Chito-oligosaccharide drug constructs

Abstract

Improved chito-oligosaccharide-drug constructs are provided. In various embodiments, the improved CO-drug constructs include CO-(m-PAB)- exatecan, CO-(m-PAB)-([β-Ala2)-exatecan, CO-(m-PAB)-duocarmycin, CO-(m-PAB)-DM1, and CO-(m-PAB)-MMAE. Also provided are methods for preparing these compounds.

Description

CHITO-OLIGOSACCHARIDE DRUG CONSTRUCTSCross-Reference to Related Applications

[0001] This applications claims priority to, and benefit of U.S. provisional patent application serial no. 63 / 656,409, filed June 5, 2024, the entirety of which is incorporated herein by reference.Background

[0002] Antibody-drug conjugates (ADCs) utilize the high physiological specificity of antibodies to deliver highly potent pharmaceutical agents to biological targets, often of onocogenic issue. ADCs consist of three components: antibody (often a monoclonal antibody, or mAb), linker (stable or self-immolative), and drug / warhead.

[0003] While ADCs such as the blockbuster breast cancer treatment trastuzumab deruxtecan (Enhertu®, Daiichi Sankyo) successfully enable treatment of metastatic and unresectable cancers, candidate ADCs often run afoul of aggregation and poor solubility that limit their utility as therapeutics. These detrimental behaviors are often attributed to the impact of the druglinker construct (“construct”) to the overall ADC hydrophobicity and surface topology. Linkers expressing solubilizing units have been developed to address these shortcomings, notably chito-oligosaccharide (Chetosensar™ technology, MilliporeSigma) as disclosed in International Patent Appl. WO2022 / 058548.Summary

[0004] Improved chito-oligosaccharide (CO) drug constructs are provided. Such embodiments include a chito-oligosaccharide, a linker, and drug useful in ADCs.

[0005] In one embodiment, the CO-drug construct is CO-(m-PAB)- exatecan of formula:

[0006] In one embodiment, the CO-drug construct is CO-(m-PAB)-([3-Ala2)- exatecan of formula:O O

[0007] In one embodiment, the CO-drug construct is CO-(m-PAB)- duocarmycin of formula:

[0008] In one embodiment, the CO-drug construct is CO-(m-PAB)-DM1 of formula:

[0009] In one embodiment, the CO-drug construct is CO-(m-PAB)-MMAE of formula:Brief Description of the Drawings

[0010] FIG. 1 shows the synthesis of intermediate lnt-12 used in the synthesis of CO-(m-PAB)-drug constructs.

[0011] FIG. 2 is the LCMS chromatogram of tert-butyl 3-(5-amino-2- (hydroxymethyl)benzamido) propanoate (2).

[0012] FIG. 3 is the1H NMR of of tert-butyl 3-(5-amino-2-(hydroxymethyl) benzamido) propanoate (2).

[0013] FIG. 4 is the LCMS chromatogram of tert-butyl (S)-3-(5-(2-((((9H- fluoren-9-yl) methoxy) carbonyl) amino)-5-ureidopentanamido)-2- (hydroxymethyl) benzamido) propanoate (4).

[0014] FIG. 5 is the LCMS chromatogram for tert-butyl (S)-3-(5-(2-amino-5- ureidopentanamido)-2-(hydroxymethyl) benzamido) propanoate (5).

[0015] FIG. 6 is the HPLC chromatogram for tert-butyl (S)-3-(5-(2-amino-5- ureidopentanamido)-2-(hydroxymethyl) benzamido) propanoate (5).

[0016] FIG. 7 is the LCMS chromatogram for tert-butyl 3-(5-((S)-2-((S)-2- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5- ureidopentanamido)-2-(hydroxymethyl)benzamido)propanoate (7).

[0017] FIG. 8 is the HPLC chromatogram for tert-butyl 3-(5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5- ureidopentanamido)-2-(hydroxymethyl)benzamido)propanoate (7).

[0018] FIG. 9 is the LCMS chromatogram for tert-butyl 3-(5-((S)-2-((S)-2- amino-3-methylbutanamido)-5-ureido pentanamido)-2-(hydroxymethyl) benzamido) propanoate (8).

[0019] FIG. 10 is the HPLC chromatogram for tert-butyl 3-(5-((S)-2-((S)-2- amino-3-methylbutanamido)-5-ureido pentanamido)-2-(hydroxymethyl) benzamido) propanoate (8).

[0020] FIG. 11 is the LCMS chromatogram for tert-butyl 3-(2- (hydroxymethyl)-5-((15S, 18S)-15-isopropyl-2,2-dimethyl-4,7, 10,13,16- pentaoxo-18-(3-ureidopropyl)-3-oxa-5,8, 11 ,14, 17-pentaazanonadecan-19- amido)benzamido)propanoate (10).

