Synthesis and in vitro antitumor activity of new deaza analogues of the nonpolyglutamatable antifolate Nα-(4-amino-4-deoxypteroyl)-Nδ- hemiphthaloyl-L-ornithine (PT523)

Article abstract of DOI:10.1021/jm010518t

Details are disclosed for the synthesis of N^α-[4-[2-(2,4-diaminoquinazolin-6-yl)ethyl]benzoyl]-N ^δ-hemiphthaloyl-L-ornithine (2) and N^α-[4-[5-(2,4-diaminoteridin-6-yl)pent-1-yn-4-yl]benzoyl]-N ^δ-hemiphthaloyl-L-

Full text of DOI:10.1021/jm010518t

1690
J . Med. Chem. 2002, 45, 1690-1696
Syn th esis a n d In Vitr o An titu m or Activity of New Dea za An a logu es of th e
Non p olyglu ta m a ta ble An tifola te
Nr-(4-Am in o-4-d eoxyp ter oyl)-Nδ-h em ip h th a loyl-L-or n ith in e (P T523)
Chitra M. Vaidya, J oel E. Wright, and Andre Rosowsky*
Dana-Farber Cancer Institute and the Department of Biological Chemistry and Molecular Pharmacology,
Harvard Medical School, Boston, Massachusetts 02115
Received November 8, 2001
Details are disclosed for the synthesis of NR-[4-[2-(2,4-diaminoquinazolin-6-yl)ethyl]benzoyl]-
Nδ-hemiphthaloyl-L-ornithine (2) and NR-[4-[5-(2,4-diaminoteridin-6-yl)pent-1-yn-4-yl]benzoyl]-
Nδ-hemiphthaloyl-L-ornithine (6) as analogues of NR-(4-amino-4-deoxypteroyl)-Nδ-hemiphthaloyl-
L-ornithine (1, PT523), a nonpolyglutamatable antifolate currently in advanced preclinical
development. In a 72 h growth inhibition assay against cultures of CCRF-CEM human leukemic
lymphoblasts, the IC50 of 2 and 6 was 0.69 ( 0.044 nM and 1.3 ( 0.35 nM, respectively, as
compared with previously reported values 4.4 ( 0.10 nM for aminopterin (AMT) and 1.5 (
0.39 nM for PT523. In a spectrophotometric assay of dihydrofolate reductase (DHFR) inhibition
using dihydrofolate and NADPH as the cosubstrates, the previously unreported compounds 2
and the mixed 10R and 10S diastereomers of 6 had Ki values of 0.21 ( 0.05 pM and 0.60 (
0.02 pM, respectively, as compared with previously reported values of 3.70 ( 0.35 pM for AMT
and 0.33 ( 0.04 pM for PT523. Thus, while they were comparable to 1 and several of its
previously studied analogues in their ability to bind to DHFR and inhibit the growth of CCRF-
CEM cells, 2 and the mixed diastereomers of 6 were several times more active than AMT despite
the fact that they cannot form γ-polyglutamylated metabolites of the type formed in cells from
AMT and other classical antifolates with a glutamate side chain.
In tr od u ction
NR-(4-Amino-4-deoxypteroyl)-Nδ-hemiphthaloyl-L-
ornithine (PT523, 1, Figure 1) is an unusually potent
antifolate originally synthesized in our laboratory1 and
currently in advanced preclinical development under the
auspices of the Rapid Access to Intervention Develop-
ment (RAID) program of the National Cancer Institute.
As noted in two recent reviews of laboratory studies
F igu r e 1. Structure of NR-(4-amino-4-deoxypteroyl)-Nδ-hemi-
published to date on the mode of action of 1,2a,b the
phthaloyl-L-ornithine (PT523, 1).
features that probably contribute most prominently to
its high potency are tight binding to dihydrofolate
reductase (DHFR) and efficient utilization of the re-
duced folate carrier (RFC), the membrane transport
protein responsible for cellular uptake of both folates
and classical antifolates. Initial interest in 1 as a
potential drug was sparked by the observation that it
could overcome a clinically relevant 10- to 30-fold level
of resistance to the classical antifolate methotrexate
(MTX) in cultured cells irrespective of whether this
resistance was associated with enhanced expression of
DHFR or diminished uptake via the RFC. In contrast
to MTX and other classical antifolates, 1 is not a
substrate for folyl polyglutamate synthetase (FPGS) and
thus resembles nonclassical antifolates such as tri-
metrexate (TMX, Figure 2) and piritrexim (PTX, Figure
2), whose action is FPGS-independent. Unlike TMX and
PTX, however, 1 is water-soluble at physiologic pH and
is able to be conveniently administered by injection.
Furthermore, 1 retains the ability to inhibit the growth
of cells that have become resistant to TMX and a range
F igu r e 2. Structures of trimetrexate (TMX) and piritrexim
(PTX).
of other lipophilic drugs by reason of increased P-
glycoprotein expression.
Of further significance in terms of the clinical poten-
tial of 1 is that tumor cells selected for resistance to a
number of the newer antifolates with a glutamate side
chain, such as the thymidylate synthase inhibitor ICI
D1694 (ZD1694, raltitrexed)3 and the multitargeted
antifolate LY231514,4 were recently found to be only
minimally cross-resistant to this drug.5 Since the effects
* To whom correspondence should be addressed. Tel: (617) 632-
3117. Fax: (617) 632-2410. E-mail: andre•rosowsky@dfci.harvard.edu.
10.1021/jm010518t CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/16/2002

Synthesis of Nonpolyglutamatable Antifolates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1691
F igu r e 4. Structures of compounds 8-11.
catalyzed coupling reaction between the previously
undescribed compounds methyl L-2-(4-iodobenzamido)-
5-phthalimidopentanoate (8) and 2,4-diamino-6-ethinyl-
quinazoline (9). As an alternative route, Pd(0)-catalyzed
coupling could also be carried out between methyl L-2-
(4-ethinylbenzamido)-5-phthalimidopentanoate (10) and
2,4-diamino-6-iodoquinazoline (11) as had been done
earlier in the published synthesis of 3.11 The structures
of 8-11 are shown in Figure 4. After considering the
merits of both routes, however, we chose the former
because 9 had not, to our knowledge, been used before
as a building block for the synthesis of DHFR inhibitors.
