N-(hydrophobe-substituted) vancosaminyl [Ψ-[C(=NH) NH] Tpg4] vancomycin and [Ψ-[CH2NH]Tpg4] vancomycin

Abstract

The total synthesis and evaluation of key analogs of vancomycin containing single atom changes in the binding pocket are disclosed as well as their peripherally modified, N-(hydrophobe-substituted) derivatives exemplified by a N-4-(4-chlorobiphenyl)-methyl derivative and their pharmaceutically acceptable salts are disclosed. Their evaluation indicates the combined pocket and peripherally modified analogs exhibit a remarkable spectrum of antimicrobial activity and truly impressive potencies against both vancomycin-sensitive and -resistant bacteria, and likely benefit from two independent and synergistic mechanisms of action. A pharmaceutical composition containing a contemplated compound or its pharmaceutically acceptable salt is disclosed, as is a method of treating a bacterial infection in a mammal by administering an antibacterial amount of a contemplated compound or its salt as above to an infected mammal in need of treatment.

Claims

1. A compound that corresponds in structure to that shown in Formula I or its pharmaceutically acceptable salt, ##STR00054## wherein X=S or NH; and R is selected from the group consisting of (C.sub.1-C.sub.16)hydrocarbyl, aryl(C.sub.1-C.sub.6)-hydrocarbyldiyl, heteroaryl-(C.sub.1-C.sub.6)hydrocarbyldiyl, (C.sub.1-C.sub.6)hydrocarbyldiylheteroaryl, halo(C.sub.1-C.sub.12)-hydrocarbyldiyl, and (C.sub.1-C.sub.16)amido substituents, wherein an aryl or heteroaryl group is itself optionally substituted with up to three substituents independently selected from the group consisting of: (i) hydroxy, (ii) halo, (iii) nitro, (iv) (C.sub.1-C.sub.6)hydrocarbyl, (v) halo(C.sub.1-C.sub.16)hydrocarbyl, (vi) (C.sub.1-C.sub.6)hydrocarbyloxy, (vii) halo(C.sub.1-C.sub.6)hydrocarbyloxy, (viii) aryl, and (ix) aryloxy, wherein an aryl or aryloxy substituent can itself be substituted with up to three substituents independently selected from the group consisting of: (i) hydroxy, (ii) halo, (iii) nitro, (iv) (C.sub.1-C.sub.6)hydrocarbyl, (v) halo(C.sub.1-C.sub.16)hydrocarbyl, (vi) (C.sub.1-C.sub.6)hydrocarbyloxy, and (vii) halo(C.sub.1-C.sub.6)hydrocarbyloxy; and R.sup.1 is CH.sub.2OH, CH.sub.2OR.sup.2, where R.sup.2 is (C.sub.1-C.sub.7)hydrocarboyl, C(O)OH [carboxyl], C(O)R.sup.3, where R.sup.3 is (C.sub.1-C.sub.6)hydrocarbyloxy, or NR.sup.4R.sup.5 where R.sup.4 and R.sup.5 are independently the same or different and are H, (C.sub.1-C.sub.6)hydrocarbyl or R.sup.4 and R.sup.5 together with the depicted nitrogen atom form a 5-7 membered ring that can contain one ring oxygen atom.

2. The compound or its pharmaceutically acceptable salt according to claim 1, wherein R is aryl(C.sub.1-C.sub.6)-hydrocarbyldiyl.

3. The compound or its pharmaceutically acceptable salt according to claim 2 that corresponds in structure to Formula II, ##STR00055##

4. The compound or its pharmaceutically acceptable salt according to claim 2 that corresponds in structure to Formula III, ##STR00056##

5. The compound or its pharmaceutically acceptable salt according to claim 2, wherein said aryl(C.sub.1-C.sub.6)-hydrocarbyldiyl R group is a 4-(4-chlorophenyl)phenylmethyldiyl group.

6. The compound or its pharmaceutically acceptable salt according to claim 2 that corresponds in structure to one or more of the formulas below ##STR00057##

7. The compound or its pharmaceutically acceptable salt according to claim 2 that corresponds in structure to one or more of the formulas below ##STR00058##

8. A pharmaceutical composition that comprises an antimicrobial amount of a compound of Formula I or a pharmaceutically acceptable salt thereof dissolved or dispersed in a physiologically acceptable diluent ##STR00059## wherein X=NH; and R is selected from the group consisting of (C.sub.1-C.sub.16)hydrocarbyl, aryl(C.sub.1-C.sub.6)hydrocarbyldiyl, heteroaryl(C.sub.1-C.sub.6)hydrocarbyldiyl, (C.sub.1-C.sub.6)hydrocarbyldiylheteroaryl, halo(C.sub.1-C.sub.12)-hydrocarbyldiyl, and (C.sub.1-C.sub.16)amido substituents, wherein an aryl or heteroaryl group is itself optionally substituted with up to three substituents independently selected from the group consisting of: (i) hydroxy, (ii) halo, (iii) nitro, (iv) (C.sub.1-C.sub.6)hydrocarbyl, (v) halo(C.sub.1-C.sub.16)hydrocarbyl, (vi) (C.sub.1-C.sub.6)hydrocarbyloxy, (vii) halo(C.sub.1-C.sub.6)hydrocarbyloxy, (viii) aryl, and (ix) aryloxy, wherein an aryl or aryloxy substituent can itself be substituted with up to three substituents independently selected from the group consisting of: (i) hydroxy, (ii) halo, (iii) nitro, (iv) (C.sub.1-C.sub.6)hydrocarbyl, (v) halo(C.sub.1-C.sub.16)hydrocarbyl, (vi) (C.sub.1-C.sub.6)hydrocarbyloxy, and (vii) halo(C.sub.1-C.sub.6)hydrocarbyloxy; and R.sup.1 is CH.sub.2OH, CH.sub.2OR.sup.2, where R.sup.2 is (C.sub.1-C.sub.7)hydrocarboyl, C(O)OH [carboxyl], C(O)R.sup.3, where R.sup.3 is (C.sub.1-C.sub.6)hydrocarbyloxy, or NR.sup.4R.sup.5 where R.sup.4 and R.sup.5 are independently the same or different and are H, (C.sub.1-C.sub.6)hydrocarbyl or R.sup.4 and R.sup.5 together with the depicted nitrogen atom form a 5-7 membered ring that can contain one ring oxygen atom.

9. The pharmaceutical composition according to claim 8, wherein R is aryl(C.sub.1-C.sub.6)-hydrocarbyldiyl.

10. The pharmaceutical composition according to claim 9, wherein said aryl(C.sub.1-C.sub.6)-hydrocarbyldiyl R group is a 4-(4-chlorophenyl)phenylmethyldiyl group.

11. The pharmaceutical composition according to claim 10, wherein the dissolved or dispersed compound or a pharmaceutically acceptable salt thereof is one or more of ##STR00060##

12. A compound or its pharmaceutically acceptable salt that corresponds in structure to one or both of the formulas below ##STR00061##

13. A pharmaceutical composition that comprises an antimicrobial amount of a compound or a pharmaceutically acceptable salt thereof according to claim 12 dissolved or dispersed in a physiologically acceptable diluent.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Contemplated Compound

(2) One aspect of the present invention is a compound or a pharmaceutically acceptable salt thereof. A preferred compound of the invention corresponds in structure to that shown in Formula I, below,

(3) ##STR00016##
wherein
XH.sub.2, S or NH; and
R is selected from the group consisting of H, (C.sub.1-C.sub.16)hydrocarbyl, aryl(C.sub.1-C.sub.6)hydrocarbyldiyl, heteroaryl(C.sub.1-C.sub.6)hydrocarbyldiyl, (C.sub.1-C.sub.6)hydrocarbyldiylheteroaryl, halo-(C.sub.1-C.sub.12)-hydrocarbyldiyl, and (C.sub.1-C.sub.16)amido substituents, wherein an aryl or heteroaryl group is itself optionally substituted with up to three substituents independently selected from the group consisting of:

(4) (i) hydroxy,

(5) (ii) halo,

(6) (iii) nitro,

(7) (iv) (C.sub.1-C.sub.6)hydrocarbyl,

(8) (v) halo(C.sub.1-C.sub.16) hydrocarbyl,

(9) (vi) (C.sub.1-C.sub.6)hydrocarbyloxy,

(10) (vii) halo(C.sub.1-C.sub.6)hydrocarbyloxy,

(11) (viii) aryl, and

(12) (ix) aryloxy, wherein an aryl or aryloxy substituent can itself be substituted with up to three substituents independently selected from the group consisting of: (i) hydroxy, (ii) halo, (iii) nitro, (iv) (C.sub.1-C.sub.6)hydrocarbyl, (v) halo(C.sub.1-C.sub.16)hydrocarbyl, (vi) (C.sub.1-C.sub.6)hydrocarbyloxy, and (vii) halo(C.sub.1-C.sub.6)hydrocarbyloxy; and
R.sup.1 is CH.sub.2OH, CH.sub.2OR.sup.2, where R.sup.2 is (C.sub.1-C.sub.7)hydrocarboyl, C(O)OH [carboxyl], C(O)R.sup.3, where R.sup.3 is (C.sub.1-C.sub.6)hydrocarbyloxy, or R.sup.3 is NR.sup.4R.sup.5, where R.sup.4 and R.sup.5 are independently the same or different and are H (hydrido), (C.sub.1-C.sub.6)hydrocarbyl or R.sup.4 and R.sup.5 together with the depicted nitrogen atom form a 5-7 membered ring that can contain one ring oxygen atom. In some preferred embodiments, R is other than hydrido.

(13) When used in a pharmaceutical composition or in a method of treating a bacterially-infected mammal in need of antibacterial treatment, the R group of an above compound is other than hydrido (H).

(14) A particularly preferred R substituent is a 4-(4-chlorophenyl)phenylmethyldiyl group, below,

(15) ##STR00017##
that can also be named a 4-(4-chlorobiphenyl)methyl group, or a 4-(4-chlorophenyl)benzyl group, and is abbreviated herein as 4-CPB.

(16) One particularly preferred compound is a thioamido vancomycin compound that corresponds in structure to Formula II, below, wherein R and R.sup.1 are as described above.

(17) ##STR00018##

(18) A still more particularly preferred compound is an amidino vancomycin that corresponds in structure to Formula III, below, wherein R and R.sup.1 are also as described above.

(19) ##STR00019##

(20) Yet another particularly preferred compound corresponds in structure to Formula IV, below, wherein R and R.sup.1 are also as described above.

(21) ##STR00020##

(22) Compounds 5, 6, and 17, whose structural formulas are shown below, are the currently most

(23) ##STR00021## ##STR00022##
preferred compounds contemplated herein. Compound 5 has surprising activity as an antibacterial in that it is inactive as the corresponding vancomycin thioamide {[[C(S) NH] Tpg.sup.4]vancomycin}, and is particularly useful as an intermediate in the formation of Compound 6. Compound 6 has surprising antibiotic activity, particularly against VanA E. faecalis and E. faecium.

(24) Compounds 26, 27 and 28, whose structural formulas are shown below, are also preferred antimicrobial compounds that compounds exhibit antimicrobial activity.

(25) ##STR00023## ##STR00024##

(26) As will be seen from data provided hereinafter, the potency of 4-CPB vancomycin (Compound 4) against a susceptible strain of S. aureus is about 83 times greater than against VanA strains of E. faecalis and E. faecium. On the other hand, the potency of Compound 6 against those same strains of VanA E. faecalis and E. faecium was found to be about 6 times greater than its potency against the susceptible strain of S. aureus.

(27) Thus, a reversal in potency toward susceptible S. aureus and the VanA strains of E. faecalis and E. faecium is observed on going from 4-CPB vancomycin to Compound 6 {(4-CPB [[C(NH)NH]Tpg.sup.4]vancomycin}. That reversal in potency includes an increase in activity against those VanA strains of about 500 times on exchanging Compound 6 for 4-CPB vancomycin (Compound 4), with both compounds having about the same potency against the susceptible strain of S. aureus.

(28) Another preferred compound that is useful as an intermediate in the synthesis of Compound 6 and similar compounds is Compound 13, whose structural formula is shown below.

(29) ##STR00025##

(30) A compound of the invention can be provided for use by itself, or as a pharmaceutically acceptable salt. A contemplated compound of Formula II is a weak base, whereas an amidine compound of Formula III is a stronger base, and a compound of Formula IV has a basicity between those of Formula II and Formula III. A carboxyl group is also present in the molecule that can be present as a carboxylate zwitterion with one of the protonated amines. That carboxyl group can also be present as part of a carboxylic ester or amide as discussed previously. At physiological pH values, a compound of Formula I, such as a compound of Formulas II, III or IV is typically present as a salt.

(31) Exemplary salts useful for a contemplated compound include but are not limited to the following: sulfate, bisulfate, hydrochloride, hydrobromide, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-ethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, mesylate and undecanoate. Salts of the carboxylate group include sodium, potassium, magnesium, calcium, aluminum, ammonium, and the many substituted ammonium salts.

(32) The reader is directed to Berge, J. Pharm. Sci. 1977 68(1):1-19 for lists of commonly used pharmaceutically acceptable acids and bases that form pharmaceutically acceptable salts with pharmaceutical compounds.

(33) In some cases, a salt can also be used as an aid in the isolation or purification of a compound of this invention. In such uses, the acid used and the salt prepared need not be pharmaceutically acceptable.

(34) In line with expectations based on the behavior of the corresponding aglycons and in stark contrast to one another, the vancomycin amidine reestablishes potent antimicrobial activity against VanA VRE, whereas vancomycin thioamide is inactive even against vancomycin sensitive bacteria. Introduction of a peripheral 4-chlorobiphenylmethyl modification into the vancomycin amidine results in a compound with a remarkable spectrum of activity and truly impressive potencies that are likely derived from cell wall biosynthesis inhibition through two independent mechanisms, indicating that such peripheral and pocket synthetic modifications are synergistic. Such analogs, like vancomycin itself, are likely to display especially durable antibiotic activity [(a) James et al., ACS Chem. Biol. 2012, 7, 797; (b) Boger, Med. Res. Rev. 2001, 21, 356] not prone to rapidly acquired clinical resistance. That 4-chlorobiphenylmethyl modification into the vancomycin thioamide also provided some antimicrobial activity to the otherwise bio-inactive compound.

(35) Composition and Treatment Method

(36) A further aspect of the invention is a method of treating a mammal infected with a microbial infection such as a bacterial infection, typically a Gram-positive infection; i.e., an infection caused by Gram-positive bacteria, and in need of antimicrobial (antibacterial) treatment. In accordance with a contemplated method, an antibacterial-effective amount of one or more compounds of Formula I (a compound of Formulas II, III or IV), such as Compound 6, or a pharmaceutically acceptable salt of such a compound is administered to an infected mammal in need.

(37) The compound can be administered as a solid or as a liquid formulation, and is preferably administered via a pharmaceutical composition discussed hereinafter. That administration can also be oral or parenteral, as are also discussed further hereinafter.

(38) It is to be understood that viable mammals are infected with bacteria and other microbes. The present invention's method of treatment is intended for use against infections of pathogenic microbes that cause illness in the mammal to be treated. Illustrative pathogenic microbes include S. aureus, methicilin-resistant S. aureus (MRSA), VanA strains of E. faecalis and E. feacium, as well as VanB strains of E. faecalis. Evidence of the presence of infection by pathogenic microbes is typically understood by physicians and other skilled medical workers.

(39) A mammal in need of treatment (a subject) and to which a pharmaceutical composition containing a Compound of Formula I or its pharmaceutically acceptable salt can be administered can be a primate such as a human, an ape such as a chimpanzee or gorilla, a monkey such as a cynomolgus monkey or a macaque, a laboratory animal such as a rat, mouse or rabbit, a companion animal such as a dog, cat, horse, or a food animal such as a cow or steer, sheep, lamb, pig, goat, llama or the like.

(40) As is seen from the data that follow, a contemplated compound is active in in vitro assay studies at less than 1 g/mL amounts, which corresponds to a molar concentration of about 6 to about 60 nanomolar (nM), using the molecular weight of Compound 6. When used in an assay such as an in vitro assay, a contemplated compound is typically present in the composition in an amount that is sufficient to provide a concentration of about 0.1 nM to about 1 M to contact microbes to be assayed.

