Acylated 1 H-1,2,4-Triazol-5-Amines Targeting Human Coagulation Factor XIIa and Thrombin: Conventional and Microscale Synthesis, Anticoagulant Properties, and Mechanism of Action

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Acylated 1H‑1,2,4-Triazol-5-amines Targeting Human Coagulation

Factor XIIa and Thrombin: Conventional and Microscale Synthesis,

Anticoagulant Properties, and Mechanism of Action

Marvin Kor ﬀ , Lukas Imberg, Jonas M. Will, Nico Buckreiß, Svetlana A. Kalinina, Benjamin M. Wenzel,

̈

Gregor A. Kastner, Constantin G. Daniliuc, Maximilian Barth, Ruzanna A. Ovsepyan, Kirill R. Butov,

Hans-Ulrich Humpf, Matthias Lehr, Mikhail A. Panteleev, Antti Poso, Uwe Karst, Torsten Steinmetzer,

Gerd Bendas, and Dmitrii V. Kalinin *

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ABSTRACT: We herein report the conventional and microscale parallel synthesis of

selective inhibitors of human blood coagulation factor XIIa and thrombin exhibiting a

1,2,4-triazol-5-amine scaﬀold. Structural variations of this scaﬀold allowed identifying

derivative 21i, a potent 29 nM inhibitor of FXIIa, with improved selectivity over other

tested serine proteases and also ﬁnding compound 21m with 27 nM inhibitory activity

toward thrombin. For the ﬁrst time, acylated 1,2,4-triazol-5-amines were proved to have

anticoagulant properties and the ability to aﬀect thrombin- and cancer-cell-induced

platelet aggregation. Performed mass spectrometric analysis and molecular modeling

allowed us to discover previously unknown interactions between the synthesized

inhibitors and the active site of FXIIa, which uncovered the mechanistic details of FXIIa

inhibition. Synthesized compounds represent a promising starting point for the

development of novel antithrombotic drugs or chemical tools for studying the role of

FXIIa and thrombin in physiological and pathological processes.

foods, have slow onset/oﬀset of action, and cause coumarin-

induced hepatitis.⁸In contrast, direct oral anticoagulants

(DOACs) such as direct thrombin inhibitors (e.g., dabigatran

(1), Figure 1) and FXa inhibitors (e.g., rivaroxaban (2), Figure

1) demonstrate rapid onset/oﬀset of action and predictable

pharmacokinetics.^9,10However, even these new drugs are

reported to induce life-threatening bleeding.¹¹Therefore, novel

anticoagulants with a new mechanism of action are needed to

control thrombosis without the risk of bleeding.

Among plasma coagulation factors, Hageman factor (FXII)

is relatively less studied and considered as an emerging target

for thrombosis prevention. Hageman factor is a serine protease

circulating in the blood as an inactive zymogen, which upon

contact with negatively charged surfaces (e.g., the collagen of

damaged vessels, artiﬁcial surfaces such as glass) undergoes the

activation (FXIIa formation). Formed FXIIa activates both

FXI, triggering the intrinsic blood coagulation cascade, and

plasma kallikrein, activating the kallikrein−kinin system.^12,13

INTRODUCTION

■

The natural process of hemostasis is essential for the

prevention of life-threatening extensive bleeding. Dysregulation

of hemostasis, however, can lead to a dangerous pathology

termed thrombosis, which is characterized by the undesired

formation of a potentially deadly blood clot inside a vein or an

artery. Deep venous and arterial thrombosis is associated with

thromboembolism, which in turn triggers major cardiovascular

disorders: myocardial infarction, ischemic stroke, and periph-

eral arterial ischemia.¹Considering the high mortality rate and

economic losses associated with thrombosis,^2,3presently

available measures in thrombosis prevention are insuﬃcient

and indicate the need for the development of novel

antithrombotic drugs.

Apart from antiplatelet drugs, the current thrombosis

therapy relies on anticoagulants, which, despite being life-

saving medications, prevent blood coagulation in situations

when the clotting is desired (e.g., trauma or surgery), thereby

causing a life-threatening side eﬀect of internal bleeding.^4,5

Besides the internal bleeding, clinically used anticoagulants

exhibit a number of drawbacks. Heparins, for instance, can only

be administered in the form of injection, have a short biological

half-life, and might elicit heparin-induced thrombocytopenia.^6,7

Vitamin K antagonists (e.g., warfarin) display a narrow

therapeutic window, interact with a number of drugs and

Received: September 18, 2020

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A

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Figure 1. Exemplary macromolecular and small-molecule inhibitors of plasma coagulation factors with their inhibitory proﬁle.

Figure 2. Molecular surface of human coagulation factor XIIa (A) and thrombin (B) near the active site (PDB: 6B74²⁸and PDB: 1A4W²⁹). The

residues are depicted as gray stick models. Substrate-binding sites are labeled (S1−S4 and S1′). The catalytic residues (His57, Asp102, Ser195) are

indicated in red. The arrows point to amino acids that form the “oxyanion hole” (Gly193 and Ser195). The surface is colored as follows: lipophilic

regions are in orange, hydrophilic in blue, and neutral in white.

Recent studies showed a fundamental role of Hageman factor

in arterial and venous thrombosis. FXII-knockout mice are

protected from arterial and deep vein thrombosis as well as

ischemic stroke.^14,15Despite its essential role in thrombosis,

the deﬁciency of FXII does not aﬀect hemostasis in animals,

and FXII-deﬁcient patients demonstrate a normal hemostatic

ability.^12,16Therefore, FXIIa represents a new, potentially safe,

and promising target for thrombosis prevention. Therapeutic

application of FXIIa inhibitors should be especially eﬀective in

the prevention of contact-mediated thrombosis triggered by

medical devices.¹⁷Devices such as vascular catheters, artiﬁcial

heart valves, and dialysis membranes are reported to induce

blood coagulation via FXII activation.¹⁸A selective inhibition

or depletion of FXII was in turn proved to prevent device-

induced thrombosis in animal models,¹⁹⁻²¹whereas antico-

agulants targeting, e.g., FXa and thrombin showed limited

ability to prevent medical-device-induced thrombosis.^22,23

Besides, as FXII serves as a connection point between

coagulation and inﬂammation cascades, inhibitors of FXIIa,

apart from being potential antithrombotics, could ﬁnd their

application in the treatment of disorders such as hereditary

angioedema,²⁴sepsis,²⁵multiple sclerosis,²⁶and Alzheimerʼs

disease.²⁷A recently published crystal structure of FXIIa gives

an insight into the enzymeʼs active site.²⁸It contains the

catalytic triad formed by Asp102, His57, and Ser195 (Figure

2A), a typical “oxyanion hole” also found in related serine

B

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a

Scheme 1. Synthesis of 3-Pyridyl-Substituted 1,2,4-Triazol-5-amines 7 and 10−13

a

Reagents and conditions: (a) aminoguanidine hydrochloride, neat, 190 °C, 5 h, 9a 58%, 9b 74%, 9c 66%; (b) tetrahydrofuran (THF)/pyridine

1:1, 0 °C to r.t., 2−5 h, 10a 42%, 7 77%, 10c 87%, 11c 57%, 12a 15%, 13c 70%.

proteases, and distinctive substrate-binding sites (S1−S4 and

S1′−S2′, Figure 2A), the structural features that could be

exploited for the rational FXIIa inhibitor design.

Historically, FXIIa was not recognized as a valuable drug

target and, therefore, only few examples of FXIIa inhibitors are

known. Among them, apart from the recently disclosed FXIIa-

blocking antibody 3F7,³⁹two macromolecular inhibitors,

insect protein infestin-4⁴⁰(3, FXIIa K_i= 0.1 nM, Figure 1)

and peptide-like macrocycle FXII801⁴¹(4, FXIIa K_i= 1.6 nM,

Figure 1), are known. Among few small molecules, coumarin

5,⁴²boronic acid 6,²⁸and also substituted aminotriazole 7⁴³

are reported as FXIIa inhibitors (Figure 1). For our study,

coumarin 5 and boronic acid 6 were disregarded as lead

structures, since 5 is a relatively weak FXIIa inhibitor (IC₅₀= 5

μM)²³and 6 is a covalent inhibitor with a problematic

selectivity proﬁle (e.g., 6 inhibits trypsin with IC₅₀= 52

nM).^28,42In contrast, acylated 1,2,4-triazol-5-amine 7 (Figure

1) with a FXIIa IC₅₀value of 210 nM⁴³represents a promising

lead structure for the development of novel FXIIa inhibitors.

The therapeutic potential of substituted 1,2,4-triazol-5-

amines remains largely underexplored, despite their synthetic

accessibility. For the aforementioned acylated aminotriazole 7,

apart from the FXIIa inhibitory activity, no information is

disclosed on its selectivity over related serine proteases,

anticoagulant activity, toxicity proﬁle, and interactions with

the FXIIa active site.

Herein, we report the synthesis of 1,2,4-triazol-5-amine-

based selective inhibitors of blood coagulation factor XIIa and

thrombin. Performed biochemical experiments revealed

anticoagulant properties of the synthesized inhibitors and

their ability to aﬀect thrombin and cancer-cell-induced platelet

aggregation, whereas mass-spectrometry-based analyses un-

covered mechanistic details of FXIIa inhibition. Experimentally

obtained data were reinforced by computational simulations,

which allowed to rationalize the interactions between

synthesized inhibitors and the active sites of the coagulation

factor XIIa and thrombin.

On the other hand, despite the risk of bleeding side eﬀects,

the therapeutic application of inhibitors of the extrinsic blood

coagulation pathway in some pathologies like cancer-associated

thrombosis is practically unavoidable. Cancer-associated

thrombosis, which develops via activation of primary and

secondary hemostasis, aﬀecting both intrinsic and extrinsic

coagulation pathways, is the second leading cause of mortality

of cancer patients.^30,31Tissue factor (TF), which is expressed

on the outer membranes of tumor cells, is suggested to be the

main contributor to thrombin generation and ﬁbrin formation

in cancer patients.³²It leaves little to no alternatives to the

inhibitors of the extrinsic or common pathway (e.g., thrombin

inhibitors) in the prevention of cancer-associated thrombosis.

Recent studies, however, suggest a signiﬁcant role of contact

system activation in the development of cancer-associated

thrombosis (recently reviewed³³). It has been found that

tumor cells trigger contact-system activating FXII by releasing

negatively charged molecules such as phosphatidylserine,

glycosaminoglycans, polyphosphate, collagen, and nucleic

acids.³³⁻³⁶For instance, breast and pancreatic cancer cells

were shown to induce TF-independent thrombin generation

via activation of FXII.³⁷Moreover, it has been proven that

prostate-cancer-associated thrombosis is driven to a signiﬁcant

extent by polyphosphate-activated FXII and that the inhibition

of this activation is suﬃcient to protect mice from lethal

pulmonary embolism without risk of causing bleeding.³⁸The

interplay between diﬀerent factors of coagulation pathways in

the cancer microenvironment is complex and not fully

understood. Therefore, compounds that could selectively

inhibit one of the pathways, as well as compounds capable of

dual inhibition, are of interest as chemical tools to study this

interplay. From this perspective, both selective and dual FXIIa

and thrombin inhibitors might be of interest.

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Figure 3. X-ray crystal structure of 21m displaying the thermal ellipsoids at the 50% probability level (left). Formation of an asymmetrical dimeric

unit (result of crystal packing) between the two crystallographically independent molecules (molecule “A” and “B”) of compound 21m involving

strong and week N−H...N interactions (right).

a

Scheme 2. Synthesis of Benzoylated 3-Phenyl-1,2,4-triazol-5-amine 16

a

Reagents and conditions: (a) S-methylisothiouronium sulfate, EtOH/H₂O 1:1, reﬂux, 16 h; (b) KOH (40%), reﬂux, 2 h, 15 57%; (c) THF/

pyridine 1:1, 0 °C to r.t., 3 h, 16 92%.

reported procedure, hydrazide 14 was treated with S-

methylisothiouronium sulfate accessing phenylamidoguanidine

14′, cyclization of which resulted in aminotriazole 15.⁴⁸

Finally, regioselective acylation of 15 allowed for the isolation

of benzoylated 1,2,4-triazol-5-amine 16 possessing nonheter-

oaromatic phenyl moiety in position 3 of the triazole core.

Finally, acylated 1,2,4-triazole-3,5-diamines 20a−c were

synthesized (Scheme 3). For this, anilines 17a−c were reacted

RESULTS AND DISCUSSION

■

Conventional Synthesis of 1,2,4-Triazol-5-amines.

The initial library of potential FXIIa inhibitors possessing the

1,2,4-triazol-5-amine scaﬀold was generated and screened for

their biological activity to get the ﬁrst insight into the

structure−activity relationship (SAR) and to identify the most

promising candidate for the further optimization. For this

purpose, syntheses shown in Schemes 1−3 were performed.

At ﬁrst, pyridinecarboxylic acids 8a−c were fused with

aminoguanidine hydrochloride at 190 °C, aﬀording amidogua-

nidines, which without the isolation were cyclized into 3-

pyridyl-substituted 1,2,4-triazol-5-amines 9a−c (Scheme 1).⁴⁴

Aminotriazoles 9 exhibit annular tautomerism and exist in

three main tautomeric forms 9, 9′, and 9″, of which the

tautomeric form 9 is prevalent (Scheme 1).^45,46Tautomerism

could complicate the subsequent acylation reactions due to the

formation of several acylated products.⁴⁷However, taking

advantage of the observed low reactivity of the primary amino

group of 1,2,4-triazol-5-amines 9a−c and the equilibrium shift

toward the tautomeric form 9, in subsequent acylation and

sulfonylation reactions employing temperature and addition

rate control, exclusively N-1-substituted products 7 and 10−13

were successfully obtained. The regioselectivity of the

mentioned reactions was unambiguously conﬁrmed by X-ray

crystallography (Figure 3).

Scheme 3. Synthesis of Acylated 3-Arylamino-1,2,4-triazol-

a

5-amines 14a−c

a

As the cyclocondensation reaction between benzoic acid and

aminoguanidine hydrochloride failed to produce correspond-

ing 3-phenyl-1H-1,2,4-triazol-5-amine 15, an alternative route

for its synthesis was utilized (Scheme 2). According to the

Reagents and conditions: (a) i-PrOH, reﬂux, overnight, 18a 47%,

18b 46%, 18c 91%; (b) NH₂NH₂·H₂O, EtOH, reﬂux, 2−16 h, 19a

74%, 19b 53%, 19c 86%; (c) THF/pyridine 1:1, 0 °C to r.t., 2−18 h,

20a 17%, 20b 51%, 20c 53%.

D

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Table 1. Initial Screening of 1,2,4-Triazol-5-amines against Selected Serine Proteases

a

Measurements were performed in triplicate; 1 h incubation time is speciﬁed as IC₅₀values were time-dependent; substrate concentration [S]₀= 25

μM; measured FXIIa K_m= 167 4 μM for the Boc-Gln-Gly-Arg-AMC substrate; measured thrombin K_m= 18 1 μM for the Boc-Val-Pro-Arg-

AMC substrate. The K_ivalues could not be directly withdrawn from the Cheng−Prusoﬀ equation in this case due to the enzyme−inhibitor covalent

interaction (see the section “Mechanism of Inhibition”).

with dimethyl N-cyanodithioimidocarbonate to form imidates

18a−c, subsequent treatment of which with hydrazine hydrate

aﬀorded cyclization products 19a−c.⁴⁷The ﬁnal acylation

reaction with benzoyl chloride yielded compounds 20a−c.

Inhibition of Serine Proteases. All synthesized 1,2,4-

triazol-5-amines of the ﬁrst round of conventional synthesis

(Schemes 1−3) were assayed in vitro against relevant serine

proteases of the blood coagulation cascade, FXIIa, thrombin,

and FXa, as well as against trypsin using ﬂuorogenic substrates

according to previously reported procedures with minor

variations.^41,43Potent thrombin and FXa inhibitors dabigatran

(1) and rivaroxaban (2), respectively, were used as positive

controls. The IC₅₀values were obtained in competition

experiments, in which the enzyme was added to the mixture

of the substrate and the inhibitor, followed by ﬂuorescence

measurements in the kinetic mode during 1 h (end-point

readout was used). The results of in vitro enzymatic assays are

summarized in Table 1.

ranging between 235 nM and 13 μM. The substitution pattern

of the 1,2,4-triazol-5-amine core signiﬁcantly inﬂuenced the

inhibitory proﬁle of the synthesized compounds. Thus, 1,2,4-

triazol-5-amines 15, 9a, 9c, exhibiting a nonacylated annular

secondary amino group, were completely inactive against the

tested serine proteases. Acylation of the secondary amino

group of the aminotriazoles with benzoyl chloride resulted in

corresponding amides 16, 10a, and 10c, which, in contrast,

showed pronounced FXIIa and thrombin inhibitory properties

(e.g., 10a: IC₅₀= 132 and 235 nM, respectively), signifying

thereby the necessity of an acyl fragment for successful

inhibition of FXIIa and thrombin. Not only the presence of an

acyl moiety but also its structure appeared as an important

factor determining the inhibitory properties of the synthesized

aminotriazoles. Thus, in terms of FXIIa inhibition, benzoylated

compounds, e.g., 10a with FXIIa IC₅₀= 132 nM, exhibited

lower IC₅₀values than their carbamate-based analogue 12a

(FXIIa IC₅₀= 617 nM), which in turn was more active than

compounds 11c and 13c possessing a cycloaliphatic acyl

moiety and a sulfonamide residue on the secondary amino

group, respectively (FXIIa IC₅₀> 1 μM, for both). The same

trend is apparent for the thrombin inhibition by the

As shown in Table 1, some of the synthesized 1,2,4-triazol-5-

amines inhibit FXIIa in the nanomolar range with IC₅₀values

varying between 106 and 617 nM, simultaneously aﬀecting the

other key blood coagulation factor thrombin with IC₅₀values

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aforementioned compounds. The structure of the acyl moiety

also determined the inhibitor selectivity. For instance, being an

FXIIa inhibitor of moderate potency, carbamate 12a exhibited

a 21-fold selectivity toward FXIIa over thrombin (Table 1).

When considering the other structural alteration of the 1,2,4-

triazol-5-amine scaﬀold, the introduction of an additional

nitrogen atom bridge (1,2,4-triazole-3,5-diamines 20a−c)

resulted in no serine protease inhibitory properties (Table

1). Notably, none of the screened 1,2,4-triazol-5-amines,

shown in Table 1, aﬀected FXa or trypsin enzymatic activity,

displaying thereby a clear preference toward FXIIa and

thrombin inhibition.

Anticoagulant Activity of Series 1. Nine representative

compounds of the synthesized 1,2,4-triazol-5-amines were

examined for their anticoagulant properties in activated partial

thromboplastin time (aPTT) and prothrombin time (PT) tests

(Figure 4). These two tests allow us to distinguish whether the

were performed with the whole blood instead of plasma to

modulate conditions resembling an in vivo situation when the

whole matrix of blood is taken into consideration.

As can be seen in Figure 4, 1,2,4-triazol-5-amines inﬂuenced

the whole blood coagulation in aPTT and PT tests to diﬀerent

extents. Speciﬁcally, synthesized compounds had almost no

inﬂuence on the extrinsic coagulation cascade only slightly

extending PT by 1−12% at 300 μM concentration. In contrast,

at the same concentration, some of the compounds showed a

stronger eﬀect on the intrinsic coagulation pathway extending

aPTT by up to 143% (aPTT for compound 10a = 108 s vs

aPTT (DMSO) = 44 s). Compounds’ preference to inhibit the

intrinsic but not the extrinsic coagulation cascade is note-

worthy and promising since extrinsic cascade triggered by TF

release is physiologically relevant, being a natural defense

against bleeding, whereas intrinsic cascade initiated by FXIIa is

implicated in thrombosis. In terms of SAR, aminotriazoles 10a,

7, and 10c, possessing 2-, 3-, and 4-pyridyl moieties in position

3 of their 1,2,4-triazole scaﬀold showed the highest ability to

prolong aPTT by 143, 53, and 80%, respectively. Pyridyl

moiety replacement with a phenyl or aminoaryl residue

resulted in anticoagulant activity reduction for compound 16

(aPTT +29%) or its complete loss for compounds 20a and

20b. This emphasizes the correlation between the superior

ability of pyridyl-substituted derivatives to inhibit FXIIa and

thrombin (7, 10a, 10c, Table 1) and, as a consequence, their

anticoagulant properties (Figure 4). Compounds exhibiting no

acyl moiety (9c), bearing a cycloaliphatic (11c) or sulfonamide

residue (13c) on the annular nitrogen atom, showed practically

no inﬂuence on the blood coagulation (Figure 4). This fact is

also in agreement with no FXIIa or thrombin inhibitory

properties of compounds 9c, 11c, and 13c (Table 1).

Microscale Parallel Synthesis and Screening. From the

screening of the initial series of synthesized compounds,

acylated aminotriazole 10a possessing the pyridin-2-yl moiety

showed both FXIIa inhibitory properties and the highest ability

to extend aPTT with only a little eﬀect on PT. However, its

FXIIa inhibitory potency, selectivity proﬁle, and anticoagulant

activity could be further improved. For this, considering the

inﬂuence of the acyl moiety structure on the biological activity

manifestation, one of the approaches would be to vary the acyl

moiety of 10a without changing its 1,2,4-triazol-5-amine core

(9a). For this purpose, conventional acylation or amide

coupling reactions could be performed, which, however, might

be time-consuming and require multiple puriﬁcation eﬀorts of

the ﬁnal products with the use of column chromatography.

Moreover, these reactions on typical laboratory scale require

considerable amounts of starting materials and solvents to

Figure 4. In vitro anticoagulant activity of selected 1,2,4-triazol-5-

amines tested at 300 μM compared to that of dabigatran (1) and

rivaroxaban (2) tested at 3 μM. The activated partial thromboplastin

time (aPTT) and prothrombin time (PT) are shown in seconds. The

percentage of aPTT and PT increase compared to the eﬀect of

dimethyl sulfoxide (DMSO) is shown under the diagram. Tests were

performed at least in triplicate, and the average with standard

deviation (SD) is given.

intrinsic (aPTT) or extrinsic (PT) pathway of blood

coagulation is aﬀected by the inhibitor. The experiments

Figure 5. Schematic representation of the microscale parallel synthetic approach combined with medium-throughput screening (MTS) toward

FXIIa and thrombin inhibitors.

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Figure 6. FXIIa and thrombin inhibition by 90 aminotriazoles obtained in microscale parallel synthesis at 1 μM (A and B) and at 100 nM (C and

D). Tests were performed in triplicate, and the average with SD is given.

1

generate libraries of test compounds, which is associated with

high expenses. Therefore, in this work, we utilized advantages

of our in-house-developed microscale parallel synthetic

methodology combined with medium-throughput screening

(MTS) collectively, allowing us to access and test the

biological activity of large series of compounds with minimum

expenses.

