-9.
The most potent inhibitor in the homopiperazine library
(Table 3) was 22 (IC50: MMP-2 = 2.88 nM, MMP-9 = 2.55 nM),
which had very low IC50 values on both MMP-2 and MMP-9, but
no selectivity. Other inhibitors such as 14 (IC50: MMP-2 = 14 nM,
MMP-9 = 13 nM), 24 (IC50: MMP-2 = 25 nM, MMP-9 = 24 nM), and
36 (IC50: MMP-2 = 24 nM, MMP-9 = 25 nM) were potent MMP-2
and MMP-9 inhibitors, but again with poor selectivity. Several

J. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4985–4999
4989
compounds were neither potent nor selective, including 19, 21, 23,
28 and 30.
Compound 22 is structurally similar to 23 but the inhibitory
potencies of the two compounds were markedly different, with
22 (IC50: MMP-2 = 2.88 nM, MMP-9 = 2.55 nM) showing nearly
field.13 The ECM by providing a physical support or barrier for cells
plays an important role in maintenance of tissue structure and cell
migration.14 During invasion, neoplastic cells detach from primary
tumours enter the lymphatic and blood vessels by crossing the epi-
thelial basement membrane, and undergo metastatic growth at
distant sites.2 MMPs are intimately connected with these pro-
cesses.2 The degradation of ECM component is necessary for cell
migration.15 MMPs have been considered to be a major regulator
of ECM composition and to be promoters for cell migration due
to their ability to degrade the ECM components. For example,
MMP-2 and MMP-9 can cleave a major protein component of base-
ment membrane-type IV collagen.16,17
Some biologically active modular breakdown products released
from the proteolytic remodelling of ECM can promote cell migra-
tion. For example, laminin-5 cleaved by MMP-2 produces a gam-
ma2 subunit, which has been shown to induce migration of
breast epithelial cells.18 Recently it has been clearly demonstrated
that MMPs are involved in regulation of cytokines, chemokines,
growth factors, and a variety of enzymes.14 Degradation of ECM
molecules results in release of various growth factors and cyto-
kines stored in the ECM molecules. For example, decroin, a small
proteoglycan, functions as a reservoir for TGF-b, and cleavage of
decroin by various MMPs results in release of TGF-b triggering its
biological functions.19
100-fold greater inhibition of MMP-2 and MMP-9 than 23 (IC50
:
MMP-2 = 215 nM, MMP-9 = 308 nM). The lower potency of 23
could be due to the size of sulfur and chlorine substitutions on
the indene ring, relative to the oxygen and methyl groups in 22.
Compound 23 with its bulky, rigid substituent attached to homo-
piperazine might be sterically excluded from the mouth of the
S20 pocket resulting in loss of binding affinity. Therefore, the size
and rigidity of the subgroup attached to homopiperazine is likely
to be important for potency. This would explain why 16 and 19
showed weak inhibition towards MMP-2 and MMP-9 compared
with 17 which has a smaller benzene ring substituent (–OCF3).
The docking indicated that the ethoxyl groups in 16 prevents
the homopiperazine substitution from entering the S20 pocket.
The most MMP-9 selective compound in the library was 39,
showing nearly ninefold selectivity for MMP-9 over MMP-2 (IC50
:
MMP-2 = 214 nM, MMP-9 = 24 nM). This may be because of the
orientation the compound can adopt in the gelatinases (Fig. 2).
When the compound was docked with MMP-9, the N-methyl
group pointed towards the sulfur on Met422. Although 39 could
be docked with a similar orientation in MMP-2, the interaction be-
tween the nitrogen and Ile222 was not as strong.
In addition, MMPs are able to cleave cell-cell adhesion proteins,
to release bioactive cell surface molecules, and to degrade signal-
transducing molecules.20 For example, the cleavage of signal-trans-
ducing molecule E-cadherin and the soluble E-cadherin fragment
by MMP-3 and MMP-7 inhibits cell aggregation and promotes cell
invasion.21
Another reason for selectivity of 39 on MMP-9 might be the size
and rigidity of subgroup attached to the homopiperazine ring, that
is, methyl indazole. When the nitrogen atom and methyl group in
the indazole pointed toward the Met422 residue, it had the best
binding affinity because the methyl indazole just fitted into the
mouth of the S20 pocket. If the methyl group turned to other direc-
tions, the walls of the S20 pocket would push the indazole group
away from the mouth of the pocket, resulting in loss of affinity.
In most of the models that we studied, the mouth of S20 pocket
of MMP-2 was slightly smaller than of MMP-9.7 Substituents of
appropriate size attached to the homopiperazine ring could go dee-
per into the S20 pocket of MMP-9 than MMP-2. This might be the
reason why 16, 31 and 35 exhibited (moderate) selectivity towards
MMP-9. These three compounds have substitutions on the benzene
ring capable more easily penetrating the mouth of the S20 pocket in
MMP-9 than the narrower MMP-2, resulting in the better binding
and inhibition of MMP-9.
Therefore, inhibition of MMP activity can counteract cancer cell
invasion by inhibiting degradation of ECM and release of cytokines
and growth factors. Homopiperazine-base barbiturates with po-
tent inhibition on MMP-2 and MMP-9 were tested in an invasion
assay to investigate the potential of inhibition of gelatinase activity
on cancer cell invasion. Many types of cells have been used in inva-
sion assays such as HT1080, MCF-7, and Caco-2 cells. Migration of
Caco-2 cells was monitored in our assay using a Matrigel mem-
brane with hepatocyte growth factor (HGF) used to stimulate
growth and invasiveness of Caco-2 cells22 (in our hands Caco-2
cells are not so aggressive and quite easy to manipulate pharmaco-
logically in this assay, whereas HT1080 cells produce a better MMP
secretion response to PMA). The negative control in this assay con-
sisted of cells untreated with HGF. Cell migration was monitored
by microscopy (Figure 3).
2.3. Inhibition of cancer cells invasiveness
Compounds 9d, 31, and 39 were assayed at 10 lM and 100 nM;
To inhibit the ability of cancer cells to undergo migration and
invasion is the one of most crucial issues in the cancer research
22 and 37 were tested at 100 nM. The inhibition of Caco-2 cells
invasiveness by these compounds was dependent on their ability
Figure 2. Pictures of 39 docked with Connolly surfaces on MMP-2 (left) and MMP-9 (right). The surfaces were generated by the programme PyMOL based on the crystal
structures of MMP-2 (pdb code: 1q1b) and MMP-9 (pdb code: 2ovx). The ligand orientations were used which had the highest predicted affinity. The nitrogen atom and
methyl group on the indazole of 39 were close to the amino acid Ile222/Met422 (yellow sulfur) interchange between MMP-2 and MMP-9. The active site zincs are shown as
red spheres.

4990
J. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4985–4999
100
80
60
40
20
0
**
**
**
A
**
**
**
**
**
B
Figure 3. Results from the invasion assay with compounds 9d, 22, 31, 38, 39 (P <0.05). Representative microscope images are shown from the invasion assays: (A) a positive
control insert, incubated with Caco-2 cells and HGF. (B) Insert incubated with Caco-2 cells, HGF, and 10 M and 100 nM 9d.
l
to inhibit activities of MMP-2 and MMP-9. The least potent com-
pound in the recombinant enzyme assay (37) exerted weakest
inhibition (20.15%) at 100 nM. Compound 39 caused greater inhibi-
class were effective inhibitors of cancer cell migration in an in vitro
model of metastasis using colon carcinoma cells (CACO-2). We are
currently using the inhibitor panel described here to probe patho-
physiological contributions of the gelatinases in inflammatory bo-
wel disease.
tion of invasion of the cells than 31 at 10
though 39 (IC50: MMP-2 = 213.79 nM, MMP-9 = 23.90 nM) had
higher IC50 values on both MMP-2 and MMP-9 than 31 (IC50
lM and 100 nM, even
:
MMP-2 = 86.50 nM, MMP-9 = 22.71 nM). In addition, 39 had better
ability to inhibit the invasiveness than 22 (IC50: MMP-2 = 2.88 nM,
MMP-9 = 2.55 nM) which possessed over 70-fold potency on MMP-
2 and ninefold on MMP-9 than 39. It is possible that the out of
trend inhibitory ability of 39 is due to its MMP-9 selectivity.
MMP-9 may play a more important functional role in facilitating
cancer cell migration than MMP-2. For example, MMP-2 and
MMP-9 can both activate latent TGF-b, but only MMP-9 coordi-
nated with TGF-b could promote cancer cell growth and invasion.23
It has also been demonstrated that tumour cell invasion is corre-
lated and regulated by MMP-2.24 Compound 9d which exerted
fourfold selectivity for MMP-2 over MMP-9 had lower potency
on MMP-9 than 22, but showed better inhibition of invasion. It
seems that inhibition of only one protein’s activity, either MMP-2
or MMP-9, may result in better inhibition of invasion. Because only
five compounds were examined in the invasion assay it is hazard-
ous to draw conclusions about relationships nevertheless the per-
centage of inhibition of migration values at 100 nM exhibited a
moderate correlation with MMP-9 IC50 values (r2 0.45). But our re-
sults illustrate that inhibition of activity of MMP-2 and MMP-9
could decrease cancer cell invasive ability, affirming that MMP-2
and MMP-9 are important for cancer cell invasion and important
targets for cancer therapy.
4. . Methods and materials
4.1. Chemistry
All chemicals were purchased from Sigma–Aldrich (Ireland), ex-
cept where stated. All the reactions were monitored using TLC.
Uncorrected melting points were measured on a Stuart Apparatus.
Infra-red (IR) spectra were performed on a Perkin Elmer FT-IR Par-
agon 1000 spectrometer. 1H and 13C nuclear magnetic resonance
(NMR) spectra were recorded at 27 °C on a Brucker DPX 400 spec-
trometer (400.13 MHz, 1H; 100.61 MHz, 13C). Coupling constants
are reported in Hertz. For 1H NMR assignments, chemical shifts
are reported: shift value (number of protons, description of absorp-
tion, coupling constant(s) where applicable). Electrospray ionisa-
tion mass spectrometry (ESI-MS) was performed in the positive
ion mode on a liquid chromatography time-of-flight mass spec-
trometer (Micromass LCT, Waters Ltd., Manchester, UK). The sam-
ples were introduced into the ion source by an LC system (Waters
Alliance 2795, Waters Corporation, USA) in acetonitrile/water
(60:40% v/v) at 200 ll/min. The capillary voltage of the mass spec-
trometer was at 3 kV. The sample cone (de-clustering) voltage was
set at 40 V. For exact mass determination, the instrument was
externally calibrated for the mass range m/z 100 to m/z 1000. A
lock (reference) mass (m/z 556.2771) was used. Mass measure-
ment accuracies of < 5 ppm were obtained. Compound purity/
homogeneity was confirmed using a combination of NMR, TLC
and HPLC.
3. Conclusion
A panel of barbiturate 5-piperazine- and homopiperazine-based
compounds were prepared to investigate interactions at the mouth
of S20 pocket of the gelatinases where this group binds. The lower
IC50 values of homopiperazine ring barbiturates and the docking
support our hypothesis that the larger ring could better fill the
S20 pocket. Several compounds exhibited moderate gelatinase sub-
type selectivity and good potency. However, selectivity in the ser-
ies was not as good as anticipated. We can speculate on possible
contributors to this observation- overlapping physicochemical
properties and binding characteristics in the Ile and Met side
chains; binding competition with water on the exposed site; fail-
ure to properly target the identified amino acid exchange site be-
cause of a lack of flexibility in the barbiturate binding mode.
Nonetheless, representative compounds from the homopiperazine
4.1.1. Methyl 2-(4-phenoxyphenyl)acetate (2)
Methyl 4-hydroxyphenylacetate (3.32 g, 20 mmol), copper (II)
acetate (3.58 g, 20 mmol), phenylboronic acid (4.88 g, 40 mmol),
powdered 4 Å molecular sieves and pyridine (8 ml) were added
into DCM (100 ml). The reaction mixture was stirred at room tem-
perature for 20 h. The resulting mixture was filtered and diethyl
ether (50 ml ꢁ 3) was used to extract the compound and purified
by column chromatography to afford the pure compounds as the
yellow oil (1.55 g, 32%). MS: calcd for C15H14O3Na = 265.0841.
(M+Na)+ = 265.0835 found. 1H NMR (CDCl3) d ppm: 3.68 (s, 2H,
CH2), 3.76 (s, 3H, CH3), 7.03–7.09 (m, 4H, Ar-H), 7.14–7.18 (t,
J = 7.53, 1H, Ar-H), 7.30–7.32 (d, J = 8.53, 2H, Ar-H), 7.37–7.41 (m,

J. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4985–4999
4991
J = 7.53, 2H, Ar-H). 13C NMR (CDCl3) d ppm: 40.2; 51.9; 115.2;
1H, CH), 1.34 (s, 9H, CH3), 1.95–2.02 (m, 2H, CH2), 2.50–2.55 (m, 2H,
CH2), 2.82–2.85 (m, 2H, CH2), 2.87–2.90 (m, 2H, CH2), 3.35–3.41 (m,
2H, CH2), 3.49–3.52 (m, 1H, CH2), 3.57–3.60 (m, 1H, CH2). 13C NMR
(CDCl3) ppm: 3.8; 7.1; 25.4; 27.8; 42.9; 43.9; 53.7; 55.2; 62.0; 79.2;
118.7; 123.1; 128.5; 129.6; 130.5; 156.2; 156.9; 172.2; IR (CH2Cl2):
m
(cmꢀ1): 3063, 1731, 1613, 1589.
4.1.2. Dimethyl 2-(4-phenoxyphenyl)malonate (3)
154.8. IR (CH2Cl2): m
(cmꢀ1): 2974, 1701, 1699, 1590.
A suspension of NaH (60% suspension in paraffin oil (paraffin oil
was removed by repeated washings with hexane)) (80 mg,
20 mmol) and dimethyl carbonate (5.4 ml, 64 mmol) in dry THF
(64 ml) was heated to 100 °C, and a solution of methyl 2-(4-phen-
oxyphenyl) acetate (2.42 g, 10 mmol) in THF (20 ml) was added
dropwise over a period of 1 h. After being refluxed for 5 h, the mix-
ture was poured onto ice water and extracted with DCM
(50 ml ꢁ 3). The combined organic layers were washed with water
and brine, dried (Na2SO4), and concentrated. The crude products
were purified by flash column chromatography using hexane and
ethyl acetate to yield the pure compounds (2.57 g, 86%) as yellow
4.1.6. General procedure for the preparation of compounds 8a–j
and 9a–d
A
solution of 5-bromo-5-(4-phenoxyphenyl)pyrimidine-
2,4,6(1H,3H,5H)-trione (1.0 mmol) in methanol (5 ml) was treated
with the substituted piperazines or homopiperazines (2 mmol) and
stirred for 24 h at ambient temperature. Compounds 8a, 8b, 8c, 8f,
and 8g precipitated. The precipitates were collected by filtration
and dried in vacuo to afford the solids. In the case of compounds
8d, 8e, 8h, 8i and 9a–d, the solvents of reactions were removed
and the residues purified by flash column chromatography and
dried to yield the pure solids.
solid. Mp: 43–45 °C. MS: calcd for
C17H16O5Na = 323.0895.