(41) The amount of a compound of Formula I or a pharmaceutically acceptable salt of such a compound that is administered to a mammal in a before-discussed method or that is present in a pharmaceutical composition discussed below, which can be used for that administration, is an antibiotic (or antibacterial or antimicrobial) effective amount. It is to be understood that that amount is not an amount that is effective to kill all of the pathogenic bacteria or other microbes present in an infected mammal in one administration. Rather, that amount is effective to kill some of the pathogenic organisms present without also killing the mammal to which it is administered, or otherwise harming the recipient mammal as is well known in the art. As a consequence, the compound usually has to be administered a plurality of times, as is discussed in more detail hereinafter.

(42) A contemplated pharmaceutical composition contains an effective antibiotic (or antimicrobial) amount of a Compound of Formula or a pharmaceutically acceptable salt thereof dissolved or dispersed in a physiologically (pharmaceutically) acceptable diluent or carrier. An effective antibiotic amount depends on several factors as is well known in the art. However, based upon the relative potency of a contemplated compound relative to that of vancomycin itself for a susceptible strain of S. aureus shown hereinafter, and the relative potencies of vancomycin and a contemplated compound against the VanA E. faecalis and E. faecium strains, a skilled worker can readily determine an appropriate dosage amount.

(43) A contemplated composition is typically administered repeatedly in vivo to a mammal in need thereof until the infection is diminished to a desired extent, such as cannot be detected. Thus, the administration to a mammal in need can occur a plurality of times within one day, daily, weekly, monthly or over a period of several months to several years as directed by the treating physician. More usually, a contemplated composition is administered a plurality of times over a course of treatment until a desired effect is achieved, typically until the bacterial infection to be treated has ceased to be evident.

(44) A contemplated pharmaceutical composition can be administered orally (perorally) or parenterally, in a formulation containing conventional nontoxic pharmaceutically acceptable carriers or diluents, adjuvants, and vehicles as desired. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques. Formulation of drugs is discussed in, for example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.; 1975 and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980.

(45) In some embodiments, a contemplated pharmaceutical composition is preferably adapted for parenteral administration. Thus, a pharmaceutical composition is preferably in liquid form when administered, and most preferably, the liquid is an aqueous liquid, although other liquids are contemplated as discussed below, and a presently most preferred composition is an injectable preparation.

(46) Thus, injectable preparations, for example, sterile injectable aqueous or oleaginous solutions or suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution, phosphate-buffered saline.

(47) Other liquid pharmaceutical compositions include, for example, solutions suitable for parenteral administration. Sterile water solutions of a Compound of Formula I or its salt or sterile solution of a Compound of Formula I (a compound of Formulas II, III or IV) in a solvent comprising water, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration. In some aspects, a contemplated Compound of Formula I is provided as a dry powder that is to be dissolved in an appropriate liquid medium such as sodium chloride for injection prior to use.

(48) In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of an injectable composition. Dimethyl acetamide, surfactants including ionic and non-ionic detergents, polyethylene glycols can be used. Mixtures of solvents and wetting agents such as those discussed above are also useful.

(49) A sterile solution can be prepared by dissolving the active component in the desired solvent system, and then passing the resulting solution through a membrane filter to sterilize it or, alternatively, by dissolving the sterile compound in a previously sterilized solvent under sterile conditions.

(50) Solid dosage forms for oral administration can include capsules, tablets, pills, powders, and granules. The amount of a contemplated Compound or salt of Formula I such as Compound 6 in a solid dosage form is as discussed previously, an amount sufficient to provide an effective antibiotic (or antimicrobial) amount. A solid dosage form can also be administered a plurality of times during a one week time period.

(51) In such solid dosage forms, a compound of this invention is ordinarily admixed as a solution or suspension in one or more diluents appropriate to the indicated route of administration. If administered per os, the compounds can be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted or encapsulated for convenient administration. Such capsules or tablets can contain a controlled-release formulation as can be provided in a dispersion of active compound in hydroxypropylmethyl cellulose. In the case of capsules, tablets, and pills, the dosage forms can also comprise buffering agents such as sodium citrate, magnesium or calcium carbonate or bicarbonate. Tablets and pills can additionally be prepared with enteric coatings.

(52) A mammal in need of treatment (a subject) and to which a pharmaceutical composition containing a Compound of Formula I or a pharmaceutically acceptable salt thereof is administered can be a primate such as a human, an ape such as a chimpanzee or gorilla, a monkey such as a cynomolgus monkey or a macaque, a laboratory animal such as a rat, mouse or rabbit, a companion animal such as a dog, cat, horse, or a food animal such as a cow or steer, sheep, lamb, pig, goat, llama or the like.

(53) Where an in vitro assay is contemplated, a sample to be assayed such as cells and tissue can be used. These in vitro compositions typically contain water, sodium or potassium chloride, and one or more buffer salts such as and acetate and phosphate salts, Hepes or the like, a metal ion chelator such as EDTA that are buffered to a desired pH value such as pH 4.0-8.5, preferably about pH 7.2-7.4, depending on the assay to be performed, as is well known.

(54) Preferably, the pharmaceutical composition is in unit dosage form. In such form, the composition is divided into unit doses containing appropriate quantities of the active compound. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation, for example, in vials or ampules.

(55) Antimicrobial Activity

(56) The pocket-modified vancomycin analogues that contain the C-terminus hydroxymethyl group (Compounds 23-25), their chlorobiphenyl derivatives (Compounds 26-28), as well as the fully functionalized vancomycin analogues [[C(S)NH]Tpg.sup.4]vancomycin, [[C(NH)NH]Tpg.sup.4]-vancomycin, and [[CH.sub.2NH]Tpg.sup.4]vancomycin (Compounds 2, 3 and 16) and their (4-chlorobiphenyl)methyl derivatives (Compounds 5, 6 and 17) were examined alongside the corresponding vancomycin (residue 4 amide) derivatives. The antimicrobial activity of the compounds was evaluated against a panel of Gram-positive bacteria that include vancomycin-sensitive S. aureus (VSSA), methicillin-resistant S. aureus (MRSA), and both VanA (E. faecalis and E. faecium) and VanB (E. faecalis) vancomycin-resistant Enterococci (VRE) of which VanA is the most stringent of the resistant organisms, with those results shown in the tables below.

(57) Notably, one VanA VRE (E. faecium, ATCC BAA-2317) tested represents an emerging challenging multidrug resistant VanA VRE that is not only resistant to vancomycin and teicoplanin, but also ampicillin, benzylpenicillin, ciprofloxacin, erythromycin, levofloxacin, nitrofurantoin, tetracycline. It is also insensitive to linezolid, but remains sensitive to tigecycline and dalfopristine.

(58) TABLE-US-00002 Antimicrobial Activity, MIC.sup.a (g/mL) sensitive MRSA VanA VanB S. aureus.sup.b S. aureus.sup.c E. faecalis.sup.d E. faecium.sup.e E. faecalis.sup.f R = H 1, X = O 0.5 0.5 250 250 8 2, X = S >32 >32 >32 >32 >32 3, X = NH nd.sup.g nd.sup.g 0.5 0.5 nd.sup.g 16, X = H.sub.2 nd.sup.g nd.sup.g 31 31 nd.sup.g R = CBP, (4-chlorobiphenyl)methyl 4, X = O 0.03 0.03 2.5 2.5 0.03 5, X = S 2 2 4 4 2 6, X = NH 0.03 0.06 0.005 0.005 0.06 17, X = H.sub.2 0.5 0.25 0.13 0.06 0.5 .sup.aMIC = Minimum inhibitory concentration. .sup.bATCC 25923. .sup.cATCC 43300. .sup.dBM 4166. .sup.eATCC BAA-2317. .sup.fATCC 51299. .sup.gnot determined.

(59) As is seen from the data in the table below, the activity of C-terminus hydroxymethyl derivatives paralleled that observed with the corresponding C-terminus carboxylic acids. However, the C-terminus hydroxymethyl compounds displayed the same general trends and near identical absolute MIC values, reinforcing the generality and significance of the conclusions.

(60) TABLE-US-00003 Antimicrobial Activity, MIC.sup.a (g/mL) sensitive MRSA VanA VanB S. aureus.sup.b S. aureus.sup.c E. faecalis.sup.d E. faecium.sup.e E. faecalis.sup.f R = H 23, X = O 0.5 0.5 250 250 8 24, X = S >32 >32 >32 >32 >32 25, X = NH nd.sup.g nd.sup.g 2 2 nd.sup.g R = CBP, (4-chlorobiphenyl)methyl 26, X = O 0.03 0.03 2 4 0.13 27, X = S 4 8 8 4 4 28, X = NH 0.13 0.13 0.02 0.02 0.06 .sup.aMIC = Minimum inhibitory concentration. .sup.bATCC 25923. .sup.cATCC 43300. .sup.dBM 4166. .sup.eATCC BAA-2317. .sup.fATCC 51299. .sup.gnot determined.

(61) Vancomycin-sensitive S. aureus (VSSA, ATCC 25923): sensitive to vancomycin, teicoplanin, oritavancin, daptomycin, linezolid, quinupristin-dalfopristin, fussidic acid, azithromycin, telithromycin, gentamycin, penicillin V, nafcillin, ampicillin, oxacillin, ciprofloxacin, levofloxacin, garenoxacin and moxifloxacin. Methicillin-resistant S. aureus (MRSA, ATCC 43300): sensitive to vancomycin, teicoplanin, daptomycin, linezolid, tigecycline and ciprofloxacin; resistant to methicillin, amoxicillin, amoxicillin with clavulanic acid, cephalexin, enrofloxacin, erythromycin, azithromycin, gentamycin, clindamycin, lincomycin-spectinomycin, neomycin, oxacillin, penicillin G, streptomycin, trimethoprim-sulfamethoxazole and tetracycline. VanA E. faecalis (VanA VRE, BM 4166): resistant to erythromycin, gentamicin, chloramphenicol, and ciprofloxacin as well as vancomycin and teicoplanin; sensitive to daptomycin. VanA E. faecium (VanA VRE, ATCC BAA-2317): resistant to ampicillin, benzylpenicillin, ciprofloxacin, erythromycin, levofloxacin, nitrofurantoin, and tetracycline as well as vancomycin and teicoplanin, insensitive to linezolid; sensitive to tigecycline and dalfopristine. VanB E. faecalis (VanB VRE, ATCC 51299): resistant to vancomycin, streptomycin, gentamicin; sensitive to teicoplanin, ampicillin, tetracycline, and ciprofloxacin.

(62) The activity of the pocket modified vancomycin analogues Compounds 2, 3 and 16 matched the in vitro antimicrobial activity of the corresponding aglycon analogue Compounds 8, 9 and 10 on which they are based. Although it is well established that the attached unmodified carbohydrate does not alter in vitro antimicrobial activity (potency) or influence target D-Ala-D-Ala or D-Ala-D-Lac binding, the vancomycin disaccharide impacts it's in vivo activity; increasing water solubility, influencing pharmacokinetic and distribution properties, and contributing a potential second mechanism of action.

(63) An analogous impact on the vancomycin analogue Compounds 2, 3 and 16 might be expected because each represents the change of a single atom in the binding pocket (residue 4 carbonyl O.fwdarw.S, NH, H.sub.2), and they would be the preferred compounds (vs Compounds 8, 9 and 10) with which to probe in vivo activity.

(64) Within this series, vancomycin displayed potent activity against VSSA and MRSA (MIC=0.5 g/mL), but was ineffective against VanA VRE (MIC=250 g/mL) and only modestly active against VanB VRE (MIC=8 g/mL) under the assay conditions employed. Consistent with its lack of binding to either D-Ala-D-Ala or D-Ala-D-Lac, the thioamide Compound 2 proved inactive as an antimicrobial agent (MICs>32 g/mL) against both sensitive and resistant bacteria.

(65) Both the amidine Compound 3 [Okano et al., J. Am. Chem. Soc. 2014, 136, 13522] and the methylene analogue Compound 16 reinstated activity against VanA VRE (BM4166) with MICs of 0.5 and 31 g/mL, respectively. This finding was precisely in line with expectations based on the relative dual D-Ala-D-Ala and D-Ala-D-Lac binding affinities of the aglycons and matching the activities observed with the corresponding aglycons Compounds 9 and 10.

(66) Of most significance, the amidine Compound 3 displayed a potency against VanA VRE that matched the activity vancomycin displays against sensitive bacteria (VSSA and MRSA, MICs=0.5 g/mL).

(67) Given the distinct origins of their impact on the antimicrobial activity of vancomycin, it was expected that incorporation of the peripheral chlorobiphenyl modification into the structure of the binding pocket-modified vancomycin analogues would further increase their antimicrobial activity against not only sensitive, but also vancomycin-resistant bacteria to truly remarkable potencies. Although this conceivably could have been demonstrated by substitution of the synthetic aglycon Compounds 7, 8, 9 and 10, the most definitive assessment of the dual impact was expected to be a direct comparison of chlorobiphenyl vancomycin (Compound 4) with Compounds 5, 6, and 17, wherein a series of key changes in a single atom in the binding pocket were introduced, despite the synthetic challenges this posed. This choice of both the site of modification and the use of the chlorobiphenyl modification proved important to understanding the behavior of such analogues and revealed unique insights into the origin of the effects.

(68) In line with reports of its impact, introduction of the (4-chlorobiphenyl)methyl group into vancomycin (Compound 4 vs Compound 1) results in 100-fold improvements in the activity against VanA and VanB VRE (MIC=2.5 vs 250 g/mL) and 20-fold improvements against VSSA and MRSA (MIC=0.03 vs 0.5 g/mL) in the strains examined. In spite of the increases in potency, it remains 100-fold less effective against VanA VRE.

(69) Both the amidine Compound 6 and the methylene analogue Compound 17 exhibited the same 100-fold increases in activity against VanA VRE, exhibiting remarkable MICs of 0.005 g/mL and 0.06-0.13 g/mL, respectively. Just as significantly, introduction of the chlorobiphenyl group into either the vancomycin amidine Compound 6 or the vancomycin methylene analogue Compound 17 resulted in compounds with remarkable spectra of activity at these impressive potencies.

(70) Both compounds were equally effective against both vancomycin-sensitive bacteria (VSSA and MRSA) and vancomycin-resistant bacteria (VanA and VanB VRE) of which VanA VRE proved especially sensitive to the analogues. Both analogues exhibit MICs below 1 g/mL across the bacterial panel, and the amidine Compound 6 was found to be on average 15-fold more potent than the methylene analogue Compound 17, precisely in line with their relative dual ligand binding affinities.

(71) Moreover, the amidine Compound 6 not only matches the activity that CBP-vancomycin (Compound 4) displays against vancomycin-sensitive bacteria (VSSA and MRSA), but it also exhibits this extraordinary potency against VanA and VanB vancomycin-resistant bacteria. In fact, the activity of Compound 6 against the most stringent of the resistant bacteria, VanA VRE, was nearly 10-fold better than the potency it displays against the sensitive bacteria, representing a 500-fold increase in activity relative to CBP-vancomycin (Compound 4) and a 50,000-fold increase in activity relative to vancomycin (Compound 1) itself. Thus, the chlorobiphenyl introduction into the pocket modified vancomycin analogues Compound 6 (MICs=0.005-0.06 g/mL) and Compound 17 (MICs=0.06-0.5 g/mL) synergistically increased their potency against both vancomycin-sensitive and vancomycin-resistant bacteria.

(72) Insights into this behavior came from the examination of the chlorobiphenyl derivative of the vancomycin thioamide (Compound 5). Introduction of the (4-chlorobiphenyl)methyl group into vancomycin thioamide (Compound 2) with Compound 7 reinstates impressive and equally potent activity (MIC=2-4 g/mL) against all vancomycin-sensitive and vancomycin-resistant strains despite its inability to bind the primary cell wall target D-Ala-D-Ala/D-Ala-D-Lac.

(73) It is unlikely such effective activity can be achieved simply by the effects of antibiotic membrane anchoring, antibiotic dimerization, or disruption of bacterial membrane integrity. Rather, it likely reflects potent antimicrobial activity derived from a second mechanism of action impacting cell wall synthesis unrelated to D-Ala-D-Ala/D-Ala-D-Lac binding.

(74) In line with observations made with CBP-vancomycin and analogues containing damaged binding pockets, this most likely involves potent transglycosylase inhibition mediated by direct binding to the enzyme [(a) Ge et al., Science 1999, 284, 507; (b) Chen et al., Proc. Natl. Acad. Sci. USA 2003, 100, 5658; and (c) Goldman et al., FEMS Microbiol. Lett. 2000, 183, 209]. Because of the insights derived from the comparative examination of the thioamide Compounds 2 and 5, the behavior of the CBP-vancomycin amidine Compound 6 and CBP-vancomycin methylene analogue Compound 17 likely represents a spectrum of activity and potency derived from bacterial cell wall synthesis inhibition through two synergistic mechanisms, one involving inhibition of transpeptidase-catalyzed cell wall cross-linking through dual substrate (D-Ala-D-Ala and D-Ala-D-Lac) binding and the second through direct inhibition of transglycosylase independent of such ligand binding.

