Synthesis and biological evaluation of N(α)-(4-amino-4-deoxy-10- methylpteroyl)-DL-4,4-difluoroornithine

Article abstract of DOI:10.1021/jm960046w

N(α)-(4-Amino-4-deoxy-10-methylpteroyl)-DL-4,4-difluoroornithine (AMPte- DL-4,4-F₂Orn, 4) was synthesized and evaluated as an inhibitor of human folylpoly-γ-glutamate synthetase (FPGS), dihydrofolate reductase (DHFR), and cell growth. Synthesis

Full text of DOI:10.1021/jm960046w

2536
J . Med. Chem. 1996, 39, 2536-2540
Syn th esis a n d Biologica l Eva lu a tion of
Nr-(4-Am in o-4-d eoxy-10-m eth ylp ter oyl)-DL-4,4-d iflu or oor n ith in e
Takashi Tsukamoto,† William H. Haile,§ J ohn J . McGuire,§ and J ames K. Coward*,†
Departments of Medicinal Chemistry and Chemistry, The University of Michigan, Ann Arbor, Michigan 48109, and
Department of Experimental Therapeutics, The Grace Cancer Drug Center, Roswell Park Cancer Institute,
Buffalo, New York 14263
Received J anuary 16, 1996X
NR-(4-Amino-4-deoxy-10-methylpteroyl)-DL-4,4-difluoroornithine (AMPte-DL-4,4-F2Orn, 4) was
synthesized and evaluated as an inhibitor of human folylpoly-γ-glutamate synthetase (FPGS),
dihydrofolate reductase (DHFR), and cell growth. Synthesis of 4 involved the use of a protected
form of DL-4,4-difluoroornithine 9 which was derived from DL-4,4-difluoroglutamic acid.
Biological activities of 4 were compared directly to those of the corresponding nonfluorinated
compound NR-(4-amino-4-deoxy-10-methylpteroyl)-L-ornithine (AMPte-L-Orn, 3). Although the
fluorinated analogue is a potent inhibitor of DHFR, it is a poor inhibitor of FPGS. However,
the compound is transported across the cell membrane and inhibits cell growth, presumably
due to the inhibition of DHFR. The data obtained with the fluorinated analogue are in contrast
to those of the corresponding nonfluorinated compound 3, which is a potent inhibitor of both
FPGS and DHFR but shows very low cytotoxicity due to poor transport.
In tr od u ction
Reduced folates play an important role as coenzymes
for folate-dependent enzymes and are required for the
one-carbon transfers involved in biosynthesis of certain
amino acids and nucleic acid precursors.1 Reduced
folates are present in cells as poly-γ-glutamate conju-
gates, which are preferred substrates for most folate-
dependent enzymes.2 These poly-γ-glutamate conju-
gates are polyanionic compounds that do not readily
cross the cell membrane and thus are well retained by
cells.3 The intracellular formation of the poly-γ-
glutamate conjugates is catalyzed by an ATP-dependent
enzyme, folylpoly-γ-glutamate synthetase (FPGS), as
shown below.
PteGlun + ATP + L-Glu f PteGlun+1 + ADP + Pi
Inhibitors of FPGS could be useful agents for studying
the physiological roles of folylpolyglutamates and could
be effective chemotherapeutic agents. Shane and co-
workers reported that NR-(tetrahydropteroyl)-L-orni-
of possible cytotoxic effect on cell growth and/or viability
thine (1) is a potent inhibitor of mammalian FPGS (Ki
elicited by specific inhibition of FPGS. The second class
of inhibitors is important because they may show
) 0.2 µM).4,5 Since their pioneering work, numerous
ornithine-containing analogues with modified pteroyl
enhanced cytotoxicity due to their two sites of action.