S95 were rapidly analyzed by H NMR spectroscopy, allowing

the determination of the conversion rate of each of the

reactions (Figure 5 and Supporting Information) and the

preparation of the samples of exact inhibitor concentration for

the MTS. The generated library of 90 compounds was

screened at 1 μM and 100 nM for the ability to inhibit

FXIIa and thrombin (Figure 6).

The microscale parallel synthetic approach was realized as

shown in Figure 5. At ﬁrst, under the optimized conditions, in

a 96-well plate, the microscale amide coupling reactions

between 3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine 9a and 95

structurally diverse carboxylic acids were performed in DMSO-

d₆(120 μL in each well) (Figure 5). The carboxylic acid

component was represented by linear aliphatic, unsaturated,

cycloaliphatic, aromatic, heteroaromatic, and amino acid-based

carboxylic acids (Supporting Information). After the com-

pletion of the reactions, formed acylated aminotriazoles S1−

Screening of aminotriazoles, generated in the microscale

reactions, revealed 37 and 29 compounds as new FXIIa and

thrombin inhibitors, respectively, the inhibitory activity of

which exceeded 50% at 1 μM concentration (Figure 6A,B).

This indicates about 30% hit-rate (active inhibitors) ﬁnding by

the approach used. In general, when the annular nitrogen atom

(1-N) of aminotriazole 9a was acylated with linear aliphatic,

unsaturated, cycloaliphatic carboxylic acids or amino acid-

based derivatives, resulted compounds showed lower ability to

inhibit both FXIIa and thrombin compared to aminotriazoles

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a

Scheme 4. Synthesis of 3-Pyridyl-Substituted 1,2,4-Triazol-5-amines 21a−v

a

Reagents and conditions: (a) oxalyl chloride, N,N-dimethylformamide (DMF, cat.), pyridine (dry), 0 °C to r.t., 1.5 h; (b) THF/pyridine 1:1, 0 °C

to r.t., 2−18 h, 21a 60%, 21b 72%, 21c 74%, 21d 25%, 21e 43%, 21f 46%, 21g 69%, 21h 64%, 21i 77%, 21j 83%; (c) EDCI, DMAP, DMF (dry), 0

°C to r.t., 3−16 h, 21k 65%, 21l 26%, 21m 55%, 21n 78%, 21o 77%, 21p 70%, 21q 89%, 21r 5%, 21s 61%, 21t 66%, 21u 75%, 21v 75%.

bearing aromatic acyl fragments (Supporting Information).

Heteroaromatic acyl moieties also showed a reduced tendency

to inhibit the activity of tested serine proteases compared to

their nonheteroaromatic analogues. In many cases, the

inhibitory activity of synthesized compounds toward FXIIa

and thrombin overlapped (Figure 6A,B). For some com-

pounds, however, the tendency for selectivity can already be

seen at 1 μM but is more apparent in the experiments with 100

nM inhibitor concentration (Figure 6C,D). Thus, amino-

triazole with screening number S15 possessing the α-naphthoyl

moiety showed noticeable 77% of FXIIa inhibition at 100 nM

while inhibiting thrombin by 13% only. In contrast, its β-

naphthoylated analogue S16 showed the inverted tendency,

inhibiting FXIIa and thrombin at 100 nM by 19 and 55%,

respectively (Figure 6C,D). Also, structurally related S-

conﬁgured 1,2,3,4-tetrahydronaphthalene derivative S36

showed selectivity toward inhibition of FXIIa (70%) over

thrombin (6%) along with 2-iodophenylacetic acid derivative

S85 inhibiting FXIIa by 47% and thrombin by 2% only (Figure

6C,D). The full table of FXIIa and thrombin inhibition rates by

90 aminotriazoles is given in the Supporting Information.

Second Round of Conventional Synthesis. Based on

the screening of compounds from the microscale parallel

synthesis, 21 acylated derivatives of 9a were selected for the

conventional synthesis and their further biological activity

evaluation. To verify the applicability of the utilized microscale

parallel synthetic approach combined with MTS as a selector

of active compounds, apart from the most promising and

potentially active aminotriazoles, several derivatives showing

low, moderate, and no inhibitory activity toward FXIIa and

FIIa were also included into the set of compounds for the

conventional synthesis.

assayed in vitro for their ability to inhibit FXIIa, thrombin,

FXa, and trypsin. The results of this evaluation are summarized

in Table 2.

Despite exhibiting a common aminotriazole scaﬀold, each

compound of series 21 displayed its unique inhibitory proﬁle

toward investigated serine proteases, which was determined by

the acyl moiety structure (Table 2). When comparing the

biological activity of compounds 21a−v to that of amino-

triazole 10a, bearing an unsubstituted phenyl ring, its mono-

(21k) and dimethylated (21a) analogues showed enhanced

FXIIa and thrombin inhibitory properties, exhibiting IC₅₀

values against both enzymes below 100 nM (e.g., for 21a

FXIIa IC₅₀= 91 nM, thrombin IC₅₀= 57 nM). However, the

introduction of a somewhat bigger para-ethyl or para-methoxy

substituent resulted in compounds 21c and 21b, respectively,

with a slightly reduced inhibitory potency against both

enzymes (Table 2). Also, a halogen atom introduction in the

para-position of the phenyl ring (21h−g) led to a further

decrease of compounds inhibitory properties toward FXIIa and

thrombin. For example, ﬂuorinated compound 21g showed

IC₅₀values of 306 and 481 nM against FXIIa and thrombin,

respectively. This eﬀect, however, was not as pronounced as for

the compounds 21d and 21f possessing para-triﬂuoromethyl-

and para-nitro-substituted aromatic acyl moieties, respectively,

which experienced a dramatic activity drop to a micromolar

level (IC₅₀= 3−5 μM and more toward FXIIa and thrombin).

Interestingly, it has been found that FXIIa tolerated the

introduction of large substituents in the para-position of the

aromatic ring better than thrombin. Thus, aminotriazoles 21p

and 21q, possessing bulky 2,2,2-triﬂuoroacetamido- and

benzamidobenzoic acid residues, showed FXIIa inhibition at

the level of IC₅₀= 142 and 109 nM, respectively, which is

comparable to the inhibitory properties of compound 10a

bearing an unsubstituted aromatic acyl moiety (Table 2).

However, in contrast to inhibitor 10a with low selectivity

proﬁle, compounds 21p and 21q showed 24-fold and at least

18-fold selectivity, respectively, toward FXIIa over thrombin,

which underwent only weak inhibition by 21p (thrombin IC₅₀

= 3.4 μM) and 21q (thrombin IC₅₀> 2 μM). These two

Acylated 1,2,4-triazol-5-amines 21a−v were accessed either

via a direct regioselective acylation of the annular nitrogen

atom (N-1) of 9a with appropriate acid chlorides or via the

amide coupling reactions between 9a and the corresponding

carboxylic acids (Scheme 4).

Inhibition of Serine Proteases by the Second Series

of Compounds. All prepared 1,2,4-triazol-5-amines 21a−v of

the second round of conventional synthesis (Scheme 4) were

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Table 2. Inhibition Properties of 1,2,4-Triazol-5-amines 10a and 21a−v Toward Selected Blood Coagulation Factors and

Trypsin

a

Measurements were performed in triplicate; 1 h incubation time is speciﬁed as IC₅₀values were time-dependent; the substrate concentration [S]₀

= 25 μM; measured FXIIa K_m= 167 4 μM for the Boc-Gln-Gly-Arg-AMC substrate; measured thrombin K_m= 18 1 μM for the Boc-Val-Pro-

Arg-AMC substrate. The K_ivalues could not be directly withdrawn from the Cheng−Prusoﬀ equation in this case due to the enzyme−inhibitor

b

covalent interaction (see the section “Mechanism of Inhibition”). A microscale reaction with 0% of conversion was used as a blank.

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Figure 7. (A) In vitro anticoagulant activity of selected 1,2,4-triazol-5-amines tested at 300 μM compared to that of dabigatran (1) and rivaroxaban

(2) tested at 3 μM. The activated partial thromboplastin time (aPTT) and prothrombin time (PT) are shown in seconds. The percentage increase

of aPTT and PT compared to the eﬀect of DMSO is shown under the diagram. (B and C) Measurement of aPTT EC_2×(concentration required to

prolong aPTT twice) for 21i (B) and 21m (C). Tests were performed at least in triplicate, and the average with SD is given.

compounds might be used as a starting point for the

development of FXIIa selective inhibitors.

As can be seen in Figure 7A, anticoagulant properties of

tested aminotriazoles varied signiﬁcantly in both aPTT and PT

tests. This activity ﬂuctuation was linked to the compounds’

selective or dual FXIIa and thrombin inhibition, which in turn

was determined by the structure of tested compounds. Thus,

α-naphthoylated compound 21i, the most potent 29 nM

inhibitor of FXIIa, aﬀected the intrinsic blood coagulation,

extending aPTT by 176%. The anticoagulant activity of 21i,

however, was lower than that of two other aminotriazoles 21a

and 21m possessing 3,4-dimethylphenyl and β-naphthoyl

residues, respectively, which prolonged aPTT even stronger

by 227 and 212%, respectively. This might look like a

discrepancy, considering a lower inhibitory activity of 21a and

21m toward FXIIa (91 and 138 nM, respectively) in

comparison to 21i. However, this contradiction is explained

by the ability of 21a and 21m to strongly inhibit thrombin (57

and 27 nM, respectively), inhibition of which is also evident by

prolonged PT for both compounds (48 and 575%,

respectively). As the aPTT test is sensitive to inhibitors of

both intrinsic and common coagulation pathways, for

compounds 21a and 21m, the synergistic eﬀect of FXIIa and

thrombin inhibition is observed. It should, however, be noted

that selective thrombin inhibition, e.g., by dabigatran, could

solely strongly prolong both PT and aPTT (Figure 7A).

Two most selective FXIIa inhibitors 21q and 21s (Table 2)

showed almost no inﬂuence on the extrinsic blood coagulation

(PT: 2 and 6%) while aﬀecting the intrinsic coagulation

pathway (aPTT was increased by 35 and 92%, respectively,

Figure 7A). Compound 21t, exhibiting no inhibitory properties

toward FXIIa or thrombin (Table 2), as expected, failed to

show anticoagulant activity, whereas more potent FXIIa and

thrombin inhibitors such as 21b, 21c, and 21h signiﬁcantly

prolonged aPTT by 108−114% (Figure 7A).

Initially noticed during the MTS (Figure 6), the remarkable

inhibitory proﬁle of two naphthoyl derivatives 21i and 21m

was now studied in detail (Table 2). Indeed, compound 21i

exhibiting the α-naphthoyl residue was found to be a potent 29

nM inhibitor of FXIIa with improved selectivity (nearly 13-

fold) over thrombin (21i: thrombin IC₅₀= 375 nM). In

contrast, its β-naphthoylated counterpart 21m was proved to

be a potent 27 nM inhibitor of thrombin with a reduced ability

to aﬀect FXIIa (IC₅₀= 138 nM).

When compared to the benzoic acid derivative 10a, its more

ﬂexible phenylacetic acid-derived analogue 21r inhibited FXIIa

and thrombin with IC₅₀values of 210 and 509 nM,

respectively, which indicates that the ﬂexibility gain resulted

in about twofold inhibitory activity loss toward both enzymes

(Table 2). Upon the structure rigidiﬁcation of compound 21r,

which resulted in chiral 1,2,3,4-tetrahydronaphthalene deriva-

tive 21v, the inhibitory potency toward FXIIa was recovered

(IC₅₀= 134 nM) and some selectivity over thrombin (IC₅₀

=

421 nM) was gained. Also, the iodine atom introduction in the

ortho-position of the aromatic ring of 21r resulted in

compound 21s, which exhibited enhanced inhibitory activity

toward FXIIa (IC₅₀= 62 nM) and 29-fold selectivity toward

FXIIa over thrombin (IC₅₀= 1.8 μM).

Finally, aminotriazoles exhibiting furanyl (21n), pyridyl

(21l), cyclohexyl (21j), tetrahydropyranyl (21o), styryl (21u),

or benzamidomethyl (21t) residues showed low to no

inhibitory properties toward FXIIa and thrombin, also

conﬁrming the results of MTS. Apart from compound 21m,

which showed 48% of FXa inhibition at 5 μM, none of the

tested 1,2,4-triazol-5-amines was able to reach or show close to

50% of FXa or trypsin inhibition at 5 μM.

Anticoagulant Activity of Series 2. Of synthesized 1,2,4-

triazol-5-amines 21a−v, 14 representative compounds were

tested for their anticoagulant properties in aPTT and PT tests.

The outcome of this evaluation is given in Figure 7.

For aminotriazoles 21i and 21m, aPTT EC_2×(concentration

required to prolong aPTT twice) was additionally measured. In

this test, β-naphthoyl derivative 21m showed about twice

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Figure 8. Time-dependent inhibition of FXIIa by aminotriazole 21i. The enzyme (FXIIa, 2.5 nM) was added to a solution of a ﬂuorogenic

substrate (Boc-Gln-Gly-Arg-AMC, 25 μM) and inhibitor (ranged 0.5 nM−132 μM) in assay buﬀer, and ﬂuorescence was read immediately in the

kinetic mode for up to 180 min. Overtime, sigmoidal IC₅₀curves undergo a shift toward the lower values of log inhibitor concentrations (left).

Measurement time plotted against calculated IC₅₀values of 21i (right). This behavior suggests the covalent mechanism of FXIIa inhibition by 21i.

Figure 9. Covalent reversible inhibition of FXIIa by aminotriazole 21m elucidated by mass spectrometry. Deconvoluted SEC/ESI-TOF mass

spectra of intact FXIIa (A), FXIIa incubated (4 min) with 21m (B), partially recovered FXIIa (C), and majorly recovered FXIIa (D). The main

peaks are labeled with the corresponding deconvoluted masses. The schematic representation of FXIIa inhibition steps is also shown.

lower EC_2×of 107 μM compared to its α-naphthoyl-derived

counterpart 21i, EC_2×of which was 236 μM (Figure 7B,C).

Mechanism of Inhibition. While studying the inhibitory

activity of synthesized 1,2,4-triazol-5-amines of both series 1

and 2 (Tables 1 and 2) toward FXIIa and thrombin, it has

been observed that the IC₅₀values for each compound change

over time signiﬁcantly. For instance, the time-dependent

behavior can be seen on the typical graph shown in Figure

8A for compound 21i incubated with FXIIa. This graph clearly

demonstrates distinct shifts of the IC₅₀sigmoidal curve during

three hours of measurement. Time-dependent inhibition is

even more apparent when measured IC₅₀values are plotted

against time (Figure 8B). For the noncovalent reversible

inhibitors such as dabigatran (1) and rivaroxaban (2), no time-

dependent inhibition of thrombin and FXa, respectively, was

observed in our experiments. These observations made us

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suspect a covalent character of FXIIa and thrombin inhibition

by the synthesized 1,2,4-triazol-5-amines.

To get insight into the mechanistic details of FXIIa

inhibition, the advantages of size-exclusion chromatography

(SEC) coupled with electrospray ionization mass spectrometry

(ESI-MS) were utilized. In a typical experiment, FXIIa was

incubated with the inhibitor of interest (molar ratio of 1:20),

followed by chromatography and the enzyme−inhibitor

complexes mass analysis. Also, the enzyme−inhibitor complex

stability was examined in additional experiments to verify the

possible reversibility of FXIIa covalent inhibition. The

outcome of this evaluation and the proposed mechanism of

FXIIa inhibition by the acylated 1,2,4-triazol-5-amines are

summarized in Figure 9.

In the experiment, native FXIIa with a mass of 29497.3 Da

(Figure 9A) changed its mass to 29651.6 Da after just 4 min of

incubation with the inhibitor 21m (Figure 9B). This signal

newly appeared on the mass spectrum was attributed to the

FXIIa-21m’ covalent complex (Figure 9B), which was

presumably formed as a result of a nucleophilic attack of

FXIIa catalytic Ser195 on the carbonyl carbon atom of 21m.

The measured mass shift of Δm = 154.3 Da indicates that

FXIIa acquired the β-naphthoic acid residue (21m’) from the

inhibitor 21m (Figure 9B). Subsequent removal of the excess

of inhibitor 21m form the sample and the washing of the

covalent complex (FXIIa-21m′) with the excess of water

allowed for the detection of at ﬁrst partial (after 5 min, Figure

9C) and then major (after 120 min, Figure 9D) recovery of

native FXIIa. The observed process of FXIIa recovery is

consistent with the general catalytic mechanism of serine

proteases, which proceeds via the hydrolysis step of the ester

intermediate.

Additionally, the covalent mechanism of FXIIa inhibition

was shown for ﬁve other acylated 1,2,4-triazol-5-amines

(Figure 10). For instance, SEC/ESI-MS analysis of the

1,2,3,4-tetrahydronaphthalene derivative 21v and benzamido-

benzoic acid derivative 21q revealed covalent complexes with

masses of 29655.8 and 29720.5 Da, respectively, corresponding

to the inhibitors’ acyl moiety adducts (158.5 and 223.2 Da,

respectively) to native FXIIa (Figure 10). Collectively, these

ﬁndings suggest that synthesized acylated 1,2,4-triazol-5-

amines are covalent reversible inhibitors of FXIIa and

presumably of FIIa.

Binding Mode Study by Molecular Modeling. To

rationalize the interactions between the synthesized inhibitors

and blood coagulation factor XIIa and thrombin, molecular

modeling studies were performed using available crystal

structures of the enzymes.^28,49The resultant binding modes

for exemplary compounds are shown in Figure 11.

Tested inhibitors demonstrated a number of interactions

with the active site of both FXIIa and thrombin sharing some

similarities upon binding. Thus, the tetrahedral intermediate,

the formation of which is suggested based on the performed

mass spectrometry assay (vide supra), in each case was

covalently bound to the oxygen atom of Ser195 (Figure 11A−

D). Apart from that, the intermediate is additionally stabilized

by two hydrogen bonds with the backbone amides of the

“oxyanion hole” (Gly193 and Ser195, Figure 11A−D) of both

FXIIa and thrombin, thereby mimicking the interactions of the

natural tetrahedral intermediate, which is formed during the

substrate cleavage. Further enzyme−inhibitor interactions are

not universal for FXIIa and thrombin and depend on the

Figure 10. Deconvoluted SEC/ESI-TOF mass spectra of intact FXIIa

(A, E) and ﬁve covalent complexes FXIIa-10a’ (B), FXIIa-21a’ (C),

FXIIa-21i’ (D), FXIIa-21v’ (F), and FXIIa-21q’ (G) formed after the

FXIIa incubation with inhibitors 10a, 21a, 21i, 21v, and 21q,

respectively. The peaks of interest are labeled with the corresponding

deconvoluted masses. The schematic representation of FXIIa and

FXIIa covalently bound to the acyl residues is also shown.

architecture of the active site of each enzyme and on the

structural features of each inhibitor.

The speciﬁc 60-insertion loop (Tyr60A-Pro60B-Pro60C-

Trp60D) of thrombin shapes its more narrow S2 pocket

(especially due to Tyr60A and Trp60D) and also a more

restricted S1′ binding site (e.g., due to Lys60F insertion),

preventing synthesized aminotriazoles from binding to the

enzyme S1′−S4′ binding sites (Figure 11B,D). Consequently,

substituted aminotriazoles bind to the active site of thrombin

in the way that their pyridyl moiety resides in S2 binding site

partially protruding toward the amino acids of the S3 site

(Figure 11B,D). Particularly, the pyridyl moiety of exemplary

aminotriazole 10a undergoes hydrophobic interactions with

Tyr60A, Trp60D, Leu99, and His57 of thrombin, whereas its

primary amino group forms a hydrogen bond with Glu192

(Figure 11B). In thrombin, the aromatic part of the inhibitor’s

acyl moiety protrudes deeper into the selectivity pocket (S1) of

the enzyme where it forms additional lipophilic interactions

with, e.g., Cys191 and the backbone of Trp215. This fact might

explain the lower tolerance of thrombin compared to that of

FXIIa toward the introduction of bulky substituents in the

para-position of the aromatic ring (e.g., low active thrombin

inhibitors 21p and 21q, Table 2).

FXIIa is characterized by more spacious S1′, S2, S3, and S4

sites compared to the corresponding sites of other serine

proteases including thrombin.⁵⁰This allows synthesized

aminotriazoles to address unrestricted S1′ site. Hence, we

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of the acyl moiety of 10a resides at the entrance of S1

selectivity pocket, forming lipophilic interactions only with

Ser195, which should permit the bigger substituents’

introduction both in the para- and meta-position of the

aromatic ring to additionally address either S1 or S3 binding

sites (Figure 11A).

Aminotriazoles possessing small acyl residues (e.g., 10a, 21a,

21k, Table 2) were found to interact eﬃciently with the active

sites of both FXIIa and thrombin (Figure 11A,B), orienting

their pyridyl residue toward either S1′ or S2 binding sites,

respectively. That is why mentioned aminotriazoles experi-

enced no selectivity, being equipotent inhibitors of FXIIa and

thrombin. In contrast, compounds exhibiting structurally

demanding acyl moieties showed a binding preference toward

either FXIIa or thrombin. For instance, the selectivity proﬁle is

evidently driven by the structure of the acyl moiety of 21i (29

nM inhibitor of FXIIa) and 21m (27 nM inhibitor of

thrombin) bearing spacious α- and β-naphthoyl residues,

respectively (Table 2). Thus, the α-naphthoyl residue due to

its geometry allows 21i to adopt the energetically favorable

conformation, facilitating compound’s binding toward the

direction of S2−S1′ binding site of FXIIa, whereas the β-

naphthoyl substituent exhibiting less geometrical restriction is

able to protrude deeper into the S1 pocket of thrombin

orienting the rest of 21m toward the S2−S3 pocket of

thrombin (Figure 11C,D). Compared to the benzoyl moiety of

10a, extended aromatic α- and β-naphthoyl residues of 21i and

21m acquire additional lipophilic interactions with, e.g.,

Cys191, Ile213 (FXIIa, Figure 11C) and Val213, Asp189,

Gly219, Gly266 (thrombin, Figure 11D). These additional

interactions could explain lower IC₅₀values of 21i and 21m

toward FXIIa and thrombin compared to 10m (Table 2).

