5-Fluorinated L-Lysine Analogues as Selective Induced Nitric Oxide Synthase Inhibitors

Article abstract of DOI:10.1021/jm030348f

5(S)-Fluoro-N6-(iminoethyl)-L-lysine (14), an analogue of the potent, selective induced nitric oxide synthase (iNOS) inhibitor iminoethyl-L-lysine (1), was synthesized and found to be a selective iNOS inhibitor.

Full text of DOI:10.1021/jm030348f

900
J . Med. Chem. 2004, 47, 900-906
5-F lu or in a ted L-Lysin e An a logu es a s Selective In d u ced Nitr ic Oxid e Syn th a se
In h ibitor s
E. Ann Hallinan,*,† Timothy J . Hagen,† Arija Bergmanis,† William M. Moore,‡ Gina M. J erome,‡
Dale P. Spangler,† Anna M. Stevens,§ Huey S. Shieh,§ Pamela T. Manning,§ and Barnett S. Pitzele†
Pfizer, 4901 Searle Parkway, Skokie, Illinois 60077, Pfizer, 800 North Lindbergh Boulevard, St. Louis, Missouri 63167, and
Pfizer, 700 Chesterfield Parkway, St. Louis, Missiouri 63198
Received J uly 18, 2003
5(S)-Fluoro-N6-(iminoethyl)-L-lysine (14), an analogue of the potent, selective induced nitric
oxide synthase (iNOS) inhibitor iminoethyl-L-lysine (1), was synthesized and found to be a
selective iNOS inhibitor.
Sch em e 1a
In tr od u ction
Elevated levels of nitric oxide (NO) generated by the
action of induced nitric oxide synthase (iNOS) on
L-arginine causes cellular cytotoxicity and tissue dam-
age and are thought to contribute to the pathophysiology
of a number of human diseases.1 During the course of
our research on inhibitors of iNOS, (iminoethyl)-L-lysine
(NIL, 1),2 an analogue of L-lysine (2), was identified as
a
(a) CuCO3, H2O, ∆, 1 h. (b) pH 9-9.5, ethyl acetimidate
hydrochloride, ion exchange, reverse phase chromatography.
Ta ble 1. Bioassay Data3
selectivity
NOS inhibition (IC50, µM) or
% inhibition at 10 µM
iNOS vs iNOS vs
compd
NIL
iNOS
eNOS
nNOS
eNOS
nNOS
4.9 ( 1.65 µM 138 µM 35 µM
7.84 µM
28
23
34
7
9
9
4
182 µM 70.4 µM
81 µM
28.2%
374 µM 311 µM
6.52% 18%
a potent, selective inhibitor of the iNOS isoform.3
Selective iNOS inhibitors, such as L-NIL,4 have been
shown to suppress the increase in plasma nitrites and/
or paw swelling associated with the overproduction of
NO in animal models of acute and chronic inflammation
including nonlethal endotoxemia,5,6 carrageenan-in-
duced paw edema,6 and adjuvant-induced arthritis.7,8
We sought to optimize the activity observed with
L-NIL. It is well-known that the introduction of fluorine
into a bioactive molecule can significantly modulate its
activity.9 In our investigation of NIL analogues, intro-
duction of fluorine at C-4 and C-5 as well as fluoroalkyl
at C-6 was of particular interest. Installing fluorine at
C-4 should have the least impact on the polar function-
alities at C-2 and C-6.9 Whereas, the introduction of
fluorine at C-5 should reduce the basicity of the C-6
amidine. However, NOS inhibitors, albeit neuronal
selective, have been reported with reduced basicity at
the δ-guanidine of arginine such as δ-nitroarginine.10
The effect of the introduction of fluorine into the lysine
side chain of NIL at C-5 on iNOS inhibition is described
here.
14 (2S,5S) 2.36 µM
15 (2S,5R) 4.0%
16 (2R,5S) 47.8 µM
17 (2R,5R) 8.9%
21.7 µM
0.00%
8
7
27
32
21.4 µM
139 µM 140 µM
57.5 µM 33.8 µM
7
7
7
4
8.75 µM
shown to have iNOS inhibition activity comparable to
that of L-NIL. For this reason, separation of the four
isomers of 5-fluorolysine was undertaken in order to
identify the active isomer.
