Design and synthesis of histidine analogues of folic acid and methotrexate as potential folylpolyglutamate synthetase inhibitors

Article abstract of DOI:10.1021/jm960250j

Folylpolyglutamate synthetase (FPGS) is responsible for the conversion of naturally occurring folates and antifolates to their poly-γ-glutamyl derivatives, which are the forms required for intracellular retention of folates and are also the preferred subs

Full text of DOI:10.1021/jm960250j

4340
J . Med. Chem. 1996, 39, 4340-4344
Design a n d Syn th esis of Histid in e An a logu es of F olic Acid a n d Meth otr exa te a s
P oten tia l F olylp olyglu ta m a te Syn th eta se In h ibitor s
Zhenmin Mao, J ianping Pan, and Thomas I. Kalman*
Department of Medicinal Chemistry, School of Pharmacy, State University of New York at Buffalo, Buffalo, New York 14260
Received March 29, 1996X
Folylpolyglutamate synthetase (FPGS) is reponsible for the conversion of naturally occurring
folates and antifolates to their poly-γ-glutamyl derivatives, which are the forms required for
intracellular retention of folates and are also the preferred substrates (cofactors) for most folate-
dependent enzymes. Folate and methotrexate analogues 6 and 4, with L-histidine in place of
L-glutamate, were designed and synthesized as potential FPGS inhibitors. Target compound
5, the Nτ-(carboxymethyl)-L-histidine derivative of 4, was also prepared. Compounds 4 and 5
inhibited the growth of L1210 cells (IC50 values: 0.091 and 0.15 µM, respectively) and were
potent inhibitors of L1210 dihydrofolate reductase. No significant inhibition of FPGS by 4, 5,
or 6 was observed at the high pH of the standard enzyme assay. This could be the consequence
of a lack of protonation of the basic side chains, which is likely to be required for FPGS inhibitory
activity. The observed cytotoxicity indicates that partial protonation of the imidazole ring
permits cellular uptake of the analogues.
Polyglutamylation is an essential process for cellular
retention and efficient utilization of folates and anti-
folates. Most folate-requiring enzymes prefer the poly-
γ-glutamate forms of folates as cofactors with a chain
of four or more glutamate residues.1-3 The enzyme
responsible for the polyglutamylation process is folylpoly-
γ-glutamate synthetase (FPGS),4-7 which requires L-
glutamic acid, ATP, and Mg2+ to carry out chain
elongation.8 The importance of this enzyme is clearly
illustrated by the auxotrophy of a Chinese hamster
ovary cell mutant (AUXB1) which has no measurable
activity of FPGS and, as a consequence, requires end
products of folate-dependent pathways for survival.9
Folate analogues targeting FPGS have been studied
widely as potential anticancer agents.10-16 The most
potent FPGS inhibitors are folate analogues in which
the glutamic acid moiety is replaced by L-ornithine, e.g.,
NR-pteroyl-L-ornithine (1).14e Ornithine derivatives of
methotrexate (MTX; see 2, 3), aminopterin, and other
antifolates have also been synthesized.14a,c,d In com-
interference with cellular uptake.12-16 It was of interest
to investigate whether a lowering of the basicity of the
amino acid side chain and consequently the positive
charge density would improve cellular transport without
compromising FPGS inhibitory activity. To that end,
analogues of folic acid and methotrexate with L-histidine
and Nτ-(carboxymethyl)-L-histidine side chains (4-6)
were synthesized and tested for inhibition of FPGS and
cell growth in vitro.
The pKa of the imidazole ring of histidine is 5.97,17a
substantially lower than that of the δ-amino group of
ornithine (10.76),17b permitting the existence of signifi-
cant fractions of both unprotonated and protonated
species of compounds 4-6 at physiological pH. Since
ionization may play opposite roles in cellular uptake and
enzyme inhibitory activity, both requirements could be
simultaneously satisfied. It was postulated that in the
enzyme-inhibitor complex the protonated δ-amino group
of mAPA-Orn may occupy the binding site of the
protonated R-amino group of the incoming glutamate.12b,c
Since L-histidine may be viewed as a conformationally
restricted analogue of L-ornithine, it is possible that its
τ-nitrogen can occupy the same FPGS binding site as
the δ-amino group of mAPA-Orn (Figure 1). The
carboxymethyl group at this position (see 5) incorporates
the R-CH-COOH moiety of the incoming glutamate and
was designed to provide additional interactions at the
substrate binding site.
parison to their acidic side chain counterparts, com-
pounds with the basic ornithine side chain have greater
affinities toward FPGS.13 It is likely that the proto-
nated δ-amino group of ornithine is responsible for
enhanced FPGS inhibitory activity. In contrast, proto-
nation of the basic side chain reduces the cytotoxicity
of ornithine derivatives of antifolates, presumably by
X Abstract published in Advance ACS Abstracts, September 15, 1996.
S0022-2623(96)00250-6 CCC: $12.00 © 1996 American Chemical Society

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4341
Sch em e 2. Synthetic Route to Compounds 4-6
F igu r e 1. Postulated analogy between the δ- and τ-nitrogens
of the ornithine and histidine derivatives of MTX, respectively,
and the R-nitrogen of glutamic acid bound to FPGS.
