Synthesis and biological activity of folic acid and methotrexate analogues containing L-threo-(2S,4S)-4-fluoroglutamic acid and DL-3,3-difluoroglutamic acid

Article abstract of DOI:10.1021/jm950515e

The stereospecific syntheses of L-threo-γ-fluoromethotrexate (1t) and L- threo-γ-fluorofolic acid (3t) are reported. Compounds 1t and 3t have no substrate activity with folylpoly-γ-glutamate synthetase isolated from CCRF- CEM human leukemia cells, and compound It inhibits human dihydrofolate reductase at similar levels as methotrexate. The synthesis of DL-3,3- difluoro-glutamic acid (6) and its incorporation into DL-β,β-difluorofolic acid (4) are also reported. Compound 4 acts as a better substrate for human CCRF-CEM folylpoly-γ-glutamate synthetase than folic acid (V/K = ca. 7-fold greater). Thus, replacement of the glutamate moiety of methotrexate and folic acid with 4-fluoroglutamic acid and 3,3-difluoroglutamic acid results in folates and antifolates with altered polyglutamylation activity.

Full text of DOI:10.1021/jm950515e

56
J . Med. Chem. 1996, 39, 56-65
Syn th esis a n d Biologica l Activity of F olic Acid a n d Meth otr exa te An a logu es
Con ta in in g L-th r eo-(2S,4S)-4-F lu or oglu ta m ic Acid a n d DL-3,3-Diflu or oglu ta m ic
Acid
Barry P. Hart,† William H. Haile,‡ Nicholas J . Licato,† Wanda E. Bolanowska,‡ J ohn J . McGuire,‡ and
J ames K. Coward*,†
Departments of Chemistry and Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, and
Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263
Received J uly 14, 1995X
The stereospecific syntheses of L-threo-γ-fluoromethotrexate (1t) and L-threo-γ-fluorofolic acid
(3t) are reported. Compounds 1t and 3t have no substrate activity with folylpoly-γ-glutamate
synthetase isolated from CCRF-CEM human leukemia cells, and compound 1t inhibits human
dihydrofolate reductase at similar levels as methotrexate. The synthesis of DL-3,3-difluoro-
glutamic acid (6) and its incorporation into DL-â,â-difluorofolic acid (4) are also reported.
Compound 4 acts as a better substrate for human CCRF-CEM folylpoly-γ-glutamate synthetase
than folic acid (V/K ) ca. 7-fold greater). Thus, replacement of the glutamate moiety of
methotrexate and folic acid with 4-fluoroglutamic acid and 3,3-difluoroglutamic acid results
in folates and antifolates with altered polyglutamylation activity.
In tr od u ction
The biosynthesis of poly-γ-glutamate “conjugates” is
an important process in one-carbon biochemistry involv-
ing folate-dependent enzymes and in the cytotoxicity
mediated by a variety of folate analogues such as
methotrexate (MTX).1,2 Previous publications from our
laboratories have documented the use of fluorinated
glutamic acids3,4 and the corresponding fluoroglutamate-
containing analogues of MTX5,6 as informative probes
to investigate the polyglutamylation process. Specifi-
cally, we have reported on the biological activity of
γ-fluoromethotrexate (γ-FMTX, 1)5,7,8 and, more re-
cently, â,â-difluoromethotrexate (â,â-F2MTX, 2).6,9 The
well-documented effects of the reduced folate, 5-formyltet-
rahydrofolic acid (leucovorin) in the rescue of cells
treated with “high-dose” MTX chemotherapy10 and the
potentiation of 5-fluorouracil cytotoxicity11 suggests that
fluoroglutamate-containing analogues of folic acid and
leucovorin might serve as useful probes of the role of
polyglutamylation in folic acid and/or leucovorin bio-
chemistry and pharmacology in intact mammalian cells.
the complex biological results when using mixtures of
stereoisomers. Therefore, we have undertaken the
With such cellular studies in mind, we have initiated a
synthetic program to obtain folic acid (FA) analogues
containing various fluoroglutamic acids (e.g., 3, 4) to
complement our ongoing research on fluoroglutamate-
containing analogues of MTX.
We have previously used racemic materials in our
research because of the paucity of stereospecific syn-
theses of the requisite amino acids, e.g., 5 and 6. Thus,
4-fluoroglutamic acid, 5, as a mixture of all four possible
isomers, is available via addition of the carbanion of
diethyl fluoromalonate to methylacetamido acrylate.5,12
This stereochemical heterogeneity of the precursor
amino acids leads to stereochemical heterogeneity in the
fluoroglutamate-containing drugs of interest in our
research (Table 1). Although in the case of MTX it has
been demonstrated that the D-isomer is biologically
inactive,13,14 it is not always a simple task to sort out
stereospecific synthesis of fluorinated glutamic acids for
use in the synthesis of several folate and MTX ana-
logues in order to investigate the role of polyglutam-
ylation in the biochemistry and pharmacology of the
parent glutamate-containing compounds. Our initial
research with racemic 4-fluoroglutamic acid, 5et (rac),
demonstrated that the L-threo (2S,4S) isomer is pref-
erentially used as a folylpoly-γ-glutamate synthetase
(FPGS, EC 6.3.2.17) chain-terminating substrate.3 On
the basis of these observations, our initial synthetic
target was L-threo-4-fluoroglutamic acid, 5t,15 and in
this paper we report on the synthesis of L-threo-γ-
† University of Michigan.
‡ Roswell Park Cancer Institute.
X Abstract published in Advance ACS Abstracts, December 1, 1995.
0022-2623/96/1839-0056$12.00/0 © 1996 American Chemical Society

Fluorinated Folic Acid and Methotrexate Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 57
These compounds were obtained through either C9-N10
bond formation or pteroyl amide bond formation. The
amide bond formation strategy is direct but does not
allow for the facile incorporation of glutamic acid
analogues into other modified pteridine ring systems.
In previous work from this laboratory, iBCF- and DEPC-
Ta ble 1. Definition of Stereochemical Parameters: 4-FGlu (5),
γ-FMTX (1), and γ-FFA (3)
mediated coupling reactions between 2,4-diamino-N10
-
methylpteroic acid and suitably protected γ-(fluoro)-
glutamyl peptides resulted in only low to moderate
yields of coupled product.20,21 The synthesis of L-t-γ-
FMTX (1t) was achieved by the coupling of 2,4-diamino-
6-(bromomethyl)pteridine (10) with 9t. In contrast, the
fluorinated folates (3t and 4) were obtained by the
coupling of N10-Tfa-pteroic acid (11), readily available
from folic acid by enzyme-catalyzed hydrolysis,22 with
the corresponding fluoroglutamate di-tert-butyl ester (7t
and 18).
Syn th esis of 4-Flu or oglu tam ate-Con tain in g An a-
logu es of MTX a n d F olic Acid . Initially, the synthe-
sis of DL-4(RS)-fluoroglutamic acid, 5et (rac), was
achieved by the method of Buchanan,12 and the dia-
stereomers of 7et (rac) were separated by silica gel
column chromatography,5 thereby providing 7e (rac) and
7t (rac). Analogues of MTX containing the two racemic
diastereomers of 5, 5e (rac), and 5t (rac), were synthe-
sized and studied extensively.5,8,14 More recently, the
method of Hudlicky19,23 has been used to effect the
stereospecific synthesis of L-4(S)-fluoroglutamic acid, 5t,
from protected L-4(R)-hydroxyproline. In the present
work, the synthesis of L-t-γ-FMTX was achieved in 50%
overall yield from 5t (Scheme 1). The coupling of 7t,
obtained from 5t in 68% yield, with N-Me,N-Cbz-PABA
provided 8t in 97% yield followed by hydrogenolysis to
afford 9t in near quantitative yield. The coupling of
6-(bromomethyl)pteridine 10 with 9t followed by TFA-
mediated deprotection and purification by DEAE-cel-
lulose chromatography provided 1t.
a
Throughout the text, including most notably the Experimental
Section and Scheme 1, a number preceding the abbreviation
indicates that the stereochemistry of the numbered compound is
as indicated in this Table; e.g., 5t is L-threo-4-fluoroglutamic acid
where the absolute stereochemistry is 2S,4S. The use of the
abbreviation rac indicates that the numbered compound is a
racemic mixture; e.g., 5t (rac) is DL-threo-4-fluoroglutamic acid
with absolute stereochemistry of 2S,4S; 2R,4R. In the glutamyl
portions of MTX or folate derivatives, the numbers 2 and 4 are
replaced by R and γ, respectively, in order to distinguish the
positions on the appended glutamate from the positions on the
pteridine or p-aminobenzoyl moieties.
fluoromethotrexate (L-t-γ-FMTX, 1t) containing only the
L-threo (2S,4S) diastereomer. In addition, we report the
synthesis of L-threo-γ-fluorofolic acid (L-t-γ-FFA, 3t)
containing the same stereochemically pure amino acid.
As expected, the 4-fluoroglutamate-containing ana-
logues, 1t and 3t, are extremely poor substrates for
FPGS.
Racemic 3,3-difluoroglutamic acid 6 can be obtained
by a multistep synthesis from a blocked 3-oxoprolinol.16
On the basis of our recent report of the synthesis and
biochemical properties of DL-3,3-difluoroglutamate-
containing MTX analogue, â,â-difluoromethotrexate
(â,â-F2MTX, 2),6,9 we have also synthesized the corre-
sponding folic acid analogue, 4. Consistent with obser-
vations on the â,â-F2MTX analogue, 2, the correspond-
ing folate, â,â-difluorofolic acid (â,â-F2FA, 4), is an
excellent FPGS substrate with V/K ) ca. 7-fold greater
than that of folic acid. Thus, the initial biochemical
results support the hypothesis17 that control of intrac-
ellular polyglutamylation of both folic acid and MTX can
be effected by the judicious use of regiospecifically
fluorinated glutamates incorporated into analogues of
the parent vitamin or drug.
