Novel water-soluble substituted pyrrolo[3,2-d]pyrimidines: Design, synthesis, and biological evaluation as antitubulin antitumor agents

Article abstract of DOI:10.1007/s11095-012-0816-3

Purpose: To study the effects of a regioisomeric change on the biological activities of previously reported water soluble, colchicine site binding, microtubule depolymerizing agents. Methods: Nine pyrrolo[3,2-d]pyrimidines were designed and synthesized. The importance of various substituents was evaluated. Their abilities to cause cellular microtubule depolymerization, inhibit proliferation of MDA-MB-435 tumor cells and inhibit colchicine binding to tubulin were studied. One of the compounds was also evaluated in the National Cancer Institute preclinical 60 cell line panel. Results: Pyrrolo[3,2-d] pyrimidine analogs were more potent than their pyrrolo[2,3-d]pyrimidine regioisomers. We identified compounds with submicromolar potency against cellular proliferation. The structure-activity relationship study gave insight into substituents that were crucial for activity and those that improved activity. The compound tested in the NCI 60 cell line is a 2-digit nanomolar (GI₅₀) inhibitor of 8 tumor cell lines. Conclusion: We have identified substituted pyrrolo[3,2-d]pyrimidines that are water-soluble colchicine site microtubule depolymerizing agents. These compounds serve as leads for further optimization.

Full text of DOI:10.1007/s11095-012-0816-3

Pharm Res
DOI 10.1007/s11095-012-0816-3
RESEARCH PAPER
Novel Water-Soluble Substituted Pyrrolo[3,2-d]pyrimidines:
Design, Synthesis, and Biological Evaluation as Antitubulin
Antitumor Agents
Aleem Gangjee & Roheeth K. Pavana & Wei Li & Ernest Hamel & Cara Westbrook & Susan L. Mooberry
Received: 30 March 2012 /Accepted: 20 June 2012
#
Springer Science+Business Media, LLC 2012
. .
KEY WORDS antitubulin colchicine-site binders drug
design microtubule depolymerizer pyrrolo[3,2-d]pyrimidines
ABSTRACT
.
.
Purpose To study the effects of a regioisomeric change on
the biological activities of previously reported water solu-
ble, colchicine site binding, microtubule depolymerizing
agents.
Methods Nine pyrrolo[3,2-d]pyrimidines were designed and
synthesized. The importance of various substituents was eval-
uated. Their abilities to cause cellular microtubule depolymeri-
zation, inhibit proliferation of MDA-MB-435 tumor cells and
inhibit colchicine binding to tubulin were studied. One of the
compounds was also evaluated in the National Cancer Institute
preclinical 60 cell line panel.
Results Pyrrolo[3,2-d]pyrimidine analogs were more potent
than their pyrrolo[2,3-d]pyrimidine regioisomers. We identified
compounds with submicromolar potency against cellular
proliferation. The structure-activity relationship study gave
insight into substituents that were crucial for activity and
those that improved activity. The compound tested in the
NCI 60 cell line is a 2-digit nanomolar (GI50) inhibitor of 8
tumor cell lines.
INTRODUCTION
Our efforts to elucidate the plausible binding modes of RTK-
inhibitors led to the discovery of highly potent water-soluble
antimitotic antitproliferative agents: N-(4-methoxyphenyl)-N, 2-
dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine 1 and N-(4-
methoxyphenyl)-N,2,6-trimethyl-6,7-dihydro-5H-cyclopen-
ta[d]pyrimidin-4-amine ( )-2•HCl (Fig. 1) (1,2). These com-
pounds bind in the colchicine site of tubulin, inhibit
microtubule assembly and cause cellular microtubule disassem-
bly. Compounds 1 and ( )-2•HCl also inhibit the growth of
cancer cells with GI50’s in the nanomolar range. They over-
come the two most clinically relevant tumor resistance mecha-
nisms that limit the utility of microtubule targeting agents (1,2):
overexpression of P-glycoprotein (3,4) and βIII-tubulin (5–9).
