Synthesis and biological activities of (R)- and (S)- N -(4-Methoxyphenyl)- N,2,6-trimethyl-6,7-dihydro-5 H -cyclopenta[ d ]pyrimidin-4-aminium chloride as potent cytotoxic antitubulin agents

Article abstract of DOI:10.1021/jm2007722

(R,S)-1 is a potent antimitotic compound. (R)-1·HCl and (S)-1·HCl were synthesized from (R)- and (S)-3-methyladipic acid. Both enantiomers were potent inhibitors of cell proliferation and caused cellular microtubule loss and mitotic arrest. They inhibited purified tubulin assembly and the binding of [³H]colchicine to tubulin, with (S)-1 being about twice as potent. Cytotoxicity against 60 tumor cell lines, however, indicated that the (S)-isomer was 10- to 88-fold more potent than the (R)-isomer.

Full text of DOI:10.1021/jm2007722

BRIEF ARTICLE
pubs.acs.org/jmc
Synthesis and Biological Activities of (R)- and (S)-N-(4-Methoxyphenyl)-
N,2,6-trimethyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-aminium
Chloride as Potent Cytotoxic Antitubulin Agents
||
||
Aleem Gangjee,*,†, Ying Zhao,† Ernest Hamel,‡ Cara Westbrook,§ and Susan L. Mooberry*,§,
†Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, 600 Forbes Avenue, Pittsburgh,
Pennsylvania 15282, United States
‡Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702, United States
§Department of Pharmacology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio,
Texas 78229, United States
S Supporting Information
b
ABSTRACT: (R,S)-1 is a potent antimitotic compound. (R)-1 HCl and (S)-1 HCl were synthesized from (R)- and (S)-3-
3
3
methyladipic acid. Both enantiomers were potent inhibitors of cell proliferation and caused cellular microtubule loss and mitotic
arrest. They inhibited purified tubulin assembly and the binding of [3H]colchicine to tubulin, with (S)-1 being about twice as potent.
Cytotoxicity against 60 tumor cell lines, however, indicated that the (S)-isomer was 10- to 88-fold more potent than the (R)-isomer.
e1,2 recently reported the discovery of a potent antitubulin
(R,S)-N-(4-methoxyphenyl)-N,2,6-trimethyl-6,7-dihydro-
’ CHEMISTRY
W
(6R)-N-(4-Methoxyphenyl)-N,2,6-trimethyl-6,7-dihydro-5H-
cyclopenta[d]pyrimidin-4-aminium chloride [(R)-1 HCl] was
5H-cyclopenta[d]pyrimidin-4-aminium chloride 1. This com-
pound is a water-soluble colchicine site binding, microtubule
depolymerizing agent that inhibited the growth of cancer cells
with GI50 in the nanomolar range. In addition, (R,S)-1 over-
comes the two most clinically relevant tumor resistance mecha-
nisms that limit activity of microtubule targeting agents: over-
expression of P-glycoprotein (Pgp)3,4 and βIII-tubulin.5À10
(R,S)-1 was the most potent among a series of analogues evaluated
and contains a chiral center at C6. It was therefore of interest to
synthesize and determine the biological activities of the (R)-1
and (S)-1 enantiomers to ascertain the contribution of the in-
dividual isomers to the antitubulin and cytotoxic properties of the
racemate and to determine if one enantiomer was more potent
than the other. While there are numerous agents reported in the
literature that target the colchicine binding site,11,12 and some of
these are currently in clinical trials as antitumor agents,13 in-
cluding combretastatin A-4 (CA4) (Figure 1) phosphate, there is
little information regarding the activities of specific enantiomers
of racemic colchicine site agents. Natural (À)-colchicine (Figure 1)
has an aS,7S-absolute configuration and is much more active than
its enantiomer, as is the case with other colchicinoids.14,15 Chinigo
et al.16 synthesized 2-biphenyl-2,3-dihydroquinazoline analogue 2
(Figure 1). Racemic 2 and each enantiomer were evaluated. Race-
mic 2 was similar in potency to the (S)-isomer, while the (R)-
isomer was 10-fold less potent as an inhibitor of tubulin assembly
and [3H]colchicine binding. The difference in activities of (S)-2
and (R)-2 was rationalized on the basis of molecular modeling.
We1 had predicted, using molecular modeling, that the (S)-
and (R)-isomers of 1 would be nearly equipotent in their effects
on tubulin.
