Replacement of the 1',4'-phenylene region in 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF) by 4,5,6,7-tetrahydrobenzo[c]thiophene and 4,5,6,7-tetrahydroisobenzofuran nuclei

Article abstract of DOI:10.1021/jo9619903

Two new analogs of DDATHF, in which the 1',4'-phenylene unit is replaced by 4,5,6,7-tetrahydrobenzo[c]thiophene and 4,5,6,7-tetrahydroisobenzofuran nuclei, have been prepared and evaluated for in vitro cytotoxicity, GARFT inhibition, and FPGS affinity as potential antitumor agents.

Full text of DOI:10.1021/jo9619903

J . Org. Chem. 1997, 62, 1599-1603
1599
Rep la cem en t of th e 1′,4′-P h en ylen e Region in
5,10-Did ea za -5,6,7,8-tetr a h yd r ofolic Acid (DDATHF ) by
4,5,6,7-Tetr a h yd r oben zo[c]th iop h en e a n d
4,5,6,7-Tetr a h yd r oisoben zofu r a n Nu clei
Edward C. Taylor* and J ames E. Dowling
Department of Chemistry, Princeton University, Princeton, New J ersey 08544
Received October 24, 1996X
Two new analogs of DDATHF, in which the 1′,4′-phenylene unit is replaced by 4,5,6,7-tetrahy-
drobenzo[c]thiophene and 4,5,6,7-tetrahydroisobenzofuran nuclei, have been prepared and evaluated
for in vitro cytotoxicity, GARFT inhibition, and FPGS affinity as potential antitumor agents.
In tr od u ction
In the de novo purine biosynthetic pathway, glycina-
mide ribonucleotide formyltransferase (GARFT) catalyzes
the transformylation of 5′-phosphoribosyl glycinamide (â-
GAR) to 5′-phosphoribosyl formylglycinamide, a reaction
which requires 10-formyl-5,6,7,8-tetrahydrofolate as a
cofactor.1 The cytotoxic2 effects of the tetrahydrofolic acid
analog 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF,
1) arise from potent and selective inhibition of GARFT.3
DDATHF exhibits pronounced in vitro activity toward a
variety of solid murine and human xenograft tumors
against which the clinically-employed dihydrofolate re-
ductase (DHFR) inhibitor methotrexate (MTX) has little
or no activity.4 Currently, the (6R) diastereomer of 1
(lometrexol, LTX)5 is being evaluated in Phase II clinical
trials in which coadministration with folic acid has been
shown to ameliorate the delayed, cumulative toxicity
observed in earlier studies conducted with the drug
alone.6 Tight-binding inhibition of GARFT requires prior
modification of DDATHF by folylpolyglutamate syn-
thetase (FPGS) which is responsible for the intracellular
conversion of folates to their γ-polyglutamyl derivatives.7
Some cancer cell lines which exhibit resistance to
DDATHF are unable to accumulate polyglutamylated
derivatives, a result that has been attributed to reduced
levels of FPGS activity and which implicates the poly-
glutamyl forms as the active intracellular metabolites.8
Acquired resistance to DDATHF has also been noted in
cell lines which exhibit an increase in γ-glutamyl hydro-
lase (γ-GH) activity and which possess impaired reduced
folate transport mechanisms.9 Recently, two antifolates
differing from DDATHF in the replacement of the 1′,4′-
F igu r e 1.
phenylene unit by a 2′,5′-thiophene ring (2, ki ) 2.1 nm)
and a 2′,5′-furan ring (3, ki ) 0.77 nm) were found to
possess inhibitory activity against the recombinant hu-
man monofunctional enzyme (hGARFT)10 without the
need for further glutamylation.11
As an extension of our earlier structure-activity
relationship (SAR) studies, we have initiated a molecular
modeling approach to aid in the design of new inhibitors
of GARFT based on 2 and 3. Visual inspection12 of the
hGARFT crystallographic ternary complex with 2 and
â-GAR revealed the existence of a large cavity in the
region which surrounds the 2′,5′-thienyl substitutent. The
presence of such a conspicuous unoccupied volume sug-
gested that substrate mimics which bear a larger or a
bicyclic ring system in place of the commonly-employed
monocyclic units might be readily accommodated.
A
similar hydrophobic cavity was observed in a molecular
modeling study conducted on analogs in the MTX series
using bacterial DHFR obtained from Lactobacillus casei.13
Subsequent replacement of the 1,4-phenylene moiety of
MTX with naphthalene-derived rings was found to have
a favorable effect on the transport of such derivatives into
X Abstract published in Advance ACS Abstracts, March 1, 1997.
(1) Caperelli, C. A. J . Biol. Chem. 1989, 264, 5053.
(2) Smith, S. G.; Lehman, N. L.; Moran, R. G. Cancer Res. 1993,
53, 5697.
(3) Taylor, E. C.; Harrington, P. M. J . Org. Chem. 1990, 55, 3222-
3227.
(4) Baldwin, S. W.; Tse, A.; Gossett, L. S.; Taylor, E. C.; Rosowsky,
A.; Shih, C.; Moran, R. G. Biochemistry 1991, 30, 1997.
(5) (a) Taylor, E. C.; Wong, G. S. K.; Fletcher, S. R.; Harrington, P.