F igu r e 3. Structures of compounds 2-7.
of raltitrexed and LY231514 are both highly dependent
on cellular FPGS levels, and resistance to them is
strongly correlated with FPGS expression, the fact that
1 is efficiently transported by the RFC6a,b and yet is not
dependent on activation by FPGS for activity can be
viewed as a very distinctive feature of its pharmacody-
namics.
The superior in vitro activity of 1 relative to a number
of other antifolates tested in the clinic over the last 10
years prompted us to undertake a series of structure
optimization studies in which the molecule was formally
dissected into five regions as shown in Figure 1. As a
result of these studies, it was determined that (i) N5
and/or N8 could be replaced by carbon (region A);7 (ii)
N10 could also be replaced by carbon (region B);8 (iii)
the 1′,4′-substituted phenyl moiety could be 3′,5′-dichlo-
rinated or replaced by a 1′,4′-substituted naphthyl
moiety (region C);8 (iv) the optimal number of CH2
groups in the amino acid side chain was three (region
D);8-10 and (v) ortho substitution was preferred over
meta or para substitution on the phthaloyl moiety
(region E).9
The improved activity of the 5,8-dideaza- and 10-
deaza analogues of 1 relative to the parent drug led us
to speculate that replacement of all three nitrogen atoms
(i.e., N5, N8, and N10) by carbon might represent an
optimal modification of regions A and B. Accordingly,
we embarked on the synthesis of the previously un-
described compound NR-[4-[2-(2,4-diaminoquinazolin-6-
yl)ethyl]benzoyl]-Nδ-hemiphthaloyl-L-ornithine (2), which
can be viewed as a nonpolyglutamatable analogue of
5,8,10-trideazaaminopterin (3).11 In addition, because
we had earlier synthesized the 10-ethyl-10-deaza de-
rivative 48 as a nonpolyglutamatable analogue of 10-
ethyl-10-deazaaminopterin (edatrexate, EDX, 5),12a,b we
also prepared NR-[4-[5-(2,4-diaminoteridin-6-yl)pent-1-
yn-4-yl)]benzoyl]-Nδ-hemiphthaloyl-L-ornithine (6), a
nonpolyglutamatable analogue of 10-propargyl-10-de-
azaaminopterin (PDX, 7).13a-d Because of a better phar-
macologic and therapeutic profile, 7 has recently been
undergoing clinical trial as a potential successor for 5.
The structures of 2-7 are shown in Figure 3.
As shown in Scheme 1, access to 9 was conveniently
achieved by reaction of 11 with trimethylsilylacetylene
to obtain 2,4-diamino-6-(trimethylsilylethinyl)quinazo-
line (12), followed by desilylation with tetrabutyl-
ammonium fluoride (TBAF). The overall two-step yield
of 9 from 11 was 62%. Compound 11 was obtained in
40% overall yield from 2-aminobenzonitrile by regio-
selective iodination with iodine monochloride, followed
by annulation with chloroformamidine hydrochloride as
described.11,14 The synthesis of 8 was accomplished by
straightforward mixed anhydride chemistry (i-BuO-
COCl/Et3N/DMF) from methyl L-2-amino-5-phthalimi-
dopentanoate9 and 4-iodobenzoic acid. A second Pd(0)-
catalyzed reaction, this time between 8 and 9, led to
the ethinyl adduct 13, which on catalytic hydrogenation
(H2/Pd-C) yielded 14. Because of the formation of several
unidentified side products during the second coupling
reaction, purification of 13 by silica gel chromatography
required careful monitoring of individual eluent frac-
tions by TLC. The presence of these side products
resulted in a yield of 13 that was somewhat low (33%
versus 67% in the case of 12). Whether this was due to
the fact that the two coupling reactions used different
iodide-alkyne pairs or different Pd catalysts and amine
bases was not determined. To complete the synthesis,
the triple bond was reduced catalytically, the methyl
ester was cleaved, and the phthalimide ring opened in
a single step with Ba(OH)2 in wet MeOH, affording 2
as a tetrahydrate after preparative reversed-phase
HPLC (C18 silica gel) followed by ion-exchange chroma-
1
tography (DEAE-cellulose). The H NMR spectrum of
2 contained the required signals for the CH2CH2 bridge
and the C7 and C5 protons. Other peaks were ap-
propriately assigned to the C8 proton, the A2B2 pattern
on the phenyl ring, the ornithine aliphatic protons, and
the protons of the hemiphthaloyl group. That the
phthaloyl ring was in the open form was confirmed from
the chemical shift of the aromatic protons, which in
Resu lts a n d Discu ssion
Retrosynthetic analysis suggested that a reasonable
approach to the preparation of 2 would be via a Pd(0)-

1692 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8
Vaidya et al.
Sch em e 1a
a
Reagents: (a) Me3SiCtCH/Pd(OAc)2/(2-tolyl)3P, CuI, piperidine/DMF; (b) TBAF; (c) methyl L-2-(4-iodobenzamido)-5-phthalimido-
pentanoate (8)/(Ph3P)4Pd, CuI, Et3N, DMF; (d) H2/5% Pd-C; (e) Ba(OH)2, MeOH-H2O.
