A Scalable Synthesis of the Difluoromethyl-allo-threonyl Hydroxamate-Based LpxC Inhibitor LPC-058

Article abstract of DOI:10.1021/acs.joc.6b00589

The difluoromethyl-allo-threonyl hydroxamate-based compound LPC-058 is a potent inhibitor of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) in Gram-negative bacteria. A scalable synthesis of this compound is described. The key step

Full text of DOI:10.1021/acs.joc.6b00589

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Note
A Scalable Synthesis of the Difluoromethyl-allo-
threonyl hydroxamate-based LpxC Inhibitor LPC-058
Xiaofei Liang, Ramesh Gopalaswamy, Frank Navas III, Eric J Toone, and Pei Zhou
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b00589 • Publication Date (Web): 29 Apr 2016
Downloaded from http://pubs.acs.org on May 2, 2016
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A Scalable Synthesis of the Difluoromethylꢀalloꢀthreonyl
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hydroxamateꢀbased LpxC Inhibitor LPCꢀ058
Xiaofei Liang1, Ramesh Gopalaswamy1*, Frank Navas III1, Eric J.Toone1,2, Pei Zhou1,2*
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1Department of Chemistry, Duke University, Durham, NC, 27708, USA
2Department of Biochemistry, Duke University Medical Center, Durham, NC 27710,
USA
Corresponding Authors
*Email: ramegop@gmail.com
*Email: peizhou@biochem.duke.edu
Abstract
The difluoromethylꢀalloꢀthreonyl hydroxamateꢀbased compound LPC-058 is a potent
inhibitor of UDPꢀ3ꢀOꢀ(Rꢀ3ꢀhydroxymyristoyl)ꢀNꢀacetylglucosamine deacetylase (LpxC)
in Gramꢀnegative bacteria. A scalable synthesis of this compound is described. The key
step in the synthetic sequence is a transition metal/baseꢀcatalyzed aldol reactions of
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methyl isocyanoacetate and difluoroacetone, giving rise to 4ꢀ(methoxycarbonyl)ꢀ5,5ꢀ
disubstituted 2ꢀoxazoline. A simple NMR based determination of enantiomeric purity is
also described.
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The alarming rise of antimicrobial resistance1ꢀ3 of Gramꢀnegative pathogens has
become a serious threat to public health and highlights the urgent need for antibiotics
with novel modes of action. The UDPꢀ3ꢀOꢀ(Rꢀ3ꢀhydroxymyristoyl)ꢀNꢀacetylglucosamine
deacetylase (LpxC) is a novel, validated antibiotic target against Gramꢀnegative
pathogens.4 The enzyme catalyzes the committed step in the biosynthesis of lipid A ꢀ the
membrane anchor of lipopolysaccharide and the predominant lipid component of the
outer leaflet of Gramꢀnegative outer membrane. We have previously reported the design
of potent LpxC inhibitors containing a difluoromethylꢀalloꢀthreonylꢀhydroxamate head
group.4 This series of compounds, represented by LPC-058, displays broadꢀspectrum
antibiotic activity against antibiotic resistant Gramꢀnegative pathogens thereby
highlighting the therapeutic potential of this class of LpxC inhibitors. Herein, we describe
a scalable and stereoselective synthesis of LPC-058, which sets the stage for preclinical
in vivo studies of this novel series.4
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The initial medicinal chemistry route for the synthesis of LPC-058 is shown in Scheme
1. The diacetylene carboxylic acid 4 was prepared in two steps from methylꢀ4ꢀethynyl
benzoate (1). The first step employed modified Glaser reaction5, 6 conditions wherein an
excess of 4ꢀethylnylbenzenamine 2 (5 equivalents) and copper (II) acetate (2 equivalents)
in the presence of piperidine in dichloromethane (DCM) provided 3 in low yield (35%).7
Subsequent alkaline hydrolysis of ester 3 provided the diacetylene carboxylic acid 4 in
quantitative yield.7
We recently published the medicinal chemistry route for (2S,3S)ꢀ3ꢀ
difluoromethylthreonine methyl ester 11, which was generated in six steps starting from
(S)ꢀ1ꢀmethylꢀ4ꢀ(methylsulfinyl)benzene (Scheme 1).4 Our synthetic strategy for amino
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ester 11 was adapted from a published procedure for the corresponding monofluoro
analog.8 The copper (I) catalyzed [2+3] cycloaddition reaction between methyl
isocyanoacetate and (S)ꢀ1,1ꢀdifluoroꢀ3ꢀ(pꢀtolylsulfinyl)propanꢀ2ꢀone (6) provided
