Imidazole-4,5-dicarboxamide derivatives with antiproliferative activity against HL-60 cells

Article abstract of DOI:10.1021/jm050160r

A series of N,N′-disubstituted imidazole-4,5-dicarboxamides (I45DCs) were prepared and tested in order to determine their antiproliferative activity against HL-60 cells. The design of the I45DCs was based in part on the structures of trisubstituted purine

Full text of DOI:10.1021/jm050160r

J. Med. Chem. 2005, 48, 5955-5965
5955
Imidazole-4,5-dicarboxamide Derivatives with Antiproliferative Activity against
HL-60 Cells
Elisabeth M. Perchellet,† Jean-Pierre Perchellet,† and Paul W. Baures*,‡
Anti-Cancer Drug Laboratory, Kansas State University, Division of Biology, Ackert Hall, Manhattan, Kansas 66506-4901, and
Department of Chemistry, Bowdoin College, 6600 College Station, Brunswick, Maine 04011
Received February 18, 2005
A series of N,N′-disubstituted imidazole-4,5-dicarboxamides (I45DCs) were prepared and tested
in order to determine their antiproliferative activity against HL-60 cells. The design of the
I45DCs was based in part on the structures of trisubstituted purines complexed with cyclin
dependent kinase 2 (cdk2), a protein important in regulating the G1/S transition in the cell
cycle, and the intramolecular hydrogen bond in I45DCs that predisposes the conformation to
one that mimics substituted adenosines. A majority of the I45DCs in this study inhibit
proliferation of HL-60 cells as measured by an MTS mitochondrial functional assay with IC50’s
in the 2.5-25 µM range. The SAR of the I45DCs is consistent with anticipated hydrogen bonding
interactions in the ATP-binding site of cdk2. Thus, the I45DCs represent a useful scaffold for
anticancer lead discovery that is both readily accessible and easily diversified.
Introduction
tors across structural classes finds that hydrogen bond-
ing between the inhibitor and backbone N-H and Cd
O functionalities is a common feature between the
inhibitor classes.11 These hydrogen bonds have been
prominently featured in structure-based drug design
projects against kinase targets.23,24 A portion of the
hydrogen bonding interactions to ATP,25 olomoucine,26
purvalanol B,27 and flavopiridol28 in the cdk2 binding
site are illustrated in Figure 1.
This paper describes derivatives of the imidazole-4,5-
dicarboxylic acid (I45DA) scaffold that exhibit antipro-
liferative activity against HL-60 cells. We reasoned that
inhibition of kinases important to the cell cycle could
be inferred through cell antiproliferative activity mea-
surements, much as recently reported for MCS-C2.29
The N,N′-disubstituted imidazole-4,5-dicarboxamides
(I45DCs) in this study are capable of forming intermo-
lecular pairwise hydrogen bonds that could serve as an
“anchor” within an ATP binding site of a kinase, in a
similar manner as established for olomoucine and
purvalanol, and knew that pairwise hydrogen bonds
were significant interactions in the observed solid-state
packing arrangements of similar I45DCs.30 We chose
substituents for our starting I45DCs on the basis of
olomoucine and purvalanol B, as well as known struc-
tural SAR patterns from the literature.31,32 Modifica-
tions to the starting set of I45DCs were designed in
order to probe the binding site for favorable interactions,
identify sterically accessible sites, and determine whether
the HL-60 antiproliferative activity data for the I45DCs
paralleled expectations of cdk2 binding.
Cancer cells share certain characteristics irrespective
of their origin, the tissue involved, or the stage of
growth, including the ability to grow unchecked.1
A
cancer cell acquires such immortality through mecha-
nisms that sidetrack those biochemical pathways that
normally check cellular viability and control growth.2
For example, the cell cycle operates through a series of
checks and balances that determine whether a cell can
proceed to the next stage or whether the cell is damaged
and should instead abort division.3 One such checkpoint
in the cell cycle transition between the initial growth
phase (G1) and the synthesis phase (S) requires the
interplay of cyclins and cyclin-dependent kinases (cdk),
including an association between cyclin E and cdk2.4-6
Kinases form one of the largest protein families in
the human genome and have an important role in signal
transduction within the cell.7,8 As noted above, kinases
are at the center of events that control the cell cycle
and as such are valued targets for cancer.9-13 There
remains an open question about whether specific or
nonspecific kinase inhibitors will ultimately prove to be
the more valuable in treating cancer.9 Thus, there is a
need for new kinase inhibitor classes to help guide
optimal selection of clinical candidates.14 This is par-
ticularly relevant as questions have arisen regarding
the ability of cells to either bypass or substitute for the
role of cdk2 in the cell cycle.15-17 On the other hand,
flavopiridol was discovered as a cdk2 inhibitor and is
showing value in the clinical treatment of cancer,18
although this may be through a combination of effects.19
Results and Discussion
Many compound classes interfere with kinase activity
by binding competitively to the ATP binding site.11,20-22
X-ray crystallography of kinase complexes with inhibi-
Chemistry. The I45DCs in this study were prepared
from 1, a compound that contains both reactive acid
chloride and acyl imidazole functionality. This com-
pound is prepared from imidazole-4,5-dicarboxylic acid
by a method previously reported.33 The synthesis of
N,N′-dissymmetrically disubstituted I45DCs with only
primary alkanamines requires the formation of a phenyl
* To whom correspondence should be addressed. Current address:
Department of Chemistry, University of Tulsa, Tulsa, OK 74104.
Phone: 918-631-3024. E-mail: paul-baures@utulsa.edu.
† Kansas State University.
‡ Bowdoin College.
10.1021/jm050160r CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/23/2005

5956 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Perchellet et al.
Scheme 1a
a (a) H2NC6H3R1R2, N,N-diethylaniline, EtOAc; (b) H2NCHR3R4,
EtOAc; (c) HNR5R6, EtOAc.
I45DCs by addition of an alkanamine or free amino acid
ester. The final products were purified by silica gel
chromatography before characterization by 1H and 13
C
NMR spectroscopy, mass spectrometry, and RP-HPLC.
Antiproliferative Activity Measurements. Com-
pounds 9-36 were tested for their antiproliferative
activity against HL-60 cells at 2 and 4 days (Table 1).
Briefly, the I45DCs were dissolved in DMSO, serially
diluted, and added to HL-60 cells in 1 µL aliquots. The
cells were incubated for 2 or 4 days before determining
their mitochondrial ability to bioreduce the tetrazolium
dye MTS to a formazan product that absorbs at 490
nm.35 The absorbance was corrected for blank values
from culture medium alone, and the results analyzed
as previously reported.36
In selecting the starting I45DC, we chose a 3,4-
dichloroaniline substituent rather than the 3-chloro-4-
carboxyaniline present in purvalanol B. This substitu-
tion seemed reasonable since the absence of the carboxy
group yields purvalanol A, a compound that is also a
kinase inhibitor.27,37 In addition, purvalanol B is inac-
tive on cells, possibly due to an inability to cross cell
membranes with the presence of a charged functional
group.38 The second I45DC substituent was then varied
in order to obtain a structure-activity relationship at
the R3 position of this scaffold. The initial compound,
9, bears an alkylamine as a partial mimic of the second
Figure 1. Comparison of hydrogen bonding interactions
between protein kinase backbone amides (labeling is shown
according to cdk2 structures) with (A) ATP (B) olomoucine,
(C) purvalanol B, and (D) flavopiridol. In all examples the
protein kinase and inhibitor associate through a combination
of strong (OH‚‚‚O, NH‚‚‚O, and NH‚‚‚N) and relatively weak
hydrogen bonds (CH‚‚‚O).
ester intermediate in order to effectively differentiate
the two different reactive functionalities of 1.33 On the
other hand, amino acid esters afford enough selectivity
between the acid chloride and acyl imidazole reactivity
in order to be useful for the synthesis of N,N′-dissym-
metrically disubstituted I45DCs bearing amino acid
esters.34 In similar fashion, anilines react selectively to
yield 2-8, intermediates that were then reacted with
alkanamines or amino acid esters to yield the product
I45DCs, 9-36, used for this study (Scheme 1).
Pyrazines 2-8 were obtained from the addition of the
appropriate aniline (2 equiv) and N,N-diethylaniline (2
equiv) that serves as a scavenger for the acid produced
in the reaction. These are heterogeneous reactions and
the product solids are isolated by vacuum filtration
before partial purification through a series of washings.
The pyrazines were then used to synthesize the desired

Cytostatic Imidazole-4,5-dicarboxamides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 5957
Table 1. Antiproliferative Activity of N,N′-Dissymmetrically Disubstituted I45DCs in HL-60 Cells
HL-60 cells: IC50 (µM)
compound
R1
R2
R3
day 2a
day 4a
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
3-ClC6H4
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH2CH(CH3)2
14.7 ( 0.9
33.1 ( 1.9
14.6 ( 1.0
11.5 ( 0.5
17.7 ( 1.3
16.2 ( 1.5
21.4 ( 0.9
24.3 ( 1.0
7.0 ( 0.4
7.2 ( 0.8
12.3 ( 1.1
7.8 ( 0.5
7.4 ( 0.4
13.2 ( 0.8
8.8 ( 0.3
15.2 ( 0.4
18.3 ( 1.6
2.9 ( 0.3
5.9 ( 0.6
5.9 ( 0.6
25.4 ( 2.8
cyclo(C6H11
(CH2)5CH3
CH2Ph
)
CH2CH2Ph
CH2-4-Cl-Ph
CH2-4-CH3-Ph
CH2-4-OCH3-Ph
CH2-4-Ph-Ph
(R)-CHCH3Ph
8.8 ( 0.3
(S)-CHCH3Ph
9.2 ( 0.4
CH2CO2C(CH3)3
49.8 ( 5.2
(R)-CHCH3CO2CH3
(R)-CHCH3CO2C(CH3)3
(S)-CHCH3CO2C(CH3)3
(R)-CHPhCO2CH3
(S)-CHPhCO2CH3
(R)-CH(CH(CH3)2)CO2CH3
(S)-CH(CH(CH3)2)CO2CH3
(R)-CHCH3CO2C(CH3)3
(R)-CHCH3CO2C(CH3)3
(R)-CHCH3CO2C(CH3)3
(R)-CHCH3CO2C(CH3)3
(R)-CHCH3CO2C(CH3)3
(R)-CHCH3CO2C(CH3)3
CH2CH2CH3
>62.5
>62.5
4.6 ( 0.5
4.9 ( 0.3b
17.3 ( 1.4
13.2 ( 0.7
8.5 ( 0.7c
2.5 ( 0.2
2.4 ( 0.1b
10.8 ( 0.7
6.8 ( 0.7
4.9 ( 0.3d
>62.5
>62.5
8.2 ( 0.7e
6.6 ( 0.5f
6.3 ( 0.4f
6.0 ( 0.5g
6.3 ( 0.5g
4.7 ( 0.4b
378.6 ( 25.1
75.5 ( 3.2
89.4 ( 3.0
2.9 ( 0.3b
3.3 ( 0.3g
3.5 ( 0.3f
3.1 ( 0.3h
3.7 ( 0.4f
2.7 ( 0.1b
38.9 ( 3.4
56.0 ( 3.4
68.8 ( 2.1
4-ClC6H4
4-CH3C6H4
4-OCH3C6H4
4-CO2CH2CH3-C6H4
C6H5
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl2C6H3
CH2CH2CH3
CH3,
CH3,
(R)-CHCH3Ph
(S)-CHCH3Ph
a Concentrations of I45DCs required to inhibit by 50% the metabolic activity of human HL-60 cells at days 2 and 4 days in vitro. Cell
proliferation results (means ( SD, n ) 3) were expressed as % of the net absorbance of MTS/formazan after bioreduction by vehicle-
treated control cells after 2 (A490 nm ) 1.192 ( 0.051, 100 ( 4.3%) and 4 days (A490 nm ) 1.235 ( 0.057, 100 ( 4.6%) in culture. The blank
values (A490 nm ) 0.368 at day 2 and 0.382 at day 4) for cell-free culture medium supplemented with MTS:PMS reagent were subtracted
from the results. IC50 values were calculated from linear regression of the slopes of the log-transformed concentration-survival curves.
