Optimization of Cyclic Plasmin Inhibitors: From Benzamidines to

Article pubs.acs.org/jmc

Optimization of Cyclic Plasmin Inhibitors: From Benzamidines to Benzylamines Stefan Hinkes, André Wuttke, Sebastian M. Saupe, Teodora Ivanova, Sebastian Wagner, Anna Knörlein, 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.

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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.1−4 In 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 graftversus-host disease.6 Moreover, enhanced plasminogen activation leading to increased plasmin activity has been linked to cancer invasion and metastasis.7,8 However, 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.3 Strongly increased plasmin, uPA, and other stratum corneum protease © XXXX American Chemical Society

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.11−13 In 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.14 Nowadays, 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 antiinflammatory properties of aprotinin. Moreover, side effects have been reported after treatment with higher doses of TXA, such as convulsive seizures.15,16 Very recently, new TXA analogues based on an isoxazolone scaffold have been reported which possess an enhanced potency and improved overall profile.17 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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DOI: 10.1021/acs.jmedchem.6b00606 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Scheme 1. Structures of Previously Described Plasmin Inhibitors

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

(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

a more rapid and stronger effect to reduce bleeding. The work on synthetic plasmin inhibitors was recently reviewed.18 Among the most potent small-molecule inhibitors are tetrapeptidic arginal derivatives19 and derivatives of trans-4-aminomethylcyclohexanecarboxylic acid.20−22 Plasmin is also inhibited by small benzamidine derivatives.23,24 Starting from the nonselective linear substrateanalogue benzamidine derivative 1 (CU-2010)25 (Scheme 1), we have recently described various types of macrocyclic analogues with improved selectivity and potency.26,27 The cyclization was performed between the side chains of a P2 residue in S-configuration and the adjacent P3 residue in Rconfiguration. 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 Ki values 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 B



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Scheme 3. Synthesis of the Cyclized Benzamidine-Derived Inhibitorsa

(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). a

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-hexahydrotyrosine-Lys-pNA), Chromozym PL (Tos-Gly-Pro-LyspNA), and S-2251 (H-DVal-Leu-Lys-pNA) possess relatively high KM values >100 μM, leading to a moderate substrate efficiency (kcat/KM). 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-ProLys-pNA in the presence of 0.9 nM plasmin, which makes it difficult to avoid tight-binding conditions28 in 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

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 subnanomolar 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.

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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.26,27 Depending 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 segment 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(pNO2)-OH was initially coupled to p-cyanobenzylamine, followed by stepwise attachment of the P3 Boc-DPhe(pC



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

(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). a

Table 1. Synthesized Fluorogenic Plasmin Substrates

a

no.

sequence

KM (μM)

kcat (s−1)

44 45 46 47 48

Mes-DSer(Bzl)-Phe-Arg-AMC Mes-DhPhe-Phe-Arg-AMCa Mes-DhPhe-Phe(4-NO2)-Arg-AMCa Mes-DArg-Phe-Arg-AMC Mes-DArg-hPhe-Arg-AMCa

8.5 7.4 3.8 16.1 9.8

2.65 2.38 0.67 9.69 1.74

kcat/KM (M−1 s−1) 3.11 3.21 1.83 6.02 1.77

× × × × ×

105 105 105 105 105

hPhe corresponds to homophenylalanine.

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

P2 residues. Therefore, phenylalanine was incorporated at this position in combination with sulfonylated P3 residues in Dconfiguration. 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)-PheArg-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 KM values, however, they suffered from reduced kcat values. The inhibition constants of the benzamidine derivatives are summarized in Table 2. In previous studies with substrate analogue thrombin,29 factor Xa,30 and uPA31 inhibitors, a benzylsulfonyl group was identified as preferred P4 residue, therefore, it was used as constant N-terminal protecting group D



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Table 2. Structures of the Cyclic Benzamidine-Derived Inhibitors

a

These compounds have been previously published; their Ki values 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 Ki values of 62 and 21 nM, respectively, which is in the same range as found for the reference compounds 2 and 3 (Table 2). E



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Table 3. Combined Modification of P1 and P4 Residues

providing the inhibition constants given in Tables 2 and 3 and for comparison in Table 4 (indicated as classical method).

