Structure-activity relationships of acyloxyamidine cytomegalovirus DNA polymerase inhibitors

Article abstract of DOI:10.1016/S0968-0896(99)00319-3

This paper describes the structure-activity relationships of a new class of cytomegalovirus DNA polymerase inhibitors having two aryl groups joined by an acyloxyamidine linker. Examination of a series of analogues in which the terminal groups are varied r

Full text of DOI:10.1016/S0968-0896(99)00319-3

Bioorganic & Medicinal Chemistry 8 (2000) 601±615
Structure±Activity Relationships of Acyloxyamidine
Cytomegalovirus DNA Polymerase Inhibitors
John A. Tucker,*,y Terrance L. Clayton, Connie G. Chidester, Martin W. Schulz,
Leigh E. Harrington, Steven J. Conrad, Yoshihiko Yagi, Nancee L. Oien,
David Yurek and Ming-Shang Kuo
Discovery Research, Pharmacia and Upjohn, Kalamazoo, MI 49001, USA
Received 27 March 1999; accepted 7 November 1999
AbstractÐThis paper describes the structure±activity relationships of a new class of cytomegalovirus DNA polymerase inhibitors
having two aryl groups joined by an acyloxyamidine linker. Examination of a series of analogues in which the terminal groups are
varied revealed a very narrow SAR around the 2,4-dichlorophenyl group of the lead compound, but a variety of replacements for
the benzothiazole ring are compatible with activity. The most notable of these is the isoxazole ring of compound 78, which provides
a 30-fold enhancement in potency compared to the lead compound. We also describe the design, synthesis and evaluation of 10
analogues in which the acyloxyamidine linker is modi®ed or replaced by an isosteric group. Structure±activity relationship studies
identi®ed the linker -NH2 group as a critical pharmacophoric element. Ab initio molecular orbital calculations combined with
qualitative estimates of steric interaction energies suggest that the lowest energy conformations of the acyloxyamidine linker are
0
characterized by an extended planar CAr±C=N±O±C arrangement and either a syn-periplanar or anti-periplanar N±O±C±CAr
arrangement. Only the anti-periplanar conformation was observed in the crystal structures of three acyloxyamidines. The most
active of the linker-modi®ed compounds designed on the basis of these studies is the amidine carbamate 20, which is approximately
one-third as potent in the cytomegalovirus DNA polymerase inhibition assay as the comparator acyloxyamidine 53. The activity of
20 suggests that acyloxyamidines may bind to the cytomegalovirus DNA polymerase via an anti-periplanar conformation similar to
that observed in the crystal structure of acyloxyamidine 36. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
mortality among the immunocompromised. The pre-
valence of CMV infection among AIDS patients is
nearly 100%, and 25±40% will develop CMV-related
morbidity.3,4 Retinitis is among the most common
manifestations of CMV infection in these patients. This
syndrome, which is characterized by retinal lesions con-
sisting of hemorrhagic exudates, will always lead to
blindness in the absence of treatment.5 Up to one third
of bone marrow transplant patients and 13±38% of
renal allograft patients will develop clinically apparent
CMV disease.6,7 The utility of currently available treat-
ments for CMV disease is limited by their toxicity, their
modest ecacy, and by the requirement of intraveneous
administration for maximum ecacy.
Cytomegalovirus (CMV) is a member of the herpesvirus
family, a group of double-stranded DNA viruses which
includes the herpes simplex viruses 1 and 2, Epstein±
Barr virus, and varicella-zoster virus.1 The members of
this virus family share the ability to establish latent
infections in otherwise healthy individuals, and to reac-
tivate following certain environmental stimuli or host
immunosuppression. Cytomegalovirus infects 40±100%
of various human populations,2 the frequency of infec-
tion varying with cultural and regional trends in breast
feeding and other forms of physical intimacy.
In contrast to the relatively benign pro®le of CMV
infection in immunocompetent individuals, CMV is
among the most common causes of morbidity and
Broad screening of the Pharmacia and Upjohn chemical
library led to the identi®cation of the acyloxyamidine 1
as a 20 mM competitive inhibitor of the CMV DNA
polymerase. This compound does not detectably inhibit
human DNA polymerase alpha at 100 mM concentra-
tion. In this report we describe a preliminary investiga-
tion into the e€ects on CMV polymerase inhibition
potency of varying the terminal aryl groups of this lead
*Corresponding author. Fax: +1-650-877-0900; e-mail: btuck@
earthlink.net
yCurrent address: Elan Pharmaceuticals, 800 Gateway Avenue, South
San Francisco, CA 94080, USA.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00319-3

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J. A. Tucker et al. / Bioorg. Med. Chem. 8 (2000) 601±615
structure. We also report molecular orbital calculations
and X-ray crystallographic investigations undertaken
to elucidate the conformational behavior of the meta-
bolically unstable acyloxyamidine linker, and the
synthesis and evaluation of a series of analogues in
which this linker is modi®ed or replaced by an isosteric
group.
Results and Discussion
Conformational analysis
Figure 1. 6-31G*//3-21G conformational energy minima of 2.
We elected to initiate our investigation into the con-
formational preferences of 1 with a series of ab initio
molecular orbital calculations on the model compound
N-(formyloxy)formamidine (2). This model system was
chosen because it represents the smallest molecular
entity which contains all the essential features of the N-
(acyloxy)amidine linker of 1. Ab initio geometry opti-
mizations were performed with the 3-21G basis set and
single point energy calculations were performed on the
optimized structures using the 6-31G* basis set.8,9
obtain an estimate of the relative energy of a cyclically
hydrogen bonded structure at the 6-31G*//3-21G level
of theory, the dihedral angle about the N±O bond was
constrained to its value in the AM1 structure (56ꢀ) and
the remaining geometrical parameters were optimized at
the 3-21G level. Single point energy calculations using
the 6-31G* basis set place this conformation (structure
E) 9.0 kcal/mol above the lowest energy conforma-
tion.10 A related structure in which the CˆN±O±C
dihedral angle is constrained to a value of 120ꢀ has a
relative energy of 3.8 kcal/mol. This structure also
minimized to structure B when the CˆN±O±C dihedral
angle constraint was removed from the calculation. A
third structure similar to E but having an E con®gura-
tion of the CˆN bond minimized to conformation D
upon geometry optimization. The energetic preference
for an antiperiplanar geometry about the N±O bond
may arise from electrostatic repulsion between the
nitrogen sp2 lone pair and the two sp3 lone pairs on
oxygen. These interactions are minimized in conforma-
tions in which the N±O bond adopts an anti-periplanar
arrangement.
The four local conformational energy minima dis-
covered in this e€ort are represented by structures A±D
in Figure 1. In these four structures the CˆN bond
adopts an E or Z geometry and the geometry about the
carboxyl C±O bond is syn-periplanar or anti-periplanar.
The observed preference for the Z con®guration of the
CˆN bond may result from attractive electrostatic
interactions between the oxime oxygen and the endo
hydrogen of the-NH2 group. The preference for a syn-
periplanar or anti-periplanar arrangement about the
carboxyl C±O bond is in accord with expectations based
on resonance interactions of the carbonyl group and a
lone pair on the single-bonded carboxyl oxygen.
In order to use the data in Figure 1 to predict the con-
formational behavior of 1, account must be taken of
steric interactions that are present in 1 and absent in the
model compound 2. Conceptually this corresponds to
replacing both C±H bonds in each of the structures of
Figure 1 with C±C(aryl) bonds. Steric interactions
involving the aryl rings which are present in some but
not in all of the resulting N-(acyloxy)amidine con-
formers can then be estimated and appropriate correc-
tion factors applied to the relative conformational
energies from Figure 1. As seen in Figure 2, all of the
steric interactions satisfying this description are 1,4
eclipsing interactions between an aryl group and either
an sp3 oxygen or an sp2 nitrogen. If these two types of
interaction are assumed to be approximately equal and
assigned a value x, the relative energies of conformers F,
G, H, I and J are ( 2.6+x), 0.0, (0.9+2x), (2.3+x),
and (6.4+x) kcal/mol, respectively. In this approxima-
tion, values of x between 1 and 4 kcal/mol lead to a
In all four local conformational energy minima the
geometry about the N±O bond is anti-periplanar. Three
structures having other torsion angles about the N±O
bond were examined in this study and found not to be
local conformational energy minima at the 3-21G level
of theory. A cyclically hydrogen bonded conformation
similar to E was found to be a local conformational
energy minimum using AM1 calculations but optimiza-
tion of this structure using the more rigorous ab initio
calculations led to the planar structure B. In order to

