Process for preparing a macrocyclic diamine, intermediates, and crystalline forms thereof

Abstract

Disclosed is a process for preparing a macrocyclic diamine and crystalline forms thereof. Further disclosed are methods for preparing intermediates in this process and the intermediate compounds and salts, hydrates and solvates thereof. Provided are methods for preparing the macrocyclic diamine of Formula (VI). Further provided are the macrocyclic diamine compound of Formula (VI) and salts, solvates, hydrates and crystalline forms thereof: (Formula (VI)).

Description

PROCESS FOR PREPARING A MACROCYCLIC DIAMINE, INTERMEDIATES, ANDCRYSTALLINE FORMS THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 717,528 filed November 7, 2024, the entire contents of which are incorporated by reference herein.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The contents of the electronic sequence listing 26062-WO-PCT_SL.xml; Size: 177 bytes; and Date of Creation: lanuary 29, 2025 are herein incorporated by reference in their entirety.FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to a process for preparing for preparing a macrocyclic diamine, its intermediates and crystalline forms thereof. In particular, the present disclosure is related to a process for preparing (3aS,27S,30S,41 aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47- ((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl-l,2,3,3a,6,7, 12,13,14,15, 16,17, 18,19, 27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8, l l-etheno-27,44-methano- 20,25:32,36-di(metheno)benzo[r]dipyrrolo[2, l-kl :2',3'-tl][l]oxa[4,14,20,27,35,38,41,44] octaazacycloheptatetracontine-4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (alternatively named (6S, 12S, 15S,18S,22S,34S,37S)-34-amino-42-fluoro-15-[(lR)-l-hydroxyethyl]-12-[(4- methoxyphenyl)methyl]-6-methyl-23-oxa-4, 10, 13,16, 19,26,36,46,53-nonaazaoctacyclo[53.2.2. 119’37. 128’32, 139’46.O6’10,018’22,040’45]dohexaconta-l(57),28(62),29,31,39(60),40,42,44,55,58- decaene-5,11, 14, 17, 25,35, 61-heptone) and its intermediates. The present disclosure also relates to crystalline forms of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l- hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl-l,2,3,3a,6,7, 12,13, 14,15, 16, 17, 18,19, 27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,l l-etheno-27,44-methano- 20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl :2',3'-tl][l]oxa[4,14,20,27,35,38,41,44] octaazacycloheptatetracontine-4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone.BACKGROUND

[0004] Peptides are ubiquitous in nature and are fundamental components of pharmaceutically active compounds; however, the practicality and economics of their chemical synthesis on manufacturing scale present significant challenges. In particular, chemo- and regioselectivity challenges arising from competing functionality of amino acids and peptides typically necessitate the use of orthogonal protecting groups and their subsequent removal, detracting from the ease and efficiency of processing. Current methodologies typically rely on the use of multiple orthogonal protecting groups, leading to additional deprotection and purification steps, thereby limiting their practical application for large-scale production.SUMMARY

[0005] The present disclosure is directed to a process for preparing a macrocyclic diamine. In particular, the process synthesizes a compound of Formula (VI):or a salt, hydrate, or solvate thereof. The synthesis comprises the steps of (a) reacting compounds of Formulae (I) and (II):or salts, hydrates, or solvates thereof to form an intermediate; and (b) cyclizing the intermediate, wherein:Ri is selected from the group consisting of: -0-(Ci-Cio)alkyl, -S-(Ci-Cio)alkyl, -S-(Ci- Cio)alkylene-NHC(0)-(Ci- Cio)alkyl, and -NH2, and each of R2and R3 is independently selected from the group consisting of: =0, -NH2, and -OH. In a further embodiment, the synthesis comprises the steps of (a) reacting compounds of Formulae (I) and (II):or salts, hydrates, or solvates thereof to form a first intermediate; and (b) cyclizing the first intermediate, wherein: Ri is selected from the group consisting of: -0-(Ci-Cio)alkyl, -S-(Ci- Cio)alkyl, -S-(Ci- Cio)alkylene-NHC(0)-(Ci- Cio)alkyl, and -NH2, and each of R2and R3 is independently selected from the group consisting of: =0, -NH2, and -OH.

[0006] The present disclosure is also directed to salts of Formula (VI), such as the compounds of Formulas (VIII-1) and (VIII-2). Further disclosed are crystalline forms of the compound of Formula (VI) and salts, hydrates, or solvatse thereof.

[0007] Following the generation of the macrocyclic diamine compound of Formula (VI), this compound can be further combined with the compound of Formula (IX):to obtain a compound of Formula (X):wherein A’ is an anion. The compound of Formula (X) corresponds to an enlicitide salt. In some embodiments, A- is caprate, or decanoate. As such, the present disclosure is directed to an efficient and scalable a process for preparing a macrocyclic diamine intermediate in a process of generating the macrocyclic peptide enlicitide decanoate.

[0008] The summary of the technology described herein is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 is an X-ray powder diffraction pattern of a crystalline form of the compound of Formula (VI).

[0010] FIG. 2 is an X-ray powder diffraction pattern of a crystalline form of the compound of Formula (VI).

[0011] FIG. 3 is an X-ray powder diffraction pattern of a crystalline form of the compound of Formula (VI).DETAILED DESCRIPTION

[0012] The present disclosure pertains to a method to synthesize the compound of Formula (VI) (macrocyclic diamine) utilizing an enzymatic, two-step, single-pot process. In an embodiment, the synthesis involves a highly evolved enzyme to facilitate a selective amide bond formation between the two starting compounds, followed by a macrocyclization catalyzed by two highly evolved enzymes and two ancillary enzymes.

[0013] This process is remarkable for the complexity of the substrates involved and the multiple transformations accomplished concurrently without need for intermediate isolation; the selectivity with which the requisite bond formations are accomplished by three highly evolved enzymes and two ancillary enzymes, without the need for protecting groups; control of oligomerization without need for highly dilute conditions; operation in aqueous media, avoiding hazardous reagents / solvents. Using traditional synthetic methods, the net transformation would undoubtedly require multiple synthetic steps, including redundant protecting group manipulations and high dilution to avoid oligomerization, together leading to vastly increased cost, hazardous waste and associated environmental impact. Furthermore, there is little precedent for the efficient macrocyclization of complex peptides via a secondary amine linkage using enzyme catalysis.

[0014] The present disclosure is directed to a novel, scalable synthesis of a compound of Formula (VI):or a salt, hydrate, or solvate thereof. The synthesis comprises the steps of (a) reacting compounds of Formulae (I) and (II):or salts, hydrates, or solvates thereof to form an intermediate; and (b) cyclizing the intermediate, wherein:Ri is selected from the group consisting of: -0-(Ci-Cio)alkyl, -S-(Ci-Cio)alkyl, -S-(Ci- Cio)alkylene-NHC(0)-(Ci-Cio)alkyl, and -NH2, and each of R2 and R3 is independently selected from the group consisting of: =0, -NH2, and -OH. In a further embodiment, the synthesis comprises the steps of (a) reacting compounds of Formulae (I) and (II):or salts, hydrates, or solvates thereof to form a first intermediate; and (b) cyclizing the first intermediate, wherein Ri is selected from the group consisting of -0-(Ci-Cio)alkyl, -S-(Ci- Cio)alkyl, -S-(Ci- Cio)alkylene-NHC(0)-(Ci- Cio)alkyl, and -NH2, and each of R2 and R3 is independently selected from the group consisting of: =0, -NH2, and -OH.

[0015] In an embodiment, the compound of Formula (I) is a compound of Formula (I- 1):

[0016] In an embodiment, the compound of Formula (II) is a compound of Formula (II-l):

[0017] In an embodiment, the first intermediate is a compound of Formula (III):or a salt, hydrate, or solvate thereof.

[0018] In an embodiment, the compound of Formula (III) is a compound of Formula (III- 1) :The compound of Formula (III-l ) is known as the “AA intermediate”.

[0019] In an embodiment, the step (a) is carried out in a presence of a thioesterase (TE) enzyme. In an embodiment, the TE enzyme comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NO: 1-39. In an embodiment, the TE enzyme comprises an amino acid sequence of any one of SEQ ID NO: 1-39. In an embodiment, the TE enzyme is an amino acid sequence of any one of SEQ ID NO: 1-39.

[0020] In an embodiment, the step (b) is carried out in a presence of a ketoreductase (KRED) enzyme. In an embodiment, the KRED enzyme comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 40-73. In an embodiment, the KRED enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 40-73. In an embodiment, the KRED enzyme is an amino acid sequence of any one of SEQ ID NOs: 40-73.

[0021] In an embodiment, the step (b) is carried out in a presence of a lactate dehydrogenase (LDH) enzyme. In an embodiment, the LDH enzyme comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 74-89. In an embodiment, the LDH enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 74-89. In an embodiment, the LDH enzyme is an amino acid sequence of any one of SEQ ID NOs: 74-89.

[0022] In an embodiment, the step (b) is carried out in a presence of an imine reductase (IRED) enzyme. In an embodiment, the IRED enzyme comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 90-151 . In an embodiment, the IRED enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 90-151. In an embodiment, the IRED enzyme is an amino acid sequence of any one of SEQ ID NOs: 90-151.

[0023] In an embodiment, the step (b) is carried out in a presence of an alcohol dehydrogenase (ADH) enzyme. In an embodiment, the ADH enzyme comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 152-153. In an embodiment, the ADH enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 152-153. In an embodiment, the ADH enzyme is an amino acid sequence of any one of SEQ ID NOs: 152-153.

[0024] In an embodiment, the step (b) comprises the steps of:(bl) reacting the first intermediate to form a second intermediate, wherein the second intermediate is a compound of Formula (IV):or a salt, hydrate, or solvate thereof;(b2) reacting the second intermediate to form a third intermediate, wherein the third intermediate is a compound of Formula (V):or a salt, hydrate, or solvate thereof; and(b3) reacting the third intermediate to form the compound of Formula (VI) or the salt, hydrate, or solvate thereof.

[0025] In an embodiment, the step (bl) is carried out in the presence of the KRED enzyme.

[0026] In an embodiment, the step (bl) is carried out in the presence of the LDH enzyme.

[0027] In an embodiment, the step (b3) is carried out in the presence of the IRED enzyme.

[0028] In an embodiment, the step (b3) is carried out in the presence of the ADH enzyme.

[0029] The present disclosure also pertains to a synthesis involving two highly evolved enzymes and two ancillary enzymes to facilitate a reductive amine bond between the two starting compounds, followed by a macrocyclization catalyzed by a highly evolved enzyme.

