Boronic acids are of paramount importance to all facets of chemical sciences.
Although their popularization and widespread utilizations can likely be attributed to the incredible utility of the Suzuki coupling, (1) to date, boronic acids have found countless applications in fields far outside of cross-coupling, such as materials science, (2) chemosensors, (3) polymer science, (2) and drug discovery. (4-5) In medicine, two alkyl boronic acids are currently approved by FDA for various oncology indications: ninlaro (1) and velcade (49). Medicinal chemists point to the bioisoteric nature of boronic acids as they can function in certain cases as a carboxylic acid surrogate. (6) Despite their popularity, boronic acids are almost entirely derived through synthesis unlike the carboxylic acids they seek to replace which are ubiquitous and inexpensive. The retrosynthetic analysis employed in such cases can itself be a deterrent to their incorporation into a drug candidate.
As illustrated with 1 (
This invention provides, in various embodiments, methods for the conversion of a carboxylic acid group to a boronic ester or acid group, when the carboxylic acid group and the boronic ester or acid group is bonded, respectively, to an alkyl, i.e., sp3 hybridized, carbon atom.
An alkyl carboxylic acid compound RCO2H, as the term is used herein, is a compound that has a carboxylic acid, —CO2H, group bonded to an alkyl carbon atom, i.e., an sp3 hybridized carbon atom. Other parts of the molecule can comprise aryl or heteroaryl rings, heteroaroms, unsaturations, and other functional groups, as well as other alkyl carbon atoms.
An alkyl boronic acid compound, RB(OH)2, as the term is used herein, is a compound that has a boronic acid, —B(OH)2, group bonded to an alkyl carbon atom, i.e., an sp3 hybridized carbon atom. Other parts of the molecule can comprise aryl or heteroaryl rings, heteroaroms, unsaturations, and other functional groups, as well as other alkyl carbon atoms.
A pinacolato ester of an alkyl boronic acid is of formula
and both the ester and the acid boronate structures can comprise the same R group unchanged from the R group of the carboxylic acid RCO2H. Consequently, the reaction is very chemoselective for a carboxylic acid substrate, tolerating a wide range of chemical functionality elsewhere in the molecule. Because a boronic acid group is a pharmaceutically interesting isosteric replacement for a carboxylic acid group, this chemoselectivity allows a wide range of pharmaceuticals that contain a carboxylic acid group to be converted to the corresponding boronic acid compounds without disruption of functional groups elsewhere in the molecule.
The invention in its various embodiments provides the following advantages in carrying out the transformation described, that replaces a carboxylic acid group with a boronic acid group under conditions that are selective, mild, and cost effective. Some of the advantages of practicing a method of the invention include: Practicality: the invention uses inexpensive reagents with minimal precaution. Therefore, it can be readily adopted in both discovery and process settings. Broad scope: carboxylic acids which are amongst the most ubiquitous functional groups in commercial chemicals and pharmaceuticals are used in this transformation. The reaction also exhibits high chemoselectivity, thereby can be readily used to diversify a broad array of substrates. Urgency: there is increasing awareness of the importance of boronic acids in drug discovery but effective and general methods of boronic acid synthesis are lacking. This invention fills up this gap in methodology.
The invention provides, in various embodiments, a method of converting an alkyl carboxylic acid compound RCO2H to a corresponding alkyl boronic pinacolato ester compound
wherein R is a hydrocarbyl group comprising an sp3 hybridized carbon atom bonded to the CO2H or the boron atom, respectively, R optionally further comprising alkyl or alkyenyl groups, both optionally comprising heteroatoms, or optionally comprising aryl, heterocyclyl, or heteroaryl groups, or any combination thereof;
the method comprising:
a) forming a redox active ester (RAE) of the alkyl carboxylic acid compound; then,
b) contacting the redox active ester of the alkyl carboxylic acid compound in an aprotic solvent, and bis(pinacolato)diboron (B2pin2), in the presence of at least 20 mole % of a Mg(II) salt and of at least one molar equivalent a lithium compound comprising a (C1-C4)alkyllithium, a (C1-C4)alkoxylithium, or lithium hydroxide, and at least 10 mole % of a Cu or Ni salt;
in the presence of a 1,3-dicarbonyl ligand forming with the Cu a compound of formula (M)
wherein R1A and R2A are each independently selected (C1-C4)alkyl, trifluoromethyl, or phenyl;
or in the presence of a ligand of formula (L) comprising a bipyridyl of formula
wherein R1 and R2 are each independently selected (C1-C4)alkyl or (C1-C4)alkoxy, n1 and n2 are each independently 0, 1, or 2, or of a 1,10-phenanthroline of formula
wherein R3 and R4 are each independently (C1-C4)alkyl, (C1-C4)alkoxy or phenyl;
to provide the corresponding alkyl boronic pincolato ester compound.
More specifically, the invention provides, in various embodiments, a method of converting the alkyl carboxylic acid compound to the corresponding alkyl boronic pinacolato ester compound, comprising:
a) forming the redox active ester (RAE) of the alkyl carboxylic acid compound; then,
b) either: 1) contacting in aprotic solvent the redox active ester, at least one molar equivalent bis(pinacolato)diboron (B2pin2), and effective amounts of a Mg(II) salt in the present of lithium hydroxide or a lithium (C1-C4)alkoxide, and in the presence of a Cu(I) or a Cu(II) complex or both of a 1,3-dicarbonyl compound, the complex being of formula (M)
wherein R1A and R2A are each independently selected (C1-C4)alkyl, trifluoromethyl, or phenyl,
or in the presence of a Cu(I) or a Cu(II) salt or both and an effective amount of a ligand (L) comprising a bipyridyl of formula
wherein R1 and R2 are each independently selected (C1-C4)alkyl or (C1-C4)alkoxy, n1 and n2 are each independently 0, 1, or 2, or of a 1,10-phenanthroline of formula
wherein R3 and R4 are each independently (C1-C4)alkyl, (C1-C4)alkoxy or phenyl;
or: 2) contacting in aprotic solution the redox active ester and effective amounts of a Ni(II) salt and a Mg(II) salt, in the presence of an effective amount of a ligand (L) comprising a bipyridyl of formula
wherein R1 and R2 are each independently selected (C1-C4)alkyl or (C1-C4)alkoxy, n1 and n2 are each independently 0, 1, or 2, or of a 1,10-phenanthroline of formula
wherein R3 and R4 are each independently (C1-C4)alkyl, (C1-C4)alkoxy or phenyl;
then, adding a premixed solution comprising at least one molar equivalent of an organolithium compound and at least one molar equivalent bis(pinacolato)diboron (B2pin2);
to provide the pinacolato ester of the corresponding alkyl boronic ester compound.
More specifically, in carrying out a method of the invention, the redox active ester of the alkyl carboxylic acid can be an N-hydroxyphthalimide (NHPI) or can be a tetrachloro-N-hydroxyphthalimide (TCNHPI) ester.
More specifically, in carrying out a method of the invention, for the Cu-catalyzed reaction the Cu(II) salt can be Cu(acac)2, or for the Ni-catalyzed reaction the Ni(II) salt can be NiCl2.
More specifically, in carrying out a method of the invention, the Mg(II) salt can be MgBr2 or Mg Cl2.
More specifically, in carrying out a method of the invention, for the Cu-catalyzed reaction the lithium compound can be LiOH or a lithium (C1-C4)alkoxide, or for the Ni-catalyzed reaction the organolithium compound can be methyllithium.
More specifically, in carrying out a method of the invention, the aprotic solvent can comprise THF or dioxane, and DMF.
The invention further provides, in various embodiments, the method disclosed above, further comprising step c) cleaving under acidic conditions the pinacolato ester of the alkyl boronic acid compound
to provide the alkyl boronic acid compound RB(OH)2. For instance, the step of cleaving the pinacolato ester of the alkyl bornic acid compound can comprise contacting the ester with BCl3 followed with methanol, or contacting the ester with trifluoroacetic acid, or contacting the ester with a boronic acid, such as phenylboronic acid or 2-methylpropylboronic acid in aqueous HCl.
In various embodiments of the invention, the ligand (L) can be a bipyridyl of formula L1-L5
wherein
R1=OMe, R2=H, L1
R1=tBu, R2=H, L2
R1=H, R2=H, L3
R1=Me, R2=H, L4
R1=OMe, R2=OMe, L5;
or the ligand (L) can be a 1,10-phenanthroline of formula L7-L9
wherein
R3=H, R4=H, L7
R3=Ph, R4=H, L8
R3=OMe, R4=H, L9.
In various embodiments of the reaction, the Cu ligand of formula (M) can be
wherein each R1A and R2A is independently selected (C1-C4)alkyl, trifluoromethyl, or phenyl.
For example, the invention provides in various embodiments a method of preparation of alkyl boronic compound ninlaro (1)
from alkyl carboxylic acid compound:
by carrying out the steps a), b), and c) as disclosed above.
For example, the invention provides in various embodiments a method of preparing a boronate ester analog of atorvastatin ketal, comprising, first, a) forming the N-hydroxyphthalimide (NHPI) ester of atorvastatin ketal to provide redox active ester
then, carrying out step b) disclosed above, to provide the boronate ester of the analog of atorvastatin ketal
The invention further provides, as a composition of matter, a boronate ester analog of atorvastatin ketal of formula
The invention further provides, as a composition of matter, a boronic acid analog, freed of the pinacolato ester group, of atorvastatin ketal of formula
For example, the invention provides in various embodiments a method of preparing a dimethyl-t-butylsilyl (TBS) hydroxyl-protected boronic acid analog of a vancomycin aglycone
comprising, first, a) converting the carboxylic acid
to the corresponding NHPI redox activated ester, then carrying out step b), above, to provide an O-protected boronate pinacolato ester of the boronic acid, then carrying out step c), above, cleaving the boronic ester group to provide the O-protected boronic acid compound of formula
The invention further provides, as a composition of matter, a hydroxyl-protected boronic acid analog of a vancomycin aglycone
wherein TBS signifies a dimethyl-t-butylsilyl O-protecting group.
The invention also provides, as a method and as a composition of matter, a boronic acid analog of vancomycin aglycone of formula
prepared by cleavage of the t-butyldimethylsilyl (TBS) esters of the two protected hydroxyl groups using standard synthetic conditions such as tris(dimethylamino)sulfonium difluorotrimethylsilicate (TASF) in DMF.
For example, the invention provides in various embodiments a method of preparing boronic acid mCBK319 elastase inhibitor compound
comprising carrying out steps a), b), and c) as described above starting with carboxylic acid compound
The invention further provides, as a composition of matter, a boronic acid mCBK319 (50) elastase inhibitor compound of formula
For example, the invention provides in various embodiments a method of preparing the Boc-protected boronic ester compound of formula
comprising carrying out step b), above, on compound
to provide the Boc-protected boronic pinacolato ester compound
In additional embodiments, the Boc-protected boronic pinolcolato ester compound
can further undergo cleaving the Boc group of the compound with trifluoroacetic acid, followed by condensation of the resulting free amino group with compound
followed by cleavage of the pinacolato boronate ester group with phenylboronic acid in aqueous HCl to provide the boronic acid mCBK320 (51) elastase inhibitor compound of formula
The invention further provides, as a composition of matter, a boronic acid mCBK320 elastase inhibitor compound of formula
In further embodiments, the above Boc-protected boronic pinolcolato ester compound can further undergo cleaving the Boc group and of the boronate ester of the compound with trifluoroacetic acid, followed by condensation of the resulting free amino group with a compound of formula
followed by cleavage of the t-Bu ester,
to provide a boronic acid compound of formula (mCBK323 elastase inhibitor, 53)
The invention further provides, as a composition of matter, a boronic acid compound of formula (mCBK323, elastase inhibitor)
The invention further provides, in various embodiments, a method of preparation of an arylomycin sidechain analog boronic acid
comprising carrying out the conversions comprising steps a), b) and c) as described above, followed by removal of the N-Boc groups with acid, starting with an arylomycin sidechain analog carboxylic acid of formula
The invention also provides, as a novel composition of matter, an arylomycin sidechain analog boronic acid of formula
The invention further provides, in various embodiments, a method of synthesis of a Cyclic Boronic Acid β-Lactamase Inhibitor (RPX7009), of formula
and related boronic acid analogs, see “Discovery of a Cyclic Boronic Acid β-Lactamase Inhibitor (RPX7009) with Utility vs Class A Serine Carbapenemases” by Scott J. Hecker, et al., DOI: 10.1021/acs.jmedchem.5b00127, J. Med. Chem. (2015), 58, 3682-3692. A key intermediate in the synthesis of β-Lactamase Inhibitor (RPX7009), Hecker-9f, and analogs as described in the cited article, is compound Hecker-12 therein,
wherein TBDMS signifies a t-butyldimethylsilyl protecting group. This key intermediate can be prepared according to a method of the present invention from a compound of formula
the preparation of which is described in racemic form in publication M. Ghosh, M. J. Miller, Tetrahedron, 1996, 52, 4225. Use of a borylation method of the invention can yield compound Hecker-12 from this carboxylic acid intermediate. Methods to effect stereoselective reduction of beta-ketoesters to yield a chiral alcohol are well known to those skilled in the art, e.g. chiral borohydride reagents, or a ruthenium catalyzed asymmetric hydrogenation reaction, providing the Ghosh carboxylic acid in chiral form. For instance, the preparation of the carboxylic acid precursor for the borylation reaction of the invention can be prepared according to the following route:
The remainder of the synthesis can be carried out as described in Hecker, where use of a chiral pinanediol in the boronate ester exchange reaction of compound Hecker—12 to yield chiral Hecker—13 (see Scheme 1 of Hecker, et al.) can provide intermediates of good enantiomeric purity. These intermediates can be used in the preparation of all of β-Lactamase Inhibitors such as RPX7009, compounds Hecker-9a through Hecker-9r (see Table 1 of Hecker et al. publication).
See also related documents: “Reaktion von a-Lithioessigsaureestern mit Bernsteinsaureanhydriden” Von Franz-Peter Montforts und Silvio Ofner, Angew. Chem. (1979) 91, no. 8, p. 656; “Highly Enantioselective Hydrogenation of β-Keto Esters under Mild Conditions” Mark J. Burk,* T. Gregory P. Harper, and Christopher S. Kalberg, (1995) J. Am. Chem. Soc., 117, 4423-4424; and “Asymmetric Hydrogenation of α-Keto Carboxylic Esters. A Practical, Purely Chemical Access to α-Hydroxy Esters in High Enantiomeric Purity” R. Noyori,* T. Ohkuma, and M. Kitamura (1987), J. Am. Chem. Soc., Vol. 109, No. 19, 5856.
While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements will be apparent to those skilled in the art without departing from the spirit and scope of the claims.
Standard abbreviations for chemical groups such as are well known in the art are used; e.g., Me=methyl, Et=ethyl, i-Pr=isopropyl, Bu=butyl, t-Bu=tert-butyl, Ph=phenyl, Bn=benzyl, Ac=acetyl, Bz=benzoyl, and the like.
In this report, a simple method for nickel-catalyzed decarboxylative borylation is presented that is mild, scalable, and general across a range of primary, secondary, tertiary, peptidic, and even naturally occurring substrates. A diverse array of boronates which would otherwise require lengthy de novo synthesis were furnished directly from the corresponding carboxylic acids. This method's capacity to directly transform native peptides into α-amino boronic acids has led to the discovery of three potent small molecule elastase inhibitors.
Recent efforts in our laboratory revealed redox active esters (RAEs, e.g., N-hydroxyphthalimide ester 2) derived from alkyl carboxylic acids as convenient surrogates for alkyl halides in nickel or iron catalyzed cross-coupling reactions. These versatile intermediates, most commonly used in amide-bond forming reactions, have enabled practical means of C—C bond formation in various modalities, including decarboxylative Negishi (22-23), Suzuki (24), and Kumada (25) couplings, as well as Giese reactions (26). Although RAEs have yet to be used in carbon-heteroatom cross-coupling reactions, our earlier discoveries, coupled with Fu's (10) pioneering work on nickel catalyzed Miyaura borylation of alkyl halides (11-14), prompted us to investigate the possibility of harnessing them for C—B bond formation, thereby achieving direct conversion of alkyl carboxylic acids into boronic acid derivatives.
Realization of this seemingly straightforward transformation required considerable experimentation.
(B2pin2);
other RAEs such as the tetrachloro-NHPI ester were less effective (entry 1). The inexpensive combination of NiCl2.6H2O and bipyridine ligand L1 emerged as the best catalyst system after an exhaustive screening—use of alternative catalysts (see SI) or ligands (entries 3-5) have deleterious effects. Choice of solvent is critical: a binary mixture of THF and DMF gave the optimal result; lower yields were observed in the absence of DMF (entry 6). Pre-mixing methyl lithium with B2pin2 is necessary to activate the diboron species toward transmetalation; numerous other organometallic reagents surveyed (e.g., entries 8-10) were less effectual, affording borylation products in lower yields if at all. Magnesium salts were also indispensable to the reaction: in the absence of the MgBr2.OEt2, virtually no products were attained (entries 11-13). Borono-ester product 3 can be accessed directly from the carboxylic in comparable yields using a one-pot procedure wherein RAE 2 is formed in situ in a similar vein to amide coupling (entry 14). Overall, the reaction proceeds smoothly at room temperature over the course of 2 hours.
With the optimized conditions in hand, the scope of this methodology was subsequently explored. RAEs derived from a broad selection of primary, secondary, and tertiary carboxylic acids were all found to be viable substrates (
As the methyl lithium was pre-mixed with B2pin2 to form ate-complexes, strongly nucleophilic/basic organometallic species were sequestered from the substrate: a gamut of functionalities such as ethers (30, 31, 35, 37, 41), esters (5, 8, 21, 22, 39, 41), carbamates/amides (8, 15, 28, 36, 37, 1), ketones (34, 38, 39, 40), olefins (39, 40, 41), and hydroxyl (40, 41) were left unscathed under the mild reaction conditions. Indeed, even the highly base-sensitive Fmoc group was tolerated (see 8). The compatibility with alkyl bromides (7) and chlorides (33) points to the orthogonality of this reaction to halide-based Miyaura borylations. Enoxolone derived boronates 39 and 40 were obtained with similar yields, suggesting that the free hydroxyl group had minimal influence on the reaction. The discrete isolation of RAEs, as alluded to earlier, is not necessary. Tertiary and secondary boronate esters can be prepared directly from carboxylic acids when RAEs are generated in situ. This one-pot procedure also pertains to primary substrates, albeit at lower yields.
Although some of the products presented herein (e.g., 4, 17, 19, 20, 23) can be synthesized from the analogous halides via Miyaura borylation reactions, the starting organohalides are oftentimes not commercially available and require extraneous steps to prepare (usually from the corresponding alcohols). Conversely, the use of readily available carboxylic acids largely circumvents this problem. A great majority of products in
The prevalence of alkyl carboxylic acids is demonstrated by their presence in over 450 approved drug molecules (30). To this end, the impressive chemoselectivity of this reaction offers the unique opportunity to pursue late-stage modifications of bioactive molecules that are densely adorned with reactive functionalities. Over 10 carboxylate containing drug molecule/natural products have been successfully converted into pinacol boronate esters (28-41) which would otherwise only be accessible through multi-step functional group interconversions or de novo syntheses.
The boronate esters can be conveniently hydrolyzed into the corresponding boronic acids (e.g., 4a, 3a, 33a, 1) (
Moreover, selective decarboxylative borylation at the C-terminus of native peptides allowed rapid access to coveted α-amino boronic acids which are privileged medicinal chemistry motifs (18, 37). Ninlaro (1), for example, was obtained in three steps from a simple peptide (
This process proceeded smoothly in the presence of four methylated phenoxy groups, two TBS-protected hydroxyls, two aryl chlorides, six secondary amides, one primary amide, one secondary amine, and seven epimerizable stereocenters. Although 44 showed less activity compared to the parent acid 43, such remarkable chemoselectivity still attests to the potential utility of this reaction.
Unpredictable stereoselectivity of radical processes oftentimes presents a hurdle to their broad adoption in late-stage modifications of drug leads or natural products. Complex α-amino boronic acid 44 was obtained as a single diastereomer in this radical-based decarboxylative borylation reaction. This result prompted us to investigate the stereoselectivity of the decarboxylative borylation on several dipeptides (
By wedding the rich medicinal potential of boronates to the ubiquity of alkyl carboxylic acids, the decarboxylative borylation reaction has the potential to open up new vistas in drug development. For example, application of the decarboxylative borylation reaction to readily available dipeptides allowed the expedient preparations of 50-52 which were formed as single diastereomers and found to be potent inhibitors of human neutrophil elastase (HNE) (
Toward this end, 52 exhibited an ICo50=15 pM (Ki=3.7 pM) while 51 exhibited an IC50=30 pM (Ki=34 pM) against purified HNE. The IC50 values were determined head-to-head with other pre-clinically and clinically validated HNE inhibitors (53-57), including BAY 85-8501 (54, a leading clinical candidate with reported Ki=80 pM) (47), 55 (POL6014, a phase I peptide-based clinical candidate for cystic fibrosis) (48) as well as 56 and 57 (reported by Chiesi Pharmaceuticals) (49). Additionally, 51 and 52 retained much of their inhibitory activities in sputum samples of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) patients, underscoring their potency in the context of a more patho-physiologically relevant environment than the traditional biochemical assay. Conversely, while dimeric compound 58 from AstraZeneca (IC50=11 pM, Ki=2.7 pM) (50), and BAY 85-8501 (54) showcased low IC50 values, their potencies diminished drastically in patient derived sputum. Comparison of the LipE values in COPD sputum revealed that the superior potency of 52 is not driven by increased lipophilicity (10.2 versus 9.45 for 57) (51).
Additionally, the IC50 value of 52 was found to remain unchanged with increasing incubation times (between 5-60 minutes) while that of 58, a non-covalent inhibitor exhibited a 55-fold increase in potency under the same conditions. These data retain the profile that is expected: compound 52 is behaving like a partial mechanism-based inhibitor (or a covalent reversible inhibitor) likely due to the potentially slow off-rate of the α-amino boronic acid. This correlates with tighter binding and potentially long residence time seen in other amino boronic acid compounds unlike the many reversible elastase inhibitors (i.e. 58) (52). Clinically, this mechanism has been proven successfully through Velcade (49), which inhibits the catalytic site of the 26S proteasome-covalent reversible bonding between the boronate and the nucleophilic oxygen results in a slow disassociation rate (53-54). As most clinical elastase inhibitors (such as 54, BAY 85-8501, one of the most potent molecules reported to date) are non-reactive, reversible, transition state inhibitors, 52's high potency and the inherent mechanism of the amino boronic acids could help address these limitations. Through this “hybrid” enzymatic inhibitory approach (based upon Fischer's Lock and Key model/Ehrlich's Pharmacophore Model), boronic acids such as 52, which combine a rapid, potent binding with a slow off-rate, may effectively restore the protease versus anti-protease balance in a clinical setting. They could therefore be tuned toward lung-specific clinical applications rapidly.
To further evaluate the therapeutic potential of 51 and 52, the in vitro ADME properties were probed to determine if any deleterious effects of the boronate replacements of the ketone would be revealed (
Procedurally, the conversion of redox active esters into boronate esters is achieved in three stages: namely, the preparation of catalyst mixture, the preparation of [B2pin2Me]Li complex and the nickel catalyzed decarboxylative borylation reaction. An abbreviated experimental protocol is presented herein with a graphical guide. Comprehensive information on the commercial source and purity of chemicals or variations in experimental details for different substrate classes can be found in the supplementary information.
Preparation of NiCl2.6H2O/Ligand Stock Solution or Suspension:
A flask charged with NiCl2.6H2O (1.0 equiv.) and ligand (L1 or L2, 1.3 equiv.) was evacuated and backfilled with argon for three times. THF (the concentration of NiCl2.6H2O was 0.025 M) or DMF (the concentration of NiCl2.6H2O was 0.050 M) was added. The resulting mixture was stirred at room temperature overnight (or until no granular NiCl2.6H2O was observed) to afford a green solution or suspension. [Note: All the solutions or suspensions kept under argon can be used for several days without appreciable deteriorations in reaction yields.]
Preparation of [B2Pin2Me]Li Complex:
To a solution of B2pin2 (1.1 equiv.) in THF (the concentration of B2pin2 was 1.1 M) was added MeLi (1.6 M in Et2O, 1.0 equiv.) at 0° C. under argon. The reaction mixture was warmed to room temperature and stirred for 1 h to afford a milky white suspension.
A flask charged with the redox-active ester (1.0 equiv.) and MgBr2.OEt2 (1.5 equiv.) was evacuated and backfilled with argon for three times. Catalyst solution or suspension (containing 10 mol % of NiCl2.6H2O and 13 mol % of ligand) was added via a syringe. When a catalyst suspension/solution in DMF was used, an additional portion of THF (twice the volume of the DMF suspension/solution needed) was added to the reaction vessel prior to the addition of the catalyst mixture [this process can be exothermic on large scales and cooling (with ice/water bath) may be necessary]. The resulting mixture was stirred vigorously until no visible solid was observed at the bottom of the reaction vessel [this was found to be accelerated by sonication]. This mixture was cooled to 0° C. before a suspension of [B2pin2Me]Li in THF (3 equiv.) was added in one portion. After stirring for 1 hour at 0° C., the reaction was warmed to room temperature and stirred for another 1 hour. When thin layer chromatography (TLC) analysis indicated the completion of the reaction, the reaction was quenched with aqueous HCl (0.1 M) or saturated aqueous NH4Cl and extracted with diethyl ether (Et2O) or ethyl acetate (EtOAc). Alternatively, on larger scales, as is the case shown in
Through the exclusive use of N-hydroxy-phthalimide redox active esters, a simple means to interconvert two functional groups of paramount importance in organic chemistry has been enabled. The practicality and chemoselectivity are illustrated through numerous complex substrates including drug molecules. The broad scope, epitomized by the ability to directly transform native peptides into borono-isosteres in good stereoselectivities, is unmatched by halide-based Miyaura-Suzuki protocols. Alkyl boronates can be introduced at any stages of a synthesis, reshaping the strategic paradigm toward their preparations. By wedding the rich medicinal potentials of boronates to the ubiquity of alkyl carboxylic acids, this method will likely open up new vistas in drug development. This is already evident from the discovery of mCBK 320, a highly potent elastase inhibitor with potentials in cancer therapy.
Preparation of mCBK319
Preparation of mCBK320 and mCBK323
All three compounds (mCBK319, mCBK320, and mCBK323) were found to possess a high propensity toward trimerization at the boronic acid motif. The bioactivities of these trimers may warrant further investigations. These novel elastase inhibitors have potential applications as therapeutics for cancer, cycstic fibrosis, and bronchiectasis.
The compounds have displayed enhanced potency compared to lead compounds screened (100-1000 fold increase compared to the analogous trifluoroketone lead compounds) as elastase inhibitors. The unique physiochemical properties of the boronic acid motif may give rise to favorable pharmacokinetic attributes. The processes for preparation disclosed herein are concise and easily amenable for scale up.
Tetrahydrofuran (THF), N,N-dimethylformamide (DMF), acetonitrile (CH3CN) dichloromethane (CH2Cl2) were obtained by passing the previously degassed solvents through activated alumina columns. Other solvents and reagents were purchased at the highest commercial quality and used without further purification, unless otherwise stated.
