Synthesis and biological evaluation of DL-4,4-difluoroglutamic acid and DL-γ,γ-difluoromethotrexate

Article abstract of DOI:10.1021/jm950514m

DL-4,4-Difluoroglutamic acid (DL-4,4-F₂Glu) and its methotrexate analogue, DL-γ,γ-difluoromethotrexate (DL-γ,γ-F₂MTX), were synthesized and evaluated as alternate substrates or inhibitors of folate-dependent enzymes. Synthesis of DL-

Full text of DOI:10.1021/jm950514m

66
J . Med. Chem. 1996, 39, 66-72
Syn th esis a n d Biologica l Eva lu a tion of DL-4,4-Diflu or oglu ta m ic Acid a n d
DL-γ,γ-Diflu or om eth otr exa te
Takashi Tsukamoto,†,‡ Tomoya Kitazume,‡ J ohn J . McGuire,§ and J ames K. Coward*,†
Departments of Medicinal Chemistry and Chemistry, The University of Michigan, Ann Arbor, Michigan 48109, Department of
Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226, J apan, and Department of
Experimental Therapeutics, The Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263
Received J uly 14, 1995X
DL-4,4-Difluoroglutamic acid (DL-4,4-F2Glu) and its methotrexate analogue, DL-γ,γ-difluo-
romethotrexate (DL-γ,γ-F2MTX), were synthesized and evaluated as alternate substrates or
inhibitors of folate-dependent enzymes. Synthesis of DL-4,4-F2Glu involved the nitroaldol
reaction of ethyl nitroacetate with a difluorinated aldehyde ethyl hemiacetal as a key step.
Attempted ligation of DL-4,4-F2Glu to methotrexate (MTX), catalyzed by human folylpoly-γ-
glutamate synthetase (FPGS), revealed that DL-4,4-F2Glu is a poor alternate substrate. DL-
γ,γ-F2MTX was synthesized by a route proceeding through N-[4-(methylamino)benzoyl]-4,4-
difluoroglutamic acid di-tert-butyl ester followed by alkylation with 6-(bromomethyl)-2,4-
pteridinediamine hydrobromide. DL-γ,γ-F2MTX was found to be neither a substrate nor an
inhibitor of human FPGS. The fluorinated analogue of MTX, however, inhibits DHFR and
cell growth with the same potency as MTX.
In tr od u ction
These findings prompted our interest in the synthesis
of DL-4,4-difluoroglutamic acid (1, DL-4,4-F2Glu) and its
MTX analogue (2, DL-γ,γ-F2MTX, AMPte(4,4-F2Glu)) for
further investigation of the effects of fluorine on bio-
chemical reactions catalyzed by folate-dependent en-
zymes and on cell growth.7
Folylpoly-γ-glutamate synthetase (FPGS) is respon-
sible for the biosynthesis of poly-γ-glutamyl conjugates
of naturally occurring folates and antifolate drugs.1
Since folylpoly-γ-glutamates are essential for one-carbon
metabolism,2 FPGS has become an important enzyme
in the study of folate biochemistry and pharmacology.
The general reaction catalyzed by FPGS is shown in eq
1. In order to distinguish the glutamyl moiety on folate
PteGlu + Glu + ATP f
PteGlu-γ-Glu + ADP + Pi (1)
Ch em istr y
(PteGlu) from free glutamic acid (Glu), it is convenient
to discuss them as the accepting and incoming glu-
tamates, respectively. In previous research from our
laboratories, we have shown that, as incoming glu-
tamates, 4-fluoroglutamic acid (4-FGlu)3 and 3,3-di-
fluoroglutamic acid (3,3-F2Glu)4 have dramatically dif-
ferent properties as FPGS substrates. Similarly, modi-
fication of the accepting glutamyl moiety of folates and
antifolates may alter their activity as substrates or
inhibitors of the FPGS-catalyzed reaction. In this
regard, we have previously synthesized a series of
methotrexate (MTX; 2,4-diamino-10-methylpteroyl-L-
glutamic acid, AMPteGlu) analogues containing differ-
ent fluoroglutamic acids. γ-Fluoromethotrexate (γ-
FMTX, AMPte(4-FGlu)) was found to be polyglutam-
ylated by FPGS at a drastically reduced rate compared
to the parent molecule, MTX.5 In contrast, enhanced
substrate activity was observed with â,â-difluorometho-
trexate (â,â-F2MTX, AMPte(3,3-F2Glu)) when compared
to MTX.6 The diametrically opposed FPGS substrate
properties observed with γ-FMTX and â,â-F2MTX are
all the more striking when it is noted that both of these
fluorinated MTX analogues inhibit dihydrofolate reduc-
tase (DHFR) with the same potency as does MTX.
A stereospecific synthesis of L-4,4-F2Glu has been
reported previously by Kitagawa and co-workers.8 Their
method, however, requires a commercially unavailable
starting material, methyl difluoroiodoacetate, which is
costly to prepare.9 In previous work, we successfully
synthesized 3,3-F2Glu from a 3-oxoproline derivative via
(diethylamido)sulfur trifluoride (DAST)-mediated fluo-
rination.10 A similar pathway utilizing an L-4-oxopro-
line derivative should afford L-4,4-F2Glu. To this end,
we found that the 4-oxoproline derivative, 3, is easily
converted into the corresponding difluorinated molecule
(4) by DAST-mediated fluorination (Scheme 1).11 Oxi-
dation of 4, however, was extremely sluggish and the
desired pyroglutamic acid derivative 5 was not obtained
on extended (up to 10 days) reaction with RuO4,
presumably as a result of the strong electron-withdraw-
ing effect of the two adjacent fluorine atoms (B. P. Hart
and J . K. Coward, unpublished results).
These results led us to investigate a more accessible
synthesis of DL-4,4-F2Glu (1) by an alternate route. The
difluorinated aldehyde ethyl hemiacetal, 6, has been
found previously to react with nitroalkanes to give nitro
alcohols in good yields.12 Reaction of 6 with ethyl
nitroacetate in the presence of diethylamine13 followed
by acid treatment afforded nitro alcohol 7 as a mixture
of diastereomers which was immediately acetylated with
acetic anhydride (Scheme 2). Due to the instability of
nitro alcohol 7 and the corresponding acetate, 8, these
† The University of Michigan.