Pyrrolo[3,2-d]pyrimidine analogs of the lead compounds
were designed to evaluate the effect of intramolecular
hydrogen bonding (if any) mediated conformational restric-
tion of the lead analogs 1 and ( )-2•HCl.
Conclusion We have identified substituted pyrrolo[3,2-d]pyr-
imidines that are water-soluble colchicine site microtubule
depolymerizing agents. These compounds serve as leads for
further optimization.
:
:
:
C. Westbrook S. L. Mooberry
A. Gangjee (*) R. K. Pavana W. Li
Division of Medicinal Chemistry
Department of Pharmacology
University of Texas Health Science Center at San Antonio
San Antonio, Texas 78229, USA
Graduate School of Pharmaceutical Sciences, Duquesne University
Pittsburgh, Pennsylvania 15282, USA
e-mail: gangjee@duq.edu
S. L. Mooberry
Cancer Therapy & Research Center
University of Texas Health Science Center at San Antonio
San Antonio, Texas 78229, USA
E. Hamel
Screening Technologies Branch
Developmental Therapeutics Program
Division of Cancer Treatment & Diagnosis
Frederick National Laboratory for Cancer Research
National Cancer Institute
Frederick, Maryland 21702, USA

Gangjee et al.
Fig. 1 Lead compounds.
OCH3
OCH3
CH3
4'
4'
OCH3
4'
1'
1'
1'
H3C
H3C
H3C
N
4
N
4
N
4
5
H
N 5
6
5
N
N
2
6
N
2
6
2
N 7
H
H3C
N
H3C
N
7
7
H3C
N
1
1
1
1
2
( )- HCl
3
Compound 3 was designed as a regioisomer of the pyr-
rolo[2,3-d]pyrimidine 1 and as an isostere of the cyclopen-
ta[d]-pyrimidine ( )-2•HCl scaffold. Compounds 4, 5 and 6
were designed to evaluate the importance of the 2-CH3, 4'-
OCH3 and 4-NCH3 moieties, respectively. Compound 7
was designed by incorporating a 2'-OCH3 group to explore
the effect of substituents in this position. Compounds 8 and
9 were synthesized to study the effect of a 6-CH3 group and
a 2-NH2 group, respectively. Compound 10 was designed to
evaluate the importance of the 4-NCH3 moiety. Compound
11 was designed as a conformationally restricted analog of
3. Figure 2 summarizes the structures of compounds 3–11.
Compounds 12 (10) and 13 (11) (Scheme 1) were chlori-
nated with phosphorus oxychloride to generate 14 and 15,
respectively. Compounds 14, 16 (12) and 17 (13) were
subjected to nucleophilic displacement reactions using
appropriately substituted anilines to afford 3–8 and 11,
which were in turn converted to their hydrochloride salts
using hydrogen chloride gas. Compound 15 was subjected
to nucleophilic displacement reactions using appropriately
substituted anilines, and the resulting compounds were sub-
sequently subjected to amide hydrolysis under basic condi-
tions to produce 9 and 10.
per million (ppm) relative to tetramethylsilane as an internal
standard: s, singlet; d, doublet; t, triplet; q, quartet; m, mul-
tiplet; br, broad singlet. Thin-layer chromatography (TLC)
was performed using Whatman Sil G/UV254 silica gel plates
with a fluorescent indicator, and the spots were visualized
under 254 and 365 nm illumination.
Proportions of solvents used for TLC are by volume. Col-
umn chromatography was performed with a 230–400 mesh
silica gel (Fisher, Somerville, NJ) column. Elemental analyses
were performed by Atlantic Microlab, Inc., Norcross, GA.
Element compositions are within 0.4% of the calculated values.
Fractional moles of water or organic solvents found in some
analytical samples of the compounds could not be prevented in
spite of 24–48 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 Aldrich Chemical Co. or
Fisher Scientific and were used as received.