Inhibition of Platelet Activation and Platelet Aggre-

gation. As the search for new antithrombotic therapy of

cancer-associated thrombosis is of high importance, the

inﬂuence of the synthesized acylated 1,2,4-triazol-5-amines

Figure 11. Calculated covalent binding conformation of inhibitor 10a

(orange stick model) in the active site of FXIIa (A) and thrombin

(B). Predicted covalent binding conformations of α- and β-naphthoyl

derivatives 21i (green stick model, C) and 21m (cyan stick model, D)

in the active sites of FXIIa (C) and thrombin (D). The residues are

depicted as gray stick models. Substrate-binding sites are labeled (S1−

S3 and S1′). The surface is colored as follows: lipophilic regions are in

orange, hydrophilic in blue, and neutral in white. PDB ID used:

6B77/FXIIa²⁸and 6CYM/FIIa.⁴⁹

observed that 10a preferably binds to the amino acid residues

of S2−S1′ binding sites of FXIIa (Figure 11A), which is in

contrast to thrombin, where 10a preferred to bind to S2−S3

sites (Figure 11B). In FXIIa, the pyridyl moiety of 10a

undergoes hydrophobic interactions not only with His57 but

also with Cys58, whereas its aminotriazole aromatic ring, being

also shifted toward S1′ site, interacts with Cys42 and Phe41

(Figure 11A). Upon binding to FXIIa, the aromatic fragment

Figure 12. Platelet aggregation induced by MDA-MB-231 cancer cells (A), thrombin (B), or TRAP-6 (C) and measured by light transmission

aggregometry in a coagulation-factor-free platelet buﬀer. In each case, platelets were preincubated with aminotriazole 21m (0.1, 1, and 10 μM),

dabigatran (1 μM), or DMSO (0.1%). Data are representative of three experiments (n = 3).

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on the cancer-cell-induced platelet activation and aggregation

was tested. Presumably, synthesized aminotriazoles might

indirectly aﬀect platelet activation via the inhibition of

thrombin, which is formed upon plasma contact with TF

expressed on the surface of cancer cells. The inhibition of

FXIIa might also be beneﬁcial in cancer-related thrombosis as

cancer cells exhibit higher levels of the phospholipid

phosphatidylserine on their outer membrane, which might

contribute to FXII and platelet activation and thrombosis

progression.⁵¹⁻⁵³However, in coagulation-independent plate-

let activation tests due to the absence of clotting factors, the

eﬀect of FXIIa inhibition is expected to be minimal.

linked to thrombin inhibition. This assumption ﬁnds further

conﬁrmation in the experiments with TRAP-6, in which 21m

irrespective of dose tested showed no inﬂuence on thrombin-

independent TRAP-6-induced platelet aggregation (Figure

12C).

In the second series of tests, platelet activation was studied

by measuring the release of adenosine triphosphate (ATP)

from platelets’ dense granules employing the bioluminescent

ﬁreﬂy luciferase assay.⁵⁴Platelets were treated with compound

21m and activated with MDA-MB-231 cancer cells, thrombin,

or TRAP-6. The luminescent signal corresponding to ATP

released from platelets activated by TRAP-6 was selected as a

functional control and set as 100% (Figure 13).

To estimate the ability of synthesized compounds to prevent

platelet activation, two tests were performed. In the ﬁrst test,

the platelet aggregation was studied in a coagulation-factor-free

buﬀer using light transmission aggregometry, where higher

transmission indicates stronger platelet aggregation.⁵⁴For this,

in the presence and the absence of selected aminotriazoles 21i,

21m, and 21q, platelets were activated with a highly aggressive

human breast cancer cell line MDA-MB-231, which expresses

high levels of TF and strongly induces thrombin generation.⁵⁴

In a typical experiment, MDA-MB-231 cells induced fast (in

about 400 s) and massive platelet aggregation (black curve,

Figure 12A). Compounds 21i and 21q exhibiting preference

toward FXIIa inhibition showed little to no inﬂuence on the

breast-cancer-cell-induced platelet aggregation (Supporting

Information). In contrast, being a 27 nM inhibitor of

thrombin, compound 21m showed a dose-dependent ability

to reduce platelet aggregation induced by MDA-MB-231

cancer cells (Figure 12A). At 100 nM, aminotriazole 21m

showed practically no inﬂuence on MDA-MB-231-induced

platelet aggregation (blue curve, Figure 12A). When, however,

the test concentration was elevated to 1 μM, the evident shift

of platelet aggregation curve (red curve) was observed,

implying partial inhibition of platelet aggregation by 21m. A

further concentration increase to 10 μM allowed for the

complete prevention of MDA-MB-231-induced platelet

aggregation by 21m during the observed time period (green

line, Figure 12A).

To verify that aminotriazole 21m inﬂuenced cancer-cell-

induced platelet aggregation via the thrombin inhibition,

additional tests with platelets activated by thrombin (Figure

12B) and by thrombin receptor activating peptide-6 (TRAP-6,

Figure 12C) were performed. Thrombin is a highly potent

platelet activator, which binds to and cleaves the amino-

terminal exodomain of the protease-activated receptors (PAR-

1 and PAR-4) on the platelet surface. This cleavage unmasks a

new receptor amino-terminus, which binds intramolecularly,

thereby activating the receptor.^55,56TRAP-6 is a peptide

fragment of PAR-1 (residues 42−47) that directly activates

PAR1 independently of the receptor cleavage, thus mimicking

the eﬀects of thrombin.⁵⁷In our experiment, both thrombin

and TRAP-6 induced complete platelet aggregation within

100−150 s (black curve, Figure 12B,C). When supplemented

to platelets, compound 21m showed partial (at 1 μM) or

complete (at 10 μM) inhibition of thrombin-induced platelet

aggregation (Figure 12B). Inactivated thrombin, now carrying

the acyl fragment of 21m on its Ser195, is probably still able to

bind to PAR-1, though without the ability to activate the

receptor due to the catalytic activity loss. This ﬁnding

correlates nicely with the compound 21m’s ability to prevent

MDA-MB-231-induced platelet aggregation (Figure 12A) and

suggests that antiaggregatory properties of 21m are likely

Figure 13. ATP release by activated platelets detected by

luminescence measurements. MDA-MB-231 cancer cells, thrombin,

and TRAP-6 strongly induce ATP release. TRAP-6-induced ATP

release is set as 100%. Aminotriazole 21m dose-dependently reduces

MDA-MB-231- and FIIa-induced ATP release. Tests were performed

in triplicate, and the average with SD is given; ** p < 0.0005; *** p <

0.0001.

MDA-MB-231 cells, thrombin, and TRAP-6 strongly

induced platelet activation as evidenced by maximal levels of

ATP released upon platelet contact with listed activators

(Figure 13). Compound 21m signiﬁcantly suppressed both

MDA-MB-231 cell- and thrombin-promoted platelet activa-

tion. This eﬀect of 21m was dose-dependent, reaching its

maximum at 10 μM (Figure 13). In contrast, compound 21m

showed little (at 10 μM) to no (at 1 μM) inﬂuence on TRAP-

6-induced platelet activation (Figure 13).

Altogether, two independent tests revealed the ability of

aminotriazole 21m to suppress MDA-MB-231 cancer-cell-

induced platelet activation. This eﬀect was linked to the

thrombin inhibitory activity of 21m. Two representative FXIIa

inhibitors 21i and 21q showed practically no inﬂuence on the

breast-cancer-cell-induced platelet aggregation.

Thrombin Generation Assay. For many anticoagulants,

thrombin generation is an important measure of their eﬃcacy.

For this, thrombin generation assay (TGA) is employed,

allowing to monitor the TF-induced thrombin generation by

measuring the ﬂuorogenic substrate cleavage. The assay

outcome is expressed as a thrombogram (e.g., Figure 14),

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which is characterized by several parameters shown in the

footnote of Table 3.

paradoxical eﬀect was reported for known thrombin inhibitors

dabigatran and melagatran, which being tested at low doses

ampliﬁed thrombin generation in TGA.^58,59This paradoxical

eﬀect is not completely understood but might be related to the

inhibition of protein C activation by thrombin inhibitors.⁶⁰

Cytotoxicity and Selectivity Proﬁle of Synthesized

Aminotriazoles. Before synthesized aminotriazoles could

further proceed into animal studies, their safety proﬁle should

be evaluated in vitro. To get the initial impression about

possible hepato- and neurotoxicity of prepared compounds, the

most potent FXIIa and thrombin inhibitors 21i and 21m,

respectively, along with the most selective FXIIa inhibitor 21q

were assayed in cytotoxicity tests employing HepG2 and CCF

cells (Figure 15).

Performed cytotoxicity tests revealed a relatively low

cytotoxicity proﬁle of exemplary aminotriazoles (Figure 15).

Thus, compounds 21i, 21m, and 21q at the highest tested dose

of 100 μM reduced the viability of HepG2 cells by only 8, 25,

and 13%, respectively. CCF cells were found to be slightly

more sensitive toward the addition of compounds 21m and

21q, which at the dose of 100 μM reduced the cell viability by

36 and 35%, respectively, which, however, was insuﬃcient to

obtain the IC₅₀values (extended cytotoxicity data are provided

in the Supporting Information).

Even though synthesized compounds inhibited FXIIa and

thrombin without aﬀecting trypsin and FXa, considering their

covalent reversible mechanism of action, other serine hydro-

lases might be aﬀected by the compounds. Therefore, to verify

that the synthesized aminotriazoles do not show aggressive

acylating behavior toward other enzymes possessing catalytic

Ser, selected compounds were assayed against the panel of

physiologically relevant serine proteases (FXIa, urokinase,

plasmin, and plasma kallikrein) and serine hydrolases MAGL

(monoacylglycerol lipase)⁶¹and FAAH (fatty acid amide

hydrolase).⁶²

Performed experiments revealed that none of the assayed

1,2,4-triazol-5-amines was able to signiﬁcantly inhibit serine

proteases implicated in the blood coagulation cascade (except

for targeted thrombin and FXIIa) as well as MAGL or FAAH

(Table 4). This ﬁnding additionally underlines the selectivity

of synthesized FXIIa and thrombin inhibitors, the inhibitory

activity of which is not only driven by the presence of catalytic

Ser in the enzymes’ active site but is rather determined by the

overall structural features of FXIIa and thrombin (Figure 11).

Figure 14. Representative thrombogram showing the TF-induced

thrombin generation in the presence of 21a, 21i, 21m, and dabigatran

(1).

Selected compounds 21a, 21i, and 21m exhibiting

anticoagulant activity (Figure 7) and showing diﬀerent

preferences toward FXIIa and thrombin inhibition (Table 2)

were assayed in TGA. As can be seen from the thrombogram

(Figure 14), at 100 μM, α-naphthoylated aminotriazole 21i

showed a limited reduction of thrombin generation, which was

expressed in the decreased amplitude of the thrombin peak

(A_maxreduction by 15% vs DMSO, Table 3) and reduced

endogenous thrombin potential (ETP reduction by 38% vs

DMSO, Table 3). The clotting time, however, was not

extended; it was even slightly shortened (T_1/2and T_max, Table

3). This limited eﬀect of 21i on thrombin generation was

expected and is linked to the preference of 21i to inhibit FXIIa

over thrombin (Table 2). In contrast, being an unselective

FXIIa and thrombin inhibitor, 3,4-dimethylbenzoic acid

derivative 21a (Table 2) showed a more pronounced reduction

of thrombin generation at 100 μM (Figure 14), decreasing

A_maxand ETP by 73 and 69% compared to DMSO,

respectively, and also extending T_1/2by 27% (Table 3).

The β-naphthoylated aminotriazole 21m exhibiting the

lowest IC₅₀value (27 nM) toward thrombin was expected to

show the strongest ability to reduce thrombin generation in

TGA. Instead, 21m demonstrated an unexpectedly paradoxical

eﬀect, amplifying thrombin generation (Figure 14). Thus,

compared to DMSO, 21m triggered higher amounts of

thrombin generation (A_maxand ETP increased 2.8- and 1.8-

fold) and also shortened thrombin generation time (21m T_1/2

= 2.3 min vs DMSO T_1/2= 3.0 min). This eﬀect is unexpected,

though not unusual, for thrombin inhibitors. A similar

CONCLUSIONS

■

This study reports on selective small-molecule inhibitors of

human blood coagulation factor XIIa and thrombin. The

combination of conventional and microscale parallel synthetic

a

Table 3. Thrombin Generation Parameters under the Inﬂuence of 21a, 21i, 21m, and Dabigatran (1)

homogeneous thrombin generation (n = 3)

T_1/2SD [min]

A_maxSD [AU/L]

T_maxSD [min]

ETP SD [AU*min/L]

21a

21i

21m

Dabig (1)

DMSO

Buﬀer

3.8 0.5

2.7 0.6

2.3 0.6

15.2 1.1

3.0 0.5

2.7 0.5

16.9 3.9

53.3 1.1

175.5 32.9

72.4 3.5

63.0 3.7

73.0 22.6

8.8 0.4

7.0 0.2

8.0 0.7

25.5 1.1

8.1 0.6

7.8 1.3

370 24

738 250

2160 278

678 110

1203 44

1737 344

a

T

_1/2, clotting time; A_max, the amplitude of the thrombin peak; T_max, time to peak; ETP, endogenous thrombin potential (the area under the

thrombogram curve).

O

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Figure 15. Cytotoxicity proﬁle of synthesized FXIIa and thrombin inhibitors 21i, 21m, and 21q tested on HepG2 and CCF-STTG-1 cell lines. T-2

toxin (10 μM) was used as a positive and DMSO (1%) as a negative control. Tests were performed in triplicate, and the average with SD is given; *

p < 0.05; ** p < 0.01.

Table 4. Selectivity Proﬁle of Acylated 1,2,4-Triazol-5-amines

a

protease IC₅₀, (nM)

10a

21a

21i

21m

21s

thrombin

FXa

FXIa

235

>5000

7

57

>5000

6

375 12

>5000

27

2

1819 155

>5000

48% @ 5 μM

>5000

FXIIa

uPA

plasmin

PK

trypsin

MAGL

FAAH

132 10

7930 2130

>5000

91

9

29

5

138 12

6762 2408

>5000

62 22

41% @ 5 μM

>5000

2942 71

25820 4880

>5000

a

Screened @ 5 μM in at least two independent determinations; for active compounds, IC₅₀measurements were performed in triplicate. Inhibition

values of reference inhibitors: MAGL inhibitor CAY10499⁶³IC₅₀= 480 nM; FAAH inhibitor URB597⁶⁴IC₅₀= 43 nM.

approaches allowed us to access a library of diverse 1,2,4-

triazol-5-amines, some of which potently inhibited FXIIa and/

or thrombin but not other tested serine proteases of the blood

coagulation cascade. Synthesized compounds preferentially

aﬀected the intrinsic blood coagulation, which is initiated by

FXIIa and is implicated in thrombosis. Structure−activity

relationship studies uncovered the inhibitors’ structural

features required for the FXIIa and thrombin successful

inhibition and for the anticoagulant activity manifestation. The

presence of a pyridyl moiety in the position 3 of the 1,2,4-

triazol-5-amine scaﬀold was found to be crucial as its

replacement with a phenyl or aminoaryl residue resulted in

activity reduction. The 1,2,4-triazol-5-amines exhibiting a

nonacylated annular secondary amino group or bearing a

sulfonamide residue on the annular nitrogen atom demon-

strated no ability to inhibit tested serine proteases as well as no

inﬂuence on blood coagulation. Instead, N1-acylated com-

pounds, especially bearing an aromatic acyl fragment, showed

the highest ability to inhibit FXIIa and thrombin. Among them,

derivative 21i exhibiting the α-naphthoyl residue was found to

be the most potent 29 nM inhibitor of FXIIa with improved

selectivity over thrombin (21i: thrombin IC₅₀= 375 nM),

whereas its β-naphthoylated analogue 21m was proved to be

the most potent 27 nM inhibitor of thrombin with a reduced

ability to aﬀect FXIIa (IC₅₀= 138 nM). More selective

inhibitors of FXIIa were also found among synthesized

aminotriazoles (e.g., 21p, 21s, and 21q), which demonstrated

almost no inﬂuence on the extrinsic blood coagulation while

disrupting the intrinsic coagulation pathway.

Mass spectrometric analysis reinforced by the molecular

modeling allowed us to discover previously unknown

interactions between the synthesized inhibitors and the active

site of FXIIa. It has been established that the carbonyl carbon

atom of acylated 1,2,4-triazol-5-amines upon contact with

FXIIa undergoes a nucleophilic attack, presumably, by the

FXIIa catalytic Ser195, which results in the acyl moiety

transfer, forming an FXIIa-Ser195-acyl covalent complex. A

single transfer of the acyl moiety to FXIIa implies this process’

speciﬁcity. Being an ester susceptible to slow hydrolytic

cleavage, the resultant covalent complex (FXIIa-Ser195-acyl)

was experimentally shown to degrade over time, recovering the

native FXIIa. These experiments along with the observed time-

dependent FXIIa and thrombin inhibition suggested acylated

1,2,4-triazol-5-amines as covalent reversible inhibitors. As

suggested by the computational modeling, apart from the

covalent interaction with Ser195, active inhibitors of FXIIa and

thrombin form two hydrogen bonds with the backbone amides

of the “oxyanion hole” (Gly193 and Ser195), thereby

mimicking the interactions of the natural tetrahedral

intermediate. Other interactions with S1−S3 and S1′

substrate-binding sites of FXIIa and thrombin are more

speciﬁc for each enzyme and also depend on the compound’s

structure.

Apart from exhibiting direct anticoagulant properties,

selected 1,2,4-triazol-5-amines reduced thrombin generation

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(1.3−2 equiv), and DMAP (1.3−2 equiv) were dissolved in DMF

(3−4 mL, dry) at 0 °C. Under stirring, the reaction mixture was

allowed to warm up to room temperature. After 3−18 h of stirring at

room temperature, the mixture was cooled down to 0 °C and diluted

with water (100 mL), and the formed precipitate was ﬁltrated oﬀ and

washed with water (10 mL) and diethyl ether (10 mL). The residue

was dried under reduced pressure.

in TGA and slowed down platelet activation and platelet

aggregation. Thus, being tested at 100 μM, 3,4-dimethylben-

zoic acid derivative 21a practically diminished thrombin

generation in TGA, whereas β-naphthoylated aminotriazole

21m suppressed MDA-MB-231 cancer-cell-induced platelet

activation. These eﬀects were linked to the ability of 21a and

21m to inhibit thrombin.

Collectively, herein disclosed 1,2,4-triazol-5-amines with

FXIIa and/or thrombin inhibitory activity, anticoagulant

properties, elucidated mechanism of inhibition, selectivity

proﬁles, and relatively low cytotoxicity represent a promising

starting point for the development of novel antithrombotic

drugs and also can ﬁnd their application as chemical tools for

studying the role of FXIIa and thrombin in physiological and

pathological processes.

(5-Amino-3-(pyridin-3-yl)-1H-1,2,4-triazol-1-yl)(phenyl)-

methanone (7). It was synthesized according to general procedure A

from 9b (195 mg, 1.2 mmol, 1 equiv) and benzoyl chloride (329 mg,

2.3 mmol, 1.9 equiv). Stirring at room temperature was continued for

4.5 h. The crude product was puriﬁed by ﬂash column

chromatography (DCM/MeOH = 1/0 → 9/1) to give 7 as a

colorless solid (247 mg, 0.93 mmol, 77%); mp = 172−174 °C. TLC:

1

0.84 (EtOAc/MeOH, 9:1). H NMR (600 MHz, DMSO-d₆) δ 7.51

(ddd, J = 8.0, 4.9, 0.9 Hz, 1H, 5′-H_pyridyl), 7.57−7.62 (m, 2H, 3″-

H_phenyl, 5″-H_phenyl), 7.67−7.72 (m, 1H, 4″-H_phenyl), 7.94 (br s, 2H,

NH₂), 8.19−8.13 (m, 2H, 2″-H_phenyl, 6″-H_phenyl), 8.25 (dt, J = 7.9, 1.9

Hz, 1H, 4′-H_pyridyl), 8.67 (dd, J = 4.9, 1.7 Hz, 1H, 6′-H_pyridyl), 9.11 (d,

J = 2.1 Hz, 1H, 2′-H_pyridyl). ¹³C NMR (151 MHz, DMSO-d₆) δ 123.9

(1C, C-5′_pyridyl), 126.0 (1C, C-3′_pyridyl), 128.1 (2C, C-3″_phenyl, C-

5″_phenyl), 130.9 (2C, C-2″_phenyl, C-6″_phenyl), 132.0 (1C, C-1″_phenyl),

133.1 (1C, C-4″_phenyl), 133.8 (1C, C-4′_pyridyl), 147.5 (1C, C-2′_pyridyl),

150.9 (1C, C-6′_pyridyl), 157.7 (1C, C-3_triazole), 159.1 (1C, C-5_triazole),

EXPERIMENTAL SECTION

■

Chemistry, General. Unless otherwise mentioned, THF was

dried with sodium/benzophenone and was freshly distilled before use.

Thin-layer chromatography (TLC): silica gel 60 F₂₅₄plates (Merck).

Flash chromatography (FC): silica gel 60, 40−63 μm (Macherey-

Nagel). Reversed-phase thin-layer chromatography (RP-TLC): silica

gel 60 RP-18 F₂₅₄S plates (Merck). Automatic ﬂash column

chromatography: Isolera One (Biotage); brackets include eluent,

cartridge-type. Melting point (m.p.): melting point apparatus SMP 3

(Stuart Scientiﬁc), uncorrected. ¹H NMR (400 MHz), ¹H NMR (600

MHz), and ¹³C NMR (100 MHz): Agilent DD2 400 and 600 MHz

spectrometers; chemical shifts (δ) are reported in ppm against the

reference substance tetramethylsilane and calculated using the solvent

residual peak of the undeuterated solvent. IR: IR Prestige-21

(Shimadzu). HRMS: MicrOTOF-QII (Bruker). The HPLC method

to determine the purity of compounds: equipment 1: pump: L-7100,

degasser: L-7614, autosampler: L-7200, UV detector: L-7400,

interface: D-7000, data transfer: D-line, data acquisition: HSMS

software (all from LaChrom, Merck Hitachi); equipment 2: pump:

LPG-3400SD, degasser: DG-1210, autosampler: ACC-3000T, UV

detector: VWD-3400RS, interface: Dionex UltiMate 3000, data

acquisition: Chromeleon 7 (Thermo Fisher Scientiﬁc); column:

LiChrospher 60 RP-select B (5 μm), LiChroCART 250−4 mm

cartridge; ﬂow rate: 1.0 mL/min; injection volume: 5.0 μL; detection

at λ = 210 nm; solvents: A: demineralized water with 0.05% (v/v)

triﬂuoroacetic acid, B: acetonitrile with 0.05% (v/v) triﬂuoroacetic

acid; gradient elution (% A): 0−4 min: 90%; 4−29 min: gradient

from 90 to 0%; 29−31 min: 0%; 31−31.5 min: gradient from 0 to

90%; 31.5−40 min: 90%. With the exception of compounds 7

(93.9%) and 21l (94.0%), the purity of all test compounds was greater

than 95%.

167.6 (CO). IR (neat): ν

̃

[cm⁻¹] = 3426, 3059, 1690, 1659, 1582,

1524, 1489, 1412, 1373, 1339, 1180, 1146, 1072, 1026, 964, 799, 748,

687, 625. HRMS (m/z): [M + H]⁺calcd for C₁₄H₁₁N₅O⁺266.1036,

found 266.1017. HPLC: t_R= 13.95 min, purity 93.9%.

