Synthesis and biological evaluation of purealin and analogues as cytoplasmic dynein heavy chain inhibitors

Article abstract of DOI:10.1021/jm051030l

Cytoplasmic dynein plays important roles in membrane transport, mitosis, and other cellular processes. A few small-molecule inhibitors of cytoplasmic dynein have been identified. We report here the first synthesis of purealin, a natural product isolated from the sea sponge Psammaplysilla purea, which is known to inhibit axonemal dynein. Also described are the first syntheses, by modular amide coupling reactions, of the natural product purealidin A (a component of purealin) and a small library of analogues. The library was examined for inhibition of cytoplasmic dynein heavy chain and cell growth. The compounds showed effective antiproliferative activity against a mouse leukemia cell line but selective activities against human carcinoma cell lines. Purealin and some of the analogues inhibited the microtubule-stimulated ATPase activity of recombinant cytoplasmic dynein heavy chain motor domain. The inhibitory effect of purealin was concentration dependent and uncompetitive, supporting the hypothesis that it does not compete with the binding of ATP.

Full text of DOI:10.1021/jm051030l

J. Med. Chem. 2006, 49, 2063-2076
2063
Synthesis and Biological Evaluation of Purealin and Analogues as Cytoplasmic Dynein Heavy
Chain Inhibitors
Guangyu Zhu,† Fanglong Yang,† Raghavan Balachandran,‡ Peter Ho¨o¨k,§ Richard B. Vallee,§ Dennis P. Curran,† and
Billy W. Day*,†,‡
Department of Chemistry, UniVersity of Pittsburgh, Pittsburgh, PennsylVania 15260, Department of Pharmaceutical Sciences,
UniVersity of Pittsburgh, Pittsburgh, PennsylVania 15261, and Department of Pathology and Cell Biology, College of Physicians and Surgeons,
Columbia UniVersity, New York, New York 10032
ReceiVed October 13, 2005
Cytoplasmic dynein plays important roles in membrane transport, mitosis, and other cellular processes. A
few small-molecule inhibitors of cytoplasmic dynein have been identified. We report here the first synthesis
of purealin, a natural product isolated from the sea sponge Psammaplysilla purea, which is known to inhibit
axonemal dynein. Also described are the first syntheses, by modular amide coupling reactions, of the natural
product purealidin A (a component of purealin) and a small library of analogues. The library was examined
for inhibition of cytoplasmic dynein heavy chain and cell growth. The compounds showed effective
antiproliferative activity against a mouse leukemia cell line but selective activities against human carcinoma
cell lines. Purealin and some of the analogues inhibited the microtubule-stimulated ATPase activity of
recombinant cytoplasmic dynein heavy chain motor domain. The inhibitory effect of purealin was
concentration dependent and uncompetitive, supporting the hypothesis that it does not compete with the
binding of ATP.
Introduction
Dyneins are protein motor complexes that generate forces
toward the minus ends of microtubules (MTs).a Among the 15
forms of dynein found in vertebrates, only two are cytoplasmic.
These are cytoplasmic dynein-1, initially described as micro-
tubule-associated protein 1C (MAP1C),2 and cytoplasmic
dynein-2.3-5 Cytoplasmic dynein-1 (hereafter, cytoplasmic dy-
nein) is a multisubunit protein complex with a heavy chain (HC)
polypeptide g500 kDa that is responsible for ATPase and motor
activities. There are also several intermediate chains (ICs), light
intermediate chains (LICs), and light chains (LCs) that are
responsible for subcellular localization and recognition and
binding of the various forms of cargo carried by dynein.2
Cytoplasmic dynein is involved in fundamental cellular
processes such as the return of vesicles to the MT organizing
center (MTOC) and the retention of the vesicles at this
subcellular locale. It is intricately involved in the maintenance
of the Golgi apparatus and in the trafficking of membrane-
Figure 1. Bromotyrosine-derived alkaloids.
encapsulated vesicles and proteins such as membrane-bound
receptors. Cytoplasmic dynein has roles in various stages of
muscle myosin without competing for the ATP sites on these
mitosis, including nuclear envelope breakdown at late pro-
motor proteins.16,17 Other small molecules that can inhibit dynein
metaphase, chromosome segregation, and mitotic spindle forma-
include the vanadate anion and several ATP analogues. Some
tion.6 Cytoplasmic dynein has also been implicated in retinitis
redox-active and thiol-perturbing agents are also inhibitors
pigmentosa,7 lissencephaly,8-10 virus transport,11,12 and neuro-
because dynein has a thioredoxin-like domain including vicinal
degenerative diseases.13,14
thiols. The structures of purealin and other related natural
Purealin (1) is a bromotyrosine-derived natural product
products are shown in Figure 1. Relatives of purealin 1 include
isolated from the sea sponge Psammaplysilla purea.15 It inhibits
lipopurealins A (2), B (3), and C (4), and purealidin A (5).18-20
the ATPase activity of isolated axonemal dynein and skeletal
Common substructures within these compounds include the
dibromotyrosine oxime (in an open form or the oxidatively
closed spiroisoxazoline form) and the aminohistamine side
* To whom correspondence should be addressed. Tel: 412-648-9706.
Fax: 412-624-1850. E-mail: bday@pitt.edu.
chain. In purealin, a spiroisoxazoline side chain is coupled to
the bromotyrosine amine, whereas in lipopurealins A, B, and
C, fatty acid side chains are coupled to the bromotyrosine amine.
The effects of the lipopurealins and purealidins on dynein
are unknown. What is known about these natural products is
that purealidin A (5) has weak inhibitory activity (22% inhibition
† Department of Chemistry, University of Pittsburgh.
‡ Department of Pharmaceutical Sciences, University of Pittsburgh.
§ Columbia University.
a Abbreviations: MTs, microtubules; MAP1C, microtubule-associated
protein 1C; HC, heavy chain; ICs, intermediate chains; LICs, light
intermediate chains; MTOC, MT organizing center; HPLC, high perfor-
mance liquid chromatography.
10.1021/jm051030l CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/17/2006

2064 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Scheme 1. Synthesis of Spiroisoxazoline Acid 13
Zhu et al.
Scheme 2. Synthesis of Protected Purealidin 20 and Attempted
Deprotection
Reagents and conditions: (a) Ba(OH)2, BnONH2‚HCl, dioxane/H2O, 60
°C, 64%. (b) TMSCH2N2, benzene/MeOH, rt, 92%. (c) H2, Pd-black, AcOH/
dioxane, rt, 81%. (d) NBS, DMF, rt. (e) Zn(BH4)2, CH2Cl2/Et2O, rt, 11:
24% in two steps; 12: 14% in two steps. (f) LiOH, MeOH/H2O, 94%.
at 100 µM) against Na,K-ATPase and exhibits cytotoxicity
against L1210 murine leukemia cells with an IC50 value of 1.9
µM.19 Lipopurealins A, B, and C exhibit inhibitory activities
against Na,K-ATPase purified from porcine brains and dog
kidneys, with lipopurealin B being the most potent inhibitor.20
We report herein the synthesis of a small bromotyrosine
library designed around the natural product purealin. The first
syntheses of purealin and purealidin A are described. Several
chemical groups were modified to examine the effects of relative
hydrophilicity in the compound library, and some alterations
in regioconnections were examined. The biological activity of
the resulting library members were examined and the results
are begining to point the way to the design of more potent
dynein inhibitors.
Reagents and conditions: (a) Br2, AcOH, NaOAc, rt, 96%. (b)
CbzNHCH2CH2CH2Br, K2CO3, DMF, 90 °C, 71%. (c) acetylglycine, Ac2O,
NaOAc, 120 °C, 96%. (d) Ba(OH)2, BnONH2‚HCl, dioxane/H2O, 60 °C,
34%. (e) aminohistamine, DCC, HOBt, Et3N, CHCl3/DMF, rt, 66%.
directly reduced with excess Zn(BH4)2 (prepared with ZnCl2
and NaBH4 as a 0.1 N solution in diethyl ether)24 to give
corresponding stereoisomers 11 and 12. These were separated
by preparative TLC or careful column chromatography. Trans
isomer 11 was obtained in 24% yield over two steps, and cis
isomer 12 was obtained in 14% yield. The modest overall yield
(38%) is probably due to the instability of 10. Characterization
data for 11 and 12 were consistent with literature.21a Ester 11
was easily saponified to requisite acid 13 in 94% yield with
LiOH,25 but hydrolysis of ester 12 under the same conditions
led to decomposition.
Results and Discussion
The synthesis of purealidin A (5) is shown in Scheme 2. The
bromination of 4-hydroxybenzaldehyde (15) afforded dibromo-
aldehyde 16 in 96% yield.26 The treatment of 16 with 3-(N-
benzyloxycarbonylamino)propyl bromide afforded 17 in 71%
yield.27 The heating of a mixture of aldehyde 17 and N-
acetylglycine to 120 °C in acetic anhydride for 4 h gave
azlactone 18 as a yellow solid in 96% yield after isolation by
filtration. Azlactone 18 was saponified with Ba(OH)2 in the
presence of O-benzylhydroxylamine in aqueous dioxane to
afford benzyloxime acid 19 in 34% yield. Acid 19 was converted
to amide 20 by coupling with aminohistamine, which was made
according to the known procedure.28 The extractive workup of
the reaction mixture containing 20 was problematic, so the
reaction solvent was simply removed by lyophilization. Amide
20 was obtained in 66% isolated yield after flash chromatog-
From a retrosynthetic perspective, purealin comprises the
spiroisoxazoline acid21 subunit and purealidin A (5). In turn,
purealidin A comprises the dibromotyrosine and the aminohis-
tamine subunits. The starting point for the synthesis of key
spiroisoxazoline acid 13 was azlactone 6 (Scheme 1), which
was prepared from 2-hydroxy-4-methoxybenzaldehyde in three
steps.22 Azlactone 6 was hydrolyzed with Ba(OH)2 in the
presence of O-benzyl hydroxylamine to provide dibenzyl oxime-
acid 7 in 64% yield. Acid 7 was treated with TMSCHN2 to
provide methyl ester 8 in 92% yield. Hydrogenolysis of 8 with
palladium-black in acetic acid and dioxane afforded O-phenolic
oxime-methyl ester (9) in 81% yield.23 Oxime-methyl ester 9
was cyclized with NBS in DMF to afford spirosioxazoline 10
in 92% crude yield. Because 10 degraded on silica gel, it was

Cytoplasmic Dynein Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2065
Scheme 5. Synthesis of Purealidin A (5) and Purealin (1)
Scheme 3. Synthesis of p-Methoxybenzylhydroxylamine 23
Reagents and conditions: (a) PMBCl, Et3N, DMF, 90 °C, 68%. (b)
hydrazine, 60 °C, then HCl, 50%.
Scheme 4. Deprotection of the PMB Group in Model System
25
Reagents and conditions: (a) Ba(OH)2, dioxane/H2O, PMBONH2‚HCl,
60 °C, 35%. (b) aminohistamine, DCC, HOBt, Et3N, DMF/CHCl3, rt, 68-
72%. (c) AlCl3, anisole, CH2Cl2/CH3NO2, rt, 94%. (d) 13, DCC, HOBt,
DMF/CH2Cl2, rt, 52%.
Scheme 6. Synthesis of Lipopurealins A (2), B (3), and C (4)
raphy by using 15:9:1:1 EtOAc/acetone/formic acid/H2O as the
eluent.
At this stage, it proved impossible to remove the protecting
groups from 20 to give 5. Attempts at hydrogenolysis of the
benzyl and Cbz protecting groups on amide 20 with palladium-
black in acetic acid and dioxane23 only returned the starting
material. Hydrogenation with Pd/C in methanol or ethanol led
to decomposition, whereas the use of acetic acid and dioxane
yielded no conversion. Treatment of amide 20 with TMSCl/
NaI29 only removed the Cbz group.
The benzyl oxime-protecting group was therefore replaced
with the more easily removable p-methoxybenzyl (PMB) group.
O-PMB hydroxyamine (23) was synthesized as shown in
Scheme 3.30 N-Hydroxyphthalimide (21) was treated with
PMBCl in DMF at 90 °C to afford PMB hydroxyphthalimide
(22) in 68% yield. This was hydrolyzed in the presence of
hydrazine to give O-PMB-hydroxyamine (23) in 50% yield.
Before the coupling reaction between acid 27 and amino-
histamine was performed, model reactions for the removal of
the PMB group were conducted (Scheme 4). Model oxime 25
was readily made from p-tolualdehyde (24) and PMB-hydroxyl-
amine (23). Attempted deprotections of 25 with TMSCl/NaI,
DDQ,31 or AlCl3 in acetonitrile were not successful. However,
treatment of 25 with AlCl3 and anisole in CH2Cl2 removed the
PMB group to give 26 in 82% yield.32 A similar reaction in 1:1
CH2Cl2/CH3NO2 gave 26 in 79% yield.
Reagents and conditions: (a) 5, DCC, HOBt, DMF/CH2Cl2, rt.
natural product.33 Racemic purealin 1 was synthesized subse-
quently in 52% yield by coupling amine 5 with spiroisoxazoline
acid 13. Again, the spectroscopic data for synthetic 1 was
identical to the literature values for the natural product.
With purealidin A (5) in hand, lipopurealins A, B, and C
2-4 were synthesized by DCC/HOBt-mediated coupling to
myristic, methylmyristic, and palmitic acids, respectively (Scheme
6). The 1H NMR spectra of these three compounds were
identical to those in the literature.34
A 16 member purealidin A library was next prepared by
coupling the 4 bromophenyl oxime acids and the 4 amines
shown in Figure 2. The oxime acids were 3,5-dibromo-4-
butoxyphenyl oxime acid (27a), 3-bromo-4-butoxyphenyl oxime
acid (27b), 3,5-dibromo-2-butoxyphenyl oxime acid (27c), and
3,5-dichloro-2-butoxyphenyl oxime acid (27d), and the amines
were phenethylamine (29a), 2-(4-methoxyphenyl) amine (29b),
2-(4-chlorophenyl) amine (29c), and tyrosine (29d).
O-PMB-protected oxime acid 27a was then prepared in 35%
yield by treatment of azlactone 18 with O-PMB-protected
hydroxyamine under basic conditions (Scheme 5). Acid 27a was
treated with aminohistamine in the presence of DCC and HOBt
to afford amide 28 in 70% yield. The PMB and Cbz groups of
28 were removed by exposure to AlCl3/anisole to afford
The synthesis of acid 27b (Scheme 7) was analogous to that
of acid 27a. 3-Bromo-4-hydroxybenzaldehyde (30) was pro-
tected by 3-(N-benzyloxycarbonylamino)propyl bromide to
afford phenyl ether 31 in 79% yield. This was treated with
acetylglycine in acetic anhydride at 120 °C for 4 h to afford
1
purealidin A (5) in 94% yield. H and 13C NMR and mass
spectra of synthetic 5 were identical to those reported for the

