Highly potent triazole-based tubulin polymerization inhibitors

Article abstract of DOI:10.1021/jm061142s

We describe the synthesis and biological evaluation of a series of tubulin polymerization inhibitors that contain the 1,2,4-triazole ring to retain the bioactive configuration afforded by the cis double bond in combretastatin A-4 (CA-4). Several of the su

Full text of DOI:10.1021/jm061142s

J. Med. Chem. 2007, 50, 749-754
749
Highly Potent Triazole-Based Tubulin Polymerization Inhibitors
Qiang Zhang,† Youyi Peng, Xin I. Wang, Susan M. Keenan,‡ Sonia Arora, and William J. Welsh*
Department of Pharmacology, UniVersity of Medicine & Dentistry of New Jersey, Robert Wood Johnson Medical School,
and Informatics Institute of the UniVersity of Medicine & Dentistry of New Jersey, Piscataway, New Jersey 08854
ReceiVed October 2, 2006
We describe the synthesis and biological evaluation of a series of tubulin polymerization inhibitors that
contain the 1,2,4-triazole ring to retain the bioactive configuration afforded by the cis double bond in
combretastatin A-4 (CA-4). Several of the subject compounds exhibited potent tubulin polymerization
inhibitory activity as well as cytotoxicity against a variety of cancer cells including multi-drug-resistant
(MDR) cancer cell lines. Attachment of the N-methyl-5-indolyl moiety to the 1,2,4-triazole core, as
exemplified by compound 7, conferred optimal properties among this series. Computer docking and molecular
simulations of 7 inside the colchicine binding site of tubulin enabled identification of residues most likely
to interact strongly with these inhibitors and explain their potent anti-tubulin activity and cytotoxicity. It is
hoped that results presented here will stimulate further examination of these substituted 1,2,4-triazoles as
potential anti-cancer therapeutic agents.
Introduction
Given their essential role in the growth and function of cells,
microtubules are among the most important molecular targets
for cancer chemotherapeutic agents.1 The formation of micro-
tubules is a dynamic process that involves the polymerization
and depolymerization of R and â tubulin heterodimers. Mol-
ecules binding to tubulins interfere with this dynamic equilib-
rium and thus induce cell cycle arrest, resulting in cell death.2
Three ligand binding sites have been identified in tubulins: the
taxane,3 vinca alkaloid,4 and colchicine sites.5 Combretastatin
Figure 1. Known SAR of CA-4 analogues.
A-4 (CA-4,a Chart 1), a natural product isolated from the South
Chart 1
African bush willow tree Combretum caffrum, binds to the
colchicine binding site and inhibits the polymerization of
microtubules.6 CA-4 has been reported to exhibit potent
cytotoxicity against a broad range of cancer cells including
multi-drug-resistant (MDR) cell lines.7,8 However, CA-4 failed
to show in vivo efficacy against murine colon 26 adenocarci-
noma, partly due to its poor water solubility.9 A disodium
phosphate derivative of CA-4 (CA-4P) has shown promising
results in human cancer clinical trials,10 thus stimulating
significant interest in a variety of CA-4 analogues.
Extensive studies have been conducted to examine the
structure-activity relationship (SAR) of CA-4 and its analogues.
The trimethoxy substitutions on the A ring and the cis-olefin
configuration at the bridge have been reported as prerequisites
for potent cytotoxicity, while the B ring is tolerant of structural
modifications (Figure 1).11 To retain the cis-olefin configuration
of CA-4, alternative bridge groups have been introduced into
CA-4 to replace the double bond. Examples of nonheterocyclic
bridges are ethers, olefins, ketones, sulfonamides, sulfonates,
amine, amide derivatives, and cyclopentanes.11,12 Heterocyclic
bridges include five-membered rings (e.g., pyrazoles, thiazoles,
triazoles, tetrazoles, oxazoles, imidazoles, furans, furanones,
* Corresponding author: phone 732-235-3234; fax 732-235-3475; e-mail
welshwj@umdnj.edu.
