Biphenyls as potent vitronectin receptor antagonists. Part 3: Squaric acid amides

Article abstract of DOI:10.1016/j.bmcl.2007.09.039

Vitronectin receptor (α_Vβ₃) antagonists have been implicated as a possible new treatment of restenosis following balloon angioplasty. In this work we investigate a series of novel arginine mimetic scaffolds leading to new insight of

Full text of DOI:10.1016/j.bmcl.2007.09.039

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6151–6154
Biphenyls as potent vitronectin receptor antagonists. Part 3:
Squaric acid amides
Klaus Urbahns,a,* Michael Ha¨rter,a Markus Albers,b Delf Schmidt,c
Beatrix Stelte-Ludwig,d Ulf Bruggemeier,c Andrea Vaupel,a Jo¨rg Keldenich,e
¨
Klemens Lustig,e Hideki Tsujishitaf and Christoph Gerdesg
aInstitute of Medicinal Chemistry, Bayer Healthcare AG, Bayer-Schering Pharma, 42096 Wuppertal, Germany
bCombinatorial Chemistry Group, Central Research, Bayer AG, 51368 Leverkusen, Germany
cInstitute of Lead Discovery, Bayer Healthcare AG, Bayer-Schering Pharma, 42096 Wuppertal, Germany
dInstitute of Protein Therapeutics, Bayer Healthcare AG, Bayer-Schering Pharma, 42096 Wuppertal, Germany
eInstitute of Drug Metabolism and Pharmacokinetics, Bayer Healthcare AG, Bayer-Schering Pharma, 42096 Wuppertal, Germany
fDepartment of Medicinal Chemistry, Bayer Yakuhin, Research Center, Kyoto, Japan
gInstitute of Cardiology and Hematology, Bayer Healthcare AG, Bayer-Schering Pharma, 42096 Wuppertal, Germany
Received 7 August 2007; revised 6 September 2007; accepted 7 September 2007
Available online 14 September 2007
Abstract—Vitronectin receptor (aVb3) antagonists have been implicated as a possible new treatment of restenosis following balloon
angioplasty. In this work we investigate a series of novel arginine mimetic scaffolds leading to new insight of the aVb3/ligand inter-
action. Squaric acid amide 10 is a subnanomolar aVb3 antagonist with improved potency on human smooth muscle cell migration.
Ó 2007 Elsevier Ltd. All rights reserved.
The integrin receptor aVb3 has been implicated in a vari-
ety of vascular-mediated disorders such as restenosis
after percutaneous transluminal coronary angioplasty,
in-stent restenosis, or transplant coronary vasculopa-
thy.1 This receptor binds to proteins and peptides con-
taining a characteristic continuous tripeptide epitope,
also referred to as the arginine–glycine–aspartic acid
(RGD) motif. The recent discovery of non-peptide
RGD mimetics as potent and selective aVb3 inhibitors
represents a hallmark in the understanding of vitronec-
tin receptor pharmacology and function.
Despite their obvious structural differences, both materi-
als share a sulfonamide and a biphenyl moiety as a com-
mon structural feature. Likewise, both compounds
exhibit urea fragments as uncharged arginine mimetics.
In an effort to broaden and compare the structure–activ-
ity relationship of the A series, we synthesized a variety
of arginine bioisosters and investigated their binding
affinity to aVb3.
O
A
OH
In preceding communications, we have described two
novel classes of biphenyl vitronectin receptor antago-
nists exemplified by the a-amino acid A and the b-amino
acid B with potencies of 2.5 and 4 nM, respectively.2,3
H
H
HN
O
O
N
N
S
O
B
H
H
H
N
N
N
Keywords: Vitronectin; Urea; Arginine; Guanidine mimetic; Squaric
acid amide; aVb3; RGD; Restenosis; Biphenyl; Smooth muscle cell.
Corresponding author at present address: AstraZeneca R&D Lund,
SE 22187, Sweden. Tel.: +46 46 33 78 25; fax: +46 46 33 71 19;
e-mail: Klaus.Urbahns@astrazeneca.com
N
O
S
O
O
NH
O
*
OH
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.039