(M+Na)+ = 323.0888 found. 1H NMR (CDCl3) d ppm: 3.79 (s, 6H,
CH3), 4.68 (s, 1H, CH), 7.01–7.07 (m, 4H, Ar-H), 7.13–7.17 (t,
J = 7.53, 1H, Ar-H), 7.35–7.41 (m, 4H, Ar-H). 13C NMR (CDCl3) d
ppm: 52.8; 56.6; 118.4; 119.2; 123.5; 126.9; 129.7; 130.6; 156.5;
4.1.6.1.
pyrimidine-2,4,6(1H,3H,5H)-trione (8a). Yield: 63% of off-white
26H31N4O4 = 463.2345.
5-(4-Cyclohexylpiperazin-1-yl)-5-(4-phenoxyphenyl)-
solids. Mp: >252 °C. MS: calcd for
C
157.4; 168.5. IR (KBr):
m
(cmꢀ1): 3064, 1734, 1611, 1590.
(M+H)+ = 463.2350 found. 1H NMR (DMSO-d6) ppm: 1.03–1.19
(m, 6H, CH2), 2.18–2.25 (m, 1H, CH), 2.51–2.57 (m, 8H, CH2),
7.00–7.06 (m, 4H, Ar-H), 7.16–7.20 (t, J = 7.53, 1H, Ar-H), 7.38–
7.43 (m, 3H, Ar-H), 7.55–7.58 (m, 1H, Ar-H),11.61 (s, 2H, NH). 13C
NMR (DMSO-d6) ppm: 25.3; 25.9; 28.2; 47.7; 49.0; 62.5; 73.9;
115.8; 118.0; 118.4; 119.3; 121.3; 124.1; 129.7; 129.8; 130.2;
4.1.3. 5-(4-Phenoxyphenyl)pyrimidine-2,4,6(1H,3H,5H)-trione (4)
Sodium (460 mg, 2 0 mmol) was dissolved in 60 ml ethanol
(HPLC grade), and urea (1.02 g, 17 mmol) was added to this solu-
tion.
A solution of dimethyl 2-(4-phenoxyphenyl)malonate
(3.00 g, 10 mmol) in ethanol was added dropwise, and the reaction
mixture was heated to reflux for 7 h. After being cooled to ambient
temperature, the mixture was poured onto ice water and adjusted
to pH 2, using dilute HCl. The precipitate was collected by suction
filtration and dried in vacuo to afford the product off-white solids
133.0; 149.5; 155.8; 157.4; 170.0. IR (KBr):
1737, 1614.
m
(cmꢀ1): 2933, 1711,
4.1.6.2.
pyrimidine-2,4,6(1H,3H,5H)-trione (8b). Yield: 57% of off-white
solids. Mp: >252 °C. MS: calcd for C23H27N4O4 = 423.2032.
5-(4-Isopropylpiperazin-1-yl)-5-(4-phenoxyphenyl)-
(2.31 g, 78%). Mp: 250–253 °C. MS: calcd for
C
16H12N2O4-
Na = 319.0695. (M+Na)+ = 319.0684 found. 1H NMR (DMSO-d6) d
ppm: 4.84 (s, 1H, CH), 6.95–6.97 (d, 2H, J = 8.56, Ar-H), 7.01–7.03
(m, 2H, Ar-H), 7.13–7.16 (t, 1H, J = 6.85, Ar-H), 7.26–7.28 (d, 2H,
J = 8.07, Ar-H), 7.37–7.41 (t, 2H, J = 7.58, Ar-H) 11.4 (s, 2H, NH).
13C NMR (DMSO-d6) d ppm: 54.4; 118.5; 119.1; 123.9; 129.3;
(M+H)+ = 423.2032 found. 1H NMR (DMSO-d6) ppm: 0.95–0.97 (d,
J = 6.52, 6H, CH3), 2.47–2.51 (m, 4H, CH2), 2.58–2.60 (m, 4H,
CH2), 2.64–2.69 (m, 1H, CH), 7.00–7.06 (m, 4H, Ar-H), 7.16–7.20
(t, J = 7.53, 1H, Ar-H), 7.39–7.43 (m, 3H, Ar-H), 7.55–7.59 (m, 1H,
Ar-H),11.61 (s, 2H, NH). 13C NMR (DMSO-d6) ppm 18.0; 47.5;
48.6; 53.9; 73.9; 115.8; 118.1; 118.4; 119.3; 121.3; 124.1; 129.7;
130.2; 131.2; 151.1; 156.4; 156.5; 169.4. IR (KBr):
3063, 1788, 1761, 1720, 1572.
m
(cmꢀ1):
129.8;130.2; 133.0;149.5; 155.8; 157.4; 170.0. IR (KBr):
3066, 1735, 1717, 1608.
m
(cmꢀ1):
4.1.4. 5-bromo-5-(4-phenoxyphenyl)pyrimidine-2,4,6(1H,3H,
5H)-trione (5)
4.1.6.3. 5-(4-(Cyclopropylmethyl)piperazin-1-yl)-5-(4-phenoxy-
phenyl)pyrimidine-2,4,6(1H,3H,5H)-trione (8c). Yield: 59% of
off-white solids. Mp: 242–246 °C. MS: calcd for C23H27N4O4 =
435.2032. (M+H)+ = 435.2032 found. 1H NMR (DMSO-d6) ppm:
0.02–0.05 (m, 2H, CH2), 0.40–0.44 (m, 2H, CH2), 0.73–0.83 (m, 1H,
CH), 2.14–2.16 (d, J = 6.53, 2H, CH2), 2.43 (s, br, 2H, CH2), 2.59 (s,
br, 2H, CH2), 7.00–7.06 (m, 4H, Ar-H), 7.15–7.20 (t, J = 7.53, 1H,
Ar-H), 7.38–7.43 (m, 3H, Ar-H), 7.55–7.58 (m, 1H, Ar-H). 13C NMR
(DMSO-d6) ppm: 3.1; 8.1; 47.3; 53.2; 62.8; 73.9; 118.1; 118.4;
119.3; 121.3; 124.1; 129.7; 129.8; 130.2; 129.8; 149.5; 155.8;
A
suspension
2,4,6(1H,3H,5H)-trione (1.185 g, 4 mmol) in 20 ml water was
cooled to 0–5 °C, and a mixture of 48% HBr (764 l, 7.1 mmol)
and bromine (328
l, 6.4 mmol) was added dropwise.8,25 After stir-
of
5-(4-phenyoxyphenyl)pyrimidine-
l
l
ring for 4–5 h at 0–10 °C the precipitate was collected by filtration
and dried in vacuo to afford off-white solids (1.22 g, 82%). Mp:
111–113 °C. 1H NMR (DMSO-d6) ppm: 6.98–7.08 (m, 4H, Ar-H),
7.15–7.18 (t, 2H, J =7.53, Ar-H), 7.38–7.45 (m, 3H, Ar-H), 7.55–
7.57 (d, 2H, J = 9.04, Ar-H), 11.56(s, 1H, NH), 11.57 (s, 1H, NH).
13C NMR (DMSO-d6) ppm: 115.6; 118.4; 118.8; 119.1; 121.1;
124.0; 127.1; 127.2; 130.2; 133.0; 149.9; 156.0; 157.3; 170.9. IR
157.4; 170.0. IR (KBr):
m
(cmꢀ1): 3000, 1735, 1709, 1607.
(KBr):
m
(cmꢀ1): 3078, 1737, 1718, 1607.
4.1.6.4. 5-(4-(Cyclopropylcarbonyl)piperazin-1-yl)-5-(4-phen-
oxyphenyl)pyrimidine-2,4,6(1H,3H,5H)-trione (8d). Yield: 38%
of off-white solids. Mp: 148–150 °C. MS: calcd for C25H27N4O5-
Na = 471.1644. (M+Na)+ = 471.1651 found. 1H NMR (acetone-d6)
ppm: 0.67–0.72 (m, 2H, CH2), 0.81–0.85 (m, 2H, CH2), 1.89–1.93
(m, H, CH), 2.72–2.81 (m, 4H, CH2); 3.58–3.74 (m, 4H, CH2);
7.04–7.10 (m, 4H, Ar-H), 7.18–7.12 (t, J = 7.53, 1H, Ar-H), 7.41–
7.45 (m, 1H, Ar-H), 7.57–7.64 (m, 3H, Ar-H) 10.49 (s, 2H, NH).
13C NMR (acetone-d6) ppm: 7.1; 10.7; 43.1; 46.5; 48.4; 48.9;
75.6; 116.5; 118.6; 119.0; 120.0; 121.9; 124.6; 129.9; 130.4;
4.1.5. tert-Butyl 4-(cyclopropylmethyl)-1,4-diazepane-1-
carboxylate (7)
A solution of Boc-homopiperazine (400 mg, 2 mmol) and trieth-
ylamine (202 mg, 2 mmol) in methanol was treated with (bromo-
methyl)cyclopropane (270 mg, 2 mmol) and stirred for 24 h at
room temperature. After reaction finished, the solvent was re-
moved and the residue was purified by flash column chromatogra-
phy to yield the pure oil compound (328 mg, 64%). MS: calcd for
C
14H27N2O2 = 255.2073. (M+H)+ = 255.2060 found. 1H NMR (CDCl3)
ppm: 0.12–0.14 (m, 2H, CH2), 0.49–0.51 (m, 2H, CH2), 0.88–0.96 (m,
130.6; 149.1; 156.7; 159.0; 170.2; 171.8. IR (KBr):
3010, 1751, 1737, 1708, 1608.
m
(cmꢀ1):

4992
J. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4985–4999
4.1.6.5.
5-(4-2(-2-Hydroxyethoxy)ethyl)piperazin-1-yl)-5-(4-
CH3), 2.29–2.35 (m, 4H, CH2), 2.57–2.61 (m, 4H, CH2), 7.02–7.08
(m, 4H, Ar-H), 7.17–7.21 (t, J = 7.53, 1H, Ar-H), 7.41–7.44 (t,
J = 7.53, 3H, Ar-H), 7.57–7.59 (m, 3H, Ar-H), 11.65 (s, 2H, NH). 13C
NMR (DMSO-d6) ppm: 45.6; 47.2; 55.2; 73.8; 118.1; 118.4;
119.4; 121.4; 124.0; 129.7; 130.2; 132.9; 149.4; 155.8; 157.4;
phenoxyphenyl)pyrimidine-2,4,6(1H,3H,5H)-trione (8e). Yield:
49% of off-white solids. Mp: 156–160 °C. MS: calcd for
C
24H29N4O6 = 469.2087. (M+H)+ = 469.2088 found. 1H NMR
(DMSO-d6) ppm: 2.42–2.47 (m, 6H, CH2), 2.56–2.59 (m, 4H, CH2),
3.36–2.50 (m, 6H, CH2), 7.00–7.06 (m, 4H, Ar-H), 7.15–7.19 (t,
J = 7.53, 1H, Ar-H), 7.37–7.43 (m, 3H, Ar-H), 7.54–7.57 (m, 1H,
Ar-H), 11.62 (s, 2H, NH). 13C NMR (DMSO-d6) ppm: 47.3; 53.7;
51.2; 62.6; 72.2; 74.0; 115.8; 118.1; 118.4; 119.3; 121.4; 124.1;
169.9. IR (KBr):
m
(cmꢀ1): 2960; 1730; 1715; 1608.
4.1.6.11. 5-(4-Phenoxyphenyl)-5-(cyclopropylmethyl)-1,4-dia-
zepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione (9a). Yield: 41%
of off-white solids. Mp: 201–204 °C. MS: calcd for C25H29N4O4 =
449.2189; found (M+H)+ = 449.2186. 1H NMR (MeOD) ppm:
0.47–0.49 (m, 2H, CH2), 0.77–0.79 (m, 2H, CH2), 1.16–1.19 (m,
1H, CH), 1.47–1.52 (m, 2H, CH2), 2.01–2.10 (m, 2H, CH2), 2.87–
2.93 (m, 2H, CH2), 3.41–3.46 (m, 2H, CH2), 3.55–3.58 (m, 1H,
CH2), 3.62–3.66 (m, 1H, CH2), 7.00–7.09 (m, 4H, Ar-H), 7.19–7.23
(t, J = 7.53, 1H, Ar-H), 7.40–7.44 (t, J = 7.53, 1H, Ar-H), 7.52–7.58
(m, 3H, Ar-H). 13C NMR (MeOD) ppm: 4.9; 9.2; 26.2; 47.8; 51.0;
53.7; 56.8; 63.2; 78.5; 119.4; 119.8; 120.9; 122.6; 125.5;
129.7; 130.2; 133.0; 149.5; 155.8; 157.4; 170.2. IR (KBr):
m
(cmꢀ1): 3066, 1737, 1708, 1605.
4.1.6.6. 5-(4-((Tetrahydrofuran-2-yl)methyl)piperazin-1-yl)-5-
(4-phenoxyphenyl)pyrimidine-2,4,6(1H,3H,5H)-trione
(8f). Yield: 52% of off-white solids. Mp: 248–250 °C. MS: calcd for
C
25H29N4O5 = 465.2138. (M+H)+ = 465.2143 found. 1H NMR
(DMSO-d6) ppm: 1.30–1.39 (m, 1H, CH2), 1.61–1.71 (m, 2H, CH2),
1.76–1.82 (m, 1H, CH2), 1.61–1.71 (d, J = 5.52, 2H, CH2), 2.32–
2.48 (m, 6H, CH2), 3.46–3.51 (m, 2H, CH2), 3.59–3.64 (m, 1H,
CH2), 3.76–3.82 (m, 1H, CH2), 6.92–6.98 (m, 4H, Ar-H), 7.07–7.11
(t, J = 7.53, 1H, Ar-H), 7.30–7.34 (t, J = 7.53, 3H, Ar-H), 7.46–7.49
(m, 1H, Ar-H), 11.50 (s, 2H, NH). 13C NMR (DMSO-d6) ppm: 25.0;
29.8; 47.3; 54.0; 62.3; 67.1; 73.9; 76.5; 115.7; 118.1; 118.4;
119.3; 121.3; 124.1; 129.7; 130.2; 133.0; 149.5; 155.8; 157.4;
130.6; 130.7; 131.2; 131.3; 150.6; 156.7; 157.2; 171.9. IR (KBr):
m
(cmꢀ1): 2962; 1768; 1711; 1604.
4.1.6.12.
5-(4-Benzyl-1,4-diazepan-1-yl)-5-(4-phenoxyphe-
nyl)pyrimidine-2,4,6(1H,3H,5H)-trione (9b). Yield: 63% of
off-white solids. Mp: 123–125 °C. MS: calcd for C28H29N4O4 =
485.2189, (M+H)+ = 485.2202 found. 1H NMR (acetone-d6): ppm:
1.80–1.86 (m, 2H, CH2), 2.07–2.10 (m, 2H, CH2), 2.75 (s, br, 2H,
CH2), 2.90–2.94 (m,4H, CH2), 3.86 (s, 2H, CH2), 7.02–7.08 (m, 4H,
Ar-H), 7.18–7.21 (t, 1H, J = 7.33, Ar-H), 7.24–7.27 (t, J = 7.33,1H,
Ar-H), 7.30–7.34 (t, 2H, J =7.33, Ar-H), 7.40–7.48 (m, 3H, Ar-H),
7.56–7.62 (m, 3H, Ar-H). 13C NMR (acetone-d6) ppm: 28.3; 50.3;
51.1; 53.5; 57.6; 61.5; 77.6; 116.5; 118.9; 120.0; 121.9; 124.6;
127.8; 128.7; 129.7; 130.2; 130.4; 130.6; 131.4; 133.5; 149.3;
170.0. IR (KBr):
m
(cmꢀ1): 3088, 1750, 1718, 1692.
