Optimization of Cyclic Plasmin Inhibitors: From Benzamidines to Benzylamines

Article

pubs.acs.org/jmc

Optimization of Cyclic Plasmin Inhibitors: From Benzamidines to

Benzylamines

Stefan Hinkes, Andre Wuttke, Sebastian M. Saupe, Teodora Ivanova, Sebastian Wagner, Anna Knorlein,

́

̈

Andreas Heine, Gerhard Klebe, and Torsten Steinmetzer*

Department of Pharmacy, Institute of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, D-35032 Marburg,

Germany

S

* Supporting Information

ABSTRACT: New macrocyclic plasmin inhibitors based on

our previously optimized P2−P3 core segment have been

developed. In the ﬁrst series, the P4 residue was modiﬁed,

whereas the 4-amidinobenzylamide in P1 position was

maintained. The originally used P4 benzylsulfonyl residue

could be replaced by various sulfonyl- or urethane-like

protecting groups. In the second series, the P1 benzamidine

was modiﬁed and a strong potency and excellent selectivity

was retained by incorporation of p-xylenediamine. Several

analogues inhibit plasmin in the subnanomolar range, and their

potency against related trypsin-like serine proteases including

trypsin itself could be further reduced. Selected derivatives

have been tested in a plasma ﬁbrinolysis assay and are more

eﬀective than the reference inhibitor aprotinin. The crystal structure of one inhibitor was determined in complex with trypsin.

The binding mode reveals a sterical clash of the inhibitor’s linker segment with the 99-hairpin loop of trypsin, which is absent in

plasmin.

activities, e.g., from various tissue kallikreins, have been found

in acute atopic dermatitis. These elevated activities were

associated with impaired barrier function, irritation, and

reduced skin capacitance, suggesting a potential use of plasmin

inhibitors for the treatment of this form of dermatitis.^9,10

Over many years, aprotinin has been clinically used for the

reduction of perioperative bleeding to avoid blood transfusions,

especially during cardiac surgery with cardiopulmonary bypass

or organ transplantations.¹¹⁻¹³In this application, aprotinin is

administered in parallel with anticoagulants such as heparins,

which minimizes the risk for thrombus formation. However,

due to side eﬀects, aprotinin was mostly withdrawn from

market.¹⁴Nowadays, the lysine mimetic tranexamic acid (TXA)

is the gold standard for the management of perioperative

bleeding. TXA inhibits plasminogen activation but has no

inhibitory eﬀect on plasmin and does not exhibit the anti-

inﬂammatory properties of aprotinin. Moreover, side eﬀects

have been reported after treatment with higher doses of TXA,

such as convulsive seizures.^15,16Very recently, new TXA

analogues based on an isoxazolone scaﬀold have been reported

which possess an enhanced potency and improved overall

proﬁle.¹⁷

INTRODUCTION

■

The trypsin-like serine protease plasmin exhibits multiple

physiological functions. It is the major ﬁbrinolytic protease in

blood and converts insoluble ﬁbrin deposits into soluble

fragments, thereby avoiding excessive thrombus formation.

Outside the vascular system, plasmin is involved in the

degradation of numerous extracellular matrix proteins and

growth factors either by direct cleavage or via activation of

matrix metalloproteases (MMPs), thereby contributing to cell

migration, tissue remodelling, and wound healing. Moreover,

plasmin modulates inﬂammatory responses via numerous

mechanisms.¹⁻⁴In a mouse model of acute colitis, the

treatment with a synthetic plasmin inhibitor reduced MMP-9

activation and subsequent release of inﬂammatory cytokines. As

a consequence, the treated mice did not develop colitis.⁵

Results from a diﬀerent mouse model revealed that plasmin

and/or MMP-9 inhibition could be a suitable strategy to

control the deadly cytokine storm in patients with acute graft-

versus-host disease.⁶Moreover, enhanced plasminogen activa-

tion leading to increased plasmin activity has been linked to

cancer invasion and metastasis.^7,8However, a long-term

treatment with plasmin inhibitors, which would be required

for tumor therapy, may disturb the well-balanced hemostasis

and ﬁbrinolysis, leading to a risk of thrombus formation.

Various clinical trials with the Kunitz-type plasmin inhibitor

aprotinin for cancer treatment have been terminated.³Strongly

increased plasmin, uPA, and other stratum corneum protease

In addition to these developments targeting the plasmin

formation, a direct and selective plasmin inhibition may provide

Received: April 20, 2016

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Scheme 1. Structures of Previously Described Plasmin Inhibitors

a

Scheme 2. Synthesis of the Linear P4−P1 Inhibitor Backbone

a

(a) 1.0 equiv of isobutyl chloroformate, 1.0 equiv of NMM, THF, −15 °C, 10 min, followed by 1.0 equiv of amino compound as salt, 1.0 equiv of

NMM, 1 h at −15 °C, rt overnight, 98% (5a), 94% (6a), 88% (7a); (b) 1 M HCl in acetic acid, precipitation in diethyl ether, 98% (5b), 97% (6b);

(c) 1.0 equiv of 6b, 1.0 equiv of cyclohexylsulfamoyl chloride, 3.0 equiv of DIPEA, THF, 2 h at 0 °C, rt overnight, followed by 2.5 equiv of

cyclohexylsulfamoyl chloride, 2.5 equiv of DIPEA, THF, 2 h at 0 °C, rt overnight, ﬂash chromatography (silica gel, DCM/MeOH 60:1), 75% (8a);

(d) 30 equiv zinc powder, 19 equiv of acetic acid, THF, rt, 90% (7b), 94% (8b).

a more rapid and stronger eﬀect to reduce bleeding. The work

on synthetic plasmin inhibitors was recently reviewed.¹⁸Among

the most potent small-molecule inhibitors are tetrapeptidic

arginal derivatives¹⁹and derivatives of trans-4-aminomethylcy-

clohexanecarboxylic acid.²⁰⁻²²

Plasmin is also inhibited by small benzamidine deriva-

tives.^23,24Starting from the nonselective linear substrate-

analogue benzamidine derivative 1 (CU-2010)²⁵(Scheme 1),

we have recently described various types of macrocyclic

analogues with improved selectivity and potency.^26,27The

cyclization was performed between the side chains of a P2

residue in S-conﬁguration and the adjacent P3 residue in R-

conﬁguration. We assumed that the cyclic inhibitor structure

should induce a steric clash with the 99-loop present in nearly

all trypsin-like serine proteases, leading to a weak inhibition of

these enzymes. However, this loop is absent in plasmin, which

enables its eﬃcient inhibition by these macrocyclic inhibitors.

Among the most potent analogues we identiﬁed compounds 2

and 3 (inhibitors 4 and 8 in our previous article²⁷), which

inhibit plasmin with subnanomolar K_ivalues of 0.68 and 0.20

nM, respectively (Scheme 1). For selectivity characterization,

both compounds were tested with additional trypsin-like serine

proteases and a negligible inhibitory potency was found for 13

of the used 14 proteases, which conﬁrmed our general design

strategy. Unexpectedly, a relatively strong nanomolar potency

was retained for trypsin, although it contains a similar 99-loop

like the other tested proteases.

Therefore, two new inhibitor series have been designed,

whereas the optimized cyclic P3−P2 core-segment containing

the shorter (n = 1) and/or longer (n = 2) bis-alkylated

piperazine linker present in inhibitors 2 and 3 was kept

constant. At ﬁrst, the previously used N-terminal P4

benzylsulfonyl residue was modiﬁed and in the second series

the P1 group was replaced. In addition, inhibitors with new P4

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a

Scheme 3. Synthesis of the Cyclized Benzamidine-Derived Inhibitors

a

(a) 1.0 equiv of 7b or 8b, 1.0 equiv of 9 or 10, 2.0 equiv of HATU, 5.0 equiv of DIPEA, DMF, 2 h at 0 °C, rt overnight, followed by 0.2 equiv of 9

or 10, 0.4 equiv of HATU, 1.0 equiv of DIPEA, 2 h 0 °C, rt overnight, ﬂash chromatography (silica gel, DCM/MeOH 15:1 with 0.5% (v/v) 25%

NH₄OH), 38% (11), 32% (12), 43% (13), 41% (14); (b) (i) 2.0 equiv of hydroxylamine hydrochloride, 2.0 equiv of DIPEA, MeOH, 6 h reﬂux, rt

overnight, (ii) 2.0 equiv of Ac₂O, acetic acid, rt, (iii) 10% Pd/C, H₂, acetic acid, rt, preparative HPLC, 21% (23), 26% (24), 25% (30), 33% yield

(31).

and P1 combinations were prepared. It was intended that most

modiﬁcations lead to a similarly reduced potency against all

trypsin-like serine proteases. However, starting from subnano-

molar plasmin aﬃnities, we expected that, despite some drop in

aﬃnity, we should still retain nanomolar plasmin inhibitors with

a suﬃcient antiﬁbrinolytic activity. We also assumed that in

parallel the same modiﬁcations should further reduce the

aﬃnities toward all other proteases, thereby moving the

inhibitory potency against trypsin into the micromolar range.

The antiﬁbrinolytic activity of selected inhibitors was conﬁrmed

in a plasma ﬁbrinolysis assay. One of the most potent

benzamidine-derived inhibitors was crystallized in complex

with trypsin. The obtained structure reveals several key

interactions, which can explain the signiﬁcant aﬃnity of this

inhibitor toward trypsin. The results of this work are

summarized in the current contribution.

NO₂)-OH residue and of the P4 group. Reduction of both

nitro groups provided the linear P4−P1 diamino intermediates

7b and 8b (Scheme 2), their subsequent cyclization with the

bisalkylated piperazine linker, and conversion of the nitrile

group into an amidine yielded the ﬁnal inhibitors 23−24 and

30−31 (Scheme 3).

In addition to the benzamidine-derived inhibitors, a few

agmatine and p-xylenediamine (p-Xda)-analogues have been

prepared. The synthesis of the p-Xda inhibitors 41 and 42 is

shown in Scheme 4.

The synthesis of all other inhibitors is described in the

Supporting Information. The structures of the synthesized

benzamidine-derived inhibitors (15−32) and their analogues

with P1 modiﬁcations (38−43) are summarized in Tables 2

and 3, respectively.

Kinetic Measurements. The widely used commercially

available plasmin substrates PefachromePL (H-^DAla-hexahy-

drotyrosine-Lys-pNA), Chromozym PL (Tos-Gly-Pro-Lys-

pNA), and S-2251 (H-DVal-Leu-Lys-pNA) possess relatively

high K_Mvalues >100 μM, leading to a moderate substrate

eﬃciency (k_cat/K_M). However, they are well suited for routine

measurements in microplate readers with classical inhibitors

possessing inhibition constants ≥1 nM. Our previous assays

have been performed with relatively high concentrations (364,

182, and 91 μM) of the chromogenic substrate Tos-Gly-Pro-

Lys-pNA in the presence of 0.9 nM plasmin, which makes it

diﬃcult to avoid tight-binding conditions²⁸in measurements

with subnanomolar inhibitors. Therefore, a set of new

ﬂuorogenic tripeptide derivatives was prepared to identify

more sensitive substrates (Table 1). On the basis of a previous

study with chromogenic tetra- and pentapeptide substrates¹⁹

composed of L-conﬁgured amino acids and from our own work

on inhibitors, it was known that plasmin prefers bulky aromatic

RESULTS

■

Synthesis. The synthesis strategy for the new plasmin

substrates (Table 1) is provided in the Supporting Information.

The previously described inhibitors including compounds 2 and

3 and a few analogues shown in Table 2 were prepared by ﬁnal

coupling of the unprotected 4-amidinobenzylamine·2HCl

(Amba) residue to the free P2 carboxyl group of the cyclized

P4−P2 segment.^26,27Depending on the coupling condition,

this strategy resulted in some amount of racemization at the P2

residue, although both diastereoisomers could be separated by

preparative HPLC. To avoid the racemization-sensitive seg-

ment condensation, a modiﬁed strategy was applied for the

preparation of some new analogues, which is shown for selected

benzamidine-derived inhibitors in Schemes 2 and 3. Boc-Phe(p-

NO₂)-OH was initially coupled to p-cyanobenzylamine,

followed by stepwise attachment of the P3 Boc-^DPhe(p-

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a

Scheme 4. Synthesis of the p-Xylenediamine-Derived Inhibitors

a

(a) 1.1 equiv of NMM, 1.0 equiv of Tfa₂O, DCM, 2 h at 0 °C, rt overnight, 99% (33); (b) 10% Pd/C, H₂, acetic acid, 3 h at rt, 87% (34); (c) (i) 1.0

equiv of Boc-Phe(p-NO₂)-OH, 1.0 equiv of NMM, 1.0 equiv of isobutyl chloroformate, THF, 10 min at −15 °C, 1.0 equiv of 34 as HCl salt, 1.0

equiv of NMM, THF, 1 h at −15 °C, rt overnight, 88%, (ii) 1 M HCl in acetic acid, 1 h at rt, precipitation in diethyl ether, 95% (35); (d) (i) 1.0

equiv of Boc-^DPhe(p-NO₂)-OH, 1.0 equiv of NMM, 1.0 equiv of isobutyl chloroformate, THF, 10 min at −15 °C, 1.0 equiv of 35, 1.0 equiv of

NMM, THF, 1 h at −15 °C, rt overnight, 97%, (ii) 30 equiv zinc powder, 19 equiv acetic acid, THF, 2 h at rt, 98% (36); (e) (i) 1.0 equiv of 10, 5.0

equiv of DIPEA, 2.0 equiv of HATU, 2 h at 0 °C, rt overnight, (ii) 1 M HCl in acetic acid, 1 h at rt, preparative HPLC, 36% (37, 2 steps); (f) (i) 1.0

equiv of cyclohexylsulfamoyl chloride or phenylsulfonyl chloride, 5.0 equiv of DIPEA, THF, 2 h at 0 °C, rt overnight, (ii) dioxane/1 M NaOH, 3 h at

rt, preparative HPLC, 14% (41, 2 steps), 54% (42, 2 steps).

