Synthesis and discovery of water-soluble microtubule targeting agents that bind to the colchicine site on tubulin and circumvent Pgp mediated resistance

Article abstract of DOI:10.1021/jm101010n

Two classes of molecules were designed and synthesized based on a 6-CH ₃ cyclopenta[d]pyrimidine scaffold and a pyrrolo[2,3-d]pyrimidine scaffold. The pyrrolo[2,3-d]pyrimidines were synthesized by reacting ethyl 2-cyano-4,4-diethoxybutanoate an

Full text of DOI:10.1021/jm101010n

8116 J. Med. Chem. 2010, 53, 8116–8128
DOI: 10.1021/jm101010n
Synthesis and Discovery of Water-Soluble Microtubule Targeting Agents that Bind
to the Colchicine Site on Tubulin and Circumvent Pgp Mediated Resistance
Aleem Gangjee,*,†, Ying Zhao,† Lu Lin,† Sudhir Raghavan,† Elizabeth G. Roberts,‡ April L. Risinger,‡
Ernest Hamel,§ and Susan L. Mooberry*,‡,
†Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, 600 Forbes Avenue, Pittsburgh,
Pennsylvania 15282, United States, ‡Department of Pharmacology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl
Drive, San Antonio, Texas 78229, United States, and 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.
These authors contributed equally to this work
Received August 4, 2010
Two classes of molecules were designed and synthesized based on a 6-CH3 cyclopenta[d]pyrimidine scaffold
and a pyrrolo[2,3-d]pyrimidine scaffold. The pyrrolo[2,3-d]pyrimidines were synthesized by reacting ethyl
2-cyano-4,4-diethoxybutanoate and acetamidine, which in turn was chlorinated and reacted with the
appropriate anilines to afford 1 and 2. The cyclopenta[d]pyrimidines were obtained from 3-methyladapic
acid, followed by reaction with acetamidine to afford the cyclopenta[d]pyrimidine scaffold. Chlorination
and reaction with appropriate anilines afforded (()-3 HCl-(()-7 HCl. Compounds 1 and (()-3 HCl had
3
3
3
potent antiproliferative activities in the nanomolar range. Compound (()-3 HCl is significantly more
3
potent than 1. Mechanistic studies showed that 1 and (()-3 HCl cause loss of cellular microtubules,
3
inhibit the polymerization of purified tubulin, and inhibit colchicine binding. Modeling studies show
interactions of these compounds within the colchicine site. The identification of these new inhibitors
that can also overcome clinically relevant mechanisms of drug resistance provides new scaffolds for
colchicine site agents.
Introduction
and its phosphorylated prodrug combretastatin A-4 phosphate
(CA4P), are a class of drugs that are in clinical trials as antitumor
agents that bind to the colchicine site.1 Colchicine itself is not
used as an antitumor agent but is used in the treatment of gout
and familial Mediterranean fever. There are no clinically ap-
proved antitumor agents that bind to the colchicine site. How-
ever, several of these agents are currrently in clinical trials,6,7
demonstrating the importance of developing colchicine site
agents both for use alone and in combination chemotherapy
protocols.
Half of all human tumors have mutations in the p53 gene,
and the most effective drugs in p53 mutant cell lines are tubulin-
binding agents.8 This further emphasizes the importance of
developing new tubulin-binding drugs that are active against
resistant tumors. Comprehensive reviews of structures and
activities for these agents are available in the literature.9-13
Despite the unprecedented success of microtubule disrupt-
ing agents in cancer chemotherapy, multidrug resistance
(MDR) is a major limitation of cancer chemotherapy, and
MDR tumors are usually resistant to tubulin-binding drugs.
Overexpression of P-glycoprotein (Pgp) has been reported in
the clinical setting in a number of tumor types, particularly
after patients have received chemotherapy.11,12 In addition, it
has also been reported that Pgp expression may be a prog-
nostic indicator in certain cancers and is associated with poor
response to chemotherapy,13,14 demonstrating the clinical
importance of this mechanism of tumor resistance. Over-
expression of Pgp appears to be more relevant in the clinical
setting than elevation of MRP1 levels.15 The overwhelming
Three major classes of microtubule active agents have been
identified (Figure 1).1-3 The vinca alkaloids vincristine, vinblas-
tine, vindesine, and vinorelbine are β-tubulin binding agents that
are important in the treatment of leukemias, lymphomas, non-
small-cell lung cancer, and other cancers. These are microtubule
polymerization inhibitors. The second group includes the tax-
oids, paclitaxel (Taxol) and docetaxel (Taxotere), as well as
the epothilones. The binding site for paclitaxel is also in the
β-subunit but is distinct from that of the vinca alkaloids.
Paclitaxel and other taxoids (and the epothilones) bind to the
interior of the microtubule,4,5 and these are designated micro-
tubule-stabilizing agents becuase they stimulate tubulin poly-
merization. The taxoids are useful in the treatment of breast, lung,
ovarian, head and neck, and prostate carcinomas among others.
The third class, typified by colchicine, is composed of a diverse
collection of small molecules that bind to the colchicine site on
β-tubulin at its interface with R-tubulin, distinct from the vinca
site. Colchicine site agents also inhibit tubulin polymerization.
Combretastatins, as exemplified by combretastatin A-4 (CA4a)
*To whom correspondence should be addressed. (A.G.) Telephone: 412-
396-6070. Fax 412-396-5593. E-mail gangjee@duq.edu. (S.L.M.) Telephone
210-567-4788. Fax 210-567-4300. E-mail mooberry@uthscsa.edu.
aAbbreviations: Combretastatin A-4, CA4; multiple drug resistance,
MDR; P-glycoprotein, Pgp; multidrug resistance protein 1, MRP1; receptor
tyrosine kinase, RTK; epidermal growth factor receptor, EGFR; platelet-
derived growth factor receptor, PDGFR; vascular endothelial growth factor
receptor, VEGFR; relative resistance, Rr; N-deacetyl-N-(2-mercaptoace-
tyl)colchicine, DAMA colchicine; 2-methoxyestradiol, 2ME2.
r
pubs.acs.org/jmc
Published on Web 10/25/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8117
Figure 1. Structures of microtubule targeting agents.
Figure 2. Lead compounds.
lack of success of Pgp inhibitors in the clinic indicates that the
ability to overcome to Pgp-mediated resistance is an important
property for new microtubule targeting agents.12,16 Such agents
will fill an unmet need in the clinic for patients that develop
resistance due to Pgp overexpression.
