Physicochemical Properties
| Molecular Formula | C30H35N5O7S |
| Molecular Weight | 609.7 |
| Exact Mass | 609.225719 |
| CAS # | 911683-33-5 |
| Related CAS # | 184770-78-3; |
| PubChem CID | 133082220 |
| Appearance | Typically exists as solid at room temperature |
| Hydrogen Bond Donor Count | 5 |
| Hydrogen Bond Acceptor Count | 10 |
| Rotatable Bond Count | 10 |
| Heavy Atom Count | 43 |
| Complexity | 963 |
| Defined Atom Stereocenter Count | 1 |
| SMILES | CN(C1CCCC1)C(=O)[C@H](CC2=CC=C(C=C2)/C(=N/N)/N)NS(=O)(=O)C3=CC4=CC=CC=C4C=C3.C(=C\C(=O)O)\C(=O)O |
| InChi Key | AERYWTWBOYIUJS-GCCVQAHUSA-N |
| InChi Code | InChI=1S/C26H31N5O3S.C4H4O4/c1-31(22-8-4-5-9-22)26(32)24(16-18-10-12-20(13-11-18)25(27)29-28)30-35(33,34)23-15-14-19-6-2-3-7-21(19)17-23;5-3(6)1-2-4(7)8/h2-3,6-7,10-15,17,22,24,30H,4-5,8-9,16,28H2,1H3,(H2,27,29);1-2H,(H,5,6)(H,7,8)/b;2-1-/t24-;/m0./s1 |
| Chemical Name | (2S)-3-[4-[(Z)-C-aminocarbonohydrazonoyl]phenyl]-N-cyclopentyl-N-methyl-2-(naphthalen-2-ylsulfonylamino)propanamide;(Z)-but-2-enedioic acid ; (S)-N-(cyclopentylmethyl)-3-(4-(hydrazinyl(imino)methyl)phenyl)-2-(naphthalene-2-sulfonamido)propanamide maleate |
| Synonyms | I51I983CDF; UNII-I51I983CDF; LB-30057; PD-172524; 911683-33-5; Benzenecarboximidic acid, 4-((2S)-3-(cyclopentylmethylamino)-2-((2-naphthalenylsulfonyl)amino)-3-oxopropyl)-, hydrazide, (2Z)-2-butenedioate (1:1); LB30057; LB 30057; |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | Thrombin (IC50 = 0.38 nM) |
| ln Vitro |
LB-30057 had excellent thrombin inhibitory activity (Ki = 1.1 nM and 0.38 nlvl against bovine and human thrombins, respectively) and excellent selectivity over other trypsin-like serine proteases (Ki = 47.9, 27.2, 1.1, and 3.2,~M against human plasmin, tPA, factor Xa, and bovine trypsin, respectively).5 Interestingly, the water solubility of LB-30057(>> 10 mg/ml) was much higher than that of 1. It is not clear how the structural difference between N-cyclopentyl-N-methylamide and hexamethyleneimide causes the large difference in water solubility. [1] In order to comprehend the structure-activity relation, the crystal structure of LB-30057-thrombin complex was determined. The structure of LB-30057 bound at the active site of thrombin is shown in Figure 2. The naphthyl group in the D-pocket is found in close contact with the side chain of Ile 174 (closest approach 3.4A). Other close contacts (under 3.7A) are with the side chains of Trp 215 and Tyr 60A. The cyclopentyl group is in the cis position of the amide bond and makes tight van der Waals interaction with Trp 60D (3.6& Tyr 60A (3.7A) and Lys 60F (3.8 A). It appears that cyclopropyl group of 2 is too small to provide this kind of interaction, while cyclohexyl group of 5 would not fit into the pocket very well. The methyl group also contributes to the binding affinity considerably, through the hydrophobic interactions with Leu 99 (3.7A) and His 57 (4.011). In contrast to the methyl group, hydrophilic groups were unfavorable substituents. Thus, this crystal structure demonstrates that LB-30057 efficiently interacts with active site of thrombin at D- and P-pockets and that the N-cyclopentyl-N-methylamide moiety is a good P-pocket binder. [1] The potential species differences of anticoagulation effects of LB-30057 and Inogatran were determined in human, rabbit, and dog plasma in vitro (Table 2). In the PT and aPTT assays, human plasma was more sensitive to LB-30057 than rabbit and dog plasma. In the TT test, the rank of order for sensitivity to LB-30057 is dog>human>rabbit. Inogatran displayed a similar pro®le to that of LB-30057 in these in vitro coagulation assays [2]. |
| ln Vivo |
The antithrombotic efficacy of LB-30057 was examined in a rat venous thrombosis model: With 5 and 1 mg/kg intravenous bolus administrations, thromboplastin-induced thrombus formation was inhibited 90% and 50%, respectively.7 In a dog arterial thrombosis model,” intravenous bolus administration of 10 mg/kg LB30057 resulted in 2-fold increase in mean occlusion time. The oral bioavailability of LB30057 was 58% with the corresponding elimination half-life of 112 min at 10 mg/ml dose in dogs.g LB-30057, a potent, selective, and orally bioavailable thrombin inhibitor, is a promising antithrombotic agent and its further pharmacological evaluation is under way. [1] LB-30057 (CI-1028) is a novel, orally bioavailable, direct thrombin inhibitor with a Ki of 0.38 nM against human thrombin. The