Synthesis of non-nucleoside analogs of toyocamycin, sangivamycin, and thiosangivamycin: The effect of certain 4- and 4,6-substituents on the antiviral activity of pyrrolo[2,3-d]pyrimidines

Article abstract of DOI:10.1021/jm950835y

A number of 4-substituted 7-(ethoxymethyl)- and 7-[(2- methoxyethoxy)methyl]pyrrolo[2,3-d]-pyrimidine-5-carbonitrile and -5- thiocarboxamide derivatives and several 7-substituted 4,6- diaminopyrrolo[2,3-d]pyrimidine-5-carbonitrile, -5-carboxamide, and -5-

Full text of DOI:10.1021/jm950835y

3470
J . Med. Chem. 1996, 39, 3470-3476
Syn th esis of Non -n u cleosid e An a logs of Toyoca m ycin , Sa n giva m ycin , a n d
Th iosa n giva m ycin : Th e Effect of Cer ta in 4- a n d 4,6-Su bstitu en ts on th e
An tivir a l Activity of P yr r olo[2,3-d ]p yr im id in es
Thomas E. Renau,† Christopher Kennedy, Roger G. Ptak, J ulie M. Breitenbach, J ohn C. Drach, and
Leroy B. Townsend*
Department of Medicinal Chemistry, College of Pharmacy, Department of Chemistry, College of Literature Sciences and Arts,
and Department of Biologic and Materials Sciences, School of Dentistry, The University of Michigan,
Ann Arbor, Michigan 48109-1065
Received November 13, 1995X
A number of 4-substituted 7-(ethoxymethyl)- and 7-[(2-methoxyethoxy)methyl]pyrrolo[2,3-d]-
pyrimidine-5-carbonitrile and -5-thiocarboxamide derivatives and several 7-substituted 4,6-
diaminopyrrolo[2,3-d]pyrimidine-5-carbonitrile, -5-carboxamide, and -5-thiocarboxamide analogs
related to the nucleoside antibiotics toyocamycin and sangivamycin were prepared and tested
for activity against human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-
1). Biologically, modifications at the 4-position were not well tolerated in cell culture, and in
almost all cases no activity against HCMV or HSV-1 was observed. Furthermore, none of the
compounds inhibited the growth of L1210 murine leukemic cells in vitro. In sharp contrast to
the 4-substituted compounds, all of the 4,6-diamino 5-nitrile and the 5-thioamide analogs were
active against HCMV, whereas the 5-carboxamides were inactive. The corresponding 4-amino
6-methylamino and 6-dimethylamino 5-nitrile analogs were inactive against HCMV, establish-
ing that an amino group at both C-4 and C-6 is a likely requirement for antiviral activity.
Overall, our results demonstrate that an amino group at C-4 and a thioamide moiety at C-5 of
a 7-substituted pyrrolo[2,3-d]pyrimidine are essential for activity against HCMV, whereas a
4,6-diamino analog does not necessarily require a thioamide group at C-5 for activity against
HCMV.
In tr od u ction
Scheme 1. Because of the good separation of antiviral
activity from toxicity with the ether-substituted com-
pounds previously described,1 two of the more active
7-ether substituents, namely, ethoxymethyl (EM) and
(methoxyethoxy)methyl (MEM) substituents, have been
chosen for this study. Aqueous diazotization of the
exocyclic amino group of 4-amino-7-(ethoxymethyl)-
pyrrolo[2,3-d]pyrimidine-5-carbonitrile (1a)1 and 4-amino-
7-[(2-methoxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine-
5-carbonitrile (1b)1 was accomplished with nitrous acid
to afford the 5-cyano 7-substituted pyrrolo[2,3-d]pyri-
midin-4-ones 2a ,b in good yields. The IR spectrum of
2a confirmed the success of the transformation showing
a strong absorbance at 1670 cm-1 for the CdO group.
Treatment of 2a ,b with hydrogen sulfide for 24 h at 100
°C furnished the thioamides 3a ,b. A lack of absorbance
in the 2200 cm-1 region of the IR spectrum of 3a
confirmed the absence of a nitrile group. The key
intermediates, 4-chloro-7-(ethoxymethyl)pyrrolo[2,3-d]-
pyrimidine-5-carbonitrile (4a ) and 4-chloro-7-[(2-meth-
oxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine-5-carboni-
trile (4b), were generated in good yields by the treatment
of 2a ,b with POCl3 at reflux for 10 min followed by silica
gel chromatography. The attempted reduction of 4a
with H2 over a Pd catalyst in EtOH and 1 equiv of 1 N
NaOH resulted in the nucleophilic substitution of an
ethoxy group at C-4 rather than a removal of the
halogen group. To avoid this, the reduction of 4a ,b was
accomplished using 1 equiv of NaHCO3 rather than
NaOH, thereby furnishing analogs 5a ,b in good yields.
As part of a study examining the potential of non-
nucleoside analogs of the nucleoside antibiotics toyoca-
mycin and sangivamycin as antiviral agents, we previ-
ously described the synthesis, cytotoxicity, and antiviral
activity of certain 7-substituted 4-aminopyrrolo[2,3-d]-
pyrimidine-5-carbonitrile, -5-carboxamide, and -5-thio-
carboxamide analogs.1 The study concentrated on the
identification of new compounds to treat infections
caused by human cytomegalovirus (HCMV) because of
the problems associated with use of the clinically
approved anti-HCMV agents ganciclovir (GCV) and
foscarnet (PFA).2-5 The results established that the
thiosangivamycin analogs, which bear a CSNH2 moiety
at C-5 of the pyrrolopyrimidine, have selective activity
against HCMV without the requirement for activation
to an active metabolite by phosphorylation of a side
chain hydroxyl group residing at the N-7 position.
To determine whether the 4-amino functionality is
necessary for antiviral activity and to extend the
structure-activity relationships, the synthesis, cyto-
toxicity, and antiviral activity of 4-substituted and 4,6-
disubstituted 5-nitrile, 5-carboxamide, and 5-thiocar-
boxamide non-nucleoside analogs related to nucleosides
toyocamycin, sangivamycin, and thiosangivamycin are
described in this report.
Ch em istr y
The synthetic route used to prepare the C-4-modified
non-nucleoside analogs 2a ,b -20b is illustrated in
Treatment of 4a ,b with various nucleophiles at room
temperature gave the 4-substituted derivatives 6a ,b-
12a ,b. We found the 4-chloro derivatives 4a ,b to be
highly reactive to a host of O, N, and S nucleophiles
† Present address: Microcide Pharmaceuticals, Mountain View, CA
94043.