Yields refer to chromatographically and spectroscopically (1H-NMR) homogeneous material, unless otherwise stated. Reactions were monitored by GC/MS, LC/MS, and thin layer chromatography (TLC). TLC was performed using 0.25 mm E. Merck silica plates (60F-254), using short-wave UV light as visualizing agent, as well as potassium permanganate (KMnO4) or ceric ammonium molybdate (CAM) and heat as developing agents. NMR spectra were recorded on Bruker DRX-600, DRX-500 or DPX-400 instruments and are calibrated using residual undeuterated solvent (1H: δ 7.26 for CDCl3, δ 3.31 for MeOH-d4, δ 3.58, 1.73 for THF-d8, δ 2.50 for DMSO-d6, δ 2.05 for acetone-d6; 13C: δ 77.16 for CDCl3, δ 49.0 for MeOH-d4, δ 67.6, 25.5 for THF-d8, δ 39.50 for DMSO-d6, δ 29.84 for acetone-d6). The following abbreviations were used to explain multiplicities: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad. Column chromatography was performed using E. Merck silica gel (60, particle size 0.043-0.063 mm), and preparative TLC was performed on 0.25 mm E. Merck silica plates (60F-254). High resolution mass spectra (HRMS) were recorded on an Agilent LC/MSD TOF mass spectrometer by electrospray ionization time of flight reflectron experiments. Preparative high performance liquid chromatography (HPLC) was performed using an Agilent SD-1 prepstar system equipped with Phenomenex Gemini 10 μm C18 column with dimension 200×50 mm. Melting points were recorded on a Fisher-Johns 12-144 melting point apparatus and are uncorrected. All X-ray diffraction data were collected and analyzed by the UCSD small molecule X-ray facility. The deactivated silica gel (35 wt % H2O) was prepared by mixing silica gel and deionized water, followed by vigorous shaking until a fluffy powder was observed.
B2pin2 is bis(pinacolato)diboron.
NHPI is N-hydroxyphthalimide. TCNHPI is tetrachloro-N-hydroxyphthalimide. Standard abbreviations for chemical groups such as are well known in the art are used; e.g., Me=methyl, Et=ethyl, i-Pr=isopropyl, Bu=butyl, t-Bu=tert-butyl, Ph=phenyl, Bn=benzyl, Ac=acetyl, Bz=benzoyl, TBS=dimethyl-t-butylsilyl, Boc=t-butoxycarbonyl, and the like.
Preparative HPLC was performed using an Agilent SD-1 prepstar system equipped with Phenomenex Gemini 10 μm C18 column with dimension 200×50 mm. Melting points were recorded on a Fisher-Johns 12-144 melting point apparatus and are uncorrected. All X-ray diffraction data were collected and analyzed by the UCSD small molecule X-ray facility. The deactivated silica gel (35 wt. % H2O) was prepared by mixing silica gel and deionized water, followed by vigorous shaking until a fluffy powder was observed.
A round bottom flask was charged with the carboxylic acid (1.0 equiv), N-hydroxyphthalimide (NHPI, 1.0 equiv) or tetrachloro-N-hydroxyphthalimide (TCNHPI, 1.0 equiv) and DMAP (0.1 equiv). CH2Cl2 (0.2 M) was added, followed by N,N′-diisopropylcarbodiimide (DIC, 1.1 equiv), both at room temperature. The mixture was allowed to stir at room temperature until all the acid was consumed (as indicated by TLC). The resulting mixture was quickly filtered and the solid residue was rinsed with more CH2Cl2. The filtrate was concentrated in vacuo and purified by flash column chromatography to afford the corresponding redox-active esters, which were used without further purification unless otherwise noted.
All reactions were screened based on 0.1 mmol scale. The optimization started with S1. TCNHPI esters were used in the initial screening since earlier conditions indicated better performance than NHPI esters (NHPI esters were used in the optimized conditions in the end.
However, under the aforementioned optimized conditions for S1, decarboxylative borylation of S2a proceeded in lower yield than the NHPI ester of S2.
In order to identify a more general set of conditions, further optimization efforts were undertaken on the NHPI ester S2.
†Isolated yield.
†Isolated yield.
This optimized set of condition for the decarboxylative borylation of S2 (1° RAE) was more general, and was also suitable for 2 (20 RAE).
Further screening indicated that employing THF as sole solvent gave the best yield for tertiary carboxylic acids (3° RAEs).
†Isolated yield.
Part I. Preparation of NiCl2.6H2O/Ligand Stock Solution or Suspension
(1) Suspension A: NiCl2.6H2O/Di-MeObipy (L1) in THF (0.025 M).
A screw-capped culture tube charged with NiCl2.6H2O (23.8 mg, 0.1 mmol) and 4,4′-dimethoxy-2,2′-bipyridine (L1, 28.1 mg, 0.13 mmol) was evacuated and backfilled with argon for three times. THF (4.0 mL) was added and the resulting mixture was stirred at room temperature overnight (or until no granular NiCl2.6H2O was observed) to afford a pale green suspension.
(2) Suspension B: NiCl2.6H2O/Di-MeObipy (L1) in DMF (0.05 M).
A screw-capped culture tube charged with NiCl2.6H2O (23.8 mg, 0.1 mmol) and 4,4′-dimethoxy-2,2′-bipyridine (L1, 28.1 mg, 0.13 mmol) was evacuated and backfilled with argon for three times. DMF (2.0 mL) was added and the resulting mixture was stirred at room temperature overnight to afford a pale green suspension.
(3) Suspension C: NiCl2.6H2O/Di-tBubipy (L2) in THF (0.025 M).
A screw-capped culture tube charged with NiCl2.6H2O (23.8 mg, 0.1 mmol) and 4,4′-di-tert-butyl-2,2′-bipyridine (L2, 34.8 mg, 0.13 mmol) was evacuated and backfilled with argon for three times. THF (4.0 mL) was added and the resulting mixture was stirred at room temperature overnight (or until no granular NiCl2.6H2O was observed) to afford a pale green suspension.
(4) Solution D: NiCl2.6H2O/Di-tBubipy (L2) in DMF (0.05 M).
A screw-capped culture tube charged with NiCl2.6H2O (23.8 mg, 0.1 mmol) and 4,4′-di-tert-butyl-2,2′-bipyridine (L2, 34.8 mg, 0.13 mmol) was evacuated and backfilled with argon for three times. DMF (2.0 mL) was added and the resulting mixture was stirred at room temperature for 2 h to afford a green solution.
Note: All the solutions or suspensions kept under argon can be used for several days without appreciable deteriorations in reaction yields.
Part II. Preparation of [B2Pin2Me]Li Suspension
To a solution of B2pin2 (168 mg, 0.66 mmol) in THF (0.6 mL) was added MeLi (0.38 mL, 1.6 M in Et2O, 0.6 mmol) at 0° C. under argon. The reaction mixture was warmed to room temperature and stirred for 1 h to afford a suspension (sometimes we also experienced to obtain this complex as a clear solution).
Note: The resulting mixture can be stored with stirring for several hours without appreciable deterioration.
A screw-capped culture tube charged with the redox-active ester (0.2 mmol, 1.0 equiv) and MgBr2.OEt2 (77 mg, 0.3 mmol, 1.5 equiv) was evacuated and backfilled with argon for three times. THF (0.8 mL) was added, and the mixture was stirred until no granular MgBr2.OEt2 was observed (ca. 10 min) before suspension B (0.4 mL, NiCl2.6H2O (10 mol %)/di-MeObipy (13 mol %) in DMF), or solution D (0.4 mL, NiCl2.6H2O (10 mol %)/di-tBubipy (13 mol %) in DMF) was added via a syringe. The resulting mixture was stirred vigorously until no visible solid was observed on the bottom of the reaction vessel (ca. 10 min). This mixture was cooled to 0° C. before a suspension of [B2pin2Me]Li in THF (3 eq, 1.1 mL) was added in one portion (note: do not add it dropwise!). After stirring for 1 h at 0° C., the reaction was warmed to room temperature and stirred for another 1 h before quenched with 0.1 N HCl (10 mL). The resulting mixture was extracted with Et2O or EtOAc (3 mL×2). The combined organic layers were concentrated in vacuo, and the crude product was purified by flash column chromatography. For acid labile substrate, the reaction was alternatively quenched with saturated aqueous NH4Cl (10 mL).
A screw-capped culture tube charged with the redox-active ester (0.2 mmol, 1.0 equiv) and MgBr2.OEt2 (77 mg, 0.3 mmol, 1.5 equiv) was evacuated and backfilled with argon for three times. Suspension A (0.8 mL, NiCl2.6H2O (10 mol %)/di-MeObipy (13 mol %) in THF) or C (0.8 mL, NiCl2.6H2O (10 mol %)/di-tBubipy (13 mol %) in THF) was added via a syringe. The mixture was stirred vigorously at room temperature until no granular MgBr2.OEt2 was observed (ca. 15 min). This suspension was cooled to 0° C. before a suspension of [B2pin2Me]Li was added in one portion (note: do not add it dropwise!). After stirring for 1 h at 0° C., the reaction was warmed to room temperature and stirred for another 1 h. The reaction mixture was diluted with Et2O (10 mL), filtered through a short pad of silica gel and celite (top layer: celite, bottom layer: silica gel, v/v celite:silica gel=1:1), washed with Et2O (50 mL). The filtrate was concentrated, and the crude product was purified by column chromatography.
For polar substrates, such as peptides, the reaction was quenched either with 0.1 N HCl (10 mL) or sat. aqueous NH4Cl (10 mL) followed by extraction with EtOAc (3 mL×2). The combined organic layers were dried over Na2SO4, concentrated in vacuo and purified by flash column chromatography.
The gram-scale procedure was slightly modified from General Procedure C. A flame-dried round bottom fask charged with B2pin2 (2.57 g, 10.1 mmol, 3.3 equiv) was evacuated and backfilled with argon for three times. THF (9.2 mL) was added, and the clear solution was cooled to 0° C. when MeLi (5.8 mL, 1.6 M in Et2O, 9.3 mmol, 3.0 equiv) was added dropwise. The reaction mixture was then warmed to room temperature and stirred for 1 h.
The NHPI redox-active ester of ibuprofen S18 (1.08 g, 3.07 mmol) and MgBr2.OEt2 (powder, 792 mg, 3.07 mmol, 1.0 equiv) were sequentially added to another flame-dried round-bottom flask. This flask was evacuated and backfilled with argon for three times and was cooled to 0° C. THF (12 mL) was added, the mixture was sonicated until no granular MgBr2.OEt2 was observed. A suspension of NiCl2.6H2O (73 mg, 0.31 mmol) and di-MeObipy (L2, 86 mg, 0.40 mmol) in THF (12 mL) was added, and the resulting mixture was sonicated again until there was no visible solid on the bottom of the flask. The mixture was then cooled to 0° C. before a suspension of [B2pin2Me]Li in THF was added in one portion. After stirring for 1 h at 0° C., the reaction mixture was warmed to room temperature and stirred for another 1 h.
The reaction mixture was then poured into Et2O (100 mL), and the flask was rinsed with additional Et2O (100 mL). The resulting mixture was filtered through a plug of silica gel and celite (top layer: celite, bottom layer: silica gel, v/v celite:silica gel=1:1), the solid residue was washed with Et2O (350 mL), and the filtrate was concentrated in vacuo. Purification by flash column chromatography (silica gel, hexanes to 1:30 Et2O:hexanes) afforded product (709 mg, 80%) as a colorless oil.
A screw-capped culture tube with a stir bar was charged with alkyl carboxylic acid (0.2 mmol), N-hydroxyphthalimide or tetrachloro-N-hydroxyphthalimide (0.2 mmol, 1.0 equiv) and N,N′-dicyclohexylcarbodiimide (0.2 mmol, 1.0 equiv). The tube was then evacuated and backfilled with argon for three times. CH2Cl2 (2.0 mL) was added and the resulting mixture was stirred at room temperature for 2 h before the volatiles were removed in vacuo. MgBr2. OEt2 (77 mg, 0.3 mmol, 1.5 equiv) was added. The tube was evacuated and backfilled with argon for three times. Suspension A (0.8 mL, NiCl2.6H2O (10 mol %)/L1 (13 mol %) in THF) or suspension C (0.8 mL, NiCl2.6H2O (10 mol %)/L2 (13 mol %) was added. The mixture was stirred vigorously at room temperature for 15 min (or until no granular MgBr2.OEt2 was observed) and was subsequently cooled to 0° C. before a suspension of [B2pin2Me]Li in THF (1.1 mL) was added in one portion (note: do not add it dropwise!). After being stirred for 1 h, the reaction was warmed to room temperature and stirred for another 1 h. The reaction mixture was then quenched with 0.1 N HCl (10 mL) and extracted with Et2O (5 mL×2). The combined organic layers were dried over Na2SO4, concentrated in vacuo and purified by column chromatography to give the desired product.
This in situ procedure was demonstrated on six alkyl carboxylic acids following General Procedure D.
General Procedure for Ni-Catalyzed Borylation of Redox-Active Esters with 2.5 Mol % Nickel
Part I. Preparation of NiCl2.6H2O/Ligand Stock Solution or Suspension
(1) Suspension E: NiCl2.6H2O/Di-MeObipy (L1) in THF (6.25 mM).
A screw-capped culture tube charged with NiCl2.6H2O (23.8 mg, 0.1 mmol) and 4,4′-dimethoxy-2,2′-bipyridine (L1, 28.1 mg, 0.13 mmol) was evacuated and backfilled with argon for three times. THF (16.0 mL) was added and the resulting mixture was stirred at room temperature overnight (or until no granular NiCl2.6H2O was observed) to afford a pale green suspension.
(2) Solution F: NiCl2.6H2O/Di-MeObipy (L1) in DMF (12.5 mM).
A screw-capped culture tube charged with NiCl2.6H2O (23.8 mg, 0.1 mmol) and 4,4′-dimethoxy-2,2′-bipyridine (L1, 28.1 mg, 0.13 mmol) was evacuated and backfilled with argon for three times. DMF (8.0 mL) was added and the resulting mixture was stirred at room temperature overnight to afford a light green solution.
(3) Suspension G: NiCl2.6H2O/Di-tBubipy (L2) in THF (6.25 mM).
A screw-capped culture tube charged with NiCl2.6H2O (23.8 mg, 0.1 mmol) and 4,4′-di-tert-butyl-2,2′-bipyridine (L2, 34.8 mg, 0.13 mmol) was evacuated and backfilled with argon for three times. THF (16.0 mL) was added and the resulting mixture was stirred at room temperature overnight (or until no granular NiCl2.6H2O was observed) to afford a pale green suspension.
Note: All the solutions or suspensions kept under argon can be used for two weeks without appreciable deteriorations in reaction yields.
Borylation of redox-active esters with 2.5 mol % nickel loading followed General Procedure B/C with Suspension E/Solution F/Suspension G.
Examples of Ni-Catalyzed Borylation of Redox-Active Esters with 2.5 Mol % Nickel
Standard Reaction conditions: Redox active NHPI ester (1.0 equiv), NiCl2.6H2O (2.5 mol %), L1 (3.3 mol %), MgBr2.OEt2 (1.5 equiv), B2pin2 (3.3 equiv), MeLi (3.0 equiv), THF/DMF (2.5:1), 0° C.-RT, 2 h.
In further embodiments of the invention, the copper-catalyzed borylation of alkyl carboxylic acids to yield alkyl boronic acids, is disclosed and claimed herein. This invention entails the conversion of carboxylate functionalities into the corresponding boronic derivatives via copper-catalyzed decarboxylative borylation of redox active esters. In various embodiments, this transformation enables late-stage modifications of pharmaceuticals or analogs thereof; it allows the expedient syntheses of boron-containing bioactive molecules, including FDA-approved drugs; it allows for the preparations of boron-containing building blocks which have been broadly used in the syntheses of pharmaceutical ingredients. Compared with other methods, it allows simple procedure and inexpensive reagents which can be readily adopted in process chemistry.
Cu(II) salts were screened for borylation catalytic activity comprising copper complexed with various ligands, including bipyridyl-type ligands of formula L (e.g., 4,4′-di-t-Bu-2,2′-bipyridyl, L2, see above), and 1,3-dicarbonyl-type ligands that form with a copper ion (Cu(I) or Cu(II)) a complex of formula M (e.g., acetonylacetonate, M1).
M1 R1A=R2A=Me, Cu(acac)2
M2 R1A=R2A=tBu
M3 R1A=R2A=iPr
M4 R1A=R2A=Ph
M5 R1A, R2A=tBu, Me
M6 R1A=R2A=CF3
M7 R1A, R2A=tBu, CF3
Table 12, below, shows initial screening results using Cu salts (Cu(I) or Cu(II)) with various bases and ligands in carrying out the borylation reaction of the invention. As can be seen, use of phosphine ligands such as PPh3 and PCy3, even in combination with tBubipy (ligand L2) resulted in zero percent product or low yields, but Cu(I) salts with tBubipy with and without MgBr2 gave a better result.
Table 13 shows a study of results using varying loads of MgBr2, and various Cu salts. As can be seen, MgBr2 appears to inhibit the reaction; best results were obtained with tBubipy as ligand, no MgBr2, and CuCl, CuCl2, CuCl2.6H2O, CuI, and Cu(OAc)2 as copper sources; both Cu(I) and Cu(II) states were catalytically active in this system.
Table 14 shows the effect of solvent on the copper-catalyzed borylation reaction using lithium t-butoxide, Cu(OAc)2 (i.e., a Cu(II) salt) in the presence of the tBubipy ligand L2. the most effective solvent mixtures seem to be DMF plus an ether, such as dioxane, THF, or glyme, diglyme or diethyl ether, although DMF mixtures with other solvents such as CH2Cl2, EtOAc, or toluene, were also effective. Interestingly, pure DMF gave a lower yield.
Table 15 shows significant increases in yield obtained using LiOH and LiOtBu, even in the presence of MgBr2, with Cu(OAc)2 as the copper source and tBubipy L2 as the ligand.
Table 16 shows the effect on yield of solvent using variants of the preferred dixoxane/DMF solvent system, with the Cu(OAc)2/tBubipy catalytic complex, in the presence of LiOH/MgBr2-Et2O. As a replacement for dioxane, other ethers and esters seem to be most effective. As a replacement for DMF, other dipolar aprotic solvents were effective, as were pyridine and acetone.
Table 17 shows the effect on yield of catalysts and copper sources using Cu(OAc)2 as a copper source in the presence of LiOH and MgBr2-Et2O in dioxane-DMF solvent system. The effective ligands were much as seen before, the bipyridyl ligand L2 being more effective than phosphine type ligands, but interestingly it was noted that adding the 1,3-dicarbonyl ligand acetonylacetonate (acac) increased the yield when tBubipy L2 was the primary ligand for the copper complex.
Tables 18 and 19 show the effect on yield using various 1,3-dicarbonyl type ligands, M1-M7, as defined above, in the dioxane-DMF solvent system in the presence of LiOH and MgBr2.Et2O. Ligand M1, copper acetonylacetonate itself, along with other 1,3-dicarbonyl ligands M2-M7, were broadly effective, while when Cu(acac)2 was the primary catalyst, the presence of other Cu salts did not bring about large changes in the yields obtained.
Table 20 shows the yields of the copper-catalyzed borylation reactions in the dioxane-DMF solvent system, in the presence of LiOH and in the absence of any bipyridyl ligand L2, with various Mg sources and LiOH loadings. It was found that MgCl2 could be substituted for the more expensive MgBr2 with no loss in yield, while LiOH loadings beyond about 15 equivalents did not produce a significant yield increase.
Table 21 shows a final optimization of the reaction using Cu(acac)2 M1 in the presence of LiOH and MgCl2 in dioxane/DMF, with production cost estimate comparisons.
To a 15 mL culture tube equipped with a stir bar were added redox-active ester (0.2 mmol), B2Pin2 (76 mg, 1.5 eq), LiOH.H2O (126 mg, 15 eq), Cu(acac)2 (10.4 mg, 20 mol %) and MgCl2 (28.5 mg, 1.5 eq). The tube was evacuated and backfilled with argon for 3 times. Degassed dioxane/DMF (from Acros extra-dry bottles, 4/1, 1.4 mL) was added and the resulting mixture was stirred under 1000 rpm at RT for 30 min before diluted with EtOAc (7 mL) and washed with saturated NH4Cl (7 mL). The organic phase was dried over anhydrous Na2SO4, evaporated and purified by silica gel chromatography to give the desired product.
To a 50 mL flask equipped with a stir bar were added redox-active ester (1.2 g, 2.5 mmol), B2Pin2 (953 mg, 1.5 eq), LiOH.H2O (1.58 g, 15 eq), Cu(acac)2 (130 mg, 20 mol %) and MgCl2 (356 mg, 1.5 eq). The flask was evacuated and backfilled with argon for 3 times. The solid in the flask was stirred for 2 min before degassed dioxane/DMF (from Acros extra-dry bottles, 4/1, 17.5 mL) was added and the resulting mixture was stirred under 1000 rpm at RT for 30 min before diluted with Et2O (50 mL) and washed with saturated NH4Cl (30 mL) and brine (30 mL) successively. The organic phase was collected, dried over anhydrous Na2SO4, evaporated and purified by silica gel chromatography to afford the borylation product (625 mg, 60%).
For the adipic acid substrate, shown above, 69% GC yield was obtained on 0.2 mmol scale while 55% isolated yield on 3.5 mmol scale. The graphic, below, shows additional substrates and yields obtained in this reaction system.
The starting carboxylic acid (50 mg, 0.044 mmol) and N-hydroxyphalmide (6.0 mg, 0.047 mmol, 1.1 eq) was placed in an oven dried culture tube fixed with a stirbar. This was evacuated and backfilled with argon 3 times. To this was added DCM (0.5 mL) via syringe, creating a suspension. While stirring, N,N′-diisopropylcarbodiimide (9.5 μL, 7.7 mg, 0.047 mmol, 1.1 eq.) was added via syringe. After consumption of starting material as monitored by TLC (˜1 h), the solvent was blown off under a stream of nitrogen, and placed on high vacuum for 2 hours. After this, Cu(acac)2 (11.5 mg, 0.044 mmol, 1.0 eq.), B2pin2 (83.8 mg, 0.33 mmol, 7.5 eq.), LiOH.H2O (55.4 mg, 1.32 mmol, 30 eq.), and MgCl2 (31.4 mg, 0.33 mmol, 7.5 eq.) were quickly added to the tube and resealed. It was evacuated and backfilled with argon 3 times. A 6:1 mixture of dioxane/DMF (0.5 mL) was added to the reaction tube. The resulting mixture was then stirred vigorously for 45 minutes at room temperature. It was then quenched with 2 mL of saturated aqueous NH4Cl. This was extracted three times with EtOAc. The combined organic layers were rinsed with brine and dried over MgSO4. The solvent was removed under reduced pressure. The crude material was purified by a swift flash chromatography column (SiO2— 3% DCM in MeOH) to provide 31.2 mg semi-pure product as an off-white solid (˜50% yield). This material was dissolved in 0.4 mL of dioxane. To this was added 3 mL of 3M HCl (aq.). The resulting mixture was stirred at room temperature for 24 hours. The reaction was concentrated on a rotary evaporator. The resulting residue was dissolved in 3 mL of 1:1 MeCN/H2O and purified by preparatory HPLC (C18; gradient of H2O to MeCN each containing 0.1% formic acid) providing 2.1 mg.
On 8.75 mmol scale, General Procedure A was followed with 2-methyl-4-phenylbutanoic acid. Purification by flash column chromatography (silica gel, 1:9 EtOAc:hexanes) furnished 2 (2.31 g, 82%).
Physical state: colorless oil;
Rf=0.60 (silica gel, 3:7 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.92-7.88 (m, 2H), 7.81-7.78 (m, 2H), 7.32-7.29 (m, 2H), 7.27-7.25 (m, 2H), 7.22-7.20 (m, 1H), 2.90-2.74 (m, 3H), 2.20-2.14 (m, 1H), 1.96-1.90 (m, 1H), 1.40 (d, J=7.2 Hz, 3H) ppm;
13C NMR (151 MHz, CDCl3): δ 172.7, 162.2, 141.3, 134.9, 129.2, 128.7, 128.6, 126.2, 124.1, 36.7, 35.7, 33.1, 17.2 ppm;
HRMS (ESI-TOF, m/z): Calcd for C19H18NO4 [M+H]+ 324.1230; found 324.1230.
On 13 mmol scale, General Procedure A was followed with 2-methyl-4-phenylbutanoic acid. Purification by flash column chromatography (silica gel, 1:10 EtOAc:hexanes) furnished a yellow product. This compound was then recrystallized from CH2Cl2/MeOH to yield S1 (4.12 g, 69%).
Physical state: white solid;
m.p.=80-81° C.;
Rf=0.63 (silica gel, 1:4 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.32-7.28 (m, 2H), 7.26-7.19 (m, 3H), 2.89-2.72 (m, 3H), 2.20-2.13 (m, 1H), 1.95-1.88 (m, 1H), 1.39 (d, J=8.4 Hz, 3H) ppm;
13C NMR (151 MHz, CDCl3): δ 172.3, 157.8, 141.1, 141.0, 130.6, 128.6, 126.3, 124.9, 36.6, 35.5, 33.1, 17.3 ppm;
HRMS (ESI-TOF, m/z): Calcd for C19H14Cl4NO4 [M+H]+ 459.9671; found 459.9659.
On 5.0 mmol scale, General Procedure A was followed with 3-(2-bromophenyl) propanoic acid. Purification by flash column chromatography (silica gel, 1:9 EtOAc:hexanes) furnished S3 (1.63 g, 87%).
Physical state: white solid;
m.p.=158-160° C.;
Rf=0.36 (silica gel, 1:4 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.90-7.87 (m, 2H), 7.80-7.77 (m, 2H), 7.56 (dd, J=1.2 Hz, 7.8 Hz, 1H), 7.34 (dd, J=7.8 Hz, 1.8 Hz, 1H), 7.28 (dt, J=7.8 Hz, 1.2 Hz, 1H), 7.12 (dt, J=7.8 Hz, 1.8 Hz, 1H), 3.21 (t, J=7.2 Hz, 2H), 3.02 (t, J=7.2 Hz, 2H) ppm;
13C NMR (151 MHz, CDCl3): δ 168.8, 162.0, 138.5, 134.9, 133.1, 130.1, 129.0, 128.7, 127.9, 124.4, 124.1, 31.2, 31.0 ppm;
HRMS (ESI-TOF, m/z): Calcd for C17H13BrNO4 [M+H]+ 374.0022; found 374.0022.
On 5.0 mmol scale, General Procedure A was followed with 6-bromohexanoic acid. Purification by flash column chromatography (silica gel, 1:10 EtOAc:hexanes) furnished S4 (1.52 g, 89%).
Physical state: white solid;
m.p.=60-62° C.;
Rf=0.45 (silica gel, 1:4 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.89-7.86 (m, 2H), 7.79-7.77 (m, 2H), 3.42 (t, J=7.2 Hz, 2H), 2.68 (t, J=7.2 Hz, 2H), 1.94-1.89 (m, 2H), 1.84-1.79 (m, 2H), 1.63-1.57 (m, 2H) ppm;
13C NMR (151 MHz, CDCl3): δ 169.4, 162.0, 134.9, 129.0, 124.1, 33.3, 32.3, 30.9, 27.5, 24.0 ppm;
HRMS (ESI-TOF, m/z): Calcd for C14H15BrNO4 [M+H]+ 340.0179; found 340.0178.
On 3.0 mmol scale, General Procedure A was followed with Fmoc-Glu-OtBu. Purification by flash column chromatography (silica gel, 1:3 EtOAc:hexanes) furnished S5 (1.53 g, 89%).