effects of LB-30057 on thrombus formation and hemostasis were evaluated in a veno-venous shunt model of thrombosis in rabbits, and compared with Inogatran, another direct inhibitor of thrombin. Each compound was studied at 5 or 6 different doses with 5 or 6 rabbits in each group. After administration as a bolus i.v. injection followed by continuous infusion, both LB-30057 and inogatran dose-dependently inhibited thrombus formation, which was measured as an increase in time to occlusion (TTO) and a decrease in thrombus weight. Both compounds also improved vena caval blood flow and reduced the overall incidence of thrombotic occlusion. LB-30057 significantly prolonged TTO from 23 +/- 4 min (before dose) to 110 +/- 10 min at the highest dose (0.7 mg/kg + 47 microg/kg/min) (p < 0.001), and reduced thrombus weight from 57 +/- 2 mg to 15 +/- 5 mg (p < 0.001). Occlusive thrombus formed in only one of six rabbits that received the highest dose of LB-30057 (vs. 13/13 in the control group, p < 0.01). At the dose that produced the maximum antithrombotic effect (0.7 mg/kg + 47 microg/kg/min), LB-30057 increased aPTT and bleeding time approximately 2-and 2.5-fold above baseline, respectively. On a gravimetric basis, LB-30057 and inogatran displayed comparable in vivo antithrombotic efficacy. When compared to equally effective anti thrombotic doses of Inogatran, LB-30057 caused less prolongation in aPTT, had no effect on PT, and tended to have less of effect on bleeding time. These results indicate that LB-30057 is an effective antithrombotic compound and it appears to have a better benefit/risk profile than inogatran in this experimental model [2]. The blood flow passing through the shunt and vena cava was continuously measured throughout the experiment. The firrst shunt was inserted before treatment in all groups, and blood flow decreased progressively and reached zero within 10 to 20 min. In the animals that received either LB-30057or inogatran, the blood flow in the second shunt was improved in a dose-dependent manner when compared to their corresponding ®rst shunt, as well as the control group (Figures 2 and 3). The AUC (area under the curve) was calculated using the average blood ¯ow over time and the duration of potency from each shunt. LB-30057 and inogatran had similar effects on blood flow (Figure 3), which indicate a comparable potency on the dynamic course of thrombus formation [2]. All of the ®rst and second shunts in the control group of rabbits formed occlusive thrombi, and there was no statistically significant difference in time to occlusion and thrombus weight between firrst and second shunts (Figures 4 and 5). As shown in Figures 4 and 5, both LB-30057 and inogatran dose-dependently prolonged the time to occlusion and reduced the incidence of occlusion in the second shunts. The net thrombus weight also was reduced by the two compounds in a dose-dependent manner (Figures, 4 and 5). The effects of LB-30057 and inogatran on time to occlusion and net thrombus weight are summarized and compared on a gravimetric basis (Figure 6). The total dose is the sum of the bolus dose and the dose infused over 140 min for each group. The dose-response curves of the two compounds were comparable at the doses tested (Figure 6) [2]. Ex vivo coagulation parameters at 60 min post-infusion were also utilized to analyze systemic anticoagulation produced by LB-30057 and inogatran. While both compounds caused comparable changes in TT, inogatran prolonged PT and aPTT to a greater extent than LB-30057 in the same dose range (Figure 7). Thus, at a given TT change, there is a greater increase of aPTT in the animals that received inogatran than LB-30057, i.e., the ratio of aPTT vs. TT is different between the two compounds. At equivalent antithrombotic doses, inogatran and LB30057 caused similar prolongation in TT, but in the inogatran group there were greater prolongations in aPTT, and a pronounced increase in PT that does not occur in the LB30057 group (Figure 7) [2]. |
| Enzyme Assay |