‡ Tokyo Institute of Technology.
§ Roswell Park Cancer Institute.
X Abstract published in Advance ACS Abstracts, December 1, 1995.
0022-2623/96/1839-0066$12.00/0 © 1996 American Chemical Society

Fluorinated Methotrexate Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 67
Sch em e 1
Sch em e 2
Sch em e 3
intermediates were carried through the synthesis in
crude form. Acetate 8 was treated with sodium boro-
hydride in dry THF14 to give the deoxygenated product
9, a key amino acid precursor. This compound was
readily purified by silica gel chromatography to afford
pure material in 68% overall yield from 6. Raney Ni-
catalyzed hydrogenation followed by hydrolysis and
anion-exchange chromatography gave DL-4,4-F2Glu, 1,
in 52% overall yield from 9. The fluorination of ketones
with DAST has been a leading method for the synthesis
of difluoromethylene-containing compounds. However,
in contrast to the reaction with alcohols, DAST-medi-
ated fluorination of ketones generally requires higher
temperatures and longer reaction times and often gives
low yields of the desired product. The synthesis de-
scribed here relies on the use of a small molecule already
bearing the difluoromethylene group as a building block.
This methodology offers an alternate route to various
difluoromethylene-containing compounds that cannot be
obtained through the DAST approach.
Synthesis of DL-γ,γ-F2MTX (2) was accomplished by
the method reported for the synthesis of MTX by Piper
and co-workers (Scheme 3).15 DL-4,4-F2Glu (1) was first
converted to the corresponding di-tert-butyl ester, 10,
by acid-catalyzed reaction with isobutylene. Condensa-
tion of 10 with an N-Cbz-protected derivative of p-(N-
methylamino)benzoic acid afforded p-(N-Cbz-N-methyl-
amino)benzoyl 4,4-difluoroglutamate (11), which was
subsequently deprotected by catalytic hydrogenation to
give N-[4-(methylamino)benzoyl]-4,4-difluoroglutamic
acid di-tert-butyl ester (12). Coupling of 12 and 6-(bro-
momethyl)-2,4-pteridinediamine hydrobromide in di-
methylacetamide followed by treatment with trifluoro-
acetic acid afforded crude DL-γ,γ-F2MTX (2). Subse-
quent DEAE-cellulose anion-exchange chromatography
provided pure DL-γ,γ-F2MTX (2) as the triethylammo-
nium salt in 61% yield from 12.
Biologica l Stu d ies
DL-4,4-F 2Glu As th e “In com in g” Glu ta m a te. The
effect of DL-4,4-F2Glu on the FPGS-catalyzed ligation
of [3H]Glu to the γ-COOH of MTX was compared
directly to the effect of L-t-4-FGlu, DL-3,3-F2Glu, and
L-Glu (isotopic dilution) (Figure 1).3,4 DL-4,4-F2Glu
showed only slight inhibition of this FPGS-catalyzed
reaction. The results indicate that DL-4,4-F2Glu is
either a poor alternate substrate or weak reversible
inhibitor of FPGS. To evaluate whether DL-4,4-F2Glu
is a poor alternate substrate that can be ligated to MTX,
[3H]MTX was employed as the pteroyl substrate with
either Glu or DL-4,4-F2Glu as incoming the amino acid.
The products formed from the FPGS-catalyzed reactions
were separated by HPLC and quantitated (Table 1).16-18
These data show that, whereas 28% of [3H]MTX was
converted to the γ-glutamyl dipeptide in the presence
of L-Glu (higher polyglutamylated derivatives were not
detected), only 5% of [3H]MTX was converted into a
glutamylated product in the presence of DL-4,4-F2Glu.
The product of FPGS-catalyzed ligation of DL-4,4-F2Glu
to [3H]MTX is postulated to be AMPteGlu-γ-(4,4-F2Glu)
on the basis of its retention time of 33.5 min (AMPteGlu-
γ-Glu, tR ) 34.5 min). These results lead to the
conclusion that, although considerably less active than
Glu, DL-4,4-F2Glu also serves as an alternate incoming
substrate for FPGS-catalyzed polyglutamylation. This
is in marked contrast to the results obtained with a
closely related mono-4-fluoroglutamate, L-threo-4-FGlu,
an excellent FPGS alternate incoming substrate, the

68 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Tsukamoto et al.
target enzyme DHFR and uptake by intact cells are two
biochemical properties critical to the mechanism of
action of MTX analogues. Inhibition of human DHFR
by DL-γ,γ-F2MTX was compared to that observed with
L-MTX (Table 4). Assuming, as shown with MTX22 and
γ-FMTX,23 that only the L-isomer of DL-γ,γ-F2MTX acts
as an inhibitor, its inhibitory activity is nearly equiva-
lent to that obtained from L-MTX. DL-γ,γ-F2MTX in-
hibits the initial velocity of uptake of 2 µM [3H]MTX by
CCRF-CEM cells more potently than L-MTX itself
(Table 4), suggesting that DL-γ,γ-F2MTX has high af-
finity for the reduced folate/MTX carrier. These proper-
ties are similar to those observed with DL-â,â-F2MTX.6
Gr ow th In h ibitor y Effects of DL-γ,γ-F 2MTX. MTX
and DL-γ,γ-F2MTX are similarly potent (based on L-
isomers)22,23 as inhibitors of growth of the CCRF-CEM
human leukemia cell line during continuous exposure
(Table 5). MTX-resistant sublines of CCRF-CEM hav-
ing elevated DHFR activity24 or decreased MTX up-
take25 are cross-resistant to DL-γ,γ-F2MTX in continuous
exposure (Table 5). The cross-resistance of the former
subline, together with the DHFR affinity data (Table
4), suggest that DHFR is the critical intracellular target
of DL-γ,γ-F2MTX. The cross-resistance of the latter
subline together with the [3H]MTX uptake inhibition
data (Table 4) suggest that DL-γ,γ-F2MTX uses the
reduced folate/MTX carrier system for uptake and does
so with at least as high an affinity as does MTX. L-MTX
and D,L-4,4-F2MTX show similar potency against these
two sublines individually instead of the ∼2-fold differ-
ence observed with the parental CCRF-CEM. This may
occur because, at the much higher drug concentrations
required for growth inhibition of resistant lines, the
D-isomer may enter cells by an alternate pathway in
sufficient concentration so that it contributes to growth
F igu r e 1. Effects of fluorinated glutamate analogues on the
ligation of [3H]Glu into methotrexate by human FPGS. CCRF-
CEM FPGS was incubated with 0.5 mM L-[3H]Glu (8.2 dpm/
pmol) in the absence (2) or presence of unlabeled DL-4,4-F2Glu
(0), L-threo-4-FGlu (9), DL-3,3-F2Glu (b), or L-Glu (O) at the
indicated concentration for 3 h. For racemic substrates, only
the concentrations of L-isomers are indicated.