Synergistic or additive effects of microtubule targeted agents
used in combination occurs, in part, due to the different target
sites of the agents on tubulin. Estramustine and paclitaxel,
which bind to different sites on tubulin, can act synergistically
or additively in combination.27 Combinations including pacli-
taxel or docetaxel plus vinorelbine are more efficacious in
combination than as single agents.28,29 CA4P is currently in
clinical trials in combination with paclitaxel (www.clinical-
trials.gov).29 As noted above, no colchicine site agent is FDA
approved for cancer chemotherapy. There are however several
colchicine site agents in clinical trials, and approval of such
a drug would allow evaluation of whether enhanced activity
could be obtained by combining it with a taxoid or vinca
alkaloid. Thus, new tubulin-binding agents that do not act at
the taxoid or vinca alkaloid site on tubulin are of consider-
able interest, both as single agents and to combine with
these established drugs.
The other clinical mechanism of resistance to microtubule
targeted agents involves the expression of specific isotypes of
β-tubulin, of which the βIII-tubulin isotype is of paramount
concern. There is unequivocal clinical evidence that βIII-tubu-
lin expression is involved in resistance to taxoids and vinca
alkaloids in multiple tumor types including non-small cell
lung,17-19 breast,20 and ovarian cancers.21,22 Excellent reviews
of the importance of βIII-tubulin in tumor resistance to micro-
tubule targeted agents are available.23 In a recent study, Stengel
et al.24 and Lee et al.25 have shown that colchicine site agents
circumvent βIII-tubulin resistance and highlight the critical
importance of developing new agents that bind to the colchicine
site as an alternative to the taxoids for the treatment of
refractory cancers. The ability to overcome resistance due to
Pgp and βIII-tubulin expression was part of the impetus for the
recent FDA approval of ixabepilone as an antitumor agent and
further attests to the importance of developing agents that
overcome these resistance mechanisms.26
An additional serious problem with several of the currently
used antitubulin agents, particularly the taxoids, is their poor
water solubility. This necessitates their formulation in polyoxy-
ethylated castor oil or polysorbate, which can cause hyper-
sensitivity reactions and require long administration times. This
is a problem, too, with ixabepilone, which also needs to be
dissolved in polyoxyethylated castor oil. Thus water-soluble
microtubule targeted agents are highly coveted, and an enor-
mous effort continues to chemically modify and/or formulate
analogues of these agents to increase their water solubility.
Synthesis of Water-Soluble Microtubule Targeting Agents
We had previously synthesized 2-amino-4-anilino-6-substi-
tuted pyrrolo[2,3-d]pyrimidines as receptor tyrosine kinase
(RTK) inhibitors of general structure A (Figure 2). Three
possible binding modes to RTK were suggested from molec-
ular modeling. To define the mode(s) of binding, we synthe-
sized the aniline N-methylated analogue (A; R1=CH3; R2=H),
the N7-methylated analogue (A; R1 = H; R2 = CH3), and
the aniline N- and N7-dimethylated analogue (A; R1 = CH3;
R2 = CH3). These results were first reported in 2005.30,31
Further, analogue design from these lead compounds afforded
the pyrrolo[2,3-d]pyrimidines 1-2 and the cyclopenta[d]pyri-
midine (()-3(Figure 2) as the free base, and 1-(()-3as the HCl,
water-soluble salts as well. Compounds 1-(()-3 were not RTK

8118 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Gangjee et al.
inhibitors (evaluated against EGFR, VEGFR-1, VEGFR-2,
and PDGFR-β), but in the preclinical screening program of
the National Cancer Institute in its 60 tumor cell line panel 1
inhibited the proliferation of most of the 60 cancer cell lines with
a GI50 of less than 500 nM (Table 1) and (()-3 HCl inhibited
3
the proliferation of the majority of the 60 cell lines with a potent
GI50 of less than 30 nM (Table 1).
The potent activities of these compounds prompted a
COMPARE analysis,32,34 which showed vincristine sulfate
to have the closest Pearsons correlation coefficient with 1 and
(()-3 HCl. Other compounds, such as vinblastine sulfate and
3
maytansine, also tubulin binding agents, were ranked as the
next closest correlation. This clearly warranted the evaluation
of 1 and (()-3 HCl as tubulin binding agents. It was also of
3
interest to synthesize and evaluate 2, the desmethyl analogue
of 1, and (()-4, the desmethyl analogue of (()-3. In addition,
due to the more potent activity of (()-3 HCl in the NCI
3
preclinical panel as compared to 1, compounds (()-5 HCl-
3
(()-7 HCl (Figure 2) were also synthesized to determine the
3
position and importance of the 40-OMe moiety to the bio-
logical activity of (()-3 HCl.
3
Chemistry
Compound 1 was synthesized as shown in Scheme 1. Ethyl
2-cyano-4,4-diethoxybutanoate 8 and acetamidine HCl, 9,
3
were reacted at reflux for 5 h and cooled to 0 °C, and then
the precipitate was collected and treated with conc. H2SO4 in
ethanol at reflux for 2 h to afford 2-methyl-4-oxo-pyrrolo[2,3-
d]pyrimidine 10 in 64% yield. Chlorination with POCl3
afforded 11 (83% yield).35 Treatment of 11 with the appro-
priate anilines 12 afforded 1 (R = CH3) (80% yield) and 2
(R = H) (41%). The water-soluble hydrochloride salt of 1
was obtained as a white precipitate (∼100%) by dissolving
1 in anhydrous ether followed by bubbling anhydrous
HCl gas.
Target compounds (()-3-(()-7 were prepared as shown in
Scheme 2. 2,6-Dimethyl-6,7-dihydro-3H-cyclopenta[d]pyri-
midin-4(5H)-one (()-14 was prepared via the reaction of
commercially available 3-methyladipic acid (()-13 and conc.
H2SO4 at reflux in ethanol and a Dieckmann condensation in
the presence of elemental sodium in toluene, followed by
treatment with acetamidine HCl 9 (21% over three steps).
3
Chlorination of (()-14 with POCl3 for 3 h at reflux afforded
(()-15 (69%).36 Compound (()-15 was unstable and easily
decomposed to (()-14 if not used immediately. Reaction of
(()-15 with the appropriate anilines in i-PrOH in the presence
of 2-3 drops of conc. HCl afforded (()-3-(()-7. Anhydrous
HCl gas was bubbled through an anhydrous ether solution of
(()-3, (()-5-(()-7 to afford the corresponding HCl salts as
white solids.