J .; Beardsley, G. P.; Shih, C. J . In Chemistry and Biology of Pteridines
1986; Cooper, B. A., Whitehead, V. M., Eds.; de Gruyter: New York,
1986; p 61. (b) Moran, R. G.; Baldwin, S. W.; Taylor, E. D.; Shih, C.
J . Biol. Chem. 1989, 264, 21047. (c) Barnett, C. J .; Wilson, T. M.
Tetrahedron Lett. 1989, 30, 6291-6294.
(6) Ray, M. S.; Muggia, F. M.; Leichman, G. C.; Nelson, R. L.; Dyke,
R. W.; Moran, R. G. J . Natl. Cancer Inst. 1993, 85, 1154-1159.
(7) Pizzorno, G.; Sokoloski, J . A.; Cashmore, A. R.; Moroson, B. A.;
Cross, A. D.; Beardsley, G. P. Mol. Pharmacol. 1991, 39, 85-89.
(8) Pizzorno, G.; Moroson, B. A.; Cashmore, A. R.; Russello, O.;
Mayer, J . R.; Galivan, J .; Bunni, M. A.; Priest, D. G.; Beardsley, G. P.
Cancer Res. 1995, 55, 566.
(9) Rhee, M. S.; Wang, Y.; Nair, G.; Galivan, J . Cancer Res. 1993,
53, 2227.
(10) Kan, C. C.; Gehring, M. R.; Nodes, B. R.; J anson, C. A.; Almassy,
R. J .; Hostomska, Z. J . Protein Chem. 1992, 11, 467.
(11) Habeck, L. L.; Leitner, T. A.; Shackelford, K. A.; Gossett, L. S.;
Schultz, R. M.; Andis, S. L.; Shih, C.; Grindey, G. B.; Mendelsohn, L.
G. Cancer Res. 1994, 54, 1021.
(12) Molecular modeling was conducted using the Molecular Simula-
tions Inc. products, Quanta 4.0 and Charmm, on a Silicon Graphics
Indigo 2 workstation.
(13) Piper, J . R.; J ohnson, C. A.; Maddry, J . A.; Malik, N. D.;
McGuire, J . J .; Otter, G. M.; Sirotnak, F. M. J . Med. Chem. 1993, 36,
4161.
S0022-3263(96)01990-1 CCC: $14.00 © 1997 American Chemical Society

1600 J . Org. Chem., Vol. 62, No. 6, 1997
Taylor and Dowling
Sch em e 1
L1210 cells, with the greatest enhancement in influx
efficiency observed for the 5,6,7,8-tetrahydronaphthoyl
derivative. Given that both DDATHF and MTX utilize
the reduced folate carrier protein (RFC),14 a similar
improvement in the rate of cellular uptake was conceiv-
able for derivatives which had been modified by fusion
of a fully-reduced six-membered ring onto the bridging
2′,5′-thienyl or 2′,5′-furanyl ring of 2 and 3. Since it is
well recognized that antifolate cytotoxicity is a conse-
quence, inter alia, of enzyme inhibition, transport ef-
ficiency, and, usually, the extent of intracellular poly-
glutamation, it is reasonable to expect that analogs which
exhibit high affinity for GARFT yet are more readily
internalized should exhibit improved cytotoxicity. In this
paper we report the synthesis of two new analogs of 1 in
which the 1′,4′-phenylene feature has been replaced by
4,5,6,7-tetrahydrobenzo[c]thiophene-1′,3′-diyl and -isoben-
zofuran-1′,3′-diyl nuclei. It was anticipated that due to
the spacious hydrophobic cavity in the target enzyme,
the larger substrates would retain the high binding
affinity for GARFT exhibited by 2 and 3 while having
the potential for improved rates of internalization via the
RFC. Although this type of structural modification was
found to have a deleterious effect on the affinity for FPGS
of the naphthoyl-derived MTX analogues13 such that the
extent of intracellular polyglutamation was reduced
relative to the benzoyl analogues, manifestation of this
characteristic in the proposed analogs of 1 may be of
lesser concern since it has been established that poly-
glutamylation of 2 and 3 is not a requirement for tight-
binding inhibition of hGARFT.11
triazine-mediated19 amino acid coupling (84%) afforded
the L-glutamic acid amide protected as its dimethyl ester
(8a ).
Palladium-catalyzed coupling of 8a with excess 2-piv-
aloyl-6-ethynyl-5-deazapterin15 (9) gave the acetylene 10a
in 65% yield. Hydrogenation using 2 wt equiv of 10%
palladium-on-carbon in a mixture of methanol and
dichloromethane at 50 °C proceeded in 95% yield to give
the penultimate intermediate 11a . Subsequent simul-
taneous deprotection of the exocyclic amino group and
glutamic acid side chain with 1 N NaOH provided the
desired material 12a in 75% yield (30% overall for the
six-step sequence beginning with 5).
Resu lts a n d Discu ssion
The synthesis of the 4,5,6,7-tetrahydroisobenzofuran
derivative 12b relies upon initial assembly of the furan
ring by treatment of 2-[bis(methylthio)methylene]cyclo-
hexanone20 (13) with LHMDS and ethyl bromoacetate to
Our synthetic strategy relies principally upon a con-
vergent, palladium-mediated approach to the preparation
of 6-substituted deazapterins initially employed in the
synthesis of 115 and subsequently in the preparation of
numerous analogs. Acid-catalyzed condensation of
2-formylcyclohexanone16 (4) with methyl thioglycolate
followed by aromatization with freshly-prepared sodium
methoxide provided methyl 4,5,6,7-tetrahydrobenzo[c]-
thiophene-2-carboxylate (5).17 Treatment of 5 with ben-
zyltrimethylammonium tribromide18 and ZnCl2 in acetic
acid gave the nuclear bromination product 6 in 85% yield.