incorporation of which into reduced folate or MTX
polyglutamates leads to termination of the ligation
process.3
DL-4,4-F 2Glu a s th e “Accep tin g” Glu ta m a te. The
ability of DL-γ,γ-F2MTX, the DL-4,4-F2Glu-containing
derivative of MTX, to act as an FPGS substrate was
compared directly to the substrate activity of MTX and
aminopterin (AMT). As shown in Table 2, no detectable
[3H]Glu was incorporated into DL-γ,γ-F2MTX under
conditions where ca. 400 pmol of Glu was incorporated
into MTX. Since inhibitors of FPGS could potentially
be useful as cytotoxic agents,19-21attention was focused
on DL-γ,γ-F2MTX for its ability to act as a FPGS
inhibitor. DL-γ,γ-F2MTX showed only very weak inhibi-
tion of FPGS-catalyzed incorporation of [3H]Glu into
AMT (IC50 > 200 µM, Table 3). Taken together, these
results indicate that DL-γ,γ-F2MTX binds only weakly
to FPGS and is not effective as either a substrate or an
inhibitor of the enzyme.
Similar to DL-γ,γ-F2MTX, neither the erythro nor the
threo isomer of DL-γ-FMTX form significant amounts of
polyglutamate products.5 In addition, neither the
γ-FMTX isomers nor DL-γ,γ-F2MTX are inhibitors of
FPGS, indicating that they do not bind to the protein.
In contrast, our recent studies on â,â-F2MTX have
shown that this compound is a better FPGS substrate
than MTX.6 The original hypothesis that led to the
synthesis of γ-FMTX focused on the effects of the fluoro
substituents on the pKa of the γ-COOH of the accepting
glutamate.3 However, our more recent findings are
inconsistent with a simple pKa effect since the γ-COOH
pKa’s of both â,â-F2MTX and γ-FMTX are quite similar
(ca. 2.5). Further studies on the mechanism of binding
of the glutamyl moiety to FPGS are required before an
explanation of the observed differences in reactivity
between different fluoroglutamate-containing folate or
antifolate substrates can be provided.
inhibition despite its weaker DHFR inhibition.
A
CCRF-CEM subline resistant to short-term, but not
continuous, MTX exposure because of reduced FPGS
activity26 is not cross-resistant to continuous exposure
to DL-γ,γ-F2MTX (Table 5), indicating that, as previously
shown,5 polyglutamyl metabolites are not required
during continuous exposure to potent DHFR inhibitors.
Con clu sion
The data presented in this paper show that 4,4-F2-
Glu (1), as either an incoming or accepting glutamate,
is a poor alternate substrate for the FPGS-catalyzed
reaction (eq 1). When activity as an incoming glutamate
is assayed directly by ligation to [3H]MTX (Table 1), 4,4-
F2Glu leads to only ca. 5% ligation compared to 28%
with Glu itself. The poor substrate activity may result
at least partially from weak binding to FPGS, as
measured indirectly by inhibition of [3H]Glu incorpora-
tion (Figure 1). In previous papers from our laboratories
exploring the incoming glutamate, we have described
the action of threo-4-FGlu as a chain-terminating al-
ternate substrate3 and 3,3-F2Glu as an elongation-
enhancing alternate substrate.4 Both of these ana-
logues have higher apparent affinity for FPGS than does
4,4-F2Glu (Figure 1). As the accepting glutamate in the
MTX analogue γ,γ-F2MTX (2), the presence of 4,4-F2-
Glu also leads to a poor FPGS ligand as either a
substrate (Table 2) or inhibitor (Table 3). In addition
to the synthesis of DL-γ,γ-F2MTX described here, we
have accomplished the synthesis of three other fluori-
nated analogues of MTX: threo-γ-FMTX, erythro-γ-
En zym e In h ibitor y Effects of DL-γ,γ-F 2MTX. In
addition to FPGS substrate activity, inhibition of the

Fluorinated Methotrexate Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 69
Ta ble 1. HPLC Analysis of FPGS-Catalyzed Polyglutamylation of [3H]MTX with L-Glu or DL-4,4-F2Glua
MTX (AMPteGlu), %
AMPteGlu-γ-Glu, %
(tR ) 34.5 min)
AMPteGlu-γ-4,4F2Glu, %
(tR ) 33.5 min)
substrate
L-Glu
DL-4,4-F2Glu
(tR ) 39.2 min)
72
95
28
5
a
FPGS, isolated from CCRF-CEM human leukemia cells, was incubated with 0.1 µM [3H]MTX (433 dpm/pmol) in the presence of
L-Glu (4 mM) or DL-4,4-F2Glu (8 mM) for 8 h under otherwise standard conditions. At the end of the incubation, samples were processed16,18
and analyzed by reversed-phase HPLC. Fractions (1 mL, 1 min) were collected, and the radioactivity was quantitated. Sufficient chemically
synthesized MTX polyglutamates to be observable by A280 were included as internal standards.
