WO2025255381A1 - Chito-oligosaccharide drug constructs - Google Patents

Chito-oligosaccharide drug constructs

Info

Publication number
WO2025255381A1
WO2025255381A1 PCT/US2025/032509 US2025032509W WO2025255381A1 WO 2025255381 A1 WO2025255381 A1 WO 2025255381A1 US 2025032509 W US2025032509 W US 2025032509W WO 2025255381 A1 WO2025255381 A1 WO 2025255381A1
Authority
WO
WIPO (PCT)
Prior art keywords
benzamido
hydroxymethyl
mmol
propanoate
tert
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/032509
Other languages
French (fr)
Inventor
Mark A MASKERI
Jamy Y. LEE
Ross BEMOWSKI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sigma Aldrich Co LLC
Original Assignee
Sigma Aldrich Co LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sigma Aldrich Co LLC filed Critical Sigma Aldrich Co LLC
Publication of WO2025255381A1 publication Critical patent/WO2025255381A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • ADCs 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.
  • 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.
  • Construct druglinker construct
  • Linkers expressing solubilizing units have been developed to address these shortcomings, notably chito-oligosaccharide (ChetosensarTM technology, MilliporeSigma) as disclosed in International Patent Appl. WO2022/058548.
  • chito-oligosaccharide (CO) drug constructs are provided. Such embodiments include a chito-oligosaccharide, a linker, and drug useful in ADCs.
  • the CO-drug construct is CO-(m-PAB)- exatecan of formula:
  • the CO-drug construct is CO-(m-PAB)-([3-Ala2)- exatecan of formula:
  • the CO-drug construct is CO-(m-PAB)- duocarmycin of formula:
  • the CO-drug construct is CO-(m-PAB)-DM1 of formula:
  • the CO-drug construct is CO-(m-PAB)-MMAE of formula:
  • FIG. 1 shows the synthesis of intermediate lnt-12 used in the synthesis of CO-(m-PAB)-drug constructs.
  • FIG. 2 is the LCMS chromatogram of tert-butyl 3-(5-amino-2- (hydroxymethyl)benzamido) propanoate (2).
  • FIG. 3 is the 1 H NMR of of tert-butyl 3-(5-amino-2-(hydroxymethyl) benzamido) propanoate (2).
  • 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).
  • FIG. 5 is the LCMS chromatogram for tert-butyl (S)-3-(5-(2-amino-5- ureidopentanamido)-2-(hydroxymethyl) benzamido) propanoate (5).
  • FIG. 6 is the HPLC chromatogram for tert-butyl (S)-3-(5-(2-amino-5- ureidopentanamido)-2-(hydroxymethyl) benzamido) propanoate (5).
  • 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).
  • FIG. 8 is the HPLC chromatogram for tert-butyl 3-(5-((S)-2-((S)-2-
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • FIG. 15 shows the scheme for the synthesis of CO-(m-PAB)-EXA.
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • FIG. 22 is the LCMS chromatogram for CO-(m-PAB)-exatecan.
  • FIG. 23 is the mass spectra for CO-(m-PAB)-exatecan.
  • FIG. 24 is the HPLC chromatogram for CO-(m-PAB)-exatecan.
  • 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.
  • Exemplary embodiments include:
  • each construct includes a chito-oligosaccharide solubility tag, a linker and a drug.
  • the chito-oligosaccharide solubility tag is selected from the following:
  • the chito-oligosaccharide solubility tag is the following, identified herein as chito-oligosaccharide CO-V:
  • 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.
  • PABC p-aminobenzyl carbamate
  • the solubility tag is covalently attached to the PABC spacer.
  • Example 1 Synthesis of Intermediate lnt-12. The synthesis scheme is shown in FIG. 1 .
  • Step 1 Synthesis of tert-butyl 3-(5-amino-2-(hydroxymethyl) benzamido) propanoate (2)
  • Step 2 Synthesis of tert-butyl (S)-3-(5-(2-((((9H-fluoren-9-yl) methoxy) carbonyl) amino)-5-ureidopentanamido)-2-(hydroxymethyl) benzamido) propanoate (4)
  • S 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.
  • Step 3 Synthesis of tert-butyl (S)-3-(5-(2-amino-5- ureidopentanamido)-2-(hydroxymethyl) benzamido) propanoate (5)
  • LCMS RT (min): 1.95; Purity (Max): 97.11 %; [M+H] + : 452.1 .
  • the LCMS chromatogram is shown in FIG. 5.
  • HPLC RT (min): 6.58; Purity (Max): 96.47 %.
  • the HPLC chromatogram is shown in FIG. 6.
  • 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)
  • LCMS RT (min): 2.80; Purity (Max): 68.79 %; [M+H] + : 773.3.
  • the LCMS chromatograph is shown in FIG 7.
  • HPLC RT (min): 10.41 ; Purity (Max): 76.30 %.
  • Step 5 tert-butyl 3-(5-((S)-2-((S)-2-amino-3-methylbutanamido)-5- ureidopentanamido)-2-(hydroxymethyl) benzamido) propanoate (8)
  • LCMS RT (min): 2.03; Purity (Max): 95.64 %; [M+H] + : 551.3.
  • the LCMS chromatogram is shown in FIG. 9.
  • 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.
  • 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)
  • LCMS RT (min): 2.32; Purity (Max): 97.68 %; [M+H] + : 822.5.
  • the LCMS chromatogram is shown in FIG. 11 .
  • 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.
  • 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)
  • HPLC RT (min): 9.81 ; Purity (Max): 85.41 %.
  • the HPLC chromatogram is shown in FIG. 14.
  • Example 2 Synthesis of CO-(m-PAB)-EXA. The synthesis scheme is shown in FIG. 15.
  • 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)
  • HPLC RT (min): 9.66; Purity (Max): 98.66 %.
  • the HPLC chromatogram is shown in FIG. 17.
  • 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)
  • 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.
  • LCMS RT (min): 2.18; Purity (Max): 96.47 %; [M+H] + : 1128.3.
  • the LCMS chromatogram is shown in FIG. 18.
  • HPLC RT (min): 7.61 ; Purity (Max): 99.45 %.
  • the HPLC chromatogram is shown in FIG. 19.
  • 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)
  • 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.
  • HPLC RT (min): 8.03; Purity (Max): 92.88 %.
  • the HPLC chromatogram is shown in FIG. 21.
  • 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-
  • 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.
  • LCMS RT (min): 2.04; Purity (Max): 93.23 %; [M+2H] + /2: 1126.7.
  • the LCMS chromatogram is shown in FIG. 22 and the mass spectra in FIG. 23.
  • HPLC RT (min): 6.96; Purity (Max): 82.75 %.