Sch em e 1. Synthetic Route to Intermediate 11
mixture, a stream of anhydrous HBr gas was passed
for 1 h to remove the BOM protecting group from the
Nπ-position of the imidazole ring to give compound 11
in good yield. The reversed order of treatment (first
passage of HBr gas in CH2Cl2 followed by TFA) resulted
in poor yield.
Pteroic acid and 4-amino-4-deoxy-N10-methylpteroic
acid (mAPA) were obtained by enzymatic hydrolysis of
folic acid and MTX, respectively, using carboxypeptidase
G1.21 The synthesis of the protected 4-amino-4-deoxy-
N10-methylpteroic acid derivatives 13 and 14 is outlined
in Scheme 2. The coupling of mAPA with both L-
histidine methyl ester dihydrochloride (7) and Nτ-
alkylated L-histidine ester 11 were achieved using
diethyl cyanophosphonate (DEPC) yielding 13 and 14,
respectively. The DEPC coupling reaction gave moder-
ate yields under a variety of conditions. Compounds 13
and 14 were hydrolyzed in aqueous NaOH solution to
yield target compounds 4 and 5.
Ch em istr y
The synthesis of NR-(4-amino-4-deoxy-N10-methyl-
pteroyl)-L-histidine (mAPA-His, 4) utilized the com-
mercially available L-histidine methyl ester (7) as start-
ing material. NR-(4-Amino-4-deoxy-N10-methylpteroyl)-
Nτ-(carboxymethyl)-L-histidine (mAPA-CMHis, 5) was
prepared from Nτ-[[(ethyloxy)carbonyl]methyl]-L-histi-
dine methyl ester (11) as shown in Scheme 1. The
synthesis of Nτ-[[(ethyloxy)carbonyl)methyl]-L-histidine
was reported by Hofmann et al. in their studies of
S-peptide antagonists.18 In their approach, ethyl bro-
moacetate was used to alkylate unprotected histidine
directly, yielding a mixture of Nπ-monoalkylated, Nτ-
monoalkylated, and Nπ,Nτ-dialkylated amino acids,
necessitating the separation of the two monoalkylated
products. In our study, an alternative method was used
to obtain exclusively the desired Nτ-monoalkylated
derivative. Nπ-(Benzyloxy)methyl (BOM)-protected L-
histidine 8 was first esterified in MeOH in the presence
of DCC. The resulting methyl ester 9 was alkylated
with ethyl bromoacetate in DMF. The yield was satis-
factory when DMF was used as the solvent. Other
solvents (e.g., THF) gave lower yields and required a
longer reaction time. To convert the hygroscopic di-
alkylated salt 10 to the deprotected intermediate 11,
both the BOM group and the t-BOC group of 10 had to
be removed. The preferred method of removal of the
BOM group is hydrogenation in the presence of a Pd/C
catalyst.19 Other procedures employed trimethylsilyl
iodide or bromide,20 but they may also cleave the ester
groups of compound 10. We found that in trifluoroacetic
acid (TFA), anhydrous gaseous hydrogen bromide cleaves
the BOM group leaving the ester group intact. Thus,
compound 10 was first treated with anhydrous TFA in
methylene chloride at 0 °C to remove the t-BOC group
on the NR-amino group, and then, in the same reaction
The synthesis of pteroyl-L-histidine (6, Pte-His) was
carried out as shown in Scheme 2, starting with N10
-
trifluoroacetyl-protected pteroic acid 15, which was
prepared by acylation of pteroic acid with trifluoroacetic
anhydride. Mixed carboxylic-carbonic anhydride cou-
pling is the method used most often to obtain derivatives
of pteroic acid protected at N10 by a trifluoroacetyl or
formyl group.12-16 This method involves one or more
cycles of addition of i-BuOCOCl and the amino acids
giving fair to poor yields. The DEPC reagent frequently
used in mAPA coupling was previously found by us to
cleave the N10-trifluoroacetyl group from the protected
pteroate.21 In the course of investigating different
coupling conditions, we found that propylphosphonic
anhydride (PPA)22 is a superior reagent for the coupling
of protected pteroic acid and an amino acid ester. The
use of this reagent has many advantages over the
traditional mixed anhydride method:12-16 It involves
simple reaction conditions and gives better yields and
a single reaction product allowing easy workup. Thus,
N10-(trifluoroacetyl)pteroic acid was coupled with L-
histidine methyl ester (7) in the presence of PPA in
N-methylpyrrolidinone at room temperature to yield the
protected compound 16. Hydrolysis of 16 in aqueous
alkaline solution gave the folic acid derivative 6. Both
compounds 4 and 6 were further purified by HPLC on

4342 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Notes
Ta ble 1. Cell Growth and Enzyme Inhibitory Activity
IC50 (µM)a
The observation that 5 is more inhibitory to DHFR than
4 but less cytotoxic seems to indicate that 5 is less
effectively transported into cells. This conclusion is
supported by the finding that in contrast to its higher
inhibitory activity in the permeabilized system, 5 is
3-fold less active than 4 as an inhibitor in the intact
cell assay21,25 (data not shown).