(75) If this is the case, it suggests that resistance is unlikely to emerge against such analogues because it would entail simultaneous bacterial changes to two distinct targets of the antibiotics, one of which is not subject to direct genetic alterations. As such, both Compound 6 and Compound 17 are superb candidates for preclinical development. Their preliminary assessments not only indicate that they address the present day emerging vancomycin resistance and exhibit remarkable spectrums of activity and superb antimicrobial potency, but also that they are endowed with a unique combination of characteristics that may allow them to display the 50 year clinical durability of vancomycin.

(76) Although at this stage still speculative, the four chlorobiphenyl derivative Compounds 4, 5, 6 and 17 are also uniquely poised to help unravel the subtleties of the mechanisms of action of such modified glycopeptide antibiotics. Due to its inability to bind either D-Ala-D-Ala or D-Ala-D-Lac, the thioamide Compound 5 (CBP-[[C(S)NH]Tpg.sup.4]-vancomycin) derives its antimicrobial activity (MIC=2-4 g/mL) exclusively through a distinct second mechanism of action that does not involve ligand binding and likely involves direct inhibition of transglycosylase [(a) Ge et al., Science 1999, 284, 507; (b) Chen et al., Proc. Natl. Acad. Sci. USA 2003, 100, 5658; and (c) Goldman et al., FEMS Microbiol. Lett. 2000, 183, 209].

(77) By virtue of its inability to bind D-Ala-D-Lac, CBP-vancomycin (Compound 4) also likely derives its similar activity against vancomycin-resistant organisms (VanA VRE, MIC=2.5 g/mL) by this same mechanism potentially involving only the direct inhibition of transglycosylase, whereas its more potent activity against vancomycin-sensitive organisms (VSSA and MRSA, MIC=0.03 g/mL) is derived from the equally potent and synergistic inhibition of both transpeptidase (via D-Ala-D-Ala binding and substrate sequestration) and transglycosylase (direct enzyme inhibition).

(78) As a result of the binding pocket redesign and ability to exhibit fully effective dual D-Ala-D-Ala and D-Ala-D-Lac binding combined with the peripheral chlorobiphenyl-mediated potential direct inhibition of transglycosylase, Compound 6 {CBP-[[C(NH)NH]Tpg.sup.4]vancomycin} picks up the ability to effectively inhibit transpeptidase in vancomycin-resistant bacteria (VanA VRE, via D-Ala-D-Lac binding), maintains the ability to inhibit transpeptidase in vancomycin-sensitive bacteria (VSSA and MRSA, via D-Ala-D-Ala binding), permits the potential indirect transglycosylase inhibition through ligand binding, and benefits potentially from an equally potent and synergistic direct inhibition of transglycosylase independent of D-Ala-D-Ala or D-Ala-D-Lac binding. The net result is an antibiotic that benefits from two equally potent, independent, and synergistic mechanisms of action and that displays the remarkable antimicrobial potencies (MIC=0.06-0.005 g/mL) against both vancomycin-sensitive and vancomycin-resistant bacteria.

(79) In contrast but similarly interestingly, the potency of CBP-[[CH.sub.2NH]Tpg.sup.4]vancomycin (Compound 17) (MIC=0.5-0.06 g/mL) suggests that the principle mechanism by which it acts is through the potential chlorobiphenyl-mediated direct inhibition of transglycosylase, but now with a second less potent contribution derived from its balanced, albeit reduced, dual ligand binding affinities for inhibition of transpeptidase in either vancomycin-sensitive and vancomycin-resistant bacteria. It is remarkable that the series appears to display the trends of two independent mechanisms, which act synergistically with one another, to provide newly predictable potency trends derived independently from the binding pocket modifications and the peripheral carbohydrate substitution.

(80) Kahne and co-workers have shown that although the potency of most lipid-linked glycopeptides or their aglycons lose activity against VanA strains when their binding pocket is chemically damaged [(a) Chen et al., Tetrahedron 2002, 58, 6585; and (b) Kerns et al., J. Am. Chem. Soc. 2000, 122, 12608-12609], indicating ligand binding is important to their activity, a small subset including CBP-vancomycin retains good antimicrobial activity even when their binding pocket is chemically damaged [(a) Ge et al., Science 1999, 284, 507; and (b) Chen et al., Proc. Natl. Acad. Sci. USA 2003, 100, 5658]. Moreover, it is such derivatives that were shown by Kahne and Walker [Chen et al., Proc. Natl. Acad. Sci. USA 2003, 100, 5658] to effectively inhibit transglycosylase without substrate or ligand binding, suggesting it directly binds and inhibits the enzyme. CBP-[[C(S)NH]Tpg.sup.4]vancomycin embodies these same characteristics, displays the same VanA VRE potency, and likely will display the same behavior toward transglycosylase. It is likely that this activity against VanA strains requires a specific positioning of the hydrophobic substituent attached to the vancomycin disaccharide. As a consequence, it is especially notable that these studies were conducted with single atom changes to the binding pocket of vancomycin and CBP-vancomycin and not conducted on simpler, more accessible aglycon derivatives.

(81) The activity of many antibiotics, especially cationic peptide antibiotics, display changes in activity with additives [Moeck, Antimicrob Agents Chemother 2008, 5, 159] including oritavancin, or can be dependent on the broth conditions [Otvos et al., In Methods in Molecular Biol., 2007, Vol 386, Fields ed.; Humana Press, Totowa, N.J., pp 309-320]. The vancomycin, [[C(S)NH]Tpg.sup.4]-vancomycin, and [[CH.sub.2NH]Tpg.sup.4]vancomycin derivatives exhibited small 2-4 fold shifts in antimicrobial activity with variations in the broth dilution, whereas [[C(NH)NH]Tpg.sup.4] vancomycin varied more.

(82) TABLE-US-00004 Antimicrobial Activity, MIC.sup.a (g/mL) sensitive MRSA Van A Van B S. aureus.sup.b S. aureus.sup.c E. faecalis.sup.d E. faecium.sup.e E. faecalis.sup.f Serum concentration 10% 25% 100% 10% 25% 100% 10% 25% 100% 10% 25% 100% 10% 25% 100% R = H 1, X = O 0.5 0.5 0.5 0.5 0.5 1 250 250 500 250 250 500 4 8 32 33, X = S >32 >32 >32 >32 >32 >32 >32 >32 >32 >32 >32 >32 >32 >32 >32 34, X = NH nd.sup.g nd.sup.g nd.sup.g nd.sup.g nd.sup.g nd.sup.g 0.5 8 >32 0.5 4 >32 nd.sup.g nd.sup.g nd.sup.g 35, X = CH.sub.2 nd.sup.g nd.sup.g nd.sup.g nd.sup.g nd.sup.g nd.sup.g 31 62 125 31 62 125 nd.sup.g nd.sup.g nd.sup.g R = CBP, (4-chlorobiphenyl)methyl 36, X = O 0.03 0.03 0.06 0.03 0.06 0.06 2.5 5 10 2.5 5 5 0.03 0.06 0.06 37, X = S 2 2 4 2 4 4 4 4 8 4 4 8 2 2 8 38, X = NH 0.03 0.12 1 0.06 0.24 1 0.005 0.06 0.5 0.005 0.06 0.5 0.06 0.12 1 39, X = CH.sub.2 0.5 1 2 0.25 0.5 1 0.13 0.26 0.5 0.06 0.13 0.5 0.5 1 2 .sup.aMIC = Minimum inhibitory concentration. .sup.bATCC 25923. .sup.cATCC 43300. .sup.dBM 4166. .sup.eATCC BAA-2317. .sup.fATCC 51299. .sup.gnot determined.
Synthetic Procedures

(83) Compounds 23-28 were prepared as described hereinafter and in (a) Xie et al., J. Am. Chem. Soc. 2011, 133, 13946; and (b) Xie et al., J. Am. Chem. Soc. 2012, 134, 1284. The general synthesis strategy is laid out in Scheme 1 hereinafter.

(84) The synthesis of Compound 3 was accomplished by enlisting two sequential enzymatic glycosylation reactions to first provide [[C(S)NH]Tpg.sup.4]vancomycin (Compound 2), followed by a final Ag(I)-promoted [Okano et al., J. Am. Chem. Soc. 2012, 134, 8790] conversion of the residue 4 thioamide to an amidine. Compound 2 was converted to Compound 5 by an additional single-step introduction of the N-4-(4-chlorobiphenyl)methyl group into [[C(S)NH]Tpg.sup.4]vancomycin, that step was followed by Ag(I)-promoted conversion of the thioamide to an amidine to afford Compound 6.

(85) In addition to providing the opportunity to assess [[C(NH)NH]Tpg.sup.4]vancomycin (Compound 3) and the impact of combining the vancomycin pocket redesign with a key peripheral structural modification, the approach was designed to shed light on the role of the chlorobiphenyl modification with the examination of [[C(S)NH]Tpg.sup.4]vancomycin (Compound 2) and its 4-(4-chlorobiphenyl)methyl derivative, which are incapable of binding D-Ala-D-Ala or D-Ala-D-Lac.

(86) The recombinant glycosyltranferases GtfE and GtfD from the vancomycin producing strain of A. orientalis (ATCC 19795) were expressed in E. coli from the corresponding constructs [(a) Losey et al., Biochemistry 2001, 40, 4745; (b) Oberthur et al., J. Am. Chem. Soc. 2005, 127, 10747; (c) Losey et al., Chem. Biol. 2002, 9, 1305; (d) Thayer et al., Chem. Asian J. 2006, 1, 445] (a gift of C. Walsh) and were purified to homogeneity (His.sub.6 tag). The two sequential glycosylations of synthetic Compound 8 [(a) Xie et al., J. Am. Chem. Soc. 2011, 133, 13946; (b) Xie et al., J. Am. Chem. Soc. 2012, 134, 1284] were conducted with the purified glycosyltransferases enzymes and the synthetic glycosyl donors (UDP-glucose [Sigma-Aldrich] for GtfE and UDP-vancosamine [(a) Nakayama et al., Org. Lett. 2014, 16, 3572; (b) Oberthur et al., Org. Lett. 2004, 6, 2873] for GtfD) under recently described conditions [(a) Nakayama et al., Org. Lett. 2014, 16, 3572; (b) Oberthur et al., Org. Lett. 2004, 6, 2873] to provide the pseudoaglycon Compound 13 (75%) and [[C(S)NH]Tpg.sup.4]-vancomycin (Compound 2, 52%) (Scheme 1).

(87) ##STR00029##

(88) Direct conversion of the thioamide to the corresponding amidine (10 equiv AgOAc, sat. NH.sub.3-MeOH, 25 C., 7 hours) [Okano et al., J. Am. Chem. Soc. 2012, 134, 8790] provided [[C(NH)NH]Tpg.sup.4]vancomycin (Compound 3). Significantly, the reaction was capable of implementation without competitive deglycosylation and the entire sequence (conversion of Compound 8 to Compound 3) was conducted without the use of intermediate protecting groups.

(89) Subsequent introduction of the 4-chlorobiphenylmethyl group into [[C(S)NH]Tpg.sup.4]-vancomycin (Compound 2) by selective reductive amination (1.5 equiv 4-(4-chlorophenyl)benzaldehyde, 5 equiv i-Pr.sub.2NEt, DMF, 70 C., 2 hours; NaCNBH.sub.3, 70 C., 5 hours) provided Compound 5 (57%) without observation of competitive reactions of either the thioamide (reduction) or the N-terminal free amine (reductive amination), using conditions modified from those disclosed for chlorobiphenyl vancomycin itself [Chen et al., Tetrahedron 2002, 58, 6585].

(90) Direct AgOAc-promoted (10 equiv. sat. NH.sub.3-MeOH, 25 C., 7 hours) conversion of the thioamide to the amidine provided Compound 6 (45%, unoptimized), the chlorobiphenylmethyl derivative of [[C(NH)NH]Tpg.sup.4]vancomycin (Compound 3), without the need for intervening protecting groups throughout the 4-step sequence. By design, the final reaction introducing the amidine functionality was conducted effectively on fully functionalized substrates (Compound 2 and Compound 5), lacking protecting groups and incorporating the vancomycin disaccharide.

(91) More particularly, for vancomycin, the carbohydrate introduction has been approached by using either chemical [(a) Ge et al., J. Am. Chem. Soc. 1998, 120, 11014; (b) Thompson et al., J. Am. Chem. Soc. 1999, 121, 1237; (c) Leimkuhler et al., Tetrahedron: Asymmetry 2005, 16, 599; (d) Nicolaou et al., Angew. Chem. Int. Ed. 1999, 38, 240; (e) Nicolaou et al., E. Chem. Eur. J. 1999, 5, 2648; (f) Ritter et al., Angew. Chem. Int. Ed. 2003, 42, 4657] or enzymatic [(a) Losey et al., Biochemistry 2001, 40, 4745; (b) Oberthur et al., J. Am. Chem. Soc. 2005, 127, 10747; (c) Losey et al., Chem. Biol. 2002, 9, 1305; (d) Dong et al., J. Am. Chem. Soc. 2002, 124, 9064; (e) Kruger et al., Chem. Biol. 2005, 12, 131; (f) Thayer et al., Chem. Asian J. 2006, 1, 445; (g) Thayer et al., Angew. Chem. Int. Ed. 2005, 44, 4596; (h) Solenberg et al., Chem. Biol. 1997, 4, 195; (i) Fu et al., Org. Lett. 2005, 7, 1513; and (j) Fu et al., Nat. Biotechnol. 2003, 21, 1467] glycosylations for sequential introduction of the glucose and vancosamine sugars located on the central residue of the aglycon or pseudoaglycon, respectively.

(92) Of these and as noted elsewhere [(a) Ge et al., J. Am. Chem. Soc. 1998, 120, 11014; (b) Thompson et al., J. Am. Chem. Soc. 1999, 121, 1237; (c) Leimkuhler et al., Tetrahedron: Asymmetry 2005, 16, 599; (d) Losey et al., Biochemistry 2001, 40, 4745; (e) Oberthur et al., J. Am. Chem. Soc. 2005, 127, 10747; (f) Losey et al., Chem. Biol. 2002, 9, 1305; (g) Dong et al., J. Am. Chem. Soc. 2002, 124, 9064; (h) Kruger et al., Chem. Biol. 2005, 12, 131; (i) Thayer et al., Chem. Asian J. 2006, 1, 445; and (j) Thayer et al., Angew. Chem. Int. Ed. 2005, 44, 4596] the enzymatic glycosylations avoid protection and the corresponding deprotection of aglycon precursors required of chemical procedures, providing the fully glycosylated products in 2-steps from the fully deprotected aglycons. As a consequence, the sequential glycosylations of the modified aglycon derivatives were examined alongside the vancomycin aglycon and its C-terminus hydroxymethyl derivative using the enzymatic approach [Nakayama et al., Org. Lett. 2014, 16, 3572].

(93) The recombinant glycosyltranferases GtfE and GtfD from the vancomycin producing strain of A. orientalis (ATCC 19795) were expressed in E. coli from the corresponding constructs [Losey et al., Biochemistry 2001, 40, 4745] and were purified to homogeneity (His.sub.6 tag). Notably and although the endogenous glycosyl donors for both enzymes are the TDP-sugars [(a) Losey et al., Biochemistry 2001, 40, 4745; (b) Oberthur et al., J. Am. Chem. Soc. 2005, 127, 10747; (c) Losey et al., Chem. Biol. 2002, 9, 1305; (d) Dong et al., J. Am. Chem. Soc. 2002, 124, 9064; (e) Kruger et al., Chem. Biol. 2005, 12, 131], UDP-sugars have been shown to be as effective co-substrates for both enzymes. Because the requisite NDP-sugar precursor UMP morpholidate is commercially available from four commercial suppliers, including Sigma-Aldrich, whose product was used herein and the corresponding activated TMP is not, UDP-vancosamine was used with GtfD [Nakayama et al., Org. Lett. 2014, 16, 3572].

(94) The UDP-vancosamine, possessing the required -anomer stereochemistry, was prepared by a procedure described in Oberthur et al., Org. Lett. 2004, 6, 2873 to access TDP-vancosamine with modifications to the synthetic route that incorporate uridine versus thymidine [Nakayama et al., Org. Lett. 2014, 16, 3572]. With use of the purified enzymes and the synthetic glycosyl donors UDP-glucose (also from Sigma-Aldrich for GtfE) and UDP-vancosamine [Nakayama et al., Org. Lett. 2014, 16, 3572] (for GtfD), conditions were optimized for the two sequential glycosylations of vancomycin aglycon (Compound 7) as well as its C-terminus hydroxymethyl derivative [Nakayama et al., Org. Lett. 2014, 16, 3572].