[0030] In an embodiment, the present disclosure is directed to a novel, scalable synthesis of a compound of Formula (VI):or a salt, hydrate, or solvate thereof. The synthesis comprises the steps of (a) reacting compounds of Formulae (I) and (II):or salts, hydrates, or solvates thereof to form a fourth intermediate; and (b) cyclizing the fourth intermediate, wherein Ri is selected from the group consisting of: -0-(Ci-Cio)alkyl, -S-(Ci- Cio)alkyl, -S-(Ci-Cio)alkylene-NHC(0)-(Ci-Cio)alkyl, and -NH2, and each of R2 and R3 is independently selected from the group consisting of: =0, -NH2, and -OH.

[0031] In an embodiment, the fourth intermediate is a compound of Formula (VII):or a salt, hydrate, or solvate thereof.

[0032] In an embodiment, the compound of Formula (VII) is a compound of Formula (VII-1):

[0033] In some aspects, the present disclosure is directed to the compound of Formula (VI) or a salt, hydrate, or solvate thereof. Further provided herein are crystalline forms of the compound of Formula (VI), or a salt, hydrate or solvate thereof. In some aspects, this crystalline form is anhydrous.

[0034] In an embodiment, the compound of Formula (VI) is a salt that is compound of Formula (VIII- 1):VIII or a hydrate or solvate thereof. This is a bis-hydrocholoride salt of the diamine.

[0035] In an embodiment, the compound of Formula (VI) is a salt that is compound of Formula(VIII-2):or a hydrate or solvate thereof. This compound is known as a diamine suberate salt

[0036] Further provided herein are crystalline forms of the compounds of Formula (VIII-1) and (VIII-2), or a hydrate or solvate thereof. In some aspects, these crystalline forms are anhydrous.

[0037] In an embodiment, the step (a) is carried out in the presence of the KRED enzyme. In an embodiment, the KRED enzyme comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 40-73. In anembodiment, the KRED enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 40- 73. In an embodiment, the KRED enzyme is an amino acid sequence of any one of SEQ ID NOs: 40-73.

[0038] In an embodiment, the step (a) is carried out in the presence of the LDH enzyme. In an embodiment, the LDH enzyme comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 74-89. In an embodiment, the LDH enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 74- 89. In an embodiment, the LDH enzyme is an amino acid sequence of any one of SEQ ID NOs: 74- 89.

[0039] In an embodiment, the step (a) is carried out in the presence of the IRED enzyme. In an embodiment, the IRED enzyme comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 90-151. In an embodiment, the IRED enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 90- 151. In an embodiment, the IRED enzyme is an amino acid sequence of any one of SEQ ID NOs: 90-151.

[0040] In an embodiment, the step (a) is carried out in the presence of the ADH enzyme. In an embodiment, the ADH enzyme comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 152-153. In an embodiment, the ADH enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 152- 153. In an embodiment, the ADH enzyme is an amino acid sequence of any one of SEQ ID NOs: 152-153.

[0041] In an embodiment, the step (b) is carried out in the presence of the TE enzyme. In an embodiment, the TE enzyme comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-39. In an embodiment, the TE enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 1-39. In an embodiment, the TE enzyme is an amino acid sequence of any one of SEQ ID NOs: 1-39.

[0042] The present disclosure is also directed to the isolation of the compound of Formula (VI) or the salt, hydrate, or solvate thereof.

[0043] In an embodiment, the process further comprises the step of: (c) isolating the compound of Formula (VI) to provide a crystalline solvate form of the compound of Formula (VI).

[0044] In an embodiment, the crystalline solvate form of the compound of Formula (VI) is a crystalline hydrochloric acid solvate.

[0045] The present disclosure is also directed to a compound of Formula (VI) or a salt, hydrate, or solvate thereof.

[0046] The present disclosure is also directed to a compound of Formula (III) or a salt, hydrate, or solvate thereof, wherein each of R2 and R3 is independently selected from the group consisting of: =0, -NH2, and -OH.

[0047] In an embodiment, the compound of Formula (III) is a compound of Formula (III- 1 ).

[0048] The present disclosure is also directed to a compound of Formula (VII) or a salt, hydrate, or solvate thereof, wherein Ri is selected from the group consisting of: -0-(Ci-Cio)alkyl, -S-(Ci- Cio)alkyl, -S-(Ci-Cio)alkylene-NHC(0)-(Ci-Cio)alkyl, and -NH2.

[0049] In an embodiment, the compound of Formula (VII) is a compound of Formula (VII-1).

[0050] In an embodiment, the present disclosure is directed to a crystalline form of the compound (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4- methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H- 8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine-4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)). In an embodiment, the present disclosure is directed to a crystalline solvate form of the compound (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4- methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H- 8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl :2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine-4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)). The compound of Formula (VI) is also referred to as (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l- hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl-l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27 ,28,30,31,37 ,38, 41a, 44a, 46, 47 ,49,50-hexacosahydro-43H-8,l l-etheno-2 ,44-methano- 20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl :2',3'-tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine-4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (alternatively named (6S,12S,15S,18S,22S,34S,37S)-34-amino-42-fluoro-15-[(lR)-l-hydroxyethyl]-12-[(4- methoxyphenyl)methyl]-6-methyl-23-oxa-4,10,13,16,19,26,36,46,53-nonaazaoctacyclo[53.2.2.119’37l28’32. l39’46.06’10.018’22.040’45]dohexaconta-l(57),28(62),29,31,39(60),40,42,44,55,58- decaene-5,11,14, 17, 25, 35, 61 -heptone).

[0051] In an embodiment, the present disclosure is directed to an isolated crystalline form of the compound (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fhroro-47-((R)-l-hydroxyethyl)- 50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'-11 ]

[0001] oxa[4, 14,20,27,35,38,41 ,44] octaazacy cl oheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)). In an embodiment, the present disclosure is directed to an isolated crystalline solvate form of the compound (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4- methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31 ,37,38,41 a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)).

[0052] In an embodiment, the present disclosure is directed to an anhydrous crystalline form of the compound (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hydroxyethyl)- 50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl :2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine-4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)). In an embodiment, the present disclosure is directed to an anhydrous crystalline solvate form of the compound(3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4- methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,l l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'-11 ]

[0001] oxa[4, 14, 20, 27, 35, 38, 41, 44] octaazacy cl oheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)).

[0053] In an embodiment, the present disclosure is directed to (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4- methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl :2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)) isolated in a form which contains at least about 40 wt.%, about 50 wt.%, about 60 wt.%, about 70 wt.%, about 80 wt.%, about 90 wt.%, about 95 wt.%, about 98 wt.%, or about 99 wt.% of the crystalline form.

[0054] In an embodiment, the present disclosure is directed to (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4- methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)) isolated in a form which contains at least about 40 wt.%, about 50 wt.%, about 60 wt.%, about 70 wt.%, about 80 wt.%, about 90 wt.%, about 95 wt.%, about 98 wt.%, or about 99 wt.% of the crystalline solvate form.

[0055] In an embodiment, the crystalline form (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30- amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl-1,2, 3, 3a, 6, 7 ,12,13,14,15,16,17 ,18,19,27 ,28,30,31,37 ,38,4 la, 44a, 46, 47 ,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)) (Form 1) is characterized by an X- ray powder diffraction pattern including peaks at about 5.7, about 17.2, about 18.9, about 19.8, and about 20.2 degrees 29, wherein the X-ray powder diffraction pattern is obtained using a Cu Ka radiation source (1.54 A).

[0056] In an embodiment, the crystalline form of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30- amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine-4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)) (Form 1) is characterized by an X- ray powder diffraction pattern including peaks at about 5.7, about 17.2, about 18.1, about 18.3, about 18.9, about 19.8, about 20.2, about 20.7, about 23.4, and about 24.6 degrees 20, wherein the X-ray powder diffraction pattern is obtained using a Cu K a radiation source (1.54 A).

[0057] In an embodiment, the X-ray powder diffraction of the crystalline form of(3aS, 27S,30S, 41 aS, 44S,44aS,47S, 50S,Z)-30-amino-23-fluoro-47-((R)-l -hydroxy ethyl)-50-(4- m ethoxyb enzy 1 )-3 a-m ethyl - l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'-11 ]

[0001] oxa[4, 14, 20, 27, 35, 38, 41, 44] octaazacy cl oheptatetracontine-4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)) (Form 1) can include one or more peaks from Table 1.

[0058] In an embodiment, the crystalline form of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30- amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12, 13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl :2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine-4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)) (Form 1) has an X-ray diffraction pattern substantially similar to that set forth in Figure 1.

[0059] In an embodiment, the crystalline form of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30- amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H- 8,l l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'-11 ]

[0001] oxa[4, 14,20,27,35,38,41 ,44] octaazacy cl oheptatetracontine-4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)) (Form 2) is characterized by an X-ray powder diffraction pattern including peaks at about 6.0, about 18.8, about 19.6, about 20.1, and about 20.6 degrees 20, wherein the X-ray powder diffraction pattern is obtained using a Cu Ka radiation source (1.54 A).

[0060] In an embodiment, the crystalline form of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30- amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H- 8,l l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'-11 ]

[0001] oxa[4, 14,20,27,35,38,41 ,44] octaazacy cl oheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)) (Form 2) is characterized by an X- ray powder diffraction pattern including peaks at about 6.0, about 12.1, about 18.3, about 18.8, about 19.6, about 20.1, about 20.6, about 21.4, about 21.9, and about 23.3 degrees 20, wherein the X-ray powder diffraction pattern is obtained using a Cu Ka radiation source (1.54 A).

[0061] In an embodiment, the X-ray powder diffraction of the crystalline form of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4- methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31 ,37,38,41 a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine-4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)) (Form 2) can include one or more peaks from Table 2.

[0062] In an embodiment, the crystalline form of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30- amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl :2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine-4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)) (Form 2) has an X-ray diffraction pattern substantially similar to that set forth in Figure 2.

[0063] In an embodiment, the crystalline form of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30- amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'-11 ]

[0001] oxa[4, 14, 20, 27, 35, 38, 41, 44] octaazacy cl oheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)) (Form 3) is characterized by an X- ray powder diffraction pattern including peaks at about 6.7, about 13.4, about 20.1, about 20.6, and about 23.4 degrees 20, wherein the X-ray powder diffraction pattern is obtained using a Cu K radiation source (1.54 A).

[0064] In an embodiment, the crystalline form of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30- amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'-11 ]

[0001] oxa[4, 14,20,27,35,38,41 ,44] octaazacy cl oheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)) (Form 3) is characterized by an X- ray powder diffraction pattern including peaks at about 6.7, about 13.4, about 17.3, about 17.7, about 19.0, about 20.1, about 20.6, about 21.6, about 21.7, and about 23.4 degrees 20, wherein the X-ray powder diffraction pattern is obtained using a Cu Ka radiation source (1.54 A).