Physical state: white foam;
Rf=0.49 (silica gel, 1:4 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.90-7.86 (m, 2H), 7.80-7.76 (m, 4H), 7.67-7.61 (m, 2H), 7.42-7.38 (m, 2H), 7.31 (dt, J=7.2 Hz, 1.2 Hz, 2H), 5.52 (br d, J=7.8 Hz, 1H), 4.50 (dd, J=10.8 Hz, 7.2 Hz, 1H), 4.39-4.36 (m, 2H), 4.23 (t, J=7.2 Hz, 1H), 2.82-2.77 (m, 1H), 2.73-2.67 (m, 1H), 2.40-2.34 (m, 1H), 2.15-2.09 (m, 1H), 1.50 (s, 9H) ppm;
13C NMR (151 MHz, CDCl3): δ 170.6, 169.1, 162.0, 156.2, 143.9, 141.5, 134.9, 129.0, 127.9, 127.2, 125.4, 125.2, 124.2, 120.1, 83.1, 67.2, 53.7, 47.4, 28.1, 28.0, 27.6 ppm;
HRMS (ESI-TOF, m/z): Calcd for C32H30N2NaO8 [M+Na]+ 593.1894; found 593.1895;
[α]D20=+5.4 (c 1.0, CHCl3).
On 5.0 mmol scale, General Procedure A was followed with 2-(4-bromophenyl)acetic acid. After completion of the reaction, reaction mixture was filtered through a short pad of silica gel and washed with EtOAc/hexanes (1:8). The filtrate was concentrated, and S6 was obtained after recrystallization with CH2Cl2/MeOH (1.52 g, 61%).
Physical state: pale yellow solid;
m.p.=212-213° C.;
Rf=0.57 (silica gel, 1:4 EtOAc:hexanes);
1H NMR (600 MHz, DMSO-d6): δ 7.61-7.59 (m, 2H), 7.37-7.35 (m, 2H), 4.25 (s, 2H) ppm;
13C NMR (151 MHz, DMSO-d6): δ 167.7, 157.5, 139.3, 131.7, 131.6, 131.6, 129.0, 125.2, 120.9, 35.8 ppm;
HRMS (ESI-TOF, m/z): Calcd for C16H7BrCl4NO4 [M+H]+ 495.8307; found 495.8323.
On 5.0 mmol scale, General Procedure A was followed with 2-methyl-3-phenylpropanoic acid. Purification by flash column chromatography (silica gel, 1:10 EtOAc:hexanes) furnished a yellow product which was recrystallized from CH2Cl2/MeOH to yield S7 (1.45 g, 65%).
Physical state: pale yellow solid;
m.p.=127-128° C.
Rf=0.63 (silica gel, 1:4 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.35-7.32 (m, 2H), 7.28-7.23 (m, 3H), 3.25 (dd, J=13.8 Hz, 6.6 Hz, 1H), 3.14-3.08 (m, 1H), 2.82 (dd, J=13.8 Hz, 7.8 Hz, 1H), 1.34 (d, J=7.2 Hz, 3H) ppm;
13C NMR (151 MHz, CDCl3): δ 171.9, 157.7, 141.2, 137.8, 130.6, 129.2, 128.8, 127.0, 124.9, 39.3, 39.0, 16.6 ppm;
HRMS (ESI-TOF, m/z): Calcd for C18H12Cl4NO4 [M+H]+ 445.9515; found 445.9516.
On 5.0 mmol scale, General Procedure A was followed with 2-phenylpropanoic acid. Purification by flash column chromatography (silica gel, 1:10 EtOAc:hexanes) furnished S8 (1.19 g, 81%).
Physical state: colorless oil;
Rf=0.21 (silica gel, 1:4 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.87-7.85 (m, 2H), 7.79-7.76 (m, 2H), 7.43-7.39 (m, 4H), 7.34-7.31 (m, 1H), 4.13 (q, J=7.2 Hz, 1H), 1.68 (d, J=7.2 Hz, 3H) pm;
13C NMR (151 MHz, CDCl3): δ 170.9, 162.0, 138.5, 134.9, 129.1, 129.1, 127.9, 127.7, 124.1, 43.1, 19.1 ppm;
HRMS (ESI-TOF, m/z): Calcd for C17H14NO4 [M+H]+ 296.0917; found 296.0920.
On 1.5 mmol scale, General Procedure A was followed with diphenylacetic acid. Purification by flash column chromatography (silica gel, 1:4 EtOAc:hexanes) furnished S9 (0.46 g, 86%).
Physical state: white solid;
m.p.=135-137° C.;
Rf=0.33 (silica gel, 1:4 EtOAc:hexanes)
1H NMR (600 MHz, CDCl3): δ 7.89-7.86 (m, 2H), 7.80-7.77 (m, 2H), 7.42-7.37 (m, 8H), 7.34-7.31 (m, 2H), 5.42 (s, 1H) ppm;
13C NMR (151 MHz, CDCl3): δ 169.2, 162.0, 136.9, 134.9, 129.1, 129.0, 128.9, 128.0, 124.1, 54.2 ppm;
HRMS (ESI-TOF, m/z): Calcd for C22H16NO4 [M+H]+ 358.1074; found 358.1078.
On 3.0 mmol scale, General Procedure A was followed with bicyclo[2.2.1]heptane-2-carboxylic acid (mixture of endo and exo). Purification by flash column chromatography (silica gel, 1:19 to 1:9 EtOAc:hexanes) furnished S10 (0.75 g, 88%) as mixture of exo/endo isomers.
Physical state: white solid;
Rf=0.41 (silica gel, 1:4 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.90-7.86 (m, 2H), 7.80-7.77 (m, 2H), 3.15-3.11 (m, 0.82H), 2.81 (br s, 0.82H), 2.77 (br d, J=4.2 Hz, 0.18H), 2.70 (dd, J=9.6 Hz, 6.0 Hz, 0.18H), 2.38 (br t, J=4.2 Hz, 0.18H), 2.35-2.33 (br, m, 0.82H), 2.00-1.96 (m, 0.18H), 1.86-1.81 (m, 0.82H), 1.74-1.70 (m, 0.82H), 1.63-1.67 (m, 3.28H), 1.51-1.44 (m, 1.64H), 1.38-1.25 (m, 1.26H) ppm;
13C NMR (151 MHz, CDCl3): δ171.5, 162.3, 134.8, 129.2, 124.0, 43.4, 41.0, 40.5, 37.0, 32.7, 29.0, 24.9 ppm (major isomer); 172.3, 162.3, 134.8, 129.2, 124.0, 43.7, 41.7, 36.7, 36.2, 34.6, 29.5, 28.6 ppm (minor isomer).
HRMS (ESI-TOF, m/z): Calcd for C16H16NO4 [M+H]+ 286.1074; found 286.1071.
On 3.0 mmol scale, General Procedure A was followed with trans-2-phenylcyclopropane-1-carboxylic acid. Upon complete consumption of starting material (TLC), the reaction mixture was filtered through celite, washed with CH2Cl2 (100 mL), and concentrated under reduced pressure. The crude product was purified by crystallization (CH2Cl2/MeOH) to furnish S11 (949 mg, 71%).
Physical state: pale yellow needle;
m.p.=203-205° C.;
Rf=0.48 ((silica gel, 1:9 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.34-7.31 (m, 2H), 7.28-7.25 (m, 1H), 7.18-7.16 (m, 2H), 2.80-2.77 (m, 1H), 2.22-2.19 (m, 1H), 1.84 (dt, J=10.2 Hz, 5.4 Hz, 1H), 1.69-1.66 (m, 1H) ppm;
13C NMR (151 MHz, CDCl3): δ 169.4, 157.7, 141.2, 138.3, 130.6, 128.8, 127.4, 126.5, 124.8, 28.8, 21.0, 18.6 ppm;
HRMS (ESI-TOF, m/z): Calcd for C18H10Cl4NO4 [M+H]+ 443.9358; found 443.9356.
On 5.0 mmol scale, General Procedure A was followed with 2,2-dimethyl-3-phenylpropanoic acid. Purification by flash column chromatography (silica gel, 1:10 EtOAc:hexanes) furnished S12 (1.36 g, 84%).
Physical state: white solid;
m.p.=70-72° C.;
Rf=0.45 (silica gel, 1:4 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.92-7.88 (m, 2H), 7.81-7.78 (m, 2H), 7.36-7.31 (m, 4H), 7.29-7.26 (m, 1H), 3.10 (s, 2H), 1.40 (s, 6H) ppm;
13C NMR (151 MHz, CDCl3): δ 173.7, 162.2, 136.5, 134.8, 130.6, 129.1, 128.3, 127.0, 124.0, 45.8, 43.3, 25.0 ppm;
HRMS (ESI-TOF, m/z): Calcd for C19H18NO4 [M+H]+ 324.1230; found 324.1232.
On 5.0 mmol scale, General Procedure A was followed with 2-phenylpropanoic acid. Purification by flash column chromatography (silica gel, 1:9 EtOAc:hexanes) furnished S13 (1.64 g, 81%).
Physical state: white solid;
m.p.=108-109° C.;
Rf=0.39 (silica gel, 1:4 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.87-7.84 (m, 2H), 7.78-7.75 (m, 2H), 7.54-7.52 (m, 2H), 7.44-7.41 (m, 2H), 7.34-7.31 (m, 1H), 2.64 (br d, J=13.2 Hz, 2H), 1.89-1.73 (m, 7H), 1.37-1.30 (m, 1H) ppm;
13C NMR (151 MHz, CDCl3): δ 171.8, 162.2, 142.3, 134.8, 129.2, 128.9, 127.7, 126.1, 124.0, 51.3, 35.5, 25.6, 23.6 ppm;
HRMS (ESI-TOF, m/z): Calcd for C21H20NO4 [M+H]+ 350.1387; found 350.1387.
On 5.0 mmol scale, general procedure A was followed with 2-methyl-2-phenylpropanoic acid. Purification by flash column chromatography (silica gel, 1:8 EtOAc:hexanes) furnished S14 (1.32 g, 85%).
Physical state: white solid;
m.p.=73-74° C.;
Rf=0.36 (silica gel, 1:4 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.88-7.85 (m, 2H), 7.79-7.75 (m, 2H), 7.51-7.49 (m, 2H), 7.44-7.41 (m, 2H), 7.34-7.31 (m, 1H), 1.79 (s, 6H) ppm;
13C NMR (151 MHz, CDCl3): δ 173.4, 162.1, 142.7, 134.8, 129.1, 128.8, 127.5, 125.9, 124.0 46.5, 27.0 ppm;
HRMS (ESI-TOF, m/z): Calcd for C18H16NO4 [M+H]+ 310.1074; found 310.1082.
On 0.44 mmol scale, General Procedure A was followed with Boc protected gabapentin. Purification by flash column chromatography (silica gel, 1:5 EtOAc:hexanes) furnished S15 (165 mg, 85%).
Physical state: white solid;
m.p.=76-79° C.;
Rf=0.32 (silica gel, 1:5 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.90-7.87 (m, 2H), 7.82-7.77 (m, 2H), 4.95 (br t, J=7.2 Hz, 1H), 3.38 (d, J=6.6 Hz, 2H), 2.63 (s, 2H), 1.65-1.43 (m, 10H), 1.44 (s, 9H) ppm;
13C NMR (151 MHz, CDCl3): δ 168.3, 162.1, 156.6, 135.0, 129.0, 124.2, 79.3, 46.9, 39.1, 37.8, 33.9, 28.5, 26.0, 21.6 ppm;
HRMS (ESI-TOF, m/z): Calcd for C22H29N2O6 [M+H]+ 417.2020; found 417.2022.
On 5.0 mmol scale, General Procedure A was followed with ibuprofen. Purification by flash column chromatography (silica gel, 1:9 EtOAc:hexanes) furnished S16 (1.48 g, 84%).
Physical state: colorless oil;
Rf=0.42 (silica gel, 1:4 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.87-7.85 (m, 2H), 7.79-7.76 (m, 2H), 7.31 (d, J=8.4 Hz, 2H), 7.17 (d, J=8.4 Hz, 2H), 4.10 (q, J=7.2 Hz, 1H), 2.48 (d, J=7.2 Hz, 2H), 1.91-1.84 (m, 1H), 1.67 (d, J=7.2 Hz, 3H), 0.91 (d, J=6.6 Hz, 6H) ppm;
13C NMR (151 MHz, CDCl3): δ 171.1, 162.0, 141.4, 135.7, 134.8, 129.8, 129.1, 127.4, 124.0, 45.2, 42.7, 30.3, 22.5, 19.2 ppm;
HRMS (ESI-TOF, m/z): Calcd for C21H22NO4 [M+H]+ 352.1543; found 352.1544.
On 1.0 mmol scale, General Procedure A was followed with gemfibrozil. Purification by flash column chromatography (silica gel, 1:25 EtOAc:hexanes) furnished S17 (0.33 g, 84%).
Physical state: white solid;
m.p.=65-67° C.;
Rf=0.50 (silica gel, 1:4 EtOAc:hexanes)
1H NMR (600 MHz, CDCl3): δ 7.90-7.87 (m, 2H), 7.80-7.77 (m, 2H), 7.01 (d, J=7.8 Hz, 1H), 6.67 (d, J=7.8 Hz, 1H), 6.66 (s, 1H), 4.02 (t, J=6.0 Hz, 2H), 2.32 (s, 3H), 2.20 (s, 3H), 1.95-2.00 (m, 4H), 1.46 (s, 6H) ppm;
13C NMR (151 MHz, CDCl3): δ 173.9, 162.2, 157.1, 136.6, 134.8, 130.4, 129.2, 124.0, 123.8, 120.8, 112.1, 67.9, 42.1, 37.5, 31.7, 25.3, 25.1, 21.5, 15.9 ppm;
HRMS (ESI-TOF, m/z): Calcd for C23H26NO5 [M+H]+ 396.1805; found 396.1803.
On 5.0 mmol scale, General Procedure A was followed with naproxen. Purification by flash column chromatography (silica gel, 1:7 EtOAc:hexanes) furnished S18 (1.65 g, 88%).
Physical state: white solid;
m.p.=110-111° C.;
Rf=0.53 (silica gel, 2:3 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.86 (br s, 2H), 7.80-7.75 (m, 5H), 7.49 (dd, J=8.4 Hz, 1.8 Hz, 1H), 7.17 (dd, J=8.4 Hz, 2.4 Hz, 1H), 7.14 (d, J=2.4 Hz, 1H), 4.26 (q, J=7.2 Hz, 1H), 3.92 (s, 3H), 1.75 (d, J=7.2 Hz, 3H) ppm;
13C NMR (151 MHz, CDCl3): δ 171.1, 162.0, 158.0, 134.9, 134.1, 133.6, 129.6, 129.1, 127.7, 126.5, 126.0, 124.1, 119.3, 105.8, 55.5, 43.1, 19.2 ppm;
HRMS (ESI-TOF, m/z): Calcd for C22H18NO5 [M+H]+ 376.1179; found 376.1183.
On 5.0 mmol scale, General Procedure A was followed with azelaic acid (5.0 mmol, 1.0 equiv), NHPI (10 mmol, 2.0 equiv), DIC (11 mmol, 2.2 equiv) and DMAP (1 mmol, 0.2 equiv). Purification by flash column chromatography (silica gel, 1:10 EtOAc:CH2Cl2) furnished S19 (1.52 g, 64%).
Physical state: white solid;
m.p.=103-105° C.;
Rf=0.55 (silica gel, 1:1 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.90-7.87 (m, 4H), 7.81-7.78 (m, 4H), 2.69 (t, J=4.8 Hz, 4H), 1.85-1.80 (m, 4H), 1.53-1.48 (m, 4H), 1.45-1.42 (m, 2H) ppm;
13C NMR (151 MHz, CDCl3): δ 169.1, 161.5, 134.3, 128.5, 123.5, 30.5, 28.1, 28.0, 24.1 ppm;
HRMS (ESI-TOF, m/z): Calcd for C25H23N2O8 [M+H]+ 479.1449; found 479.1451.
On 1.0 mmol scale, General Procedure A was followed with chlorambucil. Purification by flash column chromatography (silica gel, 1:4 EtOAc:hexanes) furnished S20 (431 mg, 96%).
Physical state: yellow oil;
Rf=0.23 (silica gel, 1:4 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.91-7.87 (m, 2H), 7.81-7.77 (m, 2H), 7.12 (d, J=8.5 Hz, 2H), 6.65 (d, J=9.0 Hz, 2H), 3.66 (AB t, J=6.7 Hz, 4H), 3.63 (BA t, J=6.7 Hz, 4H), 2.67 (dt, J=7.5 Hz, 16 Hz, 4H), 2.09-2.02 (m, 2H) ppm;
13C NMR (151 MHz, CDCl3): δ 169.6, 162.1, 144.7, 134.9, 130.0, 129.1, 124.1, 112.4, 53.8, 40.7, 33.6, 30.3, 26.6 ppm;
HRMS (ESI-TOF, m/z): Calcd for C22H23Cl2N2O4 [M+H]+ 449.1029; found 449.1009.
On 5.0 mmol scale, General Procedure A was followed with ketoprofen. Purification by flash column chromatography (silica gel, 1:3 EtOAc:hexanes) furnished S21 (1.91 g, 96%).
Physical state: white solid;
m.p.=118-120° C.;
Rf=0.45 (silica gel, 1:2 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.88-7.84 (m, 5H), 7.80-7.76 (m, 3H), 7.67 (dt, J=8.4 Hz, 1.2 Hz, 1H), 7.61-7.58 (m, 1H), 7.55-7.48 (m, 3H), 4.20 (q, J=7.2 Hz, 1H), 1.71 (d, J=7.2 Hz, 3H) ppm;
13C NMR (151 MHz, CDCl3): δ 196.4, 170.6, 161.9, 138.7, 138.4, 137.5, 134.9, 132.7, 131.7, 130.3, 129.8, 129.5, 129.1, 129.1, 128.5, 124.1, 43.0, 19.0 ppm;
HRMS (ESI-TOF, m/z): Calcd for C24H18NO5 [M+H]+ 400.1179; found 400.1181.
On 5.0 mmol scale, General Procedure A was followed with fenoprofen. Purification by flash column chromatography (silica gel, 1:8 EtOAc:hexanes) furnished S22 (1.83 g, 94%).
Physical state: colorless oil;
Rf=0.50 (silica gel, 1:4 EtOAc:hexanes)
1H NMR (600 MHz, CDCl3): δ 7.89-7.85 (m, 2H), 7.79-7.76 (m, 2H), 7.37-7.33 (m, 3H), 7.16 (dt, J=7.8 Hz, 1.2 Hz, 1H), 7.13-7.10 (m, 1H), 7.09 (t, J=2.4 Hz, 1H), 7.07-7.04 (m, 2H), 6.95 (ddd, J=7.8 Hz, 2.4 Hz, 0.6 Hz, 1H), 4.09 (q, J=7.2 Hz, 1H). 1.67 (d, J=7.2 Hz, 3H) ppm;
13C NMR (151 MHz, CDCl3): δ 170.6, 161.9, 157.8, 157.1, 140.3, 134.9, 130.3, 129.9, 129.1, 124.1, 123.5, 122.5, 119.2, 118.4, 118.2, 42.9, 19.0 ppm;
HRMS (ESI-TOF, m/z): Calcd for C23H18NO5[M+H]+ 388.1179; found 388.1178.
On 0.5 mmol scale, General Procedure A was followed with ketal ester of atorvastatin. Purification by flash column chromatography (silica gel, 1:2 EtOAc:hexanes) furnished S23 (0.35 g, 95%).
Physical state: yellow foam;
Rf=0.35 (silica gel, 1:2 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.90-7.87 (m, 2H), 7.81-7.77 (m, 2H), 7.21-7.15 (m, 9H), 7.08 (d, J=8.4 Hz, 2H), 7.02-6.97 (m, 3H), 6.88 (br s, 1H), 4.33-4.28 (m, 1H), 4.14-4.07 (m, 1H), 3.89-3.84 (m, 1H), 3.75-3.71 (m, 1H), 3.61-3.56 (m, 1H), 2.85 (dd, J=15.6 Hz, 6.6 Hz, 1H), 2.69 (dd, J=15.0 Hz, 6.6 Hz, 1H), 1.76-1.70 (m, 2H), 1.55-1.53 (m, 7H), 1.40 (s, 3H), 1.35 (s, 3H), 1.18 (dd, J=12.0 Hz, 5.4 Hz, 1H) ppm;
13C NMR (151 MHz, CDCl3): δ 166.9, 164.9, 162.4 (d, J=247.8 Hz), 161.9, 141.6, 138.5, 134.9, 134.8, 133.3 (d, J=8.0 Hz), 130.6, 129.0, 128.9, 128.8, 128.4, 128.3 (d, J=3.6 Hz), 126.7, 124.1, 123.6, 121.9, 119.7, 115.5 (d, J=21.3 Hz), 99.2, 66.4, 65.6, 40.9, 38.4, 38.1, 35.8, 29.9, 26.2, 21.9, 21.7, 19.7 ppm;
19F NMR (376 MHz, CDCl3): δ −113.91 ppm;
HRMS (ESI-TOF, m/z): Calcd for C44H43FN3O7[M+H]+ 744.3080; found 744.3061.
[α]D20=+25.1 (c 1.0, CHCl3).
On 1.0 mmol scale, General Procedure A was followed with acetyl enoxolone. Purification by flash column chromatography (silica gel, 1:5 EtOAc:hexanes) afforded S24 (0.49 g, 75%).
Physical State: white solid;
m.p.=264° C.;
Rf=0.57 (silica gel, 2:3 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.89-7.86 (m, 2H), 7.80-7.77 (m, 2H), 5.76 (s, 1H), 4.51 (dd, J=11.8, 4.6 Hz, 1H), 2.79 (dt, J=13.7, 3.7 Hz, 1H), 2.45 (ddd, J=13.7, 4.3, 1.7 Hz, 1H), 2.35 (s, 1H), 2.15-2.11 (m, 1H), 2.11-2.00 (m, 2H), 2.04 (s, 3H), 1.86 (td, J=13.7, 4.7 Hz, 1H), 1.79 (t, J=13.7 Hz, 1H), 1.74-1.55 (m, 4H), 1.51-1.40 (m, 4H), 1.43 (s, 3H), 1.37 (s, 3H), 1.20 (ddd, J=13.8, 4.6, 2.4 Hz, 1H), 1.15 (s, 3H), 1.14 (s, 3H), 1.10-1.01 (m, 3H), 0.90 (s, 3H), 0.87 (s, 6H), 0.80 (dd, J=11.9, 1.8 Hz, 1H) ppm;
13C NMR (151 MHz, CDCl3): δ 200.0, 172.7, 171.1, 168.5, 162.2, 134.8, 129.2, 129.0, 124.0, 80.8, 61.9, 55.2, 47.9, 45.5, 44.0, 43.3, 41.3, 38.9, 38.2, 37.4, 37.1, 32.9, 32.0, 31.6, 28.5, 28.2, 28.1, 26.6, 26.6, 23.7, 23.4, 21.5, 18.8, 17.5, 16.8, 16.6 ppm;
HRMS (ESI-TOF, m/z): Calcd for C40H52NO7 [M+H]+ 658.3738; found 658.3736;
[α]D20=+191.0 (c 1.0, CHCl3).
On 1.0 mmol scale, General Procedure A was followed with mycophenolic acid. Purification by flash column chromatography (silica gel, 1:4 EtOAc:hexanes) furnished S25 (0.36 g, 78%).
Physical state: white solid;
m.p.=126-128° C.;
Rf=0.40 (silica gel, 1:3 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.88-7.85 (m, 2H), 7.79-7.77 (m, 2H), 7.68 (s, 1H), 5.34 (t, J=7.2 Hz, 1H), 5.19 (s, 2H), 3.77 (s, 3H), 3.42 (d, J=6.6 Hz, 2H), 2.76 (t, J=7.8 Hz, 2H), 2.45 (t, J=7.8 Hz, 2H), 2.15 (s, 3H), 1.85 (s, 3H) ppm;
13C NMR (151 MHz, CDCl3): δ 173.0, 169.3, 163.8, 162.0, 153.8, 144.2, 134.9, 133.1, 129.0, 124.1, 123.9, 122.0, 116.8, 106.5, 70.2, 61.2, 34.1, 29.9, 22.8, 16.2, 11.7 ppm;
HRMS (ESI-TOF, m/z): Calcd for C25H24NO8 [M+H]+ 466.1496; found 466.1499.
The preparation and spectral data of the following RAEs have been reported.i 22-26
On 0.2 mmol scale, General Procedure B was followed with NHPI ester (2) and suspension B (NiCl2.6H2O/di-MeObipy in DMF). Purification by flash column chromatography (silica gel, hexanes to 1:35 Et2O:hexanes) afforded 3 (32.7 mg, 63%).
Physical state: colorless oil;
Rf=0.49 (silica gel, 1:12 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.28-7.25 (m, 2H), 7.20-7.14 (m, 3H), 2.66-2.58 (m, 2H), 1.82-1.76 (m, 1H), 1.62-1.57 (m, 1H), 1.25 (s, 12H), 1.10-1.05 (m, 1H), 1.02 (d, J=7.2 Hz, 3H) ppm;
13C NMR (151 MHz, CDCl3): δ 143.2, 128.6, 128.3, 125.6, 83.0, 35.5, 35.4, 25.0, 24.9, 15.7 ppm;
Spectroscopic data matches that reported in the literature.2
On 0.2 mmol scale, General Procedure B was followed with NHPI ester (S2) and solution B (NiCl2.6H2O/di-MeObipy in DMF). Purification by flash column chromatography (silica gel, hexanes to 1:30 Et2O:hexanes) afforded 4 (34.0 mg, 65%).
Physical state: colorless oil;
Rf=0.50 (silica gel, 1:12 EtOAc:Hexane);
1H NMR (600 MHz, CDCl3): δ 7.28-7.25 (m, 2H), 7.18-7.15 (m, 3H), 2.61 (t, J=7.8 Hz, 2H), 1.66-1.61 (m, 2H), 1.50-1.45 (m, 2H), 1.24 (s, 12H), 0.82 (t, J=7.8 Hz, 2H) ppm;
13C NMR (151 MHz, CDCl3): δ 143.1, 128.5, 128.3, 125.6, 83.0, 35.9, 34.3, 25.0, 23.9 ppm;
Spectroscopic data matches that reported in the literature.3
On 0.2 mmol scale, General Procedure B was followed with NHPI ester (S26) and solution B (NiCl2.6H2O/di-MeObipy in DMF). Purification by flash column chromatography (silica gel, hexanes to 1:100 CH2Cl2:hexanes) afforded 5 (25.2 mg, 52%).
Physical state: colorless oil;
Rf=0.55 (silica gel, 1:6 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 3.64 (s, 3H), 2.29 (t, J=7.2 Hz, 2H), 1.64-1.59 (m, 2H), 1.45-1.40 (m, 2H), 1.23 (s, 12H), 0.78 (t, J=7.8 Hz, 2H) ppm;
13C NMR (151 MHz, CDCl3): δ 174.4, 83.1, 51.6, 34.1, 27.7, 25.0, 23.8 ppm;
HRMS (ESI-TOF, m/z): Calcd for C12H24BO4 [M+H]+ 243.1762; found 243.1765.
On 0.2 mmol scale, General Procedure B was followed with NHPI ester (S3) and solution B (NiCl2.6H2O/di-MeObipy in DMF). Purification by flash column chromatography (silica gel, hexanes to 1:30 Et2O:hexanes) afforded 6 (34.3 mg, 55%).
