Novel Anthranilamide-Based FXa Inhibitors: Drug Design, Synthesis and Biological Evaluation

Factor Xa (FXa) plays a significant role in the blood coagulation cascade and it has become a promising target for anticoagulation drugs. Three oral direct FXa inhibitors have been approved by the FDA for treating thrombotic diseases. By structure-activity relationship (SAR) analysis upon these FXa inhibitors, a series of novel anthranilamide-based FXa inhibitors were designed and synthesized. According to our study, compounds 1a, 1g and 1s displayed evident FXa inhibitory activity and excellent selectivity over thrombin in in vitro inhibition activities studies. Compounds 1g and 1s also exhibited pronounced anticoagulant activities in in vitro anticoagulant activity studies.


Introduction
Thromboembolic diseases such as myocardial infarction (MI), pulmonary embolism (PE), deep vein thrombosis (DVT) and ischemic strokes are the major causes of mortality all over the world in the 21st century [1][2][3]. The typical method for treating and preventing these diseases is to use anticoagulant drugs [4]. For example, vitamin K antagonists (VKAs), low-molecular-weight heparins (LMWHs) and warfarin are validated in the prevention and treatment of these thrombotic disorders [5], but they still have many shortcomings in clinical applications including the inconvenience of frequent monitoring, interactions with many drugs and food, and slow onset action [6,7]. These shortcomings further accelerate the need for development of new oral anticoagulants with efficacy and safety.
Factor Xa (FXa), which is located at the junction of the intrinsic and extrinsic pathways in the coagulation cascade and catalyzes the conversion of prothrombin to thrombin, plays a pivotal role in the blood coagulation cascade and has become a promising target for anticoagulation effects [4,8]. It is well demonstrated that selective FXa inhibitors can decrease the generation of thrombin without affecting the existing thrombin level which means that selective FXa inhibitors can play an ideal antithrombotic effects without influencing normal hemostasis and decrease the risk of bleeding [9][10][11]. Three oral direct FXa inhibitors have been approved by the FDA for treating thrombotic diseases and several FXa inhibitors have entered the stage of clinical research or biological testing ( Figure 1) [2,[12][13][14]. However, they still have many drawbacks, such as drug interactions, narrow indications and long treatment duration [15]. Therefore, new antithrombotic drugs still need to be developed for addressing these issues. From the structures above, FXa inhibitors have three components which make up the pharmacophore: core scaffold, P1 and P4. This typical structure can help these molecules combine with FXa, P1 locates at the S1 pocket and P4 locates at the S4 pocket ( Figure 2) [16]. Presented if Figure 3 here are several examples can help us understand this more clearly [17][18][19][20][21][22][23]. Interestingly, we found that for both rivaroxaban and betrixaban employed a carboxamide group to connect the scaffold, P1 and P4. Based on analysis of the X-ray crystal structure of rivaroxaban complexed with human FXa, it was found that there are two hydrogen bonds formed between rivaroxaban and the residue Gly219 in FXa [12]. Similarly, betrixaban possesses two hydrogen bonds with residues Gly218 and Gly216 in FXa [24], so we hypothesized that