† Present address: Intra-Cellular Therapies, Inc., 3960 Broadway, New
York, NY 10032.
furazans, dioxolanes, and thiophenes)11,13 and indoles.14,15
Structural modifications on the B ring suggest that the 4-meth-
oxy group is crucial for cytotoxicity, while the 3-hydroxy group
is optional.16-18 Strategies have been pursued to improve the
cytotoxity and physicochemical properties of these compounds.
‡ Present address: School of Biological Sciences, University of Northern
Colorado, Greeley, CO 80639.
a Abbreviations: CA-4, combretastatin A-4; CA-4P, combretastatin A
4-phosphate; MDR, multi-drug-resistant; SAR, structure-activity relation-
ship; NCI-DTP, National Cancer Institute-Developmental Therapeutics
Program; EM, energy minimization; MD, molecular dynamics.
10.1021/jm061142s CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/24/2007

750 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Zhang et al.
Scheme 1a
a Reagents and conditions: (a) RNH2, triethylamine, 0 °C to rt; (b) P4S10, (CH3)3SiOSi(CH3)3, 62 °C or Lawesson’s reagent, 62 °C (for 7); (c) NH2NH2,
0 °C to rt; (d) (i) (CH3O)3CH, H2SO4 (cat.), rt; (ii) NaCNBH3, rt (for 8)
Table 1. Summary of the NCI-DTP 60-Cell-Line Screening for
Compounds 3-7
synthesized by the coupling of the indole Grignard reagent with
5-isothiocyanato-1,2,3-trimethoxybenzene. The Grignard reagent
was obtained via standard methods or by metal exchange
reaction between iodide and isopropyl magnesium chloride.21-23
All amines used in the present synthesis were obtained
through conversion of the respective nitro precursor by catalytic
hydrogenation reaction. For compounds 7 and 8, additional
N-methylation reactions were performed with methyl iodide as
the alkylation reagent (Scheme 1).
no. of
no. of cell lines
compd
cell linesa
with log10 GI50 < -4b
range of log10 GI50
3
4
5
6
7
50
53
55
54
55
49
52
53
51
55
-4.43 to -6.63
-4.00 to < -8.00
-4.04 to -7.61
-4.05 to -6.48
-4.07 to < -8.00
a Cell lines for which results were reported by NCI-DTP. b GI50 refers
to growth inhibition of 50%.
Results and Discussion
For example, Wang et al.19 reported a series of CA-4 analogues
containing five-membered ring bridges and the N-methylindole
as the B ring (1 and 2 in Chart 1) that possess potent cytotoxicity
and improved bioavailability comparable to CA-4.
Compounds 3-7 (Chart 1) were submitted for in vitro
biological testing against the ∼60 cancer cell line screen
provided by the National Cancer Institute-Developmental
Therapeutics Program (NCI-DTP). The NCI-DTP screening
procedure is described in detail elsewhere (http://dtp.nci.nih.gov/
).24,25 The results, summarized in Table 1, reveal that compounds
3-7 exhibited appreciable growth inhibition (log10 GI50 < -4)
against nearly all of the cancer cell lines reported by NCI-
DTP. As expected, the subject compounds exhibited variable
growth inhibition against different cancer cell lines of the same
tumor type (Supporting Information, Table 1). For example,
compound 7 exhibited very strong growth inhibition (log10 GI50
< -7.00) against lung cancer cell lines A549/ATCC, EKVX,
HOP-62, HOP-92, NCI-H23, NCI-H322M, NCI-H460, and
NCI-H522, whereas it showed only weak inhibition against the
lung cancer cell line NCI-H226 (log10 GI50 ) -4.07). The same
phenomenon was observed for ovarian cancer and melanoma
cell lines. Among compounds 3-7, 7 exhibited overall the most
potent cytotoxicity against the NCI-DTP cell lines. The present
results are consistent with those of Wang et al.,19 who found
that insertion of the N-methyl-5-indolyl moiety as the B ring
(Chart 1) was superior to 4-aminophenyl (4) and 3-hydroxyl-
4-methoxyphenyl (5) groups.