3
prepared from commercially available (R)-3-methyladipic acid
(R)-3 as shown in Scheme 1. (R)-4 was synthesized via the
reaction of (R)-3 and concentrated sulfuric acid at reflux in
ethanol followed by a Dieckmann condensation in the presence
of elemental sodium in toluene and then with acetamidine
hydrochloride. Chlorination of (R)-4 with POCl3 for 3 h afforded
(R)-5. Reaction of (R)-5 with 4-methoxy-N-methylaniline in the
presence of 2À3 drops of concentrated HCl gave (R)-1. Anhy-
drous hydrochloric acid gas was bubbled through the anhydrous
ether solution of (R)-1 to give the HCl salt (R)-1 HCl as a white
solid. Using the same methodology described for (R)-1 HCl in
Scheme 1, (6S)-N-(4-methoxyphenyl)-N,2,6-trimethyl-6,7-dihy-
3
3
dro-5H-cyclopenta[d]pyrimidin-4-aminiumchloride [(S)-1 HCl]
3
was synthesized from (S)-3.17
’ BIOLOGICAL EVALUATIONS
All of the biological evaluations were carried out on the HCl
salts of (R)-1 and (S)-1. The microtubule disrupting effects of
(R)-1 and (S)-1 were observed in a cell based phenotypic screen.
Both compounds caused concentration dependent loss of the
interphase microtubule network, similar to the effects of colchi-
cine, CA420 and (R,S)-1.1 The EC50 (concentration required to
cause 50% loss of cellular microtubules) (Table 1) was calculated
to be 56 nM for (R,S)-1, 23 nM for (S)-1, and 278 nM for (R)-1.
Thus, in this assay the (S)-isomer was 12-fold more potent
Received: June 14, 2011
Published: July 25, 2011
r
2011 American Chemical Society
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dx.doi.org/10.1021/jm2007722 J. Med. Chem. 2011, 54, 6151–6155
|

Journal of Medicinal Chemistry
BRIEF ARTICLE
than the (R)-isomer. Consistent with effects on microtubules,
(R)-1 and (S)-1 caused the formation of aberrant mitotic spin-
dles (not shown) and mitotic accumulation when measured
by flow cytometry. Besides the increase in the cells in G2/M,
with all three compounds, an increase in sub-G1 popula-
tion representing apoptotic cells was observed with (R,S)-1
and (S)-1 but not with (R)-1 at 18 h (see Supporting Infor-
mation).
’ ANTIPROLIFERATIVE EFFECTS
(R)-1 and (S)-1 were tested for antiproliferative effects against
the drug sensitive MDA-MB-435 cell line using the sulforhoda-
mine B assay (SRB assay).18,19 (S)-1 and (R)-1 had potent
antiproliferative effects, with IC50 (concentration required to
cause 50% inhibition of proliferation) of 12 ( 0.8 and 51 ( 4 nM.
The (S)-enantiomer was over 4-fold more potent than the (R)-
enantiomer and about 1.5 times more potent than the racemate
(Table 1).
The ability of (R)-1 and (S)-1 to circumvent Pgp-mediated
drug resistance was evaluated by using an SK-OV-3 isogenic cell
line pair (Table 2). In this cell line pair, the relative resistance
(Rr) to paclitaxel, a well-known Pgp substrate, was over 1600
while Rr values of 1.8, 3.8, 1.4, and 3.6 were obtained for (R,S)-1,
(S)-1, (R)-1, and CA4, respectively. Thus, all four compounds
are poor substrates for transport by Pgp and hence have
advantages over some clinically used tubulin-targeting drugs,
including paclitaxel.
A second mechanism of drug resistance that can lead to
chemotherapy failure with tubulin targeting agents is the expres-
sion of the βIII isotype of tubulin.5À10 An isogenic HeLa cell line
pair21 was used to evaluate the effects of βIII tubulin expression
on the activities of (R)-1 and (S)-1 in comparison with paclitaxel
and CA4. (S)-1, (R)-1, and CA4 had Rr of 0.7, 0.7, and 0.98,
respectively (Table 2), in this cell line pair, suggesting that, like
(R,S)-1, they overcome drug resistance mediated by βIII tubulin
compared with paclitaxel, which had a Rr of 6.6. These results
suggest that (R)- 1 and (S)-1 would circumvent tumor resistance
because of the overexpression of βIII tubulin.