the key factor for FXa inhibitors docked to human FXa might be the carboxamide group. Based on the structures of rivaroxaban, betrixaban and darexaban, we have designed a series of novel FXa inhibitors employing anthranilamide as the scaffold (Figure 4). Through metabolism of rivaroxaban in vivo [25], we can find that the main metabolic pathway is the hydrolysis of the amide bond. We reversed the carbonyl and amino connection order of betrixaban with the expectation that the designed compounds may display different toxicity data or participate in different metabolic pathways in humans. From the structures above, FXa inhibitors have three components which make up the pharmacophore: core scaffold, P1 and P4. This typical structure can help these molecules combine with FXa, P1 locates at the S1 pocket and P4 locates at the S4 pocket ( Figure 2) [16]. Presented if Figure 3 here are several examples can help us understand this more clearly [17][18][19][20][21][22][23]. From the structures above, FXa inhibitors have three components which make up the pharmacophore: core scaffold, P1 and P4. This typical structure can help these molecules combine with FXa, P1 locates at the S1 pocket and P4 locates at the S4 pocket ( Figure 2) [16]. Presented if Figure 3 here are several examples can help us understand this more clearly [17][18][19][20][21][22][23]. Interestingly, we found that for both rivaroxaban and betrixaban employed a carboxamide group to connect the scaffold, P1 and P4. Based on analysis of the X-ray crystal structure of rivaroxaban complexed with human FXa, it was found that there are two hydrogen bonds formed between rivaroxaban and the residue Gly219 in FXa [12]. Similarly, betrixaban possesses two hydrogen bonds with residues Gly218 and Gly216 in FXa [24], so we hypothesized that the key factor for FXa inhibitors docked to human FXa might be the carboxamide group. Based on the structures of rivaroxaban, betrixaban and darexaban, we have designed a series of novel FXa inhibitors employing anthranilamide as the scaffold (Figure 4). Through metabolism of rivaroxaban in vivo [25], we can find that the main metabolic pathway is the hydrolysis of the amide bond. We reversed the carbonyl and amino connection order of betrixaban with the expectation that the designed compounds may display different toxicity data or participate in different metabolic pathways in humans. Interestingly, we found that for both rivaroxaban and betrixaban employed a carboxamide group to connect the scaffold, P1 and P4. Based on analysis of the X-ray crystal structure of rivaroxaban complexed with human FXa, it was found that there are two hydrogen bonds formed between rivaroxaban and the residue Gly219 in FXa [12]. Similarly, betrixaban possesses two hydrogen bonds with residues Gly218 and Gly216 in FXa [24], so we hypothesized that the key factor for FXa inhibitors docked to human FXa might be the carboxamide group. Based on the structures of rivaroxaban, betrixaban and darexaban, we have designed a series of novel FXa inhibitors employing anthranilamide as the scaffold (Figure 4). Through metabolism of rivaroxaban in vivo [25], we can find that the main metabolic pathway is the hydrolysis of the amide bond. We reversed the carbonyl and amino connection order of betrixaban with the expectation that the designed compounds may display different toxicity data or participate in different metabolic pathways in humans.