2066 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Zhu et al.
Scheme 8. Sixteen Coupling Reactions of Amines with Oxime
Acids
Figure 2. Four acids and four amines used in the library synthesis.
Scheme 7. Synthesis of Bromotyrosine Acids 27b-d
which was an oil that was difficult to purify. The treatment of
acid 27b with TMSCHN2 followed by hydrolysis with LiOH
afforded acid 27b as a white solid in 32% yield over three steps.
Acids 27c and d were synthesized by similar procedures.
All of the 16 coupling reactions were achieved by the same
protocol (Scheme 8).35 A mixture of 1 equiv of the acid, 2 equiv
of the amine, 1.5 equiv of EDCI, and 0.5 equiv of DMAP in
CH2Cl2 was stirred at room temperature for 15 h. Diethyl ether
was added, and the mixture was washed with 5% HCl to remove
EDCI and DMAP. HPLC analyses showed a single peak for
each of the 16 desired amides. Table 1 shows the reaction yields
and the HPLC retention times of the 16 products. LC-MS
analyses further verified the identities and purities of the amides.
The removal of the PMB and Cbz groups from the 16 amides
provided the 16 purealidin A analogues 40a-d, 41a-d,
42a-d, and 43a-d shown in Figure 3. All 16 library compo-
nents were purified by flash chromatography followed by
preparative HPLC.
The 21 purealidins and purealin were assessed for antipro-
liferative activity against a small panel of human carcinoma
cells (MDA-MB231 (breast), PC-3 (prostate), 2008 (ovarian),
and HT-29 (colon)) as well as against the mouse L1210
leukemia cell line. The L1210 cells were used to compare data
obtained here against that reported in the literature for purealidin
A.19 The 50% growth inhibitory (GI50) values obtained (Table
2) showed purealin 1 and purealidin A (5) to be inactive against
the human cell lines. However, some of the purealidin A
analogues, for example, 43c and d, showed low micromolar
antiproliferative activity. The mouse leukemia cells, however,
were uniformly sensitive to the individual library components.
The abilities of the library components to inhibit the MT-
stimulated ATPase activity of a recombinant form of the full
motor domain fragment of the rat cytoplasmic dynein heavy
chain36 were examined. First, the library components were
screened against 50 µg/mL of rat cytoplasmic dynein motor
domain (hereafter dynein motor domain) in the presence of 5
mg/mL of paclitaxel-induced tubulin polymer. Purealin 1 and
purealidin A (5) gave IC50 values of 35 and 42 µM, respectively.
The following compounds showed some inhibitory actions, but
their IC50 values were greater than 50 µM for 2, 41c and d, 42c
and d, and 43d. Then, inhibitor/protein ratios for the best
ATPase inhibitory ranges were examined. Three compounds
Reagents and conditions: (a) CbzNHCH2CH2CH2Br, K2CO3, DMF, 100
°C, 79%. (b) acetylglycine, Ac2O, NaOAc, 125 °C, 85%. (c) Ba(OH)2,
dioxane/H2O, PMBONH2‚HCl, 60 °C. (d) TMSCHN2, benzene/MeOH. (e)
LiOH, H2O/MeOH, rt, three steps, 32%.
azlactone 32 in 85% yield. The treatment of 33 with Ba(OH)2
in the presence of PMBONH2‚HCl provided oxime acid 27b,

Cytoplasmic Dynein Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2067
Table 1. Yields and HPLC Retention Times of Sixteen Synthetic Amides Prepared as Precursors to the Test Agent Library
retention
final
product
retention
final
product
compd
yield
time (min)a
compd
yield
time (min)a
36a
36b
36c
36d
37a
37b
37c
37d
93%
93%
96%
91%
89%
95%
91%
88%
9.8
9.0
12.6
8.2
7.0
6.4
8.7
5.7
40a
40b
40c
40d
41a
41b
41c
41d
38a
38b
38c
38d
39a
39b
39c
39d
82%
90%
79%
81%
91%
88%
87%
86%
12.8
11.5
16.8
9.9
11.2
10.0
14.5
8.6
42a
42b
42c
42d
43a
43b
43c
43d
a 7:13 CH3CN/H2O (0.1% CF3CO2H), Symmetry C18 column, l mL/min.
Figure 5. Inhibition by purealin 1 of dynein motor domain catalyzed
release of free phosphate from ATP. The dynein motor domain was
used in the presence of the paclitaxel-induced tubulin polymer (1 mg/
mL) and a test agent. ATP (2 mM) was added to initiate the reaction,
and the concentration of free phosphate was determined. No inhibitor
((); 2 µM 1 (9); 10 µM 1 (2); and 50 µM 1 (b). Each point is the
mean of four determinations ( SD (some of the error bars are smaller
than the symbols used).
in Figure 5. To investigate the kinetics in the presence of
inhibitors, purealin 1, purealidin A (5), and 41c were each
incubated at 50 µM with different concentrations of the
substrate, ATP, to determine the Michaelis-Menten parameters
of the dynein motor domain (Figure 6a). The calculated Vapp
,
max
Kapp and Ki values are given in Table 3. In a typical dynein
M
motor domain ATPase kinetic assay, the basal ATPase activity
was 0.41s-1 (Vmax), whereas the MT-stimulated ATPase activity
was 2.89 s-1 (Vmax), 7-fold of the basal activity. These results
further show the inhibitory effects of purealin and analogues
on the MT-stimulated dynein motor domain ATPase activity.
The rank order of inhibition was purealin 1 > purealidin A (5)
g 41c. An examination of the kinetics in the Hanes-Woolf
format (Figure 6b), wherein no significant change in the y
intercept was noted, indicated that the compounds displayed
uncompetitive inhibition, supporting the hypothesis that
purealins do not bind to the ATP site.
Figure 3. Structures of 16 purealidin A analogues.
Conclusions
Dynein plays an important role in variety of cellular processes.
Recent work is beginning to uncover the mechanism of dynein
movement and its power stroke.36-38 A variety of molecules
involved in cell growth control, including p53, Bim, cdc2 kinase,
HDAC6, HSP70, and HSP90 have also been reported to interact
with or be under the control of cytoplasmic dynein.39-44 Motor
activity and motor-cargo interactions in dynein and other motor
proteins, such as kinesin and myosin, might represent attractive
drug targets and are certainly of interest in the development of
chemical biology tools. Recently, RNA interference technology
has been applied against dynein as well as its regulatory
polypeptides,45-49 showing that lowering the level of the
cytoplasmic dynein heavy chain (and therefore its activity in
Figure 4. Dynein motor domain ATPase inhibitory activities of
purealin 1, purealidin A (5), and compound 41c. Dynein motor domain
(5 µg/mL) and 1 mg/mL of paclitaxel-induced tubulin polymer were
used in this assay. The reaction was initiated by the addition of 2 mM
ATP: 1 (b); 5 (9); and 41c (2). Each point is the mean of four
determinations ( SD.
within the library, purealin 1, purealidin A (5), and compound
41c (the latter chosen because of its antiproliferative effects)
showed ATPase inhibitory properties (Figure 4).
The concentration-dependent inhibition of MT-stimulated
ATPase by purealin 1 against the dynein motor domain is shown

2068 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Table 2. Antiproliferative Activities of the Purealin/Purealidin Library
Zhu et al.
GI50 (µM)
compd
2008
MDA-MB231
HT29
PC3
L1210
40a
40b
40c
40d
41a
41b
41c
41d
42a
42b
42c
42d
43a
43b
43c
43d
2
29 ( 2
24 ( 3
29 ( 1
27 ( 1
30 ( 5
31 ( 12
27 ( 3
26 ( 2
19 ( 5
25 ( 2
5.6 ( 0.9
7.5 ( 0.4
8.8 ( 2.7
29 ( 1
5.4 ( 0.6
5.7 ( 0.1
28 ( 3
29 ( 1
5.7 ( 1.2
>50
27 ( 0
17 ( 2
27 ( 2
24 ( 2
>50
31 ( 3
20 ( 5
31 ( 1
26 ( 4
27 ( 4
30 ( 9
27 ( 1
30 ( 1
5.3 ( 0.2
4.9 ( 0.6
5.3 ( 0.2
6.0 ( 0.1
5.6 ( 0.2
6.2 ( 1.7
5.5 ( 1.8
6.0 ( 0.6
29 ( 2
30 ( 0
20 ( 3
>50
31 ( 3
21 ( 5
30 ( 4
26 ( 9
31 ( 6
39 ( 4
33 ( 4
32 ( 6
19 ( 6
35 ( 15
15 ( 7
31 ( 6
31 ( 7
32 ( 11
7.8 ( 0.9
5.0 ( 1.1
30 ( 4
25 ( 9
32 ( 3
>50
6.4 ( 2.2
2.8 ( 2.8
7.8 ( 2.5
5.8 ( 1.2
6.2 ( 0.9
9.9 ( 6.1
6.5 ( 2.0
7.6 ( 1.9
6.3 ( 0.4
6.2 ( 0.9
6.3 ( 0.8
5.4 ( 0.9
7.5 ( 0.9
6.9 ( 0.5
5.9 ( 0.5
5.8 ( 1.1
9.0 ( 1.9
12 ( 4
48 ( 14
26 ( 1
25 ( 1
5.7 ( 0.8
8.8 ( 0.6
5.6 ( 0.7
6.7 ( 1.0
7.6 ( 3.2
24 ( 2
5.2 ( 0.9
5.3 ( 0.0
28 ( 1
28 ( 3
7.0 ( 0.4
>50
3
4
5
1
9.7 ( 1.6
4.7 ( 1.6
3.2 ( 1.2
9.2 ( 4.3
>50
>50
40 ( 7
>50
>50
discodermolide (nM)
2.7 ( 1.7
28 ( 9
52 ( 8
Table 3. Vappmax, KappM, and Ki Values of the Microtubule-Stimulated
ATPase Activity of Recombinant Dynein Heavy Chain Motor Domain
in the Presence of 50 µM 1, 5, or 41c
Vappmax (s-1
)
KappM (mM)
Ki (mM)
no inhibitor
41c
5
2.89
2.45
2.39
1.84
0.091
0.073
0.070
0.061
N/A
0.30
0.26
0.85
1
Very little is known, however, about the structural require-
ments of small molecules to inhibit dynein. Agents that
competitively inhibit the binding of ATP to the protein are the
most studied, and redox active agents will also inhibit the
protein. The only non-ATP, noninorganic and nonredox active
agent known to inhibit the axonemal isoform of cytoplasmic
dynein is the natural product purealin 1,16 which acted as our
seminal lead in these studies. Our results showed that synthetic
purealin 1 and two of its analogues inhibit MT-stimulated
cytosolic dynein heavy chain’s ability to hydrolyze ATP. Thus,
purealins might be useful for further physiological investigation
of dynein function.
Kon and co-workers50 showed that ATP binding and its
hydrolysis only at the P1 site are essential for the motor activities
of cytoplasmic dynein and suggested that the motor activities
are also regulated by the other nucleotide-binding/hydrolysis
sites. For example, nucleotide binding at the P3 site is also
critical for MT-activated ATPase and the motile activities of
cytoplasmic dynein.50 It is known that purealin 1 inhibits the
ATPase activity of axonemal dynein but does not compete for
the ATP binding site.16 Here, we found it to inhibit cytoplasmic-
dynein motor domain ATPase activity and examined the kinetics
of inhibition by 1 and by some of its analogues (Figure 6).
Plotting the data in the Hanes-Woolf format showed no
significant change in the y intercept, which represents the value
of Km/Vmax, in the presence of purealin 1, purealidin A (5), and
41c. This indicates that the binding pattern of the purealins to
the cytoplasmic dynein motor domain is uncompetitive, meaning
the inhibitors bind to a site that becomes available only after
ATP has bound to the P1 site, giving rise to the conclusion that
purealins do not compete for the binding of ATP or its
hydrolysis at the P1 site. The binding site of purealins is still
unknown.
Figure 6. Michaelis-Menten (A) and Hanes-Woolf (B) plots of
dynein motor domain ATPase activity in the presence of purealin 1,
purealidin A (5), and compound 41c. The reaction was initiated with
different concentrations of ATP in the presence of 12.5 µg/mL of
recombinant dynein heavy chain and 1 mg/mL of paclitaxel-induced
tubulin polymer. No inhibitor (b); 50 µM 41c (O); 50 µM 5 (1); 50
µM 1 (3).
bulk) leads to a block in mitosis.45 Effective small-molecule
dynein inhibitors could therefore be useful for further investiga-
tion of the cellular function of dynein in vivo.

