Synthesis of N-{4-[2-(2-amino-5,6-dihydro-4(3H)-oxo-7H-pyrrolo[2,3-d]pyrimidin-6-y l)-ethyl]benzoyl}-L-glutamic acid: A ring-contracted analogue of 5,10-dideaza-5,6,7,8-tetrahydrofolic acid

Article abstract of DOI:10.1021/jo951471k

This paper describes the synthesis of N-{4-[2-(2-amino-5,6-dihydro-4(3H)-oxo-7H-pyrrolo[2,3-d]pyrimidin-6-y l)ethyl]benzoyl}-L-glutamic acid (4), which can be viewed as a ring-contracted analogue of 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF, 1) in which the C-7 methylene group of the latter has been excised and C-6 joined to N-8. This compound exhibits significant activity as an inhibitor of the growth of human (CCRF-CEM) lymphoblastic leukemic cells in vitro and apparently acts by blocking de novo purine biosynthesis through inhibition of glycinamide ribonucleotide formyltransferase (GAR FTase).

Full text of DOI:10.1021/jo951471k

J . Org. Chem. 1996, 61, 1261-1266
1261
Syn th esis of
N-{4-[2-(2-Am in o-5,6-d ih yd r o-4(3H)-oxo-7H-p yr r olo[2,3-d ]p yr im id in -6-yl)-
eth yl]ben zoyl}-L-glu ta m ic Acid : A Rin g-Con tr a cted An a logu e of
5,10-Did ea za -5,6,7,8-tetr a h yd r ofolic Acid †
Edward C. Taylor,* Wendy B. Young,‡ and Carsten Spanka§
Department of Chemistry, Princeton University, Princeton, New J ersey 08544
Received August 8, 1995X
This paper describes the synthesis of N-{4-[2-(2-amino-5,6-dihydro-4(3H)-oxo-7H-pyrrolo[2,3-d]-
pyrimidin-6-yl)ethyl]benzoyl}-L-glutamic acid (4), which can be viewed as a ring-contracted analogue
of 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF, 1) in which the C-7 methylene group of the
latter has been excised and C-6 joined to N-8. This compound exhibits significant activity as an
inhibitor of the growth of human (CCRF-CEM) lymphoblastic leukemic cells in vitro and apparently
acts by blocking de novo purine biosynthesis through inhibition of glycinamide ribonucleotide
formyltransferase (GAR FTase).
The synthesis of 5,10-dideaza-5,6,7,8-tetrahydrofolic
acid (DDATHF, (6RS)-1; Lometrexol, (6R)-1) by Taylor
and co-workers1 represents an important advance in
antifolate cancer chemotherapy. Lometrexol has been
shown to be an inhibitor of glycinamide ribonucleotide
formyltransferase (GAR FTase),2 the enzyme that cata-
lyzes the first of two formyl transfer reactions in de novo
purine biosynthesis. Its effectiveness in tumor chemo-
therapy (Lometrexol is now in Phase II clinical trials) is
a result of several factors which include its efficient
C-5 is deleted, C-6 is now joined to the pyrimidine ring,
intracellular conversion to polyglutamates by folylpoly-
and the B ring is aromatic and therefore planar.
A
glutamate synthetase (FPGS) and its ability to enter cells
by pathways involving both the reduced-folate and the
folate-binding membrane protein transport systems.
Since Lometrexol is not an inhibitor of dihydrofolate
reductase (DHFR), it is fully effective against tumors
resistant to methotrexate due to amplification of the
DHFR gene.3,4
primary motivation for the synthesis of 2 was removal
of the chiral center at C-6 of 1. Interestingly, although
2 (which is also currently in Phase II clinical trials) is
extremely active as an inhibitor of tumor growth in vitro
and in vivo, its activity is primarily the consequence of
inhibition of thymidylate synthase (TS), not GAR FTase.
Since the discovery of 1, many analogues have been
prepared in an attempt to uncover the structural require-
ments for GAR FTase inhibition.5 We recently prepared
2 (LY231514)6 as a ring-contracted analogue of 1 in which
† This paper is dedicated with affection and respect to Professor
Yoshifumi Maki on the occasion of his retirement from Gifu Pharma-
ceutical University, J apan.
‡ Recipient of fellowships from Eli Lilly & Company (administered
by the ACS Division of Organic Chemistry), Princeton University
(Harold W. Dodds Fellowship), The Association for Women in Science
Educational Foundation, and the J osephine De Ka´rma´n Foundation.
§ Feodore Lynen Postdoctoral Fellow of the Alexander von Humboldt
Stiftung.
In a continuation of our program directed toward the
synthesis of inhibitors of folate-dependent biochemical
processes as potential anticancer agents, we recently
prepared 37 as an isomer of 2 in which the ethylbenzoyl
glutamate moiety is connected at C-6 rather than C-5.
Since compound 3 showed only weak activity as a cell
growth inhibitor of CCRF-CEM tumor cells (IC50 > 20
µg/mL), we considered the preparation of 4, which should
possess increased flexibility in ring B due to reduction
of the 5,6 bond. In addition, compound 4 can be consid-
ered a ring-contracted analogue of 1 in which C-7 has
been deleted and C-6 joined to N-8.
X Abstract published in Advance ACS Abstracts, December 15, 1995.
(1) (a) Taylor, E. C.; Harrington, P. J .; Fletcher, S. R.; Beardsley,
G. P.; Moran, R. G. J . Med. Chem. 1985, 28, 914. (b) Taylor, E. C.;
Wong, G. S. K.; Fletcher, S. R.; Harrington, P. J .; Beardsley, G. P.;
Shih, C. In Chemistry and Biology of Pteridines; Cooper, B. A.,
Whitehead, V. M., Eds.; Walter de Gruyter: Berlin, 1986; pp 61-64.
(c) Moran, R. G.; Baldwin, S. W.; Taylor, E. C.; Shih, C. J . Biol. Chem.
1989, 264, 21047.