To quantify the cytotoxicity profile of these compounds more
precisely, MTT assays were conducted against several cancer
cell lines: colon cancer HCT-116, breast cancer ZR-75-1,
cervical cancer HeLa and KB-3-1, and P-glycoprotein-overex-
pressing multi-drug-resistant (MDR+) cancer cell line KB-V1
(Table 2). The cytotoxicity against these cell lines was 2-10-
fold higher with 4-dimethylamino substitution (4) compared with
4-methoxy (3) on the B ring (Chart 1). Introduction of hydroxyl
at the 3-position of the B ring (5) produced cytotoxicity
comparable to that of 4. However, fluorine substitution at the
3-position (6) failed to improve the cytotoxicity profile.
Compound 7, with N-methyl-5-indolyl as ring B, consistently
showed very potent cytotoxicity (IC50 < 24 nM) against the
cell lines. The cytotoxicity of 7 is comparable to that of
colchicine but slightly weaker than that of CA-4 (Table 2).
The tubulin assembly assay suggested that these new deriva-
tives function as tubulin polymerization inhibitors (Table 2).
In our efforts to discover novel tubulin polymerization
inhibitors as potential anti-cancer chemotherapeutic agents, we
selected the 1,2,4-triazole ring to retain the cis-olefin configu-
ration of CA-4. Our underlying premise was that the triazole
ring would augment bioavailability and chemical stability and,
at the same time, maintain the in vitro and in vivo efficacy of
CA-4. Additionally, insertion of N-methylindole as the B ring
would yield potent cytotoxicity and tubulin polymerization
inhibition. Here we report the synthesis and in vitro cytotoxicity
of a series of novel triazole-based tubulin polymerization
inhibitors (Chart 1, 3-9).
Chemistry. Compounds 3-8 (Chart 1) were synthesized
starting from amides that were obtained by coupling of 3,4,5-
trimethoxybenzoic chloride with different amines (Scheme 1).
The amides were converted to thioamides by reaction with
different thionation reagents. Use of P4S10 as a thionation reagent
gave reactions that were very slow at temperatures below 60
°C and failed to yield the desired product above 60 °C.
Curphey’s method in combination with P4S10 and hexamethyl-
disiloxane was found to generate thioamides in quantitative yield
with sufficient purity so that the products could be used directly
for the next step.20 This reaction is highly versatile and proceeds
smoothly with most groups as the B ring, aside from basic
groups such as indole. In the latter case, Lawesson’s reagent
was used to synthesize the thioamides. With thioamides in hand,
amidrazones could be synthesized smoothly by reaction with
anhydrous hydrazine at 0 °C. The final cyclization step was
performed with trimethyl orthoformate under acidic conditions.
Reduction of the appropriate carbon-carbon double bond in 7
by sodium cyanoborohydride (NaCNBH3) produced indoline 8
in nearly quantitative yield.
Our attempts to synthesize 9 by the coupling of acid and
amine via Scheme 1 were unsuccessful, likely due to the strong
deactivating effect of the three methoxyl groups on aniline.
Success was finally achieved via the alternative reaction pathway
depicted in Scheme 2. The key intermediate, thioamide, was

Triazole-Based Tubulin Polymerization Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 751
Scheme 2a
a Reagents and conditions: (a) (i) NaH, DMF, 0 °C; (ii) CH3I (b) i-PrMgCl, -10 °C; (c) 3,4,5-(CH3O)3C6H2NCS, -10 °C to rt; (d) NH2NH2, 0 °C to
rt; (e) (CH3O)3CH, H2SO4 (cat.), rt.
Table 2. Cytotoxicity and Tubulin Polymerization Inhibition Effects of Subject Compounds
cytotoxicity IC50, nM
HCT-116
(colon)
ZR-75-1
(breast)
HeLa
(cervix)
KB-3-1
(cervix)
KB-V1a
(cervix)
anti-tubulin activity
compd
IC50, µM
3
4
5
6
7
8
9
230
522
308
141
367
23.8
ND
ND
0.24
522
308
290
29.6
25
173
8.73
4097
18.0
0.30
1.84
ND
373
119
57.3
399
9.9
907
323
544
1479
20.8
ND
32.1
0.64
620
NDb
4.1
6.0
ND
3.0
>10
5.6
5.8
ND
ND
91.5
88.9
365
7.39
3288
24.3
0.35
2.75
ND
ND
15.0
0.78
3.99
23.3
CA-4
colchicine
paclitaxel
>10 000
a Multiple-drug-resistant positive (MDR+) cancer cell line. b ND, not determined.