Previous studies comparing the effects of DL-t-γ-FMTX
(1t (rac)) and D-t-γ-FMTX led to the conclusion that the
biological activity of DL-t-γ-FMTX resulted almost ex-
clusively from the L-isomer; however direct evidence to
support this conclusion was lacking.14 Accordingly, L-t-
γ-FMTX (1t) has now been synthesized as described
above (Scheme 1). In order to determine whether
racemization occurred during synthesis, the isomeric
composition of the amino acid moiety was determined
by limit digestion with CPG2, an enzyme which is
specific for hydrolysis of L-amino acids contained in
folate or antifolate structures.14,24 Hydrolysis by CPG2
(0.54 unit) of 10-100 nmol of L-MTX, as described in
the Experimental Section, was complete and quantita-
tive in <1 min. In a parallel experiment, L-t-γ-FMTX
was hydrolyzed to 97.5% of completion at a concentra-
tion equivalent to that of L-MTX. In contrast, DL-MTX
and DL-t-γ-FMTX were hydrolyzed to only 49.4% and
48%, respectively, the extent of L-MTX under identical
conditions. These results indicate that little, if any,
racemization occurred during synthesis and that the
product obtained, 1t, is the L-isomer.
Ch em istr y
The synthesis of MTX analogues with various replace-
ments for the glutamate moiety has been investigated
extensively.18 Recently, several fluorinated analogues
of glutamic acid, namely the four possible stereoisomers
of 4-fluoroglutamic acid, 5,19 and DL-3,3-difluoroglutamic
acid, 6,16 have been prepared via appropriate fluorinated
proline derivatives as synthetic intermediates. In the
present work, the synthesis of fluorinated glutamic acid-
containing analogues of MTX and folic acid is reported.
The synthesis of folate analogues, 3et (rac), 3e (rac),
and 3t (rac) was achieved in 31-42% yield by coupling
11, activated by isobutyl chloroformate, with the ap-
propriate di-tert-butyl ester (7et (rac), 7e (rac), or 7t
(rac)). Attempts to improve the overall yield of this
coupling and deprotection sequence involved the exami-

58 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Hart et al.
Sch em e 1
nation of several alternate coupling agents, e.g., iBCF,
DEPC, BOP, DCC/HOBt. Ultimately, the DCC/HOBt-
mediated coupling of 11 and 7t, derived from 5t,
followed by deprotection provided 3t in 56% overall yield
(Scheme 1).
Syn th esis of th e 3,3-Diflu or oglu ta m a te-Con ta in -
in g An a logu e of F olic Acid . DL-3,3-Difluoroglutamic
acid 6 was synthesized from 6-hydroxy-1-aza-3-oxa-
bicyclo[3.3.0]octan-2-one (12), a bicyclic prolinol deriva-
tive (Scheme 2), as described briefly in a previous
communication.16 The oxidation of 12 under conditions
reported by Swern25 provided 13 in 73% yield. The
(diethylamido)sulfur trifluoride (DAST)-mediated fluo-
rination of ketone 13 to the geminal difluoromethylene
compound 14 proceeded in 64% yield. Hydrolysis and
Boc protection of 14 provided 15 in 83% yield. The
alcohol 15 was readily converted to the methyl ester,
16, by sequential oxidation to the acid and esterification.
Compound 16 proved to be extremely resistant to RuO4-
mediated oxidation to lactam 17. In contrast to the
facile oxidation of appropriately protected proline de-
rivatives26 or a somewhat slower rate of oxidation of
4-fluoroproline derivatives,19,27 the conversion of 16 to
17 required 288 h of stirring at room temperature. The
reaction was monitored by NMR spectroscopy due to the
instability of 17 on silica gel TLC. In the course of
investigating the general feasibility of this oxidative
process, several isomeric fluoroprolines were subjected
Sch em e 2

Fluorinated Folic Acid and Methotrexate Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 59
Sch em e 3
F igu r e 1. Substrate activity of MTX (O) and L-t-FMTX (1t,
4) with CCRF-CEM human leukemia cell FPGS.
to the action of RuO4 under a variety of conditions.27
The overall effect of fluorine substitution is to retard
the oxidation reaction. The magnitude of this decrease
in rate is dependent on the location and extent of
fluorine substitution relative to the site of oxidation.27
DL-3,3-Difluoroglutamic acid 6 was obtained by the
hydrolysis of crude 17 followed by anion-exchange
chromatography and crystallization. The free acid 6
was converted to its di tert-butyl ester, 18, by acid-
catalyzed reaction with isobutylene. The EDC/HOBt-
mediated coupling of 18 and 11 followed by TFA and
piperidine deprotection provided 4 in 35% overall yield
for three steps (Scheme 3).
F igu r e 2. Substrate activity of indicated folates and 7,8-
dihydrofolates with rat liver FPGS: (A) L-PteGlu (b), DL-e,t-
PteFGlu (0), DL-e-PteFGlu (1), and DL-t-PteFGlu (3); (B)
L-H2PteGlu (b), DL-e,t-H2PteFGlu (0), DL-e-H2PteFGlu (1), and
DL-t-H2PteFGlu (3).
Bioch em istr y
Su bstr a te Activity of 4-F lu or oglu ta m a te-Con -
tain in g MTX an d Folate An alogu es with Folylpoly-
glu ta m a te Syn th eta se. As expected from our earlier
work with diastereomeric mixtures of γ-FMTX,5 the
stereochemically pure L-threo isomer (RS,γS) of γ-FMTX
(1t) is not a substrate for human FPGS (Figure 1) at
concentrations up to 240 µM. With the folate analogues,
the mixture of racemic erythro, threo diastereomers (3e,t
(rac)) and the individual racemates (3e (rac), 3t (rac))
were each compared to PteGlu as substrates for rat liver
FPGS (Figure 2A). All analogues were extremely poor
substrates relative to PteGlu. Similar results were
obtained with human FPGS for the diastereomeric
mixtures, 3e,t (rac), 3e (rac), and 3t (rac) (data not
shown) and for the stereochemically homogeneous ana-
logue, 3t (Figure 3). Since reduced folates are better
FPGS substrates than the fully oxidized species,1
F igu r e 3. Substrate activity of L-PteGlu (O), L-t-PteFGlu (3t,
4), and DL-Pte(3,3-F2Glu) (4, 3) with CCRF-CEM human
leukemia cell FPGS.
the 7,8-dihydro forms of the racemic mixed erythro,threo
diastereomers and the individual racemates were also
each tested for rat liver FPGS substrate activity and
compared to H2PteGlu (Figure 2B). H2PteGlu is a much
more active substrate (ca. 46-fold higher V/K) than
PteGlu for rat liver FPGS. Slight activity of the dihydro
forms of the analogues was noted; DL-e-H2PteFGlu (V/K
) 8.6 vs 232 for H2PteGlu) was the most active of the
two diastereomers, but it reached only 13% of the

60 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Hart et al.
Ta ble 2. Inhibition of Dihydrofolate Reductase Isolated from
CCRF-CEM Human Leukemia Cells by
maximal activity of the natural substrate. Similar low
substrate activity of the dihydro derivatives was noted
with human FPGS (data not shown).
Fluoroglutamate-Containing Analogues of Methotrexatea
IC50, nM
The analogues were also tested for their ability to
inhibit human CCRF-CEM FPGS activity using 30 µM
aminopterin (AMT) as the substrate. Oxidized fluoro-
folates and the 7,8-dihydro forms are weak inhibitors
of FPGS. Thus, the oxidized forms, 3e,t (rac) and 3t
(rac) with IC50 > 200 µM and 3e (rac) with IC50 > 160
µM), can be compared to the reduced forms, DL-e,t-H2-
PteFGlu (59% inhibition by 100 µM) and DL-t-H2-
PteFGlu (58% inhibition by 100 µM). Quantitative
evaluation of the inhibition data is complicated by the
weak substrate activity of the FGlu-containing ana-
logues; it is clear, however, that the poor substrate
activity reflects poor binding to the enzyme. These
analogues do not bind tightly and also fail to participate
in catalysis.
compound
L-MTX
experiment 1
experiment 2
0.72 ( 0.07
1.18 ( 0.08
0.84 ( 0.10
1.35 ( 0.05
0.60 ( 0.03
1.40 ( 0.06
DL-MTX
L-t-γ-FMTX (1t)
DL-t-γ-FMTX
DL-â,â-F2MTX (2)
1.34 ( 0.03
a
Average values are presented ( range (n ) 2).
strates; this lack of stereospecificity may also be re-
flected in the lower value of Vmax observed.
Activity of F lu or in a ted An a logu es of MTX a s
In h ibitor s of Dih yd r ofola te Red u cta se. Studies of
the inhibition of isolated DHFR by L-MTX, DL-MTX, L-t-
γ-FMTX, DL-t-γ-FMTX, and DL-â,â-F2MTX are sum-
marized by the data of Table 2. As expected, the
racemic compounds are half as potent as the L-isomers
as DHFR inhibitors, consistent with the conclusion that
only the L-isomer of glutamic acid binds to DHFR.13
Su bstr a te Activity of th e 3,3-Diflu or oglu ta m a te-
Con ta in in g F ola te An a logu e w ith F olylp olyglu ta -
m a te Syn th eta se. The activity of â,â-F2FA (4) as a
substrate for human FPGS was studied, and 4 was
found to be a better substrate than PteGlu (Figure 3).