(95) Of the two glycosylation reactions, the initial GtfE-catalyzed incorporation of glucose using UDP-glucose exhibited the greatest aglycon substrate sensitivity and those bearing a C-terminus hydroxymethyl group were established to be much less effective than the corresponding carboxylic acids. Previously reported optimization efforts focused on this glycosylation reaction and examined along with both the vancomycin aglycon (Compound 7) and the corresponding hydroxymethyl substrate (37 C.).

(96) In the case of the hydroxymethyl substrate, whose reaction proceeded at a slow rate, preparative amounts of product pseudoaglycon (55%, 48 hours) [Nakayama et al., Org. Lett. 2014, 16, 3572] were obtained by increasing the amount of enzyme used (20 vs 5 M). The residue 4 thioamide (Compound H) and the residue 4 methylene (Compound I) derivatives were capable of glycosylation using GtfE and UDP-glucose to provide the pseudoaglycons Compound 20 (35%; 65% based on recovered starting material, 25 M GtfE) and Compound 22 (HPLC scale, 22% with 5 M GtfE), whereas glycosylation of the residue 4 amidine was not sufficient to provide isolatable amounts of product.

(97) ##STR00030##
Whereas the studies with the C-terminal hydroxymethyl amide and thioamide were conducted on preparative scales, the studies with the C-terminal hydroxymethyl amidine and the more recent methylene derivative were only conducted on an analytical scale as a prelude to studies with the corresponding and more effective C-terminus carboxylic acids.

(98) The second glycosylation reaction catalyzed by GtfD using synthetic UDP-vancosamine proceeded to completion rapidly (<3 hours) independent of the substrate, displaying no impact of either the C-terminus hydroxymethyl substituent or nature of residue 4 (amide, thioamide, or methylene), and the reaction conditions required little optimization. Aside from incorporating glycerol (10% v/v) and reducing the amount of added BSA (0.2 vs 1 mg/mL), the conditions used are essentially those originally disclosed [(a) Losey et al., Biochemistry 2001, 40, 4745; (b) Oberthur et al., Am. Chem. Soc. 2005, 127, 10747; (c) Losey et al., Chem. Biol. 2002, 9, 1305; (d) Dong et al., J. Am. Chem. Soc. 2002, 124, 9064; and (e) Kruger et al., Chem. Biol. 2005, 12, 131] for use of this enzyme and provided both Compound 23 (79%) [Nakayama et al., Org. Lett. 2014, 16, 3572] and Compound 24 (84%) in excellent yields.

(99) Direct conversion of thioamide Compound 24 to the corresponding amidine [10 equiv AgOAc (Corey et al., Tetrahedron Lett. 1978, 5) sat. NH.sub.3-MeOH, 25 C., 6 hours, 50%] (Okano et al., J. Am. Chem. Soc. 2012, 134, 8790) provided Compound 25, the C-terminus hydroxymethyl analogue of vancomycin containing the residue 4 amidine modification. Importantly, this latter reaction was implemented without competitive deglycosylation and the entire 3-step sequence could be conducted without protecting groups. Most significantly, the approach defined an effective route to the key residue 4 amidine analogues despite their failure to directly participate effectively in the initial enzymatic glycosylation reaction.

(100) Subsequent introduction of the chlorobiphenyl group into Compounds 23 and 24 by selective reductive amination was conducted best with preformation of the imine (1.3-1.5 equiv 4-(4-chlorophenyl)benzaldehyde, 5 equiv i-Pr.sub.2NEt, DMF, 30 C., 9-12 hours) followed by subsequent imine reduction [100 equiv NaBH(OAc).sub.3, 30 C., 2 hours] and provided Compound 26 (67-74%) and Compound 27 (74%) using conditions modified [NaBH(OAc).sub.3 vs NaCNBH.sub.3] from those disclosed for (4-chlorobiphenyl)methyl vancomycin itself [(a) Chen et al., Tetrahedron 2002, 58, 6585; (b) Kerns et al., J. Am. Chem. Soc. 2000, 122, 12608-12609].

(101) Of most significance, the reaction of the latter compound occurs without observation of competitive reactions of either the residue 4 thioamide (reduction) or the N-terminal free amine (reductive amination). A final AgOAc-promoted (10 equiv, sat. NH.sub.3-MeOH, 22 C., 6 hours) conversion of the thioamide Compound 27 to the amidine provided Compound 28 (48%, unoptimized), the 4-chlorobiphenyl derivative of Compound 25. By design, the final reaction introducing the amidine as well as the sequential glycosylation reactions and the reductive amination could be conducted effectively on fully functionalized substrates, lacking protecting groups and incorporating the vancomycin disaccharide.

(102) Subsequent introduction of the 4-chlorobiphenyl group into Compounds 23 and 24 by selective reductive amination was conducted best with preformation of the imine (1.3-1.5 equiv 4-(4-chlorophenyl)benzaldehyde, 5 equiv i-Pr.sub.2NEt, DMF, 30 C., 9-12 hours) followed by subsequent imine reduction (100 equiv NaBH(OAc).sub.3, 30 C., 2 hours) and provided Compound 26 (67-74%) and Compound 27 (74%) using conditions modified (NaBH(OAc).sub.3 vs NaCNBH.sub.3) from those disclosed for (4-chlorobiphenyl)methyl vancomycin itself [(a) Chen et al., Tetrahedron 2002, 58, 6585; (b) Kerns et al., J. Am. Chem. Soc. 2000, 122, 12608-12609].

(103) Of most significance, the reaction of the latter compound occurs without observation of competitive reactions of either the residue 4 thioamide (reduction) or the N-terminal free amine (reductive amination). A final AgOAc-promoted (10 equiv, sat. NH.sub.3-MeOH, 22 C., 6 hours) conversion of the thioamide Compound 27 to the amidine provided Compound 28 (48%, unoptimized), the 4-chlorobiphenyl derivative of Compound 25. By design, the final reaction introducing the amidine as well as the sequential glycosylation reactions and the reductive amination could be conducted effectively on fully functionalized substrates, lacking protecting groups and incorporating the vancomycin disaccharide.

(104) Total synthesis of vancomycin, [[C(S)NH]Tpg.sup.4]-vancomycin, [[C(NH)NH]Tpg.sup.4]-vancomycin, and [[CH.sub.2NH]Tpg.sup.4]vancomycin and their (4-chlorobiphenyl)methyl derivatives

(105) The studies piloted with the C-terminus hydroxymethyl derivatives as well as vancomycin aglycon itself defined the approach taken and provided the experience needed to address the fully functionalized residue 4-modified aglycons. The two sequential glycosylations of vancomycin aglycon Compound 7 [Nakayama et al., Org. Lett. 2014, 16, 3572], the freshly prepared synthetic thioamide Compound 8 [Okano et al., J. Am. Chem. Soc. 2014, 136, 13522], amidine Compound 9 [(a) Xie et al., J. Am. Chem. Soc. 2011, 133, 13946; (b) Xie et al., J. Am. Chem. Soc. 2012, 134, 1284], and the more recently re-prepared methylene analogue Compound 10 [Crowley et al., J. Am. Chem. Soc. 2006, 128, 2885] were conducted with the recombinant glycosyl-transferases [(a) Losey et al., Biochemistry 2001, 40, 4745; (b) Oberthur et al., J. Am. Chem. Soc. 2005, 127, 10747; (c) Losey et al., Chem. Biol. 2002, 9, 1305; (d) Dong et al., J. Am. Chem. Soc. 2002, 124, 9064; and (e) Kruger et al., Chem. Biol. 2005, 12, 131] and the synthetic glycosyl donors (UDP-glucose/GtfE and UDP-vancosamine/GtfD) to provide the intermediate pseudoaglycons and subsequently, vancomycin (Compound 1, 87%) and the fully functionalized vancomycin analogues bearing single atom changes in the binding pocket, [[C(S)NH]Tpg.sup.4]-vancomycin (Compound 2, 87%, HPLC conversion >95%) and [[CH.sub.2NH]Tpg.sup.4]vancomycin (Compound 16, 76%, HPLC conversion >95%).

(106) A comparison of the relative efficiency of the initial glycosylation reaction with Compounds 3 and 5 conducted on an analytical scale alongside vancomycin aglycon (Compound 2). Unlike the significant impact of the C-terminal hydroxymethyl group, but like the well tolerated N-terminus substitutions [(a) Nakayama et al., Org. Lett. 2014, 16, 3572; and (b) Dong et al., J. Am. Chem. Soc. 2002, 124, 9064], modifications to the vancomycin binding pocket itself had a minimal impact on both the rate and overall efficiency of the initial GtfE-catalyzed reaction. However, and like the observations made with the C-terminal hydroxymethyl amidine, the amidine aglycon Compound 9 failed to undergo successful GtfE-catalyzed glycosylation. Although small amounts of product could be detected by HPLC, the aglycon itself underwent competitive conversion to several byproducts under the basic conditions (pH 9) required of the reaction.

(107) The second glycosylation reaction catalyzed by GtfD using the co-substrate UDP-vancosamine proceeded rapidly (<3 hours) regardless of the aglycon substrate, displaying no significant impact of the nature of residue 4 (amide, thioamide, or methylene) and the conditions required no further optimization. For [[C(NH)NH]Tpg.sup.4]vancomycin (Compound 3), direct conversion of thioamide Compound 2 to the corresponding amidine (10 equiv AgOAc, sat. NH.sub.3-MeOH, 25 C., 7 hours) provided Compound 3.

(108) Subsequent introduction of the chlorobiphenyl group with [[C(S)NH]Tpg.sup.4]vancomycin (Compound 2) and [[CH.sub.2NH]Tpg.sup.4]vancomycin (Compound 16) by reductive amination (1.5 equiv 4-(4-chloro-phenyl)benzaldehyde, 5 equiv i-Pr.sub.2NEt, DMF, 50-70 C., 2 hours; 100 equiv NaCNBH.sub.3, 70 C., 5 hours) provided Compound 5 (57%) and Compound 17 (41%) on the unprotected vancomycin analogues without optimization using conditions piloted with 4-chlorobiphenyl vancomycin (Compound 4, 61-74%) itself [a) Chen et al., Tetrahedron 2002, 58, 6585; and (b) Kerns et al., J. Am. Chem. Soc. 2000, 122, 12608-12609]. Direct AgOAc-promoted (10 equiv, sat. NH.sub.3-MeOH, 25 C., 7 hours) conversion of the thioamide Compound 5 to the amidine provided Compound 6 (45%), the 4-chlorobiphenyl derivative of [[C(NH)NH]Tpg.sup.4]-vancomycin (Compound 3).

(109) Significantly, the reductive amination was conducted without competing reaction of either the thioamide or the N-terminus and residue 4 secondary amines, the entire 3-4 step sequence could be conducted without protecting groups, and the amidine introduction was implemented without competitive deglycosylation.

(110) It is further worth noting that the enzymatic glycosylations were conducted on about 1-3 mg of substrate with 1 mol % enzyme and 4 equiv. of UDP-glucose or UDP-vancosamine, reflecting a piloted laboratory scale. However, the expression and purification of the enzymes and the chemical synthesis of UDP-vancosamine, along with the commercial availability of UDP-glucose, were conducted on scales that would easily support laboratory preparations on much larger scales (about 100-fold) than exemplified herein and are easily scaled beyond even this level.

(111) Specific Syntheses

(112) ##STR00031##

(113) In a total volume of 2.6 mL, 2.0 mM UDP-vancosamine (2.8 mg, 5.1 mol) and 0.5 mM Compound 13 (1.7 mg, 1.3 mol) were incubated with 75 mM Tricine-NaOH (pH 9.0), 2 mM tris-(2-carboxyethyl)phosphine, 0.2 mg/mL bovine serum albumin, 1 mM MgCl.sub.2, glycerol (10% v/v), and 10 M GtfD for 3 hours at 37 C. The reaction mixture was quenched by the addition of MeOH (23 mL) at 0 C. and was passed through a 0.45 m polyethersulfone membrane filter and concentrated by evaporation to a final volume of about 3 mL. After the addition of H.sub.2O (1.0 mL), the mixture was purified by semi-preparative reverse-phase HPLC. For the HPLC, Vydac 218TP1022-C18, 10 m, 22250 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 10 mL/minute, t.sub.R=21.3 minute) was used to afford Compound 2 (0.97 mg, 52% yield) as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 8.80 (d, J=6.0 Hz, 1H), 8.49 (s, 1H), 7.73-7.72 (m, 1H), 7.68-7.63 (m, 5H), 7.32-7.27 (m, 1H), 7.22 (d, J=1.6 Hz, 1H), 6.78 (d, J=8.4 Hz, 1H), 6.46 (d, J=1.6 Hz, 1H), 6.40 (d, J=1.6 Hz, 1H), 6.17 (s, 1H), 5.85 (s, 1H), 5.49-5.41 (m, 3H), 5.34-5.32 (m, 5H), 4.45-4.38 (br m, 1H), 4.29 (s, 1H), 4.25-4.21 (m, 1H), 4.08-4.06 (m, 2H), 3.99 (s, 1H), 3.92-3.88 (m, 1H), 3.86-3.83 (m, 2H), 3.80-3.78 (m, 1H), 3.70-3.61 (m, 2H), 3.59-3.51 (m, 2H), 3.44-3.42 (m, 1H), 3.22-3.20 (m, 1H), 3.01-2.98 (m, 1H), 2.77 (s, 3H), 2.36-2.28 (m, 2H), 2.08-2.04 (m, 2H), 1.95-1.93 (m, 1H), 1.90-1.85 (m, 1H), 1.71-1.64 (m, 1H), 1.51 (s, 3H), 1.45-1.35 (m, 3H), 1.20 (d, J=6.6 Hz, 3H), 1.19-1.12 (m, 1H), 1.00 (d, J=6.0 Hz, 3H), 0.98 (d, J=6.6 Hz, 3H); ESI-TOF HRMS m/z 1464.4131 (M+H.sup.+, C.sub.66H.sub.75Cl.sub.2N.sub.9O.sub.23S requires 1464.4152).

(114) HPLC was used to separate the crude reaction mixtures in the conversion of Compound 8 to Compound 13, and Compound 13 to Compound 2. For Compound 8 to Compound 13, Vydac 218TP1022-C18, 10 m, 22250 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 10 mL/minute, t.sub.R=23.2 minutes was used, indicating that the isolated yield (75%) underestimates the extent of the conversion (86-92% by HPLC). For Compound 13 to Compound 2, Vydac 218TP1022-C18, 10 m, 22250 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 10 mL/minute, t.sub.R=21.3 minutes was used, indicating that the isolated yield (52%) underestimates the extent of the conversion (95-100% by HPLC).

(115) Additional Synthesis

(116) In a total volume of 1.3 mL, 3.0 mM UDP-vancosamine (2.2 mg, 3.8 mol) and 0.5 mM Compound 13 (0.84 mg, 0.64 mol) were incubated with 75 mM Tricine-NaOH (pH 9.0), 2 mM tris-(2-carboxyethyl)-phosphine, 0.2 mg/mL bovine serum albumin, 1 mM MgCl.sub.2, glycerol (10% v/v), and 5 M GtfD for 1 hour at 37 C. It is noted that this reaction sequence was conducted only twice on a preparative scale and consequently is not yet optimized. The reaction mixture was quenched by the addition of MeOH (8 mL) at 0 C. and was passed through a 0.45 m polyethersulfone membrane filter and concentrated by evaporation to a final volume of about 2 mL. After the addition of H.sub.2O (1.0 mL), the mixture was purified by semi-preparative reverse-phase HPLC as discussed above to afford Compound 2 (0.81 mg, 87% yield) as a white amorphous solid.