Cytoplasmic Dynein Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2069
1
a white solid. H NMR (300 MHz, CDCl3): δ 7.42 (s, 1H), 3.94
Although purealin 1 and purealidin A (5) were inactive as
human cancer cell antiproliferative agents, their inhibition of
cytoplasmic dynein ATPase activity supports the hypothesis that
purealin/purealidin and analogues are good leads for finding
small molecules to inhibit this target. Compound 41c showed
reasonable antiproliferative activity against human carcinoma
cell lines as well as the mouse leukemia cell line and inhibited
dynein motor domain ATPase activity, providing correlative
support of the hypothesis that small molecule inhibitors of the
molecular motor can have antiproliferative effects. Compared
to the effects on human cancer cell lines, the growth of the
mouse leukemia L1210 cell line was uniformly sensitive to low
micromolar concentrations of the purealin/purealidin library
components. Future work will include more detailed cell-based
experiments to begin to determine the mechanisms by which
these bromotyrosine-containing agents exert their antiprolifera-
tive effects and whether the mechanisms include the inhibition
of cellular cytoplasmic dynein.
(s, 2H), 3.91 (s, 3H), 3.86 (s, 3H); 13C NMR (75 MHz, CDCl3):
164.5, 153.7, 151.5, 149.9, 133.4, 119.4, 107.6, 60.6, 53.5, 25.5.
Methyl 7,9-dibromo-8-methoxy-10-oxo-1-oxo-2-azospirol[4.5]-
deca-2,6,8-triene-3-carboxylate (10). A mixture of NBS (940 mg,
5.29 mmol) and O-phenolic oxime acid 9 (1.40 g, 3.53 mmol) in
DMF (15 mL) was stirred at room temperature for 3 h. After the
addition of Et2O (200 mL), the solution was washed successively
with H2O, 5% aqueous NaS2O3, and H2O, then dried over MgSO4,
and filtered. The solvent was removed under reduced pressure to
1
give spiroisoxazoline 10 (1.29 g, 92%) as a yellow oil. H NMR
(300 MHz, CDCl3): δ 6.78 (s, 1H), 4.18 (s, 3H), 3.91 (s, 3H),
3.61 (d, J ) 18.0 Hz, 1H), 3.30 (d, J ) 18.0 Hz, 1H).
Methyl 7,9-dibromo-8-methoxy-10-hydroxy-1-oxo-2-azospirol-
[4.5]deca-2,6,8-triene-3-carboxylate (trans-11 and cis-12). Zn-
(BH4)2 (0.1 N solution in Et2O, 35 mL) was added to a solution of
crude spiroisoxazoline 10 (1.1 g, 2.78 mmol) in CH2Cl2 (35 mL)
at 0 °C over a 10 min period. The reaction mixture was stirred for
another 5 min at room temperature. After the addition of saturated
aqueous NH4Cl, the mixture was extracted with Et2O (3 × 100
mL). The extracts were washed with H2O, dried over MgSO4, and
filtered. The crude product was purified by column chromatography
(2:1 hexane/EtOAc) to afford 11 (290 mg, 26.3%) and 12 as pale-
Experimental Details
Chemistry. General Procedures. All reactions were performed
under an atmosphere of argon, unless the reaction solvent contained
H2O. Benzene was distilled from Na/benzophenone. CH2Cl2, THF,
Et2O, and toluene were dried by passing through activated
alumina.51 High-resolution mass spectra were obtained on a V/G
70/70 double focusing instrument. High-performance liquid chro-
matography (HPLC) was performed using the indicated ratios of
CH3CN to H2O, both containing 0.1% CF3CO2H (TFA), flowing
at a rate of 1 mL/min through a Symmetry C18 column, and analytes
were detected by UV absorption. Percent purities given represent
the relative purity from the reactions (the purities of compounds
used for biological evaluations was >99%).
3-(2-Benzyloxy-3,5-dibromo-4-methoxyphenyl)-2-benzyloxy-
iminopropanoic Acid (7). A mixture of azlactone 6 (12 g, 25
mmol) and Ba(OH)2 (30 g, 175 mmol) in 1:1 dioxane/H2O (360
mL) was stirred for 1 h at 60 °C. O-Benzyl hydroxylamine
hydrochloride (12 g, 75 mmol) was added at 60 °C, and the mixture
was stirred vigorously at the same temperature for 14 h. The reaction
mixture was cooled to 0 °C, the solution was acidified with 15%
HCl, and a yellow solid precipitated. After filtration, the solid was
washed with cold 1:1 pentane/Et2O to yield 7 as a pale-yellow solid
(10.5 g, 75%). 1H NMR (300 MHz, CDCl3): δ 7.62-7.59 (m, 2H),
7.52-7.45 (m, 6H), 7.34-7.30 (m, 2H), 7.37 (s, 1H), 5.33 (s, 2H),
5.12 (s, 2H), 4.02 (s, 2H), 4.00 (s, 3H); 13C NMR (75 MHz,
CDCl3): 162.3, 154.5, 154.3, 149.3, 136.5, 135.6, 132.0, 128.8,
128.75, 128.5, 128.3, 128.2, 127.9, 114.7, 112.9, 78.6, 74.9, 60.7,
25.2.
1
yellow solids (170 mg, 15.5%). trans-11: H NMR (300 MHz,
acetone-d6) δ 6.53 (s, 1H), 5.42 (d, J ) 8.1 Hz, 1H), 4.21 (d, J )
8.1 Hz, 1H), 3.84 (d, J ) 18.0 Hz, 1H), 3.83 (s, 3H), 3.72 (s, 3H),
3.20 (d, J ) 18.0 Hz, 1H), 2.83 (s, 1H); 13C NMR (75 MHz,
acetone-d6) 161.2, 152.5, 148.8, 132.2, 122.2, 113.8, 92.5, 75.2,
60.2, 52.8, 40.0. cis-12: 1H NMR (300 MHz, acetone-d6) δ 6.31
(s, 1H), 5.07 (brs, 1H), 4.55 (brs, 1H), 3.82 (s, 3H), 3.73 (s, 3H),
3.46 (d, J ) 18.0 Hz, 1H), 3.39 (d, J ) 18.0 Hz, 1H); 13C NMR
(75 MHz, acetone-d6) 161.3, 152.3, 146.2, 131.7, 127.6, 124.0, 90.5,
74.47, 60.5, 52.9, 43.4.
7,9-Dibromo-8-methoxy-10-hydroxy-1-oxo-2-azospirol[4.5]-
deca-2,6,8-triene-3-carboxylic Acid (13). A mixture of spiroisox-
azoline ester 11 (198 mg, 0.5 mmol) and LiOH‚H2O (63 mg, 1.5
mmol) in 1:1 MeOH/H2O (30 mL) was stirred for 30 min at room
temperature. After the addition of 5% HCl (3 mL), the solution
was concentrated under reduced pressure. EtOAc (50 mL) was
added. The mixture was washed with H2O, dried over MgSO4, and
filtered. The solvent was removed under reduced pressure to afford
13 (189 mg, 98%) as a pale-yellow solid, mp 110-112 °C. IR (KBr,
cm-1): 3500 (broad), 1723, 1589; H NMR (300 MHz, CD3OD):
1
δ 6.44 (s, 1H), 4.91 (s, 3H), 4.11 (s, 1H), 3.75 (d, J ) 18.0 Hz,
1H), 3.73 (s, 3H), 3.10 (d, J ) 18.0 Hz, 1H); 13C NMR (75 MHz,
CD3OD) 162.7, 153.9, 149.3, 132.2, 122.8, 114.2, 93.1, 75.5, 60.4,
40.2; MS (EI) m/z: (M+•) 381 (79Br2, 12), 365 (34), 351 (57), 69-
(100); HRMS (EI) m/z: (M+•) calcd for C10H979Br2NO5, 380.8847;
found, 380.8865.
Methyl 3-(2-benzyloxy-3,5-dibromo-4-methoxyphenyl)-2-
benzyloxyiminopropanoate (8). TMSCHN2 (12 mL, 2.0 N in
hexane) was added to a solution of acid 7 (9.0 g, 16 mmol) in 3:1
benzene/MeOH (160 mL) at 0 °C. The reaction mixture was stirred
for 45 min at room temperature. 5% HCl (10 mL) was added at 0
°C. A yellow solid precipitated after most of the organic solvents
were removed. After filtration, the solid was washed with cold 1:1
pentane/Et2O to yield 8 (8.03 g, 87%). 1H NMR (300 MHz,
CDCl3): δ 7.49-7.46 (m, 2H), 7.39-7.29 (m, 6H), 7.21-7.17 (m,
2H), 7.19 (s, 1H), 5.23 (s, 2H), 4.93 (s, 2H), 3.89 (s, 2H), 3.86 (s,
3H), 3.69 (s, 3H); 13C NMR (75 MHz, CDCl3): 163.6, 154.2, 153.8,
149.9, 136.5, 136.0, 132.0, 128.6, 128.5, 128.4, 128.35, 128.2,
128.1, 127.9, 114.5, 112.7, 78.0, 74.4, 60.6, 52.8, 26.4.
Methyl 3-(2-hydroxy-3,5-dibromo-4-methoxyphenyl)-2-hy-
droxyiminopropanoate (9). A solution of benzyl ester 8 (7.4 g,
12.8 mmol) in 1:1 AcOH/dioxane (180 mL) was hydrogenated over
Pd-black (1.21 g) under H2 (1 atm) at room temperature for 24 h.
After filtration, the solvent was removed under reduced pressure.
EtOAc (200 mL) was added, and the solution was washed with
H2O, dried over MgSO4, filtered, and the solvent removed under
reduced pressure. The crude product was purified by column
chromatography (2:1 hexane/EtOAc) to afford 9 (4.19 g, 82%) as
Purealin (1). A mixture of acid 13 (11.5 mg, 0.03 mmol),
purealidin A 5 (22 mg, 0.04 mmol, free amine), DCC (9.3 mg,
0.045 mmol), and HOBt (6.1 mg, 0.045 mmol) in CH2Cl2 (3 mL)
and DMF (3 mL) was stirred at room temperature for 20 h. After
the solvent was removed under reduced pressure, the crude product
was purified by flash chromatography (15:9:1:1 EtOAc/acetone/
HCO2H/H2O). The product was dissolved in MeOH and acidified
with 0.5 N HCl. Solvents were removed to afford 1 as a pale-
yellow solid (14 mg, 54%), mp 139-141 °C. IR (KBr, cm-1): 3450
(broad), 1655, 1541; 1H NMR (300 MHz, CD3OD): δ 7.47 (s, 2H),
6.51 (s,1H), 6.42 (d, J ) 0.6 Hz, 1H), 4.08 (d, J ) 0.6 Hz, 1H),
4.04 (t, J ) 6.0 Hz, 2H), 3.83 (s, 2H), 3.78 (d, J ) 18.3 Hz, 1H),
3.72 (s, 3H), 3.58 (t, J ) 6.9 Hz, 2H), 3.46 (t, J ) 6.9 Hz, 2H),
3.09 (t, J ) 18.3 Hz, 1H), 2.70 (t, J ) 6.9 Hz, 2H), 2.10 (quin, J
) 6.6 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) 163.1, 158.9, 154.5,
150.8, 150.75, 147.1, 146.8, 136.2, 132.8, 131.2, 124.1, 120.7,
117.2, 113.1, 109.0, 90.2, 73.5, 71.2, 59.6, 37.3, 36.2, 29.3, 27.8,
24.3; MS (ESI) [M + H]+ m/z: 880 (79Br4, 6), 882 (79Br381Br, 16),
884 (79Br281Br2, 17), 886 (79Br81Br3, 13), 888 (81Br4, 5); HRMS
(ESI) [M + H]+ calcd for C27H2979Br4N7O7 m/z: 879.8884; found,
879.8935. HPLC analysis: 8:17 CH3CN (0.1% TFA)/H2O (0.1%
TFA), tR ) 19.9 min, 98% purity.

