2-Amino-4-methyl-5-phenylethyl substituted-7-N-benzyl-pyrrolo[2,3-d] pyrimidines as novel antitumor antimitotic agents that also reverse tumor resistance

Article abstract of DOI:10.1016/j.bmc.2011.05.030

Gangjee et al. recently reported a novel series of 2-amino-4-methyl-5- phenylethyl substituted-7-benzyl-pyrrolo[2,3-d]pyrimidines, some of which exhibited two digit nanomolar antitumor and antimitotic activity and were not subject to P-glycoprotein (Pgp)

Full text of DOI:10.1016/j.bmc.2011.05.030

Bioorganic & Medicinal Chemistry 19 (2011) 4355–4365
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2-Amino-4-methyl-5-phenylethyl substituted-7-N-benzyl-pyrrolo[2,3-d]pyrimi-
dines as novel antitumor antimitotic agents that also reverse tumor resistance q
Aleem Gangjee a, , Ojas A. Namjoshi a, Staci N. Keller b, Charles D. Smith b
⇑
a Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282, United States
b Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, SC 29425, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 March 2011
Revised 9 May 2011
Accepted 17 May 2011
Available online 23 May 2011
Gangjee et al. recently reported a novel series of 2-amino-4-methyl-5-phenylethyl substituted-7-benzyl-
pyrrolo[2,3-d]pyrimidines, some of which exhibited two digit nanomolar antitumor and antimitotic
activity and were not subject to P-glycoprotein (Pgp) or multidrug resistance protein 1 (MRP1) mediated
tumor resistance (unlike the Vinca alkaloids and taxanes). Some of these compounds, in addition to their
antitumor activity, had the ability to reverse the Pgp-mediated resistance to clinically used antimitotic
agents. This report consists of an attempt to optimize the various activities of the parent compounds
by synthetic variations of the phenyl ring of the 5-phenylethyl side chain. The target compounds were
synthesized via a nine-step synthesis involving a Sonogashira reaction. The substituted phenylacetylenes
as coupling partners were in turn synthesized from unactivated aryl bromides or iodides. The target com-
pounds exhibited moderate cytotoxicity against MCF-7 tumor cells. However, most of these compounds
showed improved cytotoxicity against the resistant NCI/ADR and MCF-7/VP. This study afforded an ana-
log which reversed both Pgp-mediated as well as MRP1-mediated resistance to clinically used antimitotic
agents, along with its own antimitotic mediated antitumor activity. In addition, in the NCI-60 cell line
panel one of the compounds inhibited the growth of MDA-MD-435 breast cancer cell line at submicrom-
olar concentration.
Keywords:
Antimitotic
Tumor resistance reversal
Cytotoxic
Sonogashira coupling
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
vindesine, and vinorelbine. These are b-tubulin binding agents that
interfere with proper mitotic spindle formation by preventing the
Microtubules are long, protein polymers that are formed by
normal dynamics and polymerization of spindle microtubule for-
mation. The Vinca alkaloids are important in the treatment of leu-
kemias, lymphomas, small cell lung cancer, and other cancers.4,5
These agents are also known as microtubule-destabilizing agents
or microtubule polymerization inhibitors or depolymerizers. The
second group comprises of the taxanes (Fig. 1) exemplified by
paclitaxel (Taxol) and docetaxel (Taxotere). Paclitaxel increases
microtubule polymerization and thus interferes with spindle
microtubule dynamics and prevents the dividing cell from progres-
sion. These are microtubule-stabilizing agents or polymerizing
agents. The taxanes are clinically useful in the treatment of breast,
lung, ovarian, head and neck, and bladder carcinomas among oth-
ers. The third class typified by colchicine is comprised of a diverse
collection of small molecules that bind to the colchicine binding
site. These compounds are microtubule polymerization inhibitors
similar to the Vinca alkaloids. However they bind to a different site
and their depolymerization mechanism of action is also different.
Combretastatins (Fig. 1) are a class of drugs that are in clinical
trials as antitumor agents that bind to the colchicine binding site.2
There are no clinically approved antitumor agents that bind to the
colchicine site. However, several of these agents are currently in
clinical trials.6,7
a
-tubulin and b-tubulin heterodimers.2 These heterodimers first
form a short microtubule nucleus followed by elongation and
arrangement in the form of tubes. The polymerization of microtu-
bules occurs via complex polymerization dynamics that involves
GTP hydrolysis to supply energy at the time that tubulin, with
bound GTP, adds to the microtubule ends. In mitosis, the dupli-
cated chromosomes of cells are divided into two identical sets
prior to division into two daughter cells and the polymerization
dynamics of microtubules plays a pivotal role in this process as
part of cell replication.2,3 The crucial involvement of microtubules
in mitosis makes them a target for antitumor agents.
Three distinct classes of antimitotic agents have been identified.
The Vinca alkaloids (Fig. 1) exemplified by vincristine, vinblastine,
Abbreviations: Pgp, P-glycoprotein; MDR, multiple drug resistance; MRP,
multidrug resistance protein; GTP, guanosine triphosphate; MCF-7, Michigan
Cancer Foundation-7; NCI, National Cancer Institute; PCC, Pearson correlation
coefficient; TLC, thin layer chromatography; NMR, nuclear magnetic resonance.
q
See Ref. 1.
⇑
Corresponding author. Tel.: +1 412 396 6070; fax: +1 412 396 5593.
E-mail address: gangjee@duq.edu (A. Gangjee).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.030

4356
A. Gangjee et al. / Bioorg. Med. Chem. 19 (2011) 4355–4365
Figure 1. Examples of antimitotic agents.
The clinical success of the taxanes has encouraged efforts to
clear.18 The simultaneous expression and function of both proteins
has been shown to be correlated with poor overall survival.
The clinical significance of Pgp along with its limited expression
in normal tissues makes Pgp a viable target for inhibition to reverse
MDR. Several Pgp inhibitors or modulators have been developed to
reverse MDR that occur for chemotherapeutic agents in general
and include antimitotic inhibitors.17
The other clinical mechanism of resistance to microtubule tar-
geted agents involves the expression of specific isotypes of b-tubu-
lin, of which the bIII-tubulin