Small, potent, and selective diaryl phosphonate inhibitors for urokinase-type plasminogen activator with in vivo antimetastatic properties

Article abstract of DOI:10.1021/jm700962j

A set of small nonpeptidic diaryl phosphonate inhibitors was prepared. Some of these inhibitors show potent and highly selective irreversible uPA inhibition. The biochemical and modeling data prove that the combination of a benzylguanidine moiety with a d

Full text of DOI:10.1021/jm700962j

6638
J. Med. Chem. 2007, 50, 6638–6646
Small, Potent, and Selective Diaryl Phosphonate Inhibitors for Urokinase-Type Plasminogen
Activator with In Vivo Antimetastatic Properties
Jurgen Joossens,† Omar M. Ali,† Ibrahim El-Sayed,† Georgiana Surpateanu,† Pieter Van der Veken,† Anne-Marie Lambeir,‡
Buddy Setyono-Han,§ John A. Foekens,§ Anneliese Schneider,4 Wolfgang Schmalix,4 Achiel Haemers,† and Koen Augustyns*,†
Laboratory of Medicinal Chemistry and Laboratory of Medical Biochemistry, UniVersity of Antwerp, UniVersiteitsplein 1,
B-2610 Antwerp, Belgium, Erasmus MC, Josephine Nefkens Institute, Department of Medical Oncology, Dr. Molewaterplein 50,
3015 GE Rotterdam, Netherlands, and Wilex AG, Grillparzestr. 10, 81675 Munich, Germany
ReceiVed August 2, 2007
A set of small nonpeptidic diaryl phosphonate inhibitors was prepared. Some of these inhibitors show potent
and highly selective irreversible uPA inhibition. The biochemical and modeling data prove that the
combination of a benzylguanidine moiety with a diaryl phosphonate ester results in optimized molecules
for derivatizing the serine alcohol in the uPA active site. Selected compounds show significant antimetastatic
effects in the BN-472 rat mammary carcinoma model. We report in this paper a preclinical proof of concept
that selective, irreversible uPA inhibitors could be valuable in antimetastatic therapy.
Introduction
Urokinase plasminogen activator (uPAa), a trypsin-like serine
protease and its receptor (uPAR) are essential proteins in the
proteolytic degradation process involved in cancer invasion and
metastasis. uPA is selectively bound to the membrane-anchored
uPAR and activates plasminogen into plasmin. Plasmin plays
an important role in the breakdown of extracellular matrix
(ECM). It activates several matrix metalloproteases, which in
turn degrade several components of the ECM including fibrin,
laminin, and fibronectin. Proteolytic degradation of this ECM
Figure 1. uPA inhibitor 1 is active in an in vivo tumor model, and
is a key event in tumor invasion and metastasis. Recent data
support uPA/uPAR as a valid therapeutic target.1–3 An amidine-
based, peptide-derived inhibitor (1) reduces the number of
experimental lung metastases in a fibrosarcoma model in mice,4
and two clinical phase I trials with uPA inhibitors 2a (WX-
UK1)5 and its prodrug 2b have been successfully completed
(Figure 1).6 Hence, uPA inhibition can be considered as a
promising new noncytotoxic approach in cancer therapy to
specifically block tumor metastasis in solid cancers.
Most of the published uPA inhibitors are reversible inhibi-
tors.7 Instead, we focus on irreversible uPA inhibition. Irrevers-
ible inhibition may have a stronger impact and will not lead to
mechanism-based toxicity if selective. Moreover, the physi-
ological role of the uPA system can be taken over by tissue
plasminogen activator (tPA). tPA is a related trypsin-like serine
protease which is also able to activate plasminogen but is known
to be mainly involved in fibrinolysis. Indeed, uPA-deficient mice
do not develop spontaneous phenotypic changes. This is also
true for tPA-deficient mice but not for uPA/tPA or plasminogen
deficient animals.8 The development of selective inhibitors for
uPA is hampered by its close resemblance to other trypsin-like
serine proteases. Several inhibitors suffer from poor selectivity
toward tPA, plasmin, or thrombin.9 The latter enzyme is an
inhibitors 2 are in phase 1 clinical trials.
important constituent of the blood coagulation cascade, and
therefore, high selectivity is of utmost importance in the
development of uPA inhibitors.
We recently reported the first irreversible, selective, and
potent diphenyl phosphonate peptidic inhibitors for uPA.10 These
inhibitors were designed using Z-D-Ser-Ala-Arginal 3 (Figure
2) as a lead compound.11 The aldehyde function was substituted
for a diphenyl phosphonate group (4), which is known to react
in a covalent way with the active site serine of serine proteases,
affording a phosphonylated and irreversibly inhibited enzyme.12,13
In a next step the P1 position was optimized by substituting
the arginine moiety for various linear or cyclic guanidinylated
side chains.14 A benzylguanidine afforded the most interesting
compounds (5), providing high selectivity and high potency
toward uPA.10
These inhibitors were further optimized at the P4 position.
Several amide and sulfonamide groups were linked to the
terminal amino group.11,15,16 The best compounds showed
inhibitory activities in the low nM range in specific assay
conditions and remarkable in vitro selectivity indices reflecting
ratios of inactivation rates of 3000 or more toward related
enzymes from the fibrinolytic and blood coagulation systems.
Compounds within series 5 belong to the most potent and
selective uPA inhibitors reported.16 Release of cytotoxic phenol
upon covalent binding to the target protein constitutes a potential
problem in biological settings using diphenyl phosphonates. To
circumvent this problem, a diaryl phosphonate containing a
“safe” phenol, p-acetylaminophenol (paracetamol) was pre-
pared.16
* To whom correspondence should be addressed. Tel.: +32-3-8202703.
Fax: +32-3-8202739 . E-mail: koen.augustyns@ua.ac.be.
†
Laboratory of Medicinal Chemistry, University of Antwerp.
Laboratory of Medical Biochemistry, University of Antwerp.
Erasmus MC, Josephine Nefkens Institute.
Wilex AG.
‡
§
4
aAbbreviations: uPA, urokinase-type plasminogen activator; tPA, tissue-
type plasminogen activator; uPAR, urokinase-type plasminogen activator
receptor; FXa, factor Xa; ECM, extracellular matrix.
10.1021/jm700962j CCC: $37.00
2007 American Chemical Society
Published on Web 12/01/2007

Diaryl Phosphonate Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6639
Figure 4. Structures of di-4-acetamidophenyl phosphonates 8.
Scheme 1. Synthesis of the Inhibitorsa
Figure 2. Development of small nonpeptidic irreversible uPA inhibi-
tors. 1. Conversion of the lead arginal peptide 3 into an irreversible
inhibitor 4. 2. Optimization of selectivity and activity. A benzylguani-
dine is important for activity and selectivity and the R group is important
for selectivity toward uPA (5). 3. Nonpeptidic, small, potent, and
selective uPA inhibitors 6.
a Reagents and conditions: (a) (i) di-tert-butyl dicarbonate, TEA, dioxane;
(ii) Dess-Martin oxidation; (b) benzyl carbamate, triphenyl phosphite,
Cu(OTf)2, CH2Cl2; (c) (i) trifluoroacetic acid; (ii) N,N′-bis(tert-butoxycar-
bonyl)-1-guanylpyrazole, MeCN; (d) (for 12b) H2, Pd/C, MeOH; (e) (i)
R2-Cl, pyridine; (ii) trifluoroacetic acid; (f) trifluoroacetic acid, CH2Cl2.
led to more potent and selective compounds than previously
reported. Also, in this paper we report for the first time in vivo
antimetastatic properties of diaryl phosphonate uPA inhibitors.
