3-Mercaptopropionic acids as efficacious inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa)

Article abstract of DOI:10.1016/j.bmcl.2006.11.078

A novel series of cyclic potent, selective, small molecule, thiol-based inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa) and the crystal structures of TAFIa inhibitors bound to porcine pancreatic carboxypeptidase B are described. Three series of cyclic arginine and lysine mimetic inhibitors vary significantly in their selectivity against other human basic carboxypeptidases, carboxypeptidase N and carboxypeptidase B. (-)2a displays TAFIa IC₅₀ = 3 nM and 600-fold selectivity against CPN. Inhibition of TAFIa with (rac)2a resulted in dose dependent acceleration of human plasma clot lysis in vitro and was efficacious as an adjunct to tPA in an in vivo rabbit jugular vein thrombolysis model.

Full text of DOI:10.1016/j.bmcl.2006.11.078

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1349–1354
3-Mercaptopropionic acids as efficacious inhibitors of activated
thrombin activatable fibrinolysis inhibitor (TAFIa)
Imadul Islam,a Judi Bryant,a Karen May,a Raju Mohan,a Shendong Yuan,a
Lorraine Kent,b John Morser,c Lei Zhao,c Ron Vergona,d Kathy White,d
Marc Adler,e Marc Whitlowe and Brad O. Buckmana,*
aDepartment of Medicinal Chemistry, 2600 Hilltop Drive, Richmond, CA 94804, USA
bDepartment of Molecular Pharmacology, 2600 Hilltop Drive, Richmond, CA 94804, USA
cDepartment of Cardiovascular Research, 2600 Hilltop Drive, Richmond, CA 94804, USA
dDepartment of Animal Pharmacology, 2600 Hilltop Drive, Richmond, CA 94804, USA
eDepartment of Biophysics, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, USA
Received 3 November 2006; revised 27 November 2006; accepted 30 November 2006
Available online 3 December 2006
Abstract—A novel series of cyclic potent, selective, small molecule, thiol-based inhibitors of activated thrombin activatable fibrino-
lysis inhibitor (TAFIa) and the crystal structures of TAFIa inhibitors bound to porcine pancreatic carboxypeptidase B are
described. Three series of cyclic arginine and lysine mimetic inhibitors vary significantly in their selectivity against other human basic
carboxypeptidases, carboxypeptidase N and carboxypeptidase B. (ꢀ)2a displays TAFIa IC50 = 3 nM and 600-fold selectivity against
CPN. Inhibition of TAFIa with (rac)2a resulted in dose dependent acceleration of human plasma clot lysis in vitro and was effica-
cious as an adjunct to tPA in an in vivo rabbit jugular vein thrombolysis model.
Ó 2006 Elsevier Ltd. All rights reserved.
Thrombin activatable fibrinolysis inhibitor (TAFI, plas-
ma pro-carboxypeptidase B, carboxypeptidase R, car-
We5 and others3d,e,6 have recently reported data sup-
porting small molecule TAFIa inhibitors as a treatment
for anti-thrombotic indications. Here, we report a novel
series of potent, selective TAFIa inhibitors that are effi-
cacious as an iv adjunct to tPA in an in vivo model of
thrombolysis.
boxypeptidase U, EC 3.4.17.20) is
a
zymogen
synthesized in the liver and circulating at a plasma con-
centration of about 50 nM.1 The activated enzyme (TA-
FIa) is a zinc-based basic carboxypeptidase that
suppresses fibrinolysis. Upon activation by the throm-
bin/thrombomodulin complex or plasmin, TAFIa inhib-
its the amplification of plasmin production by removing
the newly exposed C-terminal lysine residues from par-
tially degraded fibrin thus regulating fibrinolysis.2 Thus,
inhibition of TAFIa is a novel pro-fibrinolytic mecha-
nism in which fibrinolysis is enhanced without directly
affecting coagulation or platelet function. TAFIa inhibi-
tion is shown to be effective in enhancing tPA-induced
fibrinolysis in several animal models using CPI3a,b,c (a
selective peptidic TAFIa inhibitor isolated from potato)
and small molecules.3d,e The safety of TAFIa inhibition
has been demonstrated using TAFI-deficient mice.4
Our starting point for optimization was the known car-
boxypeptidase inhibitor SQ-24,798 (Fig. 1).7 However,
SQ-24,798 is equipotent for porcine pancreatic carboxy-
peptidase B (CPB) and carboxypeptidase N (CPN).