compound
L1210 growth L1210 DHFRb CEM FPGSc
mAPA-His (4)
mAPA-CMHis (5) 0.15 ( 0.020
Pte-His (6)
0.091 ( 0.020 1.3 ( 0.63
>200
>200
>200
0.37 ( 0.096
>100
>100
MTX
0.007 ( 0.0015 0.073 ( 0.020
1.30d
3.2e
F P GS In h ibition Stu d ies. None of the target
compounds showed significant inhibition of FPGS under
standard assay conditions (pH 8.5). Since we were
interested in the activity of the protonated species, a
lowering of the pH of the assay was attempted. How-
ever, at the lowest tolerated pH of 7.9, no significant
inhibitory activity was observed either. Whereas at pH
7.9-8.5 the δ-amino group of ornithine derivatives is
fully protonated (>99%), the imidazole ring of 4-6 is
not protonated to any significant extent (ca. 1%). This
may lead to weak binding of the histidine derivatives
of FPGS, since protonation of basic side chains appears
to be important for FPGS inhibitory activity.12b
a
Concentration required for 50% inhibition. Values are given
b
as mean ( standard deviation (when available). DHFR activity
was assayed in permeabilized L1210 cells in the presence of 200
µM H2folate as substrate.21 c CCRF-CEM FPGS activity was
measured at pH 8.5 and 7.9, as described.14b From ref 12b.
d
e McGuire, J . J . Unpublished results.
a C18 silica gel column with a linear water and methanol
gradient as the mobile phase. Compound 5 was purified
by cation exchange column chromatography (IR-120,
H+-form) using 5% NH4OH as solvent.
Biologica l Eva lu a tion a n d Discu ssion
Among folate analogues with altered amino acid side
chains,15 those with a basic side chain (e.g., L-ornithine
replacing L-glutamate) are the most potent inhibitors
of FPGS. However, these analogues suffer from poor
cellular uptake which limits their therapeutic utility.
We hypothesized that lowering the basicity of the side
chain, so that only partial protonation would occur at
physiological pH, may eliminate this shortcoming. Ac-
cordingly, target compounds 4-6 were evaluated as
inhibitors of the growth of L1210 murine leukemia cells
in culture and of dihydrofolate reductase (DHFR) in
permeabilized L1210 cells and as inhibitors of FPGS
from CCRF-CEM human leukemia cells. The results
are listed in Table 1.
Cell Gr ow th In h ibition Stu d ies. The histidine
analogue of MTX (4) was 13-fold less active than MTX
as an inhibitor of L1210 cell growth but 14-fold more
potent than the ornithine analogue of MTX.12b The
lower growth inhibitory potency of 4 compared to MTX
is understandable if one considers that only the unpro-
tonated species of compound 4 may be subject to cellular
transport. In contrast, compound 4 should be more
active than the fully protonated ornithine analogue 2
which cannot be transported effectively into the cell.
Compound 5 was slightly less active (IC50 ) 0.15 µM)
than its parent compound 4 (0.091 µM). The more polar
side chain of 5 may hamper membrane penetration by
the analogue resulting in lower cytotoxicity. It would
be of interest to directly compare the rate and pH
dependence of the cellular uptake of radioactive isotope-
labeled analogues, once they become available. Folic
acid derivatve 6 did not show significant cell growth
inhibition, as expected, since side chain-modified folic
acid derivatives generally lack significant cytotoxic-
ity.16,21 The results suggest that by optimizing the pKa
of potential FPGS inhibitors bearing basic side chains,
the opposite ionization requirements of cellular uptake
and enzyme inhibitory activity can be satisfied.23
In h ibition of DHF R. It is reasonable to assume
that the cytotoxicity of side chain-modified MTX ana-
logues is primarily due to inhibition of DHFR, since
structural variations at this region have relatively little
effect on enzyme inhibitory potency.15 Indeed, both 4
and 5 showed significant inhibitory activity against
DHFR with IC50 values of 1.3 and 0.37 µM, respectively.
Exp er im en ta l Section
Melting points were measured in open-ended capillary tubes
on a Mel-Temp apparatus and are uncorrected. 1H NMR
spectra were obtained on a Varian (300 MHz) or Varian EM-
390 (90 MHz) instrument. IR spectra were taken on a Nicolet
7000 Fourier Transform spectrophotometer. Elemental analy-
ses were performed by Atlantic Microlabs, Inc., Atlanta, GA.
The presence of associated solvent molecules was confirmed
by 1H NMR. Thin layer chromatography was performed on
Analtech silica gel HLF uniplates. Spots were visualized with
the aid of UV light, iodine, ninhydrin, or bromocresol green.
TLC systems were used as listed below, unless otherwise
stated: (A) 0.1 M KH2PO4/H2HPO4 (pH 7.4) or (B) 3% NH4Cl.
For silica gel column chromatography, Baker’s grade silica gel
(200 mesh) was used. HP20 was purchased from Kaser
Chemicals, Inc.; DEAE and CC31 cellulose were obtained from
Whatman Co. Most of the chemicals used were from Aldrich;
PPA (propylphosphonic anhydride) was from Hoechst Chemi-
cal, Inc. Triethylamine and N-ethylmorpholine were distilled
and stored over KOH. All commercial solvents were anhy-
drous or reagent grades. Carboxypeptidase G1 was obtained
from New England Enzyme Center, Boston, MA. Lyophiliza-
tions were carried out on a Virtis Unitrap II freeze-drier.
Folate analogues were protected from light whenever possible.
HPLC was performed on a Waters instrument with a
semipreparative upgrade. A C18 reversed phase silica gel (10
µm) column (7.8 × 30 mm) was purchased from Phenomenex.