2070 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Zhu et al.
3,5-Dibromo-4-hydroxybenzaldehyde (16). Bromine (30.2 g,
190 mmol) in AcOH (50 mL) was added to a mixture of
4-hydroxybenzaldehyde (11 g, 90 mmol) and sodium acetate (22.9
g, 279 mmol) in AcOH (200 mL) at room temperature over 20
min. The reaction mixture was stirred for 1 h at room temperature.
A solid precipitated after H2O (200 mL) was added. After filtration,
the solid was washed with H2O and dried in a vacuum desiccator
with P2O5 overnight to afford 16 as a pale-yellow solid (24.1 g,
under reduced pressure, and the crude product was purified by
column chromatography (15:9:1:1 EtOAc/acetone/HCO2H/H2O) to
afford amide 20 (245 mg, 66%) as a pale-yellow solid. H NMR
1
(300 MHz, CD3OD): δ 7.39 (s, 2H), 7.37-7.29 (m, 10H), 6.48 (s,
1H), 5.25 (s, 2H), 5.07 (s, 2H), 3.98 (t, J ) 6.0 Hz, 2H), 3.79 (s,
3H), 3.48 (t, J ) 6.1 Hz, 2H), 3.40 (t, J ) 6.1 Hz, 2H), 2.70 (t, J
) 6.1 Hz, 2H), 2.00 (quin, J ) 6.1 Hz, 2H); 13C NMR (75 MHz,
CD3OD) 164.7, 158.9, 153.0, 152.7, 148.7, 138.4, 138.0, 136.7,
134.5, 129.6, 129.5, 129.4, 128.9, 128.8, 126.2, 78.7, 72.1, 67.4,
39.0, 31.3, 29.7, 25.8; MS (ESI) m/z: [M + H]+ 741 (79Br2, 40),
743 (79Br81Br, 100); HRMS (ESI) m/z: [M + H]+ calcd for C32H35-
79Br2N6O5, 741.1018; found, 741.1030.
1
96%). H NMR (300 MHz, CDCl3): δ 9.80 (s, 1H), 8.00 (s, 2H),
6.43 (s, 1H); 13C NMR (75 MHz, acetone-d6) 189.8, 157.0, 135.0,
132.7, 112.4.
General Procedure B: Synthesis of Phenol Ethers. Benzyl
[3-(2,6-dibromo-4-formylphenoxy)propyl]carbamate (17). A mix-
ture of phenol 16 (2.8 g, 10 mmol), K2CO3 (3.45 g, 25 mmol), and
16 (3.0 g, 11 mmol) in DMF was stirred at 100 °C for 4 h. The
reaction mixture was extracted with Et2O (5 × 40 mL). The extracts
were washed with H2O, dried over MgSO4, filtered, and the solvent
removed under vacuum. The crude product was purified by column
chromatography (2:1 hexane/EtOAc) to afford 17 as a white solid
(3.32 g, 70.5%). IR (KBr, cm-1): 3311, 3063, 1693, 1544; 1H NMR
(300 MHz, CDCl3): δ 9.86 (s, 1H), 8.03 (s, 2H), 7.38-7.35 (m,
5H), 5.16 (brs, 1H), 5.13 (s, 2H), 4.15 (t, J ) 5.8 Hz, 2H), 3.56 (q,
J ) 6.3 Hz, 2H), 2.12 (quin, J ) 6.2 Hz, 2H); 13C NMR (75 MHz,
CDCl3): 188.3, 157.8, 156.4, 136.5, 134.2, 133.8, 128.4, 128.0,
119.3, 71.6, 66.6, 38.4, 29.9; MS (EI) m/z: (M+•) 468 (79Br2, 30),
277 (65), 192 (40), 91 (100); HRMS (EI) m/z: (M+•) calcd for
C18H1679Br2NO4, 467.9446; found, 467.9464.
2-(4-Methoxybenzyloxy)isoindole-1,3-dione (22). A mixture of
N-hydroxyphthalimide (26.1 g, 160 mmol), PMBCl (25 g, 160
mmol), and Et3N (53 mL, 384 mmol) in DMF (400 mL) was stirred
for 40 min at 90 °C. A solid precipitated after the mixture was
poured into ice water (500 mL). The solid was collected by
filtration. The solid was washed with H2O and dried under vacuum
1
to yield 22 as a pale-yellow solid (30.8 g, 68%). H NMR (300
MHz, CDCl3): δ 7.73-7.70 (m, 2H), 7.44 (d, J ) 8.7 Hz, 2H),
6.87 (d, J ) 8.7 Hz, 2H), 5.14 (s, 2H), 3.79 (s, 3H); 13C NMR (75
MHz, CDCl3): 163.4, 160.3, 134.3, 131.5, 128.7, 125.7, 123.3,
113.8, 79.4, 55.1.
O-(4-Methyoxybenzyl)hydroxylamine Hydrochloride (23).
Compound 22 (18.7 g, 66 mmol) in 12:25 DMF/MeOH (270 mL)
was heated to 60 °C and treated with hydrazine hydrate (10 mL).
After 5 min, the mixture was cooled to room temperature. H2O
(100 mL) was added, and most of the MeOH was removed under
reduced pressure. The solution was extracted with EtOAc (4 × 100
mL), washed with H2O, dried over MgSO4, filtered, and the solvent
removed under reduced pressure. The crude product was purified
by column chromatography (2:1 hexane/EtOAc) to provide an oil.
MeOH (30 mL) and concentrated HCl (6 mL) were added to the
oil at 0 °C, and the solvent was removed under reduced pressure
General Procedure C: Synthesis of Azlactones. Benzyl {3-
[2,6-dibromo-4-(5-oxo-2-methyloxazol-4-ylidenemethyl)phenoxy]-
propyl}carbamate (18). A mixture of aldehyde 17 (3.02 g, 6.28
mmol), sodium acetate (515 mg, 6.28 mmol), and N-acetylglycine
(735 mg, 6.28 mmol) in Ac2O (12.5 mL) was stirred at 120 °C for
4 h. A yellow solid precipitated after the reaction mixture was
cooled to room temperature. After filtration, the solid was washed
with cold 1:1 pentane/Et2O to yield 18 as a yellow solid (3.35 g,
1
to afford 23 as a white solid (6.2 g, 50%). H NMR (300 MHz,
1
97%). IR (KBr, cm-1): 3316, 3066, 1807, 1772, 1682; H NMR
CD3OD): δ 7.37 (d, J ) 6.9 Hz, 2H), 6.98 (d, J ) 6.9 Hz, 2H),
4.95 (s, 2H), 3.82 (s, 3H).
(300 MHz, CDCl3): δ 8.26 (s, 2H), 7.38-7.33 (m, 5H), 6.92 (s,
1H), 5.21 (brs, 1H), 5.13 (s, 2H), 4.12 (t, J ) 5.7 Hz, 2H), 3.55 (q,
J ) 6.3 Hz, 2H), 2.44 (s, 3H), 2.11 (quin, J ) 6.0 Hz, 2H);13C
NMR (75 MHz, CDCl3): 167.3, 167.0, 156.4, 154.7, 136.6, 135.8,
133.7, 131.8, 128.5, 128.1, 127.0, 118.6, 71.5, 66.6, 38.5, 29.9,
15.7; MS (ESI) m/z: [M + Na]+ 573 (79Br2, 50), 575 (79Br81Br,
100), 553 (35), 551 (15); HRMS (ESI) m/z: [M + Na]+ calcd for
C22H2079Br2N2O5Na, 572.9614; found, 572.9565.
4-Methylbenzaldehyde O-(4-methoxybenzyl)oxime (25). A
mixture of amine 23 (379 mg, 2 mmol) and 4-methylbenzaldehyde
24 (180 mg, 1.5 mmol) in pyridine (20 mL) was stirred for 3 h at
room temperature. EtOAc (50 mL) was added after the solvent was
removed under reduced pressure. The solution was washed suc-
cessively with H2O, saturated aqueous CuSO4 and H2O, dried over
MgSO4, filtered, and the solvent removed under reduced pressure.
The crude product was purified by column chromatography (20:1
hexane/EtOAc) to afford 25 as a white solid (350 mg, 92%). IR
(KBr, cm-1): 2931, 1612, 1584, 1511, 1441; 1H NMR (300 MHz,
CDCl3): δ 8.07 (s, 1H), 7.45 (d, J ) 8.0 Hz, 2H), 7.35 (d, J ) 8.4
Hz, 2H), 7.15 (d, J ) 8.0 Hz, 2H), 6.88 (d, J ) 8.4 Hz, 2H), 5.12
(s, 2H), 3.78 (s, 3H), 2.34 (s, 3H); 13C NMR (75 MHz, CDCl3):
159.4, 148.9, 139.9, 130.1, 129.5, 129.4, 129.3, 127.0, 113.8, 76.0,
55.2, 21.4; MS (EI) m/z: 255 (M+•, 30), 122 (100), 91 (46), 77
(65); HRMS (EI) m/z: (M+•) calcd for C16H17NO2, 255.1257; found,
255.1259.
3-[4-(3-Benzyloxycarbonylaminopropoxy)-3,5-dibromophenyl]-
2-benzyloxyiminopropanoic Acid (19). A mixture of azlactone
18 (2.2 g, 4 mmol) and Ba(OH)2 (4.8 g, 28 mmol) in 1:1 dioxane/
H2O (56 mL) was stirred at 60 °C for 1 h. O-Benzylhydroxylamine
hydrochloride (2.05 g, 12.8 mmol) was added at 60 °C, and the
mixture was stirred vigorously at the same temperature for 6 h.
The reaction mixture was cooled to 0 °C and acidified with 10%
HCl. The mixture was extracted with EtOAc. The extracts were
washed with H2O, dried over MgSO4, filtered, and the solvent
removed under reduced pressure. The crude product was purified
by column chromatography (10:1 EtOAc/MeOH) to afford 19 (850
mg, 34%) as a pale-yellow solid, mp 118-120 °C. IR (KBr, cm-1):
3437, 2933, 1763, 1721, 1513, 1455, 1369, 1231; 1H NMR (300
MHz, 1:2 CDCl3/CD3OD) δ 7.47 (s, 2H), 7.35 (brs, 10H), 5.31 (s,
2H), 5.10 (s, 2H), 4.02 (t, J ) 5.7 Hz, 2H), 3.82 (s, 2H), 3.27 (t,
J ) 6.6 Hz, 2H), 2.07 (quin, J ) 6.0, 2H); 13H NMR (75 MHz,
1:2 CDCl3/CD3OD) 164.2, 156.8, 151.2, 149.7, 136.3, 135.7, 134.5,
133.0, 128.3, 128.0, 127.6, 127.4, 117.4, 77.7, 70.8, 66.1, 38.0,
29.4; MS (ESI) m/z: [M + Na]+ 655(79Br2, 15), [M + H]+ 635-
(79Br2, 10), 591(100), 547(20), 439(15); HRMS (ESI) m/z: [M +
Na]+ calcd for C27H2679Br2N2O6Na, 655.0021; found, 655.0055.
Benzyl [3-(4-{2-[(2-amino-3H-imidazol-4-ylmethyl)carbam-
oyl]-2-benzyloxyiminoethyl}-2,6-dibromophenoxy)propyl]car-
bamate (20). A mixture of the acid 19 (320 mg, 0.5 mmol),
aminohistamine (200 mg, 1 mmol), HOBt (135 mg, 1 mmol), DCC
(192 mg, 1 mmol), and Et3N (2.8 mL) in 1:1 DMF/CHCl3 (30 mL)
was stirred for 48 h at room temperature. The solvent was removed
4-Methylbenzaldehyde Oxime (26). Anhydrous AlCl3 was
added to a solution of PMB ether 25 (25.5 mg, 0.1 mmol) in anisole
(43 mg, 0.4 mmol) and dry CH2Cl2 (4 mL) at 0 °C. The reaction
was quenched by the addition of H2O after stirring for 40 min at
room temperature. The resulting mixture was extracted with CH2-
Cl2 (10 mL), and the organic phase was washed with H2O, dried
with MgSO4, filtered, and the solvent removed. The crude product
was purified by flash chromatography (5:1 hexane/EtOAc) to afford
1
26 as a colorless oil (11 mg, 82%). H NMR (300 MHz, CDCl3):
δ 8.12 (s, 1H), 8.02 (s, 1H), 7.47 (d, J ) 8.1 Hz, 2H), 7.19 (d, J
) 8.1 Hz, 2H), 2.37 (s, 3H); 13C NMR (75 MHz, CDCl3): 150.4,
140.2, 129.5, 129.1, 127.0, 21.3.
4-[4-(3-Benzyloxycarbonylaminopropoxy)-3,5-dibromophenyl]-
2-(4-methoxybenzyloxyimino)propanoic Acid (27a). A mixture
of azlactone 18 (2.2 g, 4 mmol) and Ba(OH)2 (4.8 g, 28 mmol) in
1:1 dioxane/H2O (56 mL) was stirred at 60 °C for 1 h. O-PMB-
hydroxylamine hydrochloride (1.51 g, 8 mmol) was added at 60

