Design, Synthesis, and Preclinical Evaluation of 4-Substituted-5-methyl-furo[2,3-d]pyrimidines as Microtubule Targeting Agents That Are Effective against Multidrug Resistant Cancer Cells

Article abstract of DOI:10.1021/acs.jmedchem.6b00237

The design, synthesis, and biological evaluations of eight 4-substituted 5-methyl-furo[2,3-d]pyrimidines are reported. Synthesis involved N⁴-alkylation of N-aryl-5-methylfuro[2,3-d]pyrimidin-4-amines, obtained from Ullmann coupling of 4-amino-5

Full text of DOI:10.1021/acs.jmedchem.6b00237

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Article
Design, Synthesis and Preclinical Evaluation of 4-Substituted-5-
methyl-furo[2,3-d]pyrimidines as Microtubule Targeting Agents
that are Effective against Multidrug Resistant Cancer Cells
Ravi Kumar Vyas Devambatla, Ojas A. Namjoshi, Shruti Choudhary, Ernest Hamel,
Corena V. Shaffer, Cristina C. Rohena, Susan L. Mooberry, and Aleem Gangjee
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 23 May 2016
Downloaded from http://pubs.acs.org on May 23, 2016
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Design, Synthesis and Preclinical Evaluation of 4-Substituted-5-methyl-furo[2,3-
d]pyrimidines as Microtubule Targeting Agents that are Effective against Multidrug
Resistant Cancer Cells
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Ravi Kumar Vyas Devambatla†,#, Ojas A. Namjoshi†,#,¶, Shruti Choudhary†, Ernest Hamel£,
Corena V. Shaffer§, Cristina C. Rohena§, Susan L. Mooberry*,§,ǁ, Aleem Gangjee*,†,ǁ
† Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne
University, 600 Forbes Avenue, Pittsburgh, PA 15282,
§ Department of Pharmacology, Cancer Therapy & Research Center, University of Texas Health
Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229 and
£
Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer
Treatment and Diagnosis, National Cancer Institute, Frederick National Laboratory for Cancer
Research, National Institutes of Health, Frederick, MD 21702.
Abbreviations: National Cancer Institute (NCI); microtubule targeting agents (MTAs);
combretastatin A-1 (CA-1); combretastatin A-4 (CA-4); dimethyl sulfoxide (DMSO);
dimethylformamide (DMF); Protein Data Bank (PDB); sulforhodamine B (SRB); P-glycoprotein
(Pgp).
Abstract
The design, synthesis and biological evaluations of eight 4-substituted 5-methyl-furo[2,3-
d]pyrimidines are reported. Synthesis involved N4-alkylation of N-aryl-5-methylfuro[2,3-
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d]pyrimidin-4-amines, obtained from Ullmann coupling of 4-amino-5-methylfuro[2,3-
d]pyrimidine and appropriate aryl iodides. Compounds 3, 4 and 9 showed potent microtubule
depolymerizing activities, while compounds 6‒8 had slightly lower potency. Compounds 4, 6, 7
and 9 inhibited tubulin assembly with IC50 values comparable to that of combretastatin A-4 (CA-
4). Compounds 3, 4, and 6‒9 circumvented Pgp and βIII-tubulin mediated drug resistance,
mechanisms that can limit the efficacy of paclitaxel, docetaxel and the vinca alkaloids. In the
NCI 60-cell line panel, compound 3 exhibited GI50 values less than 10 nM in 47 of the cell lines.
In an MDA-MB-435 xenograft model, compound 3 had statistically significant antitumor effects.
The biological effects of 3 identify it as a novel, potent microtubule depolymerizing agent with
antitumor activity.
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Introduction
Microtubules are filamentous cytoskeleton protein polymers composed of -tubulin
heterodimers. Microtubules play an important role in many aspects of cellular function, including
cellular transport, protein trafficking and mitosis. Microtubule targeting agents (MTAs) are some
of the most effective drugs used to treat cancer.1-3 MTAs are the only class of cytotoxic agents
effective against p53-mutant cell lines, which constitute 39 of the 58 cell lines in the NCI 60-cell
line panel.4, 5 MTAs are widely used for the treatment of both solid tumors and hematological
malignancies.1-3 These drugs are structurally diverse and are classified into two main groups:
microtubule stabilizers and microtubule destabilizers. Microtubule stabilizers promote
microtubule polymerization and include the taxanes, paclitaxel, docetaxel and cabazitaxel and
the epothilone, ixabepilone. Microtubule destabilizers induce microtubule depolymerization and
include the vinca alkaloids and colchicine-site agents. However, at low antiproliferative
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concentrations, both types of agents suppress microtubule dynamics leading to mitotic arrest and
subsequent cell death in cells in culture.1, 2 New evidence suggests that the ability of these drugs
to inhibit interphase signaling events likely contributes to their anticancer actions.6
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Figure 1. Representative microtubule targeting agents.
MTAs are divided into 5 classes based on their interactions within the taxane, vinca,
colchicine, laulimalide/peloruside or the maytansine site on tubulin (Figure 1). Paclitaxel,
docetaxel, cabazitaxel and ixabepilone bind to -tubulin in the interior of the microtubule, a site
referred to as the taxane site.7, 8 The taxanes are widely used in the treatment of adult solid
tumors.9, 10 The vinca alkaloids, including vincristine, vinblastine, vindesine and vinorelbine, as
shown in a crystal structure with vinblastine, require two distinct tubulin αβ-heterodimers to
form a binding site. The site is formed by amino acid residues from both α- and β-tubulin, and
the vinblastine molecule was located between the two heterodimers. The vinca alkaloids are
important in the treatment of hematological malignancies, lymphoma and childhood cancers.9
Laulimalide and peloruside A bind to a distinct site on β-tubulin, referred to as the
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laulimalide/peloruside site, and both compounds cause microtubule stabilization. This site is
located on the exterior of the microtubule.11 Recently, Prota et al.12 demonstrated that
maytansine binds to a different site on β-tubulin, and they named this the maytansine site.
