Flow Platforms Useful for the Same

BACKGROUND

Every year the loss of valuable livestock/fishes to invertebrate parasites, both endoparasites (nematodes or helminths) and ectoparasites (flies, ticks, mites and sea lice) totals more than £34 billion globally. This is despite the fact that farmers spend about £4.5 billion globally on compounds to protect their animals from parasites (Parasitol Res (2005) 97:S11-S16, - Jeschke et al.). New products are continually needed as new parasites emerge and existing parasites evolve resistance to current treatments.

PF1022A is a fungally-derived, non-ribosomal peptide natural product octadepsipeptide anthelmintic agent. Emodepside, a complex semi-synthetic derivative of PF1022A, is a resistance breaking anthelmintic used exclusively for the more profitable companion animal market owing to high cost of production (Ohyama et al., Biosci, Biotechno., Biochem., 2011, 75, 1

PF1022A Emodepside

The unique and highly complex core structure of the PF1022A natural product has proven challenging for synthesis. Conversion of PF1022A to the bis-4-morpholino derivative (emodepside) entails low-yielding chemistry such as nitration of the phenyl rings followed by reduction and subsequent functionalization. In addition to the poor chemical yields arising from nitration (or acetylation, another route), the generation of regioisomers further reduces the yield of useful intermediate and necessitates expensive purification of the desired para-regioisomers. A lower cost of goods for emodepside would enable the use of the compound in livestock herds, which is an application prohibited by its present high cost of manufacture. Additionally, with the increase of insect resistance, new PF1022A derivative compounds and methods for their synthesis are needed. The recent demonstration that emodepside may have utility in the treatment of African river disease in humans only sharpens the need for new methods for the preparation of emodepside and related structures.

Semisynthetic routes to the bis-hydroxy PF1022A derivative, PF1022H, have recently been described by Scherkenbeck et al. (Bioorg. Med. Chem. 2016, 24, 873-876). One route proceeds from PF1022A by nitration of the phenyl rings followed by reduction to the amine and diazotization followed by hydrolysis to the phenol. A second route utilizes Friedel-Crafts acylation of the phenyl rings followed by Baeyer-Villiger oxidation and subsequent ester cleavage. The nature of the electrophilic substitution chemistry results in mixtures of para and meta isomers, with para predominating. The jcara-Ws-hydroxy compound PF1022H has been shown to be a useful intermediate for the preparation of lipophilic PF1022A derivatives. (Ohyama et al, Biosci., Biotechno., Biochem., 2011, 75, 1354).

There exists a need in the art for the development of an industrially feasible, cost effective, and simple process capable of providing control over the regiosomeric products of nitration of PF 1022 A.

Accordingly, as embodiments of the present invention, new methods and conditions have been developed for the synthesis of PF1022A derivatives. In addition, methods disclosed herein may be used in the preparation of new PF1022A derivatives, which may be useful as anthelmintic agents.

SUMMARY Disclosed herein is a method of producing a compound of Formula I from PF1022A, comprising: contacting a nitrating agent to a solution of PF1022A:

PF1022A

in a solvent at a temperature of about . -20 °C to about -78 °C (e.g., from about -41 °C to about -70 °C); and then

mixing the nitrating agent and the solution of PF1022A to produce the compound of Formula I:

Formula I

wherein Ri and R ₂ are each independently hydrogen or nitro, and at least one of R] and R ₂ is nitro. In some embodiments, both R ₁ and R ₂ are nitro.

In some embodiments, the nitrating agent comprises nitric acid. In some embodiments, the nitrating agent is fuming nitric acid.

In some embodiments, the solvent is dichloromethane, nitromethane, or acetic anhydride. In some embodiments, the solvent is dichloromethane.

In some embodiments, the compound of Formula I is produced as part of a mixture of compounds of Formula I, said mixture comprising aromatic positional isomers of nitro at the Ri and/or R ₂ position, and wherein 50% , 60%, 70%, 80% or greater of the aromatic positional isomers of nitro in the mixture are para isomers.

In some embodiments, the nitrating agent is added dropwise. In some embodiments, the steps of contacting the nitrating agent to the solution of PF1022A and mixing the nitrating agent and the solution of PF 1022 A are carried out in a flow reactor.

In some embodiments, the step of contacting the nitrating agent to the solution of PF1022A is carried out at a first temperature, and the step of mixing the nitrating agent and the solution of PF 1022 A is carried out at a second temperature. In some embodiments, the second temperature is higher than the first temperature.

In some embodiments, the contacting is carried out by introducing the solution of PF1022A and the nitrating agent into the flow reactor and flowing the solution of PF 1022 A and the nitrating agent along a flow path (e.g., a longitudinal direction of the flow reactor) for a predetermined residence time sufficient to produce the compound of Formula I.

In some embodiments, the introducing the solution of PF1022A and the nitrating agent into the flow reactor comprises: providing a first solution of PF1022A and the solvent; providing a second solution comprising the nitrating agent; mixing said first solution and said second solution in a mixer to produce a reaction mixture; and introducing said reaction mixture into the flow reactor. In some embodiments, the mixer is a static mixer.

In some embodiments, the introducing the solution of PF 1022 A and the nitrating agent into the flow reactor comprises: providing a first solution comprising PF1022A and the solvent; providing a second solution comprising the nitrating agent; simultaneously introducing the first solution and the second solution into the flow reactor; and introducing a system solvent into the flow reactor after a predetermined time has passed after introducing the first solution and the second solution into the flow reactor. In some embodiments, the predetermined time is from about 0.1 second to about 10 minutes, such as from 0.5 minute to 2 minutes.

In some embodiments, the predetermined residence time is from about 0.1 second to about 100 minutes.

In some embodiments of the above methods, the method further includes: reacting the compound of Formula I with a suitable reducing agent to produce a compound of Formula II:

Formula II

wherein R ₃ and R4 are each independently hydrogen or -NH ₂ , and at least one of R ₃ and R4 is -NH ₂ .

In some embodiments, the suitable reducing agent is hydrogen in the presence of a suitable catalyst. In some embodiments, the suitable catalyst is palladium on carbon.

In some embodiments, the compound of Formula II is a compound of Formula Ila, and the method further includes: reacting the compound of Formula Ila with bis-2,2'-disubstituted

wherein Z is a leaving group (e.g., chloro or bromo; or tosylate or mesylate). In some embodiments, the compound of Formula II is a compound of Formula Ila, and wherein the method further includes: reacting the compound of Formula Ila with bis-2,2 ¹ - disubstituted ethyl ether in the presence of a reducing agent to provide a compound of Formula

wherein X is an aldehyde group.

In some embodiments, the reducing agent is sodium borohydride or sodium cyanoborohy dride . In some embodiments of the above methods, the method may further include:

1) reacting the compound of Formula II with sodium nitrite in the presence of a suitable acid to form a diazonium s

Formula IV

wherein ₅ and R ₆ are each independently selected from -N=N ⁺ X ^" and hydrogen, and at least one of R ₅ and R ₆ is -N=N ⁺ X ^" ; wherein X is a counterion of the acid; and

2) converting the diazonium salt compound of Formula IV into a compound of Formula III:

Formula III

wherein R ₇ and R ₈ are each independently selected from -OH, halogen and hydrogen, and at least one of R ₇ and R ₈ is -OH or halogen. In some embodiments, both R ₇ and R ₈ are -OH. In some embodiments, both R ₇ and R ₈ are halogen.