oxazoline 7 as a mixture of diastereomers. The desired diasteroisomer of oxazoline 7 (ie
4S,5S,SS) is the less polar spot on TLC with Rf~0.35 (EtOAc:Hexanes (2:3)) and was
isolated by flash chromatography (EtOAcꢀhexanes (0ꢀ40%)) as the major product in 45%
yield. Oxazoline 7 was readily hydrolyzed in a chloroform/water mixture at room
temperature to give the amidoꢀester 8 in near quantitative yield. The sulfoxide 8 was
reduced to the corresponding sulfide 9 by using trifluoroacetic anhydride with sodium
iodide in acetone at ꢀ40 °C.9 Desulfurization of 9 with a Raney nickelꢀsodium
hypophosphite system provided the (2S,3S)ꢀ3ꢀdifluoromethylthreonine derivative (10),
which upon mild acidic hydrolysis afforded (2S,3S)ꢀ3ꢀdifluoromethylthreonine methyl
ester 11.10
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Amino ester 11 was reacted with diacetylene carboxylic acid 4 under standard coupling
conditions to give amide ester 12. Treatment of 12 with hydroxylamine under alkaline
conditions afforded the desired hydroxamate LPC-058.4
Scheme 1: Initial route to LPC-058
Synthesis of diacetylene carboxylic acid7
Synthesis of the difluoro-threonine methyl ester4
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O
HO
CF2H
CO2Me
O
S
S
O
S
O
O
S
CN
CO2Me
CF2H
CO2Me
H2O-CHCl3
5
RCO2C2H5
NHCHO
CF2H
O
LDA, THF
-78 oC, 3h
88%
rt, 24h
94%
10% Cu2O, Et2O
0 oC to rt, 16h
45%
4
N
8
5
(4S, 5S, Ss)
7
6
+ other diastereomers
HO
9
CF2H
CO2Me
OH
CF2H
O
MeO
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S
NaI,(F3CCO)2O
Raney Ni, acetate buffer (pH=5.2)
OMe
Conc HCl (1eq)
NHCHO
HF2C
HO
OHCHN
NH2.HCl
acetone, -10 oC, 20 min
aq NaH2PO2, Methanol, rt, 14h
87%
MeOH, rt, 16h
99%
O
88%
11
9
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Synthesis of LPC-0584
A number of factors made this approach unsuitable for delivering substantial quantities
(>100g) of LPC-058. The major drawback of this strategy is the tedious chromatographic
separation of oxazoline 7 from its diastereomers. Furthermore, the chiral sulfoxide 5 is
very expensive or has to be prepared.11 Finally, the copperꢀcatalyzed crossꢀcoupling of
methyl 4ꢀethynylbenzoate 1 with excess 4ꢀethylnylbenzenamine 2, is complicated by the
formation of homocoupled products which require tedious chromatographic separation.
Therefore, we directed our attention to developing efficient and scalable routes to the key
intermediates (2S,3S)ꢀ3ꢀdifluoromethylthreonine methyl ester 11 and diacetylene
carboxylic acid 4.
Synthesis of unsymmetrical diynes has recently been the subject of extensive research. A
survey of the literature indicates that diynes can be formed under various conditions and
many useful methods have been reported.12ꢀ16 In 2010, Balaraman et. al. improved the
synthesis of symmetrical and unsymmetrical 1,3ꢀdiynes by employing catalytic amounts
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(0.1 equivalent) of copper(II) acetate in the presence of a stoichiometric amount of
piperidine in 1,2ꢀdichloroethane under aerobic condition.12 With this method, the yield
of unsymmetrical 1,3 diyne 3 was over 60% (an improvement over that described above)
but unfortunately, it still involved five equivalents of 4ꢀethylnylbenzenamine 2 and
homocoupled byꢀproducts remained a problem.
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Efficient synthesis of unsymmetrical diynes using an alkynyl halide and a terminal alkyne
have been reported.14, 15 In order to reduce the need for excess 4ꢀethylnylbenzenamine 2,
we replaced methyl 4ꢀethynylbenzoate 1 with methyl 4ꢀ(bromoethynyl)benzoate 1617 as
described in Scheme 2. Treatment of methyl 4ꢀethynylbenzoate 1 with Nꢀ
bromosuccinimide in the presence of silver trifluoroacetate as a catalyst provided 4ꢀ
(bromoethynyl)benzoate 1617 in 82% yield. The reaction of 16 with near stoichiometric
amount of 4ꢀethynylbenzenamine 2 (1.05 equivalents) and copper (II) acetate (0.05
equivalent) as a catalyst in the presence of piperidine proceeded efficiently to give diyne
3 (92% yield). In the scale up process, the crude intermediate 3 was hydrolyzed to give
diacetylene acid 4 without purification.