Initially, as shown in Scheme 2, the N,N-R,ꢀ-bis-
(benzyloxycarbonyl)-5-fluorolysine 5, which was syn-
thesized under standard conditions, was subjected to
chiral chromatography using Chiralpak OJ . The initial
separation yielded three of the four isomers. The repeat
separation of two coeluting isomers gave the fourth
isomer. This process resulted in isomers 6-9. The
shortcoming of this method of chiral chromatography
was that the use of alcohol in the presence of acid as an
eluting solvent resulted in partial esterification. The
protecting groups were removed under catalytic hydro-
genation conditions, which resulted in R-amino acids
10-13. Each isomer was treated as described for 4 to
give amidines 14-17. As shown in Table 1, the S,S-
isomer 14 had much of the iNOS enzyme inhibition
activity. Because 14 had a desirable profile of enzyme
inhibition, we explored alternative routes to 14 using
different protecting group strategies. In Scheme 3, the
separation of the N,N-R,ꢀ-diphthaloyl-5-fluorolysine
methyl ester 1812 is illustrated. Using Chiralpak AD
chromatography conditions as shown in Scheme 3, the
isomers 19-22 were separated to give three of the four
isomers on the initial separation. Hydrolysis to the
Syn th esis
Commercially available 5-fluoro-D,L-lysine 311 allowed
a quick look at iNOS inhibition. As shown in Scheme 1,
6-(iminoethyl)-5-fluoro-D,L-lysine 4 was synthesized us-
ing methodology described previously.2 In Table 1, 4 is
* Corresponding author. Phone: (847) 491-9128. E-mail:
eahallinan@comcast.net.
† 4901 Searle Parkway.
‡ 800 North Lindbergh Boulevard.
§ 700 Chesterfield Parkway.
10.1021/jm030348f CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/13/2004

5-Fluorinated L-Lysines as iNOS Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 901
Sch em e 2a
a
(a) Compound 3, dibenzyl dicarbonate, NaHCO3, acetone/H2O (3:2), rt, overnight, 87%. (b) Chiralpak OJ , heptane, EtOH, MeOH,
TFA. (c) H2, 5 psi, 5% Pd/C, HOAc, 95%. (d) CuCO3, H2O, ∆, 1 h. (e) pH 9-9.5, ethyl acetimidate hydrochloride, ion exchange
chromatography, reverse phase chromatography.
Sch em e 3a
a
(a) Chiralpak AD, MeOH. (b) HCl/HOAc, ∆. (c) See Scheme 1 for reaction conditions.
Sch em e 4a
amino acids gave 10-13. By capillary electrophoresis,
the product of hydrolysis of 19 gave the desired amino
acid isomer 10, which was taken onto 14. The desired
isomer 19 was readily separated from isomers 20-22.
To improve the yield of 19, isomer 20 was treated with
DBU in CH2Cl2 to epimerize the R-amino acid stereo-
center to provide additional amounts of 19. The mixture
was rechromatographed to provide additional quantities
of 19.
a
(a) Triethyl 2-fluoro-2-phosphonoacetate, NaH, -40 °C to rt,
Another synthetic approach explored the use of as-
partyl semialdehyde.13 The synthetic strategy followed
that described by Taguchi et al.,14 in which they
synthesized 5-fluoro-L-lysine with the R-amino acid
stereocenter set by the chiral starting material. How-
ever, their synthesis commenced with mono-t-Boc-L-
homoserine methyl ester. Our concern about this start-
ing material was retaining the stereochemical integrity
at the R-amino acid stereocenter; also, the possibility
60%. (b) H2, Pd/C, EtOH. (c) NaBH4, MeOH.
of lactone formation existed. Therefore, the bis-Boc-
aspartyl semialdehyde 2313 was used to explore the
viability of the Taguchi approach. The first three steps
of the synthesis are illustrated in Scheme 4. The use of
Horner-Emmons chemistry homologated the amino
acid side chain and introduced the desired 5-fluoro
functionality and the correct chain length to give 24.

902 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Hallinan et al.
Sch em e 5a
a
(a) ethyl acetimidate hydrochloride, CuCO3, ion exchange
resin.
Sch em e 6a
a
(a) NaBH4, MeOH; (b) triflic anhydride, DCM, DMAP. (c)
3-methyl-[1,2,4]oxadiazol-5-one potassium salt, DMF. (d) 5% Pd/
C, MeOH; (e) 2 N HCl.
The alkene was reduced under catalytic hydrogenation
conditions and the ester subsequently reduced with
sodium borohydride, giving 25. This illustrates that the
bis-protected amine approach works in very much the
same fashion as the monoprotected amine described
previously and is a reasonable approach to the synthesis
of 5-fluoro-L-lysine. The appeal of this route was that
only a diastereomeric pair required separation.