(117) Second Additional Synthesis

(118) In a total volume of 1.4 mL, 3.0 mM UDP-vancosamine (2.5 mg, 4.6 mol) and 0.5 mM Compound 13 (1.1 mg, 0.84 mol) were incubated with 75 mM Tricine-NaOH (pH 9.0), 2 mM tris-(2-carboxyethyl)-phosphine, 0.2 mg/mL bovine serum albumin, 1 mM MgCl.sub.2, glycerol (10% v/v) and 10 M GtfD for 3 hours at 37 C. The reaction mixture was quenched by the addition of MeOH (10 mL) at 0 C., was passed through a 0.45 m polyethersulfone membrane filter and concentrated by evaporation to a final volume of about 2 mL. After the addition of H.sub.2O (2.0 mL), the mixture was purified by semi-preparative reverse-phase HPLC (Vydac 218TP1022-C18, 10 m, 22250 mm, 1-40% MeCN/H.sub.2O about 0.07% TFA gradient over 40 minutes, 10 mL/minute, t.sub.R=20.7 minutes) to afford Compound 2 (1.0 mg, 84%) as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 8.29 (s, 1H), 7.73-7.62 (m, 3H), 7.57 (d, 1H, J=9.0 Hz), 7.30 (d, 1H, J=9.0 Hz), 7.26 (d, 1H, J=8.4 Hz), 7.18 (s, 1H), 6.96 (d, 1H, J=9.0 Hz), 6.79 (d, 1H, J=8.4 Hz), 6.65 (s, 1H), 6.46-6.42 (m, 3H), 6.07 (s, .sup.1H), 5.77 (s, 1H), 5.44 (d, 1H, J=7.8 Hz), 5.40 (d, 1H, J=4.2 Hz), 5.31 (s, 1H), 5.28-5.25 (m, 3H), 4.40-4.39 (br m, 1H), 4.31-4.28 (m, 1H), 4.22-4.20 (br m, 2H), 4.07-4.01 (m, 2H), 3.97-3.94 (m, 1H), 3.86 (d, 1H, J=12.0 Hz), 3.82-3.79 (m, 1H), 3.76-3.73 (m, 1H), 3.68-3.55 (m, 3H), 3.52-3.50 (m, 1H), 2.97-2.94 (m, 1H), 2.76 (s, 3H), 2.07-2.04 (m, 1H), 1.92 (d, 1H, J=13.8 Hz), 1.88-1.83 (m, 1H), 1.48 (s, 3H), 1.40-1.29 (m, 2H), 1.19 (d, 3H, J=6.6 Hz), 1.02 (d, 3H, J=6.6 Hz), 1.00 (d, 3H, J=6.6 Hz); ESI-TOF HRMS m/z 1450.4375 (M+H.sup.+, C.sub.66H.sub.78Cl.sub.2N.sub.9O.sub.22S requires 1450.4353).

(119) See also, Okano et al., J. Am. Chem. Soc. 2014, 136, 13522.

(120) ##STR00032##

(121) A mixture of Compound 2 (0.27 mg, 0.18 mol) and AgOAc (0.3 mg, 1.8 mol) was treated with saturated NH.sub.3CH.sub.3OH (0.2 mL) at 0 C. The reaction mixture was stirred for 7 hours at 25 C. The reaction mixture was quenched by the addition of 50% CH.sub.3OH in H.sub.2O (0.2 mL), and the residue was purified by semi-preparative reverse-phase HPLC. For the HPLC, Zorbax SB-C18, 5 m, 9.4150 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t.sub.R=16.0 minutes was used to afford Compound 3 as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 7.8-7.68 (m, 3H), 7.43 (s, 1H), 7.20-7.11 (m, 2H), 7.04 (s, 1H), 6.89 (s, 1H), 6.49-6.45 (m, 2H), 5.59-5.51 (m, 3H), 5.42-5.38 (m, 2H), 4.31-4.16 (m, 3H), 3.82-3.76 (m, 2H), 3.67-3.47 (m, 3H), 3.19 (s, 1H), 2.95-2.82 (m, 5H), 2.45-2.34 (m, 1H), 2.11-1.99 (m, 3H), 1.90-1.75 (br m, 2H), 1.65 (s, 3H), 1.24-1.19 (m, 5H), 0.88 (d, J=6.0 Hz, 3H), 0.83 (d, J=6.0 Hz, 3H); ESI-TOF HRMS m/z 724.2307 (M+2H.sup.+, C.sub.66H.sub.76Cl.sub.2N.sub.10O.sub.23 requires 724.2304).

(122) Additional Synthesis

(123) A mixture of Compound 2 (0.38 mg, 0.26 mol) and AgOAc (0.43 mg, 2.6 mol) was treated with anhydrous saturated NH.sub.3CH.sub.3OH (0.2 mL) at 25 C. The reaction mixture was stirred for 6 hours at 25 C. before the solvent was removed under a stream of N.sub.2. The residue was dissolved in 50% CH.sub.3OH in H.sub.2O (0.2 mL) and purified by semi-preparative reverse-phase HPLC (Zorbax SB-C18, 5 m, 9.4150 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t.sub.R=16.4 minutes) to afford Compound 3 (86 g, 50% yield brsm, unoptimized) as a white film: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 7.74 (d, J=9.0 Hz, 1H), 7.74 (s, 1H), 6.90 (d, J=8.4 Hz, 1H), 6.68 (s, 1H), 6.44 (s, 1H), 5.52 (d, J=11.2 Hz, 1H), 5.45-5.37 (m, 5H), 4.33 (s, 1H), 4.13-4.06 (m, 2H), 3.85 (s, 1H), 3.77 (d, J=9.0 Hz, 1H), 3.67-3.52 (m, 2H), 2.88 (s, 3H), 2.45-2.41 (m, 1H), 2.07 (d, J=10.8 Hz, 1H), 1.86 (s, 1H), 1.61 (s, 1H), 1.51-1.39 (m, 3H), 1.30 (s, 1H), 1.28-1.20 (m, 3H), 0.89 (d, J=6.0 Hz, 3H), 0.85 (d, J=6.0 Hz, 3H); ESI-TOF HRMS m/z 1433.4760 (M+H.sup.+, C.sub.66H.sub.78Cl.sub.2N.sub.10O.sub.22 requires 1433.4742).

(124) See also, Okano et al., J. Am. Chem. Soc. 2014, 136, 13522.

(125) ##STR00033##

(126) A solution of Compound 1 (vancomycin, 0.45 mg, 0.31 mol) in anhydrous DMF (30 L) was treated with 4-(4-chlorophenyl)benzaldehyde (0.1 M in DMF, 4.7 L, 0.47 mol) and i-Pr.sub.2NEt (distilled, 0.1 M in DMF, 15.6 L, 1.56 mol) at 25 C. The reaction mixture was stirred for 2 hours at 70 C. After the reaction was complete, the mixture was treated with NaCNBH.sub.3 (1 M in THF, 31.2 L, 31.2 mol) and stirred for 5 hours at 70 C. The reaction mixture was quenched by the addition of 50% CH.sub.3OH in H.sub.2O (0.2 mL) at 25 C. and the residue was purified by semi-preparative reverse-phase HPLC. For the HPLC, Zorbax SB-C18, 5 m, 9.4150 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t.sub.R=34.3 minutes) was used to afford Compound 4 (0.31 mg, 61% yield) as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 8.98 (s, 1H), 8.71 (s, 1H), 7.76-7.70 (m, 5H), 7.62 (d, J=8.4 Hz, 2H), 7.56 (d, J=8.4 Hz, 2H), 7.45 (d, J=8.4 Hz, 2H), 7.20 (d, J=9.0 Hz, 1H), 7.08 (s, 1H), 6.71 (br s, 1H), 6.52 (d, J=2.4 Hz, 1H), 6.41 (d, J=2.4 Hz, 1H), 5.63 (s, 1H), 5.52 (s, 1H), 5.40-5.37 (m, 2H), 5.28 (d, J 2.4 Hz, 1H), 4.77 (s, 1H), 4.73 (d, J=6.0 Hz, 1H), 4.27 (s, 1H), 4.19-4.15 (m, 3H), 4.08-3.95 (m, 2H), 3.90-3.80 (m, 2H), 3.68-3.62 (m, 3H), 3.43 (s, 1H), 2.92 (d, J=12.6 Hz, 1H), 2.78 (s, 1H), 2.19 (d, J=12.0 Hz, 1H), 2.05 (d, J=13.2 Hz, 1H), 1.90-1.87 (m, 1H), 1.88 (s, 3H), 1.68-1.65 (m, 1H), 1.25 (d, J=6.6 Hz, 3H), 0.95-0.92 (m, 6H); ESI-TOF HRMS m/z 824.7421 (M+2H.sup.+, C.sub.79H.sub.84Cl.sub.3N.sub.9O.sub.24 requires 824.7420).

(127) This reaction was run on scales of 0.2-1.2 mg (55-61% yield) as part of the optimization of conditions for use with Compound 5 on the amounts available.

(128) Larger Scale Procedure:

(129) A solution of Compound 1 (vancomycin, 90.0 mg, 62.1 mol) in anhydrous DMF (8.0 mL) was treated with 4-(4-chlorophenyl)benzaldehyde (19.8 mg, 74.5 mol) and i-Pr.sub.2NEt (51.0 L, 0.32 mmol) at 25 C. The reaction mixture was stirred for 2 hours at 70 C. After the reaction was complete, the mixture was treated with NaCNBH.sub.3 (1 M in THF, 0.32 mL, 0.32 mmol) and stirred for 5 hours at 70 C. The reaction mixture was quenched by the addition of 50% CH.sub.3OH in H.sub.2O (1.0 mL) at 25 C., and the residue was purified by semi-preparative reverse-phase HPLC. For HPLC, Zorbax SB-C18, 5 m, 9.4150 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t.sub.R=34.3 minutes was used to afford Compound 4 (75.3 mg, 74% yield) as a white amorphous solid.

(130) Additional Synthesis

(131) A solution of Compound 1 (1.0 mg, 0.65 mol) in anhydrous DMF (0.1 mL) was treated with 4-(4-chlorophenyl)benzaldehyde (0.1 M in DMF, 9.7 L, 0.97 mol) and i-Pr.sub.2NEt (distilled, 0.1 M in DMF, 32.3 L, 3.23 mol) at 25 C. The reaction mixture was stirred for 12 hours at 30 C. After the reaction was complete, the mixture was treated with NaBH(OAc).sub.3 (13.7 mg, 64.6 mol) and stirred for 2 hours at 30 C.

(132) The reaction mixture was quenched by the addition of 50% CH.sub.3OH in H.sub.2O (0.2 mL) at 25 C. and the residue was purified by semi-preparative reverse-phase HPLC (Zorbax SB-C18, 5 m, 9.4150 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t.sub.R=35.2 minutes) to afford Compound 4 (0.73 mg, 67% yield) as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298K) 7.71-7.68 (m, 3H), 7.66-7.60 (m, 5H), 7.57-7.54 (m, 2H), 7.47-7.43 (m, 3H), 7.32 (d, 1H, J=8.4 Hz), 7.29 (d, 1H, J=7.8 Hz), 7.11 (d, 1H, J=8.4 Hz), 6.94 (d, 1H, J=8.4 Hz), 6.45-6.43 (m, 1H), 6.41-6.37 (m, 1H), 6.35 (d, 1H, J=2.4 Hz), 6.33 (d, 1H, J=2.4 Hz), 6.41-6.37 (m, 1H), 6.34-6.33 (m, 1H), 5.74 (d, 1H, J=11.2 Hz), 5.60 (d, 1H, J=18.0 Hz), 5.56 (d, 1H, J=7.8 Hz), 5.53 (d, 1H, J=12.0 Hz), 5.49 (d, 1H, J=4.2 Hz), 5.44-5.42 (m, 1H), 4.55-4.53 (m, 1H), 4.22-4.15 (m, 1H), 4.13-4.05 (m, 1H), 3.95-3.93 (m, 1H), 3.86-3.84 (m, 2H), 3.75-3.72 (m, 1H), 3.65 (br s, 1H), 3.62 (d, 1H, J=9.6 Hz), 3.53-3.50 (m, 1H), 2.78 (s, 3H), 2.52-2.48 (m, 1H), 2.21-2.16 (m, 1H), 2.04 (d, 1H, J=13.2 Hz), 1.67-1.64 (br m, 5H), 1.32 (d, 3H, J=6.6 Hz), 1.03-1.00 (m, 3H), 0.98-0.95 (m, 3H); ESI-TOF HRMS m/z 1634.5057 (M+H.sup.+, C.sub.79H.sub.87Cl.sub.3N.sub.9O.sub.23 requires 1634.4975).

(133) See also, Okano et al., J. Am. Chem. Soc. 2014, 136, 13522.

(134) ##STR00034##

(135) Following the procedure detailed for Compound 4 and using Compound 2 as the starting material, (0.42 mg, 0.29 mol), semi-preparative reverse-phase HPLC was used to purify the compound. For the HPLC, Zorbax SB-C18, 5 m, 9.4150 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t.sub.R=34.9 minutes afforded Compound 5 (0.27 mg, 57% yield) as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 7.72-7.66 (m, 6H), 7.62 (d, J=8.4 Hz, 2H), 7.55 (d, J=8.4 Hz, 2H), 7.34 (s, 1H), 7.27 (d, J=8.4 Hz, 2H), 7.17 (s, 1H), 6.75 (d, J=9.0 Hz, 1H), 6.52 (d, J=2.4 Hz, 1H), 6.39 (d, J=2.4 Hz, 1H), 5.52-5.46 (m, 2H), 5.37-5.31 (m, 3H), 4.59 (s, 1H), 4.24 (s, 1H), 4.16 (d, J=12.6 Hz, 1H), 4.07 (d, J=12.6 Hz, 1H), 3.92 (d, J=6.0 Hz, 1H), 3.85-3.75 (m, 2H), 3.63 (dd, J=9.0, 9.0 Hz, 1H), 3.60-3.56 (m, 2H), 3.44-3.39 (m, 2H), 3.19 (s, 1H), 2.72 (s, 3H), 2.40-2.25 (m, 1H), 2.20-2.15 (m, 1H), 2.10-1.97 (m, 2H), 1.83-1.73 (m, 2H), 1.69-1.59 (m, 5H), 1.25 (d, J=6.6 Hz, 3H), 1.00-0.94 (m, 6H); ESI-TOF HRMS m/z 832.7286 (M+2H.sup.+, C.sub.79H.sub.84Cl.sub.3N.sub.9O.sub.23S requires 832.7306).

(136) Additional Synthesis

(137) A solution of Compound 2 (0.62 mg, 0.42 mol) in anhydrous DMF (30 L) was treated with 4-(4-chlorophenyl)benzaldehyde (0.1 mM in DMF, 5.5 L, 0.546 mol) and i-Pr.sub.2NEt (distilled, 0.1 mM in DMF, 21 L, 2.1 mol) at 25 C. The reaction mixture was stirred for 9 hours at 30 C. After the reaction was complete, the mixture was treated with NaBH(OAc).sub.3 (11.2 mg, 42.0 mol) and stirred for 2 hours at 30 C. The reaction mixture was quenched with the addition of 50% CH.sub.3OH in H.sub.2O (0.2 mL) and the residue was purified by semi-preparative reverse-phase HPLC (1-40% MeCN/H.sub.2O-0.07% TFA isocratic gradient over 40 minutes) to afford Compound 5 (0.52 mg, 74%) as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz) 8.30 (d, 1H, J=6.6 Hz), 7.72-7.67 (m, 5H), 7.63 (d, 2H, J=8.4 Hz), 7.60 (d, 1H, J=6.0 Hz), 7.56 (d, 2H, J=7.8 Hz), 7.47 (d, 2H, J=8.4 Hz), 7.33 (d, 1H, J 8.4 Hz), 7.29 (d, 1H, J=8.4 Hz), 7.19 (d, 1H, J=2.4 Hz), 6.99-6.97 (m, 1H), 6.80 (d, 1H, J=8.4 Hz), 6.56 (d, 1H, J=2.4 Hz), 6.42 (d, 1H, J=1.8 Hz), 6.13-6.08 (br m, 1H), 5.80 (s, 1H), 5.51 (d, 1H, J 7.2 Hz), 5.46 (d, 1H, J=4.8 Hz), 5.33 (d, 1H, J=3.0 Hz), 5.31-5.29 (br m, 2H), 5.27 (s, 1H), 4.40-4.39 (m, 1H), 4.33-4.30 (m, 1H), 4.24 (s, 1H), 4.16 (d, 1H, J=12.0 Hz), 4.08-4.02 (m, 3H), 3.98-3.95 (m, 1H), 3.90-3.82 (m, 2H), 3.77-3.75 (m, 1H), 3.64-3.60 (m, 2H), 3.54-3.50 (m, 2H), 3.44-3.42 (m, 1H), 3.20-3.19 (m, 1H), 2.97 (d, 1H, J=13.8 Hz), 2.77 (s, 3H), 2.36-2.32 (m, 1H), 2.20-2.16 (m, 1H), 2.02 (d, 1H, J=13.8 Hz), 1.89-1.84 (m, 1H), 1.81-1.76 (m, 1H), 1.71-1.68 (m, 1H), 1.66 (s, 3H), 1.27 (d, 3H, J=6.6 Hz), 1.03 (d, 3H, J=6.0 Hz), 1.00 (d, 3H, J=6.6 Hz); ESI-TOF HRMS m/z 825.7447 (M+2H.sup.+, C.sub.79H.sub.86Cl.sub.3N.sub.9O.sub.23 requires 825.7410).

