4-Fluorinated L-lysine analogs as selective i-NOS inhibitors: Methodology for introducing fluorine into the lysine side chain

Article abstract of DOI:10.1039/b307563j

In the literature, the introduction of fluorine into bioactive molecules has been known to enhance the biological activity relative to the parent molecule. Described in this article is the synthesis of 4R-fluoro-L-NIL (12) and 4,4-difluoro-L-NIL (23) as part of our iNOS program. Both 12 and 23 were found to be selective iNOS inhibitors as shown in Table 2 below. Secondarily, methodology to synthesize orthogonally protected 4-fluoro-L-lysine and 4,4-difluoro-L-lysine has been developed.

Full text of DOI:10.1039/b307563j

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4-Fluorinated L-lysine analogs as selective i-NOS inhibitors:
methodology for introducing fluorine into the lysine side chain†‡
E. Ann Hallinan,*a Steven W. Kramer,a Stephen C. Houdek,a William M. Moore,b
Gina M. Jerome,b Dale P. Spangler,a Anna M. Stevens,c Huey S. Shieh,c Pamela T. Manningc
and Barnett S. Pitzelea
a Pharmacia, 4901 Searle Parkway, Skokie, Illinois 60077, USA.
E-mail: eahallinan@comcast.net
b Pharmacia, 800 North Lindbergh Boulevard, St. Louis, Missouri 63167, USA
c Pharmacia, 700 Chesterfield Parkway, St. Louis, Missiouri 63198, USA
Received (in Pittsburgh, PA, USA) 2nd July 2003, Accepted 1st September 2003
First published as an Advance Article on the web 24th September 2003
In the literature, the introduction of fluorine into bioactive molecules has been known to enhance the biological
activity relative to the parent molecule. Described in this article is the synthesis of 4R-fluoro--NIL (12) and
4,4-difluoro--NIL (23) as part of our iNOS program. Both 12 and 23 were found to be selective iNOS inhibitors
as shown in Table 2 below. Secondarily, methodology to synthesize orthogonally protected 4-fluoro--lysine and
4,4-difluoro--lysine has been developed.
the arginine binding site of iNOS, fluorine was introduced at
Introduction
C-4 of the lysine side chain of -NIL. The fluorine should alter
the electronic characteristics of the side chain while minimally
changing its stereochemical properties. Secondarily, we hoped
to develop methodology to synthesize orthogonally protected
4-fluoro--lysine and 4,4-difluoro--lysine. A number of strat-
egies were explored to incorporate, regioselectively, fluorine
at carbon-4 (C-4) of the lysine framework. Installing fluorine at
C-4 would have the least impact on the polar functionalities
at C-2 and C-6 of lysine; whereas, the introduction of fluorine
at C-5 would reduce the basicity of the C-6 amidine. Intro-
duction of fluorine at C-3 would impact the α-amino acid
functionalities enhancing the acidity of the carboxyl group
while attentuating the basicity of the α-amino moiety. Research
on the introduction of fluorine at C-5 will be reported else-
where.9 Synthesis of 3-fluoro--NIL proved troublesome and
has been put aside for the time being. Described, herein, are the
syntheses of 4-fluoro--NIL and 4,4-difluoro--NIL and the
effect of the introduction of fluorine at C-4 of the lysyl moiety
of -NIL on iNOS inhibition. Additionally, methods for the
syntheses of orthogonally protected 4-fluoro--lysine and 4,4-
difluoro--lysine protected are illustrated.
Since the pioneering work of Fried and Sabo,1 it has been
known that the regio- and stereo-selective introduction of
fluorine into bioactive substrates has the potential to produce
molecules that have biological properties that are significantly
more potent than the parent.1,2 The introduction of fluorine
into bioactive molecules can, in addition to profoundly enhanc-
ing biological potency such as in the case of Taxol,2b alter
pharmacodynamics and pharmacokinetics and translation
across the lipid bilayer.2 Stereochemically the carbon–fluorine
bond van der Waals radius of 1.4 Å affords a minimal change
relative to the carbon–hydrogen bond whose van der Waals
radius is 1.1 Å. Yet, the electronegativity of fluorine causes
significant modulation of the electronic characteristics of a
bioactive molecule. A recent review describes pharmaceutical
agents or potential agents in which fluorine is incorporated
illustrating the benefits of inclusion of fluorine into biologically
active molecules.2 Soloshonok et al. have reported on selective
incorporation of fluorine into α-amino acids3 and the enantio-
selective introduction of fluorine into organic molecules.4
Elevated levels of nitric oxide (NO) generated by the action
of induced nitric oxide synthase (iNOS) on -arginine and the
resulting NO derived-metabolites cause cellular cytotoxicity
and tissue damage and are thought to contribute to the patho-
physiology of a number of human diseases.5 Under normal
physiological conditions, the constitutive forms of a NOS
generate low, transient levels of NO in response to increases in
intracellular calcium concentrations. These low levels of NO
act to regulate blood pressure, platelet adhesion, gastrointest-
inal motility, bronchomotor tone and neurotransmission.6
During the course of our research on selective inhibitors
of induced nitric oxide synthase (iNOS), iminoethyl--lysine
(-NIL, 1)7 was identified as a potent selective inhibitor of the
iNOS.8 -NIL is an analog of lysine (Lys, 2) where the ε-amine
has been functionalized as an amidine (Chart 1). The amidine
moiety is a structural isostere for the guanidine group of argin-
ine which is the substrate of the NOS enzymes. Attempting to
understand the stereochemical and electronic requirements of
Chart 1 -NIL (1) and -Lys (2).
Results and discussion
In order to introduce fluorine into C-4, methodologies needed
to be developed to achieve the desired regio- and stereoselective
introduction of fluorine. 4-Fluoro--lysine has been described
once in the patent literature using drastic reaction conditions to
incorporate fluorine.10 Approaches considered for the intro-
duction of fluorine were electrophilic or nucleophilic fluorin-
ation and the use of commercially available fluorinated building
blocks.
† Electronic supplementary information (ESI) available. See http://
www.rsc.org/suppdata/ob/b3/b307563j/
‡ This article is dedicated to the memory of Professor Josef Fried of the
University of Chicago, 1914–2001.