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K. Urbahns et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6151–6154
Table 1. Structure–activity relationship of amide and guanidinium-
substituted biphenyl vitronectin receptor antagonists. Ki values are
medians of 3 dose–response curves
O
R
O
HN
H2N
COOH
X
O
R
HN
O
S
Ia: X= SO2-2,4,6-trimethylphenyl, R=Me
Ib: X= SO2-2,4,6-trimethylphenyl, R=Wang resin
Ic: X= SO2-methyl, R=Me
Compound
R
Ki (nM)
Id: X= SO2-(S)-camphoryl, R=Me
N
H
N
i), j), b)
k), l), b)
k), l), b)
k), l), b)
a), b)
c), b)
1
700
Ia
Ia
Ic
Id
9
Ia
Ia
Ib
Ib
1-4
5, 6
7
O
10-12
13
H
N
d),e)
N
2
3
4
5
6
7
8
400
>2000
650
33
f), g), e)
O
14
8
Scheme 1. Synthesis of biphenyl vitronectin receptor antagonists: (a)
carboxylic acid, di-isopropyl carbodiimide, DMF, rt; (b) LiOH,
dimethoxyethane, water (1 + 1), rt (40–90%); (c) 5H,10H-dipyrrol-
o[1,2-a:1,2-d]pyrazin-5,10-dione, pyridine (for 5) 5H,10H-diimi-
dazo[1,2-a:1,2-d]pyrazin-5,10-dione (for 6), pyridine (4 equiv), THF/
DMF (5:1), rt, 72 h, 70%; (d) HgCl2, 1,3-bis-(tert-butoxycarbonyl)-2-
methyl-2-thiopseudourea, DMF, 12 h, rt; (e) TFA/CH2Cl2 1 h, rt; (f)
thiophosgene, THF, rt, 2 h; (g) ethylenediamine, DMF, 70 °C, 12 h; (i)
3 equiv 3,4-bis(methylthio)-1,2,5-thiadiazole-1-oxide,5 n-propanol,
20 h reflux (40%), then: 10 equiv cyclopropylamine, n-propanol, 2 h,
50 °C (90%); (j) 10 equiv primary amine, ethanol reflux; (k) 5 equiv 3,4-
diethoxy-3-cyclobutene-1,2-dione, n-propanol, 20 h reflux; (l) 10 equiv
primary amine, n-propanol reflux.
H
N
N
O
O
H
N
O
NH
H
N
O
NH
H
N
1
N
All new compounds were synthesized using the central
intermediates Ia–d (Scheme 1).4 The thiadiazole oxide
9 was prepared using the corresponding bis(methyl-
thio)-substituted heterocycle as a building block.5
O
H
N
H2N
11
NH
Table 1 summarizes SAR trends observed in the class of
heterocyclic amides derived from biphenyl A. The pyri-
dine and furan amides 1–4 were only weak inhibitors,
suggesting the importance of A’s distal hydrogen bond
donor for optimal interaction with aVb3.
H
N
H
N
1.3
N
Consistently, the pyrrole and imidazole amides 5 and 6
display higher binding affinities, suggesting a direct
involvement of their heterocyclic NH fragments with
aVb3. In contrast to these two examples from the A-ser-
ies, the corresponding pyrrole and imidazole amides of
the B-series showed much weaker potency probably
indicating the need for an additional, aromatic ring to
attain activity within their structural context.2
The affinity improvement for 8, after incorporation of
an ethylene bridge into 7’s guanidine structure, has been
observed in other series as well.6 Results indicated in
Table 2 show that, whereas the thiadiazole modification
retained activity comparable to 7, the squaric acid amide
10 showed clear subnanomolar potency.
A systematic investigation of a set of squaric acid
amides displayed clear SAR within the series (Table 3).
Compound 7 has been described in an earlier communi-
cation.4 Its guanidinium group might be regarded as an
‘ideal’ atom-to-atom mimetic of the arginine side chain
in the natural RGD motif. The heterocyclic amide 6
exhibited 10-fold higher affinity to aVb3 than 7. Appar-
ently, aVb3 is tolerating the enlarged distance between
the two NH groups within 6. In an effort to take advan-
tage of this observation, we synthesized the thiadiazole
95 and the squaric acid amide 107 assuming that such
moieties would display similar geometries.
The introduction of larger, benzylic substituents into the
R1 position reduced affinity into the double-digit nano-
molar range (11 and 12). Similarly, replacing the trim-
ethylphenyl moiety with
a simple methyl group
reduces affinity (13). Consistent with observations made
earlier, camphoryl residues enhance potency (14).3
The affinities of the urea counterparts of 10, 12, 13, and
14 have been described earlier.3 When we compared the

