Interaction with the S1β-pocket of urokinase: 8-heterocycle substituted and 6,8-disubstituted 2-naphthamidine urokinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.04.030

Several 8-substituted 2-naphthamidine-based inhibitors of the serine protease urokinase plasminogen activator (uPA) are described. Direct attachment of five-membered saturated or unsaturated rings improved inhibitor performance; substitution with sulfones

Full text of DOI:10.1016/j.bmcl.2004.04.030

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3063–3068
Interaction with the S1b-pocket of urokinase:
8-heterocycle substituted and 6,8-disubstituted
2-naphthamidine urokinase inhibitors
Michael D. Wendt,a,* Andrew Geyer,a William J. McClellan,a Todd W. Rockway,a
Moshe Weitzberg,a Xumiao Zhao,a Robert Mantei,a Kent Stewart,b
Vicki Nienaber,b Vered Klinghofera and Vincent L. Girandaa
aCancer Research, Global Pharmaceutical R & D, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6101, USA
bStructural Biology, Global Pharmaceutical R & D, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6101, USA
Received 22 March 2004; revised 12 April 2004; accepted 12 April 2004
Abstract—Several 8-substituted 2-naphthamidine-based inhibitors of the serine protease urokinase plasminogen activator (uPA) are
described. Direct attachment of five-membered saturated or unsaturated rings improved inhibitor performance; substitution with
sulfones further improved binding profiles. Combination of these substituents or of previously described NH-linked heteroaromatic
rings with 6-phenyl amide substituents provided further enhancements to potency and selectivity.
Ó 2004 Elsevier Ltd. All rights reserved.
Urokinase-type plasminogen activator (uPA), or uroki-
nase, is a trypsin-family serine protease, which is
implicated in a variety of tumor-associated processes,
including extracellular matrix degradation, invasion,
angiogenesis, and metastasis.1;2 Urokinase converts
plasminogen into the active enzyme plasmin, a wide-
spectrum protease that digests various components of
the extracellular matrix, and also activates proenzymes
of matrix metalloproteinases. High levels of urokinase
are correlated with enhanced invasiveness and metasta-
sis, and poor prognosis.2 Additionally, small-molecule
urokinase inhibitors of moderate potency have been
shown to slow primary tumor growth and metastasis.3
Consequently, there is great interest in developing more
potent and selective inhibitors of urokinase as possible
cancer therapeutics.
study was the aminopyrimidine 2.5 X-ray data suggested
that as an alternative to the NH-linked pyrimidine a
small ring could be directly attached. Six-membered
rings were found to be too large (data not shown), but a
small subset of five-membered rings generated a signif-
icant increase in binding. Concurrently with that study,
6,8-disubstituted compounds were synthesized in an
effort to combine individual binding increments.6
Previously we described work starting from 2-naphth-
amidine 1a including SAR of the 6-phenyl amide 1b,4
and 8-position substitution covering NH-linked aro-
matic rings and carbamates, including X-ray structural
studies. The lead compound resulting from the latter
Several inhibitors exploring the S1b pocket were syn-
thesized with a methoxy group at the adjacent 7-posi-
tion. We determined that small groups at the 7-site
projected into solvent and had little effect on compound
inhibition profiles.5 Thus, directly linked heterocycles
were formed via the iodo compound 4 (Scheme 1), made
from the naphthol 3 by regiospecific iodination7 and
methylation. Heck reaction with cis-2-butene-1,4-diol
gave a cyclic hemiacetal,8 which was reduced to the
Keywords: Urokinase inhibitors.
* Corresponding author. Tel.: +1-847-937-9305; fax: +1-847-935-5165;
e-mail: mike.d.wendt@abbott.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.030

