Sialic acid derivatives
10472381 ยท 2019-11-12
Assignee
Inventors
Cpc classification
C07H15/04
CHEMISTRY; METALLURGY
A61P29/00
HUMAN NECESSITIES
International classification
Abstract
Sialic acid derivatives of the formula (I) ##STR00001##
Claims
1. Sialic acid derivative of the formula (I), ##STR00078## where the symbols have the following definitions: A.sup.1 is a group D.sup.1-[Y.sup.2-D.sup.2-].sub.m-; D.sup.1 is a mono- or polycyclic aromatic, partially unsaturated or saturated C.sub.3-C.sub.14 hydrocarbon radical or a mono- or polycyclic aromatic, partially unsaturated or saturated three- to twelve-membered heterocyclic radical, the stated radicals being unsubstituted or substituted one or more times by a group X; D.sup.2 is a mono- or polycyclic aromatic, partially unsaturated or saturated C.sub.3-C.sub.14 hydrocarbon radical or a mono- or polycyclic aromatic, partially unsaturated or saturated three- to eight-membered heterocyclic radical, the stated radicals being unsubstituted or substituted one or more times by a group X; Y.sup.1 is C(O), S(O).sub.2, NHC(O), (C.sub.1-C.sub.2 alkyl)-, (C.sub.1-C.sub.2 alkyl)-C(O), CHCHC(O), CCC(O), (C.sub.1-C.sub.2 alkyl)-S(O).sub.2, OC(O), (C.sub.1-C.sub.2 alkyl)-OC(O) or (C.sub.1-C.sub.2 alkyl)-NHC(O), where denotes the bond to the group A.sup.1; Y.sup.2 is O, C(O), S(O).sub.2, CH.sub.2 or a bond; A.sup.2 is a) a group OS(O).sub.2OL or b) a group N(R.sup.x)W; W is a) a group SO.sub.3L, SO.sub.2CF.sub.3 or SO.sub.2NR.sup.x.sub.2 or b) a group D.sup.3-Y.sup.3; Y.sup.3 is a bond or a group O(CO)NHS(O).sub.2, NHC(O), OC(O), CH.sub.2OC(O), S(O).sub.2, C(O), (C.sub.1-C.sub.2 alkyl)-C(O), (C.sub.1-C.sub.2 alkyl)-NHC(O) or (C.sub.1-C.sub.2 alkyl)-S(O).sub.2, where denotes the bond to the group D.sup.3; D.sup.3 is a) C.sub.1-C.sub.6 alkyl, where optionally one or more non-terminal CH.sub.2 groups are replaced by O, N(R.sup.x) and/or C(O), and where optionally one or more H atoms in the stated groups are replaced by a group X, b) is a mono- or polycyclic aromatic, partially unsaturated or saturated C.sub.3-C.sub.14 hydrocarbon radical or a mono- or polycyclic aromatic, partially unsaturated or saturated three- to eight-membered heterocyclic radical, the stated radicals being unsubstituted or substituted one or more times by a group X; A.sup.3 is a) C.sub.1-C.sub.8 alkyl, where optionally a. one or more non-terminal CH.sub.2 groups are replaced by S, O, N(R.sup.x) and/or C(O), or b. a CH.sub.2CH.sub.2CH.sub.2 group is replaced by 1,2-phenyldiyl, 1,3-phenyldiyl or 1,4-phenyldiyl, and where optionally one or more H atoms in the stated groups are replaced by a group X, or b) is a mono- or polycyclic aromatic, partially unsaturated or saturated C.sub.3-C.sub.14 hydrocarbon radical or a mono- or polycyclic aromatic, partially unsaturated or saturated three- to eight-membered heterocyclic radical, the stated radicals being substituted one or more times by a group X; X is identically or differently halogen, cyano, nitro, hydroxyl, mercapto, amino, carboxyl, carboxymethyl, hydroxylamino, azido, B(OH.sub.2), SO, SO.sub.3M, OSO.sub.3M, SO.sub.2NH.sub.2, SO.sub.2CF.sub.3, PO.sub.3M, OPO.sub.3M, cyanomethyl, haloalkyl, alkyloxy, haloalkyloxy, alkylamino, dialkylamino, trialkylamino, formyl, alkylcarbonyl, alkylsulfonyl, alkylsulfoxyl, alkylaminosulfonyl, dialkylaminosulfonyl, alkyloxycarbonyl, alkylcarbonyloxy, aminocarbonyl, alkylaminocarbonyl, alkylcarbonylamino, dialkylaminocarbonyl, oxo (O), thioxo (S), C.sub.1-C.sub.8 alkylimino (NC.sub.1-C.sub.8 alkyl) or C.sub.1-C.sub.8 alkyloximino (NOC.sub.1-C.sub.8 alkyl), the alkyl groups in these radicals containing 1 to 6 carbon atoms; m is 0, 1 or 2; Z is O, S, ONCH, ON(R.sup.x), N(R.sup.x) or 4-1H-(1,2,3)triazol-1-yl-, where denotes the bond to the group A.sup.3; R.sup.1 is C(O)OM; R.sup.2 is H, F, Cl, NR.sup.x or OR.sup.x; R.sup.3 is H, F, Cl, NR.sup.x or OR.sup.x; R.sup.4 is N(R.sup.x)C(O)CH.sub.2OH or N(R.sup.x)C(O)R.sup.x; R.sup.5, R.sup.6 are identically or differently OH or OR.sup.x; L is a cation; M is C.sub.1-C.sub.4 alkyl or a cation; R.sup.x is identically or differently H, R.sup.y or R.sup.z; R.sup.y is identically or differently C.sub.1-C.sub.4 alkyl, phenyl or benzyl, and R.sup.z is identically or differently C(O)C.sub.1-C.sub.4 alkyl, C(O)-phenyl or C(O)CH.sub.2-phenyl, where alkyl groups in each case are straight-chain, branched or cyclic, a pharmacologically tolerated salt or prodrug thereof.