K. Urbahns et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6151–6154
Table 2. Discovery of squaric acid amides. Ki values are medians of 3
dose–response curves
6153
COOH
O
S
X
HN
O
Compound
X
Ki (nM)
H
N
H
N
A
2.5
O
H
H
N
N
9
18
N
N
S
O
H
N
H
N
10
0.5
O
O
Figure 1. (a) Surface representation of the vitronectin receptor ligand-
binding site, with compounds A (green) and B2 (orange) shown as ball-
and-stick models. The Ca2+ ion in the MIDAS is indicated as a yellow
sphere. Only receptor amino acids involved in ligand binding are
shown (aV: magenta, b3: cyan). (b) Comparison of the docked
structures of urea A (green) and squaric acid 10 (orange).
Table 3. Structure–activity relationship of squaric acid amides as
vitronectin receptor antagonists. Ki values are medians of 3 dose–
response curves
COOH
H
N
H
N
HN
R1
R2
Table 4. Comparison of in vitro parameters for urea A and squaric
acid amide 10
O
O
H
H
H
H
N
Compound R1
R2
Ki (nM)
N
N
N
11
12
13
14
2-Pyridyl–CH2 SO2–2,4,6-trimethylphenyl 11
O
Ph–CH2
c-Propyl
c-Propyl
SO2–2,4,6-trimethylphenyl 25
O
O
SO2–CH3
SO2–(S)-camphoryl
10
0.1
A
10
Ki (aVb3)
2.5 nM
128 nM
85 nM
0.5 nM
47 nM
13 nM
Ki (GPIIbIIIa)2
HEK 2938
SMC2
four corresponding urea/squaric acid amide pairs, we
found that the latter are preferred in all cases by roughly
half an order of magnitude (10: 2.5 nM vs 0.5 nM, 12:
60 nM vs 25 nM, 13: 75 nM vs 10 nM, and 14: 1 nM/
0.1 nM).
390 nM
740 nM
2410-fold
10 nM
420 nM
24,000-fold
Kd (HSA)9
Membrane affinity10
In our previous report we have shown that docking our
compounds into the X-ray structure of aVb3 can assist in
rationalizing SAR trends (Fig. 1a).3 Assuming that
D218 and the Ca2+ ion are main interaction partners
for our molecules, we docked derivatives A and 10 into
aVb3 and realized that 10’s cyclopropyl ring would even
better fit into the hydrophobic cleft defined by D150,
F177, Q180, and T212 of the aV side chain, possibly
explaining its higher potency (Fig. 1b).
ently translates into improved cellular adhesive antago-
nism with aVb3-transfected HEK 293 cells.8
Interestingly, 10 also shows improved activity in terms
of smooth muscle cell (SMC) migration inhibition. The
improvement in this functional response is, to our view,
however too large (39-fold) to be explained solely by im-
proved aVb3 affinity (5-fold). We also determined the
membrane affinities and realized that 10 is about 10-fold
more lipophilic than A.9,10 While other contributing fac-
tors such as the interaction with other aV-integrins can-
not be excluded, we hypothesize that the combination of
improved receptor affinity and higher lipophilicity ac-
counts for the highly potent SMC migration inhibition
of 10.
A direct comparison of several in vitro parameters of 10
and its urea congener A is shown in Table 4. The selec-
tivity to GPIIbIIIa appears to be similar, if not better
for the squaric acid derivative (A: 51-fold, 10: 94-fold).
The improved binding affinity to isolated aVb3 appar-