Sch em e 2a
a
Reagents: (a) BrCH2CtCH/NaH/THF; (b) 2,4-diamino-6-bromomethylpteridine hydrobromide (18‚HBr)/NaH/DMF; (c) NaOH/
MeOCH2CH2OH; (d) 120 °C/DMSO; (e) methyl L-2-amino-5-phthalimidopentanoate (23)/i-BuOCOCl/DMF; (f) Ba(OH)2/aq MeOH.
characteristic fashion was at higher field than that of
the closed form. A notable aspect of the spectrum of 2,
and of its ester 14, was the consistently less deshielded
character of the aromatic protons around the CH2CH2
bridge (i.e., C5-H, C7-H, and 3′,5′-H) in comparison
with those around the CtC bridge in 13, which pre-
sumably reflects the linearity imposed on the latter by
the triple bond.
The synthesis of 6 (Scheme 2) was patterned after the
one used earlier13a,b to prepare 7 and recently adapted
in our laboratory,8 with small experimental modifica-
tions. Alkylation of the Na carbanion of dimethyl
homoterephthalate (15) with propargyl bromide in THF
solution as described was found to give mainly the
desired monopropargyl derivative 16. However, even
though we replicated as closely as possible the process
described in the literature,13ab we were unable to effect
complete consumption of the starting material 15 or
eliminate the formation of a small amount of the
unwanted dipropargyl derivative 17 as judged by careful
examination of mass spectral and 1H NMR data. Be-
cause the separation of these compounds by silica gel
chromatography on a multigram scale proved quite
difficult, the crude mixture of 15-17 was used for the
next step with the assumption that only 15 and 16
would be able to undergo further C-alkylation. A solu-
tion of the esters was added dropwise to a suspension
of NaH in dry DMF kept at -5 to 0 °C, and the resulting
mixture of carbanions was treated dropwise with a
solution of the 2,4-diamino-6-bromomethylpteridine hy-
drobromide (18‚HBr)15 in DMF while carefully main-
taining the temperature at -25 to -30 °C. The reaction
was allowed to proceed for a total of 6 h while allowing
the flask to warm first to -10 °C and then to room
temperature. Column chromatography of the product on
silica gel easily allowed 17 to be separated from the
slower-moving alkylated products, 19 and 20. However,
separation of the latter two compounds was more
difficult, requiring careful monitoring of small fractions
of the eluent from the column to be monitored by TLC
(cf. Experimental Section). The purified propargyl de-
rivative 20 was hydrolyzed to the diacid 21 with 10%
NaOH in 2-methoxyethanol, and the latter was con-
verted to the key intermediate 22 by heating in DMSO
at 120 °C for 30 min. Careful monitoring by HPLC
showed that the hydrolysis step required 24 h, and that
the decarboxylation required 30 min. For reasons we
are unable to explain, both the hydrolysis and decar-

Synthesis of Nonpolyglutamatable Antifolates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1693
Ta ble 1. Cell Growth Inhibition and DHFR Inhibition by
PT523 Analogues Modified in the B-Ring and/or 9,10-Bridge
atoms by carbon does not, in fact, lead to any further
increase in potency relative to 1. In the case of the mixed
diastereomers of 10-propargyl-10-deaza analogue 6, the
IC50 was found to be 1.3 ( 0.35 nM as compared with a
previously obtained value of 1.2 ( 0.25 nM for 4 and
3.3 ( 0.36 nM for 5.8 It thus appears that, while the
mixed diastereomers of 6 are somewhat more active
than those of 5, they are not significantly different from
those of 4 under the conditions of our assay.
cell growth
DHFR binding
Za
NH
CH CH CH2
IC50 (nM, 72 h)b,c
Ki (pM)b,d
In addition to the cytotoxicity studies, we also exam-
ined the ability of 2 and the mixed diastereomers of 6
to inhibit reduction of dihydrofolate by NADPH in the
presence of purified human recombinant DHFR. Using
the same spectrophotometric assay procedure we used
earlier to obtain Ki values for 116 and other compounds
in this series,2b we determined the Ki of 2 to be 0.21 (
0.05 pM as compared with 0.014 ( 0.005 pM for 25 and
0.35 ( 0.06 pM for 26. We conclude from these results
that, while 2 is clearly a better DHFR inhibitor than 1,
replacement of N5 and N8 by carbon does little or
nothing to enhance enzyme binding relative to 26,
whereas replacement of nitrogen by carbon at the 10-
position actually leads, somewhat suprisingly, to a 2-fold
decrease in competitive inhibition relative to 25. In the
case of 6, a Ki of 0.60 ( 0.02 pM was obtained that was
indistinguishable from that of the 10-ethyl analogue 4.
This result differed somewhat from what had been
reported previously for the glutamate analogues 5 and
7, for which there was as much as a 4-fold difference in
Ki depending on the source of the enzyme and the pH
of the assay.13a,c A possible explanation of the contrast-
ing results between 4 and 6 versus and 5 and 7 is that
the hemiphthaloylornithine derivatives come closer to
being true stoichiometric inhibitors than the glutamate
analogues, and thus may be a little less sensitive to
minor changes in molecular structure.