In some embodiments, the acid is a strong acid, optionally a strong acid selected from the group consisting of HC1, ¾S0 ₄ , and HBF ₄ .

In some embodiments, converting the diazonium salt compound of Formula IV into the compound of Formula III comprises heating the diazonium salt compound of Formula IV for decomposition.

In some embodiments, converting the diazonium salt compound of Formula IV into the compound of Formula III comprises treating (e.g., reacting) the diazonium salt compound of Formula IV with an aqueous base.

In some embodiments, the aqueous base is sodium bicarbonate in water.

Also provided is a method of producing a compound of Formula III from PF1022A, said method comprising:

reacting PF1022A: with an electrophilic halog III:

Formula III

wherein R ₇ and R ₈ are each independently selected from halo and hydrogen, and at least one of R ₇ and R ₈ is halo (e.g., bromo or chloro). In some embodiments, both of R and R ₈ are halo.

In some embodiments, the electrophilic halogenating agent is N-bromosuccinimide (e.g., N-bromosuccinimide in the presence of trifluoroacetic acid and/or N-chlorosuccinimide).

In some embodiments, reaction of PF1022A with the electrophilic halogenating agent is carried out in a suitable solvent. In some embodiments, the suitable solvent is acetonitrile.

In some embodiments, the reaction of PF1022A with the electrophilic halogenating reagent occurs from about -5°C to about 75°C. In some embodiments, the reaction of PF1022A with the electrophilic halogenating reagent occurs at about 60°C. In some embodiments, the reaction of PF1022A with the electrophilic halogenating reagent occurs at about room temperature.

In some embodiments, reaction of PF 1022 A with the electrophilic halogenating agent is carried out in a flow reactor. The foregoing and other objects and aspects of the invention are explained in greater detail below. It should be appreciated that the invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a flow platform according to some embodiments of the present invention.

FIG. 2 is a diagram of a reactant providing module and a reactor module according to some embodiments of the present invention.

FIG. 3 shows an example of a flow platform according to some embodiments of the present invention.

FIGS. 4 and 5 are schematic diagrams of flow reactors and methods for production of PF1022A derivatives using a flow reactor according to some embodiments of the present invention.

FIG. 6 Ή-NMR spectra of Compound 01.

FIG. 7 1H-NMR spectra of Compound 02.

FIG. 8 1H-NMR spectra of Compound 03.

FIG. 9 is a schematic diagram of a flow reactor according to some embodiments of the present invention employed in the production of PF1022A derivatives.

FIG. 10 HPLC Chromatogram of Compound 01 prepared as described in Example 7.

FIG. 11 HPLC Chromatogram of Compound 01 prepared according to an embodiment as described in Example 9.

FIG. 12 HPLC Chromatogram of Compound 01 prepared according to an embodiment as described in Example 9.

FIG. 13 HPLC Chromatogram of Compound 02 prepared as described in Example 10. FIG. 14 'H-NMR spectra of Compound 01 prepared as described in Example 8.

FIG. 15 HPLC Chromatogram of Compound 01 prepared as described in Example 8. FIG. 16 HPLC Chromatogram of Compound 02 prepared as described in Example 10.

FIG. 17 ^-NMR spectra showing benzylic proton signals of para-para diamino stereoisomer of Compound 02 prepared as described in Example 10.

FIG. 18 Heteronuclear Multiple Bond Correlation (HMBC) spectra of para-para diamino stereoisomer Compound 02 prepared as described in Example 10. FIG. 19 Ή-NMR spectra showing benzylic proton signals of para-meta diamino stereoisomer of Compound 02 prepared as described in Example 10.

FIG. 20 ^-NMR spectra showing benzylic proton signals of meta-meta diamino stereoisomer of Compound 02 prepared as described in Example 10.

FIG. 21 ^-NMR spectra showing benzylic proton signals of ortho-meta diamino stereoisomer of Compound 02 prepared as described in Example 10.

DETAILED DESCRIPTION OF EMBODIMENTS

Provide herein are methods useful for the preparation of cyclooctadepsipeptide compounds such as the approved animal antihelmintic compound emodepside and flow platforms useful for carrying out the methods.

A. DEFINITIONS

Methods in accordance with the present disclosure include those generally described above and below, and are further illustrated by the embodiments, sub-embodiments, and species disclosed herein. As used herein the term "and/or" includes any and all combinations of one or more of the associated listed items.

As used herein, the following definitions shall apply unless otherwise indicated.

"Nitro" refers to the group— N0 ₂ .

"Halo" refers to fluoro, chloro, bromo or iodo.

Unless otherwise stated, structures depicted herein are meant to include all enantiomeric, diastereomeric, and geometric (or conformational) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Tautomeric forms include keto-enol tautomers of a compound. In addition, unless otherwise stated, all rotamer forms of the compounds of the invention are within the scope of the invention. Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹³ C- or ¹⁴ C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or probes in biological assays.

"Isomers" refers to compounds having the same number and kind of atoms and hence the same molecular weight, but differing with respect to the arrangement or configuration of the atoms. It will be understood, however, that some isomers or racemates or others mixtures of isomers may exhibit more activity than others. "Stereoisomers" refers to isomers that differ only in the arrangement of the atoms in space. "Diastereoisomers" refers to stereoisomers that are not mirror images of each other. "Enantiomers" refers to stereoisomers that are non-superimposable mirror images of one another.

"Aromatic positional isomers" are isomers in which substituents (other than hydrogen) may differ in position along an aromatic ring in relation to each other. As known in the art, the ortho isomer has substituents that occupy positions next to each other; meta isomer substituents occupy positions 1 and 3; and para isomer substituents occupy positions 1 and 4 (e.g., opposite ends of a phenyl ring).

In some embodiments, enantiomeric compounds taught herein may be "enantiomerically pure" isomers that comprise substantially a single enantiomer, for example, greater than or equal to 90%, 92%, 95%, 98%, or 99%, or equal to 100% of a single enantiomer.

In some embodiments, enantiomeric compounds as taught herein may be stereochemically pure. "Stereochemically pure" as used herein means a compound or composition thereof that comprises one stereoisomer of a compound and is substantially free of other stereoisomers of that compound.

In some embodiments, "R" and "S" as terms describing isomers are descriptors of the stereochemical configuration at an asymmetrically substituted carbon atom. The designation of an asymmetrically substituted carbon atom as "R" or "S" is done by application of the Cahn-Ingold-Prelog priority rules, as are well known to those skilled in the art, and described in the International Union of Pure and Applied Chemistry (IUPAC) Rules for the Nomenclature of Organic Chemistry.

"Chemical reactors" or "reactors" as used herein are vessels, wherein chemical reactions may be carried out.

A "flow reactor," also known as a continuous reactor, is a chemical reactor in which materials (typically fluids such as liquids) are carried as a flowing stream (e.g, in fluid channel(s) or tube(s)). In some embodiments, reactants may be continuously fed into the reactor and emerge as a continuous stream of product. In some embodiments, the velocity, or flow rate, of the fluid in the flow reactor or a portion thereof is substantially constant across any cross-section of the radial direction of the reactor.