F igu r e 1. The refined S-configuration (in normal conventional
atom color, O ) red, N ) blue, C ) green, and F ) cyan) and
R-configuration (in silver) structures of compound 14 dock into
the unbiased, i.e., without inhibitor contribution, difference
electron density map. As indicated by the arrows, the F- and
Cb-atoms of the R-configuration are partially out of the contour
map, while those of the S-configuration are still in.
Fluoro-L-NIL 14 was a more potent inhibitor of iNOS
isozyme than L-NIL, as shown in Table 1. In addition
to 14, only 5(R)-fluoro-L-NIL 16 showed very modest
activity as an iNOS inhibitor The 5,5-difluoro-L-NIL
amidine 32 did not have the potency nor the selectivity
when compared to L-NIL. 5(S)-Monofluoro-L-NIL 14 is
more potent than L-NIL and demonstrates comparable
selectivity.
In parallel with synthesis of 5-fluoro amidine 14, the
synthesis of 5,5-difluoro amidine 27 was investigated.
The achiral synthesis of 5,5-difluorolysine 26 has been
reported previously.15 As depicted in Scheme 5, the
amidine 27 was synthesized in the manner described
in Scheme 1. Although 27 has modest inhibitory activ-
ity, we explored a chiral synthesis of 5,5-difluoro-L-lysine
as represented in Scheme 6. Our approach made use of
the electron-transfer chemistry of Taguchi et al.16
Protected L-vinylglycine sets the amino acid stereo-
chemistry and is commerically available.17 Ethyl diflu-
oroiodoacetate,18 which is used to homologate the vi-
nylglycine, adds the necessary length and the gem-
difluoro moiety, yielding 28. This reaction is somewhat
capricious and the yields are variable. Burton chemis-
try19 was attempted with results similar to that de-
scribed for Taguchi chemistry. The ester is reduced with
sodium borohydride, yielding alcohol 29. The triflate 30
is synthesized by employing triflic anhydride in the
presence of DMAP. Subsequently, the triflate is dis-
placed by the potassium salt of 3-methyl-[1,2,4]oxadia-
zol-5-one20 to give the protected amidine 31. Two
methods to expose the amidine were investigated, either
zinc in acetic acid or catalytic hydrogenation.20 Catalytic
hydrogenation was used to unmask the amidine and to
remove the benzyloxycarbonyl protecting group. The
methyl ester was removed under acid hydrolysis condi-
tions to give the chiral 5,5-difluoro-L-NIL 32.
X-r a y Cr ysta llogr a p h y
Amidine 14, a single isomer, has been cocrystallized
with the oxygenase domain of mouse iNOS (residues
66-498). The protein purification and crystal prepara-
tion were described by Tainer et al.21 The X-ray diffrac-
tion data were collected at the IMCA beamline 17ID of
the Advanced Photon Source of the Argonnne National
Laboratory. The crystals diffract to 2.34 Å resolution.
Since the absolute configuration around the fluorine
atom was not known, the inhibitor coordinates with both
configurations (S- and R-) are refined separately with
the protein. The final refinement statistics did not
suggest which isomer was in the crystal. However, the
unbiased difference electron density (Figure 1, without
the contribution of inhibitor molecule) indicated that the
S-enantiomer had a slightly better fit than the R-
configuration. On the basis of these data, amidine 14
was assigned the 2S,5S-stereochemistry.
There are several dominant features in the inhibitor-
binding pocket, as shown in Figure 2. Two strands wrap
around the inhibitor. The lower strand, FNGWYM (the
side chains of the starting residue Phe363 and the
ending residue Met368 are shown in the figure), covers
Bioa ssa y Resu lts
Enzyme inhibition assays were performed with the
human NOS isoforms as described previously.4 5(S)-

5-Fluorinated L-Lysines as iNOS Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 903
F igu r e 2. This is a ribbon representation of the inhibitor-binding pocket. The protein is shown in deep blue ribbons. The side
chains of some of the neighboring residues are shown in pink. Only the beginning residue, Phe363, and the ending residue,
Met368, of the lower strand are shown for the sake of clarity. Heme is in orange. The inhihitor, compound 14, is depicted in the
conventional color: O ) red, N ) blue, C ) green, and F ) cyan.
the amidine group. The upper strand, Pro344-Ala345-
Val346, houses a suitable hydrophobic pocket in which
to fit the fluorine atom. Another dominant feature is
the formation of three hydrogen bonds between the
inhibitor and Glu371 of the protein. This type of
hydrogen-bond formation is universal for all guanidine
or amidine inhibitors of NOS enzymes in all of the
structures published to date. In this case, the amidine
group of the inhibitor binds to the carboxylate group of
Glu371 with two hydrogen bonds. The amino acid end
of the inhibitor curves back to orient the amino group
in a proper position to form a rather strong hydrogen
bond with Glu371. The heme iron does not interact with
the inhibitor. The distance between iron and the closest
methyl group of the inhibitor is 4.23 Å. It is not shown
in the figure, but the amino acid end of the inhibitor is
F igu r e 3. This is the blow-up picture of Figure 2. NIL
molecule is added. In the NIL molecule, all its carbon atoms
are depicted as yellow.