b Not different from the respective effects of 22 at days 2 and 4. c P < 0.005, d P < 0.0005, f P < 0.01, g P < 0.025, and h P < 0.05
greater than the effects of 22 at days 2 and 4. e P < 0.01, smaller than the effects of 12 at day 2.
substituent in purvalanol B, but without an added
stereocenter. This compound was less active (IC50 ) 7.2
( 0.8 µM) on HL-60 cells than the average growth
inhibitory activity found for purvalanol A (2 µM) over
the NCI 60 tumor cell lines,21 although it was active
enough to generate optimism about improving the
bioactivity through modification.
A cyclic and linear alkanamine were tested in 10 and
11, respectively, with both compounds showing anti-
proliferative activity against HL-60 cells, but with 10
seemingly less active than either 9 or 11. A benzylamine
analogue, 12, is equipotent to 9 and 11, although a
longer chain (13) and most 4-substituted benzylamines
(14-16) yielded analogues of either weaker or compa-
rable activity. There is an exception found in 17, which
is substituted with 4-phenylbenzylamine. Although this
compound represented an improvement in biological
activity (IC50 ) 2.9 ( 0.3 µM) over 9-16, this substitu-
tion significantly increases the overall size and hydro-
phobicity of the molecule, and we chose to continue
exploring changes to the overall substituent.
of these analogues. Purvalanol A is very similar to
purvalanol B, but simply excludes the carboxyl sub-
stituent off the aromatic ring of purvalanol B. It is
interesting that the two stereoisomers of purvalanol A
inhibit the malarial parasite, P. falciparum, with nearly
identical bioactivities, but have a 15-fold difference in
bioactivity on an isolated CDK1/cyclin B complex.39 The
same antiproliferative activities of 18 and 19 can be
attributed to the lack of a distinguishing interaction
between the stereocenter and the target protein. How-
ever, it is also possible that multiple targets or other
distinctions for the two enantiomers simply yield coin-
cidental inhibition of HL-60 cell proliferation. It is not
possible to distinguish between these possibilities from
the available data.
The structure-activity development of the I45DC was
continued with a series of amino acid esters, 20-27,
having similarities to the D-valinol substituent in the
purvalanols. Of these, the I45DCs with the two stereo-
isomers of alanine tert-butyl ester, 22 and 23, are the
most cytostatic at 4 days (IC50 ≈ 2.5 µM). Glycine tert-
butyl ester is 10× less active at 4 days (IC50 ) 25.4 (
2.8 µM). Both stereoisomers of phenylglycine methyl
ester are less cytostatic (R: IC50 ) 10.8 ( 0.7 µM; S:
IC50 ) 6.8 ( 0.7 µM), as is the R-isomer of valine methyl
ester (IC50 ) 4.9 ( 0.3 µM). The R-isomer of alanine
The introduction of a chiral R-methylbenzylamine
generates a stereocenter in 18 and 19, compounds with
identical antiproliferative activity that is also compar-
able to 12. Purvalanol B contains an analogous stereo-
center and was part of the incentive for the synthesis

5958 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Perchellet et al.
bonding interactions between inhibitor and protein help
favor and direct binding. This includes hydrogen bonds
from CH groups, as recently shown in the inhibition of
glycogen synthase kinase 3.42
The similar cytotoxicities of 22, 23, and 29-33 are
likely a sign of steric and electronic tolerance within the
binding site(s). We reason that the aniline can either
accept a hydrogen bonding interaction from a Lys
ꢀ-NH3+ to its substituent(s) or form NH‚‚‚π interactions
with the ring. On the other hand, the cytotoxicities of
34-36 are 5-11 times less active than comparable
I45DCs in this study (i.e., 34 versus 9, 35 versus 18,
and 36 versus 19). These provide additional evidence
in support of this potential target and binding interac-
tion, as the antiproliferative activity differences between
35 and 18, for example, is explained in terms of the
N-methyl group blocking pairwise hydrogen bonding of
the I45DC with Leu83 in cdk2 (Figure 2). The observed
log unit difference in activity can be attributed to the
loss of a hydrogen bond to the Leu83 carbonyl oxygen,
potentially leaving 34-36 to form a single hydrogen
bond to the protein target, analogous to that observed
for the pyridinylimidazole class of P38 MAP kinase
inhibitors.20 Alkylation of the imidazole ring in 22 would
also favor a single I45DC conformation, and in this case
the intramolecular hydrogen bond donor is on the same
side as the alkylated nitrogen.41 Thus, short alkyl chains
on the same side of the imidazole ring as the aniline
substituent could provide more potent compounds, as
these chains compare with the isopropyl group in
purvalanol A/B. These substitutions will be part of
future investigations in the I45DC class.
Figure 2. Potential binding mode of 22 with the cdk2 binding
site.
methyl ester was inactive at 62.5 µM, as was the
S-isomer of valine methyl ester. We hypothesize that
this reflects hydrolysis of the methyl esters by action of
esterases or peptidases in the HL-60 cells. Although
more likely to recognize the S-amino acids as substrates,
the small side chain of the R-alanine may be tolerated
in a peptidase active site. Likewise, the nonnatural
phenylglycine side chain in 27 could reasonably preclude
productive binding. The sterically hindered tert-butyl
esters in 20 or 23 would be poor substrates regardless
of the amino acid side chain or stereochemistry.
We next made modifications to the 3,4-dichloroaniline
while retaining the R-alanine tert-butyl ester, yielding
29-33. All of these aniline variations have approxi-
mately equal antiproliferative activity on HL-60 cells
(IC50’s ) 2.7-3.5 µM) at 4 days. This suggests steric
and electronic tolerance within the binding site(s) in
order to accommodate varying substituents on the
aniline of the I45DCs used in this study, although it is
also possible that the aniline is all or partly solvent
exposed when bound. A detailed SAR around the aniline
could produce selectivity against purified targets or
enhanced potency on whole cells or both.
Comparative Antiproliferative Activity Mea-
surements. Based on their increasing ability to inhibit
HL-60 cell proliferation at 2 and 4 days, the antitumor
activity of I45DC derivatives is a combination of drug
concentration and duration of action (Table 1 and Figure
3). In contrast to olomoucine (IC50: 108.6 µM at day 2
and 77.9 µM at day 4), which is the least effective of
the reference cdk2 inhibitors tested against HL-60 cell
proliferation, 22 (IC50: 4.6 µM at day 2 and 2.5 µM at
day 4) is an interesting cytostatic agent at least as good
as, or even slightly better than, purvalanol A (IC50: 11.3
µM at day 2 and 4.8 µM at day 4), although it does not
match the superior antiproliferative activity of flavopiri-
dol (IC50: 129 nM at day 2 and 82 nM at day 4) in this
tumor cell system (Figure 3). Overall, the antiprolifera-
tive activity of 22 is about 30.5-35.6 times lower than
that of flavopiridol but 1.9-2.5 and 23.6-31.2 times
higher than those of purvalanol A and olomoucine,
respectively (Figure 3). The 842-950-fold difference
between the antiproliferative activities of olomoucine
and flavopiridol observed in our HL-60 study is in
agreement with the report that flavopiridol shows
cytostatic and cytotoxic effects at the same concentration
that is required in vitro for cdk inhibition (10-7 range),
whereas 10-100 times higher concentrations of olomou-
cine are required for cell growth suppression than those
needed to inhibit cdk activity in vitro.43
Many of the I45DCs 9-33 have two intramolecularly
hydrogen bonded conformations evident in their 1H
NMR spectra taken in either CDCl3 or DMSO-d6. The
ratio of conformers, when observable, is generally 7:3
or 8:2, and favors the aniline as the hydrogen bond
donor (Scheme 1). This ratio was assigned from the
relative integrations of the sharp singlet aniline NH
(hydrogen bonded or non-hydrogen bonded), the broad
imidazole NH, and the split amide NH (either doublets
or triplets depending upon the substitution). The NMR
data is consistent with Spartan40 calculations using the
PM3 force field that finds a -1.07 kcal mol-1 energy
difference favoring this conformer. The intramolecular
hydrogen bond for a representative I45DC has recently
been determined to be worth 14 ( 1 kcal mol-1 at 3 mM
in DMSO-d6.41 This intramolecular hydrogen bond was
integral to our hypothesis for bioactivity in this series
and yields an overall I45DC structure for 22 that, in
the less favorable conformer, is comparable to purval-
anol A. With this information, it is possible to hypoth-
esize about potential bound conformation(s) of 22, and
to use this information in subsequent designs. Our
expected interactions with a target ATP binding site,
such as found in cdk2, is shown in Figure 2. Three other
potential binding modes were discounted on the basis
of steric interference from substituents, and one formed
hydrogen bonding combinations to the protein target
that would be inconsistent with the aniline as the
intramolecular hydrogen bond donor (see Supporting
Information). The SAR data for 9-33 is consistent with
Figure 2, where the aniline is the intramolecular
hydrogen bond donor. The intermolecular hydrogen
Inhibition of DNA Synthesis. In HL-60 cells incu-
bated for 2.5 h with 22, the concentration-dependent
inhibition of DNA synthesis starts at 1.6 µM and is
maximal at 25 µM (Figure 4A). Compound 22 (IC50: 4.13
µM) inhibits DNA synthesis to a lesser degree than

Cytostatic Imidazole-4,5-dicarboxamides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 5959
Figure 3. Comparison of the abilities of serial concentrations
(plotted on a logarithmic scale) of 22 (b), flavopiridol (9),
purvalanol A (2) and olomoucine (1) to inhibit the metabolic
activity of HL-60 cells at days 2 (A) and 4 (B) in vitro. Cell
proliferation results were expressed as % of the net absorbance
of MTS/formazan after bioreduction by vehicle-treated control
cells after 2 (A490 nm ) 1.429 ( 0.055, 100 ( 3.8%, striped area
in A) and 4 (A490 nm ) 1.332 ( 0.067, 100 ( 5.1%, striped area
in B) days in culture. The blank values (A490 nm ) 0.347 at day
2 and 0.346 at day 4) for cell-free culture medium supple-
mented with MTS:PMS reagent were subtracted from the
results. Bars: means ( SD (n ) 3). aNot different from
respective controls; bP < 0.05, cP < 0.025, dP < 0.01 and eP <
Figure 4. I45DC derivatives inhibit DNA synthesis in HL-
60 cells. A, Comparison of the abilities of serial concentrations
(plotted on a logarithmic scale) of 22 (b), flavopiridol (9),
purvalanol A (2) and olomoucine (1) to inhibit the rate of
incorporation of [3H]-thymidine into DNA measured in HL-
60 cells over 30 min following a 2 h period of incubation at 37
°C in vitro. DNA synthesis in vehicle-treated control cells at
37 °C was 14,399 ( 1,526 cpm (100 ( 10.6%, striped area).