[0065] In an embodiment, the X-ray powder diffraction of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4- methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'-11 ]

[0001] oxa[4, 14, 20, 27, 35, 38, 41, 44] octaazacy cl oheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)) (Form 3) can include one or more peaks from Table 3.

[0066] In an embodiment, the crystalline form of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30- amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl :2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone (Formula (VI)) (Form 3) has an X-ray diffraction pattern substantially similar to that set forth in Figure 3.

[0067] In an embodiment, the synthesis of the compound of Formula (VI) commences with coupling the compound of Formula (I) with the compound of Formula (II) via an amide bond to provide the compound of Formula (III).

[0068] The reaction proceeds through condensation of the Ri terminus (which can be in an ester form) of the compound of Formula (I) and the A -terminus of the compound of Formula (II). The reaction can be mediated by the TE enzyme. In addition to the TE enzyme, the reaction can involve a solvent or a co-solvent. In addition to the TE enzyme, the reaction can involve other necessary reagents. The compound of Formula (III) can be isolated as an amorphous solid or a crystalline solid. The crystalline solid can be obtained via seeding or in the absence of seeding.

[0069] In an embodiment, the wild type parent of the TE enzyme originates from the Tyrocidine NRPS TE domain from Brevibacillus laterosporus and is evolved away from its natural activity on / / 7 / -ester substrates (no measurable activity on ester substrates) to effectively mediate reaction on, for example, an isopropyl ester form of the compound of Formula (I). Advantageously, such an evolution of the TE enzyme provides synthetic utility, given the hydrolytic instability of thioesters and cost / stench of thiol precursors / byproducts. In an embodiment, the TE enzyme is evolved to recognize and couple the amine of ambident nucleophile of the compound of Formula (II) with greater than about 10:1, about 20: 1, about 30: 1, about 40: 1, about 50: 1, about 60: 1, about 70: 1, about 80: 1, about 90:1, about 95: 1, about 97:1, about 98: 1, or about 99: 1 N: O selectivity as its exclusive coupling partner.

[0070] Non-limiting examples of solvents used in the synthesis of the compound of Formula (III) include DMAc, water, MeOH, EtOH, IPA, MeCN, sulfolane, NMP, DMSO, and DMF. Nonlimiting examples of bases used in the synthesis of the compound of Formula (III) include NaOH. Other non-limiting examples of reagents used in the synthesis of the compound of Formula (III) include TCEP, 2-CB, Ca(OAc)2, HEPES, DTT, organic phosphines, EPPS, MOPS, BES, tricine, bicine, CHES, bis tris, and sodium phosphate.

[0071] The synthesis of the compound of Formula (III) is carried out at a temperature ranging from about 10 °C to about 30 °C or from about 15 °C to about 23 °C, or any number therebetween. In an embodiment, the synthesis of the compound of Formula (III) is carried out at a temperature of about 20 °C. In some embodiments, the synthesis of the compound of Formula (III) is carried out at a pH ranging from about 7.5 to about 8.5. In some embodiments, the synthesis of the compound of Formula (III) is carried out at a pH ranging from about 7.6 to about 8.2. In an embodiment, the synthesis of the compound of Formula (III) is carried out at a pH of about 7.8.

[0072] In an embodiment, the compound of Formula (III) proceeds via a four-enzyme catalyzed macrocyclization to give the compound of Formula (VI). In this reaction, a KRED / IRED dual enzymatic recycling system couples the R.2 and Ra termini (which can be a primary amine and alcohol) of the compound of Formula (III) to achieve macrocyclization and generate the secondary amine of the compound of Formula (VI). In some embodiments, the R2 terminus is a primary amine and the Ra terminus is an alcohol. The reaction can be mediated by the KRED enzyme, the IRED enzyme, the LDH enzyme, and the ADH enzyme. In addition to these enzymes, the reaction can involve a solvent or in the presence of a co-solvent in aqueous media. In addition to these enzymes, the reaction can involve other necessary reagents.

[0073] Non-limiting examples of solvents used in the synthesis of the compound of Formula (VI) include DMSO, DMAc, water, and DMF. Non-limiting examples of bases used in the synthesis of the compound of Formula (III) include NaOH, KOH, and NH4OH. Other non-limiting examples of reagents used in the synthesis of the compound of Formula (VI) include NAD, NADP, sodium pyruvate, alpha-ketoglutarate, IPA, sodium formate, glucose, sodium phosphite, and buffers such as BES, bicine, bis tris, CHES, HEPES, MOPS, and tricine.

[0074] The synthesis of the compound of Formula (VI) is carried out at a temperature ranging from about 20 °C to about 40 °C, from about 30 °C to about 40 °C, from about 32 °C to about 38 °C, or any number therebetween. In some embodiments, this synthesis is carried out at a temperature of about 30 °C. In an embodiment, the synthesis of the compound of Formula (VI) is carried out at a temperature of about 32 °C or about 35 °C. In an embodiment, this synthesis is carried out at a temperature of about 35 °C.

[0075] In an embodiment, the macrocyclization proceeds via an oxidation-reduction cycle using a- ketoglutarate as the stoichiometric oxidant, isopropanol as the stoichiometric reductant, and nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) as catalytic co-factors. In various embodiments, a-ketoglutarate is added to the reaction in the a-ketoglutaric acid, disodium salt dihydrate form, or Na2-aKG.

[0076] In another embodiment, the macrocyclization proceeds via an oxidation-reduction cycle using sodium pyruvate as the stoichiometric oxidant, isopropanol as the stoichiometric reductant, and nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) as catalytic co-factors. Use of a-ketoglutarate as stoichiometric oxidant may be preferable to pyruvate, as process development revealed that pyruvate requires slow addition and may result in one or more pyruvate adduct impurities. In contrast, a-ketoglutarate may be added to the reaction

[0077] In the first step, oxidation of the benzyl alcohol of the compound of Formula (III- 1) is mediated by NAD+ and an evolved KRED enzyme. The NADH that is formed is recycled back to NAD+ by an ancillary LDH enzyme using alpha-ketoglutarate as the stoichiometric oxidant. This oxidation process provides the transient aldehyde compound of Formula (IV), which may engage with the pendant amine to establish an equilibrium with the imine form of the compound of Formula

[0078] In the second step, the reduction of the compound of Formula (V) is mediated by an evolved IRED enzyme and NADPH. The NADP+ that is formed is recycled back to NADPH by an ancillary ADH enzyme. This reduction process selectively reduces the compound of Formula (V) over the compound of Formula (IV) to generate the diamine form of the compound of Formula (VI). This transformation is surprising since it is effected using only catalytic quantities of enzymes and cofactors. In addition, this transformation is surprising for the mildness which tolerates a high degree of molecular complexity, and the selectivity of the reaction induced between just one of the multiple alcohol and amine groups of the compound of Formula (IV), and in an intramolecular fashion to provide the compound of Formula (VI) over oligomeric products which would be expected to predominate under typical chemical conditions.

[0079] In an alternate embodiment, the synthesis of the compound of Formula (VI) commences with coupling the compound of Formula (I) with the compound of Formula (II) via reductive amination to provide the compound of Formula (VII). The compounds of Formula (I) and (II) are treated with the KRED enzyme, the LDH enzyme, the IRED enzyme, the ADH enzyme, and their relevant reagents to give the compound of Formula (VII).

[0080] Then the compound of Formula (VII) is treated with the TE enzyme and its relevant reagents to give the compound of Formula (VI).

[0081] In an alternate embodiment, the compound of Formula (VI) can be synthesized by isolating the compound of Formula (III) after completion of the TE enzyme reaction. The isolated compound of Formula (III) can then be further treated with the KRED enzyme, the LDH enzyme, the IRED enzyme, the ADH enzyme, and their relevant reagents to give the compound of Formula (VI).

[0082] In an alternate embodiment, the compound of Formula (VI) can be synthesized in a single pot without isolation of the compound of Formula (III). This may be done in a stepwise fashion, in which a stream of the TE enzyme generated compound of Formula (III) is directly treated with the KRED enzyme, the LDH enzyme, the IRED enzyme, the ADH enzyme, and their relevant reagents to give the compound of Formula (VI).

[0083] In an alternate embodiment, the compound of Formula (VI) can be synthesized by directly treating the compounds of Formulae (I) and (II) with the TE enzyme, the KRED enzyme, the LDH enzyme, the IRED enzyme, the ADH enzyme, and their relevant reagents to give the compound of Formula (VI).

[0084] The skilled artisan will appreciate that the disclosed processes, and in particular any coupling step carried out in the presence of a KRED enzyme and / or an IRED enzyme, may be carried out in the absence of an LDH enzyme and / or an ADH enzyme. In some embodiments, the disclosed processes are carried out in the absence of an LDH enzyme or an ADH enzyme. Likewise, the skilled artisan will appreciate that the disclosed processes, and in particular step (b), may carried out in the absence of oxidant a-ketoglutarate (aKG) and / or reductant isopropanol (IP A). As such, in some embodiments, the compound of Formula (VI) can be synthesized in the absence of an LDH enzyme, an ADH enzyme, aKG, or IPA.

[0085] As such, in some aspects, the compound of Formula (VI) may be synthesized in the presence of a KRED enzyme and an IRED enzyme, but the absence of an LDH enzyme or an ADH enzyme, or their relevant reagents aKG and IPA.

[0086] The reactions can be performed in an aqueous environment, or in an aqueous solution containing a certain amount of an organic co-solvent. The reactions can also be performed with enzymes immobilized via His tags on metal affinity resins.

[0087] In an embodiment, the compound of Formula (VI) can be isolated from aqueous reaction streams by extraction with an solvent including but not limited to Me-THF, MeCN, and IP Ac, and combinations thereof. In some embodiments, Formula (VI) can be isolated from aqueous reaction streams by extraction with an immiscible solvent system (e.g., the combination of MeCN and IP Ac).

[0088] In an embodiment, the isolated compound of Formula (VI) can be crystallized as a free form. In an embodiment, the isolated compound of Formula (VI) can be crystallized as a hydrate. In an embodiment, the isolated compound of Formula (VI) can be crystallized as a solvate or a salt. In an embodiment, the isolated compound of Formula (VI) can be crystallized as a solvate or a salt including but not limited to hydrochloride, suberate, oxalate, imidazole-N-acetate, glutarate, alphaketoglutarate, adipate, pimelate, azelate, sebacate, 2-hydroxynicotinate, 4-hydroxynicotinate, 3- hydroxyisonicotinate, p-phenylenediacetate, o-phenylenediacetate, phthalate, terephthalate, cis- 1,2- cyclohexanedicarboxylate, cis- 1,4-cy cl ohexanedi carboxylate, trans-l,4-cyclohexanedicarboxylate,pyrazine-2, 3 -dicarboxylate, L-pyroglutamate, D-pyroglutamate, imidazo[l,5-a]pyridine-7- carboxylate, N-oxynicotinate, N-oxypicolinate, N-oxyisonicotinate, alpha-ketosuberate, O,O'-di-p- toluoyl-D-tartrate, thymine-N-acetate, 4-(2-carboxyethyl)benzoate, 1,6-hexanedisulfonate, 1,7- heptanedi sulfonate, 2,2'-(ethane-l,2-diylbis(oxy))diacetate, imidazole-N-propionate, meso-2,3- diphenylsuccinate, hippurate, and N-oxypyridine-2-oxide. In an embodiment, the isolated compound of Formula (VI) is crystallized as a bis-hydrochloride solvate.