cmpd
X
Y
1 (PT523)
N
N
1.5 ( 0.39
0.69 ( 0.04
1.2 ( 0.25
1.3 ( 0.35
0.64 ( 0.04
0.53 ( 0.07
0.33 ( 0.04e
0.21 ( 0.05
0.62 ( 0.10
0.60 ( 0.02
0.014 ( 0.005g
0.35 ( 0.06 f
2
4f
6
N
N
N
N
CHEt
CHPg
25
26
CH CH NH
CH2
N
N
a
b
Pg ) propargyl. Numbers are means ( SD for a minimum
of three independent determinations. c Assays were carried out as
in ref 8. Assays were carried out as in ref 16. e A Ki of 0.35 (
d
0.13 pM (n ) 3) was reported previously for 1 in several earlier
papers.6b,7-10,16 f Data for compound 4 are taken from ref 6b. Ki
g
values we reported originally for 25 and 26 in ref 6b were 0.09 (
0.03 pM and 0.18 ( 0.02 pM, respectively. The values listed here
were obtained after repeating the assays with a more recently
obtained batch of purified enzyme.
boxylation of 20 were found to be slower than reported
by DeGraw and co-workers.13a However, the outcome
was similar, with the overall two-step yield of 22 from
20 approaching 40%. To complete the synthesis, 22 was
condensed with methyl L-2-amino-5-phthalimidopen-
tanoate (23).10 The resulting phthalimide 24 was puri-
fied by column chromatography on silica gel and was
then treated with Ba(OH)2 in aqueous MeOH to obtain
6 as a dihydrate. The presence of a propargyl group in
1
22, 24, and 6 was confirmed by the H NMR spectra,
which all featured a broad singlet at δ 2.7-2.8 corre-
sponding to the acetylene proton. The broadness of this
signal indicated that the product was likely to be a
mixture of 10R and 10S diastereomers, as in the 10-
methyl-10-deaza and 10-ethyl-10-deaza analogues we
had made earlier.8 The yield of 24 from 21 was only 13%
despite the fact that two cycles of activation and amine
addition were used. In the reported condensation of 21
with diethyl L-glutamate by DeGraw and co-workers,13a
a total of three cycles were used, and the yield was 55%.
While it is possible that a higher conversion of 21 to 24
would have been achieved if three or more rounds had
been used, this was not investigated.
In summary it would appear, on the basis of the
results reported here for 2 and the diastereomeric
mixture 6, that further modification of 1 in the 9,10-
bridge alone (region B in Figure 1) is not likely to yield
significant changes in DHFR binding or cell growth
inhibition. However, the possibility that other analogues
of 1 with changes in more than one of the regions A, B,
and C may be more active has not been ruled out and
is being investigated in our laboratory.
Exp er im en ta l Section
IR spectra were obtained on a Perkin-Elmer 281 double-
beam spectrophotometer and UV spectra on a Perkin-Elmer
35 UV/visible instrument. Only IR peaks with ν values greater
than 1200 cm-1 are listed; weak peaks and shoulders are
omitted. 1H NMR spectra were recorded at 200 MHz with a
Varian VX200 instrument. Amino group protons did not
always give rise to a well-defined signal and thus are in some
instances omitted. Mass spectra were recorded in the Molec-
ular Biology Core Facility of the Dana-Farber Cancer Institute
using a Perkin-Elmer Voyager 4036 system (low-resolution)
and in the Mass Spectrometry Laboratory, Department of
Chemistry and Chemical Biology, Harvard University, using
a MicroMass LCT instrument in the electrospray mode (high-
resolution). TLC analyses were performed on Whatman MK6F
silica gel plates, and spots were visualized under 254 nm
illumination. The term “NH4OH” in the experimental descrip-
tion refers to a solution of ca. pH 9 as determined with
universal indicator paper. Column chromatography was done
on Baker silica gel (regular grade, 60-200 mesh; flash grade,
40 µm particle size) or Whatman DEAE cellulose (DE-52).
The ability of the 5,8,10-trideaza analogue 2 and the
10-propargyl-10-deaza analogue 6 to inhibit the growth
of CCRF-CEM human leukemic lymphoblasts in culture
during 72 h of continuous drug exposure was deter-
mined and compared with the results we had previously
obtained with other analogues of 1 modified in the
B-ring7 or 9,10-bridge.8 As shown in Table 1, the IC50
of 2 was 0.69 ( 0.04 nM as compared with 1.5 ( 0.39
nM for 1, 0.64 ( 0.04 nM for the 5,8-dideaza analogue
25, and 0.53 ( 0.07 nM for the 10-deaza analogue 26.
Thus, somewhat surprisingly, the effect of replacing N10
as well as N5 and N8 by carbon was not significantly
different than that of either replacing N5 and N8 by
carbon or replacing N10 by carbon. While we had hoped
that the combined effect of these modifications might
be greater than that of either one alone, it seems clear
from these results that replacing all three nitrogen

1694 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8
Vaidya et al.
1
Moisture-sensitive reactions were carried out in solvents that
were of Sure-Seal grade (Aldrich, Milwaukee, WI), or had been
stored over Linde 4A molecular sieves. HPLC separations were
on C18 radial compression cartridges (Millipore, Milford, MA;
analytical, 5 µm particle size, 5 × 100 mm; preparative, 15
µm particle size, 25 × 100 mm). Solids were generally dried
at 50-80 °C over P2O5 in a drying apparatus attached to a
vacuum pump. Melting points (not corrected) were determined
on a Fisher-J ohns hot-stage microscope or in open Pyrex
capillary tubes in a Mel-Temp apparatus (Cambridge Labora-
tory Devices, Cambridge, MA). 2,4-Diamino-6-iodoquinazoline
(11),14 2,4-diamino-6-bromomethylpteridine (18‚HBr),15 and
methyl 2-L-amino-5-phthalimidopentanoate (23‚HCl)10 were
prepared according to the literature. Other chemicals were
purchased from Aldrich, Milwaukee, WI, or Fisher, Boston,
MA. Microanalyses were performed by Robertson Laboratory,
Madison, NJ , or Quantitative Technologies, Whitehouse, NJ ,
and were within (0.4% of theory unless otherwise specified.
