Discovery of novel antitumor antimitotic agents that also reverse tumor resistance

Article abstract of DOI:10.1021/jm070194u

We have discovered a novel series of 7-benzyl-4-methyl-5-[(2-substituted phenyl)ethyl]-7H-pyrrolo[2,3-d]-pyrimidin-2-amines, which possess antimitotic and antitumor activities against antimitotic-sensitive as well as resistant tumor cells. These agents bi

Full text of DOI:10.1021/jm070194u

3290
J. Med. Chem. 2007, 50, 3290-3301
Discovery of Novel Antitumor Antimitotic Agents That Also Reverse Tumor Resistance1
Aleem Gangjee,*,† Jianming Yu,† Jean E. Copper,‡ and Charles D. Smith‡
DiVision of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne UniVersity, Pittsburgh, PennsylVania 15282, and
Department of Pharmaceutical Sciences, College of Pharmacy, Medical UniVersity of South Carolina, Charleston, South Carolina 29425
ReceiVed February 20, 2007
We have discovered a novel series of 7-benzyl-4-methyl-5-[(2-substituted phenyl)ethyl]-7H-pyrrolo[2,3-d]-
pyrimidin-2-amines, which possess antimitotic and antitumor activities against antimitotic-sensitive as well
as resistant tumor cells. These agents bind to a site on tubulin that is distinct from the colchicine, vinca
alkaloid, and paclitaxel binding sites and some, in addition to their antitumor activity, remarkably also
reverse tumor resistance to antimitotic agents mediated via the P-glycoprotein efflux pump. The compounds
were synthesized from N-(7-benzyl-5-ethynyl-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-2,2-dimethylpro-
panamide 11 or the corresponding 5-iodo analog 14 via Sonogashira couplings with appropriate iodobenzenes
or phenylacetylene followed by reduction and deprotection to afford the target analogs. Sodium and liquid
NH3 afforded the debenzylated analogs. The most potent analog 1 was one to three digit nanomolar against
the growth of both sensitive and resistant tumor cells in culture. Compounds of this series are promising
novel antimitotic agents that have the potential for treating both sensitive and resistant tumors.
Introduction
taxanes are clinically useful in the treatment of breast, lung,
ovarian, head and neck, and bladder carcinomas among others.
The third class typified by colchicine (Chart 1) is comprised of
a diverse collection of small molecules that bind to the
colchicine binding site. The nature of this binding site has not
been determined with certainty, however, insights into the
colchicine binding sites are available from homology models.7
These compounds are microtubule polymerization inhibitors
similar to the Vinca alkaloids, but their binding is on a different
site and their depolymerization mechanism of action is also
different. Combretastatins (Chart 1) are a class of drugs that
are in clinical trials as antitumor agents that bind to the
colchicine binding site.2 Colchicine itself is not used as an
antitumor agent, but is used in gout. It has also recently been
recognized that some antimitotic agents rapidly shut down
existing tumor vasculature.7,8 This is shown for the taxanes,
colchicine, Vinca alkaloids, as well as combretastatins. This
effect is different from the antiangiogenesis effect of inhibition
of receptor tyrosine kinases (RTKsa).
Multidrug or multiple drug resistance (MDR) is a major
drawback of cancer chemotherapy, including the clinically used
antimitotic agents. These arise from intrinsic or acquired
mechanisms of resistance. A major mechanism of MDR occurs
via an overexpression of energy dependent (ATP), unidirectional
transmembrane efflux pumps. Ultimate failure of chemotherapy
with antimitotic drugs often results due to MDR. P-glycoprotein
(Pgp) is a 170 kDa protein that belongs to the ATP-binding
cassette superfamily of transporters.9-11 A series of homologous
proteins termed multidrug-resistance proteins (MRPs) have also
been reported.12,13 The first MRP termed MRP1 was identified
in a drug resistant lung cancer cell line that does not express
Pgp.14 All these transporters bind drugs within the cell and
release them to the extracellular space using ATP.14,15 Tumor
cells pre-exposed to cytotoxic compounds often overexpress
Microtubules are long, protein polymers that are formed by
R-tubulin and â-tubulin heterodimers. These heterodimers first
form a short microtubule nucleus followed by elongation and
arrangement in the form of tubes. The polymerization of
microtubules occurs via complex polymerization dynamics that
involves GTP hydrolysis to supply energy at the time that
tubulin, with bound GTP, adds to the microtubule ends. In
mitosis, the duplicated chromosomes of cells are divided into
two identical sets prior to division into two daughter cells, and
the polymerization dynamics of microtubules plays a pivotal
role in this process as part of cell replication.2,3 The crucial
involvement of microtubules in mitosis makes them a target
for antitumor agents.
In general, antitumor agents that inhibit the function of
microtubules are known as antimitotic agents. Classification of
antimitotic agents is by their mechanisms of action and their
different binding sites on tubulin. Three distinct classes of
antimitotic agents are identified. The Vinca alkaloids exemplified
by vincristine, vinblastine, vindesine, and vinorelbine (Chart 1).
These are â-tubulin binding agents that interfere with proper
mitotic spindle formation by preventing the normal dynamics
and polymerization of spindle microtubule formation. The Vinca
alkaloids are important in the treatment of leukemias, lympho-
mas, small cell lung cancer, and other cancers.3,4 These agents
are also known as microtubule-destabilizing agents or micro-
tubule polymerization inhibitors or depolymerizers. The second
group are the taxanes exemplified by paclitaxel (Chart 1) and
docetaxel (Chart 1). The binding site for paclitaxel is in the
â-subunit as well, however, its location is different from that
of the Vinca alkaloids. Paclitaxel binds on the inside surface of
the â-subunit in microtubule, as determined in tubulin protofila-
ments in Zn sheets.5,6 Paclitaxel increases microtubule polym-
erization and thus interferes with spindle microtubule dynamics
and prevents the dividing cell from progression. These are
microtubule-stabilizing agents or polymerizing agents. The
a Abbreviations: MDR, multiple drug resistance; Pgp, P-glycoprotein;
MRP, multidrug-resistance proteins; PCC, Pearson correlation coefficients;
SRB, sulforhodamine B; RTK, receptor tyrosine kinase; MTP, microtubule
protein; TLC, thin layer chromatography; NMR, nuclear magnetic resonance;
EGFR, endothelial growth factor receptor; VEGFR, vascular endothelial
growth factor receptor; PDGFR, platelet-derived growth factor receptor;
IGF1-R, insulin growth factor 1-receptor; NCI, National Cancer Institute.
* To whom correspondence should be addressed. Telephone: 412-396-
6070. Fax: 412-396-5593. E-mail: gangjee@duq.edu.
† Duquesne University.
‡ Medical University of South Carolina.
10.1021/jm070194u CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/14/2007

NoVel Antitumor Antimitotic Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3291
Chart 1
these pumps, which allows the cells to manifest resistance in
the presence of the cytotoxic drug. MDR affects cancer patients
with leukemias as well as solid tumors. Overexpression of Pgp
has been reported in a number of tumor types, particularly after
the patient has received chemotherapy, indicating the clinical
importance of Pgp in MDR.10,16-19 In addition, it has also been
reported that Pgp expression may be a prognostic indicator in
certain cancers and is associated with poor response to
chemotherapy.20,21 MRP1 overexpression is not consistently
found in tumors even though it is expressed in a high percentage
of leukemias and solid tumors.22 It has also been shown that
the MRP1 mRNA levels in malignant melanoma,23 acute
lymphocytic leukemia,24 or chronic lymphocytic leukemia31
were not altered by chemotherapy. Thus overexpression of Pgp
appears to be more relevant in the clinical setting than elevation
of MRP1 levels.
have been developed to reverse MDR that occur for chemo-
therapeutic agents in general and include antimitotic inhibitors.
Notable among these are verapamil25 and cyclosporine11,25
(Chart 1) and, more recently, tariquidar26,27 (Chart 1) and
zosuquidar28 (Chart 1), which have improved selectivity. The
variety of structural types of agents that have MDR
inhibitory effects can be found in the comprehensive review
by Avendan˜o and Mene´ndez.29 Though the three-dimensional
(3-D) structure of Pgp is not available at high resolution,
several attempts have been made at determining structure
function relationships of MDR Pgp and predicting Pgp substrate
structure-activity relationships, the most recent of these is
described by Pajeva et al.30 and Gombar et al.,31 respectively.
In addition, structural models have also been proposed based
on the E. coli MsbA lipid transporter.32 Recently, a low-
resolution 3-D structure of mammalian Pgp at 20 Å has been
reported33 and indicates ATP binding causes a conformational
change in the structure, resulting in repacking into three compact
domains.
The clinical significance of Pgp along with its limited
expression in normal tissues makes Pgp a viable target for
inhibition to reverse MDR. Several Pgp inhibitors or modulators

3292 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Gangjee et al.
