Synthesis of thiophene-2-carboxamidines containing 2-aminothiazoles and their biological evaluation as urokinase inhibitors

Article abstract of DOI:10.1016/S0960-894X(01)00102-0

The serine protease urokinase (uPa) has been implicated in the progression of both breast and prostate cancer. Utilizing structure based design, the synthesis of a series of substituted 4-[2-amino-1,3-thiazolyl]-thiophene-2-carboxamidines is described. Further optimization of this series by substitution of the terminal amine yielded urokinase inhibitors with excellent activities.

Full text of DOI:10.1016/S0960-894X(01)00102-0

Bioorganic& Medicinal Chemistry Letters 11 (2001) 915–918
Synthesis of Thiophene-2-carboxamidines Containing 2-Amino-
thiazoles and their Biological Evaluation as Urokinase Inhibitors
Kenneth J. Wilson,* Carl R. Illig, Nalin Subasinghe, James B. Hoffman,
M. Jonathan Rudolph, Richard Soll, Christopher J. Molloy, Roger Bone, David Green,
Troy Randall, Marie Zhang, Frank A. Lewandowski, Zhao Zhou, Celia Sharp,
Diane Maguire, Bruce Grasberger, Renee L. DesJarlais and John Spurlino
3-Dimensional Pharmaceuticals, Inc., 665 Stockton Drive, Exton, PA 19341, USA
Received 4 December 2000; accepted 7 February 2001
Abstract—The serine protease urokinase (uPa) has been implicated in the progression of both breast and prostate cancer. Utilizing
structure based design, the synthesis of a series of substituted 4-[2-amino-1,3-thiazolyl]-thiophene-2-carboxamidines is described.
Further optimization of this series by substitution of the terminal amine yielded urokinase inhibitors with excellent activities.
# 2001 Elsevier Science Ltd. All rights reserved.
The progression of cancer cell invasion and metastasis is
hinged on the ability of tumor cells to produce and
recruit proteolytic enzymes. Although not well under-
stood, there is a growing body of evidence that the
plasminogen activation enzyme urokinase (uPA) and its
receptor (uPAR) exert a major role in the pathogenesis
of cancer.1À4 Thus, the correlation of metastasis with
increased levels of uPA and uPAR has initiated research
into inhibition of the plasminogen activation system as
a novel therapeutic target for cancer.5
could be increased by aryl or heterocycle substitution at
the 4-position of the thiophene ring. For example,
compound 7 was more potent with a Ki=3.90 mM.
Molecular modeling of the Eisai compounds and com-
pound 7 further supported the hypothesis that substitu-
tion at the 4-position of the thiophene would be the
most advantageous for further lead optimization. We
envisioned that substitution from the 2-aminothiazole
of the synthetically accessible thiophene 7 would enable
us to access the same proximal binding pocket as the 4-
substituted Eisai series.11 In fact, a recent disclosure of
an Eisai B428-uPA crystal structure clearly shows the
iodo-substitution vector of Eisai B428 is directed
towards the proximal pocket (referred to as S1b, see ref
6). The elaboration of the lead compound 7 is described
within.
Recently, two publications have appeared that report
the X-ray crystallographic structure of variants of uPA
bound to small molecule inhibitors.6,7 Because uroki-
nase is a trypsin-like serine protease, the majority of
uPA inhibitors reported to date are based upon either
amidine or guanidine functionality, which form a salt
bridge with the Asp189 in the S1-binding pocket of
uPA.8À10 A series of uPA inhibitors based upon a ben-
zothiophene scaffold was prepared by researchers at
Eisai.9 Those substituted at the 4-position, such as the
Eisai B428 iodo-derivative gained notable in vitro
(Ki=0.32 mM) activity (Fig. 1). Screening of amidine
libraries showed 2-amidino-5-thiomethyl thiophene to
be an active (Ki=6 mM) uPA inhibitor, whose potency
Figure 1. The structures of the Eisai benzothiophene amidine and
scaffold 4-(2-amino(1,3-thiazol-4-yl))-5-methylthiothiophene-2-car-
boxamidine 7.
*Corresponding author. Fax: +1-610-458-8249; e-mail: wilson@
3dp.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00102-0