Figure 1
Scheme 1a
Confirming the microtubule studies in the A-10 cells, we
found that (R)-1 and (S)-1, like the racemic mixture,1 strongly
inhibited the polymerization of purified bovine brain tubulin
(Table 1). The (S)-enantiomer was slightly more active than the
racemic mixture, while the (R)-enantiomer was slightly less
active. The compounds were also examined as inhibitors of
[3H]colchicine binding, and the differences between the two
enanatiomers and the racemic mixture were more convincing in
this assay. As in the assembly assay, the order of activity was (S)-1
> (R,S)-1 > (R)-1. The compounds were compared with CA4 in
both assays, and the cis-stilbene was somewhat more active than
(S)-1. These results with tubulin corroborate our molecular
modeling prediction1 that the (R)-1 and (S)-1 enantiomers
would have similar activity against tubulin.
a Conditions: (a) (1) ethanol, conc sulfuric acid, reflux, 8 h; (2) Na,
toluene, reflux, 3 h; (3) acetamidine hydrochloride, t-BuOH, t-BuOK,
25% over three steps; (b) POCl3, reflux, 3 h; (c) 4-methoxy-N-methylaniline,
i-PrOH, 2À3 drops HCl; (d) anhydrous HCl gas, anhydrous ether, 42%
over three steps.
Table 1. Effects of (R,S)-1 HCl, (S)-1 HCl, and (R)-1 HCl in cellular assays and on purified tubulina
3
3
3
cellular effects in MDA-MB-435 cells
% inhibition ( SD of colchicine binding
IC50 ( SD
EC50
% G2/M
inhibition of
5 μM
1 μM
compd
(nM)
(nM)
EC50/IC50
(3 Â IC50
)
tubulin assembly, IC50 ( SD (μM)
inhibitor
inhibitor
H2O control
6.1
(R,S)-1 HCl
17 ( 0.7
12 ( 0.8
51 ( 4
56
3
2
5
4
65.9
49.2
91.4
88.4
1.5 ( 0.04
1.3 ( 0.2
2.1 ( 0.1
1.2 ( 0.2
87 ( 2
93 ( 0.9
77 ( 2
98 ( 1
59 ( 2
3
(S)-1 HCl
23
70 ( 0.04
37 ( 3
3
(R)- 1 HCl
278
3
CA4
3.4 ( 0.6
13
85 ( 0.01
a The IC50 for inhibition of proliferation was determined in MDA-MB-435 cells using the SRB assay,18,19 n = 3. The EC50, the concentration that causes
50% loss of interphase microtubules, was determined in A-10 cells using immunofluorescence, n = 3. The ratio EC50/IC50 provides an indication of the
linkage between the microtubule depolymerizing effects and inhibition of proliferation. MDA-MB-435 cells were treated with each compound at 3 Â
IC50 for inhibition of proliferation and cell cycle distribution studied after 18 h by flow cytometry. The percentage of cells in each phase of the cell cycle
was determined using the Modfit program (Verity software). Inhibition of tubulin assembly and colchicine binding (Table 1) were carried out as
previously reported.1
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Journal of Medicinal Chemistry
BRIEF ARTICLE
Table 2. (R,S)-1 HCl, (S)-1 HCl, and (R)-1 HCl Circumvent Pgp and βIII-Tubulin Mediated Resistancea
3
3
3
IC50 ( SD (nM)
SK-OV-3 MDR-1-6/6
IC50 ( SD (nM)
WTβIII
drug
SK-OV-3
Rra
1.8
HeLa
Rra
(R,S)-1 HCl
34.5 ( 1.4
16.4 ( 1.6
85.9 ( 4.5
2.95 ( 0.07
6.05 ( 0.61
60.9 ( 4.4
62.6 ( 6.7
119.8 ( 11.3
4,875 ( 153
22.0 ( 6.9
37.3 ( 4.1
19.0 ( 0.9
92.9 ( 5.6
1.38 ( 0.13
4.09 ( 0.05
23.9 ( 1.7
13.3 ( 0.5
67.7 ( 1.3
9.05 ( 51.1
4.02 ( 0.26
0.6
0.7
0.7
6.6
0.98
3
(S)-1 HCl
3.8
3
(R)-1 HCl
1.4
3
paclitaxel
1622
3.6
CA4
a Rr: relative resistance.
