Synthesis and antitumor activity of thieno-separated tricyclic purines

Article abstract of DOI:10.1021/jm000326i

The purine ring system is undoubtedly one of the most ubiquitous heterocyclic ring systems in nature as it has the distinction of being the parent ring in countless derivatives of biological relevance. It is not surprising then that modified purines possess the potential to impact several areas, including a better understanding of the biological effects of DNA damaging agents, enzyme/substrate interactions, and in the development of more potent medicinal agents. One focus for our research at Georgia Tech has centered around the design and synthesis of a series of extended purine analogues containing a heterocyclic spacer ring, with sites set on investigations into their use as (i) potential anticancer and antiviral agents, (ii) dimensional probes for enzyme and coenzyme binding sites, and (iii) structural probes of the minor groove of DNA. The synthesis and preliminary antitumor activity of two thieno-separated purine analogues are described herein. Tricyclic 1 was synthesized in 12 steps from tribromoimidazole and with an overall yield of 7%. Tricyclic 2 was synthesized in 9 steps with an overall yield of 13%. Both 1 and 2 exhibited growth inhibitory effects on HCT116 colorectal cancer cells in vitro.

Full text of DOI:10.1021/jm000326i

J . Med. Chem. 2000, 43, 4877-4883
4877
Syn th esis a n d An titu m or Activity of Th ien o-Sep a r a ted Tr icyclic P u r in es
Katherine L. Seley,* Piotr J anuszczyk, Asmerom Hagos, and Liang Zhang
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400
Daniel T. Dransfield
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912
Received August 2, 2000
The purine ring system is undoubtedly one of the most ubiquitous heterocyclic ring systems in
nature as it has the distinction of being the parent ring in countless derivatives of biological
relevance. It is not surprising then that modified purines possess the potential to impact several
areas, including a better understanding of the biological effects of DNA damaging agents,
enzyme/substrate interactions, and in the development of more potent medicinal agents. One
focus for our research at Georgia Tech has centered around the design and synthesis of a series
of extended purine analogues containing a heterocyclic spacer ring, with sites set on
investigations into their use as (i) potential anticancer and antiviral agents, (ii) dimensional
probes for enzyme and coenzyme binding sites, and (iii) structural probes of the minor groove
of DNA. The synthesis and preliminary antitumor activity of two thieno-separated purine
analogues are described herein. Tricyclic 1 was synthesized in 12 steps from tribromoimidazole
and with an overall yield of 7%. Tricyclic 2 was synthesized in 9 steps with an overall yield of
13%. Both 1 and 2 exhibited growth inhibitory effects on HCT116 colorectal cancer cells in
vitro.
Ch a r t 1
Ba ck gr ou n d a n d Sign ifica n ce
DNA, the primary genetic material, is a key partici-
pant in a wide range of biological processes, and
disruption of its function has a detrimental effect in
many areas, in particular, on cell growth.1-3 Most
anticancer agents interact directly with DNA. Others
block the synthesis of DNA or are incorporated into
DNA, interfering with function. Furthermore, drugs
targeted toward the nucleic acids act at the earliest
possible stage of gene expression and should, therefore,
be biologically significant. Understanding the structural
interactions of DNA damaging agents and the minor
groove of DNA will give direction toward the synthesis
of more effective anticancer agents.
In that regard, understanding the structural interac-
tions between enzymes and their substrates or cofactors
is also vital to the design of more effective antiviral
agents. Leonard and co-workers4,5 designed a series of
extended ring systems (such as depicted in Chart 1) for
the purpose of testing the dimensional restrictions of
enzyme binding sites.6-9 These extended purine ana-
logues, such as lin-benzoadenine, dist-benzoadenine,
and prox-benzoadenine, have several interesting proper-
ties: (i) they exhibit strong enzyme binding, activation,
and inhibition; (ii) they act as cofactors in enzymatic
reactions; (iii) many of them exhibit fluorescent10,11
properties, which can help define their environment.
Although extended purine analogues differ electroni-
cally, which results in changes in basicity, nucleophi-
licity, and π-stacking, the similarities in the peripheral
rings allow these analogues to retain the necessary
hydrogen-bonding elements required for molecular rec-
ognition. This affords them the ability to function as
dimensional probes, thereby making it possible to
investigate the parameters available for the purine
moiety in enzyme-coenzyme binding sites, as well as
to help define the structural role of the purine ring with
respect to the minor groove of DNA. In addition, their
inherent structural resemblance to biologically signifi-
cant purines should endow them with similar medicinal
traits. Interest in extended purines has continued,12-14
but there have been few reports15-17 of tricyclic ana-
logues containing a heterocyclic spacer ring.
In that regard, the thiophene ring is considered to be
bioisosteric with the benzene ring; however, when used
as an internal spacer, it imparts an arc to the hetero-
cyclic moiety, not unlike Leonard’s dist-benzoadenine
shown in Chart 1. As noted by Leonard, the insertion
of a benzene ring expands the purine ring system by
2.4 Å at both points of attachment; the thiophene spacer,
however, causes an expansion of 2.9 and 1.5 Å between
the imidazole and pyrimidine rings.
Few examples of the use of the thiophene as a spacer
have been published,15,17-20 and several possess inter-
esting medicinal properties. To date, however, none of
these examples have contained the desired peripheral
purine ring components of adenine and guanine. On the
basis of the strong interest in structural analogues of
adenine and guanine for use in medicinal chemistry,
* To whom correspondence should be addressed. Phone: 404-894-
4013. Fax: 404-894-2295. E-mail: katherine.seley@chemistry.gatech.edu.
10.1021/jm000326i CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/11/2000

4878 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Seley et al.
Sch em e 1
Ch a r t 2
facile than other groups previously tried. This method
was initially used on the synthesis of tricyclic target 1,
but unfortunately it proved to be less than satisfactory
as well. Subsequently, during the synthesis of 2, it
became apparent that the vinyl group could not be used
due to the incompatibility with the conditions of the
deprotection method. We then altered our approach to
utilize the allyl group instead. The removal of the allyl
group has proven to be much more facile.26,27
Ch a r t 3
As shown in Scheme 1, treatment of the tribromoimi-
dazole 323 with 50% NaOH, tetrabutylammonium bro-
mide, and 1,2-dibromoethane under reflux conditions
produced 4 in 90% yield.24 Removal of the C-2 bromine
of 4 was accomplished with ethylmagnesium bromide
and H2O to form 5 in 90% yield.