The blank value (721 ( 67 cpm) for control cells incubated
and pulse-labeled at 2 °C with 1 µC of [3H]-thymidine has been
subtracted from the results. Bars: means ( SD (n ) 3). aNot
f
0.005, smaller than respective controls; P < 0.005, smaller
than control and P < 0.05, smaller than 4 µM purvalanol A;
gP < 0.005, smaller than control and P < 0.025, smaller than
1.6 µM purvalanol A; hP < 0.005, smaller than 4 µM purvalanol
A.
b
c
different from control; P < 0.01, P < 0.05 and dP < 0.005,
smaller than control. B, Comparison of the abilities of 4 (open
columns), 10 (striped columns) and 25 µM (closed columns)
concentrations of selected I45DCs to inhibit DNA synthesis
in HL-60 cells at 2.5 h. After subtracting the blank (858 ( 84
cpm), the value of the untreated control is 12,427 ( 1,056 cpm
flavopiridol (IC50: 0.96 µM) but is almost as effective
as purvalanol A (IC50: 2.14 µM) and much more potent
than olomoucine (IC50: 46.8 µM). As inhibitor of DNA
synthesis, therefore, 22 is 11.3 times more potent than
olomoucine and only 1.9 and 4.3 times less effective than
purvalanol A and flavopiridol, respectively (Figure 4A).
Although it may be somewhat misleading to compare
IC50 values for responses measured at very different
times, the abilities of 22, purvalanol A, flavopiridol, and
olomoucine to inhibit DNA synthesis at 2.5 h (Figure
4A) generally match their antiproliferative activities in
the HL-60 cell system at 2-4 days (Figure 3), suggesting
that their effectiveness as inhibitors of DNA synthesis
may play a major role in their mechanism of antitumor
activity. Interestingly, at concentrations greater than
4 µM, 22 and purvalanol A both appear to inhibit DNA
synthesis at 2.5 h (Figure 4A) and HL-60 cell prolifera-
tion at day 2 (Figure 3A) to a greater degree than
flavopiridol. Since this effect disappears at day 4 (Figure
3B), high concentrations of 22 and purvalanol A may
reach their maximal inhibitory potential more rapidly
than those of flavopiridol. In addition, because tumor
cells undergoing replicative senescence are as sensitive
to flavopiridol as rapidly dividing metastasized cancer
cells, the mechanism of flavopiridol action may not be
dependent on the activity of cyclin-dependent kinases
that regulate cell cycle progression.44
a
b
(100 ( 8.5%, striped area). Not different from control; P <
0.05, smaller than control; cP < 0.01, smaller than control and
d
P < 0.05, greater than 4 µM 22; P < 0.05, smaller than 10
µM 23; eP < 0.05, smaller than 10 µM 31 and P < 0.005,
f
greater than 10 µM 22; P < 0.0005, smaller than 25 µM 23;
gNot different from 25 µM 22 but P < 0.005, smaller than 25
µM 31.
The effectiveness of selected I45DC derivatives against
DNA synthesis in HL-60 cells (Figure 4B) was compared
to that of 22, since the antitumor effects of this lead
compound (Table 1) on DNA synthesis (Figure 4A) and
tumor cell proliferation (Figure 3) nearly match those
of the reference cdk2 inhibitor purvalanol A. With some
exception, all I45DC derivatives decrease in a concen-
tration-dependent manner the rate of incorporation of
3H-thymidine into DNA at 2.5 h in relation with their
antiproliferative activity at 2-4 days, substantiating the
link between these two inhibitory events. On an equimo-
lar concentration basis, 22 is clearly the most potent
and 33 nearly matches its effect, whereas 26 is the least
effective of the selected I45DCs tested against DNA
synthesis (Figure 4B). It should be noted that 22 and
33 are the only compounds that significantly reduce
DNA synthesis at 4 µM and totally abolish this process
at 25 µM. The other I45DCs share intermediate poten-

5960 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Perchellet et al.
The resulting solid was allowed to air-dry and was used
without further purification. In some cases the product retains
trace N,N-diethylaniline through the wash steps.
cies, although compounds 29 and 31 inhibit DNA
synthesis more effectively than compound 23 at 25 µM.
Taken together, these results suggest that synthetic
I45DCs, such as 22 and 33, which can inhibit DNA
synthesis (Figure 4) and tumor cell proliferation (Table
1 and Figure 3) in the same µM range as purvalanol A,
might be valuable to develop new means of polychemo-
therapy.
N,N′-Di-3,4-dichlorophenyl-5,10-dioxo-5H,10H-diimi-
dazo[1,5-a:1′-5′-d]pyrazine-1,6-dicarboxamide (2). Syn-
thesized from 0.750 g of 1 (2.40 mmol), 0.81 mL of N,N-
diethylaniline (5.03 mmol), and 1.00 g of 3,4-dichloroaniline
(6.17 mmol) in 10 mL of ethyl acetate, stirring 16 h to yield
1.19 g of crude 2 (88% yield) as a tan solid: mp 257-258 °C
(dec); 1H NMR (DMSO-d6) δ 11.01 (s, 2 H), 9.11, 9.10 (s, 2 H),
8.10-8.30 (m, 2 H), 7.64-7.80 (m, 4 H); 13C NMR (DMSO-d6)
δ 159.2, 158.2, 148.5, 143.9, 138.3, 135.3, 131.0, 130.6, 128.6,
125.7, 121.1, 120.9, 120.0.
Conclusion
The I45DCs tested in this study are cytostatic to HL-
60 cells at 2 and 4 days, with potencies for the most
active being comparable to those described for initial
lead compounds in other classes under investiga-
tion.27,29,37,38,45 The observed antiproliferative activities
of the I45DCs do not confirm a specific mechanism of
action or target protein. Nonetheless, the SAR of the
I45DCs hydrogen bonding features is consistent with
our hypothesis that these interactions may direct the
I45DC binding to an ATP site of either cdk2 or similar
kinases.
N,N′-Di-3-chlorophenyl-5,10-dioxo-5H,10H-diimidazo-
[1,5-a:1′-5′-d]pyrazine-1,6-dicarboxamide (3). Synthesized
from 0.163 g of 1 (0.521 mmol), 167 µL of N,N-diethylaniline
(1.04 mmol), and 110 µL of 3-chloroaniline (1.04 mmol) in 3
mL of ethyl acetate, stirring 2 h to yield 0.218 g of crude 3
1
(85% yield) as a light yellow solid: mp 250-252 °C (dec); H
NMR (DMSO-d6) δ 10.89 (s, 2 H), 9.11 (s, 2 H), 7.99, 7.93 (m,
2 H), 7.64-7.72 (m, 2 H), 7.41-7.45 (m, 2 H), 7.21-7.26 (m, 2
H); 13C NMR (DMSO-d6) δ 160.2, 159.1, 145.3, 140.6, 139.4,
138.6, 136.4, 133.9, 131.4, 129.6, 125.6, 124.9, 121.7, 120.3,
119.4.
The I45DCs are an easily accessible and useful
structural class for anticancer drug discovery. It is
expected that the I45DCs can be optimized with respect
to either antiproliferative activity against a given tumor
cell line or for inhibition against an isolated protein
target such as a kinase.
N,N′-Di-4-chlorophenyl-5,10-dioxo-5H,10H-diimidazo-
[1,5-a:1′-5′-d]pyrazine-1,6-dicarboxamide (4). Synthesized
from 0.168 g of 1 (0.537 mmol), 172 µL of N,N-diethylaniline
(1.07 mmol), and 0.137 g of 4-chloroaniline (1.07 mmol) in 3
mL of ethyl acetate, stirring 2 h to yield 0.227 g of crude 4
(85% yield) as a red solid: mp 250-252 °C (dec); 1H NMR
(DMSO-d6) δ 10.85 (s, 2 H), 9.09 (s, 2 H), 7.79-7.85 (m, 4 H),
7.45-7.47 (m, 4 H); 13C NMR (DMSO-d6) δ 159.1, 157.9, 148.6,
144.4, 138.3, 137.0, 135.9, 135.3, 128.6, 128.5, 127.8, 122.7,
121.4, 120.4.
Experimental Section
Synthesis and Purification of I45DCs. General Meth-
ods. All apparatus were oven-dried and cooled in a desiccator.
Reagent grade solvents and other reagents were purchased
from commercial suppliers and used without purification.
Thin-layer chromatography was done on 250 µm silica gel
plates containing a fluorescent indicator and were visualized
by using UV and I2 as well as ninhydrin spray for amines.
Melting points are uncorrected. 1H and 13C NMR spectra were
measured at 400 and 100.6 MHz, respectively, in either CDCl3
with CHCl3 as the internal reference for 1H (δ 7.26) and CDCl3
as the internal reference for 13C (δ 77.06) or in DMSO-d6 with
DMSO as the internal reference for 1H (δ 2.50) and DMSO-d6
as the internal reference for 13C (δ 39.50). Mass spectroscopy
was done on a HiResMALDI Fourier transform mass spec-
trometer (IonSpec, Irvine, CA) with a Cryomagnetics (Oak
Ridge, TN) 4.7 T actively shielded superconducting magnet.