Maytansine, rhizoxin F and a polyketide PM060184, initially isolated from the marine sponge
Lithoplocamia lithistoides, bind within this newly characterized site, occupancy of which
prevents the addition of new subunits to the plus ends of microtubules, resulting in microtubule
depolymerization.12 Maytansine is the cytotoxic component in the antibody drug conjugate
trastuzumab emtansine recently approved by the FDA for the treatment of HER2-positive
advanced breast cancer.13 The fifth class of MTAs comprises a variety of small molecules that
includes colchicine and combretastatins A-1 (CA-1) and A-4 (CA-4), which bind in the
colchicine site located on -tubulin at its interface with -tubulin.14, 15 Combretastatin A-4
phosphate (CA-4P, fosbretabulin) and A-1 diphosphate (CA-1P, OXi4503), 2-methoxyestradiol
and verubulin16, 17 are a few of the colchicine site binding agents which have been evaluated in
phase 1 and 2 clinical trials as anticancer agents alone and in combination with other drugs. Thus
far, no colchicine site agent has been approved as an anticancer agent.18-24 Hence, this site
provides new opportunities for drug discovery.
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Rationale and Molecular Modeling
We previously reported25 that cyclopenta[2,3-d]pyrimidine (±)-1·HCl (Figure 2) has potent
microtubule depolymerization activity (EC50 in A-10 cells = 47 nM) and in vitro cytotoxic
effects against MDA-MB-435 cancer cells (IC50 ± SD = 18.8 ± 0.4 nM).
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Figure 2. Parent 5-methyl-cyclopenta[2,3-d]pyrimidine (±)-1 and target 5-methyl-furo[2,3-
d]pyrimidines 2 and 3.
Replacement of the cyclopentyl and the 2-methyl groups of the potent MTA (±)-1 with a
furan and a 2-H moiety, respectively, afforded the 5-methyl-furo[2,3-d]pyrimidine scaffold of 2
and 3 (Figure 2). It was of interest to explore the antitumor activity of compounds with the 5-
methyl-furo[2,3-d]pyrimidine scaffold without the 2-position substitution as MTAs to determine
the contribution of the 2-position substitution and the furo ring to both inhibition of cancer cell
proliferation and microtubule depolymerization. Additionally, these lead compounds are similar
in structure to verubulin (Figure 1), which is a N4,2-dimethylquinazoline-4-amine analog with p-
methoxyphenyl substitution at N4-position.16 Similar N4-p-methoxyphenyl substitution pattern
has not been explored before in 5-methyl-furo[2,3-d]pyrimidine series of compounds.
Compounds 2 and 3 were docked in the X-ray crystal structure of colchicine in tubulin at the
colchicine site (PDB: 4O2B,26 2.30 Å) using Molecular Operating Environment MOE
2013.0801.27 Multiple low energy conformations were obtained on docking. Figure 3 shows the
docked conformation of 3 (green) as a representative example in the colchicine site of tubulin.
The furo[2,3-d]pyrimidine scaffold of 3 overlaps the C-ring of colchicine (orange) and forms
hydrophobic interactions at the αβ tubulin interface with Alaα180, Asnβ258 and Lysβ352. The 5-
Me group of 3 interacts with Leuβ255, Asnβ258 and Metβ259 of tubulin. The N-Me moiety of 3
mimics the bridged C5- and C6-positions of the B-ring of colchicine and forms hydrophobic
interactions with Leuβ248, Alaβ250 and Lysβ254. The 4'-OMe-Ph of 3 is oriented towards the
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hydrophobic pocket formed by Thrβ240, Cysβ241, Alaβ316, Ileβ318, Ileβ378 and binds in the
region occupied by the A-ring of colchicine. The phenyl ring of 3 makes hydrophobic
interactions with Leuβ248, Leuβ255, Alaβ316 and Alaβ354. Figure 3 also shows that the docked
pose of 3 at the colchicine site is similar to that of the lead compound (S)-1. The best docked
pose of 3 had a score of -5.07 kcal/mol comparable to that of colchicine (-5.54 kcal/mol) and the
lead compound (S)-1 (-5.19 kcal/mol).
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Figure 3. Superimposition of the docked pose of 3 (green) in the colchicine site (colchicine in
orange) of tubulin (PDB: 4O2B26).
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Design of target compounds 4‒9.
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The N-Me moiety of 3 forms hydrophobic interactions with Leuβ248, Alaβ250, Lysβ254
and Asnβ258 in the colchicine site (Figure 3). To optimize these hydrophobic interactions,
compounds 4‒7 (Figure 4) with larger alkyl groups at the N4-position were synthesized. In
addition, these compounds were also designed to determine the bulk tolerance at the colchicine
site for substituents at the N4-position for the furo[2,3-d]pyrimidine scaffold. The key binding
interactions indicated by the docking study of the lead compound 3 in the colchicine-binding site
of tubulin (Figure 3) are retained by target compounds 4‒7 with docking scores (-5.60 kcal/mol,
-5.11 kcal/mol, -4.35 kcal/mol, -5.12 kcal/mol respectively) similar to that of 3 (see Supporting
Information).
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Figure 4. Target compounds 4–9.