Scheme 2: Synthesis of Diyne Carboxylic Acid 4
O
O
O
R
O
OMe
OMe
2
1) , 0.05 eq Cu(OAc)2
NBS, CF3CO2Ag
3 eq piperidine, MeOH, rt, 20h
Acetone, rt, 2h
83%
2) 1N NaOH, MeOH
reflux, 1h
Br
16
3
4
R = Me
R = H
1
88%
H2N
We next turned our attention to the large scale synthesis of (2S,3S)ꢀ3ꢀ
difluoromethylthreonine methyl ester 11. The chiral sulfoxide controlled stereoselective
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synthesis of optically pure (2S,3S)ꢀ3ꢀdifluoromethylthreonine methyl ester 11 (Scheme 1)
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is efficient and useful on a small scale. However, on a large scale, this approach has
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significant drawbacks including: 1) the
high cost of (S)ꢀ1ꢀmethylꢀ4ꢀ
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(methylsulfinyl)benzene; 2) the fact that there was only a single crystalline intermediate
in the sequence (i.e. chiral difluoroacetone 6); and 3) the low boiling point of
trifluoroacetic anhydride can be a safety concern when used in large scale reactions.
Taken together, these concerns led us to reꢀevaluate our strategy. In developing an
alternative scalable route, our main objectives were to: (a) address the safety issues; (b)
enhance the robustness of the synthetic route; and (c) devise a sequence which had the
potential for additional crystalline intermediates to facilitate purification by
recrystallization. We took advantage of the well documented transition metal/base
catalyzed aldol reaction to construct oxazolines in stereoselective manner.18 It is
noteworthy to mention, the authors in that work have included entries of
monofluoroacetone and trifluoroacetone reacting with methyl αꢀisocyanoacetate to yield
corresponding oxazolines stereoselectively in ratios of 71:29 and 99:1 respectively.
Inspired by their results, we had hoped to obtain stereoselectivity somewhere in between
the aforementioned ratios when reacting difluoroacetone with methyl αꢀisocyanoacetate
as described in Scheme 3.
Scheme 3: Synthesis of Amine
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Difluoroacetone 17 was reacted with methyl αꢀisocyanoacetate 18 to give the
diastereomeric mixture of oxazolines 19 (a & b). The reaction was performed in dry 1,1ꢀ
dichloroethane (DCE) at ambient temperature in the presence of 4 mol % CuCl and 5 mol
% TEA. The ratio of oxazoline diastereomers 19a/19b is 87:13, which was determined by
NMR chemical shifts of the crude reaction mixture after removal of the catalyst and
solvent. Replacing DCE with DCM as the reaction solvent also provided similar results
in terms of chemical yield and diastereoselectivity. Hydrolysis of the diastereomeric
mixture of oxazolines 19a and 19b in methanolic HCl at room temperature afforded the
corresponding amino acid hydrochloride salts 20a and 20b. The desired diastereomer
20a was obtained in >99% purity after recrystallizaion from MeOH/MTBE at room
temperature with a chemical yield of 42%. Alternatively, recrystallization can be
accomplished from hot acetonitrile to provide 20a in 56% yield and >99% purity. In
order to get optically pure 21, the amino acid hydrochloride salt 20a was neutralized by
Na2CO3 and the racemic free base was reacted with equimolar amounts of (ꢀ)ꢀCSA at
room temperature in acetone. Upon cooling, the desired amino ester 21 was obtained in
60% yield based on single enantiomer with an optical purity of >99%. Optical purity was
1
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1
19
established using H NMR and F NMR as follows: H NMR and F NMR of racemic
hydrochloride salt in the presence of 10 fold excess of (+)ꢀtartaric acid in CD3OD,
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yielded a spectrum with wellꢀresolved peaks. Particularly, the F, αꢀCH and ester CH3
of the racemic methyl ester (Figure 1, A,D) were well resolved and readily identified
when recorded in the presence of excess tartaric acid (Figure 1, B,E). It is to be noted
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that H NMR and 19F NMR of recrystallized of optically pure sample of 21 in the
presence of 10ꢀfold excess of (+)ꢀtartaric acid in CD3OD displayed only one set of peaks
corresponding to the desired enantiomer (Figure 1, C, F). The modified route to the
chiral amino ester 21 was shorter and cheaper than that described earlier for the
corresponding hydrochloride salt 11 and was deemed suitable for large scale synthesis.