X Abstract published in Advance ACS Abstracts, April 1, 1996.
S0022-2623(95)00835-1 CCC: $12.00 © 1996 American Chemical Society

Substituent Effect on Antiviral Activity
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3471
Sch em e 1. Synthesis of 4-Substituted 5-Cyano- and
5-Thioamide Pyrrolo[2,3-d]pyrimidine Analogs Related
to Toyocamycin and Thiosangivamycina
Sch em e 2. Synthesis of 4,6-Disubstituted 5-Cyano-,
5-Thioamide, and 5-Carboxamide
Pyrrolo[2,3-d]pyrimidines Related to Toyocamycin,
Sangivamycin, and Thiosangivamycina
a
(i) NH3(l); (ii) Pd/C, H2; (iii) NH2OH‚HCl/NaOMe, EtOH/H2O;
(iv) MeOH, H2S/NaOMe. a , R7
)
CH2OCH2CH3; b ,
CH2OCH2CH2OCH3; c, R7 ) CH2OCH2C6H5; d , R7 ) CH2C6H5;
e, R7 ) CH2-3-CH3C6H5; f, R7 ) CH2-4-CH3C6H5; g, R7 ) CH2-4-
OCH3C6H5; h , R7 ) CH2CHCH2; i, R7 ) CH2CH2CH3.
a
(i) NaNO2, H2O/AcOH; (ii) MeOH, H2S/NaOMe; (iii) POCl3;
(iv) (a) Pd/C. H2, EtOH, NaHCO3, (b) NaOMe, MeOH, (c) EtOH, 1
N NaOH, (d) H2NMe, (e) H2NEt, (f) H2NC3H5, (g) HN(CH3)2, (h)
H2NCSNH2. a , R7 ) CH2OCH2CH3; b, R7 ) CH2OCH2CH2OCH3.
Sch em e 3. Synthesis of 4-Amino, 6-Substituted
Analogs of 21ba
with typical reaction times being <10 min at room
temperature. The nitriles 5a ,b-12a ,b were converted
to the corresponding thioamide derivatives 13a ,b-20b
by treatment with NaSH generated in situ. Supporting
previous work in this area, we found that the reaction
times for this conversion appeared to depend on the
substituent at the 4-position.6 For example, conversion
of the nitrile of the 4-ethoxy analog 7a to the CSNH2
derivative 15a took 3 days at 80 °C. In contrast,
reaction of the 4-HNMe analog 8a with NaSH furnished
the corresponding thioamide 16a in only 5 h at 95 °C.
Comparison of the UV spectra of analogs 2a -19a with
their corresponding ribosyl-substituted compounds dem-
onstrated similar spectral profiles.7,8
The 4,6-diamino 7-substituted pyrrolo[2,3-d]pyrimi-
dine-5-thiocarboxamide derivatives 24a -c were gener-
ated from the requisite nitriles 22a -c (Scheme 2).
Compounds 22a -h were synthesized in good yields by
the treatment of 21a -h 1 with liquid NH3 for 16 h at
100 °C. Reduction of 22h furnished the propyl deriva-
tive 22i in modest yield. Treatment of the acyclic
substituted nitriles 22a -c with hydroxylamine hydro-
chloride in sodium methoxide in methanol furnished the
carboxamide derivatives 23a -c. Comparison of the UV
spectra of analogs 22a and 24a with their corresponding
ribosyl-substituted compounds demonstrated similar
spectral profiles.6
a
(i) (a) NH2CH3, EtOH, (b) NH(CH3)2, EtOH.
3b, 5b-6b, 8b, 10b, 13b-14b, 16b, and 19b) were
examined as potential antitumor agents by determining
their effect on L1210 murine leukemic cells in vitro.
None of the compounds examined displayed any sig-
nificant inhibition with IC50’s not reached at the highest
concentration tested (100 µM).
In Vitr o An tivir a l Activity. In the series of 4-sub-
stituted 5-nitrile and 5-thioamide derivatives (com-
pounds 2-20), only compounds 16a ,b, substituted with
a methylamino group at the 4-position (R4) and a
thioamide group at the 5-position (R5), had moderate
antiviral activity (IC50 ) 17-20 µM). All of the other
compounds were inactive against HCMV with IC50’s >
100 µM. The activity of the 7-ether-substituted deriva-
tives 16a ,b was less potent than the activity for the
corresponding 4-amino, 5-thioamide derivatives (com-
pounds III and IV, Table 2). Of compounds 2-20, only
16b had moderate activity against HSV-1 (IC50 ) 30
µM). The 4-methylamino derivative of thiosangivamy-
cin6 was more active than both 16a ,b, but it was also
more toxic to uninfected HFF cells (IC50 ) 5.0 µM, CC50
) 100 µM). The 4-unsubstituted thioamide derivatives
13a ,b were inactive at concentrations up to 100 µM,
establishing that both the amino group at C-4 and a
thioamide at C-5 are essential for activity against
HCMV in this series of 7-substituted pyrrolo[2,3-d]-
pyrimidines. Like the 4-amino, 5-nitrile analogs I and
II (Table 2), none of the 4-substituted nitrile derivatives
demonstrated activity against HCMV at concentrations
The 4-amino, 6-methylamino and 4-amino, 6-di-
methylamino analogs of 22b (25 and 26, respectively)
were prepared from 21b (Scheme 3) with either meth-
ylamine or dimethylamine. The absorbance in the 2200
cm-1 region of the IR spectrum of 25 and 26 confirmed
the presence of the nitrile group. Synthetic and physical
data for all of the compounds examined in this study
are presented in Table 1.
Biologica l Resu lts a n d Discu ssion
In Vitr o An tip r olifer a tive Testin g. A number of
the 4-substituted 5-nitrile and 5-thioamide analogs (2b-

3472 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Renau et al.
Ta ble 1. Synthetic and Physical Data of the
Pyrrolo[2,3-d]pyrimidines Prepared for This Study
Interestingly, the corresponding 7-substituted (hydroxy-
ethoxy)methyl (HEM) and (dihydroxypropoxy)methyl
(DHPM) 4,6-diamino, 5-thioamide derivatives were
inactive against HCMV up to 100 µM.9 Like the HEM
and DHPM analogs, though, the corresponding carboxa-
mide derivatives 23a -c were inactive and nontoxic.