moieties have been synthesized and were found, in
Unfortunately, all of the ornithine-containing analogues
general, to be very effective inhibitors of FPGS.6-10 The
of folate and antifolates were only weakly inhibitory to
potency of the inhibitors is dependent on the nature of
cell growth. This has been attributed to poor transport
the pteroyl moiety. They are classified into two groups
of the inhibitors across the cell membrane caused by
of interest: (1) specific inhibitors of FPGS, represented
by a series of 5,8-dideazapteroyl (quinazoline) analogues
of pteroylornithine (e.g., 2),8-10 and (2) so-called “dual-
site” inhibitors that act against both FPGS and a second
folate-dependent enzyme such as dihydrofolate reduc-
tase (DHFR). The methotrexate (MTX) analogue NR-
(4-amino-4-deoxy-10-methylpteroyl)-L-ornithine (AMPte-
L-Orn, 3) and other 2,4-diamino derivatives5-7,9 belong
to this second class. The first class of inhibitors is of
interest in connection with the long-standing question
the positively charged terminal amino group (pKa
)
10.8) of ornithine.
Poor transport might be overcome by replacing the
ornithine residue in these inhibitors with 4,4-difluoro-
ornithine (4,4-F2Orn). This amino acid is presumed to
have a neutral terminal amino group at physiological
pH due to the electron-withdrawing effect of the fluorine
atoms. In fact, 5,5-difluorolysine has been synthesized
previously, and the basic dissociation constant of the
terminal amino group was determined to be 6.88.11
Assuming that 4,4-F2Orn has a similar pKa value as 5,5-
difluorolysine, 4,4-F2Orn-containing folate and anti-
† The University of Michigan.
§ Roswell Park Cancer Institute.
X Abstract published in Advance ACS Abstracts, J une 1, 1996.
S0022-2623(96)00046-5 CCC: $12.00 © 1996 American Chemical Society

Evaluation of AMPte-DL-F2Orn
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2537
Sch em e 1
Sch em e 2
folate analogues possibly could be transported more
efficiently across cell membranes.
azide 7.15 Attempted reduction16 of the amide 6 using
borane-dimethyl sulfide was nonselective based on a
19F NMR spectrum of the crude product, which appeared
to be a mixture of several fluorinated compounds, the
structures of which have not been established. Subse-
quent derivatization of the crude amine as a tert-butyl
carbamate afforded 8 in 33% yield (26% overall yield
from 5). Azide 7 was then examined as an alternate
precursor of 8. Treatment of the azide 7 with Ph3P in
THF-H2O followed by protection of the terminal amino
group afforded 8 in 41% yield (27% overall yield from
5). The best yield of 8 was obtained when the azide 7
was treated with H2 over Pd/C (1 atm) in the presence
of di-tert-butyl dicarbonate.17 This convenient one-pot
procedure gave 8 in 82% yield (54% overall yield from
5). It should be noted that the benzyl carbamate group
of 7 remained intact during the catalytic hydrogenation.
However, under H2 at higher pressure (50 psi) the Cbz
group of 8 was readily cleaved to give 9 in 87% yield.
The 4,4-difluoroornithine derivative 9 is an ideal pre-
cursor for the synthesis of 4,4-difluoroornithine-contain-
ing folate and antifolate analogues for the following
reasons: (1) the unprotected R-amino group favors
coupling to a wide variety of pteroyl moieties, (2) the
protection of both the terminal δ-amino group and
R-carboxylic acid allows easy purification, if necessary,
of synthetic intermediates, and (3) the tert-butyl ester
and the tert-butyl carbamate can be readily cleaved
together in the final step of the synthesis by treatment
with TFA.
Since there is no well-defined phenotype of FPGS-
inhibited cells, the biological evaluation of FPGS-specific
inhibitors is not straightforward and therefore, the
evaluation of transport ability of 4,4-F2Orn-containing
FPGS-specific inhibitors could be complicated. In con-
trast, inhibition of DHFR by dual-site inhibitors is
expected to result in decreased cell growth. Therefore,
4,4-F2Orn-containing analogues of the dual-site inhibi-
tors could be more straightforward probes in studying
the effect of fluorine substituent on the transport
capability of the inhibitors. Thus, as an initial target,
we have focused our effort on a potential dual-site
inhibitor and have synthesized the 4,4-F2Orn-containing
methotrexate analogue NR-(4-amino-4-deoxy-10-meth-
ylpteroyl)-DL-4,4-difluoroornithine (AMPte-DL-F2Orn, 4).