Compounds 8 and 9 were designed to improve hydrophobic interactions of the 4'-OMe of 3
within the hydrophobic pocket formed by residues Thrβ240, Cysβ241, Alaβ316, Ileβ318 and
Ileβ378 at the colchicine site as suggested by the docking scores of 8 and 9 (-5.16 kcal/mol and -
5.16 kcal/mol respectively), both of which were similar to that of 3. Thus, compounds 8 and 9
were designed to evaluate the role of the 4'-OMe group on tubulin depolymerization and
inhibition of cancer cell growth by substituting the oxygen of the 4'-OMe with a methylene and
sulfur respectively, thus decreasing the electronegative nature of the 4'-OMe substituent and
increasing the hydrophobic interactions at the colchicine site.
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Results and Discussion
Chemistry
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Scheme 1. Synthesis of target compounds 2 and 3
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Target compounds 2 and 3 were synthesized as described in Scheme 1. Compound 11 was
obtained by reacting hydroxyacetone 10 with malononitrile in the presence of triethylamine and
was used without purification for the subsequent step.28 Treatment of 11 with formamidine
hydrochloride under basic conditions provided 5-methyl-furo[2,3-d]pyrimidine 12 in 47% yield
(two steps). Ullmann coupling of 12 and 4-iodo anisole 13 using CuI and L-proline afforded
target compound 2. N4-Methylation of 2 with dimethyl sulfate furnished target compound 3 in
70% yield.
Scheme 2. Synthesis of target compounds 4–7
Compounds 4–7 were synthesized from 2 (Scheme 1) by N4-alkylation with the appropriate
alkyl iodides 14–17, respectively (Scheme 2).
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Scheme 3. Synthesis of target compounds 8 and 9
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Ullmann coupling of 12 and appropriate aryl iodides 18 and 19 (Scheme 3) using CuI and L-
proline afforded 20 and 21, respectively, which upon N4-methylation furnished target compounds
8 and 9, respectively.
Biological Results
Effects on microtubule depolymerization and cell proliferation. Compounds 2–9 were
first evaluated for their ability to depolymerize microtubules (Table 1 and Figure 5). Compounds
that caused at least 50% microtubule depolymerization at a concentration of 10 µM were further
evaluated to determine the EC50, the concentration that causes loss of 50% of cellular
microtubules in A-10 cells and for antiproliferative effects against MDA-MB-435 cancer cells.
The embryonic rat smooth muscle A-10 cells are useful to evaluate the effects of microtubule
targeting agents on interphase microtubules. Unlike cancer cells, A-10 cells do not accumulate in
mitosis in response to compounds that disrupt microtubules and allow effects on interphase
microtubules to be easily visualized and quantified. The N-H analog 2 was inactive in the
microtubule depolymerization assay and was not evaluated further. The N-Me analog 3 showed
potent microtubule depolymerization effects and also inhibited the proliferation of MDA-MB-
435 cells with potency comparable to those of CA-4 and paclitaxel. This indicates that for the
furo[2,3-d]pyrimidine scaffold the N-Me moiety is important for biological activity. Compound
3 was 2.3-fold more potent for microtubule depolymerizing effects and about 4-fold more potent
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antiproliferative activity (MDA-MB-435) than the lead compound (±)-1·HCl. Compounds 4 and
9 were equipotent with (±)-1·HCl as microtubule depolymerizers and were 1.4–2.1-fold more
active against MDA-MB-435 cells than (±)-1·HCl. These data indicate that the 2-desmethyl
furo[2,3-d]pyrimidine scaffold is conducive to both microtubule depolymerization and
antiproliferative effects.
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Table 1. IC50 values for inhibition of proliferation of MDA-MB-435 cells and EC50 values for
microtubule depolymerization.
EC50 for Microtubule
IC50 ± SD
Compd
Depolymerization
MDA-MB-435 (nM)
in A·10 Cells (nM)
(±)-1·HCla
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17.0 ± 0.7
(Lead)
2
> 10 μM
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NDb
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504 ± 28
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100 ± 6.5
183 ± 13
12.3 ± 0.9
NDb
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> 10 μM
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750
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> 10 μM
Paclitaxel
CA-4
4.5 ± 0.5
4.4 ± 0.5
9.8
aResults previously published.25 bND: Not determined.
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Compounds 4–7 with larger alkyl groups at the N4-position had lower microtubule
depolymerizing potency and higher antiproliferative IC50 values than that of the N-Me compound
3 (Table 1). The potency decreased with increased homologation and the N-Et analog 4 was 2-
fold less potent than 3 and was equipotent with 1. The N-iPr analog 5 was ineffective at
depolymerizing microtubules even at concentrations as high as 10 µM. However, compound 5
exhibited some antiproliferative activity, but it was 60-fold less potent (IC50) than the N-Et
analog 4. The N-Me analogs 8 and 9 with variations at the 4'-position had quite different
microtubule depolymerization activities in that 8 had an EC50 of 750 nM while 9 was quite
potent, with an EC50 of 45 nM (Table 1), and both were less potent than 3. The 4'-Et analog 8 had
a 30–fold lower potency for microtubule depolymerization than the 4'-OMe analog 3. In the X-
ray crystal structure (PDB: 4O2B26), there is a water mediated hydrogen bond between Cysβ241
of tubulin and the 3-OMe group on the A-ring of colchicine. The 4'-OMe group of 3 mimics the
3-OMe group on the A-ring of colchicine (Figure 3), suggesting its importance for the biological
activity of 3. The 4'-SMe analog 9 had a 2-fold lower microtubule depolymerizing potency than
the 4'-OMe analog 3. In addition, in the MDA-MB-435 cell antiproliferative assay, compounds 8
and 9 had 43- and 3-fold lower antiproliferative potencies, respectively, than 3. These data
indicate that bioisosteric replacement of the electronegative oxygen of the 4'-OMe group with
sulfur in the 2-H,5-methylfuro[2,3-d]pyrimidine scaffold is detrimental to cellular microtubule
depolymerization and to antiproliferative activity. The N4-desmethyl synthetic precursor, 21, like
2, was inactive as a MTA with EC50 >10 μM, substantiating the importance of the N4-alkyl
substitution.