2. Sialic acid derivative of the formula (I) according to claim 1, where the symbols in the formula (I) have the following definitions: A.sup.1 is a group D.sup.1-[Y.sup.2-D.sup.2-].sub.m-; D.sup.1 is a mono- or polycyclic aromatic or saturated C.sub.3-C.sub.14 hydrocarbon radical or a monocyclic aromatic, partially unsaturated or saturated four- to six-membered heterocyclic radical, the stated radicals being unsubstituted or substituted one or more times by a group X; D.sup.2 is a group phenylene-1,4-diyl, phenylene-1,3-diyl, pyridine-2,5-diyl, pyridazine-3,6-diyl, pyrimidine-2,5-diyl, pyrimidine-2,6-diyl, pyrazine-2,5-diyl, trans-cyclobutane-1,3-diyl, trans-cyclopentane-1,3-diyl, trans-cyclohexane-1,4-diyl, cubane-1,4-diyl, thiophene-2,5-diyl, pyrrole-2,4-diyl, pyrrole-2,5-diyl, pyrazole-1,3-diyl, oxazole-2,4-diyl, oxazole-2,5-diyl, 1,3,4-oxadiazole-2,5-diyl, 1,2,4-oxadiazole-3,5-diyl, isooxazole-3,5-diyl, imidazole-2,4-diyl, 2H-tetrazole-2,5-diyl, 1H(1,2,4)-triazole-2,5-diyl, 1H(1,2,3)-triazole-1,4-diyl or 1H(1,2,3)-triazole-1,5-diyl, the stated radicals being unsubstituted or substituted one or more times by a group X; Y.sup.1 is C(O), S(O).sub.2, NHC(O), CH.sub.2C(O), CH.sub.2S(O).sub.2, OC(O), CH.sub.2OC(O) or CH.sub.2NHC(O), where denotes the bond to the group A.sup.1; Y.sup.2 is O, CH.sub.2 or a bond; A.sup.2 is a) a group OS(O).sub.2OL or b) a group N(R.sup.x)W; W is a) a group SO.sub.3L, SO.sub.2CF.sub.3 or SO.sub.2NR.sup.x.sub.2 or b) a group D.sup.3-Y.sup.3; Y.sup.3 is a bond or a group NHC(O), OC(O), CH.sub.2OC(O), S(O).sub.2, C(O), CH.sub.2C(O), CH.sub.2NHC(O) or CH.sub.2S(O).sub.2, where denotes the bond to the group D.sup.3; D.sup.3 is a) a C.sub.1-C.sub.6 alkyl, where optionally one or more non-terminal CH.sub.2 groups are replaced by O and where optionally one or more H atoms in the stated groups are replaced by a group X, or b) a monocyclic or polycyclic aromatic or saturated C.sub.3-C.sub.14 hydrocarbon radical or a monocyclic aromatic, partially unsaturated or saturated four- to six-membered heterocyclic radical, the stated radicals being unsubstituted or substituted one or more times by a group X; A.sup.3 is a C.sub.3-C.sub.8 alkyl, where optionally a) one or more non-terminal CH.sub.2 groups are replaced by S or O, or b) a CH.sub.2CH.sub.2CH.sub.2 group is replaced by 1,2-phenyldiyl, 1,3-phenyldiyl or 1,4-phenyldiyl, and where optionally one or more H atoms in the stated groups are replaced by a group X; X is identically or differently halogen, hydroxyl, amino, carboxyl, carboxymethyl, SO.sub.3M, OSO.sub.3M, SO.sub.2NH.sub.2, SO.sub.2CF.sub.3, haloalkyl, alkyloxy, alkylamino, dialkylamino, trialkylamino, alkylsulfonyl, alkyloxycarbonyl, alkylcarbonyloxy, alkylaminocarbonyl, alkylcarbonylamino or oxo (O), the alkyl groups in these radicals containing 1 to 3 carbon atoms; m is 0 or 1; Z is O, S or 4-1H-(1,2,3)triazol-1-yl, where denotes the bond to the group A.sup.3; R.sup.1 is C(O)OM; R.sup.2 is H or F; R.sup.3 is H, F, Cl or OR.sup.x; R.sup.4 is NHC(O)CH.sub.2OH or NHC(O)CH.sub.3; R.sup.5, R.sup.6 are identically or differently OH or OR.sup.z; L is a cation; M is a C.sub.1-C.sub.3 alkyl or a cation; R.sup.x is identically or differently H, R.sup.y or R.sup.z; R.sup.y is identically or differently C.sub.1-C.sub.3 alkyl, phenyl or benzyl and R.sup.z is identically or differently C(O)C.sub.1-C.sub.3 alkyl or C(O)-phenyl, a pharmacologically tolerated salt or prodrug thereof.
3. Sialic acid derivative of the formula (I) according to claim 1, where the symbols in the formula (I) have the following definitions: A.sup.1 is a group D.sup.1-[Y.sup.2-D.sup.2-].sub.m-; D.sup.1 is a group phenyl, pyrimidin-5-yl, naphth-1-yl, naphth-2-yl, or thien-2-yl, the stated radicals being unsubstituted or substituted one or more times by a group X; D.sup.2 is a group phenylene-1,4-diyl, pyridine-2,5-diyl, pyrimidine-2,5-diyl or thiophene-2,5-diyl, the stated radicals being unsubstituted or substituted one or more times by a group X; Y.sup.1 is C(O) or CH.sub.2C(O), where denotes the bond to the group A.sup.1; Y.sup.2 is a bond; A.sup.2 is a) a group OS(O).sub.2OL or b) a group NHW; W is a) a group SO.sub.3L or b) a group D.sup.3-Y.sup.3; Y.sup.3 is a bond or a group CH.sub.2OC(O), OC(O), S(O).sub.2, C(O) or CH.sub.2C(O), where denotes the bond to the group D.sup.3; D.sup.3 is a group methyl, ethyl, prop-2-yl, pent-5-yl, cyclopropyl, 1,1-dimethylethyl, phenyl, thien-2-yl, furan-2-yl, imidazolidin-5-yl, pyrazin-5-yl, or naphthalin-1-yl; A.sup.3 is a group pentan-1-yl or hexan-1-yl; where one or more H atoms in the stated groups are replaced by a group X; X is identically or differently fluoro, chloro, hydroxyl, carboxyl, trifluoromethyl, methoxy, dimethylamino or oxo (O); m is 0 or 1; Z is O; R.sup.1 is C(O)OM; R.sup.2 is H; R.sup.3 is H or OH; R.sup.4 is NHC(O)CH.sub.3; R.sup.5, R.sup.6 are identically or differently OH or OC(O)CH.sub.3; L is a cation; M is methyl, ethyl or a cation, a pharmacologically tolerated salt or prodrug thereof.
4. Sialic acid derivative of the formula (I) according to claim 1, characterized by one of the formulae Ia to Ich, where the symbols have the definitions indicated in the formula (I): ##STR00079## ##STR00080## a pharmacologically tolerated salt or prodrug thereof.
5. Sialic acid derivative of the formula (I) according to claim 1, characterized by one of the formulae (Iaa)-(Iam), where the symbols have the definitions indicated in the formula (I): ##STR00081## ##STR00082## a pharmacologically tolerated salt or prodrug thereof.
6. Sialic acid derivative according to claim 1, characterized by one of the formulae (Iba)-(Ibj), where the symbols have the definitions indicated in the formula (I): ##STR00083## ##STR00084## ##STR00085## a pharmacologically tolerated salt or prodrug thereof.
7. Sialic acid derivative of the formula (I) according to claim 1, characterized by one of the formulae (Ica)-(cj), where the symbols have the definitions indicated in the formula (I): ##STR00086## ##STR00087## a pharmacologically tolerated salt or prodrug thereof.
8. Pharmaceutical preparation comprising at least one sialic acid derivative of the formula (I) or a pharmacologically tolerated salt or prodrug thereof according to claim 1, and a pharmacologically tolerated carrier.
