Phosphinanes and Azaphosphinanes as Potent and Selective Inhibitors of Activated Thrombin-Activatable Fibrinolysis Inhibitor (TAFIa)

Article abstract of DOI:10.1021/acs.jmedchem.0c02072

Selective and potent inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa) have the potential to increase endogenous and therapeutic fibrinolysis and to behave like profibrinolytic agents without the risk of major hemorrhage, since they do not interfere either with platelet activation or with coagulation during blood hemostasis. Therefore, TAFIa inhibitors could be used in at-risk patients for the treatment, prevention, and secondary prevention of stroke, venous thrombosis, and pulmonary embolisms. In this paper, we describe the design, the structure-activity relationship (SAR), and the synthesis of novel, potent, and selective phosphinanes and azaphosphinanes as TAFIa inhibitors. Several highly active azaphosphinanes display attractive properties suitable for further in vivo efficacy studies in thrombosis models.

Full text of DOI:10.1021/acs.jmedchem.0c02072

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Article
Phosphinanes and Azaphosphinanes as Potent and Selective
Inhibitors of Activated Thrombin-Activatable Fibrinolysis Inhibitor
(TAFIa)
Arnaud-Pierre Schaffner,* Patricia Sansilvestri-Morel, Nicole Despaux, Elisabeth Ruano,
Thierry Persigand, Alain Rupin, Philippe Mennecier, Marie-Odile Vallez, Eric Raimbaud, Patrice Desos,
and Philippe Gloanec
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ABSTRACT: Selective and potent inhibitors of activated thrombin
activatable fibrinolysis inhibitor (TAFIa) have the potential to increase
endogenous and therapeutic fibrinolysis and to behave like profibrino-
lytic agents without the risk of major hemorrhage, since they do not
interfere either with platelet activation or with coagulation during blood
hemostasis. Therefore, TAFIa inhibitors could be used in at-risk patients
for the treatment, prevention, and secondary prevention of stroke,
venous thrombosis, and pulmonary embolisms. In this paper, we
describe the design, the structure−activity relationship (SAR), and the
synthesis of novel, potent, and selective phosphinanes and azaphosphinanes as TAFIa inhibitors. Several highly active
azaphosphinanes display attractive properties suitable for further in vivo efficacy studies in thrombosis models.
TAFIa is activated by the thrombin/thrombomodulin (TM)
enzyme complex or by plasmin.6
INTRODUCTION
■
Hemostasis is the physiological process that stops bleeding in
all animals with a closed circulatory system while keeping the
blood in a liquid state inside the vessels. Hemostasis takes
place in three successive stages, including platelet aggregation,
coagulation, and fibrinolysis. Thrombotic diseases represent
one of the main causes of death in developed countries. Poor
nutrition, lack of physical exertion, or even smoking are
aggravating factors.
Fibrinolysis prevents the extension of the clot by destroying
the fibrin polymers. Fibrinolysis is activated by the binding of
tissue plasminogen activator (t-PA) (released by activated
endothelial cells) on the C-terminal lysine residues of fibrin,
which are high affinity receptors for t-PA. This t-PA binding
allows the enzymatic activation of the circulating plasminogen
into the proteolytic enzyme, plasmin, which breaks down the
fibrin clots. The fibrinolytic system balances breakdown of
intravascular clots formed following coagulation and preven-
tion of hemorrhages by premature dissolution of hemostatic
clots. Thus, fibrinolysis is a highly regulated process in which
endogenous fibrinolysis inhibitors slow down the fibrin
breakdown. Among these inhibitors, the carboxypeptidase
(CP) TAFIa, activated thrombin activatable fibrinolysis
inhibitor, resulting from the activation of the TAFI zymogen,
acts on fibrinolysis by cleavage of C-terminal lysine residues on
the surface of fibrin, preventing plasmin generation by t-PA.
TAFI is expressed as a 55 kDa protein synthesized primarily in
the liver. It circulates in the plasma in a form of zymogen.
TAFIa constitutes an important negative regulation of
fibrinolysis. A low level of TAFI has been associated with
chronic liver disease and an increased risk of bleeding.1 On the
other hand, high concentrations are correlated with an
increased risk of thrombosis, coronary artery disease due to
less fibrinolytic activity. Furthermore, it has been described
that venous thrombosis and thromboembolism risks are
associated with increased plasma levels of TAFIa in patients.2
The implication of TAFIa in thrombosis diseases has been
demonstrated in preclinical models. In knock out (KO) mice,
thrombus size and weight are reduced following FeCl3-induced
thrombosis3 and clot lysis is increased in a thromboembo-
lism.4,5 In recent years, consistent data have been obtained in
animal studies on the value of TAFIa inhibitors as therapy for
thrombolysis.6 Indeed, current treatments which include
thrombolytics (recombinant t-PA), antiplatelet agents, and
anticoagulants present a high risk of bleeding which might be
lesser with a TAFIa inhibitor treatment.
Received: November 29, 2020
Published: March 25, 2021
https://doi.org/10.1021/acs.jmedchem.0c02072
© 2021 American Chemical Society
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Figure 1. Holo crystal structure of human TAFI (PDB code 3D68).
TAFIa acts on fibrinolysis by cleavage of C-terminal lysine
residues on the surface of fibrin, preventing plasmin generation
by t-PA. The active site of TAFIa comprises the amino acids:
His159, Glu162, and His288 for the coordination of Zn2+ and
the charged residues Asp348, Arg235, and Arg217 for the
anchoring of the C-terminal lysine peptide substrate.7 The
penta-coordinated zinc interacts simultaneously with the
carbonyl of the substrate and a water molecule which is
essential for hydrolysis (Figure 1).
should be reinforced by the presence of a quaternary carbon
bearing a carboxylic acid function and an amino side chain of
length to be determined. To verify these hypotheses, a
preliminary series of cyclic phosphinanes were then designed
by varying the length of the basic chain as well as the size of
the cycle, prioritized by docking using the crystal structure of
human TAFI crystallized with arginine as a ligand (PDB 3D68,
Figure 3) and synthesized according to the general synthetic
RESULTS
■
It is well established in the literature that three main functions
are necessary for the recognition by TAFIa of an inhibitor
including a carboxylic acid function forming intramolecular salt
bridges with two arginine residues (Arg217 and Arg235), a
zinc-coordinating group, and a basic moiety forming a salt
bridge with Asp348.22 The carboxylic acid and the basic
function (amine) are usually separated by a 4−5 atom flexible
chain (Figure 2).
Figure 3. Docking representation of 8a, highlighting the key
interactions with TAFI.
Figure 2. General pharmacophore for TAFIa inhibitors.
pathway shown in Scheme 1. In its docked complex, as shown
in Figure 3, 8a maintaining the key interactions of the
carboxylate with Arg217, Asn234, and Arg235, the phosphi-
nane formed a strong interaction with the Zn2+ ion according
to the docked structure. The terminal basic group also
interacted with Asp348, although their distance of 4.1 Å
suggests that use of a longer linker might be beneficial.
The bromoalkylphosphinates 2a−d were obtained in good
yields via an esterification/hydrophosphinylation reaction as
described by Montchamp.19 The phosphinate ethyl ester
intermediate was generated in situ by treatment of an aqueous
solution of hypophosphorous acid with an equivalent of
tetraethylorthosilicate. The hydrophosphinylation was then
TAFIa inhibitors described in the literature have various
zinc-chelating groups including imidazoles,8 thiols,9−11 carbox-
ylic acids,12 selenols,13 phosphinic acids,14,15 sulfamides,16 and
ureas.17 To design our new inhibitors, we selected phosphinic
acid as a zinc binding motif and we hypothesized that its
incorporation in a cycle should reduce the conformational
flexibility and consequently increase the selectivity with respect
to other CPs. Indeed, other CPs such as circulating
carboxypeptidase N, which plays an important role in the
degradation of anaphylatoxins and kinins, are constitutively
active and should not be targeted.18 This greater rigidity
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a
Scheme 1. General Synthetic Scheme of Cyclic Phosphinane 8, Inhibitors of TAFIa
a
Reagents and conditions: (a) tetraethylorthosilicate, MeCN, 80 °C, 2 h; (b) ω-bromoalkene, Pd2dba3, xantphos, MeCN, reflux, 18 h; (c)
LiHMDS, THF, −78 °C; (d) LDA, DBC, THF, −70 °C, 2 h; (e) 5a−d, NaH, DMSO, 10 °C; (f) TMS-Br, DCM, 0 °C, 20 h; (g) 10% Pd/C (10
mol%), MeOH, 20 °C, 20 h.
carried out by adding the corresponding ω-bromoalkenes in
the presence of Pd2dba3 and Xantphos. The intramolecular
alkylation allowing the formation of the corresponding cyclic
phosphinanes 3a−d was performed using LiHMDS in THF. It
is important to notice that the synthetic difficulties increase
with the size of the ring, and the 9-membered ring was isolated
in only 1% yield. 3a−d were then benziloxycarbonylated with
dibenzylcarbonate and LDA while keeping the temperature of
the mixture below −65 °C to avoid decarboxylation and led to
4a−d as a mixture of four diastereoisomers. Different basic side
chains 5a−d were then introduced by alkylation using NaH in
DMSO at room temperature to give 6a−l. The simultaneous
hydrolysis of the phosphinate ethyl ester and of the two tert-
butoxycarbamate groups was performed using trimethylsilyl
bromide and followed by methanolysis of the intermediate
silylated esters to afford 7. Finally, after hydrogenolysis, the
target compounds 8a−l were isolated as racemic mixtures in
nearly quantitative yields.
These compounds were then tested for their ability to
inhibit human activated TAFI. Briefly, purified recombinant
human TAFI was activated by thrombin/thrombomodulin to
generate TAFIa. Compounds were incubated with the protein,
and hippuryl-arginine was added. The ability of compounds to
dose dependently decrease the formation of hippuric acid
(product of the reaction) was evaluated, and IC50 were
calculated (Table 1).
Although the five membered phosphinanes (n = 1) were
typically weak inhibitors of TAFIa, we observed a preference
for the longer linkers (8b, 8c). Moving to the larger rings (n >
1), this trend was recapitulated, suggesting that the chain with
three carbon atoms is too short to establish interaction with
Asp348. On comparing the effect of the chain length on the
TAFIa inhibition for the larger phosphinane cycles (n = 2−4),
the most promising results were obtained with a chain of C5
(8f, 8i, and 8k). The increase in ring size appears to improve
the affinity as illustrated with 8k and its 8-membered ring,
which represented the most active TAFIa inhibitor so far (IC50
= 1.1 μM). Further increase of the ring size (n = 5) in
combination with the optimal linker length (m = 5) led to no
further improvement (8l). The data in Table 1 suggest that we
have reached a plateau of potency in this series. Therefore,
Table 1. Inhibition of Human Activated TAFI by Cyclic
Phosphinanes 8a−l
compounds
n
m
IC50 hTAFIa (μM)
8a
8b
8c
8d
8e
8f
8g
8h
8i
1
1
1
2
2
2
2
3
3
4
4
5
3
4
5
3
4
5
6
4
5
4
5
5
>30.0
19.0
18.0
>30.0
11.7
5.0
>30.0
5.0
1.8
8j
8k
8l
2.6
1.1
1.5
other modifications were required to increase the affinity of
these inhibitors. Analysis of the docked structure of 8a (Figure
3) suggested that the incorporation of a nitrogen atom in the
cycle could lead to the formation of a salt bridge with the side
chain of Glu363. Docking of the N-benzyl-azaphosphinane 14d
into TAFI (derived from PDB 3D68, Figure 4) confirmed this
interaction. The proximity of the loop formed by Leu340 and
Tyr341 forces the benzyl group to turn away from these side
chains.