J. A. Tucker et al. / Bioorg. Med. Chem. 8 (2000) 601±615
603
Figure 2.
prediction that the conformational equilibrium of 1 will
be strongly dominated by conformers F and G. Values
of x above and below this range lead to predictions that
the conformational equilibrium will be dominated by F
or G, respectively.
Synthesis
The acyloxyamidines described in this report were pre-
pared by the acylation of hydroxyamidines with acid
chlorides in the presence of triethylamine.11 Most of the
hydroxyamidines were commercially available or were
synthesized by the reaction of a nitrile with anhydrous
methanolic hydroxylamine. The 2,6-disubstituted
hydroxybenzamidine 3 was prepared by a literature
procedure involving nucleophilic attack of ammonia on
2,6-dichloro-N-hydroxybenzimidoyl chloride.12,13 The
N-substituted hydroxyamidine 6 was synthesized by
treating 2,4-dichloro-N-hydroxybenzimidoyl chloride 5
with methylamine. The imidoyl chloride 5 was prepared
by the action of NCS on the oxime 4.14 Analogue 9 was
prepared by alkylation of the hydroxyamidine 7 with 1-
chloromethylnaphthalene. Analogue 10 was prepared
by acylating oxime 4 with benzoyl chloride (Scheme 1).
Crystallography
The X-ray crystal structure of a representative acyloxy-
amidine 36 is shown in Figure 3. Crystal structures were
also determined for two other analogues of 1, com-
pounds 48 and 55. All three of these compounds adopt a
conformation in the solid state which is similar to con-
former G of Figure 2, a result which is in accord with
the results of our ab initio calculations. The atoms of
the acyloxyamidine linker vary only slightly from
coplanarity. In each of the crystal structures the carbonyl
oxygen participates in an intermolecular hydrogen bond
with the NH2 group of an adjacent molecule. The ability
of this hydrogen bonding network to stabilize con-
formation G in the solid state by reducing electrostatic
repulsions between the imino nitrogen and the carbonyl
oxygen may explain our failure to observe conformation
F in any of these three crystal structures.
The aminoamidine 14 was prepared by the route shown
in Scheme 2.
The amidine 17 was prepared by the action of metha-
nolic ammonia on the imidate 16 (Scheme 3). Acylation
of this amidine with phenyl chloroformate gave the
amidine carbamate 20. Treating amidine 17 with isocy-
anate 19 gave the amidine urea 21.
The synthesis of the imidazole 23 (Scheme 4) was based
on a method for the synthesis of methyl 2-phenylimida-
zole-4-carboxylate reported by Heindel and Chun.15 In
this approach an intermediate hydroxyamidine vinyl
ether is obtained by conjugate addition of a hydro-
xyamidine to an electron-de®cient alkyne. Thermolysis
of this ether occurs with the loss of a molecule of water
to produce the imidazole ring. In our hands the con-
jugate addition of the hydroxyamidine 7 to phenyl eth-
ynyl ketone and the thermal rearrangement of the
resulting vinylogous ester gave the imidazole 23 in 5%
overall yield.
Figure 3. Crystal structure of compound 36.

604
J. A. Tucker et al. / Bioorg. Med. Chem. 8 (2000) 601±615
Scheme 1.
(Scheme 6). The amidine 33 was prepared by treating
the thioimidate 13 with the tetrahydrooxazine 32.16
Structure±activity relationships
The compounds of this report were screened for their
ability to inhibit CMV DNA polymerase using a scin-
tillation proximity assay (Amersham) to measure incor-
poration of nucleotide into a template/primer. The
e€ect of varying the substitution pattern of the left hand
ring on activity in the CMV polymerase assay is descri-
bed in Table 1. In this series the benzothiazole group of
the lead compound 1 was replaced by a 1-naphthyl
group because this substitution exerts a favorable e€ect
on activity and because the requisite acid chloride is
commercially available. Compound 42, which retains
the left hand 2,4-dichlorophenyl group of the lead com-
pound 1, serves as the benchmark for this series of
compounds. Among the 19 left hand ring substitution
patterns examined, none confers activity in the CMV
polymerase assay greater than the 2,4-dichloro sub-
stitution pattern of 42. The chlorine in the 2-position of
the phenyl ring can be replaced by methyl (compound
44) without signi®cantly changing the activity, suggest-
ing that the steric rather than the electronic properties
of the 2-substituent are important for activity. Exam-
ination of molecular models suggests that the presence
of a 2-substituent enforces a non-coplanar arrangement
of the left hand ring and the attached (CˆN)NH2 frag-
ment. In the absence of such a substituent, a coplanar
Scheme 2.
The tetrahydrotriazene 29 was prepared by a multistep
synthetic route involving preparation of a suitably sub-
stituted aminoalkylhydrazine 28 and cyclization onto
the imidate ester 16 (Scheme 5). The low yield in the
®nal step of this synthetic sequence results in part from
the formation of large amounts of a byproduct arising
from the reaction of 28 with two equivalents of 16. Pre-
sumably the formation of this byproduct could be
reduced by performing the cyclization under high dilu-
tion conditions.
The hydroxyamidine 31 was prepared by treating the
hydroxyimidoyl chloride 5 with 2-phenylmorpholine 30

J. A. Tucker et al. / Bioorg. Med. Chem. 8 (2000) 601±615
605
Scheme 5.
Scheme 6.
Scheme 3.
56). Examination of molecular models suggest that
ortho substituents enforce an non-coplanar arrangement
of the carboxylate group and the attached phenyl ring.
A coplanar arrangement is expected to be favored by
resonance interactions in the absence of an ortho sub-
stituent. Substitution in the para position with methyl,
methoxy, or chloro gave compounds that were less
active than 56. These results, in combination with the
activity of the lead compound 1, suggest that a steric
exclusion zone may exist in this part of the enzyme
binding pocket that will tolerate no substituent larger
than a divalent nitrogen.
Scheme 4.
Table 3 shows the structures and in vitro screening
results for eight analogues of 1 in which the benzothia-
zole moiety has been replaced by an araalkyl,
cycloalkyl, or heteroaromatic group. The benzyl and
cyclohexyl groups of compounds 72 and 73 gave com-
pounds having very modest levels of activity, as did the
relatively hydrophilic furyl and pyridyl groups of 74 and
75. The modest activity of 76 and 77 may be related to
the presence of a ring CH in the steric exclusion zone
that was hypothesized to explain the inactivity of com-
pounds 60±62. The most interesting compound of this
series is the isoxazole derivative 78, which is approxi-
mately 30 times more potent than the lead compound 1.
arrangement of these two groups is expected to be
energetically favored due to resonance interactions.
Table 2 shows the structures and in vitro screening
results for 19 analogues of 1 in which the benzothiazole
ring is replaced by a substituted or unsubstituted phenyl
ring. Replacement of the benzothiazole ring of 1 (CMV
polymerase IC50=20 mM) with phenyl gave a com-
pound of equal potency (53). Attaching a methyl,
phenyl, or chlorine substituent to the ortho position of
this phenyl ring gave compounds of enhanced potency,
the best of these substituents being phenyl (compound

606
J. A. Tucker et al. / Bioorg. Med. Chem. 8 (2000) 601±615
Table 1. Substituted phenyl rings as replacements for the 2,4-dichloro-
phenyl group of 14
Table 3. Araalkyl, cycloalkyl and aromatic groups as replacements
for the benzothiazole ring of 1
No.
R
IC50 (mM)
72
73
74
75
76
77
78
79
Benzyl
Cyclohexyl
2-Furyl
44
>100
>100
52
Substituents
No.
C-2
C-3
C-4
C-5
C-6
IC50 (mM)
4-Pyridyl
2-Benzothiophenyl
2-Quinoxalinyl
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
Cl
OCH3
CH3
>100
>100
>100
>100
50
>100
38.7
28.6
3.1
72
>100
0.7
9.5
5-Methyl-3-phenylisoxazol-4-yl
3-(2,6-Dichlorophenyl)-5-methylisoxazol-4-yl
Cl
Cl
Cl
CH3
Cl
Cl
Cl
Cl
CH3
CH3
CH3
Cl
F
Cl
Br
Cl
Br
CH3
The synthesis of this analogue was undertaken because
of its structural similarity to the active analogue 56, and
because it was hoped that the steric hinderance of the
carbonyl group in this analogue would provide
improved metabolic stability.
2.6
3.9
4.2
20.4
15.0
59.6
>100
>100
32.7
13.7
Cl
Cl
Cl
Cl
Cl
Cl
CH3
Cl
Compounds 42 and 78 were evaluated for their ability
to inhibit CMV replication in human foreskin ®broblast
(HFF) cells using standard antiviral assay conditions.
Neither of these compounds exhibited reproducible
antiviral activity at concentrations ꢁ100 mM. Because the
antiviral assay is performed in a growth medium that
includes 10% serum, we hypothesized that the lack of
antiviral activity of these compounds might be due to
rapid degradation by serum esterases. In order to test
this hypothesis, the stability of compounds 42 and 78 in
serum at 37 ꢀC was evaluated by HPLC analysis. Each
compound was rapidly degraded with the formation of
two new compounds that coelute with the hydroxy-
amidine and carboxylic acid expected from hydrolysis of
the linker C±O bond. Attempts to improve the serum
stability of these compounds by increasing the steric
hindrance around the carbonyl carbon as in compounds
68 and 69 led to reduced activity in the CMV poly-
merase assay. Based on these results our e€orts to
discover more metabolically stable compounds were
focused on the discovery of replacements for the acy-
loxyamidine linker.
Cl
OCH3
OCH3
Cl
Table 2. Substituted phenyl rings as replacements for the benzothia-
zole ring of 1
Substituents
No.
C-2
C-3
C-4
C-5
C-6
IC50 (mM)
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
22
10
6.1
5.4
11
7.7
Cl
CH3
Ph
Table 4 describes the CMV polymerase inhibition
activity of the 10 analogues of this report that incorpo-
rate structural modi®cations of the acyloxyamidine
linker. These analogues can be divided into three
structural classes. Class 1 consists of analogues in which
only incremental changes have been made to the
acyloxyamidine linker group (compounds 10, 80, 9, 14,
20 and 21). Some members of this group incorporate
structural changes which were intended to improve the
metabolic stability of the resulting analogues relative
to that of the corresponding acyloxyamidine. In others,
the incremental change was intended to elucidate the
pharmacophoric elements of the acyloxyamidine linker.
Compounds of class 2 (23 and 29) incorporate the amidine
Cl
OCH3
CH3
16
Cl
OCH3
CH3
>50
>100
>100
10
>100
2.1
6.7
Cl
OCH3
CH3
Cl
CH3
Cl
Cl
OCH3
F
Cl
F
4.4
37
32
25
5.3
Cl
CH3
CH3
Cl
OCH3
Cl
OCH3