NH2
NH
NH2
HN
HN
N
HS
HS
COOH
SQ-24,798
COOH
Cyclic Constrained
Arg & Lys Mimics
(cf 2-3)
Keywords: TAFI; Fibrinolysis; Carboxypeptidase.
*
Corresponding author. Tel.: +1 510 669 4591; fax: +1 510 669
4310; e-mail: buckmanb@yahoo.com
Figure 1. Inhibitor design scheme.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.078

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I. Islam et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1349–1354
CPN is a basic carboxypeptidase circulating in plasma
that is critical in regulating the complement system.8
Inhibition of CPN results in undesirable side effects.
Therefore, it is critical to design inhibitors that are selec-
tive for TAFIa over CPN. Little risk is anticipated from
transient inhibition of pancreatic CPB. Inhibition of
CPB alone should not lead to intestinal malabsorption
due to the presence of alternate active proteases avail-
able for digestion.
Boc protection followed by introduction of the methy-
lene group by addition of formaldehyde to the lithium
enolate produced 6. Acid hydrolysis of both the ester
and Boc group was followed by 1,4-addition of thioace-
tic acid to the double bond to provide 7. Hydrolysis of
the thioacetyl by ammonium hydroxide afforded the
unsubstituted piperidinyl thiol inhibitors 1a, c, and e.
Further N-substituted piperidines 1b, 1g, 1f–h were syn-
thesized from 7.
A crystal structure of human TAFIa would be an impor-
tant aid for drug design. Unfortunately, TAFIa has not
yet been crystallized due to its lack of stability.1 CPB is
an attractive alternative protein for structural studies.
CPB has 47% sequence identity with human TAFIa,
with >89% homology in the active site.
The series of 2-phenyl 3-mercaptopropionic acids 2 was
synthesized as follows (Scheme 2). Addition of formal-
dehyde to appropriately substituted phenylacetic esters
8 gave the key olefin intermediates 9. Ester hydrolysis
followed by 1,4-addition of thioacetic acid to the double
bond afforded 11. Para-substituted nitrile 11a was con-
verted to amidine 2f. Racemic Boc-protected aniline
11b was separated into pure enantiomers (+)12 and
(ꢀ)12 by chiral HPLC. Deprotection of (+)12, (ꢀ)12
and (rac)12 afforded the chiral aniline intermediates
and racemic aniline 2e, respectively. Treatment of
(+)12, and (ꢀ)12 with diboc-trifluoromethanesulfonyl-
guanidine followed by acid hydrolysis afforded both
enantiomers of chiral guanidine (+)2a and (ꢀ)2a.
(rac)2a was synthesized in a similar manner from 2e.
We envisioned that a series of cyclic, constrained lysine
and arginine mimics could improve potency and selec-
tivity (Fig. 1). Our structure of SQ-24,798 complexed
to CPB confirmed ample room in the active site sur-
rounding the alkyl guanidine for the introduction of cyc-
lic structures.9 Since TAFIa substrates require a basic
group to bind in the S01 pocket, our design scheme cov-
ered a series of inhibitors 1–3 containing various basic
groups substituted on phenyl or heterocyclic templates
(Fig. 2).