The biological activity of this new analogue has been
compared to the corresponding nonfluorinated com-
pound 3.
Ch em istr y
The synthetic studies first focused on preparation of
the 4,4-difluoroornithine portion of the target molecule.
Initially, we investigated DAST-mediated difluorina-
tion12 of synthetically available L-4-oxoornithine deriva-
tives.13,14 Our attempts, however, proved unsuccessful
as either aziridine formation or poor reactivity was
found in the reactions of these substrates (see Support-
ing Information for details).
We found recently that N-Cbz-protected di-tert-butyl
4,4-difluoroglutamate 5 undergoes various regioselective
nucleophilic additions at the γ-carbonyl group (Scheme
1), thus leading to a facile synthesis of amide 6 and
Incorporation of a pteroyl moiety into 9 has been
performed according to the method reported by Piper
and co-workers.18,19 Condensation of 9 with the N-Cbz-
protected derivative of N-methyl-4-aminobenzoic acid
afforded 10 (93% yield), which was subsequently depro-
tected by catalytic hydrogenation to give 11 in 88% yield
(Scheme 2). Coupling of 11 to 6-(bromomethyl)-2,4-
pteridinediamine hydrobromide in dimethylacetamide
followed by hydrolysis with TFA and precipitation from
ether gave 4 as a yellow solid in 44% overall yield for
the final two steps.

2538 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Tsukamoto et al.
for its effect on the growth of CCRF-CEM human
leukemia cells. The data in Table 1 demonstrate that
4 is more potent than is 3. The growth and DHFR
inhibitory potency data suggest that transport of the
fluorinated ornithine analogue 4 into cells is consider-
ably improved over that of the corresponding nonflu-
orinated species 3.
Con clu sion s
In order for a molecule to affect FPGS activity
intracellularly, two requirements must be satisfied as
follows: (1) The molecule is efficiently transported into
cells. (2) The molecule is an inhibitor of FPGS. Com-
pound 3 meets the second requirement but not the first,
while the opposite is true for compound 4. The data
presented here indicate that, as has been suggested by
several research groups, poor transport of ornithine-
containing analogues of folates and antifolates is due
to the positively charged terminal amino group. The
transport capability can be improved by reducing the
basicity of the terminal amino group as clearly shown
with compound 4. However, such modification results
in loss of FPGS inhibitory activity. Since the structure
of any complex involved in FPGS catalysis has not been
established, the molecular basis for the marked differ-
ence in the inhibition observed between 3 and 4 is
unclear. One possibility is that the positively charged
terminal amino moiety of 3 binds via an electrostatic
interaction with an anionic residue on FPGS. This
would explain the lack of FPGS inhibition observed with
4 since the neutral form is the predominant species at
physiological pH. The structural requirements for
transport and FPGS inhibition observed with 3 and 4
present a problem in designing ornithine-containing
analogues as inhibitors of FPGS that will be effective
both in vitro and in vivo. The present findings should
be considered in future research on the development of
FPGS inhibitors that are capable of being transported
into cells.
F igu r e 1. Inhibition of FPGS-catalyzed glutamylation of MTX
by AMPte-L-Orn (3; O) and AMPte-DL-4,4-F2Orn (4; b). For
AMPte-DL-4,4-F2Orn, only the concentration of L-isomer is
indicated.
Ta ble 1. Biological Evaluation of Ornithine-Containing
Analogues of Methotrexatea
target
3
4b
33 ( 2 >300
2.5f
1.3 ( 0
93 ( 17
MTX
FPGS,c IC50 (µM)
DHFR,d IC50 (nM)
0.82 ( 0.04
14.5 ( 0.5
cell growth,e ED50 (nM) 740f
a
All experiments were done in duplicate, and data are pre-
b
sented as average values ( range. All concentrations shown are
for the L-isomer; it has been demonstrated that the D-isomer of
MTX is biologically inactive.29,30 c FPGS isolated from CCRF-CEM
human leukemia cells.25 DHFR isolated from CCRF-CEM hu-
d
man leukemia cells.25 e 120 h continuous exposure of CCRF-CEM
human leukemia cells. f Literature data7 for DHFR inhibition and
cytotoxicity for MTX are as follows: DHFR IC50 ) 1.0 nM, ED50
) 11 nM.