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M. D. Wendt et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3063–3068
tetrahydrofuran-forming reactions and amidination
gave 16a,b, while Suzuki reactions led to 17a,b,d. Oxi-
dation to the sulfones 17c,e was easily carried out with
mCPBA, and the furan-containing nitriles were again
carried on to amidines. The syntheses of furans 17b,d
required the functionalized boronic acid 21, which was
made from 19 by a blocking/deblocking strategy
employing a TMS group. 8-Aminopyrimidines were
installed by nitration of 14, followed by reduction to the
amino compound and Pd-catalyzed amine coupling to 2-
bromopyrimidine. The resulting ester 22 was then
saponified and coupled to anilines12 to give amidonitriles,
which were converted via thioamides to the amidines
23a,b (Scheme 4).
Scheme 1. Reagents and conditions: (a) I2, aq Na2CO3, THF; (b)
NaH, MeI, DMF; (c) cis-2-butene-1,4-diol, PdCl2, NaHCO3, NMP,
130 °C, 1 h; (d) Et3SiH, BF3ÆOEt2, CH2Cl2; (e) LiN(TMS)2, THF, then
1 M HCl; (f) 2- or 3-furylboronic acid, Pd(OAc)2, dppf, Cs2CO3,
DMF.
We expected positions on heterocyclic rings adjacent to
the connecting bond to be buried by protein; conse-
quently we felt heteroatoms would be best deployed at
the more solvent exposed positions. This was immedi-
ately borne out by the furans 6 and 7. In Table 1,
binding data is shown for urokinase and a representative
panel of trypsin-like serine proteases involved in coag-
ulation and proteolysis. The 3-furyl 7 picks up a larger
increment of binding than does the 2-furyl 6. Further,
the 3-tetrahydrofuran 5 also improves affinity to uPA.
We found saturated rings attached by an NH group,
analogous to the aminopyrimidines reported earlier, to
bind very poorly to uPA (data not shown), but the
3-THF series in all cases bound with nearly identical
affinities to uPA and the other trypsin-like serine pro-
teases compared to corresponding 3-furans. Neither 5
nor 7, however, are more selective regarding the panel in
general than 1a. When combined with phenyl amides at
position 6, as in 16a and 18a, these substituents con-
tribute affinity increases in roughly additive fashion to
yield double-digit nanomolar compounds against uPA.
As the phenyl amide 1b also fails to impart a selectivity
advantage over 1a on its own, 16a and 18a likewise
maintain similar selectivity profiles to those of 5 and 7.
16b, however, possesses the additional isopropoxy
group, which interacts with the previously described
hydrophobic ‘dimple’ region of uPA.4 Thus 16b displays
improved selectivity, particularly against kallikrein and
thrombin. Moreover, 16b achieves single-digit activity
against uPA.
tetrahydrofuran and converted to the amidine 5 using
the LiN(TMS)2 amidination protocol.9 Suzuki couplings
followed by LiN(TMS)2 amidination gave 6 and 7.
Pyrazoles (Scheme 2) were made by first synthesizing the
SEM-protected boronic acid 9 via transmetalation of 8
and trapping with B(OMe)3, and protection. Suzuki
coupling to 4 and deprotection gave 10, which was
alkylated and sulfonylated through its potassium anion
in DMF. Amidines 12b and 13 were made via the thio-
amide,10 while the LiN(TMS)2 method was used for
11b.
6,8-Disubstituted compounds were made beginning with
the known cyanoester 14 (Scheme 3). Bromination at the
8-position proceeded cleanly using 1,3-dibromo-5,5-
dimethylhydantoin.11 The resulting ester was saponified,
converted to the acid chloride, and reacted with the
appropriate anilines12 to give 15a–c. A repeat of the
Additional heterocycles were studied; the pyrazole series
offered a solvent exposed heteroatom and a second
nitrogen atom easily amenable to substitution. Though
the parent pyrazole 13 and compounds with N-alkyl
substituents such as 11b offer no improvement over 1a,
the N-methylsulfonyl 12b produces not only improved
binding to uPA but also a small increase in selectivity
against the entire panel of serine proteases.
We next sought to combine the two incremental
improvements found, in the form of alkylsulfonylfurans.
The 3-furan 7 itself is threefold more potent than the
pyrazole, and substituent additivities seemed to hold for
alkylsulfonylfurans. When combined with phenyl amide
substituents at position 6, 18c and its immediate syn-
thetic precursor 18b again display greatly enhanced
binding to uPA, with 18c fivefold more potent than the
Scheme 2. Reagents and conditions: (a) NaH, SEMCl, DMF; (b)
BuLi, B(OMe)3, then 1 M HCl; (c) 4, PdCl2 (dppf), Cs2CO3, DMF; (d)
TBAF, THF, D; (e) RX, KHMDS, DMF; (f) H2S, TEA, pyridine, then
MeI, acetone, then NH4OAc, MeOH; (g) LiN(TMS)2, THF, then 1 M
HCl.