Chloro-substituted analog 2d was synthesized as follows
(Scheme 3). Reduction of the nitro group of 13 with
iron, followed by Boc protection, and conversion of
the alcohol via the mesylate gives benzylnitrile 14. Ni-
trile hydrolysis to give the acid followed by Fischer
esterification and Boc protection allowed for addition
of formaldehyde to give the key double bond intermedi-
ate 15. Ester hydrolysis, addition of thioacetic acid to
the double bond, deprotection of the aniline, and gener-
ation of the Boc-protected guanidine gave 16. Final
deprotection with TFA followed by ammonium hydrox-
ide afforded 2d.
The series of 2-piperidinyl 3-mercaptopropionic acids 1
was synthesized as follows (Scheme 1). 3 or 4-Piperidine
5a or 5b was generated from platinum oxide reduction
of the appropriate 3 or 4-pyridine acetic acid esters 4.
2-Piperidinylmethyl 3-mercaptopropionic acid analogs 3
were synthesized as follows (Scheme 4). Addition of ben-
zyl methyl malonate to 3- or 4-pyridine aldehyde fol-
lowed by platinum oxide reduction and Boc protection
of the piperidine gave 17. Mannich reaction followed
by ester hydrolysis and Boc deprotection gave key dou-
ble bond intermediates 18. Addition of thioacetic acid to
the double bond gave 19. Hydrolysis with ammonium
hydroxide provided thiol inhibitors 3a–c.
The 2-piperidine 3-mercaptopropionic acid series of
TAFIa inhibitors 1 yielded potent and selective com-
pounds against CPN and CPB (Table 1). The unsubsti-
tuted 4-piperidine analog 1a was the most potent in the
series (TAFIa IC50 = 37 nM), but showed little selectiv-
ity over CPN or CPB. To further probe binding affinity
and selectivity within the S01 pocket, substitution of the
4-piperidine nitrogen of 1a with various other basic
groups afforded less potent inhibitors (guanidine 1f,
amidine 1g, amine 1h; TAFIa IC50 = 1070, 5300,
7350 nM, respectively). The two unsubstituted dia-
stereomeric 3-piperidine analogs 1c and 1e provided
potent compounds (TAFIa IC50 = 68 and 445 nM,
respectively). Compound 1c showed the highest
Figure 2. (A) The active site of CPB/2d complex showing the hydrogen
bonds (pdb code 1ZG7). (B) The CPB/2d complex showing the
superimposed structures of SQ-24,798 (orange, pdb code 1ZG9), (ꢀ)-
2a (cyan, pdb code 1ZG8).

I. Islam et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1349–1354
1351
X
X
X
X
Y
Y
Y
Y
i
ii
iii
RS
CO2Et
CO2Et
CO2Et
CO2H
7a X = C, Y = N, R = Ac
6a X = C, Y = NBoc
5a X = C, Y = NBoc
4
7b X = N, Y=C, R = Ac
6b X = NBoc, Y = C
5b X = NBoc, Y = C
iv
1a X = N, Y = C
1c X = C, Y = N diast a
1e X = C, Y = N diast b
v
HN
NH2
NH
NH2
O
HN
NH2
N
N
N
N
HS
HS
CO2H
1b,1d
HS
HS
CO2H
CO2H
CO2H
1g
1f
1h
Scheme 1. Reagents and conditions: (i) H2, PtO2, CH3COOH; (ii) LDA, CH2O, THF, ꢀ78 °C; (iii) a—6 N HCl, dioxane, 100 °C; b—AcSH; (iv)
NH4OH; (v) for 1b, 1d, 1f: a—N,N0diBocN00trifluoromethanesulfonylguanidine, DIEA, CHCl3; b—4 N HCl, dioxane; c—NH4OH for 1g:
a—CH3C(@NH)OC2H5 HCl; b—NH4OH for 1 h: a—bocglycine N-hydroxysuccinimide ester, dioxane; b—TFA; c—NH4OH.