Table 1. Synthesized Fluorogenic Plasmin Substrates

no.

sequence

K_M(μM)

k_cat(s⁻¹

)

k_cat/K_M(M⁻¹s⁻¹

)

44

45

46

47

48

Mes-^DSer(Bzl)-Phe-Arg-AMC

8.5

7.4

2.65

2.38

0.67

9.69

1.74

3.11 × 10⁵

3.21 × 10⁵

1.83 × 10⁵

6.02 × 10⁵

1.77 × 10⁵

a

Mes-DhPhe-Phe-Arg-AMC

a

Mes-DhPhe-Phe(4-NO₂)-Arg-AMC

Mes-DArg-Phe-Arg-AMC

3.8

16.1

9.8

a

Mes-DArg-hPhe-Arg-AMC

a

hPhe corresponds to homophenylalanine.

P2 residues. Therefore, phenylalanine was incorporated at this

position in combination with sulfonylated P3 residues in D-

conﬁguration. The P3 ^DSer(Bzl) residue was selected based on

the isosteric ^D-phenylpropylglycine known from inhibitor 1.

This provided the well suited substrate Mes-^DSer(Bzl)-Phe-

Arg-AMC (44), which was later used for the inhibitor

measurements. In addition, ^D-arginine was introduced in P3

position, which provided the excellently water-soluble substrate

47 with further improved substrate eﬃciency. Substrate 47 was

used for the slow binding measurements with selected

inhibitors. For some other substrates we observed even lower

K_Mvalues, however, they suﬀered from reduced k_catvalues.

The inhibition constants of the benzamidine derivatives are

summarized in Table 2. In previous studies with substrate

analogue thrombin,²⁹factor Xa,³⁰and uPA³¹inhibitors, a

benzylsulfonyl group was identiﬁed as preferred P4 residue,

therefore, it was used as constant N-terminal protecting group

in our previous plasmin inhibitor series.^26,27Complete

elimination of the P4 group resulted in a ∼100-fold decreased

potency (15, 16). A drop in plasmin aﬃnity was also observed

by replacement of the benzylsulfonyl group with formyl or

various acyl residues, although the methoxycarbonyl- and Cbz-

inhibitors 18 and 20, both containing the longer piperazine

linker, retain subnanomolar plasmin aﬃnity. The comparison of

the Cbz-inhibitor 20 with the phenylpropionyl-analogue 24

suggests a preference for urethane-like P4 groups over acyl

residues.

The data in Table 2 reveal that the benzylsulfonyl group can

be replaced by various sulfonyl residues. An excellent potency

was determined for the phenylsulfonyl inhibitor 29 and its

cyclohexylsulfamoyl analogue 31. Moreover, most analogues

revealed a negligible aﬃnity against aPC and the clotting

proteases thrombin and factor Xa, whereas only a few

compounds inhibit plasma kallikrein (PK) in the high

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Table 2. Structures of the Cyclic Benzamidine-Derived Inhibitors

a

These compounds have been previously published; their K_ivalues are provided as reference.^25,27

nanomolar range. For selected compounds, we have also

determined the inhibition constants against trypsin, which was

used as a further reference for this protease family, although

trypsin is not present in the blood circulation. A relatively

strong trypsin inhibition was still found for the phenylsulfonyl

and cyclohexylsulfamoyl analogues 29 and 31, with K_ivalues of

62 and 21 nM, respectively, which is in the same range as found

for the reference compounds 2 and 3 (Table 2).

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Table 3. Combined Modiﬁcation of P1 and P4 Residues

The strong subnanomolar inhibitory potency of compounds

3, 29, and 31 suggested that plasmin should tolerate some P1

modiﬁcations, which lead to a generally reduced aﬃnity against

all trypsin-like serine proteases. Therefore, additional inhibitors

with the longer piperazine-dipropionyl linker have been

prepared, which provided slightly more potent inhibitors than

the shorter piperazine-diacetyl linker. Their structures are

shown in Table 3. The replacement of Amba in inhibitor 31 by

agmatine, which is known from the development of substrate-

analogue thrombin inhibitors,^32,33yielded a ∼30-fold reduced

plasmin aﬃnity for compound 39. An enhanced drop in

potency was observed after incorporation of the agmatine

precursor diaminobutane (38). Furthermore, we tested the

replacement of the P1 amidine by an aminomethyl group, a

previously applied strategy to reduce the strong basicity of the

P1 residue in the ﬁeld of thrombin and matriptase

inhibitors.³⁴⁻³⁶To our delight, the replacement of the Amba

residue in the benzylsulfonyl inhibitor 3 by p-Xda in compound

40 was relatively well accepted and only a moderate 6-fold drop

in the plasmin inhibition was observed. Further combinations

with the N-terminal cyclohexylsulfamoyl and phenylsulfonyl

groups provided the p-Xda inhibitors 41 and 42 possessing a 2-

fold stronger aﬃnity for plasmin compared with compound 40,

although also both of these derivatives were approximately 4-

and 8-fold less potent than their related Amba inhibitors 31 and

29, respectively. Interestingly, the trypsin K_ivalue of the

phenylsulfonyl derivative 42 was increased to 1.5 μM.

providing the inhibition constants given in Tables 2 and 3 and

for comparison in Table 4 (indicated as classical method).

Table 4. Comparison of K_iDeterminations for Plasmin

Using the Classical Method Neglecting the Initial Time-

Dependent Part of the Progress Curves and from Slow-

Binding Analysis, Which Provides Additional Data for the

Rate Constants k_onand k_off

K_i(nM)

inhibitor

classical

slow-binding

k_on(M⁻¹·s⁻¹

)

k_off(s⁻¹

)

18

30

31

41

42

0.77

0.32

0.13

0.52

0.56

0.85

0.42

0.15

0.43

0.54

1.74 × 10⁶

1.77 × 10⁶

1.34 × 10⁶

6.40 × 10⁵

6.00 × 10⁵

1.48 × 10⁻³

7.46 × 10⁻⁴

2.04 × 10⁻⁴

2.77 × 10⁻⁴

3.22 × 10⁻⁴

However, such biphasic progress curves also indicate a slow-

binding inhibition mechanism.²⁸Therefore, selected com-

pounds were reanalyzed as slow-binding inhibitors using the

more sensitive substrate 47. Figure 1 shows the obtained

progress curves for inhibitor 31, and the data were ﬁtted to eq

1, which provided a steady-state velocity v_sand a rate constant

k_obsfor each curve. The constant initial velocities v₀of all

progress curves indicate a slow-binding behavior according to

the one-step reversible inhibition mechanism A, lacking the

formation of a rapid pre-equilibrium.²⁸The v_svalues were ﬁtted

as a function of the inhibitor concentration according to eq 2,

providing the K_ivalues (Figure 2). These inhibition constants

are nearly identical to the values which were calculated by the

classical method when the steady-state rates have been

determined from the terminal linear parts of the progress

curves (Table 4). Moreover, k_obswas used for the calculation of

the association rate constant k_onaccording to eq 3 (Figure 3).

Slow-Binding Measurements. In a few cases, nonlinear

progress curves have been observed for some subnanomolar

plasmin inhibitors. After an initial time-dependency, normally

after 5−10 min, a linear relationship was obtained, which

indicates that the steady-state is achieved. In such cases, only

the linear part of the progress curves was used for calculation of

the steady-state velocities and subsequent K_ideterminations,

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Figure 3. k_obsvalues were ﬁtted as a function of the inhibitor

concentrations according to eq 3, providing an association rate

constant of 1.34 × 10⁶M⁻¹s⁻¹for inhibitor 31. This k_onvalue was

used to calculate the dissociation rate constant k_offusing eq 4.

Figure 1. Progress curves for the slow-binding inhibition of plasmin

(0.35 nM) by inhibitor 31 at 40 nM (solid black circles), 26.7 nM

(solid blue circles), 17.8 nM (solid green circles), 11.9 nM (solid red

circles), 7.9 nM (solid black triangles), 5.3 nM (solid blue triangles),

3.5 nM (solid green triangles), and 0 nM (solid red triangles) in the

presence of substrate 47 (50 μM). The data were ﬁtted to eq 1.

TF, Ca²⁺, and tPA concentrations, as well as to the amount of

plasma, which can lead to slight variations of the time required

for complete ﬁbrinolysis in the absence of inhibitor (range

∼10−12 min), our determined IC₅₀value for aprotinin is in the

same range as described in literature (160 nM³⁷and 327

nM²⁵). Slightly lower and therefore more potent IC₅₀values

have been determined for the benzamidine-derived inhibitors

30 (53 nM) and 31 (55 nM) and for the second tested p-Xda-

derivative 41 (100 nM).

Moreover, the rapid initial increase in OD (e.g., Figure 4A)

observed for all tested cyclic inhibitors reveals that they do not

lead to any inhibition of clotting in plasma at antiﬁbrinolytic

concentrations. In contrast, a signiﬁcant delay was reported for

the less speciﬁc linear inhibitor 1, which also inhibits the

clotting enzymes fXa and fXIa with K_ivalues of 45 and 18 nM,

respectively.²⁵

Crystal Structure of Inhibitor 31 in Complex with

Trypsin. In our previous study,²⁷we had modeled the binding

mode of the reference inhibitors 2 and 3 in plasmin and had

superimposed the complex with the crystal structures of other

trypsin-like serine proteases, for which we had also determined

the inhibition constants. The structures revealed a sterical clash

of the macrocyclic inhibitor segment with the 99-loop present

in all other proteases, including trypsin. However, we could not

explain why both inhibitors retain a signiﬁcant nanomolar

potency against trypsin. Therefore, we have cocrystallized

inhibitor 31 in complex with trypsin (K_i= 21 nM) and the

reﬁned structure is shown in Figure 5A. As expected, the 99-

loop interferes with the linker moiety and the macrocyclic P2−

P3 segment is displaced. As a consequence, the ^D-conﬁgured P3

residue cannot accommodate anymore into the characteristic

distal S3/4 pocket above Trp215, as designed for plasmin

binding. This occupancy of the S3/4 pocket is customarily

observed in most trypsin-like serine proteases with related

substrate analogue inhibitors. Despite this distorted placement

of the P2 and P3 side chains, all characteristic polar contacts of

the benzamidine group within the S1 pocket and from the

inhibitor backbone to the trypsin residues Ser214, Gly216, and

Gly219 are maintained. The complex is further stabilized by

two speciﬁc hydrogen bonds formed by the linker moiety. The

P3 side chain amide nitrogen makes a hydrogen bond to the

carbonyl of Ser96 from the 99-loop and a water-mediated

Figure 2. K_ivalue of 0.15 nM for inhibitor 31 was calculated by ﬁtting

the v_s, [I] data pairs to eq 2 using substrate 47 (50 μM).

The known K_iand k_onvalues enabled the calculation of the

dissociation rate constant k_offaccording to eq 4.

Fibrinolysis Assay in Human Plasma. The antiﬁbrino-

lytic activity of selected compounds was tested in human

plasma, as described for aprotinin.³⁷Plasma was treated with a

mixture of CaCl₂, tissue factor (TF), tissue-type plasminogen

activator (tPA), and inhibitor at diﬀerent concentrations. TF

and Ca²⁺induce clot formation, indicated by a rapid initial

increase in optical density (OD) at 405 nm leading to a plateau.

Shortly thereafter, a drop in OD can be observed due to

ﬁbrinolysis, which is caused by the concomitant tPA induced

plasmin formation. Figure 4A shows the obtained curves for

inhibitor 42 at diﬀerent concentrations and for the control in

absence of tPA (no ﬁbrinolysis). For IC₅₀determination, the

relative decrease in OD at 11 min, when complete ﬁbrinolysis

in absence of inhibitor 42 occurred, was determined for each

curve and plotted against the inhibitor concentration (Figure

4B).³⁷This provided an IC₅₀value of 81 nM for inhibitor 42, a

∼3-fold higher value of 227 nM was determined for the

reference aprotinin. Although this assay is sensitive to the used

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Figure 4. Fibrinolysis assay in human plasma. (A) TF and Ca²⁺-induced initial clotting leads to an increase in OD providing a plateau, followed by a

subsequent decrease in OD due to tPA-induced ﬁbrinolysis in the presence of various concentrations of inhibitor 42 at 200 nM (solid blue circles),

100 nM (solid green circles), 60 nM (solid red circles), 40 nM (solid black triangles), 20 nM (solid blue triangles), 10 nM (solid green triangles),

and 0 nM (solid red triangles). Complete ﬁbrinolysis in absence of inhibitor was achieved within 11 min (vertical dashed line). The control (no

ﬁbrinolysis) was measured in absence of tPA (solid black circles). (B) Plot for IC₅₀calculation showing the relative decrease of OD at the time of

complete ﬁbrinolysis as a function of the used individual inhibitor concentrations (aprotinin (solid black circles), 30 (solid blue circles), 31 (solid

green circles), 41 (solid red circles), and 42 (solid black triangles)).

contact is observed between the P2 side chain amide carbonyl

and the carbonyl of His57 from the catalytic triad. Moreover,

the Leu99 side chain is located close to the P3 residue and most

likely involved in hydrophobic contacts with the ligand’s phenyl

ring (distances 3.6 Å, 2 × 4.0 Å, and 4.1 Å, shown as green

dashed lines in Figure 5B). These interactions seem to be

speciﬁc for the structural properties of trypsin and can explain

the remaining nanomolar potency of inhibitor 31 against this

protease.