This increase in the catalytic efficiency for the L-isomer
of â,â-F2FA over PteGlu (V/K ) 0.176 vs 0.024) is
consistent with results comparing â,â-F2MTX to MTX.6
These data provide evidence that the replacement of the
glutamate potion of folates and antifolates with 3,3-F2-
Glu will provide a “generic” means of increasing the
polyglutamylation activity of these classes of com-
pounds. However, this result is complicated by the fact
that the FPGS-catalyzed polyglutamylation of â,â-F2-
FA and â,â-F2MTX terminates after the addition of a
single glutamate.6 The resulting dipeptides are not
substrates for FPGS. Further studies are underway to
better understand the unusual FPGS activities of â,â-
F2FA and â,â-F2MTX.
Activity of Red u ced F or m s of F lu or ofola tes a s
Su bstr a tes for Dih yd r ofola te Red u cta se. DL-e,t-γ-
FFA (3e,t (rac)), DL-e-γ-FFA (3e (rac)), and DL-t-γ-FFA
(3t (rac)) were reduced to the 7,8-dihydro form, and each
was tested in kinetic studies with CCRF-CEM human
leukemia cell DHFR. Inhibition by MTX, a tight-
binding inhibitor of DHFR,28 was similar (50% inhibi-
tion achieved at 0.4 nM) whether H2PteGlu or DL-e,t-
H2PteFGlu was the competing substrate, regardless of
substrate concn in the range of 1-20 µM. The Km for
NADPH, the cosubstrate for DHFR, was 3.8 ( 0.5 µM
(n ) 2) or 3.0 ( 0.3 µM (n ) 2) when determined in the
presence of 3 µM H2PteGlu or DL-e,t-H2PteFGlu, respec-
tively; thus the Km for NADPH is unchanged. The
standard spectrophotometric assay was not sensitive
enough to determine exact Km values for H2PteGlu and
DL-e,t-H2PteFGlu, but both were <4 µM, while the
apparent Vmax values were identical. A second attempt
using a more sensitive spectrophotometer and deter-
mining the Km from a single complete reaction progress
curve29 led to the conclusion that the Km values for H2-
PteGlu, DL-e,t-H2PteFGlu, DL-e-H2PteFGlu, and DL-t-H2-
PteFGlu were each <1 µM.
Con clu sion
The stereospecific synthesis of (2S,4S)-4-fluoroglutam-
ic acid and its incorporation into methotrexate and folic
acid analogues results in (RS,γS)-γ-fluoromethotrexate,
1t, and (RS,γS)-γ-fluorofolic acid, 3t, for use in studying
the role of poly-γ-glutamates in antifolate cytotoxicity
and folate-dependent one-carbon biochemistry, respec-
tively. Compound 1t was found to be essentially inac-
tive as a substrate for FPGS but is a potent inhibitor of
DHFR with IC50 values comparable to those of L-MTX
(Table 2).
The synthesis of 3 has been described previously in
the literature,30 but the reported synthesis led to a
mixture of erythro and threo isomers in very low yield
and no biological data were reported. The synthetic
research described here affords erythro, 3e (rac), and
threo, 3t (rac), diastereoisomers, separable by column
chromatography, or the stereochemically pure L-threo-
(2S,4S)-4-fluoroglutamate-containing analogue, 3t. Bio-
chemical studies show that both oxidized and reduced
fluorofolates are very poor substrates for FPGS. These
results extend previous observations with racemic γ-flu-
oromethotrexate5 that replacing the L-Glu portion of
folates and antifolates such as methotrexate with
(2S,4S)-4-FGlu will result in analogues of these com-
pounds that are not converted to the poly-γ-glutamate
conjugates. The stereochemically homogeneous com-
pounds, 1t and 3t, will facilitate ongoing research
concerning the effect of polyglutamylation of folates and
antifolates on enzymatic and cellular processes.
The synthesis of DL-3,3-difluoroglutamic acid16 and its
incorporation into methotrexate and folic acid analogues
results in DL-â,â-difluoromethotrexate, 2,6 and DL-â,â-
difluorofolic acid, 4. In agreement with our previous
observation that ligation of 3,3-difluoroglutamic acid to
either methotrexate or folic acid leads to products with
enhanced FPGS substrate properties,4 both 2 and 4 are
better substrates than MTX6 and folic acid (Figure 3),
respectively. Surprisingly, the ligation product contain-
ing â,â-difluoroglutamyl-γ-glutamate is essentially inac-
tive as an FPGS substrate.6 Thus, incorporation of 3,3-
difluoroglutamate into a growing poly-γ-glutamyl con-
jugate of folic acid or methotrexate4 or into synthetic
Substrate activity of 7,8-dihydro-DL-â,â-F2FA was
measured, and it was found that Vmax of the reduced
difluorofolate is ca. 0.74Vmax of dihydrofolate. Km values
appear to be slightly lower than for dihydrofolate,
suggesting that both the D- and L-isomers are sub-

Fluorinated Folic Acid and Methotrexate Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 61
analogues such as 2 and 46 results in enhanced ligation
of a single glutamic acid followed by chain termination;
i.e., 3,3-F2Glu mediates a position-dependent enhance-
ment or termination of FPGS-catalyzed polyglutam-
ylation.6
tained from Sigma Chemical Co. (St. Louis, MO). HPLC grade
acetonitrile (J . T. Baker) was from Fisher Scientific Co.
2-Mercaptoethanol and Norit were obtained from Kodak.
RuO2‚xH2O was obtained from AESAR (Ward Hill, MA).
DAST was from Carbolabs (Britany, CT) and was used without
further purification. The 7,8-dihydro derivatives of folates
were produced by microscale dithionite reduction and repuri-
fication by repeated acid precipitation essentially as described
by Hayman.34 The pH was adjusted to 1.8 and the solution
placed on ice for 3 h to allow precipitation. The pH is lower
than that optimal for the precipitation of dihydrofolate; this
is consistent with the report that a lower pH is required to
precipitate γ-fluorofolic acid.30 Concentrations of pteridine
solutions for all experiments were standardized using pub-
lished extinction coefficients.
(2RS,4RS)-DL-erythro,threo-4-Fluoroglutamic acid, 5e,t (rac),
was synthesized from diethylfluoromalonate and ethyl aceta-
midomalonate.5 Conversion to the di-tert-butyl ester, 7e,t
(rac), and separation of the erythro and threo diastereomeric
esters has been reported previously without details;5 complete
experimental details and spectral data are provided herein
(supporting information). (2S,4S)-L-threo-4-Fluoroglutamic
acid (5t) was synthesized as recently described by Hudlicky.19
The synthesis of DL-3,3-difluoroglutamic acid, 6, is described
below in detail; selected spectral data for 6 and intermediates
in the synthesis of 6 have been presented in a previous
communication.16 N-Me,N-Cbz-4-aminobenzoic acid,35 2,4-
diamino-6-(bromomethyl)pteridine (10),35 and N10-(trifluoro-
acetyl)pteroic acid (11)21 were synthesized as previously
described. The cyclic carbamate of 3-hydroxy-DL-prolinol, 12,
was synthesized as described by Tamaru et al.36
DEAE-cellulose chromatography general procedure: DE 53
(Whatman, 6 g) was washed with triethylammonium bicar-
bonate (TEAB) buffer (1.0 M, 2 × 50 mL), and the pH was
adjusted with CO2(g) to approximately 7.7. The DE 53 was
allowed to settle and the buffer was decanted. The resulting
DE 53 was suspended in TEAB buffer (0.025 or 0.3 M), and
the column bed (1 × 12 cm) was poured at 4 °C. The column
pH and conductivity were equilibrated with TEAB buffer
(0.025 or 0.3 M). The sample was dissolved in H2O, and the
pH and conductivity were adjusted to match the equilibrating
buffer. The sample was applied to the column, and the product
was eluted, at 4 °C, with a linear buffer gradient (500 mL total
volume, 0.025 or 0.3-1.0 M). Fractions (12 mL) were collected
and analyzed by UV absorption spectroscopy. Fractions
containing the desired product were pooled and evaporated to
dryness. The resulting solid was dissolved in H2O and
lyophilized.
HPLC chromatography general procedure: Synthetic com-
pounds were analyzed by reversed-phase HPLC (Vydac C18
column; 0.46 × 25 cm) using a Beckman/Altex system equipped
with a Rheodyne Model 7125 injector, two Model 110 A Altex
pumps, and a Hewlett-Packard Model 1040A detector set at
280 nm. The mobile phase consisted of eluent A (0.1 M sodium
acetate, pH 5.5) and eluent B (CH3CN), and the elution
proceeded as follows at 1 mL/min.37 The initial conditions were
4% B (0-12 min) and then increased linearly to 12.4% B (12-
40 min) and run at 12.4% B (40-42 min).