The inhibition potency on tubulin polymerization of 4, 5, 7,
and 9 is similar to that of CA-4. The cytotoxicity is lower for
these compounds than for CA-4, suggesting that the latter
compound may exert its cytotoxic effects through additional
targets besides tubulin. Compound 7 yielded the most potent
anti-tubulin polymerization activity and cytotoxicity against the
cancer cell lines, in concordance with the NCI-DTP screening
results.
Compound 8, in which one double bond in the indole ring
of 7 is reduced (Chart 1), lacked activity against the two cell
lines, suggesting that the aromaticity and/or planarity of the B
ring is critical for the potent activity. Quantum mechanical
calculations confirmed that the absence of this double bond in
8 renders the B ring nonplanar (see Experimental Section).
Compound 9 (Chart 1), in which the substitution pattern with
respect to the triazole ring is switched relative to 7, demonstrated
potent anti-tubulin polymerization activity (5.6 µM) and cyto-
toxicity (∼20 nM) against the cell lines. Compounds 7 and 9
also exhibited potent cytotoxicity (21 and 32 nM, respectively)
against MDR+ cancer cell line KB-V1, indicating that they are
not substrates of the MDR efflux pump. In contrast, the
inhibitory activity of both colchicine and paclitaxel diminished
substantially against KB-V1, suggesting that they are MDR
substrates. Association with MDR leads to diminished intrac-
ellular drug concentrations, thereby reducing or even abolishing
cytotoxicity.26,27
Figure 2. X-ray crystal structure of compound 7. Atoms are depicted
as 50% thermal probability ellipsoids.
colchicine binding site of tubulin. Molecular dynamics (MD)
simulations on the tubulin-7 complex indicated that 7 interacts
extensively with â tubulin but with only two residues (Ala R180
and Val R181) in R tubulin via hydrophobic interactions (Figure
3, upper panel), which is consistent with the fact that the
colchicine binding site is largely buried within â tubulin.28
More detailed analysis of the inhibitor-tubulin complex
(Figure 3, lower panel) revealed several key interactions that
appear to play a role in the binding of 7. Two methoxy O atoms
on the A ring are located within hydrogen-bond distances (3.4
and 3.5 Å) of the thiol group of Cys â241. The trimethoxyphenyl
The high-resolution crystal structure (resolution ) 3.58 Å)
of the tubulin-colchicine-stathmin-like domain complex re-
vealed the location of the colchicine binding site.28 We then
proceeded to examine the interactions of the subject compounds
with tubulin. Adopting the conformation found in its X-ray
crystal structure (Figure 2), 7 was computer docked inside the

752 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Zhang et al.
triazole ring (e.g., 7 and 9) conferred optimal bioactivity. These
compounds exhibited potent tubulin polymerization inhibitory
activity and cytotoxicity against a variety of cancer cells
including MDR cancer cell lines. Molecular docking and
dynamics simulation were performed to study the inhibitor-
protein interactions. Analysis of the inhibitor binding conforma-
tion in the colchicine binding site revealed specific residues that
may play an important role in tubulin polymerization inhibitory
activity and cytotoxicity. We are presently utilizing insights
gleaned from these molecular modeling studies to design new
tubulin inhibitors with strong affinity, potent tubulin inhibitory
activity, and high cytotoxicity.
