Synthetic analogues of the microtubule-stabilizing sponge alkaloid ceratamine A are more active than the natural product

Article abstract of DOI:10.1021/jm101012q

Desbromoceratamine A (3) exhibits significantly less potent activity than the natural product ceratamine A (1) in a cell-based assay for antimitotic activity. Synthesis of the ceratamine A analogue 4 has shown that replacing the bromine atoms in the natural product with methyl groups generates an analogue that is more active than natural ceratamine A (1). Further enhancement of the antimitotic activity of the ceratamine pharmacophore has been achieved in the synthetic analogue 33, which has both bromine atoms replaced with methyl groups and an additional methyl substituent on the amino nitrogen at C-2. An efficient synthetic route has been developed to 33 that should enable the first in vivo evaluation of the new ceratamine microtubule-stabilizing pharmacophore and has provided several additional analogues for structure-activity relationship evaluation.

Full text of DOI:10.1021/jm101012q

J. Med. Chem. 2010, 53, 7843–7851 7843
DOI: 10.1021/jm101012q
Synthetic Analogues of the Microtubule-Stabilizing Sponge Alkaloid Ceratamine A Are More
Active than the Natural Product
Matt Nodwell,† Carla Zimmerman,‡ Michel Roberge,*,‡ and Raymond J. Andersen*,†
†Departments of Chemistry and Earth & Ocean Sciences, University of British Columbia, Vancouver, BC, Canada V6T 1Z1, and
‡Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3
Received August 4, 2010
Desbromoceratamine A (3) exhibits significantly less potent activity than the natural product cerata-
mine A (1) in a cell-based assay for antimitotic activity. Synthesis of the ceratamine A analogue 4 has
shown that replacing the bromine atoms in the natural product with methyl groups generates an
analogue that is more active than natural ceratamine A (1). Further enhancement of the antimitotic
activity of the ceratamine pharmacophore has been achieved in the synthetic analogue 33, which has
both bromine atoms replaced with methyl groups and an additional methyl substituent on the amino
nitrogen at C-2. An efficient synthetic route has been developed to 33 that should enable the first in vivo
evaluation of the new ceratamine microtubule-stabilizing pharmacophore and has provided several
additional analogues for structure-activity relationship evaluation.
Introduction
in mouse models of cancer.1 To provide the material required
for further evaluation of the new ceratamine microtubule-
stabilizing pharmacophore, we embarked on a synthetic
program aimed at making ceratamine A (1) and simpler
analogues. These efforts resulted in the synthesis of several
desbromo analogues of ceratamine A (1) that confirmed the
structure proposed for the natural product and the micro-
tubule-stabilizing activity of the new pharmacophore.5,6
However, the reduced potency and efficacy of synthetic
desbromo ceratamine A (3) compared with the activity of
natural ceratamine A (1) revealed that the bromine atoms
were necessary for the full biological activity of the natural
product.
Ceratamines A (1) and B (2) are antimitotic alkaloids
isolated from extracts of the marine sponge Pseudoceratina
sp. collected in Papua New Guinea.1 The imidazo[4,5-d]-
azepine core heterocycle of the ceratamines has not been
encountered at any oxidation state in other natural products.2
Ceratamines stabilize microtubules, but they do not bind to
the same site as paclitaxel, and they generate an unusual
mitotic arrest phenotype that is characterized by the forma-
tion of pillarlike structures of tubulin.3 The recent approval of
the epothilone analogue ixabepilone for clinical use highlights
the continued interest in microtubule-stabilizing agents as
anticancer drugs.4 Ceratamines areattractiveleadcompounds
for anticancer drug development because they have novel, but
nevertheless relatively simple, chemical structures as com-
pared with other natural product microtubule-stabilizing
agents, they are achiral, they do not bind to the paclitaxcel site
on microtubules, and they generate an unprecedented mitotic
arrest phenotype.
Intriguedbythe importantrolethatthe bromineatoms play
in the antimitotic activity of the ceratamines, we set out to see
if other substituents might provide the same benefit. The
initial hypothesis was that the bromine atoms were simply
adding steric bulk to the aromatic ring that either helped to fill
a binding pocket or predefined a preferred binding conforma-
tion. Therefore, we decided to replace the bromine atoms in
ceratamine A (1) with methyl groups, reasoning that since
˚
their van der Waals radii were nearly equal (Br, 1.86 A; Me,
7
1.80 A), they would fill roughly comparable volumes of
˚
space, and the new target became the analogue 4.
Results and Discussion
The synthesis of 4 (R=Me) followed the route used in our
earlier synthesis of desbromoceratamine A (3) (R = H) as
outlined in Scheme 1.6 Vinyl bromide 7 was prepared from
3,5-dimethylanisaldehyde via a Cr(II)-mediated condensation
with tribromomethylacetate8 followed by amide formation as
previously described for the synthesis of 6.6 Stille coupling
between stannane 5 and the vinyl bromide 7 gave 9 in high
yield. Z-Vinyl bromide 11 was synthesized by reaction of the
aldehyde 9 with (bromomethyl)triphenylphosphoniumbromide
The original isolation of ceratamines A (1) and B (2) from
the Pseudoceratina sp. generated only small amounts of
material that was not adequate to support in vivo evaluation
*To whom correspondence should be addressed. Tel: 604-822-4511.
Fax: 604-822-6091. E-mail: randersn@interchange.ubc.ca (R.A.). Tel:
604-822-2304. Fax: 604-822-6091. E-mail: michelr@interchange.ubc.ca
(M.R.).
r
2010 American Chemical Society
Published on Web 10/14/2010
pubs.acs.org/jmc

7844 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Nodwell et al.