These results are informative and suggest approaches
to address more fundamental issues related to FPGS
catalysis. It is clear that the presence of CF2 in 3,3-F2-
Glu substrates vs CH2 in glutamate substrates leads to
a dramatic change in the structure of the enzyme-
substrate complex during catalysis. This change is
manifest by either a stimulation of catalytic activity
using substrates with 3,3-F2Glu at the C-terminus4,6 or
termination of the ligation reaction by substrates with
the C-terminal sequence -3,3-F2Glu-γ-Glu.6 Unfortu-
nately, very little is known about the nature of these
complexes and/or their rearrangements during chain
elongation. Since FPGS catalyzes the nonribosomal
synthesis of a polypeptide, it is important to ascertain
if the reaction proceeds via a processive or a nonproc-
essive mechanism. Steady-state kinetic data indicate
that FPGS catalysis proceeds via an ordered, sequential
mechanism with an order of substrate binding (MgATP,
folate, glutamate) and product release (ADP, folate poly-
γ-glutamate, Pi) which appear to rule out the sequential
addition of glutamate to enzyme-bound folate, i.e., a
processive mechanism. However, the published experi-
ments were carried out under conditions where only a
single glutamate was added due to the FPGS chosen
for study (Corynebacterium sp.)31 or substrate (aminop-
terin) and assay conditions used.32 Our results indicate
that 3,3-difluoroglutamate-containing conjugates of MTX
and reduced folates may be useful probes for investigat-
ing the required rearrangement of enzyme-substrate
complexes during the synthesis of a growing poly-γ-
glutamyl peptide.
Exp er im en ta l Section
Gen er a l Tech n iqu es. All reactions sensitive to moisture
were conducted under an atmosphere of nitrogen with oven-
dried glassware, unless specified otherwise. All commercial
reagents were distilled prior to use. Chloroform was distilled
from CaH2 and stored over 4 Å molecular sieves, and dichlo-
romethane was freshly distilled from CaH2. Triethylamine
was distilled from KOH and stored over molecular sieves.
Column chromatography was performed with silica gel 60
(230-400 mesh). Thin layer chromatography (TLC) was
conducted on precoated silica gel plates (Kieselgel 60 F254,
0.25-mm thickness, E. Merck & Co.) and visualized by
ultraviolet light and/or ninhydrin followed by heat. Melting
points were obtained on a Thomas-Hoover Mel-Temp ap-
paratus and are uncorrected. 1H NMR spectra were recorded
on a Bruker AM-300 or AM-360 and reported in the following
manner: chemical shift in ppm downfield from tetramethyl-
silane (multiplicity, integrated intensity, coupling constants
in hertz, assignment). 13C NMR spectra were recorded on a
Bruker AM-300 or AM-360 referenced to tetramethylsilane.
19F NMR were obtained on a GE Omega-500 or Bruker AM-
300 and referenced to trifluoroacetic acid as an external
standard. Infrared spectra were recorded on a Nicolet 5-DX
spectrometer. Mass spectra and high-resolution mass spectra
were performed on a Finnigan 4500 GC/MS-EICI system or
on a VG analytical system, Model 70-250S. Elemental analy-
ses were obtained from Atlantic Microlab Inc. (Norcross, GA)
or at the Elemental Analysis Laboratory, Department of
Chemistry, University of Michigan. Yields of fluorinated
folates and antifolates were determined on the basis of the
extinction coefficients for the analogous protio compounds.33
MTX was a generous gift of the National Cancer Institute
and Immunex (Seattle, WA). Aminopterin (AMT) was ob-
(2S,4S)-4-F lu or oglu ta m ic Acid r,γ-Di-ter t-bu tyl Ester
(7t). To a suspension of 5t (0.80 g, 4.84 mmol) in CHCl3 (50
mL) at -20 °C was added concentrated H2SO4 (0.56 mL). The
solution was maintained at -20 °C, and isobutylene gas (12
mL) was condensed into the flask. The flask was closed to
the atmosphere and stirred at room temperature for 96 h. The
mixture was cooled to -20 °C and opened to the atmosphere.
The solution was washed with 20% K2CO3 (100 mL), and the
organic layer was separated. The aqueous layer was extracted
with EtOAc (2 × 50 mL), and the combined organic layers were
dried over MgSO4, filtered, and evaporated in vacuo. The
resulting oil was purified by silica gel chromatography (hex-
anes/EtOAc, 2:1) to afford 7t (0.91 g; 68%) as a colorless oil:
[R]22 ) -8.34 (c 1.1, CHCl3); Rf 0.55 (hexanes/EtOAc, 2:1);
D
1H NMR (300 MHz, CDCl3) δ 5.10 (ddd, J ) 49.5, 10.4, 1.9
Hz, 1 H, CHF), 3.55 (dd, J ) 10.3, 3.3 Hz, 1 H, RCH), 2.40-
1.70 (m, 2 H, CH2); 13C NMR (90 MHz, CDCl3) 174.4, 169.6 (d,
J ) 20.2 Hz), 86.6 (d, J ) 183.7 Hz), 82.5, 81.4, 51.6, 37.5 (d,

62 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Hart et al.
J ) 21.2 Hz), 28.1 (6C); 19F NMR (480 MHz, CDCl3) δ -123.6
(ddd, J ) 49.2, 15.1, 14.3 Hz); MS (DCI-NH3) m/ z (relative
intensity) 278 ((M + H)+, 75), 222 (66), 166 (23); HRMS for
C13H24FNO4 (M + H)+ calcd 278.1768 found 278.1776.
178.2, 177.0 (d, J ) 20.5 Hz), 168.9, 162.6, 161.8, 153.2, 151.3,
149.0, 147.9, 128.7 (2C), 121.8, 120.1, 111.4 (2C), 88.2 (d, J )
182.3 Hz), 54.5, 52.0, 45.9, 42.4, 38.8, 35.7 (d, J ) 21.6 Hz),
8.7; 19F NMR (480 MHz, D2O) δ -115.30 (ddd, J ) 50.0, 18.0,
16.9 Hz); UV λmax (0.1 N NaOH) 258, 302, 371 nm; (0.1 N HCl)
N-[4-[[[(Ben zyloxy)ca r bon yl]m eth yl]a m in o]ben zoyl]-
(rS,γS)-γ-flu or oglu ta m ic Acid r,γ-Di-ter t-bu tyl Ester (8t).
To a solution of 4-[(N-Cbz)methylamino]benzoic acid (0.21 g,
0.72 mmol) in DMF (15 mL) at 0 °C were added DCC (0.25 g,
1.0 mmol) and HOBt (0.2 g, 1.44 mmol) followed by 7t (0.2 g,
0.72 mmol). After 60 h of stirring at room temperature, the
solution was cooled to 5 °C and filtered. The filtrate was
evaporated to dryness, and the resulting yellow semisolid was
purified by silica gel column chromatography (hexanes/EtOAc,
2:1) to afford 8t (0.38 g, 97%) as a colorless oil: [R]24D ) +11.15
(c 1.07, CHCl3); Rf 0.40 (hexanes/EtOAc, 1:1); IR (CHCl3) 1730,
307, 242, 346 (sh) nm; MS (FAB-, triethanolamine) for C20H21
-
FN8O5 m/ z (relative intensity) 471 (M - 1, 43.1); reversed
phase HPLC tR ) 37.3 min.
N-P t er oyl-(rS,γS)-γ-flu or oglu t a m ic Acid (3t). To a
solution of 11 (0.13 g, 0.32 mmol) in DMF (7 mL) cooled to 0
°C were added DCC (0.098 g, 0.48 mmol) and HOBt (0.086 g,
0.64 mmol). The mixture became homogeneous as it warmed
to room temperature. The resulting solution was cooled to 0
°C and a solution of 7t (0.089 g, 0.32 mmol) in DMF (2 mL)
was added. The reaction mixture was stirred at room tem-
perature for 68 h and filtered. The filtrate was evaporated to
dryness, and the resulting solid was dissolved in EtOAc/DMF
(90/10, 100 mL), washed with 0.5 N NaHCO3 (1 × 50 mL),
H2O (1 × 50 mL), 0.5 N H2SO4 (1 × 50 mL), H2O (1 × 50 mL),
0.5 N NaHCO3 (1 × 50 mL), and saturated NaCl (1 × 50 mL),
and dried over Na2SO4. The solution was filtered and evapo-
rated to dryness, and the resulting solid was dissolved in TFA
(10 mL) at 0 °C. After stirring for 30 min at 0 °C, the mixture
was diluted with CHCl3 (50 mL) and evaporated to dryness.
The resulting yellow solid was dissolved in 0.1 M piperidine
(25 mL) and stirred for 3 h at 15 °C. The turbid solution was
centrifuged (20 min, 1500g), the supernatant was decanted,
and the pH was adjusted to 2 with 2 N HCl. The solution
was stored at 4 °C for 12 h to provide an opaque suspension.
Centrifugation provided an orange pellet that was washed with
H2O (2 × 25 mL) and recentrifuged. The resulting yellow
pellet was dried in vacuo to afford 3t (0.083 g, 56%): 1H NMR
(360 MHz, DMSO-d6/D2O exchange) δ 8.60 (s, 1 H, C7-H), 7.65
(d, J ) 8.7, 2 H, Ar), 6.62 (d, J ) 8.7, 2 H, Ar), 4.89 (m, 1 H,
CHF), 4.50-4.40 (m, 3 H, RCH and benzylic CH2), 2.45-2.15
(m, 2 H, âCH2); 13C NMR (90 MHz, DMSO-d6) δ 173.4, 171.0
(d, J ) 22.6 Hz), 167.1, 161.6, 156.3, 153.9, 151.2, 149.0 (2C),
131.5, 129.4 (2C), 128.1, 121.3, 111.6 (2C), 86.5 (d, J ) 182.0
Hz), 48.7, 46.0, 33.6 (d, J ) 20.01 Hz); 19F NMR (500 MHz,
DMSO-d6) δ -114.8 (bm); UV λmax (0.1 N NaOH) 255, 281, 362
nm; MS (FAB-, DTT/DTE) for C19H18FN7O6 m/ z (relative
intensity) 458 (M - 1, 100); reversed phase HPLC, tR ) 24.3
min.