4-Chloro-2-methyl-5H-pyrrolo[3,2-d]pyrimidine (14)
2-Methyl-3H-pyrrolo[3,2-d]pyrimidin-4(5H)-one (10) (12,
1 g, 6.7 mmol) was added to phosphorus oxychloride
(20 mL) and heated at reflux for 4 h. The solvent was
evaporated in vacuo, and the pH of the residue was adjusted
to 8 with ammonia solution. The resulting precipitate was
filtered and purified by column chromatography (CHCl3:
MeOH; 100:1 to 50:3; v/v) to give a light yellow solid
(726 mg, 75%). TLC Rf 0.32 (MeOH: CHCl3; 1:10); Mp,
MATERIALS AND METHODS
Chemistry
1
138–139°C; H NMR, DMSO-d6: δ 2.60 (s, 3 H), 6.6 (s,
1 H), 7.9 (s, 1 H), 12.26(s, 1 H).
Analytical samples were dried in vacuo (0.2 mm Hg) in a
CHEM-DRY drying apparatus over P2O5 at 80°C. Melting
points were determined on a MEL-TEMP II melting point
apparatus with a FLUKE 51 K/J electronic thermometer and
are uncorrected. Proton nuclear magnetic resonance spectra
(1H NMR) were recorded on a Bruker WH-400 (400 MHz)
spectrometer. The chemical shift values are expressed in parts
N-(4-Chloro-5H-pyrrolo[3,2-d]pyrimidin-2-yl)pivalamide (15)
N-(4-Oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-2-yl)piv-
alamide (11) (13, 1.16 g, 4.67 mmol) was added to phos-
phorus oxychloride (30 mL) and heated at reflux for 3 h.
Fig. 2 Designed compounds.

Novel Water Soluble, Substituted Pyrrolo[3,2-d]pyrimidines
Scheme 1 Synthesis of target
compounds.
The solvent was evaporated in vacuo, and the pH of the
residue was adjusted to 8 with ammonia solution. The
resulting precipitate was filtered and purified by column
chromatography (CHCl3: MeOH; 50:1; v/v) to give a white
solid (1.1 g, 86%). TLC Rf 0.35 (MeOH: CHCl3; 1:5); Mp,
6.55 (s, H), 7.1 (d, 2 H, J08.8 Hz), 7.4 (d, 2 H, J08.7 Hz),
7.56 (s, 1 H), 8.71 (s, 1 H), 9.96 (s, 1 H), 14.39 (s, 1 H). Anal.
Calcd. for C14H14N4O∙HCl∙H2O: C, 54.46; H, 5.55; N,
18.15; Cl, 11.48. Found C, 54.21; H, 5.46; N, 17.9; Cl,
11.26.
1
162–163°C; H NMR, DMSO-d6: δ 1.19(s, 9 H), 2.47 (s,
3 H), 6.33 (s, 1 H), 9.85 (s, 1 H), 12.07 (s, 1 H). Anal. Calcd.
for C12H15ClN4O: C, 54.04; H, 5.67; N, 21.01; Cl, 13.29.
Found C, 54.06; H, 5.78; N, 20.93; Cl, 13.49.
N,2-Dimethyl-N-phenyl-5H-pyrrolo[3,2-d]pyrimidin-4-amine
(5) as Hydrochloride Salt
Compound 5 (synthesized from 14 as described for 3): TLC
1
Rf 0.42 (MeOH: CHCl3; 1:5); Mp, 253–254°C; H NMR,
N-(4-Methoxyphenyl)-N,2-dimethyl-5H-pyrrolo[3,2-d]