Chemistry. The synthesis of diphenyl phosphonates 6 is
shown in Scheme 1. Boc-protected 4-aminophenylacetaldehyde
(10), prepared from the corresponding alcohol (9) with
Dess-Martin periodinane20 was used as starting material. An
amidoalkylation reaction with the corresponding carbamate and
triphenylphosphite, using copper triflate as catalyst, afforded
the diphenyl phosphonates 11b–d.21 After acidolytic removal
of the Boc protecting group, N,N′-bis(tert-butoxycarbonyl)-1-
guanylpyrazole was used to introduce the protected guanidine
group (12b–d). Final compounds 6b-d were obtained after
deprotection with trifluoroacetic acid. Deprotection of 12b under
hydrogenolytic conditions (13) and Boc deprotection resulted
in 6k. Acylation or sulfonylation of 13 and subsequent depro-
tection afforded compounds 6e–j. The di(4-acetamidophenyl)
phosphonates 8 were prepared analogously using tri-4-acetyl-
aminophenylphosphite and TiCl4.
Amidine compounds 7 were synthesized using the corre-
sponding cyanoaldehydes (15, Scheme 2) in an amidoalkylation
reaction, followed by conversion to the amidine with a Pinner
reaction.22,23
Biochemical Evaluation. IC50 values of compounds 6–8 were
determined for uPA and for other representative trypsin-like
serine proteases (Table 1).10,16 The latter are involved in the
blood coagulation cascade and fibrinolysis: tPA, plasmin,
thrombin, and FXa.
Figure 3. Structures of diphenyl phosphonates 6 and 7.
In this paper, we report a further improvement of the drug-
like properties of these compounds. We found that substituting
the peptide tail for a small group (6) did not change the uPA
inhibitory activity nor the selectivity versus other trypsine-like
proteases. The influence of different substituents on the R-amino
group was investigated (Figure 3, 6b–k). The guanidinylated
compounds were compared with their amidine analogues (7a–c).
Finally, some di(4-acetamidophenyl)phosphonates were prepared
(Figure 4, 8a–c). Based on the activity-selectivity profile and
some physicochemical properties, two compounds were selected
for evaluation of their antitumor and antimetastatic properties
in a rat mammary carcinoma model. During the course of our
investigation,17 Oleksyszyn et al. also reported the uPA inhibi-
tory activities of 6a, 6b, and 7a, confirming our uPA inhibition
results.18,19 In contrast to the group of Oleksyszyn, we decided
not to publish preliminary data. Our more elaborated study has
Comparison of compounds 6 with different R-amino substit-
uents shows that the highest potency as uPA inhibitor is obtained
when there is no steric hindrance close to the R-amino group.
Bulky substituents such as in compounds 6h and 6i are not
favorable. The best activity-selectivity profile is observed when

6640 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Scheme 2. Synthesis of Inhibitors 7a–ca
Joossens et al.
of both compounds is more than acceptable, with more than
60% parent compound left after 3 h incubation at 37 °C. Stability
in the stock solutions used in the in vivo assays was confirmed.
LogD7.4 and the solubility also reflect their drug-like properties.
These compounds showed low cytotoxicity in an epithelial cell
line of Brown Norway rats (BN-175, unpublished data).
Compounds 6c and 8b were also tested as inhibitors of rat
and mouse enzymes. Both compounds showed low nM IC50
values (<25 nM) for rodent uPA and µM IC50 values for rodent
plasmin and thrombin. The activity against rodent enzymes
confirmed the suitability of these uPA inhibitors to be used in
vivo in rodent tumor models.
a Reagents and conditions: (a) Cu(OTf)2, CH2Cl2 (b) (i) MeOH/CHCl3
(1:1), HCl(g) (ii) 2 M NH3 in EtOH, n ) 0 to 2.
Efficacy Study in the BN472 Mammary Rat Tumor
Model. The objective of this study was to investigate the
antitumor and antimetastatic efficacy of the irreversible uPA-
inhibitors 6c and 8b in Brown Norwegian rats bearing BN-472
rat mammary tumors. In breast cancer, it is established that high
expression levels of uPA, its cellular receptor (uPAR), and its
inhibitor (PAI-1) support invasion, metastatic spread, and neo-
angiogenesis of tumors and strongly correlate with poor patient
prognosis. The BN-472 metastasizing mammary tumor that
grows in Brown Norwegian rats expresses a significant amount
of uPA, uPAR, and PAI-1 as assessed by real-time RT-PCR
analysis and represents an excellent in vivo model to study the
efficacy of newly designed drugs that interfere in the uPA-
system of plasminogen activation.5
small groups are introduced such as in methylcarbamate 6c,
acetamide 6e, and methylsulfonamide 6j. The free amine (6k)
is nearly 1000 times less-active.
The importance of the diphenyl phosphonate group in this
benzylguanidine series to obtain potent and selective nonpeptidic
inhibitors is highlighted in Figure 5. Omitting the peptide part
in 17,24 a potent and selective inhibitor without serine protease
warhead, decreases the uPA inhibitory activity about 1000-fold
(18).25 On the contrary, comparing the activity of 6b with its
corresponding peptide 5 (R ) benzylsulfonyl)16 shows no
significant decrease in uPA inhibition, and the small inhibitor
is even more selective for uPA. The importance of the
benzylguanidine group is demonstrated upon comparison of 6a
and 6b. A small diphenyl phosphonate inhibitor with ben-
zylguanidine side chain (6b) is about 100-fold more potent and
far more selective than the corresponding compound with an
arginine-like side chain (6a). The observation that the arginal
warhead in 3 is only active in a peptide structure confirms the
importance of our combination of a diphenyl phosphonate with
a benzylguanidine.10,11
As already shown for peptidic inhibitors, the diphenyl
phosphonate ester can be substituted for a di-4-acetamidophenyl
ester without loss of potency. The selectivity profile is somewhat
less favorable but these compounds (8a-c) are still highly
selective. Amidines (7a-c) are less favorable as irreversible
uPA inhibitors.
Compound 6c (UAMC-00150) and its paracetamol analogue
8b (UAMC-00251) were selected as the most promising
compounds for further investigation. Compound 6c shows the
lowest IC50 value and the best selectivity profile. Moreover, the
synthetic route is short, with a good overall yield.
Modeling Study. A molecular modeling study was performed
to offer a molecular basis for the high potency and remarkable
selectivity of the here reported nonpeptidic uPA inhibitors. The
same methodology as already reported was used.16 We presume
that the highly specific interactions of the benzylguanidine in
the S1 pocket place the diaryl phosphonate group at the optimal
position for a covalent reaction with the alcohol of Ser195
(Figure 6). Therefore, the extra interactions observed between
the peptidic part of 5 and uPA are not necessary for a good and
selective interaction with uPA.
Further Evaluation of Compounds 6c and 8b. Compounds
6c and 8b were further evaluated to determine the feasibility of
using them for in vivo evaluation (Table 2). Because these
compounds are designed with an electrophilic phosphonate
warhead for covalent interaction with the serine alcohol in the
active site, their stability might be compromised. However, they
exhibited excellent stability at different pH values, for example,
more than 2.5 days at pH 7.4. As expected, 6c and 8b are less
stable at basic pH, and the instability of the paracetamol
analogue 8b is slightly higher. Furthermore, the plasma stability
Tumors implanted orthotopically were allowed to grow for
three days. Starting on day 4 after tumor inoculation, 6c and
8b were administered i.p. daily for 17 to 18 days at 0.1 and 1
mg/kg. Control animals were treated i.p. with vehicle only. End
points of this study were the evaluation of antitumor effects
(measured by tumor size and tumor weight, Figure 7), antime-
tastatic effects (measured by number of lung foci and weight
of axillary lymph nodes, Figure 8), and side effects (measured
by body weight and weight of several organs).
Daily administration of 6a and 8b at 0.1 mg/kg and 1 mg/kg
resulted in significant inhibition of tumor growth in the range
of 10–18% considering the end point tumor size. The decrease
in tumor weight was 9–27% though the effect in the 8b low-
dose group was statistically not significant. The antitumor effect
was not dose-dependent.