(R,S)-1 HCl, (S)-1 HCl, and (R)-1 HCl were selected by
q, quartet; m, multiplet; br, broad singlet. Thin-layer chromatography
(TLC) was performed on Whatman Sil G/UV254 silica gel plates with a
fluorescent indicator, and the spots were visualized under 254 and
366 nm illumination. Proportions of solvents used for TLC are by
volume. Column chromatography was performed on a 230À400 mesh
silica gel (Fisher Scientific) column. Elemental analyses were performed
by Atlantic Microlab, Inc., Norcross, GA. Elemental compositions are
within (0.4% of the calculated values and indicate >95% purity of the
compounds. Fractional moles of water or organic solvents found in some
analytical samples could not be prevented despite 24À48 h of drying
in vacuo and were confirmed where possible by their presence in the
1H NMR spectra. All solvents and chemicals were purchased from
Sigma-Aldrich Co. or Fisher Scientific Inc. and were used as received. All
biological assays were performed as described previously.1
3
3
3
the National Cancer Institute for evaluation in their preclinical 60
cancer cell line screen.22 The GI50 values in nM are listed in Table
3 (Supporting Information). All three compounds were potent
inhibitors of almost all of the cancer cell lines. However, more so
than the previous evaluations, the (S)-1 enantiomer was clearly
superior to the (R,S)-1 racemate, which in turn was somewhat
more potent than the (R)-1 enantiomer. (S)-1 showed single
digit nanomolar GI50 against 51 of the tumors evaluated, and
(R,S)-1 and (R)-1 were at least 10-fold less potent than the (S)-
isomer in most of the tumors evaluated. Thus, chirality at the
6-position of 1 does not play a major role in inhibition or binding
to tubulin as demonstrated by the results in Table 1, and this
validates our earlier prediction from molecular modeling. How-
ever, this is not consistent with the 10- to 88-fold potency
increase of the (S)-isomer over the (R)-isomer toward cancer
cells in the NCI panel. Hence, an unknown mechanism(s), per-
haps facilitated transport and/or lack of metabolism, is respon-
sible for the much greater potency of (S)-1 over (R)-1 in most of
the NCI cancer cell lines.
General Procedure for (R)-4 and (S)-4. (R)-3-Methyladipic acid
[or (S)-3-methyladipic acid] (1.60 g, 10 mmol) was heated at reflux in
ethanol/concentrated sulfuric acid solution (35 mL, v/v = 2.5/1) for 8 h.
The solution was neutralized with ammonium hydroxide to pH 7, then
diluted with ethyl acetate (100 mL) and washed with water. The organic
phase was dried with anhydrous sodium sulfate and evaporated to afford
a light yellow liquid that was used in the next step without further
purification. The yellow liquid was dissolved in anhydrous toluene
(100 mL), and sodium (0.23 g) was added to the solution. The mixture
was heated at reflux for 3 h and cooled, neutralized with 1 N hydrochloric
acid solution, and washed with water. After drying with anhydrous
sodium sulfate, the organic phase was separated and evaporated to afford
a light brown liquid. The liquid was used in the next step without further
purification. The light brown liquid was diluted with t-BuOH. Acetami-
dine hydrochloride (1.13 g, 12 mmol) and potassium tert-butoxide
(1.34 g, 12 mmol) were added, and the mixture was heated at reflux
overnight. The mixture was cooled and the precipitate collected by
filtration. The residue was washed with warm methanol twice (30 mL Â 1,
15 mL Â 1). The filtrate and washings were combined, and then 3 g of
silica gel was added and the solvent removed in vacuo to afford a dry
plug. This plug was placed on the top of a silica gel column and eluted
with 1% methanol in chloroform. Fractions containing the product were
pooled and evaporated to afford (R)-4 [or (S)-4] as a white solid.
(R)-2,6-Dimethyl-6,7-dihydro-3H-cyclopenta[d]pyrimidin-
4(5H)-one [(R)-4]. (R)-4 (0.41 g, 25%) was synthesized from (R)-3
(1.6 g, 10 mmol) using the general procedure described above: TLC Rf =
0.30 (CHCl3/CH3OH, 10:1); mp 173.8À175.4 °C; 1H NMR (DMSO-
d6) δ 1.06(d, 3 H, J = 6.8 Hz, 3H), 2.16 (m, 1H), 2.24 (s, 3H), 2.31 (m,
1H), 2.44 (m, 1H), 2.77 (m, 1H), 2.86 (m, 1H), 12.16 (br, 1 H, OH, exch).