[0021] FIG. 12 is the HPLC chromatogram for tert-butyl 3-(2- (hydroxymethyl)-5-((15S, 18S)-15-isopropyl-2,2-dimethyl-4,7, 10,13,16- pentaoxo-18-(3-ureidopropyl)-3-oxa-5,8, 11 ,14, 17-pentaazanonadecan-19- amido)benzamido)propanoate (10).

[0022] FIG. 13 is the LCMS chromatogram for tert-butyl 3-(5-((15S,18S)- 15-isopropyl-2,2-dimethyl-4,7, 10,13,16-pentaoxo-18-(3-ureidopropyl)-3-oxa- 5,8, 11 , 14, 17-pentaazanonadecan-19-amido)-2-((((4- nitrophenoxy)carbonyl)oxy)methyl)benzamido)propanoate (lnt-12).

[0023] FIG. 14 is the HPLC chromatogram for tert-butyl 3-(5-((15S,18S)- 15-isopropyl-2,2-dimethyl-4,7, 10,13,16-pentaoxo-18-(3-ureidopropyl)-3-oxa- 5,8, 11 , 14, 17-pentaazanonadecan-19-amido)-2-((((4- nitrophenoxy)carbonyl)oxy)methyl)benzamido)propanoate (lnt-12).

[0024] FIG. 15 shows the scheme for the synthesis of CO-(m-PAB)-EXA.

[0025] FIG. 16 is the LCMS chromatogram for tert-butyl 3-(2-(((((1S,9S)-9- ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9, 10,13,15-hexahydro- 1 H, 12H-benzo[de]pyrano[3',4':6,7]indolizino[1 , 2-b]quinol in-1 -yl)carbamoyl)oxy)methyl)-5-((15S, 18S)-15-isopropyl-2,2-dimethyl- 4,7, 10, 13, 16-pentaoxo-18-(3-ureidopropyl)-3-oxa-5,8, 11 , 14, 17- pentaazanonadecan-19-amido)benzamido)propanoate (14).

[0026] FIG. 17 is the HPLC chromatogram for tert-butyl 3-(2-(((((1 S,9S)-9- ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9, 10,13,15-hexahydro- 1 H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-1- yl)carbamoyl)oxy)methyl)-5-((15S, 18S)-15-isopropyl-2,2-dimethyl- 4,7, 10, 13, 16-pentaoxo-18-(3-ureidopropyl)-3-oxa-5,8, 11 , 14, 17- pentaazanonadecan-19-amido)benzamido)propanoate (14).

[0027] FIG. 18 is the LCMS chromatogram for 3-(5-((2S,5S)-14-amino-5- isopropyl-4,7, 10, 13-tetraoxo-2-(3-ureidopropyl)-3,6,9, 12- tetraazatetradecanamido)-2-(((((1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10, 13-dioxo-2,3,9, 10,13,15-hexahydro-1 H, 12H- benzo[de]pyrano[3',4':6,7]indolizino[1 , 2-b]qu inol in-1 - yl)carbamoyl)oxy)methyl)benzamido)propanoic acid (15).

[0028] FIG. 19 is the HPLC chromatogram for 3-(5-((2S,5S)-14-amino-5- isopropyl-4,7, 10, 13-tetraoxo-2-(3-ureidopropyl)-3,6,9, 12- tetraazatetradecanamido)-2-(((((1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10, 13-dioxo-2,3,9, 10,13,15-hexahydro-1 H, 12H- benzo[de]pyrano[3',4':6,7]indolizino[1 , 2-b]qu inol in-1 - yl)carbamoyl)oxy)methyl)benzamido)propanoic acid (15).

[0029] FIG. 20 is the LCMS chromatogram for 3-(5-((2S,5S)-18-(2,5-dioxo- 2,5-dihydro-1 H-pyrrol-1 -y l)-5-isopropy I-4, 7 , 10,13,16-pentaoxo-2-(3- ureidopropyl)-3,6,9,12,15-pentaazaoctadecanamido)-2-(((((1 S,9S)-9-ethyl-5- fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9, 10, 13, 15-hexahydro-1 H, 12H- benzo[de]pyrano[3',4':6,7]indolizino[1 , 2-b]qu inol in-1 - yl)carbamoyl)oxy)methyl)benzamido) propanoic acid (17).

[0030] FIG. 21 is the HPLC chromatogram for 3-(5-((2S,5S)-18-(2,5-dioxo- 2,5-dihydro-1 H-pyrrol-1 -y l)-5-isopropy I-4, 7 , 10,13,16-pentaoxo-2-(3- ureidopropyl)-3,6,9,12,15-pentaazaoctadecanamido)-2-(((((1 S,9S)-9-ethyl-5- fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9, 10, 13, 15-hexahydro-1 H, 12H-benzo[de]pyrano[3',4':6,7]indolizino[1 , 2-b]qu inol in-1 - yl)carbamoyl)oxy)methyl)benzamido) propanoic acid (17).

