Synthesis of non-nucleoside analogs of toyocamycin, sangivamycin, and thiosangivamycin: Influence of various 7-substituents on antiviral activity

Article abstract of DOI:10.1021/jm950444j

A number of 7-substituted 4-aminopyrrolo[2,3-d]pyrimidine-5-carbonitrile, -5-carboxamide, and -5-thiocarboxamide derivatives related to the nucleoside antibiotics toyocamycin and sangivamycin were prepared and tested for their activity against human cytom

Full text of DOI:10.1021/jm950444j

J . Med. Chem. 1996, 39, 873-880
873
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 : In flu en ce of Va r iou s 7-Su bstitu en ts on An tivir a l Activity
Thomas E. Renau,† Linda L. Wotring, J ohn C. Drach, and Leroy B. Townsend*
Departments of Medicinal and Pharmaceutical 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 J une 15, 1995X
A number of 7-substituted 4-aminopyrrolo[2,3-d]pyrimidine-5-carbonitrile, -5-carboxamide, and
-5-thiocarboxamide derivatives related to the nucleoside antibiotics toyocamycin and sangi-
vamycin were prepared and tested for their activity against human cytomegalovirus (HCMV)
and herpes simplex virus type-1 (HSV-1). Treatment of 2-amino-5-bromo-3,4-dicyanopyrrole
(1) with triethyl orthoformate followed by alkylation via the sodium salt method with a variety
of alkylating agents furnished the corresponding 1-substituted pyrroles 2a -k . Ring annulation
was achieved with methanolic ammonia affording the 7-substituted 4-amino-6-bromopyrrolo-
[2,3-d]pyrimidine-5-carbonitrile derivatives 3a -k . Debromination of 3a -k , via catalytic
hydrogenation, gave the corresponding 7-substituted 4-aminopyrrolo[2,3-d]pyrimidine-5-
carbonitrile analogs 4a -j,l. A selective reduction of 4-amino-6-bromo-7-allylpyrrolo[2,3-d]-
pyrimidine-5-carbonitrile (3k ) in zinc and acetic acid furnished 4-amino-7-allylpyrrolo-
[2,3-d]pyrimidine-5-carbonitrile (4k ). Conventional functional group transformations invol-
ving the 5-cyano group of 4 furnished the 5-carboxamide derivatives 5a -l and the 5-thio-
amide analogs 6a -l. A similar transformation of the aglycone of toyocamycin (4m ) furnished
the corresponding aglycone of thiosangivamycin (6m ). Several of the new compounds
(4-6a -e,j-l) were evaluated for their ability to inhibit the growth of L1210 murine leukemic
cells. Whereas a number of the carboxamide (5) and thioamide (6) derivatives had modest
activity, the corresponding nitrile analogs (4) were all inactive. All compounds were tested for
activity against HCMV and HSV-1. The non-nucleoside nitrile analogs 4a -m and carboxamide
derivatives 5a -l were, with a few exceptions, essentially inactive against HCMV and HSV-1
and relatively nontoxic. In direct contrast, nearly all of the thioamide derivatives 6a -l,
including the aglycone of thiosangivamycin (6m ), were good inhibitors of HCMV and HSV-1.
Most were noncytotoxic in their antiviral concentration range. Cytotoxicity which was observed
appeared to be a consequence of DNA synthesis inhibition. Several of these compounds, such
as 6b,e, were particularly interesting inhibitors of HCMV with IC50’s ranging from 0.1 to 1.3
µM. The antiviral activity of both compounds was well separated from cytotoxicity in KB,
HFF, and L1210 cells.
In tr od u ction
compounds to treat HCMV infections which might
circumvent the problems associated with the use of GCV
and PFA.
Human cytomegalovirus (HCMV) infection is rela-
tively benign in immunocompetent individuals but can
be debilitating and often life or sight threatening to
immunosuppressed individuals such as organ trans-
plant recipients1,2 and AIDS patients.3,4 Moreover,
HCMV has been implicated as a potential cofactor in
the progression of human immunodeficiency virus (HIV)
infection5,6 and in coronary restenosis.7 Hence, the
ability to control replication of HCMV not only is critical
in patients with active HCMV infections but also may
be important in individuals afflicted by other life-
threatening diseases.
Prior work in our laboratory has examined analogs
of the pyrrolo[2,3-d]pyrimidine nucleosides toyocamycin
(I), sangivamycin (II), and thiosangivamycin (III) as
potential inhibitors of HCMV.18 The parent nucleosides
possess significant activity against HCMV19,20 but are
highly toxic to mammalian cells.18,20,21 It is generally
The drugs currently approved by the Food and Drug
Administration for the treatment of HCMV are ganci-
clovir (GCV) and foscarnet (PFA).8-12 Both drugs,
however, suffer from poor oral bioavailability, and their
clinical use is limited because of host toxicity.8,9,13,14 In
addition, there have been recent reports that clinical
strains of HCMV resistant to both drugs are emerg-
ing.15-17 Hence, there is a continued need to identify
believed that toxicity in uninfected cells arises because
the nucleosides are phosphorylated by cellular adeno-
sine kinase22 to afford their 5′-monophosphate deriva-
tives which ultimately are incorporated into DNA or
RNA.23-26 Our recent studies with the 5′-deoxy analog
† Present address: Parke-Davis Pharmaceutical Research, Division
of Warner-Lambert Co., Ann Arbor, MI 48105.
X Abstract published in Advance ACS Abstracts, December 1, 1995.