Similarly, the bromo-substituted analogs 21a -c1 were
also inactive and nontoxic against HCMV.
method of
yield
(%)
formula
compd
preparationa
mp (°C)
(C, H, N)b
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
10b
11a
11b
12a
12b
13a
13b
14a
14b
15a
16a
16b
17a
17b
18b
19a
19b
20b
22a
22b
22c
22d
22e
22f
c
90
73
67
74
80
63
80
76
77
86
56
73
70
77
77
94
92
37
75
93
93
61
53
72
52
46
94
64
91
85
92
23
41
27
87
74
84
20
28
41
209-211
155
240 dec
C10H10N4O2
C11H12N4O3
C10H12N4O2S
11H14N4O3S
10H9N4OCl
11H11N4O2Cl
10H10N4O
Similar to 2a
c
Similar to 3a
c
Similar to 4a
c
Similar to 5a
c
Similar to 6a
c
Similar to 7a
c
Similar to 8a
c
Similar to 9a
c
238 dec
C
C
C
C
C
125-126
105-106 dec
89
The most surprising aspect is the observed activity
against HCMV of the corresponding nitrile derivatives
22a -c. These compounds typically were more active
than the thioamide analogs 24a -c in both the plaque
reduction assay and ELISA. The activity of 22a -c
against HCMV was well separated from the toxicity in
uninfected cells. More extensive studies of the cytotox-
icity of 22b, for example, by the incorporation of labeled
precursors into protein, RNA, and DNA, supported the
cytotoxicity data in Table 2. Specifically, IC50’s for the
incorporation of [3H]Leu and [3H]Urd were >400 µM,
while the IC50 for the inhibition of [3H]dThd incorpora-
tion was 200 µM. Clearly, in the case of the 4,6-diamino
derivatives, a thioamide moiety at C-5 (R5) is not
essential. This is not the case for the 7-ether-substi-
tuted 4-aminopyrrolo[2,3-d]pyrimidines (compounds
I-IV) where a thioamide at the 5-position is required
for antiviral activity (see Table 2). This is the first case
in our study of non-nucleoside pyrrolo[2,3-d]pyrimidine
derivatives where a thioamide moiety at position 5 is
not necessary for antiviral activity. To extend these
results, we prepared and evaluated a variety of other
4,6-diamino 7-substituted derivatives of 22a -c (Table
2). Like the acyclic analogs, compounds 22d -i were all
relatively active against HCMV (0.3 µM e IC50 e 32
µM). The compounds, however, were generally more
toxic than the corresponding acyclic analogs 22a -c in
both uninfected HFF and KB cells. In general, the
activity of compounds 22a -i against HSV-1 was less
than the activity of the compounds against HCMV.
104-107
210-211
123-124
151-152
106-107
175-177
134-135
152-154
129-131
89-90
11H12N4O2
C11H12N4O2
C12H14N4O3
C12H14N4O2
C13H16N4O3
C11H13N5O
12H15N5O2
C12H15N5O
13H17N5O2
C
C
C14H17N5O2
C12H15N5O
13H17N5O2
c
90-92
Similar to 11a
c
Similar to 12a
103-104
218-220
179-181
176-177
151-153
149-150
128-129
139
C
C10H10N4OS
C
C
C
C
C
C
C
C
C
C
C
C
C
C
11H12N4O2S
10H12N4OS
11H14N4O2S
11H14N4O2S
12H16N4O3S
12H16N4O2S
11H15N5OS
12H17N5O2S
12H17N5OS
13H19N5O2S
14H19N5O2S
12H17N5OS
13H19N5O2S
11H14N4O2S2
C10H12N6O
11H14N6O2
C15H14N6O
14H12N6
C15H14N6
C15H14N6‚
0.25H2O
15H14N6O
10H10N6
C10H12N6
C10H14N6O2
C11H16N6O3
C15H16N6O2
C10H14N6OS
11H16N6O2S
15H16N6OS
12H16N6O2
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
157-158
139-141
143-144
150-152
180-182
148-150
103-105
221-224
229-230
190-192
218-219
285-288
299-301
286-289
C
C
22g
22h
22i
23a
23b
23c
24a
24b
24c
25
B
B
c
C
C
C
D
D
34
86
57
24
61
80
43
61
42
28
37
261
C
C
293-295
222-225
176
243-244
212-214
209-210
199-200
199-200
168-169
147-148
To demonstrate that an amino group at both C-4 and
C-6 is a requirement for the activity of 22b, we prepared
and evaluated two compounds (25 and 26) where the
amino group at C-6 was replaced with either a meth-
ylamino (25) or a dimethylamino (26) group (Table 2).
Both compounds were inactive against HCMV and HSV-
1, establishing that amino groups at C-4 and C-6 are
necessary for the antiviral activity of 22b and a likely
requirement for the other compounds in this series,
22a ,c-i.
C
C
C
D
c
26
Similar to 25
C13H18N6O2
a
b
See the Experimental Section for methods A-D. Letters in
parantheses refer to those elements analyzed. c See the Experi-
mental Section for the preparation of this compound.
We have recently shown that the 5-thioamide moiety
of several structurally dissimilar 7-substituted 4-ami-
nopyrrolo[2,3-d]pyrimidines, including thiosangivamy-
cin, is unstable in cell culture medium and converted
to the corresponding 5-nitrile.10 In contrast, structural
modifications of the 4-position had a definite effect on
the stability of the 5-thioamide moiety. For example,
replacement of the 4-amino group of IV with a methyl-
amino (16b) decreased the stability of the thioamide.
Specifically, the half-life decreased from 50 h for IV to
20 h for 16b. In contrast, replacement with a hydrogen
(13b) or a dimethylamino (19b) increased the stability
of the thioamide (t1/2 ) 175 and 150 h, respectively). The
5-thioamide moiety of 24a ,b was similarly unstable and
converted to the 5-nitrile following the appearance of
an unidentified peak by HPLC.11 Hence, the biological
data for compounds 3a ,b, 13a -19a , 13b-20b, and
at or below 100 µM. Of the 4-substitued derivatives,
only the 4-chloro analogs 4a ,b were cytotoxic below 100
µM (65 µM e CC50 e 95 µM). The slight toxicity of 4a ,b
may be caused by some modification of the compounds
in vitro since both compounds were amenable to mild
chemical nucleophilic substitutions.