The presence of fractional molar amounts of organic solvents
such as CHCl3 in analytical samples of lipophilic antifolate
intermediates, even after overnight drying in vacuo at 60 °C,
1580, 1520, 1400, 1360, 1200 cm-1; H NMR (CDCl3) δ 1.85-
2.02 (m, 4H, â- and γ-CH2), 3.60-3.73 (m, 2H, δ-CH2), 3.77 (s,
3H, COOMe), 4.85 (m, H, R-CH), 6.80 (br s, 1H, NH), 7.52-
7.56 (m, 2H, 2′- and 6′-H), 7.78-7.82 (m, 2H, 3′- and 5′-H),
7.69-7.84 (m, 4H, phthalimide ring); MS z/e 507, calcd (M +
1) 507. Anal. (C21H19IN2O5) C, H, N.
Meth yl 2-L-[4-[2-(2,4-Dia m in oqu in a zolin -6-yl)eth in yl]-
ben za m id o]-5-p h th a lim id op en ta n oa te (13). To a solution
of 9 (300 mg, 1.63 mmol) in dry DMF (18 mL) were added 8
(916 mg, 1.81 mmol), (Ph3P)4Pd(0) (188 mg, 0.16 mmol), CuI
(12 mg), and Et3N (0.3 mL). The mixture was stirred under
argon at room temperature for 24 h, the solvent was evapo-
rated under reduced pressure, and the residue was washed
with H2O, redissolved in a minimum volume of 10:1 CHCl3-
MeOH (system C), and chromatographed on a silica gel column
which was packed and eluted with system C. Separation was
monitored by TLC using 10:1:0.1 CHCl3-MeOH-Et3N (system
D). Fractions giving a single spot with Rf 0.3 were pooled and
evaporated to obtain 13 as a yellow solid (0.3 g, 33%); mp >150
°C dec; IR (KBr) ν 3350, 3200, 2940, 2200, 1700, 1640, 1600,
1560, 1500, 1440, 1400, 1220 cm-1 1H NMR (DMSO-d6) δ
;
1
was confirmed by H NMR whenever possible.
1.59-1.79 (m, 4H, â- and γ-CH2), 3.62 (m, 5H, COOMe and
δ-CH2), 4.45 (m, 1H, R-CH), 7.05 (br s, 2H, NH2), 7.30-7.34
(d, 1H, C8-H), 7.60-7.64 (m, 2H, 3′- and 5′-H), 7.73-7.78
(m, 1H, C7-H), 7.82-7.86 (m, 4H, phthalimide ring), 7.87-
7.92 (m, 2H, 2′- and 6′-H), 8.14 (br s, 2H, NH2), 8.39 (d, 1H,
C5-H), 8.60 (d, 1H, CONH). Anal. (C31H26N6O5‚0.3CHCl3) C,
H, N.
2,4-Dia m in o-6-(tr im eth ylsilyleth in yl)qu in a zolin e (12).
Me3SiCtCH (4.91 g, 50 mmol) was added dropwise at room
temperature to a mixture of 11 (2.86 g, 10 mmol), (o-tolyl)3P
(121 mg, 0.4 mmol), palladium(II) acetate (45 mg, 0.2 mmol),
CuI (40 mg), and piperidine (8 mL) in dry DMF (12 mL), and
the mixture was stirred under argon for 24 h. The DMF was
evaporated under reduced pressure, and the residue was
shaken with a mixture of NH4OH (150 mL) and Et2O (50 mL),
then filtered and washed with H2O until the washings were
colorless. The residue was taken up in a minimum amount of
10:1:0.1 CHCl3-MeOH-NH4OH (system A), and the solution
was applied onto a flash silica gel column packed and eluted
with the same solvent. Fractions giving a single TLC spot with
Rf 0.30 (silica gel, system A) were pooled and evaporated to
obtain 12 as a yellow solid (1.73 g, 67%); mp >240 °C, with
darkening above 200 °C; IR (KBr) ν 3340, 3260, 2950, 2160,
1650, 1640, 1620, 1550, 1500, 1480, 1450, 1370, 1320, 1280,
1250 cm-1; 1H NMR (DMSO-d6) δ 0.22 (s, 9H, Me3Si), 6.18 (br
s, 2H, NH2), 7.07-7.11 (d, 1H, C8-H), 7.34 (br s, 2H, NH2),
7.42-7.47 (dd, 1H, C7-H), 8.15-8.16 (d, 1H, C5-H); MS z/e
257, calcd (M + 1) 257. Anal. (C13H16N4Si‚0.1H2O) C, H, N.
Met h yl 2-L-[4-[2-(2,4-Dia m in oq u in a zolin -6-yl)et h yl]-
ben za m id o]-5-p h th a lim id op en ta n oa te (14). A solution of
13 (100 mg, 0.18 mmol) in a mixture of CH2Cl2 (20 mL) and
MeOH (20 mL) was shaken under H2 (50 lb/in2) in the presence
of 5% Pd-C (120 mg) in a Parr apparatus for 18 h. The reaction
was filtered (Celite), the filter cake was washed with MeOH,
and the combined filtrate and wash solution were evaporated
to dryness under reduced pressure. The residue was chro-
matographed on a column of silica gel packed and eluted with
system C. Fractions giving a single TLC spot with Rf 0.22
(silica gel, system D) were pooled and evaporated to obtain 14
as a pale-yellow solid (62 mg, 62%); mp 138-139 °C; IR (KBr)
ν 3350, 3200, 2950, 1710, 1650, 1630, 1560, 1540, 1500, 1440,
1400 cm-1; H NMR (DMSO-d6) δ 1.69-1.77 (m, 4H, â- and
1
γ-CH2), 2.98 (m, 4H, C9-C10 bridge), 3.60 (m, 5H, COOMe
and δ-CH2), 4.43 (m, 1H, R-CH), 7.28-7.32 (m, 3H, C8-H, 3′-
and 5′-H), 7.40 (br s, 2H, NH2), 7.56-7.62 (m, 1H, C7-H),
7.73-7.78 (m, 2H, 2′- and 6′-H), 7.82-7.86 (m, 4H, phthalimide
ring), 8.06 (d, 1H, C5-H), 8.50 (br s, 2H, NH2), 8.80 (d, 1H,
CONH); MS z/e 567, calcd (M + 1) 567. Anal. (C31H30N6O5‚
1.3CHCl3) C, H, N.