0022-2623/96/1839-0873$12.00/0 © 1996 American Chemical Society

874 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
Non-nucleoside Analogs of Toyocamycin and Sangivamycin
bromo-3,4-dicyanopyrrole39 (1). Treatment of 1 with
triethyl orthoformate followed by the addition of sodium
hydride and 1 equiv of the appropriate alkylating agent
gave the intermediates 2a -k . The major advantage of
alkylating a pyrrole precursor rather than the pyr-
rolopyrimidine aglycone is that the site of alkylation is
fixed thereby eliminating the possibility of producing
an isomeric mixture of alkylated products.40 Further-
more, we have found this approach28 to be amenable to
the multigram synthesis of the key intermediates 3 and
4. Reaction of 2a -k with methanolic ammonia afforded
the 7-substituted 4-amino-6-bromopyrrolo[2,3-d]pyri-
midine-5-carbonitriles 3a -k . The site of alkylation at
N-7 was confirmed by a comparison of the UV spectra
of a representative compound (3a ) to the corresponding
nucleoside, 4-amino-6-bromo-7-(â-D-ribofuranosyl)pyr-
rolo[2,3-d]pyrimidine-5-carbonitrile, synthesized by an
alternate method.41
Sch em e 1. Synthesis of 7-Substituted
4-Aminopyrrolo[2,3-d]pyrimidine Analogs Related to
Toyocamycin, Sangivamycin, and Thiosangivamycina
a
(i) (1) CH(OEt)3, CH3CN, (2) NaH, RX, CH3CN; (ii) NH3/
MeOH; (iii) H2, Pd/C, EtOAc/EtOH or Zn/AcOH (see text); (iv)
NH4OH/H2O/EtOH, H2O2; (v) MeOH, H2S/NaOMe. a ,
R )
CH2OCH2CH3; b, R ) CH2O(CH2)2OCH3; c, R ) CH2OCH2C6H5;
d , R ) CH2C6H5; e, R ) CH2C6H4-4-CH3; f, R ) CH2C6H4-3-CH3;
g, R ) CH2C6H4-2-CH3; h , R ) CH2C6H4-4-t-Bu; i, R ) CH2C6H4-
4-OCH3; j, R ) Me; k , R ) CH2CHdCH2; l, R ) CH2CH2CH3; m ,
R ) H.
Various attempts at alkylating the ethoxymethylene
derivative of 1 with propyl iodide proved unsuccessful.
Since the next step in the reaction scheme involved a
catalytic reduction, we felt that the allyl group could
substitute at this point since it would probably be
coreduced to furnish the debrominated, propyl-substi-
tuted derivative 4l. Indeed 4l was obtained from 3k
via catalytic hydrogenation. Treatment of 3a -j, in a
similar fashion, furnished the debrominated derivatives
4a -j. Attempts at selectively reducing the bromo group
of 3k without effecting a reduction of the allyl side chain
were examined. Under mild hydrogenation conditions
with 5% Pd catalyst for 30 min, two compounds were
isolated after silica gel chromatography. While one of
these was the fully reduced 4l, the other was 4-amino-
6-bromo-7-propylpyrrolo[2,3-d]pyrimidine-5-carboni-
trile. These results established that reduction of the
allyl side chain of 3k via catalytic hydrogenation oc-
curred prior to a reduction of the bromo group. Selective
reduction of 3k was accomplished in zinc and acetic acid
affording 4-amino-7-allylpyrrolo[2,3-d]pyrimidine-5-car-
bonitrile (4k ) in a yield of 75%. The IR spectrum of 4k
demonstrated that there had been no modification of
of III showed, however, that this analog also was
cytotoxic thereby establishing that phosphorylation of
the 5′-position is not necessary to produce cytotoxicity.27
In other previous studies of sugar-modified analogs
of I-III,18,21,28-31 we reported that acyclic analogs of III
possessed good activity against HCMV coupled with a
significant reduction in cytotoxicity;21,28,29 e.g., 4-amino-
7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine-5-
thiocarboxamide (IV) and 4-amino-7-[(1,3-dihydroxy-2-
p r op ox y )m e t h y l ]p y r r ol o[2 , 3 -d ]p y r i m i d i n e -5 -
thiocarboxamide (V). In contrast, similar acyclic analogs
of I and II also were nontoxic but inactive against
HCMV.21,29 Together, these results suggested that the
structural requirements of the substituent at N-7 (R7)
may have less importance than the thioamide moiety
at C-5 (R5) for activity against HCMV. If this were the
case, one might expect that activation of a side chain
hydroxyl group may not be essential for biological
activity. In fact, recent studies have established that
many non-nucleoside analogs have antiviral activity.32-38
To test this hypothesis, several model compounds were
prepared where the substituent at N-7, such as a
methyl, allyl, or propyl group, could not be phosphory-
lated.20 The data supported the hypothesis and estab-
lished that the 5-thioamide moiety confers antiviral
activity without the requirement for phosphorylation of
a side chain hydroxyl group. However, the selectivity
of these model compounds was essentially the same
when compared to the acyclic analog IV or V.
the CN since a peak was observed at 2250 cm-1
.
The carboxamides 5a -l were synthesized from the
appropriate compound 4 in yields ranging from 55% to
93% using 30% hydrogen peroxide in aqueous base. For
this step, absolute ethanol was typically added to aid
in dissolving the nitrile derivatives. The thiosangiva-
mycin analogs 6a -l were prepared in modest yields by
a reaction of the appropriate nitrile 4 with sodium
hydrogen sulfide generated in situ from methanolic
sodium methoxide and hydrogen sulfide at 95 °C in a
pressure bottle. Treatment, in a similar manner, of the
aglycone of toyocamycin42 (4m ) furnished the aglycone
of thiosangivamycin (6m ) in 56% yield. IR and UV
spectra of a representative compound (6a ) confirmed the
successful transformation of the nitrile to the thioamide.
The IR spectrum of 6a demonstrated the absence of a
CN peak, and the UV spectrum of 6a was essentially
identical with that of thiosangivamycin.43 Synthetic
and physical data for compounds 3-6 are recorded in
Table 1.