M. D. Wendt et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3063–3068
3065
Scheme 3. Reagents and conditions: (a) DBMH, TfOH, CH2Cl2; (b) LiOH, THF, MeOH, H2O; (c) (COCl)2, toluene, 55 °C; (d) aniline, toluene; (e)
cis-2-butene-1,4-diol, PdCl2, NaHCO3, NMP, 130 °C, 1 h; (f) Et3SiH, BF3ÆOEt2, CH2Cl2; (g) LiN(TMS)2, THF, then 1 M HCl; (h) 21 or furan-3-
boronic acid, PdCl2 dppf, Cs2CO3, DMF, 90 °C; (i) mCPBA, CH2Cl2; (j) LDA, TMSCl, THF, À78 °C; (k) LDA, EtSSEt, THF, À78 °C; (l) TBAF,
THF; (m) BuLi, B(OMe)3, THF, À78 °C, then 1 M HCl.
Scheme 4. Reagents and conditions: (a) KNO3, H2SO4, 0 °C; (b) H2, 10% Pd/C, EtOAc, THF; (c) 2-bromopyrimidine, Pd2dba3, BINAP, NaO-t-Bu,
toluene, 80 °C; (d) LiOH, THF, MeOH, H2O; (e) aniline, HATU, DIPEA, DMF; (f) H2S, TEA, pyridine, then MeI, acetone, then NH4OAc, MeOH.
unsubstituted furan 18a, but both 18b and 18c fail to
completely reproduce the improvements in selectivity
seen with 12b. While comparison of 13 and 12b, and 18a
and 18c, indicate that the methylsulfonyl group is
responsible for a fourfold boost in affinity, it may be
that the pyrazole is more selective than the other hetero-
cycles, and imparts the slight increase in selectivity seen
for 12b. Finally, the cyclopentyloxy-appended 18d gen-
erates improved selectivity across the panel, and was
2 nM against uPA.
shown), however the compound with the best overall
profile is 23b, resulting from addition of a para-amino-
methyl group to the phenyl amide.4
Structural aspects of the binding of inhibitors with
8-heterocycle substitution are exemplified by the co-
crystal of 12b and uPA.13 The periphery of the S1b
pocket is defined by residues Gln192, Lys143, Ser146,
and Gly218, while the Cys191-Cys220 disulfide linkage
helps form the base of the subsite. Figure 1a shows the
X-ray structure of 12b bound to uPA, with the methyl-
sulfonylpyrazole effectively filling the S1b pocket. The
methyl group extends into the farthest region of the S1b
pocket, with the oxygen atoms extended toward solvent.
Superimposed on 12b in Figure 1b is compound 2, with
the aminopyrimidine also efficiently filling the S1b
pocket.5 Amino acid residues in the vicinity of the
inhibitors are essentially identical for both X-ray struc-
tures, with the exception of Gly216 (Fig. 1b), which with
2 is angled toward the bridging NH, with a hydrogen
In spite of the gains made in the binding profile of our
inhibitors by appending heterocycles at the 8-position,
the series of NH-linked heterocycles, in particular the
aminopyrimidine, nevertheless generates greater affinity
and far greater selectivity improvements to the naph-
thamidine scaffold. Compound 2, possessing no 6-sub-
stituent, possesses better overall selectivity than 12b, and
is 18-fold more active against uPA. Addition of a simple
phenyl amide, resulting in 23a, brings the overall profile
to a level similar to 18d. Further addition of alkoxy
groups to the phenyl amide improves the binding profile
in a similar manner to the examples above (data not
ꢀ
bond distance of 3.4 A. Previous work showed that this
NH group, present in a variety of substituents, is uni-
formly worth close to 1.5 kcal/mol, or about a 13-fold

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M. D. Wendt et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3063–3068
Table 1. Inhibition profiles of 8- and 6,8-substituted 2-naphthamidines
Compound
Substituent
7
Ki (lM)a
Trypsin
7.8
6
8
uPA
5.9
P-Kall
23
tPA
Plasmin
51
Thrombin
––
1a
1b
5
>100
Ph
0.63 (0.09)
0.53
2.5 (1.1)b
3.3
32 (18)b
7.4
0.33 (0.18)c 2.0 (0.6)b
5.6 (2.2)b
0.45
O
OMe
OMe
OMe
OMe
0.32
6.0
O
6
2.1
O
7
0.82
2.3
3.7
18
1.2
9.4
1.3
N
N
NH
N
13
11b
OMe
OMe
4.6
SO2Me
N
O
O
O
N
12b
16a
16b
18a
18b
18c
18d
0.63
14
>100
0.87
0.13
0.18
3.3
2.7
8.5
7.1
Ph
0.106 (.013)
0.0085 (.0007)
0.091 (0.011)
0.040 (0.004)
0.018 (0.004)
0.40
0.30
0.27
0.069
0.019
0.03
0.40
0.19
0.32
0.053
0.056
0.038
0.36
1.1
O
0.012
0.045
0.011
0.0035
0.003
Ph
Ph
Ph
0.13
0.12
0.29
0.61
SEt
O
O
O
SO2Et
SO2Et
8.3
O
0.0021 (0.0002) 0.07
1.6
N
2
0.035 (0.007)
0.45
1.0 (0.5)
24 (17)
27
1.7 (0.2)
0.2
3.8 (1.1)
6.6
3.2 (1.1)
5.2
HN
N
24
23a
NH2
1.9
N
Ph
0.0020
0.10
0.81
0.04
0.09
0.17
HN
N
N
N
23b
0.00062
(0.00005)
0.04
0.68
0.02
0.15
0.94
H2N
HN
a Values are means of three experiments. Values in parentheses are standard deviations of two separate measurements, except where noted.
b Nine measurements.
c Eight measurements.
improvement in affinity, leaving another 10-fold
improvement, or 1.4 kcal/mol to be attributed to the
pyrimidine ring.5 The sulfonylpyrazole itself also results
in about a 10-fold improvement; thus it may appear
that, absent more specific interactions, this is roughly
the affinity gain expected from filling the S1b subsite of
uPA.
Selectivity gains from the aminopyrimidine of 2 seem to
come mostly from the pyrimidine ring itself, as seen by
comparison with the amino compound 24.5 Given the
conserved nature of Gly216, and the highly variable
identities of the S1b-forming residues 143 and 146
among the other proteases, it seems reasonable to
assume that the gain in binding for 2 results from a good