The antimetastatic effect observed by a decrease in the
number of lung foci (29–70%) and the weight of axillary lymph
nodes (46–73%) was statistically significant in all compound
and dose groups except for 0.1 mg/kg of 6c in the end point
axillary lymph node weight. The antimetastatic effect was dose-
dependent in both 6c and 8b groups.
The moderate in vivo antitumor activity with an up to 27%
inhibition in tumor weight falls within the range of 22–56%
observed previously in independent experiments with the uPA-
inhibitor 2a that was applied daily by subcutaneous or intrap-
eritoneal administration in the same tumor model.5 The anti-
metastatic activity of up to 73% was more pronounced and was
also similar to the up to 62% inhibition observed previously
following daily administrations of compound 2a.5 The higher
antimetastatic activity compared to the antitumor activity was
expected based on the function of uPA in tumor invasion and
metastasis. The Spearman rank correlations between primary
tumor weight and the metastatic end points were not statistically
significant (rs) 0.19, n ) 90, for the axial lymph node weight;
rs)0.03, n ) 90, for the number of lung foci), suggesting that
the irreversible uPA-inhibitors independently exert both anti-

Diaryl Phosphonate Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6641
IC50 (µM) or % inhibition at given concentration (µM)
Table 1. Inhibitory Activities against uPA and Related Enzymes
IC50 (nM) uPA
k
app(M-1.s-1) uPA
tPA
plasmin
thrombin
FXa
6a
6b
6c
6d
6e
6f
6g
6h
6i
840 ( 200a
7 ( 2
3.1 ( 0.5
8 ( 3
10 × 101 ( 6 × 101b
42 × 103 ( 3 × 103
62 × 103 ( 4 × 103b
40 × 103 ( 20 × 103b
14 × 105 ( 0.2 × 105
24 × 102 ( 12 × 102b
19 × 103 ( 1 × 103
73 × 102 ( 4 × 102
16 × 102 ( 1 × 102
14 × 104 ( 1 × 104
NA
44 ( 20 (50)c
12.0 ( 0.8 (1700)
23 ( 6 (7000)
57 ( 14 (7000)
36.6 ( 2.2 (3700)
125 (1000)
7.3 ( 0.5 (1000)
44 ( 10 (1600)
16 ( 1 (60)
30 ( 2 (35)
0.78 ( 0.04 (0.9)
2.4 ( 0.4 (300)
17.0 ( 2.3 (5600)
3.8 ( 0.2 (500)
32.2 ( 1.4 (3200)
65% at 125
11.9 ( 1.7 (1500)
8.1 ( 0.8 (300)
60% at 250
70% at 250
100 ( 20 (11000)
57% at 250
62% at 250
37% at 250
>250
3.0 ( 0.6 (300)
13 ( 2 (4300)
16 ( 3 (2000)
57 ( 1.3 (5700)
14 ( 1 (120)
17 ( 2 (2000)
1.2 ( 0.1 (40)
∼125 (500)
7.2 ( 0.6
114 ( 11
7.7 ( 0.7
26.5 ( 1.4
260 ( 30
6.6 ( 0.8
1600 ( 130
60000 ( 10000
67 ( 25
900 ( 80
4.2 ( 0.9
3.4 ( 0.4
3.5 ( 0.5
59% at 62
27 ( 2 (1000)
20 ( 2 (80)
∼250 (40000)
11% at 250
6.0 ( 0.2
65% at 250
13.7 ( 1.3 (15)
100 ( 24 (23000)
30 ( 6 (9000)
55% at 250
6j
8 ( 2 (1000)
32% at 250
11 ( 1 (1600)
31% at 250
∼125 (19000)
6k
7a
7b
7c
8a
8b
8c
41% at 250
43 × 100 ( 6 × 100
20 × 102 ( 1 × 102b
14 × 101 ( 1 × 101
80 × 103 ( 5 × 103b
10.00 × 105 ( 0.03 × 105
21 × 104 ( 1 × 104
66 ( 7 (1)
3.6 ( 0.4
0.288 ( 0.006
20 ( 2 (300)
17 ( 2 (20)
0.39 ( 0.03 (90)
1.13 ( 0.06 (300)
2.35 ( 0.06 (700)
4 ( 2 (60)
1.2 ( 0.1 (20)
6.2 ( 0.4 (7)
0.9 ( 0.2 (200)
2.8 ( 0.5 (800)
2.0 ( 0.4 (550)
68 ( 9 (70)
7 ( 1 (1700)
6.1 ( 0.7 (1800)
3.7 ( 0.4 (1000)
a Standard error on the fit. b Compound showed a two-step mechanism, kapp, calculated as k2/K1. c Selectivity index: IC50/IC50 uPA.
Figure 5. The small irreversible diphenyl phosphonate uPA inhibitor
6b remains nearly equally potent and selective compared to the peptidic
analogues 5 (R ) benzylsulfonyl) and 17. This is not true for the small
reversible uPA inhibitor 18. R ) R-toluenesulfonyl.
tumor and antimetastatic activities and suggesting that the
antimetastatic effects are not secondary to an effect on tumor
growth.
Daily observations during the whole treatment period of 17
to 18 days showed that both compounds were generally well-
tolerated. The body weight and the weight of different organs
Figure 6. Inhibitor 6b modeled in the active pocket of uPA after
reaction with the alcohol of Ser195. (Carbons of the inhibitor are green,
carbons of uPA are gray, nitrogens are blue, oxygens are red,
phosphorus are orange. Ionic and hydrogen bonds are shown with dotted
lines and their distances are shown in Å.) The terminal guanidine ion
is deeply inserted into the S1 pocket, and this guanidine group forms
the typical symmetrical salt bridge to the carboxylate of Asp189.
Furthermore, one nitrogen atom makes a hydrogen bond with the
carbonyl oxygen of Gly219, and another nitrogen atom forms a
hydrogen bond with the alcohol of Ser190. The phenyl ring of the
benzylguanidine is sandwiched between the planes of the amide bonds
of Trp215-Gly216 and Ser190-Cys191 and is placed in a hydrophobic
region of the active site. The phosphonate oxygen binds into the
oxyanion hole, making hydrogen bonds to the backbone NH groups of
Gly193 and Ser195. The R-amino nitrogen interacts with the backbone
carbonyl of Ser214, and the carbonyl oxygen of the carbamate forms
a hydrogen bond with the side chain of Gln192. The phenyl ring of
the remaining phenol is placed in the hydrophobic S1′ pocket.
of the animals were not affected by the treatment.
In conclusion, these results obtained with 6c and 8b show
for the first time that irreversible and selective uPA inhibitors
exert moderate effects on the primary tumor but are highly active
in the inhibition of metastatic development. These experiments
confirm the data obtained with reversible uPA inhibitors4,5,24
and prove the importance of uPA as target in the design of
antimetastatic drugs.
Conclusion
We developed a series of small irreversible uPA inhibitors
from previously reported peptidic diaryl phosphonates. Remark-
ably, removal of the peptidic tail did not influence potency nor
selectivity and resulted in irreversible uPA inhibitors with
nanomolar potency and more than 1000-fold selectivity over
highly related proteases. Two compounds of this series were
further evaluated for their suitability to be used in rodent tumor
models. We have shown for the first time that irreversible uPA
inhibitors exhibit promising antimetastatic properties in a rat
mammary carcinoma model without any signs of acute toxicity.
This further validates uPA as an interesting target in the design
of antimetastatic drugs.