916
K. J. Wilson et al. / Bioorg. Med. Chem. Lett. 11 (2001) 915–918
The highly substituted thiophene nucleus was prepared
in one step by the condensation of dinitrile 1 with
methylthioglycolate 2 to afford the multi-functionalized
aminothiophene 3 (Scheme 1).12 Deamination of the
free amino group with isoamyl nitrite in DMF solution
at 70 ꢀC cleanly gave the properly substituted precursor
4. After some experimentation, a hydrolysis of the
nitrile to the carboxylate 5, which left the ester func-
tionality intact, was achieved using the procedure of
Gribble and co-workers.13 The acid 5 could be
converted to the a-bromoketone 6 by a modified
diazotization–bromination procedure developed in our
laboratories.14 The requisite aminothiazoles were then
synthesized by reacting compound 6 with the appro-
priate 1-substituted or 1,1-disubstituted thiourea (pre-
pared via their isothiocyanates) using standard Hantzch
conditions.15À17 It is noteworthy that the thiazole
products are isolated as their hydrobromide salts by
simple filtration and no chromatographic purification is
required for any step in this synthesis. Finally, the con-
version of the esters to the amidines 7–47 were accom-
plished with the NH4Cl/AlMe3 reagent18 in parallel
fashion employing a Fisher Scientific dry bath incubator
equipped with an aluminum heating block (Scheme 2).
The replacement of the thiomethyl by methyl in the
thiophene derivatives 8, 13, 14, and 44 was also per-
formed for SAR comparison using chemistry recently
communicated from our laboratories.19
The effect of an aliphatic spacer between the amino
thiazole and either an aliphaticor an aromatictermini
was examined (see Table 1).20 While the morpholine and
piperidine derivatives 9 and 10 enhance the activity
slightly, aliphatictethers between the aminothiazole and
aryl groups result in significant improvement (see 11
and 12). The unsubstituted methyl derivative 8 shows a
moderate increase in potency over its S-methyl coun-
terpart 7. The less bulky phenyl derivative 13 is a more
active inhibitor than the trimethoxyphenyl compound
14. An improvement in activity is also achieved relative
to compound 7 by increasing the steric bulk of the
proximal substituent, such as in diphenylmethyl deriva-
tive 15.
Encouraged by these results, a library of alkyl, halogen,
and heteroatom substituted phenyl derivatives 16–33
was synthesized and assayed against urokinase (Table 2).
It is immediately apparent that single aryl substitution
on the aminothiazole results in a series of compounds
with nanomolar activity. Only compounds 20, 27, 29,
and 31 possessed measured Ki values of greater than
1 mM, but all still more potent than 7. A wide range of
substitution and functionality is tolerated, as well as
sterically demanding substrates such as 25, 31, and 32,
although the trimethoxyphenyl substituent is more
active in the thiomethyl series (compare compound 32
to the methyl derivative 14).
Scheme 1. Synthesis of thiophene nucleus. (a) triethylamine, MeOH;
(b) isoamyl nitrite, DMF, 70 ꢀC; (c) neat, 160 ꢀC, 72 h.
Scheme 2. (a) Oxalyl chloride, CH2Cl2; (b) TMSCHN2, CH3CN, 30%
HBr–acetic acid; (c) thiourea, acetone, 70 ꢀC; (d) NH4Cl, AlMe3,
toluene, 118 ꢀC.
Table 2. Measured uPA inhibition of substituted aryl aminothiazole
thiophene
Table 1. Measured uPA inhibition of alkyl-aryl substituted thiophene-
2-aminothiazoles
Compd
Aryl group
Kia Compd
Aryl group
Ki
16
17
18
19
20
21
22
23
24
Phenyl
3-Methoxy
4-Methoxy
4-Benzyloxy
2-Chloro
0.20
0.28
0.28
0.18
1.46
0.24
25
26
27
28
29
30
31
32
33
2-Isopropyl
2-Bromo
3-Oxyacetamide 1.02
2,5-Dimethoxy
2,6-Dichloro
2-Bromo-4-methyl 0.43
2,4,5-Trimethyl 1.10
0.70
0.28
Compd
R Group
Z Group
Ki (mm)
7
8
9
10
11
12
13
14
15
H
H
SCH3
CH3
3.90
1.62
1.40
1.60
0.64
0.35
0.17
1.11
1.18
0.66
1.5b
À(CH2)2-morpholine
À(CH2)2-piperidine
ÀCH2-4-tolyl
À(CH2)3-phenyl
Phenyl
3,4,5-Trimethoxyphenyl
ÀCH(C6H5)2
SCH3
SCH3
SCH3
SCH3
CH3
2-Fluoro
2-Methyl-4-chloro 0.28
2,3-Dimethyl 0.28
2-Methyl-3-chloro 0.44
3,4,5-Trimethoxy 0.16
4-Dimethylamino 0.31
CH3
SCH3
aActivity is expressed in micromolar units.
bCompound 29 possessed only limited solubility in DMSO.