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K. Urbahns et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6151–6154
Lustig, K. WO 0035864, 2000; Chem. Abstr. 2000, 133,
43809.
5. Amato, J. S.; Karady, S.; Reamer, R. A.; Schlegel, H. B.;
Springer, J. P.; Weinstock, L. M. J. Am. Chem. Soc. 1982,
1375.
When tested for degradation by rat liver microsomes, 10
showed a clearance of 3.9 L/h kg (extrapolated from
in vitro) characterizing this molecule as a high clearance
compound.11 Further, guanidine mimetics are therefore
needed to identify biphenyl vitronectin antagonists
allowing for investigations in vivo.
6. Use of imidazolines (a) Batt, D. G.; Petraitis, J. J.;
Houghton, G. C.; Modi, D. P.; Cain, G. A.; Corjay, M.
H.; Mousa, S. A.; Bouchard, P. J.; Forsythe, M. S.;
Harlow, P. P.; Barbera, F. A.; Spitz, S. M.; Wexler, R. R.;
Jadhav, P. K. J. Med. Chem. 2000, 43, 41; (b) Pitts, W. J.;
Wityak, J.; Smallheer, J. M.; Tobin, A. E.; Jetter, J. W.;
Buynitsky, J. S.; Harlow, P. P.; Solomon, K. A.; Corjay,
M. H.; Mousa, S. A.; Wexler, R. R.; Jadhav, P. K. J. Med.
Chem. 2000, 43, 27.
7. (a) Squaric acid amides have been used as a4 integrin
inhibitors before: Head, J. C.; Porter, J. R.; McKay, C.;
WO 2002-010136; (b) Chem. Abstr. 2002, 136, 167287 and
WO 2002-042264; (c) Chem. Abstr. 2002, 136, 401746
(Celltech).
In summary, we have discovered several novel arginine
mimetics, useful for the development of aVb3 inhibitors.
To the best of our knowledge, the structural elements of
9 and 10 have not been described in the context of aVb3
inhibition hitherto.12 In particular the squaric acid
amide series showed picomolar binding affinity that
translated well into improved functional activity on
SMCs.
8. Affinity for human aVb3 in a cellular context was
determined by measuring the inhibition of the adhesion
of purified human vitronectin to HEK 293 cells transfected
with human aVb3 as described: Kumar, C. S.; James, I. E.;
Wong, A.; Mwang, V.; Field, J. A.; Nuthulaganti, P.;
Conner, J. R.; Eichman, C.; Ali, F.; Hwang, S. M.;
Rieman, D. J.; Drake, F. H.; Gowen, M. J. Biol. Chem.
1997, 272, 16390.
9. The binding constant to human serum albumin (HSA) was
determined by equilibrium dialysis (a) Loidl-Stahlhofen,
A.; Schmitt, J.; Noller, J.; Hartmann, T.; Brodowsky, H.;
Schmitt, W.; Keldenich, J. Adv. Mater. 2001, 13, 1829; (b)
Sebille, B.; Zini, R.; Madjar, C. V.; Thuaud, N.; Tillement,
J. P. J. Chromatogr. 1990, 51, 531.
10. Membrane affinities were determined as described. Briefly,
the reduction of compound concentration after incubation
with liposomal egg yolk lecithin followed by ultracentri-
fugation was measured by HPLC: Loidl-Stahlhofen, A.;
Hartmann, T.; Schottner, M.; Rohring, C.; Brodowsky,
H.; Schmitt, J.; Keldenich, J. Pharm. Res. 2001, 18, 1782.
11. The prediction of hepatic clearance was performed
according to (a) Houston, J. B.; Carlile, D. J. Drug
Metab. Rev. 1997, 29, 891; (b) Briefly, compounds were
incubated (1 lM, 37 °C) with 0.2 mg/ml rat microsomal
protein. Samples were taken at 7 time points within 30 min
to calculate half-lives and extrapolate clearance. Com-
pound A showed an extrapolated clearance of 0.6 l/h/kg in
this in vitro assay.
References and notes
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2. (a) Urbahns, K.; Haerter, M.; Albers, M.; Schmidt, D.;
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analogs of 5 and 6 are examples 25 and 26 (Table 3).
3. Urbahns, K.; Haerter, A.; Vaupel, M.; Albers, M.;
Schmidt, D.; Stelte-Ludwig, B.; Brueggemeier, U.; Gerdes,
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4. For details of the syntheses and the primary assay
procedures see: Urbahns, K.; Haerter, M.; Albers, M.;
Vaupel, A.; Schmidt, D.; Stelte-Ludwig, B.; Gerdes, C.;
12. Recent review on arginine mimetics Masic, L. P. Curr.
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