Experimental Section
Reagents were obtained from Sigma-Aldrich or Acros. Charac-
terization of all compounds was done with 1H NMR and mass

6642 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Table 2. Further Evaluation of 6c and 8b
Joossens et al.
property
6c
8b
-0.08
LogD (pH 7.4)
1.1
apparent solubilitya
>200 µM
>2 weeks
>2 weeks
5 days
6 h
63% at 3 h
0.0195
0.032
>200 µM
t1/2, pH 1 (37 °C)
t1/2, pH 4 (37 °C)
t1/2, pH 7.4 (37 °C)
35 h
>2 weeks
64 h
t1/2, pH 8.8 (37 °C)
3 h
plasma stability (37 °C)b
IC50 rat-uPA (µM)
81% at 3 h
0.0245
0.008
2.2
4.8
45
IC50 mouse-uPA (µM)
IC50 rat-plasmin (µM)
IC50 mouse-plasmin (µM)
IC50 rat-thrombin (µM)
IC50 mouse-thrombin (µM)
15
25
>100
42
4.5
a In 2% DMSO in PBS, pH 7.4. b The % parent compound left.
Figure 8. Antimetastatic effects of compounds 6c and 8b are measured
by number of lung foci and axillary lymph node weight in rats bearing
BN472 breast tumors. Compounds 6c and 8b were administered i.p.
daily during days 17–18 at 0.1 and 1 mg/kg, starting on day 4 after
tumor inoculation. Control animals were treated i.p. with vehicle only.
Comparisons of the number of lung foci and the weight of axillary
lymph nodes at the end of the treatment period are presented as Box-
Whisker graphs. The boxes show the median, the 25th and 75th
percentiles as horizontal lines, the whiskers indicate the 5th and 95th
percentile ranges, and the dots represent all the individually observed
values. The unadjusted level for statistical significance was P ) 0.05
when comparing treatment groups vs control (n.s. ) not significant).
A wavelength of 214 nm was used. When necessary, the products
were purified with flash chromatography on a Flashmaster II (Jones
chromatography), with a 30 min gradient of 0–50% EtOAc in
hexane or 0–25% MeOH in EtOAc. The synthesis of compounds
10, 11b, 12b, 13, 6a, 6b, 15a–b, 16a,b, and tri-4-acetamidophenyl
phosphite was already reported.10,12,16 Slightly modified procedures
for 10, 11b, 12b, 13, and tri-4-acetamidophenyl phosphite are placed
in the Supporting Information.
Figure 7. Effects of compounds 6c and 8b on primary tumor size and
weight in rats bearing BN472 breast tumors. Compounds 6c and 8b
were administered i.p. daily during days 17–18 at 0.1 and 1 mg/kg,
starting on day 4 after tumor inoculation. Control animals were treated
i.p. with vehicle only. Comparisons of the tumor sizes and weights at
the end of the treatment period are presented as Box-Whisker graphs.
The boxes show the median, the 25th and 75th percentiles are indicated
as horizontal lines, the whiskers indicate the 5th and 95th percentile
ranges, and the dots represent all the individually observed values. The
unadjusted level for statistical significance was P ) 0.05 when
comparing treatment groups vs control (n.s. ) not significant).
Methyl 1-(Diphenoxyphosphoryl)-2-(4-(tert-butyloxycarbo-
nylamino)phenyl)ethyl-carbamate (11c). To a solution of crude
aldehyde 10 (1 equiv),10,16 triphenyl phosphite (1 equiv), and methyl
carbamate (1 equiv) in 50 mL CH2Cl2, 0.1 equiv Cu(OTf)2 was
added. The solution was stirred at room temperature for 4 h. CH2Cl2
was removed in vacuo. The crude product was dissolved in MeOH
and the solution was kept at 4 °C until precipitation of diphenyl
1
spectrometry. 1H NMR spectra were recorded on a 400 MHz Bruker
Avance DRX-400 spectrometer. ES mass spectra were obtained
from an Esquire 3000plus iontrap mass spectrometer from Bruker
Daltonics. Purity was verified using two diverse HPLC systems
using, respectively, a mass and UV-detector. Water (A) and CH3CN
(B) were used as eluents. LC-MS spectra were recorded on an
Agilent 1100 Series HPLC system using a Alltech Prevail C18
column (2.1 × 50 mm, 3 µm) coupled with an Esquire 3000plus
as MS detector and a 5–100% B, 20 min gradient was used with a
flow rate from 0.2 mL/min. Formic acid 0.1% was added to solvents
A and B. Reversed phase HPLC was run on a Gilson instrument
equipped with an Ultrasphere ODS column (4.6 × 250 mm, 5 µm).
A 10–100% B, 35 min gradient was used with a flow rate from 1
mL/min. Trifluoroacetic acid 0.1% was added to solvent A and B.
phosphonates was complete. Yield 63%; H NMR (CDCl3) δ 1.5
(s, 9H), 3.0–3.4 (m, 2H), 3.1 (s, 3H), 4.7 (m, 1H), 6.5 (s, 1H),
7.1–7.4 (m, 14H); MS (ESI) m/z 549 [M + Na]+.
Ethyl 1-(Diphenoxyphosphoryl)-2-(4-(tert-butyloxycarbonyl-
amino)phenyl)ethyl-carbamate (11d). Same procedure as for 11c.
1
Yield 57%; H NMR (CDCl3) δ 1.3 (t, 3H), 1.5 (s, 9H), 3.0–3.4
(m, 2H), 4.1 (q, 2H), 4.7 (m, 1H), 5.1 (m, 1H), 6.5 (s, 1H), 7.1–7.6
(m, 12H); MS (ESI) m/z 563 [M + Na]+.
Methyl 2-(4-(N,N′-Bis(tert-butyloxycarbonyl)guanidino)phe-
nyl)-1-(diphenoxy-phosphoryl)ethylcarbamate (12c). Compound
11c was dissolved in 50% trifluoroacetic acid in CH2Cl2. After
stirring for 3 h at room temperature, the solvent was evaporated.
The crude oil was washed with cold ether and a precipitate was
isolated. A mixture of the crude phosphonate (1 equiv), N,N′-

Diaryl Phosphonate Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6643
bis(tert-butyloxycarbonyl-1-guanyl-pyrazole (1 equiv), and triethyl-
amine (3 equiv) in chloroform (20 mL) was stirred at room
temperature for 3 days. The solvent was evaporated and the residue
was dissolved in ethylacetate and washed with 1 N HCl, saturated
solution of NaHCO3 and brine. The organic layer was dried over
Na2SO4. The solvent was evaporated and the crude product was
purified by chromatography (0–80% EtOAc in hexane) to obtain
14H). HPLC (214 nm) tr 7.2 min, 91.5%; HPLC (254 nm) tr 7.1
min, 95.1%; LC/MS tr 10.3 min, 93.1%; MS (ESI) m/z 411 [M +
H]+.
Acetylation or Sulfonylation of Compound 13 and Depro-
tection of Intermediates Leading to Compounds 6e-j and 8c.
A suspension of 1310,16 and triethylamine in dichloromethane was
cooled at -30 °C, and the corresponding acetyl chloride or
sulfonylchloride in dichloromethane was added dropwise. After 10
min, the reaction was quenched by adding 1 mL of water and the
organic layer was washed with saturated sodium bicarbonate
solution. After drying on sodium sulfate, the solvent was evaporated
and the product was purified by chromatography; ethyl acetate/
hexane (0f80%).
The intermediate was dissolved in 50% trifluoroacetic acid in
CH2Cl2 (2–5 mL). After stirring for 3 h at room temperature, the
solvent was evaporated. The crude oil was washed with cold ether
and a precipitate was isolated.
1
the protected guanidine. Yield 62%; H NMR (CDCl3) δ 1.5 (d,
18H), 3.0–3.4 (m, 2H), 3.5 (s, 3H), 4.7 (m, 1H), 7.1–7.6 (m, 14H);
HPLC (214 nm) tr 17.2 min, 99.1%; HPLC (254 nm) tr 17.2 min,
100.0%; LC/MS tr 20.8 min, 98.9%; MS (ESI) m/z 691 [M + Na]+.