(S)-2,6-Dimethyl-6,7-dihydro-3H-cyclopenta[d]pyrimidin-
4(5H)-one [(S)-4]. (S)-4 (0.17 g, 18%) was synthesized from (S)-3
(1.0 g, 6.24 mmol) using thegeneralprocedure describedabove: TLCRf =
0.30 (CHCl3/CH3OH, 10:1); mp 173.9À175.4 °C; 1H NMR (DMSO-
d6) δ 1.06 (d, J = 6.8 Hz, 3H), 2.16 (m, 1H), 2.24 (s, 3H), 2.30 (m, 1H),
2.44 (m, 1H), 2.77 (m, 1H), 2.87 (m, 1H), 12.15 (br, 1H, exch).
General Procedure for (R)-5 and (S)-5. A mixture of (R)-4
[or (S)-4] and POCl3 (10 mL) was heated at reflux for 3 h. The mixture
’ SUMMARY
The (R)- and (S)-enantiomers of the potent cytotoxic anti-
tubulin racemate (R,S)-1 were synthesized from (R)- and (S)-3-
methyladipic acid. (R)-1 and (S)-1 were potent inhibitors of
cancer cells in culture and bind to the colchicine site on tubulin.
The (S)-enantiomer is more active than the (R)-enantiomer
against cellular microtubule loss and in the inhibition of tubulin
assembly. Both enantiomers cause a G2/M cell cycle arrest. (R)-1
and (S)-1 circumvent tumor resistance because of overexpres-
sion of Pgp and βIII tubulin, thus overcoming two important
mechanisms that hamper the clinical use of the most important
antitubulin agents. Against the NCI tumor cell line panel (S)-1 is
a single digit nanomolar inhibitor of 51 cancer cell lines and is 10-
to 88-fold more potent against most of the cell lines than (R,S)-1
or (R)-1. These results suggest that the (S)-isomer has significant
potential and should be further examined in murine preclinical
studies to evaluate its antitumor efficacy.
’ EXPERIMENTAL SECTION
Analytical samples were dried in vacuo (0.2 mmHg) in a CHEM-DRY
drying apparatus over P2O5 at 50 °C. Melting points were determined
on a digital MEL-TEMP II melting point apparatus with Fluke 51 K/J
electronic thermometer and are uncorrected. Nuclear magnetic reso-
nance spectra for protons (1H NMR) were recorded on Bruker Avance
II 400 (400 MHz) and 500 (500 MHz) NMR systems. The chemical
shift values are expressed in ppm (parts per million) relative to
tetramethylsilane as an internal standard: s, singlet; d, doublet; t, triplet;
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Journal of Medicinal Chemistry
BRIEF ARTICLE
was cooled and evaporated at reduced pressure. The residue was diluted
with chloroform (50 mL), cooled in an ice bath, and neutralized carefully
with ammonium hydroxide. The organic portion was washed with water
(3 Â 30 mL) and dried with anhydrous sodium sulfate. To this was
added 1 g of silica gel, and concentration of the organic solvent afforded a
dry plug. This plug was placed on the top of a silica gel column, and the
column was eluted with 20% hexane in chloroform. Fractions containing
theproduct(TLC) were pooledandevaporatedtoafford(R)-5[or(S)-5]
as a light yellow liquid. (R)-5 and (S)-5 were unstable and were used for
the next step without further characterization.
President’s Council Research Excellence Award (S.L.M.); CTRC
Cancer Center Support Grant, CCSG (Grant CA054174)
(S.L.M.); and the Duquesne University Adrian Van Kaam Chair
in Scholarly Excellence (A.G.).
’ ABBREVIATIONS USED
CA4, combretastatin A-4; MDR, multidrug resistance; Pgp,
P-glycoprotein; Rr, relative resistance
General Procedure for (R)-1 HCl and (S)-1 HCl. (R)-5 [or
3
3
(S)-5] and N-methyl-4-methoxyaniline were dissolved in isopropanol
(5 mL). To this solution was added 37% hydrochloric acid (2À3 drops).