Initially it was thought that the nitrile intermediate
8 could be formed directly28 from displacement of the
C-5 bromine with potassium cyanide or lithium cyanide,
but all attempts failed. Dibromoimidazole 5 was once
again treated with ethylmagnesium bromide in ether
followed by addition of N-formylpiperidine to provide
the aldehyde 6 in 40% yield.24 Despite a reported24 yield
of 65%, all attempts at optimization in our hands failed
to give any better than 40%. The major product (50%)
of this reaction was 4-bromo-1-vinylimidazole. In an
attempt to improve the yield, N,N-dimethylformamide
(DMF) was then tried in place of N-formylpiperidine,
and this provided 6 in 60% yield. (Note: Although no
NMR data have been published on 6, Iddon et al.24
compared it to analogous compounds they had previ-
ously synthesized,29,30 in particular, a benzyl-protected
analogue, and found the formylation to have occurred
only at the C-5 position. This is not surprising given
the reactivity trend of imidazoles to undergo reaction
at C-5 prior to C-4.)
combined with the interesting properties discovered by
Leonard for his extended purines, we proceeded with
this modification.
Ch em istr y
The targets chosen for our study are shown in Chart
2, with initial focus on the adenine analogue 1. In
analyzing the possible approaches to 1, we contemplated
the three intermediates shown in Chart 3. The “thio-
phene pathway” (A) and the “pyrimidine pathway” (C)
were both unattractive to us.19-21 Pathway B was
chosen, however, due to a recent report22 of the synthe-
sis of an intermediate similar to the one shown for
pathway B, and we felt this could be readily adapted to
meet our needs.
Treatment of 6 with hydroxylamine hydrogen chloride
and sodium bicarbonate in ethanol produced oxime 7
in 93% yield.29,30 Subsequent dehydration of 7 with
acetic anhydride under reflux gave nitrile 8 in 95%
yield. Next, sequential addition (2 equiv, followed by an
additional 2 equiv after 1 h) of excess potassium
carbonate and thioglycolamide31 (obtainable in one step
from commercially available methyl thioglycolate with
Starting with tribromoimidazole,23 protection of the
imidazole nitrogen was first undertaken. Careful con-
sideration as to the choice of protecting group for this
step was critical, since the literature indicated that
deprotection of this imidazole nitrogen was difficult at
best. Originally we explored two reported procedures24,25
that showed deprotection of the vinyl group to be more

Thieno-Separated Tricyclic Purines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4879
Ta ble 1. Tricyclic Compound-Induced Inhibition of HCT116
Sch em e 2
Growtha
time
(h) compd
0.1 µM
1 µM
10 µM
100 µM
24
48
72
1
2
1
2
1
2
96.1 ( 3.8 106.6 ( 4.8 104.8 ( 3.8
82.0 ( 7.8
75.8 ( 8.9
71.5 ( 3.8c
98.7 ( 5.7
105.1 ( 3.3
96.8 ( 2.7
101.3 ( 7.3
96.7 ( 3.4
93.2 ( 7.1
98.3 ( 4.3 105.0 ( 2.1
95.9 ( 3.3
96.1 ( 6.6
85.0 ( 2.8b 77.0 ( 4.4b
93.8 ( 12.1 51.5 ( 10.7b
99.5 ( 1.3
106.6 ( 4.4 116.7 ( 1.9b
76.1 ( 3.5b
a
HCT116 cells were treated and growth was assessed as
described in the text. Data represent the average ( SEM as a %
b
of control-treated (DMSO) cells (n ) 3-5). p < 0.05. c p < 0.005
96% yield) afforded 9 (90%). Cyclization of 9 with
sodium ethoxide in ethanol gave the appropriately
substituted bicyclic intermediate 10 (90%). Ring closure
of the third ring was accomplished with diethoxymethyl
acetate32 to provide 11 in 80% yield.
when compared to control-treated cells.
deprotection procedure26 utilizing palladium and phen-
ylsilane, and the synthesis of 2 was then begun.
In an analogous fashion as was carried out for the
synthesis of 1, treatment of 3 with allyl bromide35
produced 16 in 99% yield (Scheme 3). Removal of the
C-2 bromine was accomplished as before to give 17
(90%). Formation of the Grignard, followed by addition
of DMF, produced aldehyde 18 (93%). Treatment of 18
gave oxime 19 (63%), which was then converted to the
nitrile 20 in 92% yield. Addition of the thiene moiety
was accomplished as previously carried out with thio-
glycolamide31 to give 21 (74%), which underwent ring
closure to afford bicyclic intermediate 22 in 92% yield.
Formation of the tricyclic 23 was accomplished with
freshly prepared chloroformamidine hydrochloride and
methyl sulfone in 58% yield.14 Removal of the allyl group
was achieved employing tetrakis(triphenylphosphine)-
palladium(0) and phenylsilane26 to give the desired
guanine analogue 2 (69%).
Several methods21,24 for removal of the vinyl protect-
ing group were then tried, but all proved poor at best.
Finally, a modification of the reported24 procedure
employing potassium permanganate and acetone under
reflux conditions was utilized. Initially the literature
workup and purification for this method proved prob-
lematic, resulting in very low yields. However, following
the reaction with potassium permanganate and acetone,
the mixture was evaporated and the residue treated
with acetic acid under reflux conditions to provide the
tricyclic hypoxanthine intermediate 12 in 60% yield.
Despite numerous efforts to improve the conversion,
60% was the optimal yield that could be achieved.
However, since the other 40% was unreacted 11 it could
be recovered. Conversion of the oxo moiety to the chloro
derivative 13 was then tried using standard conditions
with phosphorus oxychloride, but all attempts resulted
in decomposition, so an alternative route4,33,34 was then
considered. As shown in Scheme 2, conversion of 12 to
the thione derivative 14 was achieved by treatment with
phosphorus pentasulfide in dry pyridine, which was
immediately converted to its S-methyl derivative 15.
Finally, 15 was heated for 90 h with a saturated
butanolic ammonia solution to provide the adenine
analogue 1 in 12 steps and 7% overall yield.
Tricyclic 1 was synthesized in 12 steps from tribromo-
imidazole and with an overall yield of 7%. Tricyclic 2
was synthesized in 9 steps with an overall yield of 13%.
