Evaluation of substituted 6-arylquinazolin-4-amines as potent and selective inhibitors of cdc2-like kinases (Clk)

Article abstract of DOI:10.1016/j.bmcl.2009.09.121

A series of substituted 6-arylquinazolin-4-amines were prepared and analyzed as inhibitors of Clk4. Synthesis, structure-activity relationships and the selectivity of a potent analogue against a panel of 402 kinases are presented. Inhibition of Clk4 by th

Full text of DOI:10.1016/j.bmcl.2009.09.121

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6700–6705
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Evaluation of substituted 6-arylquinazolin-4-amines as potent and selective
inhibitors of cdc2-like kinases (Clk)
Bryan T. Mott a, , Cordelle Tanega a,b, , Min Shen a, David J. Maloney a, Paul Shinn a, William Leister a,
Juan J. Marugan a, James Inglese a, Christopher P. Austin a, Tom Misteli b, Douglas S. Auld a, Craig J. Thomas a,
*
a NIH Chemical Genomics Center, National Human Genome Research Institute, NIH 9800 Medical Center Drive, MSC 3370 Bethesda, MD 20892-3370, USA
b Cell Biology of Genomes, National Cancer Institute, NIH, 41 Library Drive, Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of substituted 6-arylquinazolin-4-amines were prepared and analyzed as inhibitors of Clk4. Syn-
thesis, structure–activity relationships and the selectivity of a potent analogue against a panel of 402
kinases are presented. Inhibition of Clk4 by these agents at varied concentrations of assay substrates
(ATP and receptor peptide) highly suggests that this chemotype is an ATP competitive inhibitor. Molec-
ular docking provides further evidence that inhibition is the result of binding at the kinase hinge region.
Selected compounds represent novel tools capable of potent and selective inhibition of Clk1, Clk4, and
Dyrk1A.
Received 13 August 2009
Revised 29 September 2009
Accepted 30 September 2009
Available online 3 October 2009
Keywords:
Kinase inhibition
Pre-mRNA splicing
Clk
Published by Elsevier Ltd.
Dyrk1A
The removal of intron sequences from genes occurs via the ac-
tions of the splicesome, a protein complex that removes intervening
sequences at the nuclear pre-mRNA level to afford properly coded
mRNA for translation.1,2 Many genes produce multiple mRNA iso-
forms through the actions of alternative splicing and, importantly,
numerous human diseases are caused by improper splicing.3 Exog-
enous manipulation of the splicesome is theorized to be a powerful
means to control gene translation and ultimately correct disease
phenotypes via rectification of splicing abnormalities.
The modulation of kinases provides a powerful mechanism to
control numerous aspects of cell function and offers the potential
for the management of many diseases.4 Small molecule inhibitors
of kinases (both selective and promiscuous) represent important
biochemical tools for basic research and several kinase inhibitors
have gained FDA approval as drugs.5,6 In 2002, Manning et al. re-
viewed the human kinome and found 518 putative kinase genes7
providing a roadmap to explore the consequences of kinase modu-
lation within cellular biology. The possible control of gene splicing
through the modulation of kinase activity represents an intriguing
concept.8 There are several reports of kinases that alter the func-
tion of the splicesome. Among these is the cdc2-like kinase (Clk)
family.9 A major target of Clk kinases is the prominent family of
serine- and arginine-rich (SR) splicing proteins10,11 which are in-
volved in the assembly of the splicesome and are implicated in
both constitutive and alternative splicing control and selection of
splicing sites.12,13
The Clk family contains four characterized isoforms (Clk1, Clk2,
Clk3, and Clk4). The Clks are capable of auto-phosphorylation (at
serine, threonine, and tyrosine residues) and phosphorylation of
exogenous proteins (at serine and threonine residues). Members
of the Clk family have been implicated in the regulation of alterna-
tive splicing of PKCbII14, TF15, Tau,16 and b-globin17 pre-mRNA.
These studies suggest that small molecule modulation of the Clk
family of kinases may represent an important mechanism for the
control of mRNA splicing.