Experimental Section
1H NMR spectra were recorded at 400 MHz on a Varian Gemini-
400 spectrometer and in deuterated chloroform (CDCl3) or dimethyl
sulfoxide (DMSO-d6) solution at room temperature. Tetramethyl-
silane (TMS, 0.00 ppm) was used as the internal standard, and the
spectra were reported in parts per million (ppm). Analytical thin-
layer chromatography (TLC) was performed on precoated plastic-
backed plates purchased from Aldrich (silica gel 60 F254; 0.25
mm thickness). Flash column chromatography was conducted with
silica gel 60 (230-400 mesh) (Natland Co.). Standard (MS) and
tandem (MS-MS) mass spectrometry were conducted on a Finni-
gan LCQ Duo mass spectrometer (Thermo Quest Co.). Melting
points were taken on a Mel-temp melting point apparatus in open
capillary tubes without calibration.
All reactions were carried out with anhydrous solvents in oven-
dried and argon-charged glassware. All anhydrous solvents except
as mentioned were freshly distilled and stored in 4 Å molecular
sieves. All solvents used in workup were used as received from
the commercial supplier without further purification. All reagents
were purchased from Aldrich Chemical Co., St. Louis, MO.
General Procedure for the Synthesis of 3-7. 3,4,5-Tri-
methoxybenzoyl chloride was added slowly to 1.0 equiv of amine
solution, which was dissolved in CHCl3 and mixed with 1.0 equiv
of triethylamine at 0-5 °C. After the reaction was complete, the
CHCl3 solution was extracted three times by water and concentrated
under vacuum. The resulting amide, which precipitated out im-
mediately when the concentrated syrup was cooled to rt, possessed
sufficient purity for direct use in the next step.
Figure 3. Binding conformation of compound 7 docked inside
colchicine binding site of tubulin: perspective view (upper panel) and
close-up view (lower panel) of bound ligand inside tubulin binding
site. Compound 7 is rendered in ball-and-stick mode and colored by
atom type. In the upper panel, tubulin is displayed as a flat ribbon
with R-tubulin colored green and â-tubulin colored magenta. Residues
Ala R180, Val R181, and Cys â239 are displayed in stick mode. In the
lower panel, the residues interacting with the ligand are rendered in
stick mode and labeled. Hydrogen bonds are depicted as green dotted
lines. The triazole N2 atom is labeled accordingly.
The amide was dissolved in anhydrous CHCl3, and 0.16 equiv
of P4S10 and 1.67 equiv of hexamethyldisiloxane were introduced
[for 7, 1.05 equiv of Lawesson’s reagent was added at rt]. The
solution was heated to 62 °C under inert gas. The CHCl3 solution
slowly became yellow in color. After the reaction was complete,
the product was purified by extraction by water and then concen-
trated. Gas chromatography-mass spectrometry (GC-MS) indi-
cated thioamides synthesized by this method were sufficiently pure
to be used directly for the next step. For 7, the thioamide was
purified by column chromatography with EtOAc and hexane as
eluent.
The yellow thioamide was suspended in anhydrous alcohol under
inert gas atmosphere. The reactants were cooled to 0 °C, and 5.0
equiv of anhydrous hydrazine was added slowly with vigorous
stirring. The mixture was stirred at rt overnight. Alcohol and excess
hydrazine were evaporated under vacuum, and the syrup was
dissolved in CHCl3 and extracted by water three times. The organic
layer was dried and concentrated under vacuum to yield amidrazone
as a white or off-white solid.
moiety occupies a pocket bounded by Leu â248, Val â318, Thr
â353, and Ile â358, while the indolic ring of 7 is embedded in
a hydrophobic pocket consisting of the side chains of residues
of Ala R180, Val R181, Lys â254, Asn â258, Met â259, Val
â351, and Lys â352. The protonated side-chain N atom of
residue Lys â254 remained in close proximity (e5 Å) to the
triazole N2 atom (Figure 3, lower panel) for 180 ps of the entire
200 ps MD run. Similarly, Nguyen et al.29 proposed that residues
within the loop region of nearby Lys â254 formed hydrogen
bonds with known colchicine site inhibitors (e.g., podophyllo-
toxin and methylchalcone). Also consistent with the results of
both Nguyen et al.29 and Ravelli et al.,28 our MD simulations
indicated that the side-chain N atom of Lys â254 forms
hydrogen bonds with the phosphate O atoms of R tubulin-bound
GTP. Taken together, these observations suggest that the triazole
ring in the subject compounds interacts with the Lys â254 side
chain via attractive contacts with the triazole’s N2 atom (Figure
3, lower panel).