Physical state: colorless oil;
Rf=0.55 (silica gel, 1:12 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.50 (d, J=7.8 Hz, 1H), 7.27 (d, J=7.8 Hz, 1H), 7.21 (t, J=7.8 Hz, 1H), 7.02 (t, J=7.8 Hz, 1H), 2.84 (t, J=7.8 Hz, 2H), 1.24 (s, 12H), 1.15 (t, J=7.8 Hz, 2H) ppm;
13C NMR (151 MHz, CDCl3): δ 143.7, 132.8, 129.9, 127.4, 127.4, 124.5, 83.32, 30.6, 25.0 ppm;
HRMS (ESI-TOF, m/z): Calcd for C14H21BBrO2 [M+H]+ 313.0798; found 313.0799.
On 0.2 mmol scale, General Procedure B was followed with NHPI ester (S4) and solution B (NiCl2.6H2O/di-MeObipy in DMF). Purification by flash column chromatography (silica gel, hexanes to 1:30 Et2O:hexanes) afforded 7 (36.0 mg, 65%).
Physical state: colorless oil;
Rf=0.55 (silica gel, 1:12 EtOAc:hexanes)
1H NMR (600 MHz, CDCl3): δ 3.40 (t, J=7.2 Hz, 2H), 1.88-1.83 (m, 2H), 1.45-1.42 (m, 4H), 1.24 (s, 12H), 0.80-0.77 (m, 2H) ppm;
13C NMR (151 MHz, CDCl3): δ 83.1, 34.2, 32.8, 31.0, 25.0, 23.4 ppm;
HRMS (ESI-TOF, m/z): Calcd for C11H23BBrO2 [M+H]+ 277.0969; found 277.0968.
On 0.2 mmol scale, General Procedure B was followed with NHPI ester (S5) and solution B (NiCl2.6H2O/di-MeObipy in DMF). Purification by flash column chromatography (silica gel, 1:12 EtOAc:hexanes to 1:6 EtOAc:hexanes to 1:4 EtOAc:hexanes) afforded 8 (37.6 mg, 37%).
Physical state: colorless oil;
Rf=0.40 (silica gel, 1:4 EtOAc:hexanes)
1H NMR (600 MHz, CDCl3): δ 7.76 (d, J=7.2 Hz, 2H), 7.61 (d, J=7.2 Hz, 2H), 7.41-7.38 (m, 2H), 7.33-7.30 (m, 2H), 5.53 (d, J=8.4 Hz, 1H), 4.34-4.24 (m, 2H), 4.23-4.19 (m, 2H), 1.97-1.91 (m, 1H), 1.84-1.78 (m, 1H), 1.47 (s, 9H), 1.23 (s, 12H), 0.89-0.78 (m, 2H) ppm;
13C NMR (151 MHz, CDCl3): δ 171.8, 156.2, 144.2, 144.1, 141.4, 127.8, 127.2, 125.3, 120.1, 83.5, 81.9, 67.0, 56.1, 47.4, 28.2, 27.0, 25.0, 24.9 ppm;
HRMS (ESI-TOF, m/z): Calcd for C29H38BNNaO6 [M+Na]+ 530.2684; found 530.2685;
[α]D20=+2.3 (c 0.35, CHCl3).
On 0.2 mmol scale, General Procedure C was followed with TCNHPI ester (S6) and suspension C (NiCl2.6H2O/di-tBubipy in THF). Purification by flash column chromatography (silica gel, 1:40 to 1:20 Et2O:hexanes) afforded 9 (30.5 mg, 51%).
Physical State: colorless oil;
Rf=0.30 (silica gel, 1:19 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.35-7.33 (m, 2H), 7.06-7.04 (m, 2H), 2.23 (s, 2H), 1.23 (s, 12H) ppm;
13C NMR (151 MHz, CDCl3): δ 137.8, 131.4, 130.9, 118.7, 83.7, 24.9 ppm;
Spectroscopic data matches that reported in the literature.4
On 0.2 mmol scale, General Procedure C was followed with TCNHPI ester (S7) and suspension C (NiCl2.6H2O/di-tBubipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:35 Et2O:hexanes) afforded 10 (33.1 mg, 67%).
Physical state: colorless oil;
Rf=0.53 (silica gel, 1:12 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.26-7.13 (m, 5H), 2.81 (dd, J=13.8 Hz, 7.8 Hz, 1H), 2.54 (dd, J=13.8 Hz, 7.8 Hz, 1H), 1.41-1.34 (m, 1H), 1.19 (s, 6H), 1.18 (s, 6H), 0.97 (d, J=7.8 Hz, 3H) ppm;
13C NMR (151 MHz, CDCl3): δ 142.5, 129.0, 128.1, 125.7, 83.1, 39.1, 24.9, 15.3 ppm;
Spectroscopic data matches that reported in the literature.5
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S8), MgBr2.OEt2 (52 mg, 0.2 mmol, 1 equiv) and suspension A (NiCl2.6H2O/di-MeObipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:30 Et2O:hexanes) afforded 11 (33.8 mg, 73%).
Physical State: colorless oil;
Rf=0.33 (silica gel, 1:19 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.27-7.21 (m, 4H), 7.15-7.12 (m, 1H), 2.44 (q, J=7.8 Hz, 1H), 1.33 (d, J=7.8 Hz, 3H), 1.21 (s, 6H), 1.20 (s, 6H) ppm;
13C NMR (151 MHz, CDCl3): δ 145.1, 128.4, 127.9, 125.2, 83.4, 24.8, 24.7, 17.2 ppm;
Spectroscopic data matches that reported in the literature.2
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S9) and suspension A (NiCl2.6H2O/di-MeObipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:30 Et2O:hexanes) afforded 12 (41.0 mg, 69%).
Physical State: colorless oil;
Rf=0.41 (silica gel, 1:9 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.30-7.25 (m, 8H), 7.19-7.15 (m, 2H), 3.88 (s, 1H), 1.24 (s, 12H) ppm;
13C NMR (151 MHz, CDCl3): δ 142.2, 129.2, 128.5, 125.7, 83.9, 24.7 ppm;
Spectroscopic data matches that reported in the literature.6
On 0.2 mmol scale, General Procedure C was followed with TCNHPI ester (S27) and suspension C (NiCl2.6H2O/di-tBubipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:45 Et2O:hexanes) afforded 13 (23.0 mg, 47%).
Physical state: colorless oil;
Rf=0.45 (silica gel, 1:9 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 2.02-1.95 (m, 2H), 1.82-1.78 (m, 2H), 1.75-1.58 (m, 4H), 1.23 (s, 12H), 1.00-0.96 (m, 1H) ppm;
13C NMR (151 MHz, CDCl3): δ 123.9 (t, J=239.9 Hz), 83.4, 34.5 (t, J=23.3 Hz), 24.9, 24.4 (t, J=4.6 Hz) ppm;
HRMS (ESI-TOF, m/z): High-resolution mass spec data could not be obtained for this compound.
On 0.2 mmol scale, General Procedure C was followed with TCNHPI ester (S26) and suspension C (NiCl2.6H2O/di-tBubipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:40 Et2O:hexanes) afforded 14 (25.6 mg, 57%).
Physical state: colorless oil;
Rf=0.42 (silica gel, 1:19 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 1.45-1.22 (m, 20H), 0.90-0.86 (m, 7H) ppm;
13C NMR (151 MHz, CDCl3): δ 82.9, 31.7, 30.8, 25.0, 24.4, 23.1, 14.3, 13.9 ppm;
Spectroscopic data matches that reported in the literature.
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S29) and suspension C (NiCl2.6H2O/di-tBubipy in THF). Purification by flash column chromatography (first flash column chromatography: deactivate silica gel, hexanes to 1:9 EtOAc:hexanes; second flash column chromatography (deactivated silica gel, CH2Cl2) afforded 15 (39.2 mg, 66%).
Physical State: colorless oil;
Rf=0.45 (silica gel, 1:4 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 3.42-2.99 (m, 3H), 2.09-1.65 (m, 4H), 1.43 (s, 9H), 1.26-1.22 (m, 12H) ppm;
13C NMR (151 MHz, CDCl3): δ 155.1, 83.6, 79.1, 46.1, 28.7, 27.9, 27.3 25.2, 25.0, 24.6 ppm;
HRMS (ESI-TOF, m/z): Calcd for C15H29BNO4 [M+H]+ 298.2184; found 298.2179;
[α]D20=0 (c 0.3, CHCl3).
On 0.2 mmol scale, General Procedure B was followed with NHPI ester (S10) and solution B (NiCl2.6H2O/di-MeObipy in DMF). Purification by flash column chromatography (silica gel, hexanes to 1:40 Et2O:hexanes to 1:20 Et2O:hexanes) afforded 16 (24.4 mg, 55%) as mixture of exo/endo isomers.
Physical state: colorless oil;
Rf=0.38 (silica gel, 1:19 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 2.28-2.27 (m, 1H), 2.22-2.21 (m, 1H), 1.56-1.44 (m, 3H), 1.37-1.33 (m, 1H), 1.26-1.21 (m, 14H), 1.20-1.14 (m, 2H), 0.89-0.86 (m, 1H) ppm;
13C NMR (151 MHz, CDCl3): δ 82.9, 38.9, 38.3, 36.8, 32.4, 32.3, 29.4, 24.9 ppm (exo); 83.0, 41.1, 39.1, 37.2, 32.3, 30.0, 28.0, 25.1, 25.0 ppm (endo);
Spectroscopic data matches that reported in the literature.8
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S28) and suspension A (NiCl2.6H2O/di-MeObipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:30 Et2O:hexanes) afforded 17 (30.9 mg, 59%).
Physical state: colorless oil;
Rf=0.55 (silica gel, 1:9 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 2.06-2.04 (m, 2H), 1.90-1.67 (m, 12H), 1.37-1.35 (m, 1H), 1.25 (s, 12H) ppm;
13C NMR (151 MHz, CDCl3): δ 82.9, 39.5, 37.9, 36.4, 29.5, 28.4, 28.3, 25.0 ppm;
Spectroscopic data matches that reported in the literature.2
On 0.2 mmol scale, General Procedure B was followed with TCNHPI ester (S11) and solution D (NiCl2.6H2O/di-tBubipy in DMF). Purification by flash column chromatography (silica gel, hexanes to 1:30 Et2O:hexanes) afforded 18 (11.3 mg, 23%, dr>20:1).
Physical State: colorless oil;
Rf=0.48 (silica gel, 1:9 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.25-7.22 (m, 2H), 7.15-7.11 (m, 1H), 7.09-7.06 (m, 2H), 2.10 (dt, J=7.8 Hz, 5.4 Hz, 1H), 1.25 (s, 6H), 1.24 (s, 6H), 1.17-1.14 (m, 1H), 1.02-0.99 (m, 1H), 0.32-0.29 (m, 1H) ppm;
13C NMR (151 MHz, CDCl3): δ 143.5, 128.4, 125.8, 125.7, 83.3, 24.9, 24.8, 22.0, 15.2 ppm;
HRMS (ESI-TOF, m/z): Calcd for C15H22BO2 [M+H]+ 245.1707; found 245.1714.
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S12) and suspension A (NiCl2.6H2O/di-MeObipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:30 Et2O:hexanes) afforded 19 (35.3 mg, 68%).
Physical State: colorless solid;
Rf=0.50 (silica gel, 1:12 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.24-7.19 (m, 4H), 7.17-7.14 (m, 1H), 2.61 (s, 2H), 1.21 (s, 12H), 0.94 (s, 6H) ppm;
13C NMR (151 MHz, CDCl3): δ 140.6, 130.3, 127.8, 125.8, 83.24, 46.5, 24.9 ppm.
Spectroscopic data matches that reported in the literature.2
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S31) and suspension C (NiCl2.6H2O/di-tBubipy in THF). Purification by flash column chromatography (silica gel, 1:60 Et2O:hexanes to 1:40 Et2O:hexanes) afforded 20 (29.2 mg, 56%).
Physical State: white amorphous solid;
Rf=0.60 (silica gel, 1:9 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 1.84 (br s, 3H), 1.75 (br t, J=3.6 Hz, 12H), 1.20 (s, 12H) ppm;
13C NMR (151 MHz, CDCl3): δ 82.7, 38.1, 37.6, 27.7, 24.8 ppm;
Spectroscopic data matches that reported in the literature.2
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S32) and suspension A (NiCl2.6H2O/di-MeObipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:15:15 Et2O:CH2Cl2:hexanes) afforded 21 (26.2 mg, 46%).
Physical State: white solid;
m.p.=152-155° C.;
Rf=0.45 (silica gel, 1:6 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 4.30-4.28 (m, 3H), 4.03-4.01 (m, 3H), 3.70 (s, 3H), 1.26 (s, 12H);
13C NMR (151 MHz, CDCl3): δ 172.8, 83.4, 55.4, 51.6, 47.0, 45.2, 24.9 ppm;
HRMS (ESI-TOF, m/z): Calcd for C16H22BO4 [M+H]+ 289.1606; found 289.1607.
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S34) and suspension C (NiCl2.6H2O/di-tBubipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:9 Et2O:hexanes) afforded 22 (31.1 mg, 53%).
Physical State: colorless solid; Sublimation at 100° C.;
Rf=0.39 (silica gel, 1:5 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 3.62 (s, 3H), 1.72-1.65 (m, 6H), 1.62-1.54 (m, 6H), 1.19 (s, 12H) ppm;
13C NMR (151 MHz, CDCl3): δ 179.0, 83.0, 51.7, 38.6, 27.9, 26.7, 24.8 ppm;
HRMS (ESI-TOF, m/z): Calcd for C16H28BO4 [M+H]+ 295.2075; found 295.2077.
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S33) and suspension A (NiCl2.6H2O/di-MeObipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:30 Et2O:hexanes) afforded 23 (27.8 mg, 62%).
Physical State: colorless oil;
Rf=0.50 (silica gel, 1:12 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 1.84-1.80 (m, 2H), 1.64-1.57 (m, 3H), 1.29-1.21 (m, 14H), 1.16-1.08 (m, 1H), 0.92-0.87 (m, 5H) ppm;
13C NMR (151 MHz, CDCl3): δ 82.9, 37.2, 26.6, 26.0, 25.7, 24.8 ppm;
Spectroscopic data matches that reported in the literature.2
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S13) and suspension C (NiCl2.6H2O/di-tBubipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:30 Et2O:hexanes) afforded 24 (28.5 mg, 50%).
Physical State: white solid;
m.p.=87-88° C.;
Rf=0.60 (silica gel, 1:9 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.36-7.34 (m, 2H), 7.29-7.26 (m, 2H), 7.13-7.11 (m, 1H), 2.36-2.32 (m, 2H), 1.82-1.78 (m, 2H), 1.70-1.66 (m, 1H), 1.49-1.38 (m, 4H), 1.21-1.14 (m, 1H), 1.17 (s, 12H) ppm;
13C NMR (151 MHz, CDCl3): δ 147.6, 128.2, 126.3, 125.1, 83.4, 35.0, 26.4, 25.9, 25.7 ppm;
HRMS (ESI-TOF, m/z): Calcd for C18H28BO2 [M+H]+ 287.2177; found 287.2184.
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S30) and suspension A (NiCl2.6H2O/di-MeObipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:30 Et2O:hexanes) afforded 25 (41.2 mg, 66%).
Physical State: colorless oil;
Rf=0.40 (silica gel, 1:12 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 3.39 (s, 2H), 1.22 (s, 12H), 0.90 (s, 6H), 0.88 (s, 9H), 0.01 (s, 6H) ppm;
13C NMR (151 MHz, CDCl3): δ 83.0, 72.0, 26.1, 24.9, 21.4, 18.5, −5.34 ppm;
HRMS (ESI-TOF, m/z): Calcd for C16H36BO3Si [M+H]+ 315.2521; found 315.2523.
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S14) and suspension C (NiCl2.6H2O/di-tBubipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:30 Et2O:hexanes) afforded 26 (23.3 mg, 47%).
Physical State: colorless oil;
Rf=0.51 (silica gel, 1:9 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.33-7.27 (m, 4H), 7.15-7.12 (m, 1H), 1.35 (s, 6H), 1.20 (s, 12H) ppm;
13C NMR (151 MHz, CDCl3): δ 148.8, 128.2, 126.4, 125.1, 83.4, 25.7, 24.7 ppm;
Spectroscopic data matches that reported in the literature.9
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S34) and suspension A (NiCl2.6H2O/di-MeObipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:30 Et2O:hexanes) afforded 27 (32.9 mg, 59%).
Physical State: White solid;
Rf=0.37 (silica gel, 1:19 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.19 (s, 4H), 1.21 (s, 12H), 1.11 (dd, J=6.0 Hz, 3.6 Hz, 2H), 0.87 (dd, J=6.0 Hz, 3.6 Hz, 2H) ppm;
13C NMR (151 MHz, CDCl3): δ 143.5, 131.0, 130.5, 128.2, 83.6, 24.7, 13.6 ppm;
HRMS (ESI-TOF, m/z): Calcd for C15H21BClO2 [M+H]+ 279.1318; found 279.1319.
On 0.1 mmol scale, General Procedure B was followed with NHPI ester (S15) and solution B (NiCl2.6H2O/di-MeObipy in DMF). Purification by flash column chromatography (silica gel, 1:20 EtOAc:hexanes) afforded 28 (22.5 mg, 64%).
Physical State: white solid;
m.p.=92-96° C.;
Rf=0.28 (silica gel, 1:20 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 5.32 (br s, 1H), 3.08 (d, J=6.0 Hz, 2H), 1.52-1.41 (m, 4H), 1.43 (s, 9H), 1.38-1.31 (m, 6H), 1.25 (s, 12H), 0.81 (s, 2H) ppm;
13C NMR (151 MHz, CDCl3): δ 156.5, 83.4, 78.7, 50.0, 36.7, 36.3, 28.6, 26.4, 25.0, 21.9 ppm;
HRMS (ESI-TOF, m/z): Calcd for C19H37BNO4 [M+H]+ 354.2810; found 354.2809.
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S16), MgBr2.OEt2 (52 mg, 0.2 mmol, 1 equiv) and suspension A (NiCl2.6H2O/di-MeObipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:30 Et2O:hexanes) afforded 29 (43.0 mg, 75%).
Physical State: colorless oil;
Rf=0.59 (silica gel, 1:9 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.12-7.10 (m, 2H), 7.04-7.02 (m, 2H), 2.42 (d, J=7.2 Hz, 2H), 2.40 (q, J=7.2 Hz, 1H), 1.79-1.88 (m, 1H), 1.31 (d, J=7.2 Hz, 3H), 1.21 (s, 6H), 1.20 (s, 6H), 0.89 (d, J=6.6 Hz, 6H) ppm;
13C NMR (151 MHz, CDCl3): δ 142.1, 138.4, 129.2, 127.6, 83.4, 45.2, 30.4, 24.8, 24.7, 22.6, 17.2 ppm;
Spectroscopic data matches that reported in the literature.10
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S17) and suspension A (NiCl2.6H2O/di-MeObipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:30 Et2O:hexanes) afforded 30 (36.3 mg, 55%).
Physical State: colorless solid;
m.p.=59-61° C.;
Rf=0.60 (silica gel, 1:12 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 6.99 (d, J=7.8 Hz, 1H), 6.64 (d, J=7.2 Hz, 1H), 6.62 (s, 1H), 3.92 (t, J=6.6 Hz, 2H), 2.30 (s, 3H), 2.18 (s, 3H), 1.78-1.73 (m, 2H), 1.41-1.44 (m, 2H), 1.23 (s, 12H), 0.96 (s, 6H) ppm;
13C NMR (151 MHz, CDCl3): δ 157.3, 136.5, 130.4, 123.8, 120.6, 112.2, 83.1, 68.8, 37.4, 26.6, 25.0, 24.9, 21.6, 16.0 ppm;
HRMS (ESI-TOF, m/z): Calcd for C20H34BO3 [M+H]+ 333.2595; found 333.2598.
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S18), MgBr2.OEt2 (52 mg, 0.2 mmol, 1 equiv) and suspension A (NiCl2.6H2O/di-MeObipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:25 Et2O:hexanes) afforded 31 (50.0 mg, 80%).
Physical State: white solid;
m.p.=82-84° C.;
Rf=0.62 (silica gel, 1:4 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.64-7.67 (m, 2H), 7.57 (s, 1H), 7.35 (dd, J=8.4, 1.8 Hz, 1H), 7.09-7.11 (m, 2H), 3.90 (s, 3H), 2.57 (q, J=7.2 Hz, 1H), 1.41 (d, J=7.2 Hz, 3H), 1.21 (s, 6H), 1.20 (s, 6H) ppm;
13C NMR (151 MHz, CDCl3): δ 157.1, 140.3, 132.8, 129.5, 129.1, 127.8, 126.7, 125.3, 118.5, 105.8, 83.5, 55.4, 24.8, 24.8, 17.1 ppm;
HRMS (ESI-TOF, m/z): Calcd for C19H26BO3 [M+H]+ 313.1969; found 313.1970.
On 0.2 mmol scale, General Procedure B was followed with NHPI ester (S19) and solution B (NiCl2.6H2O (20 mol %)/di-MeObipy (26% mol %) in DMF (0.8 mL)). Purification by flash column chromatography (silica gel, hexanes to 1:20 Et2O:hexanes) afforded 32 (26.5 mg, 38%).
Physical State: colorless oil;
Rf=0.45 (silica gel, 1:8 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 1.41-1.36 (m, 4H), 1.29-1.24 (m, 6H), 1.24 (s, 24H), 0.75 (t, J=7.8 Hz, 4H) ppm;
13C NMR (151 MHz, CDCl3): δ 83.0, 32.5, 29.4, 25.0, 24.2 ppm;
HRMS (ESI-TOF, m/z): Calcd for C19H39B2O4 [M+H]+ 353.3029; found 353.3030.
N, N-bis(2-chloroethyl)-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) aniline 33)
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S20) and suspension A (NiCl2.6H2O/di-MeObipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:19 EtOAc:hexanes) afforded 33 (20.7 mg, 26%).
Physical State: yellow oil;
Rf=0.36 (silica gel, 1:9 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.09-7.04 (m, 2H), 6.63-6.59 (m, 2H), 3.69 (t, J=7.1 Hz, 4H), 3.61 (t, J=7.1 Hz, 4H), 2.54-2.48 (t, J=7.8 Hz, 2H), 1.68 (p, J=7.8 Hz, 2H), 1.24 (s, 12H), 0.81 (t, J=7.8 Hz, 2H) ppm;
13C NMR (151 MHz, CDCl3): δ 144.2, 132.2, 129.9, 112.2, 83.1, 53.8, 40.7, 37.6, 26.5, 25.0 ppm;
HRMS (ESI-TOF, m/z): Calcd for C19H31BCl2NO2 [M+H]+ 386.1819; found 386.1815.
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S21), MgBr2.OEt2 (52 mg, 0.2 mmol, 1 equiv) and suspension A (NiCl2.6H2O/di-MeObipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:15 EtOAc:hexanes) afforded 34 (51.9 mg, 77%).
Physical State: colorless oil;
Rf=0.45 (silica gel, 1:6 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.83-7.81 (m, 2H), 7.66 (t, J=1.8 Hz, 1H), 7.59-7.56 (m, 2H), 7.49-7.44 (m, 3H), 7.38 (t, J=7.8 Hz, 1H), 2.51 (q, J=7.8 Hz, 1H), 1.35 (d, J=7.8 Hz, 3H), 1.21 (s, 6H), 1.21 (s, 6H) ppm;
13C NMR (151 MHz, CDCl3): δ 197.2, 145.4, 138.0, 137.7, 132.4, 132.2, 130.3, 129.7, 128.4, 128.3, 127.2, 83.6, 24.8, 24.8, 17.1 ppm;
HRMS (ESI-TOF, m/z): Calcd for C21H26BO3 [M+H]+ 337.1969; found 337.1971.
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S22), MgBr2.OEt2 (52 mg, 0.2 mmol, 1 equiv) and suspension A (NiCl2.6H2O/di-MeObipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:30 Et2O:hexanes) afforded 35 (52.6 mg, 81%).
Physical State: colorless oil;
Rf=0.50 (silica gel, 1:12 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.32 (t, J=7.8 Hz, 2H), 7.22 (t, J=7.8 Hz, 1H), 7.07 (t, J=7.8 Hz, 1H), 7.01 (d, J=7.2 Hz, 2H), 6.97 (d, J=7.2 Hz, 1H), 6.91 (t, J=1.8 Hz, 1H), 6.79 (dd, J=7.8 Hz, 2.4 Hz, 1H), 2.42 (q, J=7.8 Hz, 1H), 1.31 (d, J=7.8 Hz, 3H), 1.20 (s, 6H), 1.19 (s, 6H) ppm;
13C NMR (151 MHz, CDCl3): δ 157.7, 157.2, 147.3, 129.7, 129.6, 123.0, 123.0, 118.8, 118.7, 115.9, 83.5, 24.8, 24.7, 17.0 ppm;
HRMS (ESI-TOF, m/z): Calcd for C20H26BO3 [M+H]+ 325.1969; found 325.1970.
On 0.2 mmol scale, General Procedure B was followed with NHPI ester (S23) and solution B (NiCl2.6H2O/di-MeObipy in DMF). Purification by flash column chromatography (silica gel, hexanes to 1:9 EtOAc:hexanes) afforded 36 (77.4 mg, 57%).
Physical State: white foam;
Rf=0.52 (silica gel, 1:4 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.21-7.14 (m, 9H), 7.07 (br d, J=8.4 Hz, 2H), 7.00-6.97 (m, 3H), 6.85 (br s, 1H), 4.08-4.03 (m, 1H), 4.00-3.96 (m, 1H), 3.85-3.80 (m, 1H), 3.69-3.65 (m, 1H), 3.60-3.55 (m, 1H), 1.68-1.64 (m, 2H), 1.55 (d, J=1.8 Hz, 3H), 1.53 (d, J=1.8 Hz, 3H), 1.34 (dt, J=13.2 Hz, 1.2 Hz, 1H), 1.34 (s, 3H), 1.30 (s, 3H), 1.23 (s, 12H), 1.08-1.03 (m, 2H), 0.98-0.94 (m, 1H) ppm;
13C NMR (151 MHz, CDCl3): δ 165.0, 162.4 (d, J=247.6 Hz), 141.7, 138.6, 134.8, 134.5, 133.3 (d, J=8.2 Hz), 130.7, 128.9, 128.8, 128.5, 128.4 (d, J=3.8 Hz), 126.7, 123.8, 123.6, 121.8, 119.7, 115.4 (d, J=21.3 Hz), 98.6, 83.3, 66.7, 66.7, 41.0, 38.4, 38.3, 30.3, 26.2, 24.9, 24.9, 21.9, 21.7, 20.0 ppm;
HRMS (ESI-TOF, m/z): Calcd for C41H51BFN2O5[M+H]+ 681.3870; found 681.3870;
[α]D20=+4.0 (c 0.68, CHCl3).
On 0.1 mmol scale, General Procedure C was followed with NHPI ester (S37) and suspension A (NiCl2.6H2O/di-MeObipy in THF). Purification by flash column chromatography (silica gel, hexanes to 1:17 EtOAc:hexanes) afforded 37 (22.1 mg, 50%).