Biological Evaluations
NCI Cytotoxicity Studies and Antimicrotubule Effects in
Cells. Compounds 1 and (()-3 HCl showed potent GI50’s in
3
most of the NCI 60 cancer cell lines (Table 1). The micro-
tubule disrupting effects of 1 and (()-3 HCl were observed
(Figure 3) in a cell-based phenotypic screen. Compounds 1
3
and (()-3 HCl caused dramatic reorganization of the inter-
3
phase microtubule network, similar to the effects of colchi-
cine and CA4P. The EC50 (concentration required to cause
50% loss of cellular microtubules) (Table 2) was calculated
to be 7 nM for CA4P,25 56 nM for (()-3 HCl, and 5.8 μM
3
for 1. Consistent with effects on cellular microtubules,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8119
Figure 3. Effects of CA4P, 1, and (()-3 HCl on interphase microtubules. A-10 cells were treated with vehicle or compounds as indicated for
18 h, and then the cells were fixed and microtubules visualized by indirect immunofluorescence techniques.
3
Scheme 1. Synthesis of Pyrrolo[2,3-d]pyrimidines 1 and 2a
a Reagents and conditions: (a) (i) 2-bromo-1,1-diethoxyethane, NaOMe, anhyd. DMF, 90 °C, 4 h; (ii) acetamidine HCl, NaOEt, EtOH, reflux, 3 h;
(iii) conc. H2SO4, EtOH, reflux, 2 h. (b) POCl3, reflux, 3 h. (c) i-PrOH, HCl (1 drop), reflux, 4 h. (d) Anhyd. HCl gas, anhyd. ether.
3
Scheme 2. Synthesis of Cyclopenta[d]pyrimidines (()-3 - (()-7 and Their HCl Saltsa
a Reagents and conditions: (a) (i) conc. H2SO4, EtOH, reflux, 8 h; (ii) Na, toluene, reflux, 3 h; (iii) acetamidine HCl 9, t-BuOK, t-BuOH. (b) POCl3,
3
reflux, 3 h. (c) i-PrOH, HCl (2-3 drops), reflux, 3-6 h. (d) Anhyd. HCl gas, anhyd. ether.
Table 2. IC50 Values for Inhibition of Proliferation of MDA-MB-435
Cells and EC50s for Cellular Microtubule Loss
potent microtubule depolymerizers in cells, confirming the
COMPARE analysis results.
IC50 ( SD
(MDA-MB-435)
EC50 for microtubule
depolymerization
Antiproliferative Effects. Compounds 1 and (()-3 HCl
3
compound
were tested for antiproliferative effects against the drug
sensitive MDA-MB-435 cell line using the sulforhodamine
B assay (SRB assay).37,38 The data (Table 2) indicate that
1
2
183 ( 3.4 nM
>10 μM
17.0 ( 0.7 nM
>10 μM
5.8 μM
>40 μM
56 nM
(()-3 HCl
3
(()-3 HCl has potent antiproliferative effects with an IC50
3
(()-4
>40 μM
3.0 μM
>40 μM
>40 μM
(concentration required to cause 50% inhibition of prolifer-
ation) of 17.0 ( 0.7 nM. Compound 1 is less potent,
consistent with its effects in the phenotypic assay and by
flow cytometry, with an IC50 of 183 ( 3.4 nM.
(()-5 HCl
153 ( 11.1 nM
3
3
3
(()-6 HCl
NDa
(()-7 HCl
2.7 ( 0.3 μM
a ND: Not determined.
The ability of 1 and (()-3 HCl to circumvent Pgp-mediated
3
1 and (()-3 HCl caused the formation of aberrant mitotic
spindles and mitotic accumulation when measured by flow
cytometry (Figure 4). Compounds 1 and (()-3 HCl are
drug resistance was evaluated by using an SK-OV-3 isogenic cell
line pair (Table 3). In this cell line pair, the relative resistance
(Rr) of paclitaxel, a well-known Pgp substrate, is greater than
3
3

8120 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Gangjee et al.
800 while Rr values of 1.2 and 1.6 were obtained with 1 and
(()-3 HCl, respectively, consistent with the Rr values obtained
An initial study, shown in Figure 5, indicates that both 1 and
(()-3 HCl are potent inhibitors of purified tubulin assembly.
Both compounds were therefore compared with CA4P as
inhibitors of assembly in a quantitative study (Table 4). In
3
3
with other colchicine site agents, CA4P and 2ME2 of 1.5-2.6.
These data suggest that both 1 and (()-3 HCl are poor
3
substrates for transport by Pgp and thus have advantages
over some clinically useful tubulin-targeting drugs such as
paclitaxel.
this assay, both 1 and (()-3 HCl inhibited tubulin assembly
3
about as well as CA4 (Table 4). The data in Table 4 also
show that 1and (()-3 HCl bind at the colchicine site on tubulin,
3
A second mechanism of drug resistance that can lead
to treatment failure with tubulin-targeting drugs is the
expression of the βIII isotype of tubulin as discussed above.
An isogenic HeLa cell line pair was used to evaluate
the effect of βIII tubulin expression on the activities of 1
and (()-3 HCl.39 Compounds 1 and (()-3 HCl have Rr
since they inhibited [3H]colchicine binding to the protein,
although not as potently as CA4.
To determine the structural requirements for the 40-OCH3
and N4-CH3 moieties for activity, compounds 2 and (()-4-
(()-7 were synthesized. The superior activity of (()-3 HCl
3
suggests that the pyrrole NH of 1 is not important for
activity. Table 2 indicates that the methylation of the aniline
N is crucial for activity in both scaffolds (compare 1 with 2
3
3
values of 0.6-0.7 (Table 3) in this cell line pair, suggest-
ing that they overcome drug resistance mediated by
βIII tubulin as compared with paclitaxel, which has a Rr
and (()-3 HCl with (()-4). In addition, the 40-OCH3 moiety
3
of 4.7. Thus, both 1 and (()-3 HCl inhibit tumor cells with
is important for potent activity (compare (()-3 HCl with
3
3
equal potency without regard to their expression of Pgp or
βIII-tubulin.
Further Mechanistic Studies. Studies were conducted to
(()-7 HCl), and the optimum location for the methoxyl
3
group is at the 40-position since (()-5 HCl, which has a
3
30-OCH3, was about 9-fold less potent than (()-3 HCl,
3
determine if 1 and (()-3 HCl inhibited the polymerization of
which has a 40-OCH3. Finally, (()-6 HCl, the analogue with
3
3
purified bovine brain tubulin, as would be predicted from the
effects in cells. These biochemical studies provide an indica-
tion of a direct interaction of the compounds with tubulin.
a 20-OCH3, was inactive.