Resu lts
We initially sought to assess the effect of these
tricyclic compounds on the growth of HCT116 colorectal
cancer cells in vitro. Both 1 and 2 inhibited growth in a
dose- and time-dependent manner with 1 exhibiting
greater potency after 72 h of treatment (Table 1).
Interestingly, 2 also inhibited growth in these cells, with
significant inhibitions being observed at lower doses (10
µM) after 48 h of treatment. However, this affect was
not sustained for 72 h. In fact, lower doses (1 µM) of 2
produced a slight, but statistically significant, stimu-
latory effect on growth of HCT116 cells.
The second target 2 was envisioned in an analogous
fashion, but it was immediately apparent that the
oxidative deprotection method used to remove the vinyl
group would oxidize the ring present in the guanine
analogue. Therefore, an alternative protecting group
was sought. After considering several options, the allyl
group was selected due to the discovery of a mild
Sch em e 3

4880 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Seley et al.
4-Br om o-1-vin ylim id a zole-5-ca r ba ldeh yde (6). To a stir-
ring, room temperature solution of 5 (6.14 g, 24.3 mmol) in
dry ether (200 mL) under nitrogen was added dropwise
ethylmagnesium bromide (8.5 mL, 25.5 mmol, 3.0 M in ether)
and the mixture was stirred for 4 h, at which point DMF (2.0
mL, 25.6 mmol) was added dropwise and the solution stirred
for an additional 3 h. Following addition of H2O (400 mL), the
ether layer was separated and the aqueous layer extracted
with methylene chloride (3 × 100 mL). The organic layers were
combined, washed with brine, dried (MgSO4) and evaporated
to give a dark syrup. The residue was purified via column
chromatography eluting with hexane:ethyl acetate (2:1) to give
6 as a white crystalline solid (2.94 g, 60%): mp 92-93 °C; 1H
NMR (CDCl3) δ 5.19 (dd, 1.8 Hz, 8.5 Hz, 1H), 5.47 (dd, 1.8 Hz,
15.9 Hz, 1H), 7.56 (dd, 8.5 Hz, 15.9 Hz, 1H), 7.79 (s, 1H), 9.80
(s, 1H); 13C NMR (CDCl3) δ 107.9, 125.2, 128.6, 129.6, 140.1,
179.5. Anal. (C6H5BrN2O) C, H, N.
Discu ssion
Tricyclic 1 and 2 both exhibited growth inhibitory
effects on HCT116 colorectal cancer cells in vitro. Our
data suggest that 1 is slightly more potent at inhibiting
growth in these cells than 2, with approximately 50%
inhibition at 72 h. The reason behind these differences
in the dose responses following 72 h of treatment is
unknown at this point but may be due to the fact that
1 is a modified adenine analogue. Along these lines, we
have demonstrated that an adenine-containing nucleo-
side, 8-chloroadenosine, potently inhibits growth in
these cells.36 Coupling of 1 or 2 to a ribo-sugar to create
the thieno-separated nucleosides may increase the
growth inhibitory properties of these analogues reported
herein. We are actively pursuing this theory as well as
other possibilities at this time.
4-Br om o-1-vin ylim id a zole-5-a ld oxim e (7). A solution of
hydroxylamine hydrochloride (14.48 g, 208 mmol) and sodium
hydrogen carbonate (17.73 g, 211 mmol) in H2O (50 mL) was
added to 1-vinyl-4-bromo-5-carbaldehyde (6; 6.10 g, 30.17
mmol) in ethanol (280 mL) and the solution stirred overnight.22
The solvent was removed by rotary evaporation and H2O (50
mL) added to the residue. The aqueous layer was extracted
with ethyl acetate (50 mL × 3) and the organic layers were
combined, washed with brine (50 mL), dried (MgSO4) and
evaporated to give a colorless syrup. The residue was purified
via column chromatography eluting with methylene chloride:
ethyl acetate (95:5) to give the oxime 7 as a white crystalline
solid (6.2 g, 93%): mp 184 °C; 1H NMR (DMSO-d6) δ 5.10 (dd,
1.5 Hz, 8.7 Hz, 1H), 5.66 (dd, 1.5 Hz, 15.6 Hz, 1H), 7.39 (dd,
8.7 Hz, 15.6 Hz, 1H), 7.99 (s, 1H), 8.30 (s, 1H), 11.68 (s, 1H);
13C NMR (DMSO-d6) δ 106.0, 119.02, 121.3, 129.7, 137.1, 137.9.
Anal. (C6H6BrN3O) C, H, N.
Exp er im en ta l Section
Gen er a l. Melting points were recorded on a Meltemp II
melting point apparatus and are uncorrected. Combustion
analyses were performed by Atlantic Microlabs, Inc., Atlanta,
1
GA. H and 13C NMR spectra were recorded on a Bruker 300
spectrometer (operated at 300 and 75 MHz, respectively) all
referenced to internal tetramethylsilane (TMS) at 0.0 ppm. The
spin multiplicities are indicated by the symbols s (singlet), d
(doublet), t (triplet), m (multiplet) and b (broad). UV-vis
activity was measured on a Shimadzu 1601 UV/Vis spectro-
photometer. Reactions were monitored by thin-layer chroma-
tography (TLC) using 0.25-mm Whatman Diamond silica gel
60-F254 precoated plates with visualization by irradiation with
a Mineralight UVGL-25 lamp. Column chromatography was
performed on Whatman silica, 200-400 mesh, 60 Å, and
elution with the indicated solvent system. HPLC purification
was carried out on a Hewlett-Packard 1090 liquid chromato-
graph. Yields refer to chromatographically and spectroscopi-
cally (1H and 13C NMR) homogeneous materials. All cell culture
reagents were obtained from Media-Tech/Cellgro (Herndon,
VA), and Fisher Scientific (Pittsburgh, PA). HCT116 colorectal
cancer cells were purchased from ATCC (Bethesda, MD).