(2) (a) Moran, R. G.; Baldwin, S. W.; Taylor, E. C.; Grindey, G. B.;
Shih, C. J . Biol. Chem., 1989, 264, 21047. (b) Baldwin, S. W.; Moran,
R. G. Proc. Am. Assoc. Cancer Res., 1989, 30, A1901. (c) Beardsley, G.
P.; Moroson, B. A.; Taylor, E. C.; Moran, R. G. J . Biol. Chem. 1989,
264, 328. (d) Beardsley, G. P.; Taylor, E. C.; Shih, C.; Poore, G. A.;
Grindey, G. B.; Moran, R. G. Proc. Am. Assoc. Cancer Res. 1986, 27,
A1027.
It is obvious that target molecule 4 differs from 3 only
in the state of the reduction of the pyrrole ring. Com-
pound 3 had been synthesized as shown in Scheme 1,7
(3) Beardsley, G. P.; Taylor, E. C.; Shih, C.; Poore, G. A.; Grindey,
G. B.; Moran, R. G. Proc. Am. Assoc. Cancer Res. 1986, 27, 259.
(4) Pizzorno, G.; Moroson, B. A.; Cashmore, A. R.; Taylor, E. C.;
Beardsley, G. P. Proc. Am. Assoc. Cancer Res. 1988, 29, 281.
(5) (a) Taylor, E. C. J . Heterocycl. Chem., 1989, 27, 1. (b) Taylor, E.
C. Adv. Exper. Med. Biol. 1993, 338, 387.
(6) Taylor, E. C.; Kuhnt, D.; Shih, C.; Rinzel, S. M.; Grindey, G. B.;
Barredo, J .; J annatipour, M.; Moran, R. G. J . Med. Chem., 1992, 35,
4450.
(7) Taylor, E. C.; Young, W. B.; Chaudhari, R.; Patel, H. H.
Heterocycles 1993, 36, 1897.
0022-3263/96/1961-1261$12.00/0 © 1996 American Chemical Society

1262 J . Org. Chem., Vol. 61, No. 4, 1996
Taylor et al.
Sch em e 1
and it was anticipated that 4 should be available through
reduction of the pyrrole nucleus at some intermediate
step in the synthesis. However, since it was not clear
how or when this reduction would be accomplished,
preliminary work focused on reduction of the model
compound 5.
Literature reports on attempts to reduce pyrrolo[2,3-
d]pyrimidines to 5,6-dihydro systems were not at all
encouraging.8 Attempts in our hands to reduce 5 to 10
by catalytic hydrogenation using several different cata-
lysts (PtO2, Pd/C, Pt/C) in a variety of solvents (MeOH,
CH2Cl2, TFA) also failed; only recovered starting material
was obtained. Attempted reduction using NaBH3CN was
also unsuccessful. However, since it is well documented
that the presence of an electron-withdrawing group on
the pyrrole nitrogen greatly facilitates ring reduction,9
we turned to an examination of appropriate N-7 substi-
tuted derivatives of 5.
Sch em e 2
Treatment of 5 with 3 equiv of sodium hydride followed
by addition of 2.1 equiv of acetyl chloride led, as expected,
to the N-7 acetylated derivative 11 (90%, Scheme 2).
Unfortunately, reduction of 11 with 50 psi of hydrogen
for 12 h in a CH2Cl2/HOAc solution, using PtO2 as
catalyst, gave a mixture of deacetylated starting material
5 (47%) together with a low yield of the desired dihydro
compound 13 (30%). Extensive modifications of the
reaction conditions failed to improve the yield of 13.
Due to the ease with which 11 underwent deacetylation
during hydrogenation, the more durable tosyl protecting
(and electron-withdrawing) group was chosen for further
studies.10 Treatment of 5 with 3.3 equiv of NaH and 1.0
equiv of TsCl in DMF produced 12 in 71% yield. After
numerous attempts to reduce 12 using catalytic hydro-
genation methods with a range of catalysts (PtO2, 5% Pd/
C, 5% Pt/C) it was finally found that with Pearlman’s
catalyst (10% Pd(OH)2/C) in MeOH, under 1 atm of
hydrogen for 5 h, reduction of 12 to 14 could be ac-
complished in high (94%) yield. Conversion of 14 to the
depivaloylated/detosylated pyrrolopyrimidine 15 could
then be carried out either with methanolic NH3/metallic
Na at -78 °C11,12 or by heating at 100 °C for 18 h in
concentrated H2SO4 (Scheme 3). We then turned our
attention to the preparation of our desired target com-
pound 4.
Sch em e 3
N-7 by treatment with 3.3 equiv of NaH, followed by
addition of TsCl and acid workup to generate 16 (54%).
Pd-catalyzed coupling of methyl 4-ethynylbenzoate (17)13
with 16 in CH2Cl2 gave 18 in 85% yield.
Reduction of 18 under 1 atm of hydrogen using 20%
Pd(OH)2/C in 50% MeOH/acetone solution for 12 h gave
20 in 94% yield. With careful manipulation of the
reaction conditions, the intermediate reduction product
19 could be isolated. For instance, a 9 mM solution of
compound 18 in CH2Cl2 using Pd(OH)2 as catalyst under
1 atm of hydrogen for 12 h produced solely compound
19. Also, the partial reduction of 18 to form 19 could be
Schemes 4 and 5 illustrate the reactions utilized in the
synthesis of 4. The 6-iodo compound 67 was tosylated at
(8) Barnett, C. J .; Wilson, T. M. Heterocycles 1993, 35, 925.
(9) (a) Signaigo, F. K.; Adkins, H. J . Am. Chem. Soc., 1936, 58, 709.
(b) Rainey, J . L.; Adkins, H. J . Am. Chem. Soc. 1939, 61, 1104.
(10) Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic
Synthesis, 2nd Ed.; J ohn Wiley & Sons, NY, 1991.
(11) Roemmele, R. C.; Rapoport, H. J . Org. Chem. 1988, 53, 2367.
(12) (a) Schultz, A. G.; McCloskey, P. J .; Court, J . J . J . Am. Chem.
Soc. 1987, 109, 6493. (b) Yamazaki, N.; Kibayashi, C. J . Am. Chem.
Soc. 1989, 111, 1396.
(13) Austin, W. B.; Bilow, N.; Kelleghan, W. J .; Lau, K. S. Y. J . Org.
Chem. 1981, 46, 2280.

An Analogue of 5,10-Dideaza-5,6,7,8-tetrahydrofolic Acid
J . Org. Chem., Vol. 61, No. 4, 1996 1263
Sch em e 4
Sch em e 6
tion of 19 to 20 with PtO2 as the catalyst proved
troublesome.