Scheme 1a
Thus, we synthesized compounds 1-5 (Scheme 1) and 6-10
initially as potential RTK inhibitors.
a Reagents and conditions: (a) substituted iodobenzene, tetrakis(triph-
enylphosphine)palladium(0), triethylamine, Cul, rt, dark, dichloroethane;
(b) 5% Pd/C, 50 psi, H2; (c) 1 N NaOH, MeOH, 80 °C; (d) Na/liq NH3,
-78 °C.
Chemistry
The synthesis of the target compounds 1-10 were ac-
complished as indicated in Schemes 1 and 2. For the synthesis
of 6-9 (Scheme 1), we found that the N7-benzyl group was
necessary to increase the solubility of the intermediates and,
more importantly, facilitated the Sonogashira coupling reaction
with acetylenes. Thus, we started with the N-benzylated analog
1140 (Scheme 1). Compound 11 was coupled using the Sono-
gashira reaction with appropriately substituted iodobenzenes in
the presence of tetrakis(triphenylphosphine)palladium(0) (Pd-
(PPh3)4), copper(I) iodide (CuI), and triethylamine in dichlo-
roethane. The reaction went to completion in 24 h, and the
products 12a-d were isolated in 48-79% yield. Subsequent
catalytic hydrogenation of 12a-d, with 3-4 drops of am-
monium hydroxide (to prevent pyrrole reduction) and 5%
palladium-on-charcoal in a mixture of methanol and dichlo-
romethane (1:1), afforded 13a-d in 67-91% yield. Hydrolysis
with 1 N NaOH at 80 °C for 24 h afforded 1-4 in 79-90%
yield. Debenzylation with sodium in liquid ammonia at -78
°C was found to be optimum and afforded compounds 6-9 in
19-48% yield (Scheme 1).
Scheme 2a
a Reagents and conditions: (a) phenylacetylene, triethylamine, tetrakis(triph-
enylphosphine)palladium(0), Cul, rt, dark, dichloroethane; (b) 5% Pd/C,
50 psi, H2; (c) 1 N NaOH, MeOH, 80 °C; (d) Na/liq NH3, -78 °C.
Though antimitotic agents have shown to be some of the most
successful agents against malignancies, resistance, both intrinsic
and acquired, is of major concern and results in treatment
failures. Thus, new agents that possess antimitotic and antitumor
activities without substrate activity for Pgp are highly coveted
and would be useful antitumor agents as single agents or in
combination with chemotherapeutic agents, including antimitotic
agents.
For the synthesis of the unsubstituted phenyl analogs 5 and
10, compound 1440 was directly coupled with commercially
available phenylacetylene using Pd(PPh3)4, CuI, and triethy-
lamine to afford 15 in 78% yield (Scheme 2). The reaction
sequences described in Scheme 1 were adopted for 15 to 16
and for the target compounds 5 and 10 (Scheme 2).
We have been extensively involved in the synthesis of fused
heterocyclic analogs and the structure-based design of inhibitors
of RTK34,35 and folate metabolizing enzymes.36-38 These studies
have afforded the synthesis of substituted furo[2,3-d]pyrim-
idines, pyrrolo[2,3-d]pyrimidines, and thieno[2,3-d]pyrimidines,
among several other monocyclic, bicyclic, tricyclic, and tetra-
cyclic heterocycles. Our interest in RTK inhibitors and the vast
literature on the design and synthesis of such inhibitors,
including the clinically used agents imatinib39 (Chart 1) and
gefitinib39 (Chart 1), indicated that though the kinase binding
sites all accommodate ATP, there are subtle differences in the
ATP binding sites of the kinases such that, with rational design,
inhibitors of kinases can be obtained with selectivity and
specificity for a particular class and even a specific kinase.34,35,39
We40 had previously synthesized intermediate N-(7-benzyl-5-
ethynyl-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-2,2-dimeth-
ylpropanamide (11) and the corresponding 5-iodo analog (14;
Schemes 1 and 2). Elaborations of 11 and 14 to potential RTK
inhibitors with C5 and N7 substitutions could provide RTK
inhibitors.
Biological Evaluation and Discussion
We discovered that some of the compounds 1-5, which we
had originally synthesized as potential RTK inhibitors, had
antitumor activity against a variety of tumor cells but were not
inhibitors of the RTKs evaluated (EGFR, VEGFR1 and 2,
PDGFR-â, and IGF1-R). In the NCI41 60 cell line panel
compound 1, the 3,4,5-triOMe analog inhibited most of the cell
lines with GI50 values that ranged from single digit nanomolar
to submicromolar (Table 1). Compound 1 had a GI50 of single
or two digit nanomolar activity against 16 tumor cell lines, GI50
of submicromolar levels against 40 of the other tumor cells,
and micromolar levels against three tumor cells. Removal of
the N7-benzyl group of 1 to afford 6 decreased activity by 100
to 10 000-fold against 56 tumor cell lines evaluated (data not
shown), indicating the importance of the N7-benzyl moiety. The
3-OMe analog 3 showed GI50 values in the micromolar range
for 8 of the 56 tumor cell lines and two digit nanomolar against
a renal cancer A498 cell. The 2-OMe compound, 4, was similar

NoVel Antitumor Antimitotic Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3293
Table 1. Tumor Cell Inhibitory Activity GI50 (nM) of Compound 1 (NCI)
panel/cell line
leukemia
GI50
panel/cell line
GI50
panel/cell line
melanoma (ctd.)
GI50
panel/cell line
GI50
colon cancer (ctd.)
renal cancer (ctd.)
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
NSCLC
A549/ATCC
EKVX
58.0
64.7
84.6
285
439
31.4
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
317
286
127
281
194
236
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
381
>1000
RXF 393
SN12C
TK10
134
537
961
347
61.1
80.2
39.9
UO-31
prostate cancer
breast cancer
ovarian cancer
IGROVI
PC-3
DU-145
47.9
224
CNS cancer
61.1
28.8
821
173
424
467
195
278
581
162
47.7
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
669
41.8
74.5
273
358
144
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
MCF7
118
145
595
283
HOP-92
>1000
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
MDA-N
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
364
506
renal cancer
10.1
<10.0
melanoma
786-0
A498
ACHN
CAKI-1
254
136
728
366
LOX IMVI
MALME-3M
M14
250
283
77.0
BT-549
T-47D
>1000
colon cancer
COLO 205
563
190
Table 2. Compare Analysis Data for Compound 1a
Table 3. Cytotoxicity of Compounds 1-5a
correlation
MCF-7
drug
coefficient
cmpd
T24
IC50 (µM)
NCI/ADR
MCF-7/VP
vinblastine sulfate
paclitaxel
rhizoxin
maystansine
vincristine sulfate
0.748
0.680
0.677
0.663
0.635
1
2
3
4
5
0.28 ( 0.10
31.7 ( 1.7
25.0 ( 2.9
2.5 ( 0.3
0.30 ( 0.16
13.0 ( 3.5
8.0 ( 3.5
0.25 ( 0.12
21.7 ( 4.4
5.7 ( 0.7
0.25 ( 0.16
22.7 ( 3.7
22.0 ( 4.2
2.6 ( 0.1
2.3 ( 0.6
2.7 ( 0.4
12.7 ( 2.3
10.7 ( 4.7
18.2 ( 7.4
18.3 ( 6.0
a PCC values calculated by TGI50.
a The IC50 for each compound was determined as described in the
Experimental Section. Values are expressed as µM and represent the mean
( SEM for three experiments.
to 3, with activities (GI50s) in the micromolar to millimolar range
against the tumor cells in culture. The 4-OMe compound 2
inhibited the tumor cells at GI50 concentrations that were similar
to 6, indicating the importance of the 3- and/or 5-OMe groups.
The phenyl unsubstituted analog 5 has GI50 values in the
millimolar or less range for 52 of the tumor cells, with
micromolar GI50 values for three of the tumor cell lines.
Removal of the N7-benzyl group from any of the compounds
substantially decreased activity against the 60 cell line panel.
The tumor cell inhibitory activity from this limited 10 analog
series indicated that the 3,4,5-triOMe substitution along with
the presence of the N7-benzyl moiety afforded the most potent
inhibitory activity against tumor cells in culture.
little effect on the potency toward the other cell lines.
Conversely, 3-OMe substitution (compound 3) enhanced the
potency toward NCI/ADR cells, and a 2-OMe substitution
(compound 4) substantially increased the potency toward all of
the tested cell lines and eliminated any resistance due to
overexpression of either Pgp or MRP1. Submicromolar cytotoxic
potency was observed for the 3,4,5-triOMe compound 1, again
regardless of the transporter status of the cells. The data from
Table 3 indicates that the 3,4,5-triOMe substitution is important
for potent activity. Among the mono-OMe analogs, the 2-OMe
is better than the 3-OMe and the 4-OMe. However, the
unsubstituted phenyl analog is better than a mono-3- or -4-OMe
substitution. Thus, the triOMe analog 1 is 10-fold better than 4
(2-OMe), which in turn is about 10 times better than the 3- or
4-OMe and 5 times better than the unsubstituted phenyl
analog 5.