surrounded by a cluster of polar residues, Arg260,
Arg382, Asn257, Asp376, etc. Although the heteroatoms
(polar atoms) of the inhibitors, 14 and NIL shown in
Additional biological assessment of 14 will be described
elsewhere.
Figure 3, superimpose well in the structure, their overall
conformations are quite different. The conformational
differences of 14 and NIL are likely due to the substitu-
tion of fluorine at the middle of the chain and due to
the affinity of fluorine to the hydrophobic residues
Pro344-Ala345-Val346.
Exp er im en ta l Section
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. GC was run on a Shimadzu 9A using capillary
columns from several vendors. Preparative column chro-
matograpy using flash chromatography conditions was per-
formed on EM silica gel or on a Biotage Flash-40 system. To
successfully use the Biotage columns under flash chromatog-
raphy 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 Reverse-
phase chromatography was performed on a Rainin preparative
HPLC system with a YMC ODS-AQ (10 µm) preparative
column eluting with acetonitrile/water (0.05% HOAc) at 10 mL/
min. HPLC chromatography was performed using a Hewlitt-
Con clu sion
In this paper, we have described the synthesis of 5(S)-
fluoro-L-NIL 14 and 5,5-difluoro-L-NIL 32. The intro-
duction of two fluorines on C-5 of L-NIL yields 32, which
is not as selective as L-NIL. This would seem to suggest
that amelioration of the bascity of the amidine of 32 by
the introduction of two fluorines at C-5 results in the
loss of NOS enzyme selectivity. However, the introduc-
tion of a single fluorine into the side chain of L-NIL gave
amidine 14, which is a potent, selective iNOS inhibitor.

904 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Hallinan et al.
Packard HP 1100 with a dual absorbance detector. Both chiral
and achiral columns were acquired from several vendors.
Capillary electropheresis analyses were performed on a Beck-
man Coulter system. 1H NMR spectra were taken in com-
mercially 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
salt (D2O) as an internal standard for 1H 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) are reported upfield or
downfield relative to fluorotrichloromethane (Freon-11). 19F
NMR spectra were proton decoupled. All reagents were
purchased from Sigma-Aldrich, except where noted, and used
as is. Solvents were purchased from Burdick & J ackson.
Microanalyses and optical rotations were performed in-house.
The optical rotations were performed at concentrations of 2
mg/mL in CHCl3 for the protected amino acids and MeOH for
the amino acids.
N6-Im in oeth yl-5-flu or olysin e Hyd r och lor id e (4). Start-
ing with 5-fluorolysine 3,11 amidine 4 was prepared in the
manner described in the literature:1 1H NMR (400 MHz, CD3-
OD) δ 1.77-2.22 (m, 4 H), 2.27 (s, 3 H), 3.48-3.78 (m, 2 H),
4.02-4.12 (m, 1 H), 4.69-4.89 (m, 1 H); 13C NMR (100 MHz,
CD3OD) δ 19.4, 27.6, 29.5 (d, J ) 20.3 Hz), 47.7 (d, J ) 21.0
Hz), 53.6, 92.1 (d, J ) 173 Hz), 167.5, 171.8; 19F NMR -187.5.
5-F lu or o-N2,N6-bis(ben zoxyca r bon yl)lysin e (5). To a
stirred solution of 5-fluorolysine (3.70 g, 18.4 mmol) in 100
mL of acetone:H2O (2:3) was added dropwise a solution of
dibenzyl dicarbonate (13.20 g, 46.1 mmol) in 40 mL of acetone.
After stirring 18 h, the reaction was concentrated to remove
the acetone. The aqueous layer was washed with EtOAc (1 ×
100 mL), neutralized to pH 3, and extracted with EtOAc (2 ×
100 mL). After neutralization, the combined organic layers
were washed with saturated brine, dried over anhydrous Na2-
SO4, and concentrated to yield 8.3 g of crude product 5. After
purification on a Biotage system, 7.16 g (87%) of desired
purified 5 was recovered: 1H NMR (400 MHz, CDCl3) δ 1.52-
2.18 (m, 4 H), 3.20-3.55 (m, 2 H), 4.28-4.67 (m, 2 H), 5.02-
5.28 (m, 4 H), 7.30-7.39 (m, 10 H). Anal. (C22H25FN2O6‚0.50
H2O) C, H, N.