The introduction of fluorine at C-4 using Wadsworth–
Emmons chemistry was explored. Initially, the assembly of
the protected 4-fluoro-lysine was attempted by side chain
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 2 7 – 3 5 3 4
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Table 1 Oxidation of amino alcohol
Compound
Oxidant
Solvent
Time
Temperature
Results
9, 10
10
PDC
DMF
H2O, sonication
H2O–THF (1 : 1), sonication
H2O–CH3CN (1 : 1)
18 h
4.5 h
18 h
2.5 h
Ambient
Ambient
45–50 ЊC
Ambient
Nothing recognizable
Product, s.m, by-products
Black tar
KMnO4, MgSO4
KMnO4, MgSO4
NaIO4, RuCl3ؒH2O (cat)
10
10
77% product
homologation starting with tert-butyloxycarbonyl-glycinal11 (3)
which proved to be unstable under reaction conditions. As a
result, the yields of 4 employing Wadsworth–Emmons reaction
conditions were quite variable as illustrated in Scheme 1.
Scheme 1 Boc-Gly-ol approach to 4-fluorolysine: (i) SO3–pyr, TEA,
CH2Cl2; (ii) triethyl 2-fluoro-2-phosphonoacetate, NaH, Ϫ40 to r.t.,
25%.
The poor yields in the side chain homologation towards
4-fluoro--NIL resulted in a change in strategy. Use of the
Garner aldehyde employing Wadsworth–Emmons conditions
was explored as an appropriate starting point as shown in
Scheme 2. Initially, it was treated with the anion of triethyl
2-fluoro-2-phosphonoacetate generated either with sodium
hydride, which was quite sluggish, or lithium hexamethyl-
disilazide. The resulting olefin was hydrogenated to yield 5. The
reduction was stereoselective to give predominantly the R-
isomer of fluorine at C-4. When the olefin was reduced with
sodium borohydride in the presence of nickel() chloride both
fluorine diastereomers were obtained which could be seen in
1H NMR. The ester was reduced by sodium borohydride to
give the aldehyde and its hemiacetal. The resulting aldehyde–
hemiacetal mixture was subjected to Henry reaction conditions
using nitromethane to provide the necessary elements of the
lysine side chain as shown in Scheme 2 resulting in 6.
Scheme 3 Reduction of the nitroolefin: (i) MsCl, TEA, CH2Cl2,
DMAP cat, Ϫ70 ЊC to ambient, 2 h, 80%; (ii) H2, Pd/C, EtOH, 90%;
(iii) NaBH4, MeOH–H2O, 98%.
Scheme 4 Attempted oxidation of the amino alcohol: (i) 3 : 1 AcOH–
H2O, r.t., 3 days; (ii) see Table 1.
Scheme 5 Oxidation of the amino alcohol: (i) ZOSuc, NaHCO3, 66%;
(ii) see Table 1.
in other chemistries such as the incorporation into peptides or a
precursor in the synthesis of peptidomimetics.
The completion of the synthesis of 4R-fluoro--NIL shown
in Scheme 6 involved removal of the benzyloxycarbonyl group
of 11 by treatment under catalytic hydrogenation conditions,
followed by amidination using conditions described pre-
viously.12 As improved reaction conditions were explored, ethyl
acetimidate hydrochloride was found to be more easily handled
and a more stable amidinating reagent than methyl acetamidate
hydrochloride. For ease of purification by reverse phase separ-
ation on a YMC ODS AQ column, the tert-butyloxycarbonyl
protecting group was left on. We also found that the amidine
could be formed on the unprotected amino acid. Treatment
of the protected amidine 11 with anhydrous hydrochloric acid
yielded 4R-fluoro--NIL (12).
Scheme 2 Garner aldehyde synthesis: (i) triethyl 2-fluoro-2-phos-
phonoacetate, NaH, Ϫ40 to r.t., THF, overnight, 87%; (ii) H2, 60 psi,
Pd/C, EtOH, 90%; (iii) NaBH4, MeOH, Ϫ50–0 ЊC; (iv) CH3NO2,
Na2CO3, THF, 2 days, r.t., 100%.
The Henry product was treated with methanesulfonyl
chloride which, not surprisingly, gave directly the desired
unsaturated nitro product 7 as illustrated in Scheme 3. Catalytic
hydrogenation of 7 using Pd/C resulted in the partial loss of
fluorine. However, a two step process of sodium borohydride
reduction of the olefin followed by catalytic hydrogenation
reduction of the nitro group gave the desired amine 8.
Shown in Scheme 4, unmasking the protected amino alcohol
with aqueous acetic acid gave the desired amino alcohol 9.
Several oxidation conditions were attempted. It became neces-
sary to protect the ε-amine with the benzyloxycarbonyl group
as attempted oxidations of the alcohol were unsucessful in the
presence of the free amine. Protected 10, as shown in Scheme 5
and Table 1, was successfully oxidized using sodium periodate
with ruthenium() chloride as the catalyst. This provided an
orthogonally protected 4-fluoro-lysine 11 which could be used
In parallel with our efforts to synthesize 4-fluoro--NIL, 4,4-
difluoro--NIL was synthesized. No report in the literature on
Scheme 6 Amidine synthesis: (i) Pd/C, MeOH, 5 psi, r.t., 100 %;
(ii) ethyl acetimidate hydrochloride, NaOH, r.t., EtOH, 36 %; (iii) 4 M
HCl–dioxane, HOAc, quantitative.
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Scheme 7 Attempted synthesis of 4,4-difluoro--NIL using the Henry reaction. (i) Zn, ethyl bromodifluoroacetate, THF, ∆, 4 h, 42%; (ii) a.
thiocarbonyldiimidazole, cat. DMAP, DCM, b. Et3SiH, benzoyl peroxide, toluene, ∆, 84%; (iii) NaBH4, MeOH, Ϫ60 ЊC, 78%; (iv) nitromethane,
K2CO3, THF, 18 h, r.t., 75%; (v) ethyl nitroacetate, K2CO3, THF, 18 h, r.t., no reaction; (vi) ethyl nitroacetate; DBU, 18 h, r.t., no reaction;
(vii) triethyl Z-phosphono-glycine, base; (viii) H2, a variety of catalysts.