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The effects of compounds 3, 4 and 6–9 on cellular microtubules in A-10 cells are shown in
Figure 5. A concentration of two-times their respective EC50 values caused almost complete loss
of the microtubule cytoskeleton, with only a few remnant microtubules remaining.
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Figure 5. Effects of compounds 3, 4 and 6–9 on cellular microtubules. A-10 cells were treated
for 18 h with 2x the EC50, and cellular microtubules were visualized by indirect
immunofluorescence with a β-tubulin antibody.
Effects on mitotic spindles and cell cycle distribution. A common propensity of MTAs is their
ability to interrupt the structure and function of mitotic spindles. The disruption of microtubule
dynamics leads to the formation of aberrant mitotic spindles that are unable to align the
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chromosomes into the metaphase plate leading to the accumulation of cells arrested in mitosis.
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Figure 6. Effects of compounds 3, 4 and 6–9 on mitotic spindles. HeLa cells were treated for 18
h with a concentration five-times the respective IC50, and cellular microtubules and mitotic
spindles were visualized by indirect immunofluorescence techniques with a β-tubulin antibody.
The effects of the microtubule active compounds on mitotic spindles and cell cycle
distribution were evaluated in HeLa cells. The results showed that cells treated with these
compounds were devoid of the normal bipolar spindles that were seen in vehicle-treated cells
(Figure 6). No mitotic spindles were seen in cells treated with any of the test compounds, but
multiple spindle poles were apparent, and these effects are consistent with effects seen with other
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colchicine site agents.29 These aberrant mitotic spindles suggested that these compounds could
initiate mitotic arrest, and thus the cell cycle distribution of the HeLa cells was evaluated by flow
cytometry (Figure 7).
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Figure 7. Cell cycle analysis of HeLa cells treated with 3, 4 and 6–9. (A) Cell cycle distribution
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profiles of cells treated for 18 h with five-times the IC50 of each compound. (B) Table showing
the percent of cells in G2/M in each treatment group. Values are reported as the mean ± SD from
2 independent experiments.
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In the flow cytometry study (Figure 7), typical results were obtained for cells treated with
the MTAs 3, 4 and 6–9. These compounds cause the accumulation of cells in the G2/M phase of
the cell cycle, as was observed following treatment with CA-4, while a normal cell cycle
distribution was observed in the vehicle-treated control cells (Figure7A). HeLa cells treated with
five-times the IC50 of 3, 4 or 6–9 exhibited a significant accumulation of cells in G2/M, as with
CA-4 (Figure 7A). The percentage of cells in G2/M in each treatment group was quantified
(Figure 7B).
Inhibition of tubulin assembly and colchicine binding. On the basis of their microtubule
depolymerizing activities, compounds 3–7 and 9 were evaluated for their direct effects on tubulin
assembly and inhibition of colchicine binding (Table 2). Except for 5, all the compounds at 5 µM
inhibited [3H]colchicine binding to the protein, and the extent of inhibition was similar to that
obtained with CA-4. Straight chain N4-alkyl analogs 4 (N-Et), 6 (N-Pr) and 7 (N-Bu) inhibited
tubulin assembly about as well as CA-4, and were about 2-fold more potent than the lead
compound (±)-1·HCl. In the colchicine inhibition assay, compounds 4 (N-Et), 6 (N-Pr) and 7 (N-
Bu), at 5 M, inhibited the binding of [3H]colchicine by 87‒95%, whereas the branched alkyl N-
iPr 5 showed only a 47% inhibition of [3H]colchicine binding. The lower activity of the sterically
bulky isopropyl moiety could be attributed to limited steric tolerance in the colchicine site. The
data indicate that N4-alkylation is tolerated to an extent as predicted by molecular modeling
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(Figure 3). Compound 9 (4'-SMe) inhibited tubulin assembly with an IC50 value comparable to
that of CA-4, and it is 3-fold better than the 4'-OMe analog 3. However it is interesting to note
that 3, despite its somewhat lower activity as an inhibitor of tubulin assembly, compared to CA-
4, it was a strong inhibitor of [3H]colchicine binding to tubulin.
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Table 2. Inhibition of tubulin assembly and colchicine binding.
Inhibition of colchicine binding
Inhibition of tubulin assembly
(% inhibition ± SD)
Compd
(IC50 ± SD (M))
1 µM
5 µM
(±)-1·HCla
1.9 ± 0.01
60 ± 2
84 ± 3
(Lead)
3
3.3 ± 0.5
71 ± 6
84 ± 0.7
ND
96 ± 2
4
0.97 ± 0.09
3.0 ± 0.2
95 ± 0.5
47 ± 0.2
92 ± 0.3
87 ± 0.2
94 ± 0.01
99 ± 0.2
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6
1.1 ± 0.1
75 ± 1
61 ± 0.4
79 ± 2
90 ± 1
7
1.3 ± 0.2
9
1.2 ± 0.007
0.96 ± 0.07
CA-4
aResults previously published.25
Ability to overcome multidrug resistance mechanisms. The clinical activity of the taxanes and
the vinca alkaloids is limited by two mechanisms of drug resistance: the expression of the drug
efflux pump Pgp and the III-isotype of tubulin.30 Pgp expression is observed clinically,
particularly in patients who have received prior chemotherapy.31 The expression of Pgp was
associated with poor response to paclitaxel-based chemotherapy in patients with non-small cell
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lung and breast cancer.31, 32 While the use of Pgp inhibitors to overcome this resistance has been
explored, it has been unsuccessful due to the side effects which occur when using the high doses
required.33 Tubulin binding agents that are not substrates for Pgp represent a viable alternate
strategy for circumventing Pgp-mediated resistance. Such agents would be extremely useful for
patients that develop tumor resistance due to Pgp expression.30, 34, 35
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The expression of βIII-tubulin is involved in clinical resistance to taxanes and vinca
alkaloids in non-small cell lung,36-38 breast,39 ovarian40, 41 and gastric42 cancers. Colchicine site
agents are generally not susceptible to βIII-tubulin mediated resistance,43, 44 illustrating the
importance of developing anticancer agents that bind to the colchicine site. Development of
tubulin binding agents that are less sensitive to Pgp and/or βIII-tubulin mediated resistance could
result in broader antitumor activity and improved rates of survival with significant advantages
over other MTAs.