2,4-Dia m in o-6-eth in ylqu in a zolin e (9). A 1 M solution of
TBAF in THF (4.3 mL, 4.3 mmol) was added to a solution of
12 in THF (30 mL) under argon, and the mixture was stirred
at room temperature for 24 h. The precipitated solid was
filtered, washed with THF, and dried. The solid was then taken
up in a minimum volume of system A, and the solution was
applied onto a flash silica gel column which was packed and
eluted with the same mixture. Fractions giving a single spot
with Rf 0.24 (silica gel, system A) were pooled and evaporated
to obtain a pale-yellow solid (0.71 g, 93%); mp >240 °C,
darkening above 200 °C; IR (KBr) ν 3460, 3250, 3100, 2100,
1660, 1640, 1620, 1560, 1500, 1480, 1450, 1400, 1370, 1290,
Nr-[4-[2-(2,4-Dia m in oq u in a zolin -6-yl)et h yl]b en zoyl]-
Nδ-h em iph th aloyl-L-or n ith in e (5,8,10-Tr ideaza-P T523) (2).
Solid Ba(OH)2 (56 mg, 0.17 mmol) was added to a stirred
solution of 14 (50 mg, 0.088 mmol) in MeOH (30 mL) and H2O
(30 mL), and the mixture was stirred at room temperature
for 48 h, followed by addition of solid NH4HCO3 (100 mg), and
continued stirring for 30 min. The BaCO3 precipitate was
removed and the solvent evaporated under reduced pressure.
The residue was taken up in H2O and the solution freeze-dried
to a white solid. Preparative HPLC (C18 silica gel, 12% MeCN
in 0.1 M NH4OAc, pH 7.5, 10 mL/min), followed by anion-
exchange chromatography (DEAE-cellulose, extensive H2O
wash, then 0.2 M NH4HCO3), afforded analytically pure 2 as
a white solid (20 mg, 40%); mp >200 °C dec, with softening
ca. 180 °C; HPLC (as above except that the flow rate was
1 mL/min) 32 min; IR (KBr) ν 3340, 3200, 2950, 1650, 1600,
1530, 1490, 1380 cm-1; UV (pH 7.4) λmax 233 nm (ꢀ 55950),
1200 cm-1 1H NMR (DMSO-d6) δ 4.05 (s, 1H, CtCH), 6.15
;
(br s, 2H, NH2), 7.09-7.13 (d, 1H, C8-H), 7.34 (br s, 2H, NH2),
7.45-7.50 (dd, 1H, C7-H), 8.16 (d, 1H, C5-H). MS z/e 185,
calcd (M + 1) 185. Anal. (C10H8N4O‚0.1H2O) C, H, N.
Meth yl 2-L-(4-Iod oben za m id o)-5-p h th a lim id op en ta n -
oa te (8). To a solution of 4-iodobenzoic acid (3 g, 12.1 mmol)
in dry DMF (50 mL) at room temperature were added Et3N (5
g, 50 mmol) and i-BuOCOCl (1.65 g, 12.1 mmol), followed 1 h
later by 23‚HCl (3.77 g, 12.1 mmol). Stirring at room temper-
ature was continued for 16 h, the solvent was evaporated
under reduced pressure, the residue was taken up in CHCl3,
and the solution was washed with H2O, dried (MgSO4), and
evaporated. The residue was then taken up in 95:5 CHCl3-
MeOH (system B) and applied onto a column of silica gel (flash
grade) which was packed and eluted with appropriate volumes
of 98:2 CHCl3-MeOH followed by system B. Fractions giving
a single TLC spot with Rf 0.66 (silica gel, system B) were
pooled and evaporated to obtain 8 as a white solid (3.78 g,
62%); mp 180-181 °C; IR (KBr) ν 3310, 2950, 1745, 1710, 1640,
1
323 (4630); H NMR (DMSO-d6) δ 1.70-1.95 (m, 4H, â- and
γ-CH2), 2.89 (m, 4H, C9-C10 bridge), 3.25 (m, 2H, δ-CH2), 4.37
(m, 1H, R-CH), 7.09-7.17 (m, 3H, C8-H, 3′- and 5′-H), 7.33-
7.38 (m, 4H, phthaloyl ring), 7.55 (m, 1H, C7-H), 7.76-7.80
(m, 2H, 2′- and 6′-H), 7.86 (m, 1H, C5-H), 8.04 (br s, 2H, NH2),
8.34 (br m, 3H, NHCO and NH2); MS (high-resolution) z/e
571.2292, calcd (M + 1) 571.2305. Anal. (C30H30N6O6‚4H2O)
C, N; H: calcd, 5.97; found, 5.48.