Biologica l Eva lu a tion
The new MTX analogue 4 was first evaluated as an
inhibitor of human FPGS. As shown in Figure 1,
compound 4 was found to be devoid of inhibitory activity
at concentrations as high as 150 µM. In contrast, the
nonfluorinated analogue NR-(4-amino-4-deoxy-10-meth-
ylpteroyl)-L-ornithine, 3, was confirmed as a potent
inhibitor of FPGS (Figure 1). Assuming that the pKa
of the δ-amino group of ornithine in 3 is unchanged from
that of the free amino acid (pKa ) 10.8), the predomi-
nant species present in solution under standard FPGS
assay conditions (pH ) 8.5) would be the protonated
form. This would not be the case with 4, again assum-
ing that the pKa of 4 is unchanged from that of the free
amino acid (pKa ) ca. 6.9, vide supra). In order to
evaluate any pH dependence of the inhibition by 4,
assays of FPGS activity were run at pH ) 7.9, 8.5, and
9.0 in the presence of 4 (200 µM). There was no
significant inhibition of FPGS by 4 under any of the
assay conditions. It is not possible to evaluate 4 at lower
pH values because control enzyme activity declines
sharply below pH 7.9.
Although the lack of FPGS inhibition by 4 was
disappointing, we pursued other aspects of antifolate
activity (i.e., inhibition of DHFR and cell growth) in
order to assess the ability of 4 to cross cell membranes
and ultimately to compare the cytotoxic effects of 4 vs
3. As shown in Table 1, compound 4 is a potent
inhibitor of DHFR and comparable to both MTX and
the ornithine analogue 3. Given the potent inhibition
of DHFR by 4, this new MTX analogue was evaluated
Exp er im en ta l Section
Thin layer chromatography was performed with E. Merck
silica gel 60 F-254 plates. Column chromatography was
performed with silica gel 60 (230-400 mesh). 1H and 13C NMR
spectra were recorded on either a Bruker AM-300 or AM-360
spectrometer. 19F NMR spectra were obtained using a General
Electric AM-500 spectrometer. Chemical shifts are in parts
per million downfield from tetramethylsilane (internal stan-
1
dard for H and 13C) 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, De-
partment of Chemistry, The University of Michigan, or At-
lantic Microlabs, Atlanta, GA. UV spectra were obtained on
a Beckman Model DU-7 spectrophotometer. High-pressure
hydrogenations were carried out with a Parr hydrogenator.
Compound 3 was synthesized by the method of Piper et al.19
N2-[(Ben zyloxy)ca r b on yl]-N5-[(1,1-d im et h ylet h oxy)-
ca r bon yl]-DL-4,4-d iflu or oor n ith in e, ter t-Bu tyl Ester (8).
To a solution of 715 (304 mg, 0.79 mmol) and di-tert-butyl
dicarbonate (207 mg, 0.95 mmol) in ethyl acetate (10 mL) was
added 10% Pd/C (20 mg), and the suspension was stirred under
H2 (1 atm) for 30 min. The catalyst was removed by filtration,
and the filtrate was concentrated. The resulting oil was
purified by silica gel chromatography (hexane/EtOAc, 4/1) to
give 296 mg (82% yield) of 8 as a colorless oil: Rf 0.33 (hexane/
1
EtOAc, 4/1); IR (neat) 3353, 2981, 2938, 1736, 1694 cm-1; H
NMR (CDCl3) δ 1.46 (s, 18 H), 2.2-2.6 (m, 2 H), 3.4-3.7 (m,

Evaluation of AMPte-DL-F2Orn
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2539
2 H), 4.51 (dt, J ) 5.6, 6.6 Hz, 1 H), 4.88 (br, 1 H), 5.13 (s, 2
H), 5.69 (d, J ) 7.3 Hz, 1 H), 7.2-7.4 (m, 5 H); 13C NMR
(CDCl3) δ 28.0 (3 C), 28.4 (3 C), 35.7 (t, J ) 23.3 Hz), 45.4 (t,
J ) 30.3 Hz), 50.2, 67.1, 80.6, 82.6, 122.4 (t, J ) 244 Hz), 128.2
(2 C), 128.3, 128.7 (2 C), 136.5, 155.9, 156.0, 170.1; 19F NMR
(CDCl3) δ -27.0 (ddq, J ) 9.5, 251, 11.8 Hz, 1 F), -25.4 (ddq,
J ) 7.4, 251, 13.2 Hz, 1 F); MS (CI) m/e (rel intensity) 459
(MH+, 24.8), 420 (27.2), 403 (30.2), 393 (26.3), 364 (100), 347
(53.4), 303 (99.0); HRMS (CI) m/e calcd for C22H32F2N2O6H
(MH+) 459.2307, found 459.2322. Anal. (C22H32F2N2O6‚0.5H2O)
C, H, N.