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 substrateanalogue thrombin inhibitors,32,33 yielded 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.34−36 To 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 2fold stronger affinity for plasmin compared with compound 40, although also both of these derivatives were approximately 4and 8-fold less potent than their related Amba inhibitors 31 and 29, respectively. Interestingly, the trypsin Ki value of the phenylsulfonyl derivative 42 was increased to 1.5 μM. 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 Ki determinations,

Table 4. Comparison of Ki Determinations for Plasmin Using the Classical Method Neglecting the Initial TimeDependent Part of the Progress Curves and from SlowBinding Analysis, Which Provides Additional Data for the Rate Constants kon and koff Ki (nM) inhibitor 18 30 31 41 42

classical 0.77 0.32 0.13 0.52 0.56

slow-binding

kon (M−1·s−1)

0.85 0.42 0.15 0.43 0.54

× × × × ×

1.74 1.77 1.34 6.40 6.00

6

10 106 106 105 105

koff (s−1) 1.48 7.46 2.04 2.77 3.22

× × × × ×

10−3 10−4 10−4 10−4 10−4

However, such biphasic progress curves also indicate a slowbinding inhibition mechanism.28 Therefore, selected compounds 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 vs and a rate constant kobs for each curve. The constant initial velocities v0 of 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.28 The vs values were fitted as a function of the inhibitor concentration according to eq 2, providing the Ki values (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, kobs was used for the calculation of the association rate constant kon according to eq 3 (Figure 3). F



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Figure 3. kobs values were fitted as a function of the inhibitor concentrations according to eq 3, providing an association rate constant of 1.34 × 106 M−1 s−1 for inhibitor 31. This kon value was used to calculate the dissociation rate constant koff using 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 IC50 value for aprotinin is in the same range as described in literature (160 nM37 and 327 nM25). Slightly lower and therefore more potent IC50 values have been determined for the benzamidine-derived inhibitors 30 (53 nM) and 31 (55 nM) and for the second tested p-Xdaderivative 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 Ki values of 45 and 18 nM, respectively.25 Crystal Structure of Inhibitor 31 in Complex with Trypsin. In our previous study,27 we 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 (Ki = 21 nM) and the refined structure is shown in Figure 5A. As expected, the 99loop 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. Ki value of 0.15 nM for inhibitor 31 was calculated by fitting the vs, [I] data pairs to eq 2 using substrate 47 (50 μM).

The known Ki and kon values enabled the calculation of the dissociation rate constant koff according to eq 4. Fibrinolysis Assay in Human Plasma. The antifibrinolytic activity of selected compounds was tested in human plasma, as described for aprotinin.37 Plasma 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 IC50 determination, 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).37 This provided an IC50 value 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 G



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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 IC50 calculation 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)).

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 modifications, 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 uPA39 revealed 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 Ki values close to 0.1 μM. A considerable plasmin inhibition was also retained for the formyl- and phenylpropionyl-protected analogues 17 and 24, which possess Ki values 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 (Ki = 24 μM) compared to benzylsulfonyl-DSer(tBu)-Gly-4-Amba (Ki = 7.7 μM).30 Moreover, 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 (pKa ∼ 9.3534) with the phenylsulfonyl or cyclohexylsulfamoyl residues in P4 position still provided subnanomolar plasmin

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.

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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.38 Otherwise, 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.27 Moreover, the significance of selectivity measurements strongly depends on the number of the used enzymes of a target family, and often, especially in an H



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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).

association and dissociation rate constants in addition to the Ki values. On the basis of the kon values (Table 4), it seems that the benzamidine inhibitors bind 2−3 times more rapidly to plasmin compared to the p-Xda analogues. However, this slowbinding behavior observed in enzyme kinetic assays when using subnanomolar enzyme concentrations and inhibitor concentrations ≤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 kon × [I] in eq 3, resulting in a larger kobs value, leading to more

inhibitors. Interestingly, the drop in trypsin affinity was more pronounced for the phenylsulfonyl inhibitor 42 (Ki > 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 I



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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

rapid binding. The plasma concentration of plasminogen is ∼1.6 μM.40 It 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,42 or 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 interactions 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 60insertion 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.

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EXPERIMENTAL SECTION

General. Solvents and reagents were purchased from SigmaAldrich, Alfa Aesar, Roth, Fluka, or Merck and used without further purification. The amino acids Boc-DPhe(p-NO2)-OH and Boc-Phe(pNO2)-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, Düren, Germany). Analytical HPLC experiments were performed on a Shimadzu LC-10A system (column, Nucleodur C18 ec, 5 μm, 100 Å, 4.6 mm × 250 mm, Macherey-Nagel, Düren, 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, Düren, 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 C8 column (Nucleodur C8 ec, 5 μm, 100 Å, 32 mm × 250 mm, Macherey-Nagel, Düren, 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, Osterode 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)propionic2,2,3,3-d4 acid 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 BocPhe(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-cyanobenzylamine·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 J