Hagiwara and co-workers have reported TG003 (1) (Fig. 1) as a
small molecule with low-nanomolar IC50 values versus Clk1 and
Clk4.17 Additionally, Clk inhibitors are presented in patent reports
from Sirtris Pharmaceuticals (no structures presented) and Chro-
nogen Inc. (a series of substituted quinolines related to structure
2).18,19 The report from Hagiwara and co-workers does not define
the selectivity of TG003 beyond the Clk family and four additional
kinases (PKA, PKC, SRPK1, and SRPK2) and the patents do not dis-
close details regarding structure–activity relationships nor selec-
tivity. Thus, a need remains for small molecule probes of this
important class of enzymes. Here, we report a novel class of qui-
nazoline small molecules as potent and selective inhibitors of
Clk1 and Clk4.
We recently performed a high-throughput screen for small mol-
ecule modulators of Lamin A splicing (PubChem AID: 1487); details
of this study will be reported elsewhere. Among the actives was a
* Corresponding author. Tel.: +1 301 217 4079; fax: +1 301 217 5736.
E-mail address: craigt@nhgri.nih.gov (C.J. Thomas).
series of substituted quinazolines including
3 (Fig. 1). The
These authors contributed equally to this study.
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.09.121

B. T. Mott et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6700–6705
6701
OMe
Cl
O
N
S
N
MeO
NH
O
N
MeO
N
AcHN
OH
N
1 (TG003)
2
3
Figure 1. Structures of known Clk inhibitors 1 (TG003), 2 and the lead structure 3.
previously reported ability of quinazoline small molecules to mod-
ulate kinases through competitive inhibition at the ATP binding
site suggested potential kinase stimulated influence of splicing. Gi-
ven the association of the Clk family with splicing, we speculated
that 3 might be a Clk inhibitor and this was confirmed via a com-
mercial kinase profile.20 Based on these results, we examined se-
lected in-house compound libraries for Clk4 inhibition via two
bioluminescent, luciferase-based assays capable of visualizing sub-
strate (ATP) depletion and product (ADP) formation.21 In primary
screening, this biochemical assay revealed that 3 (Fig. 1) possessed
an IC50 value versus Clk4 of 316 nM.
Based upon this result, we examined the SAR for this chemo-
type versus Clk4. Key to this effort was the synthetic preparation
of these agents. In our hands, the most straightforward synthesis
began with 6-bromo-4-chloroquinazoline (Scheme 1). Addition of
primary and secondary amines (including aliphatic, benzylic, het-
eroaromatic and substituted analines) was accomplished in DMF
with Hunig’s base at room temperature. The resulting 6-bromo-
N-substituted-quinazolin-4-amines were then subjected to
standard Suzuki–Miyaura couplings with various aryl boronic
acids using tetrakis(triphenylphosphine)palladium(0) and sodium
carbonate in DMF and heating using microwave irradiation. This
general procedure22 resulted in the production of numerous
N-substituted-6-arylquinazolin-4-amines including 4 which were
purified via reverse-phase HPLC methods prior to testing in biolog-
ical assays.
The primary phenotypic screen and the follow-up biochemical
Clk4 assay were performed on the NIH Molecular Libraries Small
Molecule Repository and several internal NCGC compound libraries.
Each assay was performed using the quantitative high-throughput
screening method whereby each molecular entity is screened in a
dose–response format.23 This screening format provides an unprec-
edented level of detail with regards to nascent SAR from the primary
screening data. The resulting analysis of both of the aforementioned
screens provided several clues as to which substitution patterns
were likely to result in active compounds. Substitutions at the 4 po-
sition of the quinazoline core scaffold were limited to substituted
benzylic systems (for instance m-tolylmethanamine, pyridin-3-
ylmethanamine, and (3-fluorophenyl)methanamine)) and various
heterocyclic methylamines (for instance 1-(furan-3-yl)-N-meth-
ylmethanamine, (4-methylthiophen-2-yl)methanamine, and thio-
phen-2-ylmethanamine). Substitutions at the 6 position of the
quinazoline core scaffold were limited to various aryl groups (for in-
stance 3-methoxybenzene, benzo[d][1,3]dioxole, 2,3-dihydro-
benzo[b][1,4]dioxine, furan, and 3,5-dimethylisoxazole). As SAR is
often not additive, we chose to incorporate all of these substitutions
in matrix format in hopes of identifying more potent Clk4 inhibitors
and revealing as much SAR as possible. The results of this library are
listed in Figure 2. The original lead 3 was found to possess an IC50 va-
lue of 282 nM (very similar to the IC50 value determined in the pri-
mary biochemical screen). Additionally, two novel small molecules
were found with potencies of 63 nM including 6-
(benzo[d][1,3]dioxol-5-yl)-N-(thiophen-2-ylmethyl)quinazolin-4-
amine (4). The SAR revealed that the benzo[d][1,3]dioxole was con-
sistently favored in the 6 position of the quinazoline ring. The SAR of
the amine substitutions was more complex. The thiophen-2-ylme-
thanamine substitution was typically favored, but several structural
combinations favored other moieties at this position.