The mobile phases were (1) 3% CH3CN and 0.01 M NH4OAc
solution isocratic system and (2) MeOH and H2O linear
gradient system, from H2O to MeOH. Fractions were moni-
tored by a Rainin Dynemax UV-1 dual-wavelength UV detector
at λ ) 292 nm. The purity of the target compounds was
confirmed by analytical HPLC on a C18 reversed phase silica
gel column (Phenomenex, 3.9 × 300 mm). The retention time
and solvent system used for each compound are reported in
their synthesis sections.
Nr-ter t-Boc-Nπ-[(ben zyloxy)m eth yl]-L-h istid in e Meth yl
Ester (9). NR-tert-Boc-Nπ-[(benzyloxy)methyl]-L-histidine (8)
(500 mg, 1.33 mmol) and 1,3-dicyclohexylcarbodiimide (DCC;
1.0 g, 4.85 mmol) were dissolved in MeOH (30 mL). The
reaction mixture was stirred at room temperature for 18 h.
The white precipitate of 1,3-dicyclohexylurea formed during
the reaction was filtered, and the filtrate was evaporated under
reduced pressure. A sticky residue was obtained which was
subjected to flash column chromatography on silica gel (CH2-
Cl2:MeOH ) 7:1). The pooled fractions were evaporated and
dried in vacuo to yield a sticky solid (0.39 g, 79%).
To 1.70 g of this solid, 10 mL-hexane and a couple of drops
of ether were added. The material was transformed gradually
to a white precipitate over a period of hours. After filtration,
compound 9 (1.2 g, 70.5%) was obtained as a white powder:

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4343
1
mp 99-101 °C; H NMR (CDCl3) δ 7.45 (s, 1H, im-H-2), 7.25
(s, 5H, Ph), 6.85 (s, 1H, im-H-5), 5.75 (d, 1H, R-NH), 5.25 (s,
2H, Ph-CH2), 4.40 (s, 2H, O-CH2-N), 4.10 (m, 1H, R-CH), 3.75
(s, 3H, COOCH3), 3.15 (d, 2H, CH-CH2-im), 1.40 (s, 9H,
C(CH3)3). Anal. (C20H27N3O5) C, H, N.
3H, 10-CH3), 3.03 (m, 2H, CH-CH2-im). Anal. (C21H22N10O3‚
3.0H2O) C, H, N.
Nr-(4-Am in o-4-d eoxy-N10-m eth ylp ter oyl)-Nτ-(ca r boxy-
m eth yl)-L-h istid in e (5). To a solution of mAPA (12; 1.0 g,
3 mmol) in dry DMF (15 mL) at room temperature under
nitrogen were added Et3N (1.08 mL, 7.7 mmol) and DEPC (0.63
mL, 4.2 mmol). The reaction mixture was stirred for 6 h.
Compound 11 (1.3 g, 3 mmol) in DMF (3 mL) and Et3N (0.6
mL) were added to the dark red solution of activated mAPA.
The mixture was stirred at room temperature for another 72
h. MeOH (10 mL) was added, and the mixture was stirred
for 30 min. It was evaporated at 40 °C to give a dark gummy
residue to which 5% NH4OH solution (30 mL) was added. A
yellow precipitate appeared, which was collected by centrifu-
gation (8000g, 30 min, 4 °C). The residue was washed with
0.1 N NaOH (20 mL) and water (20 mL). The solid was dried
by lyophilization to give methyl ester 14 (0.63 g, 37.4%) as a
Nr-ter t-Boc-N π-[(b en zyloxy)m et h yl]-Nτ-[[(et h yloxy)-
ca r bon yl]m eth yl]-L-h istid in e Meth yl Ester (10). To a
solution of compound 9 (1.0 g, 2.75 mmol) in DMF (30 mL)
was added ethyl bromoacetate (3.0 g, 18.0 mmol). The reaction
mixture was stirred at 40 °C for 4 h and then at room
temperature for another 20 h. The solvent was evaporated,
and the residue was purified by flash column chromatography
on silica gel (CH2Cl2:MeOH from 10:1 to 5:1). The solvent of
the pooled fractions was evaporated. After drying the residue
in vacuo, the hydrobromide salt of compound 10 (1.5 g, 80.5%)
was obtained as a hygroscopic white solid: 1H NMR (CDCl3)
δ 8.05 (s, 1H, im-H-2), 7.55 (s, 1H, im-H-5), 7.30 (s, 5H, Ph),
5.85 (s, 2H, Ph-CH2), 5.35 (s, 2H, N-CH2-COOEt), 4.71 (s, 2H,
O-CH2-N), 4.20 (m, 3H, R-CH + OCH2CH3), 3.75 (s, 3H,
COOCH3), 3.35 (d, 2H, CH-CH2-im), 1.70 (s, H2O), 1.41 (s, 9H,
C(CH3)3), 1.32 (t, 3H, OCH2-CH3). Anal. (C24H33N3O‚-
HBr‚0.5H2O), C, H, N.
Nτ-[[(Eth yloxy)car bon yl]m eth yl]-L-h istidin e Meth yl Es-
ter (11). To a solution of compound 10 (1.10 g, 1.98 mmol) in
methylene chloride (30 mL) was added trifluoroacetic acid (30
mL) at 0 °C. The reaction mixture was stirred at 0 °C for 1 h.