2,4,5-Tr ibr om o-1-vin ylim id a zole (4). To a stirred, 0 °C
solution of 2,4,5-tribromoimidazole23 (3) (10.0 g, 32.8 mmol)
and tetrabutylammonium bromide (10.5 g, 32.8 mmol, 20.1 mL
50% aqueous solution) in 1,2-dibromoethane (30 mL) was
added dropwise 50% aqueous sodium hydroxide (30 mL).24 The
reaction was refluxed with vigorous stirring for 3 h. The
mixture was then cooled, diluted with H2O (50 mL) and
extracted with methylene chloride (50 mL × 3). The organic
layers were combined, washed with 10% HCl (50 mL), then
H2O (50 mL), dried (MgSO4) and evaporated under reduced
pressure. The residue was purified via column chromatography
eluting with hexane:ethyl acetate (100:3), to give the protected
imidazole 4 (9.8 g, 90%) as a white crystalline product following
4-Br om o-1-vin ylim id a zole-5-ca r bon itr ile (8). A solution
of oxime 7 (5.8 g, 26.85 mmol) in acetic anhydride22 (150 mL)
was heated under reflux for 2 h, at which point the excess
acetic anhydride was removed by rotary evaporation to give a
reddish brown oil. The residue was coevaporated with toluene
(2 × 50 mL) and then purified by column chromatography
eluting with hexane:ethyl acetate (3:1) to give nitrile 8 (5.0 g,
94%) as a white crystalline solid following recrystallization
1
with hexane: mp 83-85 °C; H NMR (CDCl3) δ 5.31 (dd, 2.4
Hz, 8.7 Hz, 1H), 5.74 (ddd, 0.9, 2.4 Hz, 15.6 Hz, 1H), 6.90 (dd,
8.7 Hz, 15.6 Hz, 1H), 7.73 (s, 1H); 13C NMR (DMSO-d6) δ 103.4,
108.4, 110.4, 127.3, 128.1, 140.4. Anal. (C6H4BrN3) C, H, N.
5-Cya n o-S-(1-vin ylim id a zol-4-yl)th ioglycola m id e (9). A
solution of nitrile 8 (123 mg, 0.62 mmol) in anhydrous DMF
(25 mL) was added with stirring to a solution of freshly
prepared thioglycolamide31 (212 mg, 2.3 mmol) that had been
treated with potassium carbonate (200 mg, 1.5 mmol) and the
mixture heated for 12 h maintaining the temperature between
50 and 55 °C. The reaction mixture was cooled, evaporated to
dryness, and the residue purified via column chromatography
eluting with chloroform:ethanol (9:1) and the resulting solid
purified further by recrystallization in ethanol to give 9 as a
white crystalline solid (116 mg, 90%): mp 139 °C; 1H NMR
(DMSO-d6) δ 3.75 (s, 2H), 5.22 (dd, 1.8 Hz, 9.0 Hz, 1H), 5.71
(dd, 1.8 Hz, 15.6 Hz, 1H), 7.05 (dd, 9.0 Hz, 15.6 Hz, 1H), 7.13
(br s, 1H), 7.53 (br s, 1H), 8.39 (s, 1H); 13C NMR (DMSO-d6) δ
35.5, 100.6, 106.9, 111.1, 128.0, 140.4, 147.9, 169.0. Anal.
(C8H8N4OS) C, H, N, S.
1
recrystallization with hexane: mp 36 °C; H NMR (CDCl3) δ
5.56 (dd, 2.1 Hz, 9.0 Hz, 1H), 5.74 (dd, 2.1 Hz, 16.0 Hz, 1H),
6.68 (dd, 9.0 Hz, 16.0 Hz, 1H); 13C NMR (CDCl3) δ 105.6, 116.8,
118.6, 118.7, 129.4. Anal. (C5H3Br3N2) C, H, N.
4,5-Dibr om o-1-vin ylim id a zole (5). To a stirring, room
temperature solution of 4 (16.55 g, 50 mmol) in dry ether (500
mL) was added dropwise ethylmagnesium bromide (16.7 mL,
3.0 M in ether, 50 mmol) over a period of 5 min.24 The solution
was stirred for 3 h at which point an additional portion of
ethylmagnesium bromide (8.3 mL, 25 mmol, 3.0 M in ether)
was added and stirred for 25 min. The solvent was then
removed by rotary evaporation, cold water added (400 mL),
and extracted with methylene chloride (3 × 150 mL). The
organic layers were combined, dried (Na2SO4) and evaporated
to afford, after recrystallization with hexane, 11.3 g (90%) of
5 as a white solid: mp 55 °C; 1H NMR (CDCl3) δ 5.14 (dd, 2.1
Hz, 9.0 Hz, 1H), 5.48 (dd, 2.1 Hz, 16.0 Hz, 1H), 6.82 (dd, 9.0
Hz, 16.0 Hz, 1H), 7.79 (s, 1H); 13C NMR (CDCl3) δ 103.1, 107.2,
117.17, 128.4, 136.3. Anal. (C5H4Br2N2) C, H, N.
6-Am in o-1-vin ylt h ien o[2,3-d ]im id a zole-5-ca r b oxa m -
id e (10). A mixture of 9 (2.33 g, 11.2 mmol) and sodium
ethoxide (220 mg, 1.0 mmol, 21% in EtOH) in absolute ethanol
(150 mL) was heated under reflux for 1.5 h. The solvent was
removed under vacuum to one-third of the original volume and
the solution was then cooled in an ice bath. The precipitate
that formed was filtered and washed with cold ethanol to afford
10 as a white crystalline solid (1.89 g, 81%): mp 179-181 °C;
1H NMR (DMSO-d6) δ 5.26 (dd, 1.8 Hz, 9.0 Hz, 1H), 5.65 (dd,
1.8 Hz, 15.6 Hz, 1H), 6.78 (br s, 1H), 6.92 (br s, 2H), 7.50 (dd,
9.0 Hz, 15.6 Hz, 1H), 8.48 (s, 1H); 13C NMR (DMSO-d6) δ 100.0,

Thieno-Separated Tricyclic Purines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4881
104.1, 125.7, 128.8, 138.6, 141.6, 143.7, 167.7. Anal. (C8H8N4-
OS) C, H, N, S.
5.82 (ddd, 5.4 Hz, 10.5 Hz, 20.4 Hz, 1H); 13C NMR (CDCl3) δ
50.0, 105.2, 116.6, 118.2, 118.9, 129.9.
4,5-Dibr om o-1-a llylim id a zole (17). To a stirring solution
of the protected imidazole 16 (50.0 g, 145 mmol) in dry ether
(500 mL) was added dropwise butylmagnesium bromide (72.5
mL, 145 mmol, 2.0 M in ether) under N2. The solution was
stirred for 4 h. The ether was then removed by rotary
evaporation, saturated NH4Cl solution added (400 mL) to the
residue, and extracted with methylene chloride (3 × 200 mL).