Article

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 NaHCO3 and 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-cyclohexylsulfamoyl 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 Ncyclohexylsulfamoyl 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 >95%). TLC: Rf = 0.32 (DCM/MeOH 20:1). 1H NMR (500 MHz, 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

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, DMSOd6): 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 BocD Phe(p-NO 2 )-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]+. 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 at 10% B (purity >95%). 1H NMR (500 MHz, DMSO-d6): δ = 9.25 (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 (purity >95%). TLC: Rf = 0.30 (DCM/MeOH 20:1). 1H NMR (500 K



Article

(purity >95%). 1H NMR (500 MHz, DMSO-d6): δ = 8.31 (t, J = 6.2 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 literature46 using 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]+. Piperazine-1,4-dipropionic Acid (10). The synthesis was performed as described in the literature27 using 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 mmol, 58.5%) as white crystals. 1H 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-phenylpropanamido)-2,8,12-triaza-5(1,4)-piperazina-1,9(1,4)-dibenzenacyclopentadecaphane-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: Rf = 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, DMSOd6): 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]+. (13S,16R)-N-(4-Cyanobenzyl)-3,9,15-trioxo-16-(3-phenylpropanamido)-2,10,14-triaza-6(1,4)-piperazina-1,11(1,4)-dibenzenacycloheptadecaphane-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

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: Rf = 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]+. (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)-dibenzenacyclopentadecaphane-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: Rf = 0.19 (DCM/MeOH 10:1 with 0.5% NH4OH (25%)). 1H 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)-dibenzenacycloheptadecaphane-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: Rf = 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]+. L



Article

(11S,14R)-N-(4-Carbamimidoylbenzyl)-3,7,13-trioxo-14-(3-phenylpropanamido)-2,8,12-triaza-5(1,4)-piperazina-1,9(1,4)-dibenzenacyclopentadecaphane-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 H2 atmosphere, 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, DMSOd6): 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]+. (13S,16R)-N-(4-Carbamimidoylbenzyl)-3,9,15-trioxo-16-(3-phenylpropanamido)-2,10,14-triaza-6(1,4)-piperazina-1,11(1,4)-dibenzenacycloheptadecaphane-13-carboxamide·3TFA (24). The synthesis 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 (purity >95%). 1H 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-cyclohexylsulfamoyl)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),

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, DMSOd6): 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-cyclohexylsulfamoyl)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 (purity >95%). 1H 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 pcyanobenzylamine·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 KHSO4 and EtOAc. The organic layer was washed thrice with 5% aq KHSO4 and 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%). 1 H NMR (400 MHz, DMSO-d6): δ = 10.12 (bs, 1H), 7.84 (d, 2H, J = 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 as white solid. HPLC: 11.6 min, start at 10% B (purity >99%). 1H 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 Bocprotected 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 solid. HPLC: 27.0 min, start at 10% B (purity 96.5%). 1H 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



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15.0 Hz, 3J = 6.2 Hz, 1H), 4.35 (dd, 2J = 15.4 Hz, 3J = 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]+. (13S,16R)-N-(4-(Aminomethyl)benzyl)-3,9,15-trioxo-16-(phenylsulfonamido)-2,10,14-triaza-6(1,4)-piperazina-1,11(1,4)-dibenzenacycloheptadecaphane-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 >99.9%). 1H NMR (600 MHz, potassium phosphate buffer 100 mM pH = 3/D2O 5:1): 10.10 (s, 1H), 10.05 (s, 1H), 8.45 (t, 3J = 6.1 Hz, 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 = 7.9 Hz, 2H), 7.24 (d, 3J = 8.6 Hz, 2H), 7.13 (d, 3J = 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 Ki values 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)27 or with fluorescence substrates in black 96-well plates using a Fluoroskan Ascent plate reader (Thermo Fisher Scientific, Vantaa, Finland) with λex 355 and λem 460 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-AMC47 was used as fluorescence substrate for human fXa (97 pM in assay, molecular weight 46 kDa, Enzyme Research South Bend, Indiana, USA, KM = 28 μM), for human PK (112 pM in assay, molecular weight 85 kDa, Enzyme Research South Bend, Indiana, USA, KM = 39 μM), and for human aPC (293 pM in assay, molecular weight 61 kDa, Kordia Laboratory Supplies, Leiden, Netherlands, KM = 26 μM), whereas Tos-Gly-ProArg-AMC was used for bovine thrombin prepared according to Walsmann48 (31 pM in assay, molecular weight 33.6 kDa, KM = 5.4 μM) and Mes-DArg-Gly-Arg-AMC47 for porcine trypsin (30 pM in assay, molecular weight 23.2 kDa, Merck, Darmstadt, Germany, KM = 8.3 μM). The provided Ki values 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 99.9%). 1H 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 (d, 3J = 7.9 Hz, 2H), 7.29 (d, 3J = 8.6 Hz, 2H), 7.22 (d, 3J = 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