Ta ble 2. FPGS-Catalyzed Polyglutamylation of Antifolatesa
Ta ble 5. Growth Inhibition of Parental CCRF-CEM and Its
Methotrexate-Resistant Sublines during Continuous (120 h)
Exposure to L-MTX or DL-γ,γ-F2MTXa
substrates
[3H]Glu incorporated (pmol)
L-AMT (100 µM)
1386, -
451, 364
2, 0
ED50 (nM)
L-MTX (100 µM)
DL-γ,γ-F2MTX (200 µM)b
CCRF-
CEM
R1
R2
R30dm
d
drug
L-MTX
(v DHFR)b (V transport)c (V Glun)
a
CCRF-CEM FPGS was incubated for 3 h, under otherwise
standard conditions, with 0.5 mM L-[3H]Glu (8.2 dpm/pmol) in the
presence of the indicated concentration of each antifolate. The
13.7 ( 0.5 655 ( 25 2550 ( 150
(n ) 6) (n ) 2) (n ) 2)
DL-γ,γ-F2MTX 29.0 ( 3.4 700 ( 20 2150 ( 350
(n ) 6) (n ) 2) (n ) 2)
15.5 ( 0.5
(n ) 2)
b
24.3 ( 4.8
(n ) 2)
data are single points from two independent experiments. DL-
γ,γ-F2MTX concentration is twice that of other antifolates, since
only the L-isomer of DL-γ,γ-F2MTX is presumed to act as a
substrate.22,23
a
Average values are presented ( range for n ) 2 and ( SD for
b
n ) 6. CCRF-CEM subline resistant to MTX solely as a result of
a 25-fold increase in wild-type DHFR protein and activity.24
c CCRF-CEM subline resistant to MTX solely as a result of
decreased MTX influx (normal levels of parental DHFR present).25
dCCRF-CEM subline resistant to MTX solely as a result of
decreased polyglutamylation; this cell line has 1% of the FPGS-
specific activity (measured with MTX as the folate substrate) of
parental CCRF-CEM.26
Ta ble 3. Effect of DL-γ,γ-F2MTX on the FPGS-Catalyzed
Polyglutamylation of L-Aminopterina
[3H]Glu incorporated, pmol (%)
L-AMT, µM DL-γ,γ-F2MTX, µM
1 h
2 h
40
40
0
0
200
200
477 (100)
343 (72)
2 (<1)
959 (100)
639 (67)
0 (0)
corded on either Bruker AM-300 or AM-360 spectrometers. 19
F
a
CCRF-CEM FPGS was incubated with 0.5 mM L-[3H]Glu (8.2
NMR spectra were obtained using a General Electric AM-500
spectrometer. Chemical shifts are in parts per million down-
dpm/pmol) and L-AMT in the presence or absence of DL-γ,γ-F2MTX
for the indicated time under otherwise standard conditions and
[3H]Glu incorporation was quantitated (single data points) as
described in the Experimental Section.
field from tetramethylsilane (internal standard for H and 13C)
1
or trifluoroacetic acid (external standard for 19F). Infrared
spectra were obtained on a Nicolet 5-DX spectrometer. Mass
spectra and high-resolution mass spectra were obtained using
a Finnegan 4500 GC/MS-EICI system or a VG Analytical
system, Model 70-250S. Elemental analyses were obtained
from the Elemental Analysis Laboratory, Department of
Chemistry, University of Michigan. UV spectra were obtained
on a Beckman Model DU-7 spectrophotometer. Radioactivity
was measured with a Packard liquid scintillation analyzer,
Model 1600 TR.
Ta ble 4. Inhibition by Antifolates of Purified Human
Dihydrofolate Reductase (DHFR) and Uptake of [3H]MTXa
IC50
antifolate
AMT
L-MTX
DL-γ,γ-F2MTX
DHFR, µM
[3H]MTX uptake, µM
nd
3.0 ( 0.3
14.5 ( 1.5
8.72 ( 0.5
0.72 ( 0.04
1.53 ( 0.03
MTX was a generous gift of the National Cancer Institute
and of Immunex (Seattle, WA). Aminopterin was purchased
from Sigma Chemical Co. (St. Louis, MO). [3H]Glutamic acid
was purchased from DuPont NEN Research Products (Boston,
MA). [3H]MTX was obtained from Moravek Biochemicals Inc.
(Brea, CA). Concentrations of all solutions of MTX and
fluoroglutamate-containing analogues, including DL-γ,γ-F2-
MTX, were determined using the extinction coefficient of
MTX.28 The synthetic methods for preparing L-threo-4-fluo-
roglutamic acid29 and DL-3,3-difluoroglutamic acid10,30 were
recently reported.
a
Uptake experiments employed human leukemia (CCRF-CEM)
cells. nd ) not determined.
FMTX, and â,â-F2MTX.5,6,30 FPGS substrate activities
of these compounds are significantly different depending
on the position or number of fluorine atoms at the
glutamyl portion (â,â-F2MTX > MTX . erythro-γ-FMTX
> threo-γ-FMTX ≈ DL-γ,γ-F2MTX). The latter two
compounds display very poor substrate activity near the
limit of detection of the FPGS assay. However, it
appears from comparison of previous data5 with those
presented here that DL-γ,γ-F2MTX is probably the
poorer substrate. All of the analogues, however, retain
other biochemical properties (e.g., DHFR inhibitory
activity) almost identical to those of MTX. Regardless
of their detailed mechanism of binding to FPGS, avail-
ability of these fluorinated analogues of MTX is of great
importance for studying the role of polyglutamylation
in antifolate cytotoxicity.
N,N-Dieth yl-4-ca r beth oxy-2,2-d iflu or o-3-h yd r oxy-4-n i-
tr obu ta n a m id e (7). Diethylamine (1.32 g, 18.0 mmol) was
added to a solution of 612 (2.63 g, 11.7 mmol) and ethyl
nitroacetate (1.60 g, 11.7 mmol) in THF (15 mL) at 0 °C, and
the mixture was stirred at ambient temperature for 5 h. The
reaction mixture was cooled to 0 °C, diluted with ethyl acetate
(30 mL), and poured into 0.5 N HCl (30 mL). The organic layer
was separated, and the aqueous layer was extracted with ethyl
acetate (30 mL × 2). The combined extracts were dried over
Na2SO4 and evaporated to give 3.98 g of a yellow oil 7 as a
mixture (53:47) of diastereomers. This compound was used
without further purification: Rf 0.21 (hexane-EtOAc, 4:1); 1H
NMR (CDCl3) δ 1.1-1.4 (m, 9 H), 3.3-3.6 (m, 4 H), 5.0-5.3
(m, 2 H), 5.52 (d, J ) 6.6 Hz, 1 H) and 5.58 (d, J ) 7.6 Hz, 1
H); 19F NMR (CDCl3) major isomer δ -37.5 (dd, J ) 15.5, 300
Hz), -30.4 (dd, J ) 6.2, 300 Hz), minor isomer δ -38.1 (dd, J
) 13.9, 300 Hz), -31.0 (dd, J ) 6.2, 300 Hz).