An additional water mediated interaction is formed between

the P3 side chain carbonyl oxygen and the carbonyl oxygen of

Lys242 of a symmetry related molecule. This symmetry related

molecule also involves its Gln243 side chain with hydrophobic

interactions to the phenyl ring of the P3 residues. However,

most likely these latter interactions are only packing artefacts

and do not contribute to binding aﬃnity.

academic environment, only a limited number can be routinely

tested. Therefore, we cannot exclude that a similar signiﬁcant

potency, as found for trypsin, could be retained for other

proteases, although we have no evidence for that so far.

Therefore, we have deliberately investigated further modiﬁca-

tions, which should lead to a slightly reduced inhibition of all

trypsin-like serine proteases but could still provide nanomolar

plasmin inhibitors. The comparison of the inhibition constants

determined for the new macrocyclic benzamidine derivatives

(Table 2) with results from previous studies when using fXa³⁰

or uPA³⁹revealed that plasmin is less sensitive to P4

substitutions than other trypsin-like serine proteases. Despite

a reduced plasmin aﬃnity for all inhibitors containing an acyl

residue in P4 position, a subnanomolar potency was maintained

for the urethane-like methoxycarbonyl- and Cbz-protected

analogues 18 and 20, although both compounds still inhibit

trypsin with K_ivalues close to 0.1 μM. A considerable plasmin

inhibition was also retained for the formyl- and phenyl-

propionyl-protected analogues 17 and 24, which possess K_i

values <5 nM, whereas their trypsin aﬃnity was further reduced

(K_i> 500 nM).

The highest potency was observed for the sulfonylated

analogues. Interestingly, plasmin is well accepting the

replacement of the N-terminal benzylsulfonyl group by the

less ﬂexible phenylsulfonyl residue. This is diﬀerent from

previous selectivity measurements performed with a series of

noncyclic substrate-analogues originally developed as inhibitors

of fXa, where we had observed a 3-fold weaker plasmin aﬃnity

for phenylsulfonyl-^DSer(tBu)-Gly-4-Amba (K_i= 24 μM)

compared to benzylsulfonyl-^DSer(tBu)-Gly-4-Amba (K_i= 7.7

μM).³⁰Moreover, the incorporation of the phenylsulfonyl

group works more easily compared to the coupling of the

alkylsulfonyl-, benzylsulfonyl-, or cyclohexylsulfamoyl chlorides.

As expected, a reduced aﬃnity was determined after

incorporation of agmatine or p-xylenediamine in P1 position,

although the combination of the less basic p-Xda residue (pK_a

∼ 9.35³⁴) with the phenylsulfonyl or cyclohexylsulfamoyl

residues in P4 position still provided subnanomolar plasmin

DISCUSSION

■

In certain contexts, e.g., in the ﬁeld of anti-infectives or cancer,

it can be advantageous to develop promiscuous ligands with

broad speciﬁcity. Such drugs are less sensitive to resistance

developments caused by mutations of the target.³⁸Otherwise, it

is a primary objective in medicinal chemistry to design highly

selective drugs, especially when the target belongs to a family of

closely related enzymes, like the trypsin-like serine proteases,

which are involved in opposing pathways such as hemostasis

and ﬁbrinolysis. This should avoid adverse eﬀects caused by the

inhibition of oﬀ-targets. All of our previously described cyclic

plasmin inhibitors contain a 4-amidinobenzylamide in P1

position and an N-terminal benzylsulfonyl residue. Both groups

strongly contribute to the potency against trypsin-like

proteases, otherwise they have only little impact on speciﬁcity.

Nevertheless, when using an optimized cyclic P3−P2 core

segment, we could already obtain relatively selective plasmin

inhibitors with exception of trypsin, which was still inhibited in

the two-digit nanomolar range.²⁷Moreover, the signiﬁcance of

selectivity measurements strongly depends on the number of

the used enzymes of a target family, and often, especially in an

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Figure 5. Structure of bovine trypsin in complex with inhibitor 31 (PDB code: 5EG4). (A) Stereo representation of the active site with the bound

inhibitor as stick model with carbon atoms in yellow, nitrogen in blue, oxygen in red, and sulfur in pale-orange. Water molecules are shown as red

spheres. The molecular surface of the trypsin loop formed by residues 95−100 is colored in lilac, of Trp215 in pale-cyan, and of all other enzyme

residues in gray. A steric clash of the inhibitor’s linker moiety with the 95−100 loop of trypsin prevents the classical binding mode, in which the side

chain of the ^D-conﬁgured P3 residue is located in the distal S3/4 binding pocket above Trp215. (B) Stereo representation of the observed polar

contacts (dashed lines in black) and of hydrophobic interactions between the P3 phenyl ring and Leu99 (dashed lines in green). The inhibitor is

shown with yellow carbon atoms, the involved trypsin residues with green carbon atoms, and waters as red spheres. The nitrogen of the P3 side chain

amide forms a hydrogen bond to the carbonyl of Ser96 and the oxygen of the P2 side chain amide interacts via a bridging water with the carbonyl of

His57. Ser96, His57, and Leu99 are shown with orange carbon atoms. The ﬁgure was prepared using PyMOL 0.98 (DeLano Scientiﬁc, San Carlos,

CA, USA).

inhibitors. Interestingly, the drop in trypsin aﬃnity was more

pronounced for the phenylsulfonyl inhibitor 42 (K_i> 1 μM)

compared to that of the benzylsulfonyl and cyclohexylsulfamoyl

analogues 40 and 41. As expected, a negligible aﬃnity was

determined for the p-Xda derivatives against all other tested

proteases. Moreover, when using relatively low inhibitor

concentrations during measurements in a plate reader, we

observed nonlinear time courses of the progress curves for the

p-Xda inhibitor 42 and a few additional potent analogues. This

slow-binding behavior enabled the determination of the

association and dissociation rate constants in addition to the

K_ivalues. On the basis of the k_onvalues (Table 4), it seems that

the benzamidine inhibitors bind 2−3 times more rapidly to

plasmin compared to the p-Xda analogues. However, this slow-

binding behavior observed in enzyme kinetic assays when using

subnanomolar enzyme concentrations and inhibitor concen-

trations ≤40 nM should be less relevant under real conditions

in blood when higher inhibitor concentrations are required for

a substantial plasmin inhibition. This would increase the term

k_on× [I] in eq 3, resulting in a larger k_obsvalue, leading to more

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rapid binding. The plasma concentration of plasminogen is

∼1.6 μM.⁴⁰It is described for aprotinin that a slightly lower

concentration of 50 kallikrein inactivator units/mL, which is

approximately ∼1 μM, is suﬃcient for a substantial plasmin

inhibition in vivo to reduce blood loss, whereas approximately 4

times higher concentrations are required to inhibit PK.⁴¹

Although we found a slightly stronger potency in the

ﬁbrinolysis assay for our synthetic inhibitors compared to

aprotinin, it might be reasonable to assume that they should be

used at a similar concentration close to 1 μM in vivo due to the

1:1 stoichiometry in the plasmin/inhibitor complex. Such a

higher inhibitor concentration would lead to a rapid onset

inhibition of plasmin.

Although we found a slightly stronger potency for the

benzamidine inhibitors 30 and 31, both tested p-xylenediamine

derivatives 41 and 42 are also eﬃcient antiﬁbrinolytic agents in

the used plasma assay. These results reveal that there is no need

to keep a benzamidine as P1 residue anymore in this cyclic

inhibitor type. This might be an advantage because some side

eﬀects have been described for certain benzamidine-derived

inhibitors, including arrhythmia caused by hERG-channel

binding,⁴²or drop in blood pressure and respiratory

disturbances.^43,44

The crystal structure of trypsin in complex with inhibitor 31

conﬁrms that the cyclization prevents the classical binding

mode, in which the ^D-conﬁgured P3 side chain is directed

toward the distal S3/4 binding pocket above Trp215. In

contrast, all interactions of the benzamidine and inhibitor

backbone are maintained and along with two speciﬁc hydrogen

bonds of the linker segment a nanomolar trypsin aﬃnity is

retained. The cyclization also may contribute to an improved

entropically driven inhibitor binding by prestabilizing the P3−

P2 backbone conformation. Although this nicely explains the

remaining trypsin aﬃnity, it is not directly evident why these

interactions should be more strongly perturbed in complexes

with other trypsin-like serine proteases, which are less potently

inhibited by this benzamidine derivative. Both H-bond

interactions of the linker are not formed to functional groups

of side chains but to unspeciﬁc backbone groups of trypsin.

Therefore, they do not suggest straightforward a preference for

trypsin. In contrast, they bind to the backbone carbonyl of

Ser96 and via a water molecule to the carbonyl group of the

highly conserved His57 of the catalytic triade. Moreover, in the

adopted conformation the side chains of Ser96, Asn97, and

Thr98 of the 99-loop are directed into the solvent and therefore

cannot interact with the inhibitor (Figure 5A). However, the

crystal structure suggests that additional hydrophobic inter-

actions of Leu99 to the P3 phenyl ring contribute to the

increased aﬃnity of these inhibitors for trypsin. The Cγ and

both Cδ atoms are located in close van der Waals distances to

two carbon atoms of the P3 phenyl ring. In contrast, the tested

plasma kallikrein possesses a Gly99, factor Xa, a sterically more

demanding Tyr99, and activated protein C, a shorter and more

polar Thr99. All of these residues are unlikely to experience

similar side chain contacts as observed for Leu99 in trypsin.

Although thrombin also contains a leucine in position 99, the

access to its active site is more restricted for such bulky

macrocyclic inhibitors due to the presence of its speciﬁc 60-

insertion loop above its S2 pocket. A sequence alignment

revealed that all other clotting and ﬁbrinolytic proteases of the

trypsin family do not contain a leucine or isoleucine in position

99.

CONCLUSION

■

In summary, two new series of highly speciﬁc macrocyclic

plasmin inhibitors have been developed. It is possible to replace

the strongly basic P1 benzamidine by a similar benzylamine

residue when it is combined with a suitable sulfonyl residue in

P4 position. The phenylsulfonyl inhibitor 42 maintains

subnanomolar plasmin aﬃnity, whereas its potency against

trypsin is reduced into the micromolar range. Although this

inhibitor shows a slightly reduced potency in a ﬁbrinolysis assay

in plasma compared with the benzamidine analogues, it is more

potent than the reference inhibitor aprotinin. The determined

crystal structure of a macrocyclic benzamidine inhibitor in

complex with trypsin reveals a conformational adaptation of the

99-loop along with the cyclic linker part of the inhibitor, which,

with some care, explains the residual potency only observed for

trypsin binding. This conﬁrms our original strategy, that a

macrocyclization between the P2 and P3 side chains enables the

design of very potent and speciﬁc plasmin inhibitors, which

could be used as new antiﬁbrinolytics to replace aprotinin.²⁶

EXPERIMENTAL SECTION

■

General. Solvents and reagents were purchased from Sigma-

Aldrich, Alfa Aesar, Roth, Fluka, or Merck and used without further

puriﬁcation. The amino acids Boc-^DPhe(p-NO₂)-OH and Boc-Phe(p-

NO₂)-OH and their unprotected analogues were purchased from

Bachem.

Analytical thin layer chromatography was performed on silica 60

plates (Alugram SIL G/UV254, Macherey-Nagel, Duren, Germany).

Analytical HPLC experiments were performed on a Shimadzu LC-10A

̈

system (column, Nucleodur C₁₈ec, 5 μm, 100 Å, 4.6 mm × 250 mm,

Macherey-Nagel, Duren, Germany). Water (A) and acetonitrile (B),

̈

both containing 0.1% TFA, were used as eluents with a linear gradient

(increase of 1% B/min) and a ﬂow rate of 1 mL/min. The detection

was performed at 220 nm. The indicated purity for all intermediates

and inhibitors is based on HPLC detection at 220 nm. Column

chromatography was performed with silica gel 60 (0.04−0.063 mm,

Macherey-Nagel, Duren, Germany). The ﬁnal inhibitors were puriﬁed

̈

via preparative HPLC (pumps, Varian PrepStar model 218 gradient

system; detector, ProStar model 320; fraction collector, Varian model

701) using a C₈column (Nucleodur C₈ec, 5 μm, 100 Å, 32 mm × 250

mm, Macherey-Nagel, Duren, Germany) using identical solvents as

̈

described for the analytical HPLC and a linear gradient (increase of

1% B in 2 min) at a ﬂow rate of 20 mL/min (detection at 220 nm).

The inhibitors were obtained as TFA salts after lyophilization on a

freeze-drier (Martin Christ Gefriertrocknungsanlagen GmbH, Oster-

ode am Harz, Germany). The molecular mass of the synthesized

compounds was determined using a QTrap 2000 ESI spectrometer

(Applied Biosystems, Foster City, CA, USA).