1
1706, 1667, 1608 cm-1; H NMR (300 MHz, CDCl3) δ 7.82 (d,
J ) 8.8 Hz, 2 H, Ar), 7.44-7.31 (m, 7 H, C6H5 and Ar), 6.85
(bd, 1 H, NH), 5.18 (s, 2 H, benzyl), 5.05-4.80 (m, 2 H, CHF
and RH), 3.35 (s, 3 H, CH3), 1.78-2.39 (m, 2 H, âCH2), 1.51 (s,
9 H, C(CH3)3), 1.45 (s, 9 H, C(CH3)3); 13C NMR (75 MHz, CDCl3)
δ 170.2, 168.1 (d, J ) 22.4 Hz), 167.8, 166.2, 154.9, 146.3,
136.3, 130.9, 128.4 (2C), 128.0 (2C), 127.7 (2C), 125.0 (2C), 86.7
(d, J ) 185.1 Hz), 83.1, 82.8, 67.6, 50.5, 37.3, 35.1 (d, J ) 20.5
Hz), 28.0 (6C); 19F NMR (500 MHz, CDCl3) δ -113 (ddd, J )
51.6, 22.5, 21.7 Hz); UV λmax (0.1 N NaOH) 267 nm; (0.1 N
HCl) 266, 226 nm; MS (EI) m/ z (relative intensity) 554 (M+,
8), 471 (3), 432 (5), 415 (12), 387 (27); HRMS for C29H37FN2O7
(M+) calcd 544.2585, found 544.2580. Anal. (C29H37FN2O7)
C, H, N.
N-[4-[Met h yla m in o]b en zoyl]-(rS,γS)-γ-flu or oglu t a m -
ic Acid r,γ-Di-ter t-bu tyl Ester (9t). To a solution of 8t (0.37
g, 0.70 mmol) in MeOH (20 mL) was added Pd(OH)2 (0.047 g),
and the mixture was shaken under hydrogen (50 psi) for 48
h. The solution was filtered over silica gel followed by
decolorizing charcoal (Norit). The filtrate was evaporated to
dryness, and the resulting semisolid was purified by silica gel
column chromatography (hexanes/EtOAc, 1:1) to afford 9t (0.26
g, 99%). 9t was crystallized from MeOH/H2O: mp 156-158
°C; [R]22 ) +19.45 (c 1.35, CHCl3); Rf 0.34 (hexanes/EtOAc,
D
1:1); IR (KBr) 3393, 1744, 1627, 1611 cm-1; 1H NMR (300 MHz,
CDCl3) δ 7.70 (d, J ) 8.7 Hz, 2 H, Ar), 6.87 (d, J ) 8.9 Hz, 1
H, NH), 6.59 (d, J ) 8.7 Hz, 2 H, Ar), 4.95 (m, 2 H, CHF and
RH), 2.87 (s, 3 H, CH3), 2.25-2.52 (m, 2 H, CH2), 1.49 (s, 9 H,
C(CH3)3), 1.45 (s, 9 H, C(CH3)3); 13C NMR (75 MHz, CDCl3) δ
170.7, 167.6 (d, J ) 20.1 Hz), 166.8, 152.0, 128.8 (2C), 121.8,
111.3 (2C), 86.6 (d, J ) 185.0 Hz), 83.0, 82.6, 50.1, 35.4 (d, J
) 20.7 Hz), 30.3, 28.0 (6C); 19F NMR (480 MHz, CDCl3) δ
-112.63 (ddd, J ) 48.9, 20.7, 19.3 Hz); UV λmax (0.1 N NaOH)
291 nm; (0.1 N HCl) 227 nm; MS (EI) m/ z (relative intensity)
410 (12, M+), 337 (3), 298 (5), 281 (9), 253 (15), 134 (100);
HRMS for C21H31FN2O5 (M+) calcd 410.2217, found 410.2209.
Anal. (C21H31FN2O5) C, H, N.
6-Oxo-1-a za -3-oxa bicyclo[3.3.0]octa n -2-on e (13). To a
solution of DMSO (0.36 mL, 5.1 mmol) in CH2Cl2 (25 mL)
cooled to -55 °C was added trifluoroacetic anhydride (0.54 mL,
3.83 mmol) in CH2Cl2 (2.1 mL), maintaining the temperature
below -50 °C. A white precipitate formed, and the mixture
was stirred for 10 min. Alcohol 12 (0.36 g, 2.55 mmol),
dissolved in CHCl3 (15 mL), was added slowly to the reaction
mixture, maintaining the temperature between -50 °C and
-55 °C. After the addition of 12 the reaction mixture was
stirred at -55 °C for 30 min. Triethylamine (1.02 mL, 7.32
mmol) was added dropwise to the mixture, maintaining the
temperature between -50 °C and -55 °C. After stirring at
room temperature for 16 h, the mixture was concentrated in
vacuo, and the resulting yellow oil was dissolved in CH2Cl2
(100 mL) and washed with H2O (50 mL). The aqueous phase
was extracted with CH2Cl2 (4 × 50 mL), and the combined
organic phases were washed with saturated NaCl (2 × 50 mL).
Additional product was obtained from the aqueous phase by
saturation with NaCl followed by extraction with CH2Cl2 (4
× 100 mL). The combined organic phases were dried over
MgSO4, filtered, and evaporated in vacuo. The crude oil was
purified by silica gel chromatography (EtOAc/hexanes/CH3CN,
3:1:1) to afford 13 (0.26 g, 73%) as a white crystalline solid:
mp 98-99 °C; Rf 0.74 (EtOAc/hexanes/CH3CN, 3:1:1); IR
N-(4-Am in o-4-d eoxy-10-m eth ylp ter oyl)-(rS,γS)-γ-flu o-
r oglu ta m ic Acid (1t). To a solution of 10 (0.12 g, 0.38 mmol)
in DMAC (2 mL) was added a solution of 9t (0.060 g, 0.16
mmol) in DMAC (2 mL) at room temperature. The mixture
was heated at 55 °C for 4 h, cooled to room temperature, and
stirred for 24 h. TLC (Rf 0.80; butanol/H2O/pyridine, B/W/P,
30:24:26) indicated the reaction was complete and the solution
was evaporated to dryness. The resulting solid was dissolved
in TFA (3 mL) and stirred at room temperature for 3 h. The
mixture was evaporated to dryness, and the resulting solid
was triturated with Et2O and filtered. The crude product was
purified by DEAE-cellulose column chromatography. The
desired product, 1t, eluted during a 0.025 M-1.0 M TEAB,
pH 7.7, linear gradient. Fractions containing pure 1t, identi-
fied by UV spectra, were pooled and evaporated to dryness.
The resulting solid was dissolved in H2O and lyophilized to
afford 1t (0.11 mmol) as the triethylamine salt in 69% yield:
1
(CHCl3) 1763, 1706 cm-1; H NMR (360 MHz, CDCl3) δ 4.55
1
(t, 1 H, J ) 9.4 Hz, C4-H), 4.40 (dd, 1 H, J ) 3.4, 3.4 Hz, C4-
H), 4.31 (m, 1 H, C8-H), 3.93 (dd, 1 H, J ) 3.3, 3.5 Hz, C5-H),
3.60 (m, 1 H, C8-H), 2.56 (m, 2 H, C7-H2); 13C NMR (90 MHz,
CDCl3) δ 212.6, 161.4, 64.5, 61.3, 43.8, 36.4; MS (EI) m/ z
(relative intensity) 142 ((M + H)+, 10), 141 (M+, 17), 113 (100),
97 (59), 86 (96); HRMS for C6H7NO3 (M+) calcd 141.0426, found
141.0434. Anal. (C6H7NO3) C, H, N.
mp >300 °C; Rf 0.57 (B/W/P, 30:24:26); H NMR (300 MHz,
D2O) δ 8.43 (s, 1 H, C-7H), 7.60 (d, J ) 8.7 Hz, 2 H, Ar), 7.08
(d, J ) 8.8 Hz, 2 H, Ar), 4.70 (s, 2 H, benzyl), 3.20 (s, 3 H,
CH3), 3.03 (q, 12 H, N(CH2CH3)3 × 2), 2.60-2.40 (m, 2 H,
âCH2), 1.1 (t, 18 H, N(CH2CH3)3 × 2), CHF and R-CH
resonances were obscured by HOD peak but were observed at
5.01 and 4.51 ppm in DMSO-d6; 13C NMR (90 MHz, D2O) δ

Fluorinated Folic Acid and Methotrexate Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 63
6,6-Diflu or o-1-a za -3-oxa bicyclo[3.3.0]octa n -2-on e (14).
To a solution of 13 (0.10 g, 0.71 mmol) in CH2Cl2 (15 mL) was
added DAST (0.30 mL, 2.10 mmol) at -78 °C and the solution
was stirred at room temperature for 36 h. Ice and CH2Cl2 (30
mL) were added to the solution, and the organic and aqueous
phases were separated. The aqueous phase was extracted with
CH2Cl2 (3 × 20 mL), and the combined organic phases were
dried over MgSO4, filtered, and concentrated in vacuo, provid-
ing 14 (0.073 g, 64%) as a yellow oil: Rf 0.90 (EtOAc/hexanes,
Hz), 28.2 (3C) and 28.1 (3C); 19F NMR (282 MHz, CDCl3) (two
rotamers) δ -18.6 (d, J ) 235.8 Hz) and -18.8 (d, J ) 236.1
Hz), -30.2 (d, J ) 236.9 Hz) and -32.6 (d, J ) 236.9 Hz); MS
(CI-NH3) m/ z (relative intensity) 283 ((M + NH4)+, 7), 266 ((M
+ H)+, 4), 227 (100), 166 (28), 136 (79); HRMS for C11H17F2-
NO4 (M + H)+ calcd 266.1204, found 266.1198. Anal. (C11H17F2-
NO4) H, N; C: calcd, 49.81; found, 49.05.