Synthesis of Nonpolyglutamatable Antifolates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1695
Meth yl 2-L-[[5-(2,4-Dia m in op ter id in -6-yl)p en t-1-yn -4-
yl)]ben za m id o]-5-p h th a lim id op en ta n oa te (24). Step 1. A
solution of 15 (3.1 g, 15.1 mmol) in dry THF (4.5 mL) was
added to a suspension of NaH (60% in mineral oil, 0.66 g, 16.6
mmol) in dry THF (11 mL) which had been precooled to 0 °C
in an ice-bath. Propargyl bromide (1.85 g, 16.6 mmol) was then
added, and stirring was continued at 0 °C for 30 min, then at
room temperature for 16 h. The reaction mixture was quenched
with 50% AcOH (1.5 mL) and poured into H2O (200 mL).
Extraction with Et2O (3 × 100 mL), drying (MgSO4), and
evaporation gave an oil, which was applied onto a silica gel
column for flash chromatography using appropriate volumes
of 95:5, 94:6, 93:7, and 90:10 hexanes-EtOAc as eluents.
Fractions were carefully monitored by TLC, and those giving
a single spot with Rf 0.35 (9:1 hexanes-EtOAc) were pooled
and evaporated to obtain a white solid (2.5 g). Although this
solid consisted mainly of the monopropargyl derivative 16
according 1H NMR and MS analysis, small amounts of
unchanged 15 and the dipropargyl derivative 17 were still
evident. Nonetheless, this material was taken directly to the
next step.
Step 4. Compound 21 (750 mg, 1.9 mmol) was dissolved in
DMSO (15 mL), and the solution was immersed in an oil bath
preheated to 120 °C. The progress of the decarboxylation was
monitored by HPLC (C18 silica gel, 1% to 25% MeCN in 0.1 M
NH4OAc, pH 7.5, 30 min gradient, 1 mL/min). The peak
corresponding to 21, at 16 min, was gradually replaced by a
slower peak at 26 min. After 30 min, the reaction mixture was
cooled to room temperature, and the solvent was removed
under high vacuum. The residue was taken up in dilute NH4-
OH, and the solution was cooled in an ice-bath and acidified
to pH 4 with 6 N HCl. The precipitated solid was filtered,
washed with H2O, and dried in vacuo to obtain 22 as a yellow
powder (400 mg, 60%); mp >200 °C dec; IR (KBr) ν 3450, 3280,
2950, 1630, 1545, 1440, 1370, 1260 cm-1; 1H NMR (DMSO-d6)
δ 2.78 (m, 1H, CtCH), 2.59-2.78 (m, 2H, CH2CtCH), 3.1 (m,
2H, 9-CH2), 3.66 (m, 1H, C10-H), 6.52 (br s, 2H, NH2), 7.38
(m, 3′- and 5′-H), 7.81 (m, 2H, 2′- and 6′-H), 8.37 (s, 1H, C7-
H); MS: z/e 349, calcd (M + 1) 349. This material was used
directly in the next step.
Step 5. i-BuOCOCl (235 mg, 1.72 mmol) was added to a
solution of 22 (300 mg, 0.86 mmol) and Et3N (353 mg, 3.5
mmol) in dry DMF (20 mL), and the solution was stirred at
room temperature for 1 h, followed by addition of 23‚HCl (536
mg, 1.72 mmol). Stirring was continued for 2 h, followed by
addition of a second portion of i-BuOCOCl (117 mg, 0.86 mmol)
and, after 30 min, a second portion of 23‚HCl (268 mg, 0.86
mmol). The reaction mixture was then stirred for 20 h,
whereupon the solvent was removed under high vacuum, and
the residue was washed with H2O and chromatographed on a
silica gel column (20 g, 2 × 20 cm) packed and eluted with
CHCl3-MeOH (98:2, then 93:7). Fractions giving a single TLC
spot with Rf 0.48 (silica gel, system E) were evaporated, the
residue was redissolved in a small volume of system E, and
the solution was added to excess Et2O. The precipitated solid
was isolated by centrifugation, washed with Et2O, and dried
in a vacuum oven at 60 °C to obtain 24 as a yellow powder
(67 mg, 13%); mp 145 °C dec; IR (KBr) ν 3460, 3360, 3280,
2950, 1760, 1710, 1700, 1600, 1550, 1520, 1430, 1400, 1350,
Step 2. A stirred suspension of NaH (60% in mineral oil,
488 mg, 12.2 mmol) in dry DMF (10 mL) was cooled to -5 °C
and treated dropwise with a solution of impure 16 from several
pooled batches (3 g, assumed to contain 12.2 mmol) in dry DMF
(10 mL). The reaction mixture was stirred at 0 °C for 30 min,
then cooled to between -25 °C and -30 °C (dry ice/ acetone
bath) while a solution of 18‚HBr (1.5 g, 4.0 mmol) in dry DMF
(20 mL) was added dropwise. The temperature was raised to
-10 °C over 2 h, and stirring at -10 °C was continued for
another 2 h. The mixture was then allowed to come to room
temperature and stirred for a final 2 h. The pH was adjusted
to 7 with small pieces of dry ice, the solvent was evaporated
under high vacuum, and the residue was washed with EtOAc
and then with H2O to obtain a yellowish-brown powder, which
was chromatographed on silica gel with 96:4 CHCl3-MeOH
as the eluent. Evaporation of fractions containing a single spot
(TLC: Rf 0.46, silica gel, 9:1 CHCl3-MeOH, system E) afforded
20 as a yellow solid (0.79 g, 46%); mp >200 °C, darkening
above 170 °C; IR (KBr) ν 3450, 3280, 3100, 2950, 1715, 1620,
1
1200 cm-1; H NMR (DMSO-d6) δ 1.67-1.74 (m, 4H, â- and
γ-CH2), 2.76 (m, 1H, CtCH), 2.56-2.74 (m, 2H, CH2CtCH),
3.17 (m, 2H, 9-CH2), 3.58 (m, 6H, COOMe, δ-CH2, C10-H),
4.38 (m, 1H, R-CH), 6.5 (br s, 2H, NH2), 7.35 (m, 2H, 3′- and
5′-H), 7.69 (m, 4H, 2′- and 6′-H, NH2), 7.80-7.85 (m, 4H,
phthalimide ring), 8.38 (d, 1H, C7-H), 8.6 (d, 1H, CONH); MS
z/e 607, calcd (M + 1) 607. Anal. (C32H30N8O5‚0.6H2O) C, H,
N.