Physical State: yellow oil;
Rf=0.5 (silica gel, 1:4 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.64 (dt, J=9.0 Hz, 1.8 Hz, 2H), 7.45 (m, dt, J=8.4 Hz, 1.8 Hz, 2H), 6.96-6.93 (m, 2H), 6.64 (dd, J=9.0 Hz, 2.6 Hz, 1H), 3.84 (s, 3H), 2.29 (s, 3H), 2.18 (s, 2H), 1.23 (s, 12H) ppm;
13C NMR (151 MHz, CDCl3): δ 168.3, 156.0, 138.8, 134.7, 133.2, 132.0, 131.2, 131.1, 129.1, 116.7, 115.0, 111.3, 101.7, 83.7, 55.8, 29.9, 25.0, 13.9 ppm;
HRMS (ESI-TOF, m/z): Calcd for C24H28BClNO4 [M+H]+ 440.1794; found 440.1794.
Compound 38
On 0.2 mmol scale, General Procedure B was followed with NHPI ester (S38) and solution B (NiCl2.6H2O/di-MeObipy in DMF). Purification by flash column chromatography (silica gel, hexanes to 1:5 EtOAc:hexanes) afforded 38 (63.0 mg, 65%).
Physical State: white solid;
Rf=0.40 (silica gel, 1:3 EtOAc:hexanes);
m.p.=230-232° C.;
1H NMR (600 MHz, CDCl3): δ 2.92-2.82 (m, 3H), 2.35-2.19 (m, 6H), 2.14-2.09 (m, 2H), 2.05-1.94 (m, 4H), 1.80-1.85 (m, 1H), 1.56-1.63 (m, 2H), 1.39 (s, 3H), 1.35-1.12 (m, 16H), 1.06 (s, 3H), 0.87-0.81 (m, 4H), 0.68-0.62 (m, 1H) ppm;
13C NMR (151 MHz, CDCl3): δ 212.1, 209.2, 208.9, 83.0, 57.1, 51.9, 49.2, 47.0, 45.8, 45.7, 45.1, 42.9, 38.8, 38.2, 36.6, 36.1, 35.4, 29.4, 27.8, 25.4, 25.0, 24.9, 22.1, 18.6, 12.0 ppm;
HRMS (ESI-TOF, m/z): Calcd for C29H45BO5 [M+H]+ 485.3433; found 485.3435.
[α]D20=+16.9 (c 0.62, CHCl3).
Compound 39
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S24) and suspension A (NiCl2.6H2O/di-MeObipy in THF). Purification by flash column chromatography (silica gel, 1:12:3 EtOAc:hexanes:CH2Cl2) afforded 39 (82.0 mg, 69%, d.r.=11.8:1).
Physical State: colorless film;
Rf=0.34 (silica gel, 1:5 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): Major isomer δ 5.57 (s, 1H), 4.51 (dd, J=11.8, 4.7 Hz, 1H), 2.79 (dt, J=13.7, 3.6 Hz, 1H), 2.35 (s, 1H), 2.20 (ddd, J=13.3, 4.4, 1.7 Hz, 1H), 2.12 (td, J=13.7, 4.6 Hz, 1H), 2.04 (s, 3H), 1.96 (t, J=13.6 Hz, 1H), 1.80 (td, J=13.7, 4.6 Hz, 1H), 1.75-1.38 (m, 7H), 1.37 (s, 3H), 1.27-1.13 (m, 5H), 1.20 (d, J=1.8 Hz, 12H), 1.15 (s, 3H), 1.12 (s, 3H), 1.02 (td, J=13.5, 3.6 Hz, 1H), 0.99 (s, 3H), 0.94 (ddt, J=13.7, 4.5, 2.2 Hz, 1H), 0.87 (s, 6H), 0.84 (s, 3H), 0.81-0.76 (m, 1H) ppm;
13C NMR (151 MHz, CDCl3): Major isomer δ 200.1, 171.1, 171.1, 128.3, 83.0, 80.8, 61.8, 55.2, 45.5, 45.3, 43.6, 38.9, 38.5, 38.2, 37.1, 34.2, 32.9, 32.7, 29.1, 28.2, 27.8, 26.7, 26.6, 24.8, 24.7, 23.7, 23.4, 21.5, 18.9, 17.7, 17.6, 16.8, 16.6 ppm;
HRMS (ESI-TOF, m/z): Calcd for C37H60BO5 [M+H]+ 595.4528; found 595.4520;
[α]D20=+65.8 (c 1.0, CHCl3).
On 0.2 mmol scale, General Procedure C was followed with NHPI ester (S39) and suspension A (NiCl2.6H2O/di-MeObipy in THF). Purification by flash column chromatography (silica gel, first flash column chromatography: 1:5.7 to 1:4 EtOAc:hexanes; second flash column chromatography, 1:6:3 to 2:6:3 EtOAc:hexanes:CH2Cl2) afforded 40 (72.1 mg, 65%, d.r.=11.3:1).
Physical State: colorless film;
Rf=0.46 (silica gel, 3:7 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): Major isomer δ 5.59 (s, 1H), 3.27-3.18 (m, 1H), 2.81 (dt, J=13.5, 3.6 Hz, 1H), 2.36 (s, 1H), 2.22 (ddd, J=13.5, 4.5, 1.7 Hz, 1H), 2.14 (td, J=13.7, 4.6 Hz, 1H), 1.99 (t, J=13.6 Hz, 1H), 1.83 (td, J=13.7, 4.7 Hz, 1H), 1.74-1.58 (m, 4H), 1.55 (td, J=13.8, 4.0 Hz, 1H), 1.51-1.35 (m, 2H), 1.41 (s, 3H), 1.33-1.15 (m, 7H), 1.22 (s, 6H), 1.22 (s, 6H), 1.15 (s, 3H), 1.15 (s, 3H), 1.02 (s, 3H), 1.01 (s, 3H), 1.00-0.94 (m, 1H), 0.86 (s, 3H), 0.82 (s, 3H), 0.71 (dd, J=11.8, 1.9 Hz, 1H) ppm;
13C NMR (151 MHz, CDCl3): Major isomer δ 200.3, 171.1, 128.3, 83.0, 79.0, 61.9, 55.2, 45.5, 45.3, 43.6, 39.3, 39.3, 38.5, 37.2, 34.2, 33.0, 32.7, 29.1, 28.3, 27.8, 27.5, 26.7, 26.6, 24.8, 24.7, 23.5, 18.9, 17.7, 16.5, 15.7 ppm;
HRMS (ESI-TOF, m/z): Calcd for C35H58BO4 [M+H]+ 553.4422; found 553.4423;
[α]D20=+73.4 (c 1.0, CHCl3).
A culture tube charged with 40 (30 mg, 0.054 mmol, 1.0 equiv.), 3,5-dinitrobenzoyl chloride (50 mg, 0.22 mmol, 4.1 equiv.), and DMAP (1.3 mg, 0.011 mmol, 0.2 equiv.). CH2Cl2 (0.3 mL) and Et3N (30 μL, 0.22 mmol, 4.1 mmol) were added, and the resulting mixture was stirred for 1 h at room temperature. The mixture was loaded directly onto a silica gel column for purification by flash column chromatography (1:11 EtOAc:hexanes) to afford 40a (39.0 mg, 96%, d.r.=11.3:1). The pure product was crystallized from hexanes/CH2Cl2.
Physical State: pale yellow solid (major isomer is a white solid);
m.p. decompose at 295° C.;
Rf=0.45 (silica gel, 1:5.7 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): Major isomer δ 9.22 (t, J=2.2 Hz, 1H), 9.13 (d, J=2.2 Hz, 2H), 5.60 (s, 1H), 4.88 (dd, J=11.9, 4.7 Hz, 1H), 2.92 (dt, J=13.7, 3.6 Hz, 1H), 2.40 (s, 1H), 2.26-2.19 (m, 1H), 2.13 (td, J=13.7, 4.5 Hz, 1H), 1.98 (t, J=13.6 Hz, 1H), 1.95-1.87 (m, 1H), 1.87-1.75 (m, 2H), 1.74-1.59 (m, 3H), 1.56-1.48 (m, 2H), 1.45 (dt, J=12.8, 3.1 Hz, 1H), 1.40 (s, 3H), 1.30-1.09 (m, 5H), 1.23 (s, 3H), 1.21 (s, 6H), 1.21 (s, 6H), 1.16 (s, 3H), 1.08 (s, 3H), 1.00 (s, 3H), 0.97 (s, 3H), 0.97-0.94 (m, 1H), 0.89 (dd, J=11.8, 1.9 Hz, 1H), 0.86 (s, 3H) ppm;
13C NMR (151 MHz, CDCl3): Major isomer δ 199.9, 171.4, 162.3, 148.8, 134.8, 129.5, 128.3, 122.4, 84.3, 83.1, 61.7, 55.3, 45.6, 45.3, 43.6, 38.9, 38.6, 38.5, 37.1, 34.2, 32.8, 32.7, 29.2, 28.5, 27.8, 26.7, 26.6, 24.8, 24.7, 23.8, 23.4, 18.9, 17.7, 17.6, 17.2, 16.6 ppm;
HRMS (ESI-TOF, m/z): Calcd for C42H60BN2O9[M+H]+ 747.4386; found 747.4385;
[α]D20=+60.5 (c 1.0, CHCl3).
On 0.2 mmol scale, General Procedure B was followed with NHPI ester (S25) and solution B (NiCl2.6H2O/di-MeObipy in DMF). Purification by flash column chromatography (silica gel, hexanes to 1:6:6 EtOAc:hexanes:CH2Cl2) afforded 41 (37.0 mg, 46%).
Physical State: white solid;
m.p.=122-124° C.;
Rf=0.40 (silica gel, 1:2 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.64 (s, 1H), 5.21-5.18 (m, 3H), 3.75 (s, 3H), 3.37 (d, J=6.6 Hz, 2H), 2.13 (s, 3H), 2.08 (t, J=7.8 Hz, 2H), 1.77 (s, 3H), 1.17 (s, 12H), 0.86 (t, J=7.8 Hz, 2H) ppm;
13C NMR (151 MHz, CDCl3): δ 173.1, 163.9, 153.8, 143.9, 137.8, 122.8, 120.6, 116.8, 106.4, 83.0, 70.1, 61.1, 33.6, 24.9, 22.7, 16.3, 11.7 ppm;
HRMS (ESI-TOF, m/z): Calcd for C22H32BO6 [M+H]+ 403.2286; found 403.2289.
Pinacol boronate ester 4 (70 mg, 0.27 mmol) was dissolved in CH2Cl2 (5 mL) under argon and the solution was cooled to −78° C. in a dry ice/acetone bath. BCl3 (0.81 mL, 1.0 M in CH2Cl2, 3.0 equiv) was added dropwise, after which the mixture was stirred for 1 h at −78° C. The mixture was then allowed to warm up to room temperature, and the volatiles were removed in vacuo. Anhydrous methanol (5 mL) was added and the resulting mixture was stirred for 10 minutes when methanol was removed in vacuo. An additional portion of methanol (5 mL) was added; the mixture was stirred for 10 minutes before it is concentrated in vacuo. This process was repeated for additional three times. The resulting crude product was then purified with preparative thin layer chromatography to afford 4a as a white solid (41.8 mg, 87%).
1H NMR (600 MHz, DMSO-d6/D2O 100/1): δ 7.28-7.22 (m, 2H), 7.15 (ddt, J=13.9 Hz, 6.9 Hz, 1.5 Hz, 3H), 2.56-2.51 (m, 2H), 1.51 (tt, J=7.8, 6.7 Hz, 2H), 1.38-1.28 (m, 2H), 0.60 (t, J=7.9 Hz, 2H).
13C NMR (151 MHz, DMSO-d6/D2O 100/1): δ 142.60, 128.32, 128.27, 125.58, 35.26, 34.21, 23.98, 15.27 (br);
HRMS (ESI-TOF) Calcd for C10H16BO2 [M+H]+ 179.1238; found 179.1236.
Pinacol boronate ester 3 (30 mg, 0.12 mmol) was dissolved in CH2Cl2 (2 mL) under argon and the solution was cooled to −78° C. in a dry ice/acetone bath. BCl3 (0.36 mL, 1.0 M in CH2Cl2, 3.0 equiv) was added dropwise, after which the mixture was stirred for 1 h at −78° C. The mixture was then allowed to warm up to room temperature, and the volatiles were removed in vacuo. Anhydrous methanol (5 mL) was added and the resulting mixture was stirred for 10 minutes before methanol was removed in vacuo. An additional portion of methanol (5 mL) was added; the mixture was stirred for 10 minutes before it was concentrated in vacuo. This process was repeated for additional three times. The resulting crude product was then purified with preparative thin layer chromatography to afford 4a as a white solid (15.4 mg, 75%).
1H NMR (600 MHz, DMSO-d6/D2O 100/1): δ 7.24 (t, J=7.6 Hz, 2H), 7.18-7.10 (m, 3H), 2.53-2.48 (m, 2H), 1.74-1.63 (m, 1H), 1.42 (ddt, J=13.0 Hz, 9.9 Hz, 6.5 Hz, 1H), 0.90 (d, J=7.2 Hz, 3H), 0.89-0.81 (m, 1H) ppm;
13C NMR (151 MHz, DMSO-d6/D2O 100/1): δ 143.03, 128.34, 125.61, 35.65, 35.05, 20.33 (br), 16.35 ppm;
HRMS (ESI-TOF) Calcd for C10H16BO2 [M+H]+ 179.1238; found 179.1232.
To a solution of pinacol boronate ester 34 (76.2 mg, 0.2 mmol) in CH2Cl2 (1 mL) was added BCl3 (0.79 mL, 1.0 M in CH2Cl2) dropwise at −78° C. The reaction mixture was stirred at −78° C. for 30 minutes followed by another 30 minutes at room temperature. The reaction was quenched with methanol (2 mL) and was concentrated in vacuo. To the residue was added MeOH (2 mL) which was subsequently removed in vacuo; this process was repeated for additional three times. Purification of the resulting residue by preparative reverse-phase HPLC (20-80% CH3CN/H2O over 30 min, both CH3CN and H2O containing 0.1% TFA) afforded 33a (27 mg, 50%) as a colorless oil.
1H NMR (600 MHz, DMSO-d6/D2O 10/1): δ 7.00 (d, J=12.6 Hz, 2H), 6.65 (d, J=12.6 Hz, 2H), 3.71-3.65 (m, 8H), 2.39 (t, J=7.8 Hz, 2H), 1.58-1.53 (m, 2H), 0.59 (t, J=8.4 Hz, 2H) ppm;
13C NMR (151 MHz, DMSO-d6/D2O 10/1): δ 144.2, 130.9, 129.3, 111.8, 52.3, 41.2, 37.2, 26.6 ppm;
HRMS (ESI-TOF) Calcd for C13H21BCl2NO2 [M+H]+ 304.1037; found 304.1030.
Deprotection of Boc:
To a solution of Boc-Gly-Leu-OMe11 (3.1 g, 10.26 mmol) in CH2Cl2 (30 mL) was added TFA (15 mL) at room temperature, the reaction mixture was stirred for 1 h before concentrated in vacuo. The residue was used directly in the next step.
Amide Bond Formation:
To a solution 2,5-dichlorobenonic acid (2.94 g, 15.4 mmol) in THF (70 mL) was added 4-methylmorpholine (4.0 mL, 35.9 mmol) at −15° C., the reaction mixture was stirred for 10 min at that temperature. To the resulting white suspension was added isobutyl chloroformate (2.0 mL, 15.4 mmol) dropwise and the mixture was stirred for another 30 min at −15° C. The crude TFA salt (from the deprotection step) in THF (35 mL) was added slowly at the same temperature. The reaction mixture was warmed up to room temperature and stirred for 6 h. The resulting mixture was diluted with EtOAc (100 mL), washed with water (100 mL), sat. aqueous NaHCO3 (100 mL), and brine (100 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. Purification by a flash column chromatography (silica, 3:2 Hexane/EtOAc) afforded the desired ester which is not very pure but can be used in next step without further purification.
Hydrolysis of Methyl Ester:
To a solution of the aforementioned ester in THF (50 mL) was added aqueous LiOH (1M, 50 mL). The reaction mixture was stirred at room temperature for 2 h and was then washed with EtOAc (60 mL). The aqueous layer was acidified with 1N HCl (65 mL) and extracted with EtOAc (100 mL). The organic layer was washed with brine (100 mL) whereby the aqueous layers were back-extracted with EtOAc (100 mL). The combined organic phase was concentrated in vacuo. To the residue was added CH2Cl2 (30 ml) when the desired product S41 precipitated out and was collected by filtration (2.31 g, 63% over 3 steps).
m.p.=137-138° C.;
1H NMR (600 MHz, MeOH-d4): δ 7.63 (dd, J=1.8 Hz, 0.6 Hz, 1H), 7.45-7.48 (m, 2H), 4.50 (dd, J=9.6 Hz, 5.4 Hz, 1H), 4.08 (dd, J=37.8 Hz, 16.8 Hz, 2H), 1.79-1.72 (m, 1H), 1.70-1.62 (m, 2H), 0.98 (d, J=6.6 Hz, 3H), 0.95 (d, J=6.6 Hz, 3H) ppm;
13C NMR (151 MHz, MeOH-d4): δ 175.8, 170.9, 168.7, 138.4, 134.0, 132.6, 132.3, 130.6, 130.2, 52.1, 43.6, 41.9, 26.0, 23.4, 21.9 ppm;
HRMS (ESI-TOF) Calcd for C15H19Cl2N2O4 [M+H]+ 361.0716; found 361.0706;
[α]D20=−14.0 (c 1.0, MeOH).
On 2.0 mmol scale, general procedure A was followed with S41. Purification by flash column chromatography (deactivated silica gel, 3:7 EtOAc:hexanes) furnished S42 (940 mg, 79%).
Physical state: white solid;
m.p.=164° C.;
Rf=0.55 (silica gel, 3:2 EtOAc:hexanes);
1H NMR (600 MHz, THF-d8): δ 8.05 (br s, 1H), 7.99-7.97 (m, 1H), 7.91-7.89 (m, 2H), 7.87-7.85 (m, 2H), 7.58 (dd, J=2.4 Hz, 0.5 Hz, 1H), 7.42-7.38 (m, 2H), 5.10-5.06 (m, 1H), 4.14 (dd, J=16.8 Hz, 6.0 Hz, 1H), 3.99 (dd, J=16.8 Hz, 6.0 Hz, 1H), 1.92-1.83 (m, 2H), 1.80-1.75 (m, 1H), 1.02 (d, J=6.0 Hz, 3H), 1.00 (d, J=6.0 Hz, 3H) ppm;
13C NMR (151 MHz, THF-d8): δ 170.5, 169.4, 166.0, 162.4, 139.2, 135.9, 133.5, 132.3, 131.5, 130.7, 130.5, 130.2, 124.7, 49.7, 43.6, 42.3, 25.8, 23.4, 22.1 ppm;
HRMS (ESI-TOF) Calcd for C23H22Cl2N3O6 [M+H]+ 506.0880; found 506.0875;
[α]D20=−1.0 (c 1.0, THF).
On 0.2 mmol scale, general procedure C was followed using suspension C (NiCl2.6H2O/di-tBubipy in THF) with S42. Flash column chromatography (silica gel, hexanes to 2:3 EtOAc:hexanes to 4:1 EtOAc:hexanes) afforded pinacol aminoboronate ester S43 which was used in the next step without further purification.
The pinacol aminoboronate ester S43 was dissolved in CH2Cl2 (5 mL) under argon and the solution was cooled to −78° C. in a dry ice/acetone bath. BCl3 (0.6 mL, 1.0 M in CH2Cl2, 3.0 equiv.) was added dropwise, after which the mixture was stirred for 1 h at −78° C. The mixture was then allowed to warm up to room temperature, and the volatiles were removed in vacuo. Anhydrous methanol (5 mL) was added and the mixture was stirred for 10 minutes when the methanol was removed in vacuo. An additional portion of methanol (5 mL) was added; the mixture was stirred for 10 minutes before it is concentrated in vacuo. This process was repeated for three times. The resulting residue was then purified by preparative reverse-phase HPLC (10-60% CH3CN/H2O over 35 min, both CH3CN and H2O containing 0.1% TFA) to afford Ninlaro (1, 23.0 mg, 32% over 2 steps).
1H NMR (600 MHz, MeOH-d4): δ 7.60 (t, J=1.5 Hz, 1H), 7.49-7.47 (m, 2H), 4.24 (s, 2H), 2.79 (t, J=7.6 Hz, 1H), 1.68 (ddt, J=14.7 Hz, 13.0 Hz, 6.4 Hz, 1H), 1.38 (tdd, J=13.8 Hz, 10.4 Hz, 5.9 Hz, 2H), 0.94 (dd, J=6.6 Hz, 1.5 Hz, 6H);
13C NMR (151 MHz, MeOH-d4): δ 175.6, 168.8, 138.0, 134.0, 132.7, 132.5, 130.7, 130.2, 44.7 (br, a to boron), 40.9, 40.2, 27.1, 23.7, 22.4.
HRMS (ESI-TOF, m/z): calc'd for C14H18BCl2N2O3 [M−H2O+H]+ 343.0782; found 343.0779;
[α]D20=−0.6 (c 1.0, MeOH).
To a solution of 36 (50 mg, 0.073 mmol) in THF/H2O (1:1, 0.73 mL) at room temperature open to air was added NaBO3.4H2O (56 mg, 0.37 mmol). The mixture was stirred vigorously for 3 h before H2O (10 mL) was added. The resulting mixture was extracted with EtOAc (10 mL×3). The combined organic extracts were dried over Na2SO4, filtered, and concentrated. Purification by flash column chromatography (silica gel, 2:3 EtOAc:hexanes) afforded 36a (40 mg, 86%).
m.p.=166-170° C.;
Rf=0.27 (silica gel, 2:3 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.21-7.15 (m, 9H), 7.07 (d, J=8.0 Hz, 2H), 7.02-6.96 (m, 3H), 6.86 (s, 1H), 4.13-4.05 (m, 1H), 3.92-3.81 (m, 2H), 3.73-3.66 (m, 1H), 3.61-3.52 (m, 2H), 3.45 (dd, J=11.4 Hz, 6.1 Hz, 1H), 1.74-1.61 (m, 2H), 1.54 (s, 3H), 1.53 (s, 3H), 1.37 (s, 3H), 1.34 (s, 3H), 1.74-1.61 (m, 2H);
13C NMR (151 MHz, CDCl3): δ 164.9, 162.4 (d, J=247.6 Hz), 141.7, 138.5, 134.8, 133.3 (d, J=8.0 Hz), 130.6, 128.9, 128.8, 128.5, 128.4 (d, J=3.5 Hz), 126.7, 123.6, 121.9, 119.7, 115.5 (d, J=21.4 Hz), 98.9, 69.4, 66.2, 66.0, 41.0, 38.3, 31.9, 30.0, 26.2, 21.9, 21.7, 20.0;
19F NMR (376 MHz, acetone-d6): δ −114.00 ppm;
HRMS (ESI-TOF, m/z): Calcd for C35H40FN2O4[M+H]+ 571.2966; found 571.2963;
[α]D20=−4.6 (c 1.0, CHCl3).
The amination was performed following the literature procedure12 with slight modifications. A solution of O-methylhydroxylamine (63 pL, 2.8 M in THF, 6.0 equiv) was diluted with THF (1 mL). n-BuLi (72 μL, 2.45 M in hexanes, 6.0 equiv) was added at −78° C., and the resulting mixture was stirred for 1 h at that temperature. A solution of pinacol boronate 36 (20 mg, 0.03 mmol) in THF (1 mL) was added dropwise at −78° C. After warming up to room temperature, the reaction mixture was heated to 65° C. and stirred for 36 h. Upon cooling to room temperature, Boc2O (66 mg, 10.0 equiv) was added. The resulting mixture was stirred at room temperature for 1 h before the volatiles were removed in vacuo. Purification of the resulting residue by preparative thin layer chromatography (silica gel, 15:1 DCM:Et2O) afforded 36b (10.7 mg, 54%) as colorless oil.
Physical state: colorless oil;
Rf=0.4 (silica gel, 1:3 hexane: EtOAc);
1H NMR (600 MHz, CDCl3): δ 7.24-7.09 (m, 9H), 7.07 (d, J=8.0 Hz, 2H), 7.04-6.94 (m, 3H), 6.86 (s, 1H), 4.84 (s, 1H), 4.07 (ddd, J=15.3 Hz, 10.7 Hz, 5.1 Hz, 1H), 3.82 (ddt, J=15.1 Hz, 10.3 Hz, 6.5 Hz, 2H), 3.66 (tt, J=8.2 Hz, 3.6 Hz, 1H), 3.57 (p, J=7.2 Hz, 1H), 3.24 (d, J=8.7 Hz, 1H), 2.98 (ddd, J=13.8 Hz, 6.8 Hz, 5.1 Hz, 1H), 1.70-1.63 (m, 2H), 1.53 (d, J=7.1 Hz, 6H), 1.44 (s, 9H), 1.34 (s, 3H), 1.31 (s, 3H), 1.27-1.20 (m, 1H), 1.07 (q, J=12.0 Hz, 1H);
13C NMR (151 MHz, CDCl3): δ 164.9, 162.4 (d, J=247.9 Hz), 156.2, 141.6, 138.5, 134.8, 133.3 (d, J=8.1 Hz), 130.6, 128.9, 128.8, 128.5, 128.4 (d, J=3.5 Hz), 126.7, 123.6, 121.9, 119.7, 115.5 (d, J=21.3 Hz), 98.8, 79.6, 68.2, 66.3, 45.4, 41.0, 38.3, 33.4, 30.5, 30.0, 28.5, 26.2, 21.9, 21.7, 20.0;
19F NMR (376 MHz, CDCl3): δ −113.93 ppm;
HRMS (ESI-TOF, m/z): Calcd for C40H49FN3O5[M+H]+ 670.3651; found 670.3646;
[α]D20=−2.0 (c 0.74, CHCl3).
To a solution of thiophene (23 μL, 0.28 mmol) in THF (1.0 mL) was added n-BuLi (0.1 mL, 2.5 M in hexanes, 0.25 mmol) at −78° C. The resulting mixture was warmed to room temperature and stirred for 1 h when some of the resulting yellow solution (0.33 mL) was transferred to a reaction tube. A solution of 36 (12.4 mg, 0.018 mmol) in THF (0.3 mL) was added dropwise at −78° C. The resulting mixture was stirred at the same temperature for 1.5 h when a solution of NBS (14.4 mg, 0.081 mmol) in THF (0.3 mL) was added. After stirring for 1 h at the same temperature, the reaction was quenched with sat. aqueous Na2S2O3 (1 mL) before warming up to room temperature. The resulting mixture was extracted with EtOAc (1 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. Purification by flash column chromatography (silica gel, 1:9 EtOAc:hexanes) and PTLC (silica gel, 1:6 EtOAc:hexanes) afforded 36c (6.5 mg, 56%).