In the present study we have expanded this work and
now report the synthesis of a diverse class of non-
nucleoside 7-substituted 4-aminopyrrolo[2,3-d]pyrimi-
dine-5-carbonitriles, -5-carboxamides, and -5-thiocar-
boxamides. 7-Substituents such as arylalkyl and alkyl
ether groups have been used to prepare a variety of
compounds which along with selected aglycones have
been evaluated for cytotoxicity and activity against two
herpes viruses.
Ch em istr y
Biologica l Resu lts a n d Discu ssion
The synthetic route used to prepare the N-7-substi-
tuted non-nucleoside analogs is illustrated in Scheme
1. Compounds 3a -k were prepared from 2-amino-5-
In Vitr o An tip r olifer a tive Testin g. A number of
the non-nucleoside pyrrolopyrimidines were evaluated

Renau et al.
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 875
Ta ble 1. Synthetic and Physical Data of the 5,7-Disubstituted 4-Aminopyrrolo[2,3-d]pyrimidines Prepared for This Study
NMR data (δ, DMSO)
compd
method of prepa
yield (%)
mp (°C)
formula (C,H,N)
H-2
H-6
3a
3b
3c
3d
3e
3f
3g
3h
3i
A
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
B
B
b
B
C
C
C
C
C
C
C
C
C
C
C
C
D
D
D
D
D
D
D
D
D
D
D
D
D
37
30
54
43
51
29
24
9
222-223
159-160
205-208
259-262
253-255
200-201
294-295
275-276
264 dec
C10H10BrN5O
8.23
8.23
8.25
8.23
8.23
8.23
8.18
8.23
8.24
8.13
8.20
8.34
8.34
8.37
8.31
8.29
8.30
8.21
8.32
8.29
8.21
8.21
8.24
8.10
8.11
8.13
8.18
8.09
8.11
8.09
8.09
8.10
8.07
8.07
8.06
8.14
8.14
8.17
8.13
8.13
8.14
8.13
8.13
8.13
8.11
8.11
8.10
8.06
C
C
C
C
C
C
C
C
11H12BrN5O2
15H12BrN5O‚H2O
14H10BrN5
15H12BrN5
15H12BrN5
15H12BrN5
18H18BrN5
15H12BrN5O
37
42
23
73
75
67
56
81
69
41
22
20
51
75
60
71
86
93
70
82
84
66
73
55
62
79
92
93
64
80
78
87
70
88
53
63
44
59
70
56
3j
275-280
243-245
175-176
169-171
170-172
218-219
244-245
238-239
256-259
242-244
220-221
275-276
188-189
178-180
222-223
184-186
231-233
255-256
273-276
285-287
265-267
165-167
259-260
300-302
244-245
236-239
207-209
150-152
180-182
192-195
225-227
225-226
248-250
145-147
222-224
250-252
201-202
186-189
>340
C8H6BrN5
3k
4a
4b
4c
4d
4e
4f
4g
4h
4i
C
C
C
C
C
C
C
C
C
C
10H8BrN5
10H11N5O
11H13N5O2
15H13N5O
14H11N5
15H13N5
15H13N5
15H13N5‚0.5H2O
18H19N5
8.24
8.24
8.26
8.22
8.22
8.26
8.16
8.23
8.22
8.16
8.18
8.20
8.07
8.07
8.12
8.10
7.97
7.99
7.91
7.99
7.96
7.91
7.94
7.97
7.85
7.85
7.91
7.93
7.83
7.85
7.72
7.85
7.82
7.75
7.76
7.80
7.71
15H13N5O
4j
4k
4l
C8H7N5
C
C
C
C
C
C
C
C
C
C
C
10H9N5‚0.25H2O
10H11N5
5a
5b
5c
5d
5e
5f
5g
5h
5i
10H13N5O2
11H15N5O3
15H15N5O2‚0.25H2O
14H13N5O
15H15N5O
15H15N5O‚0.75H2O
15H15N5O‚0.25H2O
18H21N5O‚0.25H2O
15H15N5O2‚0.25H2O
5j
5k
5l
C8H9N5O
C
C
C
C
C
C
C
C
C
C
C
10H11N5O
10H13N5O
10H13N5OS‚0.25H2O
11H15N5O2S
15H15N5OS
14H13N5S
15H15N5S
15H15N5S‚0.5H2O
15H15N5S
6a
6b
6c
6d
6e
6f
6g
6h
6i
18H21N5S
15H15N5OS‚0.5H2O
6j
6k
6l
C8H9N5S
C
C
10H11N5S
10H13N5S
6m
C7H7N5S
a
b
See the Experimental Section for methods A-D. See the Experimental Section for the preparation of 4k .