The amidrazone was dissolved in alcohol and 5.0 equiv of
trimethyl orthoformate was added at rt. Several drops of sulfuric
acid were added as catalyst, and the solution was stirred vigorously
for 2 h. After neutralization, the alcohol was evaporated, and the
triazole was purified by column chromatography.21
Conclusion
We have synthesized and biologically evaluated a series of
novel tubulin polymerization inhibitors that contain a core 1,2,4-
triazole ring to retain the cis configuration required for bioac-
tivity. Attachment of the N-methyl-5-indolyl moiety to the
4-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-4H-1,2,4-
1
triazole (3). Overall yield 86%; mp 163-165 °C; H NMR (400
MHz, CDCl3) δ 3.64 (s, 6H), 3.83 (s, 3H), 3.84 (s, 3H), 6.70 (s,

Triazole-Based Tubulin Polymerization Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 753
2H), 6.98 (d, J ) 2.0 Hz, 2H), 7.19 (d, J ) 3.8 Hz, 2H), 8.25 (s,
1H); MS (ESI) m/z ) 342.1 (M+ + 1); Anal. (C18H19O4N3) C, H,
O, N.
carcinoma cell line (HeLa), and a human cervix carcinoma cell
line (KB-3-1) and its MDR+ subclone (KB-V1). Cells in logarith-
mic phase were diluted to a density of 20 000-30 000 cells/mL in
culture medium based on growth characteristics. For each well of
a 96-well microplate, 100 µL of cell dilution was seeded, allowed
to attach overnight, and then exposed to varying concentrations
(10-5-10-12 M) of compounds for 72 h (37 °C, 5% CO2
atmosphere). The number of living cells was estimated by the MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] as-
say (Sigma, St. Louis MO). Absorbance at 595 nm was recorded
on a TECAN GENios multifunction microplate reader (TECAN
U.S. Inc., Research Triangle Park, NC). Compounds were tested
in triplicate in at least three independent assays. The IC50 values
were determined by a nonlinear regression analysis with Prism 3.03
(Graphpad Software Inc.). Average values were reported.
In Vitro Tubulin Polymerization Assay. Tubulin polymeriza-
tion assays were conducted with the CytoDYNAMIX Screen 03
assay system (Cytoskeleton Inc.) following the manufacturer’s
instructions. Tubulin (>99% pure, 3 mg/mL) in 100 µL of G-PEM
buffer was placed in a prewarmed 96-well plate in the presence of
tested compounds at varying concentrations (ranging from 0.03 to
30 µM). The OD340nm was recorded every 60 s on a TECAN
GENios multifunction microplate reader (TECAN U.S. Inc.,
Research Triangle Park, NC) at 37 °C for 1 h. Apparent IC50 values
for the compounds were determined by nonlinear regression analysis
with Prism 3.03 (Graphpad Software Inc.) by use of area under
curve (AUC) from the polymerization profiles.
Molecular Modeling. All calculations were conducted on SGI
Octane R12000 and SunFire 6900 machines. The crystal structure
of the tubulin-colchicine-stathmin-like domain complex (PDB ID
) 1SA0) was retrieved from the Research Collaboratory for
Structural Bioinformatics Protein Data Bank (http://www.rcsb.org/
pdb).30 The crystal structure of compound 7 was assigned partial
atomic charges and force constants by use of the MMFF94 force
field in Sybyl v7.1.31 Compound 7 was aligned to the structure of
colchicine in the binding site of the tubulin crystal structure by
atom-fitting the trimethoxyphenyl ring and the centroids of the B
ring (Figure 1), followed by manually docking 7 in the colchicine
binding site. Energy minimization (EM) and molecular dynamics
(MD) with AMBER832 were performed to refine the ligand-protein
interactions.