9. Sialic acid derivative of the formula (I) or a pharmacologically tolerated salt or prodrug thereof according to claim 1, as medicament.
10. Method for regulating the immune system and also for the treatment of diseases whose course or activity can be influenced by the Siglec ligands, where a person affected by the disease is administered a therapeutically effective amount of a sialic acid derivative of the formula (I) or of a pharmacologically tolerated salt or prodrug thereof according to claim 1.
11. Process for preparing sialic acid derivatives of the formula (I) according to claim 1 where R.sup.3OH, where a compound of the formula (VI), ##STR00088## in which the symbols have the same definitions as in the formula (I) in claim 1, is reacted by reduction of the azide, subsequent reaction of the resultant amine with a carboxylic acid or a sulfonyl chloride, and optional elimination of protecting groups to give a sialic acid derivative of the formula (I) where R.sup.3OH.
12. Process for preparing sialic acid derivatives of the formula (I) according to claim 1 where R.sup.3H, where a compound of the formula (IV), ##STR00089## in which the symbols have the same definitions as in the formula (I) in claim 1, is reacted by reaction of the azide, subsequent reaction of the resultant amine with an activated carboxylic acid, a sulfonyl chloride or a sulfating agent, and optional elimination of protecting groups to give a sialic acid derivative of the formula (I) where R.sup.3H.
13. Process for preparing sialic acid derivatives of the formula (I) according to claim 1, where a compound of the formula (VIII) ##STR00090## in which the symbols have the same definitions as in the formula (I) in claim 1, is reacted by reduction of the azide, subsequent reaction of the resultant amine with an activated carboxylic acid, a sulfonyl chloride or a sulfating agent, and optional elimination of protecting groups to give a sialic acid derivative of the formula (I).
14. Intermediates for the preparation of sialic acid derivatives of the formula (I) according to claim 1, selected from the group consisting of a) compounds of the formula (III), ##STR00091## b) compound of the formula (IV) ##STR00092## and c) compounds of the formula (VI), ##STR00093## where the symbols in the formulae (III), (IV) and (VI) have the same definitions as in formula (I) in claim 1.
15. A method for treatment or prevention of allergies, autoimmune disorders, chronic inflammations, paraplegia, multiple sclerosis, cancer, viral disorders, bacterial disorders, parasitic disorders, diseases in which the immune response is disrupted in the context of B cell activation, in diseases of the hematopoietic organs and of the blood, and also for regulation of the immune system, comprising administering a therapeutically effective amount of the sialic acid derivative of the formula (I) or a pharmacologically tolerated salt or prodrug thereof according to claim 1 to a person affected thereby.
16. A method producing a medicament for the regulation of the immune system, comprising, adding to a pharmacologically tolerated carrier the sialic acid derivative of the formula (I) or a pharmacologically tolerated salt or prodrug thereof according to claim 1 to produce the medicament.
17. A method for the treatment of allergies, autoimmune disorders, chronic inflammations, paraplegia, multiple sclerosis, cancer, viral disorders, bacterial disorders, parasitic disorders, diseases in which the immune response is disrupted in the context of B cell activation, and also in diseases of the hematopoietic organs and of the blood, comprising administering a therapeutically effective amount of the sialic acid derivative of the formula (I) or a pharmacologically tolerated salt or prodrug thereof according to claim 1 to a person affected thereby.
Description
EXAMPLES
(1) A. Synthesis examples are represented in Schemes 1 to 18.
(2) ##STR00018##
(3) ##STR00019##
(4) ##STR00020##
(5) ##STR00021##
(6) ##STR00022##
(7) The compounds were obtained as described below:
(8) All of the compounds used but not described were purchased or prepared by known literature protocols.
(9) For purifications with silica gel, silica gel Si60 43-60 mm was used. Around 100 g of silica gel were utilized per gram of substance. Indicated in parentheses is the eluent. For purifications on RP-18 silica gel (YMC CO LTD., YMC ODS-AQ), the gel was suspended in methanol, introduced into a column, prewashed with water, and the substance was applied as a solution or suspension with water. The column volume was about 5 cm in height and about 1 cm in diameter. The solvent was forced through the column at low pressure, generated by a hand-operated pressure ball. The eluent used was a gradient from water to ethanol, unless otherwise indicated.
(10) Solvents were removed by means of a vacuum rotary evaporator under reduced pressure with a bath temperature of 40 C. In the syntheses hereinafter, this workstep is identified as concentration.
(11) All of the substances were lyophilized from water or a water/dioxane mixture.
(12) The reactions and substances were monitored by thin-layer chromatography. This was done using aluminum plates coated with silica gel, with a fluorescence indicator (Merck TLC Silica gel 60 F.sub.254). Substance detection took place under UV light at 366 and 254 nm. The chromatograms were subsequently sprayed with dilute sulfuric acid and heated in order to visualize the substances. For their detection, amines were visualized by spraying with ninhydrin solution and heating. Details and further visualization methods are elucidated in Anfrbereagenzien fr Dnnschicht- and Papierchromatographie Merck, 1970. Spectroscopic data were recorded using Bruker ApexQe hybrid 9.4 T FT-ICR (ESI) and Varian 500 MHz or 300 MHz system (NMR) instruments.
ABBREVIATIONS
(13) DMF N,N-dimethylformamide DIPEA N,N-diisopropylethylamine EtOH ethanol HAc acetic acid HATU O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium MeOH methanol TEA triethylamine
Compound 1
(14) prepared according to: Tetrahedron Letters, 1994, 50 (25), 7445-7460
(15) Compound 4
(16) A solution of 2.2 g of compound 1 in 100 ml of MeOH was admixed with 4.45 g of triphenylphosphine and 6 ml of H.sub.2O and the suspension was stirred at room temperature for 18 h. With stirring, 20 ml of 20% strength acetic acid and 90 ml of H.sub.2O were added, the mixture was stirred for half an hour, the suspension was concentrated to 100 ml, and the concentrate was extracted with three times 100 ml of dichloromethane. The aqueous phase was lyophilized. Yield: 2.67 g of solid
(17) Compound 3
(18) A solution of 2.0 g of compound 2 in 20 ml of DMF was mixed with 1.69 g of 4-nitrophenyl 4-biphenylcarboxylate and 2.09 ml of TEA and stirred for 17 h. The solution was concentrated and the residue was purified on a silica gel column (CHCl.sub.3:MeOH gradient). Yield: 3.2 g
(19) Compound 4
(20) A solution of 2.2 g of compound 3 in 20 ml of acetic acid and 20 ml of acetic anhydride was admixed at 0 C. with 2 ml of concentrated sulfuric acid and stirred at 25 C. for 48 h. The acetic acid was removed on a rotary evaporator and the remaining solution was added slowly dropwise to 1000 ml of saturated sodium hydrogencarbonate solution. The suspension was stirred for 4 h and extracted with three times 200 ml of ethyl acetate. The combined organic phases were washed with saturated NaCl solution, dried (MgSO.sub.4), filtered and concentrated. The residue was purified on a silica gel column (CH.sub.2Cl.sub.2:MeOH gradient). Yield: 2.0 g
(21) Compound 5
(22) A solution of 1.25 g of compound 4 and 1.19 ml of trimethylsilyl azide in 20 ml of dry tert-butanol was stirred at 80 C. for 4 h. The solution was concentrated and the residue was used further without further purification.