Scheme 1
and potassium tert-butoxide (KOtBu).a9 Copper(I)-catalyzed
ringclosureof 11 to form the enamide13 followed a procedure
developed by Buchwald.10 A dilute tetrahydrofuran (THF)
solution of 11 was treated with CuI, Cs2CO3, and N,N0-
dimethylethylenediamine at elevated temperatures to yield the
5,7-bicyclic system 13 in excellent yield. Heating chloroimi-
dazole 13, triphenylsilylamine, and lithium hexamethyldisilazide
(LiHMDS) in the presence of tris(dibenzylidenacetone)di-
palladium(0) [Pd2(dba)3] and the Buchwald ligand 2-dicyclo-
hexylphosphino-20,40,60-triisopropylbiphenyl (XPhos) in toluene
(Tol) followed by acid hydrolysis of the triphenylsilyl group
gavethe primary amine 15.11 Reaction of 15withacetic formic
anhydride in THF for 24 h followed by alkylation with methyl
iodide (MeI) and K2CO3 in dimethylformamide (DMF) gave
N-methyl formamide 17 in high yield. Removal of the benzy-
loxymethyl (BOM) group from 17 with AlCl3 gave formamide
19. The desired dimethylceratamine A analogue 4 and the
Figure 1. Dose-response curves for natural and synthetic cerata-
mines in a cell-based assay for mitotic arrest.
corresponding C-11 hydroxyanalogue 21 were formed when a
stream of dry HCl(g) was bubbled through a solution of
formamide 19 in 50%1,4-dioxane and water.
As we had hoped, biological evaluation showed that replace-
ment of the bromine atoms in ceratamine A (1) with methyl
groups to give 4 produced an analogue that was slightly more
potent (IC50 ≈ 3.5 μg/mL) and significantly more efficacious
(67% cells arrested in mitosis at optimal concentration) than
the natural product (IC50 ≈ 5 μg/mL; 54% cells arrested in
mitosis at optimal concentration) as shown in Figure 1, making
it a strong candidate for testing the ceratamine antimitotic
pharmacophore in mouse models of cancer. However, the
formation of the C-11 hydroxyl byproduct 21 hampered the
a Abbreviations: BOM, benzyloxymethyl; DMAP, 4-dimethylamino-
pyridine; DMF, dimethylformamide; DMSO, dimethylsulfoxide; HRE-
SIMS, high-resolution electrospray ionization mass spectrometry; IBX,
2-iodobenzoic acid; IC50, concentration giving 50% of full biological
response; KOtBu, postassium tertiary butoxide; LiHMDS, lithium
hexamethyldisilazide; MeI, methyl iodide; NMR, nuclear magnetic
resonance; Pd2(dba)3, tris(dibenzylidenacetone)dipalladium(0); RP-HPLC,
reversed-phase high-performance liquid chromatography; SAR, structure-
activity relationship; THF, tetrahydrofuran; TLC, thin-layer chroma-
tography; Tol, toluene; XPhos, 2-dicyclohexylphosphino-20,40,60-triiso-
propylbiphenyl.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7845
Scheme 2
Scheme 3
Scheme 4
efficiency of the synthesis of 4, and we set out to circumvent
this limitation.
amine 15 could be reduced in excellent yield to give 25 using
PtO2-catalyzed hydrogenation at 400 psi. Removal of the
BOM group in 25 with AlCl3 cleanly gave intermediate 26,
which could be further hydrogenated at atmospheric pressure,
again using PtO2 as a catalyst, to give the tetrahydro cerata-
mine analogue 23 in high yield. IBX oxidation of 23 gave the
ceratamine 24 without any detectable formation of the corre-
sponding C-11 oxidized analogues. As shown in Figure 1,
compound 24 had almost no antimitotic activity in the cell-
based assay, demonstrating the critical requirement for N-2
methylation in the ceratamine antimitotic pharmacophore.
Our first attempt to incorporate N-2 methylation into the
hybrid synthetic route started with methylation of the par-
tially reduced intermediate 25 as shown in Scheme 4. Conver-
sion of the primary amine 25 into the corresponding trifluor-
acetamide followed by alkylation with methyliodide and
K2CO3 and subsequent aqueous workup gave the N-2 methyl-
ated analogue 27. Removal of the BOM protecting group in
While our work was in progress, Coleman’s group reported
the first synthesis of the natural products 1 and 2.12 The end
game of their synthesis featured an elegant 2-iodobenzoic acid
(IBX)-mediated double dehydrogenation to create the aro-
matic imidazo[4,5-d]azepine heterocycle (Scheme 2, 22 to 2),
an approach that had earlier failed in our hands.6
Coleman’s report led us to consider a hybrid synthesis of 4
shown retrosynthetically in Scheme 3, whereby heterocycle 15
would be assembled at the ceratamine oxidation state as
before, reduced to 23, and then reoxidized to the all-aromatic
tautomer 24 using Coleman’s IBX methodology, presumably
without the formation of the undesirable C-11 oxidized side
product 21. Mono N-methylation of 24 would then yield the
desired analogue 4.
Scheme 4 shows the realization of the hybrid route to cera-
tamines. The enamide functionality in BOM-protected primary

7846 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Nodwell et al.
Scheme 5
Scheme 6
Scheme 7
27 with AlCl3 followed by atmospheric pressure hydrogena-
tion with PtO2 catalysis gave 29. Unexpectedly, attempted
IBX oxidation of 29, using the conditions that readily con-
verted 23 to 24 (Scheme 4) and 22 to 2 (Scheme 2), gave no
detectable transformation of starting material to product.
Heating the reaction to 80 °C resulted in conversion of the
starting material 29 into overoxidized products. Although
these oxygenated compounds were not completely purified or
characterized because they were not the desired products, the
MS and nuclear magnetic resonance (NMR) data obtained
for the impuremixture were consistent withstructures30 or 31
(Scheme 5). We were unable to find any conditions that would
transform 29 into the desired N-2 methylated ceratamine
analogue 4.