pyrimidin-4-amine (3) as Hydrochloride Salt
DMSO-d6: δ 2.62 (s, 3 H), 3.7 (s, 3 H), 6.49 (s, H), 7.47–7.59
(m, 6 H), 10.07 (s, 1 H), 15.02 (s, 1 H). Anal. Calcd. for
C15H16N4O∙0.95 HCl∙0.25H2O: C, 60.61; H, 5.61; N,
20.1. Found C, 60.59; H, 5.6; N, 20.00.
Compound 14 (0.2 g, 1.05 mmol) and 4-methoxy-
phenylamine (0.12 g, 1.05 mmol) were dissolved in isopro-
panol (20 mL) and heated at reflux for 4 h. The solvent was
evaporated in vacuo, and the residue was purified by col-
umn chromatography (CHCl3: MeOH; 50:1; v/v) to give a
brown solid (1.1 g, 86%). TLC Rf 0.42 (MeOH: CHCl3;
1:10). The product obtained was dissolved in a minimum
amount of ethyl acetate, and diethyl ether (10 mL) was
added to the solution. Hydrogen chloride gas was bubbled
through for 2–3 mins. The precipitate obtained was col-
N-(4-Methoxyphenyl)-2-methyl-5H-pyrrolo[3,2-d]
pyrimidin-4-amine (6) as Hydrochloride Salt
Compound 6 (synthesized from 14 as described for 3): yield
81%; TLC Rf 0.4 (MeOH: CHCl3; 1:5); Mp, 260°C (decomp);
1H NMR, DMSO-d6: δ 2.61 (s, 3 H), 3.77 (s, 3 H), 6.51 (s, H),
7.0 (d, 2 H, J09 Hz), 7.82 (d, 2 H, J08.8 Hz), 7.89 (s, 1 H),
11.43 (s, 1 H), 12.94 (s, 1 H), 14.44 (s, 1 H). Anal. Calcd. For
C14H14N4O∙HCl: C, 57.83; H, 5.20; N, 19.27; Cl, 12.19.
Found C, 57.73; H, 5.38; N, 19.05; Cl, 12.07.
1
lected by filtration. Mp, 184–185°C; H NMR, DMSO-d6:
δ 2.63 (s, 3 H), 3.62 (s, 3 H), 3.84 (s, 3 H), 6.46 (s, H), 7.1 (d,
2 H, J08.9 Hz), 7.42 (d, 2 H, J08.86 Hz), 9.661 (s, 1 H),
14.996 (s, 1 H). Anal. Calcd. for C15H16N4O∙HCl∙H2O: C,
55.81; H, 5.93; N, 17.36; Cl, 10.98. Found C, 55.94; H,
5.87; N, 17.17; Cl, 10.97.
N-(2,4-Dimethoxyphenyl)-2-methyl-5H-pyrrolo[3,2-d]
pyrimidin-4-amine (7) as Hydrochloride Salt
N-(4-Methoxyphenyl)-N-methyl-5H-pyrrolo[3,2-d]
pyrimidin-4-amine (4) as Hydrochloride Salt
Compound 7 (synthesized from 14 as described for 3): yield
76%; TLC Rf 0.6 (MeOH: CHCl3; 1:10); Mp, 262–264°C;
1H NMR, DMSO-d6: δ 2.5 (s, 3 H), 3.8 (s, 6 H), 6.5 (m,
1 H), 6.59 (dd, 1 H, J08.7, 2.6), 6.7 (d, 1 H, J02.6 Hz), 7.48
(d, 1 H, J08.7 Hz), 7.85 (s, 1 H), 10.4 (s, 1 H), 12.65 (s, 1 H),
14.86 (s, 1 H). Anal. Calcd. for C15H16N4O2∙HCl∙0.2H2O:
Compound 4 (synthesized from 16 (12) as described for 3):
yield 72%; TLC Rf 0.33 (MeOH: CHCl3; 1:5); Mp, 150–
1
151°C; H NMR, DMSO-d6 δ 3.65 (s, 3 H), 3.84 (s, 3 H),

Gangjee et al.
C, 55.54; H, 5.41; N, 17.27; Cl, 10.93. Found C, 55.74; H,
5.28; N, 17.21; Cl, 10.85.