To verify this hypothesis, racemic N-benzylazaphosphinane
derivatives were prepared as illustrated in Scheme 2. Ethyl
divinylphosphinate 10 was obtained according to a previously
reported procedure by addition of 2 equiv of vinyl magnesium
bromide on ethyl dichlorophosphate 9.20 Addition of benzyl-
amine at 100 °C for 16 h afforded the desired cyclic
azaphosphinane 11 in good yield. The rest of the synthesis is
carried out in the same way as described before, that is,
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activity, 14d. To assess its absolute configuration, crystal-
lization attempts were carried out using a full-length TAFI with
quintuple mutations in the dynamic flap next to the catalytic
domain, named TAFI-CIIYQ: S327C, T347I, T351I, H355Y,
and H357Q. Crystals of the TAFI-CIIYQ-14d complex were
obtained by soaking and their X-ray structure confirmed the
(S) configuration of the quaternary carbon center (Figure 5).
Although most of the 3D68-docked and the crystal structure
of 14d bound to TAFI were similar, one could observe a major
difference in the binding site. The orientation of the benzyl
group of 14d was flipped between the two structures (Figure
5). Indeed, this benzyl group induced a pocket opening of the
TAFI binding site. In PDB 3D68, the structure used for
docking, the Tyr341 is turned toward the center of the active
site, making a H-bond with the carboxylate of the ligand that is
already making 2 H-bonds with Asn234 and Arg235. Due to
the steric clash with the N-benzyl group of our molecules, the
loop containing Leu340 and Tyr341 flips toward the outside of
the pocket, and furthermore, the whole backbone of TAFI
between Glu338 and Leu342 moves away, the largest
displacement of 4.2 Å being observed for the C alpha of
Leu340 (shown by a red arrow). This part of the backbone of
TAFI was reported to be every flexible, as it is one end of the
dynamic flap that can undergo large movements, leading to
inactivation of TAFIa.26 Tyr341 was not always visible as it
rotates freely in the solvent. To assess the contribution of the
ring nitrogen to the affinity, we synthesized the analogue of
14b replacing the ring nitrogen atom by a carbon (15). This
change led to some loss of potency with an IC50 = 0.5 μM for
the most active racemic diastereoisomer 15 compared to 0.186
μM for those of 14b.
Figure 4. Docking of 14d into TAFI (PDB 3D68) highlighting the
interaction of the ring nitrogen with Glu363.
alkoxycarbonylation reaction followed by an alkylation reaction
to introduce the basic side chain.
The resulting azaphosphinic acid ethyl ester 13a−c was then
quantitatively converted to the corresponding phosphinic acid
with TMSBr followed by methanolysis. The final deprotection
of the t-Bu ester was accomplished with 12 equiv of TFA in
DCM to afford 14a−c. The prepared cyclic azaphosphinane
derivatives were also tested in the in vitro TAFIa inhibition
assay and to our delight already the second compound
synthesized (14b) showed remarkable activity with an IC50
=
Next, we evaluated the influence of the ring size. The
corresponding 7-membered analogues were prepared accord-
ing to Scheme 3. It is important to note that the unsubstituted
azaphosphinane ring with 7 atoms is not symmetrical and two
regio-isomers (22 and 23) are therefore produced after the
carboxylation reaction with Boc-anhydride. Following separa-
tion by chromatography, these two compounds showed lower
186 nM as a racemic mixture (Table 2).
In comparison with 8e, the introduction of the N-benzyl
group into the cycle led to an impressive improvement of
nearly 2 logs in potency. Variations in chain length confirmed
the earlier findings that the optimal length is four carbons. The
two enantiomers of 14b, 14d, and 14e were then isolated. As
expected, only one of them was responsible for the inhibitory
a
Scheme 2. General Synthetic Route to N-Benzylazaphosphinane TAFIa Inhibitors 14a−c
a
Reagents and conditions: (a) vinyl magnesium bromide, THF, −78 °C, 2 h; (b) N-benzylamine, EtOH, reduced pressure, 100 °C, 16 h; (c) LDA,
Boc2O, THF, −70 °C, 2 h; (d) Boc2N−(CH2)n−Br, NaH, DMSO, 10 °C; (e) TMS-Br, DCM, 0 °C, 20 h; (f) TFA, DCM, 20 °C, 10 h.
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Table 2. Inhibition of Human Activated TAFI by N-Benzylazaphosphinanes (14a−e, 15)
compounds
basic side chains
IC50 hTAFIa (μM)
14a
14b
14c
5a
5b
5c
5b
5b
5b
>30.0
0.2
0.2
0.1
10.7
0.5
a
14d
a
14e
15
b
a
b
14d and 14e were isolated after chiral separation.21 15 was synthesized starting from 2-(2-bromoethyl) but-3-enylbenzene according to the
procedure described in the general synthetic scheme of cyclic phosphinane inhibitors and isolated as a mixture of 2 diastereoisomers (Scheme 1).
The prepared compounds were tested in the in vitro TAFIa
inhibition assay, and the results are summarized in Table 3. In
the light of these results, replacement of the benzyl moiety by
alkyl (26b,c) or cyclohexylmethyl (26d) was clearly
detrimental. Elongation of the linker between the nitrogen
and the benzene ring to 2 (26e) or 3 carbons (26f) led also to
a significant drop of potency. A similar effect was observed
when the phenyl group was replaced by heterocycles (26g−l),
although the loss of potency was sometimes moderate, and the
thiophen analogue 26l that maintained activity against TAFIa
(0.19 μM). In the pyridine series (26g−i), it appeared to us
that only the 2-position was tolerated (26i).
Having identified that the benzyl substitution was optimal
on the ring nitrogen, we evaluated the introduction of various
substituents on the phenyl group in the hope of achieving a
better occupation of the TAFIa catalytic site. Compounds
27a−n were prepared following Scheme 4 and tested in the
TAFIa inhibition assay (Table 4). Introducing a methoxy
(27a) group in the para position led to a drop of efficacy due
probably to a clash with the protein, while the hydroxy
analogue 27b showed a modest 2-fold improvement of the
potency. Data obtained for the halogen-substituted analogue
27c−h support the expectation based on the X-ray structure of
14d that the substitution in the o- and p-position should be
well tolerated. Replacement of the chlorine atom at the o-
position by a bromine led to a more than 2-fold gain in
potency (IC50 = 87 nM for 27h), while change to fluorine
resulted in a 3-fold drop underlining the importance of the size
of the substituents at this position. In order to determine if in
addition to its size, the nature of the substituents is also
important, the corresponding biphenyl derivatives 27i−k were
also synthesized and tested. While the m- (27j) and p-
biphenyls (27k) were inactive due probably to steric clash with
the protein, 27i achieved a vital 11-fold increase in potency
compared to the simple benzyl derivative 14b attaining an IC50
= 17 nM. The introduction of an oxygen between the two
benzene rings (27l) killed the inhibitory effect, while
replacement of the terminal benzene by a 3-pyridyl (27m)
or 5-pyrimidyl (27n) ring gave very potent compounds with
IC50s of 2 and 7 nM, respectively.
Figure 5. Docking (white) vs crystal (colored) structure of 14d in
complex with mutated TAFI. The red arrow shows the shift of Leu340
between 3D68 and the new X-ray structure (PDB code 7NEE).
activity compared to 14b with IC50 of 1.9 and 0.6 μM,
respectively.
Based on our observations so far, we concluded that if we
would like to improve our TAFIa inhibitors, then we should
explore the influence of the substitution on the nitrogen in the
cycle. A new set of 6-membered azaphosphinane derivatives
were designed, prioritized by docking, and synthesized
according to the synthetic pathway shown in Scheme 4.
24 was obtained by hydrogenolysis of 13b in the presence of
Pd/C in ethanol and 1 equiv of HCl in 88% yield. The
reductive amination of 24 was carried out in the presence of
1.5 equiv of aldehyde and anhydrous MgSO4 followed by in
situ reduction of the iminium intermediates by NaBH(OAc)3
to afford 25a−n. The simultaneous hydrolysis of the
azaphosphinate ethyl ester and of the two Boc protecting
groups was performed using TMS-Br followed by methanolysis
of the intermediate silylated esters. Finally, 12 equiv of TFA
were necessary to fully deprotect the t-Bu ester to lead to the
targeted compounds 26a−l as racemic mixtures of TFA salts.
The most advanced compounds tested so far were
racemates, so in the final round of optimizations, we separated
the two enantiomers by chiral chromatography and tested the
more active ones 28a−k (Table 4). In these compounds, we
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a
Scheme 3. General Synthetic Route to 7-Membered Ring Azaphosphinanes 22 and 23
a
Reagents and conditions: (a) MgSO4, 20 °C, 1 h; (b) NaBH(OAc)3, 20 °C, 16 h (c) LDA, Boc2O, THF, −70 °C, 2 h; (d) Boc2N−(CH2)n−Br,
NaH, DMSO, 10 °C; (e) TMS-Br, DCM, 0 °C, 20 h; (f) TFA, DCM, 20 °C, 10 h.
a
Scheme 4. General Synthetic Route to N-Substituted Azaphosphinanes 26a−l and 27a−n
a
Reagents and conditions: (a) 10% Pd/C (10 mol%), MeOH, 20 °C, 20 h (b) R−CHO, DCM, MgSO4, 20 °C, 1 h; (c) NaBH(OAc)3, 20 °C, 16
h; (d) TMS-Br, DCM, 0 °C, 20 h; (e) TFA, DCM, 20 °C, 10 h.
modified the benzene ring testing a variety of heterocycles with
5- or 6-membered rings, and we also modified the substituent
in the p-position of the internal benzene ring in order to
improve drug-like properties. Most of the prepared compounds
showed very similar inhibitory activity around 10 nM. We
managed to obtain the X-ray structure of 28k complexed with
TAFI (Figure 6).
A functional assay was set up to evaluate the ability of
compounds showing an IC50 ≤ 20 nM on human recombinant
TAFIa to modulate the lysis of a blood clot. For that purpose,
the lysis of rat blood clot was analyzed by thromboelastometry
(TE), and activity of compounds was evaluated by their
capacity to decrease the clot firmness (by AUC calculation), at
the fixed dose of 0.8 μM or in a dose response for EC50
calculation (Table 6). Clotting time and clot formation time
were not affected by any compounds (data not shown). Active
compounds acted only on clot lysis acceleration and thus on
clot firmness kinetics. The tested compounds at 0.8 μM dose
showed decrease of lysis time in rat blood in the 43−79%
range (Table 6). We determined the EC50 values for 28a and
28k, which were 378 and 389 nM, respectively. These EC50
values are significantly higher than the activity observed on
human TAFIa (5 and 6 nM respectively, Table 4), suggesting a
higher activity of these compounds on human TAFIa than in
rat TAFIa, as described for mouse TAFIa.22 The compounds
showing in vitro inhibition over 40% were then tested for their
duration of action in an in vivo pharmacodynamics model. Rats
were treated by IV administration of vehicle or compounds at a
0.6 mg/kg dose and blood was collected 30 and 60 min later
and percentage of inhibition of thromboelastogram AUC was
evaluated at these 2 times (Table 6).
Tyr341 is slightly rotated away from the active site making
room for the pyrazole group. The backbone between Glu338
and Leu342 deviates from 3D68 and adopts a conformation
more similar to that observed in the complex with 14d. It is
worth mentioning that the fluorine substituent on the benzene
ring points directly at the backbone carbonyl oxygen of Ser299
at 4.1 Å explaining very moderate contribution to affinity from
this interaction for the F- or HO-substituted analogues.
These cyclic azaphosphinanes have the distinction of being
relatively polar compounds in general, with a clog P < 1 and a
polar surface between 110 and 140 Å2, which give them a very
high aqueous solubility. As expected, these characteristics are
responsible for the very low permeability and oral bioavail-
ability as measured on the Caco-2 cell permeability assay. On
the other hand, these compounds present very high metabolic
stability when incubated with human or rat microsomes and
hepatocytes (>70%) and a moderate to high unbound fraction
in plasma (35−86%) as illustrated in Table 5 for 28k. Finally,
selectivity of compound 28k was evaluated on human
carboxypeptidases CPB (pancreatic) and CPN (plasmatic).