In some embodiments, the flow reactor is a microreactor. "Microreactors" as used herein are flow reactors having fluid channels ranging from about 1 μηι to about 1 mm. See also Microreactors, Ehrfeld, Hessel and Lowe, 2000.

In some embodiments, the flow reactor is a bore tube with a diameter of about or greater than 5mm.

"Flow platform" refers to a system including one or more flow reactors and one or more of: one or more pumps, one or more injection valves, one or more sample loops, one or more reagent bottles, and software for automated control of the system.

"Reagent" as used herein is a substance or compound that may participate in or otherwise may be used to carry out a chemical reaction, which substance or compound may or may not be consumed in the course of the reaction, and thus may include reactants (which are normally consumed in the reaction) and solvents.

"Residence time" as used herein is defined as reactor volume divided by flow rate of a fluid in the flow reactor.

In some embodiments, reactants are introduced simultaneously into the flow reactor. It will be understood that "introducing reactants simultaneously" (or similar language) refers to introducing reactants at approximately (but not necessarily exactly) the same time.

In some embodiments, reactants are not introduced simultaneously into the flow reactor, and at least one of the reactants is introduced into the flow reactor before the other reactants are introduced into the flow reactor. For example, one or more reactants may be introduced into the flow reactor sequentially.

It will be understood that "started simultaneously" (or similar language) refers to started at approximately (but not necessarily exactly) the same time.

B. METHODS

In some embodiments, there is provided a method of producing a compound of Formula I from PF1022A, said method comprising:

contacting a nitrating agent to a solution of PF 1022A:

PF1022A

in a solvent at a temperature of about -20 °C to about -78 °C (e.g., from about -41 °C to about -70 °C); and then mixing the nitrating agent (e.g., nitric acid such as fuming nitric acid) and the solution of PF 1022A to produce the compound of Formula I:

Formula

wherein Ri and R ₂ are each independently hydrogen or nitro, and at least one of Ri and R ₂ is nitro. In some embodiments, the solvent is dichloromethane, nitromethane, or acetic anhydride.

In some embodiments, the method is carried out in a flow reactor, which may be carried out with methods as described below.

Also provided a method of producing a compound of Formula I from PF1022A, said method comprising the steps of contacting PF1022A:

PF1022A

with a nitrating agent in a flow reactor for a time sufficient to produce the compound of Formula I:

Formula I

wherein ¾ and R ₂ are each independently hydrogen or nitro, and at least one of Rj and R ₂ is nitro.

In some embodiments, the contacting is carried out by introducing the PF1022A and the nitrating agent into the flow reactor and flowing the PF1022A and the nitrating agent along a flow path (e.g., a longitudinal direction of the flow reactor) for a predetermined residence time sufficient to produce the compound of Formula I. In some embodiments, the predetermined residence time may be from about 0.1 second to about 60 minutes.

In some embodiments, as discussed with reference to FIG. 3, PF1022A and the nitrating agent may be mixed in a mixer before entering the flow reactor. The present invention, however, is not limited thereto. In some embodiments, PF1022A and the nitrating agent may be separately provided into a mixer or to the flow reactor via separate inlets. In some embodiments of the above methods, Rj of the produced compound of Formula I is hydrogen and R ₂ of the produced compound of Formula I is nitro. In some embodiments, both Ri and R ₂ of the produced compound of Formula I are nitro.

In some embodiments of the above methods, the compound of Formula I is produced as part of a mixture of compounds of Formula I, with the mixture comprising aromatic positional isomers of nitro at the Ri and/or R ₂ position. In some embodiments, 50% or greater of the aromatic positional isomers of nitro in the mixture are para isomers (as compared to ortho and meta isomers). In some embodiments, 60% or greater of the aromatic positional isomers of nitro in the mixture are para isomers (as compared to ortho and meta isomers). In some embodiments, 70%) or greater of the aromatic positional isomers of nitro in the mixture are para isomers (as compared to ortho and meta isomers). In some embodiments, 80%) or greater of the aromatic positional isomers of nitro in the mixture are para isomers (as compared to ortho and meta isomers). In some embodiments, 90% or greater of the aromatic positional isomers of nitro in the mixture are para isomers (as compared to ortho and meta isomers). In some embodiments, 95% or greater of the aromatic positional isomers of nitro in the mixture are para isomers (as compared to ortho and meta isomers). In some embodiments, 98%> or greater of the aromatic positional isomers of nitro in the mixture are para isomers (as compared to ortho and meta isomers).

The para isomers are shown in Formula la:

Formula la

wherein Rj and R ₂ are each independently hydrogen or nitro, and at least one of R ₁ and R ₂ is nitro. Example nitrating agents include, but are not limited to, nitric acid, acetic acid or acetic anhydride with ammonium nitrate, and acetic acid or acetic anhydride with one or more metal nitrates. Nitric acid may be used as nitric acid alone or in combination with sulfuric acid, nitric acid in water, nitric acid in acetic anhydride, nitric acid in chloroform, and nitric acid in other halogenated solvents such as methylene chloride. Example metal nitrates include, but are not limited to, Mn(N0 ₃ ) ₂ , Cu(N0 ₃ ) ₂ , NaN0 ₃ , Ca(N0 ₃ ) ₂ , Mg(N0 ₃ ) ₂ , and Sr(N0 ₃ ) ₂ . Example nitrating agents also include, but are not limited to, acetonitrile, a mixture of sulpholane with N0 ₂ BF ₄ , a mixture of acetyl nitrate with acetic anhydride, and a mixture of nitric acid with H ₂ S0 ₄ , CC1 ₄ , CH ₃ N0 ₂ , HCIO4, acetic anhydride, acetic acid and/or sulpholane. Further non- limiting examples include sodium nitrite and a suitable acid, such as trifluoroacetic acid. Further non-limiting examples include nitrogen tetroxide and oxygen in the presence of a suitable catalyst.

In some embodiments, the nitrating agent is nitric acid. In some embodiments, the nitrating agent is 68%, 70% or fuming (90%, 95%, etc.) nitric acid.

In some embodiments, the nitrating agent may be a nitronium salt. In some embodiments, the nitronium salt may be tetrafluoroborate, perchlorate, hexafluorophosphate, hexafluoroarsenate, hexafluoroantimonate, trifluoromethanesulfonate, or a combination of two or more thereof.

In some embodiments, introducing PF1022A and a nitrating agent into a flow reactor may include:

1) providing a first solution of PF 1022 A in a suitable solvent;

2) providing a second solution comprising the nitrating agent;

3) mixing said first solution and said second solution in a mixer to produce a reaction mixture; and

4) introducing said reaction mixture into a flow reactor.

In some embodiments, the first solution of PF1022A may be cooled to about 0 °C before mixing. In some embodiments, the first solution of PF1022A may be cooled to less than 0 °C before mixing (e.g., to a temperature of about -20 °C to about -78 °C).

In some embodiments, the process of producing the compound of Formula I further comprises maintaining the flow reactor at a fixed temperature. In some embodiments, the fixed temperature is ambient (room) temperature. In some embodiments, the fixed temperature is from about -78°C to about 80°C. In some embodiments, the fixed temperature is from about 0°C to about 80°C. In some embodiments, the fixed temperature is less than 0 °C (e.g., about -20 °C to about -78 °C).