Figure 1. A, D: racemic hydrochloride salt 20 in CD3OD; B, E: racemic hydrochloride
salt 20 in the presence of 10 fold excess of (+)ꢀtartaric acid in CD3OD; C, F:
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recrystallized of optically pure sample of 21 in the presence of 10 fold excess of (+)ꢀ
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tartaric acid in CD3OD.
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With multigram quantities of amino ester 21 and diacetylene carboxylic acid 4 in hand,
we proceeded to the large scale synthesis of LPC-058 (Scheme 4). Amide coupling
under EDC/HOBt conditions provided the diacetylene ester 12. Treatment of 12 with
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o
hydroxylamine and sodium methoxide at 0 C provided hydroxamate LPC-058 in 67%
yield over two steps. It is noted that no epimerization at the αꢀ carbon was observed by
LCꢀMS or NMR under these conditions.
Scheme 4: Amide coupling
In order to determine the absolute configuration of 11 and 21, they were crystallized in
methanol/MTBE and acetone, respectively. The absolute configuration was determined
by the measurement of the Flack parameter, which is calculated during the structural
refinement.19, 20 If the value is near 0, the crystal is optically pure, in which case, the
absolute structure of the molecule can be determined with certainty by the Xꢀray data. In
our study, the final refinement on the Cu Kα data of the crystal of 11 and 21 resulted in a
Flack parameter of 0.0(0) and ꢀ0.0(1), allowing an unambiguous assignment of the
absolute structure as shown in figure 2. The two chiral centers of 11, Cꢀ2, Cꢀ3, and the
four chiral centers of 21, Cꢀ2, Cꢀ3, Cꢀ8, Cꢀ11, were thus determined as S, S, and S, S, R,
S, respectively.
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O
MeO
HF2C
HO
Compound 11
NH2.HCl
Figure 2. Xꢀray crystallographic structure of 11 and 21, the compounds are colored in the
following atom colors: carbon, gray; nitrogen, blue; oxygen, red; sulfur, yellow; fluorine,
green; chlorine, deep green.
Conclusion
In summary, a short and scalable synthesis of the hydroxamic acid derivative LPC-058 is
described. The syntheses of key intermediates diacetylene carboxylic acid 4 and
difluorothreonine methyl ester 21 were accomplished without any chromatography steps.
Experimental section
Methyl 4ꢀethynylbenzoate, 4ꢀethylnylbenzenamine, 1,1ꢀdifluoroacetone and methyl αꢀ
isocyanoacetate were purchased from commercial vendors and used without further
purification. LCꢀMS analysis was conducted on a HPLC instrument with a quadrapole
mass analyzer and reverse phase C18 column (4.6×50 mm, 1.8 ꢁM) with a
water/acetonitrile (each with 0.2% (v/v) formic acid) gradient at a flow rate of 0.5
mL/min. The HRMS analyses were conducted on a TOF mass spectrometer using ESI
mode. 1H, 13C and 19F spectra were recorded on 300 (or 400) MHz, 75 (or 100) MHz, and
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282.4 (or 376.5) MHz spectrometers respectively. Column chromatography was
conducted using either silica gel flash chromatography or prepacked silica gel cartridges.
All moistureꢀsensitive reactions were carried out using dry solvents and under a slight
pressure of ultraꢀpure quality argon. Glassware was dried in an oven at 140°C for at least
12h prior to use, and then assembled quickly while hot, sealed with rubber septa, and
allowed to cool under a stream of argon. Reactions were stirred magnetically using
Teflonꢀcoated magnetic stirring bars. Commercially available disposable syringes were
used for transferring reagents and solvents.
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Methyl 4-(bromoethynyl)benzoate (16).17 To a solution of methyl 4ꢀethynylbenzoate 1
(10.00 g, 62.5 mmol, 1.0 equiv) in acetone (100 mL) was added NBS (12.24 g, 68.8
mmol, 1.05 equiv) and CF3CO2Ag (0.53g, 0.31 mmol, 0.05 equiv) at room temperature
under argon. The reaction mixture was stirred at room temperature for 2h. The resulting
solution was concentrated to remove acetone. The residue was diluted with water (100
mL) and extracted with EtOAc (3×80 mL). The combined organic layers were washed
with water (2×60 mL), brine (60 mL) and dried over (anhydrous Na2SO4). Evaporation of
the solvent afforded the crude product which was purified by silica gel chromatography
(eluting with EtOAc in hexane 0ꢀ10%) to give 16 as white solid (12.4 g, yield 83%). 1H
NMR (300 MHz, CDCl3): δ 3.9 (s, 3H), 7.50 (d, J = 8.1 Hz, 2H), 7.97 (d, J = 6.9 Hz,
13
2H); C NMR (75 MHz, CDCl3): δ 52.3, 53.4, 79.4, 127.3, 129.5, 129.9, 131.9, 166.3;
MS (LC/MS; ESI) m/z 240 [M+H]+.