Ethyl 2-(4-(N,N′-Bis(tert-butyloxycarbonyl)guanidino)phenyl)-
1-(diphenoxy-phosphoryl)ethylcarbamate (12d). Same procedure
1
as for 12c. Yield 63%; H NMR (CDCl3) δ 1.3 (t, 3H), 1.5 (d,
18H), 3.0–3.4 (m, 2H), 4.0 (q, 2H), 4.7 (m, 1H), 5.6 (m, 1H),
7.1–7.6 (m, 14H); HPLC (214 nm) tr 18.0 min, 97.4%; HPLC (254
nm) tr 18.0 min, 100.0%; LC/MS tr 20.9 min, 91.3%; MS (ESI)
m/z 705 [M + Na]+.
Diphenyl 1-Acetamido-2-(4-guanidinophenyl)ethylphospho-
nate Trifluoroacetate (6e). Intermediate diphenyl 1-acetamido-2-
(4-(N,N′-bis(tert-butyloxycarbonyl)guanidino)phenyl)-ethylphos-
Methyl 1-(Bis(4-acetamidophenoxy)phosphoryl)-2-(4-(tert-bu-
tyloxycarbonylamino)phenyl)ethylcarbamate (Paracetamol Ana-
logue of 11c). Acetamidophenyl phosphonates were prepared in
the same way as the diphenyl phosphonates 11, except tri-4-
acetamidophenyl phosphite16 was used instead of triphenyl phos-
phite, MeCN was used as solvent, and TiCl4 as the catalyst. The
product did not precipitate from MeOH and flash chromatography
was necessary (EtOAC/MeOH) to isolate these compounds. Yield
1
phonate. H NMR (CDCl3) δ 1.5 (d, 18H), 1.7 (s, 3H), 3.0–3.4
(m, 2H), 5.1 (m, 1H), 5.9 (NH), 7.1–7.6 (m, 14H), 10.3 (NH), 11.6
(NH); HPLC (214 nm) tr 26.7 min, 100%; HPLC (254 nm) tr 26.7
min, 100%; LC/MS tr 20.5 min, 100.0%; MS (ESI) m/z 675 [M +
Na]+. 6e: Yield 96%; 1H NMR (CDCl3) δ 1.7 (s, 3H), 3.0–3.4 (m,
2H), 5.0 (m, 1H), 6.5 (NH), 7.0–7.5 (m, 14H), 10.0 (NH); HPLC
(214 nm) tr 15.3 min, 100%; HPLC (254 nm) tr 15.3 min, 100.0%;
LC/MS tr 12.2 min, 100.0%; MS (ESI) m/z 453 [M + H]+.
Diphenyl 1-(Benzoylamino)-2-(4-guanidinophenyl)-ethane-
phosphonate Trifluoroacetate (6f). Intermediate (diphenyl 1-(ben-
zoylamino)-2-{4-[N,N′-bis(tert-butyloxycarbonyl)guanidine]phe-
1
39%; H NMR (CDCl3) δ 1.5 (s, 9H), 2.1 (2s, 6H), 2.8–3.3 (m,
2H), 3.3 (s, 3H), 4.5 (m, 1H), 6.8 (NH), 7.1–7.4 (m, 14H); MS
(ESI) m/z 663 [M + Na]+.
Methyl 1-(Bis(4-acetamidophenoxy)phosphoryl)-2-(4-(N,N′-
bis(tert-butyloxycarbonyl)guanidino)phenyl)ethylcarbamate(Parac-
etamol Analogue of 12c). Same procedure as for 12c. Yield 51%;
1H NMR (CDCl3) δ 1.5 (d, 18H), 2.1 (2s, 6H) 3.0–3.4 (m, 2H),
3.4 (s, 3H), 4.7 (m, 1H), 6.7 (m, 1H), 7.1–7.6 (m, 12H); MS (ESI)
m/z 805 [M + Na]+.
1
nyl}ethanephosphonate. Yield 10%; H NMR (CDCl3, 400 MHz)
δ 1.5 (18H, d), 3.2 (m, 1H), 3.4 (m, 1H), 3.4 (m, 1H), 5.4, (m,
1H), 7.1–7.5 (m, 19H), 7.6 (d, 2H), 10.3 (s, 1H), 11.5 (s, 1H); MS
1
(ESI) m/z 715 [M + H]+. 6f: Yield 80%; H NMR (CD3OD, 400
MHz) δ 3.2 (m, 1H), 3.4 (m, 1H), 4.7 (m, 1H), 6.6 (m, 2H), 7.1
(m, 5H), 7.3–7.5 (m, 8H), 7.6 (m, 3H), 8.0 (d, 1H); HPLC (214
nm) tr 20.0 min, 100.0%; HPLC (254 nm) tr 21.0 min, 100.0%;
LC/MS tr 13.3 min, 91.2%; MS (ESI) m/z 515 [M + H]+.
Diphenyl 1-(o-Toluenesulfonylamino)-2-(4-guanidinophenyl)-
ethanephosphonate Trifluoroacetate (6g). Intermediate diphenyl
1-(R-toluenesulfonylamino)-2-{4-[N,N′-bis(tert-butyloxycarbon-
yl)guanidine]phenyl} ethanephosphonate. Yield 32%; 1H NMR
(CDCl3, 400 MHz) δ 1.5 (18H, d), 3.1 (m, 2H), 3.4 (m, 1H), 3.8
(d, 1H), 4.0 (d, 1H), 4.6 (m, 1H), 5.1 (d, 1H), 7.1–7.3 (m, 17H),
Diphenyl N-(Benzyloxycarbonylamino)-(3-guanidinopropyl)-
methanephosphonate Trifluoroacetate (6a). The Boc-protected
intermediate10,16 was dissolved in 50% trifluoroacetic acid in
CH2Cl2 (2–5 mL). After stirring for 3 h at room temperature, the
solvent was evaporated. The crude oil was washed with cold ether
and a precipitate was isolated. Yield 91%; 1H NMR (CD3OD, 400
MHz) δ 1.6 (m, 1H), 1.7–1.9 (m, 2H), 2.1 (m, 1H), 3.2 (m, 2H),
4.5 (m, 1H), 5.1 (q, 1H), 7.1–7.3 (m, 15H); HPLC (214 nm) tr
19.1 min, 96.2%; HPLC (254 nm) tr 19.0 min, 98.3%; LC/MS tr
13.2 min, 100.0%; MS (ESI) m/z 497 [M + H]+.
1
7.6 (d, 2H), 10.3 (s, 1H), 11.5 (s, 1H). 6g: Yield 94%; H NMR
Diphenyl 1-(Benzyloxycarbonylamino)-2-(4-guanidinophenyl)-
(CD3OD, 400 MHz) δ 3.1 (m, 1H), 3.5 (m, 1H), 4.1 (d, 1H), 4.2
(d, 1H), 4.6 (m, 1H), 7.2–7.5 (m, 19H); HPLC (214 nm) tr 20.2
min, 94.1%; HPLC (254 nm) tr 20.2 min, 100.0%; LC/MS tr 14.0
min, 100.0%; MS (ESI) m/z 565 [M + H]+.
ethanephosphonate. Trifluoroacetate (6b). Same procedure as for
1
6a. Yield 91%; H NMR (CD3OD, 400 MHz) δ 3.0 (m, 1H), 3.4
(m, 1H), 4.7 (m, 1H), 5.1 (m, 2H), 5.8 (s, 1H), 7.0–7.4 (m, 19H);
HPLC (214 nm) tr 21.1 min, 98.5%; HPLC (254 nm) tr 21.0 min,
100.0%; LC/MS tr 13.9 min, 100.0%; MS (ESI) m/z 545 [M +
H]+.