R1
R1
R1
R1
R2
R2
R2
R2
i
ii
iii
AcS
CO2Et
CO2Et
CO2H
CO2H
9a R1 = CN, R2 = H
10a R1 = CN, R2 = H
11a R1 = CN, R2 = H
8a R1 = CN, R2 = H
9b R1 = H, R2 = NHBoc 10b R1 = H, R2 = NHBoc 11b R1 = H, R2 = NHBoc
8b R1 = H, R2 = NHBoc
v for b
iv for a
NH2
HN
R2
NHBoc
vi
HS
AcS
rac 12, (+)-12, (-)-12
HS
CO2H
CO2H
CO2H
rac 2a, (+)-2a, (-)-2a R2 = NHC(NH)NH2
2e R2 = NH2
2f
Scheme 2. Reagents and conditions: (i) paraformaldehyde, K2CO3, Bu4NI, toluene, 100 °C, 4 h; (ii) NaOH, water–MeOH; (iii) AcSH, CHCl3; (iv)
HCl(g)–EtOH then NH3(g)–EtOH; (v) HPLC; (vi) a—TFA, CH2Cl2; for 2a, N,N0diBocN00trifluoromethanesulfonylguanidine, DIEA, CHCl3 then
HCl(g)–EtOH.
NHBoc
v-viii
R
NHBoc
ix-x
NO2
i-iv
xii-xiii
2d
Cl
Cl
AcS
Cl
Cl
CN
CO2H
16
R = NHC(NBoc)NHBoc
CO2Et
OH
13
14
15
Scheme 3. Reagents and conditions: (i) Fe, HCl, EtOH; (ii) (t-BuCO)2O; (iii) MsCl, DIEA, CH2Cl2; (iv) KCN, KI, 18-Crown-6, EtOH; (v) 10%
H2SO4, MeOH; (vi) HCl(g)–EtOH; (vii) (t-BuCO)2O, THF, 50 °C; (viii) paraformaldehyde, K2CO3, Bu4NF, toluene; (ix) NaOH; (x) AcSH; (xi) HCl;
(xii) N,N0diBocN00trifluoromethanesulfonylguanidine, DIEA, CHCl3; (xiii) a—TFA, CH2Cl2; b—NH4OH.
selectivity against both CPB (345-fold) and CPN (470-
fold) of the entire thiol series 1–3. Both 3-piperidine
N-substituted guanidine diastereomers 1b and 1d dis-
played good potency (IC50 = 45 and 165, respectively)
in contrast to 4-piperidine N-substituted guanidine 1f
(IC50 = 1070 nm).
The 2-piperidinylmethyl 3-mercaptopropionic acid ser-
ies of inhibitors (3a–c) showed slightly less potency than
the one carbon shorter 2-piperidine analogs 1a, c, e.
4-Piperidine 3c6c was more potent for CPN and CPB
than TAFIa. Pure diastereomer 3-piperidine 3b showed
significant selectivity against CPB (250-fold). However,

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I. Islam et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1349–1354
X
Y
X
Y
X
Y
BnO2C
CO2Me
i-iii
iv-vi
+
H
HOOC
CO2Me
COOH
17a X = NHBoc, Y = C
18a X = N, Y = C
O
17b X = C, Y = NHBoc
18b X = C, Y = N
H
N
X
Y
NH
vii
viii
AcS
HS
HS
COOH
COOH
COOH
3c
3a,b
19a X = N, Y = C
19b X = C, Y = N
Scheme 4. Reagents and conditions: (i) piperidine, benzene, Dean–Stark reflux; (ii) H2, PtO2, AcOH; (iii) (t-BuCO)2O, NaHCO3, dioxane–water; (iv)
NEt2, formalin, THF; (v) NaOH, dioxane–water; (vi) 6 N HCl, 80 °C; (vii) AcSH, i-PrOH; (viii) NH4OH.
