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Microscale Parallel Synthesis of Acylated Aminotriazoles Enabling the Development of Factor XIIa and Thrombin Inhibitors

Abstract Herein we report a microscale parallel synthetic approach allowing for rapid access to libraries of N‐acylated aminotriazoles and screening of their inhibitory activity against factor XIIa (FXIIa) and thrombin, which are targets for antithrombotic drugs. This approach, in combination with post‐screening structure optimization, yielded a potent 7 nM inhibitor of FXIIa and a 25 nM thrombin inhibitor; both compounds showed no inhibition of the other tested serine proteases. Selected N‐acylated aminotriazoles exhibited anticoagulant properties in  vitro influencing the intrinsic blood coagulation pathway, but not extrinsic coagulation. Mechanistic studies of FXIIa inhibition suggested that synthesized N‐acylated aminotriazoles are covalent inhibitors of FXIIa. These synthesized compounds may serve as a promising starting point for the development of novel antithrombotic drugs.


Introduction
Thrombosis is a life-threatening pathology characterized by a thrombus (blood clot) formation inside a vein or an artery. The formed thrombus may completely or partially block the blood supply to major organs causing their ischemia, dysfunction, and failure. Thus, acute arterial and venous thromboses are associated with myocardial infarction, ischemic stroke, and pulmonary embolism, which are the main reason of death in developed countries. [1] Anticoagulant drugs are commonly used for the prevention of thrombosis and associated cardiovascular pathologies. Among anticoagulants, injectable heparins are routinely used in clinical settings, whereas vitamin K antagonists and direct thrombin and FXa inhibitors are orally available and are among the most frequently prescribed drugs. [2] Several examples of approved and developmental anticoagulants are shown in Figure 1. [3] Despite their efficacy, all available anticoagulants exhibit side effects, of which the risk of causing major bleeding is universal and the most dangerous. [4] For example, individuals receiving oral anticoagulants have a 7-10 times higher chance to develop intracranial bleeding, which in about 50 % of cases is lethal or may result in severe disability. [5] Therefore, novel anticoagulants causing no bleeding complications are highly desired.
The ideal anticoagulant should prevent pathological thrombus formation without affecting the physiological hemostatic function. To develop such a drug, alternative targets in the blood coagulation system should be addressed. In this respect, coagulation factor XIIa (FXIIa), also known as the Hageman factor, has recently emerged as a new target for safe anticoagulation. [6] FXIIa is a serine protease ( Figure 2) initiating the intrinsic blood coagulation pathway and the kallikrein/kinin system. Consequently, the proteolytic activity of FXIIa is associated with thrombosis and inflammation. However, the deficiency of this enzyme is practically asymptomatic. [7] FXIIdeficient animals and those treated with FXIIa inhibitors are protected from thrombosis and also show no tendency to injury-induced bleeding. [6a,7] These facts characterize FXIIa as a promising target for thrombosis prevention.
Apart from FXIIa, thrombin ( Figure 2), a classical target for anticoagulation therapy, can potentially be a safe target if addressed with a specific inhibitor. For instance, it has been shown that direct thrombin inhibitor VE-1902 (no exact structure is disclosed by the authors) modulate blood coagulation preventing thrombosis in animals with only a little effect on bleeding time due to its reversible covalent mechanism of action. [3c,10] The lower bleeding risk for VE-1902 was linked to its reduced ability to inhibit thrombin-mediated platelet activation. [10] VE-1902 was attributed to a new class of precision oral anticoagulants (PROACs) and it is currently in phase 1 clinical trial.
Many examples of small molecular direct thrombin inhibitors are known, including the marketed oral drug dabigatran and injectable argatroban ( Figure 1). [3a,b] None of them, however, is similar to VE-1902 in terms of the covalent reversible mechanism of thrombin inhibition and the related reduced tendency to cause bleeding. Significantly fewer inhibitors of FXIIa are known and none of them is yet approved for clinical application. [6a] Moreover, FXIIa inhibitors mainly belong to peptides, proteins, and monoclonal antibodies. Their small molecular counterparts, which could potentially be used as highly desired oral anticoagulants, are represented by only a few examples (4-6, Figure 1). [6,11] Our recent studies and studies from other groups suggest that N-acylated aminotriazoles and aminopyrazoles can act as covalent reversible inhibitors of FXIIa and thrombin exhibiting anticoagulant properties (e. g., compounds 3 and 6, Figure 1). [3c,f,12] At that, structural variations on their N-acyl moiety as well as on their azole core allow to access inhibitors with different physicochemical properties and unique serine protease inhibitory profiles. [3f] However, the development of a potent and selective inhibitor requires the generation of big libraries of N-acylated azoles and their subsequent screening against multiple serine proteases. Using conventional methods, this is a challenging task requiring significant amounts of consumables and multiple synthetic and purification efforts. To overcome these obstacles and intensify the development of new FXIIa and thrombin inhibitors, we developed a microscale parallel synthetic approach enabling us to fast access libraries of N-acylated aminotriazoles and screen their inhibitory activity against FXIIa and thrombin.