(d, J ) 20 Hz), 52.7, 90.4 (d, J ) 171 Hz), 172.0; 19F NMR
(376 MHz, D2O, CFCl3) δ -190.8. Anal. (C6H13N2O2F‚2HCl‚
0.2H2O) C, H, N.
(5S)-5-F lu or o-D-lysin e (12). The experimental conditions
used to generate 10 were used to produce the amino acid 12:
[R]D ) +10.7 ( 3.4; see compound 11 for NMR.
(5R)-5-F lu or o-D-lysin e (13). The experimental conditions
used to generate 10 were used to produce the amino acid 13:
[R]D ) -23.4 ( 3.3; see compound 10 for NMR.
(5S)-N6-(Im in oeth yl)-5-flu or o-L-lysin e Hyd r och lor id e
(14). Amidine 14 was prepared in the same manner as
described for 4: 1H NMR (400 MHz, D2O, TSP) δ 1.69-1.89
(m, 2 H), 1.96-2.07 (m, 2 H), 2.16 (s, 3 H), 3.37-3.59 (m, 2
H), 4.03 (t, J ) 6.4 Hz, 1 H), 4.79-4.85 (m, 1 H); 13C (100 MHz,
D2O, TSP) δ 18.9, 25.9, 27.7 (d, J ) 20 Hz), 45.8 (d, J ) 20
Hz), 53.6, 91.8 (d, J ) 169 Hz), 166.2, 172.1; 19F (376 MHz,
D2O, CFCl3) δ -187.5. Anal. (C8H16N3O2F‚2HCl‚2H2O) C, H,
N.
(5R)-N6-(Im in oeth yl)-5-flu or o-L-lysin e Hyd r och lor id e
(15). Amidine 15 was prepared in the same manner as
described for compund 4: 1H NMR (400 MHz, D2O, TSP) δ
1.60-1.90 (m, 2 H), 1.90-2.02 (m, 1 H), 2.04-2.12 (m, 1 H),
2.16 (s, 3 H), 3.38-3.58 (m, 2 H), 4.02 (t, J ) 6.4 Hz, 1 H),
4.79-4.86 (m, 1 H); 13C (100 MHz, D2O, TSP) δ 18.9, 25.8, 27.5
(d, J ) 20 Hz), 45.8 (d, J ) 21 Hz), 53.7 91.7 (d, J ) 169 Hz),
166.2, 172.3; 19F (376 MHz, D2O, CFCl3) δ -187.0. Anal.
(C8H16N3O2F‚2HCl‚1.3H2O) C, H, N.
(5S)-N6-(Im in oeth yl)-5-flu or o-D-lysin e Hyd r och lor id e
(16). Amidine 16 was prepared in the same manner as
described for compound 4: see compound 15 for NMR data.
(5R)-N6-(Im in oeth yl)-5-flu or o-D-lysin e Hyd r och lor id e
(17). Amidine 17 was prepared in the same manner as
described for compound 4: see compound 14 for NMR data.
Meth yl 2,6-Bis(1,3-d ioxo-1,3-d ih yd r o-2H-isoin d ol-2-yl)-
5-flu or oh exan oic Acid (18). Diphthaloyl-5-fluorolysine meth-
yl ester 18 was prepared as described in the literature:12 1H
NMR (400 MHz, CDCl3) δ 1.58-1.94 (m, 2 H), 2.34-2.60 (m,
2 H), 3.75 (s, 5 H), 3.90-3.99 (m, 1 H), 4.87-4.91 (m, 1 H),
7.70-7.90 (m, 8 H); 13C NMR (100 MHz, CDCl3) δ 24.8 (d, J )
25 Hz), 25.0 (d, J ) 25 Hz), 30.0, 30.1, 41.9 (d, J ) 23 Hz),
42.1 (d, J ) 22 Hz), 52.2, 53.2, 90.2 (d, J ) 165 Hz), 90.8 (d,
J ) 175 Hz), 123.7, 123.9, 132.0, 132.2, 134.5, 134.7, 167.8,
167.9, 168.2, 169.6; 19F NMR (376 MHz, CDCl3) δ -187.8,
-188.3. Anal. (C23H19N2O6F) C, H, N.
