Naphthamidine urokinase plasminogen activator inhibitors with improved pharmacokinetic properties

Article abstract of DOI:10.1016/j.bmcl.2004.10.026

A series of non-amide-linked 6-substituted-2-naphthamidine urokinase plasminogen activator (uPA) inhibitors are described. These compounds possess excellent binding activities and selectivities with significantly improved pharmacokinetic profiles versus p

Full text of DOI:10.1016/j.bmcl.2004.10.026

Bioorganic & Medicinal Chemistry Letters 15 (2005) 93–98
Naphthamidine urokinase plasminogen activator inhibitors
with improved pharmacokinetic properties
Milan Bruncko,a,* William J. McClellan,a Michael D. Wendt,a Daryl R. Sauer,a
Andrew Geyer,a Christopher R. Dalton,a Michele A. Kaminski,a Moshe Weitzberg,a
Jane Gong,a Joseph F. Dellaria,a Robert Mantei,a Xumiao Zhao,a Vicki L. Nienaber,b
Kent Stewart,b Vered Klinghofer,a Jennifer Bouska,a Todd W. Rockwaya 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 4 August 2004; revised 6 October 2004; accepted 9 October 2004
Available online 28 October 2004
Abstract—A series of non-amide-linked 6-substituted-2-naphthamidine urokinase plasminogen activator (uPA) inhibitors are
described. These compounds possess excellent binding activities and selectivities with significantly improved pharmacokinetic pro-
files versus previously described amide-linked inhibitors.
Ó 2004 Elsevier Ltd. All rights reserved.
Urokinase plasminogen activator (uPA) is a trypsin
class serine protease that is involved in various biologi-
cal processes. uPA has been shown to mediate cancer
invasion and metastasis by the regulation of extracellu-
lar proteolysis through plasminogen activation.1 Addi-
tionally, recent studies have shown broader
involvement of uPA in processes involving tumour inva-
sion.2 The binding of uPA to its receptor induces cellular
events such as tumourigenesis, cell proliferation, migra-
tion, adhesion and angiogenesis. High expression levels
of uPA, uPAR (uPA receptor) and PAIs (plasminogen
activator inhibitors) typically correlate with poor prog-
nosis in cancer patients.2c,3 The involvement of uPA in
many cancer related cellular pathways makes it an
attractive target for cancer therapy.2b,4
The resulting compounds showed increased binding
affinities against uPA and good selectivities against
other proteases. However, all these compounds were
poorly bioavailable. The amide linker was identified as
a source of a potential metabolic liability,8 contributed
to high crystallinity and poor solubility, which were con-
tributors to poor intestinal absorption. This communi-
cation describes compounds designed to probe this
hypothesis.
R
NH
NH2
H
N
O
1a R = H
1b R = Br
1c R = 3-Furyl
1d R = 2-Pyrimidylamino
We have previously described 6-substituted-phenylam-
ido-2-naphthamidines 1a, and 8-substituted-2-naphtha-
midines as inhibitors of uPA.5 This series was further
elaborated to 6,8-disubstituted-2-naphthamidines 1b–
d.6 Substituents in the 8-position of such analogues
occupied the S1b binding pocket of the uPA active site.7
With the goal of developing an oral agent, an effort was
made to determine, which functional groups might be
adversely affecting bioavailability. The amide-linked
naphthamidines showed unsuitable pharmacokinetic
properties despite the excellent binding activities and
selectivities. Since amidines are generally considered to
possess poor bioavailability, we attempted to replace
the amidine functional group with less basic groups such
Keywords: Urokinase inhibitors; Naphthamidines.
*
Corresponding author. Tel.: +1 847 937 4191; fax: +1 847 935
5165; e-mail: milan.bruncko@abbott.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.026

94
M. Bruncko et al. / Bioorg. Med. Chem. Lett. 15 (2005) 93–98
as amines. However, the resulting compounds were
much less active. Then, we focused our attention on
amide linker replacements. For amide bond isosteres,9
the most appealing linker replacements were alkenes,
alkanes and cyclopropanes whose synthesis has been
described below.