The protecting groups were removed by heating 20 in
concentrated H2SO4 at 100 °C for 90 min followed by an
alkaline workup to afford 21 (93%). Coupling of 21 with
dimethyl L-glutamate was accomplished using the 6-chloro-
2,4-dimethoxy-1,3,5-triazine14 /N-methymorpholine tech-
nique, as we have described previously.7 Since attempted
isolation of 22 from the crude reaction mixture by column
chromatography on silica gel afforded a 1:1 mixture of
the desired product 22 and 2,4-dimethoxy-6(5H)-oxo-
1,3,5-triazine (23), the crude reaction mixture was stirred
with 1 N NaOH at room temperature for 12 h and then
acidified. In this way compound 4 was isolated in 32%
yield (from 21) as a 1:1 mixture of diastereomers.
A more efficient synthesis of 4 resulted from the use
of an ethoxycarbonyl N-7 protecting group rather than
an N-Ts group (Scheme 6). Thus, reaction of 2-pivaloyl-
4(3H)-oxo-7H-pyrrolo[2,3-d]pyrimidine (5) with 3 equiv
of NaH, followed by 1 equiv of ethyl chloroformate,
smoothly gave the 7-ethoxycarbonyl derivative 24. In
contrast to the behavior of 5 itself toward halogenation
reactions, 24 underwent monobromination with N-bro-
mosuccinimide at 0 °C in CH2Cl2 solution to give exclu-
sively the 6-bromo derivative 25. Standard Pd-catalyzed
coupling of 25 with dimethyl N-(4-ethynylbenzoyl)-L-
glutamate (26)15 proceeded as expected to provide 27;
catalytic reduction of the acetylenic and pyrrole systems
to give 28, followed by final deprotection with dilute
alkali, then gave target compound 4.
Sch em e 5
Biological evaluation showed that 4 was an active
inhibitor of the growth of human (CCRF-CEM) lympho-
(14) (a) Dudley, J . R.; Thurston, J . T.; Schaefer, F. C.; Holm-Hansen,
D.; Hull, C. J .; Adams, P. J . Am. Chem. Soc. 1951, 73, 2986. (b)
Kaminski, Z. J . Tetrahedron Lett. 1985, 26, 2901.
accomplished in 98% yield by exposure of 18 to 50 psi of
hydrogen over PtO2 in 5% MeOH/CH2Cl2. Further reduc-
(15) Taylor, E. C.; Wong, G. S. K. J . Org. Chem. 1989, 54, 3618.

1264 J . Org. Chem., Vol. 61, No. 4, 1996
Taylor et al.
2-(P iva loyla m in o)-4(3H )-oxo-5,6-d ih yd r o-7-t osyl-7H -
p yr r olo[2,3-d ]p yr im id in e (14). A mixture of 12 (0.10 g, 0.26
mmol), 10% Pd(OH)2/C (0.20 g), and MeOH (5 mL) was stirred
under 1 atm of H2 for 5 h. The mixture was filtered, the
filtrate was evaporated to dryness under reduced pressure, and
the product was purified by column chromatography over silica
gel with 100% CH2Cl2 to 1% MeOH/CH2Cl2 as the eluent to
yield 0.095 g (94%) of 14 as a white solid: mp > 237 °C dec;
1H NMR (CDCl3, 300 MHz) δ 11.86 (s, 1 H), 8.80 (s, 1 H), 7.84
(d, 2 H, J ) 8.1 Hz), 7.31 (d, 2 H, J ) 8.1 Hz), 3.95 (t, 2 H, J
) 9.0 Hz), 2.83 (t, 2 H, J ) 9.0 Hz), 2.43 (s, 3 H), 1.33 (s, 9 H);
IR (KBr) 3542, 3408, 3161, 2964, 2929, 1661, 1563, 1393, 1352,
1168, 1139, 654, 534 cm-1; MS m/ e (relative intensity) 390
(M+, 43), 306 (10), 151 (18), 91 (26), 78 (100); HRMS calcd for
C18H22N4O4S 390.1364, found 390.1355. Anal. Calcd for
C18H22N4O4S: C, 55.37; H, 5.68; N, 14.35. Found: C, 55.08;
H, 5.63; N, 14.13.
blastic leukemic cells in vitro (IC50 ) 0.071 µg/mL).
Thymidine (5 µM) did not protect the cells from the cell
growth inhibitory effects of 4, while hypoxanthine (100
µM) and aminoimidazolecarboxamide (300 µM) was each
able to reverse the cell growth inhibitory effects of 4.
These data indicate that the activity of 4 (presumably
following intracellular polyglutamylation) is the result
of inhibition of GAR FTase. Thus, 4 is indeed an
inhibitor of purine biosynthesis, as initially predicted.
However, since both 2 (LY231514) and its dihydro
derivative are thymidylate synthase (TS) inhibitors8 and
thus poor inhibitors of the purine biosynthetic pathway
enzymes, it can be seen that very minor changes in
structure can determine the preferred enzymatic inhibi-
tion target.
2-Am in o-4(3H)-oxo-5,6-d ih yd r o-7H-p yr r olo[2,3-d ]p yr i-
m id in e (15). Meth od A. In a 50 mL, two-neck round-bottom
flask a solution of 14 (200 mg, 0.51 mmol) in MeOH (5 mL)
was added to NH3(liq) (20 mL) at -78 °C. To this mixture was
added Na (590 mg, 25.7 mmol) in increments. The reaction
was stirred for 30 min at -78 °C, quenched with NH4Cl(aq) (2
mL), warmed to rt, and acidified to pH 4 with AcOH(gl). The
solvent was removed under reduced pressure, H2O was added
and the precipitate was filtered, washed with H2O (2 × 5 mL),
CH2Cl2 (2 × 5 mL), Et2O (2 × 5 mL), and hexanes (2 × 5 mL)
and dried overnight in a vacuum oven. The crude product was
recrystallized from H2O (5 mL) to yield 43 mg (55%) of 15 as
a white solid, mp > 272 °C dec, (lit.16 mp 278-280 °C dec); 1H
NMR (DMSO-d6, 300 MHz) δ 9.81 (s, 1 H), 6.28 (s, 2 H), 3.32
(t, 2 H, J ) 8.8 Hz), 2.54 (t, 2 H, J ) 8.8 Hz), 1.86 (s, 1 H); MS
m/ e (relative intensity) 152 (M+, 15), 78 (92); HRMS calcd for
C6H8N4O1 152.0700, found 152.0718. Anal. Calcd for
C6H8N4O: C, 47.36; H, 5.30; N, 36.82. Found: C, 47.62; H,
5.51; N, 36.60.