The organic layers were combined, dried (MgSO4), filtered over
a bed of silica gel, and then evaporated to afford 17 (35.0 g,
90%) as a white crystalline solid: mp 58-59 °C; 1H NMR
(CDCl3) δ 4.52 (dt, 1.5 Hz, 5.4 Hz, 2H), 5.06 (d, 19.0 Hz, 1H),
5.30 (d, 10.5 Hz, 1H), 5.88 (ddd, 5.4 Hz, 10.5 Hz, 18.3 Hz, 1H),
7.48 (s, 1H); 13C NMR (CDCl3) δ 49.9, 103.8, 116.6, 119.3,
130.9, 137.0. Anal. (C6H6Br2N2) C, H, N.
1-Vin ylim id a zo[4′,5′:4,5]th ien o[3,2-d ]p yr im id in -5(6H)-
on e (11). A mixture of 10 (180 mg, 0.86 mmol) and di-
ethoxymethyl acetate32 (0.5 mL, 3 mmol) was refluxed for 3 h,
the excess diethoxymethyl acetate evaporated and the residue
purified by flash chromatography eluting with ethyl acetate
to afford, following recrystallization with ethanol, 11 as a white
crystalline solid (153 mg, 81%): mp >250 °C dec; 1H NMR
(DMSO-d6) δ 5.26 (dd, 1.5 Hz, 9.0 Hz, 1H), 6.62 (dd, 1.5 Hz,
15.6 Hz, 1H), 7.45 (dd, 9.0 Hz, 15.6 Hz, 1H), 8.31 (s, 1H), 8.60
(s, 1H); 13C NMR (DMSO-d6) δ 104.8, 121.3, 127.2, 128.8, 142.8,
145.0, 146.9, 149.8, 157.7. Anal. (C9H6N4OS‚1/8EtOH) C, H, N,
S.
Im idazo[4′,5′:4,5]th ien o[3,2-d]pyr im idin -5(6H)-on e (12).
A solution of 11 (109 mg, 0.5 mmol), anhydrous acetone (50
mL) and potassium permanganate24 (158 mg, 1.0 mmol) was
heated for 6 h under reflux. To this was then added an
additional portion of potassium permanganate (79 mg, 0.5
mmol) and the mixture allowed to reflux for an additional 12
h. The solvent was removed by rotary evaporation, and the
brown residue was dissolved in glacial acetic acid (50 mL) and
refluxed for 2 h. The mixture was cooled and evaporated to
dryness and the residue purified by column chromatography
eluting with methylene chloride:ethanol (4:1) to give 12 as an
off-white solid (57 mg, 60%) following recrystallization with
ethanol: mp >250 °C dec; 1H NMR (DMSO-d6) δ 8.23 (s, 1H),
8.25 (s, 1H); 13C NMR (DMSO-d6) δ 120.2, 129.1, 143.6, 144.1,
147.1, 150.0, 158.0. Anal. (C7H4N4OS‚3/8H2O) C, H, N, S.
4-Br om o-1-a llylim id a zole-5-ca r ba ld eh yd e (18). To a
stirring solution of 17 (26.6 g, 100 mmol) in dry ether (400
mL) under nitrogen was added dropwise at -78 °C butylmag-
nesium bromide (50.0 mL, 100 mmol, 2.0 M in ether). The
mixture was allowed to warm to room temperature and then
stirred for 4 h, at which point DMF (20 mL) was added and
the solution stirred for an additional 18 h. The solvents were
then removed under reduced pressure, saturated NH4Cl solu-
tion added to the residue, and extracted with methylene
chloride. The organic layers were combined, washed with
brine, dried (MgSO4), filtered over a bed of silica gel, and
evaporated to give 18 as a pale yellow oil (20.1 g, 93%): mp
1
58-59 °C; H NMR (CDCl3) δ 4.91 (dt, 1.5 Hz, 5.4 Hz, 2H),
5.17 (d, 19.0 Hz, 1H), 5.29 (d, 10.5 Hz, 1H), 5.96 (ddd, 5.4 Hz,
10.5 Hz, 21.0 Hz, 1H), 7.58 (s, 1H), 9.76 (s, 1 H); 13C NMR
(CDCl3) δ 49.9, 119.8, 126.6, 130.6, 131.8, 142.1, 179.6. Anal.
(C7H7BrN2O) C, H, N.
I m id a z o [4′,5′:4,5]t h ie n o [3,2-d ]p y r im id in e -5(6H )-
th ion e (14). A solution of phosphorus pentasulfide4 (400 mg),
dry pyridine (30 mL) and 12 (192 mg, 1.0 mmol) was refluxed
for 24 h, cooled, evaporated to dryness under vacuum, and the
residue purified by column chromatography eluting with
methylene chloride:methanol (4:1) to afford 14 as a pale yellow
solid (180 mg, 87%: mp >250 °C dec; H NMR (DMSO-d6) δ
8.41 (s, 1H), 8.24 (s, 1H); which was used directly without
further purification.
4-Br om o-1-a llylim id a zole-5-oxim e (19). In an analogous
fashion as was employed with 7, 20.1 g (93.5 mmol) gave oxime
1
1
19 as a white crystalline solid (13.6 g, 63%): mp 172 °C; H
NMR (DMSO-d6) δ 4.85 (dt, 1.5 Hz, 5.4 Hz, 2 H), 4.93 (dd, 1.5
Hz, 17.1 Hz, 1H), 5.16 (dd, 1.5 Hz, 10.5 Hz, 1H), 5.96 (ddd,
5.4 Hz, 10.5 Hz, 21.0 Hz, 1H), 7.81 (s, 1H), 7.94 (s, 1 H), 11.47
(s, 1 H). Anal. (C7H8BrN3O) C, H, N.