[0031] FIG. 22 is the LCMS chromatogram for CO-(m-PAB)-exatecan.

[0032] FIG. 23 is the mass spectra for CO-(m-PAB)-exatecan.

[0033] FIG. 24 is the HPLC chromatogram for CO-(m-PAB)-exatecan.Detailed Description

[0034] Constructs herein disclosed exhibit higher solubility (both of construct and resulting ADC), solution stability, and mAb conjugation potential than earlier disclosed CO-containing constructs. Such enhancements position constructs as highly desirable for future ADC development.

[0035] Exemplary embodiments include:though other embodiments may be envisioned by those skilled in the art.

[0036] As noted, each construct includes a chito-oligosaccharide solubility tag, a linker and a drug. In various embodiments, the chito-oligosaccharide solubility tag is selected from the following:

[0037] In a preferred embodiment, the chito-oligosaccharide solubility tag is the following, identified herein as chito-oligosaccharide CO-V:

[0038] The synthesis of these new constructs is outlined in the examples below.

[0039] In various embodiments, the linker comprises a p-aminobenzyl carbamate (PABC) spacer that attaches to the drug, a cleavable peptide that attaches to the PABC spacer, and optionally functional group capable of binding with a ligand. In some embodiments, the solubility tag is covalently attached to the PABC spacer.Examples

[0040] Example 1 . Synthesis of Intermediate lnt-12. The synthesis scheme is shown in FIG. 1 .

[0041] Step 1 : Synthesis of tert-butyl 3-(5-amino-2-(hydroxymethyl) benzamido) propanoate (2)

[0042] tert-butyl 3-(2-(hydroxymethyl)-5-nitrobenzamido) propanoate (1 ) (2 g, 6.17 mmol) was dissolved in EtOAc (50 ml). Pd / C (5 wt%; 0.2 g, 0.094 mmol) was charged, and the resulting slurry was stirred under a H2 atmosphere at rt for 7 h. The reaction mass was filtered through a bed of Celite and washed with 300 mL of EtOAc. The filtrate was concentrated to yield crude tert-butyl 3-(5-amino-2-(hydroxymethyl)benzamido)propanoate (2) (1.9 g, 5.15 mmol, 84%) as a pale yellow gum.

[0043] Analytical data:

[0044] LCMS: RT (min): 1 .94; Purity (Max): 79.81 %; [M-OH]+: 277.1 . Method: column: XBridge C8 (50x4.6mm) 3.5 pm; mobile phase A :0.1% TFA in H2O; mobile phase B : ACN; flow Rate :1.5 ml / min. The chromatogram is shown in FIG. 2.

[0045] 1H NMR (400 MHz, DMSO-de): 5 8.41 (t, J = 5.60 Hz, 1 H), 7.06 (d, J = 8.00 Hz, 1 H), 6.66 (s, 1 H), 6.60-6.57 (m, 1 H), 5.20 (d, J = 12.80 Hz, 1 H), 5.05 (s, 1 H), 4.34 (s, 2H), 3.41 (t, J = 5.60 Hz, 2H), 2.46 (t, J = 6.80 Hz, 2H), 1.41 (s, 9H). The NMR spectrum is shown in FIG. 3.

[0046] Step 2: Synthesis of tert-butyl (S)-3-(5-(2-((((9H-fluoren-9-yl) methoxy) carbonyl) amino)-5-ureidopentanamido)-2-(hydroxymethyl) benzamido) propanoate (4)

[0047] To an ice-cooled solution of tert-butyl 3-(5-amino-2- (hydroxymethyl)benzamido)propanoate (2) (1.800 g, 6.12 mmol) in DMF (13.5 ml) was charged (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5- ureidopentanoic acid (3) (2.430 g, 6.12 mmol), HATLI (2.56 g, 6.73 mmol), and DIPEA (2.67 ml, 15.29 mmol). The reaction mixture was stirred to rt for 4 h. The reaction mass was subsequently quenched with ice cold water (30 mL) and extracted with dichloromethane (3 x 30 mL). The pooled organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to yield the crude compound as a brown liquid. This impure oil was purified by flash column chromatography using 5-10% of MeOH in DCM as eluent to yield tertbutyl (S)-3-(5-(2-((((9H-fluoren-9-yl)methoxy) carbonyl) amino)-5-ureidopentan amido)-2-(hydroxymethyl)benzamido)propanoate (4) (3.20 g, 3.67 mmol, 60%) as a pale yellow solid.