Next, we turned our attention to direct N-2 methylation of
the ceratamine analogue 24 using the conditions that had
cleanly converted the primary amine 25 to the N-2 mono-
methyl analogue 27 (Scheme 4). As shown in Scheme 6, re-
action of the N-2 primary amine 24 with trifluoracetic anhy-
dride followed by methylation with MeI/K2CO3 and aqueous
workup does indeed accomplish monomethylation at N-2 as
desired, but it also leads to tautomerization to give 32. This
unanticipated dearomatization turns out to be consistent with
all of the previous experience that we have had with the
imidazo[4,5-d]azepine ring system in the ceratamines. When
the C-2 substituent is hydrogen, chlorine, thiomethyl, formy-
lamide, or trifluoracetamide, the ceratamines prefer the non-
aromatic tautomer observed in 32.6 The only ceratamine
analogues that we have made to date that prefer the aromatic
tautomer present in 24 are compounds (1, 2, 3, 4, 24, etc.)
having a strongly electron-donating primary amine or N-
methyl amine substituent at C-2. It is interesting that reducing
theelectron-donating abilityofanaminosubsitutent at C-2 by
acylating the amine is sufficient to drive the tautomeric equilib-
rium away from aromaticity in the imidazo[4,5-d]azepine ring
system.
was not the desired monomethyl product 4, we nevertheless
tested it in the cell-based antimitotic assay. As shown in
Figure 1, the dimethyl analogue 33 had even better antimitotic
activity (33: IC50 ≈ 3.0 μg/mL; 82% cells arrested in mitosis at
optimal concentration) than the monomethyl analogue 4
(IC50 ≈ 3.5 μg/mL; 67% cells arrested in mitosis at optimal
concentration), and it is easier to make.
Our original analysis of the ceratamine microtubule-stabi-
lizing pharmacophore focused on the imidazo[4,5-d]azepine
core heterocycle as the primary structural requirement for
biological activity. This was based in part on the observation
that the 3,5-dibromo-4-methoxyphenyl substructure is com-
monly encountered in sponge metabolites, but none of the
other natural products containing this moiety are known to
target tubulin.13 As a result, our initial analogue synthesis
program was designed to make compounds that did not
contain the bromine atoms found in the natural product,
and our finding that desbromo ceratamine A was significantly
less active than the natural products was unexpected.5,6 The
current study has shown that replacing the bromine atoms in
ceratamine A (1) with methyl groups to give the analogue 4
recaptures and even exceeds the antimitotic activity of the
natural product (Figure 1 and Table 1). We assume that since
bromine atoms and methyl groups have roughly comparable
van der Walls radii,7 the effect is steric and the two subsituents
are simply filling the same space.
The current study has also revealed an additional struc-
ture-activity relationship (SAR) for the ceratamine pharma-
cophore. Compound 21, which is the C-11 hydroxy analogue
of 4, is completely inactive at the concentrations tested (Table 1).
Similarly, compound 32, which differs from 4 by being acylated
at N-2 with a trifluoracetyl group and existing in the nonaro-
matic tautomeric form, is inactive. The primary amine 24,
which just lacks the N-2 methyl substituent in 4, is essentially
inactive, while compound 33, which is dimethylated on N-2, is
significantly more active than 1 or 4.
Because methylation of the acylated N-2 amine in 24 had
failed to produce the desired result, we next attempted direct
stiochiometric alkylation. Treatment of 24 with NaH and 1 equiv
of MeI converts it to the N-2 dimethyl amine 33 (Scheme 7). It
was not possible to stop the N-2 alkylation at the momo-
methyl product 4 nor was it possible to generate the N-2
trimethylated product. Using excess MeI produces the di-
methyl compound 33in nearlyquantitative yield. Although33
The above results show that the structural requirements for
activity in the ceratamine antimitotic pharmacophore are quite
tightly constrained. There is a strict requirement for bulky
substituents at C-14 and C-16 on the phenyl ring. This steric
bulk is supplied by the bromine atoms in the natural products
1 and 2. As described above, we have found that methyl groups
at these positions in the synthetic analogues 4 and 33 produce

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7847
Table 1. Antimitotic Activity of Ceratamines in a Cell-Based Assay for Antimitotic Activity
enhanced antimitotic activity. Oxygen substituents, either
hydroxyl or ketone functionalities, at C-11 strongly attenuate
the activity. Methyl substituents are required on N-2. If there
is no methyl substituent on N-2, the compounds are inactive,
and dimethylation on N-2 enhances activity relative to mono-
methylation. All compounds tested to date that exist in the
nonaromatic tautomeric form of the imidazo[4,5-d]azepine
heterocycle are inactive.5,6 However, all of these compounds
are also missing a nonacylated methyl amine substituent at
C-2. Because these two structural changes are thus far always
interrelated, it is not possible to tell if it is just the N-2 methyl
amine susbtituent or both the N-2 methyl amine and the
aromatic tautomeric form that is required for antimitotic
activity.
The new ceratamine analogues 4 and 33 show roughly the
same potency as ceratamine A, suggesting that they bind
microtubules with comparable affinity. However, 4 and 33
arrest a higher proportion of cells at mitosis (67 and 82%,
respectively) than ceratamine A (54%). It is increasingly
recognized that cells do not remain arrested in mitosis indef-
initely in the presence of microtubule-targeting agents.14 After
a period of arrest typically lasting several hours, cells may

7848 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Nodwell et al.
enter interphase without having undergone cell division. This
process, termed mitotic slippage, can lead to the formation of
abnormal multiploid cells. We speculate that 4 and 33 may
induce a more robust mitotic arrest than ceratamine A,
resulting is a lower rate of slippage and perhaps enhanced
antitumor activity.