Effect on Pgp and βIII-tubulin mediated cancer cell resistance. The potent MTAs 3, 4 and 6‒
9 were evaluated for their activity in the SK-OV-3 ovarian carcinoma cell line and the Pgp
expressing subline SK-OV-3 MDR-1-6/6 (Table 3).45 In these cell lines, compound 3 was the
most potent compound in the series, and it had 5-fold higher potency than the lead compound
(±)-1·HCl. In addition, compound 3 had potency similar to paclitaxel and CA-4 in the SK-OV-3
cell line. Comparison of the IC50 values in the parental SK-OV-3 and genetically manipulated
SK-OV-3 MDR-1-6/6 cell line allows for the calculation of a relative resistance value,
designated Rr. This value is calculated by dividing the IC50 value obtained in the Pgp-expressing
MDR-1-6/6 cells by the IC50 obtained in the parental SK-OV-3 cells. Paclitaxel, a known Pgp
substrate has a Rr value of 240, while CA-4, a poor substrate has a Rr value of 1.3 (Table 3).
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Compound 3 had IC50 values in SK-OV-3 and SK-OV-3 MDR-1-6/6 cells comparable to that of
CA-4 and a Rr of 1.1, indicating that it is able to overcome drug resistance mediated by Pgp.
Compounds 4 and 6‒9 also had Rr values less than 2, suggesting that they are all poor substrates
for Pgp-mediated transport and have advantages over the taxanes and vinca alkaloids.
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Table 3. Target compounds 3, 4 and 6–9 circumvent Pgp and βIII-tubulin mediated resistance.
IC50 ± SD (nM)
IC50 ± SD (nM)
HeLa
Compd
SK-OV-3
SK-OV-3 MDR1-M6/6 Rr
HeLa
Rr
WTβIII
(±)-1·HCla (Lead)
38.6 ± 3.1
7.7 ± 0.8
44.4 ± 3.2
8.4 ± 0.4
1.2 37.3 ± 4.1
1.1 9.5 ± 0.8
23.9 ± 1.7
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0.6
0.9
0.9
1.0
1.0
3
4
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40.3 ± 2.6
147.8 ± 2.9
18.3 ± 1.0
71.0 ± 19
200.0 ± 17.1
1.3 15.8 ± 1.4
1.8 33.8 ± 7.4
14.6 ± 1.8
35.3 ± 9.9
6
7
1.4 111.1 ± 19.0 115.9 ± 9.8
8
155.8 ± 15.0 161.9 ± 21.5
1.0 120.5 ± 4.2 159.0 ± 17.6 1.3
9
18.6 ± 0.1
5.0 ± 0.6
5.5 ± 0.5
32.1 ± 1.5
1,200 ± 58
7.2 ± 1.1
1.7 15.7 ± 1.2
240 2.8 ± 0.36
1.3 3.3 ± 0.4
14.9 ± 1.9
24.0 ± 3.0
3.3 ± 0.3
0.9
8.6
1.0
Paclitaxel
CA-4
aResults previously published.25
Compounds 3, 4 and 6‒9 were also evaluated for the ability to overcome βIII-tubulin
mediated resistance in an isogenic HeLa cell line pair (Table 3).45 Compound 3 was the most
potent compound in the series in HeLa and HeLa WT βIII cell lines with 4-fold better activity
than the lead compound (±)-1·HCl. Similar to the Rr value in the SK-OV-3 isogenic cell line
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pair, the Rr value was calculated by dividing the IC50 of the βIII-tubulin expressing line by the
IC50 obtained in the parental HeLa cells. The expression of βIII-tubulin is known to lead to
paclitaxel resistance, and paclitaxel has a Rr value of 8.6 in this cell line pair (Table 6). The
target compounds 3, 4 and 6‒9 have Rr values ≈ 1.0 (Table 3), suggesting that they circumvent
βIII-tubulin mediated drug resistance.
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Activity of compound 3 in the NCI cancer cell line panel. The most potent compound (3) of
the series was evaluated in the NCI cancer cell line panel,46 and it had GI50 values <10 nM
against 47 of the 57 cancer cell lines (Table 4). Compound 3 had better potency than the lead
compound (±)-1·HCl25 in 29 cancer cell lines and comparable potency to (±)-1·HCl25 in 19
cancer cell lines (Table 4).