Prior to additional investigations into the SAR and optimization
of this chemotype, it was important for us to gain an appreciation
of the selectivity of these compounds towards the Clk family of ki-
nases. Quinazoline based small molecules have been reported as
potent inhibitors of numerous kinase families. Any small molecule
Clk inhibitor that is additionally capable of promiscuous inhibition
across the kinome will be of limited value as a tool compound for
evaluating Clk biochemistry. To assess the selectivity of this gen-
eral chemotype we choose to submit a representative reagent
(analogue 4) across a commercial panel of kinases. In 2008, Ambit
Biosciences reported a quantitative analysis of 38 known kinase
inhibitors across a panel of 317 kinases.24 This commercially avail-
able panel contained 402 kinases at the time of submission and is
based upon a competition binding assay of kinases fused to a pro-
prietary tag. The data is first recorded as a % of kinase bound to an
immobilized ligand in the presence and absence of the test reagent
as compared to DMSO. Activities beyond a selected threshold are
submitted for Kd determination. In addition to profiling 4 versus
this panel, we submitted the reported small molecule Clk1/4 inhib-
itor TG003 (1) to generate a comparison between both agents. The
results (tabulated in a dendrogram representation25) are shown in
Figure 3 and demonstrate that both agents are remarkably selec-
tive. TG003 (1) was determined to have Kd’s of 19 nM, 95 nM,
and 30 nM versus Clk1, Clk2, and Clk4, respectively. The Kd for
TG003 (1) versus Clk3 was 3 lM. It was also found that TG003
had activity versus CSNK1D (150 nM), CSNK1E (300 nM), Dyrk1A
(12 nM), Dyrk1B (130 nM), PIM1 (130 nM), PIM3 (280 nM), and
Ysk4 (290 nM). The novel quinazoline 4 was found to have Kd’s
of 37 nM, 50 nM and 27 nM versus Clk1, Clk4, and Dyrk1A, respec-
tively. The only other locus of relevant activity (below 500 nM) was
found for binding to the endothelial growth factor receptor (EGFR)
(230 nM).
Based upon the potency and selectivity for 4 we next aimed to
understand the binding mechanism of this chemotype at Clk4.
We explored the binding modality by examining the inhibitory
capacity of
4 in settings that varied both compound and
substrate concentrations. The results are shown in Figure 4. The
dose–response curve of 4 in the presence of three different ATP
Cl
S
S
O
O
OH
B
NH
N
NH
N
S
Br
i
ii
H2N
Br
O
O
N
HO
N
N
N
Scheme 1. Reagents and conditions: (i) DIPEA, DMF, rt, 2 h (typical yields: 80–95%); (ii) Pd(PPh3)4, Na2CO3, DMF, 150 °C (wave), 1 h (typical yields: 50–80%).

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B. T. Mott et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6700–6705
R'
R'
R
F
N
NH
NH
N
NH
NH
N
NH
S
S
O
N
Clk4 IC50 values
R
1122 nM
3550 nM
708 nM
126 nM
1000 nM
63 nM
1000 nM
178 nM
891 nM
63 nM
OMe
O
O
O
126 nM
631 nM
224 nM
282 nM
100 nM
447 nM
794 nM
891 nM
ND
ND
111 nM
ND
501 nM
ND
141 nM
250 nM
158 nM
O
141 nM
178 nM
O
O
501 nM
N
Figure 2. SAR results for matrix library of Clk4 inhibitors. Data represents the results from three separate experiments. All compounds achieved a maximum response greater
than 90%.