(E)-2-(1-((Benzyloxy)methyl)-4-((Z)-2-bromovinyl)-2-chloro-
1H-imidazol-5-yl)-3-(4-methoxy-3,5-dimethylphenyl)-N-methyl-
acrylamide (11). (Bromomethyl)triphenylphosphonium bromide
(2.5 g, 5.68 mmol) was slurried in 25 mL of dry THF. Potassium
tert-butoxide (638 mg, 5.68 mmol) was added in one portion,
and the resulting bright yellow solution was stirred at room
temperature for 30 min. The reaction mixture was then cooled
to -78 °C, and a solution of 9 (1.33 g, 2.84 mmol) in 10 mL of dry
THF was added over a period of 10 min. The solution was stirred
at -78 °C for 1.5 h, allowed to warm to room temperature, and
stirred for an additional 30 min. The reaction was quenched by
the addition of water, and the reaction mixture was extracted
with 2 ꢀ EtOAc. The organic phase was dried over Na2SO4,
filtered, and concentrated to dryness. The crude product was
purified by column chromatography to yield 11 (1.30 g, 2.39 mmol,
Conclusions
In summary, an efficient new synthetic route to the highly
methylated ceratamine analogue 33 has been developed.
Compound 33, in which the C-14 and C-16 bromine substit-
uents in the natural product ceratamine A (1) have been
replaced by methyl groups and the C-2 monomethylamino
substituent in the natural product has been replaced by a
dimethylamino substituent, is significantly more active than
the inspirational natural product. Evaluation of this highly
methylated ceratamine 33 in mouse models of cancer is on-
going, and these animal experiments will provide the first
in vivo data on the microtubule-stabilizing ceratamine phar-
macophore. The results of these in vivo experiments will be
reported elsewhere.
1
84%) as a pale yellow foam. H NMR (CD2Cl2, 400 MHz): δ
2.15 (s, 6H), 2.75 (d, J = 4.9 Hz, 3H), 3.66 (s, 3H), 4.45 (s, 2H),
4.99 (d, J = 10.8 Hz, 1H), 5.15 (d, J = 10.8 Hz, 1H), 5.79 (s, br,
1H), 6.32 (d, J = 8.2 Hz, 1H), 6.73 (m, 3H), 7.16 (m, 2H), 7.30
(m, 3H), 8.00 (s, 1H). 13C NMR (CD2Cl2, 100 MHz): δ 16.0,
26.8, 59.6, 71.1, 73.4, 106.6, 118.9, 122.1, 127.5, 128.1, 128.5, 128.6,
129.2, 130.9, 131.6, 134.4, 136.3, 136.6, 143.8, 158.9, 165.5.
HRESIMS calcd for C26H27N3O335Cl79BrNa ([M þ Na]þ),
566.0822; found, 566.0815.
(E)-3-((Benzyloxy)methyl)-2-chloro-4-(4-methoxy-3,5-dimethyl-
benzylidene)-6-methyl-4,6-dihydroimidazo[4,5-d]azepin-5(3H)-one
(13). Vinyl bromide 11 (62.3 mg, 0.114 mmol), CuI (4.4 mg,
0.23 mmol), and powdered K3PO4 (49 mg, 0.23 mmol) were
combined in 1.5 mL of Tol. N,N0-Dimethylethylenediamine
(5.0 μL, 0.46 mmol) was then added, and the reaction mixture
was heated to 55 °C for 16 h. At this point, another portion of
CuI (4.4 mg, 0.23 mmol) and N,N0-dimethylethylenediamine
(5 mL, 0.46 mmol) were added, and the reaction mixture was
stirred at 55 °C for another 5 h. The reaction mixture was cooled
to room temperature, filtered through a Celite pad, and con-
centrated to dryness. The crude product was purified by column
chromatography to yield 13 (34.4 mg, 0.074 mmol, 65%) as a
pale yellow solid. 1H NMR (CD2Cl2, 400 MHz): δ 2.16 (s, 6H),
3.29 (s, 3H), 3.68 (s, 3H), 4.36 (s, 2H), 4.52 (d, J = 11.4 Hz, 1H),
4.85 (d, J = 11.4 Hz, 1H), 6.14 (d, J = 9.2 Hz, 1H), 6.20 (d, J =
9.2 Hz, 1H), 6.60 (s, 2H), 7.27 (m, 6H). 13C NMR (CD2Cl2, 100
MHz): δ 15.9, 38.2, 59.7, 70.9, 73.2, 107.9, 122.4, 123.4, 127.8,
127.9, 128.4, 128.9, 129.5, 129.9, 131.6, 134.8, 136.1, 136.3,
136.9, 158.0, 169.2. HRESIMS calcd for C26H26N3O335ClNa
([M þ Na]þ), 486.1560; found, 486.1552.
Experimental Section
General Methods. All nonaqueous reactions were carried out
in flame-dried glassware and under an Ar atmosphere unless
otherwise noted. Air- and moisture-sensitive liquid reagents
were manipulated via a dry syringe. Anhydrous THF was obtained
from distillation over sodium. Fresh IBX was prepared according
to a literature procedure.15 All other solvents and reagents were
used as obtained from commercial sources without further puri-
1
fication. H and 13C spectra were obtained on Bruker Avance
400 direct or Bruker Avance 600 cryoprobe spectrometers. Flash
chromatography was performed using Silicycle Ultra Pure silica
gel (230-400 mesh). The purity of compounds was evaluated via
reversed-phase high-performance liquid chromatography (RP-
HPLC) on a C18 analytical column eluting with MeOH/H2O
mixtures. Samples of all compounds tested for antimitotic activ-
ity in the cell-based assay were >95% pure, as were all other
synthetic intermediates unless otherwise noted.
(Z)-2-Bromo-3-(4-methoxy-3,5-dimethylphenyl)-N-methylacryl-
amide (7). Vinylbromide 7 was prepared as described using a
Cr(II)-mediated olefination of 4-methoxy-3,5-dimethyl-benzal-
dehyde with tribromomethyl acetate, followed by functional
group interchange.6 1H NMR (CD2Cl2, 400 MHz): δ 2.32 (s,
6H), 2.94 (d, J = 4.6 Hz, 3H), 3.75 (s, 3H), 6.95 (s, broad, 1H),
7.49 (s, 2H), 8.17 (s, 1H). 13C NMR (CD2Cl2, 100 MHz): δ 16.1,
27.3, 59.7, 114.0, 129.6, 130.9, 131.1, 136.8, 158.4, 163.0. High-
resolution electrospray ionization mass spectrometry (HRESIMS)
calcd for C13H17NO279Br ([M þ H]þ), 298.0443; found, 298.0445.