Because of the effects of the compounds on microtubules
(discussed below), the cytotoxic activity of 1 was compared
with the microtubule-targeting drugs paclitaxel, vinblastine, and
vincristine (Table 4). Although each of these natural product
drugs were more potent than 1 in the sensitive cell line, each
was subject to tumor cell resistance due to the overexpression
of Pgp. Additionally, overexpression of MRP1 (MCF-7/VP) also
confers substantial resistance to vincristine. In contrast, the
observation that neither Pgp nor MRP1 affect cell sensitivity
to 1 was confirmed. Compound 1 was 35- to 40-fold more potent
than the established anticancer drug cisplatin, which is also
insensitive to transporter expression by the tumor cells.
Effects of 1 on Microtubule Structure. The morphological
effects of 1 on cultured cells and the NCI COMPARE analysis
results described above suggested that the effects of vinblastine,
paclitaxel, and 1 on microtubule structure should be examined.
For these studies, A-10 rat smooth muscle cells were used
because they grow as flat monolayers that are amenable to
imaging. None of these compounds affected the morphology
The NCI COMPARE analysis42 was also performed for 1 to
elucidate a possible mechanism of action of 1 by the similarity
responses of the 60 cell lines to known compounds. The five
compounds whose cell type selectivity profile showed the
highest Pearson correlation coefficients (PCC)43 with compound
1 were all well-known microtubule-targeting agents (Table 2).
For microtubule specific compounds, the cell type selectivity
profile in TGI level is highly indicative of the compound’s
mechanism of action. For a compound to be categorized in this
class requires that (1) the PCC values should be at least 0.6
and (2) the average GI50 should be 1 µM or less.44 Compound
1 satisfied both requirements indicating an antitubulin mecha-
nism of action for 1.
Cytotoxicity toward Sensitive and Resistant Tumor Cell
Lines. The series of analogs 1-5 was also evaluated for
cytotoxicity toward human tumor cell lines that included cell
lines resistant to anticancer drugs due to overexpression of the
transport proteins Pgp or MRP1 (Table 3). The unsubstituted
phenyl ethyl compound 5 demonstrated IC50s ranging from 11
to 18 µM. NCI/ADR cells that overexpress Pgp and MCF-7/
VP cells that overexpress MRP1 had IC50s slightly higher than
the drug-sensitive MCF-7 cells. A 4-OMe substitution of the
5-phenylethyl moiety (compound 2) decreased the cytotoxic
potency toward T24 human bladder carcinoma cells but had

3294 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Table 4. Effects of Pgp and MRP1 on Drug Sensitivitya
Gangjee et al.
MCF-7
Pgp-/MRP1
IC50
NCI/ADR
MCF-7/VP
Pgp-/MRP1+
IC50
Pgp+/MRP1
resistance
factor
resistance
factor
drug
IC50
paclitaxel (nM)
vinblastine (nM)
vincristine (nM)
cisplatin (µM)
1 (µM)
2.5 ( 0.6
2.8 ( 0.5
2700 ( 500
>375
1080
>134
167
2.8 ( 0.6
3.0 ( 0.6
9.3 ( 1.4
7.0 ( 2.9
0.18 ( 0.01
1.1
1.1
15.5
0.6
0.6 ( 0.1
100
11.5 ( 4.1
0.28 ( 0.01
12.2 ( 4.2
0.35 ( 0.04
1.1
1.3
0.6
a The IC50 for each compound was determined as described in Experimental Section. Values expressed are the mean ( SEM for three experiments.
Figure 1. Microtubule structure in A-10 cells. A-10 cells were treated for 24 h with EtOH (control), 625 nM paclitaxel, 167 nM vinblastine, or
900 nM 1 (compound 1 in Figure 2). Microtubules were then visualized by indirect immunofluoesence staining with â-tubulin antibodies. Representative
fields are shown.
Table 5. Effects of 1 and Antimicrotubule Drugs on the Cell Cycle
Distribution of HL-60 Cells
treatment
control
paclitaxel (200 nM)
vinblastine (80 nM)
colchicine (600 nM)
1 (6 µM)
G0/G1
S
G2/M
78.5 ( 0.0
67.3 ( 0.4
83.3 ( 1.3
84.1 ( 0.7
80.0 ( 2.4
16.6 ( 0.2
22.2 ( 0.4
6.6 ( 1.6
4.0 ( 1.4
10.3 ( 2.0
4.9 ( 0.2
10.5 ( 0.1
10.1 ( 0.3
11.9 ( 0.3
9.7 ( 0.4
of stress fibers composed of microfilaments (data not shown).
Control cells displayed extensive microtubule systems with
perinuclear organizing centers (Figure 1). Treatment with
paclitaxel promoted increased staining of microtubule clusters
in these cells, while vinblastine caused marked depletion of
cellular microtubules. Similarly, 1 (Figure 1) caused dose-
dependent losses of microtubules in the cells. Like paclitaxel,
many cells with aberrant nuclei were observed in cultures treated
with 1. Similar losses of microtubules were observed in cultures
of NIH/3T3 and MCF-7 cells treated with 1 (data not shown).
Cell Cycle Effects of 1. Flow cytometric analyses were used
to assess the effects of 1 and antimicrotubule drugs on the cell
cycle phase distributions of HL-60 human promyelocytic
leukemia cells. The percentages of cells in the G2/M phases
were increased approximately 2-fold by treatment of the cells
for 24 h with either paclitaxel, vinblastine, colchicine, or 1
(Table 5). Increasing the incubation time to 48 h did not
substantially increase the percentage of cells arrested in G2/M
for any of the compounds; however, the percentage of subdiploid
cells was increased in each sample, indicating the induction of
apoptosis by these compounds (data not shown).
To further characterize the cell cycle effects of 1 on human
tumor cells, MCF-7 cells were treated with 1, vinblastine, or
paclitaxel, and the expression of p53 and WAF1 were examined
by immunoblotting. Arrest of these cells in G2/M by antimi-
crotubule drugs has previously been demonstrated to correlate
with increased expression of these proteins. As predicted,
treatment with paclitaxel or vinblastine resulted in marked
increases in the levels of expression of both p53 and WAF1
(Figure 2). Similarly, treatment with 1 induced the expression
of both of these proteins. None of these treatments affected the
expression of bcl-2 or â-tubulin in the MCF-7 cells (data not
shown).
Figure 2. Expression of p53 and Waf-1 in MCF-7 cells. MCF-7 cells
were treated for 24 h with the indicated concentrations of EtOH
(control), paclitaxel, vinblastine, or 1 (compound 149 in Figure 3). Cells
were then harvested, lysed, and analyzed for levels of expression of
p53 (left) or Waf-1 (right) by Western Blot analyses. Data from one of
two similar experiments is shown.
Effects of Compounds 1-5 on Pgp-Mediated Multiple
Drug Resistance. Studies described above in Tables 3 and 4
indicated that expression of Pgp or MRP1 does not confer
resistance to the cytotoxic effects of the compounds. To
determine if this is due to inhibition of drug transport activity
by these proteins, the effects of these compounds on Pgp were
further characterized. The effects of the compounds on the
sensitivity of NCI/ADR cells to vinblastine and of MCF-7/VP
cells to vincristine were examined to evaluate antagonism of
Pgp and MRP1, respectively. As indicated in Figure 3 (left
panel), increasing doses of either 2, 3, 4, or 5 caused dose-
dependent sensitization of NCI/ADR cells to vinblastine,
indicating inhibition of Pgp. Compound 4 was approximately
10-fold more potent than the others in reversing Pgp-mediated
resistance. Apparent decreases in this activity by high doses of
the compounds are due to direct cytotoxicity of the compound,
thereby lowering the ratio of survival in the absence of
vinblastine relative to survival in the presence of vinblastine.
Compound 1 was significantly more cytotoxic than the others
in the series, but had no affect on Pgp at doses below its IC50.
In contrast with the marked effects on Pgp-mediated resistance,
none of the compounds affected the ability of vincristine to kill
MCF-7/VP cells, indicating that they are ineffective in blocking
the actions of MRP1. Overall, these data demonstrate that ortho-
OMe substitution of the 5-phenyl ethyl moiety (compound 4)
of this series greatly enhances its ability to inhibit Pgp, whereas
further substitution (3,4,5-triOMe in 1) may negate this effect

NoVel Antitumor Antimitotic Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3295
Figure 3. Compounds 1-5 reverse Pgp-mediated drug resistance but not MRP1-mediated drug resistance. Left panel: NCI/ADR cells were treated
with the indicated concentrations of 5 (9), 2 (2), 3 (1), 4 ((), or 1 (b) either in the absence or in the presence of 50 nM vinblastine. The Pgp
antagonism score is calculated as the percentage survival of cells in the absence of vinblastine/percentage survival in the presence of vinblastine.