Exp er im en ta l Section
2-(P iva loyla m in o)-4(3H)-oxo-7-a cet yl-7H-p yr r olo[2,3-
d ]p yr im id in e (11). In a 200 mL round-bottom flask, flushed
with nitrogen, NaH (80% in mineral oil, 0.16 g, 6.45 mmol) in
DMF(anh) (10 mL) was stirred at 0 °C. To this mixture was
added 2-(pivaloylamino)-4(3H)-oxo-7H-pyrrolo[2,3-d]pyrimi-
dine (2-pivaloyl-7-deazaguanine, 5) (0.50 g, 2.15 mmol) dis-
solved in DMF(anh) (15 mL) dropwise over 15 min. The mixture
was stirred an additional 30 min, and acetyl chloride (0.36 g,
4.60 mmol) was added dropwise over a period of 30 min. The
mixture was warmed to rt and stirred overnight, AcOH(gl) was
carefully added to neutralize the solution, the solvent was
removed under reduced pressure, and the crude product was
purified by column chromatography over silica gel with 0.5%
MeOH/CH2Cl2 as the eluent to yield 0.53 g (90%) of 11 as a
gum: 1H NMR (CDCl3, 300 MHz) δ 12.04 (s, 1 H), 8.06 (s, 1
H), 7.49 (d, 1 H, J ) 3.8 Hz), 6.70 (d, 1 H, J ) 3.8 Hz), 2.83 (s,
3 H), 1.35 (s, 9 H); MS m/ e (relative intensity) 276 (M+, 38),
234 (88), 150 (100); HRMS calcd for C13H16N4O3 276.1224,
found 276.1208.
2-(P iva loyla m in o)-4(3H)-oxo-5,6-d ih yd r o-7-a cetyl-7H-
p yr r olo[2,3-d ]p yr im id in e (13). A mixture of 11 (0.10 g, 0.36
mmol), PtO2 (0.05 g), and 5% MeOH/HOAc was shaken under
50 psi of hydrogen for 12 h. The mixture was filtered, the
filtrate was evaporated to dryness under reduced pressure, and
the product was purified by column chromatography over silica
gel with 100% CH2Cl2 to 1% MeOH/CH2Cl2 as the eluent to
yield 0.03 g (30%) of 13 as a white solid and 0.04 g (47%) of 5.
13: mp 230-232 °C; 1H NMR (CDCl3, 300 MHz) δ 11.84 (s, 1
H), 8.12 (s, 1 H), 4.04 (t, 2 H, J ) 9.0 Hz), 2.86 (t, 2 H, J ) 9.0
Hz), 2.52 (s, 3 H), 1.34 (s, 9 H); MS m/ e (relative intensity)
278 (M+, 50), 236 (100), 151 (60); HRMS calcd for C13H18N4O3
278.1381, found 278.1372. Anal. Calcd for C13H18N4O3: C,
56.10; H, 6.52; N, 20.13. Found: C, 55.78; H, 6.50; N, 19.89.
2-(P iva loyla m in o)-4(3H)-oxo-7-tosyl-7H-p yr r olo[2,3-d ]-
p yr im id in e (12). In a 200 mL round-bottom flask, flushed
with nitrogen, NaH (80% in mineral oil, 0.21 g, 7.08 mmol) in
DMF(anh) (5 mL) was stirred at 0 °C. To this mixture 5 (0.50
g, 2.14 mmol) dissolved in DMF(anh) (5 mL) was added dropwise
over 15 min. The mixture was stirred an additional 30 min
and TsCl (0.40 g, 2.14 mmol) in DMF(anh) (5 mL) was added
dropwise. The mixture was warmed to rt and stirred over-
night, AcOH(gl) was carefully added to neutralize the solution,
the solvent was removed under reduced pressure, and the
crude product was purified by column chromatography over
silica gel with 1% MeOH/CH2Cl2 as the eluent to yield 0.59 g
(71%) of 12 as a white powder: mp 279-280 °C. 1H NMR
(DMSO-d6, 300 MHz) δ 12.16 (s, 1 H), 10.80 (s, 1 H), 8.14 (d,
2 H, J ) 8.3 Hz), 7.40 (d, 2 H, J ) 8.3 Hz), 7.35 (d, 1 H, J )
3.8 Hz), 6.61 (d, 1 H, J ) 3.8 Hz), 2.39 (s, 3 H), 1.25 (s, 9 H);
IR (KBr) 3224, 3153, 3133, 2857, 1654, 1591, 1541, 1372, 1131,
647, 619 cm-1; MS m/ e (relative intensity) 388 (M+, 56), 232
(72), 149 (100), 107 (27), 99 (27), 85 (81); HRMS calcd for
C18H20N4O4S 388.1207, found 388.1208. Anal. Calcd for
C18H20N4O4S: C, 55.65; H, 5.19; N, 14.43. Found: C, 55.57;
H, 5.23; N, 14.40.
Meth od B. A mixture of 14 (0.043 g, 0.11 mmol) and H2-
SO4 (2 mL) was heated to 100 °C for 18 h. The mixture was
cooled, ice-water (5 mL) was added, the solution was neutral-
ized to pH 4 with 1 M NaOH, the solvent was removed under
reduced pressure, and the remaining solid was dried overnight
in a vacuum oven. The product was not further purified.
Spectral data were identical with those reported above.