In the 6-substituted series (compounds 21-26, Table
2), the thioamide derivatives 24a -c demonstrated mod-
est activity against HCMV and were essentially non-
toxic in their antiviral dose range. These three com-
pounds also were evaluated for activity in HCMV yield
reduction assays. Compound 24c was the most active
(IC90 ) 7 µM), whereas compounds 24a ,b were less
active (IC90’s ) 45 and 100 µM, respectively). Com-
pounds 24a -c were relatively inactive against HSV-1.

Substituent Effect on Antiviral Activity
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3473
Ta ble 2. Antiviral Activity and Cytotoxicity of 7-Substituted 4,6-Disubstituted 5-Cyano-, 5-Carboxamide, and 5-Thioamide
Pyrrolo[2,3-d]pyrimidines
50% inhibitory concentrationa (µM)
antiviral activity
substituent
R6
HCMV
cytotoxicity
b
compd
R5
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
R7
plaque
ELISA
HSV-1 (ELISA)
HFF (visual)
KB (growth)
21a
21b
21c
22a
22b
22c
22d
22e
22f
22g
22h
22i
23a
23b
23c
24a
24b
24c
25
Br
Br
Br
EM
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
>100
>100
>100
138
MEM
BOM
EM
MEM
BOM
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
HNCH3
N(CH3)2
H
7.2
12
9
9
68
>100
12.5
10
8.2
2.8
3.3
210
118
34
CH2C6H5
CH2-C6H4-3-CH3
CH2-C6H4-4-CH3
CH2-C6H4-4-OCH3
CH2CHCH2
CH2CH2CH3
EM
MEM
BOM
EM
MEM
32
>100
100
32
100
>100
>100
50
0.3
9
1.9
0.5
60
10
40
2.5
100
100
>100
CN
15
CONH2
CONH2
CONH2
CSNH2
CSNH2
CSNH2
CN
CN
CN
CN
CSNH2
CSNH2
>100
>100
>100
>100
45
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>32
>100
>100
>100
>320
>320
225
>100
>100
>100
>100
60
>100
>100
16
100
100
60
>100
>100
5
16
>100
>100
>100
90
20
19
BOM
MEM
MEM
EM
26
Ic
IIc
H
H
H
MEM
EM
MEM
>100
IIIc
IVc
2.7
1.3
8.7
7
1.9
22
85
3.0
3.4
>100
>100
>100
183
ganciclovir (GCV)
acyclovir (ACV)
>320
>100
>100
a
Results are the average of two or more experiments. A greater than sign (>) indicates IC50 not reached at highest concentration
tested. EM ) ethoxymethyl; MEM ) (methoxyethoxy)methyl; BOM ) (benzyloxy)methyl. c The synthesis and evaluation of compounds
I-IV are described in ref 1 and included in this table for comparative purposes.
b
Perkin-Elmer infrared (IR) spectrophotometer. Nuclear mag-
24a -c may be the result of a mixture of 5-thioamide
netic resonance (1H NMR) spectra were determined at 200,
and 5-carbonitrile derivatives. However, since several
300, or 360 MHz with a Bruker WP 200/300/360 SY instru-
ment. The chemical shift values are expressed in δ values
(ppm) relative to the internal standard tetramethylsilane.
5-thioamide pyrrolo[2,3-d]pyrimidines such as 17b and
18b are inactive against HCMV but still undergo
conversion to the nitrile,11 we feel that the conversion
Elemental analyses were performed by M-H-W Laboratories,
of the 5-thioamide to the 5-nitrile is a secondary
chemical event unrelated to the biological activity.
Certainly, the activity of the analogs 22a -i, which are
stable in cell culture medium,10,11 is due exclusively to
the 4,6-diamino, 5-nitrile-substituted derivatives.
In summary, the synthesis and biological evaluation
of 4-substituted and 4,6-disubstituted 5-cyano-, 5-car-
boxamide, or 5-thiocarboxamide non-nucleoside pyrrolo-
[2,3-d]pyrimidines related to toyocamycin and thiosan-
givamycin have revealed that an amino group at position
4 and a thioamide group at C-5 are essential for activity
against HCMV, whereas the 4,6-diamino analogs do not
necessarily require a thioamide group at C-5 for anti-
viral activity.
Phoenix, AZ, and are within (0.4% of the theoretical values.
E. Merck silica gel (230-400 mesh) was used for gravity or
flash column chromatography. Evaporations were carried out
on a rotary evaporator under reduced pressure (water aspira-
tor) with the bath temperature at 37 °C. Acetonitrile and
methanol were dried over activated molecular sieves (4 Å).
5-Cya n o-7-(et h oxym et h yl)p yr r olo[2,3-d ]p yr im id in -4-
on e (2a ). Compound 1a 1 (7.97 g, 36.7 mmol) was suspended
in distilled H2O (414 mL) and AcOH (33%, 36 mL). This
suspension was heated to 50 °C, and NaNO2 (17.73 g, 256.9
mmol) was added in six batches over a period of 4.5 h.
Following the additions of NaNO2, the reaction mixture was
heated to 70 °C for 16 h. The resulting reaction mixture was
allowed to stand at 4 °C for 24 h. The solid was collected by
filtration and dried at 60 °C for 16 h to yield 7.21 g (90%) of
pure 2a : mp 209-211 °C; IR (KBr) ν 2220 (CN), 1670 (CdO),
1590 (NH) cm-1; UV λmax [nm (ꢀ, mM)] (pH 1) 264 (12.5),
(MeOH) 263 (12.5), (pH 11) 274 (13.0); 1H NMR (DMSO-d6) δ
12.5 (1H, br s, NH, D2O exchangeable), 8.23 (1H, s, H-2), 8.09
(1H, s, H-6), 5.50 (2H, s, NCH2), 3.42-3.52 (2H, q, CH2), 1.02-
1.09 (3H, t, CH3). Anal. (C10H10N4O2) C, H, N.
Exp er im en ta l Section
Gen er a l P r oced u r es. Melting points were taken on a
Thomas-Hoover capillary melting point apparatus and are
uncorrected. Thin-layer chromatography (TLC) was run on
silica gel 60F-254 plates (Analtech, Inc.). Detection of com-
ponents on TLC was made by UV light absorption at 254 nm.