[0048] Analytical data: LCMS: RT (min): 2.71 ; Purity (Max): 77.29 %; [M+H]+: 674.3. Method: column: XBridge C8 (50x4.6mm) 3.5 pm; mobile phase A :0.1 % TFA in H2O; mobile phase B: ACN; flow rate :1.5 ml / min. The LCMS chromatogram is shown in FIG. 4.

[0049] Step 3: Synthesis of tert-butyl (S)-3-(5-(2-amino-5- ureidopentanamido)-2-(hydroxymethyl) benzamido) propanoate (5)

[0050] To a solution of tert-butyl (S)-3-(5-(2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-5-ureidopentan amido)-2- (hydroxymethyl)benzamido)propanoate (4) (3.2 g, 4.75 mmol) in DMF (16 ml) was added triethylamine (6.62 ml, 47.5 mmol), and the resulting reaction milieu was stirred at rt for 16 h. The reaction mass was diluted with petroleum ether: EtOAc (3:1 , 50m L), stirred for 5 minutes, and the solvents were decanted-off. This process was repeated twice and the residue obtained was concentrated under vacuum. The crude compound was purified by reverse phase chromatography on a C18 column using 0.1 % HCOOH in H2O and ACN as eluent. The product was eluted with 15 % ACN. The product fractions were lyophilized to afford tert-butyl (S)-3-(5-(2-amino-5-ureidopentanamido)-2- (hydroxymethyl) benzamido) propanoate (5) (1.38 g, 2.95 mmol, 62.1 %) as a white solid.

[0051] Analytical data:

[0052] LCMS: RT (min): 1.95; Purity (Max): 97.11 %; [M+H]+: 452.1 . Method: column: XBridge C8 (50x4.6mm) 3.5 pm; mobile phase A :0.1% TFA in H2O; mobile phase B : ACN; flow rate :1.5 ml / min. The LCMS chromatogram is shown in FIG. 5.

[0053] HPLC: RT (min): 6.58; Purity (Max): 96.47 %. Method: column: Atlantis T3 (150 X4.6)mm, 3pm; flow rate: 1.2 ml / min; mobile phase A: 0.1%TFA in water; mobile phase B:100% ACN; column temperature: 35°C. The HPLC chromatogram is shown in FIG. 6.

[0054] Step 4: tert-butyl 3-(5-((S)-2-((S)-2-((((9H-fluoren-9-yl) methoxy) carbonyl) am ino)-3-methylbutanam ido)-5-ureidopentanam ido)-2- (hydroxymethyl) benzamido) propanoate (7)

[0055] To an ice-cooled solution of tert-butyl (S)-3-(5-(2-amino-5- ureidopentanamido)-2-(hydroxymethyl) benzamido)propanoate (5) (1.380 g, 3.06 mmol) in DMF (9 ml) was charged (((9H-fluoren-9-yl)methoxy)carbonyl)- L-valine (1 .037 g, 3.06 mmol), HATU (1.511 g, 3.97 mmol), and DIPEA (1 .068 ml, 6.11 mmol). The resulting reaction was stirred at rt for 2 h. The milieu was subsequently quenched with ice-cold water (50 mL) and stirred for 5 minutes. The resulting solid was filtered, washed sequentially with water (3x3 mL) and dichloromethane (3x6 mL), and dried under vacuum to yield tert-butyl 3-(5- ((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)-2- (hydroxymethyl)benzamido)propanoate (7) (1.40 g, 1.382 mmol, 45.2%) as a white solid.

[0056] Analytical data:

[0057] LCMS: RT (min): 2.80; Purity (Max): 68.79 %; [M+H]+: 773.3. Method: column: XBridge C8 (50x4.6mm) 3.5 pm; mobile phase A :0.1% TFA in H2O; mobile phase B : ACN; flow rate :1.5 ml / min. The LCMS chromatograph is shown in FIG 7.

[0058] HPLC: RT (min): 10.41 ; Purity (Max): 76.30 %. Method: column: Atlantis T3 (150 X4.6)mm, 3pm; flow rate: 1 .2 ml / min; mobile phase A: 0.1%TFA in water; mobile phase B:100% ACN; Column Temperature: 35°C. The HPLC chromatogram is shown in FIG. 8.

[0059] Step 5: tert-butyl 3-(5-((S)-2-((S)-2-amino-3-methylbutanamido)-5- ureidopentanamido)-2-(hydroxymethyl) benzamido) propanoate (8)

[0060] To a solution of tert-butyl 3-(5-((S)-2-((S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3-methyl butanamido)-5-ureidopentanamido)-2- (hydroxymethyl)benzamido)propanoate (7) (1.40 g, 1.811 mmol) in DMF (4.2 ml) was charged triethylamine (2.52 ml, 18.11 mmol). The reaction mixture was stirred at rt for 16 h. The reaction mixture was subsequently diluted with 3:1 petroleum etherEtOAc (10 mL), stirred for 2 minutes, and decanted. This process was repeated twice to afford the crude compound. The crude compound was purified by reverse phase chromatography on a C18 column using 0.1 % HCOOH in H2O and ACN as eluent. The product was eluted with 15 % ACN. The product fractions were pooled and lyophilized to afford tertbutyl 3-(5-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureido pentanamido)-2- (hydroxymethyl) benzamido) propanoate (8) (0.370 g, 0.641 mmol, 35.4%) as a white solid.