2-(P ivaloylam in o)-4(3H)-oxo-6-iodo-7-tosyl-7H-pyr r olo-
[2,3-d ]p yr im id in e (16). To a mixture of NaH (95% in
mineral oil, 0.48 g, 19.1 mmol) and DMF(anh) (10 mL) at 0 °C
was added 2-(pivaloylamino)-4(3H)-oxo-6-iodo-7H-pyrrolo[2,3-
d]pyrimidine (2-pivaloyl-8-iodo-7-deazaguanine, 6)5 (2.09 g, 5.8
mmol) in DMF(anh) (15 mL) dropwise over 30 min. The mixture
was stirred an additional 30 min, and TsCl (3.3 g, 17.4 mmol)
was added dropwise. The mixture was warmed to rt and
stirred overnight, AcOH(gl) was carefully added to neutralize
the solution, the solvent was removed under reduced pressure,
and the crude product was purified by column chromatography
over silica with 100% CH2Cl2 to 1% MeOH/CH2Cl2 as the
eluent to yield 1.6 g (54%) of 16 as a white powder: mp 219-
221 °C; 1H NMR (CDCl3, 300 MHz) δ 12.12 (s, 1 H), 9.01 (s, 1
H), 7.95 (d, 2 H, J ) 8.1 Hz), 7.32 (d, 2 H, J ) 8.1 Hz), 7.02 (s,
1 H), 2.43 (s, 3 H), 1.34 (s, 9 H); IR (KBr) 3203, 2950, 2915,
2865, 2851, 1661, 1590, 1541, 1407, 1379, 1189, 1147, 1084
cm-1; MS m/ e (relative intensity) 514 (M+, 46), 388 (81), 360
(100), 276 (55), 233 (54), 149 (93); HRMS calcd for C18H19
IN4O4S 514.0174, found 514.0173.
-
Met h yl 4-[2-(2-(P iva loyla m in o)-4(3H )-oxo-7-t osyl-7H -
p yr r olo[2,3-d ]p yr im id in -6-yl)eth yn yl]ben zoa te (18). In
a 100 mL, two-neck round-bottom flask equipped with a reflux
condensor were combined 16 (0.30 g, 0.58 mmol), methyl
4-ethynylbenzoate (17)13 (0.14 g, 0.87 mmol), tetrakis(tri-
phenylphosphine)palladium(0) (0.07 g, 0.058 mmol), CuI (0.01
g, 0.058 mmol), Et3N (0.24 g, 2.32 mmol), and CH2Cl2 (15 mL),
and the mixture was heated to reflux for 3 h. The solvent was
removed under reduced pressure, and the crude product was
purified by column chromatography on silica gel with 0.5%
(16) Granik, V. G.; Glushkov, R. G., Khim.-Farm. Zh. 1967, 1, 16.

An Analogue of 5,10-Dideaza-5,6,7,8-tetrahydrofolic Acid
J . Org. Chem., Vol. 61, No. 4, 1996 1265
MeOH/CH2Cl2 as the eluent to give 0.27 g (85%) of 18 as a
white solid: mp 228-230 °C; 1H NMR (CDCl3, 300 MHz) δ
12.15 (s, 1 H), 8.81 (s, 1 H), 8.07 and 7.98 (AA′BB′, 4 H), 7.67
and 7.31 (AA′BB′) 4 H), 7.0 (s, 1 H), 3.95 (s, 3 H), 2.43 (s, 3 H),
1.36 (s, 9 H); MS m/ e (relative intensity) 546 (M+, 100), 392
(89), 307 (96), 265 (30), 91 (80); HRMS calcd for C28H26N4O6S
546.1575, found 546.1566.
reaction mixture was allowed to come to rt overnight with
stirring and then poured into ice cold water (400 mL). The
cloudy solution was extracted as rapidly as possible with
EtOAc (3 × 150 mL). The combined organic extracts were
washed with brine, dried (MgSO4), and evaporated to dryness
to leave a purple solid. Purification by column chromatogra-
phy over SiO2 with 50% EtOAc/hexanes as the eluent gave
4.7 g (77%) of 24 as a colorless white powder: mp 186-188
°C; 1H NMR (CDCl3, 300 MHz) δ 12.14 (br s, 1 H,), 8.60 (br s,
1 H), 7.23 (d, 1 H, J ) 4.1), 6.64 (d, 1 H, J ) 4.2), 4.42 (q, 2 H),
1.40 (t, 3 H, J ) 7.0), 1.28 (s, 9 H); 13C-NMR (CDCl3, 60 MHz)
δ 14.1, 26.9, 40.2, 63.9, 105.7, 108.3, 120.8, 147.7, 148.9, 157.,
180.0; IR (KBr) 3564, 3463, 3187, 3162, 2982, 2958, 2926 &
2854, 1768, 1753, 1706, 1674, 1609, 1559, 1426, 1374, 1336,
Met h yl 4-[2-(2-(P iva loyla m in o)-4(3H )-oxo-7-t osyl-7H -
p yr r olo[2,3-d ]p yr im id in -6-yl)eth yl]ben zoa te (19). A mix-
ture of 18 (0.38 g, 0.70 mmol), PtO2 (0.38 g), and 5% MeOH/
CH2Cl2 (30 mL) was shaken under 58 psi of H2 for 24 h. The
mixture was filtered, the filtrate was evaporated to dryness
under reduced pressure, and the product was purified by
column chromatography over silica gel with 2% MeOH/CH2-
Cl2 as the eluent to yield 0.38 g of 19 (98%) as an off-white
1326, 1289, 1268, 1249, 1166, 1152, 1013 (w), 784, 730 cm-1
MS m/ e (relative intensity) 306 (100%, M+), 222 (22%, M+
;
-
1
solid: mp 254 °C dec; H NMR (CDCl3, 300 MHz) δ 12.00 (s,
84, - tBuCO), 150 (85%, C6H6N4O), 133 (21%), 125 (36%), 111
(60%), 97 (90%), 83 (74%), 71 (90%); HRMS calcd for C14H18N4O4
306.1328, found 306.1330. Anal. Calcd for C14H18N4O4: C:
54.89; H, 5.92; N, 18.29. Found: C, 54.67; H, 5.85; N, 18.43.