Method Design
A new method allowing for the efficient search for novel FXIIa and thrombin inhibitors should satisfy several criteria. Firstly, it should allow fast synthesis of N-acylated aminotriazoles in a parallel fashion. Secondly, it should utilize small quantities of inexpensive consumables, including reactants, solvents, and glassware. Also, the method should avoid or minimize the purification steps. Lastly, it should allow high-or mediumthroughput screening of synthesized compounds in biological assays.
The prospective inhibitors are composed of two parts (e. g., inhibitors 3 and 6, Figure 1) namely the aminoazole fragment (FR1) and the acyl fragment (FR2), of which FR1 requires synthetic preparation, whereas FR2 is commercially available.  and 1A4W, [8] respectively. The residues are depicted as cyan stick models; the catalytic residues (His57, Asp102, Ser195, chymotrypsin numbering system) are shown in orange. For XIIa, the ligand (benzamidine) residing in the S1 selectivity pocket is shown in green. Molecular graphics were prepared with UCSF ChimeraX. [9] Having both fragments in hand, they can be assembled in a microscale coupling reaction in a 96-well plate. Using this approach, structurally diverse FR1 and FR2 can be linked to each other in a combinatorial fashion in parallel ( Figure 3). To avoid the time-and material-consuming purification steps, the microscale reaction mixtures, containing bound fragments, should be directly (without isolation) screened for the inhibitory activity against selected serine proteases. To make sure that each final product (FR1-FR2 bound) is screened for the activity in an equimolar concentration, the products should be quantified before being screened ( Figure 3). Based on the results of the screening, the hits should be selected and synthesized in a conventional reaction scale and tested for their biological activity as pure individual compounds.
The direct screening of crude microscale-reaction mixtures was proposed as a feasible approach based on the principle illustrated in Figure 4. The unbound fragments FR1 and FR2 (shown as orange and green figures) and the coupling reagents (shown as X-and square-shaped figures) are low active or inactive inhibitors of FXIIa and thrombin and do not interfere with the assay at low concentrations (Figure 4, A). We previously showed that the unbound fragments FR1 and FR2 cannot efficiently inhibit the enzymes as only connected fragments (FR1-FR2) are capable of covalent interaction with the enzymes' catalytic Ser195 (also see the "Mechanism of Inhibition" section). [3f] Accordingly, when FR1 and FR2 are bound to each other forming N-acylated aminotriazole, a significant shift in inhibitory activity is observed (toward lower doses, Figure 4, B). Therefore, when the reaction mixtures are tested at nM or low μM concentrations (of the reaction product), only the inhibitory effect of the bound fragments FR1-FR2 is detected enabling screening. This excludes the chance of false-positive results caused by unreacted fragments.

Method Development
At first, we developed a method allowing microscale parallel synthesis of N-acylated aminotriazoles in 96-well plates. For this, we selected 1H-1,2,4-triazol-5-amine 7, a non-acylated scaffold of inhibitor 6 (Figure 1), as a model aminotriazole fragment (FR1) and five structurally different carboxylic acids (8)(9)(10)(11)(12) as acyl fragments (FR2). Particularly, benzoic acid (8), nicotinic acid (9), 4-methoxybenzoic acid (10), tetrahydro-2Hpyran-4-carboxylic acid (11), and N-benzoyl-l-proline (12) were selected. To construct the N-acylated aminotriazoles (FR1-FR2), the azole's annular nitrogen atom requires to react with the carboxylic acids. For carboxylic acids being poor electrophiles, the reaction with an amine does not happen spontaneously. Currently, however, many robust methods are described for the amide bond formation, e. g., the use of activated carboxylic acid derivatives like acid chlorides and anhydrides, or the use of transition metal catalysts, microwave irradiation, as well as peptide coupling reagents. [13] For their high reactivity, acid chlorides and anhydrides are not suitable for the microscale reactions coupled with the direct biological activity screening as they could interact with the enzymes thereby interfering with the assay. Besides, many acid chlorides exhibit limited shelf stability and require synthesis under water-free conditions. Transition metal catalysts also often require anhydrous and oxygen-free reaction conditions and prior to the analytics and the inhibition assays, the metal  . The principle of the inhibitory activity testing of microscalereactions against FXIIa and thrombin. The unbound fragments FR1 and FR2 and the coupling reagents (X-and square-shaped figures) are low active inhibitors of the enzymes and do not interfere with the assay at low concentrations (A). When FR1 and FR2 are bound together, a significant shift in inhibitory activity is observed (B). When the reaction mixtures are tested at nM or low μM concentrations, only the inhibitory effect of the bound fragments FR1-FR2 is detected enabling screening. must be removed from the solution. In contrast, for the reason of reaction performance simplicity and availability of a number of bench-stable carboxylic acids and coupling reagents, the peptide coupling was considered as the most suitable method for our microscale experiments.