122.6 (t, J ) 244 Hz), 129.2 (2 C), 152.3, 156.1, 167.2, 170.6;
19F NMR (CDCl3) δ -23.9 (dtt, J ) 251, 12.6, 17.9 Hz, 1 F),
-26.7 (ddq, J ) 251, 19.3, 13.3 Hz, 1 F); MS (EI) m/e (rel
intensity) 457 (M+, 5.5), 383 (2.1), 328 (5.9), 300 (6.1), 134
(100); HRMS (EI) m/e calcd for C22H33F2N3O5 (M+) 457.2388,
found 457.2391.
N2-(4-Am in o-4-d eoxy-10-m eth ylp ter oyl)-DL-4,4-d iflu o-
r oor n ith in e (4). 6-(Bromomethyl)-2,4-pteridinediamine hy-
drobromide18 (35 mg, 0.09 mmol) was added to a solution of
11 (40 mg, 0.09 mmol) in dry DMAC (1.0 mL) at 0 °C, and the
mixture was stirred at 55 °C for 6 h. After additional stirring
(6 days) at ambient temperature, the solvent was removed by
evaporation, and the resulting yellow oil was dissolved in a
minimum amount of CH2Cl2/MeOH (20/1) and passed through
a short silica gel column (CH2Cl2/MeOH, 20/1) to remove polar
impurities. The purified di-tert-butyl ester was then dissolved
in trifluoroacetic acid (2 mL), and the resulting solution was
stirred at ambient temperature for 40 min. The reaction
mixture was poured into dry ether (10 mL); the resulting
precipitate was isolated by filtration and dried to give 28 mg
of 4 (44%) as a bright-yellow solid: Rf 0.64 (EtOH/concentrated
NH4OH, 4/1); 1H NMR (CD3OD) δ 2.6-3.0 (m, 2 H), 3.27 (s, 3
H), 3.4-3.7 (m, 2 H), 4.8-5.0 (m, 1 H), 6.85 (d, J ) 8.2 Hz, 2
H), 7.75 (d, J ) 7.9 Hz, 2 H), 8.63 (s, 1 H); 13C NMR (CD3OD)
δ 37.3 (t, J ) 22.8 Hz), 39.9, 44.5 (t, J ) 25.2 Hz), 48.7, 56.7,
112.8 (2 C), 122.1 (t, J ) 244 Hz), 122.6, 123.5, 130.4 (2 C),
147.0, 150.5, 153.2, 153.8, 157.9, 165.0, 170.0, 173.9; 19F NMR
(CD3OD) δ -29.0 (dm, J ) 245 Hz, 1 F), -27.8 (dm, J ) 245
Hz, 1 F); UV λmax (pH ) 13) 258, 303, 371; MS (FAB+) m/e (rel
intensity) 476 (MH+, 71.2), 308 (40.8), 176 (100), 103 (91.7);
HRMS (FAB+) m/e calcd for C20H23F2N9O3H (MH+) 476.1970,
found 476.1954. Anal. (C20H23F2N9O3‚2CF3COOH‚H2O) C, H;
N 5-[(1,1-Dim e t h yle t h oxy)ca r b on yl]-DL-4,4-d iflu or o-
or n ith in e, ter t-Bu tyl Ester (9). To a solution of 8 (230 mg,
0.50 mmol) in dry ethanol (10 mL) was added 10% Pd/C (20
mg), and the mixture was shaken under H2 (50 psi) for 11 h.