28k is highly selective for TAFIa with a ratio of 7000 for CPB
and more than 300,000 for CPN (Table 5).
Most of the tested compounds showed a strong activity
leading to an >50% decrease of AUC 30 min after IV
administration, but the effect was usually not lasting, and
activity decreased considerably 60 min after IV administration
except for few products (28g−k). Analyzing the inhibitory
activities, we can conclude that in general compounds having a
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Table 3. Inhibition of Human Activated TAFI by N-
Benzylazaphosphinanes 26a−l
Table 4. Inhibition of Human Activated TAFI by N-
Benzylazaphosphinanes 27a−n and 28a−k
substituent in the “para” position of the benzene ring showed
equal or better inhibitory activity after 60 min than their
counterparts (c.f. 6a−6o, 6m−6n, 6p−6q). The most efficient
compound was 28k with a remaining activity of 61% of lysis
time decreasing 60 min after IV administration. Its activity and
other properties supported its progression into preclinical and
later clinical development. PK parameters in rat were
determined for this compound (Table 5). Following single
IV administration of 28k, the terminal half-life was estimated
to 0.47 h. The compound was eliminated with a low total
clearance, almost entirely related to renal excretion. Moreover,
28k is poorly distributed in tissues which has no impact as the
targeted compartment is blood. In accordance with the low in
vitro permeability (66%, Table 5), the in vivo oral
bioavailability of 28k was very low (F = 1%, 10 mg/kg per
os in rat, Cmax 98.7 ng/mL with a Tmax at 45 min).
CONCLUSIONS
■
In conclusion, we have described here the discovery and the
optimization of original, selective, and potent human TAFIa
inhibitors. The SBDD led to the identification of 28k that
accelerates clot degradation and might increase vessel
recanalization in vivo. Acceleration of endogenous fibrinolysis
was confirmed in vivo in a murine model of pulmonary
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Table 6. Evaluation of the Activity In Vitro and Ex Vivo of
Selected TAFIa Inhibitors by Thromboelastometry (TE) in
a
Rat
TE ex vivo %
TE in vitro % inhib SEM@0.8 μM inhib@0.6 mg/kg 30/60 min
Cpds
(EC50 SEM in nM)
post IV
28a
28c
28f
28g
28h
28k
28l
28m
28n
28o
28p
28q
28r
28s
74% 3 (378 56)
66/42
62/44
66/21
75/55
76/50
NA/61
41/NA
60/13
78/30
NA/41
66/33
NA/45
63/19
80/17
66%
43%
63%
71%
2
1
0
1
57% 2 (389 72)
61%
62%
79%
75%
67%
68%
66%
61%
8
2
2
6
3
4
3
4
a
Percentage of inhibition (% inhib) represents the percentage of
thromboelastogram AUC of the tested compound vs AUC of vehicle.
Figure 6. Overlay of the TAFI bound complexes of 28k (blue, PDB
code 7NEU) and 14d (yellow, PDB code 7NEE).
over MgSO4, and concentrated in vacuo. The residue obtained is
purified by flash chromatography on silica gel using as eluant a DCM/
EtOAc gradient (90:10 to 50:50). Intermediates 13a−c are obtained
as mixture of 4 diastereoisomers.
tert-Butyl 3-{3-[bis(tert-Butoxycarbonyl)amino]propyl}-1-benzyl-
4-ethoxy-4-oxo-1,4 Azaphosphinane-3-carboxylate (13a). Inter-
mediate 13a is obtained starting from intermediates 12 and 5a in
accordance with procedure B described hereinbefore. White powder;
yield 54% (0.225 g, 0.368 mmol). 1H NMR (DMSO-d6, 400 MHz): δ
ppm 7.35−7.2 (m, 5H), 4 (m, 2H), 3.8 (m, 2H), centered at 3.52
(AB, 2H), 3−2.25 (m, 4H), 2−1.8 (m, 4H), 1.45−1.35 (2s, 27H), 1.2
(t, 3H), 1.2 (m, 2H).
Table 5. In Vitro ADME, Selectivity on CP and PK
Parameters of 28k after Single IV Administration in Rat (1
mg/kg)
clog P
0.9
Fabs (ER)
66% (1.0)
93/89/95%
100/100/100%
100/100/85%
0.00075
5.3
plasma fu (m/r/hu)
MFmic (r/m/hu)
MFhep (r/m/hu)
Ki (huTAFIa) μM
Ki (huCPB) μM
Ki (huCPN) μM
AUC (μmol·h/L)
tert-Butyl 3-{4[bis(tert-Butoxycarbonyl)amino]butyl}-1-benzyl-4-
ethoxy-4-oxo-1,4-azaphosphinane-3-carboxylate (13b). Intermedi-
ate 13b is obtained starting from intermediates 12 and 5b in
accordance with procedure B described hereinbefore. White powder;
yield 79% (13.9 g, 22.248 mmol).1H NMR (DMSO-d6, 400 MHz): δ
7.30 (m, 5H), 3.97 (m, 2H), 3.63−3.43 (dd, 2H), 3.36 (m, 2H),
2.93−2.33 (m, 2H), 2.79−2.47 (m, 2H), 1.93 (m, 2H), 1.93 (m, 2H),
1.43 (m, 2H), 1.42 (s, 18H), 1.36 (s, 9H), 1.21 (t, 3H), 0.83 (m, 2H).
IR (cm−1): 1744, 1711 cm−1 (CO), 1276 cm−1 (PO).
tert-Butyl 3-{5-bis(tert-Butoxycarbonyl)amino]pentyl}-1-benzyl-
4-ethoxy-4-oxo-1,4-azaphosphinane-3-carboxylate (13c). Inter-
mediate 13c is obtained starting from intermediates 12 and 5c in
accordance with procedure B described hereinbefore. White powder;
yield 62% (0.310 g, 0.485 mmol). 1H NMR (DMSO-d6, 400 MHz): δ
ppm 7.32−7.2 (m, 5H), 3.99 (m, 2H), 3.8 (m, 2H), centered at 3.5
(AB, 2H), 3−2.3 (m, 4H), 2−1.8 (m, 4H), 1.45−1.35 (2s, 27H),
1.35−1.15 (2m, 4H), 1.2 (t, 3H), 0.75 (m, 2H).
General Procedure C for the Synthesis of 14a−c. TMSBr
(7.92 mL, 60 mmol, 12 equiv) is added dropwise to a solution of
intermediate 13a−c (5 mmol) in DCM (40 mL) under argon and at
ambient temperature. The mixture is stirred for 16 h at ambient
temperature and then concentrated in vacuo. The residue is taken up
in MeOH (40 mL) and stirred for 20 min at ambient temperature,
before being evaporated to dryness. The evaporate is dissolved in
DCM (20 mL), and trifluoroacetic acid (44.6 mL, 60 mmol, 12 equiv)
is added. The reaction mixture is stirred for 10 h at ambient
temperature and then concentrated in vacuo. The residue obtained is
purified by reverse-phase chromatography using as eluant an H2O/
MeCN gradient. The final racemic products 14a−c (zwitterion) are
isolated as a white solid after lyophilization.
282
3.88
t1/2, z (h)
0.47
CLp (mL/min/kg)
Vss (L/kg)
9.84
0.30
CLr (mL/min/kg)
9.54
thromboembolism after IV administration of 28k.22 Treatment
with a TAFIa inhibitor, such as 28k also known as S62798,
could be a promising therapeutic option to treat patients with
pulmonary embolism or ischemic stroke by accelerating blood
vessel recanalization and improving patient outcome.
EXPERIMENTAL SECTION
General. All obtained products had a liquid chromatography
purity above 96% that was corroborated by their H NMR spectrum
■
1
unless specifically mentioned otherwise. All synthetic experimental
details including the characterization of the compounds are described
in the Supporting Information. Examples 14d, 14e, 15, 22, and 23
have been prepared according to procedures already described.21
General Procedure B for the Preparation of Intermediates
13a−c. 60% NaH (8 mmol, 1.6 equiv) is added at 10 °C in portions
to a solution of 5a−d (5 mmol, 1 equiv) in DMSO (10 mL) under
argon. Intermediate 12 (5 mmol) in solution in DMSO (5 mL) is
then added to the suspension, and the mixture is stirred for 4 h at
ambient temperature. The reaction mixture is then hydrolyzed with an
aqueous NH4Cl solution (50 mL) and extracted with EtOAc (2 × 100
mL). The organic phase is washed with H2O (2 × 100 mL), dried
3-(3-Aminopropyl)-1-benzyl-4-hydroxy-4-oxo-1,4-azaphosphi-
nane-3-carboxylic Acid (14a). 14a is obtained starting from
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Article
intermediate 13a in accordance with procedure C described
hereinbefore. White powder; yield 56% (0.091 g, 0.279 mmol). H
are filtered off over a microfiber and rinsed with DCM (100 mL). The
filtrate is then washed with water (1 × 200 mL) and then with a
saturated NaCl solution (2 × 200 mL). The organic phase is dried
over MgSO4 and concentrated under reduced pressure. The oil
obtained is then purified by flash chromatography on silica gel (330 g)
to yield intermediates 25.
Examples 26 and 27 are obtained starting from intermediates 25 in
accordance with the procedure C described hereinbefore. The yields
given are for the last 2 steps from their corresponding intermediates
25a−n.
3-(4-Aminobutyl)-4-hydroxy-4-oxo-1,4-azaphosphinane-3-car-
boxylic Acid (26a). White solid; yield 48% (0.108 g, 0.431 mmol). 1H
NMR (D2O, 400 MHZ): δ ppm 3.5−3.65 (2m, 2H), 3.2 (m, 1H),
3.05 (dd, 1H), 2.95 (m, 2H), 2.15 (m, 1H), 1.95 (m, 1H), 1.75 (m,
1H), 1.65 (m, 2H), 1.5 (m, 1H), 1.4 (m, 1H), 1.25 (m, 1H). HRMS:
calcd for [M + H]+ 251.1156; found: 251.1154. Elemental Anal.
Calcd (%) for C9H19N2O4P: C, 43.20; H, 7.65; N, 11.20. Found: C,
42.65; H, 7.23; N, 11.24.
3-(4-Aminobutyl)-4-hydroxy-1-methyl-4-oxo-1,4-azaphosphi-
nane-3-carboxylic Acid (26b). White solid; yield 65% (0.150 g, 0.567
mmol).1H NMR (D2O, 400 MHZ): δ ppm 3.7−3.5 (m, 2H), 3.3 (t,
1H), 3.2 (dd, 1H), 3 (t, 2H), 2.9 (s, 3H), 2.3 (m, 1H), 2 (m, 1H),
1.75 (m, 1H), 1.7 (m, 2H), 1.5 (m, 1H), 1.4−1.25 (2m, 2H). HRMS:
calcd for [M + H]+ 265.1313; found, 265.1300. Elemental Anal. Calcd
(%) for C10H21N2O4P: C, 45.45; H, 8.01; N, 10.60. Found: C, 45.59;
H, 7.97; N, 10.61.
3-(4-Aminobutyl)-4-hydroxy-4-oxo-1-pentyl-1,4-azaphosphi-
nane-3-carboxylic Acid (26c). White solid; yield 59% (0.141 g, 0.44
mmol). 1H NMR (D2O, 400 MHZ): δ ppm 3.7−3.5 (m, 2H), 3.3 (m,
1H), 3.15−3 (2m, 5H), 2.27 (m, 1H), 2 (m, 1H), 1.85−1.6 (m, 5H),
1.5−1.41 (2m, 2H), 1.3 (m, 5H), 0.85 (t, 3H). HRMS: calcd for [M
+ H]+ 321.1939; found, 321.1933. Elemental Anal. Calcd (%) for
C14H29N2O4P: C, 52.49; H, 9.12; N, 8.74. Found: C, 52.77; H, 8.93;
N, 9.00.