[0061] Analytical data:

[0062] LCMS: RT (min): 2.03; Purity (Max): 95.64 %; [M+H]+: 551.3. Method: column: XBridge C8 (50x4.6mm) 3.5 pm; mobile phase A :0.1% TFA in H2O; mobile phase B : ACN; flow rate :1.5 ml / min. The LCMS chromatogram is shown in FIG. 9.

[0063] HPLC: RT (min): 6.88; Purity (Max): 95.35 %. Method: column: Atlantis T3 (150 X4.6)mm, 3pm; flow rate: 1 .2 ml / min; mobile Phase A: 0.1%TFA in water; mobile Phase B:100% ACN; column temperature: 35°C. The HPLC chromatogram is shown in FIG. 10.

[0064] Step 6: tert-butyl 3-(2-(hydroxymethyl)-5-((15S, 18S)-15-isopropyl- 2,2-dimethyl-4,7, 10, 13, 16-pentaoxo-18-(3-ureidopropyl)-3-oxa-5,8, 11 ,14,17- pentaazanonadecan-19-amido)benzamido) propanoate (10)

[0065] A solution of tert-butyl 3-(5-((S)-2-((S)-2-amino-3- methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl) benzamido)propanoate (8) (0.370 g, 0.672 mmol) in DMF (1 .2 ml) was cooled with ice. To this (tert-butoxycarbonyl)glycylglycylglycine (9) (0.214 g, 0.739 mmol) was charged followed by HATLI (0.383 g, 1.008 mmol) and DIPEA (0.293 ml, 1 .680 mmol). The resulting mixture was stirred at rt for 2.5 h. The crude reaction mixture was purified by reverse phase column chromatography using 0.1 % HCOOH in H2O and ACN, eluting with 36% ACN. The product fraction was lyophilized to afford tert-butyl 3-(2-(hydroxymethyl)-5-((15S,18S)- 15-isopropyl-2,2-dimethyl-4,7, 10,13,16-pentaoxo-18-(3-ureidopropyl)-3-oxa- 5,8, 11 , 14, 17-pentaazanonadecan-19-amido)benzamido)propanoate (10) (0.230 g, 0.279 mmol, 41 .6%) as a white solid.

[0066] Analytical data:

[0067] LCMS: RT (min): 2.32; Purity (Max): 97.68 %; [M+H]+: 822.5. Method: column: XBridge C8 (50x4.6mm) 3.5 pm; mobile phase A :0.1 % TFA in H2O; mobile phase B: ACN; flow rate :1.5 ml / min. The LCMS chromatogram is shown in FIG. 11 .

[0068] HPLC: RT (min): 8.03; Purity (Max): 99.82 %. Method: column: Atlantis T3 (150 X4.6)mm, 3pm; flow rate: 1.2 ml / min; mobile phase A: 0.1 %TFA in water; mobile phase B:100% ACN; column temperature: 35°C. The HPLC chromatogram is shown in FIG. 12.

[0069] Step 7: tert-butyl 3-(5-((15S,18S)-15-isopropyl-2,2-dimethyl- 4,7, 10, 13, 16-pentaoxo-18-(3-ureidopropyl)-3-oxa-5,8, 11 , 14, 17- pentaazanonadecan-19-amido)-2-((((4-nitrophenoxy)carbonyl) oxy)methyl)benzamido)propanoate (lnt-12)

[0070] To a solution of tert-butyl 3-(2-(hydroxymethyl)-5-((15S,18S)-15- isopropyl-2,2-dimethyl-4,7, 10,13,16-pentaoxo-18-(3-ureidopropyl)-3-oxa-5,8, 11 , 14, 17-pentaazanonadecan-19-amido)benzamido) propanoate (10) (0.340 g, 0.414 mmol) in DMF (3.4 ml) at 0 °C was charged bis(4-nitrophenyl) carbonate (0.755 g, 2.482 mmol) followed by addition of DIPEA (0.578 ml, 3.31 mmol). The reaction mixture was stirred to rt for 2 h. The solvent was removed under reduced pressure and the crude residue was purified by column chromatography over silica gel using 0-100% of ethyl acetate in petroleum ether followed by 0-12% of methanol in dichloromethane to afford tert-butyl 3-(5-((15S, 18S)-15-isopropyl-2,2-dimethyl-4,7, 10, 13, 16-pentaoxo- 18-(3-ureidopropyl)-3-oxa-5,8, 11 ,14,17-pentaazanonadecan-19-amido)-2- ((((4-nitrophenoxy)carbonyl)oxy)methyl)benzamido)propanoate (lnt-12) (0.240 g, 0.208 mmol, 50.2%) as a pale yellow solid.