(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(phenyl)-

methanone (10a). It was synthesized according to general procedure

A from 9a (305 mg, 1.89 mmol, 1 equiv) and benzoyl chloride (346

mg, 2.46 mmol, 1.3 equiv). Stirring at room temperature was

continued for 4.5 h. The crude product was puriﬁed by ﬂash column

chromatography (DCM/MeOH = 1/0 → 9/1) to give 10a as a

colorless solid (210 mg, 0.79 mmol, 42%); mp = 174−175 °C. TLC:

1

0.72 (EtOAc/MeOH, 9:1). H NMR (600 MHz, DMSO-d₆) δ 7.47

(ddd, J = 7.5, 4.7, 1.2 Hz, 1H, 5′-H_pyridyl), 7.56−7.62 (m, 2H, 3″-

H_phenyl, 5″-H_phenyl), 7.70 (ddt, J = 8.7, 7.0, 1.3 Hz, 1H, 4″-H_phenyl), 7.85

(br s, 2H, NH₂), 7.92 (td, J = 7.7, 1.8 Hz, 1H, 4′-H_pyridyl), 8.00 (dt, J =

7.8, 1.1 Hz, 1H, 3′-H_pyridyl), 8.10−8.16 (m, 2H, 2″-H_phenyl, 6″-H_phenyl),

8.66 (ddd, J = 4.7, 1.8, 0.9 Hz, 1H, 6′-H_pyridyl). ¹³C NMR (151 MHz,

DMSO-d₆) δ 122.5 (1C, C-3′_pyridyl), 124.7 (1C, C-5′_pyridyl), 128.1 (2C,

3″-C_phenyl, 5″-C_phenyl), 130.7 (2C, 2″-C_phenyl, 6″-C_phenyl), 132.1 (1C,

1″-C_phenyl), 133.0 (1C, 4″-C_phenyl), 137.0 (1C, C-4′_pyridyl), 148.9 (1C,

C-2′_pyridyl), 149.7 (1C, C-6′_pyridyl), 159.0 (1C, 3-C_triazole), 159.6 (1C, 5-

C_triazole), 167.9 (CO). IR (neat): ν

̃

[cm⁻¹] = 3460, 3063, 2928,

1686, 1632, 1535, 1447, 1385, 1358, 1327, 1153, 1076, 968, 918, 799,

745, 691. HRMS (m/z): [M + H]⁺calcd for C₁₄H₁₁N₅O⁺266.1036,

found 266.1015. HPLC: t_R= 14.83 min, purity 96.6%.

General Procedure A. To a stirred solution of 1H-1,2,4-triazol-5-

amine 9a−c, 15, or 19a−c (1 equiv) in dry THF/pyridine mixture

(1:1), an acylating agent (acid chloride, sulfonyl chloride, or

chloroformate, 1−2 equiv) in dry THF was added dropwise with a

syringe pump over 30 min at 0 °C. The reaction mixture was stirred at

0 °C for 1 h and then at room temperature (for the indicated period

of time). Then, the reaction mixture was diluted with water (100 mL),

and the formed precipitate was ﬁltrated oﬀ, washed with water and

diethyl ether, and dried in vacuo. The crude product was puriﬁed, if

required, by column chromatography.

General Procedure B (Synthesis of Acid Chlorides). The

respective carboxylic acid (1 equiv) was dissolved in THF (3 mL,

dry). At 0 °C, oxalyl chloride (2.3 equiv) was added dropwise to the

reaction mixture over 3 min. One drop of DMF was added, and the

reaction mixture was allowed to warm up to room temperature. After

the mixture was stirred for 2 h, the solvent was evaporated under

reduced pressure and the residue was dissolved in low amounts of dry

THF (0.5 mL) to be used directly as an acylation agent in general

procedure A.

(5-Amino-3-(pyridin-4-yl)-1H-1,2,4-triazol-1-yl)(phenyl)-

methanone (10c). It was synthesized according to general procedure

A from 9c (499 mg, 3.1 mmol, 1 equiv) and benzoyl chloride (479

mg, 3.41 mmol, 1.1 equiv). Stirring at room temperature was

continued for 4.5 h. After the washing steps, pure product 10c was

obtained as a colorless solid (716 mg, 2.70 mmol, 87%); mp = 216−

217 °C. TLC: 0.74 (EtOAc/MeOH, 9:1). ¹H NMR (600 MHz,

DMSO-d₆) δ 7.56−7.62 (m, 2H, 3″-H_phenyl, 5″-H_phenyl), 7.68−7.74

(m, 1H, 4″-H_phenyl), 7.81−7.86 (m, 2H, 3′-H_pyridyl, 5′-H_pyridyl), 7.94

(br s, 2H, NH₂), 8.11−8.18 (m, 2H, 2″-H_phenyl, 6″-H_phenyl), 8.64−8.70

(m, 2H, 2′-H_pyridyl, 6′-H_pyridyl). ¹³C NMR (151 MHz, DMSO-d₆) δ

120.5 (2C, C-3′_pyridyl, C-5′_pyridyl), 128.1 (2C, C-3″_phenyl, C-5″_phenyl),

130.8 (2C, C-2″_phenyl, C-6″_phenyl), 131.9 (1C, C-1″_phenyl), 133.1 (1C,

C-4″_phenyl), 137.3 (1C, C-4′_pyridyl), 150.4 (2C, C-2′_pyridyl, C-6′_pyridyl),

157.8 (1C, C-3_triazole), 159.2 (1C, C-5_triazole), 167.6 (CO). IR

(neat): ν

̃

[cm⁻¹] = 3302, 3125, 1701, 1651, 1605, 1524, 1489, 1366,

1307, 922, 752, 675, 675. HRMS (m/z): [M + H]⁺calcd for

C₁₄H₁₁N₅O⁺266.1036, found 266.1051. HPLC: t_R= 13.59 min,

purity 99.1%.

(5-Amino-3-(pyridin-4-yl)-1H-1,2,4-triazol-1-yl)(cyclohexyl)-

methanone (11c). It was synthesized according to general procedure

General Procedure C. If not described otherwise, 1,2,4-triazol-5-

amine 9a (1 equiv), the respective carboxylic acid (1 equiv), EDCI

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A from 9c (300 mg, 1.86 mmol, 1 equiv) and cyclohexanecarbonyl

chloride (355 mg, 2.42 mmol, 1.3 equiv). Stirring at room

temperature was continued for 4.5 h. After the washing steps, pure

product 11c was obtained as a colorless solid (287 mg, 1.06 mmol,

57%); mp = 193−195 °C. TLC: 0.83 (EtOAc/MeOH, 9:1).¹H NMR

(600 MHz, DMSO-d₆) δ 1.18−1.29 (m, 1H, CH(CH₂CH₂)₂CH₂),

1.31−1.49 (m, 4H, CH(CH₂CH₂)₂CH₂), 1.65−1.72 (m, 1H,

CH(CH₂CH₂)₂CH₂), 1.78 (dt, J = 12.8, 3.2 Hz, 2H, CH-

(CH₂CH₂)₂CH₂), 1.98 (dd, J = 11.5, 4.3 Hz, 2H, CH-

TLC: 0.27 (EtOAc/cyclohexane, 2:8).¹H NMR (600 MHz, DMSO-

d₆) δ 7.45−7.50 (m, 3H, 3′-H_phenyl, 4′-H_phenyl, 5′-H_phenyl), 7.56−7.62

(m, 2H, 3″-H_benzoyl, 5″-H_benzoyl), 7.67−7.72 (m, 1H, 4″-H_benzoyl), 7.85

(br s, 2H, NH₂), 7.92−7.99 (m, 2H, 2′-H_phenyl, 6′-H_phenyl), 8.13−8.18

(m, 2H, 2″-H_benzoyl, 6″-H_benzoyl). ¹³C NMR (151 MHz, DMSO-d₆) δ

126.4 (2C, C-2′_phenyl, C-6′_phenyl), 128.1 (2C, C-3″_benzoyland C-

5″_benzoyl), 128.7 (2C, C-3′_phenyl, C-5′_phenyl), 130.10 (1C, C-1′_phenyl),

130.11 (1C, C-4′_phenyl), 130.8 (2C, C-2″_benzoyl, C-6″_benzoyl), 132.1 (1C,

C-1″_benzoyl), 133.0 (1C, C-2″_benzoyl), 159.0 (1C, C-5_triazole), 159.7 (1C,

C-3_triazole), 167.6 (CO). IR (neat): ν

̃

[cm⁻¹] = 3421, 3287, 3190,

(CH₂CH₂)₂CH₂), 3.42 (tt,

J

=

11.2, 3.4 Hz, 1H,

CH(CH₂CH₂)₂CH₂), 7.74 (br s, 2H, NH₂), 7.84−7.89 (m, 2H, 3′-

H_pyridyl, 5′-H_pyridyl), 8.68−8.73 (m, 2H, 2′-H_pyridyl, 6′-H_pyridyl). ¹³C

NMR (151 MHz, DMSO-d₆) δ 24.9 (2C, CH(CH₂CH₂)₂CH₂), 25.4

(1C, CH(CH₂CH₂)₂CH₂), 28.1 (2C, CH(CH₂CH₂)₂CH₂), 42.2 (1C,

CH(CH₂CH₂)₂CH₂), 120.4 (2C, C-3′_pyridyl, C-5′_pyridyl), 137.4 (1C, C-

4′_pyridyl), 150.4 (2C, C-2′_pyridyl, C-6′_pyridyl), 157.5 (1C, C-3_triazole), 158.0

1690, 1535, 1362, 1296, 737. HRMS (m/z): [M + H]⁺calcd for

C₁₅H₁₂N₄O⁺265.1084, found 265.1108. HPLC: t_R= 20.1 min, purity

98.5%.

(5-Amino-3-(pyridin-3-ylamino)-1H-1,2,4-triazol-1-yl)(phenyl)-

methanone (20a). It was synthesized according to general procedure

A from 19a (89 mg, 0.51 mmol) and benzoyl chloride (71 mg, 0.51

mmol). Stirring at room temperature was continued for 30 min. The

crude product was puriﬁed by ﬂash column chromatography (DCM/

MeOH = 1/0 → 88/12) to give 20a as a colorless solid (24.1 mg,

0.09 mmol, 17%); mp > 300 °C. TLC: 0.53 (DCM/MeOH, 92:8).

¹H NMR (400 MHz, DMSO-d₆) δ 7.23 (m, 1H, 5′-H_pyridyl), 7.54−

(1C, C-5_triazole), 176.5 (CO). IR (neat): ν

̃

[cm⁻¹] = 3421, 3105,

2935, 2854, 1717, 1647, 1605, 1524, 1489, 1416, 1362, 1331, 1277,

1234, 1138, 976, 833, 752, 675. HRMS (m/z): [M + H]⁺calcd for

C₁₄H₁₇N₅O⁺272.1506, found 272.1178. HPLC: t_R= 15.86 min,

purity 95.1%.

7.60 (m, 2H, 3′-H_phenyl, 5′-H_phenyl), 7.64−7.69 (m, 1H, 4′-H_phenyl),

7.86 (s, 2H, NH₂), 7.87−7.91 (m, 1H, 4′-H_pyridyl), 8.05 (s, 1H, 6′-

H_pyridyl), 8.10−8.16 (m, 2H, 2′-H_phenyl, 6′-H_phenyl), 8.71 (s, 1H, 2′-

H_pyridyl), 9.53 (s, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆) δ 122.8

(1C, C-4′_pyridyl), 123.4 (1C, C-5′_pyridyl), 127.9 (2C, C-3′_phenyl, C-

5′_phenyl), 130.2 (2C, C-2′_phenyl, C-6′_phenyl), 132.5 (1C, C-4′_phenyl), 132.7

(1C, C-1′_phenyl), 137.6 (1C, C-3′_pyridyl), 138.8 (1C, C-2′_pyridyl), 141.0

(1C, C-6′_pyridyl), 157.5 (1C, C-3′_triazole, C-5′_triazole), 158.0 (1C, C-

Phenyl 5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazole-1-carboxylate

(12a). It was synthesized according to general procedure A from 9a

(100 mg, 0.62 mmol, 1 equiv) and phenyl chloroformate (107 mg,

0.68 mmol, 1.1 equiv). Stirring at room temperature was continued

for 2 h. The crude product was puriﬁed by ﬂash column

chromatography (DCM/ACN = 7/3 → 0/1) to give 12a as a

colorless solid (25.9 mg, 0.092 mmol, 15%); mp = >300 °C. TLC:

0.39 (DCM/MeOH, 9:1). ¹H NMR (600 MHz, DMSO-d₆) δ 7.38 (t,

J = 7.4 Hz, 1H, 4″-H_phenyl), 7.42 (d, J = 7.9 Hz, 2H, 2″-H_phenyl, 6″-

H_phenyl), 7.49 (dd, J = 6.7, 4.9 Hz, 1H, 5′-H_pyridyl), 7.52 (t, J = 7.9 Hz,

2H, 3″-H_phenyl, 5″-H_phenyl), 7.61 (br s, 2H, NH₂), 7.93 (td, J = 7.7, 1.8

Hz, 1H, 4′-H_pyridyl), 8.03 (d, J = 7.8, 1H, 3′-H_pyridyl), 8.68 (d, J = 4.3

Hz, 1H, 6′-H_pyridyl). ¹³C NMR (151 MHz, DMSO-d₆) δ 121.7 (2C, C-

2″_phenyl, C-6″_phenyl), 122.4 (1C, C-3′_pyridyl), 124.8 (1C, C-5′_pyridyl),

126.8 (1C, C-4″_phenyl), 129.8 (2C, C-3″_phenyl, C-5″_phenyl), 137.1 (1C,

C-4′_pyridyl), 148.7 (CO), 148.9 (1C, C-2′_pyridyl), 149.7 (1C, C-

6′_pyridyl), 149.8 (1C, C-1″_phenyl), 158.6 (1C, C-5_triazole), 159.8 (1C, C-

3′_triazole, C-5′_triazole), 166.7 (1C, CO). IR (neat): ν

̃

[cm⁻¹] = 3410,

3121, 2963, 1678, 1612, 1574, 1555, 1431, 1346, 1308, 1177, 1096,

926, 799, 752, 683. HRMS (m/z): [M + H]⁺calcd for C₁₄H₁₃N₆O⁺

281.1145, found 281.1148. HPLC: t_R= 13.3 min, purity 99.2%.

(5-Amino-3-(phenylamino)-1H-1,2,4-triazol-1-yl)(phenyl)-

methanone (20b). It was synthesized according to general procedure

A from 19b (61.5 mg, 0.35 mmol) and benzoyl chloride (49 mg, 0.35

mmol). Stirring at 0 °C was continued for 2 h without further stirring

at room temperature. The crude product was puriﬁed by ﬂash column

chromatography (cyclohexane/EtOAc = 3/1 → 1/1) to give 20b as a

colorless solid (49.6 mg, 0.18 mmol, 51%); mp = 168−169 °C. TLC:

0.37 (DCM/MeOH, 95:5).¹H NMR (400 MHz, DMSO-d₆) δ 6.81−

6.85 (m, 1H, 4′-H_phenyl), 7.17−7.24 (m, 2H, 3′-H_phenyl, 5′-H_phenyl),

7.48−7.53 (m, 2H, 2′-H_phenyl, 6′-H_phenyl), 7.54−7.60 (m, 2H, 3′-

H_benzoyl, 5′-H_benzoyl), 7.62−7.71 (m, 1H, 4′-H_benzoyl), 7.80 (s, 2H,

NH₂), 8.10−8.19 (m, 2H, 2′-H_benzoyl, 6′-H_benzoyl), 9.27 (s, 1H, NH).

¹³C NMR (100 MHz, DMSO-d₆) δ 116.6 (2C, C-2′_phenyl, C-6′_phenyl),

3_triazole). IR (neat): ν

̃

[cm⁻¹] =1325, 1589, 1748, 3310, 3435. HRMS

+

(m/z): [M + H]⁺calcd for C₁₄H₁₂N₅O₂282.0986, found 282.0977.

HPLC: t_R= 14.40 min, purity 99.8%.

3-(Pyridin-4-yl)-1-tosyl-1H-1,2,4-triazol-5-amine (13c). It was

synthesized according to general procedure A from 9c (250 mg,

1.55 mmol, 1 equiv) and 4-toluenesulfonyl chloride (444 mg, 2.33

mmol, 1.5 equiv). Stirring at room temperature was continued for 4.5

h. After the washing steps, pure product 13c was obtained as a

colorless solid (342 mg, 1.08 mmol, 70%); mp = 222 °C (decomp.).

TLC: 0.80 (EtOAc/MeOH, 9:1). ¹H NMR (600 MHz, DMSO-d₆) δ

2.39 (s, 3H, CH₃), 7.48−7.54 (m, 2H, 3″-H_tosyl, 5″-H_tosyl), 7.59 (br s,

2H, NH₂), 7.70−7.79 (m, 2H, 3′-H_pyridyl, 5′-H_pyridyl), 7.92−7.99 (m,

120.0 (1C, C-4′_phenyl), 127.9 (2C, C-3′_benzoyl, C-5′_benzoyl), 128.6 (2C,

C-3′_phenyl, C-5′_phenyl), 130.3 (2C, C-2′_benzoyl, C-6′_benzoyl), 132.5 (1C, C-

4′_benzoyl), 132.8 (1C, C-1′_benzoyl), 141.0 (1C, C-1′_phenyl), 157.3 (C-

3′_triazole, C-5′_triazole), 158.2 (C-3′_triazole, C-5′_triazole), 166.6 (1C, CO).

2H, 2″-H_tosyl, 6″-H_tosyl), 8.62−8.69 (m, 2H, 2′-H_pyridyl, 6′-H_pyridyl). ¹³

C

̃

−1

IR (neat): ν [cm ] = 3379, 1670, 1582, 1555, 1450, 1358, 1200,

1084, 860, 745, 687. HRMS (m/z): [M + H]⁺calcd for C₁₅H₁₄N₅O⁺

280.1193, found 280.1202. HPLC: t_R= 19.9 min, purity 99.3%.

(5-Amino-3-((4-ethoxyphenyl)amino)-1H-1,2,4-triazol-1-yl)-

(phenyl)-methanone (20c). It was synthesized according to general

procedure A from 19c (200 mg, 0.91 mmol) and benzoyl chloride

(141 mg, 1.0 mmol). Stirring at room temperature was continued for

18 h. The crude product was puriﬁed by ﬂash column chromatog-

raphy (DCM/EtOAc = 1/0 → 45/55) to give 20c as a yellowish solid

(157 mg, 0.49 mmol, 53%); mp = 163 °C. TLC: 0.30 (DCM/MeOH,

NMR (151 MHz, DMSO-d₆) δ 21.2 (1C, CH₃), 120.3 (2C, C-3′_pyridyl

and C-5′_pyridyl), 127.6 (2C, C-2″_tosyl, C-6″_tosyl), 130.5 (2C, C-3″_tosyl, C-

5″_tosyl), 133.2 (1C, C-1″_tosyl), 136.7 (1C, C-4′_pyridyl), 146.6 (1C, C-

4″_tosyl), 150.4 (2C, C-2′_pyridyl, C-6′_pyridyl), 157.7 (1C, C-5_triazole), 159.0

(1C, C-3_triazole). IR (neat): ν

̃

[cm⁻¹] = 3429, 3144, 3117, 1717, 1647,

1562, 1520, 1485, 1415, 1377, 1169, 1076, 968, 810, 664, 602. HRMS

(m/z): [M + H]⁺calcd for C₁₄H₁₃N₅O₂S⁺316.0863, found 316.0824.

HPLC: t_R= 15.63 min, purity 99.4%.

(5-Amino-3-phenyl-1H-1,2,4-triazol-1-yl)(phenyl)methanone

(16). It was synthesized according to modiﬁed general procedure A

from 15 (100 mg, 0.62 mmol, 1 equiv) and benzoyl chloride (88 mg,

0.62 mmol, 1 equiv). Benzoyl chloride in 2 mL of dry THF was added

to the reaction mixture with a syringe pump over a period of 2 h at 0

°C, and stirring was continued at room temperature for another 2 h.

Then, H₂O (15 mL) was added and the aqueous layer was extracted

with EtOAc (3×). The combined organic layers were dried (Na₂SO₄)

and concentrated under reduced pressure. The residue was puriﬁed by

ﬂash chromatography (cyclohexane/EtOAc = 1/0 → 0/1), yielding

16 as a colorless solid (152 mg, 0.58 mmol, 92%); mp = 159 °C.

1

95:5). H NMR (400 MHz, DMSO-d₆) δ 1.28 (t, J = 7.0 Hz, 3H,

CH₃), 3.94 (q, J = 7.0 Hz, 2H, CH₂), 6.75−6.83 (m, 2H, 3′-

H_{4‑ethoxyphenyl}, 5′-H_{4‑ethoxyphenyl}), 7.39−7.44 (m, 2H, 2′-H_{4‑ethoxyphenyl}, 6′-

H_{4‑ethoxyphenyl}), 7.53−7.60 (m, 2H, 3′-H_benzoyl, 5′-H_benzoyl), 7.62−7.69

(m, 1H, 4′-H_benzoyl), 7.77 (s, 2H, NH₂), 8.14−8.19 (m, 2H, 2′-H_benzoyl

,

6′-H_benzoyl), 9.04 (s, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆) δ

14.8 (1C, CH₃), 63.1 (1C, CH₂), 114.6 (2C, C-3′_{4‑ethoxyphenyl}, C-

5′_{4‑ethoxyphenyl}), 117.9 (2C, C-2′_{4‑ethoxyphenyl}, C-6′_{4‑ethoxyphenyl}), 127.9 (2C,

C-3′_benzoyl, C-5′_benzoyl), 130.3 (2C, C-2′_benzoyl, C-6′_benzoyl), 132.5 (1C,

C-4′_benzoyl), 132.8 (1C, C-1′_benzoyl), 134.3 (1C, C-1′_{4‑ethoxyphenyl}), 152.4

R

https://dx.doi.org/10.1021/acs.jmedchem.0c01635

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(1C, C-4′_{4‑ethoxyphenyl}), 157.3 (1C, C-3′_triazole, C-5′_triazole), 158.4 (1C, C-

1362, 1323, 1188, 1153, 926, 849, 745, 691. HRMS (m/z): [M + H]⁺

calcd for C₁₆H₁₆N₅O⁺294.1349, found 294.1369. HPLC: t_R= 17.9

min, purity 96.7%.