Exp er im en ta l Section
Melting points were obtained on a Thomas-Hoover Mel-
Temp apparatus and are uncorrected. Thin layer chromatog-
raphy was performed with E. Merck silica gel 60 F-254 plates.
Column chromatography was performed with silica gel 60
(230-400 mesh). 1H NMR and 13C NMR spectra were re-
N,N-Dieth yl-3-a cetoxy-4-ca r beth oxy-2,2-d iflu or o-4-n i-
tr obu ta n a m id e (8). The crude product 7 (3.98 g) was

70 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Tsukamoto et al.
dissolved in dry dichloromethane (15 mL). To the solution at
0 °C were added acetic anhydride (1.53 g, 15.0 mmol) and three
drops of concentrated sulfuric acid, and the mixture was
stirred at ambient temperature for 12 h. The reaction mixture
was diluted with dichloromethane (30 mL) and poured into
H2O (40 mL). The organic layer was separated, and the
aqueous layer was extracted with dichloromethane (30 mL ×
2). The combined extracts were dried over MgSO4 and
evaporated to dryness to give 4.38 g of a yellow oil 8 as a
mixture (56:44) of diastereomers. This compound was used
without further purification: Rf 0.36 (hexane-EtOAc, 4:1); 1H
NMR (CDCl3) δ 1.1-1.4 (m, 9 H), 2.12 (s, 3 H), 3.2-3.5 (m, 4
H), 4.2-4.4 (m, 2 H), 5.75 (d, J ) 4.4 Hz, 1 H) and 5.84 (d, J
) 6.5 Hz, 1 H), 6.45 (dt, J ) 16.1, 6.6 Hz, 1 H) and 6.60 (ddd,
J ) 4.4, 7.8, 17.1 Hz, 1 H); 19F NMR (CDCl3) major isomer δ
-34.1 (dd, J ) 17.0, 288 Hz), -30.0 (dd, J ) 7.8, 288 Hz),
minor isomer δ -34.4 (dd, J ) 17.0, 288 Hz), -31.8 (dd, J )
7.8, 288 Hz).
N,N-Dieth yl-4-ca r beth oxy-2,2-d iflu or o-4-n itr obu ta n a -
m id e (9). The crude product 8 (750 mg) was dissolved in dry
THF (15 mL). Sodium borohydride (75 mg, 2.0 mmol) was
added to the solution at 0 °C, and the mixture was stirred at
ambient temperature for 30 min. The resulting mixture was
cooled to 0 °C, and then ethyl acetate (15 mL) and 0.5 N HCl
(15 mL) were added. The organic layer was separated, and
the aqueous layer was extracted with ethyl acetate (15 mL ×
2). The combined extracts were dried over Na2SO4 and
evaporated to dryness. The resulting oil was chromatographed
on silica gel (hexane-EtOAc, 5:1) to give a colorless oil 9 (404
mg, 1.4 mmol) in 68% yield from 6: Rf 0.30 (hexane-EtOAc,
4:1); IR (neat) 3000, 1760, 1660, 1570 cm-1; 1H NMR (CDCl3)
δ 1.17 (t, J ) 7.1 Hz, 3 H), 1.20 (t, J ) 7.1 Hz, 3 H), 1.32 (t, J
) 7.2 Hz, 3 H), 3.0-3.4 (m, 2 H), 3.39 (q, J ) 7.1 Hz, 2 H),
3.50 (tq, J ) 1.5, 7.1 Hz, 2 H), 4.32 (q, J ) 7.2 Hz, 1 H), 5.54
(dd, J ) 4.1, 8.8 Hz, 1 H); 13C NMR (CDCl3) δ 12.2, 13.8, 14.2,
35.7 (dd, J ) 24.1, 25.6 Hz), 41.6, 41.8 (t, J ) 6.1 Hz), 63.7,
82.8 (dd, J ) 4.1, 4.3 Hz), 117.6 (t, J ) 258 Hz), 161.1 (t, J )
28.0 Hz), 163.5; 19F NMR (CDCl3) δ -24.9 (ddd, J ) 11.4, 18.3,
288 Hz), -22.6 (dt, J ) 18.3, 288 Hz); MS (EI) m/e (rel
intensity) 296 (M+, 1.9), 281 (13.4), 250 (7.5), 175 (94.4), 100
(100); HRMS (EI) m/e calcd for C11H18F2N2O5 (M+) 296.1184,
found 296.1192. Anal. (C11H18F2N2O5) C, H, N.
layer was separated, and the aqueous layer was extracted with
ethyl acetate (10 mL × 2). The combined extracts were dried
over MgSO4 and evaporated. The resulting oil was purified
by silica gel column chromatography (hexane-EtOAc ) 4:1)
to give 80 mg (80% yield) of the product 10 as a yellow solid:
mp 43-45 °C; Rf 0.60 (hexane-EtOAc ) 1:1); IR (neat) 3396,
1
2979, 1763, 1748 cm-1; H NMR (CDCl3) δ 1.44 (s, 9 H), 1.51
(s, 9 H), 2.32 (dddd, J ) 9.6, 12.5, 14.6, 20.2 Hz, 1 H), 2.50
(dq, J ) 4.2, 14.6 Hz, 1 H), 3.61 (dd, J ) 4.2, 9.6 Hz, 1 H); 13
C
NMR (CDCl3) δ 27.4 (9 C), 27.7 (9 C), 38.8 (t, J ) 24.0 Hz),
50.2 (dd, J ) 3.7, 6.1 Hz), 81.8, 84.3, 115.4 (t, J ) 249 Hz),
162.8 (t, J ) 31.7 Hz), 173.3; 19F NMR (CDCl3) δ -31.7 (ddd,
J ) 13.8, 19.9, 261 Hz, 1 F), -26.6 (dt, J ) 261, 15.3 Hz, 1 F);
MS (CI/NH3) m/e (rel intensity) 296 (MH+, 100), 240 (98.0),
239 (61.3), 184 (21.2); HRMS (CI/NH3) m/e calcd for C13H23F2-
NO4H (MH+) 296.1673, found 296.1684. Anal. (C13H23F2NO4)
C, H, N.
DL-N-[4-[N′-(Ben zyloxyca r bon yl)-N′-m eth yla m in o]ben -
zoyl]-4,4-d iflu or oglu ta m ic Acid Di-ter t-bu tyl Ester (11).