Anhydrous HBr gas was passed through for 1 h at 0 °C. The
reaction mixture was then evaporated to dryness in vacuo. The
light yellow residue was purified by flash column chromatog-
raphy on silica gel (CH2Cl2:MeOH ) 5:1). After removal of
the solvents and drying in vacuo, the hydrobromide salt of
compound 11 (0.52 g, 63%) was obtained as a white hygroscopic
1
brown powder: mp 220 °C dec; H NMR (CD3OD) δ 8.59 (s,
1H, H-7), 7.80 (d, 2H, 2′,6′-H), 7.61 (s, 1H, im-H-2), 6.90 (s,
1H, im-H-5), 6.81 (d, 2H, 3′,5′-H), 4.92 (s, 2H, 9-CH2-N), 4.75
(s, 2H, N-CH2-COOEt), 4.20 (m, R-CH + OCH2CH3), 3.75 (s,
3H, COOCH3), 3.40 (d, 2H, CH-CH2-im), 3.20 (s, 3H, 10-CH3),
1.31 (t, 3H, OCH2CH3).
Compound 14 (250 mg, 0.44 mmol) was suspended in 1 N
NaOH (10 mL). The suspension was stirred at room temper-
ature for about 1 h under nitrogen. The cloudy reaction
mixture turned clear and dark red. The solution was filtered
to remove any insoluble material; then it was acidified with 2
N HCl to pH 1-2 and filtered. The precipitate was removed
by filtration, and the pH of the filtrate was adjusted to 5-6
by adding 0.1 N NaOH. A yellow precipitate appeared which
was removed by centrifugation. The solution was applied to
an ion exchange column (IR-120, H+-form), which was washed
with water and then with 5% NH4OH. Fractions containing
the product were pooled and concentrated to about 25 mL. The
pH was adjusted to 5-6 with 0.1 N HCl. The precipitated
yellow solid was allowed to sit at 4 °C overnight and then
collected by centrifugation (8000g, 30 min, 4 °C). The solid
was then washed with water (15 mL), resuspended in water,
and lyophilized to yield 5 (152 mg, 66.4%) as a yellow powder:
TLC (A) Rf ) 0.42; mp >280 °C; HPLC (30% aqueous MeOH)
retention time, 6.5 min; 1H NMR (DMSO-d6) δ 8.61 (s, 1H,
H-7), 7.68 (d, 2H, 2′,6′-H, J ) 8.9 Hz), 7.65 (s, 1H, im-H-2),
7.03 (s, 1H, im-H-5), 6.82 (d, 2H, 3′,5′-H, J ) 8.9 Hz), 6.50 (s,
1H, NH-CO), 4.82 (s, 2H, 9-CH2-N), 4.81 (s, 2H, N-CH2-COOH),
4.55 (m, 1H, R-CH), 3.33 (broad, H2O), 3.22 (s, 3H, 10-CH3),
3.02 (t, 2H, CH-CH2-im). Anal. (C23H24N10O5‚2.5H2O) C, H;
N: calcd, 24.77; found, 24.31.
1
solid: mp 267-269 °C; H NMR (CDCl3) δ 8.45 (s, 1H, im-H-
2), 7.40 (s, 1H, im-H-5), 5.10 (s, 2H, im-N-CH2-COOEt), 4.50
(m, 1H, R-CH), 4.25 (m, 2H, OCH2CH3), 3.80 (s, 3H, COOCH3),
3.30 (d, 2H, CH-CH2-im), 1.30 (t, 3H, CH2-CH3). Anal.
(C11H17N3O4‚2.5HBr) C, H, N.
Nr-(4-Am in o-4-d eoxy-N10-m et h ylp t er oyl)-L-h ist id in e
Meth yl Ester (13). 4-Amino-4-deoxy-N10-methylpteroic acid
(mAPA, 12; 330 mg, 1 mmol) was suspended in anhydrous
DMF (7 mL); Et3N (0.3 mL, 2.2 mmol) and DEPC (0.15 mL)
were added. The reaction mixture was stirred at room
temperature under nitrogen for 7 h. To the clear dark red
solution were added L-histidine methyl ester hydrochloride salt
(7) (363 mg, 1.5 mmol) in DMF (5 mL) and Et3N (0.625 mL,
4.5 mmol). The reaction mixture was kept stirring for 72 h.