K. J. Wilson et al. / Bioorg. Med. Chem. Lett. 11 (2001) 915–918
917
Table 3. Measured uPA inhibition of bicyclic substituted thiophene-
2-aminothiazoles
aminothiazole as a proximal binding pocket scaffold in
that primary, secondary, and tertiary 2-aminothiazoles
all show uPA inhibitory activity.
In conclusion, it has been demonstrated that the lead
compound 7 is an effective template for probing SARs of
the proximal binding pocket of urokinase. A significant
gain in potency can be realized by both aryl and alkyl
substitution from the 2-aminothiazole group. These
compounds can be prepared utilizing parallel synthetic
methods starting from compound 6. Also, replacement
of the thiophene S-methyl with a methyl group is well
tolerated. Presumably, the methyl and S-methyl deriva-
tives bind very similarly within the S1-binding region.
The use of larger, more rigid hydrophobicpharmaco-
phores gave the best activities. The increase in potency
observed in our 2-aminothiazole thiophene amidine
series that result from substitution at the thiophene
4-position allows a compelling argument to be made
that the proximal binding pocket can be exploited for
further inhibitor optimization.
Compd R Group: Z=SMe Ki Compd R Group: Z=SMe Ki
(mM)
(mM)
34
35
36
0.06
0.11
0.07
40
41
42
0.40
0.91
0.52
37
38
39
0.18
0.18
0.11
43
44
0.50
0.08
R Group: Z=Me
References and Notes
Table 4. Measured uPA inhibition of piperizinyl substituted thio-
phene-2-aminothiazoles
1. Reuning, U.; Magdolen, V.; Wilhelm, O.; Fisher, K.; Lutz,
V.; Graeff, H.; Schmitt, M. Int. J. Oncol. 1998, 13, 893.
2. Edwards, D. R.; Murphy, G. Nature 1998, 394, 527.
3. For an excellent review: Schmitt, M.; Wilhelm, O. G.;
Reuning, U.; Kruger, A.; Harbeck, N.; Lengyel, E.; Graeff,
H.; Gansbacher, B.; Kessler, H.; Burgle, M.; Strzebecher, J.;
Sperl, S.; Magdolen, V. Fibrinolysis Proteolysis 2000, 14, 114.
4. Hjertner, O.; Qvigstad, G.; Hjorth-Hansen, H.; Seidel, C.;
Woodliff, J.; Epstein, J.; Waage, A.; Sundan, A.; Borset, M.
Br. J. Haematol. 2000, 109, 815.
Compd
R Group
Ki (mm)
5. Magill, C.; Katz, B. A.; Mackman, R. L. Emerging Ther-
apeutic Targets 1999, 3, 109.
45
46
47
3-Methoxy
4-Methoxy
2-Fluoro
1.84
0.48
3.06
6. Nienaber, V. L.; Davidson, D.; Edalji, R.; Giranda, V. L.;
Klinghofer, V.; Henkin, J.; Magdalinos, P.; Mantei, R.; Mer-
rick, 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.
7. Zeslawska, E.; Schweinitz, A.; Karcher, A.; Sondermann,
P.; Sperl, S.; Sturzebecher, J.; Jacob, U. J. Mol. Biol. 2000,
301, 465.
8. Geyer, A. G.; McClellan, W. J.; Rockway, T. W.; Stewart,
K. D.; Weitzberg, M.; Wendt, M. D. Urokinase Inhibitors,
WO 99/05096, 1998.
9. Towle, M. J.; Lee, A.; Maduakor, E. C.; Schwartz, E.;
Bridges, A. J.; Littlefield, B. A. Cancer Res. 1993, 53, 2553.
10. Sturtzebecher, J.; Vieweg, H.; Steinmetzer, T.; Schweinitz,
A.; Stubbs, M. T.; Renatus, M.; Wikstrom, P. Bioorg. Med.
Chem. Lett. 1999, 9, 3147.
11. Preliminary disclosure: Wilson, K. J.; Illig, C. R.; Sub-
asinghe, N.; Hoffman, J. B.; Rudolph, M. J.; Soll, R.; Molloy,
C.; Bone, R.; Green, D.; Randall, T.; Zhang, M.; Lewan-
dowski, F.; Zhou, Z.; Sharp, C.; Maguire, D.; Grasberger, B.;
DesJarlais, R. L. Synthesis of Thiophene-2-carboxamidines
Containing 2-Aminothiazoles and Their Biological Evaluation
as Urokinase Inhibitors, 219th National Meeting of the
American Chemical Society, San Francisco, CA, March 26–
30, 2000; American Chemical Society: Washington, DC, 2000;
MEDI 234.
It seemed evident from these results that considerable
stericflexibility and therefore a variety of sacffolds
could be tolerated in the proximal binding region. We
next examined the effect of biaryl ether substituents and
other bicyclic proximal pocket pharmacophores (Table 3).
An improvement in activity is seen for this class of
compounds. Except for compound 41, all of the com-
pounds in this group displayed Ki values of 0.52 mM or
less with the most active compound being biaryl ether
34 (Ki=0.06 mm). The methyl derivative 44 did not show
a significant difference from 34 in activity. The boost in
potency of these compounds in relation to the simple
phenyl derivative 13 may be attributable to a more
extended, rigid conformation that allows better van der
Waals surface contacts with the proximal binding
pocket of urokinase.
Several piperazine derivatives 45–47 were also prepared
and tested (Table 4). Of the three compounds, only the
ortho-fluoro derivative 47 does not show significant
improvement over the lead compound 7. This last set
of compounds demonstrates the versatility of the
12. Tominaga, Y.; Luo, J.-K.; Castle, R. N. J. Heterocycl.
Chem. 1994, 31, 771.