NMR spectra for noncyclic compounds were recorded on a JEOL

ECX-400 or JEOL ECA-500. A Bruker DRX 400, Bruker Avance 500,

or Bruker Avance 600 was used for NMR measurements of the cyclic

derivatives. Chemical shifts (δ) are reported in ppm and calibrated on

the internal solvent signal (DMSO-d₆) or 3-(trimethylsilyl)propionic-

2,2,3,3-d₄acid sodium salt. Multiplicities are indicated as s (singlet), bs

(broad singlet), d (doublet), dd (doublet of doublets), t (triplet), and

m (multiplet). Coupling constants are reported in hertz (Hz). Water

suppression was achieved by using the Bruker Watergate pulse

sequence.

Boc-Phe(p-NO₂)-p-cyanobenzylamide (5a). To a solution of Boc-

Phe(p-NO₂)-OH (9.31 g, 30.0 mmol, 1.0 equiv) in 150 mL of THF,

N-methylmorpholine (NMM, 3.30 mL, 30.0 mmol, 1.0 equiv) and

isobutyl chloroformate (3.90 mL, 30.0 mmol, 1.0 equiv) were added at

−15 °C. After 10 min, the mixture was treated with p-cyanobenzyl-

amine·HCl (5.06 g, 30.0 mmol, 1.0 equiv) and NMM (3.30 mL, 30.0

mmol, 1.0 equiv). The suspension was stirred at −15 °C for 1 h and

then at rt overnight. The solvent was removed in vacuo, and the

remaining residue was dissolved in a mixture of EtOAc and 5% aq

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KHSO₄. The phases were separated, and the organic layer was washed

3× with 5% aq KHSO₄, one time with brine, 3× with saturated aq

NaHCO₃, and 3× with brine. The organic layer was dried over

MgSO₄, ﬁltered, and the solvent removed in vacuo. Yield: 12.4 g (29.3

mmol, 97.7%) of compound 5a as a light-yellow solid. HPLC: 47.2

min, start at 10% B (purity 95%). ¹H NMR (400 MHz, DMSO-d₆): δ

= 8.59 (t, J = 6.0 Hz, 1H), 8.11−8.18 (m, 2H), 7.72−7.79 (m, 2H),

7.50−7.57 (m, 2H), 7.36−7.43 (m, 2H), 7.15 (d, J = 8.5 Hz, 1H), 4.36

(d, J = 6.0 Hz, 2H), 4.22−4.30 (m, 1H), 3.12 (dd, J = 5.0 Hz, 13.6 Hz,

1H), 2.93 (dd, J = 10.3 Hz, 13.6 Hz, 1H), 1.30 (s, 9H) ppm. ¹³C NMR

(100 MHz, DMSO-d₆): δ = 171.3, 155.3, 146.6, 146.2, 145.2, 132.1

(2C), 130.5 (2C), 127.9 (2C), 123.1 (2C), 118.8, 109.5, 78.2, 55.4,

41.9, 37.2, 28.0 (3C) ppm. MS (ESI, negative): calcd, 424.17; m/z

423.20 [M − H]⁻.

H-Phe(p-NO₂)-p-cyanobenzylamide·HCl (5b). Compound 5a

(2.12 g, 5.00 mmol, 1.0 equiv) was dissolved in 40 mL of 1 M HCl

in acetic acid (40.0 mmol, 8.0 equiv). The yellow solution was stirred

for 1.5 h at rt. The solvent was removed in vacuo, and the remaining

residue was precipitated in diethyl ether. Yield: 1.77 g (4.91 mmol,

98.2%) of compound 5b as a light-yellow solid. HPLC: 23.2 min, start

at 10% B (purity 95%). ¹H NMR (500 MHz, DMSO-d₆): δ = 9.33 (t, J

= 5.5 Hz, 1H), 8.51 (bs, 3H), 8.08−8.18 (m, 2H), 7.69−7.76 (m, 2H),

7.49−7.57 (m, 2H), 7.31−7.38 (m, 2H), 4.27−4.42 (m, 2H), 4.16−

4.23 (m, 1H), 3.20−3.26 (m, 2H) ppm. ¹³C NMR (125 MHz, DMSO-

d₆): δ = 167.6, 146.7, 144.1, 143.1, 132.0 (2C), 130.9 (2C), 128.2

(2C), 123.4 (2C), 118.7, 109.7, 53.0, 41.8, 36.5 ppm. MS (ESI,

positive): calcd, 324.12; m/z 325.06 [M + H]⁺.

Boc-^DPhe(p-NO₂)-Phe(p-NO₂)-p-cyanobenzylamide (6a). The

synthesis was performed as described for compound 5a using Boc-

DPhe(p-NO₂)-OH (1.09 g, 3.50 mmol, 1.0 equiv), isobutyl

chloroformate (0.455 mL, 3.50 mmol, 1.0 equiv), NMM (2 × 0.385

mL, 2 × 3.50 mmol, 2 × 1.0 equiv), and compound 5b (1.26 g, 3.50

mmol, 1.0 equiv) in 50 mL of THF. Yield: 2.02 g (3.28 mmol, 93.7%)

of compound 6a as a light-yellow solid. HPLC: 53.2 min, start at 10%

B (purity >95%). ¹H NMR (500 MHz, DMSO-d₆): δ = 8.65 (t, J = 6.0

Hz, 1H), 8.54 (d, J = 8.6 Hz, 1H), 8.05−8.14 (m, 4H), 7.72−7.78 (m,

2H), 7.46−7.51 (m, 2H), 7.42−7.46 (m, 2H), 7.35−7.41 (m, 2H),

6.99 (d, J = 8.5 Hz, 1H), 4.64−4.72 (m, 1H), 4.39 (d, J = 6.0 Hz, 2H),

4.19−4.28 (m, 1H), 3.19 (dd, J = 4.9 Hz, 13.6 Hz, 1H), 2.97 (dd, J =

9.8 Hz, 13.8 Hz, 1H), 2.87 (dd, J = 4.6 Hz, 13.5 Hz, 1H), 2.68 (dd, J =

10.3 Hz, 13.9 Hz, 1H), 1.24 (s, 9H) ppm. ¹³C NMR (125 MHz,

DMSO-d₆): δ = 171.0, 170.5, 155.3, 146.3, 146.2, 146.1, 146.0, 144.9,

132.1 (2C), 130.5 (2C), 130.4 (2C), 127.8 (2C), 123.1 (2C), 122.9

(2C), 118.8, 109.5, 78.2, 55.1, 53.4, 41.8, 37.4, 37.2, 28.0 (3C) ppm.

MS (ESI, positive): calcd, 616.23; m/z 617.25 [M + H]⁺, 634.21 [M +

NH₄]⁺.

MHz, DMSO-d₆): δ = 8.68 (t, J = 6.0 Hz, 1H), 8.66 (d, J = 8.6 Hz,

1H), 8.14 (d, J = 8.0 Hz, 1H), 8.08−8.12 (m, 2H), 8.00−8.04 (m,

2H), 7.73−7.77 (m, 2H), 7.46−7.51 (m, 2H), 7.37−7.41 (m, 2H),

7.29−7.33 (m, 2H), 7.16−7.22 (m, 2H), 7.09−7.14 (m, 1H), 7.04−

7.08 (m, 2H), 4.63−4.69 (m, 1H), 4.56−4.62 (m, 1H), 4.34−4.43 (m,

2H), 3.19 (dd, J = 4.8 Hz, 13.7 Hz, 1H), 2.94 (dd, J = 9.9 Hz, 13.6 Hz,

1H), 2.87 (dd, J = 4.9 Hz, 13.7 Hz, 1H), 2.67 (dd, J = 9.4 Hz, 13.6 Hz,

1H), 2.59−2.64 (m, 2H), 2.27−2.35 (m, 2H) ppm. ¹³C NMR (125

MHz, DMSO-d₆): δ = 171.5, 170.7, 170.5, 146.2, 146.1, 146.0, 145.9,

144.9, 141.0, 132.1 (2C), 130.5 (2C), 130.3 (2C), 128.1 (2C), 128.0

(2C), 127.9 (2C), 125.7, 123.1 (2C), 122.9 (2C), 118.8, 109.6, 53.5,

53.3, 41.9, 37.4 (2C), 36.4, 30.7 ppm. MS (ESI, negative): calcd,

648.23; m/z 647.43 [M − H]⁻.

3-Phprop-^DPhe(p-NH₂)-Phe(p-NH₂)-p-cyanobenzylamide (7b).

Compound 7a (155 mg, 0.239 mmol, 1.0 equiv) was dissolved in 10

mL of THF. The solution was treated with zinc powder (469 mg, 7.17

mmol, 30.0 equiv) and acetic acid (260 μL, 4.54 mmol, 19.0 equiv).

The suspension was stirred for 1.5 h at rt and then diluted with 100

mL of EtOAc. The suspension was ﬁltered, and the ﬁltrate was washed

three times with saturated aq NaHCO₃and once with brine. The

organic layer was dried over MgSO₄, ﬁltered, and the solvent removed

in vacuo. Yield: 126 mg (0.214 mmol, 89.5%) of compound 7b as a

yellow solid. HPLC: 24.9 min, start at 10% B (purity 92.2%). ¹H NMR

(500 MHz, DMSO-d₆): δ = 8.45 (t, J = 6.0 Hz, 1H), 8.22 (d, J = 8.6

Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.69−7.76 (m, 2H), 7.19−7.29 (m,

4H), 7.12−7.18 (m, 1H), 7.07−7.12 (m, 2H), 6.83−6.89 (m, 2H),

6.75−6.81 (m, 2H), 6.45−6.50 (m, 2H), 6.41−6.45 (m, 2H), 4.85 (bs,

4H), 4.34−4.47 (m, 4H), 2.80 (dd, J = 5.7 Hz, 13.4 Hz, 1H), 2.72−

2.75 (m, 1H), 2.63−2.67 (m, 2H), 2.51−2.54 (m, 1H), 2.45 (dd, J =

9.6 Hz, 13.5 Hz, 1H), 2.27−2.36 (m, 2H) ppm. ¹³C NMR (125 MHz,

DMSO-d₆): δ = 171.5, 171.4, 171.3, 146.9 (2C), 145.1, 141.2, 132.0

(2C), 129.6 (2C), 129.5 (2C), 128.2 (2C), 128.0 (2C), 127.7 (2C),

125.7, 124.6, 124.0, 118.9, 113.7 (2C), 113.6 (2C), 109.3, 54.7, 54.6,

41.7, 36.9, 36.8, 36.6, 31.2 ppm. MS (ESI, positive): calcd, 588.28; m/z

589.27 [M + H]⁺.

Chas-^DPhe(p-NO₂)-Phe(p-NO₂)-p-cyanobenzylamide (8a). To a

solution of compound 6b (553 mg, 1.00 mmol, 1.0 equiv) and DIPEA

(525 μL, 3.00 mmol, 3.0 equiv) in 10 mL of THF, N-cyclo-

hexylsulfamoyl chloride⁴⁵(198 mg, 1.00 mmol, 1.0 equiv) was added

at 0 °C. The solution was stirred at rt overnight. Because of

incomplete conversion, the solution was treated with additional N-

cyclohexylsulfamoyl chloride (494 mg, 2.50 mmol, 2.5 equiv) and

DIPEA (425 μL, 2.50 mmol, 2.5 equiv) and stirred for 6 h. The

mixture was diluted with H₂O. The basic solution was extracted three

times with EtOAc. The organic layer was washed one time with brine,

three times with 5% aq KHSO₄, and one time with brine. The organic

phase was dried over MgSO₄, ﬁltered, and the solvent removed in

vacuo. The remaining residue was puriﬁed via ﬂash chromatography

(silica gel, DCM/MeOH 60:1). Yield: 509 mg (0.751 mmol, 75.1%) of

compound 8a as a white solid. HPLC: 56.7 min, start at 10% B (purity

H-^DPhe(p-NO₂)-Phe(p-NO₂)-p-cyanobenzylamide·HCl (6b). The

synthesis was performed as described for compound 5b using

compound 6a (1.23 g, 2.00 mmol, 1.0 equiv) in 16 mL of 1 M HCl

in acetic acid (16.0 mmol, 8.0 equiv). Yield: 1.07 g (1.93 mmol,

96.5%) of compound 6b as a light-yellow solid. HPLC: 34.2 min, start

1

>95%). TLC: R_f= 0.32 (DCM/MeOH 20:1). H NMR (500 MHz,

1

at 10% B (purity >95%). H NMR (500 MHz, DMSO-d₆): δ = 9.25

DMSO-d₆): δ = 8.57−8.63 (m, 2H), 8.09−8.16 (m, 4H), 7.72−7.77

(m, 2H), 7.50−7.54 (m, 2H), 7.46−7.50 (m, 2H), 7.35−7.40 (m, 2H),

7.03 (d, J = 9.8 Hz, 1H), 6.53 (d, J = 7.7 Hz, 1H), 4.68−4.76 (m, 1H),

4.30−4.42 (m, 2H), 3.88−3.95 (m, 1H), 3.19 (dd, J = 5.2 Hz, 13.6 Hz,

1H), 2.97 (dd, J = 9.4 Hz, 13.4 Hz, 1H), 2.72 (dd, J = 4.3 Hz, 13.5 Hz,

1H), 2.63 (dd, J = 10.0 Hz, 13.3 Hz, 1H), 2.35−2.44 (m, 1H), 1.63−

1.72 (m, 1H), 1.45−1.53 (m, 1H), 1.32−1.42 (m, 2H), 1.10−1.17 (m,

1H), 0.90−0.98 (m, 3H), 0.71−0.90 (m, 2H) ppm. ¹³C NMR (125

MHz, DMSO-d₆): δ = 171.2, 170.4, 146.3, 146.2, 146.1, 145.9, 144.8,

132.1 (2C), 130.9 (2C), 130.6 (2C), 127.9 (2C), 123.1 (2C), 122.9

(2C), 118.8, 109.5, 57.1, 53.3, 51.6, 41.8, 37.9, 37.7, 33.1, 32.8, 24.9,

24.6 (2C) ppm. MS (ESI, negative): calcd, 677.23; m/z 676.35 [M −

H]⁻.