as potential antitumor agents by determining their
effect on the growth of L1210 murine leukemic cells in
vitro. As shown in Table 2, none of the nitrile deriva-
tives (4) caused any significant inhibition. In contrast,
several of the carboxamide (5) and thioamide (6) deriva-
tives had modest inhibitory activity. Specifically, the
benzyl-substituted carboxamide analogs 5d ,e gave IC50
values of 16 and 20 µM, respectively. The corresponding
thioamide derivatives 6d ,e were less active. The ether
derivative 5c and propyl analog 5l had slight growth
inhibitory effects with IC50’s equal to 70 µM. Of the
thiocarboxamide derivatives, the 7-methyl (6j)- and
7-allyl (6k )-substituted analogs were the most active
with IC50’s of ca. 40 µM. Interestingly, the propyl
derivative 6l was inactive. Although a number of the
carboxamide- and thiocarboxamide-substituted analogs
had modest antiproliferative effects, all of the com-
pounds examined were significantly less active than the
corresponding ribonucleosides which had IC50 values in
L1210 cells ranging from 3-4 nM for toyocamycin (I)
and sangivamycin (II) to 23 nM for thiosangivamycin
(III).44
In Vitr o An tivir a l Activity. Compounds 4a -m ,
5a -l, and 6a -m were evaluated for activity against
HCMV and herpes simplex virus type-1 (HSV-1) repli-
cation. Cytotoxicity and inhibition of cell growth by
each compound were determined by several techniques
in both growing and stationary cells. The results are
presented in Table 3. Antiviral and cytotoxicity data
for the ribosyl-, (hydroxyethoxy)methyl-, and (dihy-
droxypropoxy)methyl-substituted pyrrolo[2,3-d]pyrim-
idines have been presented before20,21,29 and are in-
cluded here for comparison. The data confirm that the
major factor required for the antiviral activity of these
7-substituted 4-aminopyrrolo[2,3-d]pyrimidines is the
thioamide moiety at the 5-position (R5) and that the
hydroxyl group on the side chain of IV or V is not
necessarily required for the compound to have activity
against either HSV-1 or HCMV. Like the acyclic

876 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
Non-nucleoside Analogs of Toyocamycin and Sangivamycin
Ta ble 2. Cell Growth Inhibitory Activity of Several
7-Substituted 4-Aminopyrrolo[2,3-d]pyrimidines against L1210
Murine Leukemic Cells in Vitro
poration of labeled precursors into protein, RNA, and
DNA of CEM cells substantiated the cytotoxicity data
in Table 3. IC50’s for incorporation of [3H]Leu, [3H]Urd,
and [3H]dThd were >200 µM for the three compounds
except that the IC50 for inhibition of [3H]dThd incorpo-
ration by 6a was 27 µM. This suggests that the weak
effect on KB cell growth (Table 3) could result from
inhibition of cellular DNA synthesis. Of the three
compounds, 4-amino-7-[(2-methoxyethoxy)methyl]pyr-
rolo[2,3-d]pyrimidine-5-thiocarboxamide (6b) had good
activity against HCMV in both the plaque reduction
assay and the ELISA, and this activity was well
separated from toxicity to uninfected cells as shown in
Table 3. Also, IC50’s for incorporation of labeled precur-
sors were g400 µM for effects on protein, RNA, and
DNA synthesis in CEM cells. 6b and the other non-
phosphorylatable compounds also were as active but
considerably less cytotoxic than the 5′-deoxyribosyl
analog of thiosangivamycin (III) which is both active
and cytotoxic.27 Thus failure to be phosphorylated at
the 5′-position is not sufficient to ensure lack of cyto-
toxicity. Furthermore, 6b was more potent and less
toxic than the acyclic analogs IV and V thereby em-
phasizing that an acylic group alone is not sufficient to
reduce or eliminate cytotoxicity.
initial screen:a
growth rate, % of control
compd
IC50 (µM)b
4a
4b
4c
4d
4e
4j
76
88
83
60
64
87
75
74
91
92
0
0
2
89
66
0
4k
4l
5a
5b
5c
5d
5e
5j
70
16
20
5k
5l
73
6a
6b
6c
6d
6e
6j
111
88
52
0
92
0
100
70
>10c
38
6k
6l
0
79
38
a
The effect of each compound at 100 µM on the growth rate of
Although in most cases the N-7 benzyl-substituted
5-thioamide derivatives 6d -i were more potent inhibi-
tors of HCMV than 6a -c, they were invariably more
toxic than the ether-substituted analogs. Nonetheless
the selectivity ratios for a number of these analogs are
quite good. For example, the ratio of cytotoxicity to
activity against HCMV for the unsubstituted benzyl
derivative 6d is ca. 100 compared to 140 for the ether
analog 6b (Table 3). 6d also had only minor effects on
incorporation of [3H]Leu, [3H]Urd, and [3H]dThd (IC50’s
) 130-195 µM) in CEM cells providing additional
evidence for low cytotoxicity. The best separation
between activity and toxicity was observed with 6e
(ratio > 650; see Table 3). IC50’s for incorporation of
labeled precursors were >400 µM for [3H]Leu and [3H]-
Urd and equal to 67 µM for [3H]dThd. These results
are consistent with those in Table 3 and suggest the
observed cytotoxicity could be from effects on DNA
synthesis. There was essentially no difference in anti-
viral activity between a substituted or unsubstituted
phenyl ring. For example, the 4-methoxybenzyl deriva-
tive 6i and the methyl-substituted derivatives 6e-g
were as potent as the unsubstituted benzyl analog 6d .
Changing the site of substitution on the phenyl ring had
similar inconclusive results; eg., 6e-g were equally
active whether the CH3 group was substituted at the
para, meta, or ortho position. Interestingly, the agly-
cone 6m had a similar biological profile as the alkyl-
substituted analogs 6j-l. The antiviral activity of the
latter compounds was not well separated from their
toxicity to uninfected cells.
b
L1210 cells. IC50 is the concentration required to decrease the
growth rate to one-half of the control rate. c Highest concentration
tested.
analogs,21,29 the non-nucleoside analogs of toyocamycin
(4a -m ) and sangivamycin (5a -l) were, with a few
exceptions, nontoxic to cells and relatively inactive
against HCMV and HSV-1. The 2-methylbenzyl deriva-
tives 4g and 5g were notable exceptions with IC50’s in
the HCMV plaque reduction assay of 3.4 and 15 µM,
respectively. There was relatively little cytotoxicity in
the antiviral dose range for each compound in the two
cell lines examined. Compound 4g, however, was less
potent in an HCMV enzyme-linked immunosorbent
assay (ELISA; IC50 ) 32 µM). Although the tert-
butylbenzyl-substituted analogs 4h and 5h as well as
the 4-methoxybenzyl-substituted carboxamide deriva-
tive 5i were slightly active against HCMV, they were
also more toxic than 4g and 5g to uninfected cells. The
aglycone of toyocamycin (4m ) was also active against
HCMV with an IC50 ) 3.9 µM; however, like a number
of the benzyl-substituted analogs with activity, the
antiviral activity for 4m was not well separated from
the toxicity in uninfected cells.