[0089] In some embodiments, the isolated compound of Formula (VI) is a suberate salt (Formula (VIIL2)). In an embodiment, it is crystallized as a suberate salt solvate.

[0090] All of the steps of the above processes are optionally but preferably conducted with agitation (e.g., stirring).

[0091] An example synthesis scheme of Formula (VI) is shown in the following.Definitions

[0092] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0093] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclatureused herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0094] As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0095] As used herein, the term “about” in quantitative terms refers to plus or minus 10% of the value it modifies (rounded up to the nearest whole number if the value is not sub-dividable, such as a number of molecules or nucleotides). For example, in some instances, “about 100 [units]” can mean within ±10% of 100 (e.g., from 90 to 110).

[0096] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0097] As used herein, the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as “consisting of’ and “consisting essentially of’ the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0098] The term “substantially similar” used in reference to X-ray powder diffraction patterns means that the X-ray powder diffraction pattern of a polymorph may display “batch to batch” variations due to differences in the types of equipment used for the measurements, and fluctuations in both experimental conditions (e.g. purity and grain size of the sample) and instrumental settings (e g. X-ray wavelengths; accuracy and sensitivity of the diffractometer; and “instrumental drift”) normally associated with the X-ray diffraction measurement. Due to these variations, the same polymorph may not contain X-ray powder diffraction peaks at exactly the same positions orintensities shown in the figures disclosed herein. Accordingly, the term “about” used in reference to the peaks in an X-ray powder diffraction pattern takes into account these variations and a skilled artisan would readily appreciate the scope.

[0099] “Alkyl”, as well as other groups having the prefix “alk”, such as alkoxy, and the like, means carbon chains which may be linear or branched, or combinations thereof, containing the indicated number of carbon atoms. For instance, a Ci-Ce alkyl means an alkyl group having one (i.e., methyl) up to 6 carbon atoms (i.e., hexyl). In particular embodiments, linear alkyl groups have 1-6 carbon atoms and branched alkyl groups have 3-7 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and Zc / V-butyl, pentyl, hexyl, heptyl, octyl, nonyl and the like.

[0100] “Alkylene” means a divalent aliphatic hydrocarbon group radical. The aliphatic hydrocarbon group may be straight or branched. Two single bonds exist and each single bond attaches to a different parent group. Non-limiting examples of an alkylene group include methylene (-CH2-) and ethylene (-CH2CH2-).

[0101] “Allyl” means -(CH2)n-HC=CH2, including an alkylene (-(CH2)n~) bridge attached to a terminal vinyl group (-HC=CH2).

[0102] “Aryl” means a monocyclic, bicyclic, tricyclic, or tetracyclic carbocyclic aromatic ring or ring system containing 5-17 carbon atoms, wherein at least one of the rings is aromatic. Non-limiting examples include phenyl and naphthyl.

[0103] The compounds of the present disclosure may contain one or more asymmetric centers and can thus occur as “stereoisomers” including racemates and racemic mixtures, enantiomeric mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. Additional asymmetric centers may be present depending upon the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers, and it is intended that all of the possible optical isomers and diastereomers in mixtures and as pure or partially purified compounds are included within the scope of this disclosure. The present disclosure is meant to comprehend all such isomeric forms of these compounds. When bonds to the chiral carbon are depicted as straight lines in the formulae of the disclosure, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within a given formula. For example, Formula (X) shows the structure of the compound with the designation of specific stereochemistry. When the compounds of the present disclosure contain onechiral center, the term “stereoisomer” includes both enantiomers and mixtures of enantiomers, such as the specific 50:50 mixture referred to as a racemic mixture.

[0104] The independent syntheses of these diastereomers or their chromatographic separations may be achieved as known in the art by appropriate modification of the methodology disclosed herein. Their absolute stereochemistry may be determined by the X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing an asymmetric center of known absolute configuration. If desired, racemic mixtures of the compounds may be separated so that the individual enantiomers are isolated. The separation can be carried out by methods well known in the art, such as the coupling of a racemic mixture of compounds to an enantiomerically pure compound to form a diastereomeric mixture, followed by separation of the individual diastereomers by standard methods, such as fractional crystallization or chromatography. The coupling reaction is often the formation of salts using an enantiomerically pure acid or base. The diasteromeric derivatives may then be converted to the pure enantiomers by cleavage of the added chiral residue. The racemic mixture of the compounds can also be separated directly by chromatographic methods utilizing chiral stationary phases, which methods are well known in the art. Alternatively, any enantiomer of a compound may be obtained by stereoselective synthesis using optically pure starting materials or reagents of known configuration by methods well known in the art.

[0105] Some of the compounds described herein may exist as tautomers which have different points of attachment of hydrogen accompanied by one or more double bond shifts. For example, a ketone and its enol form are keto-enol tautomers. The individual tautomers as well as mixtures thereof are encompassed by the compounds of the present disclosure.

[0106] The individual atropisomers as well as mixtures thereof are encompassed by the compounds of the present disclosure. When resolved, individual atropisomers can be designated by established conventions such as those specified by the International Union of Pure Applied Chemistry (IUPAC) 2013 Recommendations.

[0107] In the compounds of the present disclosure, the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. The present disclosure as described and claimedherein is meant to include all suitable isotopic variations of the compounds of the present disclosure and embodiments thereof. For example, different isotopic forms of hydrogen (H) include protium (1H) and deuterium (2H, also denoted herein as D). Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements or may provide a compound useful as a standard for characterization of biological samples. Isotopically-enriched compounds can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the Schemes and Examples herein using appropriate isotopically-enriched reagents and / or intermediates.

[0108] The term “salts” refers to salts prepared from acceptable bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particular embodiments include the ammonium, calcium, magnesium, potassium, and sodium salts. Salts in the solid form may exist in more than one crystal structure, and may also be in the form of hydrates. Salts derived from organic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, / TV'-dibenzylethylene-diamine, diethylamine, 2-diethylaminoethanol, 2- dimethylaminoethanol, ethanolamine, ethylenediamine, A -ethyl morpholine, JV-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[0109] When the compound of the present disclosure is basic, salts may be prepared from acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. Particular embodiments include the citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, fumaric, and tartaric acids. If a compound of the present disclosure simultaneously contains acidic and basic groups in the molecule, the disclosure also includes, in addition to the salt forms mentioned, inner salts orbetaines (zwitterions). Salts can be obtained from the compounds of the present disclosure by customary methods which are known to the person skilled in the art, for example, by combination with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange from other salts. The present disclosure also includes all salts of the compounds which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.

[0110] Furthermore, the compounds of the present disclosure may exist in amorphous form and / or one or more crystalline forms, and as such all amorphous and crystalline forms and mixtures thereof of the compounds of the present disclosure, including the Examples, are intended to be included within the scope of the present disclosure. In addition, some of the compounds of the present disclosure may form solvates with water (i.e., a hydrate) or common organic solvents such as but not limited to ethyl acetate or isobutyl alcohol, or solvents such as but not limited to hydrochloric acid or sulfuric acid. Such solvates and hydrates, particularly the pharmaceutically acceptable solvates and hydrates, of the present compounds are likewise encompassed within the scope of this disclosure, along with unsolvated and anhydrous forms.

[0111] Throughout the synthetic schemes and examples, abbreviations and acronyms may be used with the following meanings unless otherwise indicated: 2-CB = 2-carboxybenzaldehyde; 2-Me-THF = Me-THF = 2-methyl tetrahydrofuran; aKG = a-ketoglutarate; ADH = alcohol dehydrogenase; aq = aqueous; BES = N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; bicine = N,N-bis(2- hydroxyethyl)glycine; bis tris = 2,2-bis(hydroxymethyl)-2,2 ' ,2 " -nitrilotriethanol; CHES = 2- (cyclohexylamino)ethanesulfonic acid; DMAc = N, A-dimethylacetamide; DMF = dimethylformamide; DMSO = dimethyl sulfoxide; DTT = t / w o-l,4-dimercapto-2,3-butanediol; EPPS = 4-(2-hy droxy ethyl)- 1 -piperazinepropanesulfonic acid; eq. = equivalent; HEPES = 4-(2- hy droxy ethyl)- 1 -piperazineethanesulfonic acid; zPr = isopropyl; IPA = isopropanol; IP Ac = isopropyl acetate; IRED = imine reductase; KRED = ketoreductase; LCAP = liquid chromatography area percent; LDH = lactate dehydrogenase; Me = methyl; MeCN = acetonitrile; MOPS = 3-(N- morpholino)propanesulfonic acid; N = normal; NAD+ = nicotinamide adenine dinucleotide; NADP = nicotinamide adenine dinucleotide phosphate; NMP = l-methyl-2-pyrrolidinone; NMR = nuclearmagnetic resonance; TCEP = tris-(2-carboxyethyl)phosphine; TE = thioesterase; tricine = A- [tris(hydroxymethyl)methyl]glycine.EXAMPLES

[0112] The following examples are meant to be illustrative and should not be construed as further limiting the disclosure in any way. In some embodiments, the final product may be further modified, for example, by manipulation of substituents. These manipulations may include, but are not limited to, reduction, oxidation, alkylation, acylation, and hydrolysis reactions which are commonly known to those skilled in the art. In some embodiments, the order of carrying out the foregoing reaction schemes and examples may be varied to facilitate the reaction or to avoid unwanted reaction products. The following examples are provided so that the disclosure might be more fully understood.