3′_triazole, C-5′_triazole), 166.4 (1C, CO). IR (neat): ν

̃

[cm⁻¹] = 3483,

3356, 3256, 2970, 1663, 1562, 1508, 1362, 1231, 1053, 922, 833, 748,

+

691. HRMS (m/z): [M + H]⁺calcd for C₁₇H₁₇N₅O₂324.1455,

(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(4-(triﬂuorometh-

yl)-phenyl)-methanone (21d). It was synthesized according to

general procedure A from 9a (100 mg, 620 μmol, 1 equiv) and 4-

triﬂuoromethylbenzoyl chloride (233 mg, 1.12 mmol, 1.2 equiv). The

reaction mixture was gradually heated to room temperature over 18 h.

The crude product was puriﬁed by ﬂash column chromatography

(DCM/MeOH = 1/0 → 88/12) to give 21d as a yellow solid (78.1

mg, 234 μmol, 25%); mp = 272−273 °C. TLC: 0.54 (DCM/MeOH

= 92:8). ¹H NMR (400 MHz, DMSO-d₆) δ 7.47 (ddd, J = 7.5, 4.7, 1.3

Hz, 1H, 5′-H_pyridyl), 7.86−7.95 (m, 3H, NH₂, 4′-H_pyridyl), 7.97 (d, J =

8.2 Hz, 2H, 3′-H_phenyl, 5′-H_phenyl), 7.99−8.02 (m, 1H, 3′-H_pyridyl), 8.27

(d, J = 8.2 Hz, 2H, 2′-H_phenyl, /6′-H_phenyl), 8.65 (ddd, J = 4.8, 1.8, 0.9

Hz, 1H, 6′-H_pyridyl). ¹³C NMR (151 MHz, DMSO-d₆) δ 122.6 (1C, C-

3′_pyridyl), 123.8 (q, J_C,F= 272.7 Hz, 1C, CF₃), 124.8 (1C, C-5′_pyridyl),

found 324.1479. HPLC: t_R= 20.5 min, purity 98.9%.

(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(3,4-dimethyl-

phenyl)-methanone (21a). It was synthesized according to general

procedure A from 9a (100 mg, 620 μmol, 1 equiv) and 3,4-

dimethylbenzoyl chloride, which was obtained from 3,4-dimethylben-

zoic acid (121 mg, 807 μmol, 1.3 equiv) and oxalyl chloride (236 mg,

1.86 mmol, 3 equiv), according to general procedure B. The reaction

mixture was gradually heated to room temperature over 18 h. After

the washing steps, pure product 21a was obtained as a colorless solid

(109 mg, 372 μmol, 60%); mp = 194−195 °C. TLC: 0.54 (DCM/

1

MeOH = 92:8). H NMR (600 MHz, DMSO-d₆) δ 2.32 (s, 3H, 3′-

CH₃), 2.33 (s, 3H, 4′-CH₃), 7.35 (d, J = 8.0 Hz, 1H, 5′-H_phenyl), 7.46

(ddd, J = 7.5, 4.7, 1.3 Hz, 1H, 5′-H_pyridinyl), 7.80 (s, 2H, NH₂), 7.89 (s,

1H, 2′-H_phenyl), 7.90−7.93 (m, 1H, 4′-H_pyridinyl), 7.93−7.95 (m, 1H,

6′-H_benzoyl), 8.00 (d, J = 8.0 Hz, 1H, 3′-H_pyridinyl), 8.66 (d, J = 4.7 Hz,

1H, 6′-H_pyridinyl). ¹³C NMR (151 MHz, DMSO-d₆) δ 19.4 (1C, 3′-

CH₃), 19.6 (1C, 4′-CH₃), 122.5 (1C, C-3′_pyridyl), 124.7 (1C, C-

5′_pyridyl), 128.7 (1C, C-6′_phenyl), 129.1 (1C, C-5′_phenyl), 129.5 (1C, C-

1′_phenyl), 131.5 (1C, C-2′_phenyl), 136.2 (1C, C-3′_phenyl), 137.0 (1C, C-

4′_pyridyl), 142.4 (1C, C-4′_phenyl), 149.0 (1C, C-2′_pyridyl), 149.7 (1C, C-

6′_pyridyl), 159.0 (1C, C-5′_triazole), 159.4 (1C, C-3′_triazole), 167.9 (1C,

125.0 (q, J_C,F= 3.8 Hz 2C, C-3_phenyl′, C-5′_phenyl), 131.3 (2C, C-2′_phenyl

,

C-6′_phenyl), 132.1 (q, J_C,F= 32.0 Hz 1C, C-4_phenyl′), 136.2 (1C, C-

1_phenyl′), 137.0 (1C, C-4′_pyridyl), 148.8 (1C, C-2′_pyridyl), 149.7 (1C, C-

6′_pyridyl), 158.9 (1C, C-5_triazole), 159.9 (1C, C-3_triazole), 167.0 (1C, C

O). IR (neat): ν

̃

[cm⁻¹] = 3468, 3075, 1690, 1636, 1535, 1389, 1354,

1319, 1161, 1115, 1065, 961, 922, 849, 764, 741, 687. HRMS (m/z):

[M + H]⁺calcd for C₁₅H₁₁F₃N₅O⁺334.0941, found 334.0934. HPLC:

t_R= 18.1 min, purity 98.3%.

CO). IR (neat): ν

̃

[cm⁻¹] = 3406, 3275, 3194, 3132, 1674, 1636,

(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(2-nitrophenyl)-

methanone (21e). It was synthesized according to general procedure

A from 9a (100 mg, 620 μmol, 1 equiv) and 2-nitrobenzoyl chloride

(150 mg, 807 μmol, 1.3 equiv). Stirring at room temperature was

continued for 2.5 h. The crude product was puriﬁed by

recrystallization from an EtOAc/DMSO mixture to obtain 21e as a

yellow solid (83.4 mg, 269 μmol, 43%); mp = 229−230 °C. TLC:

0.55 (H₂O/ACN = 4:6, RP). ¹H NMR (600 MHz, DMSO-d₆) δ 7.43

(t, J = 6.1 Hz, 1H, 5′-H_pyridyl), 7.82−7.85 (m, 1H, 3′-H_pyridyl), 7.86−

7.89 (m, 1H, 4′-H_pyridyl), 7.89−7.92 (m, 1H, 4′-H_phenyl), 7.92−7.98

(m, 3H, NH₂, 6′-H_phenyl), 7.99−8.03 (m, 1H, 3′-H_phenyl), 8.32 (d, J =

8.3 Hz, 1H, 5′-H_phenyl), 8.57 (d, J = 4.8 Hz, 1H, 6′-H_pyridyl). ¹³C NMR

(151 MHz, DMSO-d₆) δ 122.5 (1C, C-2′_phenyl), 124.2 (1C, C-

5′_phenyl), 124.9 (1C, C-5′_pyridyl), 129.4 (1C, C-1′_phenyl), 129.8 (1C, C-

6′_phenyl), 132.3 (1C, C-4′_pyridyl), 135.1 (1C, C-3′_phenyl), 137.0 (1C, C-

4′_pyridyl), 146.0 (1C, C-2′_phenyl), 148.4 (1C, C-2′_pyridyl), 149.7 (1C, C-

6′_pyridyl), 157.9 (1C, C-5′_triazole), 160.3 (1C, C-3′_triazole), 166.2 (1C,

1531, 1404, 1362, 1327, 1285, 1173, 1157, 1115, 976, 783, 748, 687.

HRMS (m/z): [M + H]⁺calcd for C₁₆H₁₆N₅O⁺294.1349, found

294.1368. HPLC: t_R= 17.6 min, purity 98.0%.

(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(4-methoxyphen-

yl)-methanone (21b). It was synthesized according to general

procedure A from 9a (100 mg, 620 μmol, 1 equiv) and 4-

methoxylbenzoyl chloride (138 mg, 807 μmol, 1.30 equiv). The

reaction mixture was gradually heated to room temperature over 18 h.

After the washing steps, pure product 21b was obtained as a colorless

solid (131 mg, 445 μmol, 72%); mp = 189−190 °C. TLC: 0.50

1

(DCM/MeOH = 92:8). H NMR (600 MHz, DMSO-d₆) δ 3.89 (s,

3H, CH₃), 7.13 (d, J = 8.7 Hz, 2H, 3′-H_phenyl, 5′-H_phenyl), 7.47 (ddd, J

= 7.6, 4.8, 1.2 Hz, 1H, 5′-H_pyridyl), 7.79 (s, 2H, NH₂), 7.92 (td, J = 7.7,

1.9 Hz, 1H, 4-H_pyridyl), 8.02 (d, J = 7.8 Hz, 1H, 3′-H_pyridyl), 8.26 (d, J =

8.9 Hz, 2H, 2′-H_phenyl, 6′-H_phenyl), 8.68 (d, J = 4.1 Hz, 1H, 6′-H_pyridyl).

¹³C NMR (151 MHz, DMSO-d₆) δ 55.6 (1C, CH₃), 113.6 (2C, C-

3′_phenyl, C-5′_phenyl), 122.5 (1C, C-3′pyridyl), 123.7 (1C, C-1′_phenyl),

124.7 (1C, C-5′pyridyl), 133.7 (2C, C-2′_phenyl, C-6′_phenyl), 137.0 (1C,

C-4′pyridyl), 149.0 (1C, C-2′pyridyl), 149.7 (1C, C-6′pyridyl), 159.1

(1C, C-5′triazole), 159.3 (1C, C-3′triazole), 163.2 (1C, C-4′phenyl),

CO). IR (neat): ν

̃

[cm⁻¹] = 3456, 3001, 1697, 1647, 1524, 1350,

1165, 968, 930, 791, 941, 691, 656. HRMS (m/z): [M + H]⁺calcd for

C₁₄H₁₁N₆O₃⁺311.0877, found 311.0887. HPLC: t_R= 15.0 min, purity

99.2%.

166.8 (1C, CO). IR (neat): ν

̃

[cm⁻¹] = 3433, 3063, 1674, 1636,

(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(4-nitrophenyl)-

methanone (21f). It was synthesized according to general procedure

A from 9a (100 mg, 620 μmol, 1 equiv) and 4-nitrobenzoyl chloride

(230 mg, 1.24 mmol, 2 equiv). The reaction mixture was gradually

heated to room temperature over 18 h. The crude product was

puriﬁed by recrystallization from an EtOAc/DMSO mixture to obtain

21f as a yellow solid (87.8 mg, 283 μmol, 46%); mp = 299−300 °C.

1601, 1508, 1358, 1312, 1254, 1177, 1150, 1022, 968, 922, 845, 802,

+

756, 687, 648. HRMS (m/z): [M + H]⁺calcd for C₁₅H₁₄N₅O₂

296.1142, found 296.1164. HPLC: t_R= 15.7 min, purity 97.9%.

(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(4-ethylphenyl)-

methanone (21c). It was synthesized according to general procedure

A from 9a (100 mg, 620 μmol, 1 equiv) and 4-ethylbenzoyl chloride,

which was obtained from 4-ethylbenzoic acid (121 mg, 807 μmol, 1.3

equiv) and oxalyl chloride (236 mg, 1.86 mmol, 3 equiv), according to

general procedure B. Stirring at room temperature was continued for

1 h. After the washing steps, pure product 21c was obtained as a

colorless solid (135 mg, 459 μmol, 74%); mp = 173−174 °C. TLC:

1

TLC: 0.43 (H₂O/ACN = 4:6). H NMR (600 MHz, DMSO-d₆) δ

7.47 (ddd, J = 7.6, 4.7, 1.3 Hz, 1H, 5′-H_pyridyl), 7.89−7.97 (m, 3H,

NH₂, 4′-H_pyridyl), 8.00 (d, J = 7.8 Hz, 1H, 3′-H_pyridyl), 8.29 (d, J = 8.9

Hz, 2H, 2′-H_phenyl, 6′-H_phenyl), 8.40 (d, J = 8.9 Hz, 2H, 3′-H_phenyl, 5′-

H_phenyl), 8.65 (d, J = 4.3 Hz, 1H, 6′-H_pyridyl). ¹³C NMR (151 MHz,

DMSO-d₆) δ 122.6 (1C, C-3′_pyridyl), 123.1 (2C, C-3′_phenyl, C-5′_phenyl),

124.9 (1C, C-5′_pyridyl), 131.8 (2C, C-2′_phenyl, C-6′_phenyl), 137.0 (1C, C-

4′_pyridyl), 138.0 (1C, C-1′_phenyl), 148.7 (1C, C-2′_pyridyl), 149.5 (1C, C-

4′_phenyl), 149.7 (1C, C-6′_pyridyl), 158.8 (1C, C-5′_triazole), 160.0 (1C, C-

1

0.35 (DCM/MeOH = 9:1). H NMR (600 MHz, DMSO-d₆) δ 1.24

(t, J = 7.6 Hz, 3H, CH₃), 2.72 (q, J = 7.6 Hz, 2H, CH₂), 7.43 (d, J =

8.1 Hz, 2H, 3′-H_phenyl, 5′-H_phenyl), 7.47 (ddd, J = 7.6, 4.7, 1.3 Hz, 1H,

5′-H_pyridyl), 7.82 (s, 2H, NH₂), 7.92 (td, J = 7.7, 1.8 Hz, 1H, 4′-

H_pyridyl), 8.01 (d, J = 7.8 Hz, 1H, 3′-H_pyridyl), 8.09 (d, J = 8.2 Hz, 2H,

2′-H_phenyl, 6′-H_phenyl), 8.66 (d, J = 4.3 Hz, 1H, 6′-H_pyridyl). ¹³C NMR

(151 MHz, DMSO-d₆) δ 15.2 (1C, CH₃), 28.3 (1C, CH₂), 122.5 (1C,

C-3′_pyridyl), 124.7 (1C, C-5′_pyridyl), 127.6 (2C, C-3′_phenyl, C-5′_phenyl),

129.4 (1C, C-1′_phenyl), 131.1 (2C, C-2′_phenyl, C-6′_phenyl), 137.0 (1C, C-

4′_pyridyl), 149.0 (1C, C-2′_pyridyl), 149.6 (1C, C-4′_phenyl), 149.7 (1C, C-

6′_pyridyl), 159.0 (1C, C-5′_triazole), 159.5 (1C, C-3′_triazole), 167.7 (1C,

3′_triazole), 166.6 (1C, CO). IR (neat): ν

̃

[cm⁻¹] = 3441, 3055, 1694,

1639, 1520, 1400, 1373, 1339, 1288, 1246, 1157, 968, 853, 741, 710,

+

691. HRMS (m/z): [M + H]⁺calcd for C₁₄H₁₁N₆O₃311.0877,

found 311.0891. HPLC: t_R= 15.7 min, purity 96.3%.

(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(4-ﬂuorophenyl)-

methanone (21g). It was synthesized according to general procedure

A from 9a (300 mg, 1.86 mmol, 1 equiv) and 4-ﬂuorobenzoyl chloride

(384 mg, 2.42 mmol, 1.3 equiv). Stirring at room temperature was

CO). IR (neat): ν

̃

[cm⁻¹] = 3449, 3075, 2970, 1678, 1632, 1535,

S

https://dx.doi.org/10.1021/acs.jmedchem.0c01635

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Article

continued for 3 h. After the washing steps, pure product 21g was

obtained as a colorless solid (366 mg, 1.29 mmol, 69%); mp = 202−

CH(CH₂CH₂)₂CH₂), 1.31−1.50 (m, 4H, CH(CH₂CH₂)₂CH₂),

1.69 (dtd, J = 12.7, 3.4, 1.6 Hz, 1H, CH(CH₂CH₂)₂CH₂), 1.78 (dt,

J = 13.1, 3.4 Hz, 2H, CH(CH₂CH₂)₂CH₂), 1.94−2.02 (m, 2H,

1

204 °C. TLC: 0.78 (EtOAc/MeOH = 9:1). H NMR (600 MHz,

DMSO-d₆) δ 7.41−7.46 (m, 2H, 3′-H_phenyl, 5′-H_phenyl), 7.47 (ddd, J =

7.6, 4.8, 1.2 Hz, 1H, 5′-H_pyridyl), 7.86 (s, 2H, NH₂), 7.92 (td, J = 7.7,

1.8 Hz, 1H, 4-H_pyridyl), 8.02 (dt, J = 7.9, 1.1 Hz, 1H, 3′-H_pyridyl), 8.24−

8.30 (m, 2H, 2′-H_phenyl, 6′-H_phenyl), 8.67 (ddd, J = 4.6, 1.9, 0.9 Hz, 1H,

6′-H_pyridyl). ¹³C NMR (151 MHz, DMSO-d₆) δ 115.3 (d, J_C,F= 22.0

Hz, 2C, C-3′_phenyl, C-5′_phenyl), 122.5 (1C, C-3′_pyridyl), 124.8 (1C, C-

5′_pyridyl), 128.5 (d, J_C,F= 2.9 Hz, 1C, C-1′_phenyl), 134.0 (d, J_C,F= 9.3

Hz, 2C, C-2′_phenyl, C-6′_phenyl), 137.0 (1C, C-4′_pyridyl), 148.9 (1C, C-

2′_pyridyl), 149.7 (1C, C-6′_pyridyl), 159.0 (1C, C-5′_triazole), 159.6 (1C, C-

3′_triazole), 164.8 (d, J_C,F= 252.4 Hz, 1C, C-4′_phenyl), 166.6 (1C, CO).

CH(CH₂CH₂)₂CH₂), 3.42 (tt, J = 11.3, 3.4 Hz, 1H,

CH(CH₂CH₂)₂CH₂), 7.48 (ddd, J = 7.5, 4.7, 1.2 Hz, 1H, 5′-H_pyridyl),

7.66 (br s, 2H, NH₂), 7.85 (td, J = 7.7, 1.8 Hz, 1H, 4′-H_pyridyl), 8.00

(dt, J = 7.9, 1.1 Hz, 1H, 3′-H_pyridyl), 8.68 (ddd, J = 4.7, 1.8, 0.9 Hz,

1H, 6′-H_pyridyl). ¹³C NMR (151 MHz, DMSO-d₆) δ 24.9 (2C,

CH(CH₂CH₂)₂CH₂), 25.4 (1C, CH(CH₂CH₂)₂CH₂), 28.2 (2C,

CH(CH₂CH₂)₂CH₂), 42.0 (1C, CH(CH₂CH₂)₂CH₂), 122.5 (1C, C-

3′_pyridyl), 124.7 (1C, C-5′_pyridyl), 137.0 (1C, C-4′_pyridyl), 149.0 (1C, C-

2′_pyridyl), 149.7 (1C, C-6′_pyridyl), 157.7 (1C, C-5_triazole), 159.2 (1C, C-

−1

3_triazole), 176.7 (CO). IR (neat): ν [cm ] = 3441, 3256, 3198,

3125, 2936, 2855, 1717, 1620, 1528, 1485, 1404, 1381, 1358, 1273,

1173, 1150, 1080, 976, 806, 745, 683. HRMS (m/z): [M + H]⁺calcd

for C₁₄H₁₇N₅O⁺272.1506, found 272.1467. HPLC: t_R= 15.80 min,

purity 99.5%.

̃

IR (neat): ν

̃

[cm⁻¹] = 3460, 3075, 2978, 1690, 1624, 1597, 1535,

1501, 1358, 1335, 1231, 1196, 1158, 1088, 972, 930, 845, 787, 741,

679, 610. HRMS (m/z): [M + H]⁺calcd for C₁₄H₁₀FN₅O⁺284.0942,

found 284.0919. HPLC: t_R= 19.3 min, purity 99.0%.

(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(4-bromophenyl)-

methanone (21h). It was synthesized according to general procedure

A from 9a (100 mg, 620 μmol, 1 equiv) and 4-bromobenzoyl chloride

(136 mg, 620 μmol, 1.00 equiv). Stirring at room temperature was

continued for 1 h. After the washing steps, pure product 21h was

obtained as a colorless solid (138 mg, 400 μmol, 64%); mp = 205 °C.

TLC: 0.52 (DCM/MeOH = 92:8). ¹H NMR (600 MHz, DMSO-d₆)

δ 7.47 (ddd, J = 7.6, 4.7, 1.3 Hz, 1H, 5′-H_pyridyl), 7.82 (d, J = 8.5 Hz,

2H, 3′-H_phenyl, 5′-H_phenyl), 7.87 (s, 2H, NH₂), 7.92 (td, J = 7.7, 1.8 Hz,

1H, 4′-H_pyridyl), 8.00 (d, J = 7.8 Hz, 1H, 3′-H_pyridyl), 8.07 (d, J = 8.5

(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(m-tolyl)-

methanone (21k). It was synthesized according to general procedure

C from 9a (100 mg, 621 μmol, 1 equiv) and 3-methylbenzoic acid

(84.5 mg, 621 μmol, 1 equiv), with EDCI (238 mg, 1.24 mmol, 2

equiv) and DMAP (152 mg, 1.24 mmol, 2 equiv) in DMF (4 mL).

Stirring at 0 °C was continued for 2 h, followed by 2 h at room

temperature. After the washing steps, pure product 21k was obtained

as a colorless solid (112 mg, 402 μmol, 65%); mp = 172 °C. TLC:

1

0.43 (DCM/MeOH = 9:1). H NMR (600 MHz, DMSO-d₆) δ 2.41

(s, 3H, CH₃), 7.45−7.49 (m, 2H, 5′-H_phenyl, 5′-H_pyridyl), 7.51 (d, J =

7.6 Hz, 1H, 4′-H_phenyl), 7.83 (s, 2H, NH₂), 7.87 (s, 1H, 2′-H_phenyl),

7.91 (td, J = 7.7, 1.8 Hz, 1H, 4′-H_pyridyl), 7.94 (d, J = 7.5 Hz, 1H, 6′-

H_phenyl), 7.99 (d, J = 7.7 Hz, 1H, 3′-H_pyridyl), 8.66 (d, J = 4.5 Hz, 1H,

6′-H_pyridyl). ¹³C NMR (151 MHz, DMSO-d₆) δ 20.9 (1C, CH₃), 122.5

(1C, C-3′_pyridyl), 124.7 (1C, C-5′_pyridyl), 128.0 (2C, C-5′_phenyl, C-

6′_phenyl), 130.8 (1C, C-2′_phenyl), 132.1 (1C, C-1′_phenyl), 133.6 (1C, C-

4′_phenyl), 137.0 (1C, C-4′_pyridyl), 137.5 (1C, C-3′_phenyl), 149.0 (1C, C-

2′_pyridyl), 149.7 (1C, C-6′_pyridyl), 158.9 (1C, C-5′_triazole), 159.5 (1C, C-

Hz, 2H, 2′-H_phenyl, 6′-H_phenyl), 8.66 (d, J = 4.3 Hz, 1H, 6′-H_pyridyl). ¹³

C

NMR (151 MHz, DMSO-d₆) δ 122.6 (1C, C-3′_pyridyl), 124.8 (1C, C-

5′_pyridyl), 127.0 (1C, C-4′_phenyl), 131.2 (2C, C-3′_phenyl, C-5′_phenyl), 131.2

(1C, C-1′_phenyl), 132.8 (2C, C-2′_phenyl, C-6′_phenyl), 137.0 (1C, C-

4′_pyridyl), 148.8 (1C, C-2′_pyridyl), 149.7 (1C, C-6′_pyridyl), 158.9 (1C, C-

5′_triazole), 159.7 (1C, C-3′_triazole), 167.0 (1C, CO). IR (neat): ν

[cm⁻¹] = 3453, 3098, 1682, 1636, 1582, 1535, 1396, 1366, 1339,

1288, 1157, 1072, 976, 918, 841, 791, 745, 691, 671. HRMS (m/z):

[M + H]⁺calcd for C₁₄H₁₁Br⁷⁹N₅O⁺344.0141, found 344.0165, calcd

for C₁₄H₁₁Br⁸¹N₅O⁺346.0121, found 346.0149. HPLC: t_R= 17.3 min,

purity 95.5%.