Synthesis
The synthetic routes used in this study are illustrated in Scheme 1. All the starting material are commercial available. As shown in Scheme 1, the acylation [26] of 3a-3b respectively with a series of

Synthesis
The synthetic routes used in this study are illustrated in Scheme 1. All the starting material are

Synthesis
The synthetic routes used in this study are illustrated in Scheme 1. All the starting material are commercial available. As shown in Scheme 1, the acylation [26] of 3a-3b respectively with a series of o-nitrobenzoyl chloride 2a-2d provided 4a-4h, which were subsequently reduced with Zn, NH 4 Cl to provide the corresponding anilines 5a-5h in satisfied yields. Acylation [26] of 5a-5h respectively with 6a-6c yielded the target compounds 1a-1x. provide the corresponding anilines 5a-5h in satisfied yields. Acylation [26] of 5a-5h respectively with 6a-6c yielded the target compounds 1a-1x.

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.9 nM, Ki = 22.0 nM; 1g = 23.0 nM, Ki = 16.4 nM; 1s = 76.2 nM, Ki = 54.4 nM; Km = 2.4). We also show an inhibition profile figure of the most potent compounds 1a, 1g and 1s in Figure 5.

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC 50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC 50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC 50 values at a micromole level no matter what kind of Ar 1 and Ar 2 in Table 1 they linked with. When the Ar 1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar 2 ) the anticoagulant activity was much better than in other cases (1a = 30.    was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.9 nM, Ki = 22.0 nM; 1g = 23.0 nM, Ki = 16.4 nM; 1s = 76.2 nM, Ki = 54.4 nM; Km = 2.4). We also show an inhibition profile figure of the most potent compounds 1a, 1g and 1s in Figure 5.  Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.9 nM, Ki = 22.0 nM; 1g = 23.0 nM, Ki = 16.4 nM; 1s = 76.2 nM, Ki = 54.4 nM; Km = 2.4). We also show an inhibition profile figure of the most potent compounds 1a, 1g and 1s in Figure 5.  Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.9 nM, Ki = 22.0 nM; 1g = 23.0 nM, Ki = 16.4 nM; 1s = 76.2 nM, Ki = 54.4 nM; Km = 2.4). We also show an inhibition profile figure of the most potent compounds 1a, 1g and 1s in Figure 5. from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.9 nM, Ki = 22.0 nM; 1g = 23.0 nM, Ki = 16.4 nM; 1s = 76.2 nM, Ki = 54.4 nM; Km = 2.4). We also show an inhibition profile figure of the most potent compounds 1a, 1g and 1s in Figure 5.  Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.9 nM, Ki = 22.0 nM; 1g = 23.0 nM, Ki = 16.4 nM; 1s = 76.2 nM, Ki = 54.4 nM; Km = 2.4). We also show an inhibition profile figure of the most potent compounds 1a, 1g and 1s in Figure 5. using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30. 9    All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30. 9   All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.9 nM, Ki = 22.0 nM; 1g = 23.0 nM, Ki = 16.4 nM; 1s = 76.2 nM, Ki = 54.4 nM; Km = 2.4). We also show an inhibition profile figure of the most potent compounds 1a, 1g and 1s in Figure 5.

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30. 9   All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.9 nM, Ki = 22.0 nM; 1g = 23.0 nM, Ki = 16.4 nM; 1s = 76.2 nM, Ki = 54.4 nM; Km = 2.4). We also show an inhibition profile figure of the most potent compounds 1a, 1g and 1s in Figure 5.

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2)

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.  provide the corresponding anilines 5a-5h in satisfied yields. Acylation [26] of 5a-5h respectively with 6a-6c yielded the target compounds 1a-1x.

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.  provide the corresponding anilines 5a-5h in satisfied yields. Acylation [26] of 5a-5h respectively with 6a-6c yielded the target compounds 1a-1x.

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.    provide the corresponding anilines 5a-5h in satisfied yields. Acylation [26] of 5a-5h respectively with 6a-6c yielded the target compounds 1a-1x.

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.9 nM, Ki = 22.0 nM; 1g = 23.0 nM, Ki = 16.4 nM; 1s = 76.2 nM, Ki = 54.4 nM; Km = 2.4). We also show an inhibition profile figure of the most potent compounds 1a, 1g and 1s in Figure 5.  provide the corresponding anilines 5a-5h in satisfied yields. Acylation [26] of 5a-5h respectively with 6a-6c yielded the target compounds 1a-1x.

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.9 nM, Ki = 22.0 nM; 1g = 23.0 nM, Ki = 16.4 nM; 1s = 76.2 nM, Ki = 54.4 nM; Km = 2.4). We also show an inhibition profile figure of the most potent compounds 1a, 1g and 1s in Figure 5.  . We also show an inhibition profile figure of the most potent compounds 1a, 1g and 1s in Figure 5. . We also show an inhibition profile figure of the most potent compounds 1a, 1g and 1s in Figure 5. from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.9 nM, Ki = 22.0 nM; 1g = 23.0 nM, Ki = 16.4 nM; 1s = 76.2 nM, Ki = 54.4 nM; Km = 2.4). We also show an inhibition profile figure of the most potent compounds 1a, 1g and 1s in Figure 5. from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.9 nM, Ki = 22.0 nM; 1g = 23.0 nM, Ki = 16.4 nM; 1s = 76.2 nM, Ki = 54.4 nM; Km = 2.4). We also show an inhibition profile figure of the most potent compounds 1a, 1g and 1s in Figure 5.

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several  designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.9 nM, Ki = 22.0 nM; 1g = 23.0 nM, Ki = 16.4 nM; 1s = 76.2 nM, Ki = 54.4 nM; Km = 2.4). We also show an inhibition profile figure of the most potent compounds 1a, 1g and 1s in Figure 5.