Right panel: MCF-7/VP cells were treated as indicated above in the absence or presence of 1 nM vincristine. The MRP1 antagonism score is
calculated as the percentage survival of cells in the absence of vincristine/percentage survival in the presence of vincristine. In both cases, a value
of 1 indicates that the compound has no affect on drug sensitivity, while increasing values indicate increasing antagonism of the transport protein.
Concentrations of compounds 1-5 above their IC50 toward MCF-7 cells are not indicated. Data for one of three similar experiments are shown.
the formation of these paracrystals.45 The effects of 1 on
vinblastine-tubulin paracrystal formation in aortic smooth muscle
cells (A-10) were examined. As demonstrated in Figure 5, these
cells have dense, well-organized microtubules and an elongated
morphology (panel A). Treatment of the cells with 10 µM 1
caused pronounced depolymerization of the microtubules (panel
B). Vinblastine (5 µM) also caused the loss of microtubules
but, as expected, promoted the formation of numerous vinblas-
tine-tubulin paracrystals in the cells (panel C). Pretreatment of
the cells with 10 µM 1 did not prevent paracrystals formation
(panel D), indicating that this compound does not interfere with
the vinblastine binding site on tubulin.
Figure 4. Competition for colchicine binding.
but enhance direct cytotoxicity. Thus, the 2-OMe-substituted
compound 4 is most conducive to inhibition of Pgp for reversing
Pgp-mediated resistance to vinblastine. Whether compound 1
also has this attribute cannot be determined due to its high
cytotoxicity. However, because 1 at concentrations below its
IC50 had no effect on Pgp-mediated resistance, it is possible
that it is not a substrate for Pgp but does not inhibit Pgp like
compounds 2-5.
Effects of Compound 1 on Colchicine and Vinblastine
Binding to Tubulin. Colchicine Binding. The ability of 1 to
compete with colchicine for binding to bovine brain tubulin was
assessed using a fluorescent colchicine analog (Figure 4). The
colchicine analog was incubated with tubulin either alone or in
the presence of 5 µM unlabeled colchicine or 5 or 50 µM 1.
Samples were then subjected to gel filtration chromatography
on Sephadex G-25 (Superfine) equilibrated with binding buffer,
and 0.5 mL fractions were collected and analyzed for fluores-
cence (excitation ) 485 nm, emission ) 535 nm).
Fluorescent colchicine that is bound to tubulin eluted in
fractions 6-10, whereas unbound ligand eluted in fractions 16-
24. As indicated in Figure 4, inclusion of unlabeled colchicine
in the incubation competed off approximately 90% of the
fluorescent colchicine. In contrast, neither concentration of 1
affected the binding of fluorescent colchicine to tubulin indicat-
ing that 1 does not bind at the colchicine binding site of tubulin.
Vinblastine Binding. Incubation of cells with high concen-
trations of vinblastine causes the formation of paracrystals of
tubulin. We have previously used this phenomenon to demon-
strate that compounds that interact with the vinblastine binding
site of tubulin, for example, rhizoxin and cryptophycin, prevent
Effects of 1 on Assembly of Bovine Brain Tubulin (Figure
6). Purified tubulin was incubated at 4 °C with ethanol (as a
solvent control, 5 µM colchicine, 5 µM paclitaxel, 5 µM 1, or
5 µM 1 plus 5 µM paclitaxel. GTP was added and samples
were incubated at 37 °C for the indicated times.
Colchicine (the positive control) strongly suppressed tubulin
assembly, while 1 caused a moderate reduction in the rate of
tubulin polymerization. As expected, paclitaxel promoted the
assembly of tubulin, and this effect was not inhibited by 1. These
results indicate that 1 directly interacts with tubulin, albeit with
lower affinity and/or efficacy than colchicine and does not
interfere with paclitaxel-promoted assembly of tubulin.
Further Effects of 1 on Assembly of Bovine Brain Tubulin.
Figure 7, left panel: Purified tubulin (67 µM) was incubated at
4 °C with EtOH (as a solvent control), colchicine (5 µM), or 1
(2, 5, 10 or 20 µM). GTP was added and the samples were
incubated at 37 °C for the indicated times, while the absorbance
of 340 nm was monitored. In this assay, the IC50 is ap-
proximately 20 µM (noting that the tubulin concentration in
the assay is 67 µM, indicating that it is a substoichiometric
inhibitor of microtubule assembly). Figure 7, right panel: As
before, colchicine (the positive control) strongly suppressed
tubulin assembly. Compound 1 caused a dose-dependent reduc-
tion in both the rate and the extent of tubulin polymerization.
Purified tubulin was incubated at 4 °C with EtOH, with
paclitaxel (5 µM) alone, or in the presence of 1 (2, 5, 10, or 20
µM). GTP was added, and the samples were incubated at 37
°C for the indicated times while the absorbance of 340 nm was
monitored. As before, paclitaxel promoted the assembly of
tubulin (manifested as the loss of the lag time for the initiation

3296 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Gangjee et al.
Figure 5. Tubulin paracrystal formation with vinblastin with and without compound 1.
indeed bind to tubulin at a site different from the colchicine,
Vinca, or paclitaxel binding sites. In addition, compounds 2-5
restore sensitivity of a Pgp expressing tumor cell line to
antimitotic agents. Compound 1, the most potent analog,
inhibited all of the NCIs 60 tumor cell lines with GI50s at
nanomolar to submicromolar levels. Thus, compounds in this
series 1-5 demonstrated important attributes as potential
antitumor agents and/or Pgp inhibitory agents to be used alone
or in combination with other chemotherapeutic agents (including
antimitotic agents) against sensitive as well as resistant tumors.
Experimental Section
Figure 6. Effect of 1 on the assembly of bovine brain tubulin.
General Methods for Biological Evaluations. Materials:
MCF-7 breast carcinoma cells and NCI/ADR cells, an MDR line
that overexpresses Pgp, were obtained from the Division of Cancer
Treatment of the National Cancer Institute. MCF-7/VP cells that
express MRP1 but not Pgp were provided by Drs. Schneider and
Cowan.46 Human bladder carcinoma T24 cells were from the
American Type Culture Collection. Sulforhodamine B, antibodies
against â-tubulin (T-4026), proteinase K (P2308), RNase A (R-
5503), and actomyosin (A-6394) were obtained from Sigma
Chemical Company (St. Louis, MO). RPMI-1640 and R-MEM
culture media and fetal bovine serum were from GibcoBRL (Grand
Island, NY).
of polymerization), and this effect was not inhibited by 1 at
concentrations up to at least 20 µM. Compound 1 did cause
dose-dependent reductions in the maximal extent of tubulin
polymerization, which is consistent with its ability to bind to
tubulin monomers. Thus, compound 1 directly interacts with
tubulin monomers, albeit with lower affinity and/or efficacy than
colchicine. This is independent of the paclitaxel binding site
and, from the previous binding experiments (Figures 4 and 5),
also is independent of the vinblastine and colchicine binding
sites. Thus, compound 1 has a different binding site on tubulin.
In addition to the sensitization assay shown in Figure 3, the
compounds were evaluated in a drug-accumulation assay in
which their abilities to increase the uptake of [3H]vinblastine
into NCI/ADR cells was determined according to the following
protocol. NCI/ADR cells were plated into 24-well tissue culture
dishes and allowed to grow to 90% confluency. The cells were
washed with PBS and then incubated in 0.5 mL of RPMI 1640
medium containing the test compound and 20 nM [3H]-
vinblastine sulfate (10-15 Ci/mmole) for 60 min at 37 °C. The
cultures were rapidly washed three times with ice-cold PBS.
Intracellular [3H]vinblastine was solubilized with 0.3 mL of 1%
sodium dodecylsulfate in PBS and quantified by liquid scintil-
lation counting.
The data for verapamil and compound 4 are shown in Figure
8. Compound 4 is of similar potency but more efficacious than
verapamil. The ability of compound 4 to inhibit Pgp activity
was confirmed in studies that demonstrated dose-dependent
increases in the intracellular accumulation of [3H]vinblastine
by NCI/ADR cells with potency similar to that of verapamil
(Figure 8).