[0113] Example 1: Synthesis of g-amino-H-IIl-fb-aminohexyll-S-fluoro-lH-indol-3-yl)methyl)-N-((2S,3R)-3-hydroxy-l-(((S)-l-((S)-2-((4- (hydroxymethyl)phenethyl)carbamoyl)-2-methylpyrrolidin- 1 -yl)-3 -(4-methoxy henyl)- 1 -oxopropan- 2-yl)amino)-l -oxobutan-2-yl)-4, 10, 13-trioxo-2-oxa-5, 11 -diaza- 1 (3, 1 )-pyrrolidina-7( 1 ,3)- benz

[0114] A reactor was charged water (1.6 L), DMAc (400 mL), TCEP (565 mg, 1.97 mmol, HC1 monosolvate), isopropyl ((l1S,l2S,l3S,9S,12S)-9-amino-12-((l-(6-aminohexyl)-5-fluoro-lH-indol- 3-yl)methyl)-4,10,13-trioxo-2-oxa-5,l 1 -diaza- 1(3, l)-pyrrolidina-7(l,3)-benzenacyclotridecaphane- l2-carbonyl)-L-threoninate • [oxalic acid]2 ((1-1), 100 g, 98.6 mmol), and (S)-l-((S)-2-amino-3-(4- methoxyphenyl)propanoyl)-N-(4-(hydroxymethyl)phenethyl)-2-methylpyrrolidine-2-carboxamide • [EESCLJo.s ((H-1), 50.5 g, 98.6 mmol). The process for preparing Compound 1-1 is described inInternational Patent Application No. PCT / US2025 / 037066, filed on July 10, 2025, the contents of which are expressly incorporated herein by reference. The process for preparing Compound II- 1 is described in International Patent Application No. PCT / US2024 / 059924, filed on December 13, 2024, the contents of which are expressly incorporated herein by reference.

[0115] The batch temperature was adjusted to 20 °C, then 4-(2-hy droxy ethyl)- 1- piperazineethanesulfonic acid (HEPES) buffer (1.62 g, 160 mmol) was charged, followed by 2- carboxybenzaldehyde (1.85 g, 12.3 mmol) and calcium acetate monohydrate (33 g, 187 mmol). The pH was adjusted to pH 7.5-8.0 with sodium hydroxide (NaOH) and the batch temperature was adjusted to 20 °C. The TE enzyme (5.0 g, 5 wt%) was charged and the batch was aged at 20 °C for 36-48 h to afford (l1S,l2S,l3S,9S,12S)-9-amino-12-((l-(6-aminohexyl)-5-fluoro-lH-indol-3- yl)methyl)-N-((2S,3R)-3 -hydroxy- 1-(((S)-1 -((S)-2-((4-(hydroxymethyl)phenethyl)carbamoyl)-2- methylpyrrolidin- 1 -y l)-3 -(4-methoxyphenyl)- 1 -oxopropan-2-yl)amino)- 1 -oxobutan-2-yl)-4, 10,13- trioxo-2-oxa-5,l l-diaza-l(3,l)-pyrrolidina-7(l,3)-benzenacyclotridecaphane-l2-carboxamide ((III- 1)) in 93.0% yield without isolation.

[0116] 1H NMR (600 MHz, 2: 1 v / v CDBCN / D2O) 8 = 7.32-7.27 (m, 2H), 7.24-7.19 (m 3H), 7.15 (d, 8.1 Hz, 2H), 7.13 (d, >8.8 Hz, 2H), 7.12-7.10 (m, 2H), 7.09 (d, >7.7 Hz, 1H), 6.92 (td, >9.2, 2.5 Hz, 1H), 6.81 (d, >8.6 Hz, 2H), 6.79 (t, >1.9 Hz, 1H), 4.83 (dd, >8.0, 5.8 Hz, 1H), 4.71 (dd, >8.6, 6.0 Hz, 1H), 4.58 (d, >14.4 Hz, 1H), 4.47 (s, 2H), 4.47-4.44 (m, 1H), 4.11 (d, >4.9 Hz, 1H), 4.08 (d, >15.8 Hz, 1H), 4.07-4.05 (m, 1H), 4.04-3.98 (m, 4H), 3.90 (d, >14.4 Hz, 1H), 3.81- 3.75 (m, 1H), 3.72-3.68 (m, 1H), 3.68 (s, 3H), 3.49-3.43 (m, 2H), 3.39-3.31 (m, 2H), 3.26 (td, >13.6, 7.2 Hz, 1H), 3.08 (dd, >14.9, 5.7 Hz, 1H), 2.93 (dd, >14.1, 6.0 Hz, 1H), 2.88 (dd, >14.8, 7.6 Hz, 1H), 2.85 (dd, >12.7, 3.0 Hz, 1H), 2.73-2.62 (m, 6H), 1.99-1.95 (m, 2H), 1.91-1.84 (m, 1H), 1.83-1.77 (m, 1H), 1.73-1.63 (m, 4H), 1.44 (p, >7.5 Hz, 2H), 1.35 (s, 3H), 1.29-1.22 (m, 2H), 1.21-1.15 (m, 2H), 0.98 (d, >6.3 Hz, 3H).

[0117] 13C NMR (151 MHZ, 2: 1 v / v CD3CN / D2O) 8 = 174.6, 174.3, 171.6, 170.6, 170.2, 170.0, 169.3, 158.3, 157.4 (d, . / cF=231.6 Hz), 139.3, 138.3, 138.1, 137.3, 132.8, 130.5, 129.4, 129.0, 128.9, 128.8, 128.5, 128.2 (d, >F=9.5 HZ), 127.2, 126.8, 113.9, 110.8 (d, >F=9.9 Hz), 109.3 (d, >F=26.5 Hz), 108.7 (d, >F=4.5 Hz), 103.3 (d, >F=23.2 Hz), 80.6, 67.7, 67.3, 67.0, 64.6, 63.5, 59.1, 55.8, 55.0, 52.9, 50.7, 48.4, 45.9, 45.1, 42.0, 40.6, 40.1, 39.8, 39.0, 35.7, 34.6, 30.4, 29.7, 28.4, 27.8, 26.0, 25.7, 23.1, 20.4, 18.9.

[0118] 19F NMR (565 MHz, 2: 1 v / v CD3CN / D2O) 8 = -127.2 (td, =9.7, 4.4 Hz, IF)

[0119] Example 2-1 : Synthesis of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl-1,2,3, 3a,6,7,12,13, 14,15,16,17,18,19,27,28,30,31,37,38,41a, 44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'- tH[11oxa[4, 14,20,27,35, 38,41, 44]octaazacycloheptatetracontine-4,29,39,45,48,5 l,53(5H,26H,40H,42H)-heptaone ((VI))

[0120] The slurry of (l1S,l2S,l3S,9S,12S)-9-amino-12-((l-(6-aminohexyl)-5-fluoro-lH-indol-3- yl)methyl)-N-((2S,3R)-3-hydroxy-l-(((S)-l-((S)-2-((4-(hydroxymethyl)phenethyl)carbamoyl)-2- methylpyrrolidin- 1 -y l)-3 -(4-m ethoxy phenyl)- 1 -oxopropan-2-yl)amino)- 1 -oxobutan-2-yl)-4, 10,13- trioxo-2-oxa-5,l l-diaza-l(3,l)-pyrrolidina-7(l,3)-benzenacyclotridecaphane-l2-carboxamide ((III- 1)) from the TE reaction of Example 1, containing a mixture of the compound of Formula (111-1), DMAc, TCEP, HEPES, 2-CB, Ca(OAc)2, and water, was heated to 35 °C. The pH of the slurry was adjusted to pH 7.8 at 35 °C using sodium hydroxide (NaOH). To the batch was charged IPA (22.6 mL, 296 mmol), NAD+ (0.98 g, 1.48 mmol), LDH enzyme (0.5 g), ADH enzyme (0.5 g), IRED enzyme (3 g), NADP (1.55 g, 1.97 mmol), KRED enzyme (0.7 g), and aKG (26.7 g, 118 mmol, di sodium salt dihydrate).

[0121] In some iterations of this reaction, 0.5 g of KRED was used. In some iterations, the pH of the slurry was adjusted to pH 7.8 at 30 °C, and the reaction was allowed to proceed for 20 hours. In some iterations, the pH of the slurry was adjusted to pH 7.8 at 35 °C, and the reaction was allowed to proceed for 20 hours.

[0122] The batch was aged at 35 °C for 20-24 h to afford (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4- methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12, 13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H- 8,l l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl :2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine-4,29,39,45,48,5 l,53(5H,26H,40H,42H)-heptaone ((VI)) in 86.1% yield with respect to isopropyl ((l1S,l2S,l3S,9S,12S)-9-amino-12-((l-(6-aminohexyl)-5-fluoro-lH-indol-3-yl)methyl)-4,10,13- trioxo-2-oxa-5,l l-diaza-l(3,l)-pyrrolidina-7(l,3)-benzenacyclotridecaphane-l2-carbonyl)-L- threoninate • [oxalic acid]2 ((1-1)).

[0123] Characterization data of (l1S,l2S,l3S,9S,12S)-9-amino-12-((l-(6-aminohexyl)-5-fluoro- lH-indol-3-yl)methyl)-N-((2S,3R)-l-(((S)-l-((S)-2-((4-formylphenethyl)carbamoyl)-2- methylpyrrolidin- 1 -y 1 )-3 -(4-m ethoxy phenyl)- 1 -oxopropan-2-yl)amino)-3-hydroxy- 1 -oxobutan-2-yl)- 4,10,13-trioxo-2-oxa-5,l l-diaza-l(3,l)-pyrrolidina-7(l,3)-benzenacyclotridecaphane-l2- carb oxami de (IV).

[0124] ’H NMR (600 MHz, 2: 1 v / v CD3CN / D2O) 8 = 9.83 (s, 1H), 7.78 (d, >8.2 Hz, 2H), 7.39 (d, 8.1 Hz, 2H), 7.33 (dd, 9.9, 2.5 Hz, 1H), 7.32-7.27 (m, 2H), 7.21-7.15 (m, 2H), 7.13 (s, 1H), 7.12 (d, >8.6 Hz, 2H), 6.93 (td, >9.2, 2.6 Hz, 1H), 6.81 (t, >2.0 Hz, 1H), 6.79 (d, >8.7 Hz, 2H), 4.86 (t, >6.7 Hz, 1H), 4.70 (dd, >8.8, 5.8 Hz, 1H), 4.64 (d, >14.8 Hz, 1H), 4.49 (s, 1H), 4.23 (dd, >9.4, 3.6 Hz, 1H), 4.11-4.06 (m, 2H), 4.05-3.98 (m, 5H), 3.91 (d, >14.9 Hz, 1H), 3.71-3.68 (m, 1H), 3.67 (s, 3H), 3.66-3.62 (m, 1H), 3.48 (dt, ,7=12.2, 6.1 Hz, 1H), 3.40 (dt, >13.9, 7.0 Hz, 1H), 3.32 (dt, >13.8, 7.1 Hz, 1H), 3.23 (dd, >14.0, 3.6 Hz, 1H), 3.10-2.92 (m, 4H), 2.89 (dd, >14.2, 5.7 Hz, 1H), 2.84 (t, >7.7 Hz, 2H), 2.82-2.75 (m, 2H), 2.66 (dd, >14.2, 8.8 Hz, 1H), 1.91-1.78 (m, 4H), 1.74-1.64 (m, 4H), 1.56-1.47 (m, 2H), 1.35 (s, 3H), 1.33-1.25 (m, 2H), 1.25-1.17 (m, 2H), 0.99 (d, >6.4 Hz, 3H).