Diphenyl 1-(Naphthalenesulfonylamino)-2-(4-guanidinophe-
nyl)-ethanephosphonate Trifluoroacetate (6h). Intermediate di-
phenyl 1-(N-naphthalenesulfonylamino)-2-{4-[N,N′-bis(tert-butyl-
oxycarbonyl)guanidine]phenyl}ethanephosphonate. Yield 23%; 1H
NMR (CDCl3, 400 MHz) δ 1.6 (18H, d), 3.0–3.2 (m, 2H), 4.5 (m,
1H), 6.0 (d, 1H), 6.6 (d, 2H), 6.9–7.6 (15H), 7.8 (d, 1H), 7.9 (d,
1H), 8.1 (d, 1H), 8.5 (d, 1H), 10.0 (s, 1H), 11.6 (s, 1H). 6h: Yield
93%; 1H NMR (CDCl3, 400 MHz) δ 3.0 (m, 1H), 3.4 (m, 1H), 4.4
(m, 1H), 6.7 (d, 2H), 6.9 (d, 4H), 7.1–7.4 (11H), 7.6 (t, 1H), 7.7 (t,
1H) 7.9 (m, 1H), 8.1 (d, 1H); HPLC (214 nm) tr 20.6 min, 96.3%;
HPLC (254 nm) tr 21.0 min, 100.0%; LC/MS tr 14.7 min, 100.0%;
MS (ESI) m/z 601 [M + H]+.
Methyl 1-(Diphenoxyphosphoryl)-2-(4-guanidinophenyl)eth-
ylcarbamate Trifluoroacetate (6c). Same procedure as for 6a.
1
Yield 90%; H NMR (CDCl3) δ 2.9–3.4 (m, 2H), 4.5 (s, 3H), 4.7
(m, 1H), 7.0–7.5 (m, 14H); HPLC (214 nm) tr 16.6 min, 100.0%;
HPLC (254 nm) tr 16.6 min, 100.0%; LC/MS tr 11.2 min, 100.0%;
MS (ESI) m/z 469 [M + H]+.
Ethyl 1-(Diphenoxyphosphoryl)-2-(4-guanidinophenyl)ethyl-
carbamate Trifluoroacetate (6d). Same procedure as for 6a. Yield
89%; 1H NMR (CDCl3) δ 1.2 (t, 3H), 3.0–3.4 (m, 2H), 4.3 (q,
2H), 4.7 (m, 1H), 7.0–7.5 (m, 14H); HPLC (214 nm) tr 16.7 min,
100.0%; HPLC (254 nm) tr 16.7 min, 100.0%; LC/MS tr 11.5 min,
100.0%; MS (ESI) m/z 483 [M + H]+.
Diphenyl 1-Amino-2-(4-guanidinophenyl)ethylphosphonate
2 Trifluoroacetate (6k). Intermediate 1310,16 was dissolved in 50%
trifluoroacetic acid in CH2Cl2 (2–5 mL). After stirring for 3 h at
room temperature, the solvent was evaporated. The crude oil was
washed with cold ether and a precipitate was isolated. Yield 83%;
1H NMR (CDCl3) δ 3.0–3.4 (m, 2H), 4.7 (m, 1H), 7.0–7.4 (m,
Diphenyl 1-(2,3,6-Tri-isopropylbenzenesulfonyl)amino-2-(4-
guanidinophenyl)-ethanephosphonate Trifluoroacetate (6i). In-
termediate diphenyl 1-(N-2,3,6-tri-isopropylbenzenesulfonylamino)-
2-{4-[N,N′-bis(tert-butyloxycarbonyl)guanidine]phenyl}ethanephosphonate.
1
Yield 14%; H NMR (CDCl3, 400 MHz) δ 1.2–1.4 (m, 18H), 1.5
(d, 18H), 2.9–3.0 (m, 1H), 3.3–3.5 (m, 2H), 4.1 (m, 1H), 4.2 (m,
1H), 4.4 (m, 1H), 5.0 (d, 1H), 6.7 (d, 2H), 6.9 (d, 2H), 7.1–7.3 (m,
1
8H), 7.4 (d, 2H), 7.5 (d, 2H). 6i: Yield 89%; H NMR (CD3OD,
400 MHz) δ 1.2–1.4 (m, 18H), 2.3 (m, 1H), 3.0–3.1 (m, 2H),

6644 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Joossens et al.
4.1–4.3 (3H), 7.0–7.6 (m, 16H); HPLC (214 nm) tr 26.9 min,
100.0%; HPLC (254 nm) tr 26.9 min, 100.0%; LC/MS tr 18.0 min,
99.0%; MS (ESI) m/z 677 [M + H]+.
Di-(4-acetamidophenyl) 1-(benzyloxycarbonylamino)-2-[(4-
guanidino)phenyl]ethanephosphonate Trifluoroacetate (8a).
Same procedure as for 6a. Yield 95%; 1H NMR (CD3OD, 400
MHz) δ 2.0 (s, 6H), 3.0 (m, 1H), 3.4 (m, 1H), 4.6 (m, 1H), 5.1 (m,
2H), 6.7–7.3 (m, 17H), 8.0 (d, 1H); HPLC (214 nm) tr 15.7 min,
95.0%; HPLC (254 nm) tr 15.7 min, 94.2%; LC/MS tr 12.1 min,
95.3%; MS (ESI) m/z 659 [M + H]+.
Diphenyl 1-(Methylsulfonylamino)-2-(4-guanidinophenyl)-
ethanephosphonate Trifluoroacetate (6j). Intermediate diphenyl
1-(methylsufonylamino)-2-{4-[N,N′-bis(tert-butyloxycarbonyl)guan-
idine]phenyl}ethanephosphonate. Yield 18%; 1H NMR (CDCl3, 400
MHz) δ 1.5 (d, 18H), 2.5 (s, 3H), 3.0 (m, 1H), 3.5 (m, 1H), 4.5
(m, 1H), 5.6 (d, 1H), 7.1–7.6 (m, 14), 10.3 (s, 1H), 11.6 (s, 1H).
Methyl 1-(Bis(4-acetamidophenoxy)phosphoryl)-2-(4guani-
dino-phenyl)ethylcarbamate Trifluoroacetate (8b). Same pro-
1
1
6j: Yield 92%; H NMR (CD3OD, 400 MHz) δ 3.1 (m, 1H), 3.5
cedure as for 6a. Yield 94%; H NMR (CDCl3) δ 2.1 (2s, 6H),
(m, 1H), 4.5 (m, 1H), 7.2 (m, 8H), 7.4 (m, 4H), 7.45 (d, 2H); HPLC
(214 nm) tr 18.2 min, 98.0%; HPLC (254 nm) tr 17.3 min, 100.0%;
LC/MS tr 12.8 min, 96.3%; MS (ESI) m/z 489 [M + H]+.
Diphenyl 1-(N-Benzyloxycarbonylamino)-1-(4-amidinophe-
nyl)methanephosphonate·HCl (7a). Compound was synthesized
as repoterted by Oleksyszyn et al.12 1H NMR (CD3OD, 400 MHz)
δ 2.0 (3H, s), 5.1 (q, 2H), 7.0 (m, 4H), 7.2–7.4 (m, 11H), 7.8 (s,
4H), HPLC (214 nm) tr 20.6 min, 100.0%; HPLC (254 nm) tr 20.5
min, 100.0%; LC/MS tr 13.9 min, 98.4%; MS (ESI) m/z 516 [M +
H]+.
Diphenyl 1-(N-Benzyloxycarbonylamino)-2-(4-amidinophe-
nyl) Ethanephosphonate ·HCl (7b). Compound was synthesized
as reported by Oleksyszyn et al.12 1H NMR (CD3OD, 400 MHz) δ
2.0 (4H, s), 3.1 (m, 1H), 3.5 (m, 1H), 4.7 (m, 1H), 5.0 (q, 2H),
7.1–7.4 (m, 15H), 7.5 (d, 2H), 7.7 (d, 2H); HPLC (214 nm) tr 20.2
min, 90.3%; HPLC (254 nm) tr 20.2 min, 91.3%; LC/MS tr 13.6
min, 92.2%; MS (ESI) m/z 530 [M + H]+.