4-Br om o-1-allylim idazole-5-car bon itr ile (20). In an analo-
gous fashion as was employed with conversion of 7 to 8, oxime
19 (13.0 g, 56.5 mmol) was converted to the nitrile 20 (11.5 g,
92%) as a white crystalline solid: mp 36-38 °C; 1H NMR
(CDCl3) δ 4.68 (d, 5.4 Hz, 2H), 5.32 (d, 16.8 Hz, 1H), 5.45 (d,
10.2 Hz, 1H), 5.95 (ddd, 5.4 Hz, 10.5 Hz, 23.0 Hz, 1H), 7.52 (s,
1H); 13C NMR (CDCl3) δ 50.1, 106.3, 110.0, 121.6, 126.8, 130.1,
139.9. Anal. (C7H6BrN3) C, H, N.
5-Cya n o-1-a llylim id a zol-4-ylth ioa ceta m id e (21). Con-
version of nitrile 20 (9.5 g, 45 mmol) was accomplished in the
same fashion as was used for 8 to 9 to give 21 as a white
crystalline solid (6.9 g, 74%): mp 141 °C; 1H NMR (DMSO-d6)
δ 3.72 (s, 2 H), 4.73 (dt, 1.5 Hz, 5.4 Hz, 2H), 5.11 (dd, 1.5 Hz,
17.4 Hz, 1H), 5.29 (dd, 1.5 Hz, 10.5 Hz, 1H), 5.95 (ddd, 5.4
Hz, 10.5 Hz, 21.0 Hz, 1H), 7.13 (br s, 1H), 7.53 (br s, 1H), 8.06
(s, 1H); 13C NMR (DMSO-d6) δ 49.3, 79.8, 103.9, 110.8, 119.3,
133.1, 142.7, 147.9, 169.2. Anal. (C9H10N4OS) C, H, N, S.
6-Am in o -1-a lly lt h ie n o [2,3-d ]im id a zo le -5-c a r b o x -
a m id e (22). Compound 21 (5.1 g, 22.0 mmol) was converted
to 22 in the same manner as was used for 9 to 10 to give a
white crystalline solid (4.7 g, 92%): mp 179 °C; 1H NMR
(DMSO-d6) δ 4.95 (d, 5.4 Hz, 2H), 5.01 (dd, 1.5 Hz, 17.1 Hz,
1H), 5.18 (dd, 1.5 Hz, 10.5 Hz, 1H), 6.04 (ddd, 5.4 Hz, 10.5
Hz, 18.9 Hz, 1H), 6.66 (br s, 2H), 6.82 (br s, 2H), 7.95 (s, 1H);
13C NMR (DMSO-d6) δ 48.2, 100.1, 117.8, 127.4, 135.3, 139.3,
143.7, 145.6, 168.5. Anal. (C9H10N4OS) C, H, N, S.
1-Allyl-2-am in oim idazo[4′,5′:4,5]th ien o[3,2-d]pyr im idin -
5(6H)-on e (23). To a mixture of 6-amino-1-allylthieno[2,3-d]-
imidazole-5-carboxamide (22) and methyl sulfone (5.6 g) was
added freshly prepared chloroformamidine hydrochloride14 (2.5
g, 11.3 mmol) and the mixture heated for 1.5 h at 100 °C with
stirring. The mixture was allowed to cool and H2O (5 mL) was
added. The solution was then neutralized with NH4OH and
the solvents were removed under reduced pressure. The
6-(Meth ylsu lfon yl)im id a zo[4′,5′:4,5]th ien o[3,2-d ]p yr i-
m id in e (15). To a stirring solution of 14 (104 mg, 0.5 mmol)
and potassium carbonate (100 mg, 0.72 mmol) in methanol
(20 mL) was added methyl iodide (140 mg, 1.0 mmol).4 After
10 min the solvent was removed by rotary evaporation and
the residue purified via column chromatography eluting with
methylene chloride:methanol (7:1) to give 15 (135 mg, 90%)
as a white crystalline solid following recrystallization from
anhydrous ethanol: mp 308 °C; 1H NMR (DMSO-d6) δ 2.73
(s, 3H), 3.14 (s, 1H), 8.33 (s, 1H), 8.97 (s, 1H); 13C NMR
(DMSO-d6) δ 48.6, 127.7, 144.6, 144.9, 145.8, 153.6, 153.7,
162.7. Anal. (C8H6N4S2) C, H, N, S.
6-Am in oim id a zo[4′,5′:4,5]th ien o[3,2-d ]p yr im id in e (1).
A solution of 6-(methylsulfonyl)imidazo[4′,5′:4,5]thieno[3,2-d]-
pyrimidine (15; 84 mg, 0.37 mmol) in saturated butanolic
ammonia (20 mL) was sealed in a steel bomb and heated at
160 °C for 90 h.4 The solvent was removed by rotary evapora-
tion and the residue purified on preparative TLC eluting with
methylene chloride:methanol (4:1) to give 1 (41 mg, 56%) as
an off-white solid: mp >330 °C dec; 1H NMR (DMSO-d6) δ
3.31 (s, 1H), 7.28 (s, 2H), 8.18 (s, 1H), 8.38 (s, 1H); 13C NMR
(DMSO-d6) δ 112.3, 128.2, 143.0, 145.5, 146.8, 154.3, 158.7;
λmax observed at 303 nm, ꢀ ) 1.05 × 104 M-1 cm-1. Anal.
(C7H5N5S‚1/8EtOH) C, H, N, S.
2,4,5-Tr ibr om o-1-a llylim id a zole (16). A mixture of 3 (40.0
g, 131 mmol), allyl bromide (12.6 mL, 144 mmol), and potas-
sium carbonate (19.95 g, 144 mmol) in acetone (175 mL) was
refluxed for 18 h.35 After cooling, the mixture was filtered and
the filtrate concentrated under reduced pressure. The residual
oil was dissolved in methylene chloride (100 mL), filtered
through a pad of silica gel and the silica gel washed with
additional methylene chloride (200 mL). The filtrates were
combined and evaporated to give 16 as a colorless crystalline
solid (45.0 g, 99%), which was used directly without further
1
purification: mp 30-32 °C; H NMR (CDCl3) δ 4.61 (dt, 1.8
Hz, 5.4 Hz, 2H), 5.06 (d, 17.0 Hz, 1H), 5.29 (d, 10.5 Hz, 1H),

4882 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Seley et al.
(8) Leonard, N. J . Dimensional Probes of Enzyme-Coenzyme Bind-
ing Sites. Acc. Chem. Res. 1982, 15, 128-135.
residue was purified via column chromatography eluting with
methylene chloride:methanol (15:1) to give 23 (1.6 g, 58%) as
a white solid: mp >280 °C dec; 1H NMR (DMSO-d6) δ 4.99 (d,
5.4 Hz, 2H), 5.15 (dd, 1.5 Hz, 20.4 Hz, 1H), 5.20 (dd, 1.5 Hz,
10.5 Hz, 1H), 6.16 (ddd, 5.4 Hz, 10.5 Hz, 21.0 Hz, 1H), 6.53
(br s, 2 H), 8.09 (s, 1H), 11.0 (br s, 1H); 13C NMR (DMSO-d6)
δ 48.5, 109.7, 118.8, 128.7, 134.5, 145.7, 146.4, 148.4, 155.5,
159.1. Anal. (C10H9N5OS‚1/4EtOH) C, H, N, S.
(9) Leonard, N. J .; Hiremath, S. P. Dimensional Probes of Binding
and Activity. Tetrahedron 1986, 42, 1917-1961.