3-(4-Aminobutyl)-1-(cyclohexylmethyl)-4-hydroxy-4-oxo-1,4-
azaphosphinane-3-carboxylic Acid (26d). White solid; yield 62%
(0.167 g, 0.482 mmol). 1H NMR (D2O, 400 MHZ): δ ppm 3.7−3.25
(2m, 2H), 3.55−3.15 (2dd, 2H), 3.1−2.9 (t + 2dd, 4H), 2.25 (m,
1H), 2 (m, 1H), 1.85−1.55 (m, 9H), 1.52−1.41 (2m, 2H), 1.3−1.1
(m, 4H), 1 (m, 2H). HRMS: calcd for [M + H]+ 347.2095; found,
347.2095. Elemental Anal. Calcd (%) for C16H31N2O4P: C, 55.48; H,
9.02; N, 8.09. Found: C, 55.29; H, 9.08; N, 7.95.
1
NMR (D2O, 400 MHz): δ ppm 7.5 (m, 5H), centred at 4.35 (AB,
2H), 3.75−3.35 (2m, 2H), 3.5−3.15 (2dd, 2H), 2.92 (t, 2H), 2.3−1.8
(2m, 2H), 1.95 (m, 1H), 1.6−1.4 (m, 3H). HRMS: calcd for [M + H]
327.1469; found, 327.1478. Elemental Anal. Calcd (%) for
C15H23N2O4P: C, 55.21; H, 7.10; N, 8.58. Found: C, 55.38; H,
6.85; N, 8.58.
3-(4-Aminobutyl)-1-benzyl-4-hydroxy-4-oxo-1,4-azaphosphi-
nane-3-carboxylic Acid (14b). 14b is obtained in accordance with
procedure C described hereinbefore starting from intermediate 13b.
White powder; yield 60% (1.628 g, 4.78 mmol).1H NMR (DMSO-d6,
400 MHz): δ ppm 8.2 (sl, 3H), 7.3 (m, 5H), 3.7−3.5 (2*(d, 1 + 1H),
3.05−2.4 (2*(m, 1 + 1H), 2.75−2.55 (2*(m, 1 + 1H), 2.7 (m, 2H),
1.8 (m, 1H), 1.6−1.1 (m, 7H). HRMS: calcd for [M + H]+ 341.1626;
found, 341.1628. Elemental Anal. Calcd (%) for C16H25N2O4P: C,
56.46; H, 7.40; N, 8.23. Found: C, 55.99; H, 7.14; N, 8.13.
3-(5-Aminopentyl)-1-benzyl-4-hydroxy-4-oxo-1,4-azaphosphi-
nane-3-carboxylic Acid (14c). Example 14c is obtained starting from
intermediate 13c in accordance with procedure C described
1
hereinbefore. White powder; yield 51% (0.091g, 0.256 mmol). H
NMR (D2O, 400 MHz): δ ppm 7.5 (m, 5H), centered at 4.32 (AB,
2H), 3.7−3.35 (2m, 2H), 3.5−3.1 (2dd, 2H), 2.9 (t, 2H), 2.2−1.78
(2m, 2H), 1.95−1.45 (2m, 2H), 1.6 (m, 2H), 1.3 (m, 2H), 1.1 (m,
2H). HRMS: calcdfor [M + H]+ 355.1782; found, 355.1792.
Elemental Anal. Calcd (%) for C17H27N2O4P: C, 57.62; H, 7.68; N,
7.90. Found: C, 58.04; H, 7.37; N, 7.95.
General Procedure D for the Synthesis of 26a−l, 27a−n.
tert-Butyl 3-{4-[bis(tert-Butoxycarbonyl)amino]butyl}-4-ethoxy-4-
oxo-1,4-azaphosphinane-3-carboxylate (24). Inter-mediate 13b
(73.6 g, 117.8 mmol), ethanol (1 L), Pd/C (7.36 g, 10% by mass),
and 37% HCl (7.85 mL, 0.8 equiv) are introduced in succession into a
2 L flask at ambient temperature and under a stream of argon. The
argon is then replaced by a hydrogen atmosphere. The reaction is
monitored by LC/MS. After 4 h, the reaction is complete, and the
catalyst is filtered off over a glass fiber. The filtrate is evaporated to
dryness in order to obtain a yellow oil, which is taken up in EtOAc
(400 mL) and in a 10% NaHCO3 solution (400 mL). After
decantation, the aqueous phase is extracted with EtOAc (3 × 100
mL). The organic phases are combined and then washed with a
saturated NaCl solution (400 mL), dried over MgSO4, and
concentrated to yield the expected intermediate 24 as a white solid;
1
3-(4-Aminobutyl)-4-hydroxy-4-oxo-1-(2-phenylethyl)-1,4-aza-
yield 91% (57.6 g, 107.7 mmol). H NMR (DMSO-d6, 400 MHz): δ
phosphinane-3-carboxylic Acid (26e). White solid; yield 40% (0.124
9.8/8.6 (m, 2H), 4.2/4.0 (m, 2H), 3.5−3.2 (m, 6H), 2.4−1.8 (m,
4H), 1.55−1.3 (m, 2H), 1.45 (s, 18H), 1.40 (s, 9H), 1.28 (t, 3H),
1.5/0.9 (m, 4H). IR (KBr) (cm−1): 3100−3500 cm−1 (OH), 3314
cm−1 (NH), 1712−1693 cm−1 (CO).
1
g, 0.35 mmol). H NMR (D2O, 400 MHZ): δ ppm 7.4−7.25 (m,
5H), 3.7−3.3 (2m, 2H), 3.52 (m, 1H), 3.45 (t, 2H), 3.1 (m, 3H),
2.95 (m, 2H), 2.2−1.75 (2m, 2H), 1.95 (m, 1H), 1.62 (m, 2H), 1.5−
1.1 (m, 3H). HRMS: calcd for [M + H]+ 355.1782; found, 355.1777.
Elemental Anal. Calcd (%) for C17H27N2O4P: C, 57.62; H, 7.68; N,
7.90. Found: C, 57.63; H, 7.36; N, 7.90.
The non-commercial aldehydes were prepared in accordance with
the following procedure. Ethanol (500 mL), boronic acid Ar2−
B(OH)2 (92.7 mmol, 1.2 equiv), and bromoarylaldehyde or
bromoheteroarylaldehyde Br−Ar1−CHO (77.3 mmol) are introduced
in succession into a 1 L flask under argon and at ambient temperature.
The solution is degassed with argon for 15 min. Pd(PPh3)4 (1.78 g,
1.55 mmol) and Na2CO3 (92.7 mL of a 2M solution in H2O, 185
mmol, 2.4 equiv) are then introduced in a single portion. After the
addition, the reaction mixture is heated at reflux for 5 h. The mixture
is then evaporated to dryness. The residue is taken up in DCM (1 L)
and H2O (200 mL). After decantation, the aqueous phase is extracted
with DCM (200 mL). The organic phases are combined and then
washed with a saturated NaCl solution (400 mL), dried over MgSO4
and concentrated under reduced pressure. The residue is then purified
by flash chromatography on silica gel. The expected products are
obtained in 59−94% yield.21
3-(4-Aminobutyl)-4-hydroxy-4-oxo-1-(3-phenylpropyl)-1,4-aza-
phosphinane-3-carboxylic Acid (26f). White solid; yield 66% (0.204
1
g, 0.554 mmol). H NMR (D2O, 400 MHZ): δ ppm 7.38 (t, 2H),
7.27 (m, 3H), 3.62−3.28 (2m, 2H), 3.52 (dd, 1H), 3.11 (m, 3H), 3
(td, 2H), 2.7 (t, 2H), 2.25−1.75 (2m, 2H), 2.09 (m, 2H), 1.95−1.5
(2m, 2H), 1.65 (m, 2H), 1.38−1.22 (2m, 2H). HRMS: calcd for [M
+ H]+ 369.1939; found, 369.1950. Elemental Anal. Calcd (%) for
C18H29N2O4P: C, 58.68; H, 7.93; N, 7.60. Found: C, 58.68; H, 7.45;
N, 7.54.
3-(4-Aminobutyl)-4-hydroxy-4-oxo-1-(pyridin-4-ylmethyl)-1,4-
azaphosphinane-3-carboxylic Acid (26g). White solid; yield 41%
1
(0.09 g, 0.267 mmol). H NMR (DMSO-d6, 400 MHZ): δ ppm 8.5
(d, 2H), 8.05 (m, 3H), 7.3 (d, 2H), 3.55 (AB, 2H), 3.05−2.3 (2m,
2H), 2.7 (m, 3H), 2.5 (m, 1H), 1.75 (m, 1H), 1.65−1.15 (m, 7H).
HRMS: calcd for [M + H]+ 342.1578; found, 342.1577. Elemental
Anal. Calcd (%) for C15H24N3O4P: C, 52.78; H, 7.09; N, 12.31.
Found: C, 52.56; H, 6.70; N, 12.22.
3-(4-Aminobutyl)-4-hydroxy-4-oxo-1-(pyridin-3-ylmethyl)-1,4-
azaphosphinane-3-carboxylic Acid (26h). White solid; yield 58%
(0.140 g, 0.41 mmol). 1H NMR (D2O, 400 MHZ): δ ppm 8.6 (s + d,
2H), 8 (dd, 1H), 7.52 (dd, 1H), 4.4 (AB, 2H), 3.7 (m, 1H), 3.4 (m,
24 (14 g, 26.2 mmol), anhydrous DCM (280 mL), the desired
aldehyde (39.3 mmol, 1.5 equiv), and MgSO4 (14 g) are introduced
in succession into a 500 mL three-necked flask at ambient
temperature and under a stream of argon. After stirring for 1 h,
NaBH(OAc)3 (8.32 g, 39.3 mmol, 1.5 equiv) is added in portions,
and the reaction mixture is maintained at ambient temperature for 16
h. The reaction is monitored by LC/MS. The insoluble components
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2H), 3.18 (dd, 1H), 2.9 (m, 2H), 2.28−1.8 (2m, 2H), 1.91 (m, 1H),
1.6 (m, 2H), 1.5 (m, 1H), 1.21−1.1 (2m, 2H). HRMS: calcd for [M +
H]+ 342.1578; found, 342.1568. Elemental Anal. Calcd (%) for
C15H24N3O4P: C, 52.78; H, 7.09; N, 12.31. Found: C, 52.32; H, 6.66;
N, 12.32.
7.52−7.35 (m, 4H), 4.32 (m, 2H), 3.7−3.32 (2m, 2H), 3.5−3.12
(2dd, H), 2.93 (m, 2H), 2.25−1.8 (2m, 2H), 1.95−1.48 (2m, 2H),
1.6 (m, 2H), 1.25−1.11 (2m, 2H). HRMS: calcd for [M + H]+
375.1236; found, 375.1256. Elemental Anal. Calcd (%) for
C16H24ClN2O4P: C, 51.27; H, 6.45; N, 7.47. Found: C, 51.26; H,
6.32; N, 7.43.
3-(4-Aminobutyl)-4-hydroxy-4-oxo-1-(pyridin-2-ylmethyl)-1,4-
azaphosphinane-3-carboxylic Acid (26i). White solid; yield 57%
3-(4-Aminobutyl)-1-[(2-chlorophenyl)methyl]-4-hydroxy-4-oxo-
1
(0.140 g, 0.41 mmol). H NMR (D2O, 400 MHZ): δ ppm 8.6 (dd,
1,4-azaphosphinane-3-carboxylic Acid (27e). White solid; yield
1
1H), 7.91 (t, 1H), 7.55 (d, 1H), 7.45 (dd, 1H), centered at 4.41 (AB,
2H), 3.75−3.4 (2m, 2H), 3.55−3.3 (2dd, 2H), 2.95 (m, 2H), 2.3−
1.78 (2m, 2H), 1.98 (m, 1H), 1.65 (m, 2H), 1.5 (m, 1H), 1.3−1.2
(2m, 2H). HRMS: calcd for [M + H]+ 342.1578; found, 342.1562.