J. A. Tucker et al. / Bioorg. Med. Chem. 8 (2000) 601±615
Table 4. Analogues incorporating modi®cations of the acyloxyamidine linker
607
No.
Structure
IC50
(mM)
No.
Structure
IC50
(mM)
10
>100a
21
>100b
80
>100a
23
29
31
33
174
>100a
157
9
19
14
>100
20
73
>100a
a<10% inhibition at 100 mM concentration.
b<5% inhibition at 50 mM concentration.
group into a ring structure which is intended to provide
a metabolically stable framework for maintaining the
left hand aryl group and the right hand aroyl group in
the same relative orientation that is found in the two
most energetically favored conformations of the acy-
loxyamidine parent. The design of these compounds
incorporates resonance interactions between the car-
bonyl group and the atoms of the ring. This resonance
restricts the carbonyl group to either of two coplanar
orientations with respect to the amidine group, in close
analogy with conformations F and G of Figure 2. In
class 3 (compounds 31 and 33), a six member ring con-
strains the conformation of the three atom linker
between the amidine carbon and the right hand aryl
ring. This conformational constraint, in combination
with the amidine resonance, is expected to maintain a
roughly coplanar arrangement of all the atoms of the
linker group. The amidine carbon±nitrogen single bond
in these compounds may adopt either an s-cis or s-trans
arrangement with respect to the right hand phenyl ring
and the imino nitrogen. Of these, only the s-cis con-
former closely resembles one of the preferred con-
formations of the acyloxyamidine lead compounds.
Most of the linker-modi®ed analogues described in this
report incorporate phenyl as the right-hand aryl ring.
As can be seen from data in Table 2, a variety of other
aryl groups in this position lead to acyloxyamidines that
are more potent inhibitors of CMV polymerase. Our
decision to employ phenyl as the right hand aromatic
ring in many of these analogues arose from the greater
ease of synthesis of the phenyl compounds. It was our
expectation that potency-enhancing right hand aryl
groups could be incorporated after the best isosteric

608
J. A. Tucker et al. / Bioorg. Med. Chem. 8 (2000) 601±615
we were initially encouraged that the amidine carbamate
linker of this compound might serve as a metabolically
stable replacement for the acyloxyamidine linker,
HPLC studies demonstrated that 20 is not signi®cantly
more stable in serum than the acyloxyamidine 1. Crystal
structures of other compounds having an HN±CˆN±
(CˆO)±O±C array reveal an extended, planar, cyclically
hydrogen bonded geometry for this fragment similar to
that drawn for 20 in Figure 5.17±20 This proposed con-
formation is in accord with expectations based on reso-
nance and steric e€ects, and places the two aryl rings
and the -NH2 group in relative orientations similar to
that in conformer G of Figure 2. In the hopes of
improving on the serum stability of 20 we prepared the
amidine urea 21, but this compound failed to exhibit
any activity in the CMV polymerase inhibition assay.
Figure 4.
replacement for the acyloxyamidine group had been
identi®ed.
Compounds 10 and 80 of Table 4 were designed to
evaluate the importance of the amidine -NH2 group in
binding to CMV polymerase. Replacement of the -NH2
group of 53 with a hydrogen as in compound 10 or with
an -NHCH3 group as in compound 80 resulted in a
greater than 5-fold decrease in anity for the CMV
polymerase binding site. These results can most easily
be explained by the hypothesis that the exo N H bond
of the benchmark compound 53 is required for hydro-
gen bonding interactions with the enzyme. Compound
80 might reasonably be expected to adopt a conforma-
tion similar to that shown in Figure 4, with the CH3
group occupying the exo position. This arrangement
maintains a cyclic hydrogen bond between the oxime
oxygen and the amino hydrogen while minimizing
unfavorable steric interactions involving the oxime oxy-
gen that are present in other arrangements.
The two compounds of structural class 2 that were
investigated in this study are the imidazole 23 and the
tetrahydrotriazene 29. Among these, 23 was observed to
exhibit modest activity in the CMV polymerase assay.
This compound incorporates the exo N±H, the carbonyl
group, and the sp2 hybridized nitrogen of the bench-
mark compound 53, but molecular modeling results
suggest that the 5-member imidazole ring introduces
bond angle distortions that signi®cantly alter the
relative orientation of the two aromatic rings com-
pared to that found in 53. In order to determine the
activity of a similar compound having bond angles more
similar to those in 53, the tetrahydrotriazene 29 was
prepared. The inactivity of this compound may indicate
the existence of a steric exclusion zone in the area
transversed by the two CH2 groups of the tetra-
hydrotriazene ring.
Compounds 9, 14, 20 and 21 of Table 4 were designed
to examine the e€ect on CMV polymerase inhibition
activity of changes in the molecular fragment connect-
ing the amidino group and right hand aryl ring of the
acyloxyamidine parent compounds 42, 53 and 78. When
the CˆO fragment of naphthyl compound 42 was
replaced by a CH2 group the activity of the resulting
analogue 9 was reduced about 5-fold. If one assumes
that the most energetically favored conformation of the
relatively rigid parent compound is similar to its ideal
binding geometry, the decreased binding of the more
conformationally mobile 9 can be explained by entropy
considerations. This hypothesis implies that there are no
energetically important hydrogen bonds between the
carbonyl oxygen of 42 and the enzyme.
The two members of structural class 3 that were exam-
ined are the amidines 31 and 33. The design of each of
these two analogues includes an oxygen atom attached
to one of the amidine nitrogens. The presence of this
oxygen is intended to assure that these two compounds,
like 53, will not be ionized at physiological pH.21,22 In
compound 31, the hydrogen bond donating site analo-
gous to the exo hydrogen of 53 is shifted slightly relative
to the position of the two aromatic rings because of the
formal replacement of an N±H by N±OH. This com-
pound exhibited about one-seventh the activity of 53 in
the CMV polymerase inhibition assay. In hopes of pre-
paring an analogue with greater structural similarity to
53 we prepared the tetrahydrooxazine derivative 33.
This compound proved completely inactive in the CMV
polymerase assay. The inactivity of this analogue may
The amidine carbamate 20 was found to be about 3-fold
less active than the benchmark compound 53. Although
Figure 5.
Figure 6.