(10) Leonard, N. J .; Keyser, G. E. Synthesis of Fluorescent Nucleotide
Analogues: 5′-Mono, Di-, and Triphosphates of linear-Benzogua-
nosine, linear-Benzoinosine and linear-Benzoxanthosine. Proc.
Natl. Acad. Sci. U.S.A. 1979, 76, 4262-4264.
(11) Rosendahl, M. S.; Omann, G. M.; Leonard, N. J . Synthesis and
Biological Activity of a Profluorescent Analogue of Coenzyme
B12. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 3480-3484.
(12) Zhang, J .; Nair, V. Synthesis of New Dideoxynucleosides Bearing
Ring-Extended Nucleobases. Nucleosides Nucleotides 1997, 16,
1091-1094.
(13) Zhang, J .; Neamati, N.; Pommier, Y.; Nair, V. Inhibition of HIV
Integrase by Novel Nucleotides Bearing Tricyclic Bases. Bioorg.
Med. Chem. Lett. 1998, 8, 1887-1890.
(14) Schneller, S. W.; Ibay, A. C. Synthesis of proximal-Benzoguanine
and a Simplified Synthesis of proximal-Benzohypoxanthine. J .
Org. Chem. 1986, 51, 4067-4070.
(15) Showalter, H. D. H.; Bridges, A. J .; Zhou, H.; Sercel, A. D.;
McMichael, A. et al. Tyrosine Kinase Inhibitors. 16. 6,5,6-
Tricyclic Benzothieno[3,2-d]pyrimidines and Pyrimido[5,4-b]-
and -[4,5-b]indoles as Potent Inhibitors of the Epidermal Growth
Factor Receptor Tyrosine Kinase. J . Med. Chem. 1999, 42, 5464-
5474.
(16) Schneller, S. W.; Christ, W. J . Quinazolines, Quinoxalines and
Ring-Separated Pteridines and Purines. J . Heterocycl. Chem.
1982, 19, S139-S161.
(17) Schneller, S. W.; Clough, F. W. Studies of Thieno[2,3-b]pyrazines
in Preparation of Pyrazino[2′,3′:4,5]thieno[3,2-d]pyrimidines and
Related Molecules. J . Heterocycl. Chem. 1975, 12, 513-516.
(18) Clark, J .; Hitiris, G. Heterocyclic Studies. Part 43. Thieno[2,3-
d:4,5-d]dipyrimidines. J . Chem. Soc., Perkin Trans. I 1984,
2005-2008.
(19) Athmani, S.; Iddon, B. Azoles. Part 10. Thiazolo[4′,5′:4,5]thieno-
[3,2-d]pyrimidine, a New Heterocyclic Ring System. Tetrahedron
1992, 48, 7689-7702.
2-Am in oim idazo[4′,5′:4,5]th ien o[3,2-d]pyr im idin -5(6H)-
on e (2). Phenylsilane (1.0 mL, 8.2 mmol) was added dropwise
to a mixture of the allyl protected guanine analogue 23 (200
mg, 0.81 mmol), palladium tetrakis (50 mg, 0.043 mmol) and
glacial acetic acid (5 mL) in methylene chloride (10 mL) under
Ar. The mixture was stirred overnight at 40 °C. The solvent
was removed under reduced pressure and purified via column
chromatography eluting with methylene chloride:methanol (9:
1). The fractions containing product were evaporated and
purified further via reverse-phase HPLC eluting with aceto-
nitrile/H2O to afford 2 (115 mg, 69%) as a white solid: mp
1
330 °C dec; H NMR (DMSO-d6) δ 6.74 (s, 2H), 8.10 (s, 1H),
11.0 (s, 1H); 13C NMR (DMSO-d6) δ 109.6, 128.7, 144.21, 146.3,
148.4, 155.3, 159.3; λmax observed at 297 nm, ꢀ ) 3.33 × 104
M-1 cm-1. Anal. (C7H5N5OS‚2/3H2O) C, H, N, S. HPLC analysis
on a C-18 reverse-phase column, 5 µm, 250 × 4.60 mm
(Phenomenex); detector wavelength 280 nm; flow rate ) 1 mL/
min; linear gradient 0 to 20 min, 5-20% CH3CN/H2O; reten-
tion time 8.5 min.
Cell Cu ltu r e. HCT116 cells were cultured in DMEM
supplemented with 2 mM L-glutamine, 50 IU penicillin, 50 µg/
mL streptomycin, nonessential amino acids, and 10% heat-
inactivated fetal bovine serum. All cell lines were cultured at
37 °C in 5% CO2 and media changed every 48 h.
Cell P r olifer a tion Assa ys. Cell proliferation was assessed
using the CellTiter96 system from Promega (Madison, WI)
according to the manufacturer’s instructions. HCT116 cells
were seeded in 96-well plates at a density of 500 cells/well.
After 72 h the medium was changed and the cells were treated
with fresh serum containing medium with either control
vehicle (DMSO) or test agent (at various concentrations).
Growth was assessed after 24, 48, and 72 h of treatment and
reported as a percentage (%) of the control-treated cells.
Sta tistica l An a lysis. Statistical differences were deter-
mined using a Student’s t-test (Insat 2.3, GraphPad Software,
San Diego, CA), with a p value < 0.05 considered to be
significant.
(20) Hawkins, D. W.; Iddon, B.; Longthorne, D. S.; Rosyk, P. J .
Synthesis of Thieno[2,3-b]-, [3,2-b]-, and -[3,4-b]thiophenes and
Thieno[3′,2′:4,5]-, [2′,3′:4,5]-, and [3′,4′:4,5]thieno[3,2-d]pyrimi-
din-7(6H)-ones Starting From Thiophene. J . Chem. Soc., Perkin
Trans. I 1994, 2735-2743.