In summary, we synthesized and evaluated a series of
7-benzyl-4-methyl-5-[(2-substituted phenyl)ethyl]-7H-pyrrolo-
[2,3-d]pyrimidin-2-amines 1-5 and their 7-desbenzyl analogs
6-10 (Figure 1). We have determined that compounds 1-5
are antitumor agents and antimitotic agents that cause a dose-
dependent depletion of microtubules in intact cells. Studies
indicate that they do not bind to the colchicine or Vinca or
paclitaxel binding sites and inhibit tumor cell lines that are both
sensitive and resistant (Pgp or MRP1) to clinically used
antimitotic drugs. We have confirmed that compound 1 does
Assay of Cytotoxicity and Reversals of MDR: To test for
reversal of Pgp-mediated MDR, NCI/ADR cells were placed into
96-well tissue culture plates at approximately 15% confluency and
allowed to attach and recover for 24 h. The cells were then treated
with varying concentrations (as allowed by solubility) of a test
compound in the presence of 0 or 50 nM vinblastine for 48 h
according to previously described procedures.47-53 After 48 h, cell
survival was assayed using the sulforhodamine B (SRB) binding
assay.54 The percentage of cells killed is calculated as the percentage
decrease in SRB binding as compared with control cultures and is
taken from the mean of the absorbance measurements of three
equally treated wells. Reversal of MDR is indicated if the compound
enhances the toxicity of vinblastine toward the NCI/ADR cells.
The reversal index (Pgp Antagonism Score) is calculated as the
percentage of surviving NCI/ADR cells in the absence of vinblas-
tine/the percentage of surviving NCI/ADR cells in the presence of
vinblastine. Control cultures included equivalent amounts of ethanol
(as the solvent control), which does not modulate the growth or
drug-sensitivity of these cells at the doses used in these studies.
To assess the toxicity of the compounds toward drug-sensitive cells,
the effects of the test modulators on the growth of drug-sensitive
MCF-7 cells were determined by the same methods. To test for
reversal of MRP1-mediated MDR, MCF-7/VP cells were placed
into 96-well tissue culture plates at approximately 15% confluency
and were allowed to attach and recover for 24 h. The cells were
then treated with varying concentrations (as allowed by solubility)
of a test compound in the presence of 0 or 1 nM vincristine for 48
h as above. After 48 h, cell survival was assayed using the SRB
binding assay. Reversal of MDR is indicated if the compound
enhances the toxicity of vincristine toward the MCF-7/VP cells.
The reversal index (MRP1 Antagonism Score) is calculated as the
percentage of surviving MCF-7/VP cells in the absence of vinc-
ristine/the percentage of surviving MCF-7/VP cells in the presence
of vincristine.

NoVel Antitumor Antimitotic Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3297
Figure 7. Further effects of 1 on bovine brain tubulin.
determined following the procedures of Bai et al.57 Briefly, 0.25
mL samples containing 2.5 mg of tubulin/mL (67 µM tubulin) in
1 M glutamate, pH 6.6, containing 4% DMSO were incubated with
ethanol, compound 1, paclitaxel, or colchicine at 0 °C for 15 min.
GTP (0.5 mM) was then added to the samples followed by rapid
warming to 37 °C, and the absorbance at 340 nm was continuously
monitored for approximately 60 min. Microtubule assembly is
measured as increasing A340 due to light scattering by the polymers.
Interaction of Compound 1 with the colchicine-binding site of
tubulin was examined using the competition assay kit from
Cytoskeleton, Inc. (Denver, CO), in which purified tubulin was
incubated with compound 1 or unlabeled colchicine and fluores-
cently labeled colchicine for 30 min at 37 °C. The samples were
then transferred to a column of Sephadex G-25, and 0.5 mL
fractions were collected and analyzed for fluorescence intensity with
an excitation wavelength of 485 nm and an emission wavelength
of 535 nm. In this assay, colchicine or a compound interacting at
the same site displaces the fluorescent colchicine that elutes at the
void volume of the column.
Figure 8. [3H] Vinblastine accumulation assay for verapamil (9) and
compound 4 (2).
[3H]Vinblastine Accumulation Assay. NCI/ADR cells were
plated into 24-well tissue culture dishes and allowed to grow to
90% confluency. The cells were washed with PBS and then
incubated in 0.5 mL of RPMI 1640 medium containing the test
compound and 20 nM [3H]vinblastine sulfate (10-15 Ci/mmole)
for 60 min at 37 °C. The cultures were rapidly washed three times
with ice-cold PBS. Intracellular [3H]vinblastine was solubilized with
0.3 mL of 1% sodium dodecylsulfate in PBS and quantified by
liquid scintillation counting.
Cell Cycle Analyses: HL-60 cells were treated with ethanol,
paclitaxel, vinblastine, colchicine, or compound 1 for 24 or 48 h,
and DNA was stained by a procedure similar to that of Vindelov
et al.55 Briefly, cells were harvested by centrifugation, resuspended
in citrate buffer (250 mM sucrose, 40 mM trisodium citrate, pH
7.6, and 5% dimethylsulfoxide) to a concentration of 5 × 105 cells/
200 µL, and mixed with 1.8 mL of trypsin (0.03 mg/mL) in staining
buffer (3.4 mM trisodium citrate, 0.5 mM Tris, pH 7.6, 0.1% NP-
40, and 1.5 mM spermine) for 10 min. To each sample, 1.5 mL of
trypsin inhibitor (0.5 mg/mL) and RNase A (0.1 mg/mL) in staining
buffer were added for 10 min, followed by 1.5 mL of propidium
iodide (0.4 mg/mL) and spermine (1.2 mg/mL) in staining buffer.
After 30 min at 4 °C, samples were filtered through 30 µm nylon
mesh, centrifuged at 721 × g for 5 min and resuspended in 1 mL
of staining buffer. The DNA contents of the cells were measured
using a Beckton Dickinson FACScan Flow Cytometer, and cell
cycle distribution was analyzed using the program MacCycle.
Cytoskeleton Staining: A-10 rat smooth muscle cells were
grown to near confluency on glass cover slips and treated with the
indicated compounds for 24 h. Microtubules were stained with
monoclonal anti-â-tubulin and visualized with fluorescein-conju-
gated anti-mouse IgG. For staining of microfilaments, cells were
fixed with 3% p-formaldehyde, permeabilized with 0.2% Triton
X-100, and reduced with sodium borohydride (1 mg/mL). Mi-
crofilaments were visualized by incubation with 100 nM of TRITC-
phalloidin for 45 min at 37 °C. Microtubules and microfilaments
were imaged using a Nikon Optiphot-2 microscope with a Bio-
Rad MRC 600 Confocal Laser Scanning system. The images were
reconstructed using VoxelView Ultra Software and were printed
on a Kodak Model XL 7700 digital continuous tone printer.
In Vitro Tubulin Experiments. Microtubule protein (MTP),
comprising tubulin and MAPs, was isolated from fresh bovine brain
by three cycles of assembly and disassembly, and pure tubulin was
isolated by subsequent ion-exchange chromatography, as described
by Vallee.56 The effects of compound 1 on tubulin assembly were
General Methods for Synthesis. All evaporations were carried
out in vacuo with a rotary evaporator. Analytical samples were dried
in vacuo (0.2 mmHg) in an Abderhalden drying apparatus over
P2O5 and ethanol by reflux. Thin layer chromatography (TLC) was
performed on silica gel plates with fluorescent indicator. Spots were
visualized by UV light (254 and 365 nm). All analytical samples
were homogeneous on TLC in at least two different solvent systems.
Purification by column and flash chromatography was carried out
using Merck silica gel 60 (200-400 mesh). The amount (weight)
of silica gel for column chromatography was in the range of 50-
100 times the amount (weight) of the crude compounds being
separated. Columns were dry packed unless specified otherwise.
Solvent systems are reported as volume percent mixture. Melting
points were determined on a Mel-Temp II melting point apparatus
and are uncorrected. Proton nuclear magnetic resonance (1H NMR)
spectra were recorded on a Bruker WH-300 (300 MHz) spectrom-
eter. The chemical shift (δ) values are reported as parts per million
(ppm) relative to tetramethylsilane as internal standard; s ) singlet,
d ) doublet, t ) triplet, q ) quartet, m ) multiplet, bs ) broad
singlet, exch ) protons exchangeable by addition of D2O. Elemental
analyses were performed by Atlantic Microlab, Inc., Norcross, GA.
Elemental compositions were within ( 0.4% of the calculated
values. Fractional moles of water or organic solvents frequently
found in some analytic samples could not be removed despite 24
h of drying in vacuo and were confirmed, where possible, by their
presence in the 1H NMR spectrum. All solvents and chemicals were
purchased from Aldrich Chemical Co. and Fisher Scientific and

3298 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Gangjee et al.
were used as received, except anhydrous solvents, which were
freshly dried in the laboratory.