Table 4. Comparison of cancer cell growth inhibitory activity (NCI) GI50 (nM) of 3 with (±)-
1·HCla
Panel/ Cell line GI50 (nM) Panel/ Cell line GI50 (nM) Panel/ Cell line GI50 (nM) Panel/ Cell line GI50 (nM)
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(±)-1·
(±)-1·
(±)-1·
(±)-1·
Leukemia
3
Colon Cancer
3
Melanoma
3
Renal Cancer
3
HCl
HCl
HCl
HCl
CCRF-CEM <10 16.3
HL-60(TB) <10 <10
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
<10 18.3 LOX IMVI <10 22.7
<10 24.0 MALME-3M 38.7b
786-0
A498
<10 34.8
<10 <10
22.5 14.6
<10
K-562
<10 <10
<10 31.8
<10 15.1
M14
<10 10.2
ACHN
CAKI-1
RXF 393
SN12C
TK10
MOLT-4
<10 <10 MDA-MB-435 <10 <10
<10 11.8 SK-MEL-28 <10 <10
<10 <10 SK-MEL-5 <10 <10
RPMI-8226 <10 15.7
<10 <10
<10 31.8
75.3b 292
SR
<10 <10
KM12
NSCLC
SW-620
<10 <10
UACC-62 29.6b <10
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A549/ATCC <10 24.4 CNS Cancer
Ovarian cancer
IGROVI
UO-31
<10 13.4
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
<10 19.6
18.4b 19.4
62.5 55.4
<10 31.5
<10 16.4
SF-268
SF-295
SF-539
SNB-19
SNB-75
10.1 15.2
<10 <10
<10 11.0 Prostate Cancer
OVCAR-3
<10 <10
<10 26.8
PC-3
<10 14.6
<10 21.4
<10 11.4 OVCAR-4
DU-145
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<10 36.9 OVCAR-5 12.8 38.5 Breast Cancer
<10 <10
OVCAR-8
<10 32.8
MCF7
MDA-MB-231/
ATCC
<10 <10
<10 24.6
NCI-H322M >100b 59.0
U251
<10 12.5 NCI/ADR-RES <10 <10
NCI-H460
NCI-H522
<10 23.4
<10 <10
SK-OV-3
<10 27.5
HS 578T
<10 <10
59.6 21.9
BT-549
MDA-MB-468 <10 <10
aResults previously published.25 bGI50 values in µM.
Figure 8. Conformational restriction of C4-N and N-C1' bonds in the presence of the N-Me
moiety.
1H NMR evidence for conformational restriction in compound 3. The 1H NMR spectra of the
N-H analog 2 and N-Me analog 3 in DMSO-d6 afforded additional information related to the
conformational restriction in 3 (Figures 8 and 9). For 2, the “5-Me” protons appeared at  2.40
whereas for 3, they were significantly shielded at  1.05. This shielding of the “5-Me” protons in
3 was attributed to a diamagnetic anisotropic effect in 3 arising from the proximity of the phenyl
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ring as shown in Figure 8 (more favored anti-conformation for 3 on the basis of the N-Me and
the 5-Me groups). The steric bulk of the N-Me and/or steric clash of the N-Me and the 5-Me
groups in 3 restricts the conformation and positions the phenyl group on top of the 5-Me moiety
(Figure 9B), resulting in the observed shielding effect, in the 1H NMR, on the 5-Me group in 3 as
compared to that in 2.
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Figure 9. The conformations and 1H NMR analyses of 2 (A) and 3 (B).
This shielding effect of the phenyl group on the 5-Me group (≈was also observed
for the N4-alkyl analogs 4‒7 and N4-Me analogs 8 and 9 (Figure 10).
Figure 10. The values (1H NMR) for compounds 2‒9.
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Figure 11. (A) The docked pose of 3 without the protein showing the distance (in Å) between 5-
Me and the closest carbons on the phenyl ring. (B) Stereo view. Superimposition of docked
poses of 3 (green), 4 (magenta), 5 (yellow), 6 (blue), 7 (brown), 8 (orange) and 9 (cyan) in the
colchicine site of tubulin.
1
The prediction of the possible putative bound conformation of 3 gleaned from the H NMR
was further supported by the docked pose of 3 in the colchicine site (Figure 11A, MOE
2013.080127). The phenyl ring of 3 sits on top of the 5-Me group of the furo[2,3-d]pyrimidine
scaffold and exerts its anisotropic effect on the 5-Me protons. In the docked pose of 3, the
distance of the 5-Me with the most proximal carbons on the phenyl ring were found to be 3.62 Å
and 4.08 Å. Similar mode of binding was observed for compounds 4‒9 when docked in the
colchicine site of tubulin (Figure 11B).
Antitumor activity of compound 3 in MDA-MB-435 xenografts. Compound 3 was selected
for an in vivo xenograft mouse study in light of its nanomolar potency in vitro in the NCI cancer
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cell line panel and its potent microtubule depolymerization activity. Compound 3 was tested for
in vivo antitumor effects in the MDA-MB-435 xenograft model (Figure 12). MDA-MB-435
tumor fragments were injected sc into the flank of nude mice. Once tumors reached ~200 mm3,
mice were injected 3 times ip with 3 (cumulative dose 170 mg/kg) or 6 times with paclitaxel
(120 mg/kg cumulative dose). The results show that 3 caused statistically significant inhibition
of tumor growth as compared to the untreated control and the mean tumor volume was slightly
lower than that achieved with paclitaxel.
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Figure 12. Effect of 3 and paclitaxel (PTX) on MDA-MB-435 tumor growth. Nude mice
bilaterally implanted with MDA-MB-435 tumor fragments were dosed with 60 mg/kg of 3 on
days 0 and 2; and 50 mg/kg of 3 on day 8 or PTX at 20 mg/kg on days 0, 2, 4, 6, 8 and 11 as a
positive control. (A) The average tumor volume is graphed with error bars representing the
standard deviation. (B) The final tumor volumes are plotted with lines representing the mean and
95% confidence intervals. *p = 0.0425 and **p = 0.0014
Summary
A series of eight 5-methyl-furo[2,3-d]pyrimidines were designed and synthesized as
microtubule depolymerizing agents. Synthesis of target compounds involved an Ullmann
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coupling reaction as the key step. Compounds 3, 4 and 9 showed microtubule depolymerizing
activity at concentrations less than 100 nM. Compounds 3, 4 and 6–9 demonstrated the ability to
cause G2/M cell cycle accumulation and induce the formation of abnormal mitotic spindles.