Landscapes

  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

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 CONSTRUCTS
Cross-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 the 1H 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 of
and
PCT/US2025/032509 2024-06-05 2025-06-05 Chito-oligosaccharide drug constructs Pending WO2025255381A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463656409P 2024-06-05 2024-06-05
US63/656,409 2024-06-05

Publications (1)

Publication Number Publication Date
WO2025255381A1 true WO2025255381A1 (en) 2025-12-11

Family

ID=96474452

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/032509 Pending WO2025255381A1 (en) 2024-06-05 2025-06-05 Chito-oligosaccharide drug constructs

Country Status (1)

Country Link
WO (1) WO2025255381A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020236841A2 (en) * 2019-05-20 2020-11-26 Novartis Ag Antibody drug conjugates having linkers comprising hydrophilic groups
WO2022058548A1 (en) 2020-09-17 2022-03-24 Merck Patent Gmbh Molecules with solubility tag and related methods

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020236841A2 (en) * 2019-05-20 2020-11-26 Novartis Ag Antibody drug conjugates having linkers comprising hydrophilic groups
WO2022058548A1 (en) 2020-09-17 2022-03-24 Merck Patent Gmbh Molecules with solubility tag and related methods

Similar Documents

Publication Publication Date Title
EP2678037B1 (en) Branched linker for protein drug conjugates
AU2016313228B2 (en) Piperidinobenzodiazepine compounds with anti proliferative activity
KR20250139899A (en) Exatecan derivatives and antibody-drug conjugates thereof
US9199954B2 (en) Non-ring hydroxy substituted taxanes and methods for synthesizing the same
Nagamura et al. Synthesis and antitumor activity of duocarmycin derivatives: modification of segment A of duocarmycin B2
WO2014067960A9 (en) Functionalized 9-bromo-camptothecin derivatives
WO2025255381A1 (en) Chito-oligosaccharide drug constructs
JP6898300B2 (en) Asymmetric preparation of thieno-indole derivatives
CN113439084B (en) Duocarmycin analogs
CN119591621A (en) Novel ecteinascidin derivatives, preparation method and application thereof, antibody drug conjugate and application thereof
CN103384534B (en) Branched Linkers for Protein Drug Conjugates
TW202613131A (en) Chito-oligosaccharide drug constructs
US20250154177A1 (en) Pyrrolobenzodiazepine intermediates and uses thereof
US11325888B2 (en) Method for the synthesis of monoprotected bifunctional prodrugs and antibody drug conjugates based thereon as well as a method for preparing antibody drug conjugates
JP6570034B2 (en) Novel glutamic acid derivatives and uses thereof
WO2025080748A1 (en) Chito-oligosaccharide-duocarmycin constructs
JP6453706B2 (en) Novel glutamic acid derivatives and uses thereof
JP4590198B2 (en) Method for producing folic acid derivative
HK1186668B (en) Branched linker for protein drug conjugates
HK40009733A (en) Process for the preparation of pegylated drug-linkers and intermediates thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25742768

Country of ref document: EP

Kind code of ref document: A1