2.79 (t, 2 H, J06.5 Hz), 3.8 (s, 3 H), 4.1 (t, 2 H, J06.3 Hz),
6.53 (m, 1 H, J08.7, 2.6), 6.8 (dd, 1 H, J08.8, 2.9), 6.94 (d,
1 H, J02.8 Hz), 7.15 (d, 1 H, J08.8 Hz), 7.65 (t, 1 H, J0
2 . 7 H z ) , 1 1 . 0 5 ( s , 1 H ) , 1 4 . 8 5 ( s , 1 H ) .
C16H18N4O∙HCl∙0.2H2O: C, 59.61; H, 6.06; N, 17.38;
Cl, 11.00. Found C, 59.82; H, 6.05; N, 17.25; Cl, 10.85.
N-(4-Methoxyphenyl)-N,2,6-trimethyl-5H-pyrrolo[3,2-d]
pyrimidin-4-amine (8) as Hydrochloride Salt
Compound 8 (synthesized from 17 (13) as described for 3):
yield 69%; TLC Rf 0.3 (MeOH: CHCl3; 1:10); Mp, 224–
226°C; H NMR, DMSO-d6: δ 2.38 (s, 3 H), 2.55 (s, 3 H),
Biological Evaluations
1
3.68 (s, 3 H), 3.83 (s, 3 H), 6.32 (s, H), 7.07 (d, 2 H, J0
8.9 Hz), 7.36 (d, 2 H, J08.9 Hz), 10.31 (s, 1 H), 14.55 (s,
1 H). Anal. Calcd. for C17H18N4O∙HCl∙ 0.25 H2O: C,
60.82; H, 5.86; N, 16.71; Cl, 10.57. Found C, 60.61; H,
5.91; N, 16.56; Cl, 10.80.
The effects of the compounds on interphase and mitotic
microtubules in A-10 cells were evaluated using indirect
immunofluorescence techniques, and the EC50 values (con-
centration required to cause 50% loss of cellular micro-
tubules) were calculated from a minimum of three
experiments as previously described (14).
Antiproliferative effects were evaluated against the drug
sensitive MDA-MB-435 melanoma cells using sulforhod-
amine B assay as previously described, and the IC50 values
(concentration required to cause 50% inhibition of prolifer-
ation) were calculated (15–17).
N4-(4-Methoxyphenyl)-N4,6-dimethyl-5H-pyrrolo[3,2-d]
pyrimidine-2,4-diamine (9)
Compound 15 (0.2 g, 1.05 mmol) and 4-methoxy-
phenylamine (0.12 g, 1.05 mmol) were dissolved in isopro-
panol (20 mL), followed by the addition of 2–3 drops of
conc. HCl. The mixture was heated at reflux for 45 min.
The solvent was evaporated in vacuo, 1,4-dioxane (10 mL)
and 15% aqueous KOH solution (10 mL) were added. The
resulting mixture was heated at reflux overnight. After cool-
ing, the reaction solution was neutralized with 1 N HCl, and
evaporated in vacuo to dryness and the residue was purified
by column chromatography (CHCl3: MeOH; 50:1; v/v) to
give a brown solid (1.1 g, 86%). TLC Rf 0.42 (MeOH:
The inhibition of tubulin assembly by these compounds
was studied. Tubulin polymerization was measured by
turbidimetry at 350 nm in Beckman DU7400 and
DU7500 recording spectrophotometers equipped with tem-
perature controllers. The methodology was described in
detail previously (18). In brief, 10 μM bovine brain tubulin,
purified as described previously (19), was preincubated for
15 min in a 0.24 mL volume at 30°C containing 0.75 M
monosodium glutamate (adjusted to pH 6.6 with HCl in a
2 M stock solution), varying compound concentrations, and
4% (v/v) dimethyl sulfoxide (compound solvent). Following
the preincubation, which permits detection of activity in
slow binding compounds such as colchicinoids (18), samples
were chilled on ice, and 10 μL of 10 mM GTP was added
(0.4 mM). The addition of GTP is an absolute requirement
for assembly under these reaction conditions. All concen-
trations refer to the final 0.25 mL reaction volume. Samples
were transferred to cuvettes held at 0°C in the recording
spectrophotometers, and the temperature was jumped to
30°C, which takes less than a minute. Assembly at 30°C
was followed for 20 min, and the compound concentration
required to inhibit extent of assembly after 20 min was
determined by interpolation of data obtained with individ-
ual compound concentrations. After determining the likely
range for the IC50 value, 2–4 individual determinations
were made, and the average from these determinations are
presented in Table I. The control compound was combre-
tastatin A-4 (CSA4), a potent colchicine site agent (20)
generously supplied by Dr. G. R. Pettit, Arizona State
University, Tempe AZ.