Cytoplasmic Dynein Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2071
°C, and the mixture was stirred vigorously at the same temperature
for 13 h. The reaction mixture was cooled to 0 °C and acidified
with 10% HCl. The reaction mixture was extracted with EtOAc.
The extracts were washed with H2O, dried over MgSO4, and filtered,
and the solvent was evaporated. The crude product was purified
by column chromatography (6:1 CH2Cl2/MeOH) to afford 27a (934
mg, 33.5%) as an oil. IR (KBr, cm-1): 3320 (broad), 2946, 1698,
1688, 1612, 1543, 1514, 1456, 1257; 1H NMR (300 MHz, 3:1 CD3-
OD/CDCl3) δ 7.39 (s, 2H), 7.38-7.35 (m, 5H), 7.24 (d, J ) 7.5
Hz, 2H), 6.86 (d, J ) 7.5 Hz, 2H), 5.11 (brs, 1H), 5.06 (s, 2H),
4.86 (s, 2H), 3.96 (t, J ) 5.9 Hz, 2H), 3.77 (s, 3H), 3.40 (t, J )
6.7 Hz, 2H), 2.02 (quin, J ) 6.3 Hz, 2H); 13C NMR (75 MHz,
CD3OD:CDCl3 ) 3:1) 160.7, 158.5, 152.3, 138.0, 137.2, 134.3,
130.9, 130.0, 129.2, 128.7, 128.5, 118.4, 114.8, 77.7, 71.9, 67.2,
55.7, 39.0, 31.3, 30.9: MS (ESI) m/z: [M + Na]+ 685 (79Br2, 43),
687 (79Br81Br, 100), 621 (35), 211 (46); HRMS (ESI) m/z: [M +
Na]+ calcd for C28H2879Br2N2O7Na, 685.0175; found, 685.0161.
Benzyl (3-{4-[2-[2-(2-amino-3H-imidazol-4-yl)ethylcarbam-
oyl]-2-(4-methoxybenzyloxyimino)ethyl]-2,6-dibromophenoxyl}-
propyl)carbamate (28). A mixture of acid 27a (220 mg, 0.33
mmol), aminohistamine (135 mg, 0.66 mmol), HOBt (89 mg, 0.66
mmol), DCC (192 mg, 1 mmol), and Et3N (2.8 mL) in 1:1 DMF/
CHCl3 (30 mL) was stirred for 26 h at room temperature. The
solvent was removed under reduced pressure, and the crude product
was subjected to column chromatography (15:9:1:1 EtOAc/acetone/
HCO2H/H2O) to provide 28 as a pale-yellow solid (152 mg, 68%).
IR (KBr, cm-1): 3387 (broad), 1676, 1527, 1455, 1256; 1H NMR
(300 MHz, CD3OD): δ 7.34 (s, 2H), 7.32-7.23 (m, 5H), 7.24 (d,
J ) 8.4 Hz, 2H), 6.88 (d, J ) 8.4 Hz, 2H), 6.51 (s, 1H), 3.96 (t,
J ) 5.8 Hz, 2H), 3.77 (s, 3H), 3.75 (s, 2H), 3.49 (t, J ) 6.6 Hz,
2H), 3.39 (t, J ) 6.9 Hz, 2H), 2.72 (t, J ) 6.6 Hz, 2H), 2.01 (quin,
J ) 6.5 Hz, 2H); 13C NMR (75 MHz, CD3OD) 164.8, 161.3, 158.8,
152.9, 152.5, 149.0, 138.4, 136.7, 134.5, 131.2, 130.0, 129.4, 128.9,
128.7, 126.1, 118.8, 115.1, 110.7, 39.2, 39.0, 31.3, 29.7, 25.8; MS
(ESI) m/z: [M + H]+ 771 (79Br2, 20), 773 (79Br81Br, 100), 239 (80);
HRMS (ESI) m/z: [M + H]+ calcd for C33H3779Br2N6O6, 771.1110;
found, 771.1141.
Purealidin A (5). A mixture of amide 28 (130 mg, 0.168 mmol),
AlCl3 (335 mg, 2.52 mmol) and anisole (272 mg, 2.52 mmol) in
1:1 CH3NO2/CH2Cl2 (12 mL) was stirred for 4 h at room
temperature, then H2O was added. The solvents were removed under
reduced pressure and the crude product was subjected to column
chromatography (2:1 CH2Cl2/CH3OH) to provide 5 as a pale yellow
solid (84 mg, 94%): IR (KBr, cm-1): 3387 (broad), 1679, 1661,
1529, 1458; 1H NMR (300 MHz, DMSO-d6) δ 12.11 (s, 1H), 12.09
(s, 1H), 8.18 (brs, 1H), 7.45 (s, 2H), 7.36 (s, 2H), 6.56 (s, 1H),
3.99 (t, J ) 6.6 Hz, 2H), 3.75 (s, 2H), 3.04-3.01 (m, 2H), 2.60 (t,
J ) 6.6 Hz, 2H), 2.09 (quin, J ) 6.6 Hz, 2H); 13C NMR (75 MHz,
DMSO-d6) 163.1, 150.8, 150.5, 146.8, 136.4, 132.9, 124.2, 117.2,
109.1, 70.5, 37.3, 36.3, 27.9, 27.7, 24.3; MS (ESI) m/z: [M + H]+
517 (79Br2, 30), 519(79Br81Br, 80), 521(81Br2, 64); HRMS (ESI)
[M + H]+ calcd for C17H2379Br2N6O3 m/z: 517.0174; found,
517.0198. HPLC: C18, 91%, 3:17 CH3CN (0.1% TFA)/H2O (0.1%
TFA), tR ) 11.5 min.
28.8, 27.1, 25.9, 23.7, 14.4; MS (ESI) m/z: [M + H]+ 727 (79Br2,
50), 729 (79Br81Br, 100), 731 (81Br2, 5); HRMS (ESI) m/z: [M +
H]+ calcd for C31H4979Br2N6O4, 727.2151; found, 727.2182.
HPLC: 93%, C18, 3:2 CH3CN (0.1% TFA)/H2O (0.1% TFA), tR
) 31.1 min.
Lipopurealin B (3). Using a procedure similar to that for the
preparation of compound 2, compound 3 was obtained as a pale-
yellow solid (12 mg, 53%), mp 91-93 °C. IR (KBr, cm-1): 3416
(broad), 2924, 1674, 1642, 1543, 1457; 1H NMR (300 MHz, CD3-
OD): δ 7.47 (s, 2H), 6.51 (s, 1H),4.01 (t, J ) 6.2 Hz, 2H), 3.83
(s, 2H), 3.47 (t, J ) 6.9 Hz, 2H), 3.44 (t, J ) 6.9 Hz, 2H), 2.70 (t,
J ) 6.8 Hz, 2H), 2.20 (t, J ) 7.2 Hz, 2H), 2.04 (quin, J ) 6.6 Hz,
2H), 1.61 (quin, J ) 6.9 Hz, 2H), 1.54-1.49 (m, 1H), 1.28 (brs,
16H), 1.19-1.15 (m, 2H), 0.87 (d, J ) 6.6 Hz, 6H); 13C NMR (75
MHz, CD3OD) 176.4, 165.6, 152.9, 152.1, 148.6, 137.3, 134.5,
126.1, 118.8, 110.8, 72.4, 40.2, 39.0, 37.9, 37.7, 37.2, 31.0, 30.9,
30.8, 30.7, 30.7, 30.6, 30.4, 30.3, 29.2, 29.1, 28.8, 28.5, 27.1, 25.8,
23.0; MS (ESI) m/z: 741(79Br2, 46), 743(79Br81Br, 100), 745(81Br2,
55). HPLC: 92%, C18, 3:2 CH3CN (0.1% TFA)/H2O (0.1% TFA),
tR ) 17.9 min.
Lipopurealin C (4). Using a procedure similar to that for the
preparation of compound 2, compound 4 was obtained as a pale-
yellow solid (16 mg, 55%), mp 104-105 °C; IR (KBr, cm-1): 3310
(broad), 2916, 2849, 1679, 1642, 1543, 1457; 1H NMR (300 MHz,
CD3OD): δ 7.47 (s, 2H), 6.51 (s, 1H), 4.01 (t, J ) 6.2 Hz, 2H),
3.83 (s, 2H), 3.47 (t, J ) 7.0 Hz, 2H), 3.44 (t, J ) 7.0 Hz, 2H),
2.70 (t, J ) 6.9 Hz, 2H), 2.19 (t, J ) 7.4 Hz, 2H), 2.04 (quin, J )
6.6 Hz, 2H), 1.61 (quin, J ) 6.7 Hz, 2H), 1.28 (brs, 24H), 0.90 (t,
J ) 6.9 Hz, 3H); 13C NMR (75 MHz, CD3OD) 176.5, 165.6, 152.9,
152.1, 148.7, 137.4, 134.5, 126.2, 118.8, 110.8, 72.4, 39.0, 37.8,
37.2, 33.1, 30.9, 30.8, 30.7, 30.7, 30.6, 30.5, 30.4, 30.4, 30.3, 28.8,
27.1, 25.9, 23.7, 14.4; MS (ESI) m/z: [M + H]+ 755 (79Br2, 16),
757 (79Br81Br, 32), 759 (81Br2, 16); HRMS (ESI) m/z: [M + H]+
calcd for C33H5379Br2N6O4, 755.2525; found, 755.2495. HPLC:
95%, C18, 3:2 CH3CN (0.1% TFA)/H2O (0.1% TFA), tR ) 12.2
min.
General Procedure A: Synthesis of O-PMB Oxime Acids.
3-[-4-(3-Benzyloxycarbonylaminopropoxy)-3-bromophenyl]-2-
(4-methoxybenzyloxyimino)propanoic Acid (27b). A mixture of
ester 33 (599 mg, 1 mmol) (see below) and LiOH‚H2O (126 mg,
3 mmol) in 3:1 MeOH/H2O (60 mL) was sonicated for 3 h at room
temperature. The reaction mixture was acidified with 5% HCl (2
mL). A white solid precipitated after concentrating the mixture
under reduced pressure. Filtration afforded acid 27b (567 mg, 98%)
as a white solid, mp 80-82 °C. IR (KBr, cm-1): 3321, 2940, 1701,
1
1681, 1533, 1514, 1456, 1254; H NMR (300 MHz, CDCl3): δ
7.42 (d, J ) 2.0 Hz, 1H), 7.36-7.30 (m, 5H), 7.29 (d, J ) 8.6 Hz,
2H), 7.13 (d, J ) 8.3 Hz, 1H), 6.92 (d, J ) 8.6 Hz, 2H), 6.73 (d,
J ) 8.3 Hz, 1H), 5.56 (brs, 1H), 5.24 (s, 2H), 5.12 (s, 2H), 4.03 (t,
J ) 5.7, 2H), 3.82 (s, 2H), 3.80 (s, 2H), 3.47 (q, J ) 5.7, 2H),
2.06-2.02 (m, 2H); 13C NMR (75 MHz, CDCl3): 163.2, 160.0,
156.6, 153.9, 136.7, 133.9, 130.2, 129.3, 129.2, 128.4, 128.0, 127.9,
127.8, 78.2, 67.8, 66.6, 55.3, 39.1, 29.25, 29.2.
3-[2-(3-Benzyloxycarbonylaminopropoxy)-3,5-dibromophenyl]-
2-(4-methoxybenzyloxyimino)propanoic Acid (27c). Using gen-
eral procedure A, 538 mg of the title compound was prepared in
98% yield as a white solid. IR (KBr, cm-1): 3422, 3312, 1706,
Lipopurealin A (2). A mixture of methyl myristic acid (9.7 mg,
0.04 mmol), purealidin A 5 (21 mg, 0.05 mmol), DCC (12.5 mg,
0.08 mmol), HOBt (8.1 mg, 0.08 mmol), and Et3N (0.1 mL) in 1:1
CH2Cl2/DMF (6 mL) was stirred at room temperature for 20 h.
After the solvent was removed under reduced pressure, the crude
product was purified by flash chromatography (15:9:1:1 EtOAc/
acetone/HCO2H/H2O). The product was dissolved in MeOH and
acidified with 0.5 N HCl. The solvents were removed to afford 2
as a pale-yellow solid (15 mg, 52%), mp 93-95 °C. IR (KBr,
cm-1): 3310 (broad), 2922, 2851, 1677, 1541, 1456; 1H NMR (300
MHz, CD3OD): δ 7.47 (s, 2H), 6.51 (s, 1H),4.01 (t, J ) 6.2 Hz,
2H), 3.83 (s, 2H), 3.47 (t, J ) 7.0 Hz, 2H), 3.45 (t, J ) 7.1 Hz,
2H), 2.70 (t, J ) 6.9 Hz, 2H), 2.20 (t, J ) 7.4 Hz, 2H), 2.04 (quin,
J ) 6.6 Hz, 2H), 1.61 (quin, J ) 6.9 Hz, 2H), 1.28 (brs, 20H),
0.89 (t, J ) 6.9 Hz, 3H); 13C NMR (75 MHz, CD3OD) 176.5, 165.6,
152.9, 152.1, 148.7, 137.4, 134.5, 126.2, 118.8, 110.8, 72.4, 39.0,
37.8, 37.2, 33.1, 30.9, 30.8, 30.7, 30.7, 30.6, 30.5, 30.4, 30.4, 30.3,
1
1684, 1611, 1546, 1514, 1450; H NMR (300 MHz, CDCl3): δ
7.52 (d, J ) 1.9 Hz, 1H), 7.36-7.30 (m, 5H), 7.13 (d, J ) 8.7 Hz,
2H), 7.11 (d, J ) 1.9 Hz, 1H), 6.86 (d, J ) 8.7 Hz, 2H), 5.26 (brs,
1H), 5.15 (s, 2H), 5.10 (s, 2H), 3.94 (t, J ) 5.7 Hz, 2H), 3.86 (s,
2H), 3.80 (s, 3H), 3.42 (q, J ) 6.0 Hz, 2H), 1.99 (quin, J ) 6.0
Hz, 2H); 13C NMR (75 MHz, CDCl3): 163.1, 160.0, 156.7, 153.3,
149.1, 136.6, 134.5, 132.9, 132.6, 130.2, 128.5, 128.1, 127.6, 118.1,
117.1, 114.1, 78.3, 71.2, 66.8, 55.3, 38.4, 30.1, 26.1.
3-[2-(3-Benzyloxycarbonylaminopropoxy)-3,5-dichlorophenyl]-
2-(4-methoxybenzyloxyimino)propanoic Acid (27d). Using gen-
eral procedure A, 433 mg of the title compound was prepared in
98% yield as a white solid. IR (KBr, cm-1): 3312, 2949, 1705,
1684, 1611, 1543, 1514, 1455, 1257; 1H NMR (300 MHz,
CDCl3): δ 7.35-7.30 (m, 5H), 7.22 (d, J ) 2.0, 1H), 8.3 (d, J )