[0071] Analytical data:

[0072] LCMS: RT (min): 3.00; Purity (Max): 83.50 %; [M+H]+: 987.3.Method: column: XBridge C8 (50x4.6mm) 3.5 pm; mobile phase A :0.1 % TFA in H2O; mobile phase B : ACN; flow rate :1.5 ml / min. The LCMS chromatogram is shown in FIG. 13.

[0073] HPLC: RT (min): 9.81 ; Purity (Max): 85.41 %. Method: column: Atlantis T3 (150 X4.6)mm, 3pm; flow rate: 1.2 ml / min; mobile phase A: 0.1 %TFA in water; mobile phase B:100% ACN; column temperature: 35°C. The HPLC chromatogram is shown in FIG. 14.

[0074] Example 2. Synthesis of CO-(m-PAB)-EXA. The synthesis scheme is shown in FIG. 15.

[0075] Step 8: tert-butyl 3-(2-(((((1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4- methyl-10, 13-dioxo-2,3,9, 10,13,15-hexahydro-1 H, 12H- benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-1-yl)carbamoyl)oxy) methyl)-5-((15S, 18S)-15-isopropyl-2,2-dimethyl-4,7, 10, 13, 16-pentaoxo-18-(3- ureidopropyl)-3-oxa-5,8, 11 ,14, 17-pentaazanonadecan-19- amido)benzamido)propanoate (14)

[0076] To an ice-cooled solution of tert-butyl 3-(5-((15S,18S)-15-isopropyl- 2,2-dimethyl-4,7, 10, 13, 16-pentaoxo-18-(3-ureidopropyl)-3-oxa-5,8, 11 ,14,17- pentaazanonadecan-19-amido)-2-((((4- nitrophenoxy)carbonyl)oxy)methyl)benzamido)propanoate (lnt-12) (0.240 g, 0.243 mmol) and exatecan mesylate (13) (0.129 g, 0.243 mmol) in DMF (2.4 ml) were charged 1 -(11 -oxidaneyl)-1 l4-pyridin-2-ol (0.041 g, 0.365 mmol) and DIPEA (0.127 ml, 0.729 mmol). The resulting reaction mixture was stirred at rt for 2 h. The reaction mass was directly loaded on a C18 column and purified by reverse phase chromatography using 0.1 % HCOOH in H2O and ACN. The pooled product fractions were lyophilized to afford tert-butyl 3-(2- (((((1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9, 10,13,15- hexahydro-1 H, 12H-benzo[de]pyrano[3',4':6,7]indolizino[1 , 2-b]qu inol in-1 - yl)carbamoyl)oxy)methyl)-5-((15S, 18S)-15-isopropyl-2,2-dimethyl- 4,7, 10, 13, 16-pentaoxo-18-(3-ureidopropyl)-3-oxa-5,8, 11 , 14, 17-pentaazanonadecan-19-amido)benzamido)propanoate (14) (0.200 g, 0.154 mmol, 63.2%) as a white solid.

[0077] Analytical data:

[0078] LCMS: RT (min): 2.64; Purity (Max): 97.71 %; [M+H]+: 1284.5. Method: column: XBridge C8 (50x4.6mm) 3.5 pm; mobile phase A :0.1% TFA in H2O; mobile phase B : ACN; flow rate :1.5 ml / min. The LCMS chromatogram is shown in FIG. 16.

[0079] HPLC: RT (min): 9.66; Purity (Max): 98.66 %. Method: column: Atlantis T3 (150 X4.6)mm, 3pm; flow rate: 1.2 ml / min; mobile phase A: 0.1%TFA in water; mobile phase B:100% ACN; column temperature: 35°C. The HPLC chromatogram is shown in FIG. 17.