Chas-^DPhe(p-NH₂)-Phe(p-NH₂)-p-cyanobenzylamide (8b). The

synthesis was performed as described for compound 7b using zinc

powder (946 mg, 14.5 mmol, 30.0 equiv), acetic acid (524 μL, 9.16

mmol, 19.0 equiv), and compound 8a (327 mg, 0.482 mmol, 1.0

equiv) in 10 mL of THF. Yield: 279 mg (0.452 mmol, 93.8%) of

compound 8b as a light-yellow solid. HPLC: 24.5 min, start at 10% B

(d, J = 8.6 Hz, 1H), 9.02 (t, J = 6.0 Hz, 1H), 8.34 (bs, 3H), 8.07−8.13

(m, 4H), 7.73−7.78 (m, 2H), 7.48−7.53 (m, 2H), 7.39−7.44 (m, 2H),

7.31−7.36 (m, 2H), 4.65−4.74 (m, 1H), 4.37 (d, J = 6.0 Hz, 2H),

4.13−4.20 (m, 1H), 3.20 (dd, J = 4.6 Hz, 13.8 Hz, 1H), 3.09 (dd, J =

5.4 Hz, 14.0 Hz, 1H), 2.94 (dd, J = 10.0 Hz, 13.7 Hz, 1H), 2.86 (dd, J

= 7.8 Hz, 13.9 Hz, 1H) ppm. ¹³C NMR (125 MHz, DMSO-d₆): δ =

170.0, 167.7, 146.7, 146.3, 145.8, 144.9, 142.9, 132.1 (2C), 130.8 (2C),

130.5 (2C), 128.0 (2C), 123.3 (2C), 123.1 (2C), 118.8, 109.5, 53.7,

52.9, 41.9, 37.5, 36.3 ppm. MS (ESI, positive): calcd, 516.18; m/z

517.20 [M + H]⁺.

3-Phprop-^DPhe(p-NO₂)-Phe(p-NO₂)-p-cyanobenzylamide (7a).

The synthesis was performed as described for compound 5a using

3-phenylpropionic acid (70.6 mg, 0.47 mmol, 1.0 equiv), isobutyl

chloroformate (61.6 μL, 0.47 mmol, 1.0 equiv), NMM (2 × 51.9 μL, 2

× 0.47 mmol, 2 × 1.0 equiv), and compound 6b (260 mg, 0.47 mmol,

1.0 equiv) in 10 mL of THF. Yield: 270 mg (0.42 mmol, 88.4%) of

compound 7a as a yellow solid. HPLC: 52.7 min, start at 10% B

1

(purity >95%). TLC: R_f= 0.30 (DCM/MeOH 20:1). H NMR (500

K

DOI: 10.1021/acs.jmedchem.6b00606

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1

(purity >95%). H NMR (500 MHz, DMSO-d₆): δ = 8.31 (t, J = 6.2

puriﬁed via ﬂash chromatography (silica gel, DCM/MeOH 15:1 with

0.5% NH₄OH (25%)). Yield: 50.9 mg (0.065 mmol, 31.9%) of

compound 12 as white solid. HPLC: 32.2 min, start at 10% B (purity

>95%). TLC: R_f= 0.18 (DCM/MeOH 10:1 with 0.5% NH₄OH

(25%)). ¹H NMR (500 MHz, DMSO-d₆): δ = 10.41 (s, 1H), 10.06 (s,

1H), 8.71 (t, J = 6.1 Hz, 1H), 8.60 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.3

Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 7.9 Hz, 2H), 7.35 (d, J

= 6.7 Hz, 1H), 7.13−7.28 (m, 7H), 6.98 (d, J = 8.4 Hz, 2H), 6.05 (d, J

= 7.9 Hz, 2H), 4.61−4.67 (m, 1H), 4.34−4.46 (m, 3H), 3.11−3.25 (m,

1H), 3.02−3.09 (m, 1H), 2.72−2.88 (m, 6H), 2.56−2.72 (m, 7H),

2.50−2.56 (m, 4H), 2.31−2.45 (m, 4H), 2.06−2.21 (m, 1H) ppm. ¹³C

NMR (125 MHz, DMSO-d₆): δ = 171.5, 171.0, 170.1, 169.9, 145.2,

141.2, 137.6, 136.9, 133.3, 132.2 (2C), 130.9, 129.8 (2C), 129.6 (2C),

128.3 (2C), 128.2 (2C), 127.8 (2C), 125.8, 119.8 (2C), 119.2 (2C),

118.9, 109.5, 55.3, 53.1, 52.8, 52.1, 51.3 (2C), 51.0 (2C), 41.8, 37.0,

36.9, 36.4, 32.3, 31.4, 30.9 ppm. MS (ESI, positive): calcd, 782.39; m/z

783.39 [M + H]⁺.

Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.69−7.74 (m, 2H), 7.19−7.24 (m,

2H), 6.84−6.88 (m, 2H), 6.81−6.84 (m, 2H), 6.74 (d, J = 9.2 Hz,

1H), 6.42−6.50 (m, 5H), 4.87 (bs, 2H), 4.81 (bs, 2H), 4.40−4.47 (m,

1H), 4.20−4.40 (m, 2H), 3.74−3.81 (m, 1H), 2.78 (dd, J = 6.3 Hz,

13.6 Hz, 1H), 2.67 (dd, J = 8.3 Hz, 13.7 Hz, 1H), 2.57−2.62 (m, 1H),

2.54 (dd, J = 5.1 Hz, 13.6 Hz, 1H), 2.47 (dd, J = 8.9 Hz, 13.6 Hz, 1H),

1.71−1.79 (m, 1H), 1.49−1.58 (m, 2H), 1.38−1.47 (m, 2H), 0.88−

1.08 (m, 5H) ppm. ¹³C NMR (125 MHz, DMSO-d₆): δ = 171.6,

171.0, 147.0, 146.9, 144.9, 132.0 (2C), 129.8 (2C), 129.7 (2C), 127.6

(2C), 124.4, 124.3, 118.9, 113.8 (2C), 113.6 (2C), 109.3, 58.2, 54.4,

51.5, 41.6, 37.6, 36.9, 33.2, 32.9, 25.1, 24.6 (2C) ppm. MS (ESI,

positive): calcd, 617.28; m/z 618.27 [M + H]⁺.

Piperazine-1,4-diacetic Acid (9). The synthesis was performed as

described in the literature⁴⁶using piperazine (1.15 g, 13.4 mmol, 1.0

equiv) and bromoacetic acid (3.73 g, 26.8 mmol, 2.0 equiv) in 15 mL

of 3.57 M NaOH (53.6 mmol, 4.0 equiv). Yield: 1.69 g (8.36 mmol,

62.4%) as a white solid. ¹H NMR (400 MHz, D₂O): δ = 3.49 (s, 4H),

3.19 (bs, 8H) ppm. ¹³C NMR (100 MHz, D₂O): δ = 160.3 (2C), 59.4

(2C), 50.7 (4C) ppm. MS (ESI, positive): calcd, 202.10; m/z 203.06

[M + H]⁺.

(11S,14R)-N-(4-Cyanobenzyl)-14-((N-cyclohexylsulfamoyl)-

amino)-3,7,13-trioxo-2,8,12-triaza-5(1,4)-piperazina-1,9(1,4)-diben-

zenacyclopentadecaphane-11-carboxamide (13). The synthesis was

performed as described for compound 11 with compound 8b (220 mg,

0.356 mmol, 1.0 equiv), DIPEA (372 μL, 2.14 mmol, 6.0 equiv),

compound 9 (93.6 mg, 0.427 mmol, 1.2 equiv), and HATU (325 mg,

0.854 mmol, 2.4 equiv) in 150 mL of DMF. The crude product was

puriﬁed via ﬂash chromatography (silica gel, DCM/MeOH 15:1 with

0.5% NH₄OH (25%)). Yield: 120 mg (0.153 mmol, 43.0%) of

compound 13 as white solid. HPLC: 32.8 min, start at 10% B (purity

>95%). TLC: R_f= 0.19 (DCM/MeOH 10:1 with 0.5% NH₄OH

Piperazine-1,4-dipropionic Acid (10). The synthesis was performed

as described in the literature²⁷using piperazine (1.15 g, 13.4 mmol, 1.0

equiv) and bromopropionic acid (4.10 g, 26.3 mmol, 2.0 equiv) in 15

mL of 3.57 M NaOH (53.6 mmol, 4.0 equiv). Yield: 1.81 g (7.84

1

mmol, 58.5%) as white crystals. H NMR (400 MHz, D₂O): δ = 3.81

(bs, 8H), 3.67 (t, J = 6.9 Hz, 4H), 3.01 (t, J = 6.9 Hz, 4H) ppm. ¹³C

NMR (100 MHz, D₂O): δ = 173.6 (2C), 52.4 (4C), 48.8 (2C), 28.6

(2C) ppm. MS (ESI, positive): calcd, 230.13; m/z 231.17 [M + H]⁺.

(11S,14R)-N-(4-Cyanobenzyl)-3,7,13-trioxo-14-(3-phenylpropa-

namido)-2,8,12-triaza-5(1,4)-piperazina-1,9(1,4)-dibenzenacyclo-

pentadecaphane-11-carboxamide (11). A solution of compound 7b

(140 mg, 0.238 mmol, 1.0 equiv) in 150 mL of DMF was cooled at 0

°C. DIPEA (207 μL, 1.19 mmol, 5.0 equiv), compound 9 (48.1 mg,

0.238 mmol, 1.0 equiv), and HATU (181 mg, 0.476 mmol, 2.0 equiv)

were added over a period of 1 h in 10 portions. The solution was

stirred at rt overnight. After 18 h, the reaction was detected by HPLC

and the chromatogram still showed educt 7b. To achieve a complete

reaction DIPEA (41.5 μL, 0.283 mmol, 1.0 equiv), compound 9 (9.6

mg, 0.048 mmol, 0.2 equiv) and HATU (36.2 mg, 0.095 mmol, 0.4

equiv) were added at 0 °C and the solution was stirred at rt overnight.

The solvent was removed in vacuo, and the remaining oil was treated

with 15 mL of 1 M NaOH. The resulting suspension was ﬁltered, and

the ﬁltrate was extracted thrice with DCM. The combined organic

phases were washed once with brine and dried over MgSO₄. The

solvent was removed in vacuo, and the resulting residue was combined

with the ﬁlter residue. The crude product was puriﬁed via ﬂash

chromatography (silica gel, DCM/MeOH 15:1 with 0.5% NH₄OH

(25%)). Yield: 68.6 mg (0.091 mmol, 38.2%) as white solid. HPLC:

33.4 min, start at 10% B (purity >95%). TLC: R_f= 0.21 (DCM/

MeOH 10:1 with 0.5% NH₄OH (25%)). ¹H NMR (500 MHz,

DMSO-d₆): δ = 9.96 (s, 1H), 9.92 (s, 1H), 8.81 (d, J = 7.5 Hz, 1H),

8.54 (t, J = 5.8 Hz, 1H), 8.14 (d, J = 6.4 Hz, 1H), 7.79 (d, J = 8.1 Hz,

2H), 7.40−7.49 (m, 4H), 7.23−7.32 (m, 4H), 7.18−7.22 (m, 2H),

7.09−7.17 (m, 3H), 6.88 (d, J = 7.7 Hz, 2H), 4.54−4.62 (m, 1H),

4.43−4.49 (m, 1H), 4.35−4.43 (m, 2H), 3.24 (s, 2H), 3.18 (s, 2H),

3.00−3.13 (m, 2H), 2.87−2.96 (m, 1H), 2.59−2.81 (m, 11H), 2.38−

2.47 (m, 3H), 2.19−2.29 (m, 1H) ppm. ¹³C NMR (125 MHz, DMSO-

d₆): δ = 171.8, 171.7, 171.2, 167.6, 166.8, 145.2, 141.1, 136.5, 136.4,

133.5, 132.2 (2C), 132.1, 129.0 (2C), 128.9 (2C), 128.2 (2C), 128.1

(2C), 127.7 (2C), 125.8, 118.9, 118.5 (2C), 118.1 (2C), 109.5, 60.6,

60.2, 55.0, 53.1, 51.5 (4C), 41.9, 36.4, 36.2, 36.0, 30.8 ppm. MS (ESI,

positive): calcd, 754.36; m/z 755.60 [M + H]⁺.