Molecular Modeling
In an attempt to explain the molecular basis of the potent
activity of the N-Me analogues 1 and (()-3 HCl and the
3
inactivity of the N-desmethyl analogues 2 and (()-4, we
modeled compounds 1-7 into the colchicine site. The X-ray
˚
crystal structure of tubulin at 3.58 A resolution was obtained
from the protein database (PDB ID 1SA040). This crystal
structure contains the Rβ dimers of tubulin complexed with
N-deacetyl-N-(2-mercaptoacetyl)colchicine (DAMA colchicine),
a close structural analogue of colchicine. The binding of colchi-
cine to the tubulin dimer has been described in the literature.40-42
Colchicine binds to β-tubulin at its interface with R-tubulin. The
˚
˚
˚
colchicine site has dimensions of ∼10 A ꢀ ∼10 A ꢀ ∼4-5 A
and is composed of strands S8 and S9, loop T7, and helices
H7 and H8 (nomenclature and amino acid residue positions
as described in ref 40) from the β subunit and loop T5 from the
R subunit of tubulin. ThrR179 and ValR181 in the R-tubulin
subunit form hydrogen bonds with colchicine. Additionally,
Cysβ241 forms a hydrogen bond with the oxygen atom of
the 3-OCH3 in the A-ring of colchicine. Further hydro-
phobic stabilization is afforded by side chain atoms of ValR181
and Metβ259. In addition, the carbonyl group of the A-ring
H-bonds with Lysβ352.41,42
Figure 4. Cell cycle distribution. MBA-MB-435 cells were treated
with vehicle (A), 12.5 nM paclitaxel (B), 45 nM (()-3 HCl (C), or
Docking of (R)-3. Docking studies were performed using
the docking suite of Molecular Operating Environment
software (MOE 2008.10).43 Details of the docking protocol
3
850 nM 1 (D) for 24 h. The cells were then harvested, stained with
propidium iodide, and evaluated by flow cytometry.
Table 3. 1 and (()-3 HCl Circumvent Pgp and βIII-Tubulin Mediated Resistancea
3
effect of Pgp on drug sensitivityb
effect of βIII-tubulin on drug sensitivityc
IC50 ( SD (nM)
IC50 ( SD (nM)
drug
SK-OV-3
SK-OV-3 MDR-1-6/6
Rr
HeLa
WT βIII
Rr
paclitaxel
CA4P
2ME2
1
3.0 ( 0.06
4.5 ( 0.2
867 ( 36
278 ( 19
38.6 ( 3.1
2600 ( 270
6.6 ( 1.3
2268 ( 235
435 ( 33
864
1.6 ( 0.2
4.7 ( 0.2
608 ( 55
270 ( 21
37.3 ( 4.1
7.7 ( 0.2
5.7 ( 0.4
586 ( 37
186 ( 17
23.9 ( 1.7
4.7
1.2
1
1.5
2.6
1.6
1.2
0.7
0.6
(()-3 HCl
44.4 ( 3.2
3
a Rr: Relative resistance. b Antiproliferative effects of (()-3 HCl and 1 in parental and MDR-1-transducted cell lines in comparison with other
3
microtubule disrupting agents. The IC50 values were determined using the SRB assay (n = 3 ( SD). The Rr was calculated by dividing the IC50 of the Pgp
overexpressing cell line by the IC50 of the parental cell line. c Effects of the expression of βIII-tubulin on the sensitivity of cell lines to microtubule-
targeting agents. The Rr was calculated by dividing the IC50 of the WT βIII cell line by the IC50 of the parental HeLa cells.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8121
Figure 5. Effects of microtubule disrupting agents on tubulin polymerization. Paclitaxel represents a positive control for a microtubule
stabilizer and colchicine the control for a microtubule depolymerizer. The assays were conducted using purified bovine brain tubulin from
Cytoskeleton Inc. in the presence of 10% glycerol, 3 mg/mL purified tubulin, and 10 mM GTP.
Table 4. Inhibition of Tubulin Assembly and Inhibition of Colchicine
Binding
colchicine in its X-ray crystal structure with tubulin. The
phenyl ring of (R)-3 mimics the A ring of colchicine and is
inhibition of
involved in hydrophobic interactions with amino acids
colchicine binding
from β-tubulin (Leuβ248, Alaβ250, Leuβ255, and Alaβ316).
inhibition of
Additionally, the methyl group of the 40-OCH3 moiety could
tubulin assembly
% Inhibition ( SD
interact with the side chain of Ileβ378 and/or with the side
chain of Valβ318.
cmpd
CA4
IC50 ( SD (nM)
1 μM
5 μM
We45-49 and others50 have previously demonstrated that
similar CH3 moieties dictate the biological activities predi-
cated on hydrophobic interactions and/or conformational
restriction. It was of interest to extend these findings to pro-
vide a rationale for the high potency of the N-CH3 analogues
of the present study compared with the N-desmethyl analogues.
The N-CH3 group of (R)-3 occupies a region in space in
proximity to the C5 and C6 atoms of the B-ring of DAMA
colchicine. In this orientation, the N-CH3 group is involved
in hydrophobic interactions with the side chain C atoms of
Lysβ254 and Alaβ250. An additional hydrophobic interac-
tion between the N-CH3 moiety of (R)-3 and the side chain
C-atom of Leuβ248 also occurs due to the flexible nature of
the protein (measured distance between N-CH3 and side
1.0 ( 0.09
2.6 ( 0.05
1.9 ( 0.01
88 ( 2
NDa
99 ( 0.2
70 ( 2
84 ( 3
1
(()-3 HCl
60 ( 2
3
a ND: Not determined.
used are provided in the Experimental Section. Multiple low-
energy conformations (within 1 kcal/mol of the best pose)
were obtained on docking (R)-3 and other analogues. The
multiple docked poses can be explained by the large volume
of the active site (10 A ꢀ 10 A ꢀ 4-5 A).41 Figure 6 shows the
docked conformation of (R)-3 which was arbitrarily selected
as a working model for the docking of compounds 1-7 on
the basis of their structural similarity to the bound confor-
mation of DAMA colchicine. The pose in Figure 6 for (R)-3
had a score (-6.8379 kcal/mol) within 1 kcal/mol of the best
scored pose. Comparison of the docked conformation of
(R)-3 with the crystal structure conformation of DAMA
colchicine shows overlap of the 40-OCH3 phenyl group of
(R)-3 with the trimethoxybenzene A-ring of DAMA col-
chicine (Figure 6). In this pose, the 40-OCH3 of (R)-3
overlaps with the 30-OCH3 group in the A ring of DAMA
colchicine. Similar interactions of the -OCH3 groups
with Cysβ241 have been reported in the literature.41,44
The conformation of (R)-3 depicted in Figure 6 permits
the formation of a hydrogen bond between Cysβ241 and
the oxygen atom of the 40-OCH3 of (R)-3 as is observed
between Cysβ241 and the 30-OCH3 group of DAMA
˚
˚
˚
˚
chain C of Leuβ248 is 4.21 A). These interactions could
assist in stabilization of the docked conformation of (R)-3
and could explain, in part, the observed difference in activity
of the N-desmethyl analogue, (R)-4, which would lack these
additional interactions (Figure 7). The N-CH3 also aids in
maintaining the relative conformations of the cyclopenta-
[d]pyrimidine and the phenyl ring. While similar docked
poses were observed for (R)-4, the docked poses of com-
pounds with the N-CH3 group consistently scored higher
(∼1 kcal/mol) than those of compounds that lack the N-CH3
group.