2-(P iva loyla m in o)-4(3H)-oxo-6-br om o-7-(eth oxyca r bo-
n yl)-7H-p yr r olo[2,3-d ]p yr im id in e (25). To a stirred solu-
tion of 24 (4.70 g, 15.3 mmol) in dry CH2Cl2 (150 mL) was
added over a period of 1.5 h a solution of NBS (2.73 g, 15.3
mmol, freshly recrystallized from hot H2O) in dry CH2Cl2 (200
mL) at 0 °C. To prevent deterioration of the product the
solution was protected from light by covering the flask with
aluminum foil. Stirring was continued for an additional 3 h
at 0 °C. During this time the color changed from bright yellow
to dark yellow with a green tinge. The reaction was quenched
by pouring the contents of the flask onto ice-cold 5% aqueous
sodium thiosulfate (350 mL). The organic layer was washed
(3 × H2O, 1 x brine), dried (MgSO4), and evaporated. The
remaining deep purple solid was purified by flash chromatog-
raphy (SiO2 , EtOAc/hexanes 2:1) to give 2.94 g (50%) of 25 as
a fluffy colorless solid: mp 211-213 °C; 1H NMR (CDCl3, 300
MHz) δ 12.11 (br s, 1 H), 8.50 (br s, 1 H), 6.87 (s, 1 H), 4.52 (q,
2 H), 1.47 (t, 3 H, J ) 7.2), 1.31 (s, 9 H); 13C-NMR (CDCl3, 68
MHz) δ 14.0, 26.9, 40.3, 64.9, 103.1, 107.6, 110.5, 147.6, 148.5,
149.4, 155.9, 180.2. IR (KBr) 3174 (NH), 2979 & 2919 (CH),
1771 (CdO), 1732 (w), 1663, 1609, 1557 (w), 1413 (w), 1355,
1296, 1257, 1146, 769, 760 cm-1; MS m/ e (relative intensity)
386, 384 (48%, 45%, M+); 314, 312 (53%, 51%, M+ - 72, -
EtCO2); 258, 256 (39%, 38%, M+ - 128, - H2CdCMe2 , -
EtCO2); 230, 228 (89%, 89%, M+ - 156, C6H5BrN4O); 188, 186
(28%, 32%); 187, 185 (25%, 26%); 150 (68%, C6H6N4O); 105
(23%); 85 (100%); HRMS calcd for C14H17BrN4O4 384.0432,
found 384.0449. Anal. Calcd for C14H17BrN4O4: C, 43.65; H,
4.45; N, 14.54; Br, 20.74. Found: C, 43.84; H, 4.42; N, 14.45;
Br, 20.57.
Dim eth yl N-{4-[2-(2-(P iva loyla m in o)-4(3H)-oxo-7-(eth -
oxyca r bon yl)-7H-p yr r olo[2,3-d ]p yr im id in -6-yl)eth yn yl]-
ben zoyl}-l-glu ta m a te (27). To a suspension of 25 (385 mg,
1 mmol) in dry MeCN (15 mL)17 were added freshly prepared
Pd(PPh3)4 (58 mg, 50 mmol, 5 mol %), triethylamine (1 mL),
and CuI (5 mg, 25 mmol, 2.5 mol %). The resulting mixture
was stirred for 15 min. at 50 °C, and then a solution of
dimethyl N-(4-ethynylbenzoyl)-L-glutamate (26)15 (455 mg, 1.5
mmol, 1.5 equiv) in MeCN was introduced in one portion via
syringe. After a short time all the solid material went into
solution, and the color of the mixture turned olive. The
reaction mixture was heated under reflux until 25 had been
completely consumed (TLC, ca. 6 h). To remove solid impuri-
ties the almost black mixture was filtered through a fritted
glass funnel. The filtrate was evaporated to dryness to afford
a black gum which was dissolved in EtOAc (50 mL), washed
(3 × H2O, 1 × brine), dried (MgSO4), and evaporated again to
afford a dark brown gum. Column chromatography over SiO2
with 1.5% MeOH in CH2Cl2 as the eluent gave 350 mg (57%)
of 27 as a pale yellow microcrystalline solid: mp. 91-93 °C;
1H NMR (CDCl3, 300 MHz) δ 12.17 (br s, 1 H), 8.91 (br s, 1
H), 7.80 (d, 2 H, J ) 8.2), 7.50 (d, 2 H, J ) 8.3), 7.36 (d, 1 H,
J ) 7.5), 6.99 (s, 1 H), 4.79 (ddd, 1 H, J ) 4.9, 7.7, 12.5), 4.48
1 H), 8.16 (s, 1 H), 7.97 and 7.85 (AA′BB′, 4 H), 7.30 and 7.29
(AA′BB′, 4H), 6.41 (s, 1 H), 3.91 (s, 3 H), 3.25 (t, 2 H, J ) 8.3
Hz), 3.09 (t, 2 H, J ) 8.3 Hz), 2.41 (s, 3 H), 1.34 (s, 9 H); MS
m/ e (relative intensity) 550 (M+, 9), 402 (38), 401 (100), 247
(40); HRMS calcd for C28H30N4O6S 550.1888, found 550.1893.
A mixture of 18 (0.10 g, 0.18 mmol), 20% Pd(OH)2/C (0.030
g), and CH2Cl2 (20 mL) was stirred under 1 atm of H2 for 12
h. The mixture was filtered, the filtrate was evaporated to
dryness under reduced pressure, and the product was purified
by column chromatography over silica gel with 2% MeOH/CH2-
Cl2 as the eluent to yield 0.096 g (98%) of 19 as an off-white
solid. The spectral data were identical with those obtained
for the product of the first reduction reported above.
Meth yl 4-[2-(2-(P iva loyla m in o)-4(3H)-oxo-5,6-d ih yd r o-
7-t osyl-7H-p yr r olo[2,3-d ]p yr im id in -6-yl)et h yl]b en zoa t e
(20). A mixture of 18 (0.09 g, 0.16 mmol), 20% Pd(OH)2/C (45
mg), and 50% MeOH/acetone (10 mL) was stirred under 1 atm
of H2 for 12 h. The mixture was filtered, the filtrate was
evaporated to dryness under reduced pressure, and the product
was purified by column chromatography over silica gel with
1% MeOH/CH2Cl2 as the eluent to yield 0.085 g (94%) of 20 as
a white powder: mp 120-122 °C; 1H NMR (CDCl3, 300 MHz)
δ 11.85 (s, 1 H), 8.83 (s, 1 H), 7.96 and 7.71 (AA′BB′, 4 H),
7.26 and 7.24 (AA′BB′, 4 H), 4.37-4.34 (m, 1 H), 3.91 (s, 3 H),
2.89 (dd, 1 H, J ) 10.1, 15.8 Hz), 2.77-2.69 (m, 2 H), 2.59 (dd,
1 H, J ) 3.8, 15.8 Hz), 2.41 (s, 3 H), 2.25-2.20 (m, 1 H), 2.12-
2.05 (m, 1 H), 1.32 (s, 9 H); IR (KBr) 3556, 3189, 2964, 2943,
2915, 1710, 1661, 1605, 1563, 1386, 1351, 1274, 1154 cm-1
;
MS m/ e (relative intensity) 552 (M+, 42), 389 (20), 247 (28),
235 (100), 151 (31); HRMS calcd for C28H32N4O6S 552.2045,
found 552.2026.