To a solution of 4-[N-(benzyloxycarbonyl)-N-methylamino]-
benzoic acid (80 mg, 0.27 mmol) in dry DMF (5 mL) were added
DCC (105 mg, 0.42 mmol) and HOBt (84 mg, 0.60 mmol) at 0
°C, and the mixture was stirred at ambient temperature for 5
min. A solution of 10 (80 mg, 0.27 mmol) was then added to
that mixture at 0 °C, and the mixture was stirred at ambient
temperature for 14 h (white solid appeared). The reaction
mixture was filtered, and the solvent was removed under
reduced pressure. The resulting crude oil was diluted with
hexane-EtOAc (1:1) and again filtered. The filtrate was
evaporated and the resulting oil was purified by silica gel
chromatography (hexane-EtOAc ) 4:1) to give 149 mg (98%
yield) of the product 11 as a colorless oil: Rf 0.57 (hexane-
EtOAc ) 1:1); IR (neat) 3346, 2981, 2931, 1750, 1715, 1666
1
cm-1; H NMR (CDCl3) δ 1.43 (s, 9 H), 1.49 (s, 9 H), 2.6-2.9
(m, 2 H), 3.34 (s, 3 H), 4.85 (dt, J ) 7.3, 5.6 Hz, 1 H), 5.17 (s,
2 H), 6.81 (d, J ) 7.5 Hz, 1 H), 7.30-7.50 (m, 7 H), 7.80 (d, J
) 8.7 Hz, 2 H); 13C NMR (CDCl3) δ 27.9 (3 C), 28.1 (3 C), 36.0
(t, J ) 22.9 Hz), 37.6, 49.0, 67.9, 83.4, 85.3, 115.3 (t, J ) 253
Hz), 125.2 (2 C), 127.9 (2 C), 128.0 (2 C), 128.2, 128.7 (2 C),
131.0, 136.4, 146.4, 155.2, 162.6 (t, J ) 31.7 Hz), 166.2, 169.6;
19F NMR (CDCl3) δ -28.6 (dt, J ) 265, 16.4 Hz, 1 F), -26.9
(ddd, J ) 12.4, 17.8, 265 Hz, 1 F); MS (EI) m/e (rel intensity)
562 (M+, 19.3), 489 (2.7), 433 (17.9), 405 (35.5), 268 (61.6), 91
(100); HRMS (EI) m/e calcd for C29H36F2N2O7 (M+) 562.2490,
found 562.2501. Anal. (C29H36F2N2O7) C, H, N.
DL-4,4-Diflu or oglu ta m ic Acid (1). Raney Ni31 (wet vol-
ume of 1 mL) was added to a solution of 9 (453 mg, 1.5 mmol)
in ethanol (5 mL), and the mixture was shaken under
hydrogen (40 psi) at ambient temperature for 12 h. The
catalyst was removed by filtration and washed with ethyl
acetate. The combined filtrate and washings were evaporated
to dryness and dissolved in 12 N HCl (10 mL). The mixture
was heated at 100 °C for 12 h and evaporated to dryness. The
resulting yellow solid was dissolved in H2O (3 mL) and
chromatographed on anion-exchange resin (AG1 X8, BioRad,
20 mL wet volume) with H2O and then 4 N AcOH as the
eluants. Fractions containing the product were lyophilized,
triturated with dry ether, and filtered to give 152 mg (54%
yield) of the product 1 as a white solid. The sample for
elemental analysis and bioassay was prepared by recrystalli-
zation from H2O: Rf 0.78 (EtOH-H2O, 7:3); mp 175-7 °C dec;
IR (KBr) 3424, 3143, 1708 cm-1; 1H NMR (D2O) δ 2.71 (dddd,
J ) 8.5, 13.1, 20.2 Hz, 1 H), 2.89 (ddt, J ) 13.8, 21.2, 15.5 Hz,
1 H), 4.38 (dd, J ) 3.7, 8.5 Hz, 1 H); 13C NMR (D2O) δ 34.9 (t,
J ) 26.4 Hz), 48.8, 116.3 (t, J ) 251 Hz), 168.3, 170.9; 19F
DL-N-[4-(Met h yla m in o)b en zoyl]-4,4-d iflu or oglu t a m ic
Acid Di-ter t-bu tyl Ester (12). Palladium hydroxide on
carbon (10 mg) was added to a solution of 11 (124 mg, 0.22
mmol) in dry ethanol (4 mL), and the mixture was shaken
under hydrogen (30 psi) for 8 h (Parr hydrogenator). The
mixture was filtered, and the filtrate was evaporated to
dryness. The crude product was purified by silica gel chro-
matography (hexane-EtOAc ) 4:1) to give pure 12 (64 mg,
0.15 mmol) in 69% yield: Rf 0.50 (hexane-EtOAc ) 1:1); IR
(neat) 3423, 3360, 2973, 2938, 1750, 1645; 1H NMR (CDCl3) δ
1.41 (s, 9 H), 1.47 (s, 9 H), 2.50-2.90 (m, 2 H), 2.83 (s, 3 H),
4.05-4.50 (br, 1 H), 4.84 (dt, J ) 7.2, 5.6 Hz, 1 H), 6.53 (d, J
) 8.3 Hz, 2 H), 6.68 (d, J ) 7.4 Hz, 1 H), 7.62 (d, J ) 8.3 Hz,
2 H); 13C NMR (CDCl3) δ 27.9 (3 C), 28.1 (3 C), 30.4, 36.2 (t, J
) 22.9 Hz), 48.7, 83.1, 85.1, 111.6 (2 C), 115.4 (t, J ) 252 Hz),
121.9, 129.1 (2 C), 152.4, 166.0 (t, J ) 32.6), 167.0, 170.2; 19
F
NMR (CDCl3) δ -28.6 (dt, J ) 265, 17.2 Hz), -26.5 (ddd, J )
265, 13.6, 17.0 Hz); MS (EI) m/e (rel intensity) 428 (M+, 10.3),
299 (9.8), 271 (9.6), 149 (8.8), 134 (100); HRMS (EI) m/e calcd
for C21H30F2N2O5 (M+) 428.2123, found 428.2141. Anal.
(C21H30F2N2O5) C, H, N.