(E)-2-(1-((Benzyloxy)methyl)-2-chloro-4-formyl-1H-imidazol-
5-yl)-3-(4-methoxy-3,5-dimethylphenyl)-N-methylacrylamide (9).
Stannane 5 (1.83 g, 3.39 mmol), bromide 7 (1.01 g, 3.39 mmol),
Pd(PPh3)4 (392 mg, 0.339 mmol), and CuI (646 mg, 3.39 mmol)
were combined in 12 mL of dry 1,4-dioxane. The reaction
mixture was heated to 60 °C for 2 h, cooled, filtered through a
Celite pad, and concentrated to dryness. The crude product was
purified bycolumnchromatographytoyield 9 (1.33g, 2.84 mmol,
(E)-2-Amino-3-((benzyloxy)methyl)-4-(4-methoxy-3,5-dimethyl-
benzylidene)-6-methyl-4,6-dihydroimidazo[4,5-d]azepin-5(3H)-one
(15). Chloride 13 (273.1 mg, 0.59 mmol), triphenylsilylamine
(196 mg, 0.71 mmol), Pd2(dba)3 (54 mg, 0.59 mmol), and XPhos
(67 mg, 0.14 mol) were combined in 6.5 mL of dry Tol. LiHMDS
(1.53 mmol, 1.0 M in Tol, 1.53 mmol) was added, and the re-
action mixture was heated to 90 °C for 1 h. The reaction mixture
was cooled to room temperature and diluted with EtOAc. A 1 M
concentration of HCl was added, and the biphasic mixture was
stirred rapidly for 5 min. The aqueous layer was basified by the
addition of saturated NaHCO3, and the crude reaction mixture
was extracted with 3 ꢀ EtOAc. The organic phase was dried over
Na2SO4, filtered, and concentrated to dryness. The crude product
was purified by column chromatography to yield 15 (185 mg,
1
0.42 mmol, 71%) as a bright yellow solid. H NMR (CD2Cl2,
400 MHz): δ 2.17 (s, 6H), 3.28 (s, 3H), 3.67 (s, 3H), 4.25 (d, J =
3.9 Hz, 2H), 4.51 (d, J = 3.3 Hz, 2H), 4.80 (s, br, 2H), 6.04 (d,
J = 9.1 Hz, 1H), 6.10 (d, 9.1 Hz, 1H), 6.68 (s, 2H), 7.12 (s, 1H),
7.22 (m, 2H), 7.30 (m, 3H). 13C NMR (CD2Cl2, 100 MHz): δ
15.9, 38.2, 59.7, 70.4, 72.2, 108.7, 122.9, 127.3, 128.1, 128.3,
128.5, 129.6, 130.6, 131.2, 131.3, 136.8, 157.5, 169.4. HRESIMS
calcd for C26H29N4O3 ([M þ H]þ), 445.2240; found, 445.2245.
(E)-N-(1-((Benzyloxy)methyl)-8-(4-methoxy-3,5-dimethylbenzy-
lidene)-6-methyl-7-oxo-1,6,7,8-tetrahydroimidazo[4,5-d]azepin-
2-yl)-N-methylformamide (17). To a slurry of sodium formate
1
84%) as a pale yellow solid. H NMR (CD2Cl2, 400 MHz): δ
2.13 (s, 6H), 2.80 (d, J = 4.8 Hz, 3H), 3.65 (s, 3H), 4.49 (s, 2H),
5.02 (d, J = 10.7 Hz, 1H), 5.20 (d, J = 10.7 Hz, 1H), 6.00 (d, br,
J = 4.8 Hz, 1H), 6.64 (s, 2H), 7.18 (m, 2H), 7.31 (m, 3H), 7.97
(s,1H), 9.72 (s, 1H). 13C NMR (CD2Cl2, 100 MHz): δ 16.1, 26.9,
59.6, 71.5, 73.6, 119.0, 127.6, 128.3, 128.6, 128.9, 130.8, 131.8,
136.0, 136.3, 136.7, 138.2, 143.7, 159.1, 165.2, 183.9. HRESIMS
calcd for C25H26N3O435ClNa ([M þ Na]þ), 490.1510; found,
490.1504.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7849
(215 mg, 3.16 mmol) in 3 mL of dry THF was added acetyl
chloride (150 μL, 2.1 mmol). The slurry was then heated to
45 °C for 4 h. The slurry was cooled to room temperature, and a
solution of 15 (44 mg, 0.1 mmol) in 1 mL of THF was added,
followed by a catalytic amount of 4-dimethylaminopyridine
(DMAP). The solution was then stirred at room temperature
for 24 h. A 1 M concentration of HCl was added, and the
solution was then stirred for 10 min. The solution was basified
with the addition of saturated NaHCO3, and the crude reaction
mixture was extracted into EtOAc. The organic phase was dried
over Na2SO4, filtered, and concentrated. The crude product was
thendissolved in0.5mLofDMF, andK2CO3 (33mg, 0.24 mmol)
was added to the solution, followed by MeI (50 mL, 0.15 mmol),
and the reaction mixture was stirred at room temperature for
24 h. The solution was then extracted into EtOAc and washed
with 3 ꢀ H2O. The organic phase was dried over Na2SO4,
filtered, and concentrated. The crude product was purified by
column chromatography to yield 17 (45 mg, 0.092 mmol, 92%
J = 11.3 Hz, 1H), 4.62 (d, J = 11.3 Hz, 1H), 5.04 (s, br, 1H), 6.84
(s, 1H), 6.88 (s, 2H), 7.80 (m, 5H). 13C NMR (CD2Cl2, 100
MHz): δ 15.9, 27.5, 33.6, 47.9, 59.7, 70.2, 72.3, 116.6, 125.9,
127.9, 128.3, 128.4, 129.6, 130.7, 131.0, 131.2, 135.3, 137.2,
151.5, 157.3, 172.3. HRESIMS calcd for C26H31N4O3 ([M þ H]þ),
447.2396; found, 447.2405.