Conversion to carboxylic acid 7a by saponification with
1 N NaOH followed by 2-chloro-4,6-dimethoxy-1,3,5-
(14) Pizzorno, G.; Cashmore, A. R.; Moroson, B. A.; Cross, A. D.;
Smith, A. D.; Marling-Carson, M.; Kamen, B. A.; Beardsley, G. P. J .
Biol. Chem. 1993, 268, 1017.
(15) Taylor, E. C.; Wong, G. S. K. J . Org. Chem. 1988, 54, 3618.
(16) Ainsworth, C. Org. Synth. 1963, 4, 536.
(17) Tilak, B. D.; Desai, H. S.; Gupte, S. S. Tetrahedron Lett. 1966,
1953.
(18) Okamoto, T.; Kakinami, T.; Fujimoto, H.; Kajigaeshi, S. Bull.
Chem. Soc. J pn. 1991, 64, 2566.
(19) Kaminski, Z. J . Tetrahedron Lett. 1985, 26, 2901.
(20) Arjona, O.; Cereceda, J . A.; Quiroga, M. L. Tetrahedron 1980,
36, 2137.

Replacement of the 1′,4′-Phenylene Region in DDATHF
J . Org. Chem., Vol. 62, No. 6, 1997 1601
verted to glutamate 8b via the intermediacy of acid 7b.
Subsequent coupling with excess 9 gave acetylene 10b
which, unlike its counterpart 10a , resisted all efforts to
separate it from an aromatic impurity. To overcome this
minor difficulty, the crude material was catalytically
reduced using PtO2 to give 11b which was then depro-
tected to afford 12b in 61% yield (20% overall from 15).
The results of in vitro cell culture screening of 12a and
12b against human T-cell derived lymphoblastic leuke-
mia (CCRF-CEM) cells indicated that the new compounds
were modest cytotoxic agents, with IC50 values of 0.19
and 1.6 mM, respectively. Reversal of the cytotoxicity
of 12a and 12b could be effected by the addition of
hypoxanthine (100 µM) but not by the addition of
thymidine (5 µM), indicating that the locus of activity
resides in the purine de novo biosynthetic pathway.
Subsequent evaluation of 12a and 12b against murine
trifunctional GARFT revealed the two analogs to be
competitive inhibitors with Ki’s of 25.4 and 8.7 nM,
respectively, although, surprisingly, neither was found
to have any activity against hog liver FPGS at drug
concentrations up to 300 µM in the presence of 2 µg of
enzyme. In the absence of additional data it cannot be
determined if the diminished cytotoxicity of 12a and 12b
relative to 2 and 3 (IC50 (CCRF-CEM) ) 1.3 and 15.2 nM,
respectively) is a consequence of reduced affinity of the
analogs for the RFC or if attainment of a useful level of
cytotoxicity requires that some degree of polyglutamation
be achieved even for antifolates which exhibit tight
binding inhibition of GARFT in their monoglutamated
forms.
give the methylthio derivative 14 (Scheme 1, path a).21
In our hands, 14 was consistently obtained in low yields
and was accompanied by the corresponding 4,5,6,7-
tetrahydrobenzo[c]thiophene 15. The contaminant 15 is
presumed to result from sulfur-promoted ring opening
of the Darzen’s reaction product, common intermediate
16, followed by demethylation and aromatization (Scheme
1, path b). A similar mechanism has been invoked to
rationalize the formation of thiophenes from R-oxoketene
dithioacetals under Simmons-Smith conditions.22 For-
mation of 15 was more extensive when the reaction
temperature was maintained at -78 °C for extended
periods (3 h); brief exposure (0.5 h) to the same conditions
resulted in formation of 14 as the major product (26%).
Attempted bromination of 17, obtained by Raney nickel
desulfurization of 14, using the previously-employed
conditions or bromine alone, gave complex mixtures.
Conversion to the bromo derivative 18, in 50% yield, was
finally achieved with bromine in trimethyl phosphate in
the dark to minimize the formation of additional products
which resulted from apparent benzylic bromination.
In the same fashion as described for the 4,5,6,7-
tetrahydrobenzo[c]thiophene derivative, 18 was con-
Exp er im en ta l Section
Gen er a l Meth od s. NMR spectra were recorded on a J EOL
GSX 270 FT or a Varian INOVA 500 spectrometer. Proton
and carbon chemical shifts are reported in parts per million
(ppm) and are referenced to internal solvent. Multiplicities
are abbreviated in the following manner: singlet (s), doublet
(d), triplet (t), quartet (q), multiplet (m), and broadened (br).
Coupling constants were determined by first-order approxima-
tion. High and low resolution mass spectra were obtained on
a Kratos MS50 spectrometer. Analytical thin-layer chroma-
tography (TLC) was conducted using precoated silica gel 60
plates with a fluorescent indicator. Radial chromatography
was performed with the aid of the Chromatotron, a product of
Harrison Research Inc. All reactions were carried out under
an argon atmosphere with dry, freshly-distilled solvents under
anhydrous conditions unless otherwise noted. All reagents
obtained from commercial suppliers were used without further
purification unless otherwise stated.