NMR (D2O) δ -29.6 (dd, J ) 15.1, 21.2 Hz), -28.4 (dd, J )
+
12.2, 21.4 Hz); MS (CI/NH3) m/e (rel intensity) 183 (MNH4
-
H2O, 100), 165 (9), 147 (13), 136 (21), 119 (32), 101 (36), 84
(9). Anal. (C5H7F2NO4) C, H, N.
DL-4,4-Diflu or oglu ta m ic Acid Di-ter t-bu tyl Ester (10).
A suspension of 1 (63 mg, 0.34 mmol) in dry dichloromethane
(1.5 mL) was placed in a pressure bottle. The bottle was cooled
to -30 °C, and concentrated H2SO4 (30 µL) was added.
Isobutylene (1.5 mL) was then condensed within the bottle at
the same temperature. The bottle was closed, and the mixture
was stirred at ambient temperature for 5 days as the reaction
mixture became homogeneous. The reaction solution was
cooled to -30 °C, diluted with ethyl acetate (10 mL), and
poured into 20% aqueous K2CO3 solution (10 mL). The organic
DL-γ,γ-Diflu or om eth otr exa te (2). 6-(Bromomethyl)-2,4-
pteridinediamine hydrobromide15 (101 mg, 0.30 mmol) was
added to a solution of 12 (64 mg, 0.15 mmol) in dry DMAC
(1.5 mL) at 0 °C, and the mixture was stirred at 55 °C for 6 h.
After additional stirring (18 h) at ambient temperature, the
mixture was evaporated to dryness and dissolved in trifluo-
roacetic acid (5 mL). The mixture was stirred at ambient
temperature for 40 min and evaporated to dryness. The crude
product was purified by DEAE-cellulose chromatography
(DE53, Whatman) [0.1-0.9 M triethylammonium carbonate

Fluorinated Methotrexate Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 71
(4) McGuire, J . J .; Haile, W. H.; Bey, P.; Coward, J . K. DL-3,3-
Difluoroglutamate: An enhancer of folylpolyglutamate elonga-
tion. J . Biol. Chem. 1990, 265, 14073-14079.
(5) McGuire, J . J .; Graber, M.; Licato, N.; Vincenz, C.; Coward, J .
K.; Nimec, Z.; Galivan, J . Biochemical and growth inhibitory
effects of the erythro and threo isomers of γ-fluoromethotrexate,
a methotrexate analogue defective in polyglutamylation. Cancer
Res. 1989, 49, 4517-4525.
(6) McGuire, J . J .; Hart, B. P.; Haile, W. H.; Rhee, M.; Galivan, J .;
Coward, J . K. DL-â,â-Difluoroglutamic acid mediates position-
dependent enhancement or termination of pteroylpoly (γ-
glutamate) synthesis catalyzed by folylpolyglutamate syn-
thetase. Arch. Biochem. Biophys. 1995, 321, 319-328.
(7) Coward, J . K.; McGuire, J . J .; Galivan, J . In Selective Fluorina-
tion in Organic and Bioorganic Chemistry; Welch, J . T., Ed.;
American Chemical Society: Washington, D.C., 1991; pp 196-
204.
(TEAB) buffer (pH ) 7.5) gradient] to afford 60 mg (61% yield
based on absorption spectra) of γ,γ-difluoromethotrexate 2 as
a triethylammonium salt: 1H NMR (D2O) δ 2.5-2.7 (m, 2 H),
2.95 (s, 3 H), 4.47 (s, 2 H), 4.51 (dd, J ) 4.1, 8.7 Hz, 1 H), 6.55
(d, J ) 8.7 Hz, 2 H), 7.49 (d, J ) 8.7 Hz, 2 H), 8.34 (s, 1 H);
13C NMR (D2O) δ 36.6 (t, J ) 23.6 Hz), 38.9, 50.6, 55.0, 111.9
(2 C), 117.6 (t, J ) 250 Hz), 120.8, 122.3, 129.1, 148.6, 149.4,
151.8, 153.0, 161.8, 163.0, 169.2, 170.5, 178.0; 19F NMR (D2O)
δ -28.5 (ddd, J ) 14.4, 19.9, 244 Hz), -27.7 (ddd, J ) 13.5,
18.5, 244 Hz); UV λmax (pH ) 13) 258, 302, 371; MS (FAB+)
m/e (rel intensity) 491 (MH+, 10.5), 308 (6.1), 175 (10.2), 118
(30.4), 102 (100); HRMS (FAB+) m/e calcd for C20H20F2N8O5H
(MH+) 491.1603, found 491.1607. Reversed-phase HPLC17 tR
) 13.2 min; anion-exchange HPLC18 tR ) 12.2 min.
F P GS Assa ys. CCRF-CEM FPGS was partially purified
as previously described.27,32 Unless otherwise indicated, FPGS
was assayed as previously described.16,18 Standard assay
mixtures (250 µL) which contained 100 mM Tris-HCl, pH 8.85
(at room temperature), 10 mM MgCl2, 5 mM ATP, 100 mM
BME, 20 mM KCl, glutamate or fluorinated glutamates, and
appropriate pteroyl substrates were initiated by addition of
CCRF-CEM FPGS (1170 units) and incubated at 37 °C for the
indicated time. After the addition of ice-cold 5 mM L-Glu (1
mL, pH ) 7.5) containing 25 mM 2-mercaptoethanol, the assay
mixtures were loaded onto DEAE-cellulose (DE52, Whatman)
minicolumns equilibrated with 10 mM Tris-HCl, pH 7.5,
containing 110 mM NaCl (conductivity ) 11.5 mS/cm). The
columns were washed with the same buffer to separate
unreacted [3H]glutamic acid from that incorporated into
pteroyl substrates. Minicolumns were then eluted with 0.1 N
HCl (3 mL) to obtain polyglutamate products. For HPLC
analysis, 50 mM phosphoric acid was used for elution instead
of 0.1 N HCl. One unit of FPGS catalyzes incorporation of 1
pmol of [3H]Glu/h.
(8) Kitagawa, O.; Hashimoto, A.; Kobayashi, Y.; Taguchi, T. Michael
addition of 2,2-difluoroketene silyl acetal. Preparation of 4,4-
difluoroglutamic acid and 5,5-difluorolysine. Chem. Lett. 1990,
1307-1310.