Methyl 4-((4-aminophenyl)buta-1,3-diyn-1-yl)benzoate (3).7 Copper (II) acetate
(0.45g, 0.50 mmol, 0.05 equiv) was added at room temperature and under stream of
argon to a stirred solution of methyl (4ꢀbromoethylnyl)benzoate (12.0 g, 50.0 mmol, 1.00
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equiv) 4ꢀethynylaniline 2 (6.20 g, 52.5 mmol, 1.05 equiv) and piperidine (12.77g, 150.0
mmoL, 3.00 equiv) in MeOH (200 mL, degassed by argon).The reaction mixture was
stirred at room temperature for 20h. The resulting suspension was diluted with water (400
mL) and stirred for 30 min at room temperature. The mixture was filtered and the filtered
solid was washed with water (2×200 mL). The solid obtained was dried to afford crude 3
(13.5 g) as a yellow solid which was carried on to the next step without further
purification. 1H NMR (300 MHz, DMSOꢀd6): δ 3.83 (s, 3H), 5.85 (s, 2H), 6.54 (d, J = 8.1
Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.1 Hz, 2H), 7.92 (d, J = 8.1 Hz, 2H); 13C
NMR (75 MHz, DMSOꢀd6): δ 53.0, 71.8, 78.3, 80.5, 87.0, 105.7, 114.3, 126.7, 130.1,
130.3, 133.0, 134.8, 151.6, 166.2; MS (LC/MS; ESI) m/z 276 [M+H]+.
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4-((4-Aminophenyl)buta-1,3-diyn-1-yl)benzoic acid (4).7 To a solution of crude
compound 3 (13.5 g, 50.0 mmol, 1.00 equiv) in methanol (100 mL) and was added 1N
NaOH (100 mL, 100.0 mmol, 2.00 equiv) at room temperature under a stream of argon.
The reaction mixture was heated to reflux for 1h. The solution was diluted with water
(100 mL) and acidified with concentrated HCl to pH ~3. The precipitate was filtered,
washed with water and dried under vacuum to give the crude acid (4) (11.5g, two steps,
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yield 88% for two steps), which was carried to next step without purification. H NMR
(300 MHz, DMSOꢀd6): δ 6.95 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.66 (d, J =
8.4 Hz, 2H), 7.93 (d, J = 8.4 Hz, 2H); 13C NMR (75MHz, DMSOꢀd6): δ 73.0, 77.2, 81.2,
85.0, 112.0, 118.8, 125.8, 130.2, 131.9, 133.1, 134.6, 144.2, 167.2; MS (LC/MS; ESI)
m/z 262 [M+H]+.
(4S,5S)-methyl 5-(difluoromethyl)-5-methyl-4,5-dihydrooxazole-4-carboxylate (19a)
Methyl 2ꢀisocyanoacetate (20.00 g, 201.8 mmol, 1.00 equiv) was added slowly to an ice
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cold suspension of 1,1ꢀdifluoroacetone (22.78 g, 242.20 mmol, 1.20 equiv), CuCl (0.80
g, 8.10 mmol, 0.04 equiv), and TEA (1.41 mL, 10.10 mmol, 0.05 equiv) in anhydrous
DCM (330 mL) under argon. The reaction mixture was stirred at 0 °C and gradually
warmed to room temperature overnight (20 h). The resulting solution was diluted with
DCM (150 mL). The mixture was washed with 10% aqueous ammonia (3×100 mL),
water (100 mL), brine (100 mL), and dried (anhydrous Na2SO4). Evaporation of the
solvent afforded crude mixture (86:14) of diastereomers 19 (a & b) (brown liquid, 35.5 g,
yield 91%) which was carried to the next step without further purification. 1H NMR (400
MHz, CDCl3): δ 1.35 (s, 3H), 1.54 (s, 0.5H), 3.67 (s, 0.5H), 3.74 (s, 3H), 4.47 (s, 0.17H),
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4.78 (s, 1H), 5.69 (t, JHF = 56 Hz, 1H), 5.88 (t, JHF = 56 Hz, 0.17H), 6.94 (s, 1H); C
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NMR (100 MHz, CDCl3): δ 15.4, 43.4, 52.5, 114.2 (t, JCF = 494 Hz), 168.7; F NMR
(376.5 MHz, CDCl3): δ ꢀ132.83 (d, JHF = 56 Hz, major), δ ꢀ131.40 (ABq, d, 2F, JHF = 56
Hz, JFF=300 Hz, minor); MS (LC/MS; ESI) m/z 194 [M+H]+.