Scheme 8 Synthesis of 4,4-difluoro--NIL: (i) 1 M DIBAL-H in toluene, toluene, 1.5 h, Ϫ78 ЊC, EtOH quench, 100%; (ii) nitromethane, K2CO3,
20 h, r.t., 80%; (iii) 20% Pd(OH)2/C, HOAc, 3 h, r.t., 91%; (iv) CBZ–OSuccinimide, NaHCO3, Acetone–H2O (1 : 1), 18 h, 0 ЊC to r.t., 78%;
(v) thiocarbonyldiimidazole, DMAP, DCM, 1 h, r.t., 81%; (vi) benzoyl peroxide, triethylsilane, toluene, 3 h, reflux, 71%; (vii) acetic acid–H2O (4 : 1);
(viii) NaIO4, cat. RuCl3, ACN–H2O (1 : 1), 2.5 h, r.t., 75%; (ix) Pd/C, MeOH, 5 psi, r.t.; (x) ethyl acetimidate hydrochloride, NaOH, EtOH, r.t., 35%;
(xi) glacial HOAc, 4 M HCl in dioxane, 1 h, 95%.
the synthesis of 4,4-difluoro--lysine was found. We used
published substrates, among them were 3,3-difluoro--pyro-
glutamol,13 4,4-difluoro--glutamic acid,14 and 1-benzyl
6-methyl (2S)-2-{[(benzyloxy)carbonyl]amino}-4-oxohexane-
dioate,15–17 towards the synthesis of 4,4-difluoro--NIL.
Ultimately compound 17, described by Kim and Qian18 in their
synthesis of 4,4-difluoro--arginine was the pivotal inter-
mediate needed to further homologate the fluorinated lysine
side chain. This compound was synthesized using Garner’s
aldehyde which was further elaborated under Reformatsky
reaction conditions19 followed by deoxygenation.
using Barton conditions yielded 14. With the side chain
skeleton in hand, the ester 14 was reduced using sodium boro-
hydride and quenching with ethanol to give 15. To demonstrate
that the Henry reaction would be a reasonable approach, a
model system using the hemiacetal 15 and nitromethane was
attempted. This proved successful in yielding 16. Introduction
of the incipient fragments of the amino acid was attempted
with ethyl nitroacetate as the α-amino acid presursor. This
failed to give desired nitro ester. An attempt to introduce
the amino acid fragment using triethyl N-benzyloxycarb-
onylphosphonoglycinate and 15 gave a mediocre yield of
α,β-unsaturated amino acid. What caused us to abandon
this approach was the inability to find reduction conditions,
either chiral or achiral, that did not result in the loss of one
fluorine.
With a few modifications, the synthesis of 4,4-difluoro--NIL
was completed in a similar manner to 12 as shown in Scheme 8.
The ester of oxazolidine 1718 was reduced as described
previously for 15, using either sodium borohydride or Dibal-H.
Treatment of the hemiacetal with nitromethane gave the desired
hydroxynitro adduct 18. Attempts to deoxygenate 18 under
mesylation conditions followed by elimination prior to the
reduction of the nitro group were unsuccessful. Reduction of
the nitro group and subsequent protection of the amine 19 was
necessary before successful deoxygenation to give 20. The
remaining steps were the same as described for 12 to yield the
desired amidine 23.
As described in the synthesis of 4R-fluoro--NIL, side chain
elaboration was initially attempted as shown in Scheme 7. The
Reformatsky reaction using Boc-glycinal and ethyl bromo-
difluoroacetate proceeded smoothly resulting in 13 with careful
handling needed in order to avoid loss of ester. Chromato-
graphy frequently led to lower yields. Once characterized,
chromatography of 13 was avoided after the Reformatsky
reaction because 13 was sufficiently pure to carry into the next
reaction and the yields were greatly improved. Deoxygenation
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 2 7 – 3 5 3 4
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Table 2 NOS inhibition (IC50, µM)
Compound no.
i-NOS
e-NOS
n-NOS
Selectivity e-NOS/i-NOS
Selectivity n-NOS/i-NOS
-NIL (2)
12
23
4.94 1.65
6.09
7.08
127 25
477
512
47.1 11
88.7
80.6
26
78
72
10
15
11
Fig. 1 The red nets represent the difference electron density map, calculated without including the contribution from the inhibitor. The refined
models, R-configuration in green and S-configuration in magenta, are docked into this unbiased map. The fluorine atoms in both configurations are
orange. Fig. 1a clearly indicates that the fluorine atoms in both configurations are docked nicely into the electron density. Fig. 1b is obtained by
rotating the horizontal axis a 90Њ of Fig. 1a. The CB atom of the S-configuration is a bit out of the electron density in this figure.
Bioassay
Enzyme inhibition assays were performed with the human NOS
isoforms as described previously.20 Introduction of fluorine
at C-4 of -NIL provided compounds with similar enzyme
inhibition of iNOS isozyme as -NIL as shown in Table 2.
However, both 12 and 23 showed improved selectivity for the
iNOS isozyme versus eNOS enzyme of 78 and 72 respectively
and iNOS versus nNOS enzyme of 15 and 11.
X-Ray crystallography
When miNOS became available in-house, compounds that
had previously shown promising biological activity were co-
crystallized with miNOS.
The crystal structure of the oxygenase domain (residues
66–498) of murine iNOS in complex with compound 12 has
been determined with 2.3 Å resolution. The protein production
and purification, and the crystallization and structure deter-
mination have been described by Tainer et al.21 In compound
12, there are two chiral centers at C-4 and C-2. The stereo-
chemistry at C-2 was set as ЉSЉ because the synthesis was
effected by the use of chiral Garner aldehyde and no loss of
stereochemistry was observed. As noted above, a single isomer
was prepared. Alternative chemistry that provided the saturated
fluorinated side chain produced a diastereomeric pair. Although
the unbiased difference Fourier map (without including the
inhibitor contribution) showed that the R-configuration mole-
cule fitted slightly better than the S-configuration molecule
(Fig. 1), the refinement statistics, both having same Rfree and
R, did not distinguish the two configurations.
If we consider the amino acid end of the compound 12 mole-
cule is the tail part, the chain the middle part and the amidine
group the head part, the dominant feature of the interactions is
the bi-dentate interaction of Glutamate 371 toward the amidine
group (guanidine group in arginine) of the inhibitor. Glutamate
371 also binds to the amino group of the tail part of the inhib-
itor. These three hydrogen bonds essentially tie down the head
and tail parts of the inhibitor molecule and anchor the whole
molecule in place. As shown in Fig. 2, the amino group of the
inhibitor molecule is surrounded by a cluster of polar residues
such as Arg382, Gln257, Asp376, Tyr367 and a series of water
molecules (W). The polar interactions of these arginine-like
molecules in the head and tail parts with iNOS are all very
similar. The middle part of the inhibitor molecule exhibits a
certain degree of flexibility as shown by the variations between
compound 12 and -NIL (Fig. 2). These conformational
Fig. 2 This is a ribbon representation of the arginine binding pocket
of the structure of oxygenase (residues 66–498) domain of mouse iNOS
in complex with compound 12. The -NIL molecule is also docked into
this pocket for comparison. Compound 12 has the bonds in very light
blue while -NIL in light orange and heme in magenta. Some important
adjacent residues around the inhibitor are depicted. The fluorine atom
is shown in bright orange. The three neighboring water molecules are
labeled W1, W2 and W3.
variations are summed up in the Table 3. These kinds of flex-
ibility offer opportunities for inhibitor design. So far, none of
these arginine (or -NIL) like molecules have been found to
have direct interaction with the heme molecule. In this case, the
nearest group, the methyl group of the amidine group, exhibits
a 4.2 Å distance toward the heme iron atom in compound 12
and 4.4 Å in -NIL.