The method development, as well as the subsequent screening, required a robust way of the reaction product quantification. Three quantitation methods were tested, namely HPLC-UV, HPLC-MS, and semi-quantitative 1 H-NMR (semi-q 1 H-NMR). Of them, HPLC-UV and HPLC-MS were found to be less practical due to relatively long spectra processing time, difficulties with analyzing complex reaction mixtures, and the requirement of external standard for proper reaction product quantification. In contrast, the semi-q 1 H-NMR method allowed fast reaction product quantification irrespective of the ionization and UV-absorption properties of starting materials and products. The semi-q 1 H-NMR principle used for the analysis of the microscale reaction mixtures is shown in Figure 5, Figure 6, and the Experimental Section.
The method development was performed in a 96-well plate reacting aminotriazole 7 (3 mg, 18.6 μmol, each well) with the carboxylic acids 8-12 (1 equiv. each) in DMSO-d 6 (120 μL) varying the amide coupling reaction conditions to maximize the average yield of N-acylated aminotriazoles 13 a-e (Table 1). We attempted the first three typical amide coupling conditions (entries 1-3, Table 1), of which COMU/DIPEA and HATU/DIPEA resulted in no desired products formation. In contrast, the combination of EDCI/DMAP produced N-acylated aminotriazoles with an average conversion of 38 % in 16 h at 50°C (entry 3). The average conversion was increased to 62 %, when the reaction temperature was reduced from 50°C to room temperature (entry 4). The reaction time prolongation to 92 h, however, resulted in the average conversion decrease to 32 % (entry 5). Entries 3-5 indicate that upon temperature elevation or the reaction time prolongation, lower yields are observed, which is associated with the degradation of the acylated product rather than with the low reactivity of the reacting species (see the DMAP-promoted decomposition, Supporting Information, Table S1).
The increase of EDCI/DMAP equivalents (to 1.5 each), as well as the addition of micromagnetic stirring bars into each well of Figure 5. Exemplary 1 H-NMR spectrum of the microscale reaction mixture in DMSO-d 6 , overall view (A) and a close-up view at 7.3-8.9 ppm (B) stacked against spectra of the starting material aminotriazole 7 (C) and the reaction product 13 a (D) in DMSO-d 6 . Spectrum B shows the integration of isolated characteristic signals of the starting material (7) and the product (13 a) used for the reaction conversion calculation. The use of the pyridyl moiety's 6'-H signal is the most convenient for the reaction conversion calculation as it experiences a low-field shift (from 8.59 ppm to 8.66 ppm) upon successful coupling reaction irrespective of the acyl moiety structure. The reaction conversion rate is calculated to be 36 % (13 a, entry 3, Table 1). The calculation details are shown in the Experimental Section. Figure 6. A fragment of 1 H-NMR spectrum of an exemplary microscale reaction mixture (A) stacked against spectra of the starting material carboxylic acid 30 (B) and the reaction product 33 e (C) in DMSO-d 6 . The characteristic proton signal of the methine group of 30 (B) experiences a low-field shift upon successful coupling reaction (product 33 e formation (C)) irrespective of the aminotriazole structure and, therefore, was used for the reaction conversion calculation. DMAP, which concentration remains unchanged, was used as an internal standard. The reaction conversion rate was calculated to be 57 %. The calculation details are shown in the Experimental Section. ChemMedChem Full Papers doi.org/10.1002/cmdc.202100431 the well plate, were beneficial, further increasing the reaction average conversion up to 79 % (entries [6][7][8]. Neither the excess of carboxylic acids (entry 9) nor the base (DMAP) removal or its substitution (entries 10-12) improved the reactions' yield. Also, EDCI replacement with a similar coupling reagent DCC had no positive effect on the yield of the microscale reactions (entry 13). Performing reactions under a dry inert atmosphere (the well-plate was placed into a desiccator with phosphorus pentoxide under N 2 ), resulted only in a slight improvement (81 %, entry 14) when compared to the reaction under ambient atmosphere (79 %, entry 8). Lastly, the experiments with shortened reaction time (3 h, entry 15) have been performed leading to a slightly decreased average conversion (76 %) compared to 16 h reaction time (79 %, entry 8). An extended table with additional reaction conditions is shown in the Supporting Information (Table S2).