918
K. J. Wilson et al. / Bioorg. Med. Chem. Lett. 11 (2001) 915–918
13. Rounds, W. D.; Gribble, G. W. Tetrahedron Lett. 1988,
29, 6557.
6.99–7.03 (m, 4H), 7.37–7.43 (m, 4H), 9.65 (s, 1H); MS (ESI)
m/z calcd for C13H11ClN2OS, 279.03 (M+H), found 279.4.
17. Hantzch, A. R.; Weber, J. H. Ber. 1887, 20, 3118.
18. Garigipati, R. S. Tetrahedron Lett. 1990, 31, 1969.
19. Rudolph, M. J.; Wilson, K. J.; Illig, C. R.; Subasinghe,
N.; Hoffman, J. B.; Soll, R.; Molloy, C.; Bone, R.; Green, D.;
Randall, T.; Zhang, M.; Lewandowski, F.; Zhou, Z.; Sharp,
C.; Maguire, D.; Grasberger, B.; DesJarlais, R. L. Structure
Based Design and Synthesis of 4,5-Disubstituted-thiophene-2-
amidines as Potent Urokinase Inhibitors, 219th National
Meeting of the American Chemical Society, San Francisco, CA,
March 26–30, 2000; American Chemical Society: Washington,
DC, 2000; MEDI 233.
20. Human kidney cell urokinase was purchased from Sigma
Chemical Co. (St. Louis, MO). In a typical Ki determination,
into each well of a 96-well plate is pipetted 280 mL of N-CBz-
Val-Gly-Arg-p-nitroanilide solution, 10 mL of the test com-
pound solution, and the plate allowed to thermally equilibrate
at 37 ꢀC in a molecular devices plate reader for >15 min.
Reactions were initiated by the addition of a 10 mL aliquot of
enzyme and the absorbance increase at 405 nm is recorded for
15 min. Data corresponding to less than 10% of the total
substrate hydrolysis were used in the calculations. The ratio of
rate of change in absorbance as a function of time for a sample
containing no test compound is divided by the velocity of a
sample containing test compound, and is plotted as a function
of test compound concentration. The data are fit into a linear
regression, and the value of the slope of the line calculated.
The inverse of the slope is the experimentally determined Ki
value.
14. Illig, C. R.; Subasinghe, N.; Hoffman, J. B.; Rudolph, M.
J.; Wilson, K. J.; Soll, R.; Molloy, C.; Bone, R.; Green, D.;
Randall, T.; Zhang, M.; Lewandowski, F. A.; Zhou, Z.;
Sharp, C.; Maguire, D.; Grasberger, B.; DesJarlais, R. L.
Solution-Phase Libraries for the Synthesis of Thiazolylthio-
phene-5-carboxamidines: Preparation of Urokinase Inhibitors,
219th National Meeting of the American Chemical Society,
San Francisco, CA, March 26–30, 2000; American Chemical
Society: Washington, DC, 2000; MEDI 78.
15. Uher, M.; Jendrichovsky, J. Coll. Czech 1973, 29, 289.
16. A representative synthesis of a non-commercially avail-
able thiourea (4-(4-chlorophenoxy)-phenylthiourea), precursor
for compound 35 is as follows: To a 20 mL biphasicmixture of
CHCl3/satd NaHCO3 (1:1, v/v) was added 4-phenoxy-4-
chloroaniline hydrochloride (520 mg, 2.03 mmol). Thio-
phosgene (185 mL, 2.43 mmol) was then added dropwise as a
5 mL solution in CHCl3 via an addition funnel. The mixture
was stirred vigorously for 30 min, at which time the layers
were separated. The CHCl3 layer was washed with brine
(2Â10 mL), dried (Na2SO4) and concentrated in vacuo to give
416 mg (78%) of 4-phenoxy-4-chlorophenylisothiocyanate as
a nearly colorless oil. The oil was dissolved in 3 mL of MeOH
and then treated with 3 mL of a 2 M solution of NH3 in
MeOH. The flask was sealed and allowed to stir for 12 h, at
which time the flask was cooled in an ice-bath, and the off-
white precipitate was filtered, washed with ether, and dried to
afford 326 mg (79%, 58% over 2 steps) of the title compound
as a white, crystalline solid. 1H NMR (DMSO-d6, 300 MHz) d