In some embodiments, mixing the first solution and the second solution is performed using a suitable solvent as a system solvent. In some embodiments, the system solvent may be acetonitrile. In some embodiments, the system solvent may be dilute nitric acid. In some embodiments, the system solvent may be acetic anhydride.

In some embodiments, the residence time of the reaction mixture in the flow reactor is from about 0.1 second to about 60 minutes. In some embodiments, the residence time of the reaction mixture in the flow reactor is from about 10 minutes to about 60 minutes.

In some embodiments, the reaction mixture is collected from the flow reactor at the end of the residence time into a vessel. In some embodiments, the reaction mixture is collected into a vessel using a fraction collector.

In some embodiments, the reaction is quenched using a suitable quenching agent. In some embodiments, the produced compound is extracted from the quenching mixture using a suitable extraction solvent. For example, the extraction solvent may be ethyl acetate, and the produced compound may be extracted into ethyl acetate.

In some embodiments, the solvent in the first solution is an anhydride such as acetic anhydride. In some embodiments, the solvent in the first solution is pivalic anhydride.

In some embodiments, the nitrating agent in the second solution is nitric acid. In some embodiments, the solvent in the second solution is acetic anhydride.

In some embodiments, there is provided a method of producing a compound of Formula I from PF1022A, said method comprising adding a nitrating agent to PF1022A at a first temperature, and then mixing the nitrating agent with PF1022A at a second temperature. In some embodiments, at least one of ¾ and R ₂ is nitro, and nitro is at a para position. In some embodiments, the second temperature may be higher than the first temperature. For example, the first temperature may be about 0°C, and the second temperature may be about room temperature. For example, the nitrating agent may be nitric acid (e.g., fuming nitric acid). In some embodiments, the nitrating agent may be added dropwise. In some embodiments, PF1022A may react with the nitrating agent in a reactor such as a batch reactor or a flow reactor. In some embodiments, PF1022 A may react with the nitrating agent in a microreactor.

In some embodiments, there is provided a method of preparing a compound of Formula II, comprising: reacting a compound of Formula la (e.g., as a further step of or prepared by a process disclosed herein) with a suitable reducing agent to produce a compound of Formula II:

Formula II

wherein R ₃ and R ₄ are each independently hydrogen or -NH ₂ , and at least one of R ₃ and R ₄ is -NH ₂ .

In some embodiments, R ₃ of the produced compound of Formula II is hydrogen and R4 of the produced compound of Formula II is -NH ₂ . In some embodiments, both R ₃ and R4 of the produced compound of Formula II are -NH ₂ .

In some embodiments, the suitable reducing agent is hydrogen in the presence of a suitable catalyst. In some embodiments, the catalyst is palladium on carbon.

In some embodiments, preparing a compound of Formula II may be carried out in a flow reactor.

Also disclosed herein is a process for the preparation of a compound of Formula III, comprising:

1) reacting a compound of Formula II (e.g., as a further step of or prepared by a process disclosed herein) with sodium nitrite in the presence of a suitable acid to form a diazonium salt compound of Formula I

Formula IV wherein R ₅ and R ₆ are each independently selected from -N=N ⁺ X ^" and hydrogen, and at least one of R ₅ and R ₆ is -N=N ⁺ X ^" ; and

wherein X is a counterion of the acid (e.g., a strong acid such as perchloric acid, hydrobromic acid, hydrochloric acid, sulfuric acid); and

2) converting the diazonium salt compound of Formula IV into a compound of Formula III:

Formula III wherein R ₇ and R ₈ are each independently selected from -OH, halogen and hydrogen, and at least one of R ₇ and R ₈ is -OH or halogen.

In some embodiments, converting the diazonium salt compound of Formula IV into the compound of Formula III comprises heating the diazonium salt compound of Formula IV to decomposition. In some embodiments, converting the diazonium salt compound of Formula IV into the compound of Formula III comprises reacting the diazonium salt compound of Formula IV with aqueous base. In some embodiments, the aqueous base is sodium bicarbonate in water.

Also disclosed herein is a process for preparing a compound of Formula V, comprising: reacting a compound of Formula Ila (e.g., as a further step of or prepared by a process disclosed herein) with bis-2,2'-disubstituted ethyl ether to provide a compound of Formula V:

wherein Z is a leaving group. In some embodiments, Z may be a halo or organosulfonate. For example, Z may be selected from the group consisting of chlorine, bromine, tosylate, and mesylate.

Also disclosed herein is a process for preparing a compound of Formula V, comprising: reacting a compound of Formula Ila (e.g., as a further step of or prepared by a process disclosed herein) with a suitable carbonyl compound in the presence of a reducing agent to provide the compound of Formula V:

J Formula V

In some embodiments, X is an aldehyde group. In some embodiments, the reducing agent is sodium borohydride or sodium cyanoborohydride.

In some embodiments, there is provided a method of producing a compound of Formula III from PF1022 A, said method comprising reacting PF1022A with an electrophilic halogenating agent in a suitable solvent to provide a compound of Formula III:

Formula III

wherein R ₇ and R ₈ are each independently selected from halo and hydrogen, and at least one of R ₇ and R ₈ is halo. In some embodiments, halo is bromo or chloro.

In some embodiments, at least one of R ₇ and R is halo. In some embodiments, at least one of R ⁷ and R ⁸ is bromo. In some embodiments, both R ⁷ and R ⁸ are bromo. In some embodiments, at least one of R ⁷ and R ⁸ is chloro. In some embodiments, both R ⁷ and R ⁸ are chloro.

In some embodiments, the electrophilic halogenating agent is N-bromosuccinimide. In some embodiments, the electrophilic halogenating agent is N-bromosuccinimide in the presence of trifluoroacetic acid or N-chlorosuccinimide.

In some embodiments, the solvent is acetonitrile.

In some embodiments, the reaction of PF1022A with the electrophilic halogenating reagent may be carried out at a temperature of from about -5°C to about 75°C. In some embodiments, the reaction may be carried out at a temperature of about room temperature. In some embodiments, the reaction occurs at about 60°C. In some embodiments, the reaction may be carried out in a flow reactor.

C. FLOW PLATFORMS

In some embodiments, methods as taught herein are carried out in a flow reactor. The continuous mode of operation in a flow reactor can offer possible advantages over batch processes such as simplified operations, reduced reaction times, precise process control, greater reproducibility, and in some cases enhanced reaction selectivity. It may also provide for rapid optimization, screening different reaction conditions, catalysts, ligands, bases, and solvents, mechanistic studies, and cost-effective industrial scale up. In some embodiments, the flow reactor is a component of a flow platform.

FIG. 1 is a diagram of a flow platform according to some embodiments of the present invention. Referring to FIG. 1, a flow platform 10 may include a reactant providing module 100, a reactor module 200 and a product collecting module 300. Further, the flow platform 10 may also include a control module 400 configured to control the flow platform 10 or component(s) thereof.

FIG. 2 is a diagram of a reactant providing module and a reactor module according to some embodiments of the present invention. The reactant providing module 100 may include reservoirs 110 configured to contain reactants, reagents and/or solvents therein and a pumping module 120 configured to introduce the reactants, reagents and/or solvents into a mixer 130 and/or reactors, a flow reactor 210 and additional reactors 230 (e.g., for subsequent reactions) of the reactor module 200. In some embodiments, the pumping module 120 may include a pump, an injection valve and/or a sample loop. In some embodiments, the mixer is a static mixer. In some embodiments, the mixer is a T-mixer.