A systematic conformational search carried out using
Sybyl X 1.151 for energy minimized structures of (R)-3 and

8122 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Gangjee et al.
Figure 6. Stereo view. Docked pose of (R)-3 (white) overlaid with DAMA colchicine (red) in the colchicine site of tubulin.
Figure 7. Stereo view. Superimposition of the docked poses of (R)-3 (white) and (R)-4 (magenta) in the colchicine site of tubulin.
(R)-4 returned a lower number of conformers for the N-CH3
compound (R)-3 compared with the NH compound (R)-4
using the same settings. This is indicative of restrictions on
the rotational freedom of the 40-OCH3 phenyl ring of (R)-3 in
the presence of the N-CH3 moiety and decreases the number
of low energy conformations of the 40-OCH3 phenyl ring
relative to the cyclopenta[d]pyrimidine scaffold of (R)-3.
Similar results were obtained for the conformational search
of 1 and 2.
binding poses and docked scores of (R)-3 (-6.8379 kcal/mol)
and (S)-3 (-6.9454 kcal/mol) due to the chirality of the
C6-CH3 group (Figure 8). Whether this slight predicted
difference between the two isomers is reflected in the bio-
logical activities can only be determined once the two isomers
are individually synthesized and evaluated.
While this work was in progress, studies describing a series
of quinazolines51-54 and thieno[3,2-d]pyrimidines55 as potent
apoptosis inducers were published. These reports suggest a
similar function for the N-CH3 moiety, but do not provide
molecular details about the binding modes of the quinazolines
and/or thieno[3,2-d]pyrimidines to tubulin.
Molecular modeling suggests that the binding interactions
afforded by the 40-OCH3 group on the anilino ring play an
important part and dictate the potency of these compounds
against tubulin, since deletion or moving the 40-OCH3 moiety
results in a significant loss of activity. However, molecular
modeling does not provide a reason(s) for the loss of potency of
The cyclopenta[d]pyrimidine of (R)-3 ring partially over-
laps with the C-ring of DAMA colchicine and is stabilized
by hydrophobic interactions with side chain C atoms of
Leuβ255, Asnβ258, and Lysβ252. The C7 of (R)-3 overlaps
the C9 carbonyl C of DAMA colchicine. The C2-CH3 moiety
of (R)-3 is involved in a hydrophobic interaction with
˚
AlaR180 (4.35 A), while the C6-CH3 group of (R)-3 is
involved in hydrophobic interactions with ValR181 and
Alaβ316. We did not observe a significant difference in the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8123
Figure 8. Stereo view. Superimposition of the docked poses of (R)-3 (white), (S)-3 (cyan), and DAMA colchicine (red) in the colchicine site of
tubulin.
Figure 9. Stereo view. Docking mode of (R)-3 (white) overlaid with docked poses of 15 ligands41 (blue) in the colchicine site of tubulin.
(()-6 (20-OCH3) compared with (()-3. We speculate that the
loss of potency could be explained by the loss of interactions
of the 40-OCH3 moiety of (()-3 and/or the additional con-
formational restriction of the anilino ring by a combination
of N-methylation and the 20-OCH3 moiety on the phenyl ring
or both.
As an example, (R)-3 retains the key binding interactions
exhibited by the known tubulin inhibitors and is in a confor-
mation consistent with those for the reported compounds41
(Figure 9). Similar docked poses were observed in this study for
compounds 1-7.
In summary, we report the synthesis and identification of the
The docking mode of 1 (not shown) was strikingly similar
to the docking mode of (R)-3. The pyrrole NH of 1 was not
involved in a hydrogen bond with the amino acid residues of
the tubulin binding site. Additionally, 1 lacks the 6-CH3
substitution in (R)-3 and could thus lack the additional
bonding afforded by the substituent at C6. The docked pose
of 1 had a score of -6.0330 kcal/mol, somewhat less but
comparable to that of (R)- and (S)-3.
mechanism of action of water-soluble, colchicine site microtu-
bule depolymerizing agents 1 and (()-3 HCl. These compounds
3
inhibit the growth of tumor cells with GI50 values in the nano-
molar range and overcome two clinically important tumor
resistance mechanisms that limit activity of microtubule target-
ing agents, expression of Pgp and βIII tubulin. For these
analogues, the 6-CH3 cyclopenta[d]pyrimidine scaffold is
better than the pyrrolo[2,3-d]pyrimidine scaffold, and
both the 4-N-methyl and 40-OCH3 groups on the aniline
ring are required for optimal potency. We have also
We also compared the binding mode of compounds 1-7 to
the proposed binding modes41 of 15 known tubulin inhibitors.

8124 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Gangjee et al.
provided an explanation of the importance of the N-CH3
moiety of 1 and (()-3 HCl for biological activity via mole-
was refluxed for 4 h. After evaporation of the solvents, a silica gel
plug (2 g) was made and loaded to a silica gel column. The column
was eluted sequentially with 0% and 1% methanol in chloroform.
Fractions containing the product (TLC) were pooled and evapo-
rated to afford 1 as a white solid (1.14 g, 85%). Rf 0.60 (CH3OH:
CHCl3 = 1:5); mp, 229.3-229.5 °C. 1H NMR (DMSO-d6): δ 2.45
(s, 3 H, 2-CH3), 3.45 (s, 3 H, NCH3), 3.82 (s, 3 H, OCH3), 4.52 (d, 1
H, 5-H, J = 3.50 Hz), 6.73 (d, 1 H, 6-H, J = 3.55 Hz), 7.04 (d, 2 H,
phenyl, J = 8.85 Hz), 7.27 (d, 2 H, phenyl, J = 8.85 Hz), 11.27 (s,
1 H, 7-H, D2O exchanged). HRMS m/z calcd for C15H17N4O
[M þ H]þ, 269.1397; found, 269.1385 [M þ H]þ. Anal. (C15H16-
N4O) C, H, N.
3
cular modeling. Compounds 1 and (()-3 HCl are important
3
lead analogues for the development of further compounds for
in vivo evaluation and possible advancement into clinical trials.