Replacing the N-Me group with higher alkyl groups led to a 210-fold decrease in microtubule
depolymerizing activity. N4-Alkyl-4'-OMe analogs 4, 6 and 7 and the N4-Me-4'-SMe analog 9
inhibited tubulin assembly with activity comparable to that of CA-4. Compounds 3, 4, 6 and 9, at
5 M, inhibited [3H]colchicine binding to tubulin about as well as did CA-4. Compounds 3, 4, 6‒
9 circumvented Pgp and βIII-tubulin mediated drug resistance. Proton NMR and molecular
modeling were utilized to determine the role of the N4-methyl group in tubulin inhibitory
activity. The N4-alkyl group, in addition to the 5-Me group, on the furo[2,3-d]pyrimidine
scaffold restricts the conformation about the C4-N and N-C1' bonds and contributes, in part, to
the biological activity of these analogs. The N4-H analog 2, where such a conformational
restriction is not attainable, is essentially devoid of activity as an MTA. Compound 3 displayed
nanomolar antiproliferative activity in the NCI cancer cell line panel and thus was selected for
xenograft study. In MDA-MB-435 xenografts in mice, compound 3 significantly inhibited tumor
growth. Compound 3 is an important preclinical candidate as a MTA and is currently undergoing
extensive preclinical evaluations.
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Experimental Section
Chemistry. All evaporations were carried out in vacuum with a rotary evaporator.
Analytical samples were dried in vacuo (0.2 mm Hg) in a CHEM-DRY drying apparatus over
P2O5 at 50 °C. Thin‒layer chromatography (TLC) was performed on Whatman® Sil G/UV254
silica gel plates, and the spots were visualized by irradiation with ultraviolet light (254 and 366
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nm). Proportions of solvents used for TLC are by volume. All analytical samples were
homogeneous on TLC in at least two different solvent systems. Column chromatography was
performed on a 70−230 mesh silica gel (Fisher Scientific) column. 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 wet-packed with appropriate solvent unless
specified otherwise. Melting points were determined using a digital MEL-TEMP II melting point
apparatus with FLUKE 51 K/J electronic thermometer or using an MPA100 OptiMelt automated
melting point system and are uncorrected. Nuclear magnetic resonance spectra for proton (1H
NMR) were recorded on Bruker Avance II 400 (400 MHz) and 500 (500 MHz) systems and were
analyzed using MestReC NMR data processing software. The chemical shift (δ) 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; exch, protons
exchangeable by addition of D2O.
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Elemental analyses or high-performance liquid chromatography (HPLC)/mass analysis were
used to determine the purities of the target compounds. Elemental analyses were performed by
Atlantic Microlab, Inc., Norcross, GA, USA. Elemental 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
1
vacuo and were confirmed where possible by their presence in the H NMR spectra. Mass
spectral data were acquired on an Agilent G6220AA TOF LC/MS system using the nano ESI
(Agilent chip tube system with infusion chip). HPLC analysis was performed on a Waters HPLC
system using XSelect® CSH C18 column. Peak area of the major peak versus other peaks was
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used to determine purity. All solvents and chemicals were purchased from Sigma‒Aldrich Co. or
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Fisher Scientific Inc. and were used as received.
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N-(4-Methoxyphenyl)-5-methylfuro[2,3-d]pyrimidin-4-amine (2). A 50 mL round bottom
flask was charged with CuI (66.5 mg, 0.35 mmol), anhydrous potassium carbonate (480 mg, 3.5
mmol), L-proline (80 mg, 0.7 mmol), compound 12 (150 mg, 1 mmol) and 4-iodoanisole 13 (350
mg, 3.5 mmol). The flask was connected to a vacuum for 3 min followed by addition of
anhydrous DMF (15 mL) using syringe. The flask was purged with argon for 5 min and then
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stirred at 110 C. Upon heating, the color of the suspension turned blueish grey, and this color
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persisted for about 2 h. The reaction was stirred for another 22 h at 110 C, at the end of which
the mixture was allowed to cool to room temperature. Ethyl acetate (25 mL) was added, and the
mixture was poured into water (100 mL). The product was extracted with ethyl acetate (100 mL
× 2). The combined organic extracts were washed with brine (100 mL), dried (anhydrous
Na2SO4) and concentrated under reduced pressure. Silica gel (500 mg) was added and the solvent
evaporated to obtain a plug. Purification by column chromatography using hexanes and ethyl
acetate (10:1 to 2:1) afforded 87.5 mg (33%) of 2 as a light brown solid. TLC Rf = 0.77
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(CHCl3/MeOH, 10:1); mp 99.5–101.6 C; H NMR (400 MHz, DMSO-d6):  = 2.383‒2.386 (d,
3H, 5-CH3, J = 1.2 Hz), 3.75 (s, 3H, OCH3), 6.92‒6.94 (d, 2H, Ar, J = 8.8 Hz), 7.47‒7.49 (d,
2H, Ar, J = 8.8 Hz ), 7.650‒7.653 (d, 1H, C6-CH, J = 1.2 Hz), 8.23 (s, 1H, C2-CH), 8.38 (s, 1H,
4-NH, exch). Anal. (C14H13N3O2) C, H, N.