̃

3′_triazole), 168.1 (1C, CO). IR (neat): ν

̃

[cm⁻¹] = 3460, 3063, 1856,

1636, 1537, 1464, 1389, 1362, 1325, 1288, 1179, 972. HRMS (m/z):

[M + H]⁺calcd for C₁₅H₁₄N₅O⁺280.1193, found 280.1206. HPLC:

t_R= 16.3 min, purity 98.9%.

(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(naphthalen-1-

yl)-methanone (21i). It was synthesized according to general

procedure A from 9a (100 mg, 620 μmol, 1 equiv) and 1-napthoyl

chloride (244 mg, 1.24 mmol, 2 equiv). The reaction mixture was

gradually heated to room temperature over 18 h. After the washing

steps, pure product 21i was obtained as a colorless solid (151 mg, 479

μmol, 77%); mp = 227−228 °C. TLC: 0.59 (DCM/MeOH = 92:8).

¹H NMR (600 MHz, DMSO-d₆) δ 7.40 (ddd, J = 7.3, 4.7, 1.4 Hz, 1H,

5′-H_pyridyl), 7.58−7.64 (m, 2H, 7′-H_naphthoyl, 8′-H_naphthoyl), 7.67 (dd, J =

8.3, 7.1 Hz, 1H, 3′-H_naphthoyl), 7.85 (td, J = 7.6, 1.8 Hz, 1H, 4′-H_pyridyl),

7.87−7.89 (m, 1H, 3′-H_pyridyl), 7.89−7.90 (m, 1H, 9′-H_naphthoyl),

(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(pyridin-3-yl)-

methanone (21l). It was synthesized according to general procedure

C from 9a (100 mg, 621 μmol, 1 equiv) and nicotinic acid (76.4 mg,

621 μmol, 1 equiv), with EDCI (238 mg, 1.24 mmol, 2 equiv) and

DMAP (152 mg, 1.24 mmol, 2 equiv) in DMF (4 mL). Stirring at 0

°C was continued for 2 h, followed by 4 h at room temperature. After

the washing steps, pure product 21l was obtained as a colorless solid

(42.3 mg, 159 μmol, 26%); mp = 193 °C. TLC: 0.64 (DCM/MeOH

= 85:15). ¹H NMR (600 MHz, DMSO-d₆) δ 7.48 (dd, J = 7.4, 4.7 Hz,

1H, 5′-H_pyridyl), 7.64 (dd, J = 7.9, 4.8 Hz, 1H, 5′-H_nicotinoyl), 7.87−7.96

(m, 3H, NH₂, 4′-H_pyridyl), 8.01 (d, J = 7.8 Hz, 1H, 3′-H_pyridyl), 8.48

(dt, J = 8.1, 2.0 Hz, 1H, 4′-H_nicotinoyl), 8.67 (d, J = 4.0 Hz, 1H, 6′-

H_pyridyl), 8.83 (dd, J = 4.9, 1.7 Hz, 1H, 6′-H_nicotinoyl), 9.25 (d, J = 2.2

Hz, 1H, 2′-H_nicotinoyl). ¹³C NMR (151 MHz, DMSO-d₆) δ 122.6 (1C,

C-3′_pyridyl), 123.2 (1C, C-5′_nicotinoyl), 124.8 (1C, C-5′_pyridyl), 128.5 (1C,

C-3′_nicotinoyl), 137.1 (1C, C-4′_pyridyl), 138.4 (1C, C-4′_nicotinoyl), 148.8

(1C, C-2′_pyridyl), 149.8 (1C, C-6′_pyridyl), 151.0 (1C, C-2′_nicotinoyl), 152.9

(1C, C-6′_nicotinoyl), 158.8 (1C, C-5′_triazole), 159.9 (1C, C-3′_triazole),

7.90−7.92 (m, 1H, 2′-H_naphthoyl), 7.98 (s, 2H, NH₂), 8.06−8.10 (m,

1H, 6′-H_naphthoyl), 8.19 (dt, J = 8.2, 1.1 Hz, 1H, 4′-H_naphthoyl), 8.52

(ddd, J = 4.7, 1.7, 1.0 Hz, 1H, 6′-H_pyridyl). ¹³C NMR (151 MHz,

DMSO-d₆) δ 122.5 (1C, C-3′_pyridyl), 124.6 (1C, C-9′_naphthoyl), 124.7

(1C, C-5′_pyridyl), 124.8 (1C, C-3′_naphthoyl), 126.6 (1C, C-7′_naphthoyl),

127.5 (1C, C-8′_naphthoyl), 127.6 (1C, C-2′_naphthoyl), 128.5 (1C, C-

6′_naphthoyl), 129.4 (1C, C-10′_naphthoyl), 130.8 (1C, C-1′_naphthoyl), 131.3

(1C, C-4′_naphthoyl), 132.9 (1C, C-5′_naphthoyl), 137.0 (1C, C-4′_pyridyl),

148.8 (1C, H-2′_pyridyl), 149.6 (1C, C-6′_pyridyl), 158.6 (1C, C-5′_triazole),

166.5 (1C, CO). IR (neat): ν

̃

[cm⁻¹] = 3443, 3107, 1688, 1632,

̃

[cm⁻¹] =

1589, 1530, 1464, 1366, 1329, 1290, 1248, 1155, 1094, 970, 942, 912.

HRMS (m/z): [M + H]⁺calcd for C₁₃H₁₁N₆O⁺267.0989, found

267.0994. Unstable under HPLC conditions, qNMR: purity 94%.

(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(naphthalen-2-

yl)-methanone (21m). It was synthesized according to general

procedure C from 9a (80 mg, 496 μmol, 1 equiv) and 2-naphthoic

acid (85.5 mg, 496 μmol, 1 equiv), with EDCI (190 mg, 993 μmol, 2

equiv) and DMAP (121 mg, 993 μmol, 2 equiv) in DMF (3 mL).

Stirring at 0 °C was continued for 1 h, followed by 2 h at room

temperature. The crude product was puriﬁed by ﬂash column

chromatography (DCM/ACN = 1/0 → 0/1) to give 21m as a

colorless solid (58.3 mg, 271 μmol, 55%); mp = 200−201 °C. TLC:

159.7 (1C, C-3′_triazole), 169.2 (1C, CO). IR (neat): ν

3460, 2978, 1694, 1632, 1354, 1308, 1153, 1053, 910, 779, 748, 694.

HRMS (m/z): [M + H]⁺calcd for C₁₈H₁₄N₅O⁺316.1193, found

316.1187. HPLC: t_R= 16.9 min, purity 98.4%.

(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(cyclohexyl)-

methanone (21j). It was synthesized according to general procedure

A from 9c (301 mg, 1.87 mmol, 1 equiv) and cyclohexanecarbonyl

chloride (356 mg, 2.43 mmol, 1.3 equiv). Stirring at room

temperature was continued for 4.5 h. After the washing steps, pure

product 11c was obtained as a colorless solid (418 mg, 1.54 mmol,

83%); mp = 204−206 °C. TLC: 0.76 (EtOAc/MeOH, 9:1).¹H NMR

(600 MHz, DMSO-d₆) δ 1.23 (qt, J = 12.4, 3.6 Hz, 1H,

T

https://dx.doi.org/10.1021/acs.jmedchem.0c01635

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1

0.41 (DCM/MeOH = 9:1). H NMR (600 MHz, DMSO-d₆) δ 7.47

(2,2,2-triﬂuoroacetamido)benzoic acid (145 mg, 621 μmol, 1 equiv),

with EDCI (238 mg, 1.24 mmol, 2 equiv) and DMAP (152 mg, 1.24

mmol, 2 equiv) in DMF (4 mL). Stirring at 0 °C was continued for 2

h, followed by 3 h at room temperature. After the washing steps, pure

product 21p was obtained as a yellow solid (164 mg, 436 μmol, 70%);

(ddd, J = 7.5, 4.7, 1.2 Hz, 1H, 5′-H_pyridyl), 7.65 (ddd, J = 8.1, 6.8, 1.3

Hz, 1H, 7′-H_naphthoyl), 7.72 (ddd, J = 8.2, 6.8, 1.3 Hz, 1H, 6′-

H_naphthoyl), 7.89 (br s, 2H, NH₂), 7.92 (td, J = 7.7, 1.8 Hz, 1H, 4′-

H_pyridyl), 8.02 (d, J = 7.9 Hz, 1H, 3′-H_pyridyl), 8.05 (d, J = 8.3 Hz, 1H,

5′-H_naphthoyl), 8.09 (d, J = 8.7 Hz, 1H, 4′-H_naphthoyl), 8.12−8.15 (m,

2H, 3′-H_naphthoyl, 8′-H_naphthoyl), 8.64−8.66 (m, 1H, 6′-H_pyridyl), 8.79 (s,

1H, 1′-H_naphthoyl). ¹³C NMR (151 MHz, DMSO-d₆) δ 122.5 (1C, C-

3′_pyridyl), 124.7 (1C, C-5′_pyridyl), 126.3 (1C, C-3′_naphthoyl), 127.0 (1C,

C-7′_naphthoyl), 127.5 (1C, C-4′_naphthoyl), 127.7 (1C, C-5′_naphthoyl), 128.8

(1C, C-6′_naphthoyl), 129.5 (2C, C-2′_naphthoyl, C-8′_naphthoyl), 131.6 (1C, C-

8a′_naphthoyl), 132.2 (1C, C-1′_naphthoyl), 134.7 (1C, C-4a′_naphthoyl), 137.0

(1C, C-4′_pyridyl), 149.0 (1C, C-2′_pyridyl), 149.7 (1C, C-6′_pyridyl), 159.0

(1C, C-5′_triazole), 159.7 (1C, C-3′_triazole), 168.0 (1C, CO). IR (neat):

1

mp = 230 °C. TLC: 0.65 (DCM/MeOH = 85:15). H NMR (600

MHz, DMSO-d₆) δ 7.48 (ddd, J = 7.5, 4.8, 1.3 Hz, 1H, 5′-H_pyridyl),

7.84 (s, 2H, NH₂), 7.89 (dt, J = 8.8, 1.9 Hz, 2H, 3′-H_phenyl, 5′-H_phenyl),

7.92 (td, J = 7.7, 1.8 Hz, 1H, 4′-H_pyridyl), 8.02 (dt, J = 7.9, 1.2 Hz, 1H,

3′-H_pyridyl), 8.24 (dt, J = 8.9; 1.8 Hz, 2H, 2′-H_phenyl, 6′-H_phenyl), 8.67

(ddd, J = 4.7, 1.7, 0.9 Hz, 1H, 6′-H_pyridyl), 11.62 (s, 1H, NH). ¹³C

NMR (151 MHz, DMSO-d₆) δ 115.6 (q, J = 289 Hz, 1C, CF₃), 120.0

(2C, C-3′_phenyl, C-5′_phenyl), 122.6 (1C, C-3′_pyridyl), 124.8 (1C, C-

5′_pyridyl), 128.5 (1C, C-1′_phenyl), 132.2 (2C, C-2′_phenyl, C-6′_phenyl), 137.0

(1C, C-4′_pyridyl), 140.6 (1C, C-4′_phenyl), 148.9 (1C, C-2′_pyridyl), 149.7

(1C, C-6′_pyridyl), 154.9 (q, J = 37.2 Hz, 1C, CF₃CO), 159.0 (1C, C-

ν

̃

[cm⁻¹] = 3451, 3051, 1678, 1634, 1531, 1462, 1387, 1356, 1317,

1271, 1248, 1209, 1179, 1150, 1115, 1092, 1082, 1047, 986, 932, 899,

864, 818. HRMS (m/z): [M + H]⁺calcd for C₁₈H₁₄N₅O⁺316.1193,

found 316.1188. HPLC: t_R= 18.1 min, purity 99.5%.

5′_triazole); 159.6 (1C, C-3′_triazole), 166.9 (1C, PhCO). IR (neat): ν

[cm⁻¹] = 3464, 3308, 3098, 1711, 1686, 1605, 1539, 1452, 1416,

1395, 1360, 1337, 1287, 1250, 1204, 1186, 1153, 1123, 995, 972, 922,

̃

(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(furan-2-yl)-

methanone (21n). It was synthesized according to general procedure

C from 9a (80 mg, 496 μmol, 1 equiv) and 2-furoic acid (55.0 mg,

496 μmol, 1 equiv), with EDCI (190 mg, 993 μmol, 2 equiv) and

DMAP (121 mg, 993 μmol, 2 equiv) in DMF (3 mL). Stirring at 0 °C

was continued for 1 h, followed by 2 h at room temperature. After the

washing steps, pure product 21n was obtained as a colorless solid

(98.8 mg, 387 μmol, 78%); mp = 213 °C. TLC: 0.44 (DCM/MeOH

= 92:8). ¹H NMR (600 MHz, DMSO-d₆) δ 6.88 (dd, J = 3.6, 1.7 Hz,

1H, 4′-H_furanoyl), 7.50 (ddd, J = 7.6, 4.7, 1.3 Hz, 1H, 5′-H_pyridyl), 7.84

(br s, 2H, NH₂), 7.95 (td, J = 7.7, 1.8 Hz, 1H, 4′-H_pyridyl), 8.10 (dt, J =

7.9, 1.1 Hz, 1H, 3′-H_pyridyl), 8.21−8.22 (m, 2H, 3′-H_furanoyl, 5′-

H_furanoyl), 8.72 (ddd, J = 4.6, 1.7, 0.9 Hz, 1H, 6′-H_pyridyl). ¹³C NMR

(151 MHz, DMSO-d₆) δ 113.1 (1C, C-4′_furanoyl), 122.6 (1C, C-

3′_pyridyl), 124.9 (1C, C-5′_pyridyl), 125.0 (1C, C-5′_furanoyl), 137.1 (1C, C-

4′_pyridyl), 143.9 (1C, C-2′_furanoyl), 148.8 (1C, C-2′_pyridyl), 149.7 (1C, C-

3′_furanoyl), 149.8 (1C, C-6′_pyridyl), 155.9 (1C, CO), 158.9 (1C, C-

+

901, 851. HRMS (m/z): [M + H]⁺calcd for C₁₆H₁₂F₃N₆O₂

377.0968, found 377.0965. HPLC: t_R= 16.7 min, purity 99.4%.

N-(4-(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazole-1-carbonyl)-

phenyl)benz-amide (21q). It was synthesized according to general

procedure C from 9a (80 mg, 496 μmol, 1 equiv) and 4-

benzamidobenzoic acid (112 mg, 496 μmol, 1 equiv), with EDCI

(190 mg, 993 μmol, 2 equiv) and DMAP (121 mg, 993 μmol, 2

equiv) in DMF (3 mL). Stirring at 0 °C was continued for 1 h,

followed by 2 h at room temperature. After the washing steps, pure

product 21q was obtained as a yellow solid (169 mg, 440 μmol, 89%);

1

mp = 214 °C. TLC: 0.64 (DCM/MeOH = 85:15). H NMR (600

MHz, DMSO-d₆) δ 7.48 (dd, J = 7.0, 5.2 Hz, 1H, 5′-H_pyridyl), 7.57 (t, J

= 7.6 Hz, 2H, 3′-H_benzamidyl, 5′-H_benzamidyl), 7.63 (t, J = 7.3 Hz, 1H, 4′-

H_benzamidyl), 7.82 (br s, 2H, NH₂), 7.93 (td, J = 7.7, 1.8 Hz, 1H, 4′-

H_pyridyl), 7.98−8.02 (m, 4H, 2′-H_benzamidyl, 6′-H_benzamidyl, 3′-

H_{4‑amidobenzoyl}, 5′-H_{4‑amidobenzoyl}), 8.04 (d, J = 7.9 Hz, 1H, 3′-H_pyridyl),

8.25 (0, J = 8.7 Hz, 2H, 2′-H_{4‑amidobenzoyl}, 6′-H_{4‑amidobenzoyl}), 8.68 (dd, J

= 4.8, 1.7 Hz, 1H, 6′-H_pyridyl), 10.65 (s, 1H, NH). ¹³C NMR (151

MHz, DMSO-d₆) δ 119.0 (2C, C-3′_{4‑amidobenzoyl}, C-5′_{4‑amidobenzoyl}),

122.5 (1C, C-3′_pyridyl), 124.7 (1C, C-5′_pyridyl), 126.3 (1C, C-

1′_{4‑amidobenzoyl}), 127.9 (2C, C-2′_benzamidyl, C-6′_benzamidyl), 128.5 (2C, C-

3′_benzamidyl, C-5′_benzamidyl), 132.0 (1C, C-4′_benzamidyl), 132.3 (2C, C-

5′_triazole), 160.1 (1C, C-3′_triazole). IR (neat): ν

̃

[cm⁻¹] = 3441, 3279,

3186, 3117, 3088, 1670, 1638, 1562, 1533, 1466, 1404, 1391, 1373,

1339, 1288, 1175, 1153, 1078, 1026, 970, 959, 878. HRMS (m/z):

[M + H]⁺calcd for C₁₂H₁₀N₅O₂⁺256.0829, found 256.0840. HPLC:

t_R= 13.0 min, purity 97.9%.

(5-Amino-3-(pyridin-2-yl)-1H-1, 2, 4-triazol-1-yl)-

(tetrahydropyran-4-yl)methanone (21o). It was synthesized accord-

ing to general procedure C from 9a (100 mg, 621 μmol, 1 equiv) and

tetrahydro-2H-pyran-4-carboxylic acid (80.8 mg, 621 μmol, 1 equiv),

with EDCI (238 mg, 1.24 mmol, 2 equiv) and DMAP (152 mg, 1.24

mmol, 2 equiv) in DMF (4 mL). The reaction mixture was gradually

heated from 0 °C to room temperature over 18 h. After the washing

steps, pure product 21o was obtained as a colorless solid (131 mg,

480 μmol, 77%); mp = 215 °C. TLC: 0.53 (DCM/MeOH = 92:8).

¹H NMR (600 MHz, DMSO-d₆) δ 1.70 (dtd, J = 13.3, 11.7, 4.4 Hz,

2′_{4‑amidobenzoyl}, 6′-C_{4‑amidobenzoyl}), 134.6 (1C, C-4′_benzamidyl), 137.0 (1C,

C-4′_pyridyl), 143.7 (1C, C-4′_{4‑amidobenzoyl}), 149.0 (1C, C-2′_pyridyl), 149.7

(1C, C-6′_pyridyl), 159.05 (1C, C-5′_triazole), 159.45 (1C, C-3′_triazole),

166.15 (1C, CONH), 166.93 (1C, C=ONR₂). IR (neat): ν

̃

[cm⁻¹] =

3462, 3399, 3345, 1686, 1655, 1634, 1605, 1589, 1518, 1506, 1485,

1404, 1354, 1308, 1256, 1188, 1150, 1126, 1101, 1072, 970, 928, 847.

HRMS (m/z): [M + H]⁺calcd for C₂₁H₁₇N₆O⁺385.1408, found

385.1387. HPLC: t_R= 17.0 min, purity 95.4%.

1-(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-2-phenyl-

ethan-1-one (21r). It was synthesized according to general procedure

C from 9a (80 mg, 496 μmol, 1 equiv) and phenylacetic acid (67.6

mg, 496 μmol, 1 equiv), with EDCI (190 mg, 993 μmol, 2 equiv) and

DMAP (121 mg, 993 μmol, 2 equiv) in DMF (3 mL). Stirring at 0 °C

was continued for 1 h, followed by 2 h at room temperature. After the

washing steps, pure product 21r was obtained as a colorless solid (7.0

mg, 25 μmol, 5%). TLC: 0.50 (DCM/MeOH = 9:1). ¹H NMR (600

MHz, DMSO-d₆) δ 4.43 (s, 2H, CH₂), 7.26−7.31 (m, 1H, 4′-H_phenyl),

7.32−7.40 (m, 4H, 2′-H_phenyl, 3′-H_phenyl, 5′-H_phenyl, 6′-H_phenyl), 7.49

(ddd, J = 7.6, 4.7, 1.3 Hz, 1H, 5′-H_pyridyl), 7.66 (br s, 2H, NH₂), 7.94

(td, J = 7.7, 1.8 Hz, 1H, 4′-H_pyridyl), 8.04 (d, J = 7.8 Hz, 1H, 3′-

H_pyridyl), 8.70 (d, J = 4.4 Hz, 1H, 6′-H_pyridyl). ¹³C NMR (151 MHz,

DMSO-d₆) δ 41.1 (1C, CH₂), 122.4 (1C, C-3′_pyridyl), 124.7 (1C, C-

5′_pyridyl), 127.0 (1C, C-4′_phenyl), 128.3 (2C, C-3′_phenyl, C-5′_phenyl), 130.0

(2C, C-2′_phenyl, C-6′_phenyl), 133.5 (1C, C-1′_phenyl), 137.1 (1C, C-

4′_pyridyl), 148.9 (1C, C-2′_pyridyl), 149.7 (1C, C-6′_pyridyl), 157.7 (1C, C-

5′_triazole), 159.2 (1C, C-3′_triazole), 172.1 (1C, CO). HRMS (m/z):

[M + H]⁺calcd for C₁₅H₁₄N₅O⁺280.1193, found 280.1196. HPLC:

t_R= 15.6 min, purity 98.8%.