The preparations of the amide replacement naphtham-
idines are shown in Schemes 2 and 3. Esters of
cyanonaphthalenes 5a–c were converted into Horner–
Emmons phosphonates 6a–c in three steps. The ester is
first reduced to the corresponding alcohol utilizing a cal-
cium chloride assisted sodium borohydride reduction in
ethanol. Bromination was accomplished with NBS and
triphenylphosphine followed by Arbuzov reaction to
yield phosphonates 6a–c. (E)-Alkenes 7a–h, 8a and 9a
were prepared via Horner–Emmons–Wadsworth cou-
plings of phosphonates 6a–c with the appropriate alde-
hydes 4a–h.
Dihydroisoquinoline (DHIQ) carboxaldehydes were
synthesized in three steps from bromophenethylamines
2a,b and acyl chlorides (Scheme 1). Conversion of the
resultant amides to bromo-DHIQs 3a–f was accom-
plished via an oxalyl chloride/iron chloride mediated
cyclization followed by HCl hydrolysis of the cyclic
intermediates.10 Bromides 3a–f were trans-metalated
with n-butyl lithium and subsequently quenched with
DMF providing aldehydes 4c–h.
The cyclopropyl linked amidines 19a–f, 20a and 21a
were synthesized as racemic mixtures in two steps by
treating (E)-arylalkenes 7a–f, 8a and 9a with diazometh-
ane under palladium acetate catalysis. We were able to
resolve a mixture of 16d by using chiral HPLC. The fol-
lowing amidination of nitriles 16a–f, 17a and 18a was
executed with excess LiHMDS (5equiv) and subsequent
quench with HCl.
5
H2N
6
a, b, c
Br
Br
N
7
The alkyl linked compounds 13a, 14a and 15a were pre-
pared by Pd/C catalyzed reduction with H2 followed by
standard amidination as described above.
8
2a: 3-Br
2b: 4-Br
R
d
3a 6-Br, R = i-Pr
3b 7-Br, R = i-Pr
3c 7-Br, R = c-Hex
3d 7-Br, R = Ph
3e 7-Br, R= Me
3f 7-Br, R= t-Bu
5
8
6
O
H
The arylalkene nitriles 7a, 8a and 9a were similarly
transformed to the corresponding amidines with excess
LiHMDS (5equiv) followed by quench with HCl.
N
7
4c 6-CHO, R = i-Pr
4d 7-CHO, R = i-Pr
4e 7-CHO, R = c-Hex
4f 7-CHO, R = Ph
4g 7-CHO, R= Me
4h 7-CHO, R= t-Bu
R
8-Pyrimidyl naphthamidines 25, 26, 27 were prepared by
a modified route (Scheme 3) starting with the protection
of 8-aminonaphthalene 5d6 with Cbz–Cl. The methyl
ester was converted to aldehyde 22 in three steps begin-
ning with an ester saponification with LiOH, followed
Scheme 1. Reagents and conditions: (a) RC(@O)Cl, Et3N; (b) (COCl)2,
FeCl3, chloroform; (c) HCl; (d) BuLi, DMF, THF, À78°C.