J. A. Tucker et al. / Bioorg. Med. Chem. 8 (2000) 601±615
609
be due to formation of a cyclic hydrogen bond between
the single N±H of this compound and the ring oxygen
(Fig. 6). The resulting Z geometry about the CˆN bond
would place a lone pair in the position occupied by the
exo N±H of 53. This exo N±H was was previously
hypothesized to be required for activity (vide supra). An
alternative hypothesis, that the inactivity of 33 is due to
its lack of a hydrogen bond acceptor analogous to the
carbonyl oxygen of 53, seems less attractive in light of
the observed activity of 9.
50 mL of 3 N aqueous sodium hydroxide solution. The
phases were separated and the organic phase was dried
(Na2SO4). The solvent was evaporated at reduced pres-
sure and the residue was recystallized from re¯uxing
methanol. The precipitate was collected by ®ltration,
washed with 5 mL of methanol, and dried at 20 torr/
50 ꢀC/48 h to give the title compound as 0.88 g (76%) of
1
a white powder. H NMR (300 MHz, CDCl3) d 8.91 (s,
1H), 8.11 (overlapping triplets, J=7.5, 7.5 Hz, 3H), 7.60
(tt, J=1.0, 7.5 Hz, 1H), 7.49±7.38 (m, 3H), 7.27 (dd,
J=2.0, 8.5 Hz, 1H). MS (EI) m/z (rel. intensity) 293
(M+, 1), 173 (5), 171 (7), 123 (4), 109 (4), 106 (22), 105
(100), 77 (61), 75 (4), 51 (13), 50 (5). Anal. calcd for
C14H9Cl2NO2: C, 57.17; H, 3.08; N, 4.76. Found: C,
57.06; H, 2.90; N, 4.54.
Conclusions
This report describes our preliminary SAR investigation
of a new class of CMV polymerase inhibitors char-
acterized by two aryl groups connected by an acyloxy-
amidine linker. Initial studies in which the two aryl
groups were varied revealed little tolerance for mod-
i®cation of the left hand 2,4-dichlorophenyl group, but
replacing the right hand benzothiazole ring with a dis-
ubstituted isoxazole ring as in 78 led to a 30-fold
enhancement in activity. The favorable e€ect on activity
of substituents adjacent to the linker on each aromatic
ring may re¯ect a preferred binding conformation in
which the aryl rings lie out of the plane of the adjacent
linker atoms. The metabolic instability associated with
the acyloxyamidine linker led us to undertake a research
e€ort designed to discover more stable isosteric repla-
cements for this functional group. Structure±activity
relationship studies identi®ed the linker-NH2 group as a
critical pharmacophoric element. Ab initio molecular
orbital calculations combined with qualitative estimates
of steric interaction energies suggest that the lowest
energy conformations of the acyloxyamidine linker are
characterized by an extended planar CAr±CˆN±O±C
arrangement and either a syn-periplanar or anti-peri-
2,4-Dichloro-N0 -hydroxy-N-methylbenzenecarboximid-
amide (6). A solution of 0.80 g (3.56 mmol) of 523,24 in
10 mL of methanol was added all at once to 20 mL of a
saturated solution of methylamine in methanol. After
30 min the reaction mixture was partitioned between
50 mL of dichloromethane and 50 mL of pH 12 phos-
phate bu€er. The aqueous phase was washed with
10 mL of dichloromethane and then the combined
organic extracts were washed with 50 mL of distilled
water and dried (Na2SO4). The solvent was evaporated
at reduced pressure and the residue was dissolved in
25 mL of re¯uxing methanol. Distilled water was added
to the warm solution until it became cloudy and the
solution was allowed to stand 12 h at 25 ꢀC. The pro-
duct was collected by ®ltration, yielding 0.55 g (70%) of
the title compound as a white solid. 1H NMR
(300 MHz, CDCl3) d 9.0 (broad s, 1H), 7.38 (d,
J=2.0 Hz, 1H), 7.30±7.19 (m, 2H), 5.30 (broad s, 1H),
2.54 (s, 3H). MS (EI) m/z (rel. intensity) 218 (M+, 100),
220 (64), 189 (35), 188 (31), 187 (56), 186 (40), 174 (48),
172 (74), 136 (27), 124 (26). Anal. calcd for C8H8
Cl2N2O: C, 43.86; H, 3.68; N, 12.79. Found: C, 43.87;
H, 3.78; N, 12.78.
0
planar N±O±C±CAr arrangement. Only the anti-peri-
planar conformation was observed in the crystal
structures of three acyloxyamidines. Among the linker
replacements designed on the basis of these studies,
good activity in the cytomegalovirus DNA polymerase
inhibition assay was most closely associated with the
amidine carbamate linker of 20. This compound is
approximately one-third as potent in the cytomegalo-
virus DNA polymerase inhibition assay as the com-
parator acyloxyamidine 53. The activity of 20 suggests
that acyloxyamidines may bind to the cytomegalovirus
DNA polymerase via the anti-periplanar conformation
observed in the crystal structure of 36.
[(Benzoyloxy)imino](2,4-dichlorophenyl)-N-methylmethan-
amine (80). A solution of 0.25 g (1.14 mmol) of 6 in
5 mL of tetrahydrofuran was treated successively with
0.21 mL (1.50 mmol) of triethylamine and 0.132 mL
(1.14 mmol) of benzoyl chloride. The mixture was stir-
red 20 h, then it was partitioned between 25 mL of
dichloromethane and 25 mL of 1 N aqueous sodium
hydroxide solution. The organic phase was dried
(Na2SO4) and the solvent was evaporated at reduced
pressure. The residue was recystallized from re¯uxing
methanol and water. The product was collected by ®l-
tration and washed with 9:1 methanol:distilled water.
This procedure yielded the title compound as 0.25 g
(68%) of a white powder, mp 185±186 ꢀC. An analytical
sample was dried at 20 torr/55 (C/6 h. 1H NMR
(300 MHz, CDCl3) d 8.05 (dt, J=6.0, 1.0 Hz, 2H), 7.55
(tt, J=1.5, 6.5 Hz, 1H), 7.50±7.37 (m, 4H), 7.32 (dd,
J=2.0, 8.0 Hz, 1H), 5.47 (broad q, J=indet., 1H), 2.71
(d, J=5.0 Hz, 3H). MS (EI) m/z (rel. intensity) 322
(M+, 13), 324 (8), 188 (5), 186 (8), 174 (3), 172 (4), 106
(8), 105 (100), 77 (14), 51 (3). Anal. calcd for C15H12
Cl2N2O2: C, 55.75; H, 3.74; N, 8.67. Found: C, 55.89;
H, 3.77; N, 8.75.
Experimental
Synthesis
1-{[(Benzoyloxy)imino]methyl}-2,4-dichlorobenzene (10).
A 0 ꢀC solution of 0.75 g (3.95 mmol) 2,4-dichloro-
benzaldehyde oxime22 and 0.70 mL (5.00 mmol) diiso-
propylethylamine in 15 mL of tetrahydrofuran was
treated with 0.46 mL (3.95 mmol) benzoyl chloride. The
mixture was stirred at 25 ꢀC for 18 h and then it was
partitioned between 50 mL of dichloromethane and