7.35 (m, 6 H, C6H5 & C6H4), 7.45 (d, 1 H, J ) 6.0 Hz, C6H4), 7.90
(s, 1 H, 6-H), 9.91 (s, 1 H, 2-NHPiv, exch). Anal. (C28H28N4O2)
C, H, N.
General Procedure for the Synthesis of 12a-d. To a 25-mL
round-bottom flask protected from light with aluminum foil were
added 11,40 the appropriate (substituted) iodobenzene, copper(I)
iodide, and tetrakis(triphenylphosphine)-palladium(0) dissolved in
anhydrous dichloroethane, followed by the addition of triethylamine.
The resulting, dark brown, solution was stirred at room temperature
under nitrogen for 3.5 h. Then CH2Cl2 (50 mL) was added to the
solution, and the reaction mixture was washed with brine (20 mL
× 2), and the organic layer was separated and dried over Na2SO4
and filtered. The filtrate was evaporated in vacuo. To this residue
was added silica gel (10 g) and methanol (20 mL), and the solvent
was evaporated to afford a plug. The silica gel plug obtained was
loaded onto a silica gel column and eluted with 1:1:7 ethyl acetate/
triethylamine/hexanes. Fractions containing the product (TLC) were
pooled, and the solvent was evaporated to afford analytically pure
compound.
N-{7-Benzyl-4-methyl-5-[(3,4,5-trimethoxyphenyl)ethynyl]-
7H-pyrrolo[2,3-d]pyrimidin-2-yl}-2,2-dimethylpropanamide (12a).
Compound 12a was synthesized from 11 (0.30 g, 0.88 mmol),
5-iodo-1,2,3-trimethoxybenzene (0.32 g, 1.10 mmol), copper(I)
iodide (0.01 g, 0.17 mmol), tetrakis(triphenylphosphine)-palladium-
(0) (0.13 g, 0.07 mmol), and triethylamine (0.2 mL) using the
general procedure described above to afford after purification 0.36
g (79%) as a white solid: TLC Rf 0.24 (ethyl acetate/triethylamine/
hexanes, 1:1:3); mp 130-133 °C; 1H NMR (DMSO-d6) δ 1.24 (s,
9 H, C(CH3)3), 2.85 (s, 3 H, 4-CH3), 3.68 (s, 3 H, 4′-OCH3), 3.80
(s, 6 H, 3′ & 5′-OCH3), 5.39 (s, 2 H, CH2C6H5), 6.82 (s, 2 H, C6H2),
7.31-7.35 (m, 5 H, C6H5), 7.90 (s, 1 H, 6-H), 9.91 (s, 1 H,
2-NHPiv, exch). Anal. (C30H32N4O4) C, H, N.
General Procedure for the Synthesis of 13a-d. To a Parr
hydrogenation bottle was added 12a-d dissolved in dichlo-
romethane (20 mL) and methanol (20 mL), followed by the addition
of 5% Pd/C. The mixture was hydrogenated at 50 psi at room
temperature overnight. After filtration, the catalyst was thoroughly
washed with hot methanol (20 mL × 3). The filtrate was
concentrated in vacuo and silica gel (10 g) and methanol (20 mL)
were added to the residue. The solvent was evaporated to afford a
plug. The silica gel plug obtained was loaded onto a silica gel
column and eluted with 1:1:7 ethyl acetate/triethylamine/hexanes.
Fractions containing the product (TLC) were pooled, and the solvent
was evaporated to afford analytically pure compound.
N-{7-Benzyl-5-[2-(3,4,5-trimethoxyphenyl)ethyl]-4-methyl-
7H-pyrrolo[2,3-d]pyrimidin-2-yl}-2,2-dimethylpropanamide (13a).
Compound 13a was synthesized from 12a (0.70 g, 1.37 mmol) and
5% Pd/C (0.50 g) using the general procedure described above to
afford 0.53 g (75%) as a sticky solid: TLC Rf 0.24 (ethyl acetate/
1
triethylamine/hexanes, 1:1:3); H NMR (DMSO-d6) δ 1.23 (s, 9
H, C(CH3)3), 2.70 (s, 3 H, 4-CH3), 2.86 (t, 2 H, J ) 7.4 Hz, CH2-
CH2), 3.07 (t, 2 H, J ) 7.4 Hz, CH2CH2), 3.69 (s, 3 H, 4′-OCH3),
3.82 (s, 6 H, 3′- and 5′-OCH3), 5.32 (s, 2 H, CH2C6H5), 6.53 (s, 1
H, 6-H), 7.21-7.30 (m, 7 H, C6H5 and C6H2), 9.73 (s, 1 H,
2-NHPiv, exch). Anal. (C30H36N4O4) C, H, N.
N-{7-Benzyl-5-[2-(4-methoxyphenyl)ethyl]-4-methyl-7H-pyr-
rolo[2,3-d]pyrimidin-2-yl}-2,2-dimethylpropanamide (13b). Com-
pound 13b was synthesized from 12b (0.70 g, 1.57 mmol) and 5%
Pd/C (0.50 g) using the general procedure described above to afford
0.47 g (67%) as a white foam: TLC Rf 0.42 (ethyl acetate/
1
triethylamine/hexanes, 1:1:3); H NMR (DMSO-d6) δ 1.23 (s, 9
H, C(CH3)3), 2.69 (s, 3 H, CH3), 2.84 (t, 2 H, J ) 7.9 Hz, CH2-
CH2), 3.02 (t, 2 H, J ) 7.9 Hz, CH2CH2), 3.70 (s, 3 H, OCH3),
5.31 (s, 2 H, CH2C6H5), 6.82 (d, 2 H, J ) 8.4 Hz, C6H4), 6.90-
7.62 (m, 8 H, C6H5, C6H4, and 6-H), 9.95 (s, 1 H, 2-NHPiv, exch).
Anal. (C28H32N4O2) C, H, N.
N-{7-Benzyl-4-methyl-5-[(4-methoxyphenyl)ethynyl]-7H-pyr-
rolo[2,3-d]pyrimidin-2-yl}-2,2-dimethylpropanamide (12b). Com-
pound 12b was synthesized from 11 (0.34 g, 1.11 mmol), 1-iodo-
4-methoxybenzene (0.29 g, 1.23 mmol), copper(I) iodide (0.01 g,
0.07 mmol), tetrakis(triphenylphosphine) palladium(0) (0.12 g, 0.10
mmol), and triethylamine (0.3 mL) using the general procedure
described above to afford after purification 0.79 g (53%) as a pale
yellow solid: TLC Rf 0.32 (ethyl acetate/triethylamine/hexanes,
N-{7-Benzyl-5-[2-(3-methoxyphenyl)ethyl]-4-methyl-7H-pyr-
rolo[2,3-d]pyrimidin-2-yl}-2,2-dimethylpropanamide (13c). Com-
pound 13c was synthesized from 12c (0.74 g, 1.60 mmol) and 5%
Pd/C (0.50 g) using the general procedure described above to afford
0.50 g (68%) as a sticky solid: TLC Rf 0.41 (ethyl acetate/
1
1:1:3); mp 158-160 °C; H NMR (DMSO-d6) δ 1.24 (s, 9 H,
C(CH3)3), 2.84 (s, 3 H, 4-CH3), 3.79 (s, 3 H, OCH3), 5.39 (s, 2 H,
CH2C6H5), 6.96 (d, 2 H, J ) 8.7 Hz, C6H4), 7.27 (m, 4 H, C6H5),
7.44 (d, 2 H, J ) 7.8 Hz, C6H4), 7.62 (m, 1 H, C6H5), 7.87 (s, 1 H,
6-H), 9.89 (s, 1 H, 2-NHPiv, exch). Anal. (C28H28N4O2‚0.1H2O)
C, H, N.
1
triethylamine/hexanes, 1:1:3); H NMR (DMSO-d6) δ 1.21 (s, 9
H, C(CH3)3), 2.40 (s, 3 H, 4-CH3), 2.87 (t, 2 H, J ) 7.1 Hz, CH2-
CH2), 3.04 (t, 2 H, J ) 7.1 Hz, CH2CH2), 3.70 (s, 3 H, OCH3),
6.77-7.31 (m, 10 H, C6H5, C6H4, and 6-H), 9.73 (s, 1 H, 2-NHPiv,
exch). Anal. (C28H32N4O2‚0.1MeOH) C, H, N.