1
CHCl3; 1:5); Mp, 201–202°C; H NMR, DMSO-d6: δ
2.34 (s, 3 H), 3.78 (s, 3 H), 5.33 (s, 2 H), 5.75 (s, 1 H),
6.86 (d, 2 H, J06.3 Hz), 7.72 (d, 2 H, J06.3 Hz) 8.45 (s,
1
H ) , 8 . 5 2 ( s , 1 H ) . A n a l . C a l c d . f o r
C14H15N5O∙0.71CHCl3∙0.81HCl: C, 45.93; H, 4.33; N,
18.19. Found C, 45.97; H, 4.53; N, 18.08.
N4-(4-Methoxyphenyl)-6-methyl-5H-pyrrolo[3,2-d]
pyrimidine-2,4-diamine (10)
Compound 10 (synthesized from 15 as described for 9):
yield 47%; TLC Rf 0.48 (MeOH: CHCl3; 1:5); Mp, 161–
1
163°C; H NMR, DMSO-d6: δ 2.12 (s, 3 H), 3.37 (s, 3 H),
3.78 (s, 3 H), 5.31 (s, 2 H), 5.70 (s, 1 H), 6.96 (d, 2 H, J0
5.4 Hz), 7.15 (d, 2 H, J05.4 Hz), 8.16 (s, 1 H). Anal. Calcd.
for C15H17N5O∙0.78H2O: C, 60.55; H, 6.20; N, 23.54.
Found C, 60.54; H, 5.94; N, 23.51.
6-Methoxy-1-(2-methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)-
1,2,3,4-tetrahydroquinoline (11) as Hydrochloride Salt
Compound 11 (synthesized from 14 as described for 3):
yield 79%; TLC Rf 0.36 (MeOH: CHCl3; 1:10); Mp, 230–
232°C; 1H NMR, DMSO-d6: δ 2.01 (m, 2 H), 2.63 (s, 3 H),
The abilities of these compounds to inhibit binding of
radiolabeled colchicine to tubulin were measured. The
binding of [3H]colchicine to tubulin was performed by the

Novel Water Soluble, Substituted Pyrrolo[3,2-d]pyrimidines
Table I Effects in Cellular Assays and on Purified Tubulin
EC50 Microtubule
MDA-MB-435
Inhibition of
Depolymerization (μM)
IC50 SD (nM)
Tubulin Assembly
% Colchicine Binding inhibited
IC50 SD (μM)
at 5 μM compound concentration
1
5.8
183 3.4
96.6 5.3
193 5.3
ND
3•HCl
4•HCl
5•HCl
6·HCl
7•HCl
8•HCl
9
1.2
10 0.6
1.4
Not Active Up To 10 μM
Not Active Up To 10 μM
ND
Not Active Up To 10 μM
ND
0.22
30.3 2.7
298 19.7
ND
3.1 0.3
65
62
1
2
8.4
10
Not Active Up To 40 μM
11•HCl
CSA4
0.23
42.7 3.2
3.47 0.6
3.1 0.08
1.2 0.01
0.0131
98 0.3
DEAE-cellulose filter technique (21) with a stack of two
filters, as described in detail previously (22). In brief, reac-
tion mixtures contained, in a 0.10 mL volume, 1.0 μM
purified tubulin, 5.0 μM [3H]colchicine, potential inhibitor
at 5.0 μM, 5% (v/v) dimethyl sulfoxide (the compound
solvent), and other components previously found to stabilize
the colchicine binding activity of tubulin for prolonged
periods at 37°C (23). Incubation was at 37°C for 10 min,
at which time samples were diluted with 2 mL of ice-cold
water and poured over the DEAE-filters under mild suction,
with several rinses of the reaction vessel and of the filtration
chamber. The amount of radiolabel bound to the filters was
determined by liquid scintillation counting, and samples
containing test compounds were compared to reaction mix-
tures without compound. The percent inhibition relative to
the control was determined for each compound in 2–4
independent experiments.