Ultraviolet spectra were recorded on a Kontron-Uvikon 860
spectrophotometer. Infrared (IR) spectra were taken on a
7-(Eth oxym eth yl)-4-oxopyr r olo[2,3-d]pyr im idin -5-th io-
ca r boxa m id e (3a ). NaOMe (149 mg, 2.7 mmol) in dry MeOH
(75 mL) was saturated with H2S (g) for 30 min. This solution
was transferred to a steel vessel containing 2a (300 mg, 1.4

3474 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Renau et al.
mmol) which was sealed and heated at 100 °C in an oil bath
for 24 h. After this time the solution was allowed to cool to
room temperature, and the pH was adjusted to 7 with 1 N
HCl (2 mL). The resulting solution was heated on a steam
bath and filtered and the filtrate cooled at 4 °C for 16 h. The
resulting solid was collected to yield pure 3a as a yellow
powder (235 mg) in 67% yield: mp 240 °C dec; IR (KBr) ν 3210
(NH), 1660 (CdO), 1590 (NH) cm-1; UV λmax [nm (ꢀ, mM)] (pH
1) 322 (11.7), 270 (13.4), (MeOH) 324 (13.0), 272 (16.3), (pH
11) 329 (12.1), 271 (18.4); 1H NMR (DMSO-d6) δ 12.79 (1H, br
s, NH, D2O exchangeable), 11.37 and 9.66 (1H each, br s,
CSNH2, D2O exchangeable), 8.14 (2H, m, H-2, H-6), 5.58 (2H,
s, NCH2), 3.45-3.60 (2H, q, CH2), 1.04-1.09 (3H, t, CH3). Anal.
(C10H12N4O2S) C, H, N.
4.61 (2H, q, CH2), 3.45-3.50 (2H, q, CH2), 1.38-1.42 (3H, t,
CH3), 1.03-1.07 (3H, t, CH3). Anal. (C12H14N4O2) C, H, N.
4-(Meth yla m in o)-7-(eth oxym eth yl)p yr r olo[2,3-d ]p yr i-
m id in e-5-ca r bon itr ile (8a ). Compound 4a (800 mg, 3.4
mmol) was dissolved in methylamine (100 mL, 33% in absolute
EtOH) and stirred at room temperature for 2.5 h. The solution
was cooled at 4 °C for 16 h and filtered to furnish 548 mg (70%)
of pure 8a as a white powder: mp 175-177 °C; UV λmax [nm
(ꢀ, mM)] (pH 1) 275 (19.8), 235 (17.4), (MeOH) 281 (21.0), (pH
11) 282 (20.4), 234 (10.3); 1H NMR (DMSO-d6) δ 8.33 and 8.32
(1H each, s, H-2, H-6), 6.73-6.74 (1H, br q, NH), 5.53 (2H, s,
NCH2), 3.43-3.49 (2H, q, CH2), 2.98-3.00 (3H, d, CH3), 1.01-
1.06 (3H, t, CH3). Anal. (C11H13N5O) C, H, N.
4-(Eth yla m in o)-7-(eth oxym eth yl)p yr r olo[2,3-d ]p yr im i-
d in e-5-ca r bon itr ile (9a ). Compound 4a (800 mg, 3.4 mmol)
was dissolved in EtNH2 (30 mL, 70% in H2O) and stirred at
room temperature. Within 5 min a white precipitate was
observed. The white suspension was poured into a 250 mL
extraction flask, and distilled H2O and EtOAc were added. The
organic layer (total vol ) 150 mL) was collected, dried over
Na2SO4, and filtered and the filtrate evaporated to dryness.
The resulting white solid was suspended in 100 mL of EtOH,
heated to boiling, and then stored for 16 h at 4 °C. The
resulting precipitate was filtered to collect 636 mg (77%) of
9a as a white solid: mp 152-154 °C; UV λmax [nm (ꢀ, mM)]
(pH 1) 276 (21.2), 236 (18.1), (MeOH) 283 (23.5), (pH 11) 283
(22.7), 235 (10.8); 1H NMR (DMSO-d6) δ 8.33 and 8.31 (1H
each, s, H-2, H-6), 6.66-6.68 (1H, br t, NH), 5.53 (2H, s, NCH2),
3.50-3.56 (2H, q, NCH2), 3.43-3.49 (2H, q, OCH2), 1.16-1.20
(3H, t, CH3), 1.03-1.06 (3H, t, CH3). Anal. (C12H15N5O) C,
H, N.
4-Ch lor o-7-(eth oxym eth yl)p yr r olo[2,3-d ]p yr im id in e-5-
ca r bon itr ile (4a ). Compound 2a (1.14 g, 5.2 mmol) was
dissolved in POCl3 (10 mL) and heated at reflux for 12 min.
The hot solution was poured onto ice water (100 mL), and the
pH of the resulting mixture was adjusted to 7 with NH4OH
(38%, 15 mL). The title compound was extracted into CH2Cl2
(2 × 75 mL) from distilled H2O (300 mL total) and NaHCO3
(1 × 5 mL). The organic layer was collected, dried over MgSO4,
and filtered and the filtrate evaporated to dryness to produce
a yellow solid (987 mg, 80%). A small sample (50 mg) was
recrystallized from MeOH/H2O and decolorizing charcoal to
furnish pure 4a as a white powder: mp 125-126 °C; IR (KBr)
ν 2210 (CN), 1100 (C-Cl) cm-1 1H NMR (DMSO-d6) δ 8.87
;
and 8.88 (1H each, s, H-2, H-6), 5.68 (2H, s, NCH2), 3.60-
3.45 (2H, q, CH2), 1.04-1.09 (3H, t, CH3). Anal. (C10H9N4-
OCl) C, H, N.
7-(Eth oxym eth yl)p yr r olo[2,3-d ]p yr im id in e-5-ca r bon i-
tr ile (5a ). Compound 4a (900 mg, 3.8 mmol), NaHCO3 (386
mg, 4.6 mmol), and 10% Pd/C (90 mg, 10% by wt) was dissolved
in absolute EtOH (200 mL). The reaction was hydrogenated
at room temperature at 45 psi for 4.5 h, filtered, and washed
with hot EtOH (2 × 10 mL). The filtrate was evaporated to
dryness and the residue dissolved in CH2Cl2 (2 × 50 mL) and
then extracted with distilled H2O (100 mL). The organic
fractions were collected (total vol ) 100 mL), dried over MgSO4,
filtered, and evaporated to afford 617 mg (80%) of crude 5a as
a light yellow solid. A small sample (150 mg) was recrystal-
lized from H2O/EtOH and decolorizing charcoal to furnish
pure 5a : mp 89 °C; UV λmax [nm (ꢀ, mM)] (MeOH) 271 (6.9),
(pH 11) 270 (7.1); 1H NMR (DMSO-d6) δ 9.25, 9.03, and
8.76 (1H each, s, H-2, H-4, H-6), 5.68 (2H, s, NCH2), 3.47-
3.54 (2H, q, CH2), 1.04-1.08 (3H, t, CH3). Anal. (C10H10N4O)
C, H, N.