(E)-3-((Benzyloxy)methyl)-4-(4-methoxy-3,5-dimethylbenzyli-
dene)-6-methyl-2-(methylamino)-4,6,7,8-tetrahydroimidazo[4,5-d]-
azepin-5(3H)-one (27). Primary amine 25 (42 mg, 0.094 mmol)
was dissolved in 1 mL of CH2Cl2 and cooled to 0 °C. Trifluoro-
acetic anhydride (130 μL, 0.94 mmol) was added neat, and the
solution was stirred at 0 °C for 15 min. The solution was then
concentrated to dryness, redissolved in Tol, and concentrated to
dryness again. The resulting residue was dissolved in 1 mL of
DMF, and K2CO3 (26 mg, 0.19 mmol) was added, followed by
MeI (60 μL, 0.94 mmol). This slurry was stirred rapidly at room
temperature for 1 h, then H2O (15 mL) was added, and the
solution was stirred for another 5 min. The crude product was
extracted into EtOAc and washed with 2 ꢀ H2O. The organic
phase was dried over Na2SO4, filtered, and concentrated. The
crude product was purified by column chromatography to yield
27 (35 mg, 0.076 mmol, 81%) as a pale yellow solid. 1H NMR
(CD2Cl2, 400 MHz): δ 2.21 (s, 6H), 2.74 (m, 2H), 3.11 (s, 3H),
3.34 (s, 3H), 3.45 (m, 1H), 3.71 (s, 3H), 4.05 (m, 1H), 4.37 (s, 2H),
4.50 (d, J = 11.3 Hz, 1H), 5.08 (d, J = 11.3 Hz, 1H), 6.88 (s, 2H),
7.08 (s, 1H), 7.26 (m, 5H). 13C NMR (CD2Cl2, 100 MHz): δ 15.9,
24.1, 30.8, 33.9, 46.2, 59.8, 71.7, 74.1, 118.4, 122.3, 125.6, 127.6,
127.9, 128.0, 129.5, 129.7, 132.0, 136.6, 137.2, 151.9, 154.6,
158.6, 171.3. HRESIMS calcd for C27H33N4O3 ([M þ H]þ),
461.2542; found, 461.2546.
1
yield) as a dull yellow solid. H NMR (CD2Cl2, 400 MHz): δ
2.15 (s, 6H), 3.19 (s, 3H), 3.31 (s, 3H), 3.68 (s, 3H), 4.32 (s, 2H),
4.50 (d, J=11.3 Hz, 1H), 4.58 (d, J = 11.3 Hz, 1H), 6.22 (m, 2H),
6.58 (s, 2H), 7.21 (m, 2H), 7.25 (s, 1H), 7.32 (m, 3H), 8.32 (s, 1H).
13C NMR (CD2Cl2, 100 MHz): δ 16.0, 32.6, 38.2, 59.7, 71.0,
72.6, 108.1, 122.6, 127.9, 128.1, 128.5, 129.2, 129.5, 129.8, 130.0,
131.4, 134.6, 136.6, 158.0, 162.2, 169.2. HRESIMS calcd for
C28H30N4O4Na ([M þ Na]þ), 509.2165; found, 509.2169.
4-(4-Methoxy-3,5-dimethylbenzyl)-6-methyl-2-(methylamino)-
imidazo[4,5-d]azepin-5(6H)-one (4). Compound 15 (45 mg, 0.09
mmol) was dissolved in 3 mL of CH2Cl2. Anhydrous AlCl3
(120 mg, 0.9 mmol) was added, and the resulting blood red
slurry was stirred at room temperature for 10 min. The reaction
mixture was then cooled to 0 °C and quenched with the careful
addition of H2O. EtOAc was added, and the aqueous layer was
basified by the addition of saturated NaHCO3. The biphasic
mixture was extracted with 3ꢀEtOAc, the combined organic
layers were dried over Na2SO4, filtered, and concentrated. The
crude residue was dissolved in CH2Cl2, passed through a silica
plug, and concentrated again to yield crude 19 as a pale yellow
solid. Without further purification, this product was dissolved in
5 mL of 1,4-dioxane. Five milliliters of deionized H2O was added,
and the resulting solution was sparged with nitrogen with
sonication for 10 min. The solution was then cooled to 0 °C,
and a stream of HCl (g) was passed through the solution for
5 min. The solution was then basified by addition of NaHCO3
and extracted with 3ꢀEtOAc to give a bright yellow solution.
The organic layer was dried over Na2SO4, filtered, and concen-
trated. The crude product was purified by column chromatog-
raphy to yield 4 (10 mg, 0.030 mmol, 33%) as a bright yellow
solid. Compound 21 was also isolated from the reaction mixture
but not fully purified or characterized. The NMR spectra of 4 in
dimethylsulfoxide (DMSO)-d6 showed a 3:1 ratio of two rota-
mers. Only the major isomer is described. 1H NMR (DMSO-d6,
600 MHz): δ 2.09 (s, 6H), 3.04 (d, J = 4.7 Hz, 3H), 3.51 (s, 3H),
3.54 (s, 3H), 4.19 (s, 2H), 6.38 (d, J = 9.8 Hz, 1H), 6.97 (s, 2H),
7.69 (d, J = 9.8 Hz, 1H), 8.50 (d, J = 4.7 Hz, 1H). 13C NMR
(DMSO-d6, 150 MHz): δ 29.2, 35.5, 43.7, 59.1, 100.3, 123.4,
129.2, 135.8, 142.5, 154.6, 160.7, 164.1, 169.6, 175.4. HRESIMS
calcd for C19H23N4O2 ([M þ H]þ), 339.1821; found, 339.1832.