1

(25%)). H NMR (500 MHz, DMSO-d₆): δ = 9.96 (s, 1H), 9.94 (s,

1H), 8.65 (t, J = 5.8 Hz, 1H), 8.51 (d, J = 6.9 Hz, 1H), 7.80 (d, J = 8.1

Hz, 2H), 7.46 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.1 Hz, 2H), 7.25 (d, J

= 7.6 Hz, 2H), 7.17 (d, J = 7.5 Hz, 2H), 6.91 (d, J = 7.6 Hz, 2H), 6.81

(d, J = 6.7 Hz, 1H), 6.72 (d, J = 6.1 Hz, 1H), 4.49−4.57 (m, 1H),

4.34−4.47 (m, 2H), 4.05−4.12 (m, 1H), 3.24 (s, 2H), 3.17 (s, 2H),

3.07−3.13 (m, 1H), 3.00−3.06 (m, 1H), 2.87−3.00 (m, 3H), 2.72 (bs,

4H), 2.69 (bs, 4H), 1.81−1.89 (m, 1H), 1.74−1.81 (m, 1H), 1.58−

1.66 (m, 1H), 1.50−1.57 (m, 1H), 1.41−1.49 (m, 1H), 1.14−1.22 (m,

2H), 0.96−1.14 (m, 3H) ppm. ¹³C NMR (125 MHz, DMSO-d₆): δ =

171.7, 170.5, 167.6, 166.9, 145.1, 136.7, 136.4, 133.2, 132.3 (2C),

131.9, 129.4 (2C), 129.1 (2C), 127.8 (2C), 118.9, 118.4 (2C), 118.0

(2C), 109.5, 60.6, 60.2, 55.8, 54.9, 51.9, 51.5 (4C), 41.8, 36.8, 36.4,

33.4, 33.0, 25.0, 24.6 (2C) ppm. MS (ESI, positive): calcd, 783.35; m/

z 784.40 [M + H]⁺.

(13S,16R)-N-(4-Cyanobenzyl)-16-((N-cyclohexylsulfamoyl)-

amino)-3,9,15-trioxo-2,10,14-triaza-6(1,4)-piperazina-1,11(1,4)-di-

benzenacycloheptadecaphane-13-carboxamide (14). The synthesis

was performed as described for compound 11 with compound 8b (216

mg, 0.350 mmol, 1.0 equiv), DIPEA (366 μL, 2.10 mmol, 6.0 equiv),

compound 10 (96.7 mg, 0.420 mmol, 1.2 equiv), and HATU (319 mg,

0.840 mmol, 2.4 equiv) in 150 mL of DMF. The crude product was

puriﬁed via ﬂash chromatography (silica gel, DCM/MeOH 15:1 with

0.5% NH₄OH (25%)). Yield: 116 mg (0.143 mmol, 40.9%) as white

solid. HPLC: 32.2 min, start at 10% B (purity >95%). TLC: R_f= 0.18

(DCM/MeOH 10:1 with 0.5% NH₄OH (25%)). ¹H NMR (500 MHz,

DMSO-d₆): δ = 10.45 (s, 1H), 10.16 (s, 1H), 8.70 (t, J = 6.0 Hz, 1H),

8.53 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.3 Hz, 2H), 7.44 (d, J = 8.3 Hz,

2H), 7.38 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.3 Hz, 2H), 7.06 (d, J = 8.4

Hz 2H), 6.89 (d, J = 7.5 Hz, 1H), 6.34 (d, J = 8.3 Hz, 2H), 5.68 (d, J =

6.8 Hz, 1H), 4.46−4.53 (m, 1H), 4.32−4.45 (m, 2H), 4.02−4.09 (m,

1H), 3.08−3.18 (m, 1H), 2.91−2.98 (m, 1H), 2.74−2.89 (m, 6H),

2.67−2.74 (m, 1H), 2.57−2.65 (m, 3H), 2.50−2.57 (m, 6H), 2.32−

2.41 (m, 2H), 2.08−2.17 (m, 1H), 1.79−1.88 (m, 1H), 1.65−1.73 (m,

1H), 1.59−1.65 (m, 1H), 1.46−1.55 (m, 1H), 1.39−1.46 (m, 1H),

1.11−1.19 (m, 2H), 0.97−1.08 (m, 3H) ppm. ¹³C NMR (125 MHz,

DMSO-d₆): δ = 171.2, 170.4, 170.3, 169.7, 145.1, 137.6, 137.2, 132.8,

132.2 (2C), 130.6, 130.0 (2C), 129.7 (2C), 127.9 (2C), 119.8 (2C),

119.2 (2C), 118.9, 109.5, 55.6, 55.1, 52.8, 52.2, 51.8, 51.4 (2C), 50.7

(2C), 41.8, 38.4, 37.0, 33.4, 33.0, 32.2, 31.6, 25.0, 24.6, 24.5 ppm. MS

(ESI, positive): calcd, 811.38; m/z 812.45 [M + H]⁺.

(13S,16R)-N-(4-Cyanobenzyl)-3,9,15-trioxo-16-(3-phenylpropa-

namido)-2,10,14-triaza-6(1,4)-piperazina-1,11(1,4)-dibenzenacy-

cloheptadecaphane-13-carboxamide (12). The synthesis was

performed as described for compound 11 with compound 7b (120

mg, 0.204 mmol, 1.0 equiv), DIPEA (213 μL, 1.22 mmol, 6.0 equiv),

compound 10 (56.4 mg, 0.245 mmol, 1.2 equiv), and HATU (186 mg,

0.489 mmol, 2.4 equiv) in 150 mL of DMF. The crude product was

L

DOI: 10.1021/acs.jmedchem.6b00606

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Journal of Medicinal Chemistry

Article

(11S,14R)-N-(4-Carbamimidoylbenzyl)-3,7,13-trioxo-14-(3-phe-

nylpropanamido)-2,8,12-triaza-5(1,4)-piperazina-1,9(1,4)-dibenze-

nacyclopentadecaphane-11-carboxamide·3TFA (23). Compound

11 (39.0 mg, 0.052 mmol, 1.0 equiv) was dissolved in 0.5 mL of

MeOH. The solution was treated with hydroxylamine hydrochloride

(7.23 mg, 0.104 mmol, 2.0 equiv) and DIPEA (18.1 μL, 0.104 mmol,

2.0 equiv). The solution was heated under reﬂux for 6 h and then

stirred at rt overnight. The solvent was removed in vacuo, and the

remaining residue was dissolved in 3 mL of acetic acid. The solution

was treated with acetic anhydride (9.8 μL, 0.104 mmol, 2.0 equiv) and

stirred for 40 min at rt. Pd/C (10 w%) was added to the solution.

Under H₂atmosphere, the resulting suspension was stirred for 2 d at

rt. The suspension was ﬁltered. The solvent was removed, and the

remaining oil was dissolved in MeOH and precipitated in diethyl ether.

The crude product was puriﬁed via preparative HPLC and lyophilized.

Yield: 12.3 mg (0.011 mmol, 21.2%) as white lyophilized solid. HPLC:

20.9 min, start at 10% B (purity >95%). ¹H NMR (500 MHz, DMSO-

d₆): δ = 9.95 (s, 1H), 9.92 (s, 1H), 8.72−8.83 (m, 1H), 8.48 (t, J = 5.5

Hz, 1H), 8.07−8.17 (m, 1H), 7.66−7.78 (m, 2H), 7.43 (d, J = 8.3 Hz,

2H), 7.26−7.30 (m, 2H), 7.22−7.26 (m, 4H), 7.18−7.22 (m, 2H),

7.13−7.18 (m, 3H), 6.86 (d, J = 8.3 Hz, 2H), 6.22−6.56 (bs, 3H),

4.53−4.62 (m, 1H), 4.42−4.50 (m, 1H), 4.28−4.39 (m, 2H), 3.22 (s,

2H), 3.17 (s, 2H), 2.99−3.12 (m, 2H), 2.86−2.96 (m, 1H), 2.60−2.83

(m, 11H), 2.39−2.47 (m, 1H), 2.23−2.32 (m, 1H) ppm. ¹³C NMR

(125 MHz, DMSO-d₆): δ = 171.7, 171.6, 171.0, 167.6, 166.8, 141.1,

140.9, 136.5, 136.3, 133.5, 132.2, 129.0 (2C), 128.9 (2C), 128.2 (4C),

126.5 (4C), 126.4, 125.8, 118.4 (2C), 118.0 (2C), 60.6, 60.2, 55.0,

53.1, 51.6 (4C), 41.8, 36.4, 36.3, 35.9, 30.9 ppm. MS (ESI, positive):

calcd, 771.39; m/z 772.44 [M + H]⁺.

1.79−1.89 (m, 2H), 1.61−1.68 (m, 1H), 1.54−1.61 (m, 1H), 1.43−

1.51 (m, 1H), 0.99−1.26 (m, 5H) ppm. ¹³C NMR (125 MHz, DMSO-

d₆): δ = 171.5, 170.8, 167.5, 167.4, 140.9, 137.0, 136.6, 133.3, 133.2,

129.4 (2C), 129.0 (2C), 126.7 (2C), 126.4 (2C), 126.3, 118.4 (2C),

117.9 (2C), 60.7, 60.4, 55.2, 54.8, 51.9, 51.6 (4C), 41.9, 36.7, 36.2,

33.2 (2C), 25.1, 24.7 (2C) ppm. MS (ESI, positive): calcd, 800.38; m/

z 801.55 [M + H]⁺.

(13S,16R)-N-(4-Carbamimidoylbenzyl)-16-((N-cyclohexylsulf-

amoyl)amino)-3,9,15-trioxo-2,10,14-triaza-6(1,4)-piperazina-1,11-

(1,4)-dibenzenacycloheptadecaphane-13-carboxamide·3TFA (31).

The synthesis was performed as described for inhibitor 23 using

compound 14 (113 mg, 0.139 mmol, 1.0 equiv) in 0.8 mL of MeOH,

hydroxylamine hydrochloride (19.3 mg, 0.278 mmol, 2.0 equiv),

DIPEA (48.4 μL, 0.278 mmol, 2.0 equiv), and acetic anhydride (26.3

μL, 0.278 mmol, 2.0 equiv) in 4 mL of acetic acid. The crude product

was puriﬁed via preparative HPLC. Yield: 54.0 mg (0.046 mmol,

33.2%) as white lyophilized solid. HPLC: 22.4 min, start at 10% B

1

(purity >95%). H NMR (600 MHz, potassium phosphate buﬀer 100

mM pH = 3/D₂O 5:1): δ = 10.10 (s, 1H), 10.09 (s, 1H), 8.77 (bs,

2H), 8.70 (t, J = 6.0 Hz, 1H), 8.54 (d, J = 8.3 Hz, 1H), 8.43 (bs, 2H),

7.73 (d, J = 8.5 Hz, 2H), 7.43 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 8.6 Hz,

2H), 7.21 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 6.69 (d, J = 8.6

Hz, 2H), 4.68−4.75 (m, 1H), 4.49 (dd, J = 6.0 Hz, 15.7 Hz, 1H), 4.44

(dd, J = 6.0 Hz, 15.7 Hz, 1H), 4.17−4.25 (m, 1H), 3.33−3.45 (m,

2H), 3.22−3.29 (m, 2H), 3.14−3.22 (m, 4H), 3.02−3.11 (m, 5H),

2.87−3.01 (m, 4H), 2.70−2.79 (m, 4H), 1.79−1.87 (m, 1H), 1.60−

1.70 (m, 2H), 1.44−1.56 (m, 2H), 1.15−1.24 (m, 2H), 1.01−1.03 (m,

3H) ppm. ¹³C NMR (100 MHz, potassium phosphate buﬀer 100 mM

pH = 3/D₂O 5:1): δ = 172.6, 172.4, 172.3, 172.0, 166.7, 144.3, 135.7,

135.6, 133.9, 132.7, 130.4 (2C), 130.0 (2C), 128.1 (2C), 128.0 (2C),

126.9, 122.1 (2C), 122.0 (2C), 56.9, 54.8, 52.8, 51.4, 51.3, 49.6 (2C),

49.5 (2C), 42.8, 38.4, 36.9, 33.4, 33.1, 31.6, 31.3, 24.8, 24.5 (2C) ppm.

MS (ESI, positive): calcd, 828.41; m/z 829.54 [M + H]⁺.

N-(4-(Cyano)benzyl)triﬂuoroacetamide (33). A solution of p-

cyanobenzylamine·HCl (5.058 g, 30.0 mmol, 1.0 equiv) in 50 mL of

DCM was treated with NMM (6.6 mL, 60.0 mmol, 2.0 equiv). The

suspension was cooled to 0 °C, and triﬂuoroacetic anhydride (4.23

mL, 30.0 mmol, 1.0 equiv), dissolved in 50 mL of DCM, was added

dropwise within 1 h. The mixture was stirred for 1 h at 0 °C and at rt

overnight. The solvent was evaporated, and the residue was dissolved

in a mixture of 5% aq KHSO₄and EtOAc. The organic layer was

washed thrice with 5% aq KHSO₄and brine, dried over MgSO₄, and

ﬁltered. The solvent was removed in vacuo. Yield: 6.784 g (29.7 mmol,

99%) as yellow solid. HPLC: 32.8 min, start at 10% B (purity >99%).