The mixture was heated at reflux for 3À6 h. Then the mixture was cooled
and the solvent evaporated at reduced pressure. The residue was diluted
with chloroform, neutralized with ammonium hydroxide, and then
washed with water (2 Â 30 mL). After the mixture was dried with
anhydrous sodium sulfate, 1 g of silica gel was added and the solvent
evaporated under reduced pressure to give a dry plug. This plug was
placed on the top of a silica gel column, and the column was eluted with
chloroform. Fractions containing the product (TLC) were pooled and
evaporated to afford pure (R)-1 [or (S)-1] as a liquid. (R)-1 [or (S)-1]
was dissolved in anhydrous ether (10 mL), and anhydrous hydrochloric
acid gas was bubbled into the solution until no further solid precipitated.
The white solid was collected by filtration and dried over P2O5 to afford
(R)-1 HCl [or (S)-1 HCl].
’ REFERENCES
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Risinger, A. L.; Hamel, E.; Mooberry, S. L. Corrections to Synthesis and
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3
3
(6R)-N-(4-Methoxyphenyl)-N,2,6-trimethyl-6,7-dihydro-5H-
cyclopenta[d]pyrimidin-4-aminium [(R)-1 HCl]. (R)-1 HCl
3
3
(0.22 g, 42%) was synthesized from (R)-4 (0.3 g, 1.16 mmol) using
the general procedure described above: mp 196.6À197.4 °C; 1H NMR
(DMSO-d6): δ 0.87 (d, J = 6.8 Hz, 3H), 1.43 (m, 1H), 1.95 (m, 1H),
2.30 (m, 1H), 2.46 (m, 1H), 2.62 (s, 3H), 3.04 (m, 1H), 3.52 (s, 3H),
3.81 (s, 3H), 7.04 (d, J = 8.9 Hz, 2H), 7.35 (d, J = 8.8 Hz, 2H), 15.12 (br,
1H, exch). Anal. (C17H22N3OCl 0.2H2O) C, H, N, Cl.
3
(6S)-N-(4-Methoxyphenyl)-N,2,6-trimethyl-6,7-dihydro-5H-
cyclopenta[d]pyrimidin-4-aminium [(S)-1 HCl]. (S)-1 HCl
3
3
(0.12 g, 36%) was synthesized from (S)-4 (0.2 g, 6.24 mmol) using
the general procedure described above: mp 196.7À197.6 °C; 1H
NMR (DMSO-d6): δ 0.87 (d, J = 6.8 Hz, 3H), 1.43 (m, 1H), 1.95 (m,
1H), 2.31 (m, 1H), 2.46 (m, 1H), 2.62 (s, 3H), 3.04 (m, 1H), 3.52 (s,
3H), 3.81 (s, 3H), 7.04 (d, J = 8.9 Hz, 2H), 7.35 (d, J = 8.8 Hz, 2H),
15.10 (br, 1H, exch). Anal. (C17H22N3OCl) C, H, N, Cl.
’ ASSOCIATED CONTENT
S
Supporting Information. Flow cytometry data, NCI 60
b
(9) Mozzetti, S.; Ferlini, C.; Concolino, P.; Filippetti, F.; Raspaglio,
G.; Prislei, S.; Gallo, D.; Martinelli, E.; Ranelletti, F. O.; Ferrandina, G.;
Scambia, G. Class III β-Tubulin Overexpression Is a Prominent
Mechanism of Paclitaxel Resistance in Ovarian Cancer Patients. Clin.
Cancer Res. 2005, 11, 298–305.
cancer cell line screen, Table 3, and elemental analysis results.
This material is available free of charge via the Internet at http://
pubs.acs.org.
(10) Ferrandina, G.; Zannoni, G. F.; Martinelli, E.; Paglia, A.;
Gallotta, V.; Mozzetti, S.; Scambia, G.; Ferlini, C. Class III β-Tubulin
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’ AUTHOR INFORMATION
Corresponding Author
*For A.G.: phone, 412-396-6070; fax, 412-396-5593,; e-mail,
gangjee@duq.edu. For S.L.M.: phone, 210-567-4788; fax, 210-
567-4300; e-mail, mooberry@uthscsa.edu.
Author Contributions
These authors contributed equally to this work.
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’ ACKNOWLEDGMENT
This work was supported in part by the National Institutes of
Health, National Cancer Institute Grant CA114021 (A.G.); a
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