In vitro plasma clotting assays [2] Blood samples were collected from normal human volunteers, New Zealand white rabbits, and mongrel dogs. Plasma was prepared from blood samples by centrifugation at 2000 g for 10 min. LB-30057 and Inogatran at various concentrations were mixed with plasma in vitro and incubated for 10 min at 37C before the assays. PT, aPTT, and TT were determined by using an ST4 Clot Detection System as described above. Concentrations reported are the concentrations of the drug in the plasma prior to assaying. Human thrombin was purified essentially according to the procedure described by Fenton et al. Thrombin crystals grown in the presence of hirugen and a weak inhibitor (not described here) according to the procedure reported by Qiu et al, were soaked overnight in the buffer containing 2 mM LB-30057. The completeness of inhibitor exchange was confirmed later from the electron density. X-ray diffraction data up to 2A resolution were collected using MacScience DIP-2020 imaging plate system, with mirror-focused CuK CT X-ray radiation generated by a MacScience MXO6HF rotating anode generator operating at 50kV, 90mA. The space group was C2 with cell dimensions of a = 70.9, b = 72.0, c = 73.3% and P = 100.8”. A set of 60 frames were collected from a crystal with an oscillation ange of 1.5”, and the exposure time of 50 min. A total of 61,872 diffraction spots were measured and averaged to 22,879 unique reflections with an Rym = 6.5% using the Denzo package. The protein model was taken from the entry 1HGT of the Protein Data Bank (PDB) and refined using a simulated annealing protocol. The inhibitor molecule was built into the subsequent difference density and the whole complex including 234 water molecules were refined to an R = 19.9%. The r.m.s. deviations of bond distances and angles from ideal values were O.OlOA and 1.9“, respectively [1]. |
| Animal Protocol |
Experimental protocol [2] After completion of the firrst shunt, rabbits received either IV saline (n=13), Inogatran (n=30), or LB-30057 (n=30). A bolus IV injection was administered over fifteen seconds followed by a 1 ml/kg/h continuous infusion for 140 min. The second shunt was inserted twenty minutes after the start of the infusion. Six groups of rabbits (n=5 for each group) were used for the dose-response study of Inogatran. Inogatran was given as an initial bolus followed by a maintenance infusion using the following dosing schedules: I-1, 0.04 mg/kg + 2.5 mg/kg/ min; I-2, 0.08 mg/kg + 5 mg/kg/min; I-3, 0.15 mg/kg + 10 mg/kg/min; I-4, 0.23 mg/kg + 15 mg/kg/min; I-5, 0.38 mg/kg + 25 mg/kg/min; I-6, 0.75 mg/kg + 50 mg/kg/min. Five groups of rabbits (n=6 for each group) were used for the study of LB-30057 which was administered as an initial bolus followed by a maintenance infusion: LB-I, 0.038 mg/kg + 2.5 mg/kg/min; LB-II, 0.09 mg/kg + 4.6 mg/kg/min; LB-III, 0.19 mg/kg +9.3 mg/kg/min; LB-IV, 0.38 mg/ kg + 25 mg/kg/min; LB-V, 0.7 mg/kg +46.5 mg/ kg/min. Blood concentration of LB-30057 [2] To determine the pharmacokinetic profile of LB-30057 in this rabbit model, a separate study was conducted in order to avoid excessive blood sampling in the experiments focused on efficacy. Concentrations of LB-30057 were determined in whole blood collected from four groups of rabbits (n =5 for each group). The three highest doses tested in the ef®cacy studies were used, as well as one higher dose. The rabbits received a bolus followed by a maintenance infusion of LB-30057 for three hours (mg/kg + mg/kg/min) using the following dosing regimens: 0.19 + 9.3, 0.38 + 25, 0.7 + 46.5, 1.4 + 93, respectively. The samples of blood were drawn at 0, 5, 15, 30, 60, 90, 120 and 180 min after the dosing. Previous in vitro studies have shown that LB-30057is not stable in biological matrices. To stabilize the drug immediately after sample collection, arterial blood samples (0.2 ml) were collected and added to tubes containing a mixture of methanol and aqueous 10% zinc sulfate. The tubes were vortex mixed and stored on ice until the end of the experiment. Tubes were then centrifuged and supernatants were stored at -70 C until analysis. Samples were analyzed for LB-30057 concentrations using a liquid chromatographic method with UV detection. LB-30057 was administered via the cephalic vein of left leg for i.v. study and administered via gavage for oral study to determine its pharmacokinetics in Beagle dogs. The blood was withdrawn via the cephalic vein of right leg (i.v. only) up to 720 min. The blood samples were, then, deproteinized by mixing 1.5 volume of methanol and 0.5 volume of 10% ZnSO,, and the supernatant was quantitated using HPLC on a reversed-phase column at UV 23 1 nm. [1] |
| ADME/Pharmacokinetics |