2.9–3.4 (m, 2H), 4.6 (s, 3H), 4.7 (m, 1H), 7.0–7.5 (m, 12H); HPLC
(214 nm) tr 10.9 min, 98.0%; LC/MS tr 10.7 min, 96.4%; MS (ESI)
m/z 583 [M + H]+.
Di-(4-acetamidophenyl) 1-(methylsulfonylamino)-2-(4-guani-
dinophenyl)-ethanephosphonate Trifluoroacetate (8c). Intermedi-
ate di-(4-acetamidophenyl) 1-(methylsufonylamino)-2-{4-[N,N′-
bis(tert-butyloxycarbonyl)guanidino]phenyl} ethanephosphonate.
1
Yield 18%; H NMR (CDCl3, 400 MHz) δ 1.5 (d, 18H), 2.0 (s,
3H), 2.1 (s, 3H), 2.2 (s, 3H), 2.9 (m, 1H), 3.3 (m, 1H), 4.3 (m,
1H), 7.1–7.6 (m, 12), 8.3 (s, 1H), 8.6 (s, 1H), 10.3 (s, 1H), 11.6 (s,
1H); MS (ESI) m/z 803 [M + H]+. 8c: Yield 91%; 1H NMR
(CD3OD, 400 MHz) δ 2.1 (s, 6H), 2.7 (s, 3H), 3.1 (m, 1H), 3.5
(m, 1H), 4.5 (m, 1H), 7.1 (m, 4H), 7.2 (d, 2H), 7.45 (d, 2H), 7.6
(d, 4H); HPLC (214 nm) tr 12.2 min, 93.2%; HPLC (254 nm) tr
11.6 min, 94.1%; LC/MS tr 10.7 min, 100.0%; MS (ESI) m/z 603
[M + H]+.
uPA Inhibition: In Vitro Evaluation. Enzymatic activity was
measured at 37 °C in a Spectramax 340 (Molecular Devices)
microtiter plate reader using the chromogenic substrate S-2444
(LpyroGlu-Gly L-Arg-p-NA·HCl), with a Km of 80 µM. The
substrate was obtained from Chromogenix. The human enzyme was
obtained from Sigma-Aldrich and the mouse uPA was obtained
from Molecular Innovations, Inc. (U.S.A.). The reaction was
monitored at 405 nm, and the initial rate was determined between
0 and 0.25 absorbance units in 20 min. The reaction mixture
contained 250 µM substrate and approximately 1 mU of enzyme
in 145 µL of buffer in a final volume of 200 µL. A 50 mM Tris
buffer, pH 8.8, was used. From each inhibitor concentration, 5 µL
was added, obtaining a final concentration from 0 to 250 µM in a
total volume of 0.2 mL. Activity measurements were routinely
performed in duplicate. The IC50 value is defined as the concentra-
tion of inhibitor required to reduce the enzyme activity to 50%
after a 15 min preincubation with the enzyme at 37 °C before
addition of the substrate. IC50 values were obtained by fitting the
data with the four-parameter logistics equation using Grafit 5.
Diphenyl 1-(N-Benzyloxycarbonylamino)-1-(4-amidinophen-
yl)propanephosphonate · HCl (7c). 4-Cyanobenzaldehyde (15a;
5 g, 0.038 mol) and triphenylphosphoranylidene acetic acid ethyl
ester (16 g, 0.045 mol) were dissolved in dry toluene (70 mL), and
the solution was stirred at room temperature for 12 h. Then the
solvent was removed under vacuum, and the residue was taken up
with ether. The solid formed was removed by filtration and the
organic phase was evaporated to give 3-(4-cyano-phenyl)-3-
propenoic acid ethyl ester as yellow oil. The compound was purified
with flash chromatography (hex/EtOAc). After evaporation of the
desired fractions, a white semi solid was obtained (6 g). The product
was dissolved in ethanol and added dropwise to a suspension of
sodium borohydride (11 g, 0.28 mol) in ethanol (100 mL) cooled
at 0 °C. The mixture was allowed to reach room temperature, and
stirring was maintained for 18 h. Acetone (5 mL) in 20 mL of
water was added dropwise to the mixture to destroy the remaining
NaBH4. The solvent was evaporated and water was added. This
solution was three times extracted with EtOAc. The combined
organic solutions were washed twice with 2 N HCl and once with
brine. The organic phase was dried and subsequent to evaporation
of the solvent a light yellow oil was obtained (3g, 47% yield). 3-(4-
Cyanophenyl)propanol was oxidized with the Dess-Martin reagent
to the aldehyde (15c). The crude product was immediately dissolved
in CH2Cl2 without any further purification. The same procedure as
for 11b was used to prepare the diphenyl phosphonate 16c. The
obtained product was purified with flash chromatography, 21% yield
V ) (V range)/(1 + exp(s × ln(abs(I0/IC50)) + background
where s ) slope factor, V ) rate, I0 ) inhibitor concentration, and
range ) the fitted uninhibited value minus the background. The
equation assumes the y falls with increasing x.
Inhibitor stock solutions were prepared in DMSO and stored at
-20 °C. Because the compounds described in this paper completely
inactivate uPA following pseudofirst-order kinetics, the IC50 value
is inversely correlated with the second-order rate constant of
inactivation. For a simple pseudofirst-order inactivation process,
the activity after incubation with inhibitor (Vi) varies with the
inhibitor concentration (i), as described in the following equation:
1
starting from 3-(4-cyanophenyl)propanol. H NMR (CDCl3, 400
MHz) δ 2.1 (m, 1H), 2.35 (m, 1H), 2.8 (m, 1H), 2.9 (m, 1H), 4.5
(m, 1H), 5.1 (m, 2H), 5.7 (d, 1H), 7.1–7.4 (m, 16H), 7.5 (m, 3H);
MS (ESI) m/z 527 [M + H]+.
Diphenyl 1-(N-(benzyloxycarbonyl)amino-1-(4 cyanophenyl-
)propanephosphonate was dissolved in dry chloroform/methanol
(1:1). The mixture was cooled to 0 °C and saturated with dry HCl
gas. The mixture was stirred for 3 days at 0 °C. The solvent was
evaporated, and the crude product was precipitated from dry ether.
The product was dissolved in 4 equiv of a 2 M solution of ammonia
in ethanol. The solution was stirred for 2 days at room temperature.
The solvent was evaporated. The product was washed with EtOAc
saturated with HCl. The product was precipitated from diethyl ether.
Yield 15%; 1H NMR (CD3OD, 400 MHz) δ 2.2 (m, 1H), 2.35 (m,
1H), 2.8 (m, 1H), 3.05 (m, 1H), 4.3 (m, 1H), 5.1 (q, 2H), 7.1–7.4
(m, 17H), 7.7 (d, 2H); HPLC (214 nm) tr 20.6 min, 92.0%; HPLC
(254 nm) tr 20.6 min, 93.0%; LC/MS tr 14.7 min, 97.0%; MS (ESI)
m/z 544 [M + H]+.
Vi ) V0 × e–k t, where V0 is the activity in absence of inhibitor, k is
i
the second-order rate constant of inactivation, and t is the time.
The inactivation rate constant was determined from the time course
of inhibition.