Ions were generated by MALDI with a pulsed nitrogen laser
at 337 nm and the spectra were calibrated from 2,5-dihydroxy-
benzoic acid matrix peaks.
Homogeneity of 9-36 was determined by RP-C18 HPLC
analysis. Samples were prepared at a concentration of 1 mg/
mL in methanol, except for 10 and 14-17, which were less
soluble and therefore diluted from their DMSO-d6 NMR
solutions into methanol in order to yield approximately 1 mg/
mL samples (e 10% DMSO-d6). Five or fifteen microliters of
the sample was eluted on a C18-RP analytical column with
one of two different gradients of acetonitrile/water at a 1 mL/
min flow rate. Compounds were detected at 218 nm. The two
gradients used were as follows: method A, 0 min., 1:1 CH3-
CN/H2O f 10 min., 19:1 CH3CN/H2O f 12 min., CH3CN f
14 min., CH3CN f 15 min., 1:1 CH3CN/H2O; method B, 0 min.,
1:1 CH3CN/H2O f 10 min., 19:1 CH3CN/H2O f 12 min., CH3-
CN f 29 min., CH3CN f 30 min., 1:1 CH3CN/H2O.
N,N′-Di-4-methylphenyl-5,10-dioxo-5H,10H-diimidazo-
[1,5-a:1′-5′-d]pyrazine-1,6-dicarboxamide (5). Synthesized
from 0.500 g of 1 (1.60 mmol), 0.56 mL of N,N-diethylaniline
(3.51 mmol), and 0.342 g of p-toluidine (3.19 mmol) in 10 mL
of dichloromethane, stirring 3 h to yield 0.651 g of crude 5
(90% yield) as a red solid. Dichloromethane was also used
1
instead of ethyl acetate for the first wash: mp > 270 °C; H
NMR (DMSO-d6) δ 10.64 (s, 2 H), 9.08 (s, 2 H), 7.61-7.82 (m,
4 H), 7.18-7.26 (m, 4 H), 2.28 (s, 6H); 13C NMR (DMSO-d6) δ
160.2, 136.3, 130.1, 129.6, 126.9, 21.4.
N,N′-Di-4-methoxyphenyl-5,10-dioxo-5H,10H-diimidazo-
[1,5-a:1′-5′-d]pyrazine-1,6-dicarboxamide (6). Synthesized
from 0.500 g of 1 (1.60 mmol), 0.56 mL of N,N-diethylaniline
(3.51 mmol), and 0.392 g of p-anisidine (3.19 mmol) in 10 mL
of dichloromethane, stirring 3 h to yield 0.792 g of crude 6
(102% yield) as a violet solid. Dichloromethane was also used
1
instead of ethyl acetate for the first wash: mp > 270 °C; H
NMR (DMSO-d6) δ 9.11 (s, 2 H), 8.20 (s, 2 H), 7.61-7.72 (m,
4 H), 6.92-7.00 (m, 4 H), 3.75 (s, 6H); 13C NMR (DMSO-d6) δ
159.0, 135.3, 129.4, 113.8, 113.7, 55.1.
N,N′-Di-4-(ethoxycarbonyl)phenyl-5,10-dioxo-5H,10H-
diimidazo[1,5-a:1′-5′-d]pyrazine-1,6-dicarboxamide (7).
Synthesized from 0.500 g of 1 (1.60 mmol), 0.56 mL of N,N-
diethylaniline (3.51 mmol), and 0.528 g of ethyl 4-aminoben-
zoate (3.19 mmol) in 10 mL of dichloromethane, stirring 3 h
to yield 0.848 g of crude 7 (95% yield) as a tan solid.
Dichloromethane was also used instead of ethyl acetate for
1
the first wash: mp 262-266 °C (dec); H NMR (DMSO-d6) δ
9.16 (s, 2 H), 8.35 (s, 2 H), 7.95-8.07 (m, 4 H), 4.32-4.36 (m,
4 H), 1.35-1.39 (m, 6H); 13C NMR (DMSO-d6) δ 165.1, 159.2,
141.7, 135.4, 130.0, 128.5, 120.3, 60.5, 14.1.
N,N′-Diphenyl-5,10-dioxo-5H,10H-diimidazo[1,5-a:1′-5′-
d]pyrazine-1,6-dicarboxamide (8). Synthesized from 0.500
g of 1 (1.60 mmol), 0.56 mL of N,N-diethylaniline (3.51 mmol),
and 290 µL of aniline (3.19 mmol) in 10 mL of dichlo-
romethane, stirring 3 h to yield 0.695 g of crude 8 (102% yield)
as an orange solid. Dichloromethane was also used instead of
ethyl acetate for the first wash: mp 250-254 °C (dec); 1H NMR
(DMSO-d6) δ 10.48 (s, 2 H), 8.88, 8.85 (s, 2 H), 7.51-7.56 (m,
Aniline-Substituted Pyrazines (2-8). General Proce-
dure. To a round-bottom flask was suspended 1 in either ethyl
acetate or dichloromethane. To this stirred suspension was
added all at once N,N-diethylaniline (2.0-2.2 equiv.) followed
by the primary aniline (2.0-2.6 equiv.). The reaction mixture
was stirred at room temperature for 2-16 h before collecting
the colored solid by vacuum filtration. The product was washed
with portions of solvent, cold water, and acetone, respectively.

Cytostatic Imidazole-4,5-dicarboxamides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 5961
4 H), 7.14-7.18 (m, 4 H), 6.90-6.95 (m, 2 H); 13C NMR
(DMSO-d6) δ 128.9, 120.0.
hydrogen bonded conformational isomers are observed in the
1H NMR spectrum. 1H NMR (CDCl3) δ 13.66 (s, 0.7 H), 12.12
(bs, 0.3 H), 11.65 (bs, 0.7 H), 11.33-11.37 (m, 0.3 H), 9.49 (s,
0.3 H), 8.12 (s, 0.7 H), 8.02-8.09 (m, 0.7 H), 7.94 (m, 0.3 H),
7.55-7.62 (m, 2 H), 7.31-7.43 (m, 6 H), 4.68-4.71 (m, 2 H);
13C NMR (CDCl3) δ 163.5, 156.8, 137.8, 137.3, 133.5, 130.6,
128.9, 128.7, 127.9, 127.8, 127.5, 122.0, 43.5; MALDI HR-
FTMS calcd for C18H14O2N4Cl2Na m/z 411.0386 [M + Na]+,
found 411.0388 [M + Na]+.
N,N′-Disubstituted-imidazole-4,5-dicarboxamides (9-
36). General Procedure. To a round-bottom flask was
suspended an aniline-substituted pyrazine in ethyl acetate.
To this stirred suspension was added all at once a solution of
a free amine in ethyl acetate. The free amine, in the case of
the amino acid ester salts, was obtained by extraction with a
stoichiometric amount of 5 M KOH. The extracted ethyl
acetate layer was washed with brine and dried over anhydrous
MgSO4. The reaction mixture was stirred at room temperature
14-60 h. In most cases the product was purified on SiO2 with
ethyl acetate/hexanes as the eluant.
1H-Imidazole-4,5-dicarboxylic Acid 5-[(3,4-Dichloro-
phenyl)amide] 4-Phenethylamide (13). Synthesized from
74 mg of 2 (0.13 mmol) and 41 µL of phenethylamine (0.33
mmol) in 2 mL of ethyl acetate, stirring the reaction mixture
for 16 h. The product was purified by column chromatography
on SiO2 with ethyl acetate as the eluant to yield 41 mg of 13
(38% yield) as a white solid: mp 187-190 °C; TLC Rf (ethyl
acetate/hexane 1:1) ) 0.54; HPLC (method A) 12.5 min;
Intramolecular hydrogen bonded conformational isomers are
1H-Imidazole-4,5-dicarboxylic Acid 5-[(3,4-Dichloro-
phenyl)amide] 4-(2-Methylpropyl)amide (9). Synthesized
from 65 mg of 2 (0.12 mmol) and 34 µL of isobutylamine (0.35
mmol) in 2 mL of ethyl acetate, stirring the reaction mixture
for 60 h. The product was purified by column chromatography
on SiO2 with ethyl acetate as the eluant to yield 11 mg of 9
(13% yield) as a white solid: mp 192-195 °C; TLC Rf (ethyl
acetate/hexane 1:1) ) 0.35; HPLC (method A) 12.4 min;
Intramolecular hydrogen bonded conformational isomers are
1
1
observed in the H NMR spectrum. H NMR (CDCl3) δ 13.70
(s, 0.7 H), 12.50 (bs, 0.3 H), 12.25 (bs, 0.7 H), 10.89-10.91 (m,
0.3 H), 9.40 (s, 0.3 H), 8.06 (s, 0.7 H), 7.95 (s, 0.3 H), 7.75-
7.78 (m, 0.7 H), 7.55-7.56 (m, 1 H), 7.16-7.33 (m, 7 H), 3.63-
3.71 (m, 2 H), 2.87-2.94 (m, 2 H); 13C NMR (CDCl3) δ 163.6,
161.6, 158.6, 157.1, 138.5, 137.8, 136.8, 133.8, 133.0, 132.7,
129.5, 129.2, 129.0, 128.5, 128.3, 128.0, 127.6, 121.9, 121.8,
40.8, 35.7; MALDI HR-FTMS calcd for C19H16O2N4Cl2Na m/z
425.0543 [M + Na]+, found 425.0533 [M + Na]+.
1
1
observed in the H NMR spectrum. H NMR (CDCl3) δ 13.76
(s, 0.7 H), 12.02 (bs, 0.3 H), 11.73 (bs, 0.7 H), 10.84 (t, J ) 6.0
Hz, 0.3 H), 9.51 (s, 0.3 H), 8.13 (s, 0.7 H), 7.96 (s, 0.3 H), 7.82
(t, J ) 6.0 Hz, 0.7 H), 7.40-7.68 (m, 3 H), 3.31-3.37 (m, 2 H),
1.90-2.03 (m, 1 H), 1.02-1.06 (m, 6 H); 13C NMR (CDCl3) δ
163.6, 157.0, 137.9, 136.9, 135.2, 133.8, 132.8, 130.5, 128.5,
127.6, 122.0, 119.7, 46.9, 28.6, 20.4, 20.2; MALDI HR-FTMS
calcd for C15H16O2N4Cl2Na m/z 377.0543 [M + Na]+, found
377.0552 [M + Na]+.