Catalyst was removed by filtration, and the filtrate was
concentrated. The resulting residue was purified by column
chromatography (hexane/EtOAc, 1/1) to give 142 mg (87%
yield) of 9 as a colorless oil: Rf 0.21 (hexane/EtOAc, 1/2); IR
1
(neat) 3386, 2984, 2934, 1727 cm-1; H NMR (CDCl3) δ 1.40
(s, 9 H), 1.42 (s, 9 H), 1.63 (br, 2 H), 2.02 (ddt, J ) 8.5, 18.6,
15.4 Hz, 1 H), 2.33 (dddd, J ) 4.1, 12.0, 14.6, 20.6 Hz, 1 H),
3.3-3.6 (m, 3 H), 5.28 (br, 1 H); 13C NMR (CDCl3) δ 28.1 (3
C), 28.5 (3 C), 39.1 (t, J ) 24.1 Hz), 45.5 (t, J ) 29.9 Hz), 50.6,
60.5, 65.9, 80.1, 81.8, 122.6 (t, J ) 243 Hz), 155.7, 173.6; 19F
NMR (CDCl3) δ -27.2 (ddq, J ) 13.3, 250, 19.3 Hz, 1 F), -24.3
(dp, J ) 250, 14.6 Hz, 1 F); MS (CI) m/e (rel intensity) 325
(MH+, 100), 269 (63.3), 251 (7.8), 225 (8.9), 213 (40.7); HRMS
(CI) m/e calcd for C14H26F2N2O4H (MH+) 325.1939, found
325.1934. Anal. (C14H26F2N2O4) C, H, N.
N2-[4-[[(Ben zyloxy)ca r b on yl]m et h yla m in o]b en zoyl]-
N5-[(1,1-d im et h ylet h oxy)ca r b on yl]-DL-4,4-d iflu or oor n i-
th in e, ter t-Bu tyl Ester (10). To a solution of 4-[[(benzyloxy)-
carbonyl]methylamino]benzoic acid20 (122 mg, 0.43 mmol) in
dry DMF (3 mL) were added DCC (121 mg, 0.59 mmol) and
HOBt (105 mg, 0.78 mmol) at 0 °C, and the mixture was
stirred at ambient temperature for 10 min. A solution of 9
(127 mg, 0.39 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 (DMF) was removed under reduced pressure. The
resulting crude oil was dissolved in a small volume of hexane/
EtOAc, 1/1 (7 mL), and again filtered. The filtrate was
evaporated, and the resulting oil was purified by silica gel
chromatography (hexane/EtOAc, 4/1) to give 215 mg (93%
yield) of the product 10 as a colorless oil; Rf 0.55 (hexane/
N: calcd, 17.47; found, 17.94. Reversed-phase HPLC:21 tR
26.2 min.
)
Bioch em ica l Tech n iqu es: Cell Lin es. The human T-
lymphoblastic leukemia cell line CCRF-CEM22 the primary
screen for drug effects and was the source of tumor enzymes.
Routine culture of these lines was as described.23 CCRF-CEM
was verified to be negative for mycoplasma contamination
during the course of these studies using the GenProbe test
kit.
En zym es a n d Assa ys: FPGS and DHFR from CCRF-CEM
cells22 were partially purified as described.24 The FPGS assay
method25 uses [3H]Glu to radiolabel polyglutamates of a folate-
like substrate during incubation with the enzyme. The
reaction mixture is applied 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. CCRF-CEM FPGS assay conditions26
were modified to include 50 µg of protease-free bovine serum
albumin (Miles). All reagents were made with freshly pro-
cessed deionized water, stored frozen in aliquots, and used in
only 1-2 experiments to avoid absorption of atmospheric CO2
which can lead to erroneous FPGS kinetic constants.26 DHFR
activity was assayed spectrophotometrically as described.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
increasing concentrations of an antifolate to standard FPGS
or DHFR assays and measuring the remaining activity.