Because of the high effort for moisture-free reaction conditions, compared to the low benefit (cf. entry 8 and 14), as well as the low negative impact of shortened reaction time, the reaction conditions of entry 15 have been selected for the actual microscale parallel synthesis. Utilizing optimized reaction conditions, microscale parallel synthesis of 95 N-acylated aminotriazoles based on 1H-1,2,4-triazol-5-amine 7 (FR1) and 95 structurally diverse carboxylic acids (FR2) was performed. The resultant library of compounds was screened against FXIIa and thrombin revealing potent dual and selective inhibitors of FXIIa and/or thrombin. The results of this screening have been disclosed by us earlier and will not be repeated here. [3f] The screening was limited only to the variations on the inhibitors' acyl fragment (FR2), leaving the aminotriazole core (FR1) unchanged and therefore unexplored. Now, knowing the structures of the most promising acyl moieties, we will retain them unchanged while varying the other structural features of the inhibitor, particularly its aminotriazole core.

Conventional Synthesis of 1H-1,2,4-triazol-5-amines (FR1)
To generate a library of structurally diverse 1H-1,2,4-triazol-5amines (FR1) exhibiting non-acylated triazole N-atom, syntheses shown in Schemes 1-3 were performed. The rationale behind the selection of substituents in the 3-position of the triazole core was based on the idea that the triazole core should be extended toward enzymes' S1' and S3-S4 substrate binding sites, which were previously not addressed by this class of compounds, to improve the inhibitors' binding affinity toward thrombin and/or FXIIa.
Aminotriazoles 15 a-k were synthesized in a fusion reaction between aminoguanidine hydrochloride and carboxylic acids 14 a-k at 200°C (Scheme 1). The reaction proceeds via intermediate amidoguanidines, which subsequently, without isolation undergo a cyclocondensation reaction affording desired products 15 a-k. [3f] Similarly, aminotriazoles 15 l-s were accessed reacting corresponding acid chlorides with aminoguanidine salts under basic conditions (Scheme 1). Other aminotriazoles 15 t-w were obtained via the treatment of hydrazides 18 t-w with S-methylisothiouronium sulfate, which also produces intermediate amidoguanidines followed by their [a] Reaction conditions: in one well of 96-well plate, in DMSO-d 6 (120 μL), 7 (3 mg, 18.6 μmol) reacted with a carboxylic acid (1 equiv.) in the presence of indicated coupling reagent and base at the indicated temperature and reaction time. [b] As measured by semi-q 1 H-NMR. [c] Micro magnetic stirring bars implemented into each well of the well plate were used; [d] 1.1 equiv. of carboxylic acid was used; [e] The reaction was performed under an N 2 atmosphere cyclization (Scheme 1). The necessity of having all three synthetic strategies toward aminotriazoles 15 a-w was dictated by unequal reactivity and different physicochemical properties of starting materials (carboxylic acids and their derivatives). This allowed to access aminotriazoles possessing aromatic, heteroaromatic, aliphatic, and cycloaliphatic substituents of different size and electronic properties in 3-position of the triazole core. Additionally, the exocyclic primary amino group of two aminotriazoles 15 a and 15 v was varied using a reductive amination reaction with benzaldehyde affording compounds 19 a and 19 v bearing an extended exocyclic amino group (Scheme 1).
To access 1H-1,2,4-triazol-5-amines 24 a-d exhibiting a quinoxalin-2-yl moiety as an alternative substituent in 3position of the triazole core, synthesis shown in Scheme 2 was performed. This extension of the pyridyl moiety might lead to additional interactions with S1' and S2-S3 binding sites of FXIIa or thrombin and improve the compounds' affinity or selectivity e. g., toward FXIIa over thrombin, as thrombin exhibits a more narrow S2 binding site. [3f,14] For this, commercially available βketoesters 20 a-c were brominated and then condensed with the symmetric o-phenylenediamines to afford fused heterocyclic esters 22 a-d. [15] Esters 22 a-d were then converted into hydrazides 23 a-d, the consecutive treatment of which with Smethylisothiouronium sulfate and KOH afforded 1H-1,2,4triazol-5-amines 24 a-d.
With the same purpose to access fragments capable of reaching the enzymes' S1' and/or S2-S3 binding sites, aminotriazoles 29 a-g, possessing an extended pyridyl-moiety, were synthesized as shown in Scheme 3. For this purpose, condensation of ketones 25 a-g with N,N-dimethylformamide dimethyl acetal (DMF-DMA) was performed to get enaminones 26 a-g, which then reacted in a three-component cyclocondensation reaction with a β-ketoester and ammonium acetate affording substituted pyridines 27 a-g. [16] Then, intermediates 27 a-g were treated with hydrazine monohydrate to produce hydrazides 28 a-g, which subsequently were converted into 1H-1,2,4triazol-5-amines 29 a-g using S-methylisothiouronium sulfate and KOH.