(23) Compound 6
(24) A solution of 1350 mg of compound 5 in 0.8 ml of acetonitrile and 2 ml of water was mixed at 60 C. with 538 mg of N-iodosuccinimide, stirred for 30 min and concentrated. The residue was purified on a silica gel column (CHCl.sub.3 to MeOH). Yield: 1170 mg.
(25) Compound 7
(26) A solution of 1140 mg of compound 6 in 10 ml of dry acetonitrile was mixed with 0.277 ml of 1,8-diazabicyclo[5.4.0]undec-7-ene, stirred for 15 min and concentrated. The residue was purified on a silica gel column (CHCl.sub.3 to MeOH). Yield: 750 mg
(27) Compound 8
(28) A solution of 20 mg of camphorsulfonic acid in 0.5 ml of ethyl 6-hydroxyhexanoate was mixed with 150 mg of compound 7, stirred for 20 min and diluted with 30 ml of CH.sub.2Cl.sub.2. The excess of ethyl 6-hydroxyhexanoate was removed on a silica gel column (CH.sub.2Cl.sub.2:MeOH 100:1). The product was eluted from the column (CH.sub.2Cl.sub.2:MeOH 10:1) and purified on RP18. Yield: 50 mg
(29) Compound 9
(30) A solution of 50 mg of compound 8 in 5 ml of MeOH was mixed with 0.05 ml of H.sub.2O and 51 mg of triphenylphosphine and stirred for 17 h. The solution was admixed with 0.5 ml of acetic acid (20%) and 10 ml of H.sub.2O, and the suspension was purified on RP18 (H.sub.2O, then dilute HCl pH3, then H.sub.2O to EtOH gradient). Yield: 40 mg
(31) Compounds 10
(32) A solution of 15 mg of compound 9 in 0.5 ml of DMF was admixed with 33 ml of DIPEA and 5.0 ml of propanoic anhydride, stirred for 15 min, diluted with 2 ml of H.sub.2O, admixed with 0.1 ml of 2M NaOH and stirred for 2 h. The solution was adjusted to pH 8-9 with 20% acetic acid, admixed with 0.5 ml of saturated Na.sub.2CO.sub.3 solution and purified on RP18. Yield: 9 mg; .sup.1H NMR (500 MHz, CD.sub.3OD): d ppm 7.94 (d, J=8.63 Hz, 2H), 7.73 (d, J=8.38 Hz, 2H), 7.68 (dd, J=8.19, 1.17 Hz, 1H), 7.47 (t, J=7.66 Hz, 2H), 7.38 (t, J=7.39 Hz, 1H), 4.08 (ddd, J=8.41, 7.81, 3.14 Hz, 1H), 4.03 (dd, J=10.86, 10.03 Hz, 1H), 3.98 (t, J=10.27 Hz, 1H), 3.85 (td, J=9.04, 6.85 Hz, 1H), 3.81 (dd, J=9.97, 1.89 Hz, 1H), 3.78 (dd, J=13.59, 3.02 Hz, 1H), 3.59 (dd, J=13.88, 7.80 Hz, 1H), 3.55 (td, J=9.19, 7.08 Hz, 1H), 3.50 (d, J=10.24 Hz, 1H), 3.43 (dd, J=9.00, 2.00 Hz, 1H), 2.22 (q, J=7.63 Hz, 2H), 2.17 (dd, J=8.24, 7.03 Hz, 2H), 1.90 (s, 3H), 1.66-1.57 (m, 4H), 1.44-1.35 (m, 2H), 1.13 (t, J=7.65 Hz, 3H); HRMS (ESI-neg) calculated for C33H41N3O12 [M-2Na+H].sup.: 672.2774, found: 672.2801.
(33) Compound 11
(34) A solution of 24 mg of compound 9 in 1 ml of CH.sub.2Cl.sub.2 was admixed with 21 l of TEA and 3.6 l of methanesulfonyl chloride and stirred for 15 min. The solution was admixed with 2 ml of saturated NaHCO.sub.3 solution, stirred for 15 min, admixed with 20 ml of CH.sub.2Cl.sub.2 and 20 ml of saturated NaHCO.sub.3. The organic phase was dried (MgSO.sub.4), filtered and purified on RP18. The residue was dissolved in a little EtOH. The solution was admixed with water until turbidity appeared, adjusted with 2M NaOH to pH 12-13, stirred for 2 h, neutralized with dilute acetic acid and concentrated. The residue was dissolved with a little water, admixed with 0.3 ml of saturated NaHCO.sub.3 and purified on RP18. Yield: 11 mg; .sup.1H NMR (500 MHz, CD.sub.3OD): ppm 7.94 (d, J=8.68 Hz, 2H), 7.73 (d, J=8.70 Hz, 2H), 7.68 (dd, J=8.41, 1.23 Hz, 2H), 7.47 (t, J=7.62 Hz, 2H), 7.38 (t, J=7.39 Hz, 1H), 4.08 (ddd, J=9.06, 7.59, 3.21 Hz, 1H), 3.87 (t, J=10.48 Hz, 1H), 3.85 (td, J=9.22, 6.53 Hz, 1H), 3.77 (dd, J=13.73, 3.14 Hz, 1H), 3.75 (dd, J=10.49, 2.06 Hz, 1H), 3.59 (dd, J=13.75, 7.59 Hz, 1H), 3.54 (td, J=9.08, 6.72 Hz, 1H), 3.47 (d, J=10.44 Hz, 1H), 3.44 (dd, J=8.99, 1.98 Hz, 1H), 3.34 (t, J=10.45 Hz, 1H), 3.05 (s, 3H), 2.16 (dd, J=8.22, 7.14 Hz, 2H), 1.98 (s, 3H), 1.66-1.57 (m, 4H), 1.45-1.35 (m, 2H); HRMS (ESI-neg) calculated for C31H39N3O13S [M-2Na+H].sup.: 694.2287, found: 694.2302.
(35) Compounds 12
(36) A solution of 70 mg of compound 5 in 10 ml of acetyl chloride was admixed at 0 C. with 0.5 g of LiCl, and 0.5 ml of HCl (37%) was added dropwise over the course of 1 h. The solution was stirred at 0 C. for 5 h and admixed with LiCl to saturation point. The suspension was stirred at RT for 5 days, and every 20 h it was cooled to 0 C., 0.1 ml of HCl (37%) was added dropwise and then the suspension was stirred at 0 C. for 4 h. The suspension was concentrated, the residue was admixed with CH.sub.2Cl.sub.2, the suspension was washed with cold NaHCO.sub.3 solution and with saturated NaCl solution, dried (MgSO.sub.4), filtered and concentrated. The residue was used directly without storage.