2H, 3′-H_{tetrahydropyranyl}, 5′-H_{tetrahydropyranyl}), 1.90 (ddd, J = 12.8, 4.1, 2.0

Hz, 2H, 3′-H_{tetrahydropyranyl}, 5′-H_{tetrahydropyranyl}), 3.47 (td, J = 11.7, 2.2

Hz, 2H, 2′-H_{tetrahydropyranyl}, 6′-H_{tetrahydropyranyl}), 3.69 (tt, J = 11.5, 3.9 Hz,

1H, 4′-H_{tetrahydropyranyl}), 3.92 (ddd, J = 11.5, 4.4, 2.1 Hz, 2H, 2′-

H_{tetrahydropyranyl}, 6′-H_{tetrahydropyranyl}), 7.48 (ddd, J = 7.5, 4.7, 1.2 Hz, 1H,

5′-H_pyridyl), 7.69 (br s, 2H, NH₂), 7.92 (td, J = 7.7, 1.8 Hz, 1H, 4′-

H_pyridyl), 8.01 (dt, J = 7.9, 1.1 Hz, 1H, 3′-H_pyridyl), 8.68 (ddd, J = 4.7,

1.8, 0.9 Hz, 1H, 6′-H_pyridyl). ¹³C NMR (151 MHz, DMSO-d₆) δ 27.9

(2C, C-3′_{tetrahydropyranyl}, C-5′_{tetrahydropyranyl}), 39.5 (1C, C-4′_{tetrahydropyranyl}),

66.0 (2C, C-2′_{tetrahydropyranyl}, 6′-C_{tetrahydropyranyl}), 122.5 (1C, C-3′_pyridyl),

124.7 (1C, C-5′_pyridyl), 137.0 (1C, C-4′_pyridyl), 148.9 (1C, C-2′_pyridyl),

149.7 (1C, C-6′_pyridyl), 157.7 (1C, C-5′_triazole), 159.4 (1C, C-3′_triazole),

175.3 (1C, CO). IR (neat): ν

̃

[cm⁻¹] = 3456, 3096, 2965, 2941,

2855, 1717, 1622, 1530, 1483, 1429, 1981, 1356, 1335, 1323, 1304,

1281, 1238, 1169, 1152, 1132, 1115, 1082, 1049, 1011, 982, 970, 912,

872, 829, 820, 806. HRMS (m/z): [M + H]⁺calcd for C₁₃H₁₆N₅O⁺

274.1299, found 274.1306. HPLC: t_R= 12.0 min, purity 99.1%.

N-(4-(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazole-1-carbonyl)-

phenyl)-2,2,2-triﬂuoroacetamide (21p). It was synthesized according

to general procedure C from 9a (100 mg, 621 μmol, 1 equiv) and 4-

U

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1-(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-2-(2-

iodophenyl)ethan-1-one (21s). It was synthesized according to

general procedure C from 9a (100 mg, 621 μmol, 1 equiv) and 2-(2-

iodophenyl)acetic acid (163 mg, 621 μmol, 1 equiv), with EDCI (238

mg, 1.24 mmol, 2 equiv) and DMAP (152 mg, 1.24 mmol, 2 equiv) in

DMF (4 mL). Stirring at 0 °C was continued for 1 h, followed by 2 h

at room temperature. The crude product was puriﬁed by ﬂash column

chromatography (DCM/ACN = 9/1 → 1/3) to give 21s as a

colorless solid (152 mg, 375 μmol, 61%); mp = >300 °C. TLC: 0.34

calcd for C₁₆H₁₄N₅O⁺292.1193, found 2921192. HPLC: t_R= 17.3

min, purity 99.4%.

(S)-(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(1,2,3,4-tetra-

hydronaphthalen-1-yl)methanone (21v). It was synthesized accord-

ing to general procedure C from 9a (100 mg, 621 μmol, 1 equiv) and

1,2,3,4-tetrahydro-2-naphthoic acid (109 mg, 621 μmol, 1 equiv),

with EDCI (238 mg, 1.24 mmol, 2 equiv) and DMAP (152 mg, 1.24

mmol, 2 equiv) in DMF (4 mL). Stirring at 0 °C was continued for 1

h, followed by 2 h at room temperature. After the washing steps, pure

product 21v was obtained as a yellow solid (148 mg, 463 μmol, 75%);

mp = 157−158 °C. TLC: 0.61 (DCM/MeOH = 9:1). ¹H NMR (600

MHz, DMSO-d₆) δ 1.73−1.81 (m, 1H, 3′-H_{tetrahydronaphthyl}), 1.86−1.94

(m, 1H, 3′-H_{tetrahydronaphthyl}), 2.07−2.13 (m, 1H, 2′-H_{tetrahydronaphthyl}),

2.17−2.25 (m, 1H, 2′-H_{tetrahydronaphthyl}), 2.74−2.86 (m, 2H, 4′-

H_{tetrahydronaphthyl}), 5.07 (t, J = 6.4 Hz, 1H, 1′-H_{tetrahydronaphthyl}), 7.07−

7.12 (m, 2H, 7′-H_{tetrahydronaphthyl}, 8′-H_{tetrahydronaphthyl}), 7.14−7.19 (m,

2H, 5′-H_{tetrahydronaphthyl}, 6′-H_{tetrahydronaphthyl}), 7.49 (ddd, J = 7.6, 4.7, 1.2

Hz, 1H, 5′-H_pyridyl), 7.72 (br s, 2H, NH₂), 7.94 (td, J = 7.7, 1.8 Hz,

1H, 4′-H_pyridyl), 8.05 (dt, J = 7.8, 1.0 Hz, 1H, 3′-H_pyridyl), 8.69 (ddd, J

= 4.7, 1.7, 0.7 Hz, 1H, 6′-H_pyridyl). ¹³C NMR (151 MHz, DMSO-d₆) δ

20.0 (1C, C-3′_{tetrahydronaphthyl}), 26.3 (1C, C-2′_{tetrahydronaphthyl}), 28.6 (1C,

C-4′_{tetrahydronaphthyl}), 43.2 (1C, C-1′_{tetrahydronaphthyl}), 122.6 (1C, C-

3′_pyridyl), 124.8 (1C, C-5′_pyridyl), 125.8 (1C, C-7′_{tetrahydronaphthyl}), 126.8

(1C, C-6′_{tetrahydronaphthyl}), 129.2 (1C, C-5′_{tetrahydronaphthyl}), 129.3 (1C, C-

8′_{tetrahydronaphthyl}), 133.1 (1C, C-8a′_{tetrahydronaphthyl}), 137.1 (1C, C-

4′_pyridyl), 137.5 (1C, C-4a′_{tetrahydronaphthyl}), 148.9 (1C, C-2′_pyridyl),

149.7 (1C, C-6′_pyridyl), 157.9 (1C, C-5′_triazole), 159.5 (1C, C-3′_triazole),

1

(DCM/MeOH = 9:1). H NMR (600 MHz, DMSO-d₆) δ 4.60 (s,

2H, CH₂), 7.08 (td, J = 7.6, 1.7 Hz, 1H, 4′-H_{2‑iodophenyl}), 7.41 (td, J =

7.4, 1.3 Hz, 1H, 5′-H_{2‑iodophenyl}), 7.47−7.52 (m, 2H, 5′-H_pyridyl, 6′-

H_{2‑iodophenyl}), 7.70 (br s, 2H, NH₂), 7.89 (dd, J = 7.9, 1.3 Hz, 1H, 3′-

H_{2‑iodophenyl}), 7.94 (td, J = 7.7, 1.8 Hz, 1H, 4′-H_pyridyl), 8.05 (dt, J = 7.9,

1.1 Hz, 1H, 3′-H_pyridyl), 8.70 (ddd, J = 4.7, 1.8, 0.9 Hz, 1H, 6′-H_pyridyl).

¹³C NMR (151 MHz, DMSO-d₆) δ 46.5 (1C, CH₂), 102.1 (1C, C-

2′_{2‑iodophenyl}), 122.5 (1C, C-3′_pyridyl), 124.8 (1C, C-5′_pyridyl), 128.4 (1C,

C-5′_{2‑iodophenyl}), 129.3 (1C, C-4′_{2‑iodophenyl}), 131.9 (1C, C-6′_{2‑iodophenyl}),

137.1 (1C, C-4′_pyridyl), 137.4 (1C, C-1′_{2‑iodophenyl}), 138.8 (1C, C-

3′_{2‑iodophenyl}), 148.9 (1C, C-2′_pyridyl), 149.8 (1C, C-6′_pyridyl), 157.5 (1C,

C-5′_triazole), 159.4 (1C, C-3′_triazole), 170.8 (1C, CO). IR (neat): ν

[cm⁻¹] = 3456, 2980, 1713, 1634, 1528, 1398, 1368, 1348, 1287,

1263, 1246, 1173, 1152, 1099, 1080, 1049, 995, 970. HRMS (m/z):

[M + H]⁺calcd for C₁₅H₁₃IN₅O⁺406.0159, found 406.0141. HPLC:

t_R= 17.6 min, purity 99.9%.

̃

N-(2-(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-2-

oxoethyl)benzamide (21t). It was synthesized according to general

procedure C from 9a (150 mg, 931 μmol, 1 equiv) and hippuric acid

(167 mg, 931 μmol, 1 equiv), with EDCI (232 mg, 1.21 mmol, 1.3

equiv) and DMAP (148 mg, 1.21 mmol, 1.3 equiv) in DMF (3 mL).

The reaction mixture was stirred at room temperature for 3 h. After

the washing steps, pure product 21t was obtained as a colorless solid

(198 mg, 613 μmol, 66%); mp = 278 °C. TLC: 0.62 (DCM/MeOH =

175.7 (1C, CO). IR (neat): ν

̃

[cm⁻¹] = 3447, 3426, 2932, 2866,

2835, 1713, 1628, 1533, 1464, 1447, 1391, 1368, 1352, 1271, 1256,

1169, 1150, 1090, 993, 968, 920, 876, 802. HRMS (m/z): [M + H]⁺

calcd for C₁₈H₁₈N₅O⁺320.1506, found 3201517. HPLC: t_R= 17.8

min, purity 97.5%.

X-ray Diﬀraction. Data sets for compound 21m were collected

with a D8 Venture Dual Source 100 CMOS diﬀractometer. Programs

used: data collection: APEX3 V2016.1−0 (Bruker AXS Inc., 2016);

cell reﬁnement: SAINT V8.37A (Bruker AXS Inc., 2015); data

reduction: SAINT V8.37A (Bruker AXS Inc., 2015); absorption

correction, SADABS V2014/7 (Bruker AXS Inc., 2014); structure

solution SHELXT-2015;⁶⁵structure reﬁnement SHELXL-2015⁶⁵and

graphics, XP (Version 5.1, Bruker AXS Inc., Madison, Wisconsin,

1998). R-values are given for observed reﬂections, and wR²values are

given for all reﬂections.

1

85:15). H NMR (400 MHz, DMSO-d₆) δ 4.74 (d, J = 5.7 Hz, 2H,

CH₂), 7.47−7.55 (m, 3H, 3′-H_phenyl, 5′-H_phenyl, 5′-H_pyridyl), 7.58 (tt, J

= 7.3, 2.4 Hz, 1H, 4′-H_phenyl), 7.71 (br s, 2H, NH₂), 7.91−7.98 (m,

3H, 2′-H_phenyl, 6′-H_phenyl, 4′-H_pyridyl), 8.05 (dt, J = 7.9, 1.1 Hz, 1H, 3′-

H_pyridyl), 8.70 (ddd, J = 4.8, 1.8, 1.0 Hz, 1H, 6′-H_pyridyl), 9.06 (t, J = 5.7

Hz, 1H, NH). ¹³C NMR (101 MHz, DMSO-d₆) δ 42.7 (1C, CH₂),

122.5 (1C, C-3′_pyridyl), 124.8 (1C, C-5′_pyridyl), 127.3 (2C, C-2′_phenyl, 6′-

C-6′_phenyl), 128.4 (2C, C-3′_phenyl, C-5′_phenyl), 131.6 (1C, C-4′_phenyl),

133.5 (1C, C-1′_phenyl), 137.1 (1C, C-4′_pyridyl), 148.8 (1C, C-2′_pyridyl),

149.7 (1C, C-6′_pyridyl), 157.6 (1C, C-5′_triazole), 159.8 (1C, 3′-C_triazole),

FXIIa Inhibition Assay. The inhibitory activity of synthesized

compounds toward the human coagulation factor XIIa was measured

by quantifying the hydrolysis rate of the ﬂuorogenic substrate as

reported previously.^41,43Brieﬂy, the enzymatic activity was measured

in buﬀer (10 mM Tris-Cl, 150 mM NaCl, 10 mM MgCl₂·6H₂O, 1

mM CaCl₂·2H₂O, 0.1% w/v BSA, 0.01% v/v Triton-X100, pH = 7.4)

using clear ﬂat-bottom, black polystyrene 96 well plates. The enzyme

(human β-FXIIa, HFXIIAB, >95% purity; Molecular Innovations, 2.5

nM: ﬁnal concentration) and the ﬂuorogenic substrate Boc-Gln-Gly-

Arg-AMC (Pepta Nova, 25 μM: ﬁnal concentration, K_m= 167 μM)

were employed. Dilutions of test compounds ranging from 0.5 nM to

132 μM (ﬁnal concentrations) in DMSO were prepared. The

ﬂuorogenic substrate solution was added into the wells followed by

the addition of 2 μL of test compound solution, and the reaction was

triggered by the addition of the enzyme solution (ﬁnal testing volume:

152 μL). In the case of blank (substrate + buﬀer) and control

(substrate + enzyme) wells, 2 μL of DMSO was added instead of the

test compound solution. Fluorescence intensity was measured with

Microplate Reader Mithras LB 940 (Berthold Technologies,

excitation at 355 nm, emission at 460 nm) for a period of 1 h with

a read every minute. The reactions were performed at 25 °C. To

derive 1 h IC₅₀values, end-point RFU (single ﬂuorescence reading

after 1 h) was used. Sigmoidal curves were prepared in the GraphPad

Prism software, and IC₅₀values were derived from the ﬁtted curves.

The time-dependent inhibition experiment of FXIIa was performed

similarly to the FXIIa inhibition assay. The enzyme (FXIIa, 2.5 nM)

was added to a solution of ﬂuorogenic substrate (Boc-Gln-Gly-Arg-

AMC, 25 μM) and an inhibitor (ranged 0.5 nM−132 μM, 10

166.9 (1C, CONH), 169.9 (1C, CH₂CO). IR (neat): ν

̃

[cm⁻¹] =

3453, 3302, 3055, 1736, 1636, 1528, 1485, 1462, 1385, 1354, 1312,

1285, 1250, 1177, 1153, 1084, 999, 976, 802, 760, 744, 714, 691, 625,

+

610. HRMS (m/z): [M + H]⁺calcd for C₁₆H₁₅N₆O₂323.1251,

found 323.1246. HPLC: t_R= 12.7 min, purity 99.6%.

(E)-1-(5-Amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-3-phenyl-

prop-2-en-1-one (21u). It was synthesized according to general

procedure C from 9a (100 mg, 621 μmol, 1 equiv) and cinnamic acid

(91.9 mg, 621 μmol, 1 equiv), with EDCI (238 mg, 1.24 mmol, 2

equiv) and DMAP (152 mg, 1.24 mmol, 2 equiv) in DMF (4 mL).

The reaction mixture was gradually heated from 0 °C to room

temperature over 18 h. After the washing steps, pure product 21u was

obtained as a yellow solid (136 mg, 465 μmol, 75%); mp = 215 °C.

TLC: 0.50 (DCM/MeOH = 9:1). ¹H NMR (600 MHz, DMSO-d₆) δ

7.48−7.50 (m, 1H, 5′-H_pyridyl), 7.50−7.53 (m, 3H, 3′-H_phenyl, 4′-

H_phenyl, 5′-H_phenyl), 7.75 (d, J = 16.0 Hz, 2H, PhCH=CH), 7.78 (br s,

2H, NH₂), 7.82−7.87 (m, 2H, 2′-H_phenyl, 6′-H_phenyl), 7.94 (td, J = 7.7,

1.8 Hz, 1H, 4′-H_pyridyl), 7.99 (d, J = 16.0 Hz, 1H, PhCH=CH), 8.09

(d, J = 7.8 Hz, 1H, 3′-H_pyridyl), 8.70 (d, J = 4.5 Hz, 1H, 6′-H_pyridyl). ¹³

C

NMR (151 MHz, DMSO-d₆) δ 116.7 (1C, PhCH=CH), 122.6 (1C,

C-3′_pyridyl), 124.8 (1C, C-5′_pyridyl), 128.9 (2C, C-2′_phenyl, C-6′_phenyl),

129.2 (2C, 3′_phenyl, C-5′_phenyl), 131.4 (1C, C-4′_phenyl), 133.9 (1C, C-

1′_phenyl), 137.0 (1C, C-4′_pyridyl), 147.1 (1C, PhCH=CH), 148.9 (1C,

C-2′_pyridyl), 149.7 (1C, C-6′_pyridyl), 158.1 (1C, C-5′_triazole), 159.3 (1C,

C-3′_triazole), 165.0 (1C, CO). IR (neat): ν

̃

[cm⁻¹] = 3449, 3028,

1682, 1634, 1612, 1574, 1530, 1485, 1404, 1366, 1342, 1287, 1246,

1173, 1152, 1032, 1022, 999, 968, 860, 800. HRMS (m/z): [M + H]⁺

V

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concentrations) in an assay buﬀer, and ﬂuorescence (excitation at 355

nm, emission at 460 nm) was read immediately in the kinetic mode

(readout every 87 s) for up to 180 min. The IC₅₀values were

calculated for each time point in the GraphPad Prism software and

plotted against the measurement time.

Thrombin Inhibition Assay. The inhibitory activity of

synthesized compounds toward the coagulation factor IIa (human

α-thrombin (active) protein, ABIN2127880, >95% purity; antibodies-

online, 0.25 nM: ﬁnal concentration) was determined analogously to

the described procedure for FXIIa. The ﬂuorogenic substrate Boc-Val-

Pro-Arg-AMC (Pepta Nova, 25 μM: ﬁnal concentration, K_m= 18

μM) was utilized.

FXa Inhibition Assay. The inhibitory activity of synthesized

compounds toward the coagulation factor Xa (human factor Xa,

HFXA, >95% purity; Molecular Innovations, 2.5 nM: ﬁnal

concentration) was determined analogously to the described

procedure for FXIIa. The ﬂuorogenic substrate Boc-Ile-Glu-Gly-Arg-

AMC (Pepta Nova, 25 μM: ﬁnal concentration) was utilized.

Trypsin Inhibition Assay. The inhibitory activity of synthesized

compounds toward trypsin (porcine trypsin; Merck, 3.5 nM: ﬁnal

concentration) was determined analogously to the described

procedure for FXIIa. The ﬂuorogenic substrate Z-Gly-Gly-Arg-AMC

(Sigma-Aldrich, 25 μM: ﬁnal concentration) was utilized.

Urokinase Inhibition Assay. The inhibitory activity of

synthesized compounds toward urokinase (human urokinase;

Medac GmbH, 1.68 nM: ﬁnal concentration) was determined

similarly to the described procedure for FXIIa, but 50 mM Tris

HCl buﬀer (pH 8.0), containing 154 mM NaCl, was utilized. The

ﬂuorogenic substrate Tos-Gly-Pro-Arg-AMC × TFA (synthesized in

house, 143 μM: ﬁnal concentration, K_m= 75 μM) was utilized.

Plasmin Inhibition Assay. The inhibitory activity of synthesized

compounds toward plasmin (human plasmin; Merck, 0.35 nM: ﬁnal

concentration) was determined similarly to the described procedure

for FXIIa, but 50 mM Tris HCl buﬀer (pH 8.0), containing 154 mM

NaCl, was utilized. The ﬂuorogenic substrate Mes-dArg-Phe-Arg-

AMC × 2 TFA (synthesized in house,⁶⁶100 μM: ﬁnal concentration,

K_m= 29 μM) was utilized.

FXIa Inhibition Assay. The inhibitory activity of synthesized

compounds toward FXIa (human FXIa; Molecular Innovations, 0.897

nM: ﬁnal concentration) was determined similarly to the described

procedure for FXIIa, but 50 mM Tris HCl buﬀer (pH 8.0), containing

154 mM NaCl, was utilized. The ﬂuorogenic substrate Mes-dArg-Pro-

Arg-AMC × 2 TFA (synthesized in house,⁶⁷143 μM: ﬁnal

concentration, K_m= 77 μM) was utilized.

Plasma Kallikrein Inhibition Assay. The inhibitory activity of

synthesized compounds toward plasma kallikrein (human plasma

kallikrein; Enzyme Research South Bend, 0.112 nM: ﬁnal concen-

tration) was determined similarly to the described procedure for

FXIIa, but 50 mM Tris HCl buﬀer (pH 8.0), containing 154 mM

NaCl, was utilized. The ﬂuorogenic substrate Mes-dArg-Pro-Arg-

AMC × 2 TFA (synthesized in house,⁶⁷100 μM: ﬁnal concentration,

K_m= 40 μM) was utilized.

by the addition of 100 μL of the PT assay reagent already containing

CaCl₂(prewarmed at 37 °C, Convergent Technologies, Germany),

and the clotting time was recorded.

Microscale Parallel Synthesis. A 96-well plate (clear, poly-

styrene, ﬂat bottom) was charged with 95 carboxylic acids (620

mmol/L in dry DMSO-d₆, 30 μL, 1 equiv, one acid in each well) and

95 micro magnetic stirring bars. DMAP (930 mmol/L in dry DMSO-

d₆, 30 μL, 1.5 equiv), EDCI (930 mmol/L in dry DMSO-d₆, 30 μL,

1.5 equiv), and aminotriazole 9a (620 mmol/L in dry DMSO-d₆, 30

μL, 1 equiv) were sequentially pipetted into each well. The plate was

covered with a lid, then sealed with paraﬁlm, and placed on the

magnetic stirrer. After 3 h of incubation at room temperature, the lid

was opened and 20 μL aliquot from each microscale reaction was

withdrawn, diluted with DMSO (980 μL, dry), and immediately

frozen at −20 °C* (samples for MTS). The residual 100 μL of the

reaction solution was diluted with DMSO-d₆(600 μL, dry) and

directly submitted for ¹H NMR analysis to measure the reaction

conversion rate. *: several samples (S19−S28, S34, and Table S1 in

the Supporting Information) were found to be moisture-sensitive and

were stored in a desiccator (with CaCl₂) under a N₂atmosphere

without freezing.

Determination of Microscale Reaction Conversions by ¹H

NMR. The NMR spectra of microscale parallel synthesis samples were

recorded at 25 °C using an Agilent DD2 400 MHz NMR

spectrometer and analyzed with the MestReNova software (version

12.0.3−21384, Mestrelab Research S.L.). The purest proton

resonance signals of the reaction product (S1−S95) and starting

material (aminotriazole 9a or a carboxylic acid) were identiﬁed,

integrated, and normalized. The averaged normalized integrals of

product (Int_P) protons and of the starting material (Int_Sm) protons

were used to calculate the reactions’ conversion (C, %) using the

following formula: C [%] = (Int_P/(Int_P+ Int_Sm)) × 100. When no

pure signals of either starting material (9a or carboxylic acid) could be

identiﬁed, the signals of DMAP (Int_R) were used instead with the

consideration of the used reaction equivalents (eq_R) using the

following equation: C [%] = (Int_P× eq_R/Int_R) × 100.