In contrast to the toyocamycin and sangivamycin non-
nucleoside derivatives, the non-nucleoside thiosangiva-
mycin analogs 6a -m were potent inhibitors of HCMV
and HSV-1 (Table 3). Similar results were obtained for
a number of key compounds (6a -e,i) in the HCMV
ELISA. In most cases, the potency of 6a -m against
HCMV was greater than that observed for IV or V, but
the toxicity in uninfected cells also was greater. The
separation of cytotoxicity from antiviral activity, though,
was greater for 6a -m when compared to that of
thiosangivamycin (III). Although GCV was less active
than the thioamide-substituted analogs in both the
plaque reduction assay and ELISA, it was also less toxic
in HFF and KB cells. Compounds 6a -c, containing an
acyclic ether side chain, were the least toxic of the
thioamide-substituted derivatives studied. More exten-
sive examination of the cytotoxicity of 6a -c by incor-
Taken together, the results from the 5-thioamide-
substituted analogs demonstrate that although the
substituent at N-7 is less important than the require-
ment for a thioamide moiety at C-5 for activity against
HCMV, the N-7 substituent does act to modulate the
toxicity of the compounds.
These results which establish that only the 5-thio-
amide analogs 6a -m are active against HCMV and
HSV-1 are interesting in light of our other recent

Renau et al.
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 877
Ta ble 3. Antiviral Activity and Cytotoxicity of 5,7-Disubstituted 4-Aminopyrrolo[2,3-d]pyrimidines
IC50 (µM)a,b
antiviral activity
HCMV
cytotoxicity
HFF
substituent
HSV-1
ELISA
KB
compd
R5
CN
R7
plaque
ELISA
visual
growth
4a
4b
4c
4d
4e
4f
4g
4h
4i
CH2OCH2CH3
CH2O(CH2)2OCH3
CH2OCH2C6H5
CH2C6H5
CH2C6H4-4-CH3
CH2C6H4-3-CH3
CH2C6H4-2-CH3
CH2C6H4-4-t-Bu
CH2C6H4-4-OCH3
CH3
CH2CHdCH2
CH2CH2CH3
H
CH2OCH2CH3
CH2O(CH2)2OCH3
CH2OCH2C6H5
CH2C6H5
CH2C6H4-4-CH3
CH2C6H4-3-CH3
CH2C6H4-2-CH3
CH2C6H4-4-t-Bu
CH2C6H4-4-OCH3
CH3
CH2CHdCH2
CH2CH2CH3
CH2OCH2CH3
CH2O(CH2)2OCH3
CH2OCH2C6H5
CH2C6H5
CH2C6H4-4-CH3
CH2C6H4-3-CH3
CH2C6H4-2-CH3
CH2C6H4-4-t-Bu
CH2C6H4-4-OCH3
CH3
CH2CHdCH2
CH2CH2CH3
H
>100
90
>100
>100
>100
90
>100
>100
>100
>100
>100
>100
>100
>32
>100
>100
>100
>100
10
>100
>100
>100
>32
>100
>100
>100
100
>100
>100
>100
>100
>100
>100
>100
3
>100
>100
>100
>100
18
>100
>100
>100
75
>100
>100
>100
0.9
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CSNH2
CSNH2
CSNH2
CSNH2
CSNH2
CSNH2
CSNH2
CSNH2
CSNH2
CSNH2
CSNH2
CSNH2
CSNH2
>100
>100
>100
>100
3.4
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
33
32
32
>100
>100
>100
36
4jc
4k c
4lc
4m
5a
5b
5c
5d
5e
5f
>100
3.9
>100
>100
>100
>100
>100
>100
15
28
13
>100
>100
>100
2.7
>100
>100
>100
>100
50
5g
5h
5i
35
>32
47
>100
100
100
68
183
100
5jc
5k c
5lc
6a
6b
6c
6d
6e
6f
>100
>100
>100
40
>100
>100
>100
66
>100
7
1.0
2.6
0.36
0.1
0.25
0.1
1.5
0.5
1.2
2.1
1.2
1.6
7.4
86
0.05
0.03
0.2
8.8
6.2
1.9
1.8
0.33
0.15
85
10
7.4
3.5
15
0.9
15
4
2.8
4.5
12
35
3.5
1.9
>100
>100
>32
100
66
32
65
30
6g
6h
6i
66
>17
>100
7
0.5
66
28
15
19
17
6jc
6k c
6lc
6m
25
>32
>100
>100
<0.01
<0.01
0.2
25
ganciclovir (GCV)
22
>100
>100
0.03
0.08
0.03
115
acyclovir (ACV)
Id
CN
â-D-ribofuranose
â-D-ribofuranose
â-D-ribofuranose
CH2OCH2CH2OH
CH2OCH(CH2OH)2
IId
IIIc
IVd
Ve
CONH2
CSNH2
CSNH2
CSNH2
21
>100
>100
>100
>100
a
b
Results are the average of two or more experiments with most assays except for HSV-1 ELISA’s which were done once. A greater
than sign (>) indicates IC50 not reached at highest concentration tested. Data also reported in c ref 20, ref 21, and e ref 29.
d
observations.44 We have found that 7-substituted 4-ami-
nopyrrolo[2,3-d]pyrimidine-5-thiocarboxamides, such as
6b,d , are converted to their corresponding 5-car-
bonitrile derivatives in vitro. Although we did not
examine all of the thioamides in the present study for
this phenomenon, we expect they would undergo this
conversion in cell culture media. Hence, the biological
data for compounds 6a -m may be the result of a mix-
ture of 5-thioamide and 5-carbonitrile derivatives. Be-
cause the nitrile analogs 4a -m are inactive (except for
4g), the activity of 6a -m must be greater than we ob-
served if conversion from the 5-thioamide to the 5-nitrile
occurs. Clearly, the 5-thioamide pyrrolo[2,3-d]pyrim-
idines 6a -m have completely different biological prop-
erties when compared to the 5-nitrile analogs 4a -m .