(37) Compound 13
(38) A mixture of 2 ml of CH.sub.2Cl.sub.2, 2 ml of ethyl 6-hydroxyhexanoate and 0.5 g of dry molecular sieve A4 was mixed with 50 mg of compound 12 and stirred for 5 days. CH.sub.2Cl.sub.2 was removed under reduced pressure, the suspension was stirred at 40 C. for 1 h and diluted with CH.sub.2Cl.sub.2. Ethyl 6-hydroxyhexanoate was removed on a silica gel column (CH.sub.2Cl.sub.2:MeOH 100:1), and the products were eluted from the column (CH.sub.2Cl.sub.2:MeOH 5:1) and concentrated. The residue was purified on RP18. Yield: 20 mg
(39) Compound 14
(40) A solution of 20 mg of compound 13 in 5 ml of MeOH and 0.2 ml of HAc (20%) was admixed with a catalytic amount of Pd-on-carbon and hydrogenated for 60 min. The suspension was filtered over Celite, concentrated and lyophilized. Yield: 16 mg
(41) Compound 15
(42) A solution of 16 mg of compound 14 in 1 ml of DMF was admixed with 13 mg of sulfur trioxide-pyridine (1:1) and 58 l of TEA, stirred for 20 min, admixed with MeOH and stirred for a further 20 min. The excess TEA was removed under reduced pressure and the solution was diluted with water, adjusted to pH 8 with saturated NaHCO.sub.3 and purified on RP18. The product was dissolved with a little EtOH, and the solution was diluted with water until turbidity occurred, adjusted to a pH of 13 using 2M NaOH, stirred for 2 h, diluted with 10 ml of water, adjusted to a pH of 8 with dilute acetic acid, and purified on RP18. Yield: 4 mg; .sup.1H NMR (500 MHz, CD.sub.3OD): ppm 7.94 (d, J=8.45 Hz, 2H), 7.73 (d, J=8.53 Hz, 2H), 7.68 (dd, J=8.33, 1.11 Hz, 2H), 7.47 (t, J=7.68 Hz, 2H), 7.38 (t, J=7.39 Hz, 1H), 4.08 (ddd, J=8.82, 7.94, 3.21 Hz, 1H), 3.79 (td, J=9.10, 6.96 Hz, 1H), 3.78 (dd, J=13.54, 3.23 Hz, 1H), 3.71 (dd, J=10.22, 1.90 Hz, 1H), 3.61 (t, J=10.20 Hz, 1H), 3.58 (dd, J=13.66, 7.75 Hz, 1H), 3.47 (td, J=9.05, 7.02 Hz, 1H), 3.45 (dd, J=9.05, 1.94 Hz, 1H), 3.39 (ddd, J=12.12, 10.61, 4.41 Hz, 1H), 3.07 (dd, J=12.68, 4.54 Hz, 1H), 2.16-2.12 (m, 2H), 2.01 (s, 3H), 1.65-1.52 (m, 5H), 1.40-1.32 (m, 2H); HRMS (ESI-neg) calculated for C30H36N3Na3O13S [M-3Na+2H].sup.: 680.2131, found: 680.2153.
(43) Compound 16
(44) A solution of 65 mg of compound 14 in 1 ml of DMF was admixed with 12 l of propanoic anhydride and 35 l of TEA, stirred for 1 h and concentrated. The residue was purified on RP18. Yield: 60 mg; .sup.1H NMR (500 MHz, CD.sub.3OD): ppm 7.89 (d, J=8.55 Hz, 2H), 7.65 (d, J=8.56 Hz, 2H), 7.60 (dd, J=8.30, 1.22 Hz, 2H), 7.45 (t, J=7.54 Hz, 2H), 7.38 (t, J=7.36 Hz, 1H), 7.10 (dd, J=8.03, 4.48 Hz, 1H), 5.76 (d, J=8.03 Hz, 1H), 5.69 (d, J=9.33 Hz, 1H), 5.31 (td, J=9.69, 3.31 Hz, 1H), 5.22 (dd, J=9.73, 2.13 Hz, 1H), 4.29 (ddd, J=15.04, 8.20, 3.13 Hz, 1H), 4.10 (q, J=7.13 Hz, 2H), 4.10 (dd, J=10.08, 2.24 Hz), 3.99-3.88 (m, 2H), 3.78 (td, J=9.39, 6.28 Hz, 1H), 3.77 (s, 3H), 3.21 (td, J=9.41, 6.39 Hz, 1H), 3.06 (td, J=15.07, 4.12 Hz, 1H), 2.57 (dd, J=12.82, 3.58 Hz, 1H), 2.29 (t, J=7.73 Hz, 2H), 2.22 (s, 3H), 2.13 (s, 3H), 1.89 (s, 3H), 1.72 (t, J=12.64 Hz, 1H), 1.67-1.60 (m, 2H), 1.58-1.52 (m, 2H), 1.44-1.32 (m, 2H), 1.24 (t, J=7.14 Hz, 3H), 1.09 (t, J=7.60 Hz, 3H); HRMS (ESI-pos) calculated for C40H53N3O13 [M+Na].sup.+: 806.3471, found: 806.3492.
(45) Compound 17
(46) A solution of 30 mg of compound 16 in a little EtOH was admixed with water until turbidity occurred, adjusted to a pH of 12-13 using 2M NaOH, and maintained at said pH for 2 h. The solution was diluted with 10 ml of water, adjusted to a pH of 8-9 using dilute acetic acid and purified on RP18. Yield: 24 mg; .sup.1H NMR (500 MHz, CD.sub.3OD): ppm 7.94 (d, J=8.61 Hz, 2H), 7.72 (dd, J=8.69, 1.85 Hz, 2H), 7.67 (dd, J=6.95, 1.46 Hz, 2H), 7.46 (t, J=7.61 Hz, 2H), 7.38 (t, J=7.39 Hz, 1H), 4.11-4.02 (m, 2H), 3.84-3.76 (m, 4H), 3.60 (dd, J=13.57, 7.35 Hz, 1H), 3.51-3.44 (m, 2H), 2.67 (dd, J=12.43, 4.40 Hz, 1H), 2.20-2.12 (m, 4H), 1.91 (s, 3H), 1.66 (t, J=12.56 Hz, 1H), 1.64-1.53 (m, 4H), 1.41-1.33 (m, 2H), 1.11 (t, J=7.66 Hz, 3H); HRMS (ESI-neg) calculated for C33H41N3Na2O11[M-2Na+H].sup.: 656.2825, found: 656.2815.