4.1.6.7. 5-(4-Benzylpiperazin-1-yl-5-(4-phenoxyphenyl)pyrimi-
dine-2,4,6(1H,3H,5H)-trione (8g). Yield: 57% of off-white solids.
Mp: 206–208 °C. MS: calcd for C27H27N4O4 = 471.2032. (M+H)+ =
471.2048 found. 1H NMR (DMSO-d6) ppm: 2.36 (s, br, 4H, CH2),
2.58–2.61 (m, 4H, CH2), 7.00–7.06 (m, 4H, Ar-H), 7.16–7.20 (t,
J = 7.53, 1H, Ar-H), 7.22–7.33 (m, 5H, Ar-H), 7.39–7.43 (t, J = 7.53,
3H, Ar-H), 7.55–7.58 (m, 1H, Ar-H), 11.55 (s, 2H, NH). 13C NMR
(DMSO-d6) ppm: 47.4; 53.1; 62.0; 73.9; 115.7; 118.1; 118.4;
119.3; 121.3; 124.1; 126.9; 128.2; 128.9; 129.7; 130.2; 133.0;
156.7; 158.9; 171.1. IR (KBr):
m
(cmꢀ1): 3062, 1736, 1708, 1604.
4.1.6.13. 5-(4-Acetyl-1,4-diazepan-1-yl)–5-(4-phenoxyphenyl)-
pyrimidine-2,4,6(1H,3H,5H)-trione (9c). Yield: 51% of off-white
solids. Mp: 136–139 °C. MS: calcd for C23H24N4O5 = 4459.1644.
(M+Na)+ = 459.1625 found. 1H NMR (Acetone-d6) ppm: 1.59–1.65
(m, 1H, CH2), 1.72–1.78 (m, 1H, CH2), 2.06–2.10 (m, 5H, CH2+CH3),
2.78–2.82 (m, 1H, CH2), 2.92–2.94 (m, 1H, CH2), 3.46–3.50 (m, 2H,
CH2), 4.04–4.09 (m, 2H, CH2), 7.03–7.08 (m, 4H, Ar-H), 7.18–7.21 (t,
J = 7.53, 1H, Ar-H), 7.40–7.44 (t, J = 7.33, 1H, Ar-H), 7.56–7.62 (m,
3H, Ar-H). 13C NMR (acetone-d6) ppm: 21.2; 28.4; 48.1; 50.4;
52.5; 53.8; 78.1; 116.5; 118.9; 120.0; 121.9; 124.6; 130.0; 130.5;
132.9; 149.5; 155.8; 157.4; 170.0. IR (KBr):
1708, 1609.
m
(cmꢀ1): 3064, 1731,
4.1.6.8. 5-(4-Butylpiperazin-1-yl)-5-(4-phenoxyphenyl)pyrimi-
dine-2,4,6(1H,3H,5H)-trione (8h). Yield: 39% of off-white solids.
Mp: 196–198 °C. MS: calcd for
C24H29N4O4 = 437.2189.
(M+H)+ = 437.2177 found. 1H NMR (DMSO-d6) ppm: 0.83–0.87 (t,
J = 7.28, 3H, CH3), 1.22–1.29 (m, 2H, CH2), 1.34–1.41 (m, 2H,
CH2), 2.29–2.32 (t, J = 7.03, 2H, CH2), 2.41 (s, br, 4H, CH2), 2.58–
2.61 (m, 4H, CH2), 7.00–7.06 (m, 4H, Ar-H), 7.15–7.19 (t, J = 7.53,
1H, Ar-H), 7.38–7.43 (m, 3H, Ar-H), 7.55–7.57 (m, 1H, Ar-H). 13C
NMR (DMSO-d6) ppm: 13.9; 20.0; 28.1; 47.1; 53.3; 57.3; 73.9;
76.5; 118.1; 118.4; 119.3; 121.3; 124.1; 129.6; 129.7; 130.2;
133.4; 149.2; 156.6; 158.9; 170.1; 170.9. IR (KBr):
3067, 1737, 1706, 1608.
m
(cmꢀ1):
4.1.6.14. 5-(4-Methyl-1,4-diazepan-1-yl)-5-(4-phenoxyphenyl)-
pyrimidine-2,4,6(1H,3H,5H)-trione (9d). Yield: 49% of off-white
solids. Mp: 249–251 °C. MS: calcd for C22H25N4O4 = 409.1876.
(M+H)+ = 409.1873 found. 1H NMR (DMSO-d6) ppm: 1.62–1.68
(m, 2H, CH2), 2.24 (s, 3H, CH3), 2.42–2.44 (m, 2H, CH2), 2.59–2.62
(m, 2H, CH2), 2.68–2.72 (m, 4H, CH2), 7.00–7.06 (m, 4H, Ar-H),
7.16–7.20 (t, J = 7.53, 1H, Ar-H), 7.38–7.44 (m, 3H, Ar-H), 7.55–
7.58 (m, 1H, Ar-H). 13C NMR (DMSO-d6) ppm: 28.0; 46.1; 46.7;
50.6; 55.4; 59.5; 76.2; 118.1; 118.4; 119.4; 121.4; 124.2; 129.5;
130.2; 131.1; 133.0; 149.8; 155.7; 157.5; 171.1. IR (KBr):
133.0; 149.5; 155.8; 157.4; 170.0. IR (KBr):
1705, 1605.
m
(cmꢀ1): 3057, 1734,
4.1.6.9. 5-(4-Acetyl-piperazin-1-yl)-5-(4-phenoxyphenyl)pyrim-
idine-2,4,6(1H,3H,5H)-trione (8i). Yield: 45% of off-white solids.
Mp: 176–179 °C. MS: calcd for C22H23N4O5 = 423.1668; found
(M+H)+ = 423.1617. 1H NMR (MeOD) ppm: 2.08 (s, 3H, CH3), 2.69–
2.71 (m, 2H, CH2), 2.76–2.78 (m, 2H, CH2), 3.51–3.53 (m, 2H, CH2),
3.57–3.59 (m, 2H, CH2), 6.96–7.05 (m, 4H, Ar-H), 7.16–7.20 (t,
J = 7.53, 1H, Ar-H), 7.37–7.41 (t, J = 7.53, 3H, Ar-H), 7.50–7.55 (m,
3H, Ar-H). 13C NMR (MeOD) ppm: 21.1; 43.3; 48.2; 76.3; 119.3;
119.6; 120.8; 122.4; 125.3; 130.1; 131.1; 134.1; 150.7; 156.9;
m
(cmꢀ1): 3062, 1732, 1682, 1592.
4.1.7. General procedure for the preparation of compounds 10
and 11
157.4; 171.6; 171.8. IR (KBr):
m
(cmꢀ1): 2961; 1739; 1709; 1614.
A
solution of 5-bromo-5-(4-phenoxyphenyl)pyrimidine-
4.1.6.10. 5-(4-Methyl-piperazin-1-yl)-5-(4-phenoxyphenyl)-
2,4,6(1H,3H,5H)-trione (1.0 mmol) in methanol (5 ml) was treated
with the piperazine or homopiperazine (2 mmol) and stirred for
24 h at ambient temperature. The precipitates were collected by
filtration and dried in vacuo to afford the solids.
pyrimidine-2,4,6(1H,3H,5H)-trione (8j). Yield: 38% of off-white
solids. Mp: 270–272 °C. MS: calcd for C21H23N4O4 = 395.1719;
found (M+H)+ = 395.1706. 1H NMR (DMSO-d6) ppm: 2.16 (s, 3H,

J. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4985–4999
4993
4.1.7.1.
5-(4-Phenoxyphenyl)-5-(piperazin-1-yl)pyrimidine-
4.1.8.4.
5-(4-Phenoxyphenyl)-5-(4-(cyclopropylcarbonyl)-1,4-
2,4,6 (1H,3H,5H)-trione (10). Yield: 54% of off-white solids. Mp:
diazepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione (13a). Yield:
37% of off-white solids. Mp: 147–149 °C. MS: calcd for
246–248 °C. MS: calcd for
C
20H21N4O4 = 381.1563; (M+H)+ =
381.1582 found. 1H NMR (DMSO-d6) ppm: 2.54–2.58 (m, 4H,
CH2), 2.70–2.73 (m, 4H, CH2), 7.16–7.19 (t, J = 7.53, 2H, Ar-H),
7.39–7.43 (m, 3H, Ar-H), 7.56–7.58 (m, 1H, Ar-H). 13C NMR
(DMSO-d6) ppm: 46.1; 48.6; 74.4; 118.2; 119.4; 121.4; 124.2;
C
25H36N4O5 = 485.1801. (M+H)+ = 485.1796 found. 1H NMR
(CDCl3): ppm: 0.71–0.76 (m, 2H, CH2), 1.00–1.03 (m, 2H, CH2),
1.58–1.67 (m, 1H, CH), 1.70–1.77 (m, 2H, CH2), 2.71–2.82 (m, 4H,
CH2), 3.57–3.63 (m, 2H, CH2), 3.69–3.72 (m,1H, CH2), 3.82–3.85
(m, 1H, CH2), 6.90–7.03 (m, 4H, Ar-H), 7.14–7.17 (t, 1H, J = 7.53,
Ar-H), 7.34–7.38 (t, J = 7.53, 1H, Ar-H), 7.44–7.51 (m, 3H, Ar-H),
10.03 (s, 2H, NH). 13C NMR (CDCl3): 7.5; 11.0; 29.1; 46.7; 50.3;
51.1; 53.1; 77.8; 116.6; 118.2; 119.6; 121.2; 124.1; 129.3; 129.8;
130.2; 130.4; 150.6; 156.1; 157.4; 170.8. IR (KBr):
3065, 1705, 1665, 1631.
m
(cmꢀ1):
4.1.7.2. 5-(1,4-Diazepan-1-yl)-5-(4-phenoxyphenyl)pyrimidine-
2,4,6(1H,3H,5H)-trione (11). Yield: 58% of off-white solids. Mp:
132.8; 149.1; 155.5; 158.6; 170.5; 173.0. IR (KBr):
3000, 1734, 1709, 1607, 1587.
m
(cmꢀ1):
240–244 °C. MS: calcd for
C
21H23N4O4 = 395.1719. (M+H)+ =
395.1704 found. 1H NMR (DMSO-d6) ppm: 1.52–1.58 (m, 2H,
CH2), 2.66–2.68 (m, 4H, CH2), 2.69–2.71 (m, 2H, CH2), 2.87–2.90
(m, 2H, CH2), 6.99–7.06 (m, 4H, Ar-H), 7.16–7.19 (t, J = 7.53, 1H,
Ar-H), 7.38–7.47 (m, 3H, Ar-H), 7.55–7.58 (m, 1H, Ar-H). 13C
NMR (DMSO-d6) ppm: 29.8; 45.7; 49.6; 50.7; 53.0; 76.3; 115.7;
118.0; 119.3; 121.3; 124.0; 129.4; 130.2; 150.6; 155.8; 157.3;
4.1.8.5. 5-(4-Phenoxyphenyl)-5-(4-(thiophene-2-carbonyl)-1,4-
diazepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione (13b). Yield:
35% of off-white crystalline solids. Mp: 110–113 °C. MS: calcd for
C
26H25N4O5S = 527.1365. (M+H)+ = 527.1342 found. 1H NMR
(CDCl3): ppm: 1.76–1.86 (m, 2H, CH2), 2.79–2.90 (m, 4H, CH2),
3.60–3.69 (m, 2H, CH2), 3.80–3.85 (m, 2H, CH2), 6.87–7.01 (m,
5H, CH), 7.13–7.17 (t, 1H, J = 7.53, Ar-H), 7.33–7.46 (m, 6H, CH),
9.57 (s, 2H, NH). 13C NMR (CDCl3): 30.0; 45.6; 49.2; 50.7; 53.4
77.7; 117.8; 119.4; 121.0; 123.9; 128.6; 129.0; 129.3; 129.6;
132.6; 136.1 148.7; 154.6; 155.3; 158.6; 164.4; 169.9; 171.0. IR
172.1. IR (KBr):
m
(cmꢀ1): 3065, 1702, 1665, 1625.
4.1.8. General procedure for the preparation of compounds
12a–c and 13a–d
A suspension of compounds 10 or 11 (1 mmol) in 10 ml THF
was cooled to ꢀ70 °C and triethylamine (1 mmol) was added. A
solution of the substituted acid chlorides (1 mmol) was added to
the suspension dropwise. After stirring for 3–4 h at ꢀ70 °C, the
reaction mixture was dried and purified by flash column chroma-
tography to yield the pure compounds.
(KBr):
m
(cmꢀ1): 3067, 1736, 1708, 1605, 1587.
4.1.8.6 5-(4-Phenoxyphenyl)-5-(4-(4-cyanobenzoyl)-1,4-diazepan-
1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione (13c).
Yield: 31% of yellow crystalline solids mp: 220–224 °C. MS:
calcd for C29H26N5O5 = 524.1934, (M+H)+ = 524.1934 found. 1H
NMR (CDCl3): ppm: 1.83–1.87 (m, 2H, CH2), 2.76–2.84 (m, 3H,
CH2), 3.21–3.25 (m, 1H, CH2), 3.41–3.45 (m, 1H, CH2), 3.67–3.70
(m, 1H, CH2), 3.74–3.78 (m, 1H, CH2), 3.82–3.86 (m, 1H, CH2),
6.84–7.00 (m, 4H, Ar-H), 7.15–7.19 (t, 1H, J = 7.53, Ar-H), 7.34–
7.40 (m, 2H, Ar-H), 7.44–7.48 (m, 2H, Ar-H), 7.53–7.57 (m, 2H,
Ar-H), 7.66–7.72 (m, 2H, Ar-H), 10.11 (s, 2H, NH). 13C NMR (CDCl3):
29.7; 44.7; 48.2; 50.6; 51.6; 77.6; 112.9; 116.7; 116.8; 118.2;
119.8; 121.3; 124.2; 127.1; 127.4; 129.2; 130.0; 132.3; 132.8;
4.1.8.1. 5-(4-(Thiophene-2-carbonyl)piperazin-1-yl)-5-(4-phen-
oxyphenyl)pyrimidine-2,4,6(1H,3H,5H)-trione (12a). Yield: 41%
of off-white solids. Mp: 142–144 °C. MS: calcd for C25H23N4O5 =
491.1389; found (M+H)+ = 491.1377. 1H NMR (CDCl3) ppm: 2.77–
2.82 (m, 4H, CH2), 3.77–3.79 (m, 4H, CH2), 6.94–6.96 (d, J = 8.53,
2H, Ar-H), 6.89–7.04 (m, 3H, Ar-H&CH), 7.16–7.19 (t, J = 7.53, 1H,
Ar-H), 7.35–7.39 (t, J = 7.53, 2H, Ar-H), 7.42–7.48 (m, 4H, Ar-
H&CH), 9.63 (s, 1H, NH), 9.68 (s, 1H, NH). 13C NMR (CDCl3) ppm:
47.6; 67.5; 75.1; 117.9; 119.4; 121.0; 123.9; 128.6; 129.0; 129.3;
129.6; 132.6; 136.1; 148.6; 154.7; 155.4; 158.6; 163.5; 169.1;
135.7; 140.9; 149.0; 154.7; 158.9; 169.8; 170.5. IR (KBr):
m (cm
ꢀ1): 3068, 2230, 1737, 1709, 1606, 1582.