[0125] 13C NMR (151 MHz, 2: 1 v / v CD3CN / D2O) 6 = 194.0, 174.7, 171.1, 170.8, 170.3, 170.0, 169.0, 167.1, 158.3, 157.5 (d, >F=232.3 Hz), 147.3, 138.8, 134.6, 133.8, 132.8, 130.5, 130.0, 129.8, 129.7, 129.5, 129.0, 128.8, 128.4, 128.0 (d, JCF=9.8 Hz), 127.8, 113.8, 110.9 (d, >F=9.9 Hz), 109.5 (d, >F=26.5 HZ), 108.0 (d, >F=4.8 HZ), 103.3 (d, >F=23.3 HZ), 80.6, 67.7, 67.3, 67.0, 64.3, 59.3,55.1, 53.4, 52.9, 51.1, 48.4, 45.9, 44.9, 41.8, 40.1, 39.4, 39.0, 36.4, 35.7, 35.1, 30.5, 29.6, 28.3, 26.7,25.8, 25.5, 23.2, 20.3, 19.0.

[0126] 19F NMR (565 MHz, 2: 1 v / v CD3CN / D2O) 8 = -127.0 (td, >9.9, 4.4 Hz, IF)

[0127] Characterization data of isopropyl ((1 lS,12S,13S,9S,12S)-9-amino-12-((l-(5-((4-(2-((S)-l-((S)-2-amino-3-(4-methoxyphenyl)propanoyl)-2-methylpyrrolidine-2- carboxamido)ethyl)benzyl)amino)pentyl)-5-fluoro-lH-indol-3-yl)methyl)-4,10,13-trioxo-2-oxa-5,l l-diaza-l(3,l)-pyrrolidina-7(l,3)-benzenacyclotridecaphane-12-carbonyl)-L-threoninate (VII-1):

[0128] 'HNMR (599.60 MHz, D2O:CD3CN-t732:3 v / v): 8 7.31 - 7.25 (m, 2H), 7.25 - 7.16 (m, 5H), 7.06 (d, 8.6 Hz, 2H), 6.90 (td, >9.2 and 2.5 Hz, 1H), 6.81 (td, >8.5 Hz, 2H), 6.79 (s, 1H), 4.94 (hept, >6.3 Hz, 1H), 4.81 (td, >6.8 Hz, 1H), 4.59 (d, >14.4 Hz, 1H), 4.53 (s, 1H), 4.23 (d, >3.4 Hz, 1H), 4.30 (qd, >6.30 and 3.4 Hz, 1H), 4.15 (t, >2.6 Hz, 1H), 4.11 (d, >15.8 Hz, 1H), 4.08 - 3.97 (m, 3H), 3.91 (d, >14.4 Hz, 1H), 3.83 - 3.74 (m, 3H), 3.60 (dt, >9.7 and 6.5 Hz, 1H), 3.45 (dd, >9.9 and 3.7 Hz, 1H), 3.39 - 3.28 (m, 3H), 3.28 - 3.20 (m, 1H), 3.07 (dd, >14.8 and 6.1 Hz, 1H), 2.88 (dd, >14.8 and 7.4 Hz, 1H), 2.84 (dd, >13.3 and 3.7 Hz, 1H), 2.74 (dd, >13.6 and 6.8 Hz, 1H), 2.71 - 2.62 (m, 3H), 2.60 (t, >7.8 Hz, 2H), 2.52 (dd, >13.7 and 7.3 Hz, 1H), 2.02 - 1 .97 (tn, 2H), 1 .89 - 1 .78 (m, 2H), 1 .72 - 1 .56 (tn, 4H), 1 .42 (p, J=1.6 Hz, 2H), 1 .38 (s, 3H), 1 .23 - 1.11 (m, 10H), 1.09 (d, >6.4 Hz, 3H).

[0129] 13C{1H} NMR (150.78 MHz, D2O:CD3CN-t / 32:3 v / v): 8 175.81, 175.14, 174.62, 172.29, 171.52, 171.48, 170.39, 159.08, 158.28 (d, JCF=230.5 Hz), 140.65, 139.07 138.16, 133.71, 133.19, 131.44, 130.79, 130.46, 130.38, 130.30, 129.79, 129.76, 129.43, 129.10 (d, JCF=10.5 HZ), 122.79,114.80, 111.69 (d, JCF=9.8 HZ), 110.23 (d, JCF=26.5 HZ), 109.46 (d, . / C =4,8 HZ), 104.23 (d, JCF=23.4 HZ), 81.63, 70.79, 68.46, 68.15, 67.86, 65.37, 59.39, 56.70, 55.97, 54.99, 52.04, 51.57, 49.16, 48.03, 46.75, 45.93, 42.94, 41.39, 41.02, 40.21, 39.88, 35.51, 31.24, 30.55, 28.60, 27.55, 26.88, 26.85, 23.94, 21.93, 21.86, 21.28, 20.29.

[0130] Example 2-2: Synthesis of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hvdroxyethyl)-50-(4-methoxybenzyl)-3a-methyl-1,2,3, 3a,6,7,12,13, 14,15,16,17,18,19,27,28,30,31,37,38,41a, 44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r1dipyrrolo[2,l-kl:2',3'-111 [ 11oxa[ 4, 14,20,27,35,38,41 ,441octaazacycloheptatetracontine-4,29,39,45,48,5 l,53(5H,26H,40H,42H)-heptaone ((VI))

[0131] The slurry of (l1S,l2S,l3S,9S, 12S)-9-amino-12-((l-(6-aminohexyl)-5-fluoro-lH-indol-3- yl)methyl)-N-((2S, 3R)-3 -hydroxy- 1-(((S)-1 -((S)-2-((4-(hy droxymethyl)phenethyl)carbam oyl)-2- methylpy rrolidin- 1 -y 1 ) -3 -(4-m ethoxy phenyl)- 1 -oxopropan-2-yl)amino)- 1 -oxobutan-2-y l)-4, 10,13- trioxo-2-oxa-5,l l-diaza-l(3,l)-pyrrolidina-7(l,3)-benzenacyclotridecaphane-l2-carboxamide ((III- 1)) from the TE reaction of Example 1, containing a mixture of the compound of Formula III-l, TCEP, DMAc, HEPES, 2-CB, Ca(OAc)2, and water, was heated to 32 °C. Usage of calcium acetate monohydrate [Ca(OAc)2] removes the oxalate solvate of Formula (VI) by salt metathesis or precipitation.

[0132] The pH of the batch was adjusted to 7.8 using sodium hydroxide (NaOH). To the batch was charged NAD+ (1.31 g, 1.97 mmol), NADP (1.55 g, 1.97 mmol), and IPA (22.6 mL, 296 mmol), LDH enzyme (0.5 g), ADH enzyme (0.5 g), IRED enzyme (7.5 g), and KRED enzyme (1 g). A 25wt% solution of sodium pyruvate in water (197 mmol) was then charged to the batch over 24 hours. The batch was aged at 32 °C for 20-24 h to afford (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30- amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12, 13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H- 8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl :2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone ((VI)) in 84% yield with respect to isopropyl ((l1S,l2S,l3S,9S,12S)-9-amino-12-((l-(6-aminohexyl)-5-fluoro-lH-indol-3-yl)methyl)-4,10,13- trioxo-2-oxa-5,l l-diaza-l(3,l)-pyrrolidina-7(l,3)-benzenacyclotridecaphane-l2-carbonyl)-L- threoninate • [oxalic acid]2 ((1-1)).

[0133] Example 3-1 : Isolation of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hvdroxyethyl)-50-(4-methoxybenzyl)-3a-methyl-1,2,3, 3a,6,7.12.13.14,15.16.17.18.19.27.28.30.31.37.38.41a.44a,46.47.49.50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r1dipyrrolo[2,l-kl:2',3'- tlirn oxa[ 4, 14,20,27,35, 38,41 ,441 octaazacy cl oheptatetracontine-

[0134] To the slurry of crude (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,l l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl :2',3'-tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone ((VI)) from the cyclization step at 20 °C was charged Celite (180 g), ammonium sulfate (400 g), MeCN (1.2 L), and IP Ac (0.6 L). The pH was adjusted to pH 9.0-9.5 with aqueous ammonium hydroxide. The batch was aged for 1 hour at 30 °C before being filtered and the filter cake was washed with aqueous MeCN (0.5 L). The combined filtrate and washes were then adjusted to 23 °C and the layers separated. The aqueous layer was discarded, and the organic layer washed with 25 wt% aqueous sodium chloride (2 x 0.5 L). MeCN (0.3 L) was then charged to the batch. The resulting organic stream containing (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fhioro-47-((R)-l-hydroxyethyl)-50-(4- methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H- 8,l l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl :2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone ((VI)) was carried forward in the crystallization step.

[0135] The crude organic stream containing (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino- 23-fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H- 8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'- 11 ]

[0001] oxa[4, 14, 20, 27, 35, 38, 41, 44] octaazacy cl oheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone ((VI)) was distilled under vacuum at 15-30 °C to -700 mL. The batch was then further distilled under vacuum at 15-30 °C while feeding acetonitrile (2.0 L) at a rate to maintain constant batch volume. The batch was then concentrated to -500 mL. The batch temperature was adjusted to at 20 °C. The batch was then filtered and the waste cake washed with aqueous MeCN (50 mL). MeCN (100 mL) and water (50 mL) were then charged to the batch. HC1 (5 N, 2.7 eq.) was then charged to the batch. A Form 3 seed (1.0 g) (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4- methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H- 8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl :2',3'-tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine-4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone • [HC1]2((VIII-1)) was then charged to the batch and the batch was aged at 20 °C for 18-24 h, generating a slurry containing Form 1, Form 2, or a mixture of the two. MeCN (700 mL) was then charged over 12 hours at 20 °C, and the resulting slurry aged for a further 4 hours. The batch was then filtered and the cake was washed with MeCN / water / DMAc (300 mL). The cake was washed with MeCN / water (300 mL). The cake was dried under vacuum at 30 °C - 40 °C to afford Form 3 dry (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30- amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H- 8,l l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine-4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone • [HC1]2((VIII-1), 97.3 g, 89 wt% free base basis, 73% yield).