(47) Compound 18
(48) A solution of 20 mg of compound 14 in 1 ml of DMF was admixed with 5 mg of succinic anhydride and 21 l of TEA, stirred for 30 min and concentrated. The residue was purified on RP18. Yield: 17 mg; .sup.1H NMR (300 MHz, CD.sub.3OD): ppm 7.84 (d, J=8.36 Hz, 2H), 7.71 (d, J=8.26 Hz, 2H), 7.65 (d, J=7.46 Hz, 2H), 7.46 (t, J=7.45 Hz, 2H), 7.37 (t, J=7.27 Hz, 1H), 5.44 (ddd, J=9.36, 6.87, 2.61 Hz, 1H), 5.26 (dd, J=9.25, 2.13 Hz, 1H), 4.20 (dd, J=10.13, 1.95 Hz, 1H), 4.09 (q, J=7.11 Hz, 2H), 3.95-3.73 (m, 7H), 3.37 (dd, J=14.53, 6.89 Hz, 1H), 3.23 (td, J=9.56, 6.28 Hz, 1H), 2.44 (dd, J=13.07, 2.97 Hz, 1H), 2.29 (t, J=7.39 Hz, 2H), 2.17 (s, 2H), 2.12 (s, 2H), 1.85 (s, 3H), 1.75 (t, J=12.75 Hz, 1H), 1.67-1.47 (m, 4H), 1.45-1.30 (m, 4H), 1.22 (t, J=7.12 Hz, 3H); HRMS (ESI-neg) calculated for C41H52N3NaO15 [M-Na].sup.: 826.3404, found: 826.3381.
(49) Compound 20
(50) A solution of 25 mg of compound 19 (WO 2013097942) in water was adjusted to a pH of 13 using 2M NaOH, held at that pH for 2 h, adjusted to a pH of 8 using dilute HAc and purified on RP18. The lyophilized product (20 mg) was dissolved in 0.5 ml of DMF and admixed in portions with sulfur trioxide-pyridine (1:1) until only about 20% of reactant was detectable in the TLC. MeOH was added, stirring took place for 30 min, and the MeOH was removed under reduced pressure. The solution was diluted with water, adjusted to a pH of 8 using NaHCO.sub.3 solution and purified on RP18. Yield: 5 mg (21%).sup.1H NMR (500 MHz, CD.sub.3OD): ppm 7.94 (d, J=8.63 Hz, 2H), 7.73 (d, J=8.62 Hz, 2H), 7.68 (dd, J=8.38, 1.21 Hz, 2H), 7.47 (t, J=7.66 Hz, 2H), 7.38 (t, J=7.40 Hz, 1H), 4.46 (ddd, J=11.61, 9.99, 5.15 Hz, 1H), 4.09 (ddd, J=8.84, 7.74, 3.27 Hz, 1H), 3.87 (td, J=9.47, 6.40 Hz, 1H), 3.81 (t, J=10.23 Hz, 1H), 3.79 (dd, J=13.65, 3.28 Hz, 1H), 3.73 (dd, J=10.44, 1.89 Hz, 1H), 3.58 (dd, J=13.68, 7.55 Hz, 1H), 3.58 (td, J=9.49, 6.34 Hz, 1H), 3.46 (dd, J=8.96, 1.86 Hz, 1H), 3.19 (dd, J=12.26, 5.10 Hz, 1H), 2.73 (dd, J=8.03, 7.62 Hz, 2H), 2.59 (dt, J=7.14, 5.04 Hz, 2H), 2.40 (dd, J=8.02, 7.60 Hz, 2H), 2.00 (s, 3H), 1.85-1.79 (m, 2H), 1.71 (t, J=12.00 Hz, 1H); HRMS (ESI-neg) calculated for C30H35N2Na3O14S2 [M-3Na+2H].sup.: 713.1692, found: 713.1715.
(51) Compound 22
(52) Compound 21 (3500 mg) was dissolved in 20 ml of dry allyl alcohol, admixed with 5 g of ground molecular sieve A4 and stirred for 5 h. The suspension was concentrated at 40 C. and the residue was admixed with CH.sub.2Cl.sub.2, filtered over Celite and purified on a silica gel column (CH.sub.2Cl.sub.2 to MeOH). The isolated product was recrystallized from ethyl acetate/diethyl ether. Yield: 1600 mg
(53) Compound 23
(54) A solution of 1600 mg of compound 22 in 20 ml of dry MeOH was admixed with 1 ml of 1M sodium methoxide. Following complete reaction, the solution was neutralized with ion exchange resin (Dowex H+form) in MeOH. The resin was removed by filtration and the solution was concentrated. Yield: 1200 mg
(55) Compound 24
(56) A solution of 1200 mg of compound 23 in 20 ml of MeOH and 4 ml of water was admixed with 2010 mg of zinc powder and 1670 mg of ammonium chloride and stirred for 5 h. The suspension was filtered over Celite, concentrated and used further directly. Yield: 1200 mg
(57) Compound 25
(58) A solution of 1200 mg of compound 24 in 20 ml of MeOH was admixed with 721 mg of Boc anhydride and 0.49 ml of TEA, stirred for 2 h and concentrated. The residue was purified on silica gel (ethyl acetate). Yield: 976 mg
(59) Compound 26
(60) Compound 25 (976 mg) was dissolved three times in 10 ml of dry DMF and concentrated under reduced pressure. The anhydrous residue was dissolved in 10 ml of dry DMF and admixed at 0 C. with 517 mg of dry lithium azide, 1678 mg of tetrabromomethane and 664 mg of triphenylphosphine. The solution was stirred at 25 C. for 16 h, admixed with 5 ml of MeOH and concentrated. The residue was admixed with water and extracted twice with ethyl acetate. The combined organic phases were washed with saturated NaCl, dried (MgSO.sub.4), filtered and purified on a silica gel column (ethyl acetate). Yield: 700 mg
(61) Compound 27
(62) A solution of 700 mg of compound 26 in 50 ml of DMF was admixed with 0.5 ml of water, 1130 mg of triphenylphosphine and 1375 mg of 4-nitrophenyl 4-biphenylcarboxylate, stirred for three days, concentrated and purified on a silica gel column (CH.sub.2Cl.sub.2 to MeOH). Yield: 440 mg
(63) Compound 28
(64) A solution of 30 mg of compound 27 in 5 ml of MeOH and 2 ml of water was flushed with nitrogen for 20 min, admixed with 41 l of thiopropionic acid and a catalytic amount of azaisobutyronitrile, and irradiated with UV light for 12 h. The solution was concentrated and the residue was purified on RP18. Yield: 30 mg; .sup.1H NMR (500 MHz, CD.sub.3OD): ppm 7.93 (d, J=8.62 Hz, 2H), 7.71 (d, J=8.65 Hz, 2H), 7.66 (dd, J=8.38, 1.21 Hz, 2H), 7.45 (t, J=7.62 Hz, 2H), 7.37 (t, J=7.39 Hz, 1H), 4.05 (ddd, J=8.87, 7.28, 3.21 Hz, 1H), 3.91-3.82 (m, 6H), 3.80 (dd, J=13.83, 3.23 Hz, 1H), 3.67-3.62 (m, 1H), 3.60 (dd, J=13.78, 7.35 Hz, 1H), 3.49 (td, J=9.37, 6.42 Hz, 1H), 3.45 (dd, J=8.83, 1.25 Hz, 1H), 2.71 (dd, J=8.46, 7.06 Hz, 2H), 2.62-2.51 (m, 3H), 2.39 (dd, J=8.56, 6.84 Hz, 2H), 1.90 (s, 3H), 1.82-1.70 (m, 3H), 1.42 (s, 9H); HRMS (ESI-neg) calculated for C36H48N3NaO12S [M-Na].sup.: 746.2964, found: 746.2928.