The inhibitor was mixed with the substrate (250 µM final
concentration), and the buffer solution with the enzyme was added
at the time zero. The inhibitor concentrations were chosen to obtain
total inhibition of the enzyme within 20 min. The progress curves
show the absorbance of p-nitroanilide produced as a function of
time. Initially, no inhibitor is bound to the enzyme, and the tangent
to the progress curve (dA/dt) is proportional to the concentration
of the free enzyme. The concentration of free enzyme decreases
over time due to the kinetics of inhibitor binding, as described

Diaryl Phosphonate Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6645
above. Progress curves were recorded in pseudofirst-order conditions
([I]0 >> [E]0) and with less than 10% conversion of the substrate
during the entire time course. In these conditions, dA/dt decreases
exponentially with time. The progress curves were fitted with the
integrated rate equation to yield a value for kobs, a pseudofirst-order
rate constant
3 h, 6 h, 12 h, 24 h, 48 h, 1 week, and 2 weeks. The samples (100
µL) were not diluted and were immediately analyzed by HPLC
(214 nm). The evolution of the AUC (area under the curve) of the
parent compound in function of time was taken as a parameter for
chemical stability. The following buffers were used: 125 mM
aqueous HCl, pH 1.1; 100 mM HOAc, 18 mM NaOAc, pH 4; 50
mM Tris, pH 7.4, 110 mM NaCl, and 50 mM Tris, pH 8.8, 38 mM
NaCl. The amount corresponding to the indicated molarity was
weighed/measured and dissolved/diluted in water, and the pH was
adjusted with diluted solutions of HCl or ammonium hydroxide,
depending on the required pH.
Log D. A total of 40 µL of a 10 mM stock solution in DMSO was
added to 1.960 mL of a mixture of octanol and phosphate buffer saline
(PBS), pH 7.4 (1/1). Octanol was first saturated with PBS and PBS
was first saturated with octanol before the two solvents were mixed.
After two hours of shaking at room temperature, the two layers were
separated. A total of 10 µL of each layer was added to 390 µL of
MeOH before the sample was analyzed with LC/MS/MS. The logD
is calculated from the formula: LogD ) log(AUCoctanol/AUCPBS). The
experiment was done in duplicate.42
Apparent Solubility. A total of eight different dilutions of an
initial 10 mM test compound solution were prepared in DMSO.
Each dilution was further diluted in PBS pH 7.4 to give a final
DMSO concentration of 2% and final test compound concentration
range of between 1 µM and 200 µM (4 µL DMSO solution in 196
µL buffer solution). Two replicates were prepared per concentration
in a 96 well microtiter plate. The plates were incubated for 2 h at
37 °C. Absorbance was measured at 620 nm and solubility was
determined by an increase in absorbance.
Plasma Stability. A 40 µL aliquot of the 10 mM solution in
DMSO of the test compound was added to 1.990 mL of human
plasma to obtain a 200 µM final solution. The mixture was gently
shaken for 24 h at 37 °C. Aliquots of 200 µL were taken at various
time points (0, 30 min, 1 h, 2 h, 3 h, 6 h, and 24 h) and diluted
with 400 µL of methanol. The suspension was centrifuged at 14000
rpm for 5 min at 4 °C. A total of 10 µL of the supernatant was
diluted with 990 µL of a 0.2 µM solution of the standard
UMAC00150 in MeOH and analyzed with LC/MS/MS. The
samples were analyzed in triplicate.
At ) ν0[1 - exp( - kobst)]/kobs + A0
(1)
where At ) absorbance at time t, A0 ) absorbance at time zero,
and V0 ) uninhibited initial rate. The apparent second-order rate
constant (kapp) was calculated from the slope of the linear part of
the plot of kobs versus the inhibitor concentration ([I]0). In case of
competition between the inhibitor and the substrate, kapp is smaller
than the “real” second order rate constant k discussed above because
a certain fraction of the enzyme is present as an enzyme–substrate
complex. kapp depends on the substrate concentration used in the
experiment, as described by Lambeir et al.26
For inhibitors 6a, 6c, 6d, 6f, 7b, and 8a, the kobs versus [I]0 plot
was not linear, but could be fitted with a hyperbolic function:
kobs ) k2[I]0/(K1 + [I]0) (two step mechanism).
Where K1 is the dissociation constant of the reversible en-
zyme–inhibitor complex and k2 is the first-order rate constant
associated with irreversible modification of the enzyme. A kapp could
be calculated as k2/K1.
Determination of the Selectivity for uPA. The IC50 values for
plasmin (from human plasma, Sigma), tPA (recombinant, Boe-
hringer Ingelheim), thrombin (from human plasma, Sigma), and
FXa were determined in the same way as for uPA. S-2288 (H-D-
Ile-Pro-Arg-pNa·2HCl) for tPA (Km: 1 mM), S-2366 (pyroGlu-
Pro-Arg-pNA·HCl) for plasmin (Km: 400 µM) and thrombin (Km:
150 µM), and S-2772 (Boc-D-Arg-Gly ArgpNA ·2HCl) for FXa
(Km: 1.5 mM) were used as substrates. FX (Sigma) is activated
with Russel’s viper venom (Sigma). The mixture contained 580
µM substrate for thrombin and plasmin, 1.25 mM for tPA, 522
µM for FXa, and 425 µM for trypsin, approximately 5 mU of
enzyme, and 145 µL of buffer. For tPA and thrombin, Tris buffer,
pH 8.3, was used, for FXa, Tris buffer, pH 8.3, with 2.5 mM CaCl2
was used, for plasmin, Tris buffer, pH 7.4, was used. The selectivity
index was calculated as (IC50 enzyme X)/IC50 uPA, where X is
tPA, thrombin, plasmin or FXa.
Molecular Modeling. The molecular modeling studies were
performed using MOE 2006.08 software (Chemical Computing
Group). All software necessary to build compounds and perform
minimizations, alignment, and superposition is available in the MOE
package and was used with standard settings, unless otherwise
mentioned. Crystal structures were downloaded from the Protein
Databank (PDB) and the ligands were checked and corrected when
necessary. Residues not belonging to the enzyme or ligand were
removed, including water molecules. Hydrogen atoms were added
and energy minimized with MMFF94x force field (standard
parameters) keeping all heavy atoms fixed.
Acknowledgment. This work received support from the
Fund for Scientific Research-Flanders (Belgium; FWO). P.V.d.V.
is a postdoctoral fellow of the FWO and G.S. is a visiting
postdoctoral fellow of the FWO. J.J. was a fellow of the Institute
of Promotion of Innovation in Science and Technology of
Flanders (IWT). Currently J.J. is coordinator of the Antwerp
Drug Discovery Network (ADDN) financed by the Industrial
Development Fund (IOF) of the University of Antwerp. The
excellent technical assistance of W. Bollaert is greatly appreciated.
Supporting Information Available: The experimental details
for the intermediates 10, 11b, 12b, 13, and tri-4-acetamidophenyl
phosphite and a purity table for all end compounds. Extra enzyme
kinetic data for the compounds 6a, 6c, 6f, 7b, and 8a, as well as
plasma stability of compounds 6c and 8b. Also the complete
experimental details for the BN-472 rat mammary carcinoma model
is available. This material is available free of charge via the Internet
at http://pubs.acs.org.
Following PDB structures of uPA were used: 1EJN,25 1F5K,27
1F5L,27 1F92,27 1FV9,28 1LMW,29 1SC8,4 1VJ9,4 1VJA.4
All structures were aligned and superposed. The studied com-
pounds were built using different parts of these ligands in the
mentioned enzymes followed by covalent linkage to the side chain
oxygen of Ser195. Then the complex was minimized using
MMFF94x force field with a flexible ligand in a rigid enzyme. Ser
195 residue of 1EJN was kept flexible during simulations. The
following PDB files were used to study the selectivity toward related
enzymes: tPA (1A5H,30 1RTF31), thrombin (1DWB,32 1GJ4,33
1H8I34), plasmin (1BUI35), FXa (1FAX,36 1KYE,37 1MQ638),
trypsin (1H4W,39 1EB2,40 1MAX41).
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