(138) See also, Okano et al., J. Am. Chem. Soc. 2014, 136, 13522.

(139) ##STR00035##

(140) Following the procedure detailed for Compound 3 and using Compound 5 as the starting material, (0.31 mg, 0.19 mol), semi-preparative reverse-phase HPLC was used for purification. For the HPLC, Zorbax SB-C18, 5 m, 9.4150 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t.sub.R=33.6 minutes afforded Compound 6 as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 7.82-7.72 (m, 3H), 7.64 (d, J=8.4 Hz, 2H), 7.56 (d, J=8.4 Hz, 2H), 7.49-7.33 (m, 4H), 7.07 (s, 1H), 6.91 (d, J=9.0 Hz, 1H), 6.51-6.46 (m, 2H), 5.61-5.37 (m, 5H), 4.33 (br s, 1H), 4.21-4.13 (m, 2H), 4.09-4.03 (m, 2H), 3.88-3.75 (m, 2H), 3.73-3.58 (m, 5H), 3.51-3.49 (m, 1H), 3.37 (s, 1H), 3.21 (s, 1H), 2.88 (s, 3H), 2.81-2.76 (m, 2H), 2.49-2.45 (m, 1H), 2.42-2.28 (m, 2H), 2.21-2.06 (m, 3H), 1.85-1.77 (m, 2H), 1.65 (s, 3H), 1.40-1.30 (m, 4H), 0.92 (d, J=6.6 Hz, 3H), 0.88 (d, J=6.6 Hz, 3H); ESI-TOF HRMS m/z 824.2539 (M+2H.sup.+, C.sub.79H.sub.88Cl.sub.3N.sub.10O.sub.23 requires 824.2578)

(141) Additional Synthesis

(142) A mixture of Compound 5 (0.35 mg, 0.20 mol) and AgOAc (0.33 mg, 2.0 mol) was treated with anhydrous saturated NH.sub.3CH.sub.3OH (0.2 mL) at 25 C. The reaction mixture was stirred for 6 hours at 25 C. before the solvent was removed under a stream of N.sub.2. The residue was dissolved in 50% CH.sub.3OH in H.sub.2O (0.2 mL) and purified by semi-preparative reverse-phase HPLC (Zorbax SB-C18, 5 m, 9.4150 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t.sub.R=33.2 minutes) to afford Compound 6 (86 g, 48% yield brsm, unoptimized) as a white film: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 7.79-7.68 (m, 3H), 7.61 (d, J=8.4 Hz, 2H), 7.54 (d, J=7.8 Hz, 2H), 7.46-7.44 (m, 4H), 7.08-7.02 (br m, 2H), 6.88 (d, J=9.0 Hz, 1H), 6.66 (s, 1H), 6.43 (d, J=2.4 Hz, 1H), 5.58-5.48 (m, 2H), 5.44-5.40 (m, 3H), 4.37-4.28 (m, 1H), 4.18-4.15 (m, 2H), 4.11 (d, J=4.2 Hz, 1H), 4.09-4.02 (m, 4H), 3.88-3.75 (m, 2H), 3.67-3.54 (m, 4H), 2.87 (s, 3H), 2.74 (br s, 1H), 2.41 (dd, J=14.4, 4.8 Hz, 1H), 2.19-2.16 (m, 1H), 2.06 (s, 1H), 2.03 (s, 1H), 1.85 (br s, 1H), 1.65-1.55 (m, 4H), 1.30-1.29 (m, 4H), 1.22-1.19 (m, 1H), 0.88 (d, J=6.0 Hz, 3H), 0.84 (d, J=6.6 Hz, 3H); ESI-TOF HRMS m/z 817.2614 (M+2H.sup.+, C.sub.79H.sub.88Cl.sub.3N.sub.10O.sub.22 requires 817.2604).

(143) See also, Okano et al., J. Am. Chem. Soc. 2014, 136, 13522.

(144) Conversion of Compound A to Compound B

(145) ##STR00036##

(146) The reaction was performed on scales ranging from 3.2 to about 8.0 mg (50 to about 59%, 3 steps). A representative procedure follows: A solution of Compound A [Crowley et al., J. Am. Chem. Soc. 2006, 128, 2885] (5.9 mg, 4.3 mol) in acetone (0.15 mL, degassed) and saturated aqueous NH.sub.4Cl (20 L, degassed) was treated with zinc nanoparticle (Aldrich, 11.0 mg, 0.17 mmol) at 25 C. The reaction mixture was stirred at 25 C. for 2 hours before the solvent was removed under a stream of N.sub.2.

(147) The residue was dissolved in EtOAc and purified through a short plug of silica gel (100% EtOAc then 10% CH.sub.3OHCH.sub.2Cl.sub.2) to afford the corresponding aniline as a white amorphous crude solid. This solid was dissolved in MeCN (degassed, 0.3 mL) and treated with HBF.sub.4 (0.1 M in MeCN, 43 L, 4.3 mol) at 0 C. The reaction mixture was stirred for 3 minutes before the drop-wise addition of t-butylnitrite (0.1 M in MeCN, 43 L, 4.3 mol) at 0 C. The reaction mixture was stirred at 0 C. for 3 minutes before an aqueous mixture (degassed, 0.4 mL) containing CuCl (9.0 mg, 86 mol) and CuCl.sub.2 (15.3 mg, 108 mol) was transferred to the above solution in one portion at 0 C. The heterogeneous mixture was permitted to warm to 25 C. and stirred for 45 minutes.

(148) The reaction mixture was purified by PTLC (SiO.sub.2, 10% CH.sub.3OHCH.sub.2Cl.sub.2) afforded the corresponding aryl chloride as a white amorphous solid. This solid was dissolved in anhydrous MeCN (degassed, 0.3 mL) and treated with N-methyl-N-tert-butyldimethylsilyl-trifluoroacetamide (MTBSTFA; Sigma-Aldrich, 43 L, 1.8 mmol). The reaction mixture was warmed to 55 C. and stirred for 24 hours. This protocol was repeated for a second 5.9 mg of Compound A and the batches were later combined for work-up.

(149) The reaction mixture was cooled to 25 C. and the solvent was removed under a stream of N.sub.2. The residue was diluted with EtOAc (0.5 mL), 0.1 N HCl (0.5 mL) was added, and the mixture was stirred for 30 minutes. The layers were separated, and the aqueous layer was extracted with EtOAc (30.5 mL). The combined organic layers were washed with saturated aqueous NaCl (0.5 mL), dried (Na.sub.2SO.sub.4) and the solvent was removed under reduced pressure. PTLC (SiO.sub.2, 4% CH.sub.3OHCH.sub.2Cl.sub.2) afforded Compound B (6.7 mg, 51%, 3 steps) as a white amorphous solid identical in all respects with authentic material (.sup.1H NMR, CD.sub.2OD) [Crowley et al., J. Am. Chem. Soc. 2006, 128, 2885].

(150) Improved Protocol for the Sandmeyer Chemistry Used in the Conversion of Compound C to Compound D

(151) ##STR00037##

(152) The reaction was performed on scales ranging from 3.2-4.0 mg (55-60%, 3 steps). A representative procedure follows: A solution of Compound C [(a) Xie et al., J. Am. Chem. Soc. 2011, 133, 13946; and (b) Xie et al., J. Am. Chem. Soc. 2012, 134, 1284] (3.2 mg, 2.3 mol) in acetone (0.15 mL, degassed) and saturated aqueous NH.sub.4Cl (20 L, degassed) was treated with zinc nanoparticle (Sigma-Aldrich, 8.9 mg, 0.14 mmol) at 25 C. The reaction mixture was stirred at 25 C. for 0.5 hours before the solvent was removed under a stream of N.sub.2. The residue was dissolved in EtOAc and purified through a short plug of silica gel (100% EtOAc then 12% CH.sub.3OHCH.sub.2Cl.sub.2) to afford the corresponding aniline as a white amorphous crude solid. This solid was dissolved in MeCN (degassed, 250 L) and treated with HBF.sub.4 (0.1 M in MeCN, 26 L, 2.6 mol) at 15 C. The reaction mixture was stirred for 3 minutes before the drop-wise addition of t-butylnitrite (0.1 M in MeCN, 26 L, 2.6 mol) at 15 C.

(153) The reaction mixture was stirred at 15 C. for 20 minutes before an aqueous mixture (degassed, 0.3 mL) containing CuCl (11.2 mg, 114 mol) and CuCl.sub.2 (21.0 mg, 157 mol) was transferred to the above solution in one portion at 30 C. The heterogeneous mixture was permitted to warm to 25 C. and stirred for 0.5 hours. The reaction mixture was directly purified by PTLC (SiO.sub.2, 10% CH.sub.3OHCH.sub.2Cl.sub.2) and afforded the nitro group converted to a chloro group-containing Compound C-1 as a white amorphous solid.

(154) Compound C-1 was dissolved in anhydrous MeCN (degassed, 0.3 mL) and treated with MTBSTFA (53 L, 0.22 mmol). The reaction mixture was warmed to 55 C. and stirred for 24 hours. The reaction mixture was cooled to 25 C. and the solvent was removed under a stream of N.sub.2. The residue was diluted with EtOAc (0.5 mL), 0.1 N HCl (0.5 mL) was added, and the mixture was stirred for 30 minutes.

(155) The layers were separated, and the aqueous layer was extracted with EtOAc (30.5 mL). The combined organic layers were washed with saturated aqueous NaCl (0.5 mL), dried (Na.sub.2SO.sub.4) and the solvent was removed under reduced pressure. PTLC (SiO.sub.2, 4% CH.sub.3OHCH.sub.2Cl.sub.2) afforded Compound D (2.2 mg, 60%, 3 steps) as a white amorphous solid identical in all respects with authentic material (.sup.1H NMR, acetone-d.sub.6) [Okano et al., J. Am. Chem. Soc. 2012, 134, 8790].

(156) Improved Protocol for Jones Oxidation and Global Deprotection Converting Compound E to Compound 8

(157) ##STR00038##

(158) This reaction was performed on scales ranging from 0.9-1.8 mg (44-52%, 3 steps). A representative procedure follows: A solution of CrO.sub.3 (Sigma-Aldrich 99.99%, 17.9 mg) in H.sub.2O (degassed, 340 L) was treated with conc. H.sub.2SO.sub.4 (Sigma-Aldrich 99.999%, 30 L) at 25 C. An aliquot of this stock solution (4.1 L, 2.2 mol) was added into a solution of Compound E [Crowley et al., J. Am. Chem. Soc. 2006, 128, 2885] (1.1 mg, 0.73 mol) in acetone (Sigma-Aldrich HPLC grade, degassed, 78 L) at 25 C. The reaction mixture was stirred at 25 C. for 24 hours, cooled to 0 C., and quenched by the addition of saturated NH.sub.3CH.sub.3OH (0.2 mL) and diluted with anhydrous CH.sub.2Cl.sub.2 (0.2 mL).

(159) The residue was purified through a short plug of silica gel (15% CH.sub.3OHCH.sub.2Cl.sub.2) to afford the corresponding carboxylic acid as a white amorphous crude solid. This solid was treated with TFA (neat, 0.2 mL) at 25 C. and stirred at 25 C. for 12 hours.

(160) TFA was removed under a stream of N.sub.2 and the residue was dissolved in MeOH (HPLC grade, 0.3 mL) at 25 C. The reaction mixture was stirred at 25 C. for 3 hours before the MeOH was removed under a stream of N.sub.2. The residue was treated with AlBr.sub.3 (Aldrich, 192 mg, 0.73 mmol) and EtSH (10 L) at 25 C. and stirred at 25 C. for 72 hours.

(161) The reaction mixture was quenched by the addition of 50% MeOH in H.sub.2O (1 mL) at 0 C. and purified by short reverse phase silica gel chromatography (C18-SiO.sub.2, 50% CH.sub.3CNH.sub.2O) and subsequent semi-preparative reverse-phase HPLC (Zorbax SB-C18, 5 m, 9.4150 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t.sub.R=27.3 minutes) to afford Compound 8 (0.41 mg, 48% yield, 3 steps) as a white amorphous solid identical in all respects with authentic material (.sup.1H NMR, CD.sub.3OD) [Crowley et al., J. Am. Chem. Soc. 2006, 128, 2885].

(162) ##STR00039##

(163) A solution of Compound F [(a) Xie et al., J. Am. Chem. Soc. 2011, 133, 13946; and (b) Xie et al., J. Am. Chem. Soc. 2012, 134, 1284] (2.3 mg, 1.6 mol) in DMSO (160 L) was treated sequentially with H.sub.2O.sub.2 (50% aqueous solution, 12 L, 98.4 mol) and K.sub.2CO.sub.3 (10% aqueous solution, 20 L, 16.1 mol) at 25 C. and the resulting mixture was stirred for 2 hours at 25 C. After this time, the reaction mixture was quenched by the addition of 0.1 N HCl (0.5 mL), and the aqueous phase extracted with EtOAc (30.5 mL). The combined organic layers were washed with saturated aqueous NaCl (0.5 mL), dried (Na.sub.2SO.sub.4) and the solvent was removed under reduced pressure.

(164) PTLC (SiO.sub.2, 12% CH.sub.3OHCH.sub.2Cl.sub.2) afforded Compound G (1.9 mg, 81%) as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) mixture of two rotamers (rotamer A:B=4:1) (for rotamer A) 8.42 (d, 1H, J=6.6 Hz), 7.81 (d, 1H, J=7.8 Hz), 7.65 (s, 1H), 7.59 (d, 1H, J=6.0 Hz), 7.45 (d, 1H, J=8.4 Hz), 7.34 (d, 1H, J=8.4 Hz), 7.31-7.29 (m, 3H), 7.00 (d, 1H, J=2.4 Hz), 6.97 (s, 1H), 6.94-6.92 (br m, 2H), 6.65 (d, 1H, J=2.4 Hz), 5.77 (s, 1H), 5.57-5.52 (m, 1H), 5.51 (s, 1H), 5.37 (d, 1H, J=5.4 Hz), 5.28 (s, 1H), 4.97 (d, 1H, J=9.0 Hz), 4.72 (s, 2H), 4.38-4.35 (m, 1H), 4.14 (s, 3H), 4.02 (dd, 1H, J=7.8, 7.8 Hz), 3.91 (s, 3H), 3.77-3.71 (m, 5H), 3.69 (s, 3H), 3.57 (s, 3H), 3.47 (dd, 1H, J=4.2, 4.2 Hz), 3.39 (s, 3H), 3.13 (s, 1H), 2.82 (s, 3H), 2.66-2.62 (m, 1H), 2.53-2.48 (m, 1H), 1.90-1.81 (m, 1H), 1.58 (s, 9H), 1.31 (s, 1H), 0.98 (d, 3H, J=6.0 Hz), 0.92 (d, 3H, J=6.0 Hz); ESI-TOF HRMS m/z 1417.5045 (M+H.sup.+, C.sub.68H.sub.82Cl.sub.2N.sub.8O.sub.21 requires 1417.5044).

(165) ##STR00040##

(166) A vial charged with Compound G (1.9 mg, 1.4 mol) was treated with AlBr.sub.3 (35.7 mg, 0.14 mmol) in EtSH (15 L) at 25 C. and the resulting mixture was stirred for 8 hours at 25 C. After this time, the reaction mixture was quenched with the addition of 50% CH.sub.3OH in H.sub.2O (0.5 mL) at 0 C. and the solvent was removed under a stream of N.sub.2. The residue was dissolved in 50% CH.sub.3OH in H.sub.2O (0.3 mL) and purified by semi-preparative reverse-phase HPLC (Zorbax SB-C18, 5 m, 9.4150 mm, 1-20% MeCN/H.sub.2O-0.07% TFA gradient over 10 minutes, 3 mL/minute, t.sub.R=16.2 minute) to afford Compound 10 (1.0 mg, 65%) as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 8.07 (d, 1H, J=8.4 Hz), 7.84 (d, 1H, J=8.4 Hz), 7.75 (d, 1H, J=8.4 Hz), 7.53 (s, 1H), 7.42 (d, 1H, J=8.4 Hz), 7.30 (s, 1H), 7.28 (d, 1H, J=8.4 Hz), 7.13 (d, 1H, J=6.0 Hz), 7.07 (s, 1H), 6.92 (d, 1H, J=8.4 Hz), 6.67 (s, 1H), 6.44 (s, 1H), 5.42 (s, 1H), 5.33 (s, 1H), 4.51-4.43 (m, 3H), 4.35-4.29 (br m, 1H), 4.10 (s, 1H), 4.04-4.01 (m, 1H), 3.89-3.85 (m, 1H), 2.77 (s, 3H), 2.66 (d, 1H, J=4.2 Hz), 2.03-2.02 (m, 1H), 1.84-1.77 (m, 1H), 1.65-1.62 (m, 1H), 1.31 (s, 1H), 1.00 (d, 3H, J=6.0 Hz), 0.94 (d, 3H, J=6.0 Hz); ESI-TOF HRMS m/z 1115.3313 (M+H.sup.+, C.sub.53H.sub.56Cl.sub.2N.sub.8O.sub.15 requires 1115.3315).