[0136] ’H NMR (599 MHz, 8:2 v / v CD3CN / D2O) 8 = 7.44 (dd, 7=9.9, 2.6 Hz, 1H), 7.35 (d, 7=7.9 Hz, 2H), 7.33 - 7.28 (m, 2H), 7.25 - 7.22 (m, 3H), 7.18 (d, 7=7.6 Hz, 1H), 7.10 (s, 1H), 7.08 (d, 7=8.5 Hz, 2H), 6.95 (td, 7=9.2, 2.5 Hz, 1H), 6.85 (s, 1H), 6.79 (d, 7=8.6 Hz, 2H), 4.83 (dd, 7=8.8, 4.9 Hz, 1H), 4.67 - 4.63 (m, 2H), 4.41 (s, 1H), 4.26 (dd, 7=9.9, 3.7 Hz, 1H), 4.11 - 4.01 (m, 5H), 4.01 - 3.93 (m, 4H), 3.86 (d, 7=14.6 Hz, 1H), 3.70 (s, 3H), 3.56 - 3.37 (m, 4H), 3.31 - 3.27 (m, 1H), 3.25 (dd, 7=14.0, 3.8 Hz, 1H), 3.08 - 2.98 (m, 3H), 2.89 (dd, 7=14.1, 6.0 Hz, 1H), 2.84 - 2.71 (m, 4H), 2.67 - 2.60 (m, 1H), 2.55 - 2.50 (m, 1H), 1.84 (dt, 7=12.6, 6.4 Hz, 1H), 1.79 - 1.71 (m, 2H), 1.69 - 1.61 (m, 3H), 1.61 - 1.55 (m, 2H), 1.52 (dt, 7=12.5, 7.1 Hz, 1H), 1.44 (dt, 7=12.5, 7.2 Hz, 1H), 1.26 (s, 3H), 1.24 - 1.19 (m, 4H), 0.98 (d, 7=6.4 Hz, 3H).

[0137] 13C NMR (151 MHZ, 8:2 v / v CD3CN / D2O) 8 = 174.56, 171.44, 170.71, 170.51, 170.33,169.15, 167.17, 158.61, 157.78 (d, 7=232.2 Hz), 141.12, 139.20, 134.28, 133.10, 130.77, 130.43,130.15, 129.74, 129.73, 129.32, 129.00, 128.95, 128.77, 128.38 (d, 7=9.8 Hz), 128.11, 114.10, 111.19 (d, 7=9.8 Hz), 109.81 (d, 7=26.3 Hz), 108.43 (d, 7=4.5 Hz), 103.60 (d, 7=23.7 Hz), 80.61, 68.26, 67.42, 67.20, 64.72, 59.31, 55.31, 53.97, 53.51, 51.49, 50.42, 48.69, 46.29, 46.21, 44.87, 42.00, 39.77, 39.26, 36.75, 35.82, 34.22, 30.75, 29.97, 29.36, 26.37, 25.99, 25.80, 23.21, 21.18, 19.08 ppm.

[0138] 19F NMR (151 MHz, 8:2 v / v CD3CN / D2O) 8 = -127.00 (td, 7=9.7, 4.4 Hz) ppm.

[0139] Example 3-2: Isolation of (3aS,27S.30S,41aS,44S,44aS.47S,50S.Z)-30-amino-23-fluoro-47-((R)-l-hvdroxyethyl)-50-(4-rnethoxybenzyl)-3a-methyl-1.2.3.3a.6.7.12.13.14.15.16.17.18.19.27.28.30.31.37.38,41a.44a.46.47.49,50-hexacosahydro-43H-8, l l-etheno-27,44-methano-20.25:32,36-di(metheno)benzo[r1dinyrrolor2, l-k l :2',3'- tH[11oxa[4, 14,20,27,35, 38,41, 441octaazacycloheptatetracontine-

[0140] To the slurry of crude (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47- ((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H- 8,l l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'-11 ]

[0001] oxa[4, 14,20,27,35,38,41 ,44]octaazacycloheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone ((VI)) from the cyclization step at 20 °C was charged Me-THF (2 L), sodium chloride (320 g), and Celite (240 g). The pH was adjusted to pH 3.5- 4.5 with HC1. The batch temperature was adjusted to 60 °C and was aged for 1 hour at 60 °C. The batch was then cooled to 20 °C over at least 1 hour. The pH was adjusted to pH 10.0-11.0 with sodium hydroxide (NaOH). The batch was filtered and the waste cake was washed with Me-THF (1.6 L). The combined organics were adjusted to 35 °C and the aqueous layer discarded. The organic layer was washed with water (2 x 500 m ) to provide a Me-THF solution of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4- methoxybenzyl)-3a-methyl-l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H- 8,l l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl :2',3'-11 ]

[0001] oxa[4, 14,20,27,35,38,41 ,44]octaazacycloheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone ((VI)) which was carried forward in the crystallization step.

[0141] To the crude MeTHF solution of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23- fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'-11 ]

[0001] oxa[4, 14,20,27,35,38,41 ,44]octaazacycloheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone ((VI)) was charged DMAc (100 mL). The batch was distilled under vacuum at 30 °C to -500 mL, then further distilled under vacuum at 15-30 °C while feeding acetonitrile (2.0 L) at a rate to maintain a constant batch volume. The batch was then adjusted to at 20 °C, fdtered and the cake washed with aqueous MeCN (50 ml). HC1 (5 N, 2.7 eq.) was then charged to the batch, followed by a Form 3 seed (1.0 g) of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4- methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'-11 ]

[0001] oxa[4, 14,20,27,35,38,41 ,44]octaazacycloheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone • [HC1]2((VIII-1)). MeCN (700 mL) was then charged over 12 hours at 20 °C, and the slurry aged for a further 4 hours. The slurry contained Form 1, Form 2, or a mixture of the two. The batch was then filtered and the cake washed with MeCN / water / DMAc (300 mL) followed by MeCN / water (300 mL). The cake was dried under vacuum at 30 - 40 °C to afford dry Form 3 (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23- fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl :2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine-4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone • [HC1]2((VIII-1), 76.7 g, 88.3 wt% free base basis, 59% yield).Example 4: Isolation of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l- hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13, 14,15,16,17,18,19,27,28,30,31,37,38,41a, 44a,46,47,49,50-hexacosahydro-43H- 8,1 l-etheno-27,44-methano-20,25:32,36-di(fnetheno)benzorr1dipyrrolo[2,l-kl:2',3'- tliri1oxa[4, 14,20,27,35, 38,41, 441octaazacycloheptatetracontine-4,29,39,45,48,5 l,53(5H,26H,40H,42H)-heptaone, 7-carboxyheptanoate salt (VIII-2)

[0142] To crude n-butanol solution of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23- fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H- 8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'-11 ]

[0001] oxa[4, 14,20,27,35,38,41 ,44]octaazacy cloheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone ((VI)) from the disstillation step was charged water (8.47 g), DMAc (6.27 g), suberic acid (623 mg), MTBE (6.84 g), and Type B seed (70 mg) of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l-hydroxyethyl)-50-(4- methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H- 8,11 -etheno-27,44-methano-20, 25:32, 36-di(metheno)benzo[r]dipyrrolo[2,l-kl :2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone, 7-carboxyheptanoate salt. MTBE (34.2 mL) was then charged over 6 hours at 23 °C, and the slurry aged for 30 minutes at 40 °C and was then cooled to 10 °C over 12 hours and the slurry was held for 72 hours. The batch was then filtered and the cakewashed twice with 2: 1 butanol / MTBE (300 mL). The cake was dried under vacuum at 30 - 40 °C to afford dry Type B of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23-fluoro-47-((R)-l- hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H- 8,l l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl :2',3'- tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone, 7-carboxyheptanoate salt (VIII-2), 6.36 g).

[0143] ‘HNMR (599.60 MHz, D2O:CD3CN-t / 32:3 v / v): 8 7.35 (dd, >10.0 and 2.5 Hz, 1H), 7.29 (dd, 8.9 and 4.3 Hz, 1H), 7.27 (d, >7.8 Hz, 2H), 7.24 - 7.17 (m, 3H), 7.13 (d, >7.7 Hz,lH), 7.11 - 7.05 (m, 4H), 6.93 (td, >9.2 and 2.5 Hz, 1H), 6.82 - 6.78 (m, 3H), 4.80 (dd, >7.7 and 5.9 Hz, 1H), 4.63 (dd, >8.4 and 6.4 Hz, 1H), 4.58 (d, >14.4 Hz, 1H), 4.38 (s, 1H), 4.07 (d, >5.3 Hz, 1H), 4.05 - 3.91 (m, 8H), 3.89 (d, >14.4 Hz, 1H), 3.70 (s, 3H), 3.61 (td, >10.0 and 6.8 Hz, 1H), 3.58 - 3.51 (m, 2H), 3.50 - 3.43 (m, 1H), 3.34 (ddd, >13.7, 7.7 and 4.1 Hz, 1H), 3.25 (dt, >9.8 and 6.5 Hz, 1H), 3.08 (dd, >14.6 and 7.8 Hz, 1H), 2.99 - 2.92 (m, 2H), 2.89 (dd, >13.3 and 3.7 Hz, 1H),2.85 (dd, >14.1 and 6.4 Hz, 1H), 2.80 - 2.76 (m, 1H), 2.75 - 2.59 (m, 5H), 2.04 (t, >7.6 Hz, 4H),1.86 (dd, >13.6 and 6.7 Hz, 1H), 1.82 - 1.75 (m, 1H), 1.73 (dt, >12.7 and 6.6 Hz, 1H), 1.64 (p, >7.2 Hz, 2H), 1.56 (dp, >12.8 and 6.3 Hz, 1H), 1.51 - 1.34 (m, 8H), 1.24 - 1.12 (m, 11H), 0.97 (d, >6.4 Hz, 3H).

[0144] ^CfH) NMR (150.78 MHz, D2O:CD3CN-t / 32:3 v / v): 8 138.08, 175.38, 174.52, 172.63, 171.41, 171.15, 170.92, 169.91, 159.21, 158.37 (d, >F=231.7 Hz), 141.46, 139.13, 138.02, 133.75, 131.40, 130.81, 130.74, 130.35, 130.32, 129.85, 129.72, 129.51, 129.13 (d, >F=9.8 Hz), 127.88, 114.80, 111.79 (d, JCF=9.7 Hz), 110.36 (d, >F=26.4 Hz), 109.53 (d, >F=4.8 Hz), 104.24 (d, >F=23.4 HZ), 81.42, 68.81, 68.02, 67,82, 65.59, 62.23, 59.76, 56.59, 55.99, 54.06, 51.90, 51.06, 49.30, 46.84, 46.77, 45.69, 42.93, 40.92, 40.46, 39.88, 38.53, 36.44, 35.04, 34.91, 31.21, 30.65, 29.82, 29.42, 26.98, 26.89, 26.72, 26.69, 23.79, 21.57, 19.60, 19.56, 14.17.Example 5: Method for Obtaining X-Ray Powder Diffraction Patterns

[0145] Powder X-ray Diffraction data were acquired on a Panalytical X-pert Pro PW3040 System configured in the Bragg-Brentano configuration and equipped with a Cu radiation source with monochromatization to Ka achieved using a Nickel filter. A fixed slit optical configuration wasemployed for data acquisition. Data were acquired between 2 and 40° 29. Samples of Form 1, Form 2, and Form 3 were prepared by gently pressing powdered sample onto a shallow cavity zero background silicon holder. As shown above in Examples 3-1 and 3-2, charging seeds generated a slurry of Form 1 and / or Form 2, and after drying the slurry, Form 3 was obtained.