Methyl 2-amino-4,4-difluoro-3-hydroxy-3-methylbutanoate hydrochloride (racemic
20a) To a stirred solution of oxazoline 19 (a & b) (35.30 g, 182.70 mmol, 1.00 equiv) in
methanol (180 mL) kept at room temperature in a water bath under argon was added
dropwise concentrated HCl (36 mL). The reaction mixture was stirred at room
temperature for 14h. The resulting solution was concentrated to dryness. The residue was
diluted with MTBE (400 mL) and was stirred vigorously for 2 h. The suspension was
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concentrated to afford crude 20 (a &b) as a light brown solid H NMR (400 MHz,
CD3OD): δ 1.30 (s, 3H), 1.48 (s, 0.5 H), 3.85 (s, 0.5 H), 3.86 (s, 3H), 4.15 (s, 0.17H),
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4.16 (s, 1H), 5.95 (t, JHF = 56 Hz, 0.17H), 5.99 (t, JHF = 56 Hz, 1H); F NMR (376.5
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MHz, CD3OD): δ ꢀ134.25, ꢀ135.15 (ABq, d, 2F, JHF = 56 Hz, JFF = 301 Hz); C NMR
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(100 MHz, CD3OD): δ 15.9, 52.6, 55.9, 71.2 (t, JCF = 42.5 Hz), 115.4 (t, JCF = 491Hz),
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166.7; MS (LC/MS; ESI) m/z 184 [M+H]+. The crude product was dissolved to ~55 mL
of methanol and MTBE (~270 mL) at room temperature. Then the solution was left in ꢀ20
°C for 2 days to give the racemic 20a (17.00 g, yield 42%) as an offꢀwhite solid.
Alternatively, crude mixture of 20 (a & b) can be recrystallized from hot acetonitrile
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(~200 mL) to provide racemic 20a in 56% yield. mp 142 C; H (400 MHz, CD3OD) δ
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1.30 (s, 3H), 3.87 (s, 3H), 4.16 (s, 1H), 5.99 (t, JHF = 56 Hz, 1H); F (376.5 MHz,
CD3OD) δ ꢀ134.25, ꢀ135.15 (ABq, d, 2F, JHF = 56 Hz, JFF=301 Hz); 13C (100 MHz,
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CD3OD) δ 15.9, 52.6, 56.0, 71.2 (t, JCF = 42.5 Hz), 115.4 (t, JCF = 490 Hz); F (376
MHz, CDCl3) δ ꢀ134.70; HRMS (ESI/TOF) m/z: [M+H]+ Calcd for C6H12F2NO3
184.0785; Found 184.0776.
(2S,3S)-methyl 2-amino-4,4-difluoro-3-hydroxy-3-methylbutanoate (-) CSA (21) To
an ice bath cooled solution of racemic 20 (16.70 g, 76.00 mmol, 1.00 equiv) in water
(100 mL) was added Na2CO3 (24.17 g, 228.00 mmol, 3.00 equiv) under argon. The
reaction mixture was stirred at room temperature for 1h. Then the resulting solution was
extracted with EtOAc (6×100 mL). The combined organic layers were dried over
(anhydrous Na2SO4). Evaporation of the solvent afforded the free amine (14.0 g) as a
light brown liquid. The free amine (13.50 g, 73.7 mmol, 1.00 equiv) was dissolved in
acetone (100 mL) and (1R) (ꢀ)ꢀ10 camphorsulfonic acid (17.12 g, 73.70 mmol, 1.00
equiv) was added. The mixture was stirred at room temperature overnight. The white
solid was filtered and washed with acetone (20 mL) and dried to give the (ꢀ) CSA salt 21
o
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(8.40 g, yield 28%). mp 160 C; [α]D = ꢀ24.1 (c = 0.4; MeOH); H NMR (400 MHz,
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CD3OD): δ 0.83 (s, 3H), 1.09 (s, 3H), 1.30 (s, 3H), 1.37ꢀ1.42 (m, 1H), 1.57ꢀ1.64 (m, 1H),
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1.88 (d, J = 18.4 Hz, 1H), 2.00ꢀ2.04 (m, 2H), 2.29ꢀ2.35 (m, 1H), 2.57ꢀ2.61 (m, 1H),
2.76(d, J = 14.8 Hz, 1H), 3.86 (s, 3H), 4.21 (s, 1H), 5.98 (t, JHF = 56 Hz, 1H); 19F NMR
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(376.5 MHz, CD3OD): δ ꢀ134.23, ꢀ135.13 (ABq, d, 2F, JHF = 56 Hz, JFF = 301 Hz); C
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(100 MHz, CD3OD) δ 5.8, 18.7, 19.0, 24.3, 26.4, 42.2, 42.6, 46.8, 52.6, 56.0, 58.2, 71.2
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(t, JCF = 42.5 Hz), 115.4 (t, JCF = 491 Hz), 166.7, 218.1; F (376 MHz, CDCl3) δ ꢀ
134.68 (t, JHF = 56 Hz); HRMS (ESI/TOF) m/z: [M+H]+ Calcd for C6H12F2NO3
184.0785; Found: 184.0781.
