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Type | HazMat fee for 500 gram (Estimated) |
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Structure of 7154-73-6
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The BI-3802 was designed by Boehringer Ingelheim and could be obtained free of charge through the Boehringer Ingelheim open innovation portal opnMe.com, associated with its negative control.
4.5
*For Research Use Only !
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Rhym, Luke Hyunsik ;
Abstract: mRNA lipid nanoparticles (LNP) present a promising class of therapeutics, with broad applications in protein replacement therapy, gene editing, immunotherapy, and vaccines, owing to their versatility and precise nature. While recent years have seen dramatic improvements in the safety and efficacy of mRNA therapeutics, their functional delivery to target tissues and cells in vivo remains challenging, partly due to the lack of predictive power of in vitro assays and the low-throughput and costly nature of in vivo screening approaches. Thus, there is still a need for safe, specific, and potent mRNA delivery materials, as well as higher throughput in vivo screening methods. In this work, we developed a novel in vivo nanoparticle screening platform that relies on LC-MS/MS based detection of peptide barcodes translated from barcoded mRNAs in transfected cells, allowing for a readout of functional delivery that is directly proportional to protein production effected by each nanoparticle within a pooled library. We showed that this approach has high sensitivity and accuracy in both cultured cells in vitro and in tissues in vivo and demonstrated the applicability of this approach to in vivo screening of LNPs by developing and optimizing the formulation of a biodegradable LNP, RM133-3-21, for potent mRNA delivery to the liver. We then screened a large library of ionizable lipids for their ability to deliver mRNA to the lung and optimized both the structure and formulation of the lead compound. The resulting LNP, C15-21, is highly potent and is able to transfect up to 80% of lung endothelial cells after a single dose. In addition, we demonstrated that C15-21 is able to efficiently deliver Cas9 mRNA and sgRNA for targeted gene disruption in the lung, resulting in up to 7.5% gene editing in lung endothelial cells. Finally, we also developed materials and formulations that show high specificity for splenocytes in vivo. Taken together, the work presented in this thesis contributes to the field of mRNA therapeutics by increasing the throughput of LNP testing in vivo and by introducing novel delivery materials.
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CAS No. : | 7154-73-6 |
Formula : | C6H14N2 |
M.W : | 114.19 |
SMILES Code : | NCCN1CCCC1 |
MDL No. : | MFCD00003182 |
InChI Key : | WRXNJTBODVGDRY-UHFFFAOYSA-N |
Pubchem ID : | 1344 |
GHS Pictogram: |
![]() ![]() |
Signal Word: | Danger |
Hazard Statements: | H314-H226 |
Precautionary Statements: | P501-P240-P210-P233-P243-P241-P242-P264-P280-P370+P378-P303+P361+P353-P301+P330+P331-P363-P304+P340+P310-P305+P351+P338-P310-P403+P235-P405 |
Class: | 8(3) |
UN#: | 2734 |
Packing Group: | Ⅱ |
Num. heavy atoms | 8 |
Num. arom. heavy atoms | 0 |
Fraction Csp3 | 1.0 |
Num. rotatable bonds | 2 |
Num. H-bond acceptors | 2.0 |
Num. H-bond donors | 1.0 |
Molar Refractivity | 38.36 |
TPSA ? Topological Polar Surface Area: Calculated from |
29.26 Ų |
Log Po/w (iLOGP)? iLOGP: in-house physics-based method implemented from |
1.71 |
Log Po/w (XLOGP3)? XLOGP3: Atomistic and knowledge-based method calculated by |
0.08 |
Log Po/w (WLOGP)? WLOGP: Atomistic method implemented from |
-0.34 |
Log Po/w (MLOGP)? MLOGP: Topological method implemented from |
0.21 |
Log Po/w (SILICOS-IT)? SILICOS-IT: Hybrid fragmental/topological method calculated by |
0.56 |
Consensus Log Po/w? Consensus Log Po/w: Average of all five predictions |
0.44 |
Log S (ESOL):? ESOL: Topological method implemented from |
-0.47 |
Solubility | 39.0 mg/ml ; 0.342 mol/l |
Class? Solubility class: Log S scale |
Very soluble |
Log S (Ali)? Ali: Topological method implemented from |
-0.25 |
Solubility | 64.4 mg/ml ; 0.564 mol/l |
Class? Solubility class: Log S scale |
Very soluble |
Log S (SILICOS-IT)? SILICOS-IT: Fragmental method calculated by |
-0.72 |
Solubility | 21.8 mg/ml ; 0.191 mol/l |
Class? Solubility class: Log S scale |
Soluble |
GI absorption? Gatrointestinal absorption: according to the white of the BOILED-Egg |
High |
BBB permeant? BBB permeation: according to the yolk of the BOILED-Egg |
No |
P-gp substrate? P-glycoprotein substrate: SVM model built on 1033 molecules (training set) |
No |
CYP1A2 inhibitor? Cytochrome P450 1A2 inhibitor: SVM model built on 9145 molecules (training set) |
No |
CYP2C19 inhibitor? Cytochrome P450 2C19 inhibitor: SVM model built on 9272 molecules (training set) |
No |
CYP2C9 inhibitor? Cytochrome P450 2C9 inhibitor: SVM model built on 5940 molecules (training set) |
No |
CYP2D6 inhibitor? Cytochrome P450 2D6 inhibitor: SVM model built on 3664 molecules (training set) |