169.2. IR (KBr):
m
(cmꢀ1): 2959, 1738, 1711, 1608.
4.1.8.7.
5-(4-Phenoxyphenyl)-5-(4-(6-chloropyridine-3-car-
4.1.8.2. 5-(4-(4-Cyanobenzoyl))piperazin-1-yl)-5-(4-phenoxy-
bonyl)-1,4-diazepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione
phenyl)pyrimidine-2,4,6(1H,3H,5H)-trione (12b). Yield 40% of
light yellow solids. Mp: 146–148 °C. MS: calcd for C28H24N5O5 =
510.1777; found (M+H)+ = 510.1765. 1H NMR (CDCl3) ppm: 2.71–
2.83 (m, 4H, CH2), 3.78–3.81 (m, 4H, CH2), 6.94–6.96 (d, J = 8.53,
2H, Ar-H), 7.00–7.03 (m, 2H, Ar-H), 7.17–7.21 (t, J = 7.53, 1H, Ar-
H), 7.36–7.38 (d, J = 8.03, 1H, Ar-H), 7.40–7.46 (m, 3H, Ar-H),
7.51–7.53 (d, J = 7.53H, Ar-H), 7.68–7.70 (d, J = 7.53, 2H, Ar-H),
9.63 (s, 2H, NH). 13C NMR (CDCl3) ppm: 47.5; 67.5; 75.2; 117.6;
117.9; 119.5; 121.1; 124.1; 126.7; 127.5; 129.1; 129.7; 132.0;
132.6; 135.4; 139.2; 148.3; 155.2; 158.8; 168.1; 169.2. IR (KBr):
(13d). Yield: 32% of off-white crystalline solids. Mp: 246–248 °C.
MS: calcd for C24H25ClN5O5 = 534.1544, (M+H)+ = 534.1569 found.
1H NMR (DMSO-d6): ppm: 1.48–1.54 (m, 1H, CH2), 1.62–1.68 (m,
1H, CH2), 2.73–2.85 (m, 4H, CH2), 3.19–3.23 (m, 1H, CH2), 3.40–
3.43 (m, 1H, CH2), 3.54–3.47 (m, 1H, CH2), 3.67–3.70 (m, 1H,
CH2), 6.96–7.09 (m, 4H, Ar-H), 7.15–7.19 (t, 1H, J = 7.53, Ar-H),
7.33–7.36 (m, 1H, Ar-H), 7.40–7.47 (m, 3H, Ar-H), 7.57–7.62 (m,
1H, Ar-H), 7.91–7.96 (t, 1H, J = 8.03, Ar-H), 8.49–8.50 (d, 1H,
J = 5.02, Ar-H), 11.50 (s, 2H, NH). 13C NMR (DMSO-d6): 29.5; 43.9;
47.8; 50.7; 52.6; 76.8; 116.0; 118.3; 119.5; 121.6; 124.3; 124.9;
128.0; 129.1; 129.4; 130.2; 132.2; 133.0; 138.2; 139.2; 147.8;
m
(cmꢀ1): 2959, 2231, 1738, 1711,1608.
149.4; 150.5; 155.0; 157.7; 170.7; 170.8. IR (KBr):
3068, 1736, 1708, 1609, 1586.
m
(cmꢀ1):
4.1.8.3. 5-(4-(6-Chloropyridine-3-carbonyl)piperazin-1-yl)-5-(4-
phenoxyphenyl) pyrimidine-2,4,6(1H,3H,5H)-trione (12c). Yield
42% of off-white solids. Mp: 203–206 °C. MS: (M+H)+ = 520.1390.
calcd for C26H23ClN5O5 = 520.1388. 1H NMR (CDCl3) ppm: 2.78–
2.87 (m, 4H, CH2), 3.78–3.81 (m, 4H, CH2), 6.96–7.09 (m, 4H,
Ar-H), 7.17–7.21 (t, 1H, J = 7.53, Ar-H), 7.33–7.36 (m, 1H, Ar-H),
7.41–7.47 (m, 3H, Ar-H), 7.75–7.80 (m, 1H, Ar-H), 8.46–8.49 (m,
1H, Ar-H), 8.49–8.50 (d, 1H, J = 5.02, Ar-H), 13C NMR (CDCl3) ppm:
47.5; 67.5; 75.2; 117.6; 117.9; 119.5; 121.1; 124.1; 126.7; 127.5;
129.1; 129.7; 132.0; 132.6; 135.4; 139.2; 148.3; 155.2; 158.8;
4.1.9. General procedure for the preparation of compounds 14–
38
A
suspension of 5-(1,4-diazepan-1-yl)-5-(4-phenoxyphe-
nyl)pyrimidine-2,4,6(1H,3H,5H)-trione (394 mg, 1 mmol) in 10 ml
THF was cooled to ꢀ70 °C and triethylamine (101.2 mg, 1 mmol)
was added. A solution of substituted the acid chlorides (1 mmol)
was added to the suspension dropwise. After stirring for 2 h at
ꢀ70 °C, the reaction mixture was dried and purified by flash col-
umn chromatography to yield the pure compounds.
168.1; 169.2. IR (KBr):
m
(cmꢀ1): 2956, 1739, 1706, 1614.

4994
J. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4985–4999
4.1.9.1. 5-(4-Phenoxyphenyl)-5-(4-(cyclobutanecarbonyl)-1,4-
diazepan-1-yl) pyrimidine-2,4,6(1H,3H,5H)-trione (14). Yield:
35% as off-white solids. Mp: 142–145 °C. MS: calcd for
1.65–1.69 (m, 2H, CH2), 1.71–1.77 (m, 5H, CH2 + CH), 1.86–1.90
(m, 2H, CH2), 2.33–2.39 (m, 2H, CH2), 2.73–2.80 (m, 3H, CH2),
3.40–3.43 (m, 1H, CH2), 3.59–3.62 (m, 1H, CH2), 3.64–6.67 (m,
1H, CH2), 3.70–3.73 (m, 1H, CH2), 3.77–3.80 (m, 1H, CH2), 6.92–
7.01 (m, 4H, Ar-H), 7.04–7.06 (d, J = 7.53, 1H, Ar-H), 7.37–7.41 (t,
J = 8.53, 1H, Ar-H), 7.46–7.49 (m, 3H, Ar-H), 9.36–9.54 (d, 2H,
NH). 13C NMR (CDCl3) d ppm: 25.1; 29.6; 31.5; 32.3; 32.7; 34.1;
39.8; 44.6; 46.9; 50.4; 52.3; 52.7; 77.8; 118.2; 118.4; 121.4;
124.3; 129.5; 129.9; 148.8; 155.0; 158.3; 170.2; 170.3; 173.4. IR
C
26H29N4O5 = 477.2138. (M+H)+ = 477.2133 found. 1H NMR
(CDCl3): ppm: 1.68–1.74 (m, 2H, CH2), 1.82–2.01 (m, 2H, CH2),
2.05–2.15 (m, 2H, CH2), 2.30–2.45 (m, 2H, CH2), 2.71–2.84 (m,
4H, CH2), 3.23–3.31 (m, 1H, CH), 3.52–3.60 (m, 2H, CH2), 3.69–
3.72 (m,1H, CH2), 3.77–3.80 (m, 1H, CH2), 6.91–7.05 (m, 4H,
Ar-H), 7.15–7.20 (t, 1H, J = 7.53, Ar-H), 7.36–7.40 (t, J = 7.53,1H,
Ar-H), 7.44–7.49 (m, 3H, Ar-H), 9.55 (s, 2H, NH). 13C NMR (CDCl3):
17.7; 24.9; 29.2; 36.7; 45.8; 49.4; 50.2; 53.4; 77.6; 116.5; 118.1;
119.5; 121.0; 124.0; 129.2; 129.6; 132.6; 148.7; 154.7; 158.5;
(KBr):
m
(cmꢀ1): 3065, 1750, 1731, 1697, 1598.
4.1.9.6.
5-(4-Phenoxyphenyl)-5-(4-(2,5-diethoxybenzoyl)-1,4-
170.0; 174.5. IR (KBr):
m
(cmꢀ1): 3068, 1754, 1736, 1708, 1608.
diazepan-1-yl) pyrimidine-2,4,6(1H,3H,5H)-trione (19). Yield:
35% as off-white solids. Mp: 117–119 °C. MS: calcd for
4.1.9.2. 5-(4-Phenoxyphenyl)-5-(4-(2-(phenylthio)acetyl)-1,4-
diazepan-1-yl) pyrimidine-2,4,6(1H,3H,5H)-trione (15). Yield:
38% as off-white solids. Mp: 100–103 °C. MS: calcd for
C
32H35N4O7 = 587.2506, (M+H)+ = 587.2507 found. 1H NMR
(CDCl3): ppm: 1.35–1.38 (m, 6H, CH3), 1.86–1.91 (m, 2H, CH2),
2.66–2.73(m, 1H, CH2), 2.82–2.93 (m, 3H, CH2), 3.14–3.25 (m, 1H,
CH2), 3.43–3.51 (m, 1H, CH2), 3.79–3.89 (m, 2H, CH2), 3.96–4.00
(m, 4H, CH2), 6.78–6.87 (m, 5H, Ar-H), 6.95–6.97 (d, J = 8.03, 1H,
Ar-H), 7.01–7.05 (m, 2H, Ar-H), 7.17–7.21 (t, 1H, J = 7.53, Ar-H),
7.36–7.40 (t, J = 7.28, 2H, Ar-H), 7.51–7.54 (4, J = 7.03,1H, Ar-H).
13C NMR (CDCl3): 14.9; 29.4; 47.7; 50.9; 52.3; 53.1; 64.0; 64.8;
77.8; 113.7; 113.9; 116.0; 118.2; 118.4; 119.8; 121.4; 124.3;
124.9; 127.4; 129.2; 130.0; 132.9; 148.4; 152.9; 155.6; 158.9;
C
29H29N4O5S = 545.1869, (M+H)+ = 545.1840 found. 1H NMR
(CDCl3): ppm: 1.67–1.74 (m, 2H, CH2), 2.71–2.80 (m, 3H, CH2),
2.88–2.91 (m, 1H, CH2), 3.42–3.45 (m, 1H, CH2), 3.58–3.64 (m,
2H, CH2), 3.67–3.70 (m, 1H, CH2), 3.82 (s, 2H, CH2), 6.88–6.92 (m,
4H, Ar-H), 7.01–7.03 (d, 1H, J = 8.53, Ar-H), 7.15–7.20 (m, 2H, Ar-
H), 7.23–7.29 (m, 2H, Ar-H), 7.35–7.39 (t, 1H, J = 7.53, Ar-H),
7.41–7.49 (m, 5H, Ar-H), 9.75 (s, 2H, NH). 13C NMR (CDCl3): 29.3;
36.7; 47.2; 50.7; 52.2; 53.1; 77.9; 116.8; 118.3; 119.8; 121.4;
124.3; 125.4; 126.9; 128.9; 129.5; 129.9; 130.3; 132.9; 135.0;
169.1; 169.9. IR (KBr):
1588.
m
(cmꢀ1): 3069, 2845, 1738, 1710, 1606,
149.1; 154.9; 158.3; 170.3; 170.4. IR (KBr):
1708, 1611.
m
(cmꢀ1): 3064, 1736,
4.1.9.7. 5-(4-Phenoxyphenyl)-5-(4-(2-chloro-6-methylpyridine-
4-carbonyl)-1,4-diazepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-tri-
one (20). Yield: 25% as off-white solids. Mp: 116–119 °C. MS:
calcd for C28H27ClN5O5 = 548.1701, (M+H)+ = 548.1712 found. 1H
NMR (CDCl3) ppm: 1.86–1.88 (m, 2H, CH2), 2.52–2.55 (d, 3H,
CH3), 2.82–2.94 (m, 4H, CH2), 3.19–3.24 (m, 1H, CH2), 3.42–3.46
(m, 1H, CH2), 3.66–3.70 (m, 1H, CH2), 3.78–3.84 (m, 1H, CH2),
6.89–6.91 (d, J = 8.03, 2H, Ar-H), 6.94–6.96 (d, J = 8.03, 1H, Ar-H),
6.99–7.03 (t, J = 7.53, 1H, Ar-H), 7.13–7.22 (m, 3H, Ar-H), 7.35–
7.39 (t, 2H, J = 7.53, Ar-H), 7.43–7.47 (m, 2H, Ar-H), 9.69–9.73 (m,
2H, NH). 13C NMR (CDCl3): 24.0; 29.7; 48.1; 50.8; 52.5; 54.1;
77.8; 116.9; 118.1; 119.0; 119.3; 119.8; 121.4; 124.4; 129.2;
129.9; 132.9; 147.3; 148.9; 154.8; 159.0; 160.2; 168.1; 170.1. IR
4.1.9.3.
5-(4-Phenoxyphenyl)-5-(4-(3,5-diethoxybenzoyl)-1,4-
diazepan-1-yl) pyrimidine-2,4,6(1H,3H,5H)-trione (16). Yield:
33% as off-white solids. Mp: 213–217 °C. MS: calcd for
C
32H35N4O7 = 587.2506, (M+H)+ = 587.2488 found. 1H NMR
(DMSO-d6): ppm: 1.27–1.33 (dt, J = 7.53, 6H, CH3), 1.50–1.56 (m,
1H, CH2), 1.65–1.71 (m, 1H, CH2), 2.70–2.73 (m, 3H, CH2), 2.83–
2.86 (m, 1H, CH2), 3.10–3.13 (m, 1H, CH2), 3.34–3.40 (m, 1H,
CH2), 3.51–3.64 (m, 1H, CH2), 3.63–3.66 (m, 1H, CH2), 3.95–4.06
(m, 4H, CH2), 6.44–6.50 (m, 3H, Ar-H), 6.87–6.92 (m, 1H, Ar-H),
7.01–7.10 (m, 3H, Ar-H), 7.20–7.23 (m, 1H, Ar-H), 7.35–7.47 (m,
3H, Ar-H), 7.57–7.60 (m, 1H, Ar-H), 11.57 (s, 2H, NH). 13C NMR
(DMSO-d6): 14.6; 29.7; 47.7; 50.7; 52.6; 53.7; 63.3; 76.7; 101.1;
104.6; 116.0; 118.1; 119.5; 121.6; 124.2; 124.9; 129.1; 130.2;
(KBr):
m
(cmꢀ1): 3074, 2959, 2849, 1738, 1710, 1608.