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 witha FLUKE51 K/J electronic thermometer, andare
uncorrected. Nuclear magnetic resonance spectra for proton
(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; 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 230-400 mesh silica gel (Fisher Scientific) column. Elemental
analyses were performed by Atlantic Microlab, Inc., Norcross,
GA. Element compositions are within (0.4% of the calculated
values and indicate >95% purity. Fractional moles of water or
organic solvents frequently found in some analytical samples
could not be prevented despite 24-48 h of drying in vacuo and
were confirmed where possible by their presence in the 1H NMR
spectra. Mass spectrum data were acquired on the Agilent
G6220AA TOF LC/MS system using the nano ESI (Agilent chip
tube system with infusion chip). All solvents and chemicals were
purchased from Sigma-Aldrich Co. or Fisher Scientific Inc. and
were used as received.
2-Methyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (10). The syn-
thesis of 10 utilized a reported method.35 To a solution of ethyl
2-cyanoacetate 8 (10 mmol, 1.13 g) in anhydrous dimethylforma-
mide (DMF, 20 mL) was added sodium methoxide (10 mmol,
0.54 g). After stirring for 30 min, 2-bromo-1,1-diethoxyethane
(10 mmol, 1.97 g) was added, and the reaction was heated at 90 °C
for 4 h. After cooling to room temperature, the reaction solution
was extracted with diethyl ether (2 ꢀ 20 mL). The ether layer was
collected, dried over sodium sulfate, and evaporated to give a pale
yellow liquid. The liquid was added to a solution of acetamidine
hydrochloride 9 (10 mmol, 0.94 g) and sodium ethoxide
(10 mmol, 0.68 g) in ethanol (20 mL) and refluxed for 3 h.
The solution was evaporated to dryness and extracted with ethyl
acetate and water. The organic layer was collected and evapo-
rated to afford a solid. Concentrated sulfuric acid (2 mL) in
ethanol (10 mL) was added to the solid, and the mixture was kept
at reflux for 2 h. At the end of the reaction, water (10 mL) was
added and the pH was adjusted to 8 using ammonium hydroxide.
The precipitate obtained was collected and dried to give 10 as
an off-white powder (0.85 g). Compound 10 was used without
further purification.
N-(4-Methoxyphenyl)-N,2-dimethyl-7H-pyrrolo[2,3-d]pyri-
midin-4-amine HCl (1 HCl). The HCl salt of 1 was obtained
3
3
by treating its ether/ethyl acetate solution with HCl gas in an
ice bath for 5 min. The white salt as it formed precipitated
from the solution. The salt was collected and dried to give a
white solid; mp 247.5-249.7 °C. 1H NMR (DMSO-d6): δ 2.64
(s, 3 H, 2-CH3), 3.62 (s, 3 H, NCH3), 3.81 (s, 3 H, OCH3), 4.59
(s, 1 H, 5-H), 7.00 (s, 1 H, 6-H), 7.12 (d, 2 H, phenyl, J = 8.45
Hz), 7.41 (d, 2 H, phenyl, J = 8.30), 12.32 (s, 1 H, 7-H, D2O
exchanged). Anal. (C15H16N4O) C, H, N.
N-(4-Methoxyphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-
4-amine (2). The synthesis of 2 followed the same method as for
1 using 11 (5 mmol, 0.84 g) and 4-methoxy-N-methylaniline 12
(5.5 mmol, 0.68 g). After column chromatography with 0% and
1% methanol in chloroform, fractions containing the product
(TLC) were pooled and evaporated to afford 2 as a white solid
(0.95 g, 75%). Rf 0.42 (CH3OH:CHCl3 = 1:5); mp, 258.7-
259.9 °C. 1H NMR (DMSO-d6): δ 2.42 (s, 3 H, 2-CH3), 3.73 (s, 3
H, OCH3), 6.55 (d, 1 H, 5-H, J = 3.24 Hz), 6.91 (d, 2 H, phenyl,
J = 8.96 Hz), 7.05 (d, 1 H, 6-H, J = 3.44 Hz), 7.74 (d, 2 H,
phenyl, J = 8.92 Hz), 9.02 (s, 1H, N-H), 11.41 (s, 1 H, 7-H,
D2O exchanged). HRMS m/z calcd for C14H15N4O [M þ H]þ,
255.1240; found, 255.1234 [M þ H]þ. Anal. (C14H14N4O) C, H, N.
2,6-Dimethyl-6,7-dihydro-3H-cyclopenta[d]pyrimidin-4(5H)-
one ((()-14). 3-Methyladipic acid (()-13 (1.60 g, 10 mmol) was
heated at reflux in ethanol/conc. sulfuric acid solution (35 mL,
v/v = 2.5/1) for 8 h. The solution was neutralized with ammo-
nium hydroxide to pH = 7 and 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 which 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. Acetamidine 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 reaction mixture
was cooled, and the precipitate was collected by filtration. The
residue was washed with warm methanol twice (30 mL ꢀ 1,
15 mL ꢀ 1). The filtrate and washings were combined, and then
3 g of silica gel was added and removed the solvent 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
0.35 g of (()-14 (21% yield total for three steps) as a white solid.
4-Chloro-2-methyl-7H-pyrrolo[2,3-d]pyrimidine (11). Compound
10 (5 mmol, 0.75 g) was treated with POCl3 (10 mL) and heated
under reflux for 3 h, and the remaining POCl3 was removed in
vacuo. Ammonium hydroxide was carefully added to the resulting
slurry, followed by extraction with ethyl acetate. After combining
the organic phases and evaporation, compound 11 was obtained as
a pale yellow powder (0.67 g, 81%) and used without further
purification. Rf 0.45 (CH3OH:CHCl3 = 1:5). An analytical sample
was purified for 1H NMR (DMSO-d6): δ 2.28 (s, 3 H, 2-CH3), 6.35
(d, 1 H, 5-H, J = 3.28 Hz), 6.93 (d, 1 H, 6-H, J = 3.28 Hz), 11.67
(s, 1 H, 7H, D2O exchanged).
1
TLC Rf 0.30 (CHCl3/CH3OH, 10:1); mp 173.9-175.4 °C. H
NMR (DMSO-d6): δ 1.05-1.07 (d, 3 H, CH3, J = 6.8 Hz), 2.24
(s, 3 H, CH3), 2.13-2.34, 2.73-2.90 (m, 5 H, CH2CHCH2),
12.16 (br, 1 H, OH, exchanged). Anal. (C9H12N2O) C, H, N.