Physical state: white foam;
Rf=0.61 (silica gel, 2:3 EtOAc:hexanes);
1H NMR (600 MHz, acetone-d6): δ 8.29 (br s, 1H), 7.45 (d, J=7.8 Hz, 2H), 7.30-7.27 (m, 2H), 7.24 (dd, J=5.4 Hz, 1.2 Hz, 1H), 7.20 (t, J=7.8 Hz, 2H), 7.13-7.09 (m, 6H), 7.08-7.05 (m, 1H), 6.99-6.96 (m, 1H), 6.92 (dd, J=5.4 Hz, 3.6 Hz, 1H), 6.85-6.84 (m, 1H), 4.11-4.06 (m, 1H), 4.05-4.00 (m, 1H), 3.91-3.86 (m, 1H), 3.85-3.81 (m, 1H), 3.43-3.39 (m, 1H), 2.93-2.90 (m, 1H), 2.87-2.83 (m, 1H), 1.75-1.63 (m, 2H), 1.47 (d, J=1.2 Hz, 3H), 1.45 (d, J=1.2 Hz, 3H), 1.36 (dt, J=12.6 Hz, 3.0 Hz, 1H), 1.36 (s, 3H), 1.28 (s, 3H) 1.05-0.99 (m, 1H) ppm;
13C NMR (151 MHz, acetone-d6): δ 166.4, 163.1 (d, J=245.6 Hz), 140.9, 140.6, 139.4, 136.1, 134.5 (d, J=8.2 Hz), 130.8, 129.9 (d, J=3.3 Hz), 129.3, 128.9, 128.6, 127.3, 126.7, 126.7, 124.9, 123.8, 122.4, 120.2, 118.0, 116.0 (d, J=21.6 Hz), 99.2, 70.2, 67.3, 41.3, 39.1, 37.3, 36.5, 30.5, 26.9 22.4, 22.3, 20.1 ppm;
19F NMR (376 MHz, acetone-d6): δ −114.91 ppm;
HRMS (ESI-TOF) Calcd for C39H42FN2O3S [M+H]+ 637.2895; found 637.2892;
[α]D20=+19.2 (c 0.5, CHCl3).
To a solution of 2,3-benzofuran (30 μL, 0.27 mmol) in THF (1.0 mL) was added n-BuLi (0.1 mL, 2.5 M in hexanes, 0.25 mmol) at −78° C. The resulting solution was warmed up to room temperature. The resulting mixture was warmed to room temperature and stirred for 1 h when some of the resulting yellow solution (0.33 mL) was transferred to a reaction tube. A solution of 36 (12.0 mg, 0.018 mmol) in THF (0.3 mL) was added dropwise at −78° C. The resulting mixture was stirred at the same temperature for 1 h when a solution of NBS (14.4 mg, 0.081 mmol) in THF (0.3 mL) was added. After stirring for 1 h at the same temperature, the reaction was quenched with sat. aqueous Na2S2O3 (1 mL) before warming up to room temperature. The resulting mixture was extracted with EtOAc (1 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. Purification by flash column chromatography (silica gel, 1:9 EtOAc:hexanes) and preparative thin layer chromatography (silica gel, 1:9 EtOAc:hexanes) afforded 36d (6.1 mg, 52%).
Physical state: colorless oil;
Rf=0.64 (silica gel, 2:3 EtOAc:hexanes);
1H NMR (600 MHz, acetone-d6): δ 8.29 (br s, 1H), 7.54-7.52 (m, 1H), 7.44 (d, J=7.8 Hz, 2H), 7.45-7.42 (m, 1H), 7.31-7.27 (m, 2H), 7.24-7.17 (m, 4H), 7.13-7.05 (m, 7H), 6.99-6.96 (m, 1H), 6.58 (dd, J=1.2 Hz, 0.6 Hz, 1H), 4.29-4.24 (m, 1H), 4.11-4.06 (m, 1H), 3.91-3.85 (m, 2H), 3.44-3.37 (m, 1H), 2.93 (dd, J=15.6 Hz, 6.6 Hz, 1H), 2.79 (dd, J=15.6 Hz, 6.6 Hz, 1H), 1.76-1.65 (m, 2H), 1.46 (s, 3H), 1.45 (s, 3H), 1.46-1.51 (m, 1H), 1.39 (d, J=0.6 Hz, 3H), 1.27 (d, J=0.6 Hz, 3H), 1.14-1.08 (m, 1H) ppm;
13C NMR (151 MHz, acetone-d6): δ 166.4, 163.1 (d, J=245.7 Hz), 156.5, 155.5, 140.6, 139.4, 136.1, 134.5 (d, J=8.3 Hz), 130.8, 129.9 (d, J=3.3 Hz), 129.9, 129.3, 128.9, 128.6, 126.7, 124.2, 123.8, 123.4, 122.4, 121.3, 120.2, 120.1, 118.0, 116.0 (d, J=21.6 Hz), 111.4, 104.6, 99.3, 68.1, 67.3, 41.3, 39.1, 37.0, 36.2, 30.4, 26.9, 22.4, 22.3, 20.1 ppm; 19F NMR (376 MHz, acetone-d6): 5-114.95 ppm;
HRMS (ESI-TOF) Calcd for C43H44FN2O4[M+H]+ 671.3280; found 671.3274;
[α]D20=+28.5 (c 0.5, CHCl3).
To a screw-capped culture tube was added Pd2(dba)3 (1.9 mg, 0.0022 mmol, 0.1 equiv), p-anisyldiphenylphosphine (3.7 mg, 0.0132 mmol, 0.6 equiv), 1-chloro-4-nitrobenzene (35 mg, 0.22 mmol, 10 equiv), and K3PO4 (47 mg, 0.22 mmol, 10 equiv). This tube was then evacuated and backfilled with argon for three times. 1,4-dioxane (0.4 mL) was then added via a syringe and the resulting mixture was stirred at room temperature for 5 minutes. A solution of 36 (15.0 mg, 0.022 mmol) in dioxane (0.6 mL) and degassed DI water (0.5 mL) were added sequentially. The reaction mixture was heated at 100° C. for 15 h, after which it was cooled to room temperature and treated with brine (4 mL). The resulting mixture was extracted with EtOAc (2 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. Purification by flash column chromatography (silica gel, 1:6 to 3:7 EtOAc:hexanes) and PTLC (silica gel, 1:3 EtOAc:hexanes) afforded 36e (7.9 mg, 54%).
Physical state: colorless oil;
Rf=0.38 (silica gel, 3:7 EtOAc:hexanes);
1H NMR (600 MHz, acetone-d6): δ 8.45 (dd, J=4.8 Hz, 1.8 Hz, 1H), 8.30 (br s, 1H), 7.79 (dd, J=8.4 Hz, 1.8 Hz, 1H), 7.45 (d, J=7.8 Hz, 2H), 7.30-7.25 (m, 3H), 7.20 (t, J=7.8 Hz, 2H), 7.12-7.05 (m, 7H), 6.99-6.96 (m, 1H), 4.46-4.41 (m, 1H), 4.11-4.06 (m, 1H), 3.91-3.86 (m, 1H), 3.85-3.81 (m, 1H), 3.44-3.39 (m, 1H), 3.13 (dd, J=14.4 Hz, 6.6 Hz, 1H), 2.88 (dd, J=14.4 Hz, 7.2 Hz, 1H), 1.76-1.64 (m, 2H), 1.47 (d, J=0.6 Hz, 3H), 1.45 (d, J=0.6 Hz, 3H), 1.38 (dt, J=12.6 Hz, 2.4 Hz, 1H), 1.34 (s, 3H), 1.24 (s, 3H), 1.16-1.10 (m, 1H) ppm;
13C NMR (151 MHz, acetone-d6): δ 166.4, 163.1 (d, J=245.5 Hz), 156.2, 148.3, 140.6, 139.3, 137.6, 136.2, 134.5 (d, J=8.3 Hz), 132.2, 130.8, 129.9 (d, J=3.3 Hz), 129.3, 128.9, 128.6, 126.7, 123.8, 123.8, 122.4, 120.2, 118.0, 116.0 (d, J=21.6 Hz), 99.2, 68.7, 67.3, 42.3, 41.3, 39.2, 37.0, 30.5, 26.9, 22.4, 22.3, 20.1 ppm;
19F NMR (376 MHz, acetone-d6): δ −114.92 ppm;
HRMS (ESI-TOF) Calcd for C40H42ClFN3O3[M+H]+ 666.2893; found 666.2884;
[α]D20=+26.2 (c 0.5, CHCl3).
To a screw-capped culture tube was added Pd2(dba)3 (1.9 mg, 0.0022 mmol, 0.1 equiv), p-anisyldiphenylphosphine (3.7 mg, 0.0132 mmol, 0.6 equiv), 1-chloro-4-nitrobenzene (35 mg, 0.22 mmol, 10 equiv), and K3PO4 (47 mg, 0.22 mmol, 10 equiv). This tube was evacuated and backfilled with argon for three times. 1,4-dioxane (0.4 mL) was added via a syringe and the resulting mixture was stirred at room temperature for 5 minutes. A solution of 36 (15.0 mg, 0.022 mmol) in dioxane (0.6 mL) and degassed DI water (0.5 mL) were added sequentially. The reaction mixture was heated to 100° C. for 15 h after which it was cooled to room temperature and treated with brine (4 mL). The resulting mixture was extracted with EtOAc (2 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. Purification by flash column chromatography (silica gel, 1:9 to 1:3 EtOAc:hexanes) and PTLC (silica gel, 1:4 EtOAc:hexanes) afforded 36f (10.5 mg, 72%).
Physical state: yellow oil;
Rf=0.45 (silica gel, 3:7 EtOAc:hexanes);
1H NMR (600 MHz, acetone-d6): δ 8.28 (br s, 1H), 8.15 (dt, J=9.0 Hz, 1.8 Hz, 2H), 7.51 (dt, J=9.0 Hz, 1.8 Hz, 2H), 7.45 (d, J=8.4 Hz, 2H), 7.30-7.27 (m, 2H), 7.20 (t, J=7.8 Hz, 2H), 7.14-7.10 (m, 6H), 7.09-7.05 (m, 1H), 6.99-6.96 (m, 1H), 4.17-4.13 (m, 1H), 4.11-4.06 (m, 1H), 3.92-3.87 (m, 1H), 3.85-3.81 (m, 1H), 3.44-3.37 (m, 1H), 2.87 (dd, J=13.8 Hz, 7.2 Hz, 1H), 2.81 (dd, J=13.8 Hz, 7.2 Hz, 1H), 1.73-1.65 (m, 2H), 1.46 (d, J=0.6 Hz, 3H), 1.45 (d, J=0.6 Hz, 3H), 1.36 (dt, J=12.6 Hz, 2.4 Hz, 1H), 1.32 (s, 3H), 1.25 (s, 3H), 1.09-1.03 (m, 1H) ppm;
13C NMR (151 MHz, acetone-d6): δ 166.4, 163.1 (d, J=245.5 Hz), 147.6, 147.5, 140.5, 139.4, 136.1, 134.5 (d, J=8.3 Hz), 131.4, 130.8, 129.9 (d, J=3.8 Hz), 129.3, 128.9, 128.7, 126.7, 123.9, 123.8, 122.4, 120.2, 118.0, 116.0 (d, J=21.4 Hz), 99.2, 69.8, 67.3, 42.9, 41.2, 39.1, 36.7, 30.4, 26.9, 22.4, 22.3, 20.1 ppm;
19F NMR (376 MHz, acetone-d6): δ −114.92 ppm;
HRMS (ESI-TOF) Calcd for C41H43FN3O5 [M+H]+ 676.3181; found 676.3182;
[α]D20=+10.8 (C 0.5, CHCl3).
To S44 [synthesized according to literature report (38, 62)] (600 mg, 0.43 mmol, 1.0 equiv.) in CH3CN (5.1 mL) was added N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA, 2.4 mL, 10.2 mmol, 23.7 equiv.), the resulting mixture was heated to 50° C. After 30 h, the reaction mixture was poured onto a mixture of sat. aqueous citric acid (50 mL)/EtOAc (20 mL) and stirred vigorously at room temperature for 12 h. The organic layer was separated and washed with sat. aqueous NaHCO3 (50 mL) and brine (50 mL). The aqueous layers were then back-extracted with EtOAc (20 mL×2). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. Purification by flash column chromatography (silica gel, 1:1 to 4:1 EtOAc:hexanes) and preparative TLC (7:93 MeOH/CH2Cl2) afforded the desired product S45 (440 mg, 63%).
Physical state: white film;
Rf=0.31 (silica gel, 7:93 MeOH/CH2Cl2);
1H NMR (600 MHz, acetone-d6): δ 9.43 (br s, 1H), 7.94 (d, J=6.5 Hz, 1H), 7.57 (dd, J=8.3, 1.9 Hz, 1H), 7.53 (br s, 3H), 7.49 (s, 1H), 7.46 (s, 1H), 7.45 (s, 1H), 7.43-7.33 (m, 4H), 7.26 (d, J=8.3 Hz, 1H), 7.19 (d, J=8.3 Hz, 1H), 7.08 (s, 1H), 7.03 (d, J=8.7 Hz, 1H), 6.77 (d, J=9.9 Hz, 1H), 6.73 (s, 1H), 6.67 (d, J=2.3 Hz, 1H), 6.45 (br s, 1H), 6.31 (d, J=2.3 Hz, 1H), 5.94 (br s, 1H), 5.85 (s, 1H), 5.58 (d, J=4.9 Hz, 1H), 5.54 (s, 1H), 5.51 (s, 1H), 5.39 (d, J=12.3 Hz, 1H), 5.23 (d, J=12.3 Hz, 1H), 5.20 (br s, 1H), 5.10 (br s, 1H), 4.96 (d, J=6.5 Hz, 1H), 4.67 (d, J=5.2 Hz, 1H), 4.63 (t, J=7.2 Hz, 1H), 4.42 (d, J=11.7 Hz, 1H), 4.18 (s, 3H), 3.68 (s, 3H), 3.67 (s, 3H), 3.59 (s, 3H), 2.83 (s, 3H), 2.59 (d, J=16.5 Hz, 1H), 2.42 (d, J=16.3 Hz, 1H), 2.09 (s, 1H), 1.66-1.57 (m, 2H), 1.53 (s, 9H), 1.54-1.48 (m, 2H), 1.00 (s, 9H), 0.92 (s, 9H), 0.92 (d, J=6.5 Hz, 3H) 0.86 (d, J=6.5 Hz, 3H), 0.17 (s, 6H), 0.13 (s, 3H), 0.12 (s, 3H) ppm;
13C NMR (151 MHz, acetone-d6): δ 172.3, 171.7, 171.3, 171.3, 171.1, 170.8, 168.9, 168.0, 161.1, 159.9, 158.1, 156.9, 154.5, 153.0, 151.5, 151.5, 141.5, 140.0, 138.4, 137.0, 136.9, 136.2, 135.7, 130.0, 129.3, 129.3, 129.0, 128.3, 127.9, 127.6, 126.1, 125.4, 124.7, 124.1, 122.1, 113.8, 106.5, 106.1, 105.4, 99.6, 80.3, 74.6, 74.0, 67.2, 64.3, 61.5, 60.0, 57.5, 56.5, 56.2, 56.1, 55.7, 55.4, 55.2, 52.0, 38.1, 37.2, 28.9, 28.6, 26.5, 26.3, 26.3, 25.7, 23.7, 23.3, 22.8, 19.1, 19.1, −4.4, −4.6, −4.8, −4.8.
HRMS (ESI-TOF, m/z): Calcd for C81H103Cl2N8O19Si2 [M+H]+ 1617.6249; found 1617.6248.
To a solution of S45 (600 mg, 0.37 mmol) in EtOH/EtOAc (4/1, 50 mL) was added Pd/C (240 mg, 5% Pd/C, 50% wetted powder); the resulting black suspension was stirred under a hydrogen atmosphere at room temperature for 12 h. The reaction mixture was then filtered through celite and washed with EtOH/EtOAc (4:1, 150 mL). The filtrate was concentrated under reduced pressure. The resulting residue was purified by preparative reverse-phase HPLC (85%-100% CH3CN/H2O over 30 min, 100% CH3CN for 30 min, both CH3CN and H2O containing 0.1% TFA) to afford 42 (450 mg, 79%) as a TFA salt.
Note: The Boc group was found to cleaved during the purification process.
Physical state: pale yellow film;
1H NMR (600 MHz, MeOH-d4): δ 8.68 (d, J=5.4 Hz, 1H), 7.60 (dd, J=8.4 Hz, 2.4 Hz, 1H), 7.48 (br s, 1H), 7.42 (br s, 1H), 7.37 (d, J=8.4 Hz, 1H) 7.40-7.35 (br m, 1H), 7.10 (d, J=9.0 Hz, 1H), 7.04-7.02 (m, 2H), 6.68 (d, J=2.4 Hz, 1H), 6.58 (d, J=2.4 Hz, 1H), 6.39 (br s, 2H), 5.77 (d, J=1.2 Hz, 1H), 5.65 (br s, 1H), 5.46 (s, 1H), 5.37 (s, 1H), 5.30 (br s, 1H), 4.80 (s, 1H), 4.60 (d, J=5.4 Hz, 1H), 4.23 (s, 3H), 4.10 (br s, 1H), 3.93-3.90 (m, 1H), 3.87 (s, 3H), 3.73 (s, 3H), 3.67 (s, 3H), 2.83 (s, 3H), 2.83-2.78 (m, 1H), 2.42 (dd, J=16.8, 5.4 Hz, 1H), 1.89-1.82 (m, 1H), 1.79-1.74 (m, 2H), 0.98-0.93 (m, 24H), 0.15 (s, 3H), 0.15 (s, 3H), 0.13 (s, 3H), 0.12 (s, 3H) ppm;
13C NMR (600 MHz, MeOH-d4): δ 175.3, 174.0, 172.3, 171.9, 171.8, 171.4, 170.4, 169.4, 169.2, 162.0, 160.4, 159.0, 155.2, 154.2, 153.3, 152.1, 142.2, 140.0, 139.4, 137.3, 136.8, 135.4, 130.6, 129.2, 128.5, 128.5, 128.0, 127.2, 126.1, 125.2, 124.9, 122.5, 114.1, 107.3, 106.7, 106.4, 99.4, 74.9, 65.0, 62.5, 62.4, 61.1, 58.2, 56.6, 56.1, 56.0, 55.6, 52.4, 40.8, 37.0, 33.2, 26.8, 26.5, 25.3, 23.7, 22.0, 19.7, 19.5, −4.3, −4.5, −4.7, −4.7 ppm;
19F NMR (376 MHz, MeOH-d4): δ −77.2 ppm;
HRMS (ESI-TOF, m/z): Calcd for C69H89Cl2N8O17Si2 [M+H]+ 1427.5256; found 1427.5258.
To a solution of 42 (15.0 mg, 0.0097 mmol, 1.0 equiv.) in CH3CN (1.5 mL) was added a solution of tris(dimethylamino)sulfonium difluorotrimethylsilicate (TASF) in DMF (120 μL, 1.0 M, 12.4 equiv.). The resulting mixture was stirred at room temperature for 1.5 h before it was concentrated to a final volume of ca. 0.1 mL under reduced pressure. This residue was purified by preparative reverse-phase HPLC (30%-45% CH3CN/H2O over 40 min, both CH3CN and H2O containing 0.1% TFA) to afford 43 (9.3 mg, 73%) as a TFA salt.
Physical state: white film;
1H NMR (600 MHz, MeOH-d4) δ 9.01 (d, J=6.4 Hz, 0.6H), 8.73 (d, J=5.8 Hz, 0.4H), 7.86 (d, J=8.8 Hz, 1H), 7.75 (d, J=2.1 Hz, 1H), 7.65 (d, J=8.5 Hz, 1H), 7.64 (d, J=2.1 Hz, 11H) 7.61 (ddd, J=8.5, 2.2, 0.9 Hz, 1H), 7.21 (d, J=8.5 Hz, 1H), 7.09 (d, J=2.3 Hz, 1H), 6.85 (d, J=8.8 Hz, 1H), 6.78 (d, J=8.2 Hz, 1H), 6.68 (d, J=2.3 Hz, 1H), 6.51 (d, J=2.2 Hz, 1H), 6.13 (br s, 1H), 6.06 (s, 1H), 5.87 (s, 1H), 5.40 (dd, J=2.2, 1.0 Hz, 1H), 5.37 (s, 1H), 5.27 (d, J=3.5 Hz, 1H), 4.78 (s, 1H), 4.65 (s, 1H), 4.27 (dd, J=9.6, 1.9 Hz, 1H), 4.18 (s, 1H), 4.11 (s, 3H), 4.02 (t, J=7.2 Hz, 1H), 3.86 (s, 3H), 3.66 (s, 3H), 3.63 (s, 3H), 3.03 (d, J=15.7 Hz, 1H), 2.76 (s, 3H), 2.03 (dd, J=15.7, 10.4 Hz, 1H), 1.90 (dt, J=14.0, 7.2 Hz, 1H), 1.69-1.57 (m, 2H), 0.88 (d, J=6.4 Hz, 3H), 0.85 (d, J=6.4 Hz, 3H).
13C NMR (600 MHz, MeOH-d4): δ 175.8, 174.6, 172.8, 171.7, 170.0, 169.9, 169.4, 169.0, 161.9, 160.4, 158.7, 154.2, 153.0, 152.3, 151.1, 142.7, 141.7, 138.1, 136.8, 136.7, 136.6, 130.2, 129.0, 129.0, 128.9, 128.5, 127.6, 127.3, 125.3, 125.3, 124.8, 122.4, 113.8, 109.8, 106.7, 106.3, 99.2, 74.3, 73.4, 63.9, 62.1, 61.9, 59.5, 58.5, 56.6, 56.3, 56.2, 56.0, 55.2, 53.0, 52.9, 40.2, 38.7, 36.4, 33.0, 25.5, 23.2, 22.8 ppm;
19F NMR (376 MHz, MeOH-d4): δ −76.9 ppm;
HRMS (ESI-TOF, m/z): Calcd for C57H61Cl2N8O17 [M+H]+ 1199.3526; found 1199.3521.
To a suspension of 42 (45 mg, 0.029 mmol, 1.0 equiv.), N-hydroxyphthalimide (26 mg, 0.16 mmol, 5.5 equiv.), and N,N-dimethylpyridin-4-amine (0.4 mg, 0.0033 mmol, 0.11 equiv.) in CH2Cl2 (0.5 mL) was added N,N′-diisopropylcarbodiimide (25 μL, 0.16 mmol, 5.5 equiv.). The reaction mixture was stirred at room temperature for 1 h before AcOH (10 μL) was added. The resulting mixture was stirred for another 2 h and was subjected to flash column chromatography directly (silica gel, column: d 1.6 cm×/7.5 cm, 3:2 EtOAc:hexanes (200 mL) to 1:19 MeOH:CH2Cl2 (120 mL)). The combined fractions eluted with MeOH—CH2Cl2 were concentrated under reduced pressure, and the S46 residue (31 mg) was used in next step without further purification.
(1) LC/MS indicated that the desired redox-active ester (S46) only eluted with MeOH/CH2Cl2. Nonpolar impurities, such as 1,3-diisopropylurea, were found to elute with EtOAc:hexanes.
(2) Additional amounts of DMAP or longer reaction time have deleterious effects on the reaction yield.
(3) This redox-active ester (S46) was rather unstable and should be used in next step within 3 h after purification. Alternatively, it can be stored at −20° C.
A screw-capped culture tube containing S46 (31 mg), MgBr2.OEt2 powder (38 mg, 0.15 mmol) was evacuated and backfilled with argon for three times. Suspension C (0.4 mL, NiCl2.6H2O/di-tBubipy in THF) was added next and the mixture was stirred vigorously at room temperature for 15 min (or sonicated until no granular MgBr2.OEt2 was observed). The resulting suspension was cooled to 0° C., and the suspension of [B2pin2Me]Li in THF (0.55 mL) was added in one portion. After stirring for 1 h, the reaction mixture was diluted with CH2Cl2 (5 mL), filtered through a short pad of silica gel and celite, washed with 5% MeOH/CH2Cl2 (50 mL). The filtrate was concentrated under reduced pressure, and the residue was subjected to flash column chromatography directly (silica gel, column: d 1.6 cm×17.5 cm, 1:1 EtOAc:hexanes (200 mL) to 1:19 MeOH:CH2Cl2 (120 mL)). The MeOH—CH2Cl2 elution was concentrated under reduced pressure, and the S47 residue (16 mg) was used in next step without further purification.
(1) The pinacol ester was found to hydrolyze during the reaction based on LC/MS analysis.
(2) LC/MS indicated that S47 only elutes with MeOH/CH2Cl2 based on LC/MS analysis. Nonpolar impurities, such as B2pin2, were found to elute with EtOAc:hexanes.
(3) Not all impurities can be removed through flash chromatography in this step; instead the unpure materials were carried forward to the next step.
To a solution of S47 (16 mg) in CH3CN (1.3 mL) was added a solution of tris(dimethylamino)sulfonium difluorotrimethylsilicate (TASF) in DMF (120 μL, 1.0 M). The mixture was stirred at room temperature for 1.5 h and was concentrated to a final volume of ca. 0.3 mL under reduced pressure. The residue was purified by preparative reverse-phase HPLC (20%-50% CH3CN/H2O over 30 min, both CH3CN and H2O containing 0.1% TFA) to afford 44 (4.1 mg, 11% over 3 steps) as a TFA salt.
Note: This compound was not stable in neat condition due to its propensity toward polymerization. Therefore, the purified compound was dissolved immediately. Solutions in MeOH were used for HRMS; solutions in MeOH-d4 were used for NMR study; solutions in DMSO were used for biological studies.
Physical state: white film;
1H NMR (600 MHz, MeOH-d4): δ 9.05 (d, J=6.6 Hz, 1H), 7.65-7.58 (m, 4H), 7.31 (d, J=9.0 Hz, 1H), 7.30 (d, J=9.6 Hz, 1H), 7.15 (dd, J=9.0 Hz, 1.8 Hz, 1H), 6.97-6.91 (m, 2H), 6.81 (s, 1H), 6.52 (d, J=2.4 Hz, 1H), 5.81 (d, J=5.4 Hz, 1H), 5.69 (s, 1H), 5.65 (s, 1H), 5.54 (s, 1H), 5.35 (d, J=3.6 Hz, 1H), 5.07 (br s, 1H), 5.04 (d, J=6.6 Hz, 1H), 4.43 (s, 1H), 4.32 (d, J=5.4 Hz, 1H), 4.14 (s, 3H), 4.03 (t, J=7.2 Hz, 1H), 3.87 (s, 3H), 3.68 (s, 3H), 3.65 (s, 3H), 2.96 (d, J=15.6, 1H), 2.77 (s, 3H), 2.34 (dd, J=16.2 Hz, 9.0 Hz, 1H), 1.86-1.82 (m, 1H), 1.79-1.73 (m, 1H), 1.71-1.86 (m, 1H), 1.01 (d, J=6.0 Hz, 3H), 0.98 (d, J=6.0 Hz, 3H) ppm;
11B NMR (500 MHz, MeOH-d4): δ −0.87 (s) ppm;
HRMS (ESI-TOF, m/z): Calcd for C56H62BCl2N8O17 [M+H]+ 1199.3698; found 1199.3698.
Antibiotic susceptibilities were determined using the Clinical and Laboratory Standards Institute broth microdilution method (63). Briefly, antibiotics were prepared as 2-fold dilutions in 96-well plates containing cation-adjusted Mueller-Hinton broth (S. aureus strains) or brain-heart infusion broth (Enterococcus strains). Stock solutions of antibiotics were made in dimethyl sulfoxide (DMSO). Wells were inoculated from a fresh plate scrape diluted to a final concentration of 5×105 CFU/mL and incubated at 37° C. Growth observed visually at 20 h. All MICs are an average of at least three independent determinations.
