4-[[5-(Aminosulfonyl)-1,3,4-thiadiazol-2-yl]amino]-4-oxobutanoic acid

To solution of compound 3 (0.40 g, 2.22 mmol) in N, N-dimethylformamide was added succinic anhydride (0.22 g, 2.22 mmol). The reaction mixture was heated at 100 EC3105 (178 mg, 0.988 mmol) was dissolved in DMF (1.6 mL). To this solution was added succinic anhydride (98.8 mg, 1 eq.) The solution was heated to 50 Acetazolamide (10.0 g, 45 mmol) was dispersed in 56 mL of 4N HCl solution and heated to 100 C for 4 h.After the reaction was completed, the reaction solution was cooled to room temperature, and 25 mL of 9N NaOH (aq) was added thereto under ice-water bath to pH 3.The solid which precipitated in the reaction liquid was filtered, and dried to obtain 6.8 g of a white solid, yield: 84.0%.The white solid was then dispersed with succinic anhydride (6.67 g, 66.6 mmol) in acetonitrile and heated to reflux for 12 h.After the reaction is completed, the obtained solid is suction filtered and washed with acetonitrile.White solid 7 g, yield 66.2%.Compound 13 (200 mg, 0.25 mmol) and 16 (105 mg, 0.38 mmol)After dissolving 10 mL of DMF, DCC (77.25 mg, 0.38 mmol), TEA (0.10 mL, 0.75 mmol) was added thereto, and the mixture was reacted at room temperature for 12 h.After the reaction was completed, the reaction was quenched with water and ethyl acetate was evaporated.Separation of pure by column chromatography160 mg of a white solid was obtained in a yield of 52.4%.To solution of compound 4 (0.30 g, 1.07 mmol) in N, N-dimethylformamide were added 2-(2-(2-Azidoethoxy)ethoxy)ethan-1-amine (5) (0.21 g, 1.18 mmol), (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent) (0.47 g, 1.07 mmol) and iPrNEt2 (0.42 g, 3.21 mmol). This reaction mixture was stirred at room temperature for 1 hr. To this reaction mixture was added1N-HCl and compound was extracted by ethyl acetate. Ethyl acetate layer was washed with brine, dried over Na2SO4 and concentrated. Residue obtained was purified on flash column by DCM:MeOH 95:5 to obtained title compound 6 in 53 % yield (0.25 g). 1H NMR (400 MHz, DMSO-d6) delta 8.31 (s, 2H), 7.99 (t, J = 5.5 Hz, 1H), 3.60 (t, J = 5.0 Hz, 2H), 3.57-3.48 (m, 4H), 3.46-3.37 (4H), 3.19 (q, J = 5.6 Hz, 2H), 2.73 (t, J = 6.9 Hz, 2H), 2.53 (s, 2H); 13C NMR (100 MHz, DMSO-d6) delta 172.45, 171.30, 164.64, 161.80, 70.12, 69.79, 69.63, 50.51, 37.00, 30.85, 29.92; HRMS (ESI+): m/z Calcd for C12H21N8O6S2 [M+H]+: 437.1026, Found: 437.1011.H-Cys(Trt)-2-Cl-Trt-resin (760 mg, 0.658 mmol/g, 0.5 mmol) was loaded into a peptide synthesis vessel and placed on the peptide synthesizer. Following standard Fmoc-solid phase peptide synthesis protocols, Fmoc-Asp(Ot-Bu)-OH (412 mg, 2eq.) were added four times followed by Fmoc-6-aminohexanoic acid (353mg, 2eq.) PyBop and DIPEA were used during each coupling step (1 hour duration) and 20% piperidine/DMF was used for each Fmoc deprotection step (2X 20 minutes duration). A portion of this resin (184mg, 0.09 mmol) was treated with EC3108 (30 mg, 1.2 eq.), DIPEA (39 mL, 2.5 eq.) and PyBop (56 mg, 1.2 eq.), after the final Fmoc protecting group had been removed, for 1 hour. After washing and drying, the peptide was cleaved from the resin with TFA/H2O/TIPS/DTT (92.5: 2.5:2.5:2.5). The peptide was precipitated from the cleavage solution upon addition of Et2O and was recovered by centrifugation. The crude peptide was re- dissolved in DMSO and purified by Biotage C18 column (0.1% TFA/ACN) to give, after recovery and lyophilization, purified peptide EC3156 (45mg, 52% yield). ESI-MS [M+H]+ = 957.6 Representative peaks 1H NMR (500 MHz, DMSO-d6/D2O): delta 4.6-4.4 (m, 4H), 3.8 (m, 1H), 2.99 (t, 2H), 2.9-2.6 (m, 8H), 2.08 (t, 2H), 1.45 (dt, 2H), 1.35 (dt, 2H), 1.2 (dt, 2H).H-Cys(Trt)-2-Cl-Trt-resin (608 mg, 0.658 mmol/g, 0.4 mmol) was loaded into a peptide synthesis vessel. Following standard Fmoc-solid phase peptide synthesis protocols, Fmoc- Lys(Boc)-OH (374 mg, 2eq.) and Fmoc-Asp(Ot-Bu)-OH (329 mg, 2eq.) were added alternately, in sequence, until three Lys residues and two Asp residues were added to the peptide chain. PyBop (416 mg, 2 eq.) and DIPEA (278 muL, 4 eq.) were used during each coupling step (1 hour coupling time) and 20% piperidine/DMF was used for each Fmoc deprotection step (2X 20 minutes deprotection time). To of this resin (0.2 mmol) was then added 6-aminohexanoic acid (141 mg, 2 eq.) with PyBop (208 mg, 2 eq.) and DIPEA (139 muL, 4 eq.), after the Fmoc protecting group was removed. To of this resin (0.1 mmol), after a final Fmoc deprotection, was added EC3108 (42 mg, 1.5 eq.), PyBop (78 mg, 1.5 eq.), and DIPEA (35 muL, 3 eq.). After washing and drying, the peptide was cleaved from the resin with TFA/H2O/TIPS/DTT (92.5: 2.5:2.5:2.5). The peptide was precipitated from the cleavage solution upon addition of Et2O and was recovered by centrifugation. The crude peptide was re-dissolved in DMSO and purified by Biotage C18 column (0.1% TFA/ACN) to give, after recovery and lyophilization, purified cysteine terminating-CA IX targeting peptide EC3109 (72mg, 60% yield). ESI-MS [M+2H]2+ = 556.3.A mixture of DIPEA (0.11 mL, 0.65 mmol), Compound 8 (0.18 g, 0.65 mmol), HATU (0.25 g, 0.65 mmol) in DMF (2 mL) was stirred at room temperature for 20 minutes.Then propargylamine (0.042 mL, 0.65 mmol) and triethylamine (0.18 mL, 1.3 mmol) were added at room temperature and the mixture was stirred for an additional 6 hours at room temperature.After removing the solvent under reduced pressure, the crude product was purified on silica gel usingCH2Cl2/ MeOH (v / v, 95: 5) as eluent to afford compound 7 as a white solid (0.14 g, 70%).

Molecular Weight:280.3
XLogP3:-0.8
Hydrogen Bond Donor Count:3
Hydrogen Bond Acceptor Count:9
Rotatable Bond Count:5
Exact Mass:279.99361172
Monoisotopic Mass:279.99361172
Topological Polar Surface Area:189
Heavy Atom Count:17
Complexity:405
Covalently-Bonded Unit Count:1
Compound Is Canonicalized:Yes