1550, 1440, 1280 cm-1 1H NMR (DMSO-d6) δ 2.84 (m, 1H,
;
CtCH), 2.84-2.93 (m, 2H, CH2CtCH), 3.59 (s, 3H, aliphatic
COOMe), 3.72 (m, 2H, 9-CH2), 3.83 (s, 3H, aromatic COOMe),
7.5-7.9 (m, 4H, phenyl protons), 8.45 (s, 1H, C7-H); MS z/e
421, calcd (M + 1) 421.
Nr-[4-[5-(2,4-Dia m in op ter id in -6-yl)p en t-1-yn -4-yl]ben -
zoyl]-Nδ-h em ip h t h a loyl-L-or n it h in e (10-p r op a r gyl-10-
d ea za -P T523) (6). Compound 24 (30 mg, 49 mmol) was
dissolved in MeOH (20 mL) to which were then added H2O
(20 mL) and Ba(OH)2 (46 mg, 1.46 mmol). The mixture was
stirred at room temperature for 24 h, treated with NH4HCO3
(100 mg), and stirred for another 30 min. The BaCO3 precipi-
tate was removed, and the solvents were evaporated under
reduced pressure. The residue was taken up in a small volume
of H2O, and the solution lyophilized. The resulting product was
dried in vacuo at 60 °C to obtain 6 as a yellow solid (26 mg,
87%); mp >180 °C dec; HPLC 25.5 min (C18 silica gel, 1% to
25% MeCN in 0.1 M NH4OAc, pH 7.5, 30 min gradient, 1 mL/
min); IR (KBr) ν 3400, 2950, 1635, 1530, 1500, 1440, 1370,
1300, 1210 cm-1; UV (pH 7.4) λmax 254 (ꢀ 37000), 371 (7050);
1H NMR (DMSO-d6) δ 1.58-1.92 (m, 4H, â- and γ-CH2), 2.76
(m, 1H, CtCH), 2.56-2.74 (m, 2H, CH2CtCH), 3.20 (m, 4H,
9-CH2, δ-CH2), 3.63 (m, 1H, 10-CH), 4.39 (m, 1H, R-CH), 6.58
(br s, 2H, NH2), 7.40 (m, 2H, 3′- and 5′-H), 7.45-7.65 (m, 4H,
phthaloyl ring), 7.60 (br s, 2H, NH2), 7.76 (m, 2H, 2′- and 6′-
H), 8.30 (m, 1H, phthaloyl CONH), 8.39 (d, 1H, C7-H), 8.47
(d, 1H, CONH); MS z/ e 611, calcd (M + 1) 611. Anal.
(C31H30N8O6‚2H2O) C, H, N.
A slightly slower-moving, but resolvable band on the silica
gel column was also isolated and identified as the nonpro-
pargylated compound 19; TLC: Rf 0.44 (silica gel, system E);
MS z/e 383; calcd (M + 1), 383.
Step 3. To a suspension of 20 (0.78 g, 1.9 mmol) in
2-methoxyethanol (10 mL) was added H2O (10 mL) followed
by 10% NaOH (7.8 mL). The mixture was stirred at room
temperature for 24 h, while monitoring the progress of the
reaction by HPLC (C18 silica gel, 1% to 25% MeCN in 0.1 M
NH4OAc, pH 7.5, 30 min gradient, 1 mL/min). Analysis after
4 h showed the diacid 21 as a peak at 16 min [MS: z/e 393,
calcd (M + 1), 393] and a second product, probably consisting
of a mixture of monoacids, as a slower peak at 26 min [MS:
z/e 407, calcd (M + 1) 407]. After 24 h, when only the fast peak
could be seen, AcOH was added dropwise until the pH
decreased to ca. 8. The solvents were removed under high
vacuum, the residue was taken up in H2O (25 mL), the solution
was cooled in an ice-bath, and the pH was adjusted to 5.5 with
6 N HCl. The precipitate was collected, washed with H2O, and
dried in vacuo to obtain 21 as a beige solid (470 mg, 64%); mp
>200 °C dec, darkening above 180 °C; IR (KBr) ν 3350, 2950,
1640, 1550, 1490, 1420, 1350, 1250 cm-1; 1H NMR (DMSO-d6)
δ 2.82 (m, 1H, CtCH), 2.73-2.97 (m, 2H, CH2CtCΗ), 3.67
(m, 2H, 9-CH2), 6.66 (br s, 2H, NH2), 7.5-7.9 (m, 4H, phenyl
protons), 8.45 (s, 1H, C7-H); MS z/e 393, calcd (M + 1) 393.
An additional 250 mg of 21 was recovered by lyophilization of
the mother liquor, and the two batches were combined and
used directly in the next step.
Ack n ow led gm en t. This work was supported in part
by Grants CA25394 and CA70349 from the National
Cancer Institute (DHHS). Technical assistance in ob-
taining cytotoxicity data against CCRF-CEM cells was

1696 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8
Vaidya et al.
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