Microscale Reaction Product Screening in the Enzyme

Inhibition Assay. After the reaction conversion determination (by

¹H NMR), frozen samples of the microscale parallel synthesis were

defrosted at room temperature and 50 μL aliquot was withdrawn from

each sample and diluted with the calculated amount of dry DMSO to

set the reaction product concentration to 76 μmol/L. For this, the

actual product concentration c_P[mmol/L] in the microscale reaction

was calculated using theoretical product concentration (1.55 mmol/

L) and the actual reaction conversion rate of each sample C [%]

applying the following formula: c_P[mmol/L] = 1.55 mmol/L × C

[%]/100. Resultant equimolar samples (90 acylated 1,2,4-triazol-5-

amines) were appropriately diluted to be directly screened at 1 μM

and 100 nM in the enzyme inhibition assays (FXIIa and thrombin)

using the ﬂuorogenic substrates. The results of these measurements

(triplicate) were expressed as the percentage of inhibition at 1 μM

and 100 nM for each sample.

In Vitro Blood Coagulation Assays (aPTT and PT). All

measurements were performed using citrated (3.8%) human whole

̈

blood (University Hospital Munster) on a semiautomated coagul-

Analysis of Covalent FXIIa-Inhibitor Complexes by SEC/ESI-

MS. Materials. Formic acid was obtained from Th. Geyer

(Renningen, DE); sodium hydroxide and ammonium acetate were

obtained from Merck (Darmstadt, DE); and isopropanol, acetic acid,

and ammonia solution were obtained from VWR International

(Radnor, PA). All chemicals were of analytical grade. Human plasma

β-FXIIa was purchased from Molecular Innovations (Novi, MI).

Aqueous solutions were prepared using an Aquatron A4000D water

still (Stuart, Staﬀordshire, GB). All pH values were adjusted using a

FiveEasy pH meter from Mettler Toledo (Greifensee, DE).

Equipment: The herein used HPLC system from Shimadzu (Kyoto,

JP) was equipped with a system controller (CBM-20A), a degasser

(DGU-14A), two pumps (LC-10ADvp), an autosampler (SIL-HTA),

and a column oven (CTO-10Avp). Mass spectrometric detection was

carried out using a micrOTOF time-of-ﬂight mass spectrometer from

Bruker Daltonics (Bremen, DE) equipped with an electrospray

ionization interface. Mass calibration was performed prior to the

measurement using a solution of 0.2% formic acid and 5 mM sodium

ometer (Thrombotimer-2, Behnk Elektronik, Germany) according to

the manufacturer instructions. For aPTT measurements, whole blood

(100 μL) was placed into the incubation cuvettes of the instrument

and incubated for 2 min at 37 °C. Then, the test compound solution

(10 μL) or solvent (DMSO, 10 μL) was added with a pipette. After 1

min of incubation, 100 μL of prewarmed (37 °C) aPTT reagent

(Convergent Technologies, Germany) was added and incubated for

additional 2 min. The cuvettes were transferred to a measuring

position, the coagulation was initiated by the addition of 100 μL of a

CaCl₂solution (25 mM, prewarmed at 37 °C, Convergent

Technologies, Germany), and the clotting time was recorded. For

PT assays, whole blood (100 μL) was incubated for 2 min at 37 °C.

Then, the test compound solution (10 μL) or solvent (DMSO, 10

μL) was added with a pipette. After 3 min of incubation, the cuvettes

were transferred to a measuring position, the coagulation was initiated

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hydroxide in a mixture of puriﬁed water and isopropanol (1:1, v/v),

which was directly introduced into the mass spectrometer using a

syringe pump. The system was controlled using micrOTOF control

(Version 3.0) and HyStar (Version 3.2), and post-processing was

carried out using DataAnalysis (Version 5.3). Size-exclusion

chromatography (SEC) was performed using a MAbPac SEC-1

analytical column (150 mm × 4 mm, 5 μm particle size, 300 Å pore

size) from Thermo Fisher Scientiﬁc (Bremen, DE).

Sample Preparation. The protein stock solution (37.67 μM) was

diluted by adding 90 μL of puriﬁed water. An aliquot of 46.3 μL was

mixed with 3.7 μL of inhibitor solution (1 mM in DMSO) to prepare

the enzyme−inhibitor solution (molar ratio of 1:20), which was then

analyzed via SEC/ESI-MS.

Enzyme−Inhibitor Covalent Complex Stability Measurement

(Desalination Procedure). The prepared enzyme−inhibitor solution

(50 μL) was diluted with 450 μL of puriﬁed water. The mixture was

transferred into the vial equipped with Amicon Ultra 0.5 mL

Centrifugal Filters (Ultracel − 3K) (Merck Millipore) and centrifuged

at 14000 rpm for 30 min. The residue was again diluted with puriﬁed

water to 500 μL volume, and the centrifugation process was repeated.

Last, the residue was diluted to the initial volume of 50 μL with

puriﬁed water and analyzed by SEC/ESI-MS.

SEC/ESI-MS Analysis. For chromatographic analysis of the sample

solutions, isocratic elution was carried out using an aqueous

ammonium acetate solution (10 mM, pH 6.8) as the mobile phase

for 15 min. The ﬂow rate was 150 μL/min, the column oven

temperature was set to 30 °C, and the injection volume was 10 μL.

The LC outlet was directly coupled to the ESI source of the mass

spectrometer. The enzyme−inhibitor complexes were analyzed in the

positive ion mode using the following ESI-MS parameters. The mass

range was set to m/z 1000−5000, nebulizer gas (nitrogen) was 1.2

bar, dry gas (nitrogen) was 9.0 L/min, the dry gas temperature was

200 °C, hexapole RF was 600 V, pre pulse storage time was 30 μs, and

transfer time was 100 μs. Deconvoluted mass spectra were obtained

by averaging frames from 6.8 min to 7.6 min retention time and

deconvolution using the charge states 9+ to 11+ of the respective

protein species.

2.6 mM KCl, 1 mM MgCl₂, 13.8 mM NaHCO₃, 0.36 mM NaH₂PO₄,

5.5 mM ^D-glucose), which results in a remaining plasma protein

content of about 1%. Prior to activation, platelets were preincubated

with the indicated inhibitors or left untreated for control.

Light Transmission Aggregometry. The measurement of

tumor-cell-induced platelet aggregation was performed by light

transmission aggregometry using an APACT-4004 aggregometer

(Haemochrom Diagnostica, Essen, Germany). Platelets were prepared

and coincubated with the indicated inhibitors as described. Platelet

aggregation was induced by either 1 × 10⁴tumor cells/mL or 20.5

μM TRAP-6 or 0.2 U thrombin. The measurement was performed at

37 °C in adequate cuvettes, stirred continuously at 1000 rpm.

Aggregate formation was measured by light transmission, with buﬀer

set as 100% and platelets in the buﬀer as 0% reference values.

ATP Release Assay. Platelets were coincubated with inhibitors for

20 min or phosphate-buﬀered saline (PBS). MDA-MB-231 cells were

detached with EDTA and resuspended in PBS. Platelets (400 × 10⁶

Plts/mL) were activated with 1 × 10⁴tumor cells/mL for 30 min,

with 20.5 μM TRAP6 for 10 min or 0.2 U thrombin for 10 min.

Platelets’ ATP secretion from dense granules was assessed by

luminescence measurements using a luciferin-based ATP-Determi-

nation Kit (Thermo Fisher Scientiﬁc, Waltham) and a FLUOstar

Optima plate reader (BMG Labtech, Ortenberg, Germany).

Thrombin Generation Assay. Reagents. The following regents

were uses: relipidated recombinant TF (Instrumentation Laboratory,

Bedford, MA); and Thrombodynamics-4D kits consisting of a corn

trypsin inhibitor with the ﬂuorogenic substrate Z-Gly-Gly-Arg-AMC,

calcium acetate, and phospholipids in the form of a lyophilized

suspension of phospholipid vesicles (HemaCore Labs, Moscow,

Russia).

Plasma Preparation. Blood was taken from apparently healthy

individuals after obtaining an informed consent. We used Vacuette

3.2% citrate tubes (Greiner BioOne, Kremsmunster, Austria) with

straight 21G x 1 1/2 needles. The ﬁrst tube after the venipuncture was

discarded. Blood was immediately processed by two sequential

centrifugations: 15 min at 1 600g and 5 min at 10 000g at room

temperature. Platelet-free plasma (PFP) was mixed from n = 3 donors

(1:1:1) and was aliquoted and snap-frozen in liquid nitrogen, stored

at −80 °C.

Experimental Design. Frozen samples of plasma were thawed in a

water bath at 37 °C for 15 min and then incubated for 15 min at room

temperature. Thawed plasma (110 μL) was added to the dried corn

trypsin inhibitor (ﬁnal concentration 0.2 mg/mL) with a dried

substrate (ﬁnal concentration 400 μM), and 5 μL of phospholipids

(prepared according to manufacturer’s instructions, ﬁnal concen-

tration 4 μM) was added. Plasma was spiked with 4 μL of 21a, 21i,

21m, dabigatran (1), or DMSO or diluted from the stock solution

with buﬀer (20 mM HEPES, 140 mM NaCl, pH 7.2−7.4). The ﬁnal

21a, 21i and 21m concentration was 100 μM and that of dabigatran

was 1 μM, and the DMSO concentration in plasma did not exceed 1%

(except for “buﬀer” sample, which was spiked only with 5 μL of

buﬀer). The sample was incubated for 15 min at 37 °C and then

supplemented with dried calcium acetate (ﬁnal concentration 20

mM). Clotting was activated using 5 μL of TF diluted in distilled

water (stored at −80 °C, ﬁnal concentration 5 pM) poured directly

into the plasma, and the sample (120 mL) was placed in a plastic

cuvette.

Binding Mode Study by Molecular Modeling. Structures of

small molecules were prepared using the LigPrep module of the

̈

Schrodinger suite (Schrodinger Release 2019−4: Schrodinger, LLC,

New York, NY, 2019) employing the OPLS3e force ﬁeld. The

experimental structures of studied proteins were obtained from the

RCSB database (PDB IDs: 6B77/FXIIa, 6CYM/thrombin). Struc-

tures were processed with the protein preparation wizard of

̈

Schrodinger Maestro suite 2019−4 (Schrodinger Release 2019−4)

to optimize the hydrogen-bonding network, followed by constrained

minimization (heavy-atom RMSD converge of 0.3 Å) to remove

crystallographic artifacts. Covalent docking was carried out using the

̈

Schrodinger Covalent Docking procedure with Nucleophilic Addition

to a Double Bond protocol. In both cases, active serine residues (195/

FXIIa and 219/thrombin) were selected as attachment points and

centers for Glide Grid. Input and output ﬁles are available upon

request.

Cell Lines. The human breast cancer cell line MDA-MB-231 was

grown in Dulbecco’s modiﬁed Eagle’s Medium (DMEM) (Sigma-

Aldrich, St. Louis) with 10% (v/v) FCS, 1% ^L-glutamine, 100 U/mL

penicillin, 100 μg/mL streptomycin, and 1% sodium pyruvate. Cells

were incubated at 37 °C in a humidiﬁed atmosphere containing 5%

CO₂. Cells were detached at 90% conﬂuence using a solution of

ethylenediamine tetraacetic acid (EDTA, 0.2 g/L EDTA × 4 Na) for

10 min at 37 °C. All reagents were from Thermo Fisher Scientiﬁc Inc.

(Waltham, MA). Cell identity was evaluated using an STR proﬁle

analysis.

Preparation of Platelets. Platelet-rich-plasma was obtained from

the Institute for Experimental Hematology and Transfusion Medicine,

University of Bonn, Medical Centre, in accordance with the

declaration of Helsinki. Isolated human platelets (Plts) in buﬀer

were prepared from platelet-rich-plasma as described⁵⁴by centrifu-

gation (670 g, 10 min, 22 °C) and resuspension (400 × 10⁶Plts/mL)

in recalciﬁed (1 mM) platelet buﬀer (10 mM HEPES, 137 mM NaCl,

Thrombodynamics-T2T analyzer (HemaCore Labs LLC, Moscow,

Russia) allows detecting a ﬂuorescence signal from AMC generated

during clot formation as well as light scattering from the ﬁbrin clot

(Thrombodynamics-4D Analyzer; HemaCore Labs, as described⁶⁸).

The ﬂuorogenic substrate added to the plasma was cleaved by

thrombin and produced a ﬂuorescent AMC molecule. Its generation

rate was proportional to the thrombin concentration. The plasma

sample was illuminated in turn by red (625 nm) and ultraviolet (365

nm) light-emitting diodes. The ﬁbrin clot formation was detected by

red light scattering, and the ﬂuorescence of AMC was excited by

ultraviolet emission (440 nm). Scattered light and ﬂuorescence passed

through a multiband ﬁlter and were detected by a digital camera every

30 s for 90 min.

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Parameters of ﬁbrin clot growth and thrombin generation were

calculated from a series of images as described before.⁶⁹The light

scattering of ﬁbrin clots and thrombin concentrations were plotted as

functions of time, and the following parameters were calculated:

clotting time (the time to reach one-half maximum light scattering,

containing EDTA (1 mM). Then, 10 volume parts of this dilution

were mixed with 86 volume parts of 0.2% Triton X-100 in phosphate-

buﬀered saline (prepared from tablets from Sigma-Aldrich, P4417:

one tablet dissolved in 200 mL of deionized water yields 0.01 M

phosphate buﬀer, 0.0027 M potassium chloride, and 0.137 M sodium

chloride, pH 7.4, at 25 °C). The assay was started by adding 2 μL of

the substrate solution in DMSO (5 mM) to 2 μL of a DMSO solution

of the inhibitor or to 2 μL of DMSO in the case of the controls. The

mixture was preincubated for 5 min at 37 °C. Then, the enzymatic

reaction was started by adding 96 μL of the prepared enzyme solution

in Triton X-100/phosphate-buﬀered saline. In the ﬁnal incubation

volume of 100 μL, the pyrenylbutanamide substrate concentration

was 100 μM. After incubation at 37 °C for 60 min, the enzyme

reaction was terminated by the addition of 200 μL of acetonitrile/

methanol (1:1, v/v) supplemented with the internal standard 6-pyren-

1-ylhexanoic acid (0.025 μg/200 μL). After being cooled in an ice

bath for 10 min, the samples were centrifuged at 12 000g at 4 °C for 5

min. Blank incubations in the absence of the enzyme were carried out

in parallel. FAAH inhibition was determined by measuring the

amount of 4-pyren-1-ylbutanoic acid released by the enzyme in the

absence and the presence of a test compound (corrected by the blank

value) by reversed-phase HPLC with ﬂuorescence detection as

described recently.⁷⁵Under the conditions applied, for the reference

FAAH inhibitor URB597, an IC₅₀value of 0.043 μM was measured.

T_1/2), the amplitude of the thrombin peak (A_max), the time to peak

(T_max), and endogenous thrombin potential (ETP, the area under the

curve). For each compound were done n = 3 measurements. Six

curves of one representative experiment for each compound are

shown in Figure 14. Mean values and standard deviation of calculated

parameters are shown in Table 3.

Cytotoxicity. To examine the cytotoxicity, 10 mM solutions of

each test compound were prepared in DMSO. Human liver cancer

cells (HepG2, HB-8065) (ATCC, Manassas, VA) were cultivated in

Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with

10 mM N-2-hydroxyethylpiperazine-N′-2-ethane-sulfonic acid

(HEPES buﬀer), 100 U/mL penicillin, 100 μg/mL streptomycin, 2

mM ^L-glutamine, and 10% (v/v) fetal calf serum (FCS) using

standardized culture conditions (37 °C, 5% CO₂, saturated humidiﬁed

atmosphere). The human astrocytoma cell line (CCF-STTG-1)

(ATCC, Manassas, VA) was cultivated in Roswell Park Memorial

Institute medium (RPMI 1640) supplemented with 100 U/mL

penicillin, 100 μg/mL streptomycin, 2 mM ^L-glutamine, and 10% (v/

v) FCS. For the subcultivation, both cell types were trypsinated when

reaching a microscopic conﬂuence of 80%. Media were changed two

times in 7 days. Cytotoxic eﬀects of the tested compounds were

evaluated with the Alamar blue assay. The assay was performed

according to previous studies.⁷⁰⁻⁷²Brieﬂy, the cells were seeded in

96-well plates of a suspension of 25 000 cells/well for HepG2 and

10 000 cells/well for CCF. Cells were incubated for 24 h after the

medium was replaced by serum-free medium and cultivated for

another 24 h. Afterward, the cells were treated with 21i, 21m, and

21q in a concentration range of 0.05−300 μM (HepG2) and 0.5−300

μM (CCF) and incubated for 48 h. After compound exposure, the

dye solution of resazurin was added to the cells, followed by

incubation for 1.5 h at 37 °C. The ﬂuorescence of reduced resazurin

(resoruﬁn) was measured at λ = 590 nm with a microplate reader

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01635.

■

sı

¹H and ¹³C NMR spectra of all synthesized compounds;

experimental procedures and analytical data for the

compounds 9a−c, 18a−c, 15, and 19a−c; conversion

rates in microscale parallel synthesis and inhibitory

activity screening of 1,2,4-triazol-5-amines S1−S95;

extended cytotoxicity data for 21i, 21m, and 21q; X-

ray crystal structure analysis of 21m (PDF)

̈

(Inﬁnite M200 pro, Tecan, Mannendorf, Switzerland). Cytotoxicity

tests were repeated three times from three independent passages (n ≥

9). The data are shown as the mean standard deviation (SD). As a

positive control, T-2 toxin, which was previously isolated by the

working group of Prof. Humpf,⁷⁰was used at a concentration of 10

μM. The statistical analysis of the data was performed using one-way

analysis of variance (ANOVA) and Tukey’s post-hoc test; *

statistically signiﬁcant (p ≤ 0.05), ** statistically highly signiﬁcant

(p ≤ 0.01).

Molecular formula strings (CSV)

CCDC-1997967 for 21m contains the crystallographic

data; data can be obtained free of charge from the

Cambridge Crystallographic Data Centre via www.ccdc.

cam.ac.uk/data_request/cif; coordinates for the inhib-

itors 21i docked in FXIIa (PDB)

Coordinates for the inhibitor 21m docked in FIIa

Inhibition of MAGL (Monoacylglycerol Lipase). The eﬀect of

selected compounds on the activity of the endocannabinoid-degrading

enzyme monoacylglycerol lipase (MAGL) was evaluated as previously

described using human recombinant MAGL.^73,74Brieﬂy, the substrate

1,3-dihydroxypropan-2-yl 4-pyren-1-ylbutanoate was solubilized with

Triton X-100 (0.2 %). The enzyme reaction was terminated after 45

min by the addition of a mixture of acetonitrile/methanol (1:1, v/v)

supplemented with the internal standard 6-pyren-1-ylhexanoic acid.

Blank incubations in the absence of the enzyme were carried out in

parallel. MAGL inhibition was determined by measuring the amount

of 4-pyren-1-ylbutanoic acid released by the enzyme in the absence

and the presence of a test compound (corrected by the blank value)

by reversed-phase HPLC with ﬂuorescence detection. Under the

conditions applied, for the reference MAGL inhibitor CAY10499, an

IC₅₀value of 0.48 μM was measured.

Inhibition of FAAH (Fatty Acid Amide Hydrolase). Inhibition

of FAAH by selected compounds was measured in a similar manner as

described recently.⁷³The substrate N-(2-hydroxyethyl)-4-pyren-1-

ylbutanamide⁷⁵was dissolved in methanol (2.5 mg/mL). An aliquot

of this solution was thoroughly dried under a stream of nitrogen. The

residue was dissolved in such an amount of DMSO that a 5 mM

substrate solution was obtained. The enzyme solution was prepared

by diluting 50 μL of the rat brain homogenate obtained as described

recently⁴with 350 μL of potassium phosphate buﬀer (0.1 M, pH 7.4)

(PDB)

AUTHOR INFORMATION

Corresponding Author

■

Dmitrii V. Kalinin − Institute of Pharmaceutical and Medicinal

Chemistry, University of Munster, 48149 Munster, Germany;

̈ ̈

orcid.org/0000-0003-2717-5364; Phone: +49-251-

8333372; Email: d_kali01@uni-muenster.de

Authors

Marvin Korﬀ − Institute of Pharmaceutical and Medicinal

Chemistry, University of Munster, 48149 Munster, Germany

Lukas Imberg − Institute of Pharmaceutical and Medicinal

Chemistry, University of Munster, 48149 Munster, Germany

Jonas M. Will − Institute of Inorganic and Analytical Chemistry,

University of Munster, 48149 Munster, Germany

Nico Bu

̈

ckreiß − Pharmaceutical Institute, University of Bonn,

53121 Bonn, Germany

Svetlana A. Kalinina − Institute of Food Chemistry, University

of Mu

0001-7564-8213

̈

nster, 48149 Mu

̈

nster, Germany; orcid.org/0000-

Y

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Article

Benjamin M. Wenzel − Department of Pharmacy, Institute of

Pharmaceutical Chemistry, Philipps University Marburg, 35032

Marburg, Germany

Gregor A. Kastner − Institute of Pharmaceutical and Medicinal

Chemistry, University of Munster, 48149 Munster, Germany

̈ ̈

plastin time; DCM, dichloromethane; DMAP, 4-dimethylami-

nopyridine; DOACs, direct oral anticoagulants; ESI-MS,

electrospray ionization mass spectrometry; ETP, endogenous

thrombin potential; FAAH, fatty acid amide hydrolase; MAGL,

monoacylglycerol lipase; MTS, medium-throughput screening;

PARs, protease-activated receptors; PK, plasma kallikrein; PT,

prothrombin time; SAR, structure−activity relationship; SEC,

size-exclusion chromatography; TGA, thrombin generation

assay; THF, tetrahydrofuran; TF, tissue factor; TOF, time-of-

ﬂight; TRAP-6, thrombin receptor activating peptide-6; uPA,

urokinase-type plasminogen activator (urokinase)

Constantin G. Daniliuc − Institute for Organic Chemistry,

University of Munster, 48149 Munster, Germany; orcid.org/

0000-0002-6709-3673

Maximilian Barth − Institute of Pharmaceutical and Medicinal

Chemistry, University of Munster, 48149 Munster, Germany

̈

Ruzanna A. Ovsepyan − Laboratory of Translational Medicine,

Dmitriy Rogachev National Medical Research Center of

Pediatric Hematology, Oncology, and Immunology, 117997

Moscow, Russia; Center for Theoretical Problems of

Physicochemical Pharmacology, Russian Academy of Sciences,

119991 Moscow, Russia

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Author Contributions

The manuscript was written through contributions of all

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Notes

The authors declare no competing ﬁnancial interest.

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ABBREVIATIONS

■

ACN, acetonitrile; ADP, adenosine diphosphate; AMC, 7-

Amino-4-methylcoumarin; aPTT, activated partial thrombo-

Z

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Acylated 1 H-1,2,4-Triazol-5-Amines Targeting Human Coagulation Factor XIIa and Thrombin: Conventional and Microscale Synthesis, Anticoagulant Properties, and Mechanism of Action

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