In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity, using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several  designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted scaffold (1j-1l, 1v-1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group was pyridin-2(1H)-one, the compounds (1a-1l) demonstrated that the affinity with FXa ranking was electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant activity was much better than in other cases (1a = 30.    To evaluate the inhibitory activity against FXa of compounds 1a, 1g and 1s more accurately, these compounds were chosen to assess degree of selectivity versus thrombin and the extension of the prothrombin time (PT). Compounds 1a, 1g and 1s showed no inhibition effect on thrombin, with IC 50 values far higher than 10 µM. They showed similar selectivity against thrombin as rivaroxaban which is far more than 6.9 µM [27]. The prothrombin time (PT) assay results are shown in Table 2, where compounds 1g and 1s also show good anticoagulant activity, judging by their 2ˆPT value of 19.7 and 24.2 µM in rat plasma and 12.8 µM and 10.4 µM in human plasma. Computer-based docking simulation studies were used to analyze the binding mode of compounds 1g and 1s at the active site of FXa. As shown in Figure 6, the pyridine moiety of 1g and morpholino unit of 1s are located at the sides of the phenyl groups of Tyr99 and Phe174 of the S4 aryl binding pocket, while its aryl ring is oriented perpendicularly, extending across the face of Trp215. Two hydrogen bonds are formed between 1g and the residue Gly216 and Gly218 in FXa and one hydrogen bond is formed between 1s and the amino acid Gly218 of FXa. In the S1 pocket of FXa, there is an interaction between the chlorine substituent of the thiophene moiety and the aromatic ring of Tyr228 at the bottom of the S1 pocket. They thus show similar interactions with FXa as rivaroxaban reported before [12]. Computer-based docking simulation studies were used to analyze the binding mode of compounds 1g and 1s at the active site of FXa. As shown in Figure 6, the pyridine moiety of 1g and morpholino unit of 1s are located at the sides of the phenyl groups of Tyr99 and Phe174 of the S4 aryl binding pocket, while its aryl ring is oriented perpendicularly, extending across the face of Trp215. Two hydrogen bonds are formed between 1g and the residue Gly216 and Gly218 in FXa and one hydrogen bond is formed between 1s and the amino acid Gly218 of FXa. In the S1 pocket of FXa, there is an interaction between the chlorine substituent of the thiophene moiety and the aromatic ring of Tyr228 at the bottom of the S1 pocket. They thus show similar interactions with FXa as rivaroxaban reported before [12].

General Information
Reagents and solvents were obtained from commercial suppliers and used as received without further purification. All reactions were monitored by thin layer chromatography. 1 H-NMR spectra (400 MHz) were recorded for DMSO-d6 solutions on an AV400 NMR (Bruker, Billerica, MA, USA), MS were measured on a Finnigan LCQ Mass (Thermo Fisher Scientific, Cambridge, MA, USA), HRMS were measured on a miorOTOF-QII instrument (Bruker Daltonics, Billerica, MA, USA) and melting

General Information
Reagents and solvents were obtained from commercial suppliers and used as received without further purification. All reactions were monitored by thin layer chromatography. 1 H-NMR spectra (400 MHz) were recorded for DMSO-d 6 solutions on an AV400 NMR (Bruker, Billerica, MA, USA), MS were measured on a Finnigan LCQ Mass (Thermo Fisher Scientific, Cambridge, MA, USA), HRMS were measured on a miorOTOF-QII instrument (Bruker Daltonics, Billerica, MA, USA) and melting points (uncorrected) were determined on a YRT-3 Melting Point Tester (Precision Instrument of Tianjin University, Tianjin, China).