610
J. A. Tucker et al. / Bioorg. Med. Chem. 8 (2000) 601±615
2,4-Dichloro-N0-(1-naphthylmethoxy)benzenecarboximid-
amide (9). To a 0 ꢀC solution of 0.400 g (1.95 mmol) 2,4-
dichlorobenzamidoxime (7) in 3 mL of tetrahydrofuran
was added over 1 min 2.0 mL (2.0 mmol) of a 1.0 M
solution of potassium tert-butoxide in tetrahydrofuran.
To the resulting thick suspension was added 0.91 mL
(1.95 mmol) of neat 1-(chloromethyl)napthalene over
1 min, followed by 1.0 mL of DMPU. The mixture was
stirred 19 h at 25 ꢀC and then it was partitioned between
30 mL of distilled water and 30 mL of dichloromethane.
The organic phase was dried (MgSO4) and the solvent
was evaporated at reduced pressure. The residue was
puri®ed by chromatography (55% dichloromethane in
hexanes) followed by recrystallization from dichlor-
omethane and hexane. This procedure yielded the pro-
duct as 170 mg (25%) of a white solid, mp 78±80 ꢀC. A
sample was dried at 40 ꢀC/20 torr/18 h for elemental
analysis. 1H NMR (300 MHz, CDCl3) d 8.18 (d,
J=7.0 Hz, 1H), 7.92-7.80 (m, 2H), 7.60±7.40 (m, 6H),
7.28 (dd, J=2.0, 8.0 Hz, 1H), 5.57 (s, 2H), 4.86 (broad s,
2H). MS (FAB) m/z (rel. intensity) 345 (M+H, 71), 349
(7), 348 (11), 347 (46), 346 (28), 344 (21), 191 (7), 189
(12), 142 (13), 141 (100). Anal. calcd for C18H14N2OCl2:
C, 62.62; H, 4.09; N, 8.11. Found: C, 62.43; H, 4.08; N,
8.01.
H). MS (FAB) m/z (rel. intensity) 220 (M+H, 100), 376
(3), 374 (4), 224 (13), 223 (7), 222 (69), 221 (11), 186 (2),
174 (3), 172 (4). Anal. calcd for C8H8Cl2INS: C, 27.60;
H, 2.32; N, 4.02. Found: C, 27.53; H, 2.22; N, 3.89.
N0 -[(2,4-Dichlorophenyl)(imino)methyl]benzohydrazide
hydroiodide (14).
A
25 ꢀC suspension of 0.196 g
(1.44 mmol) benzoic hydrazide in 1.0 mL of absolute
ethanol was added all at once to a 0 ꢀC solution of
0.50 g (1.44 mmol) 13 in 2.0 mL of ethanol. The mixture
was stirred 26 h at 5 ꢀC and then it was diluted with
30 mL of diethyl ether and 10 mL of cyclohexane. The
mixture was allowed to stand 35 min and then the pre-
cipitate was collected by ®ltration. It was washed with
4:1 diethyl ether:absolute ethanol. The solid was dried
in a stream of air and then further dried at 20 torr/80 ꢀC
for 4 h to give the title compound as 0.25 g (40%) of a
1
white solid, mp 190±191.5 (C. H NMR (CD3OD) d
8.01 (d, J=7.5 Hz, 2H), 7.84 (d, J=2.0 Hz, 1H), 7.80 (d,
J=8.5 Hz, 1H), 7.73±7.62 (m, 2H), 7.56 (t, J=7.5 Hz,
2H). MS (FAB) m/z (rel. intensity) 308 (M+H, 100),
312 (11), 311 (6), 310 (63), 174 (3), 172 (5), 105 (13).
.
Anal. calcd for C14H11Cl2N3O HI 0.5 H2O: C, 37.78;
H, 2.95; N, 9.27. Found: C, 37.70; H, 2.94; N, 9.27.
Phenyl amino(2,4-dichlorophenyl)methylidenecarbamate
(20). A solution of 3.7 g (25 mmol) trimethyloxonium
tetra¯uoroborate and 4.75 g (25 mmol) of 2,4-dichloro-
benzamide in 20 mL of anhydrous dichloromethane was
stirred three days at 25 ꢀC. The reaction mixture was
diluted with 30 mL of anhydrous diethyl ether and the
mixture was ®ltered. The ®ltrant was washed anhydrous
diethyl ether and dried in a stream of air for 10 min.
This procedure gave the imidate hydrotetra¯uoroborate
(16) as 5.5 g of a white powder (75%). 1H NMR
(300MHz, DMSO) d 7.97 (d, J=2.0Hz, 1H), 7.82 (d,
J=8.5Hz, 1H), 7.76 (dd, J=2.0, 8.5 Hz, 1H), 4.23 (s, 3H).
2,4-Dichlorobenzenecarbothioamide (12). A ¯ask was
loaded with a solution of 25g (145 mmol) 2,4-dichloro-
benzonitrile (11) in a mixturre of 100 mL of pyridine
and 28.0 mL (200 mmol) of triethhylammine. Hydrogen
sul®de gas was bubbled through the reaction mixture at
a moderately vigorous rate for 20 min, then the mixture
was stirred 20 h at 25 ꢀC. A vigorous ¯ow of nitrogen
was passed through the reaction mixture for 20 min, and
then the reaction mixture was diluted with 500 mL of
distilled water and 500 mL of 9:1 ethyl ether:ethyl ace-
tate. The aqueous phase was extracted with 100 mL of
ethyl ether and the combined organic extracts were
washed successively with two 300 mL portions of 3.0 N
hydrochloric acid and one 100 mL portion of brine. The
organic phase was dried (MgSO4), and the solvent was
evaporated at reduced pressure. The residue was stirred
in hexanes, and the resulting yellow solid was collected
by ®ltration. It was then recrystallized from cyclohexane
and collected by ®ltration to give 16.6 g (56%) of the
title compound as bright yellow needles, mp 86±87 ꢀC.
1H NMR (300 MHz, CDCl3) d 8.09 (broad s, 1H), 7.65
(d, J=8.5 Hz, 1H), 7.39 (d, J=2.0 Hz, 1H), 7.27 (dd,
J=8.5, 2.0 Hz, 1H), 7.22 (broad s, 1H). MS (EI) m/z
(rel. intensity) 205 (M+, 75), 207 (51), 174 (19), 172 (63),
170 (100), 136 (20), 75 (25), 74 (22), 60 (64), 50 (21).
Anal. calcd for C7H5Cl2NS: C, 40.80; H, 2.45; N, 6.80.
Found: C, 40.70; H, 2.42; N, 6.68.
A solution of 2.2 g (7.53 mmol) of this imidate in 25 mL
of anhydrous methanol was treated with 25 mL of
anhydrous ammonia-saturated methanol. The mixture
was stirred 6 days at 25 ꢀC and then it was partitioned
between 100 mL of ethyl acetate and 50 mL of 10%
aqueous sodium hydroxide solution. The aqueous phase
was washed with 25mL of ethyl acetate and the combined
organic extracts were dried (MgSO4). The solvent was
evaporated at reduced pressure and the oily residue was
dissolved in 10 mL of isopropanol. The solution was
treated with 15 mL of 1.0 N hydrogen chloride in diethyl
ether. After standing 15 min, the white solid precipitate
was collected by ®ltration and washed with 1:1 diethyl
ether:isopropanol. This procedure gave the amidine 17
as 0.69 g (41%) of a white solid, mp 252±254 ꢀC. H
1
NMR (300 MHz, DMSO-d6) d 9.61 (s, 2H), 9.57 (s, 2H),
7.95 (d, J=2 Hz, 1H), 7.74 (d, J=8.0 Hz, 1H), 7.68 (dd,
J=2.0, 8.0 Hz, 1H).
Methyl 2,4-dichlorobenzenecarbimidothioate hydroiodide
(13). A mixture of 5.0 g (24.3 mmol) of 12, 2.3 mL
(36.0 mmol) of methyl iodide and 35 mL of acetone was
stirred 3 days at 25 ꢀC. The white solid precipitate
which formed during this period was collected by ®ltra-
tion and washed with acetone. The product was
obtained as 6.5 g (76%) of a white powder. H NMR
(300 MHz, CD3OD) d 7.80 (d, J=2.0 Hz, 1H), 7.72 (d,
J=8.0 Hz, 1H), 7.63 (dd, J=8.0, 2.0 Hz, 1H), 2.86 (s, 3
A solution of 300 mg (1.33 mmol) of 17 in 6 mL of 2:1
acetone:deionized water was cooled to 0 ꢀC and treated
with 0.175 mL of 15 M aqueous sodium hydroxide
solution. After 3 min at 0 ꢀC phenyl chloroformate
(0.167 mL, 1.33 mmol) was added and the solution was
stirred 1 h at 0 ꢀC and 20 min at 25 ꢀC. The mixture was
1