R
R
R
CN
CN
CN
d
O
P
a,b,c
7a-h R = H, Ar
EtO
EtO
8a
9a
R = Br, Ar = Ph
MeO2C
Ar
R = 3-Furyl, Ar = Ph
5a R = H
5b R = Br
6a R = H
6b R = Br
5c R = 3-Furyl
6c R = 3-Furyl
g
e
f,e
R
NH
NH2
R
R
NH
NH2
R
NH
NH2
e
CN
Ar
Ar
16a-h R = H, Ar
Ar
Ar
19a-f R = H, Ar
13a R = H, Ar = Ph
14a R = Br, Ar = Ph
15a R = 3-Furyl, Ar = Ph
10a R = H, Ar = Ph
11a R = Br, Ar = Ph
20a
21a
R = Br, Ar = Ph
17a R = Br, Ar = Ph
R = 3-Furyl, Ar = Ph
18a R = 3-Furyl, Ar = Ph
12a R = 3-Furyl, Ar = Ph
O
N
N
N
N
Ar =
N
-f
-a
-b
-c
-d
-e
-g
-h
N
Scheme 2. Reagents and conditions: (a) NaBH4, CaCl2, EtOH; (b) PPh3, NBS, dichloromethane; (c) P(OEt)3, 130°C; (d) LiHMDS, ArCHO 4a–h,
THF; (e) LiHMDS, THF, HCl; (f) Pd/C, H2, MeOH; (g) CH2N2, Pd(OAc)2, THF, ether.

M. Bruncko et al. / Bioorg. Med. Chem. Lett. 15 (2005) 93–98
95
N
NH2
NHCbz
NHCbz
N
NH
g,h
CN
CN
CN
CN
e,f
a,b,c,d
H
MeO2C
Ph
O
5d
22
23
24
j,i
k,i
i
N
N
N
N
NH
NH
NH2
N
NH
NH
NH2
N
NH
NH
NH2
27
26
25
Scheme 3. Reagents and conditions: (a) Cbz–Cl, i-PrNEt2, THF; (b) LiOH, dioxane, H2O; (c) (COCl)2, THF; (d) LiAl(OtBu)3H, THF, À78°C;
(e) Ph3PCH3Br, NaHMDS, THF; (f) PhN2BF4, Pd2(dba)3, DMF; (g) Ba(OH)2, DME, 70°C; (h) 2-Br-pyrimidine, Pd2(dba)3, NaOtBu, toluene;
(i) AlMe3, NH4Cl, toluene; (j) CH2N2, Pd(OAc)2, THF, ether; (k) Pd/C, H2, MeOH.
by formation of the acid chloride with oxalyl chloride,
and reduction to aldehyde 22 with LiAl(OtBu)3H at
À78°C. The formation of alkene 23 was achieved via a
two-step process, a Wittig olefination to provide the
vinyl intermediate followed by Pd-catalyzed coupling
with phenyldiazonium species. The Cbz group was
cleaved with Ba(OH)2 in DME at 70°C. The pyrimidyl
group was introduced under Buchwald amination condi-
tions with 2-bromopyrimidine and 8-aminonaphthalene
to yield alkene 24.
From previous studies, it was known that the amide lin-
ker is quite rigid and the NH participates in a water
mediated hydrogen bond to Ser214.5 By implementing
carbon–carbon replacements, we would lose this interac-
tion and possibly some activity, however, this might be
acceptably offset by an increase in the oral bioavailabil-
ity of the compounds. The alkene linker might be
expected to be most structurally similar to an amide
linker since it has sp2-hybridized atoms, thus the orien-
tation and vectors of aromatic substituents would be
in close proximity. In fact, we found from the X-ray
(E)-Arylalkene 24 was converted to the corresponding
arylcyclopropane and arylalkane in the same fashion
as described above (Scheme 2). All nitriles in the pyrim-
idyl series were transformed to amidines 25, 26 and 27
by treatment with AlMe3 and NH4Cl in toluene.
Finally, racemic tetrahydroisoquinolines (THIQ) 29a–d
were obtained via a one pot imine reduction/reductive
amination of the corresponding DHIQ (16d,e,g,h) with
formaldehyde and followed by standard amidine forma-
tion with LiHMDS (Scheme 4).
CN
a
CN
R
R
N
N
b
e
16g R = M
28a R = Me
28b R = t-Bu
28c R = i-Pr
28d R = c-Hex
16h R = t-Bu
16d R = i-Pr
16e R = c-Hex
NH
NH2
R
N
Figure 1. Crystal structure of compound 19a (pink, cyclopropyl link,
PDB entry 1U6Q) bound to urokinase is shown overlaid with
compound 1a (grey, amide link, PDB entry 1OWE). His57, Asp189,
Gln192 and Ser195 are shown in thick bonds. The surface of the S1
pocket is shown. The inset shows a close-up view of the cyclopropyl/
amide overlay.