(21) Iddon, B.; Khan, N.; Lim, B. L. Azoles. Part 4. Nucleophilic
Substitution Reactions of Halogenoimidazoles. J . Chem. Soc.,
Perkin Trans. I 1987, 1437-1443.
(22) Hawkins, D. W.; Iddon, B.; Longthorne, D. S. Azoles. 12.
Synthesis of Thieno-Extended Purines - First Example of the
Imidazo[4′,5′/4,5]Thieno[3,2-d]Pyrimidine Ring-System. Tetra-
hedron 1995, 51, 12807-12820.
(23) Stensio¨, K.-E.; Wahlberg, K.; Wahren, R. Synthesis of Bromi-
nated Imidazoles. Acta Chem. Scand. 1973, 27, 2179-2183.
(24) Hartley, D. J .; Iddon, B. Use of the vinyl group as an efficient
protecting group for azole N-atoms: Synthesis of polyfunction-
alized imidazoles and thieno[2,3-d]T[3,2-d]imidazole. Tetrahe-
dron Lett. 1997, 38, 4647-4650.
(25) Chen, Y. L.; Hedberg, K. G.; Guarino, K. J . Vinyl Protecting
Group for Benzimidazole Nitrogen: Synthesis of Benzimidazole-
Penam Alcohol. Tetrahedron Lett. 1989, 30, 1067-1068.
(26) Kimbonguila, A. M.; Boucida, S.; Guibe, F.; Loffet, A. On the
Allyl Protection of the Imidazole Ring of Histidine. Tetrahedron
1997, 53, 12525-12538.
(27) Lemaire-Audoire, S.; Savignac, M.; Geneˆt, J . P.; Bernard, J .-M.
Selective Deprotection of Allylamines using Palladium. Tetra-
hedron Lett. 1995, 36, 1267-1270.
(28) Babaev, E. V.; Bush, A. A.; Orlova, I. A.; Rybakov, V. B.; Zhukov,
S. G. Novel Route to b-Fused Thiazoles Starting from a 2-Chloro-
1-Phenacylpyridinium Salt and KSCN. Crystal Structures of
Thiazolo- and Oxazolo[3,2-a]pyridinium Thiocyanates. Tetrahe-
dron Lett. 1999, 40, 7553-7556.
(29) Iddon, B.; Khan, N. Azoles. 5. Metal-Halogen Exchange-
Reactions of Polybromoimidazoles. J . Chem. Soc., Perkin Trans.
I 1987, 1445-1451.
(30) Iddon, B.; Khan, N.; Lim, B. L. Azoles. Part 7. A Convenient
Synthesis of Thieno[2,3-d]imidazoles. J . Chem. Soc., Perkin
Trans. I 1987, 1457-1463.
(31) Sokol, H.; Ritter, J . J . The Reaction of Thioglycolamide with
Halomethyl Ketones. J . Am. Chem. Soc. 1948, 70, 3517-3518.
(32) Cosstick, R.; Li, X.; Tuli, D. K.; Williams, D. M.; Connolly, B.
A., et al. Molecular Recognition in the Minor Groove of the DNA
Helix. Studies on the Synthesis of Oligonucleotides and Poly-
nucleotides Containing 3-Deaza-2′-Deoxyadenosine. Nucleic Ac-
ids Res. 1990, 18, 4771-4778.
(33) Leonard, N. J .; Henderson, T. R. Purine Ring Rearrangements
Leading to the Development of Cytokinin Activity. Mechanism
of the Rearrangement of 3-Benzyladenine to N6-Benzyladenine.
J . Am. Chem. Soc. 1975, 97, 4990-4999.
Ackn owledgm en t. We thank Ms. Nadia Boguslavsky
and Ms. J ulie Ha for their help in the HPLC purification
of 2. The initial work on this project was funded by the
Emory/Georgia Tech Biomedical Technology Research
Center, and this assistance is appreciated.
Refer en ces
(1) Murray, V. A Survey of the Sequence-Specific Interaction of
Damaging Agents with DNA: Emphasis on Antitumor Agents.
Progress in Nucleic Acid Research and Molecular Biology;
Academic Press: New York, 2000; pp 367-415.
(2) Denny, W. A. DNA-Intercalating Ligands as Anti-cancer
Drugs: Prospects for Future Design. Anti-Cancer Drug Des.
1989, 4, 241-263.
(3) Sielechi, T. M.; Boylan, J . F.; Benfield, P. A.; Trainor, G. L.
Cyclin-Dependent Kinase Inhibitors: Useful Targets in Cell
Cycle Regulation. J . Med. Chem. 2000, 43, 1-18.
(4) Leonard, N. J .; Morrice, A. G.; Sprecker, M. A. Linear Benzo-
adenine. A Stretched-Out Analogue of Adenine. J . Org. Chem.
1975, 40, 356-366.
(5) Leonard, N. J .; Kazmierczak, F.; Rykowski, A. A Convenient
Synthesis of lin-Benzopurines through a Common Intermediate.
J . Org. Chem. 1987, 52, 2933-2935.
(6) Leonard, N. J .; Scopes, D. I. C.; VanDerLijn, P.; Barrio, J . R.
Dimensional Probes of the Enzyme Binding Sites of Adenine
Nucleotides. Biological Effects of Widening the Adenine Ring by
2.4 Å. Biochemistry 1978, 17, 3677-3685.
(7) Leonard, N. J .; Sprecker, M. A.; Morrice, A. G. Defined Dimen-
sional Changes in Enzyme Substrates and Cofactors. Synthesis
of lin-Benzoadenosine and Enzymatic Evaluation of Derivatives
of the Benzopurines. J . Am. Chem. Soc. 1976, 98, 3987-3994.

Thieno-Separated Tricyclic Purines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4883
(34) Morrice, A. G.; Sprecker, M. A.; Leonard, N. J . The Angular
Benzoadenines. 9-Aminoindiazo[4,5-f]quinazoline and 6-Ami-
noimidazo[4,5-h]quinazoline. J . Org. Chem. 1975, 40, 363-366.
(35) Pissiotas, G. 2,4,5-Trichloro- and 2,4,5-Tribromoimidazole De-
rivatives and Pesticidal Compositions Containing Them; Ciba-
Geigy AG: Switzerland, United States, 1976.
(36) Carlson, C. C.; Chinery, R.; Burnham, L. L.; Dransfield, D. T.
8-Cl-Adenosine Inhibits Colorectal Cancer Growth In Vitro and
In Vivo. Neoplasia, in press.
J M000326I