Elemental Anal. Calcd (%) for C15H24N3O4P: C, 52.78; H, 7.09; N,
12.31. Found: C, 52.72; H, 6.92; N, 12.25.
64% (0.224 g, 0.598 mmol). H NMR (D2O, 400 MHZ): δ ppm
7.58−7.53 (2d, 2H), 7.48−7.4 (2m, 2H), 4.44 (m, 2H), 3.71−3.41
(2m, 2H), 3.51−3.28 (2m, 2H), 2.94 (m, 2H), 2.22−1.78 (2m, 2H),
1.95−1.61 (2m, 2H), 1.6−1.49 (2m, 2H), 1.28−1.11 (2m, 2H). 31P
NMR: (D2O, 400 MHZ): δ ppm −25.5. HRMS: calcd for [M + H]+
375.1236; found, 375.1235. Elemental Anal. Calcd (%) for
C16H24ClN2O4P: C, 51.27; H, 6.45; N, 7.47. Found: C, 51.58; H,
6.22; N, 7.65.
3-(4-Aminobutyl)-1-(furan-2-ylmethyl)-4-hydroxy-4-oxo-1,4-
azaphosphinane-3-carboxylic Acid (26j). White solid; yield 57%
1
(0.12 g, 0.363 mmol). H NMR (D2O, 400 MHZ): δ ppm 7.6 (sl,
3-(4-Aminobutyl)-1-[(4-fluorophenyl)methyl]-4-hydroxy-4-oxo-
1H), 6.7 (tf, 1H), 6.5 (tf, 1H), 4.45−4.32 (2d, 2H), 3.7−3.3 (2m,
2H), 3.52−3.12 (2m, 2H), 2.98 (m, 2H), 2.25−1.8 (2m, 2H), 1.95−
1.5 (2m, 2H), 1.62 (m, 2H), 1.32−1.2 (2m, 2H). HRMS: calcd for
[M + H]+ 331.1419; found, 331.1422. Elemental Anal. Calcd (%) for
C14H23N2O5P: C, 50.91; H, 7.02; N, 8.48. Found: C, 50.48; H, 6.48;
N, 8.37.
1,4-azaphosphinane-3-carboxylic Acid (27f). White solid; yield 72%
1
(0.21 g, 0.586 mmol). H NMR (D2O, 400 MHZ): δ ppm 7.5 (dd,
2H), 7.2 (t, 2H), 4.41−4.21 (2d, 2H), 3.7−3.31 (2m, 2H), 3.45−3.1
(2dd, 2H), 2.95 (m, 2H), 2.25−1.78 (2m, 2H), 1.95−1.5 (2m, 2H),
1.6 (m, 2H), 1.25−1.1 (2m, 2H). HRMS: calcd for [M + H]+
359.1532; found, 359.1532. Elemental Anal. Calcd (%) for
C16H24FN2O4P: C, 53.65; H, 6.75; N, 7.82. Found: C, 53.65; H,
6.20; N, 7.83.
3-(4-Aminobutyl)-1-[(2-fluorophenyl)methyl]-4-hydroxy-4-oxo-
1,4-azaphosphinane-3-carboxylic Acid (27g). White solid; yield
73% (0.243 g, 0.678 mmol).1H NMR (D2O, 300 MHZ): δ ppm 7.5
(m, 2H), 7.26 (m, 2H), 4.41 (sl, 2H), 3.7−3.35 (2m, 2H), 3.52−3.21
(2m, 2H), 2.94 (m, 2H), 2.22−1.77 (2m, 2H), 1.95−1.49 (2m, 2H),
1.61 (quint., 2H), 1.28−1.14 (2m, 2H). 19F NMR: (D2O, 300 MHZ):
δ ppm −115. 31P NMR: (D2O, 300 MHZ): δ ppm 25.6. HRMS: calcd
for [M + H]+ 359.1532; found, 359.1530. Elemental Anal. Calcd (%)
for C16H24FN2O4P: C, 53.65; H, 6.75; N, 7.82. Found: C, 53.34; H,
6.46; N, 7.77.
3-(4-Aminobutyl)-1-[(2-bromophenyl)methyl]-4-hydroxy-4-oxo-
1,4-azaphosphinane-3-carboxylic Acid (27h). White solid; yield
48% (0.188 g, 0.449 mmol). 1H NMR (D2O, 300 MHZ): δ ppm 7.75
(dl, 1H), 7.53 (dd, 1H), 7.45 (tl, 1H), 7.39 (td, 1H), 4.44 (AB, 2H),
3.74−3.43 (2m, 2H), 3.5−3.28 (2m, 2H), 2.94 (m, 2H), 2.24−1.79
(2m, 2H), 1.95−1.5 (2m, 2H), 1.6 (m, 2H), 1.26−1.1 (2m, 2H).
HRMS: calcd for [M + H]+ 419.0731; found, 419.0732. Elemental
Anal. Calcd (%) for C16H24BrN2O4P: C, 45.84; H, 5.77; N, 6.68.
Found: C, 45.33; H, 5.29; N, 7.00.
3-(4-Aminobutyl)-4-hydroxy-4-oxo-1-[(2-phenylphenyl)-methyl]-
1,4-azaphosphinane-3-carboxylic Acid (27i). White solid; yield 70%
(0.274 g, 0.658 mmol).1H NMR (DMSO-d6, 400 MHZ): δ ppm
7.65−7.3 (m, 6H), 4.33 (AB, 2H), 3.43−3 (m, 3H), 2.92 (dd, 1H),
2.82 (m, 2H), 2.06−1.57 (2m, 2H), 1.83−1.33 (2m, 2H), 1.57 (m,
2H), 1.08 (m, 2H). HRMS: calcd for [M + H]+ 417.1939; found,
417.1945. Elemental Anal. Calcd (%) for C22H29N2O4P: C, 63.68; H,
7.02; N, 6.73. Found: C, 63.68; H, 6.84; N, 6.85.
3-(4-Aminobutyl)-1-(furan-3-ylmethyl)-4-hydroxy-4-oxo-1,4-
azaphosphinane-3-carboxylic Acid (26k). White solid; yield 38%
1
(0.105 g, 0.318 mmol). H NMR (D2O, 400 MHZ): δ ppm 7.71 (sl,
1H), 7.59 (sl, 1H), 6.58 (sl, 1H), 4.22 (m, 2H), 3.7−3.35 (2m, 2H),
3.55−3.08 (2dd, 2H), 2.95 (m, 2H), 2.25−1.8 (2m, 2H), 1.95−1.5
(2m, 2H), 1.62 (m, 2H), 1.31−1.18 (2m, 2H). HRMS: calcd for [M
+ H]+ 331.1419; found, 331.1431. Elemental Anal. Calcd (%) for
C14H23N2O5P: C, 50.91; H, 7.02; N, 8.48. Found: C, 50.45; H, 6.72;
N, 8.39.
3-(4-Aminobutyl)-4-hydroxy-4-oxo-1-(thiophen-3-ylmethyl)-1,4-
azaphosphinane-3-carboxylic Acid (26l). White solid; yield 31 %
1
(0.201 g, 0.58 mmol). H NMR (D2O, 400 MHZ): δ ppm 7.61 (d,
1H), 7.54 (dd, 1H), 7.2 (d, 1H), 4.42−4.28 (2*d, 2H), 3.71−3.32
(m, 2H), 3.48−3.06 (m, 2H), 2.94 (m, 2H), 2.25−1.77 (m, 2H),
1.93−1.46 (m, 2H), 1.6 (m, 2H), 1.26−1.13 (m, 2H). HRMS: calcd
for [M + H]+ 347.1190; found, 347.1182. Elemental Anal. Calcd (%)
for C14H23N2O4PS: C, 48.55; H, 6.69; N, 8.09; S, 9.26. Found: C,
48.55; H, 6.55; N, 7.87; S, 8.65.
3-(4-Aminobutyl)-4-hydroxy-1-[(4-methoxyphenyl)methyl]-4-
oxo-1,4-azaphosphinane-3-carboxylic Acid (27a). White solid;
1
yield 59% (0.180 g, 0.486 mmol). H NMR (D2O, 400 MHZ): δ
ppm 7.41 (d, 2H), 7.02 (d, 2H), 4.25 (m, 2H), 3.8 (s, 3H), 3.7−3.3
(2m, 2H), 3.45−3.08 (2dd, 2H), 2.95 (m, 2H), 2.25−1.75 (2m, 2H),
1.95−1.45 (2m, 2H), 1.6 (m, 2H), 1.25−1.1 (2m, 2H). HRMS: calcd
for [M + H]+ 371.1732; found, 371.1719. Elemental Anal. Calcd (%)
for C17H27N2O5P: C, 55.13; H, 7.35; N, 7.56. Found: C, 54.93; H,
7.41; N, 7.56.
3-(4-Aminobutyl)-4-hydroxy-1-[(4-hydroxyphenyl)methyl]-4-
oxo-1,4-azaphosphinane-3-carboxylic Acid (27b). White solid;
1
yield 56% (0.135 g, 0.379 mmol). H NMR (D2O, 400 MHZ): δ
3-(4-Aminobutyl)-4-hydroxy-4-oxo-1-[(3-phenylphenyl)methyl]-
1,4-azaphosphinane-3-carboxylic Acid (27j). White solid; yield 40%
(0.157 g, 0.377 mmol). H NMR (D2O, 400 MHZ): δ ppm 7.79−7.4
(m, 9H), 4.49 (m, 1H), 4.29 (d, 1H), 3.76 (m, 1H), 3.51 (dd, 1H),
3.39 (m, 1H), 3.13 (dd, 1H), 2.89 (m, 2H), 2.27 (m, 1H), 1.92 (m,
1H), 1.79 (m, 1H), 1.58 (m, 2H), 1.48 (m, 1H), 1.21 (m, 1H), 1.09
(m, 1H). HRMS: calcd for [M + H]+ 417.1939; found, 417.1937.
Elemental Anal. Calcd (%) for C22H29N2O4P: C, 63.68; H, 7.02; N,
6.73. Found: C, 63.45; H, 6.72; N, 6.86.
ppm 7.38 (d, 2H), 6.95 (d, 2H), 4.25 (m, 2H), 3.7−3.3 (2m, 2H),
3.48−3.05 (2dd, 2H), 2.95 (m, 2H), 2.21−1.75 (2m, 2H), 1.95−1.5
(2m, 2H), 1.6 (m, 2H), 1.25−1.11 (2m, 2H). HRMS: calcd for [M +
H]+ 371.1732; found, 371.1712. Elemental Anal. Calcd (%) for
C16H25N2O5P: C, 53.93; H, 7.07; N, 7.86. Found: C, 53.24; H, 6.89;
N, 7.57.
3-(4-Aminobutyl)-1-[(4-chlorophenyl)methyl]-4-hydroxy-4-oxo-
1,4-azaphosphinane-3-carboxylic Acid (27c). White solid; yield
68% (0.205 g, 0.545 mmol). 1H NMR (D2O, 400 MHZ): δ ppm 7.5−
7.45 (2d, 4H), 4.41−4.21 (2d, 2H), 3.7−3.33 (2m, 2H), 3.45−3.1
(2dd, 2H), 2.95 (m, 2H), 2.25−1.78 (2m, 2H), 1.95−1.5 (2m, 2H),
1.6 (m, 2H), 1.25−1.1 (2m, 2H). HRMS: calcd for [M + H]+
375.1236; found, 375.1242. Elemental Anal. Calcd (%) for
C16H24ClN2O4P: C, 51.27; H, 6.45; N, 7.47. Found: C, 50.89; H,
6.01; N, 7.44.