2072 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Zhu et al.
8.3 Hz, 2H), 6.93 (brs, 1H), 8.3 (d, J ) 8.3 Hz, 2H), 5.23 (brs,
1H), 5.14 (s, 2H), 5.11 (s, 2H), 3.96 (t, J ) 5.6 Hz, 2H), 3.85 (s,
2H), 3.81 (s, 3H), 3.42 (quin, J ) 6.0 Hz, 2H); 13C NMR (75 MHz,
CDCl3): 162.8, 160.0, 156.7, 151.8, 149.1, 136.6, 132.4, 130.2,
129.3, 129.1, 128.9, 128.6, 128.5, 128.1, 127.6, 114.1, 78.3, 71.3,
66.8, 55.3, 38.4, 30.1, 26.0.
Benzyl [3-(2,4-dichloro-6-formylphenoxy)propyl]carbamate.
Using general procedure B, 4.03 g of the title compound was
prepared in 78% yield as a white solid, mp 56 °C. IR (KBr, cm-1):
1
3319, 3066, 1687, 1678, 1534; H NMR (300 MHz, CDCl3): δ
10.22 (s, 1H), 7.69 (d, J ) 2.7 Hz, 1H), 7.61 (d, J ) 2.7 Hz, 1H),
7.35-7.32 (m, 5H), 5.27 (brs, 1H), 5.10 (s, 2H), 4.09 (t, J ) 6.0
Hz, 2H), 3.49 (q, J ) 6.0 Hz, 2H), 2.07 (quin, J ) 6.0 Hz, 2H);
13C NMR (75 MHz, CDCl3): 187.6, 156.4, 156.2, 136.4, 135.6,
131.2, 130.5, 129.9, 128.4, 128.0, 127.2, 126.8, 74.1, 66.2, 38.1,
30.1; MS (ESI) m/z: [M + H]+ 382 (35Cl2, 20), 338 (14), 192 (28),
91 (100); HRMS (EI) m/z: [M + H]+ calcd for C18H1835Cl2NO4,
382.0613; found, 382.0619.
Benzyl [3-(2-bromo-4-formylphenoxy)propyl]carbamate (31).
Using general procedure B, 7.8 g of the title compound was
prepared in 79% yield as a white solid, mp 70 °C. IR (KBr, cm-1):
1
3423, 3310, 1684, 1595, 1542; H NMR (300 MHz, CDCl3): δ
9.81 (s, 1H), 8.04 (d, J ) 1.9 Hz, 1H), 7.77 (dd, J ) 8.4, 1.7 Hz,
1H), 7.34 (brs, 5H), 6.97 (d, J ) 8.4 Hz, 1H), 5.49 (brs, 1H), 5.10
(s, 2H), 4.18 (t, J ) 5.7 Hz, 2H), 3.48 (q, J ) 6.0 Hz, 2H), 2.10
(quin, J ) 6.0 Hz, 2H); 13C NMR (75 MHz, CDCl3): 189.5, 156.0,
156.4, 136.5, 134.3, 131.1, 130.6, 128.4, 127.9, 127.8, 112.7, 112.1,
67.7, 66.4, 38.5, 28.9; MS (EI) m/z: 391 (M+•, 79Br, 0.2), 300 (0.3),
192 (30), 91 (100).
Benzyl {3-[2,4-dibromo-6-(2-methyl-5-oxooxazol-4-ylidene-
methyl)phenoxy]propyl}carbamate. Using general procedure C,
6.98 g of the title compound was prepared in 85% yield as a yellow
solid, mp 167 °C. 1H NMR (300 MHz, CDCl3): δ 8.74 (d, J ) 2.4
Hz, 1H), 7.74 (d, J ) 2.4 Hz, 1H), 7.36-7.26 (m, 6H), 5.27 (brs,
1H), 5.11 (s, 2H), 3.97 (t, J ) 5.8 Hz, 2H), 3.53 (q, J ) 6.0 Hz,
2H), 2.43 (s, 3H), 2.10 (quin, J ) 6.0 Hz, 2H); 13C NMR (75 MHz,
CDCl3): 167.9, 167.1, 156.5, 155.2, 137.8, 136.5, 134.7, 134.1,
129.9, 128.5, 128.0, 122.2, 118.5, 117.9, 73.3, 66.6, 38.2, 29.9,
15.7.
Benzyl {3-[2,4-dichloro-6-(2-methyl-5-oxooxazol-4-ylidene-
methyl)phenoxy]propyl}carbamate. Using general procedure C,
4.23 g of the title compound was prepared in 92% yield as a yellow
solid, mp 159 °C. 1H NMR (300 MHz, CDCl3): δ 8.58 (d, J ) 2.5
Hz, 1H), 7.44 (d, J ) 2.5 Hz, 1H), 7.36-7.32 (m, 6H), 5.24 (brs,
1H), 5.11 (s, 2H), 4.0 (t, J ) 6.0 Hz, 2H), 3.53 (q, J ) 6.3 Hz,
2H), 2.45 (s, 3H), 2.10 (quin, J ) 6.0 Hz, 2H); 13C NMR (75 MHz,
CDCl3): 167.8, 167.1, 156.5, 153.7, 136.5, 134.6, 132.3, 130.4,
130.1, 129.5, 129.0, 128.5, 128.0, 122.2, 73.2, 66.6, 38.2, 30.0,
15.7.
Benzyl {3-[2-bromo-4-(2-methyl-5-oxo-oxazol-4-ylidenemethyl)-
phenoxy]propyl}carbamate (32). Using general procedure C, 6.4
g of the title compound was prepared in 85% yield as a white solid.
1H NMR (300 MHz, CD3OD): δ 8.41 (d, J ) 1.8 Hz, 1H), 7.94
(dd, J ) 8.6, 1.8 Hz, 1H), 7.37-7.32 (m, 5H), 7.00 (s, 1H), 6.91
(d, J ) 8.6 Hz, 1H)H, 5.44 (brs, 1H), 5.12 (s, 2H), 4.17 (t, J ) 5.7
Hz, 2H), 3.49 (q, J ) 6.0 Hz, 2H), 2.41 (s, 3H), 2.13-2.08 (m,
2H); 13C NMR (75 MHz, CDCl3): 167.6, 165.8, 157.1, 156.6,
136.8, 133.2, 131.7, 129.4, 128.5, 128.0, 128.0, 127.7, 67.8, 66.7,
38.9, 29.2, 15.6.
General Procedure D: Synthesis of O-PMB Oxime Methyl
Esters. Methyl 3-[2-(3-benzyloxycarbonylaminopropoxy)-3,5-
dibromophenyl]-2-(4-methoxybenzyloxyimino)propanoate. A mix-
ture of benzyl {3-[2,4-dibromo-6-(2-methyl-5-oxooxazol-4-ylidene-
methyl)phenoxy]propyl}carbamate (1.89 g, 4 mmol) (see below)
and Ba(OH)2 (4.8 g, 28 mmol) in 1:1 dioxane/H2O (56 mL) was
stirred at 60 °C for 1 h. O-PMB-Hydroxylamine hydrochloride (1.52
g, 8 mmol) was added at 60 °C, and the mixture was stirred
vigorously at the same temperature for 13 h. The reaction mixture
was cooled to 0 °C and acidified with 10% HCl. The reaction
mixture was extracted with EtOAc. The extracts were washed with
H2O, dried over MgSO4, filtered, and the solvent evaporated. The
crude product in dry benzene (40 mL) and MeOH (15 mL) was
treated with TMSCHN2 (3 mL, 2.0 N in hexane) at 0 °C. The
mixture was stirred for 40 min at room temperature. The organic
solvents were removed after the addition of saturated aqueous NH4-
Cl. The residue was extracted with Et2O, and the extracts were
dried over MgSO4, filtered, and the solvent evaporated. The crude
product was purified by column chromatography (2:1 hexane/
EtOAc) to afford the title compound (752 mg, 32%) as a white
Methyl
3-[2-(3-benzyloxycarbonylaminopropoxy)-3,5-di-
chlorophenyl]-2-(4-methoxybenzyloxyimino)propanoate. Using
general procedure D, 770 mg of the title compound was prepared
1
in 33% yield as a white solid. H NMR (300 MHz, CDCl3): δ
7.37-7.32 (m, 5H), 7.22 (d, J ) 2.0 Hz, 1H), 7.17 (d, J ) 8.3 Hz,
2H), 6.90 (d, J ) 2.0 Hz, 1H), 6.86 (d, J ) 8.3 Hz, 2H), 5.26 (brs,
1H), 5.21 (s, 2H), 5.11 (s, 2H), 3.95 (t, J ) 5.9 Hz, 2H), 3.90 (s,
2H), 3.84 (s, 2H), 3.81 (s, 3H), 3.43 (q, J ) 6.0 Hz, 2H) 1.97
(quin, J ) 6.0 Hz, 2H); MS (ESI) m/z: 611 [M + Na]+ (35Cl2,
100), 589 [M + H]+ (35Cl2, 6).
General Procedure E. 3-[4-(3-Aminopropoxy)-3,5-di-
bromophenyl]-2-hydroxyimino-N-phenethylpropanamide (40a).
A mixture of amide 36a (71 mg, 0.095 mmol), AlCl3 (134 mg, 1
mmol), and anisole (108 mg, 1 mmol) in 1:1 CH3NO2-CH2Cl2
(20 mL) was stirred for 4 h at room temperature. The mixture was
stirred after H2O was added. The solvents were removed under
reduced pressure, and the crude product was subjected to flash
chromatography (10:6:1:1 EtOAC/acetone/H2CO2H/H2O) to afford
an oil, which was acidified with 5% HCl to give the hydrochloride
1
solid. H NMR (300 MHz, CDCl3): δ 7.52 (d, J ) 2.4 Hz, 1H),
7.36-7.32 (m, 5H), 7.18 (d, J ) 8.7 Hz, 2H), 7.07 (d, J ) 2.4 Hz,
1H), 6.87 (d, J ) 8.7 Hz, 2H), 5.26 (brs, 1H), 5.20 (s, 2H), 5.10
(s, 2H), 3.93 (t, J ) 6.0 Hz, 2H), 3.89 (s, 2H), 3.82 (s, 3H), 3.80
(s, 3H), 3.43 (q, J ) 6.0 Hz, 2H), 1.99-1.95 (m, 2H).
1
salt of 40a (50 mg, 95%) as a pale-yellow solid. H NMR (300
Methyl 3-[4-(3-benzyloxycarbonylaminopropoxy)-3-bromo-
phenyl]-2-(4-methoxybenzyloxyimino)propanoate. Using general
procedure D, 752 mg of the title compound was prepared in 32%
MHz, CD3OD): δ 7.50 (s, 2H), 7.27-7.16 (m, 5H), 4.11 (t, J )
5.7 Hz, 2H), 3.84 (s, 2H), 3.46 (t, J ) 7.2 Hz, 2H), 3.31 (t, J ) 7.2
Hz, 2H), 2.80 (t, J ) 7.2 Hz, 2H), 2.21 (quin, J ) 6.3 Hz, 2H);
13C NMR (75 MHz, CDCl3): 165.2, 152.2, 152.0, 140.3, 137.9,
134.6, 129.8, 129.5, 127.4, 118.6, 71.6, 42.0, 38.9, 36.5, 29.0, 28.8;
MS (ESI) m/z: [M + H]+ 512 (79Br2, 53), 514 (79Br81Br, 100), 516
(81Br2, 50); HRMS (ESI) m/z: [M + H]+ calcd for C20H2479Br2N3O3,
512.0814; found, 512.0815. HPLC: C18, 99%, 7:13 CH3CN (0.1%
TFA)/H2O (0.1%TFA), tR ) 4.0 min.
1
yield as a white solid. H NMR (300 MHz, CDCl3): δ 7.42 (d, J
) 1.8 Hz, 1H), 7.36-7.28 (m, 5H), 7.29 (d, J ) 8.6 Hz, 2H), 7.11
(dd, J ) 8.4, 1.8 Hz, 1H), 6.91 (d, J ) 8.6 Hz, 2H), 6.71 (d, J )
8.4 Hz, 1H), 5.50 (brs, 1H), 5.25 (s, 2H), 5.10 (s, 2H), 4.05 (t, J )
5.7 Hz, 2H), 3.84 (s, 2H), 3.81(s, 5H), 3.47 (q, J ) 5.7 Hz, 2H),
2.07-2.03 (m, 2H).
Benzyl [3-(2,4-dibromo-6-formylphenoxy)propyl]carbamate.
Using general procedure B, 9.5 g of the title compound was
prepared in 80% yield as a white solid, mp 60 °C. IR (KBr, cm-1):
3-[4-(3-Aminopropoxy)-3,5-dibromophenyl]-2-hydroxyimino-
N-[2-(4-methoxyphenyl)-ethyl]propanamide (40b). Using general
procedure E, 39 mg of the title compound was obtained in 85%
yield as a pale-yellow solid. 1H NMR (300 MHz, CD3OD): δ 7.51
(s, 2H), 7.08 (d, J ) 5.0 Hz, 2H), 6.80 (d, J ) 5.0 Hz, 2H), 4.12
(t, J ) 5.7 Hz, 2H), 3.84 (s, 2H), 3.75 (s, 3H), 3.43 (t, J ) 7.5 Hz,
2H), 3.31 (t, J ) 7.5 Hz, 2H), 2.73 (t, J ) 7.5 Hz, 2H), 2.13 (quin,
J ) 7.5 Hz, 2H); 13C NMR (75 MHz, CDCl3): 165.3, 159.8, 152.3,
152.2, 137.9, 134.6, 132.4, 130.7, 118.6, 115.0, 71.7, 55.7, 42.1,
39.0, 35.6, 29.0, 28.8; MS (ESI) m/z: [M + H]+ 542 (79Br2, 60),
1
3297, 3059, 1691, 1574, 1551; H NMR (300 MHz, CDCl3): δ
10.18 (s, 1H), 7.91 (d, J ) 2.4 Hz, 1H), 7.87 (d, J ) 2.4 Hz, 1H),
7.35-7.29 (m, 5H), 5.26 (brs, 1H), 5.10 (s, 2H), 4.07 (t, J ) 6.0
Hz, 2H), 3.51(q, J ) 6.0 Hz, 2H), 2.08 (quin, J ) 6.0 Hz, 2H);
13C NMR (75 MHz, CDCl3): 187.6, 157.6, 156.4, 141.2, 136.4,
131.6, 131.2, 128.5, 128.1, 126.9, 119.5, 118.2, 74.4, 66.7, 38.1,
30.1.