The crystal structure determination did not distinguish the
absolution configuration of the inhibitor. On one hand, it may
be due to the limitation of the data resolution. Although
the fluorine atom points to different direction for each
diastereomer, the pocket is large enough to accommodate both
R and S fluorine atoms as shown in Fig. 2.
Conclusion
The introduction of fluorine into the lysine side chain at C-4
has yielded highly selective iNOS inhibitors with potencies
comparable to -NIL and greater selectivity than NIL versus
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 2 7 – 3 5 3 4
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Table 3 The formula, numbering system and torsion angles for compound 12 and -NIL
N=8–7–6
8–7–6–5
7–6–5–4
6–5–4–3
5–4–3–2
4–3–2–1
3–2–1=O
12
NIL
159
162
173
116
Ϫ66
67
94
Ϫ102
87
Ϫ178
125
161
60
122
eNOS and nNOS. The X-ray crystallography data shows the
arginine binding site can accommodate modification of the
lysine side chain. In addition, we have developed methods to
synthesize orthogonally protected 4-fluoro and 4,4-difluoro-
-lysine.
The residue was filtered through a pad of silica gel (90 × 50
mm). The aldehyde was eluted from the silica gel with 20%
EtOAc in DCM. The yield of aldehyde 3 was 78%. The alde-
hyde is not particularly stable and needs to be either stored at
Ϫ20 ЊC or used immediately.
5. tert-Butyl (4S )-4-(3-ethoxy-2-fluoro-3-oxopropyl)-2,2-
dimethyl-1,3-oxazolidine-3-carboxylate
Experimental
Thin layer chromatograms were run on 0.25 mm EM precoated
plates of silica gel 60 F254. Visualization was achieved by
exposure to I2 or phosphomolybdic acid or Ca(ClO)2 followed
by spraying with a 0.5% KI–0.5% potato starch solution. G.C.
were run on a Shimadzu 9A using capillary columns from
several vendors. Preparative column chromatograpy using flash
chromatography conditions was performed on EM silica gel or
a Biotage Flash-40 system. To successfully use the Biotage
columns under flash chromatography conditions the optimal Rf
needs to be 0.15–0.2 in contrast to an Rf of 0.35–0.4 as
described by Still.22 In general, eluting solvents for the normal
phase columns were combinations of EtOAc–hexane (heptane
for larger columns), EtOAc–DCM and EtOH–DCM. The
solvent choice was made based on the compound lipophilicity
and Rf. Hydrophilic compounds were purified by reverse phase
column chromatography. Reverse phase chromatography was
performed on a Rainin preparative HPLC system with a YMC
ODS-AQ (10 µ) preparative column eluting with acetonitrile–
water (0.05% HOAc) at 10 mL minϪ1. HPLC chromatography
was performed using a Hewlitt-Packard HP 1100 with a dual
absorbance detector. Both chiral and achiral columns were
acquired from several vendors. 1H NMR spectra were taken in
commercially available deuterated solvents on a Bruker 400
MHz Ultrashield. All chemical shifts are reported in parts
per million (δ) downfield (positive) relative to Me4Si (organic
solvents) or 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium
5a: To a stirred cooled (15–20 ЊC) suspension of NaH (6.6
mmol, 0.264 g) in 40 mL of THF was added triethyl 2-fluoro-2-
phosphonacetate (6.6 mmol, 1.59 g) in 5 mL of THF. After
stirring for 18 h at ambient temperature, the reaction was
cooled to Ϫ40 ЊC to which was added Garner’s aldehyde (4.4
mmol, 1.00 g) in 5 mL THF. After stirring for 2 h at 5 ЊC and 3 h
at room temperature, an additional 2.2 mmol of the phosphon-
ate anion was added to the reaction mixture. After 18 h, MTBE
(50 mL) was added to the reaction. The organic layer was
washed with brine. After drying over MgSO4, the reaction
mixture was filtered and concentrated under vacuum. The
residue was purified by flash chromatography to yield 1.20 g
(85%) of desired 5a. δH (400 MHz; CDCl3) 1.27 (6 H, s, 2 CH3),
1.34 (3 H, t, J = 7.1 Hz, OCH2CH3), 1.45 (9 H, s, C(CH3)3), 4.16
(2 H, br t, CH2O), 4.32 (2 H, q, J = 7.1 Hz, OCH2CH3), 4.93
(1 H, br s, NCHCH ), 6.01 (1 H, dt, J = 7.2, 19.3 Hz, CH᎐CF).
᎐
2
δC (100 MHz, CDCl3) 14.5 (OCH2CH3), 24.1 (CH3), 27.0
(CH3), 28.8 ((CH3)3C)), 53.8 (CH), 62.1 (OCH2), 69.1 (OCH2),
80.2 ((CH3)3CO)), 92.9 (OC(CH3)2N), 117.4, 129.0 (d, J = 81.0
Hz, CH᎐CF), 149.2 (d, J = 261.4 Hz, CH᎐CF), 160.9 (d,
᎐
᎐
J = 34.0 Hz, CH᎐CFC᎐O).
᎐
᎐
5: To a solution of 5a (1.57 mmol, 0.50 g) in EtOH was added
5% Pd/C. After 18 h under H2 at 60 psi at ambient temperature,
the Pd/C was filtered from the reaction and the reaction mixture
1
was concentrated under vacuum to quantitatively yield 5. H
NMR confirmed reduction of olefin. δH (400 MHz; CDCl3)
1.32 (3 H, t, J = 7.0 Hz, OCH2CH3), 1.49 (9 H, s, C(CH3)3), 1.55
(3 H, s, CH3), 1.60 (3 H, s, CH3), 2.01–2.38 (2 H, m, CH2CHF);
3.77–4.20 (3 H, m, NCHCH2, CH2O), 4.27 (2 H, q, J = 7.0 Hz,
OCH2CH3), 5.00 (1 H, ddd, J = 8.2, 35.2, 46.0 Hz, CHF).