The Hawkins-Cramer-Truhlar33,34 pairwise generalized Born
model was used for the EM and MD calculations of the tubulin
system. The standard force-field parameter set parm99 in AM-
BER832 was selected with dielectric constant ꢀ ) 1, and cutoff
distance ) 12.0 Å was applied for both electrostatic and van der
Waals interactions. The concentration of mobile counterions in
solution was set to 0.2 M35 and surface area was computed by use
of the linear combination of pairwise overlaps (LCPO) model.36
The system was energy-minimized in two steps: (i) 1000 iterations
of steepest descent and (ii) 4000 iterations of conjugate gradient
optimization. MD simulations were then run on the resulting system
with the same force-field parameters as EM. The SHAKE algo-
rithm37 was implemented for bonds involving hydrogen atoms. The
system was coupled to a Berendsen bath at 300 K by using a
coupling constant τT ) 1.0 ps.38 A 2.0 fs time step was applied
during MD simulations. Restrained MD was first run for 10 ps to
equilibrate the tubulin system by freezing the protein and ligand.
Unconstrained MD was then applied to the entire system for 200
ps; the time-averaged structure derived from the final 150 ps was
energy-minimized by 500 iterations of steepest descent and 500
iterations of conjugate gradient optimization. The hydrogen bonds
and hydrophobic interactions between the ligand and protein were
identified by LIGPLOT 4.439 and displayed in DSviewerPro5.0.40
Compound 8 was constructed in Sybyl7.1, based on the crystal
structure of 7, and energy-minimized with the MMFF force field
and charges. Ab initio molecular orbital calculations were performed
on 7 and 8 at the HF/6-31G** level of theory, accessed through
the Spartan ’02 program (Wavefunction, Inc., Irvine CA). By
comparison of the resulting structures, it was found that the B ring
was essentially planar (0°) in 7, consistent with the extended
N,N-Dimethyl-4(3-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-
1
4-yl)benzenamine (4). Overall yield 81%; mp 145-147 °C; H
NMR (400 MHz, CDCl3) δ 2.99 (s, 6H), 3.65 (s, 6H), 3.83 (s,
3H), 6.71 (d, J ) 3.8 Hz, 2H), 6.76 (s, 2H), 7.09 (d, J ) 6.0 Hz,
2H), 8.22 (s, 1H); MS (ESI) m/z ) 355.2 (M+ + 1); Anal.
(C19H20O3N4) C, H, O, N.
2-Methoxy-5-[3-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-4-
yl]phenol (5). Overall yield 83%; mp 197-198 °C; 1H NMR (400
MHz, CDCl3) δ 3.66 (s, 6H), 3.83 (s, 3H), 3.94 (s, 3H), 6.18 (s,
1H), 6.74 (m, 3H), 6.89 (m, 2H), 8.24 (s, 1H); MS (ESI) m/z )
358.3 (M+ + 1); Anal. (C18H19O5N3) C, H, O, N.
4-(3-Fluoro-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-
1
4H-1,2,4-triazole (6). Overall yield 88%; mp 151-152 °C; H
NMR (400 MHz, CDCl3) δ 3.67 (s, 6H), 3.89 (s, 3H), 3.93 (s,
3H), 3.69 (s, 2H), 7.05 (m, 3H), 8.25(s, 1H); MS (ESI) m/ z )
360.3 (M+ + 1); Anal. (C18H18O4N3F) C, H, O, N, F.
1-Methyl-5-[3-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-4-yl]-
1H-indole (7). Overall yield 69%; mp 157-159 °C; 1H NMR (400
MHz, CDCl3) δ 3.52 (s, 6H), 3.80 (s, 3H), 3.84 (s, 3H), 6.51 (s,
1H), 6.73 (s, 2H), 7.04 (d, 1H), 7.17 (s, 1H), 7.37 (d, J ) 1.6 Hz,
1H), 7.559 (s, 1H), 8.30 (s, 1H); MS (ESI) m/z ) 365.3 (M+ + 1);
Anal. (C20H20O3N4) C, H, O, N.