J. A. Tucker et al. / Bioorg. Med. Chem. 8 (2000) 601±615
611
partitioned between 50 mL of diethyl ether and 50 mL
of deionized water. The organic phase was washed with
10 mL of 2 M aqueous NaHSO4 solution, then with
10 mL of 3.0 N aqueous sodium hydroxide solution, and
®nally with 10 mL of brine. The solution was dried
(MgSO4), and the solvent was evaporated at reduced
pressure. The residue was puri®ed by chromatography
on silica gel (15±20% ethyl acetate in cyclohexane). This
material was recrystallized from dichloromethane and
cyclohexane. The white needles which formed were col-
lected by ®ltration and dried at 45 ꢀC/20 torr for 5 h.
The yield is 40 mg (10%), mp 112±113 ꢀC. 1H NMR
(300 MHz, CDCl3) d 7.72 (d, J=8.5 Hz, 1H), 7.41 (d,
J=2.0 Hz, 1H), 7.37±7.27 (m, 4H), 7.20±7.09 (m, 4H).
MS (FAB) m/z (rel. intensity) 309 (M+H, 100), 313
(11), 312 (11), 311 (69), 310 (17), 217 (24), 215 (35), 152
50 mL of dichloromethane. The aqueous phase was
washed with 25 mL of dichloromethane and the com-
bined organic extracts were dried (Na2SO4). The solvent
was evaporated at reduced pressure and the residue was
chromatographed on 1500 mL of silica gel (15±20%
ethyl acetate:cyclohexanes). Fractions containing the
product were combined to give the addition product as
0.77 g (30%) of a slightly impure orange gum. 1H NMR
(CDCl3) d 8.05 (d, J=12.0 Hz, 1H), 7.92 (d, J=7.5 Hz,
2H), 7.60±7.42 (m, 5H), 7.35 (dd, J=2.0, 8.0 Hz, 1H),
6.83 (d, J=12.0 Hz, 1H), 5.23 (broad s, 2H).
A mixture of 0.63 g (1.88 mmol) of this gum and 4.0 mL
of diphenyl ether were combined under a nitrogen
atmosphere. The reaction mixture was immersed in a
preheated 250 ꢀC oil bath and stirred for 15 min. The
mixture was cooled to 25 ꢀC. The mixture was chroma-
tographed on 100 mL of silica gel (0±20% ethyl acetate
in cyclohexane). Fractions corresponding to the more
polar of the two major products were combined and the
solvent was evaporated at reduced pressure. The residue
was dissolved in 20 mL of 3:1 methanol:distilled water
and decanted from a small volume of insoluble dark oil.
The supernatent was partitioned between distilled water
and dichloromethane. The organic phase was dried
(MgSO4) and the solvent was evaporated at reduced
pressure. The residue was recrystallized from cyclohex-
ane, and the product was collected by ®ltration to give
the title compound as 100 mg (17%) of a tan powder,
.
(4), 95 (6), 77 (5). Anal. calcd for C14H10Cl2N2O2 0.25
CH2Cl2: C, 51.80; H, 3.20; N, 8.48. Found: C, 51.75; H,
2.94; N, 8.49. The presence of 0.25 mol of dichloro-
1
methane in the crystals was con®rmed by H NMR.
N-[Amino(2,4-dichlorophenyl)methylidene]-N0-(5-methyl-
3-phenyl-4-isoxazolyl)urea (21). Two millilitres of dis-
tilled water were added to a suspension of 1.7 g
(7.7 mmol) of 18 and 0.52 g (8.0 mmol) of NaN3 in
10 mL of acetone. After 16 h the mixture was parti-
tioned between 1,2-dichloroethane and distilled water.
The organic phase was washed with brine and then it
was dried (MgSO4). The volume of the solution was
reduced 50% by evaporation at reduced pressure. The
residue was re¯uxed for 45 min and then the solvent was
evaporated at reduced pressure. Kugelrohr distillation
(0.1 torr, 125 ꢀC) of the residue gave 230 mg of colorless
mp 171.5±174 ꢀC. H NMR (300 MHz, CDCl3) d 8.29
1
(d, J=8.5 Hz, 1H), 7.95 (d, J=7.5 Hz, 2H), 7.79 (s, 1H),
7.63 (d, J=7.5 Hz, 1H), 7.58±7.42 (m, 4H), 7.39 (dd,
J=8.5, 2.0 Hz, 1H). MS (EI) m/z (rel. intensity) 316
(M+, 100), 318 (65), 317 (23), 241 (28), 239 (42), 186
(19), 184 (29), 145 (56), 105 (31), 77 (43). Anal. calcd for
C16H10Cl2N2O: C, 60.59; H, 3.18; N, 8.83. Found: C,
60.44; H, 3.25; N, 8.58.
1
oil. This oil gave a strong isocyanate peak at 2264 cm
in its IR spectrum.
A suspension of 200 mg (0.89 mmol) of the amidine 17
in 2 mL of absolute ethanol was treated with 0.57 mL of
1.56 M sodium ethoxide in ethanol. After 5 min 180 mg
(0.89 mmol) of the isocyanate was added and the mix-
ture was stirred 18 h. The mixture was diluted with
15 mL of distilled water and the precipitate was col-
lected by ®ltration. It was washed with three 3 mL por-
tions of distilled water and dried in a stream of air. It
was chromatographed on 50 mL of silica gel eluting
with 10% EtOAc in CH2Cl2. Fractions containing the
desired product were combined and the solvent was
evaporated at reduced pressure. The solid was dissolved
in 5 mL of methanol and precipitated with 10 mL of
distilled water. The product was collected by ®ltration
and dried in a stream of air. The product ꢀwas obtained
tert-Butyl 2-{-2-[(tert-butoxycarbonyl)amino]ethylidene}-
1-hydrazine-carboxylate (25). To a freshly prepared
solution of 2.6 g (16.4 mmol) Boc-glycinal25,26 in 50 mL
of cyclohexane as added 1.94 g (14.7 mmol) of solid t-
butyl carbazate. The solution was rapidly heated to
re¯ux. After 1 h it was allowed to slowly cool to 25 ꢀC
and stirred overnight. The resulting thick suspension
was diluted with 75 mL of hexanes and ®ltered. The
product was obtained as 3.5 g (79%) of a white solid,
mp 107±108 ꢀC. A sample was dried at 20 torr/60 ꢀC for
1
16 h prior to elemental analysis. H NMR (300 MHz,
DMSO) d 10.53 (s, 1H), 7.16 (broad s, 1H), 7.09 (broad
t, J=indet., 1H), 3.62 (broad t, J=indet., 2H), 1.42 (s,
9H), 1.38 (s, 9H). MS (EI) m/z (rel. intensity) 200 (M+±
(CH3)3CO, 5%), 161 (36), 144 (14), 117 (12), 101 (6),
100 (5), 74 (4), 59 (4), 58 (6), 57 (100), 56 (4). Anal. calcd
for C12H23N3O4 0.15 C6H12: C, 54.18; H, 8.74; N, 14.69:
Found: C, 54.13; H, 8.50; N, 14.55. The persistence of
cyclohexane in the crystal after heating the compound
1
as 0.185 g of a white powder, mp 112±113 C. H NMR
(CDCl3) d 7.8±7.62 (m, 3H), 7.52±7.30 (m, 7H), 2.47 (s,
3H). MS (ESI+) for C18H14Cl2N4O2 m/z 389 (M+H)+.
Anal. calcd for C18H14Cl2N4O2: C, 55.54; H, 3.63; N,
14.39; Cl, 18.22. Found: C, 55.29; H, 3.62; N, 14.25.
1
[2-(2,4-Dichlorophenyl)-1H-imidazol-4-yl](phenyl)meth-
anone (23). A solution of 1.57 g (7.69 mmol) of 7, 1.0 g
(7.69 mmol) phenylethynyl ketone, and 50 mL of acetic
acid in 16 mL of methanol was stirred in the dark under
a nitrogen atmosphere for 20 h. The reaction mixture
was partitioned between 75 mL of distilled water and
at reduced pressure was con®rmed by H NMR.
tert-Butyl 2-{2-[(tert-butoxycarbonyl)amino]ethyl}-1-hy-
drazinecarboxylate (26). A mixture of 2.0 g (7.33 mmol)
of hydrazone 25, 0.4 g 10% Pd/C and 75 mL of 95%
ethanol was agitated under 50 psi hydrogen until 25