1H-Imidazole-4,5-dicarboxylic Acid 4-Chlorobenzyl-
amide 5-[(3,4-Dichlorophenyl)amide] (14). Synthesized
from 110 mg of 2 (0.20 mmol) and 53 µL of 4-chlorobenzy-
lamine (0.43 mmol) in 2 mL of ethyl acetate, stirring the
reaction mixture for 16 h. The product solid was collected by
vacuum filtration and washed with ethyl acetate. Crystalliza-
tion from ethyl acetate/methanol provided 85 mg of 14 (52%
yield) as a white solid: mp 218-219 °C; TLC Rf (ethyl acetate/
hexane 1:1) ) 0.47; HPLC (method B) 13.0 min; 1H NMR
(DMSO-d6) δ 13.82 (bs, 1 H), 9.53 (bs, 1 H), 8.08 (bs, 1 H),
7.98 (s, 1 H), 7.34-7.62 (m, 7 H), 4.52-4.53 (m, 2 H); 13C NMR
(DMSO-d6) δ 137.9, 133.5, 133.1, 131.4, 130.7, 129.1, 128.5,
128.3, 43.8; MALDI HR-FTMS calcd for C18H13O2N4Cl3Na m/z
444.9996 [M + Na]+, found 444.9998 [M + Na]+.
1H-Imidazole-4,5-dicarboxylic Acid 5-[(3,4-Dichloro-
phenyl)amide] 4-Methylbenzylamide (15). Synthesized
from 90 mg of 2 (0.16 mmol) and 45 µL of 4-methylbenzylamine
(0.35 mmol) in 2 mL of ethyl acetate, stirring the reaction
mixture for 16 h. The product solid was collected by vacuum
filtration and washed with ethyl acetate. Crystallization from
ethyl acetate/methanol provided 55 mg of 15 (43% yield) as a
white solid: mp 213-225 °C (dec); TLC Rf (ethyl acetate/
hexane 1:1) ) 0.55; HPLC (method A) 13.0 min; 1H NMR
(DMSO-d6) δ 13.90 (s, 1 H), 9.20 (s, 1 H), 8.04-8.06 (m, 1 H),
7.98 (s, 1 H), 7.05-7.66 (m, 7 H), 4.49-4.51 (m, 2 H), 2.07 (s,
3 H); 13C NMR (DMSO-d6) δ 137.8, 135.9, 135.7, 135.3, 130.9,
129.1, 129.0, 128.8, 128.7, 127.7, 127.1, 42.0; MALDI HR-
FTMS calcd for C19H16O2N4Cl2Na m/z 425.0543 [M + Na]+,
found 425.0545 [M + Na]+.
1H-Imidazole-4,5-dicarboxylic Acid 4-Cyclohexyl-
amide 5-[(3,4-Dichlorophenyl)amide] (10). Synthesized
from 79 mg of 2 (0.14 mmol) and 40 µL of cyclohexylamine
(0.35 mmol) in 2 mL of ethyl acetate, stirring the reaction
mixture for 14 h. The solids were dissolved with methanol and
heat. The off-white solid obtained following crystallization was
recrystallized from ethyl acetate and methanol. Vacuum
filtration gave 46 mg of 10 (43% yield) as an amorphous white
solid: mp 235-237 °C; TLC Rf (ethyl acetate/hexane 1:1) )
0.41; HPLC (method B) 14.4 min; 1H NMR (DMSO-d6) δ 13.50
(bs, 1 H), 8.75 (bs, 1 H), 8.12 (s, 1 H), 7.95 (s, 1 H), 7.60-7.66
(m, 2 H), 3.78-3.90 (m, 1 H), 1.78-1.88 (m, 2 H), 1.67-1.76
(m, 2 H), 1.55-1.63 (m, 1 H), 1.28-1.47 (m, 4 H), 1.12-1.24
(m, 1 H); 13C NMR (DMSO-d6) δ 138.5, 136.0, 131.1, 130.8,
125.3, 123.0, 122.5, 47.8, 31.9, 25.0, 24.5; MALDI HR-FTMS
calcd for C17H18O2N4Cl2Na m/z 403.0699 [M + Na]+, found
403.0707 [M + Na]+.
1H-Imidazole-4,5-dicarboxylic Acid 5-[(3,4-Dichloro-
phenyl)amide] 4-n-Hexylamide (11). Synthesized from 94
mg of 2 (0.17 mmol) and 55 µL of 1-hexylamine (0.42 mmol)
in 2 mL of ethyl acetate, stirring the reaction mixture for 16
h. The product was purified by column chromatography on
SiO2 with ethyl acetate/hexanes as the eluant to yield 84 mg
of 11 (65% yield) as a white solid: mp 143-146 °C; TLC Rf
(ethyl acetate/hexane 1:1) ) 0.49; HPLC (method A) 15.3 min;
Intramolecular hydrogen bonded conformational isomers are
1H-Imidazole-4,5-dicarboxylic Acid 5-[(3,4-Dichloro-
phenyl)amide] 4-Methoxybenzylamide (16). Synthesized
from 89 mg of 2 (0.16 mmol) and 51 µL of 4-methoxybenzy-
lamine (0.39 mmol) in 2 mL of ethyl acetate, stirring the
reaction mixture for 16 h. The product solid was collected by
vacuum filtration. Crystallization from ethyl acetate/methanol
provided 87 mg of 16 (66% yield) as a white solid: mp 208-
209 °C; TLC Rf (ethyl acetate/hexane 1:1) ) 0.37; HPLC
1
1
observed in the H NMR spectrum. H NMR (CDCl3) δ 13.85
(s, 0.8 H), 12.82 (bs, 0.2 H), 12.69 (bs, 0.8 H), 10.86-10.88 (m,
0.2 H), 9.51 (s, 0.2 H), 8.12 (s, 0.8 H), 7.96 (s, 0.2 H), 7.75-
7.78 (m, 0.8 H), 7.60-7.62 (m, 1 H), 7.32-7.45 (m, 2 H), 3.40-
3.49 (m, 2 H), 1.62-1.68 (m, 2 H), 1.26-1.41 (m, 6 H), 0.89-
0.92 (m, 3 H); 13C NMR (CDCl3) δ 163.4, 161.7, 157.1, 137.9,
137.8, 134.0, 132.6, 129.6, 128.3, 127.9, 127.5, 121.7, 39.7, 31.5,
29.4, 22.6, 14.5, 13.5; MALDI HR-FTMS calcd for C17H20O2N4-
Cl2Na m/z 405.0856 [M + Na]+, found 405.0847 [M + Na]+.
1
(method A) 11.5 min; H NMR (DMSO-d6) δ 13.90 (bs, 1 H),
10.60 (bs, 1 H), 9.45 (bs, 1 H), 8.12 (s, 1 H), 7.98 (s, 1 H), 7.56-
7.62 (m, 2 H), 7.28-7.30 (m, 1 H), 6.89-6.99 (s, 1 H), 7.48-
7.50 (m, 2 H), 3.72, 3.76 (s, 3 H); 13C NMR (DMSO-d6) δ 159.1,
158.1, 135.2, 130.6, 130.1, 128.5, 126.0, 113.7, 113.5, 59.5, 55.0,
54.8, 20.5, 13.8; MALDI HR-FTMS calcd for C19H16O3N4Cl2-
Na m/z 441.0492 [M + Na]+, found 441.0497 [M + Na]+.
1H-Imidazole-4,5-dicarboxylic Acid 4-Benzylamide
5-[(3,4-Dichlorophenyl)amide] (12). Synthesized from 64
mg of 2 (0.11 mmol) and 37 µL of benzylamine (0.34 mmol) in
2 mL of ethyl acetate, stirring the reaction mixture for 18 h.
The product was purified by column chromatography on SiO2
with ethyl acetate as the eluant to yield 17 mg of 12 (19% yield)
as a white solid: mp 204-205 °C; TLC Rf (ethyl acetate/hexane
1:1) ) 0.53; HPLC (method A) 11.9 min; Intramolecular
1H-Imidazole-4,5-dicarboxylic Acid 5-[(3,4-Dichloro-
phenyl)amide] 4-Phenylbenzylamide (17). Synthesized
from 79 mg of 2 (0.14 mmol) and 54 mg of 4-phenylbenzyl-

5962 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Perchellet et al.
amine (0.30 mmol) in 2 mL of ethyl acetate, stirring the
reaction mixture for 16 h. The product solid was collected by
vacuum filtration and washed with ethyl acetate to provide
61 mg of 17 (47% yield) as a white solid: mp 242-244 °C; TLC
Rf (ethyl acetate/hexane 1:1) ) 0.58; HPLC (method A) 14.3
min; Intramolecular hydrogen bonded conformational isomers
are observed in the 1H NMR spectrum. 1H NMR (DMSO-d6) δ
13.90 (bs, 0.7 H), 13.59 (bs, 1 H), 9.53 (bs, 0.7 H), 8.89 (s, 0.3
H), 8.11 (m, 0.7 H), 8.00 (s, 1 H), 7.33-7.65 (m, 12 H), 4.60-
4.62 (m, 2 H); 13C NMR (DMSO-d6) δ 139.9, 138.9, 138.1, 128.9,
127.9, 127.3, 126.7, 126.6, 42.0; MALDI HR-FTMS calcd for
C24H18O2N4Cl2Na m/z 487.0699 [M + Na]+, found 487.0703 [M
+ Na]+.
132.8, 130.6, 130.5, 128.9, 128.7, 128.1, 127.7, 121.9, 121.8,
119.6, 119.4, 52.7, 52.5, 49.1, 48.2, 18.3, 17.6; MALDI HR-
FTMS calcd for C15H14O4N4Cl2Na m/z 407.0284 [M + Na]+,
found 407.0290 [M + Na]+.
(R)-2-{[5-(3,4-Dichlorophenylcarbamoyl)-1H-imidazole-
4-carbonyl]amino}propionic Acid tert-Butyl Ester (22).