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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 Ki calculation according to eq 2, and the kobs values were fitted against the inhibitor concentrations to provide the association rate constant kon (eq 3). The dissociation rate constants were calculated from eq 4 using the known Ki and kon values. [P] = vSt + (v0 − vS)[1 − exp(− kobst )]/kobs + d

(1)

vs = Vmax ·[S]/[K m· (1 + [I ]/K i) + [S]]

(2)

kobs = kon[I ]/(1 + [S]/K m) + koff

(3)

K i = koff /kon

(4)

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.

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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.

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ABBREVIATIONS USED aPC, activated protein C; Boc, tert-butyloxycarbonyl; Bzls, benzylsulfonyl; Cbz, benzyloxycarbonyl; Chas, cyclohexylsulfamoyl; DIPEA, N,N-diisopropylethylamine; DCM, dichloromethane; DMF, N,N-dimethylformamide; EtOAc, ethyl acetate; fXa, factor Xa; HATU, 2-(7-aza-1H-benzotriazol-1-yl)1,1,3,3-tetramethyluronium-hexafluorophosphate; MeCN, acetonitrile; Mes, mesyl; MS, mass spectrometry; NMM, 4methylmorpholine; Phe(p-NO 2 ), p-nitrophenylalanine; Phprop, 3-phenylpropyl; PK, plasma kallikrein; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; p-Xda, p-xylenediamine; tBu, tert-butyl; TFA, trifluoroacetic acid; Tos, tosyl; TXA, tranexamic acid

37

Fibrinolysis Assay. Measurements 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, Germany) 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 CaCl2 solution 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. 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.50 A table containing the data collection and refinement statistics for the complex (PDB ID: 5EG4) is given in the Supporting Information.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00606. 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) Molecular formula strings (CSV) Accession Codes

Authors will release the atomic coordinates and experimental data for 31 in complex with trypsin (5EG4) upon article publication.

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REFERENCES

(1) Draxler, D. F.; Medcalf, R. L. The fibrinolytic system-more than fibrinolysis? Transfus. Med. Rev. 2015, 29, 102−109. (2) Castellino, F. J.; Ploplis, V. A. Structure and function of the plasminogen/plasmin system. Thromb. Haemostasis 2005, 93, 647− 654. (3) Deryugina, E. I.; Quigley, J. P. Cell surface remodeling by plasmin: a new function for an old enzyme. J. Biomed. Biotechnol. 2012, 2012, 564259. (4) Swedberg, J. E.; Harris, J. M. Natural and engineered plasmin inhibitors: applications and design strategies. ChemBioChem 2012, 13, 336−348. (5) Munakata, S.; Tashiro, Y.; Nishida, C.; Sato, A.; Komiyama, H.; Shimazu, H.; Dhahri, D.; Salama, Y.; Eiamboonsert, S.; Takeda, K.; Yagita, H.; Tsuda, Y.; Okada, Y.; Nakauchi, H.; Sakamoto, K.; Heissig, B.; Hattori, K. Inhibition of plasmin protects against colitis in mice by suppressing matrix metalloproteinase 9-mediated cytokine release from myeloid cells. Gastroenterology 2015, 148, 565−578. (6) Sato, A.; Nishida, C.; Sato-Kusubata, K.; Ishihara, M.; Tashiro, Y.; Gritli, I.; Shimazu, H.; Munakata, S.; Yagita, H.; Okumura, K.; Tsuda, Y.; Okada, Y.; Tojo, A.; Nakauchi, H.; Takahashi, S.; Heissig, B.; Hattori, K. Inhibition of plasmin attenuates murine acute graft-versushost disease mortality by suppressing the matrix metalloproteinase-9dependent inflammatory cytokine storm and effector cell trafficking. Leukemia 2015, 29, 145−156. (7) Phipps, K. D.; Surette, A. P.; O’Connell, P. A.; Waisman, D. M. Plasminogen receptor S100A10 is essential for the migration of tumorpromoting macrophages into tumor sites. Cancer Res. 2011, 71, 6676− 6683. (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.

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