As illustrated in FIG. 2, in some embodiments, reactants, reagents and/or solvents may be mixed in the mixer 130 and then may be introduced into the reactors, and/or reactants. However, in some embodiments, reagents and/or solvents may be introduced into the reactors 210 and 230 without mixing (and mixer 103 may or may not be present). In some embodiments, reactants, reagents and/or solvents may be simultaneously introduced into the reactors. In some embodiments, reactants, reagents, and solvents may not be simultaneously introduced into the reactors, and at least one of the reactants, reagents and solvents may be introduced into the reactors before the other reactants, reagents and solvents are introduced into the reactors.

The reactor module 200 may also include a quenching module 220 configured to introduce a quenching agent into the flow reactor 210. In some embodiments, a volume of the flow reactor 210 may be determined to provide a predetermined residence time.

Examples of suitable pumps of the pumping module 120 include, but are not limited to, High Performance Liquid Chromatography-type (HPLC-type) pumps (e.g., acid resistant HPLC- type pump), peristaltic pumps, syringe pumps, etc., or a combination thereof.

Although FIG. 2 shows a single flow reactor 210, in some embodiments, the flow platform 120 may include multiple flow reactors 210.

In some embodiments, the reservoirs 1 10 of the flow platform 10 may be reagent bottles.

In some embodiments, the pumping module 120 may include a liquid handler, which is used to automatically introduce reagents into the mixer 130 and/or the reactors 210 and 230. In some embodiments, the pumping module 120 may also include an injection valve, a syringe pump and a sample loop, and the liquid handler may be connected to the injection valve via the syringe pump to load the sample loop.

In some embodiments, the flow platform 10 may optionally include a fraction collector. The fraction collector may be in a product collecting module 300.

In some embodiments, the flow platform 10 may optionally include a UV detector and/or an IR detector, which in some embodiments may be placed at the exit of one or more of the reactors 210 and 230.

In some embodiments, the mixer 130 may be a static mixer or a T-mixer.

In some embodiments, the control module 400 of the flow platform 10 may include a computer for automated control of the flow platform 10. In some embodiments, the control module 400 of the flow platform 10 may use software (e.g., FlowCommander™ of Vapourtec) to control reactions by control of pump flow, reactant, reagent and/or solvent delivery, etc.

FIG. 3 shows an example of a flow platform according to some embodiments of the present invention. Shown in this example embodiment are two acid resistant HPLC-type pumps, one injection loop (i.e., sample loop), a static mixer, a flow reactor, a backpressure regulator and a collection vessel. As an example, a solution of PF1022A in a suitable solvent (e.g., acetic anhydride) may be placed in the injection loop (e.g., 1 mL injection loop). A reservoir is filled with a stock solution of 70% nitric acid (10-20ml). These two solutions are pumped at appropriate flow rates into the static mixer and are mixed in the static mixer before entering the flow reactor, which may be held at a pre-determined temperature. The product exits the flow reactor and is collected. The whole system may be placed under a pressure of approximately 8 bar. The reaction may be quenched upon collection, and the product of the reaction extracted and purified (e.g., using column chromatography). The product may then be analyzed using various analytical methods such as HPLC and proton NMR.

FIG. 4 shows another example of a flow platform according to some embodiments of the present invention. Shown in this example embodiment are two pumps, one injection loop (i.e., sample loop), a static t-mixer, a flow reactor, and a collection vessel. As an example, a solution of PF1022A in a suitable solvent (e.g., dichloromethane, nitromethane, or acetic anhydride) may be placed in the injection loop (e.g., 1 mL injection loop). A reservoir is filled with a stock solution of 70% nitric acid (10-20ml). These two solutions are pumped with the system solvent (e.g., acetonitrile) at appropriate flow rates into the static mixer and are mixed in the static mixer before entering the flow reactor, which may be held at a pre-determined temperature (e.g. room temperature). The product exits the flow reactor and is collected. The whole system may be placed under a pressure of approximately 8 bar. The reaction may be quenched upon collection, and the product of the reaction extracted and purified (e.g., using column chromatography). The product may then be analyzed using various analytical methods such as HPLC and proton NMR.

FIG. 5 shows another example of a flow platform according to some embodiments of the present invention. Shown in this example embodiment are two pumps, one injection loop (i.e., sample loop), a first flow reactor at a reduced temperature, a static t-mixer at a reduced temperature, a second flow reactor, and a collection vessel. As an example, a solution of PF1022A in a suitable solvent (e.g., dichloromethane, nitromethane, or acetic anhydride) may be placed in the injection loop (e.g., 1 mL injection loop) and flowed through the first flow reactor to cool the solution. A reservoir is filled with a stock solution of fuming nitric acid (10-20 ml) and the solution is pumped with the system solvent (e.g., acetonitrile) at appropriate flow rates into the static mixer and are mixed in the static mixer at a reduced temperature before entering the second flow reactor, which may be held at a pre-determined temperature (e.g. room temperature). The product exits the flow reactor and is collected. The whole system may be placed under a pressure of approximately 8 bar. The reaction may be quenched upon collection, and the product of the reaction extracted and purified (e.g., using column chromatography). The product may then be analyzed using various analytical methods such as HPLC and proton NMR.

FIG. 9 shows another example of a flow platform according to some embodiments of the present invention. Shown in this example embodiment are three pumps, a first flow reactor at a reduced temperature, a static mixer at a reduced temperature, a second flow reactor at a reduced temperature, and a collection vessel. As an example, a solution of PF1022A in a suitable solvent (e.g., dichloromethane, nitromethane, or acetic anhydride) may flowed through the first flow reactor to cool the solution. A reservoir is filled with a stock solution of fuming nitric acid (10-20 ml) and the solution is pumped with the system solvent (e.g., acetonitrile) at appropriate flow rates into the static mixer and is mixed in the static mixer at a reduced temperature before entering the second flow reactor, which may also be held at the reduced temperature. The product exits the flow reactor and is collected. The whole system may be placed under a pressure of approximately 8 bar. The reaction may be quenched upon collection, and the product of the reaction extracted and purified (e.g., using column chromatography). The product may then be analyzed using various analytical methods such as HPLC and proton NMR.

The present invention is further illustrated by the following non-limiting examples.

EXAMPLE 1

The system was configured substantially as shown in FIG. 4.

The following solutions were made.

a) 5.7 mg of PF1022A was made up to 0.5 ml in Ac ₂ 0 (0.012 M) and was injected to the 0.5 mL sample loop.

b) Nitrating agent: HNO ₃ 70% was employed directly from a bottle as reagent in the second pump (B).

All reagents were completely soluble in acetic anhydride (Ac ₂ 0).

Acetonitrile (MeCN) was used as the system solvent to push the reagents through the system. FlowCommander™ was used to determine flow rates based on concentrations (Starting material 0.012M) and also volumetric ratios (PF1022A/Ac ₂ O:HNO ₃ equals 1 :0.6). The residence time in the 10 mL reactor was set to be 5 min so the total flow rate should be 2.0 mL/min. Both pumps started simultaneously while the sample loop was switched inline. Flow rates in pump A (Starting material): 1.25 mL/min and pump B nitrating agent (HN0 ₃ ): 0.750 mL/min.