612
J. A. Tucker et al. / Bioorg. Med. Chem. 8 (2000) 601±615
could no longer be detected by TLC. At this time (ca.
5 h) the mixture was ®ltered through Celite and the
solvent was evaporated from the ®ltrate at reduced
pressure. The residue was chromatographed on silica gel
(35±40% ethyl acetate in cyclohexane). The product was
obtained as a colorless oil which solidi®ed on standing
to give 1.52 g (75%) of a white solid, mp 158±159 ꢀC. A
sample was dried at 20 torr/50 ꢀC for 4 h prior to ele-
mental analysis. 1H NMR (300 MHz, CDCl3) d 6.37
(broad s, 1H), 5.23 (broad s, 1H), 3.86 (broad s, 1H),
3.15 (dt, J=5.5 Hz, indeterminate, 2H), 2.83 (t, J=5.5
Hz, 2H), 1.39 (s, 9H), 1.37 (s, 9H). MS (FAB) m/z (rel.
intensity) 276 (M+H, 57), 275 (16), 220 (41), 176 (13),
164 (100), 163 (29), 120 (34), 57 (78), 41 (13). Anal.
calcd for C12H25N3O4: C, 52.35; H, 9.15; N, 15.26;
Found: C, 52.41; H, 9.02; N, 15.22.
(2.0 mmol) triethylamine. The mixture was stirred 18 h
at 25 ꢀC and then 18 h at re¯ux. The mixture was cooled
to 25 ꢀC and partitioned between 15 mL of diethyl ether
and 15 mL of water. The organic phase was washed with
15 mL of saturated sodium bicarbonate solution and
then with 10 mL of brine. The solution was dried
(MgSO4), and the solvent was removed by evaporation
at reduced pressure. The residue was chromatographed
on silica gel (4±8% absolute ethanol in dichloro-
methane). Three fractions were obtained having Rf
values (silica gel, 6% absolute ethanol in dichloro-
methane, I2 visualization) of 0.57, 0.45 and 0.36,
respectively. The least polar material was further pur-
i®ed by silica gel chromatography (15% ethyl acetate in
dichloromethane). This material was recrystallized from
dichloromethane and hexanes. The white solid was col-
lected by ®ltration and dried in a stream of air to give
the title compoundꢀas 18 mg (11%) of a white solid
7.76 (d, J=8 Hz, 2H), 7.60±7.20 (m, 7H), 4.10 (broad s,
2H), 3.73 (broad s, 2H). IR 3276 (m), 1615 (s), 1575 (m),
1476 (m), 1450 (m), 1426 (s). MS (FAB) m/z (rel. inten-
sity) 333 (M+, 40), 338 (12), 337 (16), 336 (66), 335 (43),
334 (100), 332 (11), 330 (11), 105 (54), 77 (7). HRMS
(EI) calcd for C16H13Cl2N3O: 333.0436, found:
333.0441. Anal. calcd for C16H13Cl2N3O : C, 57.50; H,
3.92; N, 12.57. Found: C, 57.69; H, 3.90; N, 12.43.
tert-Butyl 2-benzoyl-2-{2-[(tert-butoxycarbonyl)amino]-
ethyl}-1-hydrazinecarboxylate (27). Benzoyl chloride
(0.49 mL, 4.22 mmol) was added to a 0 ꢀC solution of
1.16 g (4.22 mmol) 26 and 1.21 mL (15 mmol) pyridine in
50 mL of anhydrous THF. After 1.5 h the mixture was
partitioned between 100 mL of dichloromethane and
100 mL of deionized water. The aqueous phase was
washed with 10 mL of dichloromethane and the com-
bined organic extracts were washed with 50 mL of 1 M
aqueous KH2PO4 solution and then with 50 mL of
saturated aqueous sodium bicarbonate solution. The
solution was dried (Na2SO4). The solvent was evapo-
rated reduced pressure. The residue was puri®ed by
silica gel chromatography (25±30% ethyl acetate in cy-
clohexane) followed by recrystallization from 1 mL of
dichloromethane and the solution was diluted with
7 mL of hexanes. The product was collected by ®ltration
to give 1.09 g (68%) of a white solid, mp 126±127.5 ꢀC.
1H NMR (300 MHz, CDCl3) d 7.48 (d, J=7.7 Hz, 2H),
7.40±7.25 (m, 3H), 4.0±2.7 (broad, 4H), 1.43 (s, 9H),
1.25 (broad s, 9H). MS (EI) m/z (rel. intensity) 379
(M+, 0.1), 279 (9), 250 (9), 223 (28), 149 (24), 145 (9),
106 (8), 105 (100), 101 (39), 77 (21), 57 (83). Anal. calcd
for C19H29N3O5: C, 60.14; H, 7.70; N, 11.07. Found: C,
60.26; H, 7.76; N, 11.04.
1
having mp 160±162 C. H NMR (300 MHz, CDCl3) d
(2,4-Dichlorophenyl)(2-phenyl-4-morpholinyl)methanone
oxime (31). This compound was prepared using 3-phe-
nylmorpholine in place of methylamine in the procedure
for the preparation of 6. Mp 202±204 ꢀC (dec.). 1H
NMR (300 MHz, DMSO-d6) d 7.79 (d, J=8.0 Hz, 1H),
7.65±7.25 (m, 7H), 4.65±4.55 (m, 1H), 4.05±3.78 (m,
1H), 3.78±3.65 (m, 1H), 3.65±3.45 (m, 1H), 3.45±3.25
(m, 1H), 3.15±2.97 (m, 1H), 2.97±2.82 (m, 1H). MS
(FAB) m/z (rel. intensity) 351 (M+H, 100), 355 (10),
354 (14), 353 (64), 352 (26), 350 (10), 337 (14), 336 (5),
.
335 (23), 162 (5). Anal. calcd for C17H16Cl2N2O2 HCl:
C, 52.67; H, 4.42; N, 7.22. Found: C, 52.67; H, 4.60; N,
7.18.
(2,4-Dichlorophenyl)(6-phenyl-1,2-oxazinan-2-yl)metha-
nimine hydrochloride (33). Tetrahydrooxazine hydro-
chloride 32 (0.150 g, 0.752 mmol) was dissolved in
15 mL of water and treated with a large excess of 3.0 M
sodium hydroxide solution. The free base was extracted
into diethyl ether and the solvent was evaporated at
reduced pressure. The residue was dissolved in 1.5 mL
of methanol and treated with 0.257 g (0.740 mmol) of
the isothiouronium hydroiodide salt 13. After 15 min at
25 ꢀC the reaction mixture was partitioned between pH
10 phosphate bu€er and ethyl acetate. the organic phase
was dried (MgSO4) and the solvent was evaporated at
reduced pressure. The residue was puri®ed by silica gel
chromatography (1:1 ethyl acetate:hexanes). The resi-
due was dissolved in 10 mL of isopropanol and treated
with 2 mL of 3 N hydrochloric acid. The solvent was
evaporated in a stream of nitrogen and the residue was
dissolved in 3 mL of ethyl acetate. Diluting this solution
with diethyl ether gave a precipitate which was collected
by ®ltration and washed with 2:1 ethyl ether:ethyl ace-
tate. After drying at 70 ꢀC/20 torr for 16 h the product
N-(2-Aminoethyl)benzohydrazide dihydrochloride (28).
To a 50 mL round bottom ¯ask loaded with 0.45 g
(1.1 mmol) of solid 27 was added 15 mL of 25% metha-
nolic HCl. After stirring for 5 min a heavy white pre-
cipitate formed. After 3 additional hours the mixture
was diluted with 15 mL of diethyl ether and the solid
was collected by ®ltration. After drying at 20 torr/45 ꢀC
the product was obtained as 0.27 g (98%) of a white
solid, mp 175±178 ꢀC. 1H NMR (300 MHz, D2O) d
7.62±7.45 (m, 5H), 3.91 (broad s, 2H), 3.33 (broad s,
2H). MS (FAB) m/z (rel. intensity) 180 (M+H, 100),
360 (5), 359 (20), 334 (3), 181 (12), 179 (7), 165 (3), 163
.
(13), 148 (7), 105 (16). Anal. calcd for C9H13N3O
.
2
HCl 0.7 H2O: C, 40.83; H, 6.24; N, 15.87. Found: C,
40.74; H, 6.33; N, 16.07.
[3-(2,4-Dichlorophenyl)-5,6-dihydro-1,2,4-triazin-1(4H)-yl]-
(phenyl)methanone (29). A suspension of 0.120 g
(0.476 mmol)of 28 and 0.139 g (0.476 mmol) of 16 in
2 mL of anhydrous methanol was treated with 0.279 mL

J. A. Tucker et al. / Bioorg. Med. Chem. 8 (2000) 601±615
613
was obtained as 61 mg (23%) of a white solid, mp 219±
220 ꢀC. 1H NMR (DMSO-d6). The spectrum of this
compound suggests that two slowly interconverting
conformers are present in approximately equal
amounts. The integrations in the peak list have been
normalized to a single molecule. d 9.99 (s, 0.5H), 9.96 (s,
0.5H), 9.55 (s, 0.5H), 9.54 (s, 0.5H), 8.01 (dd, J=2.0,
6.5 Hz, 1H), 7.94 (d, J=8.5 Hz, 0.5H), 7.82 (d,
J=6.5 Hz, 0.5H), 7.75 (dd, J=2.0, 8.5 Hz, 0.5H), 7.73
(dd, J=2.0, 8.5 Hz, 0.5H), 7.57±7.38 (m, 5H), 5.38 (d,
J=8.8 Hz, 0.5H), 5.16 (d, J=10.6 Hz, 0.5H), 3.70±3.55
(m, 2H), 2.20±1.75 (m, 4H). MS (FAB) m/z (rel. inten-
sity) 335 (M+H, 100), 671 (3), 669 (2), 340 (2), 339 (11),
338 (13), 337 (66), 336 (21), 301 (2), 131 (10). HRMS
(FAB) calcd for C17H16Cl2N2O+H1 335.0718, found
acid chloride was prepared from the corresponding
carboxylic acid in the following manner. One drop of
anhydrous dimethylformamide was added to
a
mixture of the appropriate carboxylic acid (2.44 mmol),
anhydrous methylene chloride (5 mL), and oxalyl
chloride (2.44 mmol). Within a few seconds of this
addition, gas evolution was observed. The mixture was
stirred 4 h, and the resulting clear solution was then
added to a solution of a hydroxyamidine (2.44 mmol)
and triethylamine (6.0 mmol) in anhydrous tetra-
hydrofuran (10 mL). The mixture was stirred 24 h at
25 ꢀC, and then the product was isolated as described in
Procedure B.
Crystallography
.
335.0714. Anal. calcd for C17H16Cl2N2O HCl: C,
54.93; H, 4.61; N, 7.54. Found: C, 54.58; H, 4.66; N,
7.48.
Crystal data for 36. C19H16N2O2; formula wt.=304.4;
monoclinic; space group P 21 Z=2; a=5.194(5),
b=11.115(13), c=13.209(17)A, b=91.67(6), V=762(1)A3;
calculated density=1.33 g cm 3; absorption coecient
m=0.62 mm 1; clear, thick plate 0.08Â0.23Â0.23±0.28
mm crystallized from acetone and H2O and mounted on
a glass ®ber. The total number of re¯ections measured
was 6649; there were 1624 unique re¯ections; 1307 had
intensities >3s; Rint was 0.056.
Acyloxyamidines 34±79 were prepared by the general
1
procedures below. Mass spectral data, H NMR data
and combustion analysis results were compatible with
the assigned structures.
Conversion of nitriles to hydroxyamidines (Procedure A).
A solution of the nitrile in anhydrous 3.1 M methanolic
NH2OH (2.0 molar equivalents) was heated at re¯ux
overnight. After cooling to room temperature, the solu-
tion was concentrated to dryness. The residual solid was
dissolved in 3 M hydrochloric acid and the solution was
®ltered to remove insoluble material. The acid solution
was washed with ether and then neutralized with solid
K2CO3, which caused a solid to precipitate. The solu-
tion was allowed to stand for at least 20 min, after which
the solid was collected via vacuum ®ltration and washed
with water. The product was dried overnight in a 60 ꢀC
vacuum oven. The product obtained was used in the
next step without further characterization.
Crystal data for 55. C15H12N2O2Cl2; formula wt.=
323.2; monoclinic; space group
P 21 /c, Z=4;
a=5.225(1), b=21.739(2), c=13.372(2)A, b=106.08(6),
3
V=1459(1)A3; calculated density=1.47 g cm
;
absorption coecient m=0.45 mm 1; clear needle 0.08Â
0.13Â0.62 mm crystallized from acetone and H2O and
mounted on a glass ®ber. The total number of re¯ec-
tions measured was 7899; there were 2552 unique
re¯ections; 1754 had intensities >3s; Rint was 0.061.
Crystal data for 48. C18H12N2O2Cl2; formula wt.=
359.2; monoclinic; space group P 21, Z=2; a=7.275(6),
b=9.559(3), c=11.449(7)A, b=92.55(9), V=798(1)A3;
calculated density=1.50 g cm 3; absorption coecient
m=0.37 mm 1; clear prism 0.19Â0.11Â0.4 mm crystal-
lized from chloroform and mounted on a glass ®ber.
The total number of re¯ections measured was 7724;
there were 1837 unique re¯ections; 1600 had intensities
>3s; Rint was 0.047.
Acylation of hydroxyamidines (Procedure B). To a mix-
ture of a hydroxyamidine (2.44 mmol) and triethylamine
(3.0 mmol) in anhydrous tetrahydrofuran (8 mL) was
added the appropriate acid chloride (2.44 mmol) all at
once. The mixture was stirred 24 h at 25 ꢀC and then it
was diluted with an organic solvent (50 mL of ethyl
acetate, dichloromethane, or chloroform) and a satu-
rated aqueous solution of sodium bicarbonate (50 mL).
The organic phase was washed with brine and then it
was dried with magnesium sulfate. The solution was ®l-
tered and the solvent was evaporated at reduced pres-
sure. Except where otherwise noted, the following
procedure was used to purify the product. The material
was dissolved in a minimum volume of warm acetone
(typically 15 mL) and cyclohexane was added until the
solution became cloudy. The resulting mixture was
stored at 5 ꢀC for 12±48 h, and the crystals which
formed were collected by ®ltration. The solid was
washed with cyclohexane (5 mL), and dried in a stream
of air.
General. Intensity data for all three structures were
measured at low temperature, 120 ꢀC, on a Mar ima-
ging plate area detector from Area Detector Systems
Corp., Poway, CA with a molybdenum long ®ne focus
sealed tube X-ray source and graphite monochromator,
(loK)=0.7107A). The resolution was 0.77A; 2ymax=55.
Data from 300 mm images using 5ꢀ oscillations in phi at
a distance of 100.75 mm were reduced and corrected
using the Denzo data processing package.27 Exposure
times were 3 min/image for 48, and 4 min/image for 36
and 55. Cell parameters are means and standard devia-
tions of determinations from 36 independent images.
All three structures were solved by direct methods using
SHELXS86.28 Least squares re®nements included coor-
dinates for all atoms and anisotropic thermal para-
meters for nonhydrogen atoms. Temperature factors for
Acylation of hydroxyamidines (Procedure C). This pro-
cedure was used in cases in which the required acid
chloride is not commercially available. The required