Compound 3 was evaluated in the National Cancer
Institute preclinical 60 cell line panel, as described in detail
previously (24).
Table II Tumor Cell Growth Inhibitory Activity GI50 (10−8 M) of 3•HCl in NCI 60 Cell Line Panel
Panel/Cell line
GI50
Panel/Cell line
GI50
Panel/Cell line
GI50
Panel/Cell line
GI50
Leukemia
CCRF-CEM
HL-60(TB)
K-562
3•HCl
33.9
21.7
8.69
40.2
30.1
4.47
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
3•HCl
13.4
12.9
29.2
36.3
18.9
7.56
16.5
Melanoma
LOX IMVI
MALME-3 M
M14
3•HCl
51.2
13.6
16.7
2.79
18.5
405
Renal Cancer
786-0
3•HCl
44.6
4.45
71.2
37.2
9.65
86.3
56.6
43.5
A498
ACHN
MOLT-4
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROVI
CAKI-1
RPMI-8226
SR
RXF 393
KM12
SN12C
NSCLC
A549/ATCC
EKVX
SW-620
CNS Cancer
SF-268
17.1
37.0
47.4
TK10
27.1
35.2
33.7
295
UO-31
58.6
13.0
25.3
48.9
24.1
26.0
Prostate Cancer
PC-3
HOP-62
SF-295
25.9
21.2
HOP-92
SF-539
29.5
16.0
252
DU-145
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
55.9
32.8
37.2
19.1
4.56
SNB-19
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Breast Cancer
MCF7
SNB-75
8.67
46.6
17.9
24.7
11.6
U251
45.4
39.2
66.9
23.3
MDA-MB-231/ATCC
HS 578 T
BT-549
MDA-MB-468

Gangjee et al.
RESULTS AND DISCUSSION
overcome two clinically important tumor resistance
mechanisms that limit the activity of microtubule targeting
agents, expression of P-glycoprotein and βIII tubulin (1,2).
Compounds 3, 8 and 11 serve as leads for further structural
modifications to potentially identify a clinical lead
candidate.
All target compounds were synthesized in yields varying
from 47% to 86%, and effects on cellular microtubule
depolymerization in A-10 cells was used as an initial screen-
ing assay (Table I). In addition, selected compounds were
evaluated for cytotoxicity in a human melanoma cancer cell
line (MDA-MB-435 cells), and the two most active com-
pounds (8•HCl and 11•HCl) were evaluated for interactions
with purified tubulin (inhibition of assembly and inhibition
of colchicine binding). Compound 3•HCl was evaluated in
the National Cancer Institute preclinical 60 cancer cell line
screen. The GI50 against different cell lines is shown in
Table II, and the mean GI50 in the cell screen was 44.1×
ACKNOWLEDGMENTS AND DISCLOSURES
National Cancer Institute for performing the in vitro anti-
tumor evaluation in their 60 tumor preclinical screening
program.
Grant from the National Institute of Health, National Cancer
Institute, CA142868 (AG,SLM).
NSF equipment grant for the NMR (NMR: CHE 0614785)
10−8 M, with eight cell lines showing less than 10×10−8
M
GI50 values and three cell lines showing micromolar GI50
values. These data indicate that 3•HCl has varied GI50
values across the 60 tumor cell line panel as well as within
a particular tumor type, indicating that the compound does
have selectivity for certain tumor cells in culture and is not a
general cell-poison.
and the CTRC Cancer Center Support Grant, P30
CA054174
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