4-[2-(2-Am in o-4(3H)-oxo-5,6-d ih yd r o-7H-p yr r olo[2,3-d ]-
p yr im id in -6-yl)eth yl]ben zoic Acid (21). A solution of 20
(0.31 g, 0.56 mmol) and H2SO4(conc) (4 mL) was heated at 100
°C for 90 min and cooled, ice-water (3 mL) was added, and
the precipitate was removed by filtration. The collected solid
was placed in H2O (1 mL), 1 N NaOH was added until the
solution went clear and the mixture was filtered. To the
filtrate was added AcOH(gl) (1 mL), and the precipitate was
collected by filtration, washed with H2O (2 × 5 mL), Et2O (2
× 10 mL), and hexanes (2 × 10 mL) and dried in a vacuum
oven overnight to yield 0.16 g (93%) of 21 as a white solid:
1
mp > 257 °C slow dec; H NMR (DMSO-d6, 270 MHz) δ 13.0
(s, 1 H), 9.65 (s, 1 H), 7.79 and 7.28 (AA′BB′, 4 H), 6.59 (s, 1
H), 6.21 (s, 2 H), 3.67-3.61 (m, 1 H), 2.71 (dd, 1 H, J ) 14.2,
10.0 Hz), 2.64-2.53 (m, 2 H), 2.25 (dd, 1 H, J ) 14.2, 6.9 Hz),
1.87-1.67 (m, 2H); IR (KBr) 3408, 3338, 3140, 2922, 1640,
1598, 1570, 1252, 1168 cm-1; MS m/ e (relative intensity) 300
(M+, 34), 163 (45), 151 (100); HRMS calcd for C15H16N4O3
300.1224, found 300.1240.
2-(P iva loyla m in o)-4(3H )-oxo-7-(et h oxyca r b on yl)-7H -
p yr r olo[2,3-d ]p yr im id in e (24). A solution of 2-(pivaloy-
lamino)-4(3H)-oxo-7H-pyrrolo[2,3-d]pyrimidine (5 , 4.68 g, 20
mmol) in DMF(anh) (200 mL) was placed into an ice bath and
treated with NaH (420 mg, 80% suspension in mineral oil, 22
mmol, 1.1 equiv). The mixture was stirred vigorously until
the evolution of H2 ceased completely. To make certain that
deprotonation was complete, the purple-colored suspension
was stirred for an additional 1 h. Ethyl chloroformate (2.1
mL, 2.39 g, 22 mmol) was added in one portion, and the
(17) Acetonitrile was purchased from Aldrich (sure seal grade). This
coupling step failed with standard-grade J . T. Baker acetonitrile.

1266 J . Org. Chem., Vol. 61, No. 4, 1996
Taylor et al.
(q, 2 H, J ) 7.2), 3.75 (s, 3 H), 3.63 (s, 3 H), 2.48 (m, 2 H), 2.30
(m, 1 H), 2.16 (m, 1 H),1.40 (t, 3 H, J ) 7.2), 1.28 (s, 9 H). 13C
NMR (CDCl3, 75 MHz) δ 14.8, 27.5, 27.6, 30.9, 41.0, 52.6, 53.0,
53.3, 65.0, 83.9, 94.3, 107.9, 115.0, 116.2, 128.0, 131.9, 132.6,
133.9, 149.3, 149.6, 150.8, 156.9, 167.0, 173.0, 174.3, 181.1;
IR (KBr) 3440 (NH), 2954, 2920, 1737, 1676, 1605, 1556, 1540,
1484 (w), 1447 (w), 1420 (w), 1372 (w), 1303, 1245, 1150, 1106
(w), 775 (w) cm-1; MS m/ e (relative intensity) 607 (17%, M+),
591 (33%, M+ - 16), 577 (23%, M+ - 30), 563 (100%), 549
(14%), 535 (15%), 479 (18%), 461 (24%), 389 (17%), 305 (14%),
304 (17%), 303 (13%), 277 (27%), 276 (27%), 249 (10%). Anal.
Calcd for C30H33N5O9: C, 59.30; H, 5.47; N, 11.53; O, 23.70.
Found: C, 59.59; H, 5.51; N, 11.64.
crude product was purified by column chromatography on
silica gel with 5% MeOH/CH2Cl2 as the eluent to yield 92 mg
of a 1:1 mixture of 22 [1H NMR (DMSO-d6, 270 MHz) δ 9.56
(s, 1 H), 8.64 (d, 1 H, J ) 7.3 Hz), 7.76 (d, 2 H, J ) 8.3 Hz),
7.28 (d, 2 H, J ) 8.3 Hz), 6.60 (s, 1 H), 6.17 (s, 2 H), 4.42-4.39
(m, 1 H), 3.63 (m, 1 H), 3.60 (s, 3 H), 3.54 (s, 3 H), 2.71 (dd, 1
H, J ) 14.2, 9.9 Hz), 2.66-2.60 (m, 2 H), 2.42-2.38 (m, 2 H),
2.25 (dd, 1 H, J ) 14.2, 6.6 Hz), 2.08-1.94 (m, 2 H), 1.75-
1.67 (m, 2 H); FABMS calcd MH+ for C22H28N5O6 458.2040,
found 458.2073] and 2,4-dimethoxy-6(5H)-oxo-1,3,5-triazine
(23) (37%).