All the system was at room temperature. After the reactor, a back-pressure regulator of eight bar was placed.

After 0.4 min, when 0.3mL of HN0 ₃ was introduced to the system, pump B switched to

MeCN bottle. After 5 min, collection was started. The collection was carried on for another 3 min. The long collection time was chosen to make sure that all the dispersed material was collected.

MeCN was evaporated from the collected material and the crude mixture was diluted with ethyl acetate (EtOAc) and extracted with water (x3). Then, the organic layer was dried over sodium sulfate, filtered and evaporated in vacuum to deliver nitrated PF 1022 A.

EXAMPLE 2

The use of fuming nitric acid was investigated in a flow reactor. In the experiments, the substrate (PF1022A) was initially cooled to 0°C before mixing with fuming nitric acid and entering the reactor where nitration occurs. Different temperatures (room temperature or 40°C) were investigated, as well as different residence times in the reactor (1 or 0.5 minutes). The ratio of regioisomers in the nitrated product was measured using proton NMR. Substrate was dissolved in either acetic anhydride or nitromethane, and acetonitrile was used as the system solvent. Results of experiments varying these parameters are shown in Table 1. The ratio column gives the ratio of b is-par -nitro compound to all other isomers.

TABLE 1

Procedure for Experiment 4

The system was configured substantially as shown in FIG. 5. All reagents were completely soluble in Ac ₂ 0.

a) 5.0 mg of PF1022A was made up to 0.5 ml in acetic anhydride (0.01 M) and was injected to the 0.5 mL sample loop.

b) Fuming HN0 ₃ (90%) was employed as reagent directly from a bottle in the second pump (B).

MeCN was used as the system solvent to push the reagents through the system. Flowcommander™ was used to determine flow rates based on concentrations (Starting material 0.01M) and also volumetric ratios (PF1022A/Ac ₂ O: HN0 ₃ 1.0:1.0). The residence time is set to be 1.0 min in the second reactor (2 mL) so a total flow rate of 2 mL/min is required. The first reactor was only used to cool the solution of PF 1022 A.

Both pumps started simultaneously while the sample loop was switched inline. Flow rates in pump A (Starting material): 1.000 mL/min and pump B nitrating agent (HN0 ₃ ): 1.000 mL/min. The T-mixer and the first reactor were at 0°C and the rest of the system was at room temperature. After the second reactor, a back-pressure regulator of eight bar was placed. After 0.5 min, when 0.5 mL of fuming ΗΝΌ ₃ was introduced to the system, pump B switched to MeCN bottle. After 3.0 min, collection was started over 7 mL of a saturated solution of NaHC0 ₃ (to neutralize the nitric acid). The collection was carried on for another 1.6 min. The crude was diluted with EtOAc and washed with water (x3) and brine (xl). Then, the organic layer was dried over sodium sulfate, filtered and evaporated until dryness in vacuum. Analysis of the material obtained by NMR showed 71% bis-para-mtio PF1022A plus 29% other isomers.

EXAMPLE 3

In a flask containing PF1022A (500 mg, 0.527 mmol) at 0°C fuming HNO ₃ (5 mL) was added dropwise during 15 min, and the mixture stirred at room temperature for 1 hour.

After 1 hour cold saturated NaHC0 ₃ solution (25 mL) was added with vigorous stirring. The reaction was then extracted with EtOAc and washed with water (x3) and brine (xl). The crude organics were dried over sodium sulfate, filtered and evaporated under vacuum.

The crude was purified by flash chromatography, employing hexane/ethyl acetate (3:7) as eluent, to provide 487 mg (0.469 mmol, 89%) of Compound 01 as a white/pale yellow foam (FIG. 6). 1H NMR (600 MHz, Chloroform-c/) δ 8.25 - 7.30 (m, 8H, arom.), 5.73 - 5.00 (m, 7H, 2xCH ^a -Phl, 2xH ^a -Lac, 3xH ^a -Leu), 4.57 - 4.41 (m, 1H, CH ^a -Leu), 3.33 - 3.15 (m, 4H, 2xCH ₂ ^p - Phi), 3.14 - 2.75 (m, 12H,m 4xNMe), 1.77 - 1.60 (m, 8H, 4xCH ₂ ^p -Leu), 1.49 - 1.35 (m, 2 ^p -Lac), 1.06 - 0.80 (m, 28H, 4xCH ^Y -Leu, 8xC ⁶ -Leu). m/z (M ⁺ +l) 1039.52.

Compound 01

EXAMPLE 4

A solution of Compound 01 (487 mg, 0.469 mmol) in dry MeOH (5 mL) was hydrogenated for 2 hours at room temperature under a hydrogen atmosphere (balloon), using Pd/C 10% as catalyst (162 mg, 1/3 w/w of palladium respect to substrate). The reaction was then filtered over celite to remove the catalyst and the solution evaporated.

The crude was purified by flash chromatography, employing hexane/ethyl acetate/ethanol (15:80:5) as eluent, to provide 307 mg (0.313 mmol, 67%) of Compound 02 as a white/pale yellow foam (FIG. 7). Ή NMR (600 MHz, Chloroform-c ) δ 7.09 - 6.97, 6.65 - 6.51 (m, 8H, arom.), 5.65 - 5.00 (m, 7H, 2xCH ^a -Phl, 2xH ^a -Lac, 3xH ^a -Leu), 4.50 - 4.43 (m, 1H, CH ^a -Leu), 3.11 - 2.94 (m, 7H, 2xCH ₂ ^p -Phl, NMe), 2.85 - 2.74 (m, 9H, 3xNMe), 1.79 - 1.62 (m, 8H, 4xCH ₂ ^p -Leu), 1.45 - 1.32 (m, 6H, 2xCH ₃ ^p -Lac), 1.05 - 0.82 (m, 28H, 4xCH ^Y -Leu, 8xCH ₃ ⁸ -Leu).

+ +1) 979.58.

Compound 01 Compound 02 EXAMPLE 5

Over a solution of PF1022A (5 mg, 0.005 mmol) in MeCN (0.15 mL), N- Bromosuccinimide (2.1 mg, 0.012 mmol) was added and stirred until total solubilisation. Next trifluoroacetic acid (0.15 mL) was added and the reaction stirred at room temperature for 2 days.

Next the reaction was stopped by pouring into water. The reaction was then extracted with EtOAc and washed with water (x3) and brine (xl). The crude was dried over sodium sulfate, filtered and evaporated in vacuum.

The crude was purified using flash chromatography, employing hexane/ethyl acetate (3 :7) as eluent, to provide 5 mg (0.0045 mmol, 90%) of Compound 03 as a pale yellow oil (FIG. 8). 1H NMR (600 MHz, Chloroform- ) δ 7.58 - 7.09 (m, 8H, arom.), 5.82 - 5.05 (m, 7H, 2xCH ^a - Phl, 2xH ^a -Lac, 3xH ^a -Leu), 4.51 - 4.44 (m, 1H, CH ^a -Leu), 3.30 - 2.70 (m, 16H, 2xCH ₂ ^p -Phl, 4xNMe), 1.73 - 1.60 (m, 8H, 4xCH ₂ ^p -Leu), 1.44 - 1.36 (m, 6H, 2xCH ₃ ^p -Lac), 1.05 - 0.78 (m, 28H, 4xCH ^Y -Leu, 8xCH ₃ ⁵ -Leu). m/z (M ⁺ +l) 1107.99.