S. aureus
a
E. faecium
c
E. faecalis
d
E. faecalis
e
a
Staphylococcus aureus (ATCC 25923)
b
Staphylococcus aureus (methicillin resistant, ATCC 43300)
c
Enterococcus faecium (Van A, ATCC BAA-2317)
d
Enterococcus faecalis (VanA, BM4166)
e
Enterococcus faecalis (VanB, ATCC 51299)
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, 422 mg, 2.2 mmol, 1.1 equiv.) was added into a solution of Boc-L-Ala-L-Val-OH (2.0 mmol, 1.0 equiv.) and NHPI (359 mg, 2.2 mmol, 1.1 equiv.) in CH2Cl2 (30 mL) at −10° C. After stirring for 1 h at room temperature, the mixture was washed with water and the aqueous phase was extracted with CH2Cl2 for three times. The combined organic phases were dried over anhydrous Na2SO4 and concentrated in vacuo. Purification by flash column chromatography (silica gel, 3:7 EtOAc:hexanes to EtOAc) afforded S48 (591 mg, 62%).
Physical state: white foam;
Rf=0.36 (silica gel, 2:3 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.88 (dd, J=5.5, 3.1 Hz, 2H), 7.79 (dd, J=5.5, 3.1 Hz, 2H), 6.93-6.79 (br, 1H), 5.02-4.88 (m, 2H), 4.28-4.14 (br, s, 1H), 2.48-2.32 (m, 1H), 1.44 (s, 9H), 1.38 (d, J=7.0 Hz, 3H), 1.10 (d, J=6.9 Hz, 3H), 1.08 (d, J=6.9 Hz, 3H) ppm;
13C NMR (151 MHz, CDCl3): δ 172.6, 168.4, 161.6, 155.9, 135.0, 129.0, 124.2, 80.5, 55.6, 50.0, 31.8, 28.4, 18.9, 17.5 ppm;
HRMS (ESI-TOF, m/z): Calcd for C21H28N3O7 [M+H]+ 434.1922; found 434.1930.
On 0.2 mmol scale, General Procedure C was followed with suspension C (NiCl2.6H2O/di-tBubipy in THF). Flash column chromatography (silica gel, 3:7 EtOAc:hexanes) afforded 45 as a mixture of inseparable diastereomers (50 mg, dr=1:1, 67%)
Physical state: colorless oil;
Rf=0.22 (silica gel, 3:7 EtOAc:hexanes);
1H NMR (600 MHz, C6D6): δ 6.77 (s, 1H), 6.73 (s, 1H), 5.56 (s, 1H), 5.37 (s, 1H), 4.23 (s, 1H), 4.11 (s, 1H), 3.05 (s, 2H), 2.10-2.04 (m, 2H), 1.41 (s, 9H), 1.40 (s, 9H), 1.25-0.92 (m, 42H) ppm;
13C NMR (151 MHz, C6D6): δ 174.7, 174.2, 156.0, 155.9, 82.8, 82.6, 79.4, 74.7, 49.1, 49.0, 37.0, 30.3, 28.4, 25.3, 25.3, 25.2, 25.0, 20.7, 20.7, 20.0, 19.9, 17.9, 17.7 ppm;
HRMS (ESI-TOF, m/z): Calcd for C18H36BN2O5[M+H]+ 371.2712; found 371.2710.
On a 2.0 mmol scale, General Procedure A was followed with Boc-L-Val-L-Val-OH (S49). Purification by flash column chromatography (silica gel, 1:3 EtOAc:hexanes) afforded S50a (187 mg, 20%) and S50b (395 mg, 43%).
Physical state: white foam;
Rf=0.40 (silica gel, 1:2 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.90-7.88 (m, 2H), 7.81-7.79 (m, 2H), 6.48 (br d, J=8.8 Hz, 1H), 5.09 (br d, J=8.4 Hz, 1H), 4.98 (dd, J=8.8, 5.1 Hz, 1H), 3.91 (dd, J=8.7, 6.8 Hz, 1H), 2.43-2.38 (m, 1H), 2.14 (br s, 1H), 1.43 (s, 9H), 1.11 (t, J=6.3 Hz, 6H), 0.97 (dd, J=16.5, 6.8 Hz, 6H) ppm;
13C NMR (151 MHz, CDCl3): δ 171.9, 168.4, 161.6, 156.1, 135.0, 129.0, 124.2, 80.2, 60.4, 55.6, 31.7, 30.6, 28.4, 19.4, 18.9, 18.2, 17.7 ppm;
HRMS (ESI-TOF, m/z): Calcd for C18H24N3O5 [M-Boc+H]+ 362.1710; found 362.1705;
[α]D20=−31.8 (c 0.96, CHCl3).
Physical state: white foam;
Rf=0.4 (silica gel, 1:2 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.87-7.85 (m, 2H), 7.79-7.77 (m, 2H), 6.60 (br d, J=8.8 Hz, 1H), 5.15 (d, J=8.9 Hz, 1H), 4.96 (dd, J=8.8, 5.2 Hz, 1H), 3.92 (dd, J=8.8, 6.8 Hz, 1H), 2.41-2.36 (m, 1H), 2.10 (br s, 1H), 1.42 (s, 9H), 1.09 (dd, J=6.9, 4.6 Hz, 6H), 0.97 (d, J=6.8 Hz, 3H), 0.94 (d, J=6.8 Hz, 3H) ppm;
13C NMR (151 MHz, CDCl3): δ 172.0, 168.4, 161.6, 156.1, 134.9, 128.9, 124.1, 80.1, 60.3, 55.6, 31.6, 30.6, 28.4, 19.4, 18.9, 18.2, 17.7 ppm;
HRMS (ESI-TOF, m/z): Calcd for C18H24N3O5 [M-Boc+H]+ 362.1710; found 362.1714;
[α]D20=−31.2 (c 1.0, CHCl3).
On 0.2 mmol scale, General Procedure C was followed with suspension C (NiCl2.6H2O/di-tBubipy in THF) from S50a (1.0 equiv. of MgBr2.Et2O was used in this case). Purification by flash column chromatography (silica gel, 1:3 EtOAc:hexanes) afforded 46 as a mixture of inseparable diastereomers (37.1 mg, d.r.=1.7:1, 47%)
On 0.2 mmol scale, General Procedure C was followed with suspension C (NiCl2.6H2O/di-tBubipy in THF) from S50b (1.0 equiv. of MgBr2.Et2O was used in this case). Purification by flash column chromatography (silica gel, 1:3 EtOAc: hexanes) afforded 46 as a mixture of inseparable diastereomers (36.5 mg, d.r.=1.7:1, 46%).
Physical state: colorless oil;
Rf=0.30 (silica gel, 1:2 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 6.30 (br d, J=5.5 Hz, 0.78H), 6.22 (br s, 0.22H), 5.10 (br d, J=8.7 Hz, 1H), 3.92-3.86 (m, 1H), 3.03 (br s, 1H), 2.10 (br s, 1H), 1.96-1.90 (m, 1H), 1.42 (s, 9H), 1.28-1.16 (m, 12H), 0.95 (d, J=6.7 Hz, 3H), 0.93 (d, J=6.7 Hz, 3H), 0.92 (d, J=6.7 Hz, 6H) ppm;
13C NMR (151 MHz, CDCl3): δ 172.48 (minor), 172.46, 155.92, 83.37, 79.91, 59.57 (minor), 59.25, 44.96 (br), 31.10, 31.02 (minor), 30.01, 29.91 (minor), 28.51, 28.44, 25.18, 25.12, 25.10 (minor), 25.03 (minor), 24.97, 20.42, 20.37 (minor), 20.12, 20.03, 19.35, 19.21 (minor), 18.13 (minor), 17.90 ppm;
HRMS (ESI-TOF, m/z): Calcd for C20H40BN2O5[M+H]+ 399.3025; found 399.3028.
On 1.0 mmol scale (based on Boc-L-Pro-L-Leu-OH), the same procedure as in the synthesis of S48 was used. Purification by flash column chromatography (silica gel, 1:2 EtOAc:hexanes) afforded S51 (308 mg, 67%).
Physical state: white foam;
Rf=0.4 (silica gel, 1:1 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): δ 7.88-7.85 (m, 2H), 7.87 (br s, 0.6H), 7.79-7.77 (m, 2H), 6.63 (s, 0.4H), 4.99-4.88 (m, 1H), 4.37-4.30 (m, 1H), 3.61-3.21 (m, 2H), 2.47 (br s, 0.4H), 2.43-2.37 (m, 1H), 2.16 (br s, 0.6H), 2.03-1.76 (m, 3H), 1.51-1.39 (m, 9H), 1.12-1.03 (m, 6H); (complex spectrum was observed due to mixture of rotamers);
13C NMR (151 MHz, CDCl3): δ 172.7, 172.0, 168.4, 161.6, 156.3, 154.9, 140.9, 137.2, 134.9, 130.1, 129.0, 124.1, 115.6, 110.4, 81.4, 80.7, 61.3, 59.5, 55.7, 55.1, 47.0, 31.5, 28.5, 27.1, 24.8, 19.0, 17.5; (complex spectrum was observed due to mixture of rotamers);
HRMS (ESI-TOF, m/z): Calcd for C18H22N3O5 [M-Boc+H]+ 360.1554; found 360.1554.
On 0.28 mmol scale, General Procedure C was followed with S51 and suspension C (NiCl2. 6H2O/di-tBubipy in THF). Purification by flash column chromatography (silica gel, 2:1 EtOAc:hexanes) afforded 47 as a mixture of diastereomers (70.5 mg, d.r.=2.6:1, 63%). Diastereomeric ratio was determined by 1H NMR and NOESY in DMSO-d6 at 65° C.
Physical state: colorless oil;
Rf=0.30 (silica gel, 2:1 EtOAc:hexanes);
1H NMR (500 MHz, DMSO-d6): δ 8.37 (s, 0.72H), 8.28 (s, 0.28H), 4.25 (dd, J=8.5, 2.8 Hz, 1H), 3.44-3.35 (m, 1H), 3.34-3.27 (m, 1H), 2.46 (t, J=5.3 Hz, 0.28H), 2.40 (t, J=4.7 Hz, 0.72H), 2.19-2.05 (m, 1H), 1.89-1.74 (m, 4H), 1.39 (s, 9H), 1.13 (s, 3.36H), 1.12 (s, 8.64H), 0.93-0.85 (m, 6H) ppm;
13C NMR (126 MHz, DMSO-d6): δ 174.9, 153.0, 80.6 (minor), 80.4, 78.5, 57.5 (minor), 57.3, 46.2, 28.9 (minor), 28.7, 27.8, 27.7, 24.9 (minor), 24.8, 24.7, 20.1, 20.0 (minor), 19.2 ppm;
HRMS (ESI-TOF, m/z): Calcd for C20H38BN2O5[M+H]+ 397.2868; found 397.2864.
On 3.0 mmol scale, General Procedure A was followed with Boc-L-Phe-L-Leu-OH (64) (S62). Purification by flash column chromatography (deactivated silica gel, 1:5.6 EtOAc:hexanes) afforded S53 as a mixture of inseparable diastereomers (1.42 g, d.r.=3:2, 90%). Diastereomeric ratio was determined by 1H NMR and NOESY.
Physical state: White foam;
Rf=0.50 (silica gel, 2:3 EtOAc:hexanes);
1H NMR (600 MHz, MeOH-d4): Minor isomer: δ 7.94-7.89 (m, 4H), 7.29-7.15 (m, 5H), 4.78 (dd, J=9.5 Hz, 5.8 Hz, 1H), 4.39-4.35 (m, 1H), 3.05 (dd, J=13.7 Hz, 7.2 Hz, 1H), 2.90 (dd, J=13.6 Hz, 8.1 Hz, 1H), 1.76-1.70 (m, 2H), 1.54-1.50 (m, 1H), 1.38 (s, 9H), 0.94 (d, J=6.6 Hz, 3H), 0.89 (d, J=6.6 Hz, 3H) ppm; Major isomer: δ 7.94-7.89 (m, 4H), 7.29-7.15 (m, 5H), 4.92 (dd, J=9.6 Hz, 6.0 Hz, 1H), 4.39-4.35 (m, 1H), 3.13 (dd, J=14.4 Hz, 5.4 Hz, 1H), 2.84 (dd, J=13.8 Hz, 9.0 Hz, 1H), 1.89-1.83 (m, 3H), 1.37 (s, 9H), 1.02 (d, J=6.0 Hz, 3H), 0.99 (d, J=6.0 Hz, 3H) ppm;
13C NMR (151 MHz, MeOH-d4): Minor isomer: δ 174.4, 170.4, 163.1, 157.3, 138.3, 136.3, 135.5, 130.4, 130.1, 129.5, 127.7, 124.9, 124.0, 80.7, 57.4, 50.2, 41.2, 39.6, 28.6, 28.4, 25.7, 25.5, 23.2, 21.7 ppm; Major isomer: δ 174.6, 170.4, 163.1, 157.6, 138.4, 136.4, 135.5, 130.4, 130.1, 129.4, 127.6, 124.9, 124.0, 80.6, 57.1, 50.2, 41.5, 39.1, 28.6, 28.4, 25.7, 25.5, 23.2, 21.8 ppm;
HRMS (ESI-TOF, m/z): Calcd for C23H26N3O5 [M-Boc+H]+ 424.1867; found 424.1871.
On 0.6 mmol scale, General Procedure C was followed with suspension C (NiCl2. 6H2O/di-tBubipy in THF) and S53. The reaction was started from □15° C. and warmed to room temperature over 3 h. Flash column chromatography (silica gel, 1:9 EtOAc:hexanes to 1:4 EtOAc:hexanes) afforded 48, which was dissolved in hexanes and filtered through celite. The filtrate was concentrated in vacuo to afford 48 as a mixture of inseparable diastereomers (151 mg, d.r.=5.1:1, 55%).
Physical state: Pale yellow oil;
Rf=0.50 (silica gel, 2:3 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3): Major isomer: δ 7.31-7.26 (m, 2H), 7.24-7.21 (m, 3H), 6.19 (br s, 1H), 5.00 (br s, 1H), 4.35 (q, J=7.3 Hz, 1H), 3.10-3.02 (m, 2H), 2.98 (ddd, J=8.8 Hz, 6.3 Hz, 4.4 Hz, 1H), 1.49-1.42 (m, 1H), 1.39 (s, 9H), 1.37-1.35 (m, 2H), 1.24 (s, 6H), 1.23 (s, 6H), 0.86 (d, J=6.6 Hz, 3H), 0.84 (d, J=6.6 Hz, 3H) ppm;
13C NMR (151 MHz, CDCl3): Major isomer: δ 172.6, 155.5, 134.4, 129.6, 128.8, 127.1, 83.0, 80.3, 54.8, 39.9, 38.3, 28.4, 25.6, 25.1, 25.0, 23.3, 22.0 ppm;
HRMS (ESI-TOF, m/z): Calcd for C25H42BN2O5[M+H]+ 461.3181; found 461.3179.
Bortezomib (49) was synthesized from 48 using the literature procedure (19) with slight modifications.
Boc Deprotection:
To a screw-capped culture tube charged with 48 (151 mg, 0.33 mmol) was added HCl in EtOAc (14 wt %) at 0° C., and the reaction mixture was stirred at 0° C. for 3 h and room temperature for an additional 1 h. The reaction mixture was concentrated to dryness and the resulting solid was washed with hexanes. The desired product was afforded as a white solid and was used in next step without further purification.
Esterification:
CH2Cl2 (1.2 mL, 0.5 M) was added to a screw-capped culture tube containing the hydrochloride salt obtained from the previous step. The mixture was cooled to 0° C. Diisopropylethylamine (0.15 mL, 0.86 mmol) was added dropwise, and the reaction mixture was stirred for 5 min. 2-Pyrazine carboxylic acid (56 mg, 0.45 mmol) was then added to the solution in one portion. O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU, 118 mg, 0.37 mmol) was then added to the reaction mixture which was stirred at 0° C. for 2 h and room temperature for additional 1 h. The reaction mixture was then concentrated in vacuo. The crude residue was dissolved in EtOAc (10 mL) and transferred to a separatory funnel. The organic layer was washed with deionic H2O (2×10 mL), 1% phosphoric acid (2×10 mL), 2% K2CO3 (2×10 mL), and brine (2×10 mL) successively. Each aqueous layer was back-extracted with EtOAc (2×10 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The resulting pale yellow foam was carried on to the next step without further purification.
Boronate Ester Exchange:
Pentane (0.8 mL) and MeOH (0.8 mL) were added to a screw-capped culture tube containing the pinacol boronate obtained from the previous step. 2-Methylpropaneboronic acid (125 mg, 1.2 mmol) was then added to the solution. 1 N aq. HCl (0.6 mL) was added to the reaction mixture, and the resulting biphasic solution was stirred vigorously for 16 h. Stirring was then stopped and the biphasic mixture was allowed to separate. The aqueous layer was washed with pentane (2×10 mL) and was then concentrated in vacuo. The resulting film was partitioned between CH2Cl2 and 1 N aq. NaOH (10 mL). The aqueous layer was washed with CH2Cl2 (3×10 mL) and the organic phase was back-extracted with 1 N aq. NaOH (2×10 mL). 1 N aq. HCl was added to the combined aqueous layers until the pH=6 when the desired product was extracted into the organic layer with CH2Cl2 (3×10 mL). The combined organic phase was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The resulting residue was dissolved in EtOAc (2 mL), and the solution was subsequently concentrated in vacuo. To the residue was then added hexanes (2 mL), and the suspension was concentrated in vacuo to afford the product 49 (64 mg, d.r.=5.1:1, 51% over 3 steps).
Physical state: white solid;
1H NMR (600 MHz, CD3CN:D2O=4:1): Major isomer: δ 9.10 (d, J=1.8 Hz, 1H), 8.74 (d, J=2.4 Hz, 1H)), 8.61 (dd, J=2.4 Hz, 1.8 Hz, 1H), 7.26-7.22 (m, 4H), 7.20-7.17 (m, 1H), 4.78 (dd, J=8.4 Hz, 6.0 Hz, 1H), 3.19 (dd, J=13.8 Hz, 6.0 Hz, 1H), 3.07 (dd, J=13.8 Hz, 8.2 Hz, 1H), 2.93 (dd, J=10.2 Hz, 5.4 Hz, 1H), 1.44-1.33 (m, 2H), 1.26-1.21 (m, 1H), 0.80 (d, J=6.6 Hz, 3H), 0.78 (d, J=6.6 Hz, 3H) ppm;
13C NMR (151 MHz, CD3CN:D2O=4:1): Major isomer: 172.4, 164.5, 148.7, 145.0, 144.7, 144.4, 137.7, 130.4, 129.5, 127.8, 54.9, 40.2, 40.2 (br s), 38.5, 25.9, 23.6, 22.0 ppm;
HRMS (ESI-TOF, m/z): Calcd for C19H24BN4O3[M−H2O+H]+ 367.1936; found 367.1950.
Cbz deprotection: A 100 mL flask equipped with a stirrer bar was charged with Z-L-Pro-L-Val-OtBu (65) (S54, 2.55 g, 6.3 mmol), 10% Pd/C (128 mg, 5 wt %), and MeOH (30 mL). The flask was then evacuated and backfilled with H2 from a balloon for three times. The mixture was stirred at room temperature for 6 h and was filtered through a short pad of celite which was then rinsed with MeOH (10 mL). The filtrate was concentrated in vacuo to give the corresponding amine as colorless oil.
Amide Bond Formation:
The aforementioned amine was treated successively with S55 (1.1 g, 6.3 mmol, 1.0 equiv.), HOBt H2O (96 mg, 0.07 mmol, 0.11 equiv.), and CH2Cl2 (25 mL). The resulting solution was cooled to 0° C. before DCC (1.43 g, 6.9 mmol, 1.1 equiv.) was added. The reaction mixture was allowed to stir at 0° C. for 30 min and then at room temperature overnight. The reaction mixture was filtrered through a pad a celite; the filtrate was redissolved in EtOAc and washed with 0.1 N aq. HCl, 0.1 M aq.NH4OH, and brine successively. The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo to give S56 (2.2 g) as colorless oil, which was used in the next step without further purification.
tBu Deprotection:
In a 25 mL flask equipped with a stirrer bar, S56 (428 mg, 1.0 mmol) was dissolved in CH2Cl2 (3 mL). TFA (3 mL) was added and the resulting solution was allowed to stir at room temperature for 5 h. After the volatiles were removed in vacuo, the crude mixture was purified by flash column chromatography (silica gel, 2:1 EtOAc:hexanes) furnished 50a (359 mg, 80% over 3 steps).
Physical state: white foam;
Rf=0.35 (silica gel, 1:2 hexanes: EtOAc);
1H NMR (600 MHz, CDCl3): δ 7.43 (br d, J=8.4 Hz, 2H), 6.17 (d, J=9.0 Hz, 1H), 4.64 (dd, J=7.8 Hz, 3.0 Hz, 1H), 4.48 (dd, J=8.4 Hz, 4.0 Hz, 1H), 4.29 (t, J=8.4 Hz, 1H), 3.84 (dd, J=16.8 Hz, 8.4 Hz, 1H), 3.69-3.63 (m, 4H), 2.33-2.29 (m, 1H), 2.20-2.10 (m, 2H), 2.03-1.92 (m, 3H), 0.97 (d, J=6.6 Hz, 3H), 0.95 (d, J=6.6 Hz, 3H), 0.91 (d, J=7.2 Hz, 3H), 0.89 (d, J=7.2 Hz, 3H) ppm;
13C NMR (151 MHz, CDCl3): δ 174.5, 173.5, 171.1, 157.8, 60.6, 58.1, 57.8, 52.5, 48.3, 31.4, 31.2, 27.7, 25.2, 19.4, 19.0, 18.1, 17.8 ppm;
HRMS (ESI-TOF, m/z): Calcd for C17H30N3O6 [M+H]+ 372.2129; found 372.2126;
[α]D20=−62.9 (c 0.79, CHCl3)
On 2.34 mmol scale, General Procedure A was followed with (methoxycarbonyl)-L-valyl-L-prolyl-L-valine. Purification by flash column chromatography (silica gel, 1:1 EtOAc:hexanes) furnished S57 (640 mg, 53%).
Physical state: white foam;
Rf=0.40 (silica gel, 1:2 hexanes: EtOAc);
1H NMR (600 MHz, CDCl3) δ 7.88-7.84 (m, 2H), 7.78-7.75 (m, 2H), 7.50 (d, J=8.4 Hz, 1H), 5.61 (d, J=9.2 Hz, 1H), 4.84 (dd, J=8.5, 5.0 Hz, 1H), 4.61 (dd, J=8.1, 3.0 Hz, 1H), 4.29 (dd, J=9.3, 6.9 Hz, 1H), 3.79-3.72 (m, 1H), 3.63 (s, 3H), 3.64-3.61 (m, 1H), 2.41-2.30 (m, 2H), 2.17 (dt, J=12.3, 9.1 Hz, 1H), 2.01-1.96 (m, 2H), 1.95-1.89 (m, 1H), 1.07 (d, J=7.2 Hz, 3H), 1.06 (d, J=6.6 Hz, 3H), 0.97 (d, J=6.7 Hz, 3H), 0.93 (d, J=6.7 Hz, 3H) ppm;
13C NMR (151 MHz, CDCl3) δ 172.5, 171.2, 168.4, 161.7, 157.3, 134.9, 129.0, 124.1, 60.0, 57.7, 56.0, 52.4, 48.0, 31.6, 31.4, 27.4, 25.3, 19.5, 18.8, 17.8, 17.7 ppm;
HRMS (ESI-TOF, m/z): Calcd for C25H33N4O8 [M+H]+ 517.2293; found 517.2289;
[α]D20=−61.0 (c 1.0, CHCl3).
On 0.33 mmol scale, General Procedure C was followed with suspension C (NiCl2.6H2O/di-tBubipy in THF). MgBr2.Et2O (1.0 equiv.) was used in this case. Purification by flash column chromatography (silica gel, 2:3 EtOAc:hexanes to 20:1 CH2Cl2:MeOH) furnished S58 (72 mg, 48%) as slightly yellow oil.
Aminoboronate ester S58 (24 mg, 0.053 mmol) was dissolved in CH2Cl2 (2 mL) under argon; the solution was cooled to −78° C. with a dry ice/acetone bath when BCl3 (0.16 mL, 1.0 M in CH2Cl2, 3.0 equiv.) was added dropwise, after which the mixture was stirred for 1 h at −78° C. The reaction was then allowed to warm up to room temperature, and the volatiles were removed in vacuo. Anhydrous methanol (4 mL) was added and the resulting mixture was stirred for 10 minutes prior to concentration in vacuo. The resulting residue was treated with methanol (4 mL) for 10 minutes and was concentrated in vacuo. This process was repeated for three times. The resulting crude product was then purified by preparative reverse-phase HPLC (10-40% CH3CN/H2O over 25 min, both CH3CN and H2O containing 0.1% TFA) and lyophilized to afford 50 as a white floppy powder (15.0 mg, 76%).
Physical state: white powder;
1H NMR (600 MHz, MeOH-d4): δ 4.61 (dd, J=8.4 Hz, 6.0 Hz, 1H), 4.17 (d, J=7.8 Hz, 1H), 3.97-3.93 (m, 1H), 3.75-3.71 (m, 1H), 3.64 (s, 3H), 2.33-2.24 (m, 2H), 2.19-2.13 (m, 1H), 2.08-1.98 (m, 3H), 1.80-1.74 (m, 1H), 1.05 (d, J=6.6 Hz, 3H), 1.00 (d, J=6.6 Hz, 3H), 0.96 (d, J=6.6 Hz, 3H), 0.92 (d, J=6.6 Hz, 3H) ppm;
13C NMR (151 MHz, MeOH-d4): δ 179.3, 173.5, 159.4, 59.7, 57.9, 52.7, 31.7, 31.0, 29.8, 26.2, 21.4, 21.2, 19.6, 18.8 ppm;
HRMS (ESI-TOF, m/z): Calcd for C16H29BN3O5[M−H2O+H]+ 354.2195; found 354.2189;
[α]D20=−81.1 (c 0.44, MeOH).
Synthesis of Elastase Inhibitors mCBK320 (51) and mCBK323(52)
Cbz Deprotection:
A 100 mL flask equipped with a stirrer bar was charged with Z-L-Pro-L-Val-OMe (66) (S59, 1.95 g, 5.4 mmol), 10% Pd/C (98 mg, 5 wt %), and MeOH (25 mL). This flask was then evacuated and backfilled with H2 from a balloon for three times. The reaction mixture was stirred at room temperature for 6 h and was filtered through a thin pad of celite which was then rinsed with MeOH (10 mL). The filtrate was concentrated in vacuo to give the corresponding amine as colorless oil.
Amide Bond Formation:
The aforementioned amine was treated sequentially with Boc-L-Valine (1.17 g, 5.4 mmol, 1.0 equiv.), HOBt.H2O (83 mg, 0.61 mmol, 0.11 equiv.), and CH2Cl2 (25 mL). The resulting solution was cooled to 0° C. before DCC (1.23 g, 6.0 mmol, 1.1 equiv.) was added. The reaction mixture was allowed to stir at 0° C. for 30 min and then at room temperature overnight. The resulting mixture was filtered through a pad of celite; the filtrate was concentrated in vacuo, redissolved in EtOAc, and washed with 0.1N aq. HCl, 0.1 M aq. NH4OH, and brine successively. The organic layer was dried over anhydrous Na2SO4, concentrated in vacuo, and purified by flash column chromatography (silica gel, 2:1 EtOAc:hexanes) to give Boc-L-Val-L-Pro-L-Val-OMe (1.32 g) as a colorless oil.