Meth yl (2S,5S)-2,6-Bis(1,3-d ioxo-1,3-d ih yd r o-2H-isoin -
d ol-2-yl)-5-flu or oh exa n oa te (19). Compound 18 (4.3 g) was
separated into four isomers using chiral chromatography on
Chiralpak AD in 100% MeOH. This resulted in isomers 19-
22. The yield of isomer 19 was 0.90 g: [R]D ) 4.3 ( 3.4.
Meth yl (2R,5S)-2,6-Bis(1,3-d ioxo-1,3-d ih yd r o-2H-isoin -
d ol-2-yl)-5-flu or oh exa n oa te (20). See the method for com-
pound 19. The yield of isomer 20 was 0.28 g: [R]D ) -33.1 (
3.4.
(5S)-5-F lu or o-N2,N6-bis(ben zoxyca r bon yl)-L-lysin e (6).
The product 5 (6.05 g) was subjected to chiral chromatography
employing Chiralcel OJ and eluting with a quaternary mixture
of heptane/MeOH/EtOH/TFA to yield 0.48 g (8%): [R]D ) +6.8
( 3.5.
(5R)-5-F lu or o-N2,N6-bis(ben zoxyca r bon yl)-L-lysin e (7).
See the method for compound 6, which yielded 0.97 g (16%) of
7: [R]D ) -7.5 ( 3.5; 1H NMR (400 MHz, CDCl3) δ 1.51-2.18
(m, 4 H), 3.18-3.55 (m, 2 H), 4.28-4.65 (m, 2 H), 5.04-5.18
(m, 4 H), 7.28-7.40 (m, 10 H).
(5S)-5-F lu or o-N2,N6-bis(ben zoxyca r bon yl)-D-lysin e (8).
See the method for compound 6, which yielded 0.28 g (5%) of
8.
Meth yl (2S,5R)-2,6-Bis(1,3-d ioxo-1,3-d ih yd r o-2H-isoin -
d ol-2-yl)-5-flu or oh exa n oa te (21). See the method for com-
pound 19. The yield of isomer 21 was 0.25 g: [R]D ) +23.7 (
3.4. Anal. (C23H19N2O6F‚2H2O) C, H, N.
Meth yl (2R,5R)-2,6-Bis(1,3-d ioxo-1,3-d ih yd r o-2H-isoin -
d ol-2-yl)-5-flu or oh exa n oic Acid (22). See the method for
compound 19. The yield of isomer 22 was 0.45 g: [R]D ) -12.3
( 3.4. Anal. (C23H19N2O6F‚0.3H2O) C, H, N.
(5R)-5-F lu or o-N2,N6-bis(ben zoxyca r bon yl)-D-lysin e (9).
See the method for compound 6, which yielded 0.53 g (9%) of
9. [R]D) -13.3 ( 3.5. Anal. (C22H25FN2O6‚1.1H2O) C, H, N.
(5S)-5-F lu or o-L-lysin e (10). The protecting groups were
removed under catalytic hydrogenation conditions using 5%
Pd/C in EtOH/HOAc to give a quantitative yield of the amino
acid 10: [R]D ) +9.3 ( 3.5; 1H NMR (400 MHz, D2O) δ 1.63-
1.98 (m, 2 H), 2.02 (q, J ) 8.0 Hz, 2 H), 3.04-3.30 (m, 2 H),
4.04 (t, J ) 6.5 Hz, 1 H), 4.85 (tt, J ) 3.4, 9.2 Hz, 1 H); 13C
NMR (100 MHz, D2O) δ 25.6 (d, J ) 6 Hz), 27.7 (d, J ) 20
Met h yl 2-[Bis(ter t-b u t oxyca r b on yl)a m in o]-4-oxob u t -
a n oa te (23). Bis-Boc-aspartyl semialdehyde 23 was prepared
as described in the literature.15
1-Eth yl 6-Meth yl (2Z,5S)-5-[bis(ter t-bu toxyca r bon yl)-
a m in o]-2-flu or oh ex-2-en ed ioa te (24). Intermediate 24 was
prepared in the same manner described by Taguchi et al.14
with a yield of 60%: 1H NMR (400 MHz, CDCl3) δ 1.34 (t, J )
7.0 Hz, 3 H), 1.48 (s, 9 H), 1.49 (s, 9 H), 3.26 (dt, J ) 1.5, 8.3
Hz, 2 H), 3.74 (s, 3 H), 4.29 (dq, J ) 1.5, 7.0 Hz, 2 H), 5.02 (t,
J ) 7.5 Hz, 1 H), 5.93 (td, J ) 8.3, 20.7 Hz, 1 H); 13C NMR
(100 MHz, CDCl3) δ 14.5, 27.0, 28.3, 52.7, 57.9, 61.9, 83.7, 119.0
(d, J ) 21.4 Hz), 149.6, 153.0, 161.9, 171.6. Anal. (C19H30NO8F)
C, H, N.