Synthesized from 81 mg of 2 (0.14 mmol) and 83 mg of (R)-
alanine tert-butyl ester hydrochloride (0.46 mmol, neutralized)
in 2 mL of ethyl acetate, stirring the reaction mixture for 16
h. The product was purified by column chromatography on
SiO2 with ethyl acetate/hexanes as the eluant to yield 33 mg
of 22 (27% yield) as a white solid: mp 189-191 °C; TLC Rf
(ethyl acetate/hexane 1:1) ) 0.45; HPLC (method A) 12.8 min;
Intramolecular hydrogen bonded conformational isomers are
(R)-1H-Imidazole-4,5-dicarboxylic Acid 5-[(3,4-Dichlo-
rophenyl)amide] 4-[(1-Phenylethyl)amide] (18). Synthe-
sized from 46 mg of 2 (0.08 mmol) and 23 µL of (R)-R-
methylbenzylamine (0.18 mmol) in 2 mL of ethyl acetate,
stirring the reaction mixture for 16 h. The product was purified
by column chromatography on SiO2 with ethyl acetate as the
eluant to yield 42 mg of 18 (64% yield) as a white solid: mp
196-199 °C; TLC Rf (ethyl acetate/hexane 1:1) ) 0.28; HPLC
(method A) 12.6 min; Intramolecular hydrogen bonded con-
1
1
observed in the H NMR spectrum. H NMR (CDCl3) δ 13.57
(s, 0.8 H), 12.42 (bs, 0.2 H), 12.11 (bs, 0.8 H), 11.30 (d, J ) 8.0
Hz, 0.2 H), 9.55 (s, 0.2 H), 8.21 (d, J ) 8.0 Hz, 0.8 H), 8.10 (s,
0.8 H), 7.96 (s, 0.2 H), 7.26-7.70 (m, 3 H), 4.63-4.66 (m, 1.6
H), 4.08-4.11 (m, 0.4 H), 1.26-1.60 (m, 12 H); 13C NMR
(CDCl3) δ 171.4, 171.3, 163.2, 161.5, 157.0, 137.8, 136.8, 135.5,
133.5, 133.0, 132.8, 130.6, 130.5, 129.0, 128.8, 127.6, 121.9,
121.8, 119.6, 82.5, 82.0, 48.9, 28.1, 28.0, 18.5; MALDI HR-
FTMS calcd for C18H20O4N4Cl2Na m/z 449.0754 [M + Na]+,
found 449.0760 [M + Na]+.
1
formational isomers are observed in the H NMR spectrum.
1H NMR (CDCl3) δ 13.72 (s, 0.7 H), 12.70 (bs, 0.3 H), 12.32
(bs, 0.7 H), 11.43-11.44 (m, 0.3 H), 9.53 (s, 0.3 H), 8.12 (s, 0.7
H), 7.97-8.02 (m, 1 H), 7.25-7.50 (m, 8 H), 5.29-5.35 (m, 0.7
H), 5.19-5.22 (m, 0.3 H), 1.63-1.67 (m, 3 H); 13C NMR (CDCl3)
δ 162.8, 161.8, 158.1, 157.1, 142.5, 137.8, 136.8, 133.8, 133.0,
132.7, 130.7, 130.5, 129.4, 128.9, 128.7, 128.0, 127.7, 126.0,
122.0, 119.7, 50.4, 49.0, 23.0, 22.4; MALDI HR-FTMS calcd
for C19H16O2N4Cl2Na m/z 425.0543 [M + Na]+, found 525.0534
[M + Na]+.
(S)-2-{[5-(3,4-Dichlorophenylcarbamoyl)-1H-imidazole-
4-carbonyl]amino}propionic Acid tert-Butyl Ester (23).
Synthesized from 71 mg of 2 (0.13 mmol) and 69 mg of (S)-
alanine tert-butyl ester hydrochloride (0.38 mmol, neutralized)
in 2 mL of ethyl acetate, stirring the reaction mixture for 16
h. The product was purified by column chromatography on
SiO2 with ethyl acetate/hexanes as the eluant to yield 14 mg
of 23 (13% yield) as a white solid: The physical and spectral
data was identical with enantiomer 22.
(R)-2-{[5-(3,4-Dichlorophenylcarbamoyl)-1H-imidazole-
4-carbonyl]amino}phenylethanoic Acid Methyl Ester
(24). Synthesized from 82 mg of 2 (0.15 mmol) and 70 mg of
(R)-phenylglycine methyl ester hydrochloride (0.35 mmol,
neutralized) in 2 mL of ethyl acetate, stirring the reaction
mixture for 16 h. The product was purified by column chro-
matography on SiO2 with ethyl acetate/hexanes as the eluant
to yield 27 mg of 24 (23% yield) as a white solid: mp 199-203
°C; TLC Rf (ethyl acetate/hexane 1:1) ) 0.37; HPLC (method
A) 11.0 min; Intramolecular hydrogen bonded conformational
isomers are observed in the 1H NMR spectrum. 1H NMR
(CDCl3) δ 13.34 (s, 0.7 H), 12.33 (bs, 0.3 H), 11.97-11.99 (m,
1 H), 9.49 (s, 0.3 H), 8.59 (d, J ) 8.0 Hz, 0.7 H), 8.08 (s, 0.7
H), 7.94 (s, 0.3 H), 7.26-7.64 (m, 8 H), 5.74-5.79 (m, 1 H),
3.78, 3.81 (s, 3 H); 13C NMR (CDCl3) δ 170.7, 170.6, 163.1,
161.5, 156.8, 137.6, 135.8, 133.0, 132.7, 129.0, 128.5, 127.7,
121.9,; MALDI HR-FTMS calcd for C20H16O4N4Cl2Na m/z
469.0441 [M + Na]+, found 469.0440 [M + Na]+.
(S)-1H-Imidazole-4,5-dicarboxylic Acid 5-[(3,4-Dichlo-
rophenyl)amide] 4-[(1-Phenylethyl)amide] (19). Synthe-
sized from 55 mg of 2 (0.10 mmol) and 27 µL of (S)-R-
methylbenzylamine (0.22 mmol) in 2 mL of ethyl acetate,
stirring the reaction mixture for 16 h. The product was purified
by column chromatography on SiO2 with ethyl acetate as the
eluant to yield 43 mg of 19 (55% yield) as a white solid:
Physical and spectral data identical to enantiomer 18.
2-{[5-(3,4-Dichlorophenylcarbamoyl)-1H-imidazole-4-
carbonyl]amino}ethanoic Acid tert-Butyl Ester (20).
Synthesized from 62 mg of 2 (0.11 mmol) and 55 mg of glycine
tert-butyl ester hydrochloride (0.33 mmol, neutralized) in 2 mL
of ethyl acetate, stirring the reaction mixture for 18 h. The
product was purified by column chromatography on SiO2 with
ethyl acetate as the eluant to yield 14 mg of 20 (15% yield) as
a white solid: mp 212-213 °C; TLC Rf (ethyl acetate/hexane
1:1) ) 0.32; HPLC (method A) 11.6 min; Intramolecular
hydrogen bonded conformational isomers are observed in the
1H NMR spectrum. 1H NMR (CDCl3) δ 13.44 (s, 0.7 H), 12.00
(bs, 0.3 H), 11.67 (bs, 0.7 H), 11.20-11.23 (m, 0.3 H), 9.50 (s,
0.3 H), 8.13-8.16 (m, 0.7 H), 8.08 (s, 0.7 H), 8.00 (s, 0.3 H),
7.26-7.69 (m, 3 H), 4.15-4.18 (m, 2 H), 1.52, 1.53 (s, 9 H);
13C NMR (CDCl3) δ 168.5, 164.0, 138.2, 135.4, 133.1, 130.9,
129.2, 128.0, 124.1, 122.2, 119.9, 83.2, 42.4, 28.5; MALDI HR-
FTMS calcd for C17H18O4N4Cl2Na m/z 435.0597 [M + Na]+,
found 435.0588 [M + Na]+.
(R)-2-{[5-(3,4-Dichlorophenylcarbamoyl)-1H-imidazole-
4-carbonyl]amino}propionic Acid Methyl Ester (21).
Synthesized from 74 mg of 2 (0.13 mmol) and 56 mg of (R)-
alanine methyl ester hydrochloride (0.33 mmol, neutralized)
in 2 mL of ethyl acetate, stirring the reaction mixture for 16
h. The product was purified by column chromatography on
SiO2 with ethyl acetate as the eluant to yield 36 mg of 21 (36%
yield) as a white solid: mp 186-190 °C; TLC Rf (ethyl acetate/
hexane 1:1) ) 0.18; HPLC (method A) 9.3 min; Intramolecular
hydrogen bonded conformational isomers are observed in the
1H NMR spectrum. 1H NMR (CDCl3) δ 13.47 (s, 0.7 H), 12.48
(bs, 0.3 H), 12.16 (bs, 0.7 H), 11.35 (d, J ) 8.0 Hz, 0.3 H), 9.52
(s, 0.3 H), 8.18 (d, J ) 8.0 Hz, 0.7 H), 8.10 (s, 0.7 H), 7.96 (s,
0.3 H), 7.26-7.70 (m, 3 H), 4.65-4.88 (m, 2 H), 3.79, 3.82 (s,
3 H), 1.58-1.65 (m, 3 H); 13C NMR (CDCl3) δ 172.8, 172.6,
163.2, 161.6, 156.9, 137.7, 136.7, 135.6, 133.5, 133.2, 133.0,
(S)-2-{[5-(3,4-Dichlorophenylcarbamoyl)-1H-imidazole-
4-carbonyl]amino}phenylethanoic Acid Methyl Ester
(25). Synthesized from 80 mg of 2 (0.14 mmol) and 69 mg of
(S)-phenylglycine methyl ester hydrochloride (0.34 mmol,
neutralized) in 2 mL of ethyl acetate, stirring the reaction
mixture for 16 h. The product was purified by column chro-
matography on SiO2 with ethyl acetate/hexanes as the eluant
to yield 24 mg of 25 (20% yield) as a white solid: The physical
and spectral data was identical with enantiomer 24.
(R)-2-{[5-(3,4-Dichlorophenylcarbamoyl)-1H-imidazole-
4-carbonyl]-amino}-3-methylbutyric Acid Methyl Ester
(26). Synthesized from 77 mg of 2 (0.14 mmol) and 66 mg of
(R)-valine methyl ester hydrochloride (0.39 mmol, neutralized)
in 2 mL of ethyl acetate, stirring the reaction mixture for 16
h. The product was purified by column chromatography on
SiO2 with ethyl acetate/hexanes as the eluant to yield 47 mg
of 26 (41% yield) as a white solid: mp 130-135 °C; TLC Rf
(ethyl acetate/hexane 1:1) ) 0.27; HPLC (method A) 11.4 min;
Intramolecular hydrogen bonded conformational isomers are
1
1
observed in the H NMR spectrum. H NMR (CDCl3) δ 13.51
(s, 0.7 H), 12.50 (bs, 0.3 H), 12.24 (s, 0.7 H), 11.36 (d, J ) 8.0
Hz, 1 H), 9.54 (s, 0.3 H), 8.16 (d, J ) 8.0 Hz, 0.7 H), 8.13 (s,
0.7 H), 7.93 (s, 0.3 H), 7.26 - 7.68 (m, 3 H), 4.70-4.74 (m, 1

Cytostatic Imidazole-4,5-dicarboxamides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 5963
H), 3.79, 3.81 (s, 3 H), 2.32-2.45 (m, 1 H), 1.04 - 1.15 (m, 6
H); 13C NMR (CDCl3) δ 171.9, 171.7, 163.6, 161.5, 156.9, 137.8,
136.9, 135.5, 133.3, 132.9, 132.8, 130.5, 128.8, 128.0, 127.7,
122.0, 121.8, 119.6, 119.4, 57.4, 52.4, 52.2, 31.4, 19.2, 18.1, 18.0;
MALDI HR-FTMS calcd for C17H18O4N4Cl2Na m/z 435.0597
[M + Na]+, found 435.0585 [M + Na]+.