Hydrolysis of Ester:
A 25 mL flask equipped with a stirrer bar was charged with Boc-L-Val-L-Pro-L-Val-OMe (1.32 g) and THF (3 mL). LiOH (4 mL, 1 M aqueous solution) was added and the resulting solution was allowed to stir vigorously at room temperature for 12 h. 1 N HCl was added to the reaction mixture until pH=2-3 and the mixture was extracted with EtOAc. The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo to give S60 (1.24 g, 53% over 3 steps) as a white foam, which was used in the next step without further purification.
On 3.0 mmol scale, General Procedure A was followed with Boc-L-valyl-L-prolyl-L-valine (S60). Purification by flash column chromatography (silica gel, 1:1 EtOAc:hexanes) furnished S61 (920 mg, 55%).
Physical state: white foam;
Rf=0.50 (silica gel, 1:2 hexane: EtOAc);
1H NMR (600 MHz, CDCl3): δ 7.88-7.85 (m, 2H), 7.79-7.76 (m, 2H), 7.50 (d, J=8.4 Hz, 1H), 5.28 (d, J=9.6 Hz, 1H), 4.84 (dd, J=8.4, 4.8 Hz, 1H), 4.62 (dd, J=7.8, 3.0 Hz, 1H), 4.28 (dd, J=9.6, 6.6 Hz, 1H), 3.71-3.77 (m, 1H), 3.60 (dt, J=8.4, 3.6 Hz, 1H), 2.43-2.39 (m, 1H), 2.37-2.31 (m, 1H), 2.11-2.19 (m, 1H), 1.88-2.02 (m, 3H), 1.41 (s, 9H), 1.08 (d, J=6.6 Hz, 3H), 1.07 (d, J=6.6 Hz, 3H), 0.98 (d, J=7.2 Hz, 3H), 0.92 (d, J=7.2 Hz, 3H) ppm;
13C NMR (151 MHz, CDCl3): δ 172.9, 171.1, 168.3, 161.7, 156.0, 134.9, 129.0, 124.1, 79.7, 60.0, 57.0, 56.1, 47.9, 31.6, 31.6, 28.5, 27.1, 25.4, 19.7, 18.9, 17.8, 17.6 ppm;
HRMS (ESI-TOF, m/z): Calcd for C28H39N4O8 [M+H]+ 559.2762; found 559.2757.
[α]D20=−86.2 (c 1.0, CHCl3).
On 1.1 mmol scale, General Procedure C was followed with S61 and suspension C (NiCl2.6H2O/di-tBubipy in THF), 1.0 equiv. of MgBr2.Et2O was used in this case. Flash column chromatography (silica gel, 2:3 EtOAc:hexanes to 3:1 EtOAc:hexanes) furnished S62 (257 mg, 47%) as a slightly yellow oil.
Physical state: slight yellow oil;
Rf=0.65 (silica gel, 1:2 hexanes: EtOAc);
1H NMR (600 MHz, CDCl3): δ 7.08 (br s, 1H), 5.22 (d, J=9.3 Hz, 1H), 4.66 (dd, J=8.2, 2.6 Hz, 1H), 4.28 (dd, J=9.3, 6.0 Hz, 1H), 3.70 (q, J=8.7 Hz, 1H), 3.56 (ddd, J=9.7, 8.1, 3.7 Hz, 1H), 2.97-2.86 (m, 1H), 2.41-2.38 (m, 1H), 2.19-2.11 (m, 1H), 2.01-1.94 (m, 2H), 1.94-1.80 (m, 2H), 1.43 (s, 9H), 1.25 (d, J=5.4 Hz, 12H), 0.97 (d, J=6.8 Hz, 3H), 0.95 (d, J=6.8 Hz, 3H), 0.93 (d, J=6.8 Hz, 3H), 0.91 (d, J=6.7 Hz, 3H) ppm;
13C NMR (151 MHz, CDCl3): δ 172.8, 171.8, 156.0, 83.3, 79.8, 59.0, 56.9, 47.7, 31.6, 29.8, 28.5, 27.0, 25.3, 25.2, 25.1, 20.6, 20.3, 19.7, 17.5 ppm;
HRMS (ESI-TOF, m/z): Calcd for C25H47BN3O6[M+H]+ 496.3552; found 496.3550.
[α]D20=−73.6 (c 1.0, CHCl3).
Boc deprotection: In a culture tube equipped with a stir bar, S62 (55 mg, 0.11 mmol) was dissolved in CH2Cl2 (1 mL). TFA (1 mL) was added at 0° C. and the resulting solution was allowed to stir at 0° C. for 2 h. The volatiles were removed in vacuo using a rotary evaporator (water bath temperature <25° C.), and the residue was used in next step without purification.
Esterification:
Benzoic acid S63 (45 mg, 0.13 mmol, 1.2 equiv.) and PyBOP (69 mg, 0.13 mmol, 1.2 equiv.) were then added and the mixture was dissolved in DMF (2.0 mL). N-methyl morpholine (49 μL, 0.45 mmol, 4.0 equiv.) was added and the reaction was allowed to stir at room temperature for 3 h. The mixture was then diluted with EtOAc, washed with brine, dried over anhydrous Na2SO4, concentrated in vacuo, and purified by flash column chromatography (silica gel, 10:1 CH2Cl2:MeOH) to give the pinacol boronate of 51 (69 mg) contaminated with some tripyrrolidinophosphine oxide. This mixture was used in the next step without further purification.
Boronate Ester Exchange:
In a culture tube equipped with a stir bar, the aforementioned mixture (53 mg) and PhB(OH)2 (14 mg) was dissolved in Et2O (3 mL). 2 N HCl (3 mL) was added and the resulting biphasic mixture was allowed to stir vigorously at room temperature for 36 h when it was extracted with EtOAc (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4, concentrated in vacuo. The resulting residue was purified by preparative reverse-phase HPLC (20-80% CH3CN/H2O over 35 min, both CH3CN and H2O containing 0.1% TFA) and lyophilized to afford 51 (14.0 mg, 26% for 3 steps).
Physical state: white powder;
1H NMR (600 MHz, MeOH-d4): δ 8.10 (d, J=8.4 Hz, 2H), 7.94-7.90 (m, 4H), 7.66 (d, J=9.0 Hz, 2H), 4.64 (dd, J=8.4 Hz, 3.6 Hz, 1H), 4.61 (d, J=9.6 Hz, 1H), 4.12 (dt, J=9.6 Hz, 6.6 Hz, 1H), 3.82 (dt, J=9.6 Hz, 6.6 Hz, 1H), 2.38-2.31 (m, 2H), 2.25-2.19 (m, 2H), 2.14-2.02 (m, 2H), 1.82-1.77 (m, 1H), 1.16 (d, J=6.6 Hz, 3H), 1.10 (d, J=7.2 Hz, 3H), 0.98 (d, J=6.6 Hz, 3H), 0.94 (d, J=6.6 Hz, 3H) ppm;
13C NMR (151 MHz, MeOH-d4): δ 179.2, 173.0, 169.1, 166.9, 141.3, 139.5, 139.4, 135.9, 131.2, 130.3, 129.5, 128.9, 59.0, 57.9, 31.8, 31.1, 29.8, 26.3, 21.4, 21.2, 19.5, 19.5 ppm;
HRMS (ESI-TOF, m/z): Calcd for C28H35BClN4O7S [M−H2O+H]+ 617.2003; found 617.2002.
[α]D20=−72.2 (c 0.36, MeOH).
Boc deprotection: In a culture tube equipped with a stir bar, S62 (55 mg, 0.11 mmol) was dissolved in CH2Cl2 (1 mL). TFA (1 mL) was added at 0° C. and the resulting solution was allowed to stir at 0° C. for 2 h. The volatiles were removed in vacuo using a rotary evaporator (water bath temperature <25° C.), and the residue was used in next step without purification.
Esterification:
Benzoic acid S64 (37 mg, 0.13 mmol, 1.2 equiv.) and PyBOP (69 mg, 0.13 mmol, 1.2 equiv.) were then added and the mixture was dissolved in DMF (2.0 mL). N-methyl morpholine (49 μL, 0.45 mmol, 4.0 equiv.) was added and the reaction was allowed to stir at room temperature for 3 h. The mixture was then diluted with EtOAc, washed with brine, dried over anhydrous Na2SO4, concentrated in vacuo and purified by flash column chromatography (silica gel, 10:1 CH2Cl2: MeOH) to give the pinacol boronate (63 mg, 86%) which was used in the next step without further purification.
Global Deprotection:
In a culture tube equipped with a stir bar, the aforementioned pinacol boronic ester (32 mg) was dissolved in CH2Cl2 (1 mL). TFA (1 mL) was added at 0° C. and the resulting solution was allowed to stir at room temperature overnight. The volatiles were removed in vacuo using a rotary evaporator (water bath temperature <25° C.), and the residue was purified by preparative reverse-phase HPLC (20-80% CH3CN/H2O over 40 min, both CH3CN and H2O containing 0.1% TFA) and lyophilized to afford 52 (13.0 mg, 52% over 3 steps).
Physical state: white powder;
1H NMR (600 MHz, Methanol-d4): δ 7.96-7.90 (m, 4H), 4.66-4.59 (m, 2H), 4.16-4.07 (m, 3H), 3.83 (dt, J=10.1, 6.8 Hz, 1H), 2.40-2.29 (m, 2H), 2.26-2.16 (m, 2H), 2.14-2.02 (m, 2H), 1.82-1.72 (m, 1H), 1.17 (d, J=6.7 Hz, 3H), 1.12 (d, J=6.7 Hz, 3H), 0.98 (d, J=6.6 Hz, 3H), 0.95 (d, J=6.6 Hz, 3H) ppm;
13C NMR (151 MHz, Methanol-d4): δ 179.3, 173.1, 173.0, 169.50, 169.48, 138.1, 138.0, 128.8, 128.6, 59.0, 58.0, 42.3, 31.8, 31.1, 29.8, 26.3, 21.4, 21.2, 19.6, 19.5 ppm;
HRMS (ESI-TOF, m/z): Calcd for C24H34BN4O7[M−H2O+H]+ 501.2515; found 501.2516; [α]D20=−97.3 (c 0.26, MeOH).
The pinacol α-amino boronate S66a/S66b was prepared using the literature procedure (67) with slight modifications.
A culture tube equipped with a stirrer bar was charged sequentially with PCy3.HBF4 (12 mg, 0.033 mmol, 1.2 mol %), toluene (0.55 mL), aqueous CuSO4 (1.1 mL, 0.03 M, 1.2 mol %) and benzylamine (15.3 μL, 0.14 mmol, 5 mol %). The mixture was stirred for 10 min at the room temperature when a solution of aldimine S65 (480 mg, 2.74 mmol, 1.0 equiv.) in toluene (5.0 mL) was added, followed by B2pin2 (1.39 g, 5.5 mmol, 2.0 equiv.). The mixture was stirred vigorously for 14 h, diluted with EtOAc and filtered through a silica gel plug eluting with EtOAc. The filtrate was concentrated and purified by flash column chromatography (silica gel, 1:3 EtOAc:hexanes) to give S66a (1.07 g, d.r.>20:1) that was contaminated with impurities originating from B2pin2 which could be removed in the next step.
To a culture tube equipped with a stirrer bar were added a solution of P(OPh)3 (0.33 mL, 0.1 M in toluene, 1.2 mol %), aqueous CuSO4 (1.1 mL, 0.03 M, 1.2 mol %), and benzylamine (15.3 μL, 0.14 mmol, 5 mol %) sequentially. The mixture was stirred for 10 min, after which a solution of aldimine S65 (480 mg, 2.74 mmol, 1.0 equiv.) in toluene (5.0 mL) and B2pin2 (1.39 g, 5.5 mmol, 2.0 equiv.) were added sequentially. The mixture was stirred vigorously for 14 h, diluted with EtOAc, and filtered through a silica gel plug eluting with EtOAc. The filtrate was concentrated in vacuo, and purified by flash column chromatography (silica gel, 1:3 EtOAc:hexanes) to give S66b (857 mg, d.r.=6.1:1) contaminated with impurities originating from B2pin2 which could be removed in the next step.
The α-boronic amine hydrochloride S67a/S67b was prepared using the literature procedure (19)
S66a (190 mg, contaminated with B2pin2 impurities) was dissolved in 1,4-dioxane (1.2 mL) and methanol (0.1 mL) under argon. HCl (80 μL, 4.0 M in 1,4-dioxane) was added at room temperature and the resulting mixture was stirred at the same temperature before the volatiles were removed in vacuo. The resulting solid was triturated with a 2:1 mixture of hexanes and Et2O to give S67a (48 mg, 42% over 2 steps).
Physical state: white solid;
1H NMR (600 MHz, CDCl3) δ 8.23 (s, 3H), 2.79 (br s, 1H), 2.26 (pd, J=6.9, 4.8 Hz, 1H), 1.28 (br s, 12H), 1.11 (d, J=7.0 Hz, 3H), 1.10 (d, J=7.0 Hz, 3H).
13C NMR (151 MHz, CDCl3) δ 85.2, 44.4 (br), 29.3, 25.2, 24.8, 20.4, 19.9.
HRMS (ESI-TOF, m/z): Calcd for C10H23BNO2 [M+H]+ 200.1816; found 200.1812.
[α]D20=−3.0 (C 1.0, CHCl3).
S66b (350 mg, contaminated with Bpin impurities) was dissolved in 1,4-dioxane (2.4 mL) and methanol (0.2 mL) under argon. HCl (0.16 mL, 4.0 M in 1,4-dioxane) was added at room temperature and the resulting mixture was stirred at the same temperature before the volatiles were removed in vacuo. The resulting solid was triturated with a 2:1 mixture of hexanes and Et2O to give S67b (94 mg, 37% over 2 steps).
Physical state: white solid;
1H NMR (600 MHz, CDCl3) δ 8.25 (s, 3H), 2.80 (q, J=5.6 Hz, 1H), 2.26 (pd, J=6.9, 4.9 Hz, 1H), 1.28 (br s, 12H), 1.12 (d, J=7.2 Hz, 3H), 1.11 (d, J=7.2 Hz, 3H);
13C NMR (151 MHz, CDCl3) δ 85.2, 44.5 (br), 29.3, 25.2, 24.8, 20.4, 19.9;
HRMS (ESI-TOF, m/z): Calcd for C10H23BNO2 [M+H]+ 200.1816; found 200.1817;
[α]D20=+2.7 (c 1.0, CHCl3).
To a culture tube charged with Boc-L-Val-L-Pro-OH (S68, 34 mg, 0.11 mmol, 1.2 equiv.) and 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU, 44 mg, 0.12 mmol, 1.3 equiv.) was added DMF (0.5 mL), followed by diisopropylethylamine (45 μL, 0.26 mmol, 2.9 equiv.). S67a (21 mg, 0.089 mmol) in DMF (1.0 mL) was added dropwise at 0° C. After the completion of addition, the reaction was kept stirring at room temperature for 1 h. The mixture was diluted with Et2O, washed with brine, dried over anhydrous Na2SO4, concentrated in vacuo. The resulting residue was purified by flash column chromatography (silica gel, 1:1 EtOAc:hexanes to 3:1 EtOAc:hexanes) to give S62a (32.3 mg, 73%) as a colorless oil.
The NMR spectra of S62a are in agreements with those of S62 prepared via decarboxylative borylation. This confirms the configuration of the stereocenter a to boron in S62 to be R.
To a culture tube charged with Boc-L-Val-L-Pro-OH (S68, 26 mg, 0.083 mmol, 1.2 equiv.) and 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU, 34 mg, 0.089 mmol, 1.3 equiv.) was added DMF (0.5 mL), followed by diisopropylethylamine (35 μL, 0.2 mmol, 2.9 equiv.). S67b (16 mg, 0.068 mmol, 1.0 equiv.) in DMF (1.0 mL) was added dropwise at 0° C. After the completion of addition, the reaction was kept stirring at room temperature for 1 h. The mixture was diluted with Et2O, washed with brine, dried over anhydrous Na2SO4, concentrated in vacuo, and purified by flash column chromatography (silica gel, 1:1 EtOAc:hexanes to EtOAc) to give S62b (22 mg, 65%) as a colorless oil.
Physical state: colorless oil;
Rf=0.60 (silica gel, 1:2 EtOAc:hexanes);
1H NMR (600 MHz, CDCl3) δ 7.08 (br s, 1H), 5.21 (d, J=9.3 Hz, 1H), 4.65 (dd, J=8.2 Hz, 2.3 Hz, 1H), 4.28 (dd, J=9.4 Hz, 6.1 Hz, 1H), 3.70 (td, J=9.4 Hz, 7.1 Hz, 1H), 3.56 (ddd, J=9.6 Hz, 8.1 Hz, 3.4 Hz, 1H), 2.94 (td, J=5.7 Hz, 2.6 Hz, 1H), 2.39 (ddd, J=12.8 Hz, 6.1 Hz, 2.6 Hz, 1H), 2.14-2.05 (m, 1H), 1.98 (dtd, J=12.3 Hz, 6.8 Hz, 3.5 Hz, 2H), 1.88 (tdd, J=11.3 Hz, 9.0 Hz, 5.8 Hz, 2H), 1.42 (s, 9H), 1.21 (d, J=6.3 Hz, 12H), 1.00 (d, J=6.8 Hz, 3H), 0.94 (d, J=6.8 Hz, 3H), 0.91 (d, J=6.8 Hz, 6H) ppm;
13C NMR (151 MHz, CDCl3) δ 172.9, 171.8, 156.0, 83.1, 79.8, 59.0, 56.9, 47.7, 45.5, 31.6, 29.8, 28.5, 27.2, 25.2, 25.12, 25.10, 20.4, 20.3, 19.9, 17.7 ppm.
The NMR spectra of S62b differ from those of S62.
For chemical structures, see
Compounds 50-58, 50a, 50b, and 51a were subjected to this assay.
Serially diluted compounds in DMSO were dispensed into a 384-well black opaque plate by Echo dispenser. 0.1 μg/mL human neutrophil elastase (EPC, Catalog # SE563, Owensville, Miss.) or human sputum diluted with assay buffer (100 mM HEPES, 500 mM NaCl, 0.02% Tween 20) was added into the 384-well plate, and was incubated with different compounds at different concentrations for 30 minutes at room temperature. The final concentration of DMSO in the reaction was 0.1%. Elastase substrate MeOSuc-AAPV-AMC (Bachem, Catalog #1-1270, Torrance, Calif.) of 100 μM final concentration was then added into the reaction system just before enzyme kinetics were read on PheraSTAR plate reader at excitation of 380 nm and emission of 460 nm with a 3-minutes interval for 30 minutes in total. Slope of fluorescence intensity vs. time representing the Vmax of enzyme activity was calculated with MARS software. % relative inhibition was calculated as:
IC50 was calculated based on the % relative inhibition curve using log(agonist) vs. response (three parameters) method with Prism software. All experiments were performed in triplicate for at least three independent times. The IC50 results of all experiments are shown as the average of triplicates with error bar indicating standard deviation as indicated in individual figures. For compounds 51, 52, and 58, the assay above was repeated with 2.5, 25, 50 and 100 μM of elastase substrate (MeOSuc-AAPV-AMC) and Ki/nM values were calculated based on these results using the mixed model (68).
Human sputum was purchased from Discovery Life Sciences (Los Osos, Calif.). Human sputum was diluted 1:10 in volume with assay buffer (100 mM HEPES, 500 mM NaCl, 0.02% Tween 20) followed by vigorous vortexing. The 1:10 diluted human sputum was further diluted into 1:30, 1:90, 1:270, 1:810, and 1:2430. The elastase concentration was determined by elastase inhibition assay as described above. Specifically, a series of standards of human neutrophil elastase (starting at 2 μg/mL and further diluted 1:2 in volume) were prepared in the assay buffer. The samples and standards were plated in a 384-well black solid bottom plate; the substrate MeOSuc-AAPV-AMC of 100 μM final concentration was then added into the reaction system just before enzyme kinetics were read on PheraSTAR plate reader as mentioned above. The slope of enzymatic kinetic reading was calculated by MARS software. The elastase levels of the human sputum were calculated based on the standard curve.
The procedure for the elastase inhibition assay was followed with slight modifications. 0.1 μg/mL human neutrophil elastase was incubated with a range of concentrations of inhibitors for 5, 15, 30 and 60 minutes before substrate MeOSuc-AAPV-AMC of 100 μM final concentration was added. Enzyme kinetics were read on PheraSTAR plate reader and IC50 was calculated with the method described above.
The frozen plasma was thawed in a water bath at 37° C. prior to the experiments. The plasma was centrifuged at 4000 rpm for 5 min and the clots were removed if necessary. The pH was adjusted to 7.4±0.1 as necessary. An intermediate solution (1 mM) was prepared and a 100 μM dosing solution was prepared by diluting 10 μL of the intermediate solution with 90 μL 45% MeOH/H2O. Duplicate of test samples were made by mixing 98 μL of blank plasma with 2 μL of dosing solution (100 μM) to achieve the final concentration of 2 μM. Samples were incubated at 37° C. At each time point (0, 10, 30, 60, and 120 min), 400 μL of stop solution (consisting of 200 ng/mL tolbutamide and 20 ng/mL buspirone in 50% MeOH/CH3CN) was added to precipitate protein under thorough mixing. The sample plates were then centrifuged at 4,000 rpm for 10 min. An aliquot of supernatant (100 μL) was transferred from each well and mixed with 200 of μL ultrapure water. The samples were shaken at 800 rpm for about 10 min before LC-MS/MS analysis.
The % remaining of test compound after incubation in plasma was calculated using following equation:
% Remaining=100×(PAR at Tn/PAR at T0)
where PAR is the peak area ratio of analyte versus internal standard (IS) and The appointed incubation time points are T0 (0 min), Tn (n=0, 10, 30, 60, 120 min).
Each compound was analyzed by LC/MS using an ACE 5-phenyl 50×2.1 mm column (Part No. ACE-125-0502) with 0.1% formic acid in water and 0.1% formic acid in acetonitrile as the mobile phases. Tobultamide was used as the internal standard. Data collected were processed by Analyst 1.6.2 software and MultiQuant 3.0.2 software.
1) Compounds 50, 50a, 51, 51a, and 52 were tested in this assay. Testosterone, Dichlofenac, and Propafenone were used as control.
1. 100 mM potassium phosphate buffer, pH 7.4.
2. 10 mM MgCl2
Intermediate solution was prepared by diluting 5 μL of compound or control stock solution (10 mM in DMSO) with DMSO (45 μL) and 1:1 Methanol/Water (450 μL) (concentration=100 μM, 45% MeOH). Working solution was prepared by diluting 50 μL of the intermediate solution with 450 μL of 100 mM potassium phosphate buffer, pH=7.4 (centration=10 μM, 4.5% MeOH).
4) NADPH regenerating system (final Isocitric dehydrogenase concentration=1 unit/mL at incubation) comprised:
β-Nicotinamide adenine dinucleotide phosphate acquired from Sigma (Catalog # N0505), isocitric acid from Sigma (Cat. No. 11252) and isocitric dehydrogenase from Sigma (Catalog #12002).
5) Liver microsome solution (final concentration of 0.5 mg protein/mL) was prepared using Mouse liver microsomes from Xenotech (Catalog # M1000, Lot #1310028).
6) Stop solution: Cold acetonitrile containing 100 ng/mL Tolbutamide and 100 ng/mL Labetalol as internal standards (IS)
10 μL/well of compound working solution or control working solution was added to all plates (T0, T5, T10, T20, T30, T60, NCF60) except the matrix blank. 80 μL/well of microsome solution was added to every plate. The mixtures of microsome solution and compound were incubated at 37° C. for about 10 min. 10 μL/well of NADPH regenerating system (pre-warmed to 37° C.) was then added to every plate to start the reaction. The plates were incubated for the durations indicated (matrix blank: 1 h; T60: 1 h; T30: 31 min; T20: 40 min; T10 50 min; T5: 55 min). For NCF60 (abbreviation of no co-factor) no NADPH regenerating system was added, but was replaced by 10 μL/well of potassium phosphate buffer (100 mM, pH 7.4); the resulting mixture was incubated at 37° C. for 1 h.
The reactions were then terminated with the stop solution (cold at 4° C.) containing 100 ng/mL Tolbutamide and 100 ng/mL Labetalol (300 μL/well). The sampling plates were shaken for approximately 10 minutes, then were centrifuged at 4000 rpm for 20 min at 4° C. While centrifuging, 8 new 96 well plates were loaded with 300 μL of HPLC grade water. 100 μL of supernatant was finally added to 300 μL of HPLC grade water and mixed for LC/MS/MS analysis.
Apricot pipetting robot was used for all additions, mixing, and transformations described above in 96-well plate format.
The equation of first order kinetics was used to calculate T1/2 and Clint(mic):
Whereby Qh(mL/min/Kg liver) = 90.0 mL/min/Kg for mouse liver
Compounds 50, 50a, 51, 51a, and 52 were tested.
The stock solution of each compound (10 μL; 10 mM in DMSO) was diluted with phosphate buffer solution (490 μL; 50 mM, pH 6.8). The resulting mixture was shaken for 24 h. Samples were then filtered. Kinetic solubility was then determined by UV spectroscopy [calibrated by a standard curve (1, 20, and 200 μM)].
The transport buffer used in the study was HBSS with 10 mM HEPES at pH 7.40±0.05. Compounds were tested at 2 μM bi-directionally in duplicates. Digoxin was tested at 10 μM bi-directionally in a duplicate, while fenoterol and propranolol were tested at 2 μM in A (apical) to B (basolateral) direction in duplicates. The final DMSO concentration was adjusted to less than 1%. The plate was incubated for 2 hours in a CO2 incubator at 37±1° C., with 5% CO2 at saturated humidity without shaking. All samples, after mixing with acetonitrile containing internal standard, were centrifuged at 4000 rpm for 20 min. Subsequently, 100 μL supernatant solution was diluted with 100 μL distilled water for LC/MS/MS analysis. Concentrations of test and control compounds in starting solution, donor solution, and receiver solution were quantified by LC/MS/MS methodologies, using peak area ratio of analyte/internal standard. After transport assay, lucifer yellow rejection assay was applied to determine the Caco-2 cell monolayer integrity. All data presented herein have passed this test.
The apparent permeability coefficient Papp (cm/s) was calculated using the equation:
is the cumulative concentration of compound in the receiver chamber as a function of time (μM/s); Vr is the solution volume in the receiver chamber (0.075 mL on the apical side, 0.25 mL on the basolateral side); A is the surface area for the transport, i.e. 0.0804 cm2 for the area of the monolayer; Co is the initial concentration in the donor chamber (μM).
The efflux ratio was calculated using the equation:
Efflux ratio=Papp(BA)/Papp(AB)
Percent recovery was calculated using the equation:
% Recovery=100×[(Vr·Cr)+(Vd·Cd)]/(Vd·C0)
Where Vd is the volume in the donor chambers (0.075 mL on the apical side, 0.25 mL on the basolateral side); Cd and Cr are the final concentrations of transport compound in donor and receiver chambers, respectively.
Each compound was analyzed by LC/MS using an ACE 5-phenyl 50×2.1 mm column (Part No. ACE-125-0502) with 0.1% formic acid in water and 0.1% formic acid in acetonitrile as the mobile phases. Tobultamide was used as the internal standard. Data collected were processed by Analyst 1.6.2 software and MultiQuant 3.0.2 software.
All patents and publications referred to herein are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.
This invention was made with government support under GM-118176 awarded by the National Institutes of Health. The government has certain rights in the invention.
Filing Document | Filing Date | Country | Kind |
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PCT/US2018/022394 | 3/14/2018 | WO | 00 |
Number | Date | Country | |
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62474181 | Mar 2017 | US |