(21) Datta, A.; Pooranchand, D.; Ila, H.; J unjappa, H. Tetrahedron
1989, 45, 7631.
(22) Thomas, A.; Singh, G.; Ila, H.; J unjappa, H. Tetrahedron Lett.
1989, 30, 3093.

1602 J . Org. Chem., Vol. 62, No. 6, 1997
Taylor and Dowling
Met h yl 4,5,6,7-Tet r a h yd r ob en zo[c]t h iop h en e-1-ca r -
boxyla te (5). To a magnetically stirred, neat mixture of
2-oxocyclohexanecarboxaldehyde (3.6 g, 28 mmol) and methyl
thioglycolate (6.0 g, 56 mmol) was added 3 drops of concen-
trated H2SO4. The resulting yellow solution was stirred at rt
for 12 h, diluted with 25 mL of ice-water, and extracted with
CH2Cl2 (25 mL). The aqueous phase was reextracted with an
additional 25 mL portion of CH2Cl2, and the combined organic
phases were washed with 50 mL of a saturated aqueous NaCl
solution and dried over Na2SO4. Removal of the drying agent
by filtration followed by evaporation in vacuo gave a viscous
yellow oil which was dissolved in 25 mL of MeOH and added
dropwise over 1 h to a freshly-prepared solution of NaOMe
(from 1.7 g, 2.5 equiv of sodium metal) in 100 mL of MeOH.
The deep orange solution was allowed to stir overnight (12 h),
concentrated to one-quarter volume in vacuo, and partitioned
between CH2Cl2 (50 mL) and water (50 mL). The aqueous
phase was reextracted with an additional 25 mL portion of
CH2Cl2, and the combined organic phases were washed with
50 mL of a saturated aqueous NaCl solution and dried over
Na2SO4. The drying agent was removed by filtration and the
filtrate concentrated in vacuo to afford a yellow oil which was
purified by chromatography on silica using 5% EtOAc in
hexanes as eluent to give 1.5 g (27%) of a clear, colorless liquid.
1H NMR (CDCl3, 300 MHz) δ 1.73 (m, 4 H), 2.68 (t, 2 H, J )
6.2 Hz), 3.02 (t, 2 H, J ) 6.2 Hz), 3.83 (s, 3 H), 7.05 (s, 1 H);
13C NMR (CDCl3, 75.6 MHz) 22.5, 22.5, 26.1, 26.6, 51.2, 125.1,
125.1, 139.8, 146.4, 162.9; IR (NaCl) 3088, 2935, 2848, 1698,
1538 cm-1; MS m/e (relative intensity) 196 (99), 181 (15), 165
(55), 137 (92); HRMS calcd for C10H12O2S: 196.0557, found:
196.0557.
Meth yl 3-Br om o-4,5,6,7-tetr a h yd r oben zo[c]th iop h en e-
1-ca r boxyla te (6). To a solution of 5 (0.40 g, 2.0 mmol) in 5
mL of HOAc was added benzyl trimethylammonium tribro-
mide (0.84 g, 2.1 mmol) followed by zinc chloride (1.1 g, 8.0
mmol). The resulting orange suspension was stirred at rt until
a fine precipitate had developed and a pale yellow coloration
persisted (5 h). The reaction mixture was partitioned between
CH2Cl2 (25 mL) and water (25 mL). The organic phase was
washed with an aqueous NaCl solution (25 mL), dried over
Na2SO4, filtered, and evaporated. The resulting solid residue
was purified by radial chromatography on a 2 mm plate using
5% EtOAc in hexanes as eluent to give a white solid (0.47 g,
85%) which was recrystallized from hexanes, mp 85-86 °C.
1H NMR (CDCl3, 270 MHz) δ 1.55 (m, 4 H), 2.35 (br t, 2 H, J
) 6.1 Hz), 2.82 (br t, 2 H, J ) 6.1 Hz), 3.65 (s, 3 H); 13C NMR
(CDCl3, 75.6 MHz) δ 23.3, 23.4, 27.3, 28.1, 52.8, 117.1, 126.7,
141.0, 148.3, 163.4; IR (KBr) 2929, 2851, 1675, 1534 cm-1; MS
m/e (relative intensity) 276 (88), 274 (92), 261 (88), 259 (90),
(15 mL) and water (15 mL). The aqueous phase was extracted
with CH2Cl2 (15 mL), and the combined organic extracts were
washed with an aqueous NaCl solution (15 mL), dried over
Na2SO4, filtered, and evaporated in vacuo. The resulting oily
residue was purified by radial chromatography (2 mm plate)
using 20% EtOAc in hexanes as eluent to give 120 mg (84%)
of a clear, colorless oil. 1H NMR (CDCl3, 500 MHz) δ 1.73 (m,
4 H), 2.09 (m, 1 H), 2.28 (m, 1 H), 2.37-2.51 (m, 4 H), 2.94
(m, 2 H), 3.64 (s, 3 H), 3.76 (s, 3 H), 4.73 (dt, 1 H, J ) 5.1, 7.2
Hz), 6.50 (d, 1 H, J ) 7.2 Hz); 13C NMR (CDCl3, 75.6 MHz) δ
21.9, 22.3, 26.0, 26.7, 27.0, 29.8, 51.6, 51.9, 52.4, 113.0, 130.3,
139.6, 141.6, 161.6, 172.0, 173.0; IR (NaCl) 3309, 2936, 1731,
1632 cm-1; MS m/e (relative intensity) 419 (6), 417 (16), 246
(14), 245 (92), 244 (100), 243 (95), 242 (94); HRMS calcd for
C16H20BrNO5S: 417.0245, found: 417.0252. Anal. Calcd for
C16H20BrNO5S: C, 45.94; H, 4.82; N, 3.35; S, 7.66. Found: C,
46.17; H, 4.79; N, 3.32; S, 7.92.