In summary, the design, synthesis, and antiviral
evaluation of several non-nucleoside pyrrolo[2,3-d]-
pyrimidines were performed. The antiviral activity and
cytotoxicity of several compounds reported herein are
significant improvements over the biological properties
of the initial model compounds.20 The data reported in
the present study have unequivocally established that
the 5-thioamide moiety can provide selective antiviral
activity without the requirement for phosphorylation of
a side chain hydroxyl group.
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

878 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
Non-nucleoside Analogs of Toyocamycin and Sangivamycin
silica gel 60F-254 plates (Analtech, Inc.). Detection of com-
ponents on TLC was made by UV light absorption at 254 nm.
Ultraviolet (UV) spectra were recorded on a Kontron-Uvikon
860 spectrophotometer. Infrared (IR) spectra were taken on
a Perkin-Elmer 1310 infrared spectrophotometer. Nuclear
magnetic resonance (1H NMR) spectra were determined at 200,
300, or 360 MHz with a Bruker WP 200/300/360 SY spectrom-
eter. The chemical shift values are expressed in δ values
(parts per million) relative to the standard chemical shift of
the solvent (DMSO-d6). Elemental analyses were performed
by M-H-W Laboratories, 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. All
evaporations were carried out on a rotary evaporator under
reduced pressure (water aspirator) with the bath temperature
at 37 °C. Acetonitrile was dried over activated molecular
sieves (4 Å).
Gen er a l Meth od A for th e Syn th esis of 7-Su bstitu ted
4-Am in o-6-b r om op yr r olo[2,3-d ]p yr im id in e-5-ca r b on i-
t r ile Der iva t ives 3a -k : 4-Am in o-6-b r om o-7-(et h oxy-
m eth yl)p yr r olo[2,3-d ]p yr im id in e-5-ca r bon itr ile (3a ). A
mixture of 2-amino-5-bromo-3,4-dicyanopyrrole39 (1; 30.0 g, 141
mmol) and freshly distilled triethyl orthoformate (42.77 g, 282
mmol) in dry acetonitrile (500 mL) under argon was heated
at reflux for 2.5 h and cooled to room temperature, and the
solvent evaporated in vacuo. The resulting residue was
coevaporated with toluene (4 × 50 mL) and evaporated in
vacuo until a dry powder was obtained. The crude powder
was dissolved in dry acetonitrile (250 mL) and then treated
with sodium hydride (80%, w/w, 6.36 g, 212 mmol) at room
temperature. The mixture was stirred for 0.5 h; then 1 equiv
of chloromethyl ethyl ether (13.3 g, 141 mmol) was added. The
alkylation was followed closely by monitoring the reaction with
TLC. The reaction mixture was stirred at room temperature
for 30 min and then filtered. The filtrate was evaporated, and
the resulting oil (2a ) was transferred to a pressure bottle
containing a saturated solution of NH3 in MeOH (150 mL).
The bottle was sealed and the solution stirred for 24 h at room
temperature. The resulting suspension was cooled at 4 °C for
16 h and then filtered and dried for 16 h at 60 °C to yield
14.93 g (37%) of 3a . This compound was used without fur-
ther purification. A sample was recrystallized from H2O
containing a small amount of MeOH to yield pure 3a : mp
222-223 °C; UV λmax [nm (ꢀ, mM)] (pH 1) 282 (19.1), (MeOH)
284 (22.1), (pH 11) 284 (19.1); 1H NMR (DMSO-d6) δ 8.23 (1H,
s, H-2), 7.00 (2H, br s, NH2), 5.58 (2H, s, NCH2), 3.46-3.52
(2H, q, OCH2), 1.03-1.07 (3H, t, CH3). Anal. (C10H10BrN5O)
C, H, N.
solution was filtered and the filtrate adsorbed onto 500 mg of
silica gel and applied to a column prepacked with silica.
Compound 4k was eluted from the column with CHCl3/MeOH
(98:2, v:v) to give 60 mg (75%) of an off-yellow solid which was
recrystallized from H2O/MeOH: mp 188-189 °C; IR (KBr) ν
2250 (CN) cm-1; 1H NMR (DMSO-d6) δ 8.21 (1H, s, H-2), 8.18
(1H, s, H-6), 6.82 (2H, br s, NH2), 5.90-6.15 (1H, m, CH),
5.15-5.20 and 4.95-5.03 (1H each, dd, dCH2), 4.78-4.81 (2H,
d, NCH2). Anal. (C10H9N5‚0.25H2O) C, H, N.
Gen er a l Meth od C for th e Syn th esis of 7-Su bstitu ted
4-Am in op yr r olo[2,3-d ]p yr im id in e-5-ca r b oxa m id e De-
r iva tives 5a -l: 4-Am in o-7-(eth oxym eth yl)p yr r olo[2,3-d ]-
p yr im id in e-5-ca r boxa m id e (5a ). Compound 4a (200 mg,
0.9 mmol) was added to 10 mL of distilled water and dissolved
with NH4OH (10 mL, 38%) (or 1:1:1 H2O/NH4OH/EtOH, v:v).
Hydrogen peroxide (2 mL, 30%) was added to the solution, and
the reaction mixture was stirred at room temperature for 1 h,
at which time no starting material was present as determined
by TLC. The solution was evaporated in vacuo; the residue
was triturated with EtOH (2 × 10 mL) and recrystallized from
1
H2O/EtOH to yield 153 mg (71%) of 5a : mp 222-223 °C; H
NMR (DMSO-d6) δ 8.10 (1H, s, H-2), 8.07 (1H, s, H-6), 7.94
(2H, br s, NH2), 7.36 (2H, br s, CONH2), 5.51 (2H, s, NCH2),
3.42-3.48 (2H, q, OCH2), 1.04-1.08 (3H, t, CH3). Anal.
(C10H13N5O2) C, H, N.