DL-3,3-Diflu or oglu ta m ic Acid (6). A solution of 16 (0.25
g, 0.94 mmol) in EtOAc (10 mL) was added to a solution of
RuO2‚xH2O (0.050 g, catalytic) in aqueous 10% NaIO4 (8.6 mL)
at room temperature. The biphasic solution was stirred
vigorously at room temperature to allow for thorough mixing
between phases. Additional aliquots of 10% NaIO4 (5 × 2 mL)
were added to maintain a yellow-colored solution, and the
reaction mixture was stirred at room temperature for 288 h.
The organic and aqueous phases were separated, and the
aqueous phase was extracted with EtOAc (3 × 50 mL). i-PrOH
(5 mL) was added to the combined organic phases, and the
mixture was stirred at room temperature for 3 h, dried over
MgSO4, filtered through a plug of Celite, and evaporated in
vacuo, providing a dark oil 17 (0.17 g). A portion of the crude
pyroglutamate 17 was crystallized for analysis: mp 62-63 °C;
4:1, developed by I2); IR (CHCl3) 1760 cm-1 1H NMR (300
;
MHz, CDCl3) δ 4.52 (m, 2 H, C4-H2), 4.08 (m, 1 H, C5-H2),
3.89-3.47 (m, 2 H, C8-H2), 2.46 (m, 2 H, C8-H2); 13C NMR (90
MHz, CDCl3) δ 160.6, 126.7 (t, J ) 256.6 Hz), 62.2 (t, J ) 26.1
Hz), 61.6, 43.2, 34.5 (t, J ) 24.9 Hz); MS (EI) m/ z (relative
intensity) 163 (M+, 25), 119 (12), 99 (19); HRMS for C6H7F2-
NO2 (M+) calcd 163.0445, found 163.0452. Compound 14 was
converted to 15 without further purification.
DL-N-(ter t-Bu t oxyca r b on yl)-3,3-d iflu or o-2-(h yd r oxy-
m eth yl)pyr r olidin e (DL-(N-Boc)-3,3-diflu or opr olin ol) (15).
A solution of difluorooxazolidinone 14 (0.10 g, 0.61 mmol) in
6 N HCl (3 mL) was heated at reflux temperature for 18 h.
The solution was evaporated in vacuo, providing crude 3,3-
difluoroprolinol as a light brown oil (0.10 g). To a suspension
of the crude oil in CHCl3 (1.5 mL) was added 0.36 M NaHCO3
(1.5 mL), NaCl (0.06 g), and (Boc)2O (0.16 g, 0.70 mmol). The
solution was heated at reflux temperature for 3 h and cooled
to room temperature. The aqueous and organic phases were
separated, and the aqueous phase was extracted with CHCl3
(4 × 30 mL). The combined organic phases were dried over
MgSO4, filtered, and evaporated in vacuo to give 15 as a yellow
oil (0.13 g, 83% from 14): Rf 0.90 (CHCl3/EtOAc, 9:1); IR
(CHCl3) 3473, 1686 cm-1; 1H NMR (300 MHz, CDCl3) δ 4.00-
3.65 (m, 3 H, C2-H and C5-H2), 3.5 (m, 2 H, CH2O), 2.44-
2.21 (m, 2 H, C4-H2), 1.45 and 1.60 (two singlets, 9 H, C(CH3)3);
13C NMR (90 MHz, CDCl3) δ 155.3, 127.3 (t, J ) 269.9 Hz),
81.2, 64.2 (t, J ) 28.1 Hz), 61.3, 43.4, 33.2 (t, J ) 23.7 Hz),
28.5 (3C); 19F NMR (282 MHz, CDCl3) δ (two rotamers) -17.5
(dm, J ) 231.5 Hz) and -22.4 (d, J ) 235.9 Hz), -35.9 (d, J
) 234.6 Hz) and -36.4 (d, J ) 236.7 Hz); MS (CI-NH3) m/ z
(relative intensity) 238 ((M + H)+, 9), 199 (37), 182 (100), 138
(26); HRMS for C10H17F2NO3 (M + H)+ calcd 238.1247, found
238.1249. Compound 15 was converted to 16 without further
purification. An analytical sample was obtained by silica gel
column chromatography (hexanes/EtOAc, 3:1). Anal. (C10H17F2-
NO3‚0.1H2O) C, H, N.
DL-N-(ter t-Bu toxyca r bon yl)-3,3-d iflu or op r olin e Meth yl
Ester (16). A solution of 15 (0.13 g, 0.41 mmol) dissolved in
EtOAc (3 mL) was added to a solution of RuO2‚xH2O (0.020 g,
catalytic) in aqueous 10% NaIO4 (3.5 mL, 1.6 mmol) at room
temperature. The biphasic solution was stirred vigorously at
room temperature for 3 h, allowing thorough mixing between
phases. The organic and aqueous phases were separated, and
the aqueous phase was extracted with EtOAc (4 × 30 mL).
i-PrOH (5 mL) was added to the combined organic phases, and
the mixture was stirred at room temperature for 3 h and was
dried over MgSO4, filtered through a plug of Celite, and
evaporated in vacuo. The resulting crude acid (0.13 g) was
not purified. 1H NMR indicated no starting material remained
and was consistent with the formation of the corresponding
acid, Rf 0.10 (CHCl3/EtOAc, 9:1, bromocresol green-positive).
To a solution of the crude acid in Et2O (15 mL) was added
CH2N2 (3.0 mL, 1.53 mmol), and the mixture was stirred at 0
°C for 1 h. Glacial acetic acid (0.05 mL) was added to the
reaction mixture and the solution stirred at room temperature
for 25 min. The solvent was removed under a weak vacuum
(20 °C, 340 mbar) (CAUTION! 16 is volatile under high
vacuum) and the residue was purified by silica gel column
chromatography (hexanes/EtOAc, 2:1) to afford 16 (0.10 g, 93%
from 15) as a colorless oil: Rf 0.70 (hexanes/EtOAc, 2:1); IR
(CHCl3) 1754, 1706, 1704 (sh) cm-1; 1H NMR (300 MHz, CDCl3)
δ 4.53 (m, 2 H, RCH), 3.70-3.50 (m, 5 H, δCH2 and CH3), 2.50-
2.35 (m, 2 H, γCH2), 1.50 and 1.45 (two singlets, 9 H, C(CH3)3);
13C NMR (90 MHz, CDCl3) (two rotamers) δ 167.8, 153.6 and
153.0, 126.5 (t, J ) 254.2 Hz) and 125.9 (t, J ) 260.0 Hz),
81.0, 65.0 (t, J ) 30.3 Hz) and 64.4 (t, J ) 30.3 Hz), 52.8 and
52.6, 43.3 and 42.8, 33.3 (t, J ) 29.9 Hz) and 32.7 (t, J ) 23.1
Rf 0.40 (hexanes/EtOAc, 2:1); IR (KBr) 2950, 1721, 1658 cm-1
;
1H NMR (300 MHz, CDCl3) δ 4.90-4.80 (d, J ) 17.4 Hz, 1 H,
RCH), 3.88 (s, 3 H, CH3), 3.29-2.98 (m, 2 H, γCH2), 1.45 (s, 9
H, C(CH3)3); 13C NMR (90 MHz, CDCl3) δ 166.1, 165.6, 148.0,
118.8 (t, J ) 252.5 Hz), 85.2, 66.4 (t, J ) 31.0 Hz), 53.4, 41.3
(t, J ) 24.5 Hz), 27.8 (3C); 19F NMR (282 MHz, CDCl3) δ -13.6
(dm, J ) 240.8 Hz), -31.1 (dd, J ) 240.4 Hz, J ) 12.0 Hz);
MS (CI-NH3) m/ z (relative intensity) 297 ((M + NH4)+, 36),
280 ((M + H)+, 2), 197 (49), 177 (42), 136 (100); HRMS for
C11H15F2NO5 (M + NH4)+ calcd 297.1262, found 297.1259.
Anal. (C11H15F2NO5) C, H, N.
A solution of crude 17 (0.032 g) in 12 N HCl (3 mL) was
heated at reflux temperature for 2 h. The solution was
evaporated to dryness, providing 0.030 g of an off-white solid.
The crude solid 6 was dissolved in ddH2O and purified by
anion-exchange column chromatography (BioRad AG 1-X8
200-400 mesh, acetate form, 11 mL of wet resin ) 18.7
mequiv). The column was eluted with ddH2O (110 mL), 1 N
HOAc (110 mL), and 2 N HOAc (110 mL), and fractions (10
mL) were collected. Fractions 16-18 (1N HOAc) were pooled,
concentrated in vacuo, and crystallized (EtOH/H2O) to afford
6 (0.013 g, 40% from 16): mp 171-174 °C, Rf 0.35 (B/W/P,
30:24:26); 1H NMR (300 MHz, D2O) δ 4.65-4.51 (dd, J ) 3.8,
3.7 Hz, 1 H, RCH), 3.60-3.36 (m, 2 H, γCH2); 13C NMR (90
MHz, D2O) δ 170.7 (d, J ) 11.8 Hz), 167.2 (d, J ) 7.1 Hz),
119.2 (t, J ) 248.3 Hz), 57.5 (dd, J ) 19.9, 19.7 Hz), 39.9 (t, J
) 24.9 Hz); 19F NMR (282 MHz, D2O) δ -22.1 (dm, J ) 253.3
Hz), -27.1 (d, J ) 256.4 Hz). Anal. (C5H7F2O4) C, H, N.