1-Methyl-5-[3-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-4-yl]-
indoline (8). Compound 7 (25 mg) was dissolved in 2 mL of HOAc
at rt. NaCNBH3 (1.5 equiv) was added in one portion while stirring,
and the solution was stirred overnight. TLC indicated that the
reaction was complete. The acidic solution was neutralized by
adding NaOH solution dropwise and extracted by CHCl3. After
concentration, the residue was purified by column chromatography
with CHCl3/MeOH (20:1) as eluent to obtain 20 mg of 7 as a white
1
solid: mp 158-160 °C; H NMR (400 MHz, CDCl3) δ 2.83 (s,
3H), 3.00 (t, J ) 8.0 Hz, 2H), 3.49 (t, J ) 9.0 Hz, 2H), 3.67 (s,
6H), 3.87 (s, 3H), 6.46 (d, J ) 8.0 Hz, 1H), 6.75 (s, 1H), 6.92 (s,
1H), 7.01 (d, J ) 7.0 Hz, 1H), 8.65 (s, 1H); MS (ESI) m/z ) 367.3
(M+ + 1); Anal. (C20H22O3N4) C, H, O, N.
1-Methyl-5-[4-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl]-
1H-indole (9). 5-Iodo-1-methyl-1H-indole (1.0 g, 3.89 mmol) was
dissolved in THF (10 mL) and cooled to -10 °C. To the solution
was added isopropylmagnesium chloride (2.5 mL, 2.0 M in THF),
and the reaction mixture was kept at -10 °C for 30 min.
5-Isothiocyanato-1,2,3-trimethoxybenzene (1.0 equiv) was added
dropwise, and the resulting brown solution was stirred at rt
overnight. Water was added to quench the reaction, and the mixture
was extracted by CHCl3. After drying and concentration, the product
was purified by column chromatography (EtOAc/hexane 1:4) to
give thioamide 1.0 g as a yellow solid, yield 75%.
The thioamide (0.5 g, 1.37 mmol) was suspended in anhydrous
alcohol under inert gas atmosphere. The reactants were cooled to
0 °C and 5.0 equiv of anhydrous hydrazine was added slowly with
vigorous stirring, after which the thioamide was stirred at rt
overnight. The alcohol and excess hydrazine were evaporated under
vacuum, and the syrup was dissolved in CHCl3 and extracted by
water three times. The organic layer was dried and concentrated
under vacuum to yield 0.4 g of amidrazone as a white solid.
The amidrazone was dissolved in alcohol and 5.0 equiv of
trimethyl orthoformate was added at rt. Several drops of sulfuric
acid were added as catalyst, and the solution was stirred vigorously
for 2 h. After neutralization, the alcohol was evaporated and the
triazole was purified by column chromatography (EtOAc/hexane
4:1) to obtain 366 mg of triazole with a yield of 71% from
thioamide: mp 157-159 °C; 1H NMR (400 MHz, CDCl3) δ 3.20
(s, 6H), 3.26 (s, 3H), 3.36 (s, 3H), 5.92-5.94 (m, 3H), 6.55 (d, J
) 3.0 Hz, 1H), 6.72-6.75 (m, 3H), 6.83 (d, J ) 1.5 Hz, 1H), 7.77
(s, 1H); MS (ESI) m/z ) 365.1 (M+ + 1); Anal. (C20H20O3N4) C,
H, O, N.
In Vitro Cytotoxicity Assay. Cytotoxic effects were examined
in a human breast carcinoma cell line (ZR-75-1), a human colon
carcinoma cell line (HCT-116), a human cervix epithelial adeno-

754 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Zhang et al.
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aromatic character of the indolic moiety, but was distinctly
nonplanar (14°) in 8, reflecting a loss of aromatic character.
Acknowledgment. Access to the computational facilities at
the UMDNJ Informatics Institute, supported in part by the
National Library of Medicine (Grant G08 LM6230-07), is
gratefully acknowledged. We thank Dr. Nigam Rath, Depart-
ment of Chemistry & Biochemistry, University of Missouris
St. Louis, St. Louis, MO, for providing the X-ray crystal
structure of 7.
Supporting Information Available: Results from the NCI-
DTP 60 cell-line assays on compounds 3-7 and tabulated X-ray
crystal structure data for compound 7. This material is available
free of charge via the Internet at http://pubs.acs.org.
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