Gen er a l Meth od D for th e Syn th esis of 7-Su bstitu ted
4-Am in opyr r olo[2,3-d]pyr im idin e-5-th iocar boxam ide De-
r iva t ives 6a -m : 4-Am in o-7-(et h oxym et h yl)p yr r olo-
[2,3-d ]p yr im id in e-5-th ioca r boxa m id e (6a ). Dry H2S was
passed through a solution of sodium methoxide (97 mg, 1.8
mmol) in dry methanol (20 mL) for 0.5 h. The nitrile 4a (200
mg, 0.9 mmol) was added in one portion, and the mixture was
stirred in a sealed pressure tube at 95 °C for 2 h. The resulting
solution was allowed to cool to room temperature and then
adjusted to pH 7 with 1 N HCl. The solvent was rotary
evaporated to dryness, and the resulting compound was
recrystallized from H2O containing a small amount of EtOH
to yield 210 mg (93%) of 6a : mp 207-209 °C; UV λmax [nm (ꢀ,
mM)] (pH 1) 241 (15.4), 294 (11.8), (MeOH) 242 (12.9), 289
(10.8), (pH 11) 242 (9.8), 279 (11.4); IR (KBr) ν no cyano
1
absorption; H NMR (DMSO-d6) δ 9.46 and 9.65 (1H each, s,
CSNH2), 8.14 (1H, s, H-2), 7.92 (2H, br s, NH2), 7.85 (1H, s,
H-6), 5.51 (2H, s, NCH2), 3.46-3.52 (2H, q, OCH2), 1.05-1.09
(3H, t, CH3). Anal. (C10H13N5OS‚0.25H2O) C, H, N.
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.45 L1210 murine leukemic cells
were grown in Fischer’s medium supplemented with 10% heat
inactivated (56 °C, 30 min) horse serum and subcultured by
serial dilution. Growth rates were calculated from determina-
tions 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 cells. Growth rate
was calculated as the slope of a semilogarithmic plot of cell
number against time for the treated culture as a percent of
the control.
An tivir a l Eva lu a tion : (a ) Cells a n d Vir u ses. Human
foreskin fibroblast (HFF) cells and MRC-5 cells, a human
embryonic lung cell line, were grown in minimal 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.19 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 Stinksi, University
of Iowa. The S-148 strain of HSV-1 was provided by Dr. T.
W. Schafer of Schering Corp. Stock preparations of HCMV
and HSV-1 were prepared as described elsewhere.19
Gen er a l Meth od B for th e Syn th esis of 7-Su bstitu ted
4-Am in op yr r olo[2,3-d ]p yr im id in e-5-ca r bon itr ile Der iva -
t ives 4a -j,l: 4-Am in o-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 (4a ). To a mixture containing
EtOAc/EtOH (600 mL, 2:1, v:v) was added 3a (14.9 g, 50.0
mmol) with 10% Pd/C (1.49 g, 10% by wt) and 1 N NaOH (50
mL, 50.0 mmol). The mixture was hydrogenated at 50 psi at
room temperature and the reaction closely monitored by TLC.
After 30 min the mixture was filtered and washed with hot
EtOAc (2 × 25 mL) and the filtrate evaporated to dryness.
The resulting solid was suspended in H2O/MeOH (450 mL, 3:1,
v:v) and heated to boiling. To this solution was added
decolorizing charcoal (1.5 g) which was filtered over Celite,
and the filtrate was cooled for 16 h at 4 °C. The resulting
solid was collected by filtration and dried for 16 h at 60 °C to
yield pure 4a (7.97 g, 73%): mp 175-176 °C; UV λmax [nm (ꢀ,
mM)] (pH 1) 233 (22.2), 272 (18.1), (MeOH) 278 (19.3), (pH
1
11) 277 (21.1); H NMR (DMSO-d6) δ 8.34 (1H, s, H-2), 8.24
(1H, s, H-6), 6.87 (2H, s, NH2), 5.53 (2H, s, NCH2), 3.42-3.52
(2H, q, OCH2), 1.01-1.08 (3H, t, CH3). Anal. (C10H11N5O) C,
H, N.
4-Am in o-7-a llylp yr r olo[2,3-d ]p yr im id in e-5-ca r b on i-
tr ile (4k ). Compound 3k (100 mg, 0.4 mmol) was dissolved
in AcOH (99.7%, 20 mL), and Zn dust (209 mg, 3.2 mmol) was
added in one portion. The reaction mixture was stirred for
1.5 h at room temperature and filtered and the filtrate
evaporated to dryness. The resulting solid was triturated with
toluene (2 × 10 mL) and dissolved in hot EtOAc (50 mL). This
(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

Renau et al.
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 879
(8) Shepp, D. H.; Dandliker, P. S.; de Miranda, P.; Burnette, T. C.;
Cederberg, D. M.; Kirk, L. E.; Meyers, J . D. Activity of 9-[2-
hydroxy-1-(hydroxymethyl)ethoxymethyl]guanine in the treat-
ment of cytomegalovirus pneumonia. Ann. Intern. Med. 1985,
103, 368-373.
(9) Collaborative DHPG treatment study group. Treatment of
serious cytomegalovirus infections with 9-(1,3-dihydroxy-2-pro-
poxymethyl)guanine in patients with AIDS and other immuno-
deficiencies. N. Engl. J . Med. 1986, 314, 801-805.
(10) Palestine, A. G.; Polis, M. A.; De Smet, M. D.; Baird, B. F.;
Falloon, J .; Kovacs, J . A.; Davey, R. T.; Zurlo, J . J .; Zunich, K.
M.; Davis, M.; Hubbard, L.; Brothers, R.; Ferris, F. L.; Chew,
E.; Davis, J . L.; Rubin, B. I.; Mellow, S. D.; Metcalf, J . A.;
Manischewitz, J .; Minor, J . R.; Nussenblatt, R. B.; Masur, H.;
Lane, H. C. A randomized, controlled trial of foscarnet in the
treatment of cytomegalovirus retinitis in patients with AIDS.