Table 1. Inhibition of human TAFIa, human CPN, and porcine pancreatic CPB
R1
X
R1
R2
R2
X
Y
Y
R3
HS
HS
HS
COOH
COOH
COOH
1
2
3
a
TAFIa IC50 (nM)
a
CPN IC50 (nM)
a
CPB IC50 (nM)
Compound
X
Y
R1
R2
R3
SQ-24,798
1a
1b
—
N
—
C
—
H
—
H2
—
—
—
—
—
—
—
—
—
H
8
37
6
5
10
7
2
1
195 21
38 14
60 17
38
23,500 4950
58
C
N
H2
H2
H2
H2
C(NH)NH2
H
C(NH)NH2
45 18
68 10
165 21
0
1c
1d
C
N
>30,000
3
C
N
2
6
1e
C
N
H
445 106
1070 325
5300 266
7350 3040
19,000 5660
45
1750 778
3100 707
1f
1g
N
C
C(NH)NH2
C(NH)CH3
C(O)CH2NH2
H
6
N
C
H
1040 14
>30,000
29,000 4240
4950 2470
1h
N
C
H
NHC(NH)NH2
(ꢀ)2a
(rac)2a
(+)2a
2d
—
—
—
—
—
—
NH
NH
CH2
—
—
—
—
—
—
CH2
CH2
NH
H
3
4
1
1
1
3
1720 239
137 15
6
3
8
7
4
H
NHC(NH)NH2
NHC(NH)NH2
NHC(NH)NH2
H
0.2
2
H
H
5
11
36
5
H
Cl
H
4650 81
>30,000
1
2e
H
C(NH)NH2
NH2
H
785 353
905 695
92 33
357 110
1000 7070
420 99
2f
3a
H
845 219
4150 354
1950 778
29 12
—
—
—
—
—
—
—
—
3b
3c6c
129 72
139 52
>30,000
—
61 25
a Values are mean of more than 2 experiments standard deviation. For assay descriptions see Ref. 11.
pure diastereomer 3-piperidine 3a showed 45-fold selec-
tivity against CPN.
aryl guanidine 2a affords 2d, a potent and very selective
inhibitor against CPN (TAFIa IC50 = 11 nM, 420-fold
selective against CPN).
A key discovery was replacing the guanidinopropyl
chain of SQ-24,798 by an aryl guanidine (2). This
2-phenyl 3-mercaptopropionic acid series of inhibitors
2 yielded the most potent and CPN-selective TAFIa
inhibitors; however, the series is equipotent against
CPB. Both enantiomers of aryl guanidine 2a show nano-
molar potency against TAFIa with (ꢀ)2a being slightly
more potent (TAFIa IC50 = 3 nM). (ꢀ)2a is about 270-
fold more potent than aniline 2e and 300-fold more po-
tent than amidine 2f. Compound (ꢀ)2a shows increased
selectivity of nearly 600-fold over CPN, compared to
(+)2a (7-fold). Introduction of a chloro substituent to
X-ray structures of SQ-24,798, (ꢀ)2a, and 2d bound to
porcine pancreatic CPB reveal a common binding
mode.9 (Fig. 2) The thiol chelates the zinc, and the car-
boxylic acid and guanidine group form salt bridges to
Arg145 and Asp255, respectively. These inhibitors are
close mimics of arginine and probably bind in a manner
similar to the natural substrates.
The high selectivity of (ꢀ)2a and 2d for TAFIa over
CPN could be explained based upon comparisons of
our structures9 to the published structure of carboxy-

I. Islam et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1349–1354
1353
Table 2. Efficacy of (rac)2a in rabbit jugular vein thrombolysis model
0.4
0.35
0.3
Dosea
(mg/kg + mg/kg h)
Thrombus
weight (mg)
SEMb
% reduction
in thrombus wt
-thrombomodulin
+thrombomodulin
2 uM rac-2a
PBS
tPA
141
145
82
15
19
21
11
—
0.5 uM rac-2a
—
0.125 uM rac-2a
0.0625 uM rac-2a
tPA + CPI
tPA + (rac)2a
42c
31c
97
a Dose: tPA = 0.01 mg/kg + 0.07 mg/kg h; CPI = 0.5 mg/kg + 0.3 mg/
kg h; (rac)2a = 15 mg/kg + 15 mg/kg h; n = 8–9/group.
b Standard error of the mean.