Dim et h yl N-[3-[[2-(P iva loyla m in o)-4-oxo-3,4-p yr id o-
[2,3-d ]p yr im id in -6-yl]eth yn yl]-4,5,6,7-tetr a h yd r oben zo-
[c]th ien oyl]-L-glu ta m a te (10a ). A mixture of 9 (210 mg, 0.77
mmol), Pd(OAc)2 (3.8 mg, 4 mol %), tri-o-tolylphosphine (9.4
mg, 8 mol %), Et3N (117 mg, 1.16 mmol), and 8a (162 mg, 0.39
mmol) in 10 mL of MeCN was heated at reflux under an argon
atmosphere for 24 h. At this point an additional 53 mg of 9
was added, and the reaction was allowed to continue for 24 h.
After cooling to rt, the solvent was removed in vacuo, and the
resulting black residue was passed through a short column of
silica gel using 5% MeOH in CH2Cl2 as eluent. Further
purification by radial chromatography on a 2 mm plate using
80% EtOAc in hexanes as eluent afforded the coupled product
as a pale yellow solid (141 mg, 60%). 1H NMR (CDCl3, 270
MHz) δ 1.33 (s, 9 H), 1.76 (m, 4 H), 2.24 (m, 2 H), 2.47 (m, 2
H), 2.76 (m, 2 H), 2.95 (m, 2 H), 3.65 (s, 3 H), 3.77 (s, 3 H),
4.76 (dt, 1 H, J ) 5.1, 7.2 Hz), 6.75 (d, 1 H, J ) 7.3 Hz), 8.53
(d, 1 H, J ) 2.3 Hz), 8.56 (br s, 1 H), 8.87 (s, 1 H), 12.12 (br s,
1 H); MS m/e (relative intensity) 607 (21), 564 (50), 550 (20),
432 (100), 389 (51), 375 (50); HRMS calcd for C30H33N5O7S:
607.2111, found: 607.2100.
Dim eth yl N-[3-[2-[2-(P iva loyla m in o)-4-oxo-3,4,5,6,7,8-
h exa h yd r op yr id o[2,3-d ]p yr im id in -6-yl]eth yl]-4,5,6,7-tet-
r a h yd r oben zo[c]th ien oyl]-L-glu ta m a te (11a ). A mixture
of 10a (220 mg, 0.36 mmol) and 10% Pd/C (440 mg, 2 wt eq)
in 25 mL of MeOH and 2 mL of CH2Cl2 was hydrogenated at
48 psi and 50 °C for 24 h. The mixture was filtered through
Celite with the aid of additional MeOH, and the filtrate was
concentrated in vacuo to afford a pale yellow oil which was
purified by radial chromatography, using 5% MeOH in CH2-
Cl2 as eluent, to give a white solid (210 mg, 95%), mp 178-
180 °C dec.1H NMR (CDCl3, 500 MHz) δ 1.28 (s, 9 H), 1.58-
1.72 (m, 6 H), 1.90 (m, 1 H), 2.13 (m, 2 H), 2.33 (m, 1 H), 2.47
(m, 2 H), 2.57 (br t, 2H, J ) 6.1 Hz), 2.80 (m, 2 H), 3.01 (m, 4
H), 3.39 (d, 1H, J ) 11.9 Hz), 3.62 (s, 3 H), 3.74 (s, 3 H), 4.76
(dt, 1 H, J ) 5.1, 7.3 Hz), 5.11 (s, 1 H), 6.44 (d, 1 H, J ) 7.3
Hz), 8.47 (s, 1 H), 11.25 (s, 1 H); 13C NMR (CDCl3, 75.6 MHz)
δ 22.8, 23.1, 25.3, 25.4, 25.7, 27.2 27.4, 27.8, 30.3, 30.8, 34.2,
40.4, 46.3, 52.1, 52.1, 52.8, 89.8, 126.2, 136.2, 141.8, 143.2,
148.4, 158.2, 160.7, 163.0, 172.8, 173.5, 179.9. MS m/e (relative
intensity) 615 (34), 583 (7), 441 (25), 440 (13), 413 (40), 412
(33), 250 (90), 249 (100); HRMS calcd for C30H41N5O7S:
615.2726, found: 615.2718.