EtOAc, 1/1); IR (neat) 3346, 2981, 2931, 1736, 1652, 1610 cm-1
;
1H NMR (CDCl3) δ 1.45 (s, 9 H), 1.47 (s, 9 H), 2.4-2.7 (m, 2
H), 3.34 (s, 3 H), 3.3-3.8 (m, 2 H), 4.88 (dt, J ) 4.6, 7.4 Hz, 1
H), 5.07 (t, J ) 6.5 Hz, 1 H), 5.17 (s, 2 H), 7.2-7.4 (m, 7 H),
7.27 (d, J ) 7.4 Hz, 1 H), 7.86 (d, J ) 8.4 Hz, 2 H); 13C NMR
(CDCl3) δ 28.0 (3 C), 28.4 (3 C), 34.9 (t, J ) 24.5 Hz), 37.5,
45.3 (t, J ) 30.7 Hz), 49.2, 67.7, 80.7, 82.8, 122.4 (t, J ) 244
Hz), 125.1 (2 C), 128.0 (2 C), 128.1, 128.2 (2 C), 128.6 (2 C),
131.0, 136.4, 146.3, 155.2, 156.2, 166.5, 170.2; 19F NMR (CDCl3)
δ -27.0 (dm, J ) 252 Hz, 1 F), -22.9 (dm, J ) 252 Hz, 1 F);
MS (EI) m/e (rel intensity) 591 (M+, 0.9), 518 (0.6), 462 (4.9),
434 (9.8), 285 (34.3), 268 (36.0), 91 (100); HRMS (EI) m/e calcd
for C30H39F2N3O7 (M+) 591.2756, found 591.2757. Anal.
(C30H39F2N3O7‚0.5H2O) C, H, N.
N5-[(1,1-Dim eth yleth oxy)ca r bon yl]-N2-[4-(m eth yla m i-
n o)b en zoyl]-DL-4,4-d iflu or oor n it h in e, ter t-Bu t yl E st er
(11). Palladium hydroxide on carbon (19 mg) was added to a
solution of 10 (147 mg, 0.25 mmol) in dry ethanol (7 mL), and
the mixture was shaken under H2 (40 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 chromatography (hexane/EtOAc, 4/1) to give 99 mg
of 11 (88%) as a colorless oil: Rf 0.38 (hexane/EtOAc, 1/1); IR
Ack n ow led gm en t. This research was supported by
grants from the National Cancer Institute, CA28097
(J .K.C.), CA43500 (J .J .M.), and CA16056 (J .J .M.). We
thank Scott N. VanderWel and Robert N. Sibley for the
synthesis of key intermediates. We thank Ms. Carol
Capelle for careful preparation of the manuscript.
(neat) 3360, 2931, 1729, 1645, 1609, 1518 cm-1 1H NMR
;
(CDCl3) δ 1.45 (s, 9 H), 1.47 (s, 9 H), 2.4-2.7 (m, 2 H), 2.86 (s,
3 H), 3.4-3.7 (m, 2 H), 4.1-4.4 (br, 1 H), 4.88 (dt, J ) 5.0, 7.1
Hz, 1 H), 5.00 (t, J ) 6.2 Hz, 1 H), 6.56 (d, J ) 8.6 Hz, 2 H),
7.10 (d, J ) 7.5 Hz, 1 H), 7.72 (d, J ) 8.6 Hz, 2 H); 13C NMR
(CDCl3) δ 28.0 (3 C), 28.4 (3 C), 30.4, 35.5 (t, J ) 23.9 Hz),
45.4 (t, J ) 30.3 Hz), 48.9, 60.6, 80.6, 82.6, 111.5 (2 C), 121.9,
Su p p or tin g In for m a tion Ava ila ble: Details of DAST-
mediated difluorination of 4-oxoornithine derivatives (1 page).
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2540 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Tsukamoto et al.
(15) Tsukamoto, T.; Coward, J . K. Facile Synthesis of DL-4,4-
difluoroornithine, DL-4,4-difluoroglutamine, and γ-DL-4,4-di-
fluoroglutamyl-containing peptides: Regiospecific addition of
nucleophiles to N-Cbz-di-tert-butyl-DL-4,4-difluoroglutamate. J .
Org. Chem. 1996, 61, 2497-2500.
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