Cytoplasmic Dynein Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2073
544 (79Br81Br, 100), 546 (81Br2, 50); HRMS (ESI) m/z: [M + H]+
calcd for C21H2679Br2N3O4, 542.0290; found, 542.0290. HPLC: C18,
98%, 7:13 CH3CN (0.1% TFA)/H2O (0.1%TFA), tR ) 3.5 min.
3-[4-(3-Aminopropoxy)-3,5-dibromophenyl]-N-[2-(4-chloro-
phenyl)ethyl]-2-hydroxyiminopropanamide (40c). Using general
procedure E, 44 mg of the title product was obtained in 89% yield
3-[4-(3-Aminopropoxy)-3-bromophenyl]-2-hydroxyimino-N-
[2-(1H-indol-3-yl)ethyl]propanamide (41d). Using general pro-
cedure E, 40 mg of the title compound was obtained in 87% yield
1
as a pale-yellow solid. H NMR (300 MHz, CD3OD): δ 7.54 (d,
J ) 7.9 Hz, 1H), 7.48 (d, J ) 1.9 Hz, 1H), 7.32 (d, J ) 8.1 Hz,
1H), 7.21 (dd, J ) 8.1 Hz, 1.8 Hz, 1H), 7.07 (t, J ) 7.2 Hz, 1H),
7.04 (s, 1H), 6.97 (t, J ) 7.2 Hz, 1H), 6.92 (d, J ) 8.4 Hz, 1H),
4.14 (t, J ) 5.6 Hz, 2H), 3.83 (s, 2H), 3.53 (t, J ) 7.3 Hz, 2H),
3.20 (t, J ) 7.1 Hz, 2H), 2.94 (t, J ) 7.2 Hz, 2H), 2.16 (quin, J )
6.0 Hz, 2H); 13C NMR (75 MHz, CDCl3): 165.5, 154.6, 152.9,
138.1, 134.7, 132.6, 130.6, 128.7, 123.4, 122.3, 119.6, 119.3, 114.4,
113.1, 112.5, 112.2, 67.6, 41.4, 38.9, 28.2, 26.3; MS (ESI) m/z:
[M + H]+ 473 (79Br, 92), 475 (81Br, 100); HRMS (ESI) m/z: [M
+ H]+ calcd for C22H2679BrN3O3, 473.1188; found, 473.1194.
HPLC: C18, 98%, 7:13 CH3CN (0.1% TFA)/H2O (0.1%TFA), tR
) 2.4 min.
1
as a pale-yellow solid. H NMR (300 MHz, CD3OD): δ 7.48 (s,
2H), 7.23 (d, J ) 8.4 Hz, 2H), 7.15 (d, J ) 8.4 Hz, 2H), 4.08 (t,
J ) 5.7 Hz, 2H), 3.82 (s, 2H), 3.46 (t, J ) 6.9 Hz, 2H), 3.17 (t, J
) 4.8 Hz, 2H), 2.79 (t, J ) 7.0 Hz, 2H), 2.13 (quin, J ) 6.9 Hz,
2H); 13C NMR (75 MHz, CDCl3): 165.3, 152.3, 152.1, 139.2,
137.9, 134.6, 133.2, 133.4, 129.5, 118.6, 71.7, 41.6, 39.0, 35.7,
29.0, 28.8; MS (ESI) m/z: [M + H]+ 546 (79Br2, 43), 548 (79Br-
81Br, 100), 550 (81Br2, 70); HRMS (ESI) m/z: [M + H]+ calcd for
C20H23Br2N3O3Cl, 545.9795; found, 545.9802. HPLC: C18, 100%,
7:13 CH3CN (0.1% TFA)/H2O (0.1%TFA), tR ) 3.6 min.
3-[4-(3-Aminopropoxy)-3,5-dibromophenyl]-2-hydroxyimino-
N-[2-(1H-indol-3-yl)ethyl]propanamide (40d). Using general
procedure E, 26 mg of the title compound was obtained in 50%
yield as a pale-yellow solid. 1H NMR (300 MHz, CD3OD): δ 7.54
(d, J ) 8.1 Hz, 1H), 7.53 (s, 2H), 7.32 (d, J ) 8.1 Hz, 1H), 7.07
(t, J ) 8.1 Hz, 1H), 7.06 (s, 1H), 6.98 (t, J ) 8.1 Hz, 1H), 4.10 (t,
J ) 5.7 Hz, 2H), 3.85 (s, 2H), 3.29 (t, J ) 7.4 Hz, 2H), 2.96 (t, J
) 7.5 Hz, 2H), 2.10 (quin, J ) 6.6 Hz, 2H); 13C NMR (75 MHz,
CDCl3): 165.3, 152.3, 152.1, 138.2, 138.0, 134.6, 128.8, 123.5,
122.6, 119.7, 119.3, 118.6, 117.3, 113.2, 112.2, 71.7, 41.4, 39.0,
29.0, 28.9, 16.3; MS (ESI) m/z: [M + H]+ 551 (79Br2, 68),
553(79Br81Br, 100), 555 (81Br2, 80); HRMS (ESI) m/z: [M + H]+
calcd for C22H2579Br2N4O3, 551.0293; found, 551.0289. HPLC: C18,
92%, 7:13 CH3CN (0.1% TFA)/H2O (0.1%TFA), tR ) 3.6 min.
3-[4-(3-Aminopropoxy)-3-bromophenyl]-2-hydroxyimino-N-
phenethylpropanamide (41a). Using general procedure E, 45 mg
of the title compound was obtained in 89% yield as a pale-yellow
solid. 1H NMR (300 MHz, CD3OD): δ 7.46 (d, J ) 1.8 Hz, 1H),
7.46-7.15 (m, 6H), 6.94 (d, J ) 8.4 Hz, 2H), 4.15 (t, J ) 5.5 Hz,
2H), 3.82 (s, 2H), 3.44 (t, J ) 7.0 Hz, 2H), 3.21 (t, J ) 7.0 Hz,
2H), 2.78 (t, J ) 7.2 Hz, 2H), 2.18 (quin, J ) 6.0 Hz, 2H); 13C
NMR (75 MHz, CDCl3): 165.6, 154.7, 153.0, 140.4, 134.7, 132.8,
130.6, 129.8, 127.3, 114.7, 112.7, 67.9, 41.9, 39.0, 36.5, 28.8, 28.3;
MS (ESI) m/z: [M + H]+ 434 (79Br, 90), 436(81Br, 100); HRMS
(ESI) m/z: [M + H]+ calcd for C20H2579BrN3O3, 434.1079; found,
434.1079. HPLC: C18, 99%, 7:13 CH3CN (0.1% TFA)/H2O
(0.1%TFA), tR ) 2.6 min.
3-[2-(3-Aminopropoxy)-3,5-dibromophenyl]-2-hydroxyimino-
N-phenethylpropanamide (42a). Using general procedure E, 44
mg of the title compound was obtained in 89% yield as a pale-
1
yellow solid. H NMR (300 MHz, CD3OD): δ 7.62 (d, J ) 2.3
Hz, 1H), 7.24-7.14 (m, 6H), 4.09 (t, J ) 6.0 Hz, 2H), 3.95 (s,
2H), 3.47 (t, J ) 6.9 Hz, 2H), 3.10 (t, J ) 7.0 Hz, 2H), 2.80 (t, J
) 7.2 Hz, 2H), 2.10 (quin, J ) 6.6 Hz, 2H); 13C NMR (75 MHz,
CDCl3): 165.4, 154.3, 152.3, 140.4, 135.6, 135.1, 133.5, 129.8,
129.5, 127.4, 119.2, 118.4, 72.1, 42.0, 39.1, 36.5, 29.0, 24.7; MS
(ESI) m/z: [M + H]+ 512 (79Br2, 50), 514(79Br81Br, 100), 516(81-
Br2, 50); HRMS (ESI) m/z: [M + Na]+ calcd for C20H2379Br2N3O3-
Na, 534.0004; found, 534.0012. HPLC: C18, 91%, 7:13 CH3CN
(0.1% TFA)/H2O (0.1%TFA), tR ) 9.0 min.
3-[2-(3-Aminopropoxy)-3,5-dibromophenyl]-2-hydroxyimino-
N-[2-(4-methoxyphenyl)-ethyl]propanamide (42b). Using general
procedure E, 45 mg of the title compound was obtained in 90%
yield as a pale-yellow solid. 1H NMR (300 MHz, CD3OD): δ 7.62
(d, J ) 2.3 Hz, 1H), 7.25 (d, J ) 2.3 Hz, 1H), 7.07 (d, J ) 8.6 Hz,
2H), 6.78 (d, J ) 8.6 Hz, 2H), 4.11 (t, J ) 5.9 Hz, 2H), 3.96 (s,
2H), 3.74 (s, 3H), 3.44 (t, J ) 7.0 Hz, 2H), 3.28 (t, J ) 7.1 Hz,
2H), 2.73 (t, J ) 7.2 Hz, 2H), 2.21 (quin, J ) 6.3 Hz, 2H); 13C
NMR (75 MHz, CDCl3): 165.3, 159.7, 154.3, 152.3, 135.6, 135.0,
133.4, 132.3, 130.8, 119.2, 118.4, 114.9, 71.9, 55.7, 42.2, 38.9,
35.6, 29.0, 24.6; MS (ESI) m/z: [M + H]+ 542 (79Br2, 50), 544(79-
Br81Br, 100), 546(81Br2, 50); HRMS (ESI) m/z: [M + H]+ calcd
for C21H2679Br2N3O4, 542.0290; found, 542.0291. HPLC: C18,
100%, 7:13 CH3CN (0.1% TFA)/H2O (0.1%TFA), tR ) 7.7 min.
3-[4-(3-Aminopropoxy)-3-bromophenyl]-2-hydroxyimino-N-
[2-(4-methoxyphenyl)ethyl]propanamide (41b). Using general
procedure E, 49 mg of the title compound was obtained in 95%
yield as a pale-yellow solid. 1H NMR (300 MHz, CD3OD): δ 7.47
(d, J ) 2.0 Hz, 1H), 7.20 (dd, J ) 8.4 Hz, 2.0 Hz, 1H), 7.07 (d,
J ) 8.5 Hz, 2H), 6.94 (d, J ) 8.5 Hz, 1H), 6.80 (d, J ) 8.6 Hz,
2H), 4.16 (t, J ) 6.6 Hz, 2H), 3.82 (s, 2H), 3.75 (s, 3H), 3.41 (t,
J ) 6.9 Hz, 2H), 3.22 (t, J ) 7.2 Hz, 2H), 2.72 (t, J ) 7.2 Hz,
2H), 2.18 (quin, J ) 6.0 Hz, 2H); 13C NMR (75 MHz, CDCl3):
165.5, 159.7, 154.6, 152.9, 134.7, 132.6, 132.3, 130.8, 130.6, 114.9,
114.4, 112.5, 67.6, 55.6, 42.1, 38.9, 35.6, 28.7, 28.2; MS (ESI)
m/z: [M + H]+ 464 (79Br, 55), 466 (81Br, 50); HRMS (ESI) m/z:
[M + H]+ calcd for C21H2779BrN3O4, 464.1185; found, 464.1187.
HPLC: C18, 99%, 7:13 CH3CN (0.1% TFA)/H2O (0.1%TFA),
tR ) 2.6 min.
3-[4-(3-Aminopropoxy)-3-bromophenyl]-N-[2-(4-chloro-
phenyl)ethyl]-2- hydroxyiminopropanamide (41c). Using general
procedure E, 45 mg of the title product was obtained in 96% yield
a pale-yellow solid. 1H NMR (300 MHz, CD3OD): δ 7.43 (s, 1H),
7.23-7.12 (m, 6H), 6.95 (d, J ) 8.2 Hz, 1H), 4.16 (t, J ) 6.9 Hz,
2H), 3.81 (s, 2H), 3.44 (t, J ) 6.9 Hz, 2H), 3.22 (t, J ) 6.9 Hz,
2H), 2.77 (t, J ) 6.9 Hz, 2H), 2.18 (brs, 2H); 13C NMR (75 MHz,
CDCl3): 165.5, 154.6, 152.8, 139.2, 134.7, 133.0, 132.4, 131.5,
130.5, 129.4, 114.4, 112.5, 106.5, 67.6, 41.6, 38.8, 35.7, 28.7, 28.2;
MS (ESI) m/z: [M + H]+ 468 (79Br, 70), 470 (81Br, 100); HRMS
(ESI) m/z: [M + H]+ calcd for C20H2479BrN3O3Cl, 468.0690; found,
468.0692. HPLC: C18, 97%, 7:13 CH3CN (0.1% TFA)/H2O
(0.1%TFA), tR ) 4.30 min.
3-[2-(3-Aminopropoxy)-3,5-dibromophenyl]-N-[2-(4-chloro-
phenyl)ethyl]-2-hydroxyiminopropanamide (42c). Using general
procedure E, 46 mg of the title compound was obtained in 90%
yield as a pale-yellow solid. 1H NMR (300 MHz, CD3OD): δ 7.63
(d, J ) 2.2 Hz, 1H), 7.22 (d, J ) 2.2 Hz, 1H), 7.20 (d, J ) 8.7 Hz,
2H), 7.13 (d, J ) 8.6 Hz, 2H), 4.11 (t, J ) 5.7 Hz, 2H), 3.95 (s,
2H), 3.47 (t, J ) 7.1 Hz, 2H), 3.28 (t, J ) 7.2 Hz, 2H), 2.78 (t, J
) 7.0 Hz, 2H), 2.21 (quin, J ) 6.3 Hz, 2H); 13C NMR (75 MHz,
CDCl3): 165.4, 154.3, 152.3, 139.2, 135.5, 135.1, 133.4, 131.4,
129.5, 129.4, 119.2, 118.4, 72.0, 41.6, 39.0, 35.8, 29.0, 24.7; MS
(ESI) m/z: [M + H]+ 546 (79Br2, 50), 548 (79Br81Br, 100), 550
(81Br2, 70); HRMS (ESI) m/z: [M + H]+ calcd for C20H2379Br2N3O3-
Cl, 545.9795; found, 545.9774. HPLC: C18, 98%, 7:13 CH3CN
(0.1% TFA)/H2O (0.1%TFA), tR ) 15.7 min.
3-[2-(3-Aminopropoxy)-3,5-dibromophenyl]-2-hydroxyimino-
N-[2-(1H-indol-3-yl)ethyl]propanamide (42d). Using general
procedure E, 26 mg of the title compound was obtained in 87%
yield as a pale-yellow solid. 1H NMR (300 MHz, CD3OD): δ 7.62
(d, J ) 2.3 Hz, 1H), 7.54 (d, J ) 7.8 Hz, 1H), 7.32 (d, J ) 7.8 Hz,
1H), 7.29 (d, J ) 2.3 Hz, 1H), 7.09-7.07 (m, 1H), 7.04 (s, 1H),
7.00-6.95 (m, 1H), 4.08 (t, J ) 5.7 Hz, 2H), 3.96 (s, 2H), 3.56 (t,
J ) 7.2 Hz, 2H), 3.25 (t, J ) 7.1 Hz, 2H), 2.96 (t, J ) 7.2 Hz,
2H), 2.19 (quin, J ) 6.3 Hz, 2H); 13C NMR (75 MHz, CDCl3):
165.3, 154.2, 152.1, 138.0, 135.6, 134.9, 133.5, 128.6, 123.5, 122.3,
119.5, 119.3, 119.2, 118.3, 113.0, 112.2, 71.9, 46.0, 38.9, 29.0,
26.2, 24.8; MS (ESI) m/z: [M + H]+ 551 (79Br2, 54), 553 (79Br-
81Br, 100), 555 (81Br2, 42); HRMS (ESI) m/z: [M + H]+ calcd for