N-[3-[2-(2-Am in o-4-oxo-3,4,5,6,7,8-h exa h yd r op yr id o-
[2,3-d ]p yr im id in -6-yl)eth yl]-4,5,6,7-tetr a h yd r oben zo[c]-
th ien oyl]-L-glu ta m ic Acid (12a ). A suspension of 11a (245
mg, 0.400 mmol) in 2 mL of 1 N NaOH was stirred at rt for 48
h. Acidification with 3 M HCl gave a yellow gum which was
stirred overnight. The resulting pale yellow solid was col-
lected, washed with water, and dried to afford the glutamic
acid derivative (150 mg, 75%), mp 195-200 °C dec. 1H NMR
(DMSO-d6, 500 MHz) δ 1.52-1.66 (m, 6 H), 1.85-1.91 (m, 2
H), 2.05 (m, 1 H), 2.31 (m, 2 H), 2.50 (m, 2 H, obscured by
residual solvent peak), 2.54 (m, 2 H), 2.75 (m, 2 H), 2.81-2.93
(m, 3 H), 3.24 (m, 1 H), 4.31 (dt, 1 H, J ) 5.1, 7.3 Hz), 6.49 (s,
2 H), 6.61 (s, 1 H), 7.76 (d, 1 H, J ) 7.8 Hz), 12.50 (br s, 1 H).
HRMS (FAB) calcd for C23H30N5O6S: 504.1917 (MH+), found:
504.1924.
245 (45), 243 (66), 217 (27), 215 (36); HRMS calcd for C10H11
BrO2S: 273.9663, found: 273.9663. Anal. Calcd for C10H11
BrO2S: C, 43.65; H, 4.03. Found: C, 43.94; H, 4.05.
-
-
3-Br om o-4,5,6,7-t et r a h yd r ob en zo[c]t h iop h en e-1-ca r -
boxylic Acid (7a ). A suspension of 6 (300 mg, 1.09 mmol) in
6 mL of 1 N NaOH was heated at 80 °C for 6 h. The resulting
clear solution was cooled with the aid of an ice bath and
carefully acidified using a 3 M HCl solution. The milky white
suspension was extracted with EtOAc (2 × 10 mL), and the
combined organic phases were dried over MgSO4, filtered, and
evaporated to afford a white solid (255 mg, 90%), mp 220 °C
dec. 1H NMR (CDCl3, 270 MHz) δ 1.65 (m, 4 H), 2.47 (br t, 2
H, J ) 6.1 Hz), 2.95 (br t, 2 H, J ) 6.1 Hz); IR (KBr) 1661
cm-1; MS m/e (relative intensity) 262 (100) 260 (96) 215 (69)
217 (71); HRMS calcd for C9H9BrO2S: 259.9507, found:
259.9507. Anal. Calcd for C9H9BrO2S: C, 41.40; H, 3.47.
Found: C, 41.33; H, 3.52.
Dim eth yl N-(3-Br om o-4,5,6,7-tetr a h yd r oben zo[c]th ien -
oyl)-L-glu ta m a te (8a ). To a solution of 7 (90 mg, 0.34 mmol)
in 5 mL of MeCN and 5 mL of THF at rt was added
4-methylmorpholine (NMM, 45 µL, 0.41 mmol, 1.2 equiv) neat,
via syringe. After 10 min, 2-chloro-4,6-dimethoxy-1,3,5-triaz-
ine (66 mg, 0.37 mmol, 1.1 equiv) was added in one portion,
and the resulting mixture was stirred for 1 h. An additional
portion of NMM (1.2 equiv) was added followed by L-glutamic
acid dimethyl ester hydrochloride (106 mg, 0.41 mmol). After
stirring for 4 h, the mixture was partitioned between CH2Cl2
Eth yl 3-(Meth ylth io)-4,5,6,7-tetr a h yd r oisoben zofu r a n -

Replacement of the 1′,4′-Phenylene Region in DDATHF
J . Org. Chem., Vol. 62, No. 6, 1997 1603
1-ca r boxyla te (14) a n d Eth yl 3-(Meth ylth io)-4,5,6,7-tet-
r a h yd r oben zo[c]th iop h en e-1-ca r boxyla te (15). A solution
of LHMDS was prepared by the addition of n-BuLi (3.8 mL of
a 2.5 M solution in hexanes) to a solution of HMDS (1.51 g,
9.4 mmol) in 10 mL of THF at 0 °C. The solution was then
cooled to -78 °C and stirred at this temperature for 0.5 h.
Ethyl bromoacetate (1.05 mL, 9.4 mmol) in 7 mL of THF was
added over a period of 10 min. After the reaction mixture had
been allowed to stir for 0.5 h, a solution of 2-[bis(methylthio)-
methylene]cyclohexanone (950 mg, 4.7 mmol) in 10 mL of THF
was added over 15 min. The reaction mixture was maintained
at -78 °C for 3 h, allowed to stir at rt for 12 h, and partitioned
between water (50 mL) and ether (50 mL). The aqueous phase
was extracted with ether (25 mL), and the combined organic
phases were then washed with brine (25 mL) and dried over
MgSO4. Filtration and evaporation in vacuo gave 1.8 g of a
red oil which was purified by chromatography on silica using
5% EtOAc in hexanes as eluent. Further purification by radial
chromatography (2 mm plate), using 2% EtOAc in hexanes as
eluent afforded 14 (0.20 g, 0.83 mmol, 18%) and 15 (0.20 g,
0.78 mmol, 16%), mp 41 °C (from hexanes). Spectral and
analytical data for 15 are as follows: 1H NMR (CDCl3, 500
MHz) δ 1.35 (t, 3 H, J ) 7.3 Hz), 1.74 (m, 4 H), 2.51 (s, 3 H),
2.55 (m, 2 H), 2.99 (m, 2 H), 4.30 (q, 2 H, J ) 7.3, 14.6 Hz);
13C NMR (CDCl3, 75.6 MHz) δ 14.6, 19.1, 22.7, 22.8, 25.7, 27.3,
60.6, 124.9, 138.9, 139.2, 147.5, 162.4; MS m/e (relative
intensity) 256 (100), 227 (87), 211 (31), 183 (28), 136 (55);
HRMS calcd for C12H16O2S2: 256.0592. Found: 256.0598.