All solvents were removed in vacuo, and the residue was
subjected to CC31 cellulose column chromatography (2 × 6
cm). The column was washed with cold 0.1 N NaOH followed
by H2O (100 mL) and eluted with MeOH (150 mL). The
combined MeOH fractions were evaporated to dryness. The
residue was further purified by flash column chromatography
on silica gel (CHCl3:MeOH ) 7:1). The pooled fractions were
collected, concentrated, and dried in vacuo to afford 13 (165
mg, 34.7%) as a yellow solid: mp >230 °C dec; 1H NMR
(DMSO-d6) δ 8.60 (s, 1H, H-7), 7.75 (d, 2H, 2′,6′-H), 7.40 (s,
1H, im-H-2), 6.85 (d, 2H, 3′,5′-H), 6.60 (s, 1H, im-H-5), 4.81
(s, 2H, 9-CH2-N), 4.10 (m, 1H, R-CH), 3.75 (s, 3H, COOCH3),
Nr-[N10-(Tr iflu or oacetyl)pter oyl]-L-h istidin e Meth yl Es-
ter (16). N10-(Trifluoroacetyl)pteroic acid (15; 408 mg, 1 mmol)
was dissolved in anhydrous N-methylpyrrolidinone (3 mL) and
N-ethylmorpholine (1.15 g, 10 mmol). L-Histidine methyl ester
hydrochloride (7) (266 mg, 1.1 mmol) was added, and the
reaction mixture was cooled to 0 °C in an ice bath. PPA (1.26
g, 6 mequiv) in ethyl acetate (50%) was added. The reaction
mixture was stirred at room temperature under nitrogen for
72 h and cooled in an ice bath; then H2O (30 mL) was added
dropwise. A yellow precipitate formed. The pH was adjusted
to 9 with 5% aqueous sodium bicarbonate solution; then the
precipitate was filtered and washed with H2O (10 mL) and
ether (15 mL). The solid was dissolved in 0.5 N HCl (5 mL),
and the solution was filtered to remove any insoluble material.
The filtrate was adjusted to pH 5.5 with 1 N NaOH. The
resulting yellow precipitate was collected, washed with cold
water (5 mL), and dried in vacuo to give 16 (220 mg, 39.4%)
3.25 (s, 3H, 10-CH3), 3.15 (t, 2H, CH-CH2-im). Anal. (C22H25
N10O3‚1.5H2O) C, H, N.
-
Nr-(4-Am in o-4-d eoxy-N10-m eth ylp ter oyl)h istid in e (4).
Compound 13 (50 mg, 0.01 mmol) was suspended in 0.5 N
NaOH aqueous solution (1.5 mL). The suspension was stirred
at 40 °C under nitrogen for 2 h. The reaction mixture became
a clear dark brown solution. This solution was subjected to
HPLC on a C18 reversed phase silica gel column with a linear
gradient of H2O and MeOH as mobile phase. Pooled pure
fractions were freeze-dried after the solvent was removed by
rotary evaporation under reduced pressure. Compound 4 (22
mg, 47.8%) was obtained as a yellow solid: TLC (A) Rf ) 0.26;
mp >280 °C; HPLC (30% aqueous MeOH) retention time, 6.61
min; 1H NMR (DMSO-d6) δ 8.57 (s, 1H, H-7), 7.88 (s, 1H, im-
H-2), 7.66 (d, 2H, 2′,6′-H, J ) 8.9 Hz), 6.95 (s, 1H, im-H-5),
6.82 (d, 2H, 3′,5′-H, J ) 8.9 Hz), 6.70 (s, 1H, NH-CO), 4.78 (s,
2H, 9-CH2-N), 4.57 (m, 1H, R-CH), 3.37 (broad, H2O), 3.21 (s,
1
as a pale yellow solid: mp 235 °C dec; H NMR (DMSO-d6) δ
8.6 (s, 1H, H-7), 7.6 (d, 2H, 2′,6′-H), 7.4 (s, 1H, im-H-2), 7.0 (d,
2H, 3′,5′-H), 6.65 (s, 1H, im-H-5), 5.05 (s, 2H, 9-CH2-N), 4.4
(m, 1H, R-CH), 3.75 (s, 3H, COOCH3), 3.1 (m, 2H, CH-CH2-
im).
Nr-P ter oyl-L-h istid in e (6). To compound 16 (90 mg, 0.16
mmol) was added 1 N NaOH (2 mL). The reaction mixture
was stirred at 60 °C under nitrogen until most of the starting
material disappeared. Some remaining insoluble material was
removed by filtration, and the filtrate was adjusted to pH 5.5
by 1 N HCl. The yellow precipitate formed was collected by

4344 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Notes
(12) (a) Clark, L.; Rosowsky, A.; Waxman, D. J . Inhibition of Human
Liver Folylpolyglutamate Synthetase by Non-γ-Glutamylatable
Antifolate Analogs. Mol. Pharmacol. 1987, 31, 122-127. (b)
Rosowsky, A.; Freisheim, J . H.; Moran, R. G.; Solan, V. C.; Bader,
H.; Wright, J . E.; Radike-Smith, M. Methotrexate Analogues 26.
Inhibition of Dihydrofolate Reductase and Folylpolyglutamate
Synthetase Activity and In Vitro Tumor Cell Growth by Meth-
otrexate and Aminopterin Analogues Containing a Basic Amino
Acid Side Chain. J . Med. Chem. 1986, 29, 655-660. (c) Moran,
R. G.; Colman, P. D.; Harvison, P. J .; Kalman, T. I. Evaluation
of Pteroyl-S-alkylhomocysteine Sulfoximines as Inhibitors of
Mammalian Folylpolyglutamate Synthetase. Biochem. Phar-
macol. 1988, 37, 1997-2003.
(13) Rosowsky, A.; Forsch, R. A.; Reich, V. E.; Freisheim, J . H.;
Moran, R. G. Side Chain Modified 5-Deazafolate and 5-Deaza-
tetrahydrofolate Analogues as Mammalian Folylpolyglutamate
Synthetase and Glycinamide Ribonucleotide Formyltransferase
Inhibitors: Synthesis and in Vitro Biological Evaluation. J .