Chemistry
2-Nitro-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (4a). To a stirred solution of 1-(4-aminophenyl) pyridin-2(1H)-one (3a) (0.90 g, 4.8 mmol), K 2 CO 3 (0.80 g, 5.8 mmol) and DMAP (0.05 g, 0.4 mmol) in THF (20 mL), solution of 2-nitrobenzoyl chloride (2a) (1.15 g, 6.24 mmol) in THF (5 mL) was added at room temperature and the mixture was refluxed for 2 h. The reaction mixture was cooled down to room temperature and concentrated under reduced pressure. Then water (100 mL) was added to the mixture and stirred for 10 min at room temperature. The resulting precipitate was collected by filtration.  2-Amino-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (5a). To a 250 mL round bottom flask, 4a (1.40 g, 3.9 mmol), zinc powder (2.05 g, 31.2 mmol), NH 4 Cl (2.11 g, 39 mmol), methanol (30 mL), THF (30 mL) and water (15 mL) were added. The mixture stirred at 40˝C for 2 h. The reaction mixture was filtered, washed with DMF and the filtrate was concentrated under reduced pressure. Then water (200 mL) was added to the mixture and stirred for 0.5 h. The residue was filtered and washed with water to yield the title compound as a white solid product (1.13 g, 89%).  After incubated at 37˝C for 5 min, 8 µL of FXa substrate solution (3.5 mM) was added and then incubated at 37˝C for 25 min. The FXa activity was measured at 405 nm using a SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA). The IC 50 was calculated by the software named SPSS (IBM, North Castle, NY, USA) and the Probit function in it. Vitro of 1a, 1g and 1s The inhibition of thrombin was measured using human FIIa (Hyphen BioMed, Paris, France) and chromogenic substrate CS-01(38) (Hyphen BioMed, Paris, France) in 384-well microtiter plates at room temperature. The compounds 1a, 1g and 1s and Rivaroxaban were dissolved in DMSO to a concentration of 10 mM and then serially diluted to spanning a range of 10 µM to 100 µM, respectively. 2 µL of FIIa (3 NIH/mL), 20 µL of Tris buffer (adjust to pH 7.4 with HCl) containing 0.3 M NaCl and 50 mM Tris and 2 µL of test compound were added to the well, respectively. The negative control was composed of the same mixed solutions except replacing test compound with DMSO. The positive control was composed of the same mixed solutions except replacing test compound with Rivaroxaban. After incubated at 37˝C for 5 min, 3 µL of FIIa substrate solution (4 mM) was added and then incubated at 37˝C for 25 min. The FIIa activity was measured at 405 nm using a SpectraMax M5 (Molecular Devices).

Prothrombin Time (PT) Assay
A commercially available automatic coagulometer (Steellex Science Instrument Co., Ltd., Beijing, China) was employed to measure PT. The clotting times were also measured using the instrument itself, in accordance with the manufacturer's instructions. Increasing concentrations of inhibitor or solvent were added to rat (Sprague-Dawley rats, Shanchuanhong Experimental Animals Co., Ltd., Tianjin, China) and human (29 Years old, male, Chinese) plasma and incubated for 3 min at 37˝C. Prothrombin time (PT) was determined by automatic coagulometer.

Docking Simulation Study
FXa structure was selected from the protein data bank (PDB code: 2xbv) and prepared using Protein Preparation Wizard in Schrödinger package, including assigning bond orders, adding hydrogen atoms, deleting water molecules, creating disulfide bonds and capping terminals. The original ligand of the protein structure-XBV was used as the docking center to generate the receptor grid parameters. The box size was set as 12 Å. Compounds 1g and 1s were prepared using the LigPrep module in Schrödinger. Epik method was used to determine possible ionization state of ligands at pH 7.0˘2.0 and low-energy conformers were produced using OPLS-2005 force field. Molecular docking calculations were performed by using Glide module with default parameters at standard precision in Schrödinger.

Conclusions
In this study, we designed and synthesized a series of novel potent FXa inhibitors based on the anthranilamide scaffold. In vitro inhibition activity studies showed that compounds 1a, 1g and 1s displayed evident FXa inhibition and excellent selectivity over thrombin. Compounds 1g and 1s also exhibited pronounced anticoagulant activity in in vitro anticoagulant activity studies. Further docking simulation study also disclosed that the interaction of compounds 1g and 1s with FXa was very similar to that of rivaroxaban. Therefore, compounds 1g and 1s with an anthranilamide scaffold could be considered as lead compounds for exploring new FXa inhibitors with better medicinal effects and further modification of this structure for better antithrombotic activity is still in progress.