133.0; 139.2; 149.4; 155.4; 157.8; 159.5; 169.9; 170.6. IR (KBr):
m
4.1.9.8. 5-(4-Phenoxyphenyl)-5-(4-(2-oxoimidazolidine-1-car-
bonyl)-1,4-diazepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione
(21). Yield: 35% as off-white solids. Mp: 186–190 °C. MS: calcd for
(cmꢀ1): 3063, 2851, 1736, 1706, 1590.
4.1.9.4.
zoyl)-1,4-diazepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione
(17). Yield: 32% as off-white solids. Mp: 183–187 °C. MS: calcd for
29H26F3N4O6 = 583.1804, (M+H)+ = 583.1823 found.1H NMR
5-(4-Phenoxyphenyl)-5-(4-(4-(trifluoromethoxy)ben-
C
25H27N6O6 = 529.1838, (M+H)+ = 529.1812 found. 1H NMR
(MeOD) ppm: 1.76–1.82 (m, 2H, CH2), 2.80–2.83 (m, 2H, CH2),
2.91–2.94 (m, 2H, CH2), 3.46–3.50 (m, 4H, CH2), 3.62–3.68 (m,
2H, CH2), 3.84–3.87 (m, 2H, CH2), 5.51 (s, 1H, NH), 6.99–7.01 (d,
J = 8.04, 2H, Ar-H), 7.05–7.07 (d, J = 8.03, 2H, Ar-H), 7.18–7.21 (t,
J = 7.53, 1H, Ar-H), 7.40–7.43 (t, J = 8.03, 2H, Ar-H), 7.50–7.53 (m,
2H, Ar-H). 13C NMR (MeOD) ppm: 29.4; 30.7; 38.9; 48.5; 51.8;
53.4; 54.8; 78.8; 118.5; 119.1; 120.8; 125.3; 130.6; 131.1; 150.9;
C
(MeOD): ppm: 1.61–1.67 (m, 1H, CH2), 1.81–1.87 (m, 1H, CH2),
2.84–2.87 (m, 3H, CH2), 3.00–3.02 (m, 1H, CH2), 3.27–3.30 (m,
1H, CH2), 3.52–3.55 (m, 1H, CH2), 3.68–3.71 (m, 1H, CH2), 3.81–
3.84 (m, 1H, CH2), 6.84–6.89 (m, 1H, Ar-H), 6.92–6.95 (m, 1H, Ar-
H), 6.98–7.02 (m, 1H, Ar-H), 7.03–7.07 (m, 1H, Ar-H), 7.17–7.21
(t, 1H, J = 7.53, Ar-H), 7.37–7.46 (m, 4H, Ar-H), 7.51–7.54 (m, 1H,
Ar-H), 7.55–7.57 (m, 1H, Ar-H), 7.58–7.63 (m, 2H, Ar-H). 13C
NMR (MeOD): ppm: 28.8; 48.4; 51.4; 52.6; 53.7; 78.7; 117.4;
118.8; 119.2; 120.5; 122.0; 122.3; 125.1; 125.9; 129.4; 129.9;
155.8; 157.5; 157.8; 160.4; 172.5. IR (KBr):
1735, 1641, 1588.
m
(cmꢀ1): 3068, 2958,
4.1.9.9. 5-(4-Phenoxyphenyl)-5-(4-(3-methylbenzofuran-2-car-
bonyl)-1,4-diazepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione
(22). Yield: 29% as off-white solids. Mp: 134–137 °C. MS: calcd for
130.9; 136.8; 150.9; 156.7; 159.4; 160.0; 172.1; 172.4. IR (KBr):
m
(cmꢀ1): 3067, 2852, 1752, 1732, 1708, 1603.
C
31H29N4O6 = 533.2093, (M+H)+ = 533.2087 found. 1H NMR
(MeOD) ppm: 1.76–1.82 (m, 2H, CH2), 2.43 (s, 3H, CH3), 2.79–
2.83 (m, 1H, CH2), 2.90–2.94 (m, 1H, CH2), 3.45–3.48 (m, 1H,
CH2), 3.58–3.63 (m, 1H, CH2), 3.70–3.73 (m, 1H, CH2), 3.79–3.85
(m, 2H, CH2), 3.95–3.98 (m, 2H, CH2), 6.88–7.02 (m, 3H, Ar-H),
7.14–7.17 (t, J = 7.53, 1H, Ar-H), 7.23–7.26 (t, J = 7.53, 1H, Ar-H),
7.30–7.37 (m, 3H, Ar-H), 7.41–7.44 (m, 1H, Ar-H), 7.47–7.54 (m,
4.1.9.5.
5-(4-Phenoxyphenyl)-5-(4-(3-cyclopentylpropanoyl)-
1,4-diazepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione
(18). Yield: 38% as off-white solids. Mp: 139–141 °C. MS: calcd for
C29H35N4O5 = 519.2607, (M+H)+ = 19.2609 found. 1H NMR (CDCl3)
d
ppm: 1.50–1.52 (m, 2H, CH2), 1.58–1.61 (m, 2H, CH2),

J. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4985–4999
4995
2H, Ar-H), 7.58–7.60 (m, 1H, Ar-H), 7.63–7.65 (m, 1H, Ar-H). 13C
NMR (MeOD) ppm: 8.8; 28.6; 48.7; 51.9; 52.8; 54.3; 78.9; 112.4;
119.0; 119.2; 120.7; 121.3; 121.5; 122.4; 125.3; 130.5; 131.1;
29.8; 36.5; 45.8; 49.2; 51.0; 53.2; 77.8; 117.8; 119.4; 121.2; 123.9;
124.3; 125.4; 126.1; 129.0; 129.3; 129.6; 130.3; 139.1; 148.7;
155.3; 158.6; 159.3; 169.9. IR (KBr):
1730, 1706, 1601.
m
(cmꢀ1): 3068, 2957, 2766,
134.1; 145.8; 150.9; 155.0; 157.3; 159.4; 160.3; 172.4. IR (KBr):
m
(cmꢀ1): 3065, 2954, 2846, 1737, 1711, 1608, 1588.
4.1.9.14. 2-(4-(2,4,6-Trioxo-5-(4-phenoxyphenyl)hexanhydro-
pyrimidin-5-yl)-1,4-diazepane-1-carbonyl)benzyl benzoate
4.1.9.10.
5-(4-Phenoxyphenyl)-5-(4-(3-chloro-6-fluorobenzo-
[b]-thiophene-2-carbonyl)-1,4-diazepan-1-yl)pyrimidine-2,4,6-
(1H,3H,5H)-trione (23). Yield: 27% as off-white solids. Mp: 138–
142 °C. MS: calcd for C30H24ClFN4O5SNa = 629.1609, (M+Na)+ =
629.1601 found. 1H NMR (CDCl3) ppm: 1.70–1.76 (m, 1H, CH2),
1.92–1.98 (m, 1H, CH2), 2.85–2.91 (m, 3H, CH2), 3.00–3.05 (m,
1H, CH2), 3.43–3.46 (m, 1H, CH2), 3.68–3.72(m, 1H, CH2), 3.80–
3.85 (m, 1H, CH2), 3.93–3.98 (m, 1H, CH2), 6.84–6.90 (t, J = 8.03,
1H, Ar-H), 6.95–7.13 (m, 3H, Ar-H), 7.23–7.34 (m, 2H, Ar-H),
7.40–7.57 (m, 5H, Ar-H), 7.77–7.88 (m, 1H, Ar-H), 9.45–9.57 (m,
2H, NH). 13C NMR (CDCl3): 29.4; 47.8; 50.5; 52.3; 53.4; 77.6;
108.6; 114.5; 116.6; 117.9; 118.3; 119.5; 121.1; 123.5; 124.1;
128.4; 129.4; 131.7; 137.9; 148.7; 154.3; 155.0; 158.1; 158.7;
(27). Yield: 36% as off-white solids. Mp: 120–123 °C. MS: calcd
for C36H33N4O7 = 633.2349, (M+H)+ = 633.2344 found. 1H NMR
(CDCl3) d ppm: 1.87–1.90 (m, 2H, CH2), 2.71–2.74 (m, 2H, CH2),
2.78–2.81 (m, 1H, CH2), 2.92–2.96 (m, 1H, CH2), 3.18–3.27 (m,
1H, CH2), 3.35–3.40 (m, 1H, CH2), 3.55–3.61 (m, 2H, CH2), 3.78–
3.81 (m, 1H, CH2), 5.47 (s, 2H, CH2), 6.83–6.85 (d, J = 8.53, 1H, Ar-
H), 6.89–6.94 (t, J = 8.53, 1H, Ar-H), 6.99–7.03 (t, J = 8.53, 2H, Ar-
H), 7.17–7.21 (m, 2H, Ar-H), 7.35–7.46 (m, 9H, Ar-H), 7.52–7.56
(m, 2H, Ar-H), 8.04–8.06 (d, J = 8.33, 2H, Ar-H), 9.49–9.54 (d, 2H,
NH). 13C NMR (CDCl3): 29.6; 47.8; 50.9; 52.5; 53.4; 64.1; 77.8;
118.0; 118.2; 119.8; 121.4; 124.3; 125.5; 126.5; 128.0; 128.3;
128.4; 128.9; 129.0; 129.2; 129.4; 129.6; 129.8; 129.9; 132.9;
133.1; 136.0; 148.9; 155.8; 158.9; 166.2; 170.2; 170.4; 170.6.. IR
162.4; 169.9. IR (KBr):
1588.
m
(cmꢀ1): 3071, 2953, 1736, 1708, 1606,
(KBr):
m
(cmꢀ1): 3065, 2953, 1738, 1708, 1607, 1588.
4.1.9.11. 5-(4-Phenoxyphenyl)-5-(4-(2,5-dimethylfuran-3-car-
bonyl)-1,4-diazepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione
(24). Yield: 37% as off-white solids. Mp: 122–126 °C. MS: calcd for
4.1.9.15. 5-(4-Phenoxyphenyl)-5-(4-(6-(trifluoromethyl)nicoti-
noyl)-1,4-diazepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione
(28). Yield: 31% as light yellow solids. Mp: 162–165 °C. MS: calcd
C
28H28N4O6Na = 539.1923, (M+Na)+ = 539.1907 found. 1H NMR
for C
28H24F3N5O5Na = 590.1627, (M+Na)+ = 590.1622 found. 1H
(CDCl3) ppm: 1.78–1.82 (m, 1H, CH2), 1.93–1.99 (m, 1H, CH2),
2.28–2.33 (d, 3H, CH3), 2.39–2.44 (d, 3H, CH3), 2.86–2.91 (m, 3H,
CH2), 2.99–3.04 (m, 1H, CH2), 3.51–3.55 (m, 1H, CH2), 3.72–
3.78(m, 2H, CH2), 3.85–3.89 (m, 1H, CH2), 6.03–6.06 (d, 1H, CH),
7.01–7.03 (m, 4H, Ar-H), 7.26–7.31 (t, J = 7.53, 1H, Ar-H), 7.49–
7.58 (m, 4H, Ar-H), 9.33–9.48 (m, 2H, NH). 13C NMR (CDCl3):
12.8; 13.2; 27.5; 47.8; 51.4; 52.7; 53.5; 77.7; 105.8; 116.6;
118.2; 119.8; 121.4; 124.3; 129.4; 130.0; 149.6; 150.9;
NMR (CDCl3) d ppm: 1.87–1.90 (m, 2H, CH2), 2.83–2.90 (m, 2H,
CH2), 3.03–3.05 (m, 1H, CH2), 3.31–3.35 (m, 1H, CH2), 3.50–3.54
(m, 1H, CH2), 3.77–3.81 (m, 2H, CH2), 3.88–3.92 (m, 1H, CH2),
6.92–6.94 (d, J = 8.53, 2H, Ar-H), 6.96–7.06 (m, 2H, Ar-H), 7.20–
7.23 (t, J = 7.53, 1H, Ar-H), 7.39–7.43 (m, 2H, Ar-H), 7.48–7.51 (t,
J = 7.53, 1H, Ar-H), 7.75–7.81(t, J = 7.53, 1H, Ar-H), 8.01–8.03 (d,
J = 7.53, 2H, Ar-H), 8.34–8.36 (d, J = 7.53, 2H, Ar-H), 8.84 (s,
1H,Ar-H), 13C NMR (CDCl3): 29.6; 48.5; 51.6.52.3; 52.9; 78.1;
118.2; 118.4; 119.8; 120.0; 120.3; 121.6; 124.6; 125.5; 128.2;
129.1; 129.5; 130.0; 133.0; 138.8; 143.3; 147.9; 151.5; 155.5;
155.6;158.0; 158.9; 166.9; 170.1. IR (KBr):
1737, 1708, 1588.
m
(cmꢀ1): 3068, 2952,
159.4; 167.7; 169.9. IR (KBr):
1610, 1588.
m
(cmꢀ1): 3090, 2954, 1730, 1702,
4.1.9.12. 5-(4-Phenoxyphenyl)-5-(4-(quinoxaline-2-carbonyl)-
1,4-diazepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione
(25). Yield: 42% as yellow solids. Mp: 186–188 °C. MS: calcd for
C
4.1.9.16. 5-(4-Phenoxyphenyl)-5-(4-(4-methylbenzoyl)-1,4-dia-
zepan-1-yl) pyrimidine-2,4,6(1H,3H,5H)-trione (29). Yield: 38%
as off-white solids. Mp: 206–210 °C. MS: calcd for C29H29N4O5 =
513.2137, (M+H)+ = 513.2138 found. 1H NMR (DMSO-d6) ppm:
1.48–1.53 (m, 1H, CH2) 1.67–1.71 (m, 1H, CH2), 2.27 (s, 3H, CH3),
2.69–2.74 (m, 3H, CH2), 2.83–2.87 (m, 1H, CH2), 3.17–3.20 (m,
1H, CH2), 3.37–3.43 (m, 1H, CH2), 3.52–3.55 (m, 1H, CH2), 3.65–
3.69 (m, 1H, CH2), 6.84–6.88 (m, 1H, Ar-H), 7.00–7.09 (m, 3H, Ar-
H), 7.21–7.28 (m, 5H, Ar-H), 7.33–7.35 (d, J = 7.35, 1H, Ar-H),
7.40–7.47 (m, 2H, Ar-H), 7.57–7.59 (d, J = 7.53, 1H, Ar-H), 11.56
(s, 1H, NH), 11.62 (s, 1H, NH). 13C NMR (DMSO-d6) ppm: 20.8;
29.7; 47.6; 50.9; 52.8; 53.6; 76.7; 117.9; 119.5; 121.6; 124.2;
124.9; 126.3; 128.7; 129.1; 130.2; 133.0; 138.4; 149.4; 155.5;
30H26N6O5Na = 573.1862, (M+Na)+ = 578.7877 found. 1H NMR
(CDCl3) d ppm: 1.86–1.89 (m, 2H, CH2), 2.89–2.92 (m, 2H, CH2),
2.93–2.96 (m, 1H, CH2), 3.01–3.04 (m, 1H, CH2), 3.60–3.63 (m,
1H, CH2), 3.80–3.83 (m, 2H, CH2), 3.96–3.99 (m, 1H, CH2), 6.85–
6.87 (d, J = 8.53, 1H, Ar-H), 6.93–6.98 (m, 2H, Ar-H), 7.03–7.05 (d,
J = 8.53, 1H, Ar-H), 7.15–7.18 (t, J = 8.03, 1H, Ar-H), 7.34–7.37 (m,
1H, Ar-H), 7.42–7.45 (m, 2H, Ar-H), 7.50–7.52 (d, J = 8.03, 1H, Ar-
H), 7.79–7.85 (m, 2H, Ar-H), 8.05–8.09 (t, J = 7.53, 1H, Ar-H),
8.17–8.22 (t, J = 8.03, 1H, Ar-H), 9.27 (s, 1H, Ar-H), 9.38 (s, 1H,
NH), 9.46 (s, 1H, NH). 13C NMR (CDCl3) d ppm: 29.7; 48.8; 51.1;
51.9; 53.3; 77.7; 117.8; 117.9. 119.6. 121.1; 124.0; 125.2; 128.6;
128.8; 129.1; 129.3; 129.6; 130.3; 130.8; 132.6; 140.0; 141.8;
144.6; 148.5; 155.3; 158.7; 166.7; 169.8; 169.9. IR (KBr):
m
(cm
157.7; 170.5; 170.7. IR (KBr):
1736, 1709, 1597.
m
(cmꢀ1): 3068, 2952, 3068, 2952,
ꢀ1): 3066, 2953, 1729, 169, 1578.