Anal. Calcd for C12H16O2S2: C, 56.22; H, 6.29; S, 25.01.
Found: C, 56.00; H, 6.20; S, 24.87.
Eth yl 3-Br om o-4,5,6,7-tetr a h yd r oisoben zofu r a n -1-ca r -
boxyla te (18). Ethyl 4,5,6,7-tetrahydroisobenzofuran-1-car-
boxylate21 (425 mg, 2.20 mmol) was dissolved in 12 mL of 1,2-
dichloroethane, and the reaction flask was then shrouded with
aluminum foil. After extinguishing the hood lights, bromine
(2.2 mL of a 1.0 M solution in trimethyl phosphate) was added
via syringe over 10 min. The resulting solution was stirred
at rt for 20 min. The reaction mixture was partitioned
between CH2Cl2 (15 mL) and ice-water (15 mL). The organic
phase was washed with 15 mL of a 20% Na2S2O3 aqueous
solution, dried over Na2SO4, filtered, and evaporated in vacuo
to give a clear yellow oil. Purification of the crude material
by radial chromatography (2 mm plate) using 2% EtOAc in
hexanes gave 269 mg (50%) of a white powder, mp 89-90 °C.
1H NMR (CDCl3, 300 MHz) δ 1.28 (t, 3 H, J ) 7.3 Hz), 1.63
(m, 4 H), 2.30 (m, 2 H), 2.70 (m, 2 H), 4.24 (q, 2 H, J ) 7.3 ,
14.3 Hz); 13C NMR (CDCl3, 75.6 MHz) δ 14.2, 20.2, 21.8, 21.9,
22.0, 60.3, 122.9, 123.8, 134.0, 140.1, 158.3; IR (KBr) 1710,
1520, cm-1; MS m/e (relative intensity) 274 (21), 272 (22), 246
(28), 245 (33), 244 (29), 243 (32), 229 (13), 227 (20), 78 (100);
HRMS calcd for C11H13BrO3: 272.0048, found: 272.0036.
Anal. Calcd for C11H13BrO3: C, 48.37; H, 4.80; Br, 29.26.
Found: C, 48.49; H, 4.82; Br, 29.07.
3-Br om o-4,5,6,7-t et r a h yd r oisob en zofu r a n -1-ca r b ox-
ylic Acid (7b). A suspension of 18 (200 mg, 0.73 mmol) in 6
mL of 1 N NaOH was heated at 80 °C for 6 h. The resulting
clear solution was cooled with an ice bath and carefully
acidified using a 3 M HCl solution. The milky white suspen-
sion was extracted with EtOAc (2 × 10 mL), and the combined
organic phases were then dried over MgSO4, filtered and
evaporated to afford a white solid (160 mg, 89%), mp 145-
150 °C dec. 1H NMR (CDCl3, 300 MHz) δ 1.57 (m, 4 H), 2.36
(m 2 H), 2.84 (m, 2 H); IR (KBr) 1661, 1597, cm-1; MS m/e
(relative intensity) 246 (77) 245 (24) 244 (78) 243 (17); HRMS
calcd for C9H9BrO3: 243.9736. Found: 243.9742. Anal. Calcd
for C9H9BrO3: C, 44.11; H, 3.70. Found: C, 43.80; H, 3.55.
Dim eth yl N-(3-Br om o-4,5,6,7-tetr a h yd r oisoben zofu r -
oyl)-L-glu ta m a te (8b). Acid 7b (89 mg, 0.36 mmol) was
dissolved in 10 mL of THF, and NMM (47 µL, 0.43 mmol) was
added neat, via syringe. After stirring the solution for 10 min,
2-chloro-4,6-dimethoxy-1,3,5-triazine (69 mg, 0.40 mmol) was
added in one portion, and the resulting mixture was allowed
to stir for 40 min as a fine white precipitate deposited.
Additional NMM (47 µL, 0.43 mmol) and L-glutamic acid
dimethyl ester hydrochloride (91 mg, 0.43 mmol) were added
sequentially, and after 2 h the reaction mixture was parti-
tioned between EtOAc (15 mL) and water (15 mL). The
organic phase was dried over MgSO4, filtered, and evaporated
in vacuo to give a clear oil. Purification by radial chromatog-
raphy (2 mm plate), using 25% EtOAc in hexanes as eluent,
provided 120 mg (83%) of a clear, colorless oil which darkened
upon prolonged exposure to air. 1H NMR (CDCl3, 270 MHz)
δ 1.47 (m, 4 H), 1.81-2.26 (m, 6 H), 2.60 (m, 2 H), 3.44 (s, 3
H), 3.54 (s, 3 H), 4.53 (dt, 1 H, J ) 4.9, 8.1 Hz), 6.70 (d, 1 H,
J ) 8.1 Hz); 13C NMR (CDCl3, 75.6 MHz) δ 20.6, 22.1, 22.2,
22.3, 27.7, 30.3, 51.1, 51.8, 52.6, 120.5, 124.4, 132.0, 142.1,
158.3, 172.2, 173.1; IR (NaCl) 1731, 1647, 1541 cm-1; MS m/e
(relative intensity) 403 (8), 401 (8); Anal. Calcd for C16H20
-
BrNO6: C, 47.78; H, 5.01; N, 3.48. Found: C, 47.36; H, 4.86;
N, 3.05.