Med. Chem. 1992, 35, 1578-1588.
centrifugation (7000g, 30 min, 4 °C) and washed with H2O (2
× 5 mL), MeOH (5 mL), and ethyl ether (15 mL) to yield 6 (38
mg, 53.1%) as a yellow solid: TLC (A) Rf ) 0.35; mp >280 °C;
HPLC (H2O/30% aqueous MeOH gradient) retention time, 18.2
1
min; H NMR (D2O) δ 8.67 (s, 1H, H-7), 7.71 (s, 1H, im-H-2),
7.64 (d, 2H, J ) 9.0 Hz), 6.98 (s, 1H, im-H-5), 6.87 (d, 2H,
3′,5′-H, J ) 9.0 Hz), 4.65 (m, 3H, 9-CH2-N + R-CH), 3.22 (m,
2H, CH-CH2-im). Anal. (C20H19N9O4‚1.5H2O‚0.5MeOH) H, N;
C: calcd, 50.00; found, 50.48.
In h ibition of Tu m or Cell Gr ow th . Duplicate cultures
of L1210 murine leukemia cells were exposed to increasing
concentrations of test compounds. After 48 h incubation at
37 °C in RPMI 1640 medium supplemented with 10% fetal
calf serum, cell numbers were counted with a hemocytometer,
and concentrations corresponding to 50% inhibition of control
growth (IC50 values) were determined. Viability was g90%
as determined by Trypan blue dye exclusion.
En zym e Assa ys. DHFR activity in permeabilized cells was
assayed in situ in the presence and absence of inhibitors by
measuring the ability of dihydrofolate to support the thymidy-
late synthase-catalyzed release of tritium into water from
[5-3H]dUMP14f and processed essentially as described.21,25
FPGS from CCRF-CEM cells was prepared as previously
described24 and assayed6,24 at pH 8.5, unless stated otherwise.
(14) (a) Singh, S. K.; Singer, S. C.; Ferone, R.; Waters, K. A.; Mullin,
R. J .; Hynes, J . B. Synthesis and Biological Evaluation of NR-
(5-Deaza-5,6,7,8-Tetrahydropteroyl)-L-Ornithine. J . Med. Chem.
1992, 35, 2002-2006. (b) McGuire, J . J .; Hseih, P.; Franco, C.
T.; Piper, J . R. Folylpolyglutamate Synthetase Inhibition and
Cytotoxic Effects of Methotrexate Analogs containing 2,ω-
Diamino-alkanoic Acid. Biochem. Pharmacol. 1986, 35, 2607-
2613. (c) 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. (d) Patil, S. A.; Shane, B.;
Freisheim, J . H.; Singh, S. K.; Hynes, J . B. Inhibition of
Mammalian Folylpolyglutamate Synthetase and Human Dihy-
drofolate Reductase by 5,8-Dideaza Analogues of Folic Acid and
Aminopterin Bearing a Terminal L-Ornithine. J . Med. Chem.
1989, 32, 1559-1565. (e) George, S.; Cichowicz, D. J .; Shane,
B. Mammalian Folylpoly-γ-glutamate Synthetase. 3. Specificity
for Folate Analogues. Biochemistry 1987, 26, 522-529. (f)
Kalman, T. I. Polyglutamylation of Folates and Antifolates as a
Target of Chemotherapy. In Molecular Aspects of Chemotherapy;
Borowski, E., Shugar, D., Eds.; Plenum Press: New York, 1990;
pp 139-149.
Ack n ow led gm en t. This work was supported in part
by Research Grant CA35212 from the National Cancer
Institute, USPHS, Department of Health and Human
Services. The authors thank Dr. J . J . McGuire for the
FPGS data (work supported by NIH Grant CA43500).
The expert technical assistance of Mrs. M. C. Hsiao in
carrying out the cellular experiments is appreciated.
Refer en ces
(1) Ratnam, M.; Freisheim, J . H. Proteins Involved in the Transport
of Folates and Antifolates by Normal and Neoplastic Cells. In
Folic Acid Metabolism in Health and Disease; Picciano, M. F.,
Stokstad, E. L. R., Gregory, J . F., III, Eds.; Wiley-Liss: New
York, 1990; pp 91-120.
(2) McGuire, J . J .; Coward, J . K. Pteroylpolyglutamates: Biosyn-
thesis, Degradation, and Function. In Folates and Pterins, Vol.
1; Blakley, R. L., Benkovic, S. J ., Eds.; J ohn Wiley & Sons: New
York, 1984; pp 135-190.
(3) Moran, R. G. Characterization of the Function of Mammalian
Folylpolyglutamate Synthetase. In Folyl and Antifolyl Poly-
glutamates; Goldman, I. D., Chabner, B. A., Bertino, J . R., Eds.;
Plenum Press: New York, 1983; pp 327-339.
(4) J ones, C.; Kao, F. T.; Taylor, R. T. Chromosomal Assignment of
the Gene for Folylpolyglutamate Synthetase to Human Chromo-
some 9. Cytogenet. Cell Genet. 1980, 28, 181-194.
(5) Griffin, M. J .; Brown, G. M. The Biosynthesis of Folic Acid. III.
Enzymatic Formation of Dihydrofolic Acid from Dihydropteroic
Acid and of Tetrahydropteroylpolyglutamic Acid Compound from
Tetrahydrofolic Acid. J . Biol. Chem. 1964, 239, 310-316.