4.1.9.13. 5-(4-Phenoxyphenyl)-5-(4-(2-(thiophen-2-yl)acetyl)-
1,4-diazepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione (26). Yield:
32% as off-white solids. Mp: 107–109 °C. MS: calcd for
4.1.9.17. 5-(4-Phenoxyphenyl)-5-(4-(4-bromobenzoyl)-1,4-dia-
zepan-1-yl) pyrimidine-2,4,6(1H,3H,5H)-trione (30). Yield: 43%
as off-white solids. Mp: 258–260 °C. MS: calcd for C28H25BrN4O5-
Na = 599.0906, (M+Na)+ = 599.0880 found. 1H NMR (DMSO-d6)
ppm: 1.49–1.53 (m, 1H, CH2) 1.68–1.72 (m, 1H, CH2), 2.68–2.74
(m, 3H, CH2), 2.82–2.87 (m, 1H, CH2), 3.17–3.20 (m, 1H, CH2),
3.35–3.41 (m, 1H, CH2), 3.50–3.54 (m, 1H, CH2), 3.65–3.69 (m,
1H, CH2), 6.91–6.94 (m, 2H, Ar-H), 7.00–7.09 (m, 2H, Ar-H), 7.19–
7.23 (t, J = 7.53, 1H, Ar-H), 7.27–7.32 (m, 4H, Ar-H), 7.45–7.49 (m,
2H, Ar-H), 7.59–7.61 (d, J = 7.53, 2H, Ar-H). 13C NMR (DMSO-d6)
ppm: 29.6; 47.7; 50.9; 52.7; 53.8; 76.9; 118.0; 118.4; 119.3;
C
27H27N4O5S = 519.1702, (M+H)+ = 519.1721 found. 1H NMR (CDCl3)
d ppm: 1.63–1.68 (m, 1H, CH2), 1.73–1.77 (m, 1H, CH2), 2.70–2.73
(m, 1H, CH2), 2.74–2.77 (m, 1H, CH2), 2.80–2.84 (m, 2H, CH2),
3.40–3.43 (m, 1H, CH2), 3.61–3.63 (m, 1H, CH2), 3.67–3.70 (m, 1H,
CH2), 3.72–3.75 (m, 1H, CH2), 3.93 (s, 1H, CH2), 3.95 (s, 1H, CH2),
6.90–6.95 (m, 4H, Ar-H), 7.01–7.05 (t, J = 7.53, 2H, Ar-H), 7.136–
7.19 (m, 2H, Ar-H), 7.36–7.40 (m, 2H, Ar-H), 7.42–7.48 (m, 2H, Ar-
H), 9.44 (s, 1H, NH), 9.51 (s, 1H, NH). 13C NMR (CDCl3) d ppm:

4996
J. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4985–4999
121.3; 124.5; 128.5; 128.9; 129.1; 129.6; 129.8; 130.2; 131.5;
77.5; 117.9. 118.1; 119.4; 119.5; 121.0; 127.9; 128.9; 129.0;
131.6; 135.4; 150.3; 155.7; 168.9; 170.7. IR (KBr):
3086, 2956, 1738, 1709, 1588.
m
(cmꢀ1):
129.2; 129.6; 148.8; 151.1; 155.4; 158.5; 169.9; 170.0. IR (KBr):
m
(cmꢀ1): 3066, 2956, 1730, 1711, 1590.
4.1.9.18. 5-(4-Phenoxyphenyl)-5-(4-(2,6-dichlorobenzoyl)-1,4-
diazepan-1-yl) pyrimidine-2,4,6(1H,3H,5H)-trione (31). Yield:
40% as off-white solids. Mp: 158–160 °C. MS: calcd for
4.1.9.22. 5-(4-Phenoxyphenyl)-5-(4-(2,6-dichloro-5-fluoronicot-
inoyl)-1,4-diazepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione
(35). Yield: 32% as off-white solids. Mp: 154–156 °C. MS: calcd for
C
28H25Cl2N4O5Na = 589.1021, (M+Na)+ = 589.1019 found. 1H NMR
C
27H22ClN5O5Na = 608.0880, (M+Na)+ = 608.0881 found. 1H NMR
(CDCl3) d ppm: 1.88–1.92 (m, 2H, CH2), 2.86–2.90 (m, 2H, CH2),
2.96–2.98 (m, 1H, CH2), 3.15–3.18 (m, 1H, CH2), 3.41–3.44 (m,
1H, CH2), 3.78–3.81 (m, 2H, CH2), 3.97–3.99 (m, 1H, CH2), 6.89–
6.91 (d, J = 8.03, 2H, Ar-H), 6.95–6.97 (d, J = 8.03, 1H, Ar-H), 7.00–
7.05 (t, J = 8.03, 1H, Ar-H), 7.16–7.25 (m, 2H, Ar-H), 7.27–7.33 (m,
2H, Ar-H), 7.36–7.39 (m, 2H, Ar-H), 7.46–7.49 (m, 2H, Ar-H),
9.49–9.54 (d, 2H, NH). 13C NMR (CDCl3): 28.9; 47.3; 50.3;
52.1;52.8; 77.9; 116.8; 118.4; 119.8; 121.4; 124.3; 127.9; 128.1;
128.2; 129.0; 129.3; 131.6; 132.9; 135.3; 135.6; 148.9; 154.9;
(CDCl3) d ppm: 1.87–1.90 (m, 2H, CH2), 2.75–2.81 (m, 1H, CH2),
2.83–2.87 (m, 1H, CH2), 2.96–2.98 (m, 1H, CH2), 3.18–3.23 (m,
1H, CH2), 3.43–3.46 (m, 1H, CH2), 3.78–3.81 (m, 1H, CH2), 3.95–
3.99 (m, 1H, CH2), 6.91–6.94 (m, 2H, Ar–H), 6.96–6.98 (m, 1H,
Ar-H), 7.04–7.06 (m, 1H, Ar-H), 7.19–7.23 (t, J = 7.53, 1H, Ar-H),
7.37–7.41 (m, 2H, Ar-H), 7.43–7.45 (d, J = 8.03, 1H, Ar-H), 7.48–
7.51 (m, 1H, Ar-H), 9.13 (s, 1H, Ar–H), 9.24 (s, 1H, NH), 9.28 (s,
1H, NH). 13C NMR (CDCl3) d ppm: 29.5; 48.0; 50.5; 50.7; 52.6;
78.1; 118.1; 118.2; 120.0; 121.6; 124.6; 125.7; 128.2; 129.1;
129.3; 130.0; 132.6; 133.0; 148.5; 148.7; 152.7; 155.3; 159.2;
155.6; 158.9; 165.3; 170.2. IR (KBr):
1702, 1579.
m
(cmꢀ1): 3105, 2958, 1728,
164.6; 170.2. IR (KBr):
m
(cmꢀ1): 3066, 2958, 1735, 1640, 1588.
4.1.9.19.
5-(4-Phenoxyphenyl)-5-(4-(3-phenylpropanoyl)-1,4-
4.1.9.23. 5-(4-Phenoxyphenyl)-5-(4-(morpholine-4-carbonyl)-
1,4-diazepan-1-yl) pyrimidine-2,4,6(1H,3H,5H)-trione (36). Yield:
41% as off-white solids. Mp: 135–138 °C. MS: calcd for C26H29N5O6-
Na = 530.2016, (M+Na)+ = 530.1994 found. 1H NMR (CDCl3) d ppm:
1.79–1.83 (m, 1H, CH2), 1.86–1.89 (m, 1H, CH2), 2.76–2.78 (m, 2H,
CH2), 2.87–2.90 (m, 1H, CH2), 3.22–3.24 (m, 4H, Ar–H), 3.36–3.39
(m, 2H, CH2), 3.48–3.52 (m, 2H, CH2), 3.3.67–3.69 (m, 4H CH2),
3.78–3.80 (m, 1H, CH2), 6.91–6.96 (m, 3H, Ar-H), 7.00–7.04 (m, 1H,
Ar-H), 7.15–7.19 (t, J = 7.53, 1H, Ar-H), 7.35–7.39 (m, 1H, Ar-H),
7.46–7.50 (m, 3H, Ar-H) 9.81 (s, 1H, NH), 9.87 (s, 1H, NH). 13C
NMR (CDCl3) d ppm: 29.2; 47.4; 47.6; 50.6; 51.1; 52.2; 66.6; 77.2;
118.1; 118.3; 119.7; 121.3; 124.4; 128.2; 129.3; 129.4; 130.0;
diazepan-1-yl) pyrimidine-2,4,6(1H,3H,5H)-trione (32). Yield:
45% as off-white solids. Mp: 121–124 °C. MS: calcd for C30H30N4O5-
Na = 549.2114, (M+Na)+ = 549.2110 found. 1H NMR (CDCl3) d ppm:
1.61–1.67 (m, 1H, CH2), 1.70–1.76 (m, 1H, CH2), 2.71–2.76 (m, 2H,
CH2), 2.80–2.84 (m, 2H, CH2), 2.99–3.04 (m, 2H, CH2), 3.35–3.38
(m, 1H, CH2), 3.57–3.60 (m, 1H, CH2), 3.61–3.64 (m, 1H, CH2),
3.71–3.74 (m, 1H, CH2), 6.92–6.96 (m, 2H, Ar-H), 7.02–7.05 (m,
2H, Ar-H), 7.19–7.29 (m, 6H, Ar-H), 7.36–7.40 (t, J = 7.53, 2H, Ar-
H), 7.44–7.48 (m, 2H, CH2), 9.36 (s, 1H, NH), 9.50 (s, 1H, NH). 13C
NMR (CDCl3) d ppm: 29.0; 31.4; 34.1; 48.3; 50.7; 52.2; 53.4;
77.8; 118.2; 119.8; 121.5; 124.3; 126.1; 128.4; 128.6; 129.1;
129.3; 129.9; 130.0; 141.4; 148.9; 155.6; 158.9; 170.2; 172.5. IR
149.2; 155.7; 158.7; 164.2; 170.3; 170.4. IR (KBr):
2978, 1711, 1610, 1580.
m
(cmꢀ1): 3065,
(KBr):
m
(cmꢀ1): 3068, 2955, 1730, 1711, 1590.
4.1.9.20. 5-(4-Phenoxyphenyl)-5-(4-(2-(4-chlorophenyl)acetyl)-
1,4-diazepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione (33). Yield:
32% as off-white solids. Mp: 138–140 °C. MS: calcd for
4.1.9.24.
5-(4-Phenoxyphenyl)-5-(4-((8-(dimethylamino)nap-
ththalen-4-yl)sulfonyl)-1,4-diazepan-1-yl)pyrimidine-
2,4,6(1H,3H,5H)-trione (37). Yield: 52% as yellow solids. Mp:
138–140 °C. MS: calcd for
C
29H27ClN4O5Na = 569.1568, (M+Na)+ = 569.1571 found. 1H NMR
C
33H34N5O6S = 628.2230, (M+H)+ =
(MeOD) d ppm: 1.55–1.59 (m, 1H, CH2), 1.66–1.72 (m, 1H, CH2),
2.67–2.73 (m, 2H, CH2), 2.85–2.89 (m, 1H, CH2), 2.94–2.98 (m, 1H,
CH2), 3.33–3.39 (m, 1H, CH2), 3.52–3.55 (m, 1H, CH2), 3.60–3.63
(m, 1H, CH2), 3.71–3.74 (m, 1H, CH2), 3.89–3.93 (m, 1H, CH2),
3.99–4.02(m, 1H, CH2), 6.85–6.91 (m, 1H, Ar-H), 6.93–6.97(m, 2H,
Ar-H), 7.01–7.05 (m, 1 H, Ar-H), 7.18–7.25 (m, 2H, Ar-H), 7.28–7.3
(m, 1H, Ar-H), 7.33–7.38 (m, 3H, Ar-H), 7.41–7.45 (m, 1H, Ar-H),
7.49–7.57 (m, 2H, Ar-H). 13C NMR (MeOD) d ppm: 30.8; 45.4;
51.4; 52.3; 53.7; 54.8; 78.7; 119.0; 119.5; 120.7; 121.0 l 122.7;
125.5; 129.4; 129.9; 130.4; 130.6; 130.7; 130.9; 131.1; 131.2;
628.2249 found. 1H NMR (CDCl3) ppm: 1.75–1.78 (m, 2H, CH2),
2.86–2.89 (m, 4H, CH2), 2.91 (s, 6H, CH3), 3.34–3.38 (m, 2H, CH2),
3.63–3.66 (m, 2H, CH2), 6.89–6.92 (m, 3H, Ar-H), 7.02–7.04 (d,
J = 7.53, 1H, Ar-H), 7.16–7.22 (t, J = 7.53, 2H, Ar-H), 7.36–7.43 (m,
4H, Ar-H), 7.51–7.59 (m, 2H, Ar-H), 8.16–8.18 (d, J = 7.53, 1H, Ar-
H), 8.34–8.36 (d, J = 7.03, 1H, Ar-H), 8.54–8.56 (m, 1H, Ar-H),
8.86 (s, 1H, N–H), 8.90 (s, 1H, N–H). 13C NMR (CDCl3) ppm: 29.6;
46.7; 50.0; 50.8; 51.6; 53.8; 78.1; 118.2; 118.3; 119.9; 121.4;
123.2. 123.4; 128.0; 128.8; 129.0; 129.2; 129.4; 130.0; 132.9;
m
(cmꢀ1):
144.3; 148.5; 151.3; 155.5; 159.1; 169.9; 170.0. IR (KBr):
m
134.0; 134.4; 150.8; 156.1; 159.2; 160.4; 170.3. IR (KBr):
3096, 2956, 1738, 1710, 1588.
(cmꢀ1): 3170, 1736, 1713, 1609, 1588.
4.1.9.25.
1,4-diazepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione
(38). Yield: 32% as off-white solids. Mp: 240–244 °C. MS: calcd for
29H29N4O5S = 545.1841, (M+H)+ = 545.1859 found. 1H NMR
5-(4-Phenoxyphenyl)-5-(4-(4-(methylthio)benzoyl)-
4.1.9.21.