[0080] Step 9: 3-(5-((2S,5S)-14-amino-5-isopropyl-4,7, 10, 13-tetraoxo-2-(3- ureidopropyl)-3,6,9,12-tetraazatetradecanamido)-2-(((((1 S,9S)-9-ethyl-5- fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9, 10, 13, 15-hexahydro-1 H, 12H- benzo[de]pyrano[3',4':6,7]indolizino[1 , 2-b]qu inol in-1 - yl)carbamoyl)oxy)methyl)benzamido)propanoic acid (15)

[0081] tert-buty l-3-(2-((((( 1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9, 10,13,15-hexahydro-1 H, 12H- benzo[de]pyrano[3',4':6,7]indolizino[1 , 2-b]qu inol in-1 - yl)carbamoyl)oxy)methyl)-5-((15S, 18S)-15-isopropyl-2,2-dimethyl- 4,7, 10, 13, 16-pentaoxo-18-(3-ureidopropyl)-3-oxa-5,8, 11 , 14, 17- pentaazanonadecan-19-amido)benzamido)propanoate (14) (0.200 g, 0.156 mmol) was suspended in water (2 ml) and 85 % phosphoric acid (2 ml, 29.6 mmol). The reaction mixture was stirred at rt for 2 h. (Note: another 50 mg batch of the reaction was performed using the above condition and combined for purification.) The reaction mixture was purified by reverse phase column chromatography on C18 snap cartridge using 0.1 % HCOOH in H2O and ACN. The product was eluted with 52 % ACN. The product fractions were lyophilized to afford 3-(5-((2S,5S)-14-amino-5-isopropyl-4,7,10,13-tetraoxo-2- (3-ureidopropyl)-3,6,9,12-tetraazatetradecanamido)-2-(((((1 S,9S)-9-ethyl-5- fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9, 10, 13, 15-hexahydro-1 H, 12H-benzo[de]pyrano[3',4':6,7]indolizino[1 , 2-b]qu inol in-1 - yl)carbamoyl)oxy)methyl)benzamido)propanoic acid (15) (0.185 g, 0.163 mmol, 71 .1 %) as a pale yellow solid.

[0082] Analytical data:

[0083] LCMS: RT (min): 2.18; Purity (Max): 96.47 %; [M+H]+: 1128.3. Method: column: XBridge C8 (50x4.6mm) 3.5 pm; mobile phase A :0.1 % TFA in H2O; mobile phase B : ACN; flow rate :1.5 ml / min. The LCMS chromatogram is shown in FIG. 18.

[0084] HPLC: RT (min): 7.61 ; Purity (Max): 99.45 %. Method: column: Atlantis T3 (150 X4.6)mm, 3pm; flow rate: 1.2 ml / min; mobile phase A: 0.1 %TFA in water; mobile phase B:100% ACN; column temperature: 35°C. The HPLC chromatogram is shown in FIG. 19.

[0085] Step 10: 3-(5-((2S,5S)-18-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-5- isopropyl-4,7, 10,13,16-pentaoxo-2-(3-ureidopropyl)-3,6,9, 12,15- pentaazaoctadecanamido)-2-(((((1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10, 13-dioxo-2,3,9, 10,13,15-hexahydro-1 H, 12H- benzo[de]pyrano[3',4':6,7]indolizino[1 , 2-b]qu inol in-1 - yl)carbamoyl)oxy)methyl)benzamido) propanoic acid (17)

[0086] To a solution of 3-(5-((2S,5S)-14-amino-5-isopropyl-4,7,10,13- tetraoxo-2-(3-ureidopropyl)-3,6,9,12-tetraazatetradecanamido)-2-(((((1 S,9S)- 9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9, 10,13,15-hexahydro- 1 H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-1 - yl)carbamoyl)oxy)methyl)benzamido)propanoic acid (15) (150 mg, 0.133 mmol) and 2,5-dioxopyrrolidin-1 -yl 3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)propanoate (16) (70.9 mg, 0.266 mmol) in DMF (3 ml) was charged N- methylmorpholine (0.06 ml, 0.532 mmol). The resulting reaction mixture was stirred at rt for 2 h. The crude reaction mixture was purified by reverse phase chromatography on a C18 column using 0.1 % HCOOH in H2O and ACN as eluent. The product was eluted with 53 % ACN. The product fraction was lyophilized to afford 3-(5-((2S,5S)-18-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-5- isopropyl-4,7, 10,13,16-pentaoxo-2-(3-ureidopropyl)-3,6,9, 12,15-pentaazaoctadecanamido)-2-(((((1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10, 13-dioxo-2,3,9, 10,13,15-hexahydro-1 H, 12H- benzo[de]pyrano[3',4':6,7]indolizino[1 , 2-b]qu inol in-1 - yl)carbamoyl)oxy)methyl)benzamido)propanoic acid (17) (130 mg, 0.094 mmol, 71.0%) as an off-white solid.

[0087] Analytical data:

[0088] LCMS: RT (min): 2.27; Purity (Max): 96.61 %; [M+H]+: 1279.3.Method: column: XBridge C8 (50x4.6mm) 3.5 pm; mobile phase A :0.1 % TFA in H2O; mobile phase B : ACN; flow rate :1.5 ml / min. The LCMS chromatogram is shown in FIG. 20.