[0146] Tables 1-3 provide the major 29 peaks and d-spacings for each of the isolated crystalline forms (Form 1, Form 2, and Form 3) of (3aS,27S,30S,41aS,44S,44aS,47S,50S,Z)-30-amino-23- fluoro-47-((R)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl- l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,1 l-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'-11 ]

[0001] oxa[4, 14,29,27,35,38,41 ,44]octaazacycloheptatetracontine- 4,29,39,45,48,51,53(5H,26H,40H,42H)-heptaone ((VI)) disclosed herein.Table 1: Diffraction peaks and corresponding d-spacings for Form 1 (wet phase solvate)Table 2: Diffraction peaks and corresponding d-spacings for Form 2 (wet phase solvate)Table 3: Diffraction peaks and corresponding d-spacings for Form 3 (dry phase solvate)

[0147] It will be appreciated that various of the above-discussed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. It will also be appreciated that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.

[0148] The disclosed subject matter is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0149] All figures and references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.

[0150] The amino acid sequences encoding engineered enzymes useful in the disclosed methods for preparing macrocyclic diamines are provided in Table 4 below. Each of these sequences are synthetic constructs.

Claims

1. CLAIMSWhat is claimed is:1 . A process for preparing a compound of Formula (VI):or a salt, hydrate, or solvate thereof, comprising the steps of:(a) reacting compounds of Formulae (I) and (II):or salts, hydrates, or solvates thereof to form an intermediate; and(b) cyclizing the intermediate, wherein:Ri is selected from the group consisting of: -0-(Ci-Cio)alkyl, -S-(Ci-Cio)alkyl, -S-(Ci-Cio)alkylene-NHC(0)-(Ci-Cio)alkyl, and -NH2, and each of R2 and R3 is independently selected from the group consisting of: =0, -NH2, and -OH.

2. The process of claim 1, wherein the compound of Formula (I) is a compound of Formula (I-1):

3. The process of claim 1 or 2, wherein the compound of Formula (II) is a compound of Formula (II- 1):

4. The process of any one of claims 1-3, wherein the intermediate is a first intermediate.

5. The process of any one of claims 1-4, wherein the first intermediate is a compound of Formula (III):or a salt, hydrate, or solvate thereof.

6. The process of claim 5, wherein the compound of Formula (III) is a compound of Formula (III-l):

7. The process of any one of claim 1-6, wherein the step (a) is carried out in a presence of a thioesterase (TE) enzyme.

8. The process of claim 7, wherein the TE enzyme comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-39.

9. The process of claim 7, wherein the TE enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 1-39.

10. The process of any one of claims 1-9, wherein the step (b) is carried out in a presence of a ketoreductase (KRED) enzyme.

11. The process of claim 10, wherein the KRED enzyme comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 40-73.

12. The process of claim 10, wherein the KRED enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 40-73.

13. The process of any one of claims 1-12, wherein the step (b) is carried out in a presence of a lactate dehydrogenase (LDH) enzyme.

14. The process of claim 13, wherein the LDH enzyme comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 74-89.

15. The process of claim 13, wherein the LDH enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 74-89.

16. The process of any one of claims 1-15, wherein the step (b) is carried out in a presence of an imine reductase (IRED) enzyme.

17. The process of claim 16, wherein the IRED enzyme comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 90-151.

18. The process of claim 16, wherein the IRED enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 90-151.

19. The process of any one of claims 1-18, wherein the step (b) is carried out in a presence of an alcohol dehydrogenase (ADH) enzyme.

20. The process of claim 19, wherein the ADH enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO 152 or 153.

21. The process of claim 20, wherein the ADH enzyme comprises the amino acid sequence of SEQ ID NO 152 or 153.

22. The process of any one of claims 1-21, wherein the step (b) comprises the steps of:(bl) reacting the first intermediate to form a second intermediate, wherein the second intermediate is a compound of Formula (IV):or a salt, hydrate, or solvate thereof;(b2) reacting the second intermediate to form a third intermediate, wherein the third intermediate is a compound of Formula (V):or a salt, hydrate, or solvate thereof; and(b3) reacting the third intermediate to form the compound of Formula (VI) or the salt, hydrate, or solvate thereof.

23. The process of claim 22, wherein the step (bl) is carried out in the presence of the KRED enzyme.

24. The process of claim 22 or 23, wherein the step (bl) is carried out in the presence of the LDH enzyme.

25. The process of any one of claims 22-24, wherein the step (b3) is carried out in the presence of the IRED enzyme.

26. The process of any one of claims 22-25, wherein the step (b3) is carried out in the presence of the ADH enzyme.

27. The process of any one of claims 1-3, wherein the intermediate is a fourth intermediate, which is a compound of Formula (VII):or a salt, hydrate, or solvate thereof.

28. The process of claim 27, wherein the compound of Formula (VII) is a compound of Formula(VII-1):

29. The process of any one of claims 1-3 or 27-28, wherein the step (a) is carried out in a presence of a ketoreductase (KRED) enzyme.

30. The process of claim 29, wherein the KRED enzyme comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 40-73.

31. The process of claim 29, wherein the KRED enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 40-73.

32. The process of any one of claims 1-3 or 27-31, wherein the step (a) is carried out in a presence of a lactate dehydrogenase (LDH) enzyme.

33. The process of claim 32, wherein the LDH enzyme comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 74-89.

34. The process of claim 32, wherein the LDH enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 74-89.

35. The process of any one of claims 1-3 or 27-34, wherein the step (a) is carried out in a presence of an imine reductase (IRED) enzyme.

36. The process of claim 35, wherein the IRED enzyme comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 90-151.

37. The process of claim 35, wherein the IRED enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 90-151.

38. The process of any one of claims 1-3 or 27-37, wherein the step (a) is carried out in a presence of an alcohol dehydrogenase (ADH) enzyme.

39. The process of claim 38, wherein the ADH enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO 152 or 153.

40. The process of claim 38, wherein the ADH enzyme comprises the amino acid sequence of SEQ ID NO 152 or 153.

41. The process of any one of claims 1-3 or 27-40, wherein the step (b) is carried out in a presence of a thioesterase (TE) enzyme.

42. The process of claim 41, wherein the TE enzyme comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-39.

43. The process of claim 41, wherein the TE enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 1-39.

44. The process of any one of claims 1-43, further comprising the step of:(c) isolating the compound of Formula (VI) to provide a crystalline solvate form of the compound of Formula (VI).

45. The process of claim 44, wherein the crystalline solvate form of the compound of Formula (VI) is a crystalline bis-hydrochloric acid solvate.

46. A compound of Formula (VI):or a salt, hydrate, or solvate thereof.

47. A compound of Formula (III):or a salt, hydrate, or solvate thereof, wherein each of R2 and R3 is independently selected from the group consisting of: =0, -NH2, and -OH.

48. The compound of claim 47, wherein the compound of Formula (III) is a compound ofFormula (III-l):

49. A compound of Formula (VII):or a salt, hydrate, or solvate thereof, wherein Ri is selected from the group consisting of: -0-(Ci-Cio)alkyl, -S-(Ci-Cio)alkyl, -S-(Ci- Cio)alkylene-NHC(0)-(Ci-Cio)alkyl, and -NFb.

50. The compound of claim 49, wherein the compound of Formula (VII) is a compound ofFormula (VII-1):

51. A crystalline form of a compound of Formula (VI):

52. The crystalline form of claim 51, wherein the crystalline form is a crystalline solvate form of the compound of Formula (VI).

53. The crystalline form of claim 51 or 52, wherein the crystalline solvate form of the compound of Formula (VI) is a crystalline bis-hydrochloride solvate form of the compound of Formula (VI).

54. The crystalline form of any one of claims 51-53, characterized by an X-ray powder diffraction pattern including peaks at about 5.7, about 17.2, about 18.9, about 19.8, and about 20.2 degrees 20, wherein the X-ray powder diffraction pattern is obtained using a Cu Ka radiation source (1.54 A).

55. The crystalline form of any one of claims 51-54, characterized by an X-ray powder diffraction pattern including peaks at about 5.7, about 17.2, about 18.1, about 18.3, about 18.9, about 19.8, about 20.2, about 20.7, about 23.4, and about 24.6 degrees 20, wherein the X-ray powder diffraction pattern is obtained using a Cu Ka radiation source (1.54 A).

56. The crystalline form of any one of claims 51-55, having an X-ray diffraction pattern substantially similar to that set forth in FIG. 1.

57. The crystalline form of any one of claims 51-53, characterized by an X-ray powder diffraction pattern including peaks at about 6.0, about 18.8, about 19.6, about 20.1, and about 20.6 degrees 20, wherein the X-ray powder diffraction pattern is obtained using a Cu Ka radiation source (1.54 A).

58. The crystalline form of any one of claims 51-53 or 57, characterized by an X-ray powder diffraction pattern including peaks at about 6.0, about 12.1, about 18.3, about 18.8, about 19.6, about 20.1, about 20.6, about 21.4, about 21.9, and about 23.3 degrees 20, wherein the X-ray powder diffraction pattern is obtained using a Cu Ka radiation source (1.54 A).

59. The crystalline form of any one of claims 51-53 or 57-58, having an X-ray diffraction pattern substantially similar to that set forth in FIG. 2.

60. The crystalline form of any one of claims 51-53, characterized by an X-ray powder diffraction pattern including peaks at about 6.7, about 13.4, about 20.1, about 20.6, and about 23.4 degrees 20, wherein the X-ray powder diffraction pattern is obtained using a Cu Ka radiation source (1.54 A).

61. The crystalline form of any one of claims 51-53 or 60, characterized by an X-ray powder diffraction pattern including peaks at about 6.7, about 13.4, about 17.3, about 17.7, about 19.0, about 20.1, about 20.6, about 21.6, about 21.7, and about 23.4 degrees 20, wherein the X-ray powder diffraction pattern is obtained using a Cu Ka radiation source (1.54 A).

62. The crystalline form of any one of claims 51-53 or 60-61, having an X-ray diffraction pattern substantially similar to that set forth in FIG. 3.

63. The crystalline form of any one of claims 51-62, wherein the crystalline form is a compound of F ormul a (VIII- 1 ) :

64. A compound of Formula (VIII- 1 ).or a hydrate thereof.

65. A compound of Formula (VIII-2):or a hydrate thereof.

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