29a R = Me
29b R = t-Bu
29c R = i-Pr
29d R = c-Hex
Scheme 4. Reagents and conditions: (a) NaBH3CN, CH2O, MeOH;
(b) LiHMDS, THF, HCl.

Table 1. Phenyl substituted naphthamidines with variable linker
R
NH
NH2
X
t1/2 (h) Clb (L/hkg) Vdb (L/kg) AUCb (lMh) F d (%)
b
Compd
X
R
Kia (lM)
Plasmin
uPA
tPA
Thrombin
Kallikrein
Trypsin (porcine)
H
N
1a
H
H
0.631 0.086 31.8 17.8(9) 5.64 2.16(9) 1.95 0.58(9) 2.50 1.14(9) 0.325 0.175(8)
2.1e
2.8
8.5
8.2
3
O
O
c
—
c
—
c
—
c
10a
1.20
34.564
69.11
5.269
1.89
1.216
3.523
0.991
2.482
0.346
0.720
—
19a
13a
H
H
1.31
7.4e
1.0
2.16
6.75
23.0
9.8
11.6
1.14
30
60
c
—
c
c
—
c
—
c
—
3.183
—
H
N
1b
Br
Br
0.281
0.820
5.59
2.655
1.015
0.992
0.064
1.05
12.3
18.7
0.6
0
c
—
c
—
c
—
c
—
c
11a
13.38
0.92
1.35
0.77
0.480
0.334
—
20a
14a
Br
Br
0.622
1.37
30.783
0.503
0.239
0.379
4.71
3.3
2.1
3.8
14.3
18
2.72
1.75
17
c
—
c
c
—
c
—
c
—
—
>32
H
N
1c
3-Furyl
0.091 0.011
0.176
4.465
0.132
0.237
0.321
0.134
0.265
0.083
0.045
0.096
3
5.0
21.7
5
0
O
12a
21a
15a
3-Furyl
3-Furyl
0.168
0.198
0.731
0.62
4.2
1.3
1.2c
14.5
17.6
5.8c
2.0
0
10
18
c
—
c
c
—
c
—
c
—
—
2.4
c
—
c
c
—
c
—
c
—
3-Furyl
—
3.31
1.53
1.83
H
N
N
1d
25
27
26
0.020
0.810
>50
0.170
0.395
0.048
0.191
0.090
0.277
0.243
0.570
0.100
0.042
0.054
0.156
0.040
0.084
0.054
0.167
0.6
42.9
37.1
0.14
0
N
N
N
N
NH
O
N
N
N
c
—
c
—
c
—
c
—
c
0.010
—
NH
NH
NH
0.005
5.049
5.786
0.33
21.1
4.7
10.0
13.6
0.23
4.2
0
0
0.047 0.001
2e
a
Standard deviation (number of determinations if more than two). All determinations are triplicate values.
b Half life, clearance, volume distribution and area under the curve (AUC) in Sprague Dawley rat, IV dosing 3mg/kg, vehicle 10% DMSO, 20% EtOH, 27.5% PEG400, 42.5% saline.
c Not tested.
d Oral bioavailability in rat, dosing 10mg/kg.
e IV dosing 10mg/kg.