δF (376 MHz; CDCl3) Ϫ156.2.
1
salt (D2O) as an internal standard for H and 13C NMR. 13C
NMR spectra were obtained on a Bruker 400 MHz Ultrashield
spectrometer at an observation frequency of 100 MHz. The
observation frequency for 19F NMR was 376 MHz and the
chemical shifts in parts per million (δ) were reported upfield
or downfield relative to fluorotrichloromethane (Freon-11).
19F NMR spectra were proton decoupled. All reagents were
purchased from Sigma-Aldrich and used as is. Solvents were
purchased from Burdick & Jackson. Microanalyses and optical
rotations were performed in-house.
6. tert-Butyl (4S )-4-[(2S )-2-fluoro-3-hydroxy-4-nitrobutyl]-2,2-
dimethyl-1,3-oxazolidine-3-carboxylate
6a: To a stirred solution cooled to Ϫ50 ЊC of 5 (1.57 mmol,
0.50 g) in 3.5 mL of MeOH was added NaBH4 (3.13 mmol,
0.118 g) in four portions at 15 min intervals. After stirring for
an additional hour, KHSO4 (1 M, 0.5 mL) was added and the
reaction was stirred with warming to 0 ЊC. To the reaction was
added EtOAc (50 mL), which was washed with KHSO4 (1 M,
50 mL), and brine (1 × 100 mL). The organic layer was dried
over MgSO4, filtered, and concentrated under vacuum. The
yield of product was 0.46 g which was a mixture of hemiacetal
and aldehyde.
6: To a stirred suspension of K2CO3 (0.75 g, 5.4 mmol) in
50 mL of THF cooled to 5 ЊC was added a solution ot 6a
(24.0 mmol, 7.85 g) and nitromethane (5.0 mL, 92.3 mmol) in
100 mL of THF. After warming to ambient temperature, the
reaction was stirred overnight. To the reaction was added 0.5 M
3. Boc-glycinal
To an ice cooled stirred solution of Boc-glycinol (50.0 mmol,
8.06 g) in 150 mL of DCM was added TEA (150 mmol, 20.9
mL) and SO3ؒpyridine (150 mmol, 23.96 g) in 100 mL of
DMSO. The reaction changed from clear to yellow upon
addition of the oxidant. After removal from the icebath, the
reaction was stirred for 20 min. The reaction mixture was
poured into 450 mL of ice cold brine. The aqueous layer was
extracted with Et2O (2 × 500 mL). The combined organic
washes were washed with cold 1 M KHSO4 (100 mL) and with
cold brine (2 × 100 mL). The organic layer was dried over
Na2SO4 anhydrous, filtered, and concentrated under vacuum.
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KHSO4 (50 mL) and MTBE (100 mL). The layers were
separated and the organic layer was washed with saturated
brine (2 × 100 mL). The organic layer was dried over MgSO4,
filtered, and concentrated under vacuum to yield 7.93 g (99%)
of a white solid. (Found: C, 50.35; H, 7.86; N, 8.05. Calc. for
C19H25F1N2O6: C, 50.00; H, 7.49; N, 8.33%.) δH (400 MHz;
CDCl3) 1.50 (9 H, s, C(CH3)3), 1.52–1.64 (6 H, m, 2 CH3), 1.92
(1 H, hept, J = 6.7 Hz, CH2CHF), 2.07–2.38 (1 H, m,
CH2CHF), 3.75–3.92 (1 H, m, NCHCH2), 3.91–4.09 (2 H, m,
CH2O), 4.21–4.49 (1 H, m, CHF), 4.46–4.75 (4 H, m, CH2NO2,
CHF, OH ).
11. (4R)-N-6-[(benzyloxy)carbonyl]-N-2-(tert-butoxycarbonyl)-
4-fluoro-3-methyl-L-lysine
To a solution of 10 (0.92, 2.4 mmol) in 72 mL of CH3CN : H2O
(1 : 1) was added Ru()Cl3ؒH2O. The reaction turned a greenish
brown. To the stirred reaction was added NaIO4 (2.08 g, 9.6
mmol). The stirring was stopped once the addition was com-
plete. After 20 min, a white precipitate had formed. After 2 h,
EtOAc : MTBE (1 : 1, 60 mL) and 10% NaHSO3 solution
(60 mL) were added to the reaction. The organic layer was
washed with additional 10% NaHSO3 (60 mL) and saturated
brine (60 mL). The organic layer was dried over Na2SO4
anhydrous, filtered, and concentrated under vacuum to yield
0.74 g (77%) of the desired acid. δH (400 MHz; CDCl3) 1.43
(9 H, s, (CH3)3C), 1.48–1.55 (2 H, m, CH2CHF), 1.58–1.92 ((2
H, m, CH2CHF), 3.01–3.21 (2 H, m, NHCH2), 4.24–4.36 (1 H,
7. tert-Butyl (4S )-4-[(2S,3E )-2-fluoro-4-nitrobut-3-enyl]-2,2-
dimethyl-1,3-oxazolidine-3-carboxylate
To a stirred solution of 6 (13.3 mmol, 4.48 g) in 70 mL of DCM
cooled to Ϫ70 ЊC was added TEA (3.7 mL, 26.6 mmol),
methanesulfonyl chloride (1.07 mL, 13.8 mmol), and DMAP
(5 mg). After stirring for 1 h, the cloudy yellow solution was
allowed to warm to room temperature. The reaction mixture
was filtered through a thin pad of EM silica gel and washed
with 20% EtOAc–DCM. The filtrate was concentrated under
vacuum yielding 4.14 g (99%) of product.
m, NHCHC᎐O), 5.10 (2 H, CH O), 5.16–5.40 (1 H, m, CHF),
᎐
2
6.40 (1 H, br d, NH ), 7.28–7.39 (5 H, m, Ph). δC (100 MHz,
CDCl3) 28.7 ((CH3)3C), 29.7 (CH2CHF), 32.0 (CH2CHF), 41.0
(NHCH2), 53.5 (NHCH), 67.1 (PhCH2O), 81.5 ((CH3)3CO),
128.5 (Ph), 128.6 (Ph), 128.9 (Ph), 137.0 (Ph), 156.0 (OC᎐O),
᎐
157.0 (OC᎐O), 176.6 (COOH).