(167) ##STR00041##
See, Nakayama et al., Org. Lett. 2014, 16, 3572.

(168) ##STR00042##

(169) In a total volume of 1.0 mL, 4.0 mM UDP-glucose (2.3 mg, Sigma-Aldrich, 4.0 mol) and 0.5 mM Compound 8 (0.58 mg, 0.50 mol) were incubated with 75 mM Tricine-NaOH (pH 9.0), 2 mM tris-(2-carboxyethyl)phosphine, 1 mM MgCl.sub.2, glycerol (10% v/v) and 10 M GtfE for 42 hours at 37 C. The reaction mixture was quenched by the addition of MeOH (9.0 mL) at 0 C. and the residue was passed through a 0.45 m polyethersulfone membrane filter and concentrated by evaporation to a final volume of about 1.5 mL. After the addition of H.sub.2O (0.5 mL), the mixture was purified by semi-preparative reverse-phase HPLC. For HPLC, Vydac 218TP1022-C18, 10 m, 22250 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 10 mL/minute, t.sub.R=23.2 minutes was used to afford Compound 13 (0.48 mg, 75% yield) as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 8.83 (d, J=6.0 Hz, 1H), 8.42 (br s, 1H), 7.74-7.72 (m, 2H), 7.69-7.65 (m, 2H), 7.64-7.60 (m, 2H), 7.30 (d, J=8.4 Hz, 1H), 7.24 (br s, 1H), 6.78-6.73 (m, 1H), 6.45 (d, J=1.6 Hz, 1H), 6.40 (d, J=1.6 Hz, 1H), 6.26 (s, 1H), 5.95 (s, 1H), 5.41-5.36 (m, 3H), 5.32-5.28 (m, 1H), 4.41 (d, J=9.0 Hz, 1H), 4.30 (s, 1H), 4.23 (dd, J=4.8, 4.8 Hz, 1H), 4.07-4.04 (m, 1H), 3.92 (d, J=11.4 Hz, 1H), 3.82-3.81 (br m, 1H), 3.68-3.64 (m, 1H), 3.57-3.49 (m, 2H), 3.44-3.42 (m, 2H), 3.21-3.20 (m, 1H), 2.79 (s, 3H), 2.78 (s, 1H), 2.67 (s, 1H), 1.90-1.84 (m, 2H), 1.74-1.62 (m, 1H), 1.46-1.43 (m, 1H), 1.40-1.30 (m, 3H), 0.97 (d, J=6.0 Hz, 3H), 0.95 (d, J=6.0 Hz, 3H); ESI-TOF HRMS m/z 1321.3245 (M+H.sup.+, C.sub.59H.sub.62Cl.sub.2N.sub.8O.sub.21S requires 1321.3206).

(170) Additional Synthesis

(171) In a total volume of 10.3 mL, 2.0 mM UDP-glucose (21.7 mg, Sigma-Aldrich, 38.3 mol) and 0.5 mM Compound 8 (5.5 mg, 4.8 mol) were incubated with 75 mM Tricine-NaOH (pH 9.0), 2 mM tris(2-carboxy-ethyl)phosphine, 1 mM MgCl.sub.2, glycerol (25% v/v) and 25 M GtfE for 38 hours at 37 C. The reaction mixture was quenched by the addition of MeOH (90 mL) and the residue was passed through a 0.45 m polyethersulfone membrane filter and concentrated by evaporation to a final volume of about 2 mL. After the addition of H.sub.2O (1.0 mL), the mixture was purified by semi-preparative reverse-phase HPLC (Vydac 218TP1022-C18, 10 m, 22250 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 10 mL/minute, t.sub.R=22.7 minutes) to afford Compound 13 (2.3 mg, 35%) as a white amorphous solid and recovered starting material Compound 8 (2.5 mg, 46%) as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 8.35 (d, 1H, J=6.6 Hz), 7.67-7.66 (m, 2H), 7.64-7.60 (m, 2H), 7.35-7.30 (m, 2H), 7.18 (d, 1H, J=2.4 Hz), 6.74 (d, 1H, J=9.0 Hz), 6.65 (d, 1H, J=2.4 Hz), 6.44-6.43 (m, 1H), 6.41 (d, 1H, J=2.4 Hz), 6.16 (s, 1H), 5.86 (s, 1H), 5.38 (d, 1H, J=7.8 Hz), 5.32-5.29 (m, 3H), 5.26 (br s, 1H), 4.40 (d, 1H, J=6.6 Hz), 4.30-4.27 (m, 1H), 4.23-4.21 (m, 2H), 4.04-4.01 (m, 2H), 3.97-3.95 (m, 1H), 3.88-3.86 (m, 1H), 3.77-3.74 (m, 1H), 3.64-3.62 (m, 1H), 3.50-3.48 (m, 2H), 3.42-3.38 (m, 3H), 3.34 (s, 1H), 3.19-3.18 (m, 2H), 3.05-3.00 (m, 1H), 2.75 (s, 3H), 2.30-2.22 (m, 1H), 1.90-1.85 (m, 1H), 1.76-1.65 (m, 3H), 1.46-1.40 (m, 1H), 1.38-1.36 (m, 1H), 1.00 (d, 3H, J=6.0 Hz), 0.97 (d, 3H, J=6.0 Hz); ESI-TOF HRMS m/z 1307.3420 (M+H.sup.+, C.sub.59H.sub.65Cl.sub.2N.sub.8O.sub.20S requires 1307.3407).

(172) See also, Okano et al., J. Am. Chem. Soc. 2014, 136, 13522.

(173) ##STR00043##

(174) In a total volume of 2.8 mL, 2.0 mM UDP-glucose (3.22 mg, Sigma-Aldrich, 5.7 mol) and 0.5 mM of Compound 10 [Okano et al., J. Am. Chem. Soc. 2012, 134, 8790] (1.5 mg, 1.3 mol) were incubated with 75 mM Tricine-NaOH (pH 9.0), 2 mM tris-(2-carboxy-ethyl)phosphine, 1 mM MgCl.sub.2, glycerol (25% v/v) and 25 M GtfE for 48 hours at 37 C. The reaction mixture was quenched by the addition of MeOH (26 mL) and the residue was passed through a 0.45 m polyethersulfone membrane filter and concentrated by evaporation to a final volume of about 1.5 mL. After the addition of H.sub.2O (1.0 mL), the mixture was purified by semi-preparative reverse-phase HPLC (Zorbax SB-C18, 5 m, 9.4150 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 3 mL/minutes, t.sub.R=16.8 minutes) to afford Compound 15 (1.1 mg, 66%; typically 66-72%) as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 8.69 (d, 1H, J=8.4 Hz), 8.53 (d, 1H, J=7.2 Hz), 7.82 (dd, 1H, J=8.4, 2.4 Hz), 7.75 (dd, 1H, J=8.4, 1.8 Hz), 7.60 (d, 1H, J=1.8 Hz), 7.41 (d, 1H, J=8.4 Hz), 7.32 (d, 1H, J=2.4 Hz), 7.28 (d, 1H, J=8.4 Hz), 7.17-7.13 (m, 3H), 6.95 (dd, 1H, J=8.4, 2.4 Hz), 6.48 (d, 1H, J=4.8 Hz), 6.43 (d, 1H, J=2.4 Hz), 5.45-5.44 (m, 2H), 5.40 (d, 1H, J=7.8 Hz), 5.38 (d, 1H, J=5.4 Hz), 5.16 (d, 1H, J=7.8 Hz), 5.11 (d, 1H, J=9.6 Hz), 5.06 (s, 1H), 4.99-4.98 (m, 2H), 4.59 (dd, 1H, J=5.4, 5.4 Hz), 4.36 (dd, 1H, J=8.4, 6.0 Hz), 4.27-4.25 (m, 2H), 3.89 (dd, 1H, J=4.8, 2.4 Hz), 3.80-3.76 (m, 1H), 3.69-3.65 (m, 1H), 3.57-3.52 (m, 3H), 3.47-3.41 (m, 1H), 2.81 (s, 3H), 2.76-2.68 (m, 1H), 2.64 (dd, 1H, J=4.8, 2.4 Hz), 2.32-2.30 (m, 1H), 1.82 (dd, 1H, J=8.4, 8.4 Hz), 1.66-1.64 (m, 3H), 0.97 (d, 3H, J=6.0 Hz), 0.92 (d, 3H, J=6.0 Hz); ESI-TOF HRMS m/z 1291.3625 (M+H.sup.+, C.sub.59H.sub.64Cl.sub.2N.sub.8O.sub.21 requires 1291.3636).

(175) ##STR00044##

(176) In a total volume of 1.9 mL, 3.0 mM UDP-vancosamine (3.1 mg, 5.7 mol) and 0.5 mM Compound 15 (1.1 mg, 0.85 mol) were incubated with 75 mM Tricine-NaOH (pH 9.0), 2 mM tris-(2-carboxyethyl)-phosphine, 0.2 mg/mL bovine serum albumin, 1 mM MgCl.sub.2, glycerol (10% v/v) and 10 M GtfD for 1 hour at 37 C. The reaction mixture was quenched by the addition of MeOH (10 mL) at 0 C. and was passed through a 0.45 m polyethersulfone membrane filter and concentrated by evaporation to a final volume of about 2 mL.

(177) After the addition of H.sub.2O (2.0 mL), the mixture was purified by reverse-phase HPLC (Zorbax SB-C18, 5 m, 9.4150 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t.sub.R=14.0 minutes) to afford Compound 16 (0.93 mg, 76%) as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 8.65 (d, 1H, J=7.8 Hz), 8.49 (d, 1H, J=7.8 Hz), 7.84 (d, 1H, J=9.6 Hz), 7.77 (d, 1H, J=8.4 Hz), 7.62 (d, 1H, J=1.6 Hz), 7.41 (d, 1H, J=8.4 Hz), 7.33 (d, 1H, J=1.8 Hz), 7.22 (d, 1H, J=8.4 Hz), 7.15-7.14 (m, 2H), 7.09 (d, 1H, J=7.8 Hz), 6.94 (d, 1H, J=8.4 Hz), 6.48 (d, 1H, J=2.4 Hz), 6.42 (d, 1H, J=2.4 Hz), 5.47-5.43 (m, 5H), 5.39 (d, 1H, J=5.4 Hz), 5.14 (d, 1H, J=6.0 Hz), 5.10 (d, 1H, J=9.0 Hz), 4.59 (br s, 1H), 4.39 (d, 1H, J=7.2 Hz), 4.31-4.26 (m, 2H), 3.88-3.82 (m, 2H), 3.77-3.74 (m, 1H), 3.71-3.62 (m, 2H), 3.54 (dd, 1H, J=9.0, 9.0 Hz), 3.13-3.06 (m, 1H), 2.87-2.84 (m, 1H), 2.82 (s, 3H), 2.76-2.74 (m, 1H), 2.64-2.61 (m, 1H), 2.33-2.30 (m, 1H), 2.10-2.06 (m, 2H), 1.98-1.96 (m, 1H), 1.80 (dd, 1H, J=7.2, 7.2 Hz), 1.64-1.62 (m, 3H), 1.56 (s, 3H), 1.31 (s, 1H), 1.22 (d, 3H, J=6.6 Hz), 0.97 (d, 3H, J=6.0 Hz), 0.92 (d, 3H, J=6.0 Hz); ESI-TOF HRMS m/z 1434.4581 (M+H.sup.+, C.sub.66H.sub.77Cl.sub.2N.sub.9O.sub.23 requires 1434.4582.

(178) ##STR00045##

(179) A solution of Compound 16 (0.68 mg, 0.48 mol) in anhydrous DMF (60 L) was treated with 4-(4-chlorophenyl)benzaldehyde (0.1 M in DMF, 7.2 L, 0.72 mol) and i-Pr.sub.2NEt (distilled, 0.1 M in DMF, 24.0 L, 2.4 mol) at 25 C. The reaction mixture was stirred for 2 hours at 50 C. After the reaction was complete, the mixture was treated with NaCNBH.sub.3 (1 M in THF, 47.4 L, 47.4 mol) and stirred for 5 hours at 70 C.

(180) The reaction mixture was quenched by the addition of 50% CH.sub.3OH in H.sub.2O (0.2 mL) at 25 C. and the residue was purified by semi-preparative reverse-phase HPLC (Zorbax SB-C18, 5 m, 9.4150 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t.sub.R=34.2 minutes) to afford Compound 17 (0.31 mg, 41% yield) as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 8.07 (s, 1H), 8.02 (d, 1H, J=7.8 Hz), 7.98 (s, 1H), 7.92 (d, 1H, J=7.2 Hz), 7.79 (d, 1H, J=9.0 Hz), 7.76 (d, 1H, J=7.2 Hz), 7.70 (d, 2H, J=8.4 Hz), 7.63 (d, 2H, J=8.4 Hz), 7.56 (d, 2H, J=8.4 Hz), 7.47 (d, 2H, J=8.4 Hz), 7.43 (s, 1H), 7.33 (s, 1H), 7.22 (d, 1H, J=9.0 Hz), 7.12 (br s, 1H), 6.95-6.90 (m, 1H), 6.48 (d, 1H, J=2.4 Hz), 6.40 (d, 1H, J=2.4 Hz), 5.55 (d, 1H, J=4.2 Hz), 5.49-5.46 (m, 3H), 5.44-5.34 (m, 2H), 4.37-4.31 (m, 2H), 4.20-4.16 (m, 2H), 4.13-4.07 (m, 2H), 3.88-3.82 (m, 2H), 3.74-3.72 (m, 1H), 3.68-3.61 (m, 1H), 3.54-3.50 (m, 2H), 3.00 (s, 1H), 2.86 (s, 1H), 2.83 (s, 3H), 2.71-2.64 (m, 1H), 2.55-2.50 (m, 1H), 2.23 (dd, 1H, J=7.8, 7.8 Hz), 2.22-2.12 (m, 2H), 1.93 (s, 1H), 1.86-1.77 (m, 2H), 1.74 (s, 3H), 1.27 (d, 3H, J=6.6 Hz), 1.00 (d, 3H, J=6.0 Hz), 0.95 (d, 3H, J=6.6 Hz); ESI-TOF HRMS m/z 817.7525 (M+2H.sup.+, C.sub.79H.sub.86Cl.sub.3N.sub.9O.sub.23 requires 817.7524).

(181) ##STR00046##

(182) In a total volume of 10.3 mL, 2.0 mM UDP-glucose (21.7 mg, Sigma-Aldrich, 38.3 mol) and 0.5 mM Compound 8 [Xie et al., J Am Chem Soc 2012 134:1284-1297 Compound 44] (5.5 mg, 4.8 mol) were incubated with 75 mM Tricine-NaOH (pH 9.0), 2 mM tris(2-carboxyethyl)-phosphine, 1 mM MgCl.sub.2, glycerol (25% v/v) and 25 M GtfE for 38 hours at 37 C. The reaction mixture was quenched by the addition of MeOH (90 mL) and the residue was passed through a 0.45 m polyethersulfone membrane filter and concentrated by evaporation to a final volume of about 2 mL. After the addition of H.sub.2O (1.0 mL), the mixture was purified by semi-preparative reverse-phase HPLC (Vydac 218TP1022-C18, 10 m, 22250 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 10 mL/minute, t.sub.R=22.7 minutes) to afford Compound 20 (2.3 mg, 35%) as a white amorphous solid and recovered starting material (2.5 mg, 46%) as a white amorphous solid.