DL-3,3-Diflu or oglu t a m ic Acid r,γ-Di-ter t-b u t yl E st er
(18). To a suspension of 6 (0.047 g, 0.26 mmol) in freshly
distilled CHCl3 (3 mL) at -20 °C was added concentrated H2-
SO4 (0.028 mL) slowly by pipette. The solution was main-
tained at -20 °C, and isobutylene gas (3 mL) was condensed
into the flask. The vessel was sealed, and after stirring at
room temperature for 72 h the solution became homogeneous.
After 120 h the mixture was cooled to -20 °C, opened, and
washed with 20% K2CO3 (50 mL). The aqueous phase was
extracted with EtOAc (3 × 50 mL), and the combined organic
phases were dried over MgSO4, filtered, and evaporated in
vacuo. The resulting oil was purified by silica gel column
chromatography (hexanes/EtOAc, 10:3) to afford 18 (0.068 g,
89% yield) as a colorless oil: Rf 0.33 (hexanes/EtOAc, 10:3);
1H NMR (300 MHz, CDCl3) δ 4.06 (t, J ) 13.6 Hz, 1 H, RCH),
3.02 (m, 2 H, γCH2), 1.48 (s, 9 H, C(CH3)3), 1.45 (s, 9 H,
C(CH3)3); 13C NMR (90.0 MHz, CDCl3) δ 168.7, 166.1, 120.6
(t, J ) 248.3 Hz), 82.6, 81.9, 58.4 (t, J ) 26.1 Hz), 40.6 (t, J )
26.7 Hz), 27.9 (6C); 19F NMR (470 MHz, CDCl3) δ -27.2 (ddd,
J ) 253.2, 15.1, 11.8 Hz), -27.8 (ddd, J ) 253.2, 15.1, 12.2
Hz); MS (DCI-NH3) m/ z (relative intensity) 296 ((M + H)+,
25), 240 (32), 184 (14); HRMS for C13H23F2NO4 (M + H)+ calcd
296.1673 found 296.1674.
N-P ter oyl-DL-â,â-d iflu or oglu ta m ic Acid (4). To a solu-
tion of 11 (0.080 g, 0.20 mmol) in DMF (2 mL) was added HOBt
(0.053 g, 0.39 mmol), EDC (0.075 g, 0.39 mmol), and 18 (0.058

64 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Hart et al.
g, 0.20 mmol) in DMF (2 mL) at room temperature. After 72
h the solution was evaporated to dryness, and the resulting
solid was dissolved in EtOAc/DMF (90/10, 100 mL), washed
with 0.5 N NaHCO3 (1 × 50 mL), H2O (1 × 50 mL), 0.5 N
H2SO4 (1 × 50 mL), H2O (1 × 50 mL), 0.5 N NaHCO3 (1 × 50
mL), and saturated NaCl (1 × 50 mL), and dried over Na2-
SO4. The solution was filtered and evaporated to dryness, and
the resulting solid was dissolved in TFA (10 mL) at 0 °C. After
stirring for 30 min at 0 °C the mixture was diluted with CHCl3
(50 mL) and evaporated to dryness. The resulting yellow solid
was dissolved in 0.1 M piperidine (25 mL) and stirred for 3 h
at 15 °C. The pH of the solution was adjusted to 2 with 2 N
HCl and was stored at 4 °C for 12 h to provide an opaque
suspension. Centrifugation provided an orange pellet that was
washed with H2O (2 × 25 mL) and the suspension recentri-
fuged. The resulting pellet was dried in vacuo to afford crude
4. The solid was purified by DEAE-cellulose column chroma-
tography. The product eluted during a 0.3-1.0 M TEAB, pH
7.7, linear gradient. Fractions containing pure 4 (0.5-0.8M
TEAB), identified by UV spectra, were pooled and evaporated
to dryness. The resulting solid was dissolved in H2O and
lyophilized to afford 4 (0.069 mmol) as the triethylamine salt
in 35% yield: mp >280 °C dec; 1H NMR (360 MHz, D2O) δ
8.46 (s, 1 H, C7-H), 7.42 (d, J ) 8.7 Hz, 2 H, Ar), 6.36 (d, J )
8.7 Hz, 2 H, Ar), 4.75 (t, 1 H, RCH), 4.29 (s, 2 H, C9-H2), 3.11
(q, H, N(CH2CH3)), 2.90 (t, 2 H, γCH2), 1.1 (t, N(CH2CH3));
13C NMR (90 MHz, D2O) δ 174.3, 172.4, 169.1, 164.6, 154.0,
152.7, 150.5, 148.7, 148.4, 129.1 (2C), 126.6, 121.1 (t, J ) 248.1
Hz), 120.2, 115.1, 111.6 (2C), 59.2 (t, J ) 26.3 Hz), 58.6, 47.2,
46.6, 45.0, 42.6 (t, J ) 24.4 Hz), 42.3, 18.3, 10.6, 8.2; 19F NMR
(470 MHz, D2O) -24.67 (m); UV λmax (0.1 M NaOH) 364, 286,
255 nm; MS (FAB-, DTT/DTE) for C19H17F2N7O6 m/ z (relative
intensity) 476 ((M - H)-, 7); MS (FAB+, DTT/DTE) (relative
as a factor of 2.44 DHFR activity was assayed spectrophoto-
metrically as described.41 Standard assays contained 100 mM
Tris-HCl, pH 7.0, 150 mM KCl, 20 µM dihydrofolate, 20 mM
2-mercaptoethanol, and 50 µM NADPH. CPG2 was assayed
spectrophotometrically using MTX as the substrate.22
Kin etic Da ta An a lysis. FPGS kinetic data were quanti-
tated using the nonlinear curve-fitting program of SigmaPlot
(J andel Scientific, Corte Madera, CA). Kinetic constants for
FPGS substrates were determined by fitting to the rectangular
hyperbola function; initial estimates of parameters were based
on visual inspection of kinetic data. Inhibitory potency was
measured by adding increasing concentrations of an antifolate
to standard DHFR assays and measuring the remaining
activity.
Ack n ow led gm en t. This work was supported by
research grants CA28097 (J .K.C.), CA 13038 (J .J .M.),
and CA16056 (J .J .M.) from the National Cancer Insti-
tute. We thank Professor Milos Hudlicky for providing
details of his stereospecific synthesis of (2S,4S)-4-
fluoroglutamic acid prior to publication. We thank Dr.
R. F. Sherwood at the PHLS Centre for Applied Micro-
biology and Research, Salisbury, Wiltshire, U.K., for a
generous gift of carboxypeptidase G2. We also thank
J ane MacDonald for careful preparation of initial ver-
sions of the manuscript.
Su p p or tin g In for m a tion Ava ila ble: Synthesis of 3e,t
(rac), 3e (rac), and 3t (rac) with full experimental details (2
pages). Ordering information is given on any current mast-
head page.
Refer en ces
intensity) 588 ((M + Et3N)+, 8); reversed phase HPLC tR
)
(1) McGuire, J . J .; Coward, J . K. In Folates and Pterins; Blakley,
R. L., Benkovic, S. J ., Eds.; J ohn Wiley & Sons: New York, 1984;
Vol. 1, pp 135-190.
(2) Matherly, L. H.; Seither, R. L.; Goldman, I. D. Metabolism of
the diaminoantifolates: Biosynthesis and pharmacology of the
7-hydroxyl and polyglutamyl metabolites of methotrexate and
related antifolates. Pharmacol. Ther. 1987, 35, 27-56.
(3) McGuire, J . J .; Coward, J . K. DL-threo-4-Fluoroglutamic acid:
A chain terminating inhibitor of folylpolyglutamate synthetase.
J . Biol. Chem. 1985, 260, 6747-6754.
(4) McGuire, J . J .; Haile, W. H.; Bey, P.; Coward, J . K. DL-3,3-Di-
fluoroglutamate: An enhancer of folylpolyglutamate elongation.
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) Galivan, J .; Inglese, J .; McGuire, J . J .; Nimec, Z.; Coward, J . K.
γ-Fluoromethotrexate: Synthesis and biological activity of a
potent inhibitor of dihydrofolate reductase with greatly dimin-
ished ability to form poly-γ-glutamates. Proc. Natl. Acad. Sci.,
U.S.A. 1985, 82, 2598-2602.
(8) McGuire, J . J .; Haile, W. H.; Coward, J . K. Interaction of DL-
erythro- and DL-threo-γ-fluoromethotrexate with human leuke-
mia cell dihydrofolate reductase. Biochem. Pharmacol. 1989, 38,
4321-4325.
29.3 min.