(E)-2-Amino-3-((benzyloxy)methyl)-4-(4-methoxy-3,5-dimethyl-
benzylidene)-6-methyl-4,6,7,8-tetrahydroimidazo[4,5-d]azepin-
5(3H)-one (25). Primary amine 15 (56.8 mg, 0.13 mmol) was
dissolved in 1.0 mL of MeOH. PtO2 (20 mg) was added, and the
resulting slurry was hydrogenated at 400 psi for 16 h. The slurry
was then filtered through a 0.4 mm membrane, and the filtrate
was washed with 3 ꢀ MeOH. The resulting solution was concen-
trated to dryness to yield 25 (58 mg, 0.13 mmol, quant yield) as a
pale yellow solid. 1H NMR (CD2Cl2, 400 MHz): δ 2.19 (s, 6H),
2.72 (d, br, J = 17.4 Hz, 1H), 2.87 (td, J = 13.3, 3.2 Hz, 1H),
3.07 (s, 3H), 3.34 (dt, J = 14.8, 2.7 Hz, 1H), 3.67 (s, 3H), 4.04
(t, br, J = 13.3 Hz, 1H), 4.22 (d, J = 2.7 Hz, 2H), 4.55 (d,
(E)-4-(4-Methoxy-3,5-dimethylbenzylidene)-6-methyl-2-(methyl-
amino)-4,6,7,8-tetrahydroimidazo[4,5-d]azepin-5(3H)-one (28).
Secondary amine 27 (62 mg, 0.13 mmol) was dissolved in 10 mL
of CH2Cl2. Anhydrous AlCl3 (173 mg, 1.4 mmol) was added,
and the slurry was stirred rapidly for 10 min. The slurry was
cooled to 0 °C and quenched carefully with H2O. The aqueous
phase was basified by addition of saturated NaHCO3, and the
product was extracted with 3ꢀEtOAc. The organic phase was
dried over Na2SO4, filtered, and concentrated. The crude pro-
duct was purified by column chromatography to yield 28 (46 mg,
1
0.13 mmol, 93%) as a pale yellow solid. H NMR (CD2Cl2,
400 MHz): δ 2.24 (s, 6H), 2.83 (t, J = 5.2 Hz, 2H), 3.10 (s, 3H),
3.50 (s, 3H), 3.74 (s, 3H), 3.78 (t, J = 4.5 Hz, 2H), 7.06 (s, 2H),
7.34 (s, 1H), 10.9 (s, br, 1H). 13C NMR (CD2Cl2, 100 MHz): δ
15.9, 24.2, 28.9, 34.6, 46.7, 59.6, 115.7, 123.2, 125.6, 129.1, 129.9,
131.8, 137.1, 149.5, 158.1, 164.3, 169.9. HRESIMS calcd for
C19H25N4O2 ([M þ H]þ), 341.1978; found, 341.1985.
4-(4-Methoxy-3,5-dimethylbenzyl)-6-methyl-2-(methylamino)-
4,6,7,8-tetrahydroimidazo[4,5-d]azepin-5(3H)-one (29). Secondary
amine 28 (33.6 mg, 0.099 mmol) was dissolved in 2 mL of EtOH.
PtO2 (10 mg) was added, and the resulting slurry was hydro-
genated under balloon pressure for 4 h, until thin-layer chro-
matography (TLC) analysis (5% HOAc/CH2Cl2) showed the
reaction to be complete. The slurry was filtered through a 0.4
mm membrane, and the filtrate was washed with 3 ꢀMeOH.
The resulting solution was concentrated to dryness to yield 29
(33 mg, 0.096 mmol, 97%) as a colorless solid. This product was
used without further purification. The NMR spectra of this
compound showed the presence of two isomers. HRESIMS
calcd for C19H27N4O2 ([M þ H]þ), 343.2134; found, 343.2140.
2-Amino-4-(4-methoxy-3,5-dimethylbenzyl)-6-methyl-4,6,7,8-
tetrahydroimidazo[4,5-d]azepin-5(3H)-one (23). Primary amine
25 (40 mg, 0.090 mmol) was dissolved in 2 mL of CH2Cl2.
Anydrous AlCl3 (120 mg, 0.9 mmol) was added, and the slurry
was stirred at room temperature for 15 min. The slurry was
cooled to 0 °C and quenched carefully with H2O. The aqueous
layer was basified by the addition of NaHCO3, and the crude
product was extracted with 5ꢀEtOAc. The combined organic
layers were dried over Na2SO4, filtered, and concentrated to yield
26, which was used without further purification. HRESIMS calcd

7850 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Nodwell et al.
for C18H23N4O2 ([M þ H]þ), 327.1821; found, 327.1825. Crude
26 was slurried in 5 mL of 1:1 MeOH/EtOH. PtO2 (10 mg) was
added, and the solution was hydrogenated at 400 psi for 18 h.
The slurry was then filtered through a 0.4 mm membrane, and
the filtrate was washed with 2 ꢀ MeOH and 2 ꢀ EtOAc. The
resulting solution was concentrated to dryness to yield 23 (15 mg,
0.045 mmol, 50%) as a colorless solid. This material was used
was then added, and the reaction mixture was stirred at room
temperature for 1 h. After this time, the reaction was quenched
by the careful addition of H2O, and the product was extracted
with EtOAc. The organic layer was dried over Na2SO4, filtered,
and concentrated. The crude product was purified by column
chromatography to yield 33 (10 mg, 0.028 mmol, 90%) as a
bright yellow solid. 1H NMR (DMSO-d6, 600 MHz): δ 2.11 (s,
6H), 3.34 (s, 6H), 3.55 (s, 3H), 3.56 (s, 3H), 4.20 (s, 2H), 6.48 (d,
J = 9.9 Hz, 1H), 6.99 (s, 2H), 7.78 (d, J = 9.9 Hz, 1H). 13C NMR
(DMSO-d6, 150 MHz): δ 15.9, 35.6, 37.7, 43.9, 59.1, 100.5,
123.3, 129.3, 135.8, 143.1, 154.6, 160.3, 164.1, 169.9, 175.1.