(183) .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 8.35 (d, 1H, J=6.6 Hz), 7.67-7.66 (m, 2H), 7.64-7.60 (m, 2H), 7.35-7.30 (m, 2H), 7.18 (d, 1H, J=2.4 Hz), 6.74 (d, 1H, J 9.0 Hz), 6.65 (d, 1H, J=2.4 Hz), 6.44-6.43 (m, 1H), 6.41 (d, 1H, J=2.4 Hz), 6.16 (s, 1H), 5.86 (s, 1H), 5.38 (d, 1H, J=7.8 Hz), 5.32-5.29 (m, 3H), 5.26 (br s, 1H), 4.40 (d, 1H, J=6.6 Hz), 4.30-4.27 (m, 1H), 4.23-4.21 (m, 2H), 4.04-4.01 (m, 2H), 3.97-3.95 (m, 1H), 3.88-3.86 (m, 1H), 3.77-3.74 (m, 1H), 3.64-3.62 (m, 1H), 3.50-3.48 (m, 2H), 3.42-3.38 (m, 3H), 3.34 (s, 1H), 3.19-3.18 (m, 2H), 3.05-3.00 (m, 1H), 2.75 (s, 3H), 2.30-2.22 (m, 1H), 1.90-1.85 (m, 1H), 1.76-1.65 (m, 3H), 1.46-1.40 (m, 1H), 1.38-1.36 (m, 1H), 1.00 (d, 3H, J=6.0 Hz), 0.97 (d, 3H, J=6.0 Hz); ESI-TOF HRMS m/z 1307.3420 (M+H.sup.+, C.sub.59H.sub.65Cl.sub.2N.sub.8O.sub.20S requires 1307.3407).

(184) ##STR00047##

(185) In a total volume of 1.4 mL, 3.0 mM UDP-vancosamine (2.5 mg, 4.6 mol) and 0.5 mM Compound 20 (1.1 mg, 0.84 mol) were incubated with 75 mM Tricine-NaOH (pH 9.0), 2 mM tris-(2-carboxyethyl)-phosphine, 0.2 mg/mL bovine serum albumin, 1 mM MgCl.sub.2, glycerol (10% v/v) and 10 M GtfD for 3 hours at 37 C. The reaction mixture was quenched by the addition of MeOH (10 mL) at 0 C. and was passed through a 0.45 m polyethersulfone membrane filter and concentrated by evaporation to a final volume of about 2 mL. After the addition of H.sub.2O (2.0 mL), the mixture was purified by semi-preparative reverse-phase HPLC (Vydac 218TP1022-C18, 10 m, 22250 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 10 mL/minute, t.sub.R=20.7 minutes) to afford Compound 24 (1.0 mg, 84%) as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 8.29 (s, 1H), 7.73-7.62 (m, 3H), 7.57 (d, 1H, J=9.0 Hz), 7.30 (d, 1H, J=9.0 Hz), 7.26 (d, 1H, J=8.4 Hz), 7.18 (s, 1H), 6.96 (d, 1H, J=9.0 Hz), 6.79 (d, 1H, J=8.4 Hz), 6.65 (s, 1H), 6.46-6.42 (m, 3H), 6.07 (s, 1H), 5.77 (s, 1H), 5.44 (d, 1H, J=7.8 Hz), 5.40 (d, 1H, J=4.2 Hz), 5.31 (s, 1H), 5.28-5.25 (m, 3H), 4.40-4.39 (br m, 1H), 4.31-4.28 (m, 1H), 4.22-4.20 (br m, 2H), 4.07-4.01 (m, 2H), 3.97-3.94 (m, 1H), 3.86 (d, 1H, J=12.0 Hz), 3.82-3.79 (m, 1H), 3.76-3.73 (m, 1H), 3.68-3.55 (m, 3H), 3.52-3.50 (m, 1H), 2.97-2.94 (m, 1H), 2.76 (s, 3H), 2.07-2.04 (m, 1H), 1.92 (d, 1H, J=13.8 Hz), 1.88-1.83 (m, 1H), 1.48 (s, 3H), 1.40-1.29 (m, 2H), 1.19 (d, 3H, J=6.6 Hz), 1.02 (d, 3H, J=6.6 Hz), 1.00 (d, 3H, J=6.6 Hz); ESI-TOF HRMS m/z 1450.4375 (M+H.sup.+, C.sub.66H.sub.78Cl.sub.2N.sub.9O.sub.22S requires 1450.4353).

(186) ##STR00048##

(187) A mixture of Compound 24 (0.38 mg, 0.26 Mmol) and AgOAc (0.43 mg, 2.6 mol) was treated with anhydrous saturated NH.sub.3CH.sub.3OH (0.2 mL) at 25 C. The reaction mixture was stirred for 6 hours at 25 C. before the solvent was removed under a stream of N.sub.2. The residue was dissolved in 50% CH.sub.3OH in H.sub.2O (0.2 mL) and purified by semi-preparative reverse-phase HPLC (Zorbax SB-C18, 5 m, 9.4150 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t.sub.R=16.4 minutes) to afford Compound 25 (86 g, 50% yield brsm, unoptimized) as a white film: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 7.74 (d, J=9.0 Hz, 1H), 7.74 (s, 1H), 6.90 (d, J=8.4 Hz, 1H), 6.68 (s, 1H), 6.44 (s, 1H), 5.52 (d, J=11.2 Hz, 1H), 5.45-5.37 (m, 5H), 4.33 (s, 1H), 4.13-4.06 (m, 2H), 3.85 (s, 1H), 3.77 (d, J=9.0 Hz, 1H), 3.67-3.52 (m, 2H), 2.88 (s, 3H), 2.45-2.41 (m, 1H), 2.07 (d, J=10.8 Hz, 1H), 1.86 (s, 1H), 1.61 (s, 1H), 1.51-1.39 (m, 3H), 1.30 (s, 1H), 1.28-1.20 (m, 3H), 0.89 (d, J=6.0 Hz, 3H), 0.85 (d, J=6.0 Hz, 3H); ESI-TOF HRMS m/z 1433.4760 (M+H.sup.+, C.sub.66H.sub.78Cl.sub.2N.sub.10O.sub.22 requires 1433.4742).

(188) ##STR00049##

(189) A solution of hydroxymethylvancomycin [Nakayama et al., Org. Lett. 2014, 16, 3572] (1.0 mg, 0.65 mol) in anhydrous DMF (0.1 mL) was treated with 4-(4-chlorophenyl)benzaldehyde (0.1 M in DMF, 9.7 L, 0.97 mol) and i-Pr.sub.2NEt (distilled, 0.1 M in DMF, 32.3 L, 3.23 mol) at 25 C. The reaction mixture was stirred for 12 hours at 30 C. After the reaction was complete, the mixture was treated with NaBH(OAc).sub.3 (13.7 mg, 64.6 mol) and stirred for 2 hours at 30 C. The reaction mixture was quenched by the addition of 50% CH.sub.3OH in H.sub.2O (0.2 mL) at 25 C. and the residue was purified by semi-preparative reverse-phase HPLC (Zorbax SB-C18, 5 m, 9.4150 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t.sub.R=35.2 minutes) to afford Compound 26 (0.73 mg, 67% yield) as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298K) 7.71-7.68 (m, 3H), 7.66-7.60 (m, 5H), 7.57-7.54 (m, 2H), 7.47-7.43 (m, 3H), 7.32 (d, 1H, J=8.4 Hz), 7.29 (d, 1H, J=7.8 Hz), 7.11 (d, 1H, J=8.4 Hz), 6.94 (d, 1H, J=8.4 Hz), 6.45-6.43 (m, 1H), 6.41-6.37 (m, 1H), 6.35 (d, 1H, J=2.4 Hz), 6.33 (d, 1H, J=2.4 Hz), 6.41-6.37 (m, 1H), 6.34-6.33 (m, 1H), 5.74 (d, 1H, J=11.2 Hz), 5.60 (d, 1H, J=18.0 Hz), 5.56 (d, 1H, J=7.8 Hz), 5.53 (d, 1H, J=12.0 Hz), 5.49 (d, 1H, J=4.2 Hz), 5.44-5.42 (m, 1H), 4.55-4.53 (m, 1H), 4.22-4.15 (m, 1H), 4.13-4.05 (m, 1H), 3.95-3.93 (m, 1H), 3.86-3.84 (m, 2H), 3.75-3.72 (m, 1H), 3.65 (br s, 1H), 3.62 (d, 1H, J=9.6 Hz), 3.53-3.50 (m, 1H), 2.78 (s, 3H), 2.52-2.48 (m, 1H), 2.21-2.16 (m, 1H), 2.04 (d, 1H, J=13.2 Hz), 1.67-1.64 (br m, 5H), 1.32 (d, 3H, J=6.6 Hz), 1.03-1.00 (m, 3H), 0.98-0.95 (m, 3H); ESI-TOF HRMS m/z 1634.5057 (M+H.sup.+, C.sub.79H.sub.87Cl.sub.3N.sub.9O.sub.23 requires 1634.4975).

(190) This reaction was run on scales of 0.2-1.1 mg (67-74%) as part of the optimization of conditions for use with Compound 27 on the amounts available.

(191) ##STR00050##

(192) A solution of Compound 24 (0.62 mg, 0.42 mol) in anhydrous DMF (30 L) was treated with 4-(4-chlorophenyl)benzaldehyde (0.1 mM in DMF, 5.5 L, 0.546 mol) and i-Pr.sub.2NEt (distilled, 0.1 mM in DMF, 21 L, 2.1 mol) at 25 C. The reaction mixture was stirred for 9 hours at 30 C. After the reaction was complete, the mixture was treated with NaBH(OAc).sub.3 (11.2 mg, 42.0 mol) and stirred for 2 hours at 30 C.

(193) The reaction mixture was quenched with the addition of 50% CH.sub.3OH in H.sub.2O (0.2 mL) and the residue was purified by semi-preparative reverse-phase HPLC (1-40% MeCN/H.sub.2O-0.07% TFA isocratic gradient over 40 minutes) to afford Compound 27 (0.52 mg, 74%) as a white amorphous solid: .sup.1H NMR (CD.sub.3OD, 600 MHz) 8.30 (d, 1H, J=6.6 Hz), 7.72-7.67 (m, 5H), 7.63 (d, 2H, J=8.4 Hz), 7.60 (d, 1H, J=6.0 Hz), 7.56 (d, 2H, J=7.8 Hz), 7.47 (d, 2H, J=8.4 Hz), 7.33 (d, 1H, J=8.4 Hz), 7.29 (d, 1H, J=8.4 Hz), 7.19 (d, 1H, J=2.4 Hz), 6.99-6.97 (m, 1H), 6.80 (d, 1H, J=8.4 Hz), 6.56 (d, 1H, J=2.4 Hz), 6.42 (d, 1H, J=1.8 Hz), 6.13-6.08 (br m, 1H), 5.80 (s, 1H), 5.51 (d, 1H, J 7.2 Hz), 5.46 (d, 1H, J=4.8 Hz), 5.33 (d, 1H, J=3.0 Hz), 5.31-5.29 (br m, 2H), 5.27 (s, 1H), 4.40-4.39 (m, 1H), 4.33-4.30 (m, 1H), 4.24 (s, 1H), 4.16 (d, 1H, J=12.0 Hz), 4.08-4.02 (m, 3H), 3.98-3.95 (m, 1H), 3.90-3.82 (m, 2H), 3.77-3.75 (m, 1H), 3.64-3.60 (m, 2H), 3.54-3.50 (m, 2H), 3.44-3.42 (m, 1H), 3.20-3.19 (m, 1H), 2.97 (d, 1H, J=13.8 Hz), 2.77 (s, 3H), 2.36-2.32 (m, 1H), 2.20-2.16 (m, 1H), 2.02 (d, 1H, J=13.8 Hz), 1.89-1.84 (m, 1H), 1.81-1.76 (m, 1H), 1.71-1.68 (m, 1H), 1.66 (s, 3H), 1.27 (d, 3H, J=6.6 Hz), 1.03 (d, 3H, J=6.0 Hz), 1.00 (d, 3H, J=6.6 Hz); ESI-TOF HRMS m/z 825.7447 (M+2H.sup.+, C.sub.79H.sub.86Cl.sub.3N.sub.9O.sub.23 requires 825.7410).

(194) ##STR00051##

(195) A mixture of Compound 27 (0.35 mg, 0.20 mol) and AgOAc (0.33 mg, 2.0 mol) was treated with anhydrous saturated NH.sub.3CH.sub.3OH (0.2 mL) at 25 C. The reaction mixture was stirred for 6 hours at 25 C. before the solvent was removed under a stream of N.sub.2. The residue was dissolved in 50% CH.sub.3OH in H.sub.2O (0.2 mL) and purified by semi-preparative reverse-phase HPLC (Zorbax SB-C18, 5 m, 9.4150 mm, 1-40% MeCN/H.sub.2O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t.sub.R=332 minutes) to afford Compound 28 (86 g, 48% yield brsm, unoptimized) as a white film: .sup.1H NMR (CD.sub.3OD, 600 MHz, 298 K) 7.79-7.68 (m, 3H), 7.61 (d, J=8.4 Hz, 2H), 7.54 (d, J=7.8 Hz, 2H), 7.46-7.44 (m, 4H), 7.08-7.02 (br m, 2H), 6.88 (d, J=9.0 Hz, 1H), 6.66 (s, 1H), 6.43 (d, J=2.4 Hz, 1H), 5.58-5.48 (m, 2H), 5.44-5.40 (m, 3H), 4.37-4.28 (m, 1H), 4.18-4.15 (m, 2H), 4.11 (d, J=4.2 Hz, 1H), 4.09-4.02 (m, 4H), 3.88-3.75 (m, 2H), 3.67-3.54 (m, 4H), 2.87 (s, 3H), 2.74 (br s, 1H), 2.41 (dd, J=14.4, 4.8 Hz, 1H), 2.19-2.16 (m, 1H), 2.06 (s, 1H), 2.03 (s, 1H), 1.85 (br s, 1H), 1.65-1.55 (m, 4H), 1.30-1.29 (m, 4H), 1.22-1.19 (m, 1H), 0.88 (d, J=6.0 Hz, 3H), 0.84 (d, J=6.6 Hz, 3H); ESI-TOF HRMS m/z 817.2614 (M+2H.sup.+, C.sub.79H.sub.88Cl.sub.3N.sub.10O.sub.22 requires 817.2604).

(196) ##STR00052##

(197) This compound was described in Nakayama et al., Org. Lett. 2014, 16:3572.

(198) ##STR00053##

(199) This compound was described in Okano et al., J. Am. Chem. Soc. 2014, 136, 13522.

(200) Antimicrobial Assays

(201) Assays were carried out following the methods of Clinical and Laboratory Standards Institute; Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, 7th ed.; CLSI document M07-A8; Clinical and Laboratory Standards Institute: Wayne, Pa., 2009.

(202) One day before studies were carried out, fresh cultures of vancomycin-sensitive Staphylococcus aureus (VSSA strain ATCC 25923), methicillin and oxacillin-resistant Staphylococcus aureus subsp. aureus (MRSA strain ATCC 43300), vancomycin-resistant Enterococcus faecalis (VanA VRE, BM4166), Enterococcus faecium (VanA VRE, ATCC BAA-2317) and vancomycin-resistant Enterococcus faecalis (VanB VRE, strain ATCC 51299), were inoculated and grown in an orbital shaker at 37 C. in 100% Mueller-Hinton broth (VSSA, MRSA and VanB VRE) or 100% Brain-Heart Infusion broth (VanA VRE). After 24 hours, the bacterial stock solutions were serial diluted with the culture medium (10% Mueller-Hinton broth for VSSA, MRSA and VanB VRE or 10% Brain-Heart Infusion broth for VanA VRE) to achieve the turbidity equivalent of 1:100 dilution of 0.5 M Macfarland solution. This diluted bacterial stock solution was then inoculated into a well of a V-shaped 96-well glass coated microtiter plate, supplemented with serial diluted aliquots of the antibiotic solution DMSO (4 L), to achieve a total assay volume of 0.1 mL. The plate was then incubated at 37 C. for 18 hours, after which minimal inhibitory concentrations (MICs) were determined by monitoring the cell growth (observed as a pellet) in the wells. The lowest concentration of antibiotic (in g/mL) capable of eliminating the cell growth in the wells is reported as the MIC. The reported MIC values for the new antibiotics were determined against vancomycin as a standard in the first well, which have well-established MIC values.

(203) Each of the patents, patent applications and articles cited herein is incorporated by reference.

(204) The foregoing description and the examples are intended as illustrative and are not to be taken as limiting. Still other variations within the spirit and scope of this invention are possible and will readily present themselves to those skilled in the art.