0.25
0.2
c p < 0.05 Fisher’s PLSD compared to vehicle and tPA.
When either CPI or (rac)2a was given together with the
inefficacious sub-threshold dose of tPA, the effect of tPA
was enhanced with a significant reduction in thrombus
weights being observed when compared to the vehicle
or tPA treated group (Table 2). The assessment of bleed-
ing risk was evaluated by measuring the weight of blood
loss from toe nail clips performed prior to thrombolytic
treatment, and at the end of the study. No significant
differences in bleeding were observed in any of the treat-
ment groups. Thus, TAFIa inhibition with (rac)2a in
combination with tPA enhances fibrinolysis in vivo.
0.15
0.1
0
10
20
30
40
50
60
70
Time (min)
Figure 3. Clot lysis time exhibits dose dependent acceleration by
(rac)2a.
peptidase D (CPD) bound with the related arginine
mimic GEMSA.10 CPD has 50% sequence homology
with CPN and the homology is >87% in the active site.
GEMSA binds Asp192 in CPD via terminal (Ng) guani-
dine nitrogen atoms. SQ-24,798, (ꢀ)2a, and 2d bind
Asp255 in CPB via internal (Ne)and terminal (Ng) gua-
nidine nitrogen atoms. Since replacement of the alkyl
chain with an aromatic ring eliminates the possibility
of a terminal guanidine nitrogen binding conformation,
the selectivity of our inhibitors may stem from the
hydrogen bonds formed by the guanidine moiety.9
In conclusion, the data shown describe the discovery of
a series of cyclic, constrained arginine and lysine mimet-
ics as potent and selective TAFIa inhibitors. Several
inhibitors show significant selectivity against CPN and
CPB. (rac)2a dose dependently accelerates clot lysis in
human plasma in vitro and is efficacious in an in vivo
thrombolysis model as an iv adjunct to tPA. These data
provide further support for TAFIa inhibitors as a treat-
ment for managing thrombosis in the clinic.
References and notes
TAFIa inhibition by aryl guanidine (rac)2a was tested in
an in vitro human plasma clot lysis assay to show that
the inhibition of activated TAFI observed in the enzyme
inhibition assay translated to an effect in human plas-
ma.3b Addition of thrombin, thrombomodulin, and cal-
cium to human plasma leads to activation of TAFI and
subsequent prolongation of clot lysis time. Clot lysis was
monitored by measuring turbidity at 405 nm. Lysis time
is accelerated by (rac)2a in a dose dependent manner
(Fig. 3).
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An in vivo rabbit jugular vein thrombolysis model was
used to test the hypothesis that TAFIa inhibition with
(rac)2a in combination with tPA would enhance fibrino-
lysis.3b A segment of the jugular vein was isolated with
ligatures and injected with a mixture of autologous
citrated blood and thromboplastin to induce thrombus
formation. Doses of tPA, CPI, (rac)2a or phosphate-
buffered saline (PBS) vehicle were administered as iv
bolus injections, followed by a constant infusion for
90 min via the right marginal ear vein. Fibrinolysis
was assessed by measuring clot weight at the end of
the study. An inefficacious sub-threshold dose of tPA
was combined with either the TAFIa inhibitor (rac)2a
or the peptidic TAFIa inhibitor CPI. CPI was used as
a positive control to confirm the effect of TAFIa inhibi-
tion in this model.
4. (a) Nagashima, M.; Yin, Z.-F.; Zhao, L.; White, K.; Zhu,
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