O
S
N
S
O
O
NH
N
TKL
TKL
MeO
N
1 (TG003)
STE
4
STE
TK
TK
Clk1-4
Clk1-4
CK1
CK1
AGC
AGC
CMGC
CMGC
Kd: 1 nM
100 nM
1000 nM
CAMK
CAMK
Kd: 100 nM
Figure 3. Dendrogram representation of the selectivity profile for kinase binding by TG003 (1) and 4 within a panel of 402 kinases. Activity for 1: Clk1 = 19 nM, Clk2 = 95 nM,
Clk3 = 3000 nM, Clk4 = 30 nM, CSNK1D = 150 nM, CSNK1E = 300 nM, CSNK1G2 = 270 nM, CSNK1G3 = 290 nM, Dyrk1A = 12 nM, Dyrk1B = 130 nM, PIM1 = 130 nM,
PIM3 = 280 nM, Ysk4 = 290 nM. Activity for 4: Clk1 = 37 nM, Clk2 = 680 nM, Clk3 = 470 nM, Clk4 = 50 nM, Dyrk1A = 27 nM, Dyrk1B = 430 nM, EGFR = 230 nM. Note: 4 was
active (>250 nM) versus numerous EGFR mutants.
concentrations demonstrates a loss in potency when ATP levels
rise (Fig. 4A). Conversely, the % activity of 4 in the presence of vary-
ing concentrations of the peptide substrate has no affect on the
compound potency (Fig. 4B). Additionally, while maintaining 4 at
a constant concentration (70 nM), an examination of the dose–re-
sponse of ATP demonstrated a sharp decline in enzyme inhibition
at high ATP concentrations (>200 lM) while an increase in the
dose of the peptide did not effect the potency of 4 (data not
shown).
Following confirmation that this chemotype inhibits Clk4 via an
ATP competitive mechanism, it was of interest to explore docking of
4 at a Clk kinase. As previously mentioned, quinazoline based small
molecules have precedence as kinase inhibitors.26 Among these re-
agents is the clinically approved drug erlotinib (TarcevaÒ).27 Erloti-
nib is currently indicated for treatment of non-small cell lung and
pancreatic cancer and its actions are mediated through inhibition
of the EGFR tyrosine kinase.28 In 2002, Stomos et al. reported the
structure of erlotinib bound to the ATP binding domain of EGFR

B. T. Mott et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6700–6705
6703
Figure 4. (A) Inhibitory dose–response of 4 versus Clk4 in the presence of three different ATP concentrations [1
lM (filled circles), 50
l
M (empty circles), 100
lM (empty
squares)]. (B) Inhibitory dose–response of 4 in the presence of three different peptide concentrations [50
lM (filled circles), 100
lM (empty circles), 200 lM (empty squares)].
(PDB code: 1M17).29 We theorized that the relationship of the 4-ani-
linoquinazoline structure of erlotinib to our newly discovered Clk
family inhibitors may provide significant insight into the binding
modality and mechanism of action for this class of compounds.
There are no published X-ray structures of Clk4. There are structures
of Clk1 (PDB code: 1Z57) and Clk3 (PDB code: 2EU9). Clk1 and Clk4
are highly homologous enzymes (>85% sequence identity) while
Clk2 and Clk3 also share a high degree of sequence homology
(>70% sequence identity). Based upon this, we utilized the X-ray
structure of Clk1 as the template to derive a homology model of
Clk4 (Fig. 5A) using MOE molecular modeling software.30 Molecular
docking was performed on 4 within the ATP binding domain of Clk1
and Clk4 to achieve an optimal binding pose using FRED31 (Fig. 5B).
In the crystal structure of erlotinib and EGFR, the N1 of the quinaz-
oline heterocycle makes a critical H-bond to an amide NH of the
hinge region of the ATP binding pocket. This interaction is mimicked
within our docking analysis of 4 at Clk1 and Clk4. The thiophen-2-
ylmethanamine moiety is oriented to fill an open pocket formed
by the gatekeeper Phe241 (Phe239 in Clk4) while the
benzo[d][1,3]dioxole extends toward the solvent exposed face of
the hinge region. While this model partially accounts for various as-
pects of the SAR found to date, more extensive exploration of this
binding modality is required to fully understand the potency and
remarkable selectivity associated with this chemotype.