5-(4-Phenoxyphenyl)-5-(4-(2-cyclohexylacetyl)-1,4-
diazepan-1-yl) pyrimidine-2,4,6(1H,3H,5H)-trione (34). Yield:
34% as off-white solids. Mp: 121–124 °C. MS: calcd for
29H35N4O5 = 519.2607, (M+H)+ = 519.2597 found. 1H NMR (CDCl3)
C
C
(DMSO-d6) ppm: 1.48–1.55 (m, 1H, CH2) 1.64–1.70 (m, 1H, CH2),
2.46 (s, 3H, CH3), 2.68–2.73 (m, 3H, CH2), 3.19–3.23 (m, 1H, CH2),
3.41–3.41 (m, 1H, CH2), 3.51–3.54 (m, 1H, CH2), 3.64–3.68 (m,
1H, CH2), 6.90–6.95 (m, 1H, Ar-H), 7.00–7.03 (m, 3H, Ar-H), 7.18–
7.21 (t, J = 7.53, 1H, Ar-H), 7.27–7.35 (m, 5H, Ar-H), 7.39–7.47 (m,
2H, Ar-H), 7.56–7.58 (m, 1H, Ar-H), 11.57 (s, 2H, NH). 13C NMR
(DMSO-d6) ppm: 14.3; 29.8; 47.7; 50.8; 52.9; 53.5; 76.7; 118.0;
119.4; 121.5; 124.2; 125.1; 127.0; 127.4; 129.0; 129.4; 130.0;
d ppm: 1.22–1.25 (m, 2H, CH2), 1.28–1.32 (m, 2H, CH2), 1.66–1.70
(m, 4H, CH2), 1.74–1.79 (m, 3H, CH2 + CH), 1.86–1.91 (m, 2H, CH2),
2.20–2.22 (d, J = 6.84, 1H, CH2), 2.24–2.26 (d, J = 6.84, 1H, CH2),
2.73–2.76 (m, 1H, CH2), 2.79–2.82 (m, 2H, CH2), 2.86–2.89 (m,
1H, CH2), 3.41–3.44 (m, 1H, CH2), 3.60–3.63 (m, 1H, CH2), 3.64–
3.67 (m, 1H, CH2), 3.71–3.74 (m, 1H, CH2), 3.78–3.81 (m, 1H,
CH2), 6.94–6.97 (m, 2H, Ar-H), 7.04–7.06 (d, J = 8.03, 2H, Ar-H),
7.16–7.20 (t, J = 7.53, 1H, Ar-H), 7.37–7.40 (t, J = 7.53, 1H, Ar-H),
7.47–7.51 (m, 3H, Ar-H), 9.60 (s, 2H, NH). 13C NMR (CDCl3) d
ppm: 25.8; 27.4; 29.0; 33.0; 34.6; 40.5; 48.0; 50.3; 52.4; 53.1;
133.0; 133.3; 139.6; 149.5; 155.5; 157.6; 170.0; 170.7. IR (KBr):
m
(cmꢀ1): 3058, 2951, 2763, 1729, 1708, 1589.

J. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4985–4999
4997
4.1.10. General procedure for the preparation of compounds
39–46
3.18–3.21 (m, 1H, CH2), 3.42–3.45 (m, 1H, CH2), 3.68–3.71(m, 1H,
CH2), 3.81–3.83 (m, 1H, CH2), 6.2 (s, 1H, CH), 6.80–6.83 (d,
J = 8.53, 1H, Ar-H), 6.94–6.95 (m, 1H, Ar-H), 6.97–7.00 (m, 1H,
Ar-H), 7.03–7.06 (m, 1H, Ar-H), 7.18–7.21 (t, J = 7.03, 1H, Ar-H),
7.36–7.41 (m, 1H, Ar-H), 7.51–7.55 (m, 1H, Ar-H), 7.56–7.59 (m,
1H, Ar-H), 7.63–7.68 (m, 1H, Ar-H), 7.74–7.76 (m, 1H, Ar-H),
7.96–7.98 (m, 1H, Ar-H), 8.01–8.03 (m, 2H, Ar-H). 13C NMR (MeOD)
d ppm: 12.3; 28.9; 45.6; 51.6; 52.4; 54.3; 78.8; 96.5; 117.7;
119.0; 119.3; 119.6; 120.9; 122.6; 125.4; 129.2; 129.5; 130.3;
130.5; 130.8; 131.1; 132.2; 136.2; 141.3; 150.8; 152.8; 156.7;
To
a
solution of 5-(1,4-diazepan-1-yl)-5-(4-phenoxyphe-
nyl)pyrimidine-2,4,6(1H,3H, 5H)-trione (394 mg, 1 mmol) in
10 ml THF was added DCC (206 mg, 1 mmol), DMAP (132 mg,
1 mmol) and the substituted carboxylic acids (1 mmol). The mix-
ture was stirred at room temperature for 24 h before filtering.
The solvents of the filtrate were removed in vacuo and the result-
ing crude products purified by flash column chromatography to
yield pure products.
158.9; 171.8; 172.1; 172.5. IR (KBr):
1613.
m
(cmꢀ1): 2929, 1738, 1712,
4.1.10.1. 5-(4-Phenoxyphenyl)-5-(4-(1-methyl-1H-indazole-3-
carbonyl)-1,4-diazepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione
(39). Yield: 49% as off-white solids. Mp: 185–187 °C. MS: calcd for
4.1.10.5. 5-(4-Phenoxyphenyl)-5-(4-(5-fluoro-2-(methylsulfo-
namido) benzoyl)-1,4-diazepan-1-yl)pyrimidine-2,4,6(1H,3H,
C
30H29N6O6 = 553.2199. (M+H)+ = 553.2193 found. 1H NMR
(DMSO-d6) d ppm: 1.76–1.78 (m, 2H, CH2), 2.71–2.75 (m, 2H,
CH2), 2.82–2.88 (m, 2H, CH2), 3.60–3.63 (m, 2H, CH2), 3.77–3.80
(m, 2H, CH2), 4.11 (s, 3H, CH2), 7.03–7.10 (m, 3H, Ar-H), 7.21–
7.26 (m, 2H, Ar-H), 7.30–7.34 (t, J = 7.53, 1H, Ar-H), 7.41–7.49 (m,
4H, Ar-H), 7.59–7.62 (m, 1H, Ar-H), 7.70–7.72 (d, J = 8.53, 1H, Ar-
H), 8.00–8.03 (d, J = 8.03, 1H, Ar-H), 11.55 (s, 2H, NH). 13C NMR
(DMSO-d6) d ppm: 29.9; 35.8; 48.4; 51.2; 52.1; 53.3; 76.7; 110.0;
117.6; 118.5; 119.6; 121.4; 123.4; 124.9; 126.4; 128.0; 129.1;
129.3; 130.2; 133.0; 139.2; 140.0; 149.4; 155.4; 157.7; 162.8;
5H)-trione (43). Yield: 38% as off-white solids. Mp: 200–204 °C.
MS: calcd for C29H29N5O7S = 610.1772, (M+H)+ = 610.1799 found.
1H NMR (MeOD) d ppm: 1.82–1.89 (m, 2H, CH2), 2.85–2.90 (m,
3H, CH2), 3.00–3.03 (m, 1H, CH2), 3.06 (s, 3H, CH3), 3.21–3.24 (m,
1H, CH2), 3.44–3.50 (m, 1H, CH2), 3.68–3.71 (m, 1H, CH2), 3.81–
3.85 (m, 1H, CH2), 6.87–6.91 (m, 1H, Ar-H), 6.93–6.96 (m, 2H, Ar-
H), 7.00–7.06 (m, 2H, Ar-H), 7.17–7.22 (m, 1H, Ar-H), 7.25–7.28
(m, 1H, Ar-H), 7.34–7.39 (m, 1H, Ar-H), 7.74–7.44 (m, 1H, Ar-H),
7.45–7.50 (m, 1H, Ar-H), 7.51–7.55 (m, 2H, Ar-H). 13C NMR (MeOD)
d ppm: 29.4; 41.0; 45.7; 51.1; 52.5; 53.6; 79.0; 113.5; 115.6; 116.3;
117.9; 118.1; 119.2; 119.6; 120.8; 122.5; 129.5; 130.4; 130.6;
130.9; 131.1; 139.2; 150.8; 152.3; 156.8; 159.6; 169.8; 172.4. IR
170.7. IR (KBr):
m
(cmꢀ1): 2929, 1737, 1710, 1625.
4.1.10.2. 5-(4-Phenoxyphenyl)-5-(4-(2-(2,3-dihydrobenzo[b]-
(KBr):
m
(cmꢀ1): 2931, 1738, 1712, 1613.
[1,4]dioxine-6-sulfonamido)3-methylbutyryl)-1,4-diazepan-1-yl)-
pyrimidine-2,4,6(1H,3H,5H)-trione (40). Yield: 31% as off-white
solids. Mp: 143–146 °C. MS: calcd for C34H38N5O9S = 692.2390,
(M+H)+ = 692.2383 found. 1H NMR (CDCl3) d ppm: 0.84–0.89 (m,
3H, CH3), 1.01–1.04 (m, 3H, CH3), 1.88–1.93 (m, 2H, CH2), 2.39–
2.43 (m, 1H, CH), 2.68–2.74 (m, 2H, CH2), 2.82–2.84 (m, 1H, CH2),
3.24–3.26 (m, 1H, CH2), 3.51–3.54 (m, 2H, CH2 + CH), 3.76–3.80
(m, 1H, CH2), 3.84–3.87 (m, 1H, CH2), 3.92–3.94 (m, 1H, CH2),
4.27–4.31 (m, 4H, CH2), 6.91–7.00 (m, 6H, Ar-H), 7.04–7.07 (t,
J = 7.53, 1H, Ar-H), 7.18–7.22 (m, 1H, CH), 7.29–7.32 (m, 3H, Ar–
H), 7.37–7.47 (m, 1H, Ar–H), 7.47–7.51 (m, 2H, Ar–H), 8.87 (s,
1H, NH), 9.07 (s, 1H, NH). 13C NMR (CDCl3) d ppm: 19.5; 29.0;
30.8; 46.1; 49.6; 50.1; 51.5; 53.2; 57.5; 77.7; 116.4; 116.8;
117.4; 117.9; 118.1; 119.6; 120.6; 121.2; 124.2; 128.9; 129.1;
129.7; 132.0; 146.9; 151.2; 154.5; 155.2; 158.3; 169.4; 170.5. IR
4.1.10.6. 5-(4-Phenoxyphenyl)-5-(4-(3-(N-(4-methoxyphenyl)-
sulfamoyl)-4-methylbenzoyl)-1,4-diazepan-1-yl)pyrimidine-
2,4,6(1H,3H,5H)-trione (44). Yield: 26% as off-white solids. Mp:
147–149 °C. MS: calcd for
C
36H26N5O8S = 698.2285, (M+H)+ =
698.2290 found. 1H NMR (MeOD) d ppm: 1.70–1.76 (m, 2H, CH2),
2.67 (s, 3H, CH3) 2.70–2.73 (m, 1H, CH2), 2.75–2.78 (m, 2H, CH2),
2.91–2.96 (m, 1H, CH2), 3.33 (s, 3H, CH3), 3.47–3.50 (m, 1H, CH2),
3.56–3.58 (m, 1H, CH2), 3.63–3.65 (m, 1H, CH2), 3.70–3.73 (m,
1H, CH2), 6.53–6.55 (d, J = 8.53, 1H, Ar-H), 6.70–6.73 (d, J = 8.53,
1H, Ar-H), 6.77–6.80 (m, 2H, Ar-H), 6.90–6.94 (m, 2H, Ar-H),
6.97–7.03 (m, 2H, Ar-H), 7.20–7.23 (t, J = 7.03, 1H, Ar-H), 7.32–
7.37 (m, 1H, Ar-H), 7.40–7.46 (m, 2H, Ar-H), 7.52–7.55 (m, 1H,
Ar-H), 7.59–7.61 (d, J = 8.03, 1H, Ar-H), 7.66–7.68 (d, J = 8.03, 1H,
Ar-H), 8.11 (s, 1H, Ar-H). 13C NMR (MeOD) d ppm: 20.5; 28.9;
45.5; 51.2; 52.7; 53.8; 55.9; 78.9; 115.3; 115.4; 118.8; 119.1;
119.5; 119.7; 120.8; 121.0; 122.7; 124.3; 125.5; 125.7; 129.5;
129.6; 130.4; 130.5; 130.6; 130.7; 130.8; 135.4; 138.5; 139.2;
(KBr):
m
(cmꢀ1): 2929, 1736, 1710, 1615.
4.1.10.3. 5-(4-Phenoxyphenyl)-5-(4-(3-(benzo[d]thiazol-2-yl)-2-
methyl-2-phenylpropanoyl)-1,4-diazepan-1-yl)pyrimidine-
2,4,6(1H,3H,5H)-trione (41). Yield: 33% as off-white solids. Mp:
141–144 °C. MS: calcd for C38H36N5O5S = 674.2437, (M+H)+ =
674.2426 found. 1H NMR (MeOD) d ppm: 1.82–7.89 (m, 2H, CH2),
2.24 (s, 3H, CH3), 2.93–2.96 (m, 3H, CH2), 3.32–3.35 (m, 2H, CH2),
3.45–3.51 (m, 2H, CH2), 3.73–3.76 (m, 2H, CH2), 6.93–6.97 (m,
6H, Ar–H), 6.99–7.02 (m, 2H, Ar-H), 7.14–7.18 (t, J = 7.53, 1H, Ar-
H), 7.35–7.39 (m, 2H, Ar-H), 7.48–7.52 (m, 3H, Ar-H), 7.54–7.57
(m, 2H, Ar-H), 7.80–7.82 (d, J = 8.53, 1H, Ar-H), 7.87–7.89 (d,
J = 8.53, 1H, Ar-H). 13C NMR (MeOD) d ppm: 21.4; 29.0; 39.7;
47.3; 48.4; 52.3; 55.7; 78.7; 117.4; 119.1; 119.4; 120.7; 122.3;
122.5; 122.9; 125.2; 126.1; 127.0; 129.5; 130.3; 130.4; 130.7;
130.9; 131.1; 133.0; 139.1; 150.9; 154.0; 157.0; 159.2; 170.4;
150.9; 152.9; 155.8; 157.2; 169.4; 172.2. IR (KBr):
2932, 1731, 1708, 1607.
m
(cmꢀ1):
4.1.10.7. 5-(4-Phenoxyphenyl)-5-(4-(4-methyl-2-(methylsulfo-
namido)benzoyl)-1,4-diazepan-1-yl)pyrimidine-2,4,6(1H,3H,5H)-
trione (45). Yield: 40% as off-white solids. Mp: 203–206 °C. MS:
calcd for C30H31N5O7SNa = 628.1842, (M+Na)+ = 628.1821 found.