(2S,3S)-Methyl 2-(4-((4-aminophenyl)buta-1,3-diyn-1-yl)benzamido)-4,4-difluoro-3-
hydroxy-3-methylbutanoate (12).4 To a solution of diacetylene carboxylic acid 4 (3.00
g, 12.49 mmol) in anhydrous DMF (25 mL) was added amino ester 21 (5.40 g,
13.11mmol, 1.05 equiv), EDC.HCl (2.64 g, 13.80 mmol, 1.2 equiv), HOBt (1.87 g, 13.80
mmol, 1.2 equiv) at room temperature under argon. The mixture was cooled to 0 °C and
DIEA (8.1 mL, 45.90 mmol, 4.00 equiv) was added. The reaction mixture was stirred at 0
°C for 2 hours, and then was allowed to warm to room temperature for 14 hours. The
yellow solution was then concentrated to dryness. The residue was treated with water
(100 mL), extracted with EtOAc (3×80 mL). The combined extracts were washed with
water (80 mL), brine (80 mL), and dried (anhydrous Na2SO4). The crude product was
purified by silica gel chromatography (eluting with MeOH in DCM 0ꢀ2.5 %) to give 12
o
as a yellow solid (4.23 g, 80%). mp 167ꢀ168 C (decomp); [α]D = +28.2 (c = 0.2;
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MeOH); H NMR (300 MHz, CD3OD): δ 1.37 (d, J = 2.1 Hz, 3 H), 3,76 (s, 3H), 4.267
(dd, J = 9.3, 19.5 Hz, 1H), 4.42 (dd, J = 9.3, 19.8 Hz, 1H), 4.81 (s, 1H), 5.85 (t, JHF = 56
Hz, 1H), 6.61 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.1 Hz, 2H), 7.82
(d, J = 8.1 Hz, 2H); 19F NMR (400 MHz, CD3OD): δ ꢀ131.06, ꢀ137.61 (ABq, d, 2F, JHF =
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56 Hz, JFF = 300 Hz); 13C NMR (75 MHz, CD3OD): δ 20.5, 51.7, 70.9, (71.9, 72.2), 76.9,
79.1, 84.7, (85.5, 87.8), 108.3, 114.3, 126.2, 127.2, 127.6, 132.2, 133.6, 133.9, 150.2,
168.2, 170.6; MS (LC/MS; ESI) m/z 427 [M+H]+; HRMS (ESI/TOF) m/z: [M]+ Calcd
for C23H20F2N2O4 426.1391; Found: 426.1392.