Hz), 43.0 (d, J ) 20 Hz), 52.7, 90.4 (d, J ) 171 Hz), 172.0; 19
NMR (376 MHz, D2O, CFCl3) δ -190.8.
F
(5R)-5-F lu or o-L-lysin e (11). The experimental conditions
used to generate 10 were used to produce the amino acid 11:
[R]D ) -23.4 ( 3.4; 1H NMR (400 MHz, D2O) δ 1.58-1.88 (m,
2 H), 1.89-2.01 (m, 1 H), 2.02-2.15 (m, 1 H), 3.03-3.25 (m, 2
H), 4.01 (t, J ) 6.4 Hz, 1 H), 4.65-4.89 (m, 1 H); 13C NMR
(100 MHz, D2O) δ 25.6 (d, J ) 6 Hz), 27.7 (d, J ) 20 Hz), 43.0

5-Fluorinated L-Lysines as iNOS Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 905
Met h yl N,N-Bis(ter t-b u t oxyca r b on yl)-5-flu or o-6-h y-
d r oxy-L-n or leu cin a te (25). Step a . The alkene diester
product 24 was reduced under catalytic hydrogenation condi-
tions using 5% Pd/C as a catalyst. The yield of 25a was 86%.
25.2 (t, J ) 4 Hz), 29.8 (t, J ) 24 Hz), 53.2, 53.4, 67.7, 73.0 (t,
J ) 36 Hz), 119.5 (t, J ) 243 Hz), 128.6, 128.8, 129.0, 136.4,
156.2, 172.3; 19F NMR (376 MHz, CDCl3) δ -71.5 (3 F), -105.7
(2 F).
Meth yl N-(Ben zyloxyca r bon yl)-5,5-d iflu or o-6-(3-m eth -
yl-5-oxo-1,2,4-oxa d ia zol-4(5H)-yl)-L-n or leu cin a te (31). To
a solution of 30 (0.55 g, 1.2 mmol) in 20 mL of DMF was added
potassium 3-methyl-[1,2,4]oxadiazol-5-one (0.18 g, 1.3 mmol).
After heating for 20 h at 50 °C, the reaction was concentrated
under high vacuum. The residue was dissolved in 25 mL of
EtOAc, washed with brine (2 × 25 mL), dried MgSO4, and
concentrated under vacuum. The crude residue was purified
by using a Flash-40 chromatography system. The yield of 31
was 0.20 g (42%): 1H NMR (400 MHz, CDCl3) δ 1.88-2.08
(m, 3 H), 2.10-2.24 (m, 1 H), 2.28 (s, 3 H), 3.78 (s, 3 H), 3.80-
3.91 (m, 2 H), 4.35-4.46 (m, 1 H), 5.11 (s, 2 H), 5.46 (br s, 1
H), 7.30-7.50 (m, 5 H); 13C NMR (100 MHz, CDCl3) δ 10.9,
30.8 (t, J ) 20 Hz), 46.1 (t, J ) 20 Hz), 53.2, 53.4, 67.4, 128.6,
128.8, 129.0; 19F NMR (376 MHz, CDCl3) δ -100.4, -100.7.
Anal. (C18H21N3O6F2) C, H, N.
Step b. To a rapidly stirred solution of NaBH4 (0.044 g, 1.2
mmol) in 5 mL of MeOH was added dropwise the product 25a
(0.42 g, 1.2 mmol) in 20 mL of MeOH. After stirring for 3 h at
room temperature, additional NaBH4 (0.016 g) was added.
After 4 h, the reaction was concentrated under vacuum. The
residue was dissolved in 25 mL of EtOAc and 25 mL of H2O,
the layers were separated, the aqueous layer was back-
extracted with EtOAc (2 × 25 mL), and the combined organic
layers dried over MgSO4, filtered, and evaporated. The residue
was purified on a Flash-40 system to yield 0.12 g (27%) of 25.
In addition, an equal amount of aldehyde hemiacetal was
isolated: 1H NMR (400 MHz, CDCl3) δ 1.43 (s, 18 H), 1.45-
1.78 (m, 2 H), 1.82-2.03 (m, 1 H), 1.82-2.03 (m, 1 H), 2.12-
2.30 (m, 1 H), 3.58-3.62 (m, 1 H), 3.65 (s, 5 H), 4.43-4.64 (m,
1 H), 4.81 (dt, J ) 4.8, 10.8 Hz, 1 H); 13C NMR (100 MHz,
CDCl3) δ 25.8, 25.9, 28.0 (d, J ) 9 Hz), 28.3 (d, J ) 9 Hz),
28.4, 52.6, 57.9, 58.3, 64.9 (d, J ) 15 Hz), 65.2 (d, J ) 15 Hz),
63.8, 94.8 (d, J ) 169 Hz), 95.1 (d, J ) 169 Hz), 152.4, 172.5.