Ann. Intern. Med. 1991, 115, 665-673.
(11) Manischewitz, J . F.; Quinnan, G. V., J r.; Lane, H. C.; Wittek,
A. E. Synergistic effect of ganciclovir and foscarnet on cytome-
galovirus replication in vitro. Antimicrob. Agents Chemother.
1990, 34, 373-375.
(12) Mortality in patients with the acquired immunodeficiency
syndrome treated with either foscarnet or ganciclovir for cy-
tomegalovirus retinitis. N. Engl. J . Med. 1992, 326, 213-220.
(13) J acobson, M. A.; Gambertoglio, J . G.; Aweeka, F. T.; Causey, D.
M.; Portale, A. A. Foscarnet-induced hypocalcemia and effects
of foscarnet on calcium metabolism. J . Clin. Endocrinol. Metab.
1991, 72, 1130-1135.
(14) Deray, G.; Martinez, F.; Katlama, C.; Levaltier, B.; Beaufils, H.;
Danis, M.; Rozenheim, M.; Baumelou, A.; Dohin, E.; Gentilini,
M.; J acobs, C. Foscarnet nephrotoxicity: mechanism, incidence
and prevention. Am. J . Nephrol. 1989, 9, 316-321.
(15) Field, A. K.; Biron, K. K. "The end of innocence" revisited:
resistance of herpesviruses to antiviral drugs. Clin. Microbiol.
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(16) Erice, A.; Chou, S.; Biron, K. K.; Stanat, S. C.; Balfour, H. H.;
J ordan, M. C., J r. Progressive disease due to ganciclovir-
resistant cytomegalovirus in immunocompromised patients. N.
Engl. J . Med. 1989, 320, 289-293.
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. HSV-1 ELISA assays were performed
using a procedure described by Prichard and Shipman.46
HCMV was also assayed using an ELISA in MRC-5 cells
by the modification of a procedure previously described to
assay HSV-1.46 MRC-5 cells were incubated at 37 °C overnight
and the cells infected with HCMV (moi ) 0.002 PFU/cell).
Following a 1 h adsorption, up to 10 concentrations of drugs
were applied in triplicate. After a 6.5 day incubation, cells
were fixed with 95% EtOH. The ELISA was performed in the
wells containing the infected cell sheets. Wells were blocked
and then treated with a 1:400 dilution of monoclonal mouse
antibody to HCMV. After 1 h, a 1:1000 dilution of peroxidase-
conjugated rabbit anti-mouse antibody was added to each well,
the wells were incubated for 2 h, and plates were developed
and read at 450/570 nm in a microplate kinetics reader.
Background was subtracted using control wells.
(c) Cytotoxicity Assa ys. Two basic tests for cellular
toxicity were employed for compounds examined in antiviral
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 exponentially growing KB cells was
determined by a staining method previously described.47 For
selected compounds, cytotoxicity also was measured by deter-
mining incorporation of labeled precursors into protein, RNA,
and DNA of uninfected cells. Exponentially growing CEM cells
were incubated with 1 µCi/mL [3H]Leu, [3H]Urd, or [3H]dThd,
and the amount of incorporation into acid-precipitable material
was determined as described previously.19
(17) Sullivan, V.; Coen, D. M. Isolation of foscarnet-resistant human
cytomegalovirus patterns of resistance and sensitivity to other
antiviral drugs. J . Infect. Dis. 1991, 164, 781-784.
(d ) Da ta An a lysis. Dose-response relationships were used
to quantify 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) and acyclovir (ACV) were
used as positive controls in HCMV and HSV-1 assays, respec-
tively.
(18) Townsend, L. B.; Drach, J . C.; Wotring, L. L.; Vittori, S.; Pudlo,
J . S.; Swayze, E. E.; Gupta, P.; Maruyama, T.; Saxena, N.;
Coleman, L. A.; Westerman, A. C.; Spurr, J .; Nassiri, M. R.;
Turk, S. R.; Krawczyk, S. H. Design, synthesis and studies of
the structure activity relationships of certain pyrrolo[2,3-d]-
pyrimidine nucleosides and structurally related analogs as
potential antineoplastic and antiviral agents. Farmaco 1991,
46, 113-139.
(19) Turk, S. R.; Shipman, C., J r.; Nassiri, M. R.; Genzlinger, G.;
Krawczyk, S. H.; Townsend, L. B.; Drach, J . C. Pyrrolo[2,3-d]-
pyrimidine nucleosides as inhibitors of human cytomegalovirus.
Antimicrob. Agents Chemother. 1987, 31, 544-550.
(20) Renau, T. E.; Ludwig, M. S.; Drach, J . C.; Townsend, L. B.
Design, synthesis and activity against human cytomegalovirus
of non-phosphorylatable analogs of toyocamycin, sangivamycin
and thiosangivamycin. Bioorg. Med. Chem. Lett. 1992, 2, 1755-
1760.
(21) Gupta, P. K.; Daunert, S.; Nassiri, M. R.; Wotring, L. L.; Drach,
J . C.; Townsend, L. B. Synthesis, cytotoxicity and antiviral
activity of some acyclic analogues of the pyrrolo[2,3-d]pyrimidine
nucleoside antibiotics tubercidin, toyocamycin, and sangivamy-
cin. J . Med. Chem. 1989, 32, 402-408.
Ack n ow led gm en t. We thank J ack Hinkley for the
large-scale preparation of the pyrrole 1. We also thank
Dr. Mary Ludwig, J ulie Breitenbach, Cathy Yeung, and
Roger Ptak for confirmation of the biological data. This
work was supported by the Department of Health and
Human Services Research contracts N01-AI72641 and
U19-AI-31718 from NIAID, American Cancer Society
Research Grant No. DHP-36, and The University of
Michigan research funds.
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