(65) Compound 29
(66) A solution of 30 mg of compound 28 in a little MeOH was admixed with water until turbidity occurred, adjusted to a pH of 13 using 2M NaOH, stirred at the pH for 2 h, adjusted to a pH of 8-9 using dilute acetic acid, and purified on RP18. Yield: 25 mg; .sup.1H NMR (500 MHz, CD.sub.3OD): ppm 7.93 (d, J=8.61 Hz, 2H), 7.71 (d, J=8.63 Hz, 2H), 7.66 (dd, J=8.37, 1.19 Hz, 2H), 7.45 (t, J=7.63 Hz, 2H), 7.36 (t, J=7.39 Hz, 1H), 4.06 (ddd, J=8.96, 7.60, 3.17 Hz, 1H), 3.87 (td, J=9.48, 6.39 Hz, 1H), 3.80-3.66 (m, 4H), 3.57 (dd, J=13.22, 6.98 Hz, 1H), 3.54 (td, J=9.48, 6.25 Hz, 1H), 3.44 (dd, J=8.99, 1.24 Hz, 1H), 2.72 (dd, J=8.51, 6.99 Hz, 12H), 2.68 (dd, J=12.56, 3.65 Hz, 1H), 2.63-2.52 (m, 2H), 2.40 (dd, J=8.51, 7.12 Hz, 2H), 1.92 (s, 3H), 1.83-1.76 (m, 2H), 1.61 (t, J=12.16 Hz, 1H), 1.42 (s, 9H); HRMS (ESI-neg) calculated for C36H45N3Na2O12S [M-2Na+H].sup.: 732.2808, found: 732.2763.
(67) Compound 30
(68) A solution of 3500 mg of compound 21 in 4 ml of CH.sub.2Cl.sub.2 was stirred together with 1.9 ml of ethyl 6-hydroxyhexanoate and 5 g of dry molecular sieve A4, and CH.sub.2Cl.sub.2 was removed on a rotary evaporator. The suspension was stirred at 40 C. for 6 h, diluted with CH.sub.2Cl.sub.2 and filtered over Celite. Ethyl 6-hydroxyhexanoate was removed on a silica gel column (CH.sub.2Cl.sub.2:MeOH 100:1). The products were eluted from the column with (CH.sub.2Cl.sub.2:MeOH 5:1) and again purified on silica gel (CH.sub.2Cl.sub.2 to MeOH). Yield 2300 mg of unclean product, which was used further without additional purification.
(69) Compound 31
(70) A solution of 2300 mg of compound 30 in 20 ml of dry MeOH was admixed with fresh sodium methoxide in MeOH until pH indicator paper indicated a pH of 8-9. After 1 h the solution was neutralized with ion exchange resin (Dowex H+form) in MeOH. The resin was removed by filtration, the solution was concentrated and the residue was purified on a silica gel column (CH.sub.2Cl.sub.2 to MeOH). The fractions containing product were purified on RP18. Yield: 374 mg
(71) Compound 32
(72) A solution of 370 mg of compound 31 in 10 ml of MeOH was admixed with 0.5 ml of acetic acid (20%) and a catalytic amount of Pd-on-carbon and hydrogenated for 2 h. The suspension was filtered over Celite, the solution was concentrated and the residue was lyophilized. Yield: 395 mg
(73) Compound 33
(74) A solution of 395 mg of compound 32 in 10 ml of DMF was admixed with 142 mg of benzyloxycarbonyl chloride and 0.31 ml of TEA, stirred for 2 h and concentrated. The residue was purified on a silica gel column (CH.sub.2Cl.sub.2 to MeOH). Yield: 245 mg
(75) Compound 34
(76) Compound 33 (245 mg) was dissolved three times in 10 ml of dry DMF and concentrated under reduced pressure. The anhydrous residue was dissolved in 10 ml of dry DMF and admixed at 0 C. with 100 mg of dry lithium azide, 325 mg of tetrabromomethane and 139 mg of triphenylphosphine. The solution was stirred at 25 C. for 16 h, admixed with 5 ml of MeOH and concentrated. The residue was admixed with water and was extracted twice with toluene and twice with ethyl acetate. The combined ethyl acetate phases were dried (MgSO.sub.4), filtered and purified on RP18. Yield: 176 mg
(77) Compound 35
(78) A solution of 175 mg of compound 34 in 10 ml of MeOH and 2 ml of water was admixed with 182 mg of zinc powder and 151 mg of ammonium chloride and stirred for 2 h. The suspension was filtered over Celite, concentrated and used further directly. Yield: 270 mg
(79) Compound 36
(80) A solution of 55 mg of compound 35 and 28 mg of 4-(4-fluorophenyl)benzoic acid in 0.5 ml of DMF was admixed with 49 mg of O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium and 0.146 ml of DIPEA. The solution was stirred for 10 min and concentrated and the residue was purified over RP18. Yield 40 mg
(81) Compound 37
(82) A solution of 40 mg of compound 36 in 5 ml of MeOH and 0.5 ml of HAc (20%) was admixed with a catalytic amount of Pd-on-carbon and hydrogenated for 60 min. The suspension was filtered over Celite, concentrated and lyophilized. The residue (30 mg) was dissolved in 2 ml of DMF and admixed with 20 mg of sulfur trioxide-pyridine (1:1) and 59 l of TEA. The solution was stirred for 60 min, admixed with 5 ml of water and 0.5 ml of 2M NaOH and stirred for a further 40 min, neutralized with HAc and concentrated. The residue was dissolved in water, admixed with 1 ml of saturated Na.sub.2CO.sub.3 and purified on RP18. Yield: 8 mg; .sup.1H NMR (500 MHz, CD.sub.3OD): ppm 7.92 (d, J=8.54 Hz, 2H), 7.71-7.67 (m, 4H), 7.19 (t, J=8.84 Hz, 2H), 4.06 (ddd, J=9.19, 8.27, 3.20 Hz, 1H), 3.80-3.73 (m, 2H), 3.69 (dd, J=10.20, 1.94 Hz, 1H), 3.60 (dd, J=10.84, 9.69 Hz, 1H), 3.56 (dd, J=13.66, 7.80 Hz, 1H), 3.46 (td, J=8.94, 6.86 Hz, 1H), 3.43 (dd, J=8.96, 1.93 Hz, 1H), 3.37 (ddd, J=12.25, 10.61, 4.49 Hz, 1H), 3.05 (dd, J=12.71, 4.53 Hz, 1H), 2.13 (dd, J=8.02, 7.36 Hz, 2H), 2.00 (s, 3H), 1.62-1.51 (m, 5H), 1.38-1.31 (m, 2H); HRMS (ESI-neg) calculated for C30H35FN3Na3O13S [M-2Na+H].sup.: 720.1889, found: 720.1856.