614
J. A. Tucker et al. / Bioorg. Med. Chem. 8 (2000) 601±615
hydrogens were assigned as one-half unit higher than
the equivalent isotropic temperature factors for the
attached carbon. The function minimized in the re®ne-
poration was linear (30 min). Ten microlitres of strepta-
vidin±SPA beads (20 mg/mL in PBS/10% glycerol) were
added following termination of the reaction. Plates were
incubated 10 min at 37 ꢀC, then equilibrated to room
temperature, and counted on a Packard Topcount.
Linear regressions were performed and IC50's calculated
using computer software.
Ã2
2
ment was ÆwꢂFo2 Fc † , where weights w were
Ã2
2
1=ꢀꢂ ꢂFo2† and Fc was as de®ned by Larson,29 except
for 55 (no secondary extinction parameter was con-
sidered necessary for that re®nement). In the ®nal cycles
all shifts were <0.3s. Atomic form factors were from
Doyle and Turner,30 and, for hydrogen, from Stewart,
Davidson and Simpson.31 The CRYM32 system of
computer programs was used for the re®nements. All
three structures exhibit a similar hydrogen bonding
pattern involving one hydrogen of the primary amine
substituent and the carbonyl oxygen in a symmetry
related molecule.
References and Notes
1. Levine, A. J. Viruses; Scienti®c American Library: New
York, 1992; pp 67±86.
2. Krech, U.; Jung, M.; Jung, F. Cytomegalovirus Infections of
Man; Karger A.G: New York, 1971.
3. Mustafa, M. M. Pediatr. Infect. Disease J. 1994, 13, 249.
4. Jacobson, M. A.; Mills, J. Ann. Intern. Med. 1988, 108, 585.
5. Jabs, D. A.; Enger, C.; Barlett, A. G. Arch. Ophthalmol.
1989, 107, 75.
Final agreement parameters for 36 were: R=0.050 for
1623 re¯ections, (one low angle re¯ection was given zero
weight in the re®nement), and 0.040 for the 1307 re¯ec-
tions having Fo2ꢃ3s; standard deviation of ®t=2.0.
Translations in x relate hydrogen bonded molecules;
N±O distance 2.983(3)A.
6. Glenn, J. Rev. Infectious Dis. 1981, 3, 1151.
7. Rubin, R. H. Rev. Infectious Dis. 1990, 12 (Suppl. 7), S754.
8. Frisch, M. J.; Head-Gordon, M.; Defrees, D. J.; Fox, D. J.;
Whiteside, R. A.; Seeger, R.; Melius, C. F.; Baker, J.; Martin,
R.; Kahn, L. R.; Stewart, J. J. P.; Fluder, E. M.; Topiol, S.;
Pople, J. A. Gaussian 88. Pittsburg, PA: Gaussiann, 1988.
9. Hehre, W. J.; Radom, L.; Schleyer, P. V. R.; Pople, J. A. Ab
Initio Molecular Orbital Theory. John Wiley and Sons: New-
York, 1986.
Final agreement parameters for 55 were: R=0.073 for
2550 re¯ections, (two low angle re¯ections were given
zero weight in the re®nement), and 0.047 for the 1754
re¯ections having Fo2ꢃ3s; standard deviation of
®t=2.0. Translations in x relate hydrogen bonded
molecules; N±O distance 2.947(3)A.
10. The distance between the carbonyl oxygen and the endo
hydrogen of the -NH2 group is 1.92 A in this structure. The
H±N±CˆN and OˆC±O±N torsion angles are distorted from
0ꢀ in a way which brings the carbonyl oxygen and endo
hydrogen closer together ( 20ꢀ and 24ꢀ, respectively), sug-
gesting that the interaction between these two atoms is attrac-
tive and not repulsive at this separation. The -NH2 group is
also slightly pryamidalized in a way that suggests an attractive
Final agreement parameters for 48 were: R=0.041 for
all 1837 re¯ections, and 0.035 for the 1600 re¯ections
having Fo2ꢃ3s; standard deviation of ®t=1.82. In this
case, the primary amine hydrogen forms a bifurcated
hydrogen bond. It is approximately equidistant from
the carbonyl oxygen and the imino nitrogen in the
symmetry related molecule at 1-x, y-0.5, 2.0-z. (N±O
distance: 3.018(3) A; N±N distance: 2.968(3)A); H±O
distance: 2.16(3)A; H±N distance: 2.28(3)A.
...
O
H interaction.
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13. Hackman, J. T.; Harthoorn, P. A. British Patent 949371.
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1980, 45, 3918.
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1951, 16, 3.
17. Travlos, S. D.; White, J. Acta Cryst. C. 1994, 50, 1631.
18. Chern, J.-W.; Wise, D. S.; Butler, W.; Townsend, L. B. J.
Org. Chem. 1988, 53, 5622.
Biology
The HCMV polymerase inhibition assay was performed
using a scintillation proximity assay (SPA). Reactions
were performed in 96-well plates. The assay was
conducted in 100 mL volume with 5.4 mM HEPES
(pH 7.5), 11.7 mM KCl, 4.5 mM MgCl2, 0.36 mg/mL
19. Debitus, C.; Cesario, M.; Guilhem, J.; Pascard, C; Pais, M.
Tetrahedron Lett. 1989, 1535.
3
BSA, and 90 nM H-dTTP and 2 mM (3-[(3-cholamido-
propyl)-dimethylammonio]-1-propane-sulfonate. HCMV
polymerase was diluted in enzyme dilution bu€er con-
taining 50% glycerol, 250 mM NaCl, 10 mM HEPES
(pH 7.5), 100 mg/mL BSA, 0.01% sodium azide and
added at 10% (or 10 mL) of the ®nal reaction volume.
Compounds were diluted in 50% DMSO and 10 mL
were added to each well. Control wells contained an
equivalent concentration of DMSO. Reactions were
initiated via the addition of 6 nM biotinylated poly(dA)-
oligo(dT) template/primer to reaction mixtures con-
taining the enzyme, substrate, and compounds of inter-
est. Plates were incubated in a 25 or 37 ꢀC H2O bath and
terminated via the addition of 40 mL/reaction of 0.5 M
EDTA (pH 8) per well. Reactions were terminated
within the time-frame during which substrate incor-
20. Sokol, V. I.; Davidov, V. V.; Porai-Koshits, M. A.; Zait-
sev, B. E.; Palishkin, M. V.; Sheban, G. V.; Pakhomov, V. I.;
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