4-(Cyclop r op yla m in o)-7-[(2-m et h oxyet h oxy)m et h yl]-
p yr r olo[2,3-d ]p yr im id in e-5-ca r bon itr ile (10b). Compound
4a (500 mg, 1.9 mmol) was dissolved in EtOH (50 mL), and
cyclopropylamine (749 mg, 13.1 mmol) was added. The
reaction mixture was heated at reflux for 1.5 h and then cooled
to room temperature. The solution was evaporated to dryness
and the resultant oil dissolved in water and extracted from
CH2Cl2. The organic layer (total vol ) 150 mL) was collected,
dried over Na2SO4, filtered, and evaporated to dryness. The
resulting oil was triturated with hexanes to reveal 10b as a
1
white solid (500 mg, 92%): mp 89-90 °C; H NMR (DMSO-
d6) δ 8.35 (2H, m, H-2, H-6), 6.90 (1H, m, NH), 5.57 (2H, s,
NCH2), 3.52-3.57 (2H, m, OCH2CH2), 3.32-3.37 (2H, m,
OCH2CH2), 3.17 (3H, s, OCH3), 2.89-2.93 (1H, m, CH), 0.79-
0.81 (2H, m, CH2), 0.61 (2H, m, CH2). Anal. (C14H17N5O2) C,
H, N.
4-Meth oxy-7-(eth oxym eth yl)p yr r olo[2,3-d ]p yr im id in e-
5-ca r bon itr ile (6a ). Compound 4a (100 mg, 0.4 mmol) was
added to dry MeOH (20 mL) and stirred under argon. NaOMe
(114 mg, 2.1 mmol) was then added, and the reaction mixture
was heated at reflux for 1.5 h. At this time it was allowed to
cool to room temperature, the pH adjusted to 7 with AcOH,
and the solution evaporated to dryness. The resulting solid
was dissolved in EtOAc (30 mL) and extracted with distilled
H2O (50 mL). The organic layer (50 mL) was collected, dried
over MgSO4, and filtered and the filtrate evaporated in vacuo
to yield 72 mg (77%) of a tan solid. This solid was recrystal-
lized from H2O/MeOH to furnish pure 6a : mp 210-211 °C;
UV λmax [nm (ꢀ, mM)] (pH 1) 264 (12.5), (MeOH) 264 (12.3),
4-(Dim eth yla m in o)-7-(eth oxym eth yl)p yr r olo[2,3-d ]p y-
r im id in e-5-ca r bon itr ile (11a ). Compound 4a (500 mg, 2.1
mmol) was dissolved in dimethylamine (30 mL, 33% in
absolute EtOH). The reaction mixture was stirred at room
temperature for 30 min, at which time no starting material
was observed by TLC. The solution was evaporated in vacuo,
and the resultant brown solid was suspended in H2O (50 mL)
and MeOH (5 mL) and heated to boiling. To this mixture was
added decolorizing charcoal. The boiling solution was filtered
over Celite and the filtrate cooled for 16 h at 4 °C. The
resulting white solid was collected by vacuum filtration and
dried for 16 h at 60 °C to yield pure 11a (193 mg, 37%): mp
90-92 °C; UV λmax [nm (ꢀ, mM)] (pH 1) 281 (15.3), 244 (11.0),
(MeOH) 290 (19.8), (pH 11) 290 (18.5); 1H NMR (DMSO-d6) δ
8.46 and 8.30 (1H each, s, H-2, H-6), 5.56 (2H, s, NCH2), 3.44-
3.51 (2H, q, CH2), 3.31 (6H, s, CH3 × 2), 1.03-1.08 (3H, t, CH3).
Anal. (C12H15N5O) C, H, N.
1
(pH 11) 264 (12.4); H NMR (DMSO-d6) δ 8.60 and 8.55 (1H
each, s, H-2, H-6), 5.62 (2H, s, NCH2), 4.10 (3H, s, OCH3),
3.45-3.51 (2H, q, CH2), 1.03-1.07 (3H, t, CH3). Anal.
(C11H12N4O2) C, H, N.
4-E t h oxy-7-(et h oxym et h yl)p yr r olo[2,3-d ]p yr im id in e-
5-ca r bon itr ile (7a ). Compound 4a (100 mg, 0.4 mmol) was
dissolved in absolute EtOH (30 mL), and to this solution was
added 1 N NaOH (400 µL, 0.4 mmol). The reaction mixture
was stirred at room temperature for 2 h 45 min, at which time
no starting material was observed by TLC. The solution was
cooled at 4 °C for 2 days, at which time the solid was collected
by filtration to furnish 55 mg (56%) of 7a as a white powder:
mp 151-152 °C; UV λmax [nm (ꢀ, mM)] (pH 1) 264 (13.8),
(MeOH) 264 (14.4), (pH 11) 264 (14.2); 1H NMR (DMSO-d6) δ
8.57 and 8.54 (1H each, s, H-2, H-6), 5.61 (2H, s, NCH2), 4.55-
5-Cya n o-7-(eth oxym eth yl)p yr r olo[2,3-d ]p yr im id in e-4-
th ion e (12a ). Compound 4a (800 mg, 3.3 mmol) and thiourea
(515 mg, 6.8 mmol) were dissolved in absolute EtOH (110 mL)
and the reaction mixture heated at reflux for 60 min. The
reaction mixture was allowed to cool to room temperature and
then evaporated at 37 °C to 1/4 vol. The resulting suspension
was cooled at 4 °C for 16 h and then filtered to furnish 12a as
an off-white solid (720 mg, 93%): mp 218-220 °C; UV λmax
[nm (ꢀ, mM)] (pH 1) 330 (24.0), (MeOH) 331 (23.0), (pH 11)
1
321 (21.5), 231 (13.8); H NMR (DMSO-d6) δ 13.8 (1H, br s,
NH), 8.40 and 8.22 (1H each, s, H-2, H-6), 5.52 (2H, s, NCH2),

Substituent Effect on Antiviral Activity
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3475
3.43-3.50 (2H, q, CH2), 1.03-1.08 (3H, t, CH3). Anal.
br s, NH2, D2O exchangeable), 5.49 (2H, s, CH2), 3.45-3.52
(2H, q, CH2), 1.06-1.10 (3H, t, CH3). Anal. (C10H14N6OS) C,
H, N.