(83) Compounds 38, 39, 40, 41, 42
(84) General procedure: A solution of 0.20 mg of compound 9 in 50 l of DMF was admixed with a 25-fold excess of the corresponding anhydride and 100-fold excess of DIPEA. The solution was stirred at 20 C. for 10 min. The reaction was monitored by thin-layer chromatography and was quantitative for all the experiments. The solution was diluted with 500 l of water, admixed with 50 l of 2M NaOH and, after 2 h, was admixed with 50 l of HAc (20%) and lyophilized. Following the addition of acetic acid, the reaction was monitored by thin-layer chromatography and was quantitative for all the experiments.
(85) Compounds 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64
(86) General procedure: A solution of 0.20 mg of compound 9 in 50 l of DMF was admixed with a 25-fold excess of the corresponding carboxylic acid, a 15-fold excess of HATU and 100-fold excess of DIPEA and the solution was stirred at 20 C. for 10 min. The reaction was monitored by thin-layer chromatography and was quantitative for all the experiments. The solution was diluted with 500 l of water, admixed with 50 l of 2M NaOH and, after 2 h, was admixed with 50 l of HAc (20%) and lyophilized. Following the addition of acetic acid, the reaction was monitored by thin-layer chromatography and was quantitative for all the experiments.
(87) Compounds 65
(88) A solution of 0.20 mg of compound 9 in 50 l of DMF was admixed with a 25-fold excess of dansyl chloride and 100-fold excess of DIPEA and the solution was stirred at 20 C. for 10 min. The reaction was monitored by thin-layer chromatography and was quantitative. The solution was diluted with 500 l of water, admixed with 50 l of 2M NaOH and, after 2 h, was admixed with 50 l of HAc (20%) and lyophilized. Following the addition of acetic acid, the reaction was monitored by thin-layer chromatography and was quantitative.
(89) Compounds 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76
(90) General procedure: A solution of 0.20 mg of compound 35 in 50 l of DMF was admixed with a 25-fold excess of the corresponding carboxylic acid, a 15-fold excess of HATU and 100-fold excess of DIPEA and the solution was stirred at 20 C. for 10 min. The reactions were monitored by thin-layer chromatography and was quantitative for all the experiments. The solution was diluted with 500 l of water, admixed with 50 l of 2M NaOH and, after 2 h, was admixed with 50 l of HAc (20%) and lyophilized. Following the addition of acetic acid, the reaction was monitored by thin-layer chromatography and was quantitative for all the experiments.
(91) Compound 77
(92) A solution of 40 mg of compound 27 in 5 ml of MeOH and 2 ml of water was flushed with nitrogen for 20 min, admixed with 705 mg of cysteamine hydrochloride and a catalytic amount of azaisobutyronitrile, and irradiated with UV light for 12 h. The solution was concentrated and the residue was purified on RP18. Yield: 407 mg; .sup.1H NMR (500 MHz, CD.sub.3OD): ppm 7.93 (d, J=8.64 Hz, 2H), 7.72 (d, J=8.66 Hz, 2H), 7.66 (dd, J=8.38, 1.23 Hz, 2H), 7.46 (t, J=7.60 Hz, 2H), 7.38 (t, J=7.38 Hz, 1H), 4.07 (ddd, J=8.67, 7.73, 3.08 Hz, 1H), 3.93 (dd, J=13.76, 2.91 Hz, 1H), 3.92 (ddd, J=9.63, 7.03, 5.02 Hz, 1H), 3.88-3.84 (m, 1H), 3.86 (s, 3H), 3.82 (dd, J=10.34, 1.28 Hz, 1H), 3.47 (dd, J=8.76, 1.42 Hz, 1H), 3.47-3.43 (m, 1H), 3.43 (dd, J=13.83, 7.64 Hz, 1H), 3.14 (t, J=6.96 Hz, 1H), 3.14 (t, J=6.56 Hz, 1H), 2.80 (t, J=6.75 Hz, 2H), 2.64 (t, J=6.97 Hz, 2H), 2.55 (dd, J=12.94, 4.00 Hz, 1H), 1.93 (s, 3H), 1.86-1.71 (m, 3H), 1.43 (s, 9H); HRMS (ESI-neg) calculated for C36H48N3NaO12S [M-Na].sup.: 719.3320, found: 719.3335.
(93) Biological Tests
(94) The biological activity of the substances was determined in an ELISA-based inhibition assay as described in Eur. J. Immunol. 2012, 42, 2792-2802. The sialic acid derivatives of the formula (I) inhibit the binding of soluble CD22 to immobilized IgM in proportion to their affinity for CD22. The soluble CD22 protein was expressed in the CHO-Lec1 cell line and isolated as described by Eur. J. Immunol. 2012, 42, 2792-2802. The known substance BPCNeu5Ac (J. Exp. Med. 2002, 195, 1207-1213) was co-tested in the assays as a reference. The increase in affinity is reported as rIP (relative inhibitory affinity).
(95) Table I shows the affinity or relative inhibitory potency (rIP) of the sialic acid derivatives of the formula (I).
(96) TABLE-US-00001 TABLE I IC50 No. Structure microM rIP BPC Neu5 Ac
(97) Further sialic acid derivatives of the formula (I) for which rIP values were determined are listed in Table II. As a reference, compound 10, a sialic acid derivative of the formula (I) with surprisingly high affinity, was co-tested in the same assay. The sialic acid derivatives of the formula (I) in Table II show an affinity level comparable with that of reference compound 10.
(98) TABLE-US-00002 TABLE II No. Structure rIP 10
(99) Further sialic acid derivatives of the formula (I) for which rIP values were determined are listed in Table III. As a reference, compound 17, a sialic acid derivative of the formula (I) with surprisingly high affinity, was co-tested in the same assay. The sialic acid derivatives of the formula (I) in Table III show an affinity level comparable with that of reference compound 17.
(100) TABLE-US-00003 TABLE III No. Structure rIP 17
(101) Further sialic acid derivatives of the formula (I) for which rIP values were determined are listed in Table IV. As a reference, compound 66 was co-tested in the same assay. The sialic acid derivatives of the formula (I) in Table IV show an affinity level comparable with that of reference compound 66.
(102) TABLE-US-00004 TABLE IV No. Structure rIP 66
(103) Table V shows sialic acid derivatives of the formula (I) which are suitable as prodrugs, their affinity for CD22 having not been determined, since the active molecule is liberated only in vivo by enzymatic cleavage.
(104) TABLE-US-00005 TABLE V No. Structure 16