2074 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Zhu et al.
C22H2579Br2N4O3, 551.0293; found, 551.0283. HPLC: C18, 98%,
7:13 CH3CN (0.1% TFA)/H2O (0.1%TFA), tR ) 7.6 min.
3-[2-(3-Aminopropoxy)-3,5-dichlorophenyl]-2-hydroxyimino-
N-phenethylpropanamide (43a). Using general procedure E, 38
mg of the title compound was obtained in 91% yield as a pale-
yellow solid: 1H NMR (300 MHz, CD3OD): δ 7.34 (d, J ) 2.5
Hz, 1H), 7.26-7.15 (m, 5H), 7.07 (d, J ) 2.5 Hz, 1H), 4.12 (t, J
) 5.7 Hz, 2H), 3.96 (s, 2H), 3.48 (t, J ) 6.9 Hz, 2H), 3.27 (t, J )
7.1 Hz, 2H), 2.80 (t, J ) 7.2 Hz, 2H), 2.04 (quin, J ) 6.0 Hz, 2H);
13C NMR (75 MHz, CDCl3): 165.4, 152.8, 152.3, 140.4, 135.2,
130.8, 129.8, 129.7, 129.5, 129.3, 127.4, 72.0, 41.9, 39.0, 36.5,
29.1, 24.6; MS (ESI) m/z: [M + H]+ 424 (35Cl2, 100), 426 (35Cl-
37Cl, 65); HRMS (ESI) m/z: [M+Na]+ calcd for C20H2435Cl2N3O3,
424.1195; found, 424.1198. HPLC: C18, 99%, 7:13 CH3CN (0.1%
TFA)/H2O (0.1%TFA), tR ) 7.0 min.
3-[2-(3-Aminopropoxy)-3,5-dichlorophenyl]-2-hydroxyimino-
N-[2-(4-methoxyphenyl)ethyl]propanamide (43b). Using general
procedure E, 37 mg of the title compound was obtained in 88%
yield as a pale-yellow solid. 1H NMR (300 MHz, CD3OD): δ 7.34
(d, J ) 2.3 Hz, 1H), 7.09 (d, J ) 2.3 Hz, 1H), 7.07 (d, J ) 8.6 Hz,
2H), 6.78 (d, J ) 8.5 Hz, 2H), 4.12 (t, J ) 5.7 Hz, 2H), 3.95 (s,
2H), 3.74 (s, 3H), 3.44 (t, J ) 7.0 Hz, 2H), 3.27 (t, J ) 7.1 Hz,
2H), 2.73 (t, J ) 7.2 Hz, 2H), 2.20 (quin, J ) 6.3 Hz, 2H); 13C
NMR (75 MHz, CDCl3): 165.4, 159.8, 152.8, 152.3, 135.2, 132.3,
130.7, 129.8, 129.7, 129.3, 115.0, 72.0, 55.7, 42.1, 39.0, 35.6, 29.0,
24.6; MS (ESI) m/z: [M + H]+ 454 (35Cl2, 100), 456 (35Cl37Cl,
70); HRMS (ESI) m/z: [M + H]+ calcd for C21H2635Cl2N3O4,
454.1300; found, 454.1313. HPLC: C18, 99%, 7:13 CH3CN (0.1%
TFA)/H2O (0.1%TFA), tR ) 6.1 min.
wells of medium, and each agent concentration was tested in
quadruplicate. Cell viability was determined with the MTS assay,
and 50% growth-inhibitory concentrations (GI50) were calculated.52
Discodermolide was used as a positive control in these assays.
Recombinant Cytoplasmic Dynein Motor Domain Expression
and Purification. The gene encoding the 380 kDa motor domain
fragment [Gly(1286)-Glu(4644)] of rat cytoplasmic dynein with a
C-terminal in-frame hexahistidine tag was inserted into the bacu-
lovirus expression vector pVL1393 (BD biosciences) as described.36
The Hi5 cells were infected with the virus for 40 h. The cells were
washed and resuspended in PBS, and the recombinant motor domain
fragment was extracted from the cells by homogenization in the
extraction buffer described above supplemented with 1 mM DTT
and a protease inhibitor cocktail (Sigma-Aldrich). The cytosolic
extract was spun at 5000g for 10 min and 100 000g for 30 min.
The supernatant was applied on a Ni2+-affinity column (Ni-NTA
superflow, Qiagen) equilibrated in an extraction buffer supple-
mented with 20 mM imidazole. Unbound material was washed out
with 16 volumes of the extraction buffer supplemented with 10
mM imidazole, and the protein was eluted in 5 volumes of the
elution buffer (50 mM Pipes + 50 mM Hepes, pH 7.2, containing
2 mM MgSO4, 2 mM EGTA, 125 mM imidazole, and 1 mM DTT).
The eluted protein was dialyzed extensively against the extraction
buffer to remove the imidazole. Protein concentration was deter-
mined by the Bradford method using albumin as a standard.53
A
typical batch of six dishes of confluent Hi5 cells produced 2-4
mg of recombinant protein that had no visible sign of contaminants
or degradation. Peak fractions were pooled, flash-frozen in liquid
nitrogen and subsequently stored at -80 °C until use.
Dynein Motor Domain ATPase Inhibition Assay. The ability
of the purealin analogues to inhibit the MT-stimulated ATPase
activity of the recombinant motor domain was determined colori-
metrically with the malachite green assay.54 Paclitaxel-induced
tubulin polymer (5 mg/mL final concentration based on soluble
tubulin incubated with 1 mM paclitaxel), dynein motor domain (50
µg/mL final concentration), and the test agents (50, 10, 2, 0.4, and
0.08 µM) as well as the positive control (EHNA, an adenosine
analogue known to inhibit dynein ATPase activity, 5 mM final
concentration) and the negative control (DMSO only), were
premixed in a reaction buffer (10 mM PIPES and 10 mM HEPES,
pH 7.0, containing 0.4 mM MgCl2 and 0.2 mM EDTA). ATP (2
mM) was added to initiate the reaction. The other controls, including
a no enzyme control (all but dynein motor domain), no substrate
control (all but ATP), and no enzyme/substrate control (all but ATP
and dynein motor domain), were also prepared. The reaction mixture
was incubated at 37 °C for 30 min. The malachite green solution
(1 mM malachite green, 8.5 mM ammonium molybdate ,and 0.8
M HCl) and 34% aqueous sodium citrate were added. The
absorbance at 655 nm was determined in a 96-well microtiter plate
reader. Absorbance values from the control wells were subtracted
from those from wells containing the replete set of reaction
components. The amount of free phosphate released by the dynein
heavy chain was calculated from a sodium phosphate standard
curve. The percentage of control was calculated from the ratio of
the ATPase activity of cytoplasmic dynein motor domain with a
specific concentration of an inhibitor and the ATPase activity of
cytoplasmic dynein motor domain only.
3-[2-(3-Aminopropoxy)-3,5-dichloro-phenyl]-N-[2-(4-
chlorophenyl)ethyl]-2-hydroxyiminopropanamide (43c). Using
general procedure E, 38 mg of the title compound was obtained in
87% yield as a pale-yellow solid: 1H NMR (300 MHz, CD3OD):
δ 7.34 (d, J ) 2.4 Hz, 1H), 7.20 (d, J ) 8.7 Hz, 2H), 7.14 (d, J )
8.7 Hz, 2H), 7.05 (d, J ) 2.4 Hz, 1H), 4.11 (t, J ) 5.7 Hz, 2H),
3.94 (s, 2H), 3.47 (t, J ) 7.2 Hz, 2H), 3.27 (t, J ) 7.1 Hz, 2H),
2.79 (t, J ) 7.0 Hz, 2H), 2.20 (quin, J ) 6.3 Hz, 2H); 13C NMR
(75 MHz, CDCl3): 165.4, 152.8, 152.4, 139.2, 135.1, 133.1, 131.5,
130.7, 129.8, 129.7, 129.4, 71.9, 41.6, 38.9, 35.8, 29.0, 24.5; MS
(ESI) m/z: [M + H]+ 458 (35Cl3, 100), 460 (35Cl237Cl, 100); HRMS
(ESI) m/z: [M+Na]+ calcd for C20H2235Cl3N3O3Na, 480.0624;
found, 480.0627. HPLC: C18, 99%, 7:13 CH3CN (0.1% TFA)/H2O
(0.1%TFA), tR ) 13.0 min.
3-[2-(3-Aminopropoxy)-3,5-dichlorophenyl]-2-hydroxyimino-
N-[2-(1H-indol-3-yl)ethyl]propanamide (43d). Using general
procedure E, 32 mg of the title compound was obtained in 64%
yield as a pale-yellow solid. 1H NMR (300 MHz, CD3OD): δ 7.54
(d, J ) 7.8 Hz, 1H), 7.34 (d, J ) 2.5 Hz, 1H), 7.31 (d, J ) 7.8 Hz,
1H), 7.11 (d, J ) 2.5 Hz, 1H), 7.07 (t, J ) 7.8 Hz, 1H), 7.04 (s,
1H), 6.97 (t, J ) 7.8 Hz, 1H), 4.08 (t, J ) 5.7 Hz, 2H), 3.96 (s,
2H), 3.56 (t, J ) 7.1 Hz, 2H), 3.23 (t, J ) 7.1 Hz, 2H), 2.96 (t,
J ) 7.2 Hz, 2H), 2.17 (quin, J ) 6.3 Hz, 2H); 13C NMR (75 MHz,
CDCl3): 165.6, 153.0, 152.5, 138.4, 135.4, 130.9, 130.1, 129.5,
123.7, 122.5, 119.8, 119.5, 113.3, 112.4, 72.2, 41.5, 39.1, 29.2,
26.4, 24.8; MS (ESI) m/z: [M + H]+ 463 (35Cl2, 100), 465 (35Cl-
37Cl, 75); HRMS (ESI) m/z: [M + H]+ calcd for C22H2535Cl2N4O3,
463.1304; found, 463.1310. HPLC: C18, 99%, 7:13 CH3CN (0.1%
TFA)/H2O (0.1%TFA), tR ) 6.1 min.
Biology. Antiproliferative Assay. The cells were maintained
as exponentially growing cultures in an RPMI 1640 medium with
10% FBS, 1% penicillin, and 1% glutamine. For the human cancer
cell lines, the cells were trypsinized and washed, and a cell
suspension in RPMI-1640 plus additives was plated into 96-well
microtiter plates at 500-2000 cells/well (depending on the cell line).
The cells were allowed to attach and grow for 72 h before treatment
with vehicle (DMSO) or test agent for an additional 72 h. For the
L1210 line, the cells were plated into 96-well plates at 7500 cells/
well and incubated with vehicle or test agents for 48 h. In each
case, one plate consisted entirely of cells in medium and medium
alone to determine time zero cell numbers. The other plates in a
given determination contained eight wells of control cells and eight
Kinetics of Dynein Inhibition. Dynein motor domain (12.5 µg/
mL final concentration) and paclitaxel-induced tubulin polymer
(1 mg/mL final concentration with 10 µg/mL paclitaxel) were
premixed with the test agents in the extraction buffer as described.
Different concentrations of ATP (1000, 500, 250, 125, and 62.5
µM) were added to the system to initiate the reaction. The mixtures
were incubated at 37 °C for 30 min. The reaction was stopped by
the addition of the malachite-green solution, and the absorbance at
655 nm was determined in a 96-well microtiter plate reader.
Acknowledgment. This work was supported in part by NIH
grants CA078039 and GM047434.
Supporting Information Available: Representative HPLC
chromatograms for key compounds subjected to biological testing.

Cytoplasmic Dynein Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2075
(23) Murakata, M.; Yamada, K.; Hoshino, O. Oxidative cyclisation of
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