Dim eth yl N-[3-[[2-(P iva loyla m in o)-4-oxo-3,4-d ih yd r o-
p yr id o[2,3-d ]p yr im id in -6-yl]e t h yn yl]-4,5,6,7-t e t r a h y-
d r oisoben zofu r oyl]-L-glu ta m a te (10b). A mixture of 9 (270
mg, 1.0 mmol), Pd(OAc)2 (4.5 mg, 4 mol %), tri-o-tolylphosphine
(12 mg, 8 mol%), Et3N (103 mg, 1.0 mmol), and 8b (205 mg,
0.51 mmol) in 10 mL of MeCN was heated at reflux under an
argon atmosphere for 24 h. After cooling to rt, the solvent
was removed in vacuo, and the resulting black residue was
passed through a short column of silica gel using 5% MeOH
in CH2Cl2 as eluent. Further purification by radial chroma-
tography on a 2 mm plate using 80% EtOAc in hexanes as
eluent afforded the coupled product as a pale yellow solid (195
mg, 65%) which was used directly in the next step. 1H NMR
(CDCl3, 270 MHz) δ 1.33 (s, 9 H), 1.76 (m, 4 H), 2.24 (m, 2 H),
2.47 (m, 2 H), 2.76 (m, 2 H), 2.95 (m, 2 H), 3.65 (s, 3 H), 3.77
(s, 3 H), 4.76 (dt, 1 H, J ) 4.9, 8.2 Hz), 6.75 (d, 1 H, J ) 7.9
Hz), 8.53 (d, 1 H, J ) 2.3 Hz), 8.56 (br s, 1 H), 8.87 (s, 1 H),
12.12 (br s, 1 H).
Dim eth yl N-[3-[2-[2-(P iva loyla m in o)-4-oxo-3,4,5,6,7,8-
h exa h yd r op yr id o[2,3-d ]p yr im id in -6-yl]eth yl]-4,5,6,7-tet-
r a h yd r oisoben zofu r oyl]-L-glu ta m a te (11b). A mixture of
10b (195 mg, 0.33 mmol) and PtO2 (4 mg) in 25 mL of MeOH
and 2 mL of CH2Cl2 was hydrogenated at 48 psi for 24 h. The
mixture was filtered through Celite with the aid of additional
MeOH, and the filtrate was concentrated in vacuo to afford a
pale yellow solid. Purification by radial chromatography (2
mm plate), using 5% MeOH in CH2Cl2 as eluent, afforded a
white solid (188 mg, 95%) .1H NMR (CDCl3, 270 MHz) δ 1.27
(s, 9 H), 1.66 (m, 4 H), 1.62-1.80 (m, 2 H), 1.92 (m, 1 H), 2.11
(m, 2 H), 2.28 (m, 1 H), 2.39 (m, 2 H), 2.45 (m, 2 H), 2.62-
2.77 (m, 2 H), 2.79 (m, 3 H), 2.97 (t, 1 H, J ) 10.2 Hz), 3.35
(m, 1 H), 3.63 (s, 3 H), 3.75 (s, 3 H), 4.76 (dt, 1 H, J ) 5.1, 8.0
Hz), 4.84 (s, 1 H), 6.70 (d, 1 H, J ) 8.0 Hz), 8.09 (s, 1 H), 11.27
(s, 1 H); HRMS calcd for C30H41N5O8: 599.2954. Found:
599.2933.
N-[3-[2-(2-Am in o-4-oxo-3,4,5,6,7,8-h exa h yd r op yr id o-
[2,3-d ]p yr im id in -6-yl)eth yl]-4,5,6,7-tetr a h yd r oisoben zo-
fu r oyl]-L-glu ta m ic Acid (12b). A suspension of 11b (35 mg,
58 µmol) in 1 mL of 1 N NaOH was stirred at rt for 48 h.
Acidification with 3 M HCl gave a pale yellow solid which was
collected and dried (17 mg, 61%). 1H NMR (DMSO-d6, 300
MHz) δ 1.65 (m, 6 H), 2.00 (m, 3 H), 2.34 (m, 2 H), 2.46 (m, 2
H), 2.72-2.87 (m, 7 H), 3.25 (m, 1 H, partially obscured by
residual water peak), 4.35 (dt, 1 H, J ) 5.0, 8.0 Hz), 5.99 (s, 1
H), 6.33 (br s, 2 H), 7.92 (d, 1H, J ) 8.1 Hz), 9.71 (br s,1 H),
12.55 (br s, 1 H). HRMS (FAB) calcd for C23H30N5O7: 488.2145
(MH+). Found: 488.2140.
Ack n ow led gm en t . The authors owe a debt of
gratitude to Chuan Shih, Laurane Mendelsohn, and
Lillian Habeck for obtaining the biological data for
compounds 12a and 12b and to Eli Lilly and Company
for financial support of this work.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra for
compounds 10a , 11a , 11b, 12a , and 12b (5 pages). This
material is contained in libraries on microfiche, immediately
follows this article in the microfilm version of the journal, and
can be ordered from the ACS; see any current masthead page
for ordering information.
J O9619903