An interesting aspect of these agents is the inhibitory activity of
this chemotype versus Dyrk1A. The Dyrk1A [dual specificity tyro-
sine (Y)-phosphorylation-regulated kinase 1A] gene is located on
chromosome 21 and is known to be highly expressed in CNS tis-
sues. Dyrk1A knock-out mice are embryonic lethal and transgenic
mice overexpressing Dyrk1A display learning and memory defi-
ciencies.32,33 The Dyrk1A gene is located on the Down’s syndrome
(DS) critical region of chromosome 21 and trisomy-driven overex-
Figure 5. (A) Ribbon representation of the catalytic clefts in the Clk1 and Clk4
kinase domain. Clk4 kinase domain is a homology model derived from the X-ray
structure of Clk1 (PDB code: 1Z57). Protein kinase structural elements are labeled
and the key residues are colored: Clk1 in cyan, Clk4 in green. This figure was
prepared with the program VIDA (OpenEye Scientific Software). (B) Docking model of
4 in the Clk1 catalytic cleft. The binding pocket is depicted by molecular surface in
mesh grey. This figure was prepared with the program VIDA (OpenEye Scientific
Software).
pression in DS patients has been demonstrated.34 Further evidence
has been presented that hyperphosphorylation of Tau by Dyrk1A is
a causative factor in the early onset of Alzheimer disease in DS pa-
tients.35 To date, a class of pyrazolidine-3,5-diones discovered via
in silico screening have been reported as Dyrk1A inhibitors.36,37
The most potent of these agents were demonstrated to have an
IC50 value of 0.6 lM and selectivity versus a panel of 15 selected
kinases highly suggested that these agents were fairly promiscu-
ous. The binding of 4 to Dyrk1A with a potency of 27 nM suggests
between these two enzyme classes. Clk and Dyrk are both mem-
bers of the CMCG branch of the kinome, however, Dyrk1A and
Clk1 are only 32.8% homologous. A sequence comparison is
provided in Figure 6. While each kinase retains several key amino
acids residues that seem to be fundamental to forming the ATP
binding domain (including Glu206, Lys191 and matched hydro-
phobic residues at positions 243 and 244) there are significant
differences that likely confer divergent structural aspects between
the Clks and Dryk1A. A concerted effort to correlate compound SAR
at each enzyme will be required to better understand the relation-
ship between these kinases.
that
4 and related 6-arylquinazolin-4-amines may represent
important new tool compounds for exploration of Dyrk1A bio-
chemistry. We have confirmed that 4 and related analogues are po-
tent inhibitors of Dyrk1A (data not shown). Interestingly, Dyrk1A
has been implicated as an important modulator of pre-mRNA splic-
ing via several molecular interactions including the phosphoryla-
tion of the SR protein cyclin L2.38
The fact that both 4 and TG003 were highly selective for the Clk
family and Dyrk1A leads to questions regarding the relationship

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B. T. Mott et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6700–6705
Figure 6. Multiple sequence alignment of the catalytic domain of protein kinase for all four human Clk isozymes (Clk1, Clk2, Clk3, and Clk4) and human Dyrk1A. The amino
acid residues that are within 10 Å of the ATP binding site are highlighted: red for negatively charged, cyan for positively charged, yellow for hydrophobic and purple for
hydrophilic. The numbering of amino acid residues is taken from Clk1 crystal structure (PDB code: 1Z57). Multiple sequence alignment was prepared by MOE molecular
modeling software.
In conclusion, we report a novel class of Clk inhibitors based
upon a core 6-arylquinazolin-4-amine scaffold. Selected agents
were screened versus Clk4 to gain an appreciation of this chemo-
types SAR and selected agents were found to inhibit this enzyme
with potencies below 100 nM. One agent (analogue 4) was profiled
against a panel of over 400 kinases and found to be remarkably
selective for Clk1, Clk4, and Dyrk1A. The only other reported inhib-
itor of the Clk family [TG003 (1)] was also profiled and found to
bind selectively to Clk1, Clk2, Clk4, and Dyrk1A. Analysis of the
mechanism of action highly suggests that this chemotype inhibits
Clk4 via competition with ATP binding. Molecular modeling also
suggests that 4 and related agents inhibit the Clk isozymes through
binding at the ATP binding domain. These agents provide useful
tools for the study of Clk1, Clk4, and Dyrk1A and their respective
roles in pre-mRNA splicing. Efforts to expand on the SAR of this
chemotype in hopes of finding small molecules with divergent
SAR for each isozyme of the Clk family and Dyrk1A are currently
underway.