M. Bruncko et al. / Bioorg. Med. Chem. Lett. 15 (2005) 93–98
Table 2. SAR and oral bioavailability for cyclopropyl linked naphthamidines
97
NH
NH2
Ar
Clb
Vdb
AUCb F d (%)
b
Compd
Ar
Kia (lM)
t1/2
(h)
(L/hkg) (L/kg) (lMh)
uPA
tPA
Thrombin Plasmin Kallikrein Trypsin
(porcine)
O
19b
19c
0.263
21.924 1.099
2.454
1.402
0.141
5.91
2.26
19.3
7.9d
3.48
23
c
—
c
c
—
c
—
c
—
0.33 16.4d
0.27d
0d
N
0.610 0.045
0.139 0.006
—
19d
4.74
2.139
1.79
1.046
1.48
0.525
0.64
0.053
0.03
1.33
9.67
18.6
0.51
26
—
—
N
N
N
c
c
—
c
c
—
c
c
19d (S,S)
19d (R,R)
0.065 0.005 10.43
—
—
c
—
c
c
—
c
—
c
—
c
—
c
—
c
c
—
0.38 0.01
—
—
19e
0.050
4.017 1.136
0.594
0.955
0.206
0.336
0.428
0.165
0.27 11.49
4.5
0.57
22
N
N
c
—
c
—
c
c
—
c
19f
29a
29b
29c
0.142
7.038 2.124
—
—
c
c
c
c
c
—
c
—
c
—
0.293 0.021
—
—
—
—
0.28
7.28
2.9
0.69
0
N
c
—
c
—
c
—
c
—
c
—
c
c
—
c
0.548
—
—
N
N
0.047
6.23
1.796
1.302
0.811
0.576
0.295
0.090
0.031
0.55 12.95
10.3
18.7
0.37
0.53
55
29d
0.028 0.001 18.943 0.996
1.53
8.49
27
N
a
Standard deviation (number of determinations if more than two). All determinations are triplicate values.
b Half life, clearance, volume distribution and area under the curve (AUC) in Sprague Dawley rat, IV dosing 3mg/kg, vehicle 10% DMSO, 20%
EtOH, 27.5% PEG400, 42.5% saline.
c Not tested.
d Oral bioavailability in rat, dosing 10mg/kg.
data of compounds 1a and 19a11 bound to urokinase
that aryl substituents on the cyclopropane linker overlay
and are oriented closely with substituents on the amide
linker (Fig. 1).

98
M. Bruncko et al. / Bioorg. Med. Chem. Lett. 15 (2005) 93–98
The PK profiles of the compounds with modified linkers
demonstrated significant improvement. In comparison
to the corresponding amides (1a–c), the cyclopropanes
(19a, 20a, 21a) and the alkanes (14a, 15a) had signifi-
cantly lower clearance rates and longer half lives. The
oral bioavailability of alkanes 13a, 14a and cyclopro-
pane 19a were particularly good (30–60%) (Table 1).
In summary, we have developed a new group of uPA
inhibitors by replacing the amide linker in the aryl sub-
stituted naphthamidines with a cyclopropyl moiety.
These novel compounds possess improved pharmaco-
kinetic profiles with good oral bioavailabilities and
extended half lives in rats.
Oral bioavailability decreased with introduction of the
substituent at the 8-position of naphthamidine. 2-Pyrim-
idylamino substituted compounds (1d, 25, 26 and 27)
entirely lacked oral bioavailability. The alkene linked
amidine 12a was not also orally bioavailable with a
shorter half life compared to cyclopropyl 18a or alkyl
15a linked amidines.
Acknowledgements
We thank Dr. Christopher L. Lynch for his help in
preparing this manuscript.
References and notes
Based on Pk data and binding activities we chose the
cyclopropane as the most suitable amide bond replace-
ment. We also eliminated the substituent at the 8-posi-
tion of naphthamidine moiety, since majority of the 8-
substituted compounds with both polar and non-polar
groups lacked oral bioavailability. Our attention was
focused on substituted phenyl analogues (Table 2). Pre-
vious work indicated that this series was likely to provide
compounds with good activities. 3-Isopropoxyphenyl
analogue (19b) is fivefold more active against uPA com-
pared to unsubstituted phenyl analogue. The compound
19b possesses a good half life, lower clearance and is
23% orally bioavailable.