Other 1H-1,2,4-triazol-5-amines used for the subsequent microscale parallel synthesis were available from our in-house library of aminoazoles prepared as previously reported. [
The carboxylic acids 30 and 31 were selected due to their unique structural features allowing, on the one hand, to access inhibitors with an improved selectivity toward FXIIa [3f,18] and, on the other hand, to simplify the analysis of the microscale reaction mixtures by semi-q 1 H-NMR. The proton signals of the methine group of 30 and of the methylene bridge of 31 are characteristic, do not overlap with other signals, and experience a low-field shift upon successful coupling reaction (e. g., the exemplary microscale reaction mixture shown in Figure 6). Moreover, both carboxylic acids being connected to the aminotriazoles, add three-dimensional character to the resulting molecules thereby improving their drug-like properties.
Right after the reaction was finished (3 h), 20 μL of the reaction mixture was taken and diluted 1/100 with DMSO (solution A). Solution A was stored at À 20°C until the subsequent biological activity screening, whereas the rest of the reaction mixture (100 μL) was diluted with 600 μL DMSO-d 6 (solution B) and submitted directly for semi-q 1 H-NMR to determine the reaction conversion. The % of each aminotriazole conversion (94 microscale reactions) into the corresponding amide is shown in Figure 7. In general, the majority of aminotriazoles were successfully converted into desired N-acylated products with only a few showing low conversion under applied conditions. The average conversion of aminotriazoles into the corresponding amides was higher for tetrahydronaphthalene-1-carboxylic acid compared to 2-iodophenylacetic acid ( Figure 7). Sufficient conversion was observed in 90 of 94 microscale reactions allowing further biological activity screening of 90 formed acylated aminotriazoles. Reaction mixtures with a conversion rate < 10 % were excluded.
After the reaction conversion was determined by the analysis of the NMR spectrum of each reaction mixture, the concentration of the acylated product in solution A was calculated. The frozen samples (solution A) were thawed and diluted a second time with a calculated amount of DMSO to set the concentration of each acylated product to 76 μmol/L. This allowed screening of the generated library of 90 acylated products against FXIIa and thrombin at two certain concentrations 1 μM and 100 nM. For this purpose, the % of enzymes' inhibition was estimated utilizing fluorogenic substrates (Figure 8).

Conventional Synthesis and Structure of Acylated Aminotriazoles
Based on the screening results of the microscale reaction mixtures, the acylated 1H-1,2,4-triazol-5-amines were clustered according to their ability to inhibit FXIIa and thrombin into four groups, namely high-, medium-, low-, and inactive inhibitors. To validate the parallel synthesis-based screening approach, apart from the most promising hit compounds, representatives of each activity cluster have been selected and synthesized, using the conventional synthetic method shown in Scheme 4. For this, aminotriazoles 15, 24, and 29 were subjected to amide coupling reactions with carboxylic acids 30 and 31 to afford desired N-acylated products 32 a-e and 33 a-j.
Before further progressing to the biological activity testing of pure compounds, their structure should have been explicitly confirmed. Exhibiting annular tautomerism, 1,2,4-triazol-5amines exist in three tautomeric forms. [17a] Therefore, the acylation of 1,2,4-triazol-5-amines may theoretically result in four acylated regioisomers, three of which arise from the annular tautomers and one from the possible acylation of the primary exocyclic amino group. To unambiguously prove the regioselectivity of the acylation process, we performed the Xray crystallographic analysis of the reaction product 33 g ( Figure 9).
The X-ray analysis confirmed that the synthesized acylated aminotriazole 33 g possess the acyl moiety in the N1-position of its triazole core. Also, the absolute configuration of the asymmetric C-atom of the acyl moiety was confirmed to be (S)configured. Concerning other structural features, it is worth mentioning that the aromatic quinoline and aminotriazole moieties of 33 g are practically coplanar, whereas the compound's (S)-1,2,3,4-tetrahydronaphthoyl fragment is out of their plane. The cyclohexene ring adopts a half-chair conformation and adds certain three-dimensional properties to the molecule. Interestingly, in the crystal packing, two different half-chair conformers of 33 g were observed (Supporting Information, Figure S1).  Figure 7. The extent of the enzymes' inhibition was quantified in percentage of residual protease activity. Crossed out cells indicate that no acylated product was formed (< 10 %) according to semi-q 1 H-NMR and the mixture was not tested. Dabigatran (Dabi) was used as a positive control for thrombin inhibition and as a negative control for FXIIa inhibition.

Serine Protease Inhibition by Pure Acylated Aminotriazoles
Selected from the microscale reactions screening, pure acylated 1H-1,2,4-triazol-5-amines (Scheme 4) were tested for their ability to inhibit the proteolytic activity of two main targeted enzymes FXIIa and thrombin (Table 2). Also, the compounds' selectivity against two structurally related serine proteases namely FXa and trypsin was studied ( Table 2).