᎐
12. N-6-iminoethyl-4-fluoro-L-lysine dihydrochloride
8. tert-Butyl (4S )-4-[(2R)-4-amino-2-fluorobutyl]-2,2-dimethyl-
1,3-oxazolidine-3-carboxylate
12a: Removal of the benzyloxycarbonyl protecting group of 11
(0.43 g, 1.1 mmol) was achieved under catalytic hydrogenation
conditions at 5 psi and Pd/C to quantitatively yield the free
ε-amine.
8a: To a stirred solution of 7 (3.62 g, 11.5 mmol) in 115 mL of
THF was added H2O (23 mL) followed by NaBH4 resulting in
the evolution of gas. After 3 h, the reaction was concentrated
under vacuum. The residue was treated with EtOAc (200 mL)
and 1 M KHSO4 (100 mL). After the layers were separated, the
organic layer was washed with saturated brine (1 × 100 mL).
The organic layer was dried over MgSO4, filtered, and concen-
trated to yield 3.77 g (100%) of a pale yellow oil. δH (400 MHz;
CDCl3) 1.49 (9 H, s, C(CH3)3), 1.56 (3 H, s, CH3), 1.59 (3 H, s,
CH3),1.96 (2 H, hept, J = 6.7 Hz, CH2CHF), 2.20–2.49 (2 H, m,
CH2CHF), 3.78–3.88 (1 H, m, NCHCH2), 3.90–4.08 (2 H, m,
CH2O), 4.47–4.59 (2 H, m, CH2NO2), 4.60–4.88 (1 H, m, CHF).
8: The product from 8a (3.61 g, 11.3 mmol) in EtOH was
treated with H2 at 60 psi and 5% Pd/C for 24 h. After the
catalyst was filtered, the filtrate was concentrated under
vacuum to give 3.61 g of 8.
12b: The α-N-Boc-4R-fluoro--lysine (0.37 g, 1.1 mmol, 12a)
in 5 mL of H2O was treated with 1 M NaOH to pH 9.5. In three
portions, ethyl acetimidate hydrochloride (0.424 g, 3.3 mmol)
was added to the stirred lysine solution. After each addition the
pH was adjusted to pH 9.5. After 15 min of stirring, the pH was
adjusted to pH 2. The reaction mixture was concentrated under
vacuum. The solids were triturated with EtOH and filtered. The
concentrated EtOH filtrate was chromatographed on a YMC
ODS AQ column to yield 0.133 g (36%) of Boc-protected
amidine.
12. The above 12b (012 g, 0.35 mmol) in 1.5 mL of HOAc
was treated with 4 M HCl–dioxane (0.5 mL, 2 mmol) for
30 min. The reaction mixture was concentrated followed by
lyophilization to yield 0.11 g (quantitative) of 12 as a white
glassy solid. (Found: C, 32.21; H, 6.63; N, 13.72; Cl, 21.23.
Calc. for C8H16F1N3O2ؒ1.8 HClؒ1.5 H2O: C, 32.26; H, 7.04; N,
14.11; Cl, 21.42%). δH (400 MHz; D2O) 1.82–2.00 (2 H, m,
CH2CH2CF), 2.11 (3 H, s, CH3), 2.20–2.31 (2 H, m,
CFCH2CH), 3.33 (2 H, t, J = 6.9 Hz, NHCH2), 4.00 (1 H, dd,
9. 1,1-Dimethylethyl [(1S,3R)-5-amino-3-fluoro-1-
(hydroxymethyl)pentyl]carbamate
A solution of 8 (3.30 g, 11.4 mmol) in 120 mL HOAc : H2O
(5 : 1) was stirred for 3 d at ambient temperature. The reaction
mixture was concentrated under vacuum to quantitatively
recover product.
J = 3.9, 7.1 Hz, NHCHC᎐O). δ (100 MHz; D2O) 18.7
᎐
C
(CH3), 32.4 (d, J = 25 Hz, CH2CHF), 35.0 (d, J = 25 Hz,
CH2CHF,), 38.4 (CH2NH), 51.7 (CHNH), 90.2 (d, J = 205 Hz,
10. Phenylmethyl [(3R,5S )-5-[[(1,1-dimethylethoxy)carbonyl]-
amino]-3-fluoro-6-hydroxyhexyl]carbamate
CHF), 165.3 (NHC᎐NH), 173.0 (COOH). δ (376 MHz; D O)
᎐
F
2
Ϫ185.1.
To a stirred icebath cooled solution of 9 (3.60 g, 11.4 mmol) in
120 mL of acetone:H2O (1 : 1) was added KHCO3 (2.28 g, 22.8
mmol) and benzyloxycarbonylsuccinimde (2.84 g, 11.4 mmol).
After 30 min, additional KHCO3 (1.14 g, 5.7 mmol) was added
to the reaction. The reaction was allowed to warm to room
temperature and was stirred for 1 h. After concentrating the
reaction under vacuum, EtOAc : MTBE (1 : 1, 100 mL) was
added to reaction mixture. The organic layer was washed with 1
M KHSO4 (100 mL), saturated solution of KHCO3 (100 mL),
and brine (100 mL). Following the washes the organic layer
was dried over MgSO4, filtered, and concentrated. The crude
product was purified using flash chromatography conditions
yielding 2.90 g (66%) of a pale yellow solid. δH (400 MHz;
CDCl3) 1.42 (9 H, s, C(CH3)3), 1.64–1.90 (4 H, m, CH2CHF),
3.24–3.38 (2 H, m, NHCH2), 3.53–3.68 (2 H, m, CH2O), 3.75–
3.90 (1 H, m, NHCH ), 4.58–4.80 (1 H, m, CHF), 5.04–5.15
(2 H, m, PhCH2O), 7.29–7.38 (5 H, m, Ph).
13. Ethyl 4-[(tert-butoxycarbonyl)amino]-2,2-difluoro-3-
hydroxybutanoate
To a solution of 3 (0.56 g, 3.5 mmol) in 15 mL of THF was
added etched Zn (0.46 g, 7.0 mmol) and ethyl bromo-
difluoroacetate (1.42 g, 7.0 mmol). The reaction mixture was
refluxed for 2 h. The reaction solution was decanted from
unreacted Zn. To the reaction mixture was added a saturated
solution of 1 M KHSO4 (15 mL) and 15 mL of Et2O. The
organic layer was washed with NH4Cl solution (2 × 15 mL).