Bioch em ica l Tech n iqu es. An a lytica l HP LC of Bio-
syn th etic P r od u cts. Performed on a Rainin Instruments
HPLC system using the Dynamax controller and data capture
module run on a Macintosh computer.4 Eluant was monitored
at 280 and 254 nm. RP-HPLC was performed on a C18 column
(0.4 × 25 cm; Rainin Microsorb, 5 µm) at ambient temperature;
the column was eluted isocratically at 1 mL/min using 0.1 M
sodium acetate, pH 5.5, containing 7.5% acetonitrile according
to Nimec and Galivan37 unless otherwise noted. Anion-
exchange HPLC was performed on an Ultrasil AX column (0.4
× 25 cm; Beckman) at 45 °C eluted isocratically at 0.8 mL/
min with 35 or 85 mM sodium phosphate, pH 3.3; the
concentration required to obtain a constant elution time (7.5
min) for MTX depended on column age. Separation of poly-
glutamate products was performed on the anion-exchange
column using a linear gradient.4
Cell Lin es. The human T-lymphoblastic leukemia cell line
CCRF-CEM38 was the primary screen for drug effects and was
the source of tumor enzymes. Routine culture of these lines
was as described.39 CCRF-CEM was verified to be negative
for Mycoplasma contamination during the course of these
studies using the GenProbe test kit.
E n zym es a n d Assa ys. Rat liver FPGS was purified as
described.40 FPGS and DHFR from the human T-lymphoblas-
tic leukemia cell line CCRF-CEM38 were partially purified as
described.41,42 CPG2 was obtained from Dr. Roger Sherwood
(PHLS Centre for Applied Microbiology and Research, Porton
Down, England). The FPGS assay method40,43 uses [3H]Glu
to radiolabel polyglutamates of a folate-like substrate during
incubation with the enzyme. The reaction mixture is applied
(9) Hart, B. P.; McGuire, J . J .; Haile, W. H.; Coward, J . K.
3,3-Difluoromethotrexate (F2MTX): Enhanced poly-γ-glutamy-
lation as a result of substitution of DL-3,3-difluoroglutamate (F2-
Glu) for glutamate (Glu). Proc. Am. Assoc. Cancer Res. 1994,
35, 302.
(10) Bertino, J . R. “Rescue” techniques in cancer chemotherapy: Use
of leucovorin and other rescue agents after methotrexate treat-
ment. Seminars Oncol. 1977, 4, 203-216.
(11) The Expanding Role of Folates and Fluoropyrimidines in Cancer
Chemotherapy; Rustum, Y., McGuire, J . J ., Eds.; Plenum Press:
New York, 1988; Advances in Experimental Medicine and
Biology, Vol. 244.
to a DEAE-cellulose minicolumn that is washed with
a
buffered NaCl solution to remove unligated [3H]Glu. [3H]-
Polyglutamate products are retained on the minicolumn during
the wash and are then eluted quantitatively with acid. Rat
liver FPGS assay conditions were exactly as described.40
CCRF-CEM FPGS assays conditions44 were modified to include
50 µg of protease-free bovine serum albumin (Miles). All
reagents were made with freshly processed deionized water,
stored frozen in aliquots, and used in only one or two
experiments to avoid absorption of atmospheric CO2 which can
lead to FPGS kinetic constants that can be in error by as much
(12) Buchanan, R. L.; Dean, F. H.; Pattison, F. L. M. γ-Fluoroglutamic
acid. Can. J . Chem. 1962, 40, 1571-1575.
(13) 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.

Fluorinated Folic Acid and Methotrexate Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 65
(14) 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.
(15) During the course of this research, a communication appeared
which described the stereospecific synthesis of 5t; Hudlicky, M.;
Merola, J . S. Tetrahedron Lett. 1990, 31, 7403-7406. We thank
Professor Hudlicky for providing details on the synthetic route,
which we had also investigated independently, and for an
authentic sample of 5t.
(16) 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.
(17) 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.
(18) Rosowsky, A. Chemistry and Biological Activity of Antifolates.
Prog. Med. Chem. 1989, 26, 1-252.
(19) Hudlicky, M. Stereospecific syntheses of all four stereoisomers
of 4-fluoroglutamic acid. J . Fluorine Chem. 1993, 60, 193-210.
(20) Balinska, M.; Galivan, J .; Coward, J . K. Efflux of Methotrexate
and Its Polyglutamate Derivatives from Hepatic Cells In Vitro.
Cancer Res. 1981, 41, 2751-2756.
(21) Licato, N. J .; Coward, J . K.; Nimec, Z.; Galivan, J .; Bolanowska,
W. E.; McGuire, J . J . Synthesis of N-[N-(4-deoxy-4-amino-10-
methylpteroyl)-4-fluoroglutamyl]-γ-glutamate, an unusual sub-
strate for folylpoly-γ-glutamate synthetase and γ-glutamyl
hydrolase. J . Med. Chem. 1990, 33, 1022-1027.
(22) McCullough, J . L.; Chabner, B. A.; Bertino, J . R. Purification
and properties of carboxypeptidase G1. J . Biol. Chem. 1971, 246,
7207-7213.
(23) Hudlicky, M.; Merola, J . S. New stereospecific syntheses and
X-ray diffraction structures of (-)-D-erythro- and (+)-L-threo-4-
fluoroglutamic acid. Tetrahedron Lett. 1990, 31, 7403-7406.
(24) Sherwood, R. F.; Melton, R. G.; Alwan, S. M.; Hughes, P.
Purification and properties of carboxypeptidase G2 from
Pseudomonas sp. strain RS-16. Use of a novel triazine dye
affinity method. Eur. J . Biochem. 1985, 148, 447-453.
(25) Huang, S. L.; Omura, K.; Swern, D. Oxidation of Sterically
Hindered Alcohols to Carbonyls with Dimethyl Sulfoxide-Trif-
luoroacetic Anhydride. J . Org. Chem. 1976, 41, 3329-3331.
(26) Yoshifuji, S.; Tanaka, K.; Kawai, T.; Nitta, Y. Chem. Pharm.
Bull. 1986, 34, 3873-3878.
(27) Hart, B. P. Ph.D. Thesis, The University of Michigan, Ann Arbor,
May 1995.
(28) Werkheiser, W. C. Specific binding of 4-amino folic acid ana-
logues by folic acid reductase. J . Biol. Chem. 1961, 236, 888-
893.
(29) Delcamp, T. J .; Susten, S. S.; Blankenship, D. T.; Freisheim, J .
H. Purification and characterization of dihydrofolate reductase
from methotrexate-resistant human lymphoid cells. Biochemistry
1983, 22, 633-639.
(30) Bergmann, E. D.; Chun-Hsu, L. Organic fluorine compounds;
Part XLVI. γ-Fluoroglutamic acid and fluorofolic acid. Synthesis
1973, 1, 44-46.
(31) Shane, B. Pteroylpoly(γ-glutamate) Synthesis by Corynebacte-
rium Species. Studies on the Mechanism of Folylpoly(γ-
glutamate) Synthetase. J . Biol. Chem. 1980, 255, 5663-5667.
(32) Cichowicz, D. J .; Shane, B. Mammalian Folylpoly-γ-glutamate
Synthetase. 2. Substrate Specificity and Kinetic Properties.
Biochemistry 1987, 26, 513-521.
(33) Temple, J ., C.; Montgomery, J . A. In Folates and Pterins; Blakley,
R. L. Benkovic, S. J ., Eds.; J ohn Wiley & Sons: New York, 1984;
Vol. 1, pp 61-120.
(34) Hayman, R.; McGready, R.; Van der Weyden, M. B. A rapid
radiometric assay for dihydrofolate reductase. Anal. Biochem.
1978, 87, 460-465.
(35) Tang, K.-C.; Coward, J . K. Synthesis of keto phosphonate analogs
of biologically important acyl phosphates. J . Org. Chem. 1983,
48, 5001-5006.
(36) Tamaru, Y.; Kawamura, S.-I.; Bando, T.; Tanaka, K.; Hojo, M.;
Yoshida, Z.-C. Stereoselective intramolecular haloamidation of
N-protected 3-hydroxy-4-pentenylamines and 4-hydroxy-5-hex-
enylamines. J . Org. Chem. 1988, 53, 5491-5501.
(37) Nimec, Z.; Galivan, J . Regulatory aspects of the glutamylation
of methotrexate in cultured hepatoma cells. Arch. Biochem.
Biophys. 1983, 226, 671-680.
(38) Foley, G. F.; Lazarus, H.; Farber, S.; Uzman, B. G.; Boone, B.
A.; McCarthy, R. E. Continuous culture of lymphoblasts from
peripheral blood of a child with acute leukemia. Cancer 1965,
18, 522-529.
(39) McCloskey, D. E.; McGuire, J . J .; Russell, C. A.; Rowan, B. G.;
Bertino, J . R.; Pizzorno, G.; Mini, E. Decreased folylpoly-
glutamate synthetase activity as a mechanism of methotrexate
resistance in CCRF-CEM human leukemia sublines. J . Biol.
Chem. 1991, 266, 6181-6187.
(40) 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.
(41) McGuire, J . J .; Russell, C. A.; Bolanowska, W. E.; Freitag, C.
M.; J ones, C. S.; Kalman, T. I. Biochemical and growth inhibition
studies of methotrexate and aminopterin analogues containing
a tetrazole ring in place of the γ-carboxyl group. Cancer Res.
1990, 50, 1726-1731.
(42) McGuire, J . J .; Bergoltz, V. V.; Heitzman, K. J .; Haile, W. H.;
Russell, C. A.; Bolanowska, W. E.; Kotake, Y.; Haneda, T.;
Nomura, H. Novel 6,5-fused-ring heterocyclic antifolates: Bio-
chemical and biological characterization. Cancer Res. 1994, 54,
2673-2679.
(43) 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.
(44) Bolanowska, W. E.; Russell, C. A.; McGuire, J . J . Activation of
mammalian folylpolyglutamate synthetase by sodium bicarbon-
ate. Arch. Biochem. Biophys. 1990, 281, 198-203.
J M950515E