Acknowledgments
We thank Ms. Allison Mandich for critical reading of this Letter.
We thank Mr. Dac-Trung Nguyen for generation of the dendrogram
representations of kinase activity. This research was supported by

B. T. Mott et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6700–6705
6705
amine (0.1 g, 0.31 mmol) in DMF (3 mL) were added benzo[d][1,3]dioxol-5-
ylboronic acid (0.078 g, 0.47 mmol), sodium carbonate (0.066 g, 0.63 mmol),
and tetrakis(triphenylphosphine)palladium(0) (0.036 g, 0.03 mmol). The
reaction mixture was heated in a Biotage InitiatorÒ microwave at 150 °C for
1 h. Upon completion, the reaction mixture was diluted with EtOAc (75 mL)
and washed with NaHCO3 (50 mL), three times with 3 N LiCl (3 Â 30 mL), and
brine (30 mL). The organic layer was collected, filtered through a pad of Celite,
and concentrated in vacuo. The residue was purified directly on silica column.
the Molecular Libraries Initiative of the National Institutes of
Health Roadmap for Medical Research and the Intramural Research
Program of the National Human Genome Research Institute at the
National Institutes of Health. TM was supported by a grant from
the Progeria Research Foundation.
Gradient elution with ethyl acetate (5–65%) in hexanes provided
4 as a
References and notes
colorless solid: yield (0.065 g, 0.18 mmol, 58 %). 1H NMR (DMSO-d6) d 4.97 (d,
J = 5.53 Hz, 2H), 6.09 (s, 2H), 6.92–7.01 (m, 1H), 7.02–7.18 (m, 2H), 7.29–7.41
(m, 2H), 7.44 (s, 1H), 7.73 (d, J = 8.56 Hz, 1H), 8.08 (d, J = 8.46 Hz, 1H), 8.51 (d,
J = 4.70 Hz, 2H), 8.99 (d, J = 4.89 Hz, 1H); 13C NMR (DMSO-d6) d 54.88, 101.28,
107.14, 108.70, 115.04, 119.52, 120.54, 125.11, 125.79, 126.54, 127.99, 131.04,
133.21, 137.02, 142.18, 147.14, 148.11, 148.26, 154.75, 159.05. HRMS (ESI) m/z
362.0957 (M+H)+ (C20H16N3O2S requires 362.0958). Purity determination via
LC–MS was performed using an Agilent Diode Array Detector using a 3 min
gradient of 4–100% acetonitrile (containing 0.025% trifluoroacetic acid) in
water (containing 0.05% trifluoroacetic acid) with a 4.5 min run time at a flow
rate of 1 mL/min and a 7 min gradient of 4–100% acetonitrile (containing
0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid)
with an 8 min run time at a flow rate of 1 mL/min. Three minutes gradient
retention time = 3.050; 7 min gradient retention time = 4.531 min. Purity was
determined to be >95% in both methods.
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22. Procedure for the preparation of 6-bromo-N-(thiophen-2-ylmethyl)quinazolin-4-
amine: To a solution of 6-bromo-4-chloroquinazoline (0.3 g, 1.23 mmol) in
DMF (8 mL)were added thiophen-2-ylmethanamine (0.139 g, 1.23 mmol) and
Hunig’s base (0.21 mL, 1.23 mmol). The reaction mixture was stirred at rt for
2 h. Upon completion, the reaction mixture was diluted with EtOAc (100 mL)
and washed with 10% KHSO4 (25 mL) and three times with 3 N LiCl
(3 Â 30 mL). The organic layer was extracted, dried on MgSO4, filtered, and
concentrated in vacuo. The residue was purified directly on silica column.
Gradient elution with ethyl acetate (15–75%) in hexanes provided the title
compound as a colorless solid: yield (0.39 g, 1.22 mmol, 99%). Procedure for the
preparation of 6-(benzo[d][1,3]dioxol-5-yl)-N-(thiophen-2-ylmethyl)quinazolin-4-
amine (4): To a solution of 6-bromo-N-(thiophen-2-ylmethyl)quinazolin-4-
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37. Koo, K. A.; Kim, N. D.; Chon, Y. S.; Jung, M.-S.; Lee, B.-J.; Kim, J. H.; Song, W.-J.
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