M. D. Wendt et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3063–3068
3067
Figure 1. (a) Compound 12b and surface of S1b pocket with key residues highlighted and labeled; (b) 12b and 2 with surface of S1b pocket cut away
to allow view of pocket-forming residues. Compound 2 and the corresponding location of Gly216 are overlaid in gray. Other protein residues in the
structure with 2 have an near-identical orientation to that with 12b. Unlabeled Cys191 is behind the 8-substituent groups.
fit of the ring with the S1b pocket of uPA, whereas the
fit with corresponding regions of other proteases neither
adds to nor subtracts from affinity. Only trypsin suffers a
drop in affinity to 2 due to the pyrimidine, probably
resulting from an asparagine substitution for Lys143.
posed. In general, we saw only rotations of the naphthyl
group of a few degrees, and translations within the S1
subsite toward or away from Asp189 of no more than a
few tenths of an Angstrom. Further, these movements
did not appear to be systematically related to any sub-
stituent type.
ꢀ
However, while the overlap of the 8-substituents of 12b
and 2 in Figure 1b is substantial, the selectivities
imparted by the methylsulfonylpyrazole of 12b, or by
the ethylthiofuran of 18b or the ethylsulfonylfuran of
18c, do not follow the pattern set by 2. Again, the direct-
linked heterocycles themselves seem to impart little or
no selectivity, while the alkylsulfonyl group only seems
to impact the affinity toward tPA. In the S1b-forming
region, tPA is particularly similar to uPA, differing only
in possessing an Ala146 rather than a serine. It is
important to note that the backbone structures of all the
proteins are similar to uPA in this region. This is par-
ticularly so for tPA, thus it is difficult to rationalize the
decrease in affinity to tPA upon appending an
alkylsulfonyl group or even an alkylthio group to an
8-heterocycle. We note that there appears be a weak
hydrogen bond between the Ser146 hydroxyl group of
In summary, a new group of uPA inhibitors have been
developed by combining structural units interacting with
the S1b pocket and additional subsites described in
detail earlier. These interactions, illustrated by a wealth
of structural information, are substantially additive in
nature, and have resulted in selective and extremely
potent inhibitors and an increased understanding of
ꢀ
uPA and a sulfonyl oxygen with a distance of 3.2 A, but
this interaction is missing for all of the other serine
proteases, and not just tPA. Thus, it seems evident that
selectivity changes in this region depend on more subtle
factors.
A last point relating to binding in Figure 1b is the ori-
entation of the inhibitor cores. Throughout our studies,
we assumed that substituent effects at the 6- and
8-positions would be additive, and further, that indi-
vidual segments of a single substituent would have
similarly additive effects. Our binding data confirmed
this to within experimental error; more convincingly,
our collected X-ray structures attest to the substantial
invariance of inhibitor binding conformations. A further
illustration is shown as Figure 2, where the crystal
structures of 1b and 23b bound to uPA are superim-
Figure 2. Compound 1b (gray) and 23b (pink) overlaid. H57, D60,
D189, Q192, and S195 are shown in thick bonds. The surface of the S1
pocket is shown.

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effective inhibitors.
5. Nienaber, V. L.; Davidson, D.; Edalji, R.; Giranda, V. L.;
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Walter, K.; Wang, J.; Wendt, M.; Weitzberg, M.; Zhao,
X.; Rockway, T. Structure 2000, 8, 553.
6. (a) Rockway, T. W.; Nienaber, V.; Giranda, V. L. Curr.
Pharm. Des. 2002, 8, 2541; (b) Rockway, T. W.; Giranda,
V. L. Curr. Pharm. Des. 2003, 9, 639.
7. Edgar, K. J.; Falling, S. N. J. Org. Chem. 1990, 55, 5287.
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9. Boere, R. T.; Oakley, R. T.; Reed, R. W. J. Orgmet. Chem.
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