Performed enzymatic assays showed that those compounds, which significantly reduced the enzymatic activity in the screening, appeared to be active inhibitors of FXIIa and/or thrombin. E.g., aminotriazoles 32 a-c, and 33 a,c,g,h, which inhibited FXIIa activity by more than 50 % at 100 nM in crude mixtures, showed the IC 50 values toward FXIIa close to 100 nM or lower when tested as pure compounds (Table 2). Among them, aminotriazole 33 g acylated with tetrahydronaphthalene-1-carboxylic acid and possessing quinolinyl moiety in 3-position of its triazole core was found to be the most potent FXIIa inhibitor with the IC 50 = 33 nM, which is in a good agreement with its 94 % of FXIIa inhibition (at 100 nM) in screening. Similarly, isoquinoline derivative 33 i, being the most potent thrombin inhibitor in the screening (62 % at 100 nM), indeed showed the lowest IC 50 value of 38 nM toward thrombin ( Table 2).
Tests of pure compounds also confirmed that the developed screening method allows not only to find potent compounds but also to differentiate and discard low active and inactive inhibitors. For instance, aminotriazoles 32 d,e, and 33 e,f,j showed low activity in the microscale reactions screening and subsequently were proved to be only micromolar inhibitors or no inhibitors of FXIIa and thrombin when tested as pure compounds (Table 2). Additionally, tests of individual compounds proved that the developed screening method can efficiently predict compounds' selectivity. Thus, for instance, aminotriazoles 32 c and 33 g potently inhibited FXIIa at 100 nM in the screening by 75 % and 94 %, respectively, showing subsequently the IC 50 values of 65 nM and 33 nM, respectively ( Table 2). At the same time, being screened against thrombin as crude mixtures, these two compounds inhibited thrombin by 15 % and 5 %, respectively, subsequently showing thrombin inhibition in the micromolar range as pure compounds (Table 2). Also, aminotriazole 33 c, which inhibited both FXIIa and thrombin during the screening (80 % and 36 % at 100 nM, respectively), was proved to be a dual inhibitor with IC 50 = 106 nM (FXIIa) and 143 nM (thrombin) ( Table 2).
Interestingly, 2-iodophenylacetic acid derivatives 32 a-d appeared to be more potent and selective FXIIa inhibitors than their counterparts 33 a-c possessing S-configured tetrahydronaphthoyl moiety (Table 2). This finding once again underlines that the structure of both fragments, aminotriazole core (FR1) and the acyl moiety (FR2) influences the compounds' inhibitory properties. It should also be noted that synthesized acylated aminotriazoles showed no off-targeting effect against two other serine proteases (IC 50 > 5 μM), FXa and trypsin, despite these enzymes' structural similarity with FXIIa and thrombin. [14,19]

Post-Screening Optimization
Although compounds identified in the screening demonstrated promising inhibitory activity toward FXIIa and thrombin, there was still room for improvement, especially in terms of compounds' selectivity. We, therefore, designed post-screening structural modifications of the most promising N-acylated aminotriazoles. Particularly, the necessity of the methyl group on 3-methylquinoxalin-2-yl moiety of compound 33 h (122 nM inhibitor of FXIIa) was questioned. Its formal removal was suggested to be beneficial as structurally related compounds 33 c and 33 g having no such substituent were active FXIIa inhibitors (Table 2). Also, the analogs of aminotriazole 33 h, bearing the phenyl (A2) or the ethyl (A4) moiety in 3-position of Figure 9. X-ray crystal structure of FXIIa inhibitor N-acylated 1H-1,2,4-triazol-5-amine 33 g displaying the thermal ellipsoids at the 50 % probability level.  [c] inhibition % at 1 μM; [d] inhibition % at 5 μM.
their quinoxaline scaffold instead of the methyl group, were low active FXIIa inhibitors ( Figure 8). Apart from that, the FXIIa inhibitory activity of the quinoline derivative 33 g (FXIIa IC 50 = 33 nM) might be further improved upon its acyl moiety replacement with a more suitable variant. For instance, α-naphthoyl moiety, previously found as a promising acyl fragment, [3f] could increase the compound's ability to inhibit FXIIa. Similarly, the thrombin inhibitory activity of isoquinoline-derived compound 33 i (FIIa IC 50 = 33 nM) might be improved upon its acyl moiety replacement with a fragment better fitting into the active site of thrombin. E.g., benzoyl and β-naphthoyl acyl fragments were previously shown to be favorable for thrombin inhibition. [3f] To perform the formal removal of the methyl group of 33 h and access compounds exhibiting a quinoxaline scaffold, synthesis shown in Scheme 5 was performed. For this purpose, ophenylenediamine was condensed with D-fructose giving substituted quinoxaline 35, the tetraol side chain of which was then oxidized accessing quinoxaline-2-carboxylic acid (36). [20] The acid was esterified yielding ester 37, which was then converted into corresponding aminotriazole 38 using aminoguanidine hydrochloride. Finally, 38 was coupled with two carboxylic acids affording N-acylated aminotriazoles 39 a and 39 b (Scheme 5).