4-Chloro-2,6-dimethyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine
((()-15). A mixture of (()-14 (0.25 g, 1.5 mmol) and POCl3 (10
mL) was heated at reflux for 3 h. The reaction mixture was cooled
and evaporated at reduced pressure. The residue was diluted with
N-(4-Methoxyphenyl)-N,2-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-
4-amine (1). To a solution of compound 11 (5 mmol, 0.84 g) in
isopropanol (4 mL) was added 4-methoxy-N-methylaniline 12
(5.5 mmol, 0.75 g) and a drop of conc. HCl. The reaction mixture

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8125
chloroform (50 mL) and neutralized with ammonium hydroxide
slowly in an ice bath. The organic portion was washed with water
(3 ꢀ 30 mL) and dried with anhydrous sodium sulfate. Concen-
tration of the organic solvent with 1 g of silica gel afforded a dry
plug. This plug was placed on the top of a silica gel column and
eluted with 20% hexane in chloroform. Fractions containing the
product were pooled and evaporated to afford 0.14 g of (()-15 as
a colorless liquid (69%). Compound (()-15 was unstable and was
used for the next step without further characterization.
procedure described above to afford after purification as a
colorless liquid. Rf 0.37 (CHCl3/CH3OH, 10:1). Compound
(()-6 HCl was synthesized from (()-6 as a white solid (0.23 g,
3
1
55% for two steps); mp 148-149 °C. H NMR (DMSO-d6): δ
0.83-0.85 (d, 3 H, CH3, J = 6.8 Hz), 1.31-1.39 (m, 1 H, CH),
1.82-1.93 (m, 1 H, CH), 2.26-2.31 (m, 1 H, CH), 2.43-2.46 (m,
1 H, CH), 2.99-3.07 (m, 1 H, CH), 2.62 (s, 3 H, 2-CH3), 3.45 (s, 3
H, NCH3), 3.79 (s, 3 H, OCH3), 7.03-7.51 (m, 4 H, Ph-H), 15.09
(br, 1 H, HCl, exchanged). Anal. (C17H22N3OCl 0.1H2O) C, H,
3
General Procedure for (()-3-(()-7. Compound (()-15 and
aniline or the appropriate substituted N-methyl anilines were
dissolved in isopropanol (5 mL). To this solution was added
37% hydrochloric acid (2-3 drops). The mixture was heated at
reflux for 3-6 h. Then the reaction was cooled and evaporated
at reduced pressure. The residue was diluted with chloroform,
neutralized with ammonium hydroxide, and then washed with
water (2 ꢀ 30 mL). After drying with anhydrous sodium sulfate,
organic solvent with 1 g of silica gel was evaporated under
reduced pressure to give a dry plug. This plug was placed on the
top of a silica gel column and eluted with chloroform. Fractions
containing the product were pooled and evaporated to afford
pure compounds (()-3-(()-7. Compounds were dissolved in
anhydrous ether (10 mL), and then anhydrous hydrochloric acid
gas was bubbled into the solution until no more solid precipi-
tated out. The white solid was collected by filtration and dried
over P2O5 to afford (()-3 HCl, (()-5 HCl-(()-7 HCl.
N, Cl.
N-Phenyl-N,2,6-trimethyl-6,7-dihydro-5H-cyclopenta[d]pyri-
midin-4-aminium chloride ((()-7 HCl). Compound (()-7 was
3
synthesized from (()-15 (0.18 g, 0.99 mmol) and N-methylani-
line (0.11 g, 0.99 mmol) using the general procedure described
above to afford after purification 0.21 g (83%) as a white solid.
Rf 0.36 (CHCl3/CH3OH, 10:1); mp 112.6-113.2 °C. 1H NMR
(DMSO-d6): δ 0.81-0.83 (d, 3 H, CH3), 1.40-1.44, 1.86-2.26,
2.74-2.80 (m, 5 H, CH2CHCH2), 2.41 (s, 3 H, 2-CH3), 3.37
(s, 3 H, NCH3), 7.18-7.41 (m, 4 H, Ph-H). Anal. (C16H19N3) C,
H, N. Compound (()-7 HCl was synthesized from (()-7 (0.17 g,
3
0.65 mmol) as a white solid (0.15 g, 79%); mp 263.2-
264.6 °C. 1H NMR (DMSO-d6): δ 0.83-0.85 (d, 3 H, CH3, J =
6.8 Hz), 1.35-1.42 (m, 1 H, CH), 1.81-1.91 (m, 1 H, CH), 2.24-
2.32 (m, 1 H, CH), 2.42-2.45 (m, 1 H, CH), 2.99-3.05 (m, 1 H,
CH), 2.61 (s, 3 H, 2-CH3), 3.55 (s, 3 H, NCH3), 7.41-7.53 (m,
4 H, Ph-H), 15.09 (br, 1 H, HCl, exch). Anal. (C16H20ClN3) C,
H, N, Cl.
3
3
3
N-(4-Methoxyphenyl)-N,2,6-trimethyl-6,7-dihydro-5H-cyclo-
penta[d]pyrimidin-4-aminium chloride ((()-3 HCl). Compound
Molecular Modeling and Computational Studies. The X-ray
3
˚
(()-3 was synthesized from (()-15 (0.19 g, 1.01 mmol) and
4-methoxy-N-methyl aniline (0.17 mg, 1.22 mmol) using the
general procedure described above to afford after purification
0.18 g (61%) as a light yellow liquid. TLC Rf 0.24 (CHCl3/
crystal structure of tubulin at 3.58 A resolution was obtained
from the protein database (PDB ID 1SA040). This crystal
structure contains the Rβ dimers of tubulin complexed with
N-deacetyl-N-(2-mercaptoacetyl) colchicine (DAMA colchicine),
a close structural analogue of colchicine.
CH3OH, 10:1). Compound (()-3 HCl was synthesized from
3
(()-3 as a white solid (0.16 g, 79%); mp 196-198 °C. 1H NMR
(DMSO-d6): δ 0.85-0.86 (d, 3 H, CH3, J = 6.8 Hz), 1.37-1.47
(m, 1 H, CH), 1.89-1.99 (m, 1 H, CH), 2.26-2.35 (m, 1 H, CH),
2.43-2.44 (m, 1 H, CH), 2.98-3.05 (m, 1 H, CH), 2.60 (s, 3 H,
2-CH3), 3.51 (s, 3 H, NCH3), 3.80 (s, 3 H, OCH3), 7.01-7.03 (d,
2 H, Ph-H, J = 8.8 Hz), 7.33-7.35 (d, 2 H, Ph-H, J = 8.8 Hz),
14.88 (br, 1 H, HCl, exch). Anal. (C17H22N3OCl) C, H, N, Cl.