The organic layer was dried over anhydrous Na2SO4, filtered,
and concentrated under vacuum. The reaction mixture was
purified using flash chromatography conditions to yield 0.42 g
(42%) of yellow oil 13. Chromatography is not ideal for 13.
(Found: C, 46.40; H, 6.62; N, 5.26. Calc for C11H19F2NO5:
C, 46.64; H, 6.76; N, 4.94%.)
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14. Ethyl 4-[(tert-butoxycarbonyl)amino]-2,2-difluoro-3-
[(1H-imidazol-1-ylcarbonothioyl)oxy]butanoate
solution of 18a (5.0 mmol, 1.70 g) and nitromethane (7.5 mmol,
0.45 g, 0.4 mL) in 15 mL of THF. After stirring for 20 h with
the icebath removed, 25 mL of Et2O was added to the reaction.
The organic layer was washed with 1 M KHSO4 (15 mL) and
saturated brine (2 × 15 mL). After treating the organic layer as
described in 18a, the residue was chromatographed using flash
chromatography conditions. Although the diastereomers were
separated, they were recombined to yield 1.42 g (80%) of 18.
δH (400 MHz; CDCl3) 1.47 (3 H, s, CH3), 1.49 (9 H, s (CH3)3C),
1.55 (3 H, s, CH3), 2.22–2.38 (1 H, m, CH2CF2), 2.54 (1 H, ddd,
J = 9.1, 11.1, 24.0 Hz, CH2CF2), 3.82–3.88 (1 H, m, NCH ),
4.01–4.07 (2 H, m, CH2O), 4.55–4.65 (m, 2 H, CH2NO2), 4.81
(1 H, dhext, J = 4.0, 33.6 Hz, CHOH), 5.52 (1 H, br d, OH ). δF
(376 MHz, CDCl3) Ϫ108.6, Ϫ109.8.
14a: To a stirred solution of 13 (0.65 g, 2.3 mmol) in 10 mL was
added 1,1-thiocarbonyldiimidazole (0.61 g, 3.4 mmol) followed
by DMAP (0.024 g, 0.2 mmol). The yellow of the reaction
changed upon the addition of DMAP. After 1 h, the reaction
was filtered through a 30 × 20 mm pad of silica gel and was
washed with 20% EtOAc–DCM. The solvent was removed
under N2 to yield 14a (0.86 g, 96%), a pale yellow oil. (Found:
C, 46.40; H, 6.62; N, 5.26. Calc. for: C15H21F2N3O5S: C, 46.64;
H, 6.76; N, 4.94.)
14: The product of 14a was dissolved in 10 mL of toluene
which was concentrated to 5 mL under vacuum. A solution of
14a (0.79 g, 2.2 mmol) in 20 mL of toluene:Et3SiH (1 : 1) was
heated to reflux. To this solution was added benzoyl peroxide
(0.53 g, 2.2 mmol) in 5 mL of toluene in four portions at 15 min
intervals. After heating for 2 h, the reaction mixture cooled to
room temperature was filtered through a 65 × 30 mm pad of
silica gel washed with DCM followed by 20% EtOAc–DCM
to yield 0.40 g (84%) of 14. δH (400 MHz; CDCl3) 1.37 (3 H, t,
J = 7.1 Hz, OCH2CH3), 1.43 (9 H, s, (CH3)3C), 2.24–2.39 (2 H,
m, CH2CF2), 3.38 (2 H, br q, OCH2), 4.34 (2 H, q, J = 7.1 Hz,
OCH2CH3), 4.71 (1 H, br s, NCH ). δF (376 MHz, CDCl3)
Ϫ105.3.
19. tert-Butyl (4S )-4-(4-{[(benzyloxy)carbonyl]amino}-2,2-
difluoro-3-hydroxybutyl)-2,2-dimethyl-1,3-oxazolidine-3-
carboxylate
19a: Under catalytic hydrogenation conditions at 60 psi, EtOH–
HOAc and ambient temperature with 20% Pd(OH)2/C, 18 (1.7
mmol, 0.62 g) was reduced to give 0.50 g (91%) of the desired
amine 19a. δH (400 MHz; DOAc) 1.50 (3 H, s, CH3), 1.52 (9 H,
s, (CH3)3C), 1.60 (3 H, s, CH3), 2.18–2.62 (2 H, m, CH2CF2),
3.20–3.49 (2 H, m, CH2NH), 3.93–4.11 (2 H, m, CH2O), 4.2–4.4
(2 H, m, CHNH, CHOH). δC (100 MHz, DOAc) (major
diastereomer) 24.7 (CH3), 27.8 (CH3), 28.7 ((CH3)3C), 36.7 (t,
J = 25 Hz, CH2CF2), 40.6 (CH2NH), 52.8 (NCH), 68.2
(CH2O), 72.3 (t, J = 25 Hz, CHOH), 81.5 ((CH3)3CO), 93.4
15. tert-Butyl 4-ethoxy-3,3-difluoro-4-hydroxybutylcarbamate
To a solution cooled to Ϫ60 ЊC of 14 (0.27 g, 1.0 mmol) in 2 mL
was added NaBH4 (0.75 g, 2.0 mmol) in 4 equal portions 15 min
apart. After 1 h, 1 M KHSO4 (1 mL) and 15 mL Et2O were
added. When the reaction had warmed to 5 ЊC, 15 mL of
saturated brine was added. The aqueous layer was extracted
with Et2O (2 × 5 mL). The combined organic layers were
washed with saturated brine (2 × 20 mL). The organic layer was
treated with Na2SO4, filtered, and stripped to recover 15 (0.21 g,
78%). δH (400 MHz; CDCl3) δ 1.25 (3 H, t, J = 7.1 Hz,
OCH2CH3), 1.44 (9 H, s, (CH3)3C), 2.07–2.30 (2 H, m,
CH2CF2), 2.96 (1 H, br s, OH ), 3.28–3.48 (2 H, m, NHCH2),
3.49–3.58 (1 H, m, OCH2CH3), 3.85–3.94 (1 H, m, OCH2CH3),
4.66 (1 H, ddd, J = 5.0, 7.8, 9.9 Hz, CHOH), 4.80 (1 H, br s,
NH ).
(NCO), 124.0 (t, CF ), 152.7 (C᎐O). δ (376 MHz, DOAc)
᎐
2
F
Ϫ108.6, Ϫ109.1.