To the 1:1 mixture of 22 and 23 (50 mg), 1 N NaOH (10
mL) was added, and the mixture was stirred at rt for 12 h,
HCl(concd) (1 drop) was added, the precipitate was filtered, and
the crude product was washed with H2O (2 × 2 mL), Et2O (2
× 10 mL), and hexanes (2 × 10 mL) and dried in a vacuum
oven overnight to yield 32 mg (32% from 21) of 4 as a white
solid: mp 241-243 °C; 1H NMR (DMSO-d6, 300 MHz) δ 12.30
(br s, 2 H), 9.65 (br s, 1 H), 8.47 (d, 1 H, J ) 7.6), 7.77 (d, 2 H,
J ) 7.9), 7.29 (d, 2 H, J ) 7.9), 6.66 (br s, 1 H), 6.24 (br s, 2
H), 4.35 (dd, 1 H, J ) 8.8, 12.8), 3.65 (m, 1 H), 2.73 (dd, 1 H,
J ) 10.1, 14.1), 2.63 (m, 2 H), 2.31 (m, 3 H), 2.03 (m, 1 H),
1.91 (m, 1 H), 1.70 (m, 2 H); 13C-NMR (DMSO-d6, 75 MHz) δ
27.1, 31.2, 31.5, 32.2, 39.8 , 53.0, 56.4, 85.4, 128.6, 129.2, 132.5,
146.7, 157.8, 159.8, 167.4, 170.2, 174.6, 175. IR (KBr) 3368,
2980, 2851, 1696 (sh), 1640, 1541, 1504 (w), 1445, 1319, 1259,
1189, 1155, 762, 654 cm-1; FABMS, calcd for MH+ C20H24N5O6
430.1728, found 430.1727. Anal. Calcd for C20H23N5O6: C,
55.94; H, 5.40; N, 16.31. Found: C, 55.66; H, 5.38; N, 16.00.
Meth od B. A solution of 28 (92 mg, 0.15 mmol) in 1 N
NaOH (3 mL) was stirred for 48 h at rt. The material went
almost immediately into solution. At the end of the reaction
time a clear pale yellow liquid was obtained. The product was
precipitated by careful addition of 1 N HCl. The gel thus
obtained was stirred for a short time to facilitate crystalliza-
tion. The resulting powder was collected by filtration, washed
with ice-water (3 × 2 mL), and dried at rt (high vacuum) to
afford 55 mg (85%) of 4 as a off-white microcrystalline powder,
mp. 241-243 °C. Its spectral properties were identical with
those observed for the product obtained by Method A above.
Dim eth yl N-{4-[2-(2-(P iva loyla m in o)-4(3H)-oxo-5,6-d i-
h yd r o-7-(eth oxyca r bon yl)-7H-p yr r olo[2,3-d ]p yr im id in -6-
yl)eth yl]ben zoyl}-L-glu ta m a te (28). A solution of the cou-
pling product 27 (150 mg, 0.25 mmol) and 10% Pd/C (150 mg)
in methanol (50 mL) was hydrogenated in a Parr shaker at
50 psi of H2 and at rt for 5 h. The catalyst was removed by
means of a fritted glass filter, and the collected filtrate was
evaporated to dryness. The residual greenish gum was
purified by chromatography over SiO2 with 5% MeOH in CH -
Cl2 as the eluent to give 120 mg (78%) of 28 as a colorles2s
1
glassy syrup, mp 105-108 °C; H NMR (CDCl3, 300 MHz) δ
11.95 (br s, 1 H), 9.40 (br s, 1 H), 7.73 (d, 2 H, J ) 7.8), 7.22
(d, 2 H, J ) 7.7), 7.07 (d, 1 H, J ) 7.5), 4.80 (dd, 1 H, J ) 7.2,
12.3), 4.23 (m, 2 H), 4.06 (m, 1 H), 3.75 (s, 3 H), 3.62 (s, 3 H),
3.07 (dd, 1 H, J )10.2, 15.9), 2.72 (m, 3 H), 2.56 (m, 2 H), 2.32
(m, 1 H),1.50-2.25 (m, 3 H), 1.28 (s, 9 H), 1.27 (t, 3 H, J )
7.1); 13C NMR (CDCl3, 75 MHz) δ 15.0, 27.3, 27.1, 27.6, 28.4,
30.9, 36.2, 40.7, 52.5, 52.8, 53.2, 59.2, 91.3, 100.7, 128.0, 129.0,
132.2 , 145.6, 152.1, 160.5, 167.7, 173.2, 174.2, 181.3; IR (KBr)
3420, 3240, 2981, 1744, 1673, 1608, 1556, 1373, 1305, 1246
(w), 1214 (w), 1153, 1108 (w), 777 (w) cm-1; MS m/ e (relative
intensity) 613 (15%, M+), 584 (100%, M+ - 29, - OMe), 570
t
(55%, M+ - 43), 556 (78%, M+ - 57, - Bu), 409 (26%), 395
(27%), 381 (47%); HRMS calcd for C30H39N5O9 613.2787, found
613.2767. Anal. Calcd for C30H39N5O9: C, 58.72; H, 6.41; N,
11.42. Found: C, 58.47; H, 6.36; N, 11.35.
N-{4-[2-(2-Am in o-4(3H )-oxo-5,6-d ih yd r o-7H -p yr r olo-
[2,3-d ]p yr im id in -6-yl)eth yl]ben zoyl}-L-glu ta m ic Acid (4).
Meth od A. To a solution of 21 (130 mg, 0.43 mmol) in DMF
(8 mL) was added 6-chloro-2,4-dimethoxy-1,3,5-triazine (0.08
g, 0.45 mmol) and N-methylmorpholine (0.052 g, 0.52 mmol)
at 0 °C. After the mixture was stirred at 0 °C for 2 h,
N-methylmorpholine (0.052 g, 0.52 mmol) and dimethyl L-
glutamate hydrochloride (0.095 g, 0.45 mmol) were added all
at once. The mixture was stirred at 0 °C for 2 h, at rt for 12
h, the solvent was removed under reduced pressure, and the
Ack n ow led gm en t. We are deeply indebted to the
late Dr. G. B. Grindey of Eli Lilly & Co. for the biological
evaluations reported herein. Gratitude is expressed to
Drs. Chuan Shih and Charles J . Barnett of Lilly for
many stimulating discussions, and to Lilly for financial
support of this work.
J O951471K