The oral bioavailability of LB30057 was 58% with the corresponding elimination half-life of 112 min at 10 mg/ml dose in dogs. [1] Summarized in Table 1 are the values for blood LB-30057 concentration at various time points during the 3 h of IV infusion. There was good linearity between the increment in doses and blood concentration of LB-30057 in the four groups of rabbits. Blood LB-30057 concentration was >0.3 mg/ml at the dose that produces a signi®cant antithrombotic effect (0.19 mg/kg + 9.3 mg/kg/min). [1] |
| References |
[1]. Discovery of LB30057, a benzamidrazone-based selective oral thrombin inhibitor. Bioorg Med Chem Lett. 1998 Mar 17;8(6):631-4. [2]. Antithrombotic effect of LB-30057 (CI-1028), a new synthetic thrombin inhibitor, in a rabbit model of thrombosis: comparison with inogatran. J Thromb Thrombolysis. 2001 Feb;11(1):19-31. |
| Additional Infomation |
Systematic variation of the so-called P-pocket moiety of benzamidrazone-based selective thrombin inhibitors led to the discovery of LB-30057. It is potent (Ki = 0.38 nM for human thrombin), selective (Ki = 3290 nM for bovine trypsin), and orally bioavailable (58% oral bioavailability in dogs). LB-30057 was efficacious in thrombosis animal models. [1]
The objective of the present study is to describe the effects of a novel, small molecule inhibitor of thrombin, LB-30057 (CI-1028), in a rabbit model of experimental thrombosis. Because of its low molecular weight (493.6 Dalton) and selective binding to the active site of thrombin, synthetic thrombin inhibitors such as LB-30057 may inhibit clot-bound thrombin more effectively than large molecules such as hirudin, hirulog, or heparin [16±18], potentially translating into improved in vivo antithrombotic efficacy [2]. A dose-dependent increase in blood LB-30057 concentration was observed in the pharmacokinetic studies. Because of the minimal species differences between the rabbit and human plasma in response to LB-30057, the information derived from the present study may be useful for clinical trials in humans. In addition to the different animal species, the experimental protocol and model used in this study have several differences in comparison with the clinical settings in which antithrombotic agents are administered. First, in clinical settings, the antithrombotic therapy is applied to patients who have pre-existing conditions, i.e. those who have been diagnosed with DVT or acute coronary syndromes. In the present study, the test compounds were administered to the animals before thrombosis was initiated. These two situations represent treatment vs. primary prevention of thrombotic diseases, respectively. Nevertheless, the two regimens target the same underlying pathophysiology, i.e., inhibition of the enhanced activation of coagulation and platelets, thus the process of thrombus formation. For example, in clinical trials with antithrombotic agents in patients who had acute coronary syndromes, the end point includes new myocardial infarction and recurrent angina, which are associated with new thrombus formation. Another difference between this experimental model and the clinical setting is that in this model, thrombus forms inside a polyethylene tube, and cotton threads were used as a thrombogenic surface. In humans, thrombosis develops in the injured vessel wall due to atherosclerotic lesions or in¯ammation. When the coagulation cascade is activated by the contact reaction in this model, thrombin is generated subsequently by similar biochemical reactions as in the vasculature. Therefore, a compound that inhibits thrombin should be able to inhibit fibrin formation and platelet activation, and thus prevent thrombus formation in both systems. In summary, the results of the present study demonstrate the in vivo antithrombotic efficacy of LB-30057, a novel, speci®c, direct inhibitor of thrombin. LB-30057 dose-dependently inhibited thrombus formation in a rabbit model of venous shunt thrombosis and was at least as potent as Inogatran on a gravimetric basis. At the doses required for equivalent antithrombotic effects, LB-30057 caused less prolongation in aPTT, PT compared to Inogatran. The mechanisms that contributed to this difference and their implications on bleeding risk warrant further investigation.[2] |
Solubility Data
| Solubility (In Vitro) | May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples |
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6402 mL | 8.2008 mL | 16.4015 mL | |
| 5 mM | 0.3280 mL | 1.6402 mL | 3.2803 mL | |
| 10 mM | 0.1640 mL | 0.8201 mL | 1.6402 mL |