(C10H10N4OS) C, H, N.
Gen er a l Meth od A for th e Syn th esis of Com p ou n d s
13a -20b: 7-(Eth oxym eth yl)p yr r olo[2,3-d ]p yr im id in e-5-
th ioca r boxa m id e (13a ). H2S(g) was passed through a solu-
tion of NaOMe (162 mg, 3.0 mmol) in MeOH (75 mL) for 30
min. This solution was transferred to a steel vessel containing
5a (300 mg, 1.5 mmol). The vessel was sealed, stirred at room
temperature for 24 h, at which time the pH was adjusted to 7
with 1 N HCl (1 mL), heated on a steam bath for 5 min, and
then evaporated to dryness. The crude solid was directly
recrystallized from H2O and a small amount of MeOH to
furnish 214 mg (61%) of 13a as a light yellow powder: mp
176-177 °C; UV λmax [nm (ꢀ, mM)] (MeOH) 266 (16.2), (pH
4-Am in o-6-(m eth yla m in o)-7-[(2-m eth oxyeth oxy)m eth -
yl]p yr r olo[2,3-d ]p yr im id in e-5-ca r b on it r ile (25). Com-
pound 21b (300 mg, 0.9 mmol) was dissolved in methylamine
(33% in absolute alcohol) and stirred for 24 h in a pressure
bottle at room temperature. The solution was evaporated to
dryness and the crude solid recrystallized from MeOH. The
white solid was collected by filtration to furnish 71 mg (28%)
of pure 25: mp 168-169 °C; IR (KBr) ν (cm-1) 2175; 1H NMR
(DMSO-d6) δ 8.02 (1H, s, H-2), 7.3-7.4 (1H, m, NH), 6.14 (2H,
s, NH2), 5.41 (2H, s, NCH2), 3.52-3.56 (2H, m, OCH2CH2),
3.30-3.40 (2H, m, OCH2CH2), 3.19 (3H, s, OCH3), 3.12-3.10
(3H, d, NCH3). Anal. (C12H16N6O2) C, H, N.
1
11) 268 (14.9); H NMR (DMSO-d6) δ 9.80 (1H, s), 9.49 and
9.27 (1H each, br s, CSNH2), 8.90 (1H, s), 8.39 (1H, s), 5.67
(2H, s, NCH2), 3.46-3.52 (2H, q, CH2), 1.04-1.08 (3H, t, CH3).
Anal. (C10H12N4OS) C, H, N.
An tivir a l Eva lu a tion : (a ) Cells a n d Vir u ses. Human
foreskin fibroblasts (HFF cells) and MRC-5 cells, a human
embryonic lung cell line, were grown in minimum essential
medium (MEM) with Earle’s salts [MEM(E)] supplemented
with 10% fetal bovine serum (FBS). KB cells, an established
human cell line derived from an epidermoid oral carcinoma,
were grown in MEM with Hank's salts [MEM(H)] supple-
mented with 10% calf serum (CS). These cell lines were
subcultured according to conventional procedures as described
previously.10 All cell lines were screened periodically for
mycoplasma contamination and were negative. A plaque-
purified isolate, Po, of the Towne strain of HCMV was used in
all experiments and was a gift of Dr. Mark Stinski, University
of Iowa. Stock preparations of HCMV and HSV-1 were
prepared as described elsewhere.12
Gen er a l Meth od B for th e Syn th esis of Com p ou n d s
22a -h : 4,6-Dia m in o-7-(eth oxym eth yl)p yr r olo[2,3-d ]p y-
r im id in e-5-ca r bon itr ile (22a ). A 125 mL steel vessel con-
taining 21a 1 (2.5 g, 8.4 mmol) was charged with 75 mL of
NH3(l). The vessel was sealed, and the reaction mixture was
heated to 100 °C for 16 h. At this time, the vessel was allowed
to cool to room temperature and further cooled to -75 °C, at
which time the vessel was vented. The resulting solid was
suspended in H2O and heated to boiling. Following filtration
the filtrate was cooled at 4 °C for 16 h, and pure 22a was
collected (1.70 g, 87%): mp 229-230 °C; UV λmax [nm (ꢀ, mM)]
1
(pH 1) 293 (15.9), (MeOH) 295 (16.0), (pH 11) 290 (19.9); H
NMR (DMSO-d6) δ 8.00 (1H, s, H-2), 7.22 (2H, br s, NH2, D2O
exchangeable), 6.10 (2H, br s, NH2, D2O exchangeable), 5.42
(2H, s, NCH2), 3.44-3.48 (2H, q, CH2), 1.03-1.07 (3H, t, CH3).
Anal. (C10H12N6O) C, H, N.
(b) Assa ys for An tivir a l Activity. HCMV plaque-reduc-
tion assays were performed using monolayer cultures of HFF
cells by a procedure similar to that referenced above for
titration of HCMV, with the exception that the virus inoculum
(0.2 mL) contained ca. 100 plaque-forming units (PFU) of
HCMV and the compounds to be assayed were dissolved in
the overlay medium. HCMV also was assayed using an
enzyme immunoassay in MRC-5 cells by a procedure described
by Prichard and Shipman to assay HSV-113 and modified by
us.1 HSV-1 ELISA assays were performed as previously
described.13 Protocols for HCMV yield reduction experiments
have been previously described.14 Briefly, monolayer cultures
of HFF cells in 96-well culture dishes (Costar, Cambridge, MA)
were infected at an moi of 0.5, 0.05, or 0.005 PFU/cell and
incubated in the presence of the test compounds for 7 days.
Following one cycle of freezing at -76 °C and thawing at 36
°C, the resulting lysates were diluted, and the amount of
infectious virus was quantified on new cultures of HFF cells.14
(c) Cytotoxicity Assa ys. Two basic tests for cellular
cytotoxicity were employed for compounds examined in anti-
viral assays. Cytotoxicity produced in HFF cells was estimated
by visual scoring of cells not affected by virus infection in the
plaque-reduction assay described above. Drug-induced cyto-
pathology was estimated at 35-fold magnification and scored
on a zero to four basis on the day of staining for plaque
counting. Cytotoxicity in KB cells was determined by a
staining method previously described.15
(d ) Da ta An a lysis. Dose-response relationships were used
to quantitate drug effects. These were constructed by linearly
regressing the percent inhibition of parameters derived in the
preceding sections against log drug concentrations. The 50%
inhibitory (IC50) concentrations were calculated from the
regression lines. Ganciclovir (GCV) was used as a positive
control in all antiviral assays.