(6) McGuire, J . J .; Hsieh, P.; Coward, J . K.; Bertino, J . R. Enzymatic
Synthesis of Folylpolyglutamates. Characterization of the Reac-
tion and Its Products. J . Biol. Chem. 1980, 255, 5776-5788.
(7) Pristupa, Z. B.; Vickers, P. J .; Sephton, G. B.; Scrimgeour, K.
G. Folylpolyglutamate Synthetase from Beef Liver: Assay,
Stabilization and Characterization. Can. J . Biochem. Cell Biol.
1984, 62, 495-506.
(8) Shane, B. Pteroylpoly(γ-glutamate) Synthesis by Corynebacte-
rium Species. Studies on the Mechanism of Folylpoly(γ-
glutamate) Synthetase. J . Biol. Chem. 1967, 242, 1466-1475.
(9) (a) Taylor, R. T.; Hanna, M. L. Folate-Dependent Enzymes in
Cultured Chinese Hamster Cells: Folylpolyglutamate Syn-
thetase and Its Absence in Mutants Auxotrophic for Glycine-
Adenosine-Thymidine. Arch. Biochem. Biophys. 1977, 181, 331-
334. (b) McBurney, M. W.; Whitmore, G. F. Isolation and
Biochemical Characterization of Folate Deficient Mutants of
Chinese Hamster Cells. Cell 1974, 2, 173-182.
(10) Rosowsky, A.; Moran, R. G.; Forsch, R.; Colman, P.; Wick, M.
Methotrexate Analogues. XVII. Antitumor Activity of 4-Amino-
4-deoxy-N10-methylpteroyl-D,L-homocysteic Acid and Its Dual
Inhibition of Dihydrofolate Reductase and Folylpolyglutamate
Synthetase. Biochem. Pharmacol. 1984, 33, 155-161.
(11) Rosowsky, A.; Forsch, R. A.; Moran, R. G.; Freisheim, J . H.
Methotrexate Analogues. 32. Chain Extension, R-Carboxyl
Deletion, and γ-Carboxyl Replacement by Sulfonate and Phos-
phonate: Effect on Enzyme Binding and Cell-Growth Inhibition.
J . Med. Chem. 1988, 31, 1326-1331.
(15) Rosowsky, A. Chemistry and Biological Activity of Antifolates.
Prog. Med. Chem. 1989, 26, 1-252.
(16) (a) Rosowsky, A.; Forsch, R. A.; Bader, H.; Freisheim, J . H.
Synthesis and In Vitro Biological Activity of New Deaza Ana-
logues of Folic Acid, Aminopterin, and Methotrexate with an
L-Ornithine Side Chain. J . Med. Chem. 1991, 34, 1447-1454.
(b) Hynes, J . B.; Singh, S. K.; Fetzer, O.; Shane, B. Inhibition of
Hog Liver Folylpolyglutamate Synthetase by 5-Substituted 5,8-
Dideaza Analogues of Folic Acid Bearing a Terminal L-Ornithine
Residue. J . Med. Chem. 1992, 35, 4078-4085.
(17) (a) Merck Index, 11th ed.; Merck & Co., Inc.: Rahway, NJ , 1989;
p 746. (b) Ibid. p 1086.
(18) Hofmann, K.; Visser, J . P.; Finn, F. M. Studies on Polypeptides.
XLIV. Potent Synthetic S-Peptide Antagonists. J . Am. Chem.
Soc. 1970, 92, 2900-2909.
(19) Bergman, M.; Zervas, L. The General Process For the Synthesis
of Peptides. Ber. Dtsch. Chem. Ges. 1932, 65B, 1192-1201.
(20) Schmidt, A. H. Bromotrimethylsilane and Iodotrimethylsilane.
Versatile Reagents for Organic Synthesis. Aldrichim. Acta 1981,
14, 31-38.
(21) Harvison, P. J .; Kalman, T. I. Synthesis and Biological Activity
of Novel Folic Acid Analogues: Pteroyl-S-alkylhomocysteine
Sulfoximines. J . Med. Chem. 1992, 35, 1227-1233.
(22) Ueda, M.; Honma, T. Synthesis of Polyamides by Direct Poly-
condensation with Propylphosphonic Anhydride as an Activating
Agent. Polym. J . 1988, 20, 477-483.
(23) McGuire, J . J .; Tsukamoto, T.; Hart, B. P.; Coward, J . K.;
Kalman, T. I.; Galivan, J . Exploitation of Folate and Antifolate
Polyglutamylation to Achieve Selective Anticancer Chemo-
therapy. Invest. New Drugs 1996, in press.
(24) McGuire, J . J .; Russell, C. A.; Bolanowska, W. A.; Freitag, C.
M.; J ones, C. S.; Kalman, T. I. Biochemical and Growth Inhibi-
tion Studies of Methotrexate and Aminopterin Analogues Con-
taining a Tetrazole Ring in Place of the γ-Carboxyl Group.
Cancer Res. 1990, 50, 1726-1731.
(25) Yalowich, J . C.; Kalman, T. I. Rapid Determination of Thymidy-
late Synthetase Activity and Its Inhibition in Intact L1210
Leukemia Cells in Vitro. Biochem. Pharmacol. 1985, 34,
2319-2324.
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