Anal. (C17H30NO7F) C, H, N.
N6-(Im in oeth yl)-5,5-L-d iflu or olysin e Dih yd r och lor id e
(32). Step a . The benzyloxycarbonyl protecting groups were
removed as described for compound 10.
Step b. The methyl ester was removed in refluxing 2 N HCl.
After 20 h, the reaction was concentrated under vacuum and
subsequently lyophilized. 1H NMR (400 MHz, D2O) δ 1.99-
2.16 (m, 4 H), 2.18 (s, 3 H), 3.73 (t, J ) 15 Hz, 2 H), 4.03 (t, J
) 5 Hz, 1 H); 13C NMR (100 MHz, D2O, TSP) δ 19.0, 22.5,
29.9 (t, J ) 24 Hz), 46.0 (t, J ) 27 Hz), 52.6, 122.2 (t, J ) 242
Hz), 167.5, 172.0; 19F NMR (376 MHz, D2O, CFCl3) δ -105.7.
Anal. (C8H15N3O2F2) C, H, N.
5,5-Diflu or olysin e (26). 5,5-Difluorolysine was synthesized
according to the literature procedure.15
N6-(Im in oeth yl)-5,5-diflu or olysin e Dih ydr och lor ide (27).
Amidine 27 was prepared as described for compound 4: 1H
NMR (400 MHz, D2O) δ 1.99-2.16 (m, 4 H), 2.18 (s, 3 H), 3.73
(t, J ) 15 Hz, 2 H), 4.03 (t, J ) 5 Hz, 1 H); 13C NMR (100
MHz, D2O, TSP) δ 19.0, 22.5, 29.9 (t, J ) 24 Hz), 45.9 (t, J )
27 Hz), 52.6, 122.2 (t, J ) 242 Hz), 167.5, 172.0; 19F NMR (376
MHz, D2O, CFCl3) δ -105.7
Ack n ow led gm en t. The authors would like to thank
Lawrence Miller and his colleagues for their work on
chiral preparative separations of the fluorolysine iso-
mers and Yuri Zelechonok and his colleagues for the
analytical chromatography of the fluorolysine isomers.
7-Eth yl 1-Meth yl 2-[(Ben zyloxyca r bon yl)a m in o]-5,5-
d iflu or oh ep ta n ed ioa te (28). To a stirred solution of methyl
benzyloxycarbonyl-L-vinylglycinate (0.50 g, 2.0 mmol) in 10 mL
of ultrapure DMF was added ethyl difluoroiodoacetate (0.65
g, 2.6 mmol) and Cu (0.04 g, 0.6 mmol). After 24 h, EtOAc (50
mL) was added to the reaction. The organic layer was washed
with brine solution (5 × 50 mL), dried over MgSO4, filtered,
and concentrated under vacuum. The residue was dissolved
in 20 mL of HOAc/EtOH (1:3) and treated with Zn (0.26 g, 3.9
mmol). After heating at 80 °C for 4 h, the reaction mixture
was cooled to room temperature and concentrated under
vacuum. To the residue was added 25 mL of EtOAc, which
was washed with saturated NaHCO3 (25 mL), brine (25 mL),
and H2O (25 mL). The organic layer was dried over MgSO4,
filtered, and concentrated to yield 0.54 g of crude product. After
purification on a Flash-40 system, 0.25 g (32%) of 28 was
isolated: 1H NMR (400 MHz, CDCl3) δ 1.35 (t, J ) 7.5 Hz, 3
H), 1.81-1.90 (m, 1 H), 2.05-2.28 (m, 3 H), 3.78 (s, 3 H), 4.33
(q, J ) 7.5 Hz, 2 H), 5.12 (s, 2 H), 5.36 (br d, 1 H), 7.30-7.40
(m, 5 H); 13C NMR (100 MHz, CDCl3) δ 14.3, 25.2, 30.9 (t, J )
23 Hz), 53.1, 63.4, 67.5, 116.0 (t, J ) 251 Hz), 128.5, 128.8,
129.0, 136.4, 156.3, 164.2, 172.4; 19F NMR (376 MHz, D2O,
CFCl3) δ -105.6. Anal. (C17H21NO6F2) C, H, N.
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