In Vitr o An tip r olifer a tive Stu d ies. The in vitro cyto-
toxicity against L1210 was determined in an L1210 cell growth
assay described previously.16 L1210 murine leukemic cells
were grown in Fischer's medium supplemented with 10% heat-
inactivated (56 °C, 30 min) horse serum and were subcultured
by serial dilution. Growth rates were calculated from deter-
minations of the number of cells at 0, 24, 48, 72, and 96 h in
the presence of selected concentrations of the test compound.
The IC50 was defined as the concentration required to decrease
the growth rate to 50% of the untreated control rate. Growth
inhibition was calculated as the slope of a semilogarithmic plot
4,6-Dia m in o-7-p r op ylp yr r olo[2,3-d ]p yr im id in e-5-ca r -
bon itr ile (22i). Compound 22h (300 mg, 1.41 mmol) was
dissolved in 100 mL of EtOAc/EtOH (2:1, v:v), and 10% Pd/C
(10% by wt, 30 mg) was added. The reaction mixture was
hydrogenated at 50 psi for 1 h and the resultant red solution
filtered through Celite. The filtrate was evaporated and the
crude solid recrystallized from water and charcoal to furnish
170 mg (57%) of 22i: 222-225 °C; 1H NMR (DMSO-d6) δ 7.98
(1H, s, C-2), 7.13 (2H, s, 4-NH2), 6.02 (2H, s, 6-NH2), 3.96 (2H,
t, NCH2), 1.60 (2H, sextet, CH2), 0.82 (3H, t, CH3). Anal.
(C10H12N6) C, H, N.
Gen er a l Meth od C for th e Syn th esis of Com p ou n d s
23a -c: 4,6-Dia m in o-7-(eth oxym eth yl)p yr r olo[2,3-d ]p y-
r im id in e-5-ca r boxa m id e (23a ). NH2OH hydrochloride (382
mg, 5.5 mmol) and NaOMe (297 mg, 5.5 mmol) were suspended
in EtOH (40 mL), and the suspension was stirred for 30 min.
Compound 22a (250 mg, 1.1 mmol) and 10 mL of distilled H2O
were then added. This mixture was heated at reflux for 9 h
and then evaporated to dryness. The resulting solid was
suspended in MeOH and allowed to stand at 4 °C for 16 h.
The solid was collected by filtration and recrystallized from
aqueous EtOH to furnish pure 23a (67 mg, 24%): mp 176 °C;
1H NMR (DMSO-d6) δ 7.92 (1H, s, H-2), 7.42, 6.97, and 6.22
(2H each, br s, 2 × NH2, CONH2), 5.48 (2H, s, NCH2), 3.42-
3.49 (2H, q, CH2), 1.04-1.09 (3H, t, CH3). Anal. (C10H14N6O2)
C, H, N.
Gen er a l Meth od D for th e Syn th esis of Com p ou n d s
24a -c: 4,6-Dia m in o-7-(eth oxym eth yl)p yr r olo[2,3-d ]p y-
r im id in e-5-th ioca r boxa m id e (24a ). NaOMe (194 mg, 3.6
mmol) in dry MeOH (60 mL) was saturated with H2S(g) for
30 min. This solution was transferred to a steel vessel
containing 22a (400 mg, 1.8 mmol) which was sealed and
heated to 100 °C in an oil bath for 16 h. After this time the
solution was allowed to cool to room temperature and the pH
adjusted to 7 with 1 N HCl (2.5 mL). The resulting solution
was evaporated to dryness and recrystallized from H2O/MeOH
and decolorizing charcoal to furnish pure 24a (204 mg, 43%):
mp 209-210 °C; UV λmax [nm (ꢀ, mM)] (pH 1) 366 (13.6), 277
(15.5), (MeOH) 364 (11.7), 279 (14.6), (pH 11) 360 (12.3), 269
(17.4); 1H NMR (DMSO-d6) δ 8.15 and 8.02 (2H each, br s,
4-NH2, CSNH2, D2O exchangeable), 8.06 (1H, s, H-2), 6.39 (2H,

3476 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Renau et al.
(7) Hinshaw, B. C.; Leonoudakis, O.; Schram, K.; Townsend, L. B.
Pyrrolopyrimidine nucleosides. Part X. Synthesis of certain 4,5-
disubstituted 7-(â-D-ribofuranosyl)-pyrrolo[2,3-d]pyrimidines re-
lated to toyocamycin and sangivamycin. J . Chem. Soc., Perkin
Trans. I 1975, 1248-1253.
of cell number against time for the treated culture as a percent
of the control.
Ack n ow led gm en t. We thank Dr. Linda Wotring
and J ames Lee for conducting the antitumor portion of
this work and Cathy Yeung for performing the cytotox-
icity assays with labeled precursors. These studies were
supported by National Institute of Allergy and Infec-
tious Diseases Research Contract N01-AI72641 and
Grant U19-AI31718 for a National Cooperative Drug
Discovery Group for Infectious Diseases, by American
Cancer Society Research Grant No. DHP-36, and by
research funds from the University of Michigan.
(8) Hinshaw, B. C.; Gerster, J . F.; Robins, R. K.; Townsend, L. B.
Pyrrolopyrimidine nucleosides. V.
A study on the relative
chemical reactivity of the 5-cyano group of the nucleoside
antibiotic toyocamycin and desaminotoyocamycin. The synthesis
of analogs of sangivamycin. J . Org. Chem. 1970, 35, 236-241.
(9) Swayze, E. E.; Shannon, W. M.; Buckheit, R. W.; Wotring, L.
L.; Drach, J . C.; Townsend, L. B. Synthesis, antiproliferative and
antiviral evaluation of certain acyclic 6-substituted pyrrolo[2,3-
d]pyrimidine nucleoside analogs related to sangivamycin and
toyocamycin. Nucleosides Nucleotides 1992, 11, 1507-1527.
(10) Renau, T. E.; Lee, J . S.; Kim, H.; Young, C. G.; Wotring, L. L.;
Townsend, L. B.; Drach, J . C. Relationship between cytotoxicity
and conversion of thiosangivamycin analogs to toyocamycin
analogs in cell culture medium. Biochem. Pharmacol. 1994, 48,
801-807.
Refer en ces
(1) Renau, T. E.; Wotring, L. L.; Drach, J . C.; Townsend, L. B.
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