PF1022A Compound 03

EXAMPLE 6

This example is similar to Example 5, but a higher temperature and more material was used. Over a solution of PF1022A (50 mg, 0.053 mmol) in MeCN (0.5 mL), N-

Bromosuccinimide (NBS, 28 mg, 0.158 mmol) was added and stirred until total solubilisation.

Next trifluoroacetic acid (TFA, 0.5 mL) was added and the reaction stirred at 60 °C overnight.

After that time, an additional portion of NBS (other 28 mg, 3 eq) was added and the reaction stirred for a further 18 hours.

Next the reaction was stopped by pouring into water. The reaction was then extracted with EtOAc and washed with water (x3) and brine (xl). The crude was dried over sodium sulfate, filtered and evaporated in vacuum. The crude was checked by HPLC where an 81% of bis Bromo derivative was observed. Isolated mass of clean bis-para material (Compound 03) was (11.9mg, 20%). ¹ H NMR (600 MHz, Chloroform-;/) δ 7.58 - 7.09 (m, 8H, arom.), 5.82 - 5.05 (m, 7H, 2xCH ^a -Phl, 2xH ^a -Lac, 3xH ^a -Leu), 4.51 - 4.44 (m, 1H, CH ^a -Leu), 3.30 - 2.70 (m, 16H, 2xCH ₂ ^p -Phl, 4xNMe), 1.73 - 1.60 (m, 8H, 4xCH ₂ ^p -Leu), 1.44 - 1.36 (m, 6H, 2xCH ₃ ^p -Lac), 1.05 - 0.78 (m, 28H, 4xCH ^Y -Leu, 8xCH ₃ ⁵ -Leu). m/z (M ⁺ +l) 1107.99.

EXAMPLE 7

The system was configured substantially as shown in FIG. 9. The following solutions were made:

a) 5000 mg of PF1022A was added to 28.0 ml in dichloromethane (DCM, 0.19M) and placed in the first pump (pump A).

b) Fuming HN0 ₃ (90%) was employed as reagent in the second pump (pump B).

A solution of PF1022A (5000 mg) in dichloromethane (DCM, 28.0 ml) was prepared. Acetonitrile (MeCN) was used as the system solvent. Flowcommander™ was used to determine flow rates based on concentrations (starting material 0.19 M) and also volumetric ratios (starting material/DCM 1.0 and HN0 ₃ 1.0). The residence time was set to be 100 min in the second reactor (20 mL), The first reactor was only used to cool the solution of starting material/DCM. Flow rates were 0.100 mL/min in pump A (starting material), and 0.100 mL/min in pump B (fuming HN0 ₃ ). The whole system was at -41°C. No back pressure regulator (BPR) was placed after the second reactor. Pump B was started, to pump the HN0 ₃ bottle and after 48.86 pump A started. After 98.95 min collection was started over 100 mL of a saturated solution of NaHC0 ₃ (to neutralize the nitric acid). After 263.36 min, pump A started to pump the MeCN bottle and after 322.88 min, pump B started to pump the MeCN bottle. The collection was carried out for another 347 min.

The crude collection was diluted with EtOAc and washed with water (x3) and brine (xl). Then, the organic layer was dried over sodium sulfate, filtered and evaporated until dryness in vacuum to provide 4.05 g (3.9 mmol, 91%) of a yellow foam.

Once the reaction was finished, the HPLC chromatogram showed two signals eluting at 4.27 min and 4.59 minutes (Fig. 10) in a ratio of 78:22. The major signal (based on the calculated area under the curve of the signal) was determined to be a mixture of three dinitrated stereoisomers of Compound 01 with the following nitro group orientations: para-para, para- meta, and meta-meta. The minor signal in the HPLC chromatogram was determined to be a para- ortho dinitrated stereoisomer of Compound 01. (Methods used for identifying the various dinitrated stereoisomers and their corresponding signal designations in the HPLC chromatogram of Fig. 10 are discussed in more detail in Example 10.) The para-para dinitrated stereoisomer of Compound 01 was identified as being the major stereoisomer contributing to the signal intensity

Compound 01

EXAMPLE 8

Over a flask that contains PF1022A at -10°C, fuming HN0 ₃ was added dropwise until PF1022A is totally solubilized. Next, the reaction mixture was stirred at RT for 1 hour. After that time, the reaction was stopped by adding the reaction mixture slowly (dropwise) over a cold NaOH (10 mL, 5 M) solution while stirring vigorously. The reaction mixture was extracted with EtOAc and the resulting organic layer was washed with water (x3) and brine (x2). The crude material was dried over sodium sulfate, filtered and evaporated in vacuum to obtain 5.43g (5.22 mmol, 99%) of a brown/dark yellow foam.

T 00 mg of this foam was dissolved in the minimum amount of Acetone and Hexane was added slowly while shaking the mixture. A white powder precipitated out of solution and was collected {see HPLC chromatogram in Fig. 15). When the same conditions were tried on a 5.3 g scale of the foam no precipitated formed and only a gel was obtained. 1H NMR analysis (Fig. 14) of the precipitated solid identified two sets of signals in the aromatic region in a ratio of 81 : 19, wherein the stronger set of signals represents a mixture of para-para, para-meta, and meta-meta dinitrated stereoisomers of Compound 01 and the weaker set of signal represents the ortho-meta dinitrated stereoisomers of Compound 01 (Methods used for identifying the various dinitrated stereoisomers and their corresponding signal designations in the Ή NMR are discussed in more detail in Example 10.)

Compound 01

EXAMPLE 9

Into a flask with PF1022A (100 mg; 0.105 mmol) and DCM (2.0 mL) at -70 °C was added fuming HN0 ₃ dropwise (0.5 mL), and the mixture was stirred at the same conditions and checked every hour. With this volume of DCM, the mixture was not solidified ("frozen"), but some solid drops of HN0 ₃ were observed in the bottom of the flask.

After 4 hours the reaction was finished by HPLC. The reaction was quenched by adding slowly (dropwise) a cold aqueous 5M NaOH solution until the reaction mixture had a pH ~7 or was mildly basic. The reaction was extracted with EtOAc and washed with water (x3) and brine (xl). The crude material was dried over sodium sulfate, filtered and evaporated in vacuo.

Once the reaction was finished, the HPLC chromatogram showed two signals eluting at 4.22 min and 4.54 minutes in a ratio of 83:17 (Fig. 11). The major signal (based on the calculated area under the curve of the signal) was determined to be a mixture of three dinitrated stereoisomers of Compound 01 with the following nitro group orientations: para-para, para- meta, and meta-meta. The minor signal in the HPLC chromatogram was determined to be a para- ortho dinitrated stereoisomer of Compound 01. (Methods used for identifying the various dinitrated compounds and their corresponding signal designations in the HPLC chromatogram of Fig. 11 are discussed in more detail in Example 10) The para-para dinitrated stereoisomer of Compound 01 was identified as being the major stereoisomer contributing to the signal intensity of the major signal eluting at 4.22 min. in the HPLC chromatogram of Fig. 11.