N-{7-Benzyl-4-methyl-5-[(3-methoxyphenyl)ethynyl]-7H-pyr-
rolo[2,3-d]pyrimidin-2-yl}-2,2-dimethylpropanamide (12c). Com-
pound 12c was synthesized from 11 (0.91 g, 2.6 mmol), 1-iodo-
3-methoxybenzene (0.77 g, 3.30 mmol), copper(I) iodide (0.04 g,
0.20 mmol), tetrakis(triphenylphosphine) palladium(0) (0.36 g, 0.30
mmol), and triethylamine (0.3 mL) using the general procedure
described above to afford after purification 0.74 g (62%) as a pale
yellow solid: TLC Rf 0.27 (ethyl acetate/triethylamine/hexanes, 1:1:
3); mp 110-112 °C; 1H NMR (DMSO-d6) δ 1.24 (s, 9 H, C(CH3)3),
2.85 (s, 3 H, 4-CH3), 3.78 (s, 3 H, OCH3), 5.39 (s, 2 H, CH2C6H5),
6.96-7.11 (m, 3 H, C6H4), 7.32 (m, 6 H, C6H5 & C6H4), 7.93 (s,
1 H, 6-H), 9.90 (s, 1 H, 2-NHPiv, exch). Anal. (C28H28N4O2‚
0.1MeOH) C, H. N.
N-{7-Benzyl-4-methyl-5-[(2-methoxyphenyl)ethynyl]-7H-pyr-
rolo[2,3-d]pyrimidin-2-yl}-2,2-dimethylpropanamide (12d). Com-
pound 12d was synthesized from 11 (0.87 g, 2.50 mmol), 1-iodo-
2-methoxybenzene (0.64 g, 2.75 mmol), copper(I) iodide (0.03 g,
0.17 mmol), tetrakis(triphenylphosphine) palladium(0) (0.12 g, 0.10
mmol), and triethylamine (0.3 mL) using the general procedure
described above to afford after purification 0.54 g (48%) as a pale
yellow solid: TLC Rf 0.23 (ethyl acetate/triethylamine/hexanes, 1:1:
3); mp 153-155.5 °C; 1H NMR (DMSO-d6) δ 1.24 (s, 9 H,
C(CH3)3), 2.88 (s, 3 H, 4-CH3), 3.85 (s, 3 H, OCH3), 5.39 (s, 2 H,
CH2C6H5), 6.97 (t, 1 H, C6H5), 7.09 (d, 1 H, J ) 8.3 Hz, C6H5),
N-{7-Benzyl-5-[2-(2-methoxyphenyl)ethyl]-4-methyl-7H-pyr-
rolo[2,3-d]pyrimidin-2-yl}-2,2-dimethylpropanamide (13d). Com-
pound 13d was synthesized from 12d (0.50 g, 1.10 mmol) and 5%
Pd/C (0.60 g) using the general procedure described above to afford
0.46 g (91%) as an oil: TLC Rf 0.42 (ethyl acetate/triethylamine/
1
hexanes, 1:1:3); H NMR (DMSO-d6) δ 1.23 (s, 9 H, C(CH3)3),
2.71 (s, 3 H, 4-CH3), 2.86 (t, 2 H, J ) 8.0 Hz, CH2CH2), 2.97 (t,
2 H, J ) 8.0 Hz, CH2CH2), 3.75 (s, 3 H, OCH3), 5.32 (s, 2 H,
CH2C6H5), 6.83-7.34 (m, 10 H, C6H5, C6H4, and 6-H), 9.72 (s, 1
H, 2-NHPiv, exch). When placed in a vial, the oil solidifies. Anal.
(C28H32N4O2‚0.1H2O) C, H, N.
General Procedure for the Synthesis of 1-4. To a round-
bottom flask was added 13a-d dissolved in methanol (10 mL),
followed by the addition of 1 N NaOH (2 mL). The reaction mixture
was heated at reflux at 80 °C for 24 h. The reaction was then cooled
and the MeOH evaporated under vacuum. The precipitated solid
obtained was filtered, washed with cold water and a mixture of
1:2 ethyl acetate/hexanes, and dried to afford analytically pure
compound.
7-Benzyl-4-methyl-5-[2-(3,4,5-trimethoxyphenyl)ethyl]-7H-
pyrrolo[2,3-d]pyrimidin-2-amine (1). Compound 1 was synthe-
sized from 13a (0.46 g, 0.89 mmol) using the general procedure
described above to afford 0.33 g (86%) as a pale yellow solid: TLC
Rf 0.40 (ethyl acetate/triethylamine/hexanes, 5:1:3); mp 168.5-

NoVel Antitumor Antimitotic Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3299
171 °C; 1H NMR (DMSO-d6) δ 2.52 (s, 3 H, 4-CH3), 2.84 (t, 2 H,
J ) 8.0 Hz, CH2CH2), 3.00 (t, 2 H, J ) 8.0 Hz, CH2CH2), 3.70 (s,
3 H, 4′-OCH3), 3.81 (s, 6 H, 3′- and 5′-OCH3), 5.17 (s, 2 H, CH2
C6H5), 6.08 (s, 2 H, 2-NH2, exch), 6.52 (s, 2H, C6H2), 7.09 (s, 1
H, 6-H), 7.27 (m, 5 H, C6H5). Anal. (C25H28N4O3) C, H, N.
3.72 (s, 3 H, OCH3), 5.87 (s, 2 H, NH2, exch), 6.68-7.19 (m, 5 H,
6-H and C6H4), 10.71 (s, 1 H, 7-NH, exch). Anal. (C16H18N4O) C,
H, N.
5-[2-(2-Methoxyphenyl)ethyl]-4-methyl-7H-pyrrolo[2,3-d]py-
rimidin-2-amine (9). Compound 9 was synthesized from 4 (0.22
g, 0.59 mmol) using the general procedure described above to afford
0.08 g (48%) as a white solid: TLC Rf 0.48 (ethyl acetate/ethanol/
7-Benzyl-5-[2-(4-methoxyphenyl)ethyl]-4-methyl-7H-pyrrolo-
[2,3-d]pyrimidin-2-amine (2). Compound 2 was synthesized from
13b (0.45 g, 0.98 mmol) using the general procedure described
above to afford 0.29 g (79%) as a white solid: TLC Rf 0.34 (ethyl
1
acetone/water, 20:2:2:1); mp 218.5-223 °C; H NMR (DMSO-
d6) δ 2.52 (s, 3 H, 4-CH3), 2.85 (s, 4 H, CH2CH2), 3.78 (s, 3 H,
OCH3), 5.88 (s, 2 H, NH2, exch), 6.69 (s, 1 H, 6-H), 6.88 (t, 1 H,
J ) 7.2 Hz, C6H4), 6.96 (t, 1 H, J ) 8.0 Hz, C6H4), 7.17 (m, 2 H,
C6H4), 10.73 (s, 1 H, 7-NH, exch). Anal. (C16H18N4O‚0.2MeOH)
C, H, N.
1
acetate/triethylamine/hexanes, 5:1:3); mp 153-156 °C; H NMR
(DMSO-d6) δ 2.53 (s, 3 H, 4-CH3), 2.81 (t, 2 H, CH2CH2), 2.90 (t,
2 H, CH2CH2), 3.71 (s, 3 H, OCH3), 5.16 (s, 2 H, CH2C6H5), 6.07
(s, 2 H, 2-NH2, exch), 6.74 (s, 1 H, 6-H), 6.83 (d, 2 H, J ) 8.6 Hz,
C6H4),7.08-7.32(m,7H,C6H5andC6H4).Anal.(C23H24N4O‚0.25CH3-
COOCH2CH3) C, H, N.
N-[7-Benzyl-4-methyl-5-(phenylethynyl)-7H-pyrrolo[2,3-d]py-
rimidin-2-yl]-N-(2,2-dimethylpropanoyl)-2,2-dimethylpropana-
mide (15). To a 50-mL round-bottom flask covered with aluminum
foil were added 1440 (0.27 g, 0.42 mmol), ethynyl benzene (0.15
g, 1.5 mmol), copper(I) iodide (0.02 g, 0.1 mmol) and tetrakis-
(triphenylphosphine)palladium(0) (0.06 g, 0.05 mmol) dissolved in
anhydrous dichloroethane (5 mL) and triethylamine (0.14 mL). The
resulting dark brown solution was stirred at room temperature under
nitrogen for 24 h. The solvents were removed in vacuo, and the
crude residue was flash chromatographed on silica gel and eluted
with 1:1:10 ethyl acetate/triethylamine/hexanes to afford 0.20 g
(78%) of 15 as a white solid: TLC Rf 0.45 (ethyl acetate/
triethylamine/hexanes, 1:1:7); mp 125-127 °C; 1H NMR (DMSO-
d6) δ 1.17 (s, 18 H, C(CH3)3), 2.90 (s, 3 H, 4-CH3), 5.42 (s, 2 H,
CH2C6H5), 7.19-7.57 (m, 10 H, C6H5), 8.18 (s, 1 H, 6-H). Anal.
(C32H34N4O2) C, H, N.