3-(4-Aminobutyl)-4-hydroxy-4-oxo-1-[(4-phenylphenyl)-methyl]-
1,4-azaphosphinane-3-carboxylic Acid (27k). White solid; yield
1
50% (0.175 g, 0.42 mmol). H NMR (D2O, 400 MHZ): δ ppm 7.8
(d, 2H), 7.7 (d, 2H), 7.55 (d, 2H), 7.5 (t, 2H), 7.41 (t, 1H), 4.45−
4.28 (2d, 2H), 3.75−3.35 (2m, 2H), 3.5−3.15 (2dd, 2H), 2.9 (m,
2H), 2.25−1.8 (2m, 2H), 1.95−1.45 (2m, 2H), 1.6 (m, 2H), 1.25−
1.1 (2m, 2H). HRMS: calcd for [M + H]+ 417.1939; found, 417.1932.
Elemental Anal. Calcd (%) for C22H29N2O4P: C, 63.68; H, 7.02; N,
6.73. Found: C, 63.25; H, 6.64; N, 6.55.
3-(4-Aminobutyl)-1-[(3-chlorophenyl)methyl]-4-hydroxy-4-oxo-
1,4-azaphosphinane-3-carboxylic Acid (27d). White solid; yield
1
43% (0.123 g, 0.328 mmol). H NMR (D2O, 400 MHZ): δ ppm
3906
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Article
3-(4-Aminobutyl)-4-hydroxy-4-oxo-1-[(2-phenoxyphenyl)-meth-
yl]-1,4-azaphosphinane-3-carboxylic Acid (27l). White solid; yield
61% (0.249 g, 0.515 mmol). 1H NMR (D2O, 300 MHZ): δ ppm 7.47
(t, 1H), 7.41 (t, 2H), 7.27−7.09 (m, 4H), 7.05 (d, 2H), 4.27 (m,
2H), 3.68−3.3 (2m, 2H), 3.46−3.07 (2m, 2H), 2.91 (m, 2H), 2.23−
1.76 (2m, 2H), 1.93−1.46 (2m, 2H), 1.6 (quint., 2H), 1.15 (m, 2H).
31P NMR: (D2O, 300 MHZ): δ ppm 25.8. HRMS: calcd for [M + H]+
433.1888; found, 433.1894. Elemental Anal. Calcd (%) for
C22H29N2O5P: C, 61.51; H, 6.76; N, 6.48. Found: C, 61.51; H,
6.57; N, 6.67.
2H), 1.05 (m, 2H). HRMS: calcd for [M + H]+ 485.1159; found,
485.1172. Elemental Anal. Calcd (%) for C22H27Cl2N2O4P: C, 54.44;
H, 5.61; N, 5.77. Found: C, 55.11; H, 5.26; N, 5.87. RP: −17.410
(589 nm, T = 19 °C, C = 1.0).
(3S)-3-(4-Aminobutyl)-1-[(4-fluoro-2-phenylphenyl)-methyl]-4-
hydroxy-4-oxo-1,4-azaphosphinane-3-carboxylic Acid (28e). White
solid; yield 41% (0.141 g, 0.324 mmol). 1H NMR (D2O, 300 MHZ):
δ ppm 7.6 (dd, 1H), 7.49 (m, 3H), 7.34 (m, 2H), 7.22 (td, 1H), 7.15
(dd, 1H), 4.31 (AB, 2H), 3.32−3.06 (2m, 2H), 3.16−2.8 (2m, 2H),
2.92 (m, 2H), 2.05−1.6 (2m, 2H), 1.82−1.33 (2m, 2H), 1.56 (m,
2H), 1.07 (m, 2H) | NMR 19F (D2O, 300 MHZ): δ ppm −111.5.
HRMS: calcd for [M + H]+ 435.1845; found, 435.1842. Elemental
Anal. Calcd (%) for C22H28FN2O4P: C, 60.82; H, 6.50; N, 6.45.
Found: C, 60.79; H, 6.07; N, 6.19.
(3S)-3-(4-Aminobutyl)-1-[(4-fluoro-2-phenylphenyl)-methyl]-4-
hydroxy-4-oxo-1,4-azaphosphinane-3-carboxylic Acid (28f). White
solid; yield 72% (0.348 g, 0.797 mmol). 1H NMR (D2O, 300 MHZ):
δ ppm 7.6 (dd, 1H), 7.49 (m, 3H), 7.34 (m, 2H), 7.22 (td, 1H), 7.15
(dd, 1H), 4.31 (AB, 2H), 3.32−3.06 (2m, 2H), 3.16−2.8 (2m, 2H),
2.92 (m, 2H), 2.05−1.6 (2m, 2H), 1.82−1.33 (2m, 2H), 1.56 (m,
2H), 1.07 (m, 2H). 19F NMR: (D2O, 300 MHZ): δ ppm −111.5.
HRMS: calcd for [M + H]+ 437.1750; found, 437.1754. Elemental
Anal. Calcd (%) for C20H26FN4O4P: C, 60.82; H, 6.50; N, 6.45.
Found: C, 60.82; H, 6.07; N, 6.19.
3-(4-Aminobutyl)-4-hydroxy-4-oxo-1-[(2-pyridin-3-ylphenyl)-
methyl]-1,4-azaphosphinane-3-carboxylic Acid (27m). White solid;
1
yield 67% (0.2 g, 0.479 mmol). H NMR (D2O, 400 MHZ): δ ppm
8.58 (dd, 1H), 8.51 (d, 1H), 7.86 (dt, 1H), 7.67 (m, 1H), 7.58 (m,
1H), 7.58 (m, 2H), 7.43 (m, 1H), 4.42−4.3 (2*d, 2H), 3.46−3.11
(m, 2H), 3.2−2.87 (m, 2H), 2.94 (m, 2H), 2.11−1.65 (m, 2H),
1.86−1.36 (m, 2H), 1.59 (m, 2H), 1.08 (m, 2H). MS HRMS: calcd
for [M + H]+ 418.1891; found, 418.1898. Elemental Anal. Calcd (%)
for C21H28N3O4P: C, 60.42; H, 6.76; N, 10.07; Found: C, 60.58; H,
6.51; N, 10.09.
3-(4-Aminobutyl)-4-hydroxy-4-oxo-1-[(2-pyrimidin-5-ylphenyl)-
methyl]-1,4-azaphosphinane-3-carboxylic Acid (27n). White solid;
yield 67% (0.187 g, 0.447 mmol). 1H NMR (D2O, 400 MHZ): δ ppm
9.19 (s, 1H), 8.85 (s, 2H), 7.7 (m, 1H), 7.68−7.59 (m, 2H), 7.45 (m,
1H), 4.38 (dd, 2H), 3.52−3.15 (2m, 2H), 3.25−2.9 (2m, 2H), 2.9
(m, 2H), 2.1−1.7 (2m, 2H), 1.85−1.4 (2m, 2H), 1.6 (m, 2H), 1.1
(m, 2H). HRMS: calcd for [M + H]+ 419.1844; found, 419.1856.
Elemental Anal. Calcd (%) for C20H27N4O4P: C, 57.41; H, 6.50; N,
13.39. Found: C, 57.94; H, 6.37; N, 13.40.
General Procedure E for the Synthesis of 28a−s. Examples
28a−s are obtained starting from (S)-24 in accordance with the
procedure D described hereinbefore. The yields given are for the last
2 steps of their synthesis that is from their corresponding enantiopure
intermediates (S)-25a−s.
(3S)-3-(4-Aminobutyl)-4-hydroxy-1-[2-(6-methoxypyridin-3-yl)-
benzyl]-4-oxo-1,4-azaphosphinane-3-carboxylic Acid (28a). White
solid; yield 91% (6.11 g, 13.654 mmol). 1H NMR (D2O, 400 MHZ):
δ ppm 8.05 (d, 1H), 7.75 (dd, 1H), 7.6 (m, 1H), 7.5 (m, 2H), 7.4 (m,
1H), 7 (d, 1H), 4.3 (dd, 2H), 3.9 (s, 3H), 3.45−3.15 (2m, 2H), 3.2−
2.85 (2dd, 2H), 2.9 (m, 2H), 2.1−1.7 (2m, 2H), 1.8−1.35 (2m, 2H),
1.6 (m, 2H), 1.1 (m, 2H). HRMS: calcd for [M + H]+ 448.1997;
found, 448.2006. Elemental Anal. Calcd (%) for C22H30N3O5P: C,
59.05; H, 6.76; N, 9.39. Found: C, 58.81; H, 6.79; N, 9.31. RP:
−11.510 (589 nm, T = 19 °C, C = 0.9).
(3S)-3-(4-Aminobutyl)-4-hydroxy-1-[[2-[2-methyl-5-
(trifluoromethyl)pyrazol-3-yl]phenyl]methyl]-4-oxo-1,4-azaphos-
phinane-3-carboxylic Acid (28b). White solid; yield 90% (1.304 g,
2.67 mmol). 1H NMR (D2O, 300 MHZ): δ ppm 7.63−7.46 (m, 4H),
6.81 (s, 1H), 4.21 (m, 2H), 3.65 (s, 3H), 3.5−3.19 (2m, 2H), 3.26−3
(2m, 2H), 2.91 (m, 2H), 2.15−1.69 (2m, 2H), 1.87−1.4 (2m, 2H),
1.58 (m, 2H), 1.11 (m, 2H) | NMR 19F (D2O, 300 MHZ): δ ppm
−61.8. HRMS: calcd for [M + H]+ 489.1874; found, 489.1903.
Elemental Anal. Calcd (%) for C21H28F3N4O4P: C, 51.64; H, 5.78; N,
11.25. Found: C, 51.66; H, 5.61; N, 11.25. RP: −11.510 (589 nm, T =
19 °C, C = 0.9).
(3S)-3-(4-Aminobutyl)-4-hydroxy-1-[(4-hydroxy-2-phenyl-
phenyl)methyl]-4-oxo-1,4-azaphosphinane-3-carboxylic Acid
(28c). White solid; yield 76% (0.78 g, 1.083 mmol). 1H NMR
(D2O, 400 MHZ): δ ppm 7.54−7.44 (m, 3H), 7.54−7.44 (m, 1H),
7.34 (d, 2H), 6.97 (dd, 1H), 6.86 (df, 1H), 4.34/4.19 (2d, 2H),
3.33−3.05 (2m, 2H), 3.19−2.78 (2m, 2H), 2.95 (m, 2H), 2.07−1.63
(2m, 2H), 1.85−1.35 (2m, 2H), 1.59 (m, 2H), 1.2−1 (2m, 2H).
HRMS: calcd for [M + H]+ 433.1888; found, 433.1888. Elemental
Anal. Calcd (%) for C22H29N2O5P: C, 61.10; H, 6.76; N, 6.48. Found:
C, 60.81; H, 6.31; N, 6.49.
(3S)-3-(4-Aminobutyl)-1-[(4-fluoro-2-pyrimidin-5-ylphenyl)-
methyl]-4-hydroxy-4-oxo-1,4-azaphosphinane-3-carboxylic Acid
1
(28g). White solid; yield 86% (2.7 g, 6.02 mmol). H NMR (D2O,
400 MHZ): δ ppm 7.61 (dd, 1H), 7.36 (d, 2H), 7.26 (d, 2H), 7.23
(td, 1H), 7.15 (dd, 1H), 4.41/4.26 (2*d, 2H), 3.35−3.09 (m, 2H),
3.17−2.81 (m, 2H), 2.95 (m, 2H), 2.37 (s, 3H), 2.08−1.64 (m, 2H),
1.85−1.35 (m, 2H), 1.6 (m, 2H), 1.13−1.05 (m, 2H). HRMS: calcd
for [M + H]+ 449.2001; found, 449.2005. Elemental Anal. Calcd (%)
for C23H30FN2O4P: C, 61.60; H, 6.74; N, 6.25. Found: C, 61.57; H,
6.53; N, 6.45. RP: −15.640 (589 nm, T = 20 °C, C = 1.0).