(S)-2-{[5-(3,4-Dichloro-phenylcarbamoyl)-1H-imidazole-
4-carbonyl]-amino}-3-methylbutyric Acid Methyl Ester
(27). Synthesized from 81 mg of 2 (0.14 mmol) and 80 mg of
(S)-valine methyl ester hydrochloride (0.47 mmol, neutralized)
in 2 mL of ethyl acetate, stirring the reaction mixture for 16
h. The product was purified by column chromatography on
SiO2 with ethyl acetate/hexanes as the eluant to yield 21 mg
of 27 (18% yield) as a white solid: The physical and spectral
data was identical with enantiomer 26.
17.9, 17.2; MALDI HR-FTMS calcd for C19H24O4N4Na m/z
395.1690 [M + Na]+, found 395.1700 [M + Na]+.
(R)-2-{[5-(4-Methoxyphenylcarbamoyl)-1H-imidazole-
4-carbonyl]amino}propionic Acid tert-Butyl Ester (31).
Synthesized from 99 mg of 6 (0.20 mmol) and 92 mg of (R)-
alanine tert-butyl ester hydrochloride (0.51 mmol, neutralized)
in 3 mL of ethyl acetate, stirring the reaction mixture for 24
h. The product was purified by column chromatography on
SiO2 with ethyl acetate/hexanes as the eluant to yield 90 mg
of 31 (58% yield) as a white solid: mp 146-151 °C; TLC Rf
(ethyl acetate/hexane 1:1) ) 0.58; HPLC (method A) 8.5 min;
Intramolecular hydrogen bonded conformational isomers are
1
1
observed in the H NMR spectrum. H NMR (CDCl3) δ 13.21
(s, 0.7 H), 12.79 (bs, 0.3 H), 12.67 (s, 0.7 H), 11.55 (d, J ) 8.0
Hz, 1 H), 9.35 (s, 0.3 H), 8.15 (d, J ) 8.0 Hz, 0.7 H), 7.52-7.65
(m, 3 H), 6.81-6.84 (m, 2 H), 4.55-4.65 (m, 1 H), 3.73, 3.72
(s, 3 H), 1.40-1.51 (m, 12 H); 13C NMR (CDCl3) δ 171.5, 171.4,
163.3, 161.7, 161.3, 158.8, 156.6, 156.5, 134.1, 133.0, 131.5,
131.4, 130.4, 129.4, 128.4, 122.1, 121.9, 121.8, 121.7, 114.8,
114.7, 113.9, 113.5, 82.2, 82.2, 81.7, 56.1, 55.8, 55.7, 55.2, 55.1,
54.8, 54.6, 49.2, 48.3, 28.6, 28.2, 27.8, 27.3, 18.3, 17.8; MALDI
HR-FTMS calcd for C19H24O5N4Na m/z 411.1639 [M + Na]+,
found 411.1651 [M + Na]+.
(R)-2-{[5-(3-Chlorophenylcarbamoyl)-1H-imidazole-4-
carbonyl]amino}propionic Acid tert-Butyl Ester (28).
Synthesized from 43 mg of 3 (0.087 mmol) and 47 mg of (R)-
alanine tert-butyl ester hydrochloride (0.26 mmol, neutralized)
in 2 mL of ethyl acetate, stirring the reaction mixture for 16
h. The product was purified by column chromatography on
SiO2 with ethyl acetate/hexanes as the eluant to yield 37 mg
of 28 (54% yield) as a white solid: mp 168-171 °C; TLC Rf
(ethyl acetate/hexane 1:1) ) 0.30; HPLC (method A) 11.2 min;
Intramolecular hydrogen bonded conformational isomers are
(R)-4-{[5-(1-tert-Butoxycarbonyl-ethylcarbamoyl)-3H-
imidazole-4-carbonyl]amino}benzoic Acid Ethyl Ester
(32). Synthesized from 83 mg of 7 (0.15 mmol) and 67 mg of
(R)-alanine tert-butyl ester hydrochloride (0.37 mmol, neutral-
ized) in 3 mL of ethyl acetate, stirring the reaction mixture
for 24 h. The product was purified by column chromatography
on SiO2 with ethyl acetate/hexanes as the eluant to yield 62
mg of 32 (48% yield) as a white solid: mp 128-130 °C; TLC
Rf (ethyl acetate/hexane 1:1) ) 0.62; HPLC (method A) 10.4
min; Intramolecular hydrogen bonded conformational isomers
are observed in the 1H NMR spectrum. 1H NMR (CDCl3) δ
13.67 (s, 0.7 H), 12.77 (bs, 0.3 H), 12.45 (s, 0.7 H), 11.40 (d, J
) 8.0 Hz, 1 H), 9.71 (s, 0.3 H), 8.25 (d, J ) 8.0 Hz, 0.7 H),
8.04-8.07 (m, 2 H), 7.82-7.87 (m, 2 H), 7.69-7.70 (m, 1 H),
4.62-4.72 (m, 1 H), 4.35-4.41 (m, 2 H), 1.52-1.61 (m, 9 H),
1.38-1.43 (m, 3 H); 13C NMR (CDCl3) δ 171.5, 171.3, 166.2,
166.1, 163.2, 161.6, 158.4, 157.2, 142.4, 141.4, 133.6, 133.3,
130.8, 130.7, 128.9, 126.3, 126.1, 119.5, 82.2, 82.3, 60.9, 28.6,
28.2, 27.7, 27.3, 17.8; MALDI HR-FTMS calcd for C18H22O4N4-
Na m/z 381.1533 [M + Na]+, found 381.1531 [M + Na]+.
(R)-2-{[5-(phenylcarbamoyl)-1H-imidazole-4-carbonyl]-
amino}propionic Acid tert-Butyl Ester (33). Synthesized
from 65 mg of 8 (0.15 mmol) and 69 mg of (R)-alanine tert-
butyl ester hydrochloride (0.38 mmol, neutralized) in 3 mL of
ethyl acetate, stirring the reaction mixture for 24 h. The
product was purified by column chromatography on SiO2 with
ethyl acetate/hexanes as the eluant to yield 74 mg of 33 (67%
yield) as a white solid: mp 166-169 °C; TLC Rf (ethyl acetate/
hexane 1:1) ) 0.66; HPLC (method A) 9.2 min; Intramolecular
hydrogen bonded conformational isomers are observed in the
1H NMR spectrum. 1H NMR (CDCl3) δ 13.42 (s, 0.7 H), 12.93
(bs, 0.3 H), 12.75 (s, 0.7 H), 11.58 (d, J ) 8.0 Hz, 1 H), 9.55 (s,
0.3 H), 8.26 (d, J ) 8.0 Hz, 0.7 H), 7.67-7.82 (m, 3 H), 7.15-
7.40 (m, 3 H), 4.65-4.74 (m, 1 H), 1.49-1.60 (m, 9 H), 1.38-
1.43 (m, 3 H); 13C NMR (CDCl3) δ 171.5, 171.4, 163.3, 161.5,
158.7, 157.0, 138.2, 137.2, 134.0, 133.2, 129.4, 129.2, 128.8,
128.6, 120.4, 120.3, 82.2, 81.8, 49.2, 48.4, 28.6, 28.2, 27.8, 27.3,
18.3, 17.8; MALDI HR-FTMS calcd for C21H26O6N4Na m/z
453.1745 [M + Na]+, found 453.1751 [M + Na]+.
1
1
observed in the H NMR spectrum. H NMR (CDCl3) δ 13.53
(s, 0.7 H), 12.65 (bs, 0.3 H), 12.40 (s, 0.7 H), 11.37 (d, J ) 8.0
Hz, 1 H), 9.54 (s, 0.3 H), 8.21 (d, J ) 8.0 Hz, 0.7 H), 8.01 (s,
0.7 H), 7.91 (s, 0.3 H), 7.11-7.70 (m, 4 H), 4.58-4.70 (m, 1
H), 1.51-1.60 (m, 12 H); 13C NMR (CDCl3) δ 171.4, 171.3,
163.2, 161.6, 158.5, 157.1, 139.5, 138.5, 135.6, 135.1, 134.8,
134.6, 133.7, 133.4, 130.0, 128.9, 124.7, 124.5, 120.4, 120.3,
118.4, 118.2, 82.4, 82.0, 49.9, 48.9, 28.1, 28.0, 18.5, 17.9;
MALDI HR-FTMS calcd for C18H21O4N4ClNa m/z 415.1144
[M + Na]+, found 415.1144 [M + Na]+.
(R)-2-{[5-(4-Chlorophenylcarbamoyl)-1H-imidazole-4-
carbonyl]amino}propionic Acid tert-Butyl Ester (29).
Synthesized from 53 mg of 4 (0.11 mmol) and 58 mg of (R)-
alanine tert-butyl ester hydrochloride (0.32 mmol, neutralized)
in 2 mL of ethyl acetate, stirring the reaction mixture for 16
h. The product was purified by column chromatography on
SiO2 with ethyl acetate/hexanes as the eluant to yield 28 mg
of 29 (33% yield) as a white solid: mp 174-175 °C; TLC Rf
(ethyl acetate/hexane 1:1) ) 0.26; HPLC (method A) 11.0 min;
Intramolecular hydrogen bonded conformational isomers are
1
1
observed in the H NMR spectrum. H NMR (CDCl3) δ 13.47
(s, 0.7 H), 12.46 (bs, 0.3 H), 12.05 (s, 0.7 H), 11.37 (d, J ) 8.0
Hz, 1 H), 9.47 (s, 0.3 H), 8.20 (d, J ) 8.0 Hz, 0.7 H), 7.65-7.75
(m, 3 H), 7.26-7.34 (m, 2 H), 4.60-4.70 (m, 1 H), 1.51-1.59
(m, 12 H); 13C NMR (CDCl3) δ 171.4, 171.3, 163.2, 161.6, 158.5,
157.1, 139.5, 138.5, 135.6, 135.1, 134.8, 134.6, 133.7, 133.4,
130.0, 128.9, 124.7, 124.5, 120.4, 120.3, 118.4, 118.2, 82.4, 82.0,
49.9, 48.9, 28.1, 28.0, 18.5, 17.9; MALDI HR-FTMS calcd for
C18H21O4N4ClNa m/z 415.1144 [M + Na]+, found 415.1155 [M
+ Na]+.