N-(4-Methoxyphenyl)-2,6-dimethyl-6,7-dihydro-5H-cyclopenta-
[d]pyrimidin-4-amine ((()-4). Compound (()-4 was synthesized
from (()-15 (0.17 g, 0.94 mmol) and 4-methoxyaniline (0.14 g,
1.13 mmol) using the general procedure described above to afford
after purification 0.16 g (61%) as a white solid. TLC Rf 0.32
(CHCl3/CH3OH, 10:1); mp 130.5-131.3 °C. 1H NMR (DMSO-
d6): δ 1.09-1.12 (d, 3 H, CH3, J = 6.8 Hz), 2.35 (s, 3 H, 2-CH3),
1.36-1.48 (m, 1 H, CH), 1.88-2.01 (m, 1 H, CH), 2.25-2.35 (m, 1
H, CH), 2.41-2.45 (m, 1 H, CH), 2.98-3.05 (m, 1 H, CH), 3.73 (s,
3 H, OCH3), 6.86-6.89 (d, 2 H, Ph-H, J = 8.8 Hz), 7.60-7.63 (d,
2 H, Ph-H, J = 8.8 Hz), 8.41 (br, 1 H, NH, exchanged). Anal.
(C16H19N3O) C, H, N.
Docking Procedure. Docking studies were performed using
the docking suite of Molecular Operating Environment soft-
ware (MOE 2008.10).43 The PDB file was downloaded (www.
pdb.org) and imported into MOE. The A and B subunits of the
protein, along with the crystallized ligand, were retained, while
the C, D, and E subunits, GTP, GDP, and Mg ions were deleted.
After addition of hydrogen atoms, the protein was then “pre-
pared” using the LigX function in MOE. LigX is a graphical
interface and collection of procedures for conducting interactive
ligand modification and energy minimization in the active site of a
flexible receptor. In LigX calculations, the receptor atoms far from
the ligand are held fixed (constrained not to move) while receptor
atoms near the ligand (in the active site) are allowed to move but
are subject to tether restraints that discourage gross movement.
The procedure was performed with the default settings.
Ligands were built using the molecule builder function in
MOE and were energy minimized to its local minima using the
MMF94X forcefield to a constant (0.05 kcal/mol).
Ligands were docked into the active site of the prepared
protein using the docking suite as implemented in MOE. The
docking was restricted to the active site pocket residues using the
Alpha triangle placement method. Refinement of the docked
poses was carried out using the Forcefield refinement scheme
and scored using the Affinity dG scoring system. Around 30
poses were returned for each compound at the end of each
docking run. The docked poses were manually examined in the
binding pocket to ensure quality of docking and to confirm
absence of steric clashes with the amino acid residues of the
binding pocket.
N-(3-Methoxyphenyl)-N,2,6-trimethyl-6,7-dihydro-5H-cyclo-
penta[d]pyrimidin-4-aminium chloride ((()-5 HCl). Compound
3
(()-5 was synthesized from (()-15 (0.23 g, 1.3 mmol) and
3-methoxy-N-methylaniline (0.20 g, 1.4 mmol) using the general
procedure described above to afford after purification as a light
yellow liquid. TLC Rf 0.33 (CHCl3/CH3OH, 10:1). Compound
(()-5 HCl was synthesized from (()-5 as an off white solid
3
(0.27 g, 66% for two steps); mp 209.6-210.7 °C. 1H NMR
(DMSO-d6): δ 0.85-0.87 (d, 3 H, CH3, J = 6.8 Hz), 1.44-1.50
(m, 1 H, CH), 1.95-2.01 (m, 1 H, CH), 2.26-2.35 (m, 1 H, CH),
2.45-2.48 (m, 1 H, CH), 3.00-3.07 (m, 1 H, CH), 2.62 (s, 3 H,
2-CH3), 3.55 (s, 3 H, NCH3), 3.78 (s, 3 H, OCH3), 6.69-7.42 (m,
To validate the utility of MOE 2008.10 for docking ligands
into the active site, DAMA colchicine, the native ligand in the
crystal structure (PDB: 1SA0), was built using the molecule
builder, energy minimized, and docked into the active site using
the above parameters. The best docked pose of DAMA colchi-
4 H, Ph-H), 15.21 (br, 1 H, HCl, exchanged). Anal. (C17H22N3OCl
0.1H2O) C, H, N, Cl.
N-(2-Methoxyphenyl)-N,2,6-trimethyl-6,7-dihydro-5H-cyclo-
3
˚
cine displayed an rmsd of 0.9531 A compared with the crystal
structure pose of DAMA colchicine. MOE 2008.10 was thus
validated for our docking studies. Docking studies of the lead
penta[d]pyrimidin-4-aminium chloride ((()-6 HCl). Compound
3
(()-6 was synthesized from (()-15 (0.24 g, 1.31 mmol) and
2-methoxy-N-methylaniline (0.18 g, 1.31 mmol) using the general

8126 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Gangjee et al.
compounds and selected proposed molecules were performed
using the same procedure.
Grant CA098850 (AG). We thank Desiree LeBouf and Cara
Westbrook for excellent technical assistance. A.L.R. was sup-
ported by a Cowles Fellowship, and support from the CTRC
Cancer Center Support Grant, CCSG (CA054174) (S.L.M.) is
acknowledged.
Coordinates of the binding models of the 15 known tubulin
inhibitors reported by Nguyen et al.41 were retrieved from the
Supporting Information. The coordinates as well as the docked
pose of (R)-3 were imported and visualized using MOE 2008.10.
A systematic conformational search was carried out using Sybyl
X 1.151 using 5° increments. Molecules were built using the
molecule builder function in MOE 2008.10 and were imported
into Sybyl X 1.1 and energy minimized using the Tripos force-
field with a distance-dependent dielecetric and the Powell con-
jugate gradient with a convergence criterion of 0.01 kcal/mol.
Cellular Studies. Effects of Compounds on Cellular Microtu-
bules. A-10 cells were used to evaluate the effects of the compounds
on interphase and mitotic microtubules using indirect immuno-
fluorescence techniques, and the EC50 values were calculated from
a minimum of three experiments as described previously.25
Sulforhodamine B (SRB) Assay. The SRB assay37,38 was used
to evaluate the antiproliferative and cytotoxic effects of the
compounds against cancer cells as previously described.39 MDA-
MB-435 cells were obtained from the Lombardi Cancer Institute
(Washington D.C.). SK-OV-3 and HeLa cells were purchased
from the American Type Culture Collection (Manassas, VA).
The methods used to generate and characterize the SK-OV-3
MDR-1-6/6 and WTβIII cell lines have been described.39 The
IC50 represents the mean of at least three independent experi-
ments using triplicate points in each experiment.
Supporting Information Available: Results from elemental
analysis: high resolution mass spectra (HRMS) (EI); high
resolution mass spectra (HRMS) (ESI); and representation of
cell cycle distribution data (Figure 4). This material is available
free of charge via the Internet at http://pubs.acs.org.
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