Quinoline and isoquinoline-derived aminotriazoles possessing alternative acyl fragments in N1-position of their core scaffold were obtained as shown in Scheme 6. For this, either a direct acylation reaction with acid chlorides or an amide coupling reaction was utilized affording desired N-acylated compounds 40 a,b and 41 a,c (Scheme 6).
Acylated aminotriazoles 39-41 obtained in the post-screening optimization step were tested against FXIIa, thrombin, FXa, and trypsin ( Table 3). The performed structural modifications generally improved parent compounds' inhibitory properties against targeted enzymes. Particularly, the formal removal of the methyl group of 33 h resulted in a quinoxaline 39 a showing a 3.7-fold improved FXIIa inhibitory activity (122 nM vs. 33 nM) without losing selectivity (Table 3). Further FXIIa inhibitory activity enhancement was reached when the (S)-tetrahydronaphthoyl fragment of 39 a was replaced with the α-naphthoyl moiety (39 b). The resulting compound 39 b demonstrated the lowest IC 50 value of 7 nM against FXIIa without off-targeting other tested serine proteases (IC 50 > 5 μM, Table 3). Likewise, the replacement of the tetrahydronaphthoyl moiety of 33 g (FXIIa IC 50 = 33 nM) with the benzoyl (40 a) or the α-naphthoyl moiety (40 b) decreased the parent compound's FXIIa IC 50 value to 13 nM and 8 nM, respectively. All three quinolines 33 g, 40 a, and 40 b, however, showed some off-targeting effect toward thrombin thus being dual FXIIa and thrombin inhibitors ( Table 2 and Table 3). The acyl moiety variation was also beneficial for the isoquinoline-derived thrombin inhibitor 33 i (FIIa IC 50 = 33 nM). The replacement of the original acyl moiety of 33 i by the β-naphthoyl fragment (41 c) slightly improved the compound's inhibitory activity toward thrombin (41 c, FIIa IC 50 = 25 nM) and made it a selective thrombin inhibitor (Table 3). It should, however, be noted that the β-naphthoyl fragment despite being a good fit for thrombin inhibition, forms the enzyme-acyl complex with a rather short half-life.

Anticoagulant Activity of Acylated Aminotriazoles
Selected acylated aminotriazoles were screened for their anticoagulant properties in two tests namely activated partial thromboplastin time (aPTT) and prothrombin time (PT). These tests allow distinguishing whether the intrinsic or the extrinsic blood coagulation pathway is affected by test compounds. The measurement results are shown in Figure 10.
In general, compounds showing the FXIIa and/or FIIa inhibitory activity influenced the plasma coagulation time in the aPTT test ( Figure 10). Among them, dual FXIIa/thrombin (IC 50 = 106/143 nM) inhibitor 33 c possessing a pyrazin-2-yl moiety in 3-position of its aminotriazole core and the Sconfigured tetrahydronaphthoyl residue as an acyl fragment showed the highest (2.7-fold) prolongation of aPTT. Two quinoline-derived FXIIa inhibitors 40 a and 40 b (FXIIa IC 50 = 13 nM and 8 nM, respectively) with moderate thrombin inhibitory properties (124 nM and 348 nM, respectively) also extended coagulation time 1.7-1.8-fold in aPTT test. Except for thrombin inhibitor 41 c, tested aminotriazoles had practically no influence on the PT. This implies that compounds predominantly affected the intrinsic pathway of the blood coagulation cascade having little to no influence on the extrinsic pathway. This finding is interesting and might be considered as a beneficial property of acylated aminotriazoles as inhibition of intrinsic pathway was recently recognized as a potentially safe approach in therapy of thrombosis. [6a]

Mechanism of Inhibition
Based on the previous studies, synthesized N-acylated aminotriazoles were expected to inhibit FXIIa and thrombin via a covalent reversible mechanism. [3c,f] To prove that the com-pounds undergo a covalent interaction with the targeted enzymes, we performed a mass-shift experiment with FXIIa and inhibitor 33 g (Figure 11).
In the experiment, native FXIIa was incubated for 15 min with the excess of inhibitor 33 g followed by size-exclusion chromatography (SEC) and the enzyme-inhibitor complex mass analysis using electrospray ionization mass spectrometry (ESI-MS). The measurement revealed that after the incubation, the initial mass of 29496.5 Da of FXIIa was shifted to 29654.7 Da. This mass shift (Δm = 158.2 Da) corresponds to the acyl moiety adduct of inhibitor 33 g to FXIIa confirming the covalent mechanism of FXIIa inhibition. As only a single acyl moiety transfer was observed, this process is limited to a specific site of the enzyme. Most likely, this site is the serine protease active site comprising the activated Ser195 capable of a nucleophilic attack on the inhibitor's carbonyl C-atom ( Figure 11). Being acylated, serine protease temporarily (shown previously [3f] ) loses its proteolytic activity.