1H NMR (DMSO-d6) d ppm: 1.64–1.70 (m, 2H, CH2), 2.27 (s, 3H,
CH3), 2.70–2.75 (m, 2H, CH2), 2.84–2.87 (m, 1H, CH2), 2.98 (s, 3H,
CH3), 3.09–3.13(m, 1H, CH2), 3.31–3.36 (m, 2H, CH2), 3.70–3.73
(m, 1H, CH2), 6.88 (s, 1H, Ar-H), 6.94–6.96 (d, J = 8.03, 1H, Ar-H),
7.02–7.04 (d, J = 8.53, 1H, Ar-H), 7.06–7.09 (m, 1H, Ar-H), 7.16–
7.23 (m, 2H, Ar-H), 7.23–7.31 (m, 2H, Ar-H), 7.34–7.39 (m, 2H,
Ar-H), 7.42–7.44 (m, 1H, Ar-H), 7.57–7.59 (d, J = 7.53, 1H, Ar-H).
13C NMR (DMSO-d6) d ppm: 21.0; 29.4; 42.3; 47.3; 50.5; 50.9;
52.3; 76.7; 116.0; 117.7; 118.2; 118.5; 119.5; 119.6; 121.7;
127.9; 128.4; 129.4; 129.9; 130.1; 130.6; 139.2; 141.1; 149.4;
172.6; 178.7. IR (KBr):
m
(cmꢀ1): 2929, 1736, 1710, 1615.
4.1.10.4. 5-(4-Phenoxyphenyl)-5-(4-(3-(N-(5-methylisoxazol-3-
yl)sulfamoyl) benzoyl)-1,4-diazepan-1-yl)pyrimidine-2,4,6(1H,
3H,5H)-trione (42). Yield: 36% as off-white solids. Mp: 145–
147 °C. MS: calcd for C32H31N6O8S = 659.1924, (M+H)+ = 659.1931
found. 1H NMR (MeOD) d ppm: 1.83–1.88 (m, 2H, CH2), 2.24 (s,
3H, CH3), 2.81–2.85 (m, 3H, CH2), 2.98–3.00 (m, 1H, CH2),
151.5; 155.2; 157.3; 168.1; 170.7. IR (KBr):
1706, 1602.
m
(cmꢀ1): 2933, 1744,

4998
J. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4985–4999
4.1.10.8.
5-(4-Phenoxyphenyl)-5-(4-(2-(4-oxo-2-(phenylimi-
Reaction rates were measured from the initial 10 min of the
reaction profile where product release was linear with time and
plotted as a function of inhibitor dose. From the resulting inhibition
curves, the IC50 value for each inhibitor was calculated by nonlinear
regression analysis using GraphPad Prism 4.0 (La Jolla, CA, USA).
no)thizolidin-5-yl)acetyl)-)-1,4-diazepan-1-yl)pyrimidine-2,4,6-
(1H,3H,5H)-trione (46). Yield: 34% as off-white solids. Mp: 167–
169 °C. MS: calcd for
C
32H30N6O6SNa = 649.1845, (M+Na)+ =
649.1827 found.1H NMR (MeOD) d ppm: 1.82–7.89 (m, 2H, CH2),
2.75–2.79 (m, 2H, CH2), 2.83–2.86 (m, 1H, CH2), 2.90–2.94 (m,
2H, CH2), 3.42–3.47 (m, 2H, CH2), 3.50–3.55 (m, 1H, CH2), 3.61–
3.67 (m, 2H, CH2), 4.53–4.55 (m, 1H, CH2), 6.91–6.97 (m, 3H,
Ar-H), 7.01–7.02 (d, J = 7.53, 2H, Ar-H), 7.15–7.19 (t, J = 7.03, 2H,
Ar-H), 7.30–7.32 (m, 1H, Ar-H), 7.34–7.37 (t, J = 7.03, 2H, Ar-H),
7.46–7.54 (m, 4H, Ar-H). 13C NMR (MeOD) d ppm: 29.0; 35.7;
46.3; 47.7; 50.4; 52.5; 53.6; 79.0; 117.5; 119.3; 119.8; 120.7;
122.2; 122.4; 122.5; 127.2; 129.5; 130.0; 130.5; 130.8; 131.1;
4.2.4. Invasion assay
Caco-2 cells were cultured until 80% confluence and removed
from the flask using DPBS-EDTA. The cells were collected in ser-
um-free medium and counted in a cell-counting machine (Z1 coul-
terÒ Particle counter). The inserts containing matrigel membranes
with 8.0 lm pore size (BD Bioscience, UK) were treated with 500 ll
serum-free medium. After incubation for 2 h, the blank inserts
were treated with 250,000 Caco-2 cells and the positive control in-
serts contained 250,000 Caco-2 cells and 75 ng HGF.15 All the
remaining inserts were treated with 250,000 Caco-2 cells, 75 ng
HGF, and the tested inhibitors (with final concentrations at
150.8; 152.3; 156.9; 157.4; 159.5; 171.6; 172.4; 178.0. IR (KBr):
m
(cmꢀ1): 2930, 1737, 1710, 1618.
4.2. Biological methods
10 lM and/or 100 nM). Medium with 2% FCS was added to each in-
4.2.1. Cell culture and treatment
Human fibrosarcoma HT-1080 cells were cultivated in Eagle’s
minimal essential medium (MEM) containing 10% fetal bovine ser-
serts to make the total volume 1 ml. The inserts were then trans-
ferred to the assay chambers containing 750 ml of 20% FCS
medium and left in cell culture incubator for 48 h. After incubation,
supernatants were taken out and the non-migratory cells on the
upper surface of the membranes were removed with serum-free
medium using cotton swabs. The inserts were treated with the
Diff-Quik kit (BD Bioscience, USA) for fixing and staining the invade
cells on the membranes. The inserts were left to dry and the mi-
grated cells were counted under using microscopy (Zeiss Axiovert
200 M). The experiments were carried out in duplicate and re-
peated three times for each concentration inhibitor.
um (FBS), 100 IU/ml penicillin, and 50 lg/ml streptomycin. The
cultures were incubated at 37 °C in a humidified 5% CO2 atmo-
sphere until confluence. Before experiments, cultures were washed
twice with serum-free medium and then incubated under serum-
free conditions for 6 h. 12-O-Tetradecanoylphorbol-13-acetate
(PMA) was added into the cell and final concentration of PMA
was 5 nM. After incubation for 24 h, the supernatants from the cell
cultures were taken out for assay.26 Caco-2 cells (Sigma–Aldrich,
Ireland) were cultivated in Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% FBS, 1 ꢁ nonessential amino acids, 2 mM
4.3. Molecular modelling
L-glutamine, 100 IU/ml penicillin, and 50
lg/ml streptomycin.
The crystal structure of MMP-2 (PDB code 1QIB) was used for
the study. Since this is a co-crystallised structure complexed with
a hydroxamate inhibitor, the hydroxamate inhibitor was removed.
The structure of MMP-9 (PDB code 2VOX) used was an MMP-9 ac-
tive site mutant with barbiturate inhibitor, so the barbiturate
inhibitor was removed. In addition water molecules were removed.
Docking calculations were carried out using AutoDock version 4.0,
with the Lamarckian genetic algorithm (LGA).28 The molecular
models of each inhibitor were built using the builder function of
MOE and minimised with MOPAC 7 (AM1 method) interfaced to
MOE. The zinc parameters were changed to zinc radius: 0.87 Å;
well depth: 0.35 kcal/mol; and zinc charges: +0.95e.29 The 3D affin-
ity grid box was designed to include the full active site and possible
residues. The setting of the centre of grid boxes was based on the
value of active zinc atom. Docking calculations were set to 20 runs.
Energy (250,00,000) evaluations were allowed as a maximum in
each run. At the end of the calculation, AutoDock performed cluster
analysis. Docking solutions with ligand all-atom root mean square
deviation (RMSD) within 2.0 Å of each other were clustered
together and ranked by the lowest energy representative.
4.2.2. Zymography
The enzymatic activities of MMP-2 and MMP-9 were assayed by
gelatin zymography in the absence of serum.27 Cell supernatants
were electrophoresed on an 8% SDS–PAGE containing 2% gelatin.
The gels were washed with 0.25% triton three times. Then the gels
were cut and incubated in the zymography buffer (0.15 M NaCl,
5 mM CaCl2, 0.05% NaN3 and 50 mM Tris–HCl buffer, pH 7.5) at
37 °C for 48 h, which contained the compounds at the final concen-
tration of 0.5–10 lM. After incubation, the gels were stained with
0.25% Coomassie Brilliant Blue R250, and destained with acetic
acid, methanol and water. MMP activity was represented by a
white band of gelatin digestion. Remaining enzymatic activity in
the presence of test inhibitor was quantitated by densitometry
on the gel reader (Bio-RAD, Universal hood II).
4.2.3. MMP-2 and MMP-9 fluorogenic assay
Recombinant MMP-2 and MMP-9 (R&D Systems, Ireland) were
activated by treatment with APMA (p-aminophenylmercuric
acetate) at 37 °C for 1 h and 24 h, respectively.1 The synthetic
broad-spectrum fluorogenic substrate (7-methoxycoumarin-4-
yl)-acetyl-pro-Leu-Gly-Leu-(3-(2,4-dinitrophenyl)-
L
-2,3-diamino-
4.4. Statistical analysis
propionyl)-Ala-Arg-NH2 (R&D Systems, UK) was used to assay
MMP-2 and MMP-9 activity. The inhibition of human active
MMP-2 and MMP-9 was assayed by preincubating MMP-2 (2 nM)
or MMP-9 (2 nM) and the inhibitory compounds at varying concen-
All date are presented as group of means with standard error of
the mean of n P 3. Statistical analysis of the mean difference
between multiple groups was determined by one-way ANOVA fol-
lowed by Tukey-Kramer multiple comparison post test. A P value
<0.05 was considered to be statistically significant. All statistical
analyses were performed using GraphPad Prism 4.0.
trations (10 pM to 10
0.2 M NaCl, 5 mM CaCl2, 20
for 30–45 min. An aliquot of substrate (10
was then added to 90 l of the preincubated MMP/inhibitor mix-
l
M) in 50 mM Tris–HCl, pH 7.5, containing
M ZnSO4, and 0.05% Brij 35, at 37 °C
l of a 50 M solution)
l
l
l
l
ture, and the activity was determined at 37 °C by following product
release with time. The fluorescence changes were monitored using
a plate reader (Fluostar Optima, BMG Labtech) with excitation and
emission wavelengths set to 330 and 405 nm, respectively.
Acknowledgements
This work was partly funded by Science Foundation Ireland
(SFI). C.M. is an SFI Stokes Lecturer.

J. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4985–4999
15. Friedl, P.; Wolf, K. Nat. Rev. Cancer 2003, 3, 362.
4999
References and notes
16. Yu, A. E.; Murphy, A. N.; Stetler-Stevenson, W. G. In Matrix Metalloproteinases;
Parks, W. C., Mecham, R. P., Eds.; Academic Press: San Diego, 1998; p 85.
17. Vu, T. H.; Shipley, J. M.; Bergers, G.; Berger, J. E.; Helms, J. A.; Hanahan, D.;
Shapiro, S. D.; Senior, R. M.; Werb, Z. Cell 1998, 93, 411.
18. Giannelli, G.; Flak-Marziller, J.; Schiraldi, O.; Stetler-Stevenson, W. G.;
Quaranta, V. Science 1997, 277, 225.
1. Deryugina, E. I.; Quigley, J. P. Cancer Metastasis Rev. 2006, 25, 9.
2. Nagase, H.; Woessner, J. F., Jr. Biol. Chem. 1999, 274, 21491.
3. Rao, B. G. Curr. Pharm. Des. 2005, 11, 295.
4. Brandstetter, H.; Grams, F.; Glitz, D.; Lang, A.; Huber, R.; Bode, W.; Krell, H. W.;
Engh, R. A. J. Biol. Chem. 2001, 276, 17405.
19. Imai, K.; Hiramatsu, A.; Fukushima, D.; Pierschbacher, M. D.; Okada, Y. Biochem.
J. 1997, 322, 809.
5. Grams, F.; Brandstetter, H.; D’Alò, S.; Geppert, D.; Krell, H. W.; Leinert, H.; Livi,
V.; Menta, E.; Oliva, A.; Zimmermann, G. Biol. Chem. 2001, 382, 1277.
6. Drummond, A. H.; Beckett, P.; Brown, P. D.; Bone, E. A.; Davidson, A. H.;
Galloway, W. A.; Gearing, A. J.; Huxley, P.; Laber, D.; McCourt, M.; Whittaker,
M.; Wood, L. M.; Wright, A. Ann. N. Y. Acad. Sci. 1999, 878, 228.
7. Tochowicz, A.; Maskos, K.; Huber, R.; Oltenfreiter, R.; Dive, V.; Yiotakis, A.;
Zanda, M.; Pourmotabbed, T.; Bode, W.; Goettig, P. J. Mol. Biol. 2007, 371, 989.
8. Breyholz, H. J.; Schäfers, M.; Wagner, S.; Höltke, C.; Faust, A.; Rabeneck, H.;
Levkau, B.; Schober, O.; Kopka, K. J. Med. Chem. 2005, 48, 3400.
9. Faust, A.; Waschkau, B.; Waldeck, J.; Höltke, C.; Breyholz, H. J.; Wagner, S.;
Kopka, K.; Heindel, W.; Schäfers, M.; Bremer, C. Bioconjug. Chem. 2008, 19, 1001.
10. Breyholz, H. J.; Wagner, S.; Faust, A.; Riemann, B.; Höltke, C.; Hermann, S.;
Schober, O.; Schäfers, M.; Kopka, K. ChemMedChem 2010, 5, 777.
11. Evans, D. A.; Katz, J. L.; West, T. R. Tetrahedron Lett. 1998, 39, 2937.
12. Kopka, K.; Breyholz, H. J.; Wagner, S.; Law, M. P.; Riemann, B.; Schroer, S.; Trub,
M.; Guilbert, B.; Levkau, B.; Schober, O.; Schafers, M. Nucl. Med. Biol. 2004, 31,
257.
20. Sternlicht, M. D.; Werb, Z. Annu. Rev. Cell Dev. Biol. 2001, 17, 463.
21. Noe, V.; Fingleton, B.; Jacobs, K.; Crawford, H. C.; Vermeulen, S.; Steelant, W.;
Bruyneel, E.; Matrisian, L. M.; Maree, l. M. J. Cell Sci. 2001, 114, 111.
22. Kermorgant, S.; Aparicio, T.; Dessirier, V.; Lewin, M. J.; Lehy, T. Carcinogenesis
2001, 22, 1035.
23. Yu, Q.; Stamenkovic, I. Gemes Dev. 2000, 14, 163.
24. Deryugina, E. I.; Luo, G. X.; Reisfeld, R. A.; Bourdon, M. A.; Strongin, A.
Anticancer Res. 1997, 17, 3201.
25. Meusel, M.; Ambrozak, A.; Hecker, T. K.; Gütschow, M. J. Org. Chem. 2003, 68,
4684.
26. Lohi, J.; Keski-Oja, J. J. Biol. Chem. 1995, 270, 17602.
27. Herron, G. S.; Banda, M. J.; Clark, E. J.; Gavrilovic, J.; Werb, Z. J. Biol. Chem. 1986,
261, 2814.
28. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 2009, 30, 2785.
29. Hu, X.; Shelver, W. H. J. Mol. Graphics Modell. 2003, 22, 115.
13. Friedl, P.; Brocker, E. B. Cell. Mol. Life Sci. 2000, 57, 41.
14. Stamenkovic, I. J. Pathol. 2003, 200, 448.