HRESIMS calcd for C20H25N4O2 ([M þ H]þ), 353.1978; found,
353.1982.
1
without further purification. H NMR (CD2Cl2, 400 MHz): δ
2.20 (s, 6H), 2.63 (m, 2H), 2.95 (s, 3H), 3.11 (m, 2H), 3.53 (m,
1H), 3.66 (s, 3H), 3.86 (m, 1H), 4.10 (m, 1H), 6.87 (s, 2H). 13C
NMR (CD2Cl2, 100 MHz): δ 15.9, 24.9, 29.9, 34.8, 38.9, 47.2,
59.6, 122.3, 128.3, 129.1, 129.4, 130.5, 135.4, 148.0, 155.4, 173.2.
HRESIMS calcd for C18H25N4O2 ([M þ H]þ), 329.1978; found,
329.1973.
Cell-Based Assay for Compounds Causing Mitotic Arrest. We
used a modification of a literature protocol.16 MCF-7mp53 cells
were seeded in 96-well plates (PerkinElmer Viewplate) and
treated in triplicate with compounds at 1-50 μg/mL for 20 h
at 37 °C. The plates were centrifuged at 50g for 2 min to increase
adherence of mitotic cells. The cells were then fixed and permea-
bilized with 3.7% formaldehyde and 0.1% Triton X-100 in TBS
(10 mM Tris-HCl, pH 7.4, 150 mM NaCl) for 15 min. Protein-
binding sites were blocked using 1% bovine serum albumin in
TBS for 30 min. The cells were then incubated with 1:50 TG-3
antibody17,18 for 1 h, washed in TBS, and incubated with 1:500
goat antimouse AlexaFluor 568 (Invitrogen) for 1 h at room
temperature. The wells were rinsed once with TBS, and nuclei
were stained with 500 ng/mL Hoechst 33342 (Invitrogen) in TBS
for 10 min and rinsed three times with TBS. The plates were
analyzed with a Cellomics ArrayScan VTI using the Target
Activation protocol. The cells were identified by their nuclear
staining in the first channel, and the number of cells in each field
with the strong TG-3 staining indicative of a mitotic cell was
quantified in the second red channel, allowing calculation of the
percentage of cells in mitosis.
7- or 8-Hydroxy-4-(4-methoxy-3,5-dimethylbenzoyl)-6-methyl-
2-(methylamino)imidazo[4,5-d]azepin-5(6H)-one (30/31). Com-
pound 29 (10.3 mg, 0.03 mmol) was dissolved in 0.5 mL of
DMSO. IBX (33.7 mg, 0.12 mmol) was added, and the solution
was heated to 80 °C for 16 h. The yellow reaction mixture was
cooled to room temperature, diluted with saturated NaHCO3,
and extracted with EtOAc. The organic phase was dried over
Na2SO4, filtered, and concentrated to yield a dull yellow residue.
This mixture was purified by column chromatography to yield
30/31 (2 mg, 0.005 mmol, 17%) as the only isolable product. 1H
NMR (CD2Cl2, 600 MHz): δ 2.27 (s, 6H), 3.34 (s, 3H), 3.37 (s,
3H), 3.74 (s, 3H), 6.22 (s, 1H), 7.51 (s, 2H). 13C NMR (CD2Cl2,
150 MHz): δ 15.7, 28.7, 31.9, 59.4, 105.1, 129.8, 131.6, 132.2,
162.7, 163.9, 192.6. HRESIMS calcd for C19H20N4O4Na ([M þ
Na]þ), 391.1382; found, 391.1378.
2-Amino-4-(4-methoxy-3,5-dimethylbenzyl)-6-methylimidazo-
[4,5-d]azepin-5(6H)-one (24). Reduced ceratamine 23 (15 mg,
0.045 mmol) was dissolved in 0.5 mL of DMSO. IBX (50 mg,
0.18 mmol) was added, and the slurry was heated to 40 °C for 15
min, after which the solution turned homogeneous. The result-
ing dark yellow solution was diluted with saturated NaHCO3
and extracted with 3 ꢀ EtOAc. The organic phase was dried over
Na2SO4, filtered, and concentrated. The crude product was
purified by column chromatography to yield 24 (8.7 mg, 0.027
mmol, 60%) as a bright yellow solid. 1H NMR (DMSO-d6, 600
MHz): δ 2.08 (s, 6H), 3.52 (s, 3H), 3.54 (s, 3H), 4.16 (s, 2H), 6.43
(d, J = 9.8 Hz, 1H), 6.88 (s, 2H), 7.74 (d, J = 9.8 Hz, 1H), 8.04
(s, 1H), 8.18 (s, 1H). 13C NMR (DMSO-d6, 150 MHz): δ 15.8,
35.7, 43.8, 59.1, 100.3, 123.2, 129.0, 129.3, 135.6, 142.6, 154.5,
160.8, 164.0, 170.1, 176.3. HRESIMS calcd for C18H21N4O2
([M þ H]þ), 325.1665; found, 325.1670.
Acknowledgment. Financial support was provided by
the Canadian Cancer Society (R.J.A. and M.R.) and NSERC
(R.J.A.).
Supporting Information Available: NMR spectra for com-
pounds 4, 7, 9, 11, 13, 15, 17, 23-25, and 27-33. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(E)-2,2,2-Trifluoro-N-(8-(4-methoxy-3,5-dimethylbenzylidene)-
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N-methylacetamide (32). Amine 24 (3 mg, 0.009 mmol) was
dissolved in 0.3 mL of CH2Cl2. Trifluoroacetic anhydride (13 μL,
0.09 mmol) was added, and the solution was stirred at room
temperature for 30 min. The reaction mixture was then concen-
trated to dryness, redissolved in Tol, and dried again. The residue
was dissolved in 0.2 mL of DMF, and K2CO3 (5 mg, 0.036 mmol)
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(s, 3H), 3.26 (s, 3H), 3.53 (s, 3H), 3.71 (s, 3H), 6.00 (d, J = 9.1
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