No |
CYP3A4 inhibitor? Cytochrome P450 3A4 inhibitor: SVM model built on 7518 molecules (training set) |
No |
Log Kp (skin permeation)? Skin permeation: QSPR model implemented from |
-6.94 cm/s |
Lipinski? Lipinski (Pfizer) filter: implemented from |
0.0 |
Ghose? Ghose filter: implemented from |
None |
Veber? Veber (GSK) filter: implemented from |
0.0 |
Egan? Egan (Pharmacia) filter: implemented from |
0.0 |
Muegge? Muegge (Bayer) filter: implemented from |
1.0 |
Bioavailability Score? Abbott Bioavailability Score: Probability of F > 10% in rat |
0.55 |
PAINS? Pan Assay Interference Structures: implemented from |
0.0 alert |
Brenk? Structural Alert: implemented from |
0.0 alert: heavy_metal |
Leadlikeness? Leadlikeness: implemented from |
No; 1 violation:MW<1.0 |
Synthetic accessibility? Synthetic accessibility score: from 1 (very easy) to 10 (very difficult) |
1.0 |
* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
A library of compounds in which R4 was various groups having the formula [CONHR »] was prepared by the process described above using 4-fluoro-3-nitrobenzoic acid, as follows: [72] Aldehyde resin was mixed with a primary amine (R17-NH2) in [DICHLOROETHANE] (DCE), triethylorthoformate (TEOF), and DMF (containing [1%] acetic acid) in a 1: 1: 1 ratio. After shaken overnight, sodium triacetoxyborohydride (20 eq. ) dissolved in DMF was added (Abdel-Magid, A. F. , et al., Tetrahedron Lett, 3 1: 5595-5598 (1990) ). After the mixture was shaken at room temperature overnight, the resin was filtered and washed with DMF (3 x 5 mL), [MEOH] [(3 X 5] mL), DMF [(3 X 5] mL), [MEOH] [(3 X 5] mL), and [CH2CL2] [(3 X 5] mL). The resin was washed twice with 5 mL DMF containing [1%] Hunig's base. To the filtered resin was added a mixture of 4-fluoro-3-nitrobenzoic acid (FNBA, 10 eq. ) and diisopropylcarbodiimide (DIC, 5 eq. ) in 2: 1 DMF : DCM. After shaking at room temperature overnight, the resin was filtered and washed with DMF (3 x 5 mL) and [CH2C12] (3 x 5 mL). [73] The resin was shaken with a primary amine [(R2-NH2)] in DMF for 8 hrs, filtered, and washed with DMF (6 x 5 mL), [MEOH] [(3 X 5] mL), and CH2C12 (3 x 5 mL). The aryl nitro group was reduced by the addition of tin (II) chloride dihydrate (20 eq. , >2 M) and N-methyl morpholine (NMM, 20 eq. ) in N-methyl pyrrolidinone (NMP). After shaken at room temperature overnight, the resin was filtered and washed with NMP (3 x 5 mL), [MEOH] (3 x 5 mL), and [CH2CI2 (3 X 5] mL). The resulting resin was shaken at room temperature with cyanogen bromide (5 eq. ) overnight, filtered, and washed with CH2Cl2 (3 x 5 mL), [MEOH] (3 x 5 mL), and CH2CI2 (3 x 5 mL). To produce a free amine, the resin was shaken for 30 min. in CHCl2 with the addition of sodium methoxide in methanol, filtered, and washed with CH2Cl2 [(4 X 5] mL). [[74]] In the final diversification step, the resin was heated at 500 C in DMF with a mono- substituted epoxide [[RLCH (-CH2O-)].] After shaking for 2 to 4 days the resin was filtered and washed with DMF (5 x 5 mL), [MEOH] [(3 X 5] mL), and CH2Cl2 (3 x 5 mL). T he resin-bound benzimidazole was cleaved from the solid-support by treatment with TFA: [CH2C12] (2: 3) for 1 hour at room temperature. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With benzotriazol-1-yloxyl-tris-(pyrrolidino)-phosphonium hexafluorophosphate; triethylamine; In N,N-dimethyl-formamide; at 20℃; for 2h; | General procedure: Method 2: to the solution of Indomethacin (100 mg, 0.28 mmol), the corresponding amine (0.28 mmol), and PyBOP (145.7 mg, 0.28 mmol) in anhydrous dimethylformamide was added triethylamine (56.5 mg, 0.56 mmol) and the mixture was stirred at room temperature for 2 h. Then saturated sodium chloride solution was added. The reaction mixture was extracted using ethyl acetate (3 x 50 ml). The combined organic layers werewashed successively with water, 5% aqueous sodium bicarbonate, and then concentrated under vacuum. The obtained crude product was purified by recrystallization or silica gel column chromatography. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
92% | With benzotriazol-1-yloxyl-tris-(pyrrolidino)-phosphonium hexafluorophosphate; triethylamine; In dichloromethane; acetonitrile; at 20℃; | General procedure: A mixture of 1 mmol (1 equiv.) of carboxylic acids, 1.4 mmol (1.4 equiv.) of amine, 552 mg (1.8 equiv.) of PyBOP and 2 mL of triethylamine was stirred overnight at room temperature in 40 ml of a 1:1 mixture of CH2Cl2 and CH3CN. After solvent evaporation, the crude product was purified by column chromatography on silica (CHCl3:CH3CH2OH = 9:1) resulting in yellow thick oil that slowly crystallized. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
78% | In N,N-dimethyl-formamide; at 20℃; | General procedure: Compound 11a (5mmol, 2.14g) and N1,N1-dimethylethane-1,2-diamine (15mmol, 1.32g) in 15mL DMF were stirred at room temperature overnight. The solvents were removed under vacuum and the residue was purified by silica gel flash chromatography (DCM: MeOH=10: 1) to give compound 14a (1.9g, yield 79%). |
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