[0089] HPLC: RT (min): 8.03; Purity (Max): 92.88 %. Method: column: Atlantis T3(150X4.6 )mm, 3pm, mobile phase A: 0.1 % TFA in MilliQ water, mobile phase B: acetonitrile, flow: 1.2mL / min, temperature: 35°C. The HPLC chromatogram is shown in FIG. 21.

[0090] Step 11 : 2-((3-(((2S,3R,4R,5S,6R)-2-(((2R,4R,5R,6S)-5-acetamido- 6-(((2R,4R,5R,6S)-5-acetamido-6-(((2R,4R,5R,6S)-5-acetamido-6-(((2R,4R,5R)-5-acetamido-4,6-dihydroxy-2-(hydroxymethyl)tetrahydro-2H- pyran-3-yl)oxy)-4-hydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-4- hydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-4-hydroxy-2- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-3-oxopropyl)carbamoyl)-4- ((2S,5S)-18-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -y l)-5-isopropy I-4, 7, 10,13,16- pentaoxo-2-(3-ureidopropyl)-3,6,9, 12, 15-pentaazaoctadecanamido)benzyl ((1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9, 10,13,15- hexahydro-1 H, 12H-benzo[de]pyrano[3',4':6,7]indolizino[1 , 2-b]qu inol in-1 - yl)carbamate [CO-(m-PAB)-EXA]

[0091] To a solution of 3-(5-((2S,5S)-18-(2,5-dioxo-2,5-dihydro-1 H-pyrrol- 1 -y l)-5-isopropy I-4, 7, 10, 13, 16-pentaoxo-2-(3-ureidopropyl)-3,6,9, 12,15- pentaazaoctadecanamido)-2-(((((1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10, 13-dioxo-2,3,9, 10,13,15-hexahydro-1 H, 12H- benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-1 -yl)carbamoyl)oxy)methyl)benzamido)propanoic acid (17) (130 mg, 0.102 mmol) and N-((2S,3R,4R,6R)-2-(((2R,4R,5R)-5-acetamido-4,6-dihydroxy-2- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-5-(((2S,3R,4R,6R)-3- acetamido-5-(((2S,3R,4R,6R)-3-acetamido-5-(((2S,3R,4R,5S,6R)-3-amino- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (18) (101 mg, 0.102 mmol) in DMSO (3 mL) was charged HATLI (58 mg, 0.153 mmol) and DIPEA (0.04 mL, 0.254 mmol). The reaction mixture was stirred at rt for 2 h. The reaction mass was directly loaded onto a C18 reverse phase column and purified using 0.1 % HCOOH in H2O and ACN as eluent. The product was eluted with 53 % ACN. The product fractions were collected and lyophilized to afford 2-((3-(((2S,3R,4R,5S,6R)-2-(((2R,4R,5R,6S)-5-acetamido-6- (((2R,4R,5R,6S)-5-acetamido-6-(((2R,4R,5R,6S)-5-acetamido-6- (((2R,4R,5R)-5-acetamido-4,6-dihydroxy-2-(hydroxymethyl)tetrahydro-2H- pyran-3-yl)oxy)-4-hydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-4- hydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-4-hydroxy-2- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-3-oxopropyl)carbamoyl)-4- ((2S,5S)-18-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -y l)-5-isopropy I-4, 7, 10,13,16- pentaoxo-2-(3-ureidopropyl)-3,6,9, 12, 15-pentaazaoctadecanamido)benzyl ((1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9, 10,13,15- hexahydro-1 H, 12H-benzo[de]pyrano[3',4':6,7]indolizino[1 , 2-b]qu inol in-1 - yl)carbamate [CO-(m-PAB)-EXA] (187.6 mg, 0.069 mmol, 67.8%) as an off- white solid.

[0092] Analytical data:

[0093] LCMS: RT (min): 2.04; Purity (Max): 93.23 %; [M+2H]+ / 2: 1126.7. Method: column: XBridge C8 (50x4.6mm) 3.5 pm; mobile phase A :0.1 % TFA in H2O; mobile phase B : ACN; flow rate :1.5 ml / min. The LCMS chromatogram is shown in FIG. 22 and the mass spectra in FIG. 23.

[0094] HPLC: RT (min): 6.96; Purity (Max): 82.75 %. Method: column: Atlantis T3(150X4.6 )mm, 3pm; mobile phase A: 0.1 % TFA in MilliQ water; mobile phase B: acetonitrile; flow:1.2mL / min; temperature: 35°C. The HPLC chromatogram is shown in FIG. 24.

[0095] The other disclosed compounds may be prepared using analogous methods.

[0096] The examples included herein are for illustrative purposes only and are not meant to limit the scope of the invention as defined by the claims.

Claims

We claim:1 . A chito-oligosaccharide-drug construct selected from the group consisting ofand

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