Compound 01

Additional nitration reactions of PF1022A were carried out under the same reactions conditions with an about 5.5 fold increase in PF1022A reaction mixture concentration to afford a mixture of dinitrated stereoisomers of Compound 01, wherein HPLC analysis of this mixture showed two signals eluting at 4.20 min (major) and 4.53 min (minor) in a ratio of 72:18 (Fig. 12). The major signal (based on the calculated area under the curve of the signal) was determined to be a mixture of para-para, para-meta, and meta-meta dinitrated stereoisomers of Compound 01 and the minor signal was determined to be a para-ortho dinitrated stereoisomer of Compound 01. This ratio between signals in the HPLC chromatogram is slightly less selective compared to the ratio of signals obtained in Example 7 (i.e., about 78:22) when the reaction was carried out in a flow system at -41 °C (Table 2). However, essentially the same ratio was obtained when the nitration was carried out under batch reaction conditions at -10 °C compared to -70 °C.

TABLE 2

EXAMPLE 10

A solution of Compound 01 (5.05 g, 3.898 mmol) from Example 7 in MeOH (30 mL) was hydrogenated for 2 h. at RT under H ₂ atmosphere (2 balloons), using Pd/C 10% (1.01 g) as a catalyst (1/4 w/w of palladium respect to substrate). Once the reaction was finished, the crude was filtered over celite to remove the catalyst. The solvent was removed under vacuum to yield a peptide product as a crude material. HPLC analysis of the crude material showed four different signals in the chromatogram eluting at 4.62, 6.22, 8.17, and 9.79 min. with intensities in a ratio of 63: 15: 1 :21, respectively {see Fig. 13 as representative HPLC chromatogram).

The crude material was diluted with TFA (10 mL) and then added over cold Et ₂ 0 until the product peptide was precipitated. The liquid was decanted and more cold Et ₂ 0 was added and the mixture was shaken again. The liquid was decanted one more time. Then, the white solid was analyzed by HPLC to investigate the ratios of the various diamino stereoisomers of the product peptide present in the solid material after the 1 ^st precipitation eluting as four different signals in the HPLC chromatogram. HPLC analysis of the four signals in the chromatogram eluting at 4.49, 6.09, 7.99, and 9.73 min. showed intensities in a ratio of 60: 16:1 :23, respectively, and a recovery of 87 % (3.7 g, 3.07 mmol).

From that first precipitated solid, 1 gram was diluted again with TFA (10 mL) and then added cold Et ₂ 0 until the peptide was precipitated. The liquid was decanted and more cold Et ₂ 0 was added and shaken again. The liquid was decanted one more time. Then, the white solid was analyzed by HPLC to know the ratio of the diamino stereoisomers of the product peptide present in the solid material after the 2 ^nd precipitation eluting as four different signals in the HPLC chromatogram. HPLC analysis of the four signals in the chromatogram eluting at 4.43, 6.07, and 9.68 min showed intensities in a ratio of 68: 15:0: 17, respectively, and a recovery of 48 % (479 mg, 0.40 mmol).

Compound 01 Compound 02 Preparative HPLC chromatography of the crude peptide product (Betasil CI 8 Dim (mm) 250x10, particle size (μ) 5 (Thermoscientific); flow rate 1 mL/min; injection volume 1000 μ ,, solvent system: acetonitrile with 0.1% formic acid: water with 0.1% formic acid with a gradient of 50:50 to 70:30 over 20 min, with 1%/min increments of increasing acetonitrile with 0.1% formic acid) was carried out to collect sufficient material of each of the four eluting diamino stereoisomers. Nuclear magnetic resonance (NMR) studies (e.g., 1H, ¹³ C) and two-dimensional NMR studies (e.g., HMBC) were performed to elucidate the chemical structure of each diamino stereoisomer present in each of the four eluting signals, which are summarized in Table 3. In l ' · · particular, H NMR analysis of the benzylic region in the spectra of each diamino stereoisomer aided in the structural identification of each diamino stereoisomer, which was also supported by the ¹³ C NMR spectra obtained for each diamino stereoisomer.

TABLE 3

171.1, 170.4,

169.7, 168.8,

168.7, 130.3,

129.5, 129.4,

1 16.8, 1 16.0,

115.1 , 71.1, 68.6,

66.8, 54.1 , 36.2,

29.4, 25.1, 24.7,

23.4, 23.3, 21.1,

20.8, 17.1 , 15.8

3 8.1 1 20 Meta-meta

4 9.8 21 21 178.0, 174.8, Ortho-meta

170.9, 170.4,

169.8, 147.5,

145.3, 145.2,

130.3, 115.1 ,

69.1 , 68.6, 68.4,

67.0, 66.9, 59.9,

59.6, 57.2, 54.2,

54.1 , 53.9, 31.2,

31.2, 30.7, 30.5,

29.4, 24.7, 24.6,

23.6, 23.4, 23.3,

23.2, 21.3, 21.2,

17.1 , 16.9, 15.9,

15.8

Based on the signal intensity and elution times of the four characterized diamino stereoisomers of the product peptide shown in Table 3, we extrapolated these results to characterize the two eluting signals obtained in the HPLC chromatograms of Figs. 10-12 showing signals for a mixture of dinitrated stereoisomers of Compound 01 prepared in Examples 7, 8 and 9 (see Table 2). Thus, the first eluting signal in the HPLC chromatogram of Fig. 10 contains a mixture of three dinitrated stereoisomers, wherein the orientation of the dinitro groups in each of the three dinitrated stereoisomer corresponds to the orientation of the diamino groups in the diamino stereoisomers eluting as the first (para-para), second (para-meta), and third signal (meta-meta) in the HPLC chromatogram of Fig. 13 as is also summarized in Table 3. The para-para diamino stereoisomer exhibits the highest signal intensity in the HPLC chromatogram of Fig. 13 as is also noted in Table 3, and thus, one can conclude that the majority of the signal intensity in the first eluding peak in the HPLC chromatogram of Fig. 10 is derived from the para- para dinitro stereoisomer of Compound 01 as is also noted in Table 2.

Likewise, the signal intensity of the fourth peak (21% of ortho-para stereoisomer) in the HPLC chromatogram of Fig. 13 (see Table 3) is about the same as the signal intensity of the second peak (22%) eluting the HPLC chromatogram of Fig. 10 (see Table 2) and thus, it is concluded that the second peak in the HPLC chromatogram of Fig. 10 is the para-ortho stereoisomer of Compound 01 (see Table 2).

EXAMPLE 11

A solution of Compound 01 in MeOH (c = 0.09 M) was introduced in an H-Cube Pro™ (ThalesNano, Hatboro, PA) system provided with a CatCart® disposable catalyst cartridge of Pd/C (70 mm long, THS 01131) (ThalesNano, Hatboro, PA). The conditions were configured to 60 °C, a pressure of 24 bar and a flow rate of 0.5 mL/min. With this set up, the reaction took 12 min to be completed. The solvent was removed under vacuum and HPLC analysis (Fig.16) of the crude material provided a ratio of 60:17:0:23 of para-para: para-meta: meta-meta: ortho-meta diamino stereoisomers of Compound 02.

Compound 01 Compound 02

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.