Conclusion
Collectively, we report a microscale parallel synthetic approach allowing for fast access to libraries of structurally diverse Nacylated aminotriazoles and screening of their inhibitory activity against selected serine proteases. This approach is simple, purification-free, and relies on inexpensive materials. Utilizing this method, a series of FXIIa and/or thrombin inhibitors were successfully identified. Subsequently performed post-screening structural optimization allowed to obtain more potent FXIIa and thrombin inhibitors, e. g. aminotriazoles 39 b (FXIIa IC 50 = 7 nM) and 41 c (FIIa IC 50 = 25 nM), which also showed no off-target effect against other physiologically relevant serine proteases. Some of the synthesized N-acylated aminotriazoles exhibited anticoagulant properties in vitro influencing the intrinsic blood  coagulation pathway but not the extrinsic coagulation. This is notable as extrinsic cascade triggered by tissue factor is physiologically important to prevent bleeding, whereas intrinsic pathway initiated by FXIIa is associated with thrombosis. Performed herein mechanistic studies of FXIIa inhibition suggested that synthesized N-acylated aminotriazoles are covalent inhibitors of FXIIa. Despite their prominent in vitro inhibitory activity against FXIIa and thrombin, synthesized compounds demonstrate weaker anticoagulant properties in plasma coagulation tests compared to dabigatran and rivaroxaban. Further structural variations on the 1H-1,2,4-triazol-5amine-scaffold are required to obtain compounds with more potent anticoagulant properties before moving toward their in vivo tests.

Determination of Microscale Reaction Conversions by semi-q 1 H-NMR.
The NMR spectra of microscale parallel synthesis samples were recorded at 25°C using Agilent DD2 400 MHz NMR spectrometer and analyzed with MestReNova software (version 12.0.3-21384, Mestrelab Research S.L.). For (S)-1,2,3,4-tetrahydronaphthalene-1carboxylic acid (30) amides (N-acylated products), the characteristic proton resonance signal of the methine group (t, J = 6.3 Hz, 1H, typically found between 4.60 and 5.10 ppm) was used for the calculations. For 2-iodophenylacetic acid (31) amides (N-acylated products), the characteristic proton resonance signal of the methylene bridge (s, 2H, typically found between 4.10 and 4.65 ppm) was used for the calculations. These proton signals of the product and the signals of the aromatic protons of DMAP (m, 2H, at 6.72 ppm or m, 2H, at 8.13 ppm) were identified, integrated, and normalized. The normalized integrals of the product protons (Int P ) and of DMAP (Int R ) were used to calculate the reactions' conversion (C,%) considering the used reaction equivalents (eq R ) according to the following formula: C [%] = (Int P × eq R /Int R ) × 100. For the method development (Table 1), the purest proton resonance signals of the reaction product (13 a-e) and starting material (aminotriazole 7 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 (7 or a carboxylic acid) could be identified, the signals of DMAP (Int R ) were used instead as mentioned above.

Microscale Reactions Products Screening in the Enzymes Inhibition Assay.
After the reaction conversion was determined by semiq 1 H-NMR, frozen samples of the microscale parallel synthesis were defrosted at room temperature, 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 (1.25 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 N-acylated acylated 1,2,4triazol-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 % of inhibition at 1 μM and 100 nM for each sample. [3f] Analysis of the Covalent FXIIa-Inhibitor Complex by SEC/ESI-MS. The analysis of covalent FXIIa-inhibitor complex was performed utilizing SEC/ESI-MS as reported previously. [3f] Briefly, 10 μL of the stock solution (37.67 μM) of human plasma β-FXIIa (HFXIIAB, > 95 % purity; Molecular Innovations) were diluted with 90 μL of purified water. An aliquot of 46.3 μL was mixed with 3.7 μL of inhibitor 33 g solution (1 mM in DMSO) to prepare the enzyme-inhibitor solution (molar ratio of 1 : 20), which was then analyzed via SEC/ESI-MS. Sizeexclusion chromatography (SEC) was performed using a MAbPac SEC-1 analytical column (150 x 4 mm, 5 μm particle size, 300 Å pore size) from Thermo Fisher Scientific (Bremen, DE). 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. For chromatographic analysis of the sample solutions, isocratic elution was carried out using an aqueous ammonium acetate solution (10 mM, pH 6.8) as mobile phase for 15 min. The enzyme-inhibitor complex was 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, 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.