19: On a 1.5 mmol scale, 19 was prepared in the same manner
as described in example 10 starting with 19a. δH (400 MHz;
CDCl3) 1.45 (3 H, s, CH3), 1.48 (9 H, s, (CH3)3C), 1.54 (3 H, s,
CH3), 2.2–2.5 (2 H, m, CH2CF2), 3.2–3.4 (2 H, m, CH2NH),
3.4–4.0 (4 H, m, CH2O, CHNH, CHOH), 5.1–5.3 (2 H, m,
OCH2Ph), 7.3–7.4 (5 H, m, Ph).
20. tert-Butyl (4S )-4-(4-{[(benzyloxy)carbonyl]amino}-2,2-
difluorobutyl)-2,2-dimethyl-1,3-oxazolidine-3-carboxylate
On a 1.4 mmol scale, compound 20 was prepared as described
in example 14.
16. tert-Butyl 3,3-difluoro-4-hydroxy-5-nitropentylcarbamate
δH (400 MHz; CDCl3) δ 1.42 (6 H, s, CH3), 1.53 (9 H, s,
C(CH3)3), 1.97–2.29 (4 H, m, CH2CF2CH2), 3.39–3.52 (2 H, m,
CH2NH), 3.88–4.02 (2 H, m, CH2O), 4.08–4.20 (m, 1 H,
NCH ), 5.10 (2 H, br s, PhCH2O), 7.36 (5 H, br s, Ph).
On a 7.9 mmol scale, 16 was prepared as described in example 6
starting with the product of example 15.
17. tert-Butyl (4S )-4-(3-ethoxy-2,2-difluoro-3-oxopropyl)-2,2-
21. N-6-[(benzyloxy)carbonyl]-N-2-(tert-butoxycarbonyl)-4,4-
difluoro-L-lysinol
dimethyl-1,3-oxazolidine-3-carboxylate16
Compound 17 was synthesized as described in literature
reference 16.
On a 1.2 mmol scale, compound 21 was synthesized as
exemplified in example 10 starting with 20.
18. tert-Butyl (4S )-4-(2,2-difluoro-3-hydroxy-4-nitrobutyl)-2,2-
dimethyl-1,3-oxazolidine-3-carboxylate
22. N-6-[(benzyloxy)carbonyl]-N-2-(tert-butoxycarbonyl)-4,4-
difluoro-L-lysine
18a: A solution of 17 in 10 mL of toluene was concentrated to
5 mL under vacuum. To a stirred solution of 17 (5.0 mmol,
1.69 g) at Ϫ70 ЊC was added Dibal-H in toluene (1.5 M,
10.5 mmol, 7.0 mL) while maintaining the temperature at or
below Ϫ65 ЊC. After stirring the reaction for 30 min, the
reaction was quenched with 2 mL of EtOH and warmed to Ϫ20
ЊC, to which was added 25 mL of a Rochelle salt solution. The
reaction mixture was stirred for 30 min. To the reaction was
added 25 mL of EtOAc. Once the emulsion had dissipated, the
layers were separated. The aqueous layer was extracted with
EtOAc (25 mL). The combined organic layers were washed with
a saturated brine solution (3 × 25 mL). The organic layer was
dried over anhydrous Na2SO4, filtered and concentrated under
vacuum to yield quantitatively 18a.
On a 0.42 mmol scale, compound 22 was synthesized as
described in example 11 starting with 21. δH (400 MHz; CDCl3)
1.46 (9 H, s, (CH3)3C), 2.02–2.20 (2 H, m, CH2CF2), 2.24–2.58
(2 H, m, CH2CF2), 3.30–3.46 (2 H, m, NHCH2), 4.45–4.59 (1 H,
m, NHCH ), 5.16 (2 H, br s, PhCH2O), 7.37 (5 H, br s, Ph).
δC (100 MHz; CDCl3) 28.8 ((CH3)3C), 35.2 (CH2NH), 37.1 (t,
J = 25 Hz, CH2CF2), 38.5 (t, J = 25 Hz, CH2CF2), 49.5
(NHCH), 80.9 (CH3)3CO), 128.8 (Ph), 128.9 (Ph), 130.5 (Ph),
136.7 (Ph), 156.0 (C᎐O), 157.0 (C᎐O), 175.4 (COOH).
᎐
᎐
23. N-6-iminoethyl-4,4-difluoro-L-lysine dihydrochloride
On a 0.26 mmol scale, compound 23 was synthesized following
the method of example 12 starting with 22. (Found: C, 30.17;
H, 6.60; N, 13.41; Cl, 22.33. Calc. for C8H15F2N3O2ؒ2 HClؒ1.4
H2O: C, 29.90; H, 6.21; N, 13.08; Cl, 22.06%). δH (400 MHz;
18: To an ice bath cooled stirred suspension of K2CO3
(1.0 mmol, 0.14 g) in 10 mL of THF was added dropwise a
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8 W. M. Moore, R. K. Webber, G. M. Jerome, F. S. Tjoeng, T. P. Misko
and M. G. Currie, J. Med. Chem., 1994, 37, 3886–88.
D O) 1.96 (3 H, s, C᎐NHCH ), 2.10–2.60 (4 H, m, CH CF -
᎐
2
3
2
2
CH2), 3.57 (2 H, t, J = 6.8 Hz, CH2NH), 4.08 (1 H, dd, J = 3.7,
8.3 Hz, NHCH ). δC (100 MHz, D2O) 18.8 (CH3), 34.2 (t, J = 24
Hz, CH2CF2), 36.0 (CH2NH), 37.2 (t, J = 24 Hz, CH2CF2),
9 E. A. Hallinan, T. J. Hagen, A. Bergmanis, W. M. Moore,
G. M. Jerome, D. P. Spangler, A. M. Stevens, H. S. Shieh,
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10 P. E. Curley, R. F. Hirschmann, USP 4,427,661, 1984.
11 Y. Hamada, M. Shibata, T. Sugiura, S. Kata and T. Shioiri, J. Org.
Chem., 1987, 52, 1252–1255.
49.8 (NHCH), 123.9 (t, J = 241.5 Hz, CF ), 165.3 (C᎐NH),
᎐
2
173.4 (COOH). δF (376 MHz, D2O) Ϫ98.2.
12 E. A. Hallinan, S. Tsymbalov, C. R. Dorn, B. S. Pitzele,
D. W. Hansen, Jr., W. M. Moore, G. M. Jerome, J. R. Connor,
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16 C. A. M. Afonso, M. T. Barros, L. S. Godinho and C. D. Maycock,
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17 R. A. Berglund and P. L. Fuchs, Synth. Commun., 1989,
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