1. (a) Rabbani, S. A.; Xing, R. H. J. Int. Oncol. 1998, 12,
911; (b) Bell, W. R. Semin. Thromb. Hemost. 1996, 22, 459;
(c) Dano, K.; Andreasen, P. A.; Grondahl-Hansen, K.;
Kristensen, P.; Nielsen, L. S.; Skriver, L. Adv. Cancer Res.
1985, 44, 139.
2. (a) Duffy, M. J. Curr. Pharm. Des. 2004, 10, 39; (b)
Reuning, U.; Sperl, S.; Kopitz, C.; Kessler, H.; Kruger,
A.; Schmitt, M.; Magdolen, V. Curr. Pharm. Des. 2003, 9,
1529; (c) Sidenius, N.; Blasi, F. Cancer Metast. Rev. 2003,
22, 205.
3. (a) Andreasen, P. A.; Kjoller, L.; Christensen, L.; Duffy,
M. J. J. Int. Cancer 1997, 72, 1; (b) Duffy, M. J. Clin.
Chem. 2002, 48, 1194; (c) Fernebro, E.; Madsen, R. R.;
Ferno, M.; Brunner, N.; Bendahl, P.; Christensen, I. J.;
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6, 138.
A significant increase in potency was observed by intro-
duction of the DHIQ moiety (19c–f).
5. Wendt, M. D.; Rockway, T. W.; Geyer, A.; McClellan, W.
J.; Weitzberg, M.; Zhao, X.; Mantei, R.; Nienaber, V. L.;
Stewart, K.; Klinghofer, V.; Giranda, V. L. J. Med. Chem.
2004, 47, 303.
6. Wendt, M. D.; Geyer, A.; McClellan, W. J.; Rockway, T.
W.; Weitzberg, M.; Zhao, X.; Mantei, R.; Stewart, K.;
Nienaber, V. L.; Klinghofer, V.; Giranda, V. L. Bioorg.
Med. Chem. Lett. 2004, 14, 3063.
7. Nienaber, V. L.; Davidson, D.; Edalji, R.; Giranda, V. L.;
Klinghofer, V.; Henkin, J.; Magdalinos, P.; Mantei, R.;
Merrick, S.; Severin, J. M.; Smith, R. A.; Stewart, K.;
Walter, K.; Wang, J.; Wendt, M.; Weitzberg, M.; Zhao,
X.; Rockway, T. Structure 2000, 8, 553.
8. The half lives in the rat plasma at pH = 7.4, 37°C for:
19a > 1000min, 1b = 40min, 1c = 51.5min, 1d = 17.2min,
26 > 500min.
The 7-linked DHIQs were more active compared to 6-
linked analogues, so further SAR continued in this ser-
ies. These efforts yielded 1-c-hexyl-substituted analogue
(19f) that possessed 50nM potency against uPA and
22% oral bioavailability. The potency of cyclopropyl
analogues can be possibly increased by resolving a race-
mic mixture or preparing a single enantiomer as was
shown in example 19d. Compound 19d (S,S) enantiomer
is six times more potent against uPA than 19d (R,R).
The THIQ and DHIQ analogues were significantly more
potent than phenyl substituted cyclopropyl naphthamid-
ines (19a,b) however they suffered shorter half lives and
higher clearances.
9. Ahn, J.-M.; Boyle, N. A.; MacDonald, M. T.; Janda,
K. D. Mini-Rev. Med. Chem. 2002, 2(5), 463.
10. Larsen, R. D.; Reamer, R. A.; Corley, E. G.; Davis, P.;
Grabowski, E. J. J.; Reider, P. J.; Shinkai, I. A. J. Org.
Chem. 1991, 56, 6034.
The better substituents at the 1-position of the THIQ
proved to be a secondary branched alkyl group. Methyl
(29a) or t-butyl (29b) were less active. The isopropyl
(29c) and the c-hexyl (29d) analogues were highly bio-
available (F = 55% and 27%) and possessed excellent
potencies (47 and 28nM).
11. Crystallographic data for structures are on file with
CCDC. Deposition numbers: 1a 1OWE; 9a 1U6Q.