(3S)-3-(4-Aminobutyl)-1-[[4-fluoro-2-(4-methoxyphenyl)-
phenyl]methyl]-4-hydroxy-4-oxo-1,4-azaphosphinane-3-carbox-
1
ylic Acid (28h). White solid; yield 84% (0.492 g, 1.059 mmol). H
NMR (D2O, 300 MHZ): δ ppm 7.57 (dd, 1H), 7.3 (d, 2H), 7.19 (td,
1H), 7.12 (dd, 1H), 7.08 (d, 2H), 4.32 (AB, 2H), 3.84 (s, 3H), 3.33−
3.07 (2m, 2H), 3.13−2.8 (2m, 2H), 2.92 (m, 2H), 2.05−1.62 (2m,
2H), 1.83−1.32 (2m, 2H), 1.57 (m, 2H), 1.05 (m, 2H). HRMS: calcd
for [M + H]+ 465.1950; found, 465.1960. Elemental Anal. Calcd (%)
for C23H30FN2O5P: C, 59.48; H, 651; N, 6.03. Found: C, 59.41; H,
6.76; N, 6.09. RP: −15.640 (589 nm, T = 20 °C, C = 1.0).
(3S)-3-(4-Aminobutyl)-1-[[2-fluoro-6-(4-methoxyphenyl)-
phenyl]methyl]-4-hydroxy-4-oxo-1,4-azaphosphinane-3-carbox-
1
ylic Acid (28i). White solid; yield 74% (0.240 g, 0.517 mmol). H
NMR (D2O, 300 MHZ): δ ppm 7.54 (m, 1H), 7.3 (d, 2H), 7.28 (m,
1H), 7.21 (d, 1H), 7.11 (d, 2H), 4.42 (dd, 2H), 3.86 (s, 3H), 3.46−
3.09 (m, 2H), 3.2−2.9 (m, 2H), 2.95 (m, 2H), 2.11−1.67 (m, 2H),
1.87−1.37 (m, 2H), 1.6 (m, 2H), 1.11 (m, 2H). HRMS: calcd for [M
+ H]+ 465.1950; found, 465.1955. Elemental Anal. Calcd (%) for
C23H30FN2O5P: C, 59.48; H, 6.51; N, 6.03. Found: C, 59.31; H, 6.75;
N, 6.10.
(3S)-3-(4-Aminobutyl)-1-[[2-(3,4-dimethoxyphenyl)-4-
fluorophenyl]methyl]-4-hydroxy-4-oxo-1,4-azaphosphinane-3-car-
boxylic Acid (28j). White solid; yield 87% (0.235 g, 0.475 mmol). 1H
NMR (D2O, 300 MHZ): δ ppm 7.6 (dd, 1H), 7.2 (td, 1H), 7.1 (m,
1H), 7 (dd, 1H), 6.9 (dd, 1H), 4.4/4.25 (2 d, 2H), 3.8 (2 s, 6H), 3.35
(m, 1H), 3.2−2.75 (m, 5H), 2.05 (m, 1H), 1.8 (m, 1H), 1.6 (m, 3H),
1.3 (m, 1H), 1 (m, 2H). HRMS: calcd for [M + H]+ 495.2056; found,
495.2056. Elemental Anal. Calcd (%) for C24H32FN2O6P: C, 58.29;
H, 6.52; N, 5.67. Found: C, 58.64; H, 6.44; N, 5.72. RP: −23.170
(589 nm, T = 21 °C, C = 0.8).
(3S)-3-(4-Aminobutyl)-1-[4-fluoro-2-(1-methyl-1H-imidazole-5-
yl)benzyl]-4-hydroxy-4-oxo-1,4-azaphosphinane-3-carboxylic Acid
(3S)-3-(4-Aminobutyl)-1-[[4-chloro-2-(4-chlorophenyl)-phenyl]-
methyl]-4-hydroxy-4-oxo-1,4-azaphosphinane-3-carboxylic Acid
(28d). White solid; yield 66% (0.267 g, 0.55 mmol). 1H NMR
(D2O, 400 MHZ): δ ppm 7.55−7.5 (2d, 2H), 7.5 (d, 2H), 7.4 (d,
1H), 7.28 (d, 2H), 4.3 (dd, 2H), 3.35−3.05 (2m, 2H), 3.15−2.8 (2m,
2H), 2.9 (m, 2H), 2.05−1.65 (2m, 2H), 1.8−1.3 (2m, 2H), 1.55 (m,
1
(28k). White solid; yield 72% (6.1g, 13.913 mmol). H NMR (D2O,
400 MHZ): δ ppm 7.8 (s, 1H), 7.68 (dd, 1H), 7.33 (td, 1H), 7.22
(dd, 1H), 7.1 (s, 1H), 4.25/4.18 (AB, 2H), 3.6−3.2 (2m, 2H), 3.45
(s, 3H), 3.3−3 (2m, 2H), 2.95 (m, 2H), 2.12−1.71 (2m, 2H), 1.9−
1.42 (2m, 2H), 1.6 (m, 2H), 1.3−1 (m, 2H).19F NMR: (D2O, 400
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MHZ): δ ppm −109.75. HRMS: calcd for [M + H]+ 439.1906; found,
439.1905. Elemental Anal. Calcd (%) for C20H28FN4O4P: C, 54.79;
H, 6.44; N, 12.78. Found: C, 54.41; H, 6.12; N, 12.74. RP: −27.720
(589 nm, T = 20 °C, C = 0.7).
2H), 1.88−1.37 (2m, 2H), 1.61 (m, 2H), 1.11 (m, 2H). 19F NMR:
(D2O, 300 MHZ): δ ppm −111). HRMS: calcd for [M + H]+
436.1797; found, 436.1794. Elemental Anal. Calcd (%) for
C21H27FN3O4P: C, 57.93; H, 6.25; N, 9.65. Found: C, 57.69; H,
5.69; N, 9.60. RP: −19.680 (589 nm, T = 20 °C, C = 0.9).
(3S)-3-(4-Aminobutyl)-1-[[2-(4-chlorophenyl)-4-fluoro-phenyl]-
methyl]-4-hydroxy-4-oxo-1,4-azaphosphinane-3-carboxylic Acid
(28s). White solid; yield 90% (0.374 g, 0.798 mmol). 1H NMR
(D2O, 400 MHZ): δ ppm 7.62 (dd, 1H), 7.54 (d, 2H), 7.35 (d, 2H),
7.25 (td, 1H), 7.17 (dd, 1H), 4.39/4.28 (2d, 2H), 3.4−3.09 (2m,
2H), 3.13−2.84 (2m, 2H), 2.95 (m, 2H), 2.09−1.65 (2m, 2H), 1.86−
1.36 (2m, 2H), 1.6 (m, 2H), 1.09 (m, 2H). HRMS: calcd for [M +
H]+ 469.1455; found, 469.1452. Elemental Anal. Calcd (%) for
C21H27FN3O4P: C, 56.35; H, 5.80; N, 5.97. Found: C, 56.19; H, 5.57;
N, 5.97. RP: −12.560 (589 nm, T = 20 °C, C = 0.9).
(3S)-3-(4-Aminobutyl)-4-hydroxy-1-[[2-(4-methylphenyl)-
phenyl]methyl]-4-oxo-1,4-azaphosphinane-3-carboxylic Acid (28l).
1
White solid; yield 68% (0.272 g, 0.632 mmol). H NMR (D2O, 400
MHZ): δ ppm 7.6−7.35 (m, 4H), 7.33−7.22 (2d, 4H), 4.33 (AB,
2H), 3.19−2.8 (2m, 2H), 2.91 (m, 2H), 2.35 (s, 3H), 2.06−1.58 (2m,
2H), 1.82−1.32 (2m, 2H), 1.57 (m, 2H), 1.07 (m, 2H). HRMS: calcd
for [M + H]+ 431.2095; found, 431.2097. Elemental Anal. Calcd (%)
for C23H31N2O4P: C, 64.17; H, 7.26; N, 6.51. Found: C, 64.03; H,
6.92; N, 6.45. RP: −13.150 (589 nm, T = 20 °C, C = 0.9).
(3S)-3-(4-Aminobutyl)-4-hydroxy-4-oxo-1-[[2-(2-oxo-1H-pyridin-
4-yl)phenyl]methyl]-1,4-azaphosphinane-3-carboxylic Acid (28m).
1
White solid; yield 84% (0.321 g, 0.741 mmol). H NMR (D2O, 300
Pharmacology Materials and Methods. IC50 Evaluation on
Human TAFIa. Human TAFI (4 nM, Calbiochem) was incubated
with human thrombin (10 nM, Sigma) and human thrombomodulin
(5 nM, American Diagnostica) in the presence of calcium (10 mM).
After incubation for 20 min, the activation reaction was stopped by
addition of PPACK (1 μM final), an irreversible thrombin inhibitor.
The reactions took place in Hepes buffer (25 mM Hepes, 137 mM
NaCl, 3.5 mM KCl) + 0.1% bovine albumin, pH = 7.4 at 28 °C and
with stirring. The test compound was added to the solution of TAFIa
(2 nM) and incubated for 45 min in the presence of hippuryl-arginine
(5 mM). The reaction was stopped by addition of hydrochloric acid
(1 M) neutralized subsequently with sodium hydroxide (1 M), and
then the mixture was buffered with disodium hydroxyphosphate (1 M
pH = 7.4). The reaction producthippuric acidwas revealed by
addition of cyanuric chloride (6%). The reaction mixture was stirred
(vortex) and then centrifuged. The supernatant was transferred to a
96-well microplate, and the absorbance was measured using a
spectrophotometer at 405 nm (Spectramax plus, Molecular Devices).
The OD value of a well containing the reagents without TAFI was
subtracted from all the measured OD values. The percentage
inhibition of TAFIa at a given concentration of the tested compound
was determined by means of the following formula: % inhibition =
100 − [(OD compound × 100)/OD carrier]. Data were analyzed
according to a one-phase exponential decay function to evaluate IC50
(n = 2/compound).
Ki Evaluation on Human TAFIa, CPN, and CPB. All the reactions
were performed at 25 °C in Hepes buffer (hepes 25 mM, NaCl 137
mM, KCl 3.5 mM) + 0.1% bovine serum albumin at pH = 7.4 and
under stirring. Human pancreatic CPB (2 nM, Sigma) and 28k (0,
2.5, 5, 10 μM) or human plasma CPN (20 nM, Elastin Product
Company) and 28k (0, 100, 200, 400 μM) or activated TAFI (as
previously described) and 28k (0, 1, 2, 4 nM) were mixed in the
presence of different concentrations of hippuryl-Arginine (from 0.1 to
12 mM, Sigma). The reaction was performed for 25 (for CPB and
CPN) or 45 min (for TAFIa) at 25 °C, then stopped by addition of
HCl (1 M), and then neutralized by addition of sodium hydroxide (1
M). The reaction was then buffered by addition of di-sodium
hydroxyphosphate (1 M, pH = 7.4). Hippuric acid was detected by
addition of cyanuric chloride. After centrifugation (13,200 rpm), the
supernatant was recovered and transferred in a 96-well plate. The
absorbance (405 nm) of the wells without CPB or CPN (blank) was
subtracted to samples for each concentration of the substrate. The
calculation of Ki (n = 5) was evaluated by the method of apparent
Km.23,24
MHZ): δ ppm 7.65−7.3 (m, 4H), 7.6 (d, 1H), 6.55 (s, 1H), 6.5 (d,
1H), 4.35 (dd, 2H), 3.5 (2m, 2H), 3.25−2.9 (2m, 2H), 2.9 (m, 2H),
2.15−1.7 (2m, 2H), 1.85−1.4 (2m, 2H), 1.6 (m, 2H), 1.1 (m, 2H).
MS HRMS: calcd for [M + H]+ 434.1841; found, 434.1838.
Elemental Anal. Calcd (%) for C21H28N3O5P: C, 58.19; H, 6.51; N,
9.69. Found: C, 58.88; H, 6.31; N, 9.95. RP: −10.530 (589 nm, T =
19 °C, C = 1.0).