(9) Yang, Z.-Y.; Burton, D. J . A new approach to R,R-difluoro-
functionalized esters. J . Org. Chem. 1991, 56, 5125-5132.
(10) Hart, B. P.; Coward, J . K. The synthesis of DL-3,3-difluoro-
glutamic acid from a 3-oxoprolinol derivative. Tetrahedron Lett.
1993, 34, 4917-4920.
(11) Sufrin, J . R.; Balasubramanian, T. M.; Vora, C. M.; Marshall,
G. R. Synthetic approaches to peptide analogues containing 4,4-
difluoro-L-proline and 4-keto-L-proline. Int. J . Pept. Protein Res.
1982, 20, 438-442.
(12) Tsukamoto, T.; Kitazume, T. Preparation and reaction of diflu-
orinated malonaldehydic acid derivatives: A new route to
functionalized R,R-difluorinated esters and amides. J . Chem.
Soc., Perkin Trans. 1 1993, 1177-1181.
(13) Martynov, V. F.; Grebenkina, V. M. Fluorine-containing carbonyl
compounds in the knoevenagel reaction. J . Gen. Chem. USSR
1968, 38, 999-1001.
(14) Wollenberg, R. H.; Miller, S. J . Nitroalkane synthesis.
A
convenient method for aldehyde reductive nitromethylation.
Tetrahedron Lett. 1978, 35, 3219-3222.
(15) Piper, J . R.; Montgomery, J . A. Preparation of 6-(bromomethyl)-
2,4-pteridinediamine hydrobromide and its use in improved
syntheses of methotrexate and related compounds. J . Org. Chem.
1977, 42, 208-211.
(16) McGuire, J . J .; Hsieh, P.; Coward, J . K.; Bertino, J . R. Enzymatic
synthesis of folylpolyglutamates. Characterization of the reaction
and its products. J . Biol. Chem. 1980, 255, 5776-5788.
(17) Nimec, Z.; Galivan, J . Regulatory aspects of the glutamylation
of methotrexate in cultured hepatoma cells. Arch. Biochem.
Biophys. 1983, 226, 671-680.
(18) McGuire, J . J .; Hsieh, P.; Coward, J . K.; Bertino, J . R. In vitro
methotrexate polyglutamate synthesis by rat liver folylpoly-
glutamate synthetase and inhibition by bromosulfophthalein.
Adv. Exp. Med. Biol. 1983, 163, 199-214.
(19) Cichowicz, D. J .; Cook, J . D.; George, S.; Shane, B. In Proceedings
of the Second Workshop on Folyl and Antifolylpolyglutamates;
Goldman, I. D., Ed.; Praeger: New York, 1985; pp 7-13.
(20) George, S.; Cichowicz, D. J .; Shane, B. Mammalian folylpoly-γ-
glutamate synthetase. 3. Specificity for folate analogues. Bio-
chemistry 1987, 26, 522-529.
(21) McGuire, J . J .; Bolanowska, W. E.; Piper, J . R. Structural
specificity of inhibition of human folylpolyglutamate synthetase
by ornithine-containing folate analogs. Biochem. Pharmacol.
1988, 37, 3931-3939.
(22) Cramer, S. M.; Schornagel, J . H.; Kalghatgi, K. K.; Bertino, J .
R.; Horvath, C. Occurrence and significance of D-methotrexate
as a contaminant of commercial methotrexate. Cancer Res. 1984,
44, 1843-1846.
HP LC An a lysis of F P GS Rea ction P r od u cts.16-18 The
50 mM phosphoric acid elution of the FPGS assay columns
was filtered, and the filtrate injected into a C18 reversed-phase
column (0.46 × 25 cm, Vydac 218TP54) previously equilibrated
with 0.1 M sodium acetate (pH ) 5.5) containing 4% acetoni-
trile. The column was washed isocratically with this buffer
at 1 mL/min for 10 min and then with a linear gradient from
4 to 12.4% acetonitrile. Fractions were collected at a rate of
1 min/fraction, and radioactivity in each fraction was quan-
titated by liquid scintillation counting.
DHF R In h ibition . CCRF-CEM DHFR was partially puri-
fied and assayed at 37 °C as described previously.27 Standard
assays contained 100 mM Tris-HCl, pH 7.0, 150 mM KCl, 20
µM dihydrofolate, 20 mM 2-mercaptoethanol, and 50 µM
NADPH. Inhibitory potency was measured by adding increas-
ing concentrations of antifolate to standard DHFR assays and
measuring the remaining activity. The IC50 values for DHFR
inhibition were determined graphically from plots of net
activity versus antifolate concentration.
Cell Gr ow th In h ibition . Inhibition of the growth of
CCRF-CEM cells and its MTX-resistant sublines in continuous
(120 h) drug exposure was measured as previously described.26
EC50 values were determined graphically from plots of per-
centage growth relative to untreated control growth versus the
logarithm of drug concentration.
(23) McGuire, J . J .; Bolanowska, W. E.; Coward, J . K.; Sherwood, R.
F.; Russell, C. A.; Felschow, D. M. Biochemical and biological
properties of methotrexate analogs containing D-glutamic acid
or D-erythro,threo-4-fluoroglutamic acid. Biochem. Pharmacol.
1991, 42, 2400-2403.
(24) Mini, E.; Srimatkandada, S.; Medina, W. D.; Moroson, B. A.;
Carman, M. D.; Bertino, J . R. Molecular and karyological
analysis of methotrexate-resistant and -sensitive human leuke-
mic CCRF-CEM cells. Cancer Res. 1985, 45, 317-325.
(25) Rosowsky, A.; Lazaraus, H.; Yuan, G. C.; Beltz, W. R.; Mangini,
L.; Abelson, H. T.; Modest, E. J .; Frei, E. I. Effects of methotr-
exate esters and other lipophilic antifolates on methotrexate-
resistant human leukemic lymphoblasts. Biochem. Pharmacol.
1980, 29, 648-652.
Ack n ow led gm en t. This work was supported by
research grants CA28097 (J .K.C.), CA13038 (J .J .M.),
and CA16056 (J .J .M.) from the National Cancer Insti-
tute. We gratefully acknowledge the technical as-
sistance of Holly and Robert Bogaudo, Wanda Bolanows-
ka, J ill Galvin, and William Haile.
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