(R)-2-{[5-(4-Methylphenylcarbamoyl)-1H-imidazole-4-
carbonyl]amino}propionic Acid tert-Butyl Ester (30).
Synthesized from 90 mg of 5 (0.20 mmol) and 90 mg of (R)-
alanine tert-butyl ester hydrochloride (0.50 mmol, neutralized)
in 3 mL of ethyl acetate, stirring the reaction mixture for 24
h. The product was purified by column chromatography on
SiO2 with ethyl acetate/hexanes as the eluant to yield 65 mg
of 30 (46% yield) as a white solid: mp 165-168 °C; TLC Rf
(ethyl acetate/hexane 1:1) ) 0.67; HPLC (method A) 10.2 min;
Intramolecular hydrogen bonded conformational isomers are
1H-Imidazole-4,5-dicarboxylic Acid 5-[(3,4-Dichloro-
phenyl)amide] 4-Di-n-propylamide (34). Synthesized from
64 mg of 2 (0.11 mmol) and 47 µL of di-n-propylamine (0.34
mmol) in 2 mL of ethyl acetate, stirring the reaction mixture
for 48 h. The product was purified by column chromatography
on SiO2 with ethyl acetate/hexanes as the eluant to yield 21
mg of 34 (25% yield) as a white solid: mp 163-166 °C; TLC
Rf (ethyl acetate/hexane 1:1) ) 0.52; HPLC (method A) 12.7
min; 1H NMR (CDCl3) δ 13.22 (s, 1 H), 11.68 (s, 1 H), 8.10 (s,
1 H), 7.28-7.72 (m, 3 H), 3.87-3.91 (m, 2 H), 3.51-3.55 (m, 2
H), 1.69-1.77 (m, 4 H), 0.87-1.04 (m, 6 H); 13C NMR (CDCl3)
δ 165.1, 157.6, 137.9,135.6, 134.4, 132.7, 130.5, 129.9, 127.5,
1
1
observed in the H NMR spectrum. H NMR (CDCl3) δ 13.35
(s, 0.7 H), 12.80 (bs, 0.3 H), 12.67 (s, 0.7 H), 11.58 (d, J ) 8.0
Hz, 1 H), 9.47 (s, 0.3 H), 8.21 (d, J ) 8.0 Hz, 0.7 H), 7.66-7.70
(m, 3 H), 7.17-7.19 (m, 2 H), 4.61-4.71 (m, 1 H), 2.35, 2.34
(s, 3 H), 1.50-1.59 (m, 12 H); 13C NMR (CDCl3) δ 171.5, 171.4,
163.3, 161.4, 158.8, 156.8, 135.7, 134.4, 134.2, 134.1, 133.0,
130.1, 129.7, 129.3, 129.0, 128.5, 120.5, 120.4, 120.3, 120.2,
82.2, 81.7, 49.2, 48.4, 28.7, 28.6, 28.2, 27.5, 21.1, 19.1, 18.3,

5964 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Perchellet et al.
122.0, 119.7, 51.4, 49.6, 22.5, 20.8, 11.6, 11.1; MALDI HR-
FTMS calcd for C17H20O2N4Cl2Na m/z 405.0856 [M + Na]+,
found 405.0856 [M + Na]+.
DNA Synthesis. To estimate the rate of DNA synthesis,
HL-60 cells were resuspended in fresh FCS-containing RPMI
1640 medium at a density of 7.5 × 105 cells/0.5 mL, incubated
at 37 °C for 2 h in the presence or absence (control) of drugs
and then pulse-labeled for an additional 30 min with 1 µCi of
[methyl-3H]thymidine (50 Ci/mmol; Amersham, Arlington
Heights, IL). The incubations were terminated by the addition
of 0.5 mL of 10% trichloroacetic acid (TCA). After holding on
ice for 15 min, the acid-insoluble material was recovered over
Whatman GF/A glass microfiber filters and washed thrice with
2 mL of 5% TCA and twice with 2 mL of 100% EtOH. After
drying the filters, the radioactivity bound to the acid-precipi-
table material was determined by liquid scintillation counting
in 6 mL of Bio-Safe NA (Research Products International,
Mount Prospect, IL).36 Data of all biochemical experiments
were analyzed using Student’s t-test with the level of signifi-
cance set at P < 0.05.
Molecular Modeling: Spartan Calculations. The two
conformers of 22 were built independently in silico with an
intramolecular hydrogen bond in each and minimized with the
PM3 force field to their equilibrium geometries. This experi-
ment was repeated a total of 10 times for each conformation,
where the starting point for minimization was slightly varied
each time as a result of the building process. The conformer
with the aniline as the intramolecular hydrogen bond donor
was -1.07 ( 0.04 kcal mol-1 lower in energy, by this analysis,
than the conformer with the aniline as the hydrogen bond
acceptor. The amino acid torsion angles for each conformer in
all runs approximated the extended conformation.
(R)-1H-Imidazole-4,5-dicarboxylic Acid 5-[(3,4-Dichlo-
rophenyl)amide] 4-[Methyl(1-phenylethyl)amide] (35).
Synthesized from 76 mg of 2 (0.13 mmol) and 78 µL of (R)-
N,R-dimethylbenzylamine (0.54 mmol) in 2 mL of ethyl acetate,
stirring the reaction mixture for 60 h. The product was purified
by column chromatography on SiO2 with ethyl acetate/hexanes
as the eluant to yield 35 mg of 35 (31% yield) as a white solid:
mp 186-188 °C; TLC Rf (ethyl acetate/hexane 1:1) ) 0.27;
HPLC (method A) 11.9 min; Conformational isomers are
1
1
observed in the H NMR spectrum. H NMR (CDCl3) δ 13.30
(s, 0.5 H), 13.18 (s, 0.5 H), 12.09 (s, 1 H), 8.16 (s, 0.5 H), 8.16
(s, 0.5 H), 7.28-7.76 (m, 3 H), 6.82-6.84 (m, 0.5 H), 6.22-
6.24 (m, 0.5 H), 3.17 (s, 1.5 H), 2.84 (s, 1.5 H), 1.67-1.79 (m,
3 H); 13C NMR (CDCl3) δ 165.6, 165.4, 157.6, 140.1, 139.7,
137.8, 135.5, 132.8, 130.6, 130.3, 130.2, 128.7, 128.6, 127.7,
127.6, 127.5, 127.4, 127.1, 122.0,121.9,119.6, 60.4, 55.8, 52.6,
32.6, 29.5, 21.1, 17.0, 15.6; MALDI HR-FTMS calcd for
C20H18O2N4Cl2Na m/z 439.0699 [M + Na]+, found 439.0706 [M
+ Na]+.
(S)-1H-Imidazole-4,5-dicarboxylic Acid 5-[(3,4-Dichlo-
rophenyl)amide] 4-[Methyl(1-phenylethyl)amide] (36).
Synthesized from 72 mg of 2 (0.13 mmol) and 75 µL of (R)-
N,R-dimethylbenzylamine (0.51 mmol) in 2 mL of ethyl acetate,
stirring the reaction mixture for 60 h. The product was purified
by column chromatography on SiO2 with ethyl acetate/hexanes
as the eluant to yield 38 mg of 36 (36% yield) as a white solid:
The physical and spectral data was identical with enantiomer
35.
Cell Cultures and Drug Treatments. Solutions of syn-
thetic imidazole-4,5-dicarboxamide (I45DC) derivatives, pur-
valanol A, olomoucine (both purchased from Sigma, St. Louis,
MO), and flavopiridol (a generous gift from Dr. Gunda Georg,
University of Kansas, Lawrence, KS) were dissolved and
serially diluted in dimethyl sulfoxide (DMSO). Suspended
cultures of human HL-60 promyelocytic leukemia cells (from
American Type Culture Collection, Manassas, VA) were
maintained in continuous exponential growth by twice-a-week
passage in RPMI 1640 medium supplemented with 8.25% fetal
bovine calf serum (FCS; Atlanta Biologicals, Norcross, GA) and
penicillin (100 IU/mL)-streptomycin (100 µg/mL) and incu-
bated in the presence or absence (control) of drugs at 37 °C in
a humidified atmosphere containing 5% CO2. Since drugs were
supplemented to the culture medium in 1-µL aliquots, the
concentration of vehicle (0.2% DMSO) in the final incubation
volume (0.5 mL) was shown to not affect the growth and
metabolic activity of control HL-60 cells incubated in the
absence of drugs.36
Acknowledgment. This study was supported in
part by grants from the National Institutes of Health
(National Cancer Institute 1R01 CA86842-05 and Cen-
ter of Biomedical Research Excellence 1P20 RR15563-
04 with matching funds from the State of Kansas), the
Howard Hughes Medical Institute (Biological Sciences
Education Grant), Kansas State University (Terry C.
Johnson Center for Basic Cancer Research), Bowdoin
College (Faculty Research Fund), and the National
Science Foundation (NSF-MRI #0116416). We thank
Elizabeth A. Stemmler (Bowdoin College) for mass
spectrometry assistance and Gunda Georg (University
of Kansas) for providing us with flavopiridol.
Supporting Information Available: NMR spectra, HPLC
traces, theorized binding modes. This material is available free
of charge via the Internet at http://pubs.acs.org.”
References
Cell Proliferation Assay. HL-60 cells suspended in FCS-
containing RPMI 1640 medium were grown at 37 °C in 48-
well Costar cell culture plates for up to 4 days in the presence
or absence (control) of I45DCs in order to evaluate their
antiproliferative activity. Decreasing concentrations of cells,
such as 12.5 × 104 and 3.75 × 104 cells/0.5 mL/well, were
initially plated in triplicate at time 0 in order to collect control
samples with approximately equal cell densities after 2 and 4
days in culture, respectively. The proliferation of drug-treated
cells was assessed from their mitochondrial ability to bioreduce
the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent (Promega,
Madison, WI) in the presence of phenazine methosulfate (PMS;
Sigma, St. Louis, MO) into a water-soluble formazan product
that absorbs at 490 nm.46 After 2 or 4 days in culture, control
and drug-treated cell samples (about 106/0.5 mL/well for
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in the presence of 0.1 mL of MTS:PMS (2:0.1) reagent and their
relative metabolic activity was estimated by recording the
absorbance at 490 nm, using a Cambridge model 750 auto-
matic microplate reader (Packard, Downers Grove, IL). Blank
values for culture medium supplemented with MTS:PMS
reagent in the absence of cells were subtracted from the
results.36
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