N-(4-Methoxyphenyl)-N,5-dimethylfuro[2,3-d]pyrimidin-4-amine (3). In a 25 mL round
bottom flask, compound 2 (510 mg, 2 mmol) was dissolved in DMF (10 mL). The flask was
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purged with argon for 5 min and cooled to 0 C using an ice bath. NaH (144 mg, 6 mmol) was
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added to the solution at 0 oC, and the reaction mixture was stirred for 30 min at 0 oC under argon
atmosphere. Dimethyl sulfate (757 mg, 6 mmol) was injected into the reaction mixture, and the
flask was warmed to room temperature. The mixture was stirred for 3 h, and 1 N HCl (5 mL)
was added carefully to quench the reaction. Water (20 mL) was added, resulting in formation of
a precipitate. The product was extracted with ethyl acetate (50 mL × 3). The combined organic
extracts were washed with brine (10 mL), dried (anhydrous Na2SO4) and concentrated under
reduced pressure. Silica gel (2 g) was added, and the solvent was evaporated to yield a plug.
Column chromatography by elution with hexanes and ethyl acetate (5:1) afforded 3 (416 mg,
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70%) as an off-white solid. TLC Rf = 0.87 (CHCl3/MeOH, 10:1); mp 87‒88 C; H NMR (400
MHz, DMSO-d6):  = 1.05 (s, 3H, 5-CH3), 3.44 (s, 3H, OCH3), 3.77 (s, 3H, NCH3), 6.96‒6.98
(d, 2H, Ar, J = 9.0 Hz), 7.19‒7.22 (d, 2H, Ar, J = 9.0 Hz), 7.52 (s, 1H, C6-CH), 8.45 (s, 1H, C2-
CH). Anal. (C15H15N3O2) C, H, N.
General procedure for the synthesis of substituted furo[2,3-d]pyrimidines 4-7. In a 25 mL
round bottom flask, compound 2 (1 equivalent) was dissolved in DMF (2 mL). The flask was
purged with argon for 5 min and cooled to 0 oC using an ice bath. NaH (3 equivalents) was added
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to the solution at 0 C, and the reaction mixture was stirred for 20 min under argon atmosphere.
The appropriate alkyl iodide (3‒4 equivalents) was injected into the reaction mixture, and the
flask was warmed to room temperature. The mixture was stirred at room temperature until TLC
showed disappearance of the reactant 2. Aqueous 1 N HCl was added dropwise to quench the
reaction. Water (4 mL) was added, resulting in formation of a precipitate. The product was
extracted with ethyl acetate (10 mL × 3). The combined organic extracts were washed with brine
(10 mL), dried (anhydrous Na2SO4) and concentrated under reduced pressure. Silica gel (200
mg) was added and the solvent was evaporated under reduced pressure. The resulting plug was
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loaded onto a silica gel column in hexanes (the silica was twenty times the weight of the plug),
and the column was eluted with hexanes and ethyl acetate (5:1) to obtain 4‒7 in yields of 57‒
85%.
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N-Ethyl-N-(4-methoxyphenyl)-5-methylfuro[2,3-d]pyrimidin-4-amine (4). Using the general
procedure described above, ethyl iodide 14 (61.70 mg or 0.1 mL, 1.2 mmol) was added to an ice-
cold solution of 2 (100 mg, 0.4 mmol) and NaH (28.8 mg, 1.2 mmol) in 2 mL of DMF, and the
reaction was run at room temperature for 2 h to provide 94 mg (85%) of 4 as an off-white solid.
TLC Rf = 0.88 (CHCl3/MeOH, 15:1); mp 77.8–78.3 oC; 1H NMR (400 MHz, CDCl3):  = 1.064‒
1.067 (d, 3H, 5-CH3, J = 1.2 Hz), 1.22 (t, 3H, CH3, J = 7 Hz), 3.82 (s, 3H, OCH3), 4.06‒4.11 (q,
2H, N-CH2, J = 7 Hz), 6.88‒6.91 (d, 2H, Ar, J = 8.9 Hz), 7.074‒7.077 (d, 1H, C6-CH, J = 1.2
Hz), 7.09‒7.11 (d, 2H, Ar, J = 8.9 Hz), 8.48 (s, 1H, C2-CH). Anal. (C16H17N3O2) C, H, N.
N-Isopropyl-N-(4-methoxyphenyl)-5-methylfuro[2,3-d]pyrimidin-4-amine (5). Using the
general procedure described above, isopropyl iodide 15 (266.36 mg, 1.6 mmol) was added to an
ice-cold solution of 2 (100 mg, 0.4 mmol) and NaH (38.4 mg, 1.6 mmol) in 2 mL of DMF, and
the reaction was run at room temperature for 6 h to provide 75 mg (63%) of 5 as an off-white
solid. TLC Rf = 0.88 (CHCl3/MeOH, 15:1); mp 95.8–96.6 oC; 1H NMR (400 MHz, CDCl3):  =
1.02 (d, 3H, 5-CH3, J = 1.3 Hz), 1.14‒1.16 (d, 6H, two CH3, J = 6.8 Hz), 3.79 (s, 3H, OCH3),
5.30‒5.37 (m, 1H, N-CH), 6.85‒6.87 (d, 2H, Ar, J = 8.9 Hz), 7.000‒7.004 (d, 1H, C6-CH, J =
1.3 Hz), 7.02‒7.04 (d, 2H, Ar, J = 8.9 Hz), 8.42 (s, 1H, C2-CH). Anal. (C17H19N3O2) C, H, N.
N-(4-Methoxyphenyl)-5-methyl-N-propylfuro[2,3-d]pyrimidin-4-amine (6). Using the
general procedure described above, propyl iodide 16 (199 mg or 0.12 mL, 1.2 mmol) was added
to an ice-cold solution of 2 (100 mg, 0.4 mmol) and NaH (28.8 mg, 1.2 mmol) in 2 mL of DMF,
and the reaction was run at room temperature for 2 h to provide 80 mg (69%) of 6 as an off-white
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