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4-((4-Aminophenyl)buta-1,3-diyn-1-yl)-N-((2S,3S)-4,4-difluoro-3-hydroxy-1-
(hydroxyamino)-3-methyl-1-oxobutan-2-yl)benzamide (LPC-058).4 To an iceꢀcold
solution of 12 (4.00 g, 9.4 mmol) dissolved in anhydrous MeOH (15 mL) and THF (15
mL) was added hydroxylamine hydrochloride (3.27 g, 46.9 mmol, 5.0 equiv) followed by
25% sodium methoxide in methanol solution (16.0 mL, 70.50 mmol, 7.5 equiv). The
reaction mixture was stirred under argon and at 0°C for 2h, then allowed to warm to
ambient temperature with stirring continued overnight (14h). The resulting yellow
suspension was condensed to dryness with a rotary evaporator, and the residue obtained
was treated water (200 mL) and saturated NH4Cl (80 mL), and extracted with EtOAc
(3×100 mL). The combined extracts were washed with water (80 mL), brine (80 mL),
and dried over anhydrous Na2SO4. Evaporation of the solvent afforded the crude product,
which was purified by silica gel chromatography (eluting with 0ꢀ5% MeOH in DCM) to
o
afford the title compound as a yellow solid (3.29 g, 82% yield). mp 155ꢀ157 C
(decomp); [α]D25 = +61.2 (c = 0.4; MeOH). 1H NMR (300 MHz, CD3OD): δ 1.36 (s, 3H),
4.73 (s, 1H), 5.80 (t, JHF = 56 Hz, 1H), 6.61 (d, J = 8.7 Hz, 2H), 7.24 (d, J = 8.7 Hz, 2H),
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7.57 (d, J = 8.1 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H); F NMR (400 MHz, CD3OD): δ ꢀ
130.00, ꢀ137.91 (ABq, d, 2F, JHF = 56 Hz, JFF = 300 Hz); 13C NMR (75 MHz, CD3OD): δ
16.5, 54.9, 70.8, (72.6, 72.9, 73.2), 76.8, 79.0, 84.7, 108.4, (112.7, 116.0, 119.2), 114.3,
126.3, 127.5, 132.2, 133.5, 133.8, 150.2, 166.5, 167.5; MS (LC/MS; ESI) m/z 428.2
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[M+H]+; HRMS (ESI/TOF) m/z: [M]+ Calcd for C22H19F2N3O4 427.1344; Found
427.1346.
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Supporting Information
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Crystallographic data for 11 and 21 (CIF)
1H, 13C and wherever applicable 19F NMR spectra of all compounds synthesized
Acknowledgements
This work was supported by National Institutes of Health grants AI055588, AI094475,
and GM115355 awarded to P.Z. We thank Dr. Kalpathy Santhosh for experimental
assistance.
References
1.
2.
Vergidis, P. I.; Falagas, M. E., Curr. Opin. Invest. Drugs 2008, 9, 176.
Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E.; Gilbert, D.; Rice, L.
B.; Scheld, M.; Spellberg, B.; Bartlett, J. Clin. Infect. Dis. 2009, 48, 1.
3.
4.
Cooper, M. A. S., D., Nature 2011, 472, 32.
Lee, C. J. Lliang, X.F.; Wu, Q.L.; Najeeb, J.; Zhao, J.; Gopalaswamy, R.; Titecat,
M.; Sebbane,F.; Lemaitre, N.; Toone, E. J.; Zhou, P., Nature Communications 2016,
10.1038/ncomms10638.
5.
6.
7.
Glaser, C., Ber. Dtsch. Chem. Ges. 1869, 2, 422.
Nicolaou, K. C.; Zipkin, R. E.; Petasis, N. A., J. Am. Chem. Soc. 1982, 104, 5558.
Liang, X. F. Lee., C.J.; Chen, X.; Chung, H.S.; Zeng, D.N.; Raetz, C.; Li, Y.;
Zhou, P.; Toone, E. J. , Bioorganic & Medicinal Chemistry 2011, 19, 852.
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3
4
8.
Arnone, A.; Gestmann, D.; Meille, S.V.; Resnati ; Sidoti, G., Chemical
5
6
Communications 1996, 22, 2569.
7
8
9
9.
Drabowicz, J.; Oae, S. , Synthesis-Stuttgart 1977, 6, 404.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10.
Nishide, K.; Shigeta, Y.; Obata, K., Inoue, T.; Node, M. , Tetrahedron Letters
1996, 37, 2271.
11.
Zhao, S. H.; Samuel, O.; Kagan, H. B., Organic Syntheses 1993, 8, 464.
12.
Balaraman, K.; Kesavan, V., Synthesis-Stuttgart 2010, 2010, 3461.
Nye, S. A.; Potts, K.T., Synthesis 1988, 5, 375.
13.
14.
Weng, Y.; Cheng, B.; He, C.; Lei, A., Angew. Chem., Int. Ed.Engl. 2012, 51,
9547.
15.
Jiang, H.F. Wang, A.Z., Synthesis 2007, 11, 1649.
16.
Paixão, M. W.; Weber, M.; Braga, A. L.; de Azeredo, J. B.; Deobald, A. M.;
Stefani, H. A., Tetrahedron Letters 2008, 49, 2366.
17.
18.
Kara, S. N.; Liu, R.S., Angew. Chem., Int. Ed.Engl. 2014, 53, 9072.
Soloshonok, V. A.; Kacharov., A. D.; Avilov, D. V.; Ishikawa, K.; Nagashima, N.
J. , Org. Chem. 1997, 62, 3470.
19.
20.
Flack, H. D.; Bernardinelli., G. , Chirality 2008, 20, 681.
Flack, H. D., Acta Crystallographica Section A 1983, 39, 876.
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