7-Benzyl-5-[2-(3-methoxyphenyl)ethyl]-4-methyl-7H-pyrrolo-
[2,3-d]pyrimidin-2-amine (3). Compound 3 was synthesized from
13c (0.45 g, 0.98 mmol) using the general procedure described
above to afford 0.32 g (87%) as a white solid: TLC Rf 0.46 (ethyl
acetate/triethylamine/hexanes, 5:1:3); mp 126-127.5 °C; 1H NMR
(DMSO-d6) δ 2.53 (s, 3 H, 4-CH3), 2.85 (t, 2 H, J ) 8.0 Hz, CH2-
CH2), 2.97 (t, 2 H, J ) 8.0 Hz, CH2CH2), 3.70 (s, 3 H, OCH3),
5.17 (s, 2 H, CH2 C6H5), 6.08 (s, 2 H, 2-NH2, exch), 6.73-7.31
(m, 10 H, C6H5, C6H4, and 6-H). Anal. (C23H24N4O‚0.10MeOH)
C, H, N.
7-Benzyl-5-[2-(2-methoxyphenyl)ethyl]-4-methyl-7H-pyrrolo-
[2,3-d]pyrimidin-2-amine (4). Compound 4 was synthesized from
13d (0.40 g, 0.88 mmol) using the general procedure described
above to afford 0.29 g (90%) as a pale pink solid: TLC Rf 0.50
(ethyl acetate/triethylamine/hexanes, 5:1:3); mp 138.5-140 °C; 1H
NMR (DMSO-d6) δ 2.50 (s, 3 H, 4-CH3), 2.85-2.91 (m, 4 H, CH2-
CH2), 3.76 (s, 3 H, OCH3), 5.17 (s, 2 H, CH2 C6H5), 6.08 (s, 2 H,
2-NH2, exch), 6.78-7.32 (m, 9H, C6H5 and C6H4). Anal. (C23H24N4O)
C, H, N.
General Procedure for the Synthesis of 6-9. To a 25-mL
round-bottom flask cooled to -78 °C were added 1-4 and liquid
NH3 (20 mL), followed by the addition of sodium metal (one and
one-half the weight of 1-4). The reaction was maintained at
-78 °C for 4.5 h and then quenched with NH4Cl (same as the
weight of 1-4). The mixture was allowed to warm to room
temperature and the liquid NH3 was evaporated. The residue was
purified by column chromatography on silica gel and eluted with
20:2:2:1 ethyl acetate/ethanol/acetone/water to afford analytically
pure compound.
N-[7-Benzyl-4-methyl-5-(2-phenylethyl)-7H-pyrrolo[2,3-d]py-
rimidin-2-yl]-N-(2,2-dimethylpropanoyl)-2,2-dimethylpropana-
mide (16). To a Parr hydrogenation bottle was added 15 (0.27 g,
0.50 mmol) dissolved in CH2Cl2 (25 mL) and MeOH (25 mL),
followed by the addition of 5% Pd/C (0.20 g). The mixture was
then hydrogenated at 50 psi for 3.5 h. The catalyst was filtered
and washed thoroughly with hot MeOH (20 mL), and the filtrate
was evaporated in vacuo. The residue obtained was flash chro-
matographed on silica gel and eluted with 1:1:7 ethyl acetate/
triethylamine/hexanes to afford 0.27 g (100%) of 16 as a white
solid: TLC Rf 0.21 (ethyl acetate/triethylamine/hexanes, 1:1:7); mp
1
105.5-107 °C; H NMR (DMSO-d6) δ 1.15 (s, 18 H, C(CH3)3),
2.73 (s, 3 H, 4-CH3), 2.93 (t, 2 H, J ) 8.1 Hz, CH2CH2), 3.11 (t,
2 H, J ) 8.1 Hz, CH2CH2), 5.33 (s, 2 H, CH2C6H5), 7.09-7.31
(m, 10 H, C6H5), 7.42 (s, 1 H, 6-H). Anal. (C32H38N4O2) C, H, N.
7-Benzyl-4-methyl-5-(2-phenylethyl)-7H-pyrrolo[2,3-d]pyri-
midin-2-amine (5). To a round-bottom flask was added 16 (0.23
g, mmol) suspended in MeOH (10 mL) and 1 N NaOH (5 mL).
The reaction mixture was heated at reflux at 80 °C for 24 h. The
reaction was cooled and the MeOH evaporated under vacuum. The
precipitated solid was filtered, washed with cold water, and dried
to afford 0.11 g (79%) of 5 as a white solid: TLC Rf 0.37 (ethyl
acetate/triethylamine/hexanes, 5:1:3); mp 121.5-123 °C; 1H
NMR (DMSO-d6) δ 2.54 (s, 3 H, 4-CH3), 2.88-2.97 (m, 4 H, CH2-
CH2), 5.17 (s, 2 H, CH2Ph), 6.09 (s, 2 H, 2-NH2, exch), 6.77 (s, 1
H, 6-H), 7.08-7.30 (m, 10 H, C6H5). Anal. (C22H22N4‚0.3H2O) C,
H, N.
4-Methyl-5-[2-(3,4,5-trimethoxyphenyl)ethyl]-7H-pyrrolo[2,3-
d]pyrimidin-2-amine (6). Compound 6 was synthesized from 1
(0.27 g, 0.63 mmol) using the general procedure described above
to afford 0.06 g (28%) as a pale yellow solid: TLC Rf 0.33 (ethyl
1
acetate/ethanol/acetone/water, 20:2:2:1); mp 175.8-178.8 °C; H
NMR (DMSO-d6) δ 2.51 (s, 3 H, 4-CH3), 2.79-2.96 (m, 4 H, CH2-
CH2), 3.72 (s, 3 H, 4′-OCH3), 3.81 (s, 6 H, 3′ & 5′-OCH3), 5.89 (s,
2 H, NH2, exch), 6.07 (s, 1 H, 6-H), 6.53 (s, 2 H, C6H2), 10.73 (s,
1 H, 7-NH, exch). Anal. (C18H22N4O3) C, H, N.
5-[2-(4-Methoxyphenyl)ethyl]-4-methyl-7H-pyrrolo[2,3-d]py-
rimidin-2-amine (7). Compound 7 was synthesized from 2 (0.24
g, 0.68 mmol) using the general procedure described above to afford
0.05 g (28%) as a pale yellow power: TLC Rf 0.52 (ethyl acetate/
ethanol/acetone/water, 20:2:2:1); mp 201-204.6 °C; 1H NMR
(DMSO-d6) δ 2.50 (s, 3 H, 4-CH3), 2.83-2.90 (m, 4 H, CH2CH2),
3.70 (s, 3 H, OCH3), 5.89 (bs, 2 H, NH2, exch), 6.66 (s, 1 H, 6-H),
6.81-6.84 (d, 2 H, J ) 9.1 Hz, C6H4), 7.13-7.16 (d, 2 H, J ) 9.1
Hz, C6H4), 10.71 (bs, 1 H, 7-NH, exch). Anal. (C16H18N4O‚
0.3MeOH) C, H, N.
5-[2-(3-Methoxyphenyl)ethyl]-4-methyl-7H-pyrrolo[2,3-d]py-
rimidin-2-amine (8). Compound 8 was synthesized from 3 (0.20
g, 0.54 mmol) using the general procedure described above to afford
0.03 g (19%) as a pale yellow solid: TLC Rf 0.47 (ethyl acetate/
ethanol/acetone/water, 20:2:2:1); mp 166-169 °C; 1H NMR
(DMSO-d6) δ 2.49 (s, 3 H, 4-CH3), 2.92 (m, 4 H, CH2CH2),
4-Methyl-5-(2-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-
amine (10). To a 25-mL round-bottom flask cooled to -78 °C
were added 5 (0.24 g, 0.70 mmol) and liquid NH3 (20 mL), followed
by the addition of sodium metal (0.18 g). The reaction was
maintained at -78 °C for 4.5 h and then quenched with NH4Cl
(0.30 g). The mixture was allowed to warm to room temperature,
and the liquid NH3 was evaporated. The residue was purified by
column chromatography on silica gel and eluted with 20:2:2:1 ethyl
acetate/ethanol/acetone/water to afford 0.05 g (30%) of 10 as a pale
yellow solid: TLC Rf 0.45 (ethyl acetate/ethanol/acetone/water, 20:
1
2:2:1); mp 202.5-206 °C; H NMR (DMSO-d6) δ 2.50 (s, 3 H,
CH3), 2.88-2.97 (m, 4 H, CH2CH2), 5.88 (s, 2 H, NH2, exch),
6.67 (s, 1 H, 6-H), 7.16-7.27 (m, 5 H, C6H5), 10.72 (s, 1 H, 7-NH,
exch). Anal. (C15H16N4‚0.1H2O) C, H, N.

3300 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Gangjee et al.
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Supporting Information Available: Results from elemental
analyses. This material is available free of charge via the Internet
at http://pubs.acs.org.
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