(3S)-3-(4-Aminobutyl)-1-[[4-fluoro-2-(2-oxo-1H-pyridin-4-yl)-
phenyl]-methyl]-4-hydroxy-4-oxo-1,4-azaphosphinane-3-carbox-
1
ylic Acid (28n). White solid; yield 86% (0.480 g, 1.063 mmol). H
NMR (D2O, 400 MHZ): δ ppm 7.64 (dd, 1H), 7.62 (d, 1H), 7.29
(td, 1H), 7.18 (dd, 1H), 6.57 (d, 1H), 6.53 (dd, 1H), 4.3 (dd, 2H),
3.51−3.15 (2m, 2H), 3.24−2.95 (2m, 2H), 2.92 (m, 2H), 2.13−1.7
(2m, 2H), 1.86−1.38 (2m, 2H), 1.58 (m, 2H), 1.1 (m, 2H). 19F
NMR: (D2O, 400 MHZ): δ ppm −109.6. HRMS: calcd for [M + H]+
452.1746; found, 452.1745. Elemental Anal. Calcd (%) for
C21H27FN3O5P: C, 55.87; H, 6.03; N, 9.31. Found: C, 56.06; H,
5.96; N, 9.21. RP: −10.270 (589 nm, T = 19 °C, C = 0.9).
(3S)-3-(4-Aminobutyl)-1-[[4-fluoro-2-(6-methoxypyridin-3-yl)-
phenyl]methyl]-4-hydroxy-4-oxo-1,4-azaphosphinane-3-carbox-
1
ylic Acid (28o). White solid; yield 73% (0.306 g, 0.657 mmol). H
NMR (D2O, 400 MHZ): δ ppm 8.2 (d, 1H), 7.6 (dd, 1H), 7.3 (td,
1H), 7.2 (dd, 1H), 7.0 (dd, 1H), 6.9 (s, 1H), 4.3 (dd, 2H), 3.9 (s,
3H), 3.4−3.1 (2m, 2H), 3.2−2.8 (2m, 2H), 2.95 (m, 2H), 2.1−1.7
(2m, 2H), 1.85−1.35 (2m, 2H), 1.6 (m, 2H), 1.07 (m, 2H). HRMS:
calcd for [M + H]+ 466.1903; found, 466.1903. Elemental Anal. Calcd
(%) for C22H29FN3O5P: C, 56.77; H, 6.28; N, 9.03. Found: C, 57.18;
H, 6.17; N, 9.05. RP: −11.850 (589 nm, T = 20 °C, C = 0.9).
(3S)-3-(4-Aminobutyl)-1-[[2-(2,3-dimethylimidazol-4-yl)-phenyl]-
methyl]-4-hydroxy-4-oxo-1,4-azaphosphinane-3-carboxylic Acid
(28p). White solid; yield 35% (0.157 g, 0.361 mmol). 1H NMR
(D2O, 400 MHZ): δ ppm 7.75−7.45 (m, 4H), 7.45 (s, 1H), 4.25 (m,
2H), 3.5−3.2 (2m, 2H), 3.4−3.1 (m, 2H), 3.4 (s, 3H), 2.9 (m, 2H),
2.65 (s, 3H), 2.1−1.7 (2m, 2H), 1.9−1.45 (2m, 2H), 1.6 (m, 2H),
1.25−1.1 (2m, 2H). HRMS: calcd for [M + H]+ 435.2157; found,
435.2179. Elemental Anal. Calcd (%) for C21H31N4O4P: C, 50.37; H,
5.88; N, 10.21. Found: C, 49.57; H, 5.92; N, 9.92. RP: −23.310 (589
nm, T = 20 °C, C = 0.9).
(3S)-3-(4-Aminobutyl)-1-[2-(1,2-dimethyl-1H-imidazole-5-yl)-4-
fluorobenzyl]-4-hydroxy-4-oxo-1,4-azaphosphinane-3-carboxylic
1
Acid (28q). White solid; yield 56% (0.2 g, 0.442 mmol). H NMR
(D2O, 400 MHZ): δ ppm 7.75 (dd, 1H), 7.55 (s, 1H), 7.5 (m, 1H),
7.3 (dd, 1H), 4.25 (s, 2H), 3.55 (m, 1H), 3.45 (s, 3H), 3.4 (m, 1H),
3.25 (m, 1H), 3.15 (dd, 1H), 2.95 (t, 2H), 2.65 (s, 3H), 2.15 (m,
1H), 1.9 (m, 1H), 1.75 (m, 1H), 1.65 (m, 2H), 1.45 (m, 1H), 1.3 (m,
a1H), 1.15 (m, 1H). HRMS: calcd for [M + H]+ 453.2063; found,
453.2058. Elemental Anal. Calcd (%) for C21H30FN4O4P: C, 47.29;
H, 5.86; N, 10.50. Found: C, 46.18; H, 5.24; N, 10.02. RP: −28.200
(589 nm, T = 20 °C, C = 0.9).
Animals. Experiments were performed on adult male Sprague
Dawley (SD) or Wistar rats (Charles River). All experiments were
conducted in accordance with local ethical laws and local ethics
committees in accordance with the French regulations (Decree n°
2013-118 from 01 February 2013 relative to the protection of animals
used for scientific purposes and 4 orders of 01 February 2013).
In Vitro Fibrinolytic Activity of Compounds by Thrombo-
Elastometry (TE) on SD Rat Blood. Fresh rat whole blood (480
μL) was preincubated for 2 min at 37 °C with 0.8 μM or different
concentrations of compounds (500 μL final) in a plastic cup. A
mixture of 20 μL of CaCl2 (0.2M, starTEM, ROTEM) and 20 μL of
(3S)-3-(4-Aminobutyl)-1-[(4-fluoro-2-pyridin-3-ylphenyl)-meth-
yl]-4-hydroxy-4-oxo-1,4-azaphosphinane-3-carboxylic Acid (28r).
1
White solid; yield 82% (0.303 g, 0.696 mmol). H NMR (D2O, 300
MHZ): δ ppm 8.62 (dd, 1H), 8.54 (d, 1H), 7.89 (dt, 1H), 7.7 (dd,
1H), 7.6 (dd, 1H), 7.34 (td, 1H), 7.23 (dd, 1H), 4.35 (m, 2H), 3.46−
3.12 (2m, 2H), 3.18−2.87 (2m, 2H), 2.97 (m, 2H), 2.12−1.67 (2m,
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Article
tissue factor (ex-TEM, ROTEM) was then added. Human tPA [16
nM, (Alteplase, Boehringer Ingelheim)] was added, and the cups were
placed in the rotational thromboelastometer (ROTEM). ROTEM
continuously records viscoelastic profiles of blood clot formation/
dissolution by measuring the viscoelastic changes by a pin which
oscillates into the blood. Fibrin formation and fibrinolysis were
followed for 3 h at 37 °C. The clot firmness was measured as the area
under the curve (AUC) of the amplitude in function of time
(thrombo-elastogram). The AUC was computed using the trapezoid
rule (software GraphPad Prism 5). The percentage of inhibition of
AUC (vs vehicle) was evaluated for 0.8 μM of compounds, n = 2/
compound. For dose response experiments, the concentration which
decreases by 50% the AUC (“EC50”) was calculated (n = 1−5).
Ex Vivo Fibrinolytic Activity of Compounds by TE in Rat.
Experiments were performed on adult male SD rats (350−450 g).
Rats were anesthetized [ketamine (75 mg/kg IP) (Merial)/xylazine
(5 mg/kg IP) (Bayer Healthcare)] and maintained anesthetized all
along the experiment by an IV infusion (ketamine 50 mg/kg/h,
xylazine 3 mg/kg/h). The corporal temperature was maintained by a
thermo-regulated coverage. Catheters were placed in jugular veins and
tail vein (for anesthesia and treatments). Rats were treated with
vehicle (0.9% NaCl) or compounds (0.6 mg/kg). The treatments
were administered IV. Thirty and 60 min after IV treatment, arterial
blood (from carotid artery) was collected on sodium citrate. For 0.5
mL of blood, 20 μL of CaCl2 (0.2M, starTEM, ROTEM), 20 μL of
tissue factor (ex-TEM, ROTEM), and 16 nM of tPA (Alteplase,
Boehringer Ingelheim) were mixed in a cup. The cups were placed in
the rotational thromboelastometer (ROTEM). Fibrin formation and
fibrinolysis were followed for 1 h at 37 °C. The AUC was calculated
for each condition (software GraphPad Prism 5). The percentage of
inhibition (vs AUC of group vehicle) was then calculated at 30 and/or
60 min post IV treatment (n = 1−2/compound).
Thierry Persigand − Institut de Recherches Servier, 92150
Suresnes, France
Alain Rupin − Institut de Recherches Servier, 92150 Suresnes,
France
Philippe Mennecier − Institut de Recherches Servier, 92150
Suresnes, France
Marie-Odile Vallez − Institut de Recherches Servier, 92150
Suresnes, France
Eric Raimbaud − Institut de Recherches Servier, 92150
Suresnes, France
Patrice Desos − Institut de Recherches Servier, 92150
Suresnes, France
Philippe Gloanec − Institut de Recherches Servier, 92150
Suresnes, France
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c02072
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank co-workers at the Analytical Division of the
Institut de Recherches Servier for providing the detailed
chemical analysis of the compounds, Philippe Baumy at
Technology Servier for the DMPK experiments, Andras
Kotschy and Laurent-Michel Vuillard for their advices in
preparing the manuscript.
ABBREVIATIONS
Structure Determination. X-ray structure determinations were
performed following methods described25 (PDB entry 3D68, 7NEE
and 7NEU) and are further described in the associated content
section.
■
A, apical; ADME, absorption, distribution, metabolism,
elimination; AUC, area under curve; B, basal; br s, broad
signal; n-BuLi, n-butyllithium; CLp, plasma clearance; CLr,
renal clearance; CP, carboxypeptidase; DBC, dibenzyl
carbonate; DMF, N,N-dimethylformamide; DMSO, dimethyl
sulfoxide; EC50, half maximal effective concentration; ER,
efflux ratio; Fabs, prediction of oral absorption; fu, fraction
unbound; h, hour; hep, hepatocyte; hu, human; IC50, half
maximal inhibitory concentration; inhib, inhibition; IP,
intraperitoneal; IV, intravenous; Ki, inhibition constant; Km,
Michaelis constant; m, mouse; MF, metabolic bioavailability;
mic, microsome; min, minute; mp, melting point; OD, optical
density; Papp, apparent permeability coefficient; PK, Pharma-
cokinetics; PPACK, Hippuryl-D-phenylalanyl-L-prolyl-L-argi-
nine chloromethyl ketone; r, rat; rpm, rotation per minute;
rt, room temperature; SD, sprague dawley; SEM, standard
error of mean; TAFIa, activated thrombin activatable
fibrinolysis inhibitor; TE, thromboelastometry; THF, tetrahy-
drofuran; TMSBr, trimethylsilyl bromide; tPA, tissue plasmi-
nogen activator; t1/2, terminal half-life; Vss, apparent volume of
distribution at steady state
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02072.
■
sı
Molecular formula strings of TAFIa inhibitors (CSV)
General synthetic procedures, characterizations of key
compounds, 1H NMR spectra, molecular formula
strings, chiral separation procedure, ADME and
pharmacokinetic data, protein production, crystallization
conditions, X-ray diffraction data and docking protocol
(PDF)
Atomic coordinates of TAFIa 8a (PDB)
Atomic coordinates of TAFIa 14d (PDB)
AUTHOR INFORMATION
Corresponding Author
■
Arnaud-Pierre Schaffner − Institut de Recherches Servier,
92150 Suresnes, France; orcid.org/0000-0002-2848-
6554; Phone: +33-6 02 06 63 91; Email: arnaud-
pierre.schaffner@servier.com
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