¹H NMR (400 MHz, DMSO-d₆): δ = 10.12 (bs, 1H), 7.84 (d, 2H, J =

(13S,16R)-N-(4-Carbamimidoylbenzyl)-3,9,15-trioxo-16-(3-phe-

nylpropanamido)-2,10,14-triaza-6(1,4)-piperazina-1,11(1,4)-diben-

zenacycloheptadecaphane-13-carboxamide·3TFA (24). The syn-

thesis was performed as described for inhibitor 23 using compound

12 (50.0 mg, 0.064 mmol, 1.0 equiv) in 0.6 mL of MeOH,

hydroxylamine hydrochloride (8.9 mg, 0.128 mmol, 2.0 equiv),

DIPEA (22.3 μL, 0.128 mmol, 2.0 equiv), and acetic anhydride (12.1

μL, 0.128 mmol, 2.0 equiv) in 3 mL of acetic acid. The crude product

was puriﬁed via preparative HPLC. Yield: 18.9 mg (0.017 mmol,

25.9%) as white lyophilized solid. HPLC: 22.2 min, start at 10% B

1

(purity >95%). H NMR (600 MHz, potassium phosphate buﬀer 100

mM pH = 3/D₂O 5:1): δ = 10.13 (s, 1H), 10.11 (s, 1H), 8.69 (bs,

2H), 8.64 (t, J = 6.0 Hz, 1H), 8.53 (d, J = 8.3 Hz, 1H), 8.38 (bs, 2H),

7.68−7.74 (m, 3H), 7.40 (d, J = 8.5 Hz, 2H), 7.22−7.32 (m, 5H),

7.13−7.19 (m, 4H), 6.96 (d, J = 8.5 Hz, 2H), 6.43 (d, J = 8.6 Hz, 2H),

4.57−4.64 (m, 1H), 4.50−4.57 (m, 1H), 4.39−4.47 (m, 2H), 3.35−

3.38 (m, 2H), 3.25−3.31 (m, 2H), 3.17−3.24 (m, 4H), 3.04−3.14 (m,

5H), 2.92 (dd, J = 9.5 Hz, 13.9 Hz, 1H), 2.71−2.85 (m, 8H), 2.49−

2.55 (m, 2H) ppm. ¹³C NMR (100 MHz, potassium phosphate buﬀer

100 mM pH = 3/D₂O 5:1): δ = 175.3, 173.0, 172.3, 172.1, 171.9,

166.5, 144.3, 140.4, 135.6, 135.5, 134.3, 132.8, 130.1 (2C), 129.9 (2C),

128.7 (2C), 128.5 (2C), 128.0 (2C), 127.8 (2C), 126.8, 126.5, 122.2

(2C), 122.1 (2C), 55.1, 54.4, 51.3, 51.2, 49.3 (2C), 49.2 (2C), 42.8,

36.8, 36.7, 36.3, 31.5, 31.2, 31.0 ppm. MS (ESI, positive): calcd,

799.42; m/z 800.45 [M + H]⁺.

(11S,14R)-N-(4-Carbamimidoylbenzyl)-14-((N-cyclohexylsulf-

amoyl)amino)-3,7,13-trioxo-2,8,12-triaza-5(1,4)-piperazina-1,9-

(1,4)-dibenzenacyclopentadecaphane-11-carboxamide·3TFA (30).

The synthesis was performed as described for inhibitor 23 using

compound 13 (59.0 mg, 0.075 mmol, 1.0 equiv) in 0.6 mL of MeOH,

hydroxylamine hydrochloride (10.5 mg, 0.151 mmol, 2.0 equiv),

DIPEA (25.7 μL, 0.151 mmol, 2.0 equiv), and acetic anhydride (14.3

μL, 0.151 mmol, 2.0 equiv) in 3.5 mL of acetic acid. The crude

product was puriﬁed via preparative HPLC. Yield: 21.1 mg, (0.018

mmol, 24.5%) as white lyophilized solid. HPLC: 21.0 min, start at 10%

B (purity >95%). ¹H NMR (500 MHz, DMSO-d₆): δ = 9.93 (bs, 2H),

8.31−8.84 (m, 2H), 7.67−7.78 (m, 2H), 7.42 (d, J = 7.7 Hz, 2H), 7.29

(d, J = 8.2 Hz, 2H), 7.25 (d, J = 7.8 Hz, 2H), 7.16 (d, J = 7.9 Hz, 2H),

6.93 (d, J = 7.7 Hz, 2H), 6.11−6.74 (m, 5H), 4.44−4.54 (m, 1H),

4.26−4.39 (m, 2H), 3.97−4.10 (m, 1H), 3.21 (s, 2H), 3.17 (s, 2H),

2.95−3.11 (m, 3H), 2.84−2.94 (m, 2H), 2.71 (bs, 4H), 2.69 (bs, 4H),

8.0 Hz), 7.46 (d, 2H, J = 8.2 Hz), 4.48 (d, 2H, J = 6.0 Hz) ppm. MS

(ESI, positive): calcd, 228.05; m/z 246.11 [M + NH₄]⁺.

N-(4-(Aminomethyl)benzyl)triﬂuoroacetamide·HCl (H-p-Xda-Tfa·

HCl (34)). Compound 33 (9.282 g, 40.7 mmol, 1.0 equiv) was

dissolved in 300 mL of 90% acetic acid. After addition of 10% Pd/C

(900 mg), the compound was hydrogenated at ambient pressure for 3

h. The catalyst was removed by ﬁltration, and the solvent was

evaporated. The remaining residue was dissolved in 1 M HCl, and the

solvent was removed in vacuo. Yield: 9.544 g (35.5 mmol, 87%) of 34

1

as white solid. HPLC: 11.6 min, start at 10% B (purity >99%). H

NMR (400 MHz, D₂O): δ = 7.33−7.43 (m, 4H), 4.49 (s, 2H), 4.07 (s,

2H) ppm. MS (ESI, positive): calcd, 232.08; m/z 233.11 [M + H]⁺.

H-Phe(p-NO₂)-p-Xda-Tfa·HCl (35). The synthesis was performed as

described for compound 5a using Boc-Phe(p-NO₂)-OH (6.21 g, 20.0

mmol, 1.0 equiv), isobutyl chloroformate (2.60 mL, 20.0 mmol, 1.0

equiv), NMM (2 × 2.20 mL, 2 × 20.0 mmol, 2 × 1.0 equiv), and

compound 34 (5.37 g, 20.0 mmol, 1.0 equiv) in 100 mL of THF.

Yield: 9.215 g of Boc-protected intermediate as white solid (17.6

mmol, 88%). HPLC: 50.1 min, start at 10% B (purity: 93.2%). MS

(ESI, positive): calcd, 541.21; m/z 542.25 [M + NH₄]⁺. The Boc-

protected intermediate (4.99 g, 9.51 mmol, 1.0 equiv) was treated as

described for compound 5b. Yield: 4.177 g (9.06 mmol, 95%) as white

1

solid. HPLC: 27.0 min, start at 10% B (purity 96.5%). H NMR (400

MHz, DMSO-d₆): δ = 10.00 (bs, 1H), 8.98 (bs, 1H), 8.38 (bs, 3H),

8.16 (d, 2H, J = 8.2 Hz), 7.50 (d, 2H, J = 8.2 Hz), 7.11−7.19 (m, 4H),

M

DOI: 10.1021/acs.jmedchem.6b00606

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

3

2

3

4.18−4.38 (m, 4H), 4.10 (m, 1H), 3.15−3.22 (m, 2H) ppm. MS (ESI,

positive): calcd, 424.13; m/z 425.14 [M + H]⁺.

15.0 Hz, J = 6.2 Hz, 1H), 4.35 (dd, J = 15.4 Hz, J = 5.6 Hz, 1H),

4.23−4.07 (m, 4), 3.46−3.32 (m, 2H), 3.30−3.13 (m, 6H), 3.12−3.00

(m, 5H), 2.99−2.85 (m, 4H), 2.81−2.66 (m, 4H), 1.86−1.78 (m, 1H),

1.75−1.60 (m, 2H), 1.59−1.44 (m, 2H), 1.22−1.00 (m, 5H). MS

(ESI, positive): calcd, 815.41; m/z 816.63 [M + H]⁺.

Boc-^DPhe(p-NH₂)-Phe(p-NH₂)-p-Xda-Tfa (36). The synthesis was

performed as described for compound 5a using Boc-^DPhe(p-NO₂)-

OH (135.7 mg, 0.44 mmol, 1.0 equiv), isobutyl chloroformate (57 μL,

0.44 mmol, 1.0 equiv), NMM (2 × 48 μL, 2 × 0.44 mmol, 2 × 1.0

equiv), and compound 35 (201.5 mg, 0.44 mmol, 1.0 equiv) in 5 mL

of THF. Yield: 306.5 mg of crude Boc-^DPhe(p-NO₂)-Phe(p-NO₂)-p-

Xda-Tfa as yellow solid. HPLC: 54.8 min, start at 10% B (purity:

89.5%). MS (ESI, positive): calcd, 716.24; m/z 734.28 [M + NH₄]⁺.

The reduction of the nitro groups and subsequent washing procedure

was performed as described for compound 7b using zinc powder (844

mg, 12.9 mmol, 30.0 equiv) and acetic acid (468 μL, 8.17 mmol, 19.0

equiv) in 20 mL of THF. Yield: 272.0 mg (0.41 mmol, 95%) as white

solid. HPLC: 24.4 min, start at 10% B (purity: 86.6%). A small sample

(13S,16R)-N-(4-(Aminomethyl)benzyl)-3,9,15-trioxo-16-(phenyl-

sulfonamido)-2,10,14-triaza-6(1,4)-piperazina-1,11(1,4)-dibenzena-

cycloheptadecaphane-13-carboxamide·3TFA (42). The synthesis

was performed as described for inhibitor 41 using compound 37

(67.9 mg, 0.062 mmol, 1.0 equiv), 53 μL of DIPEA (0.31 mmol, 5.0

equiv), and 10 μL of phenylsulfonyl chloride (0.079 mmol, 1.3 equiv)

in 10 mL of THF. The Tfa-protected intermediate was puriﬁed via

preparative HPLC. Yield: 50.8 mg (0.041 mmol, 66%) as white,

lyophilized solid. HPLC: 31.0 min, start at 10% B (purity >99.9%). MS

(ESI, positive): calcd, 890.34; m/z 891.74 [M + H]⁺. According to the

synthesis of inhibitor 41, cleavage of the Tfa group was achieved by

dissolving the intermediate in a mixture of dioxane/1 M NaOH (9:1,

v/v) and stirring for 3 h at rt. The crude product was puriﬁed by

preparative HPLC. Yield: 25.1 mg of inhibitor 42 (0.022 mmol, 54%)

as white, lyophilized solid. HPLC: 18.4 min, start at 10% B (purity

1

was puriﬁed by preparative HPLC for NMR analysis. H NMR (400

MHz, DMSO-d₆): δ = 9.95 (bs, 1H), 8.27 (bs, 1H), 8.09 (d, 1H, J =

8.0 Hz), 7.14−7.21 (m, 4H), 6.78−6.94 (m, 4H), 6.67−6.69 (m, 1H),

6.57−6.60 (m, 3H), 6.40−6.45 (m, 3H), 4.60 (bs, 6H), 4.24−4.39 (m,

6H), 2.55−2.87 (m, 4H), 1.29 (s, 9H) ppm. MS (ESI, positive): calcd,

656.29; m/z 657.41[M + H]⁺.

1

>99.9%). H NMR (600 MHz, potassium phosphate buﬀer 100 mM

3

pH = 3/D₂O 5:1): 10.10 (s, 1H), 10.05 (s, 1H), 8.45 (t, J = 6.1 Hz,

(13S,16R)-16-Amino-3,9,15-trioxo-N-(4-((2,2,2-triﬂuoroacet-

amido)methyl)benzyl)-2,10,14-triaza-6(1,4)-piperazina-1,11(1,4)-di-

benzenacycloheptadecaphane-13-carboxamide·3TFA (37). The

synthesis was performed as described for compound 11 using

compound 36 (240.0 mg, 0.37 mmol, 1.0 equiv), compound 10

(110.8 mg, 0.37 mmol, 1.0 equiv), DIPEA (255 μL, 1.46 mmol, 4.0

equiv), and HATU (278.0 mg, 0.73 mmol, 2.0 equiv) in 240 mL of

DMF. The solvent was removed in vacuo, and the residue was

dissolved in EtOAc. The organic layer was washed thrice with

saturated aq NaHCO₃, once with brine, and then dried over MgSO₄

and ﬁltered. The solvent was removed in vacuo. Yield: 435.7 mg of

crude Boc-protected intermediate containing impurities as yellow

solid. HPLC: 33.1 min, start at 10% B. MS (ESI, positive): calcd,

850.40; m/z 851.65 [M + H]⁺. Then 311 mg of the crude intermediate

was treated with 5 mL of 1 M HCl in acetic acid (5.0 mmol, 19.2

equiv) as described for compound 5b. The product was puriﬁed via

preparative HPLC. Yield: 101.4 mg (0.093 mmol, 36%, 2 steps) of

compound 37 as white, lyophilized solid. HPLC: 19.2 min, start at

1H), 8.13 (d, ³J = 8.6 Hz, 1H), 7.72 (d, ³J = 8.3 Hz, 2H), 7.54 (t, ³J =

7.6 Hz, 1H), 7.45 (d, ³J = 8.0 Hz, 2H), 7.43−7.38 (m, 2H), 7.34 (d, ³J

3

= 7.9 Hz, 2H), 7.24 (d, J = 8.6 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H),

6.79 (d, ³J = 8.6 Hz, 2H), 6.57 (d, ³J = 8.5 Hz, 2H), 4.39 (dd, ²J = 14.8

Hz, ³J = 6.0 Hz, 1H), 4.29 (dd, ²J = 15.4 Hz, ³J = 5.7 Hz, 1H), 4.22−

4.10 (m, 4H), 3.49−3.38 (m, 1H), 3.38−3.30 (m, 1H), 3.30−3.13 (m,

6H), 3.13−3.02 (m, 4H), 2.98−2.86 (m, 2H), 2.82−2.64 (m, 6H). ¹³C

NMR (150 MHz, potassium phosphate buﬀer 100 mM pH = 3/D₂O

5:1): 174.9, 174.5, 174.0, 173.3, 141.5, 141.2, 138.7, 138.4, 136.4,

136.2, 135.2, 134.9, 133.4, 132.8, 132.2,132.1, 131.4, 129.5, 124.7,

124.6, 59.8, 57.0, 54.1, 53.8, 52.2, 51.9, 45.9, 45.7, 41.1, 40.1, 34.4,

34.0. MS (ESI, positive): calcd, 794.36; m/z 795.20 [M + H]⁺.

Enzyme Kinetics. All measurements were performed at room

temperature in 50 mM Tris·HCl buﬀer pH 8.0, containing 154 mM

NaCl. The K_ivalues for PK, thrombin, factor Xa, aPC, and trypsin

were determined with chromogenic p-nitroanilide substrates in a

microplate reader (Labsystems, iEMS reader MF) at 405 nm (details

described in Supporting Information)²⁷or with ﬂuorescence substrates

in black 96-well plates using a Fluoroskan Ascent plate reader

(Thermo Fisher Scientiﬁc, Vantaa, Finland) with λ_ex355 and λ_em460

nm. The ﬂuorescence measurements were performed with 100 μL of

buﬀer containing the inhibitor, 20 μL of substrate dissolved in water

and were started by addition of 20 μL of enzyme solution (total assay

volume 140 μL). Mes-^DArg-Pro-Arg-AMC⁴⁷was used as ﬂuorescence

substrate for human fXa (97 pM in assay, molecular weight 46 kDa,

Enzyme Research South Bend, Indiana, USA, K_M= 28 μM), for

human PK (112 pM in assay, molecular weight 85 kDa, Enzyme

Research South Bend, Indiana, USA, K_M= 39 μM), and for human

aPC (293 pM in assay, molecular weight 61 kDa, Kordia Laboratory

Supplies, Leiden, Netherlands, K_M= 26 μM), whereas Tos-Gly-Pro-

Arg-AMC was used for bovine thrombin prepared according to

Walsmann⁴⁸(31 pM in assay, molecular weight 33.6 kDa, K_M= 5.4

μM) and Mes-^DArg-Gly-Arg-AMC⁴⁷for porcine trypsin (30 pM in

1

10% B (purity 94.1%). H NMR (500 MHz, DMSO-d₆): δ = 9.94−

10.02 (m, 3H), 9.05 (d, 1H, J = 8.2 Hz), 8.78 (t, 1H, J = 5.7 Hz), 7.93

(bs, 3H), 7.02−7.51 (m, 12H), 6.18 (bs, 2H), 4.08−4.54 (m, 10H),

2.76−3.06 (m, 16H) ppm. MS (ESI, positive): calcd, 750.34; m/z

751.45 [M + H]⁺.

(13S,16R)-N-(4-(Aminomethyl)benzyl)-16-((N-cyclohexylsulf-

amoyl)amino)-3,9,15-trioxo-2,10,14-triaza-6(1,4)-piperazina-1,11-

(1,4)-dibenzenacycloheptadecaphane-13-carboxamide·3TFA (41).

A solution of compound 37 (80.0 mg, 0.073 mmol, 1.0 equiv) in 10

mL of THF was cooled to 0 °C and treated with 64 μL of DIPEA

(0.365 mmol, 5.0 equiv). After addition of 14.4 mg of cyclohexyl

sulfamoyl chloride (0.073 mmol, 1.0 equiv), the solution was stirred

for 2 h at 0 °C, then at rt overnight. The solvent was removed in

vacuo, and the remaining residue was dissolved in a mixture of

saturated aq NaHCO₃and EtOAc and then extracted 3× with EtOAc.

The combined organic phase was washed 3× with brine, dried over

MgSO₄, and ﬁltered. The solvent was removed in vacuo. HPLC: 34.3

min, start at 10% B. MS (ESI, positive): calcd, 911.40; m/z 912.25 [M

+ H]⁺. The Tfa-protected intermediate was converted to the ﬁnal

inhibitor by dissolving the residue in a mixture of dioxane/1 M NaOH

(9:1, v/v). The reaction mixture was stirred for 3 h at rt, then

neutralized with 1 M HCl (pH 7). The solvent was removed in vacuo,

and the residue was dissolved with small amounts of MeOH then

precipitated in cold diethyl ether. The product was puriﬁed by

preparative HPLC. Yield: 3.81 mg (3.3 μmol, 14%, 2 steps) of

inhibitor 41 as white, lyophilized solid. HPLC: 21.2 min, start at 10%

assay, molecular weight 23.2 kDa, Merck, Darmstadt, Germany, K_M

=

8.3 μM). The provided K_ivalues were obtained from Dixon plots and

are the average of at least two measurements. The inhibition constants

for human plasmin (molecular weight 78 kDa, Chromogenix,

Lexington, USA) shown in Tables 2 and 3 were obtained from

Dixon plots and performed with an enzyme concentration of 0.7 nM

using the substrate Mes-^DSer(Bzl)-Phe-Arg-AMC (compound 44,

prepared as 20 mM stock solution in DMSO, which was further

diluted with water, used concentrations in assay: 50, 25 μM and 12.5

μM, ﬁnal DMSO content <1% in all measurements). The slow-binding

measurements (Table 4) were performed with 0.3 nM plasmin in the

presence of the substrate Mes-^DArg-Phe-Arg-AMC (47) at a single

concentration of 50 μM over a period of 20 min. The obtained

progress curves were ﬁtted to eq 1,^28,49where v₀, v_s, k_obs, and d

represent the initial velocity in the absence of inhibitor, the steady-

1

B (purity >99.9%). H NMR (600 MHz, potassium phosphate buﬀer

100 mM pH = 3/D₂O 5:1): 10.11 (s, 1H), 10.09 (s, 1H), 8.62 (t, ³J =

6.1 Hz, 1H), 8.51 (d, ³J = 8.4 Hz, 1H), 7.42 (d, ³J = 8.0 Hz, 2H), 7.33

3

(d, J = 7.9 Hz, 2H), 7.29 (d, J = 8.6 Hz, 2H), 7.22 (d, J = 8.6 Hz,

2H), 6.95 (d, ³J = 8.6 Hz, 2H), 6.71 (d, ³J = 8.6 Hz, 2H), 4.42 (dd, ²J =

N

DOI: 10.1021/acs.jmedchem.6b00606

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

state velocity, the apparent ﬁrst-order rate constant, and the

displacement of the ﬂuorescence signal from zero at t = 0, respectively.

The steady-state velocities were used for K_icalculation according to eq

2, and the k_obsvalues were ﬁtted against the inhibitor concentrations to

provide the association rate constant k_on(eq 3). The dissociation rate

constants were calculated from eq 4 using the known K_iand k_onvalues.

Author Contributions

All authors have given approval to the ﬁnal version of the

manuscript. S.H., A.W., and S.M.S. contributed equally.

Notes

The authors declare no competing ﬁnancial interest.

[P] = v_St + (v₀− v_S)[1 − exp(−k_obst)]/k_obs+ d

(1)

ACKNOWLEDGMENTS

■

We gratefully acknowledge the beamline staﬀ at BESSY II

(Helmholtz-Zentrum Berlin) in Berlin, Germany for providing

us outstanding support during the data collection. We also

thank the Helmholtz-Zentrum Berlin for travel support.

v = V_max·[S]/[K_m·(1 + [I]/K_i) + [S]]

(2)

(3)

(4)

s

k_obs= k_on[I]/(1 + [S]/K_m) + k_off

K_i= k_off/k_on

ABBREVIATIONS USED

Fibrinolysis Assay.³⁷Measurements have been performed over a

period of 40 min at 37 °C in transparent Cellstar 96-well-plates

(Greiner Bio One, Frickenhausen, Germany) using a microplate reader

ELx808 (BioTek Instruments, Winooski, Vermont, USA) at 405 nm

(OD₄₀₅). Inhibitors were prepared as 10 mM stock solutions in

DMSO and further diluted with 0.9% aq NaCl. The reference inhibitor

aprotinin was purchased from Carl Roth GmbH (Karlsruhe, Germany)

and dissolved in 0.9% aq NaCl without DMSO. Tissue factor (Dade

Innovin-reagent, Siemens Healthcare Diagnostics, Eschborn, Ger-

many) was dissolved in 10 mL of water, and the stock solution was

stored at 4 °C and further diluted (1/100, v/v) using 0.9% aq NaCl

directly before the measurements. Recombinant tPA (Actilyse,

Boehringer Ingelheim, Biberach, Germany) was dissolved in water at

a concentration of 1 mg/mL and was further diluted (1/125, v/v) with

0.9% NaCl directly before the measurements. Moreover, a 125 mM

CaCl₂solution was used. The total assay volume was 200 μL

consisting of 20 μL of inhibitor, 20 μL of CaCl₂(12.5 mM in assay),

20 μL of Actilyse (12.3 nM tPA in assay), 40 μL of Innovin reagent

(stock 1/500 diluted in assay), and 100 μL of citrated human plasma

(German Red Cross, Kassel, Germany). Solutions of inhibitor, CaCl₂,

Actilyse, and Innovin reagent were mixed and preincubated at 37 °C

for 5 min in the plate reader. The measurement was started by

addition of plasma, which was also preincubated at 37 °C for 5 min in

a water bath.

■

aPC, activated protein C; Boc, tert-butyloxycarbonyl; Bzls,

benzylsulfonyl; Cbz, benzyloxycarbonyl; Chas, cyclohexylsulfa-

moyl; DIPEA, N,N-diisopropylethylamine; DCM, dichloro-

methane; DMF, N,N-dimethylformamide; EtOAc, ethyl ac-

etate; fXa, factor Xa; HATU, 2-(7-aza-1H-benzotriazol-1-yl)-

1,1,3,3-tetramethyluronium-hexaﬂuorophosphate; MeCN, ace-

tonitrile; Mes, mesyl; MS, mass spectrometry; NMM, 4-

methylmorpholine; Phe(p-NO₂), p-nitrophenylalanine;

Phprop, 3-phenylpropyl; PK, plasma kallikrein; PyBOP,

benzotriazol-1-yl-oxytripyrrolidinophosphonium hexaﬂuoro-

phosphate; p-Xda, p-xylenediamine; tBu, tert-butyl; TFA,

triﬂuoroacetic acid; Tos, tosyl; TXA, tranexamic acid

REFERENCES

■

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Crystal Structure Analysis. Cocrystallization of bovine β-trypsin

(Sigma, no. T8003) with inhibitor 31, data collection and processing,

structure determination, and reﬁnement was performed as described

recently.⁵⁰A table containing the data collection and reﬁnement

statistics for the complex (PDB ID: 5EG4) is given in the Supporting

Information.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.6b00606.

■

S

The synthesis procedures for all other inhibitors, their

analytical data, as well as information for enzyme kinetics

with chromogenic substrates, and data collection and

reﬁnement statistics of the trypsin/inhibitor 31 complex

(PDF)

(7) Phipps, K. D.; Surette, A. P.; O’Connell, P. A.; Waisman, D. M.

Plasminogen receptor S100A10 is essential for the migration of tumor-

promoting macrophages into tumor sites. Cancer Res. 2011, 71, 6676−

6683.

Molecular formula strings (CSV)

Accession Codes

(8) Ishihara, M.; Nishida, C.; Tashiro, Y.; Gritli, I.; Rosenkvist, J.;

Koizumi, M.; Okaji, Y.; Yamamoto, R.; Yagita, H.; Okumura, K.;

Nishikori, M.; Wanaka, K.; Tsuda, Y.; Okada, Y.; Nakauchi, H.;

Heissig, B.; Hattori, K. Plasmin inhibitor reduces T-cell lymphoid

tumor growth by suppressing matrix metalloproteinase-9-dependent

CD11b(+)/F4/80(+) myeloid cell recruitment. Leukemia 2012, 26,

332−339.

(9) Voegeli, R.; Rawlings, A. V.; Breternitz, M.; Doppler, S.; Schreier,

T.; Fluhr, J. W. Increased stratum corneum serine protease activity in

acute eczematous atopic skin. Br. J. Dermatol. 2009, 161, 70−77.

Authors will release the atomic coordinates and experimental

data for 31 in complex with trypsin (5EG4) upon article

publication.

AUTHOR INFORMATION

Corresponding Author

*Phone: +49 6421 2825900. Fax: +49 6421 2825901. E-mail:

steinmetzer@uni-marburg.de.

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DOI: 10.1021/acs.jmedchem.6b00606

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

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14756366.2016.1172574.

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DOI: 10.1021/acs.jmedchem.6b00606

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Optimization of Cyclic Plasmin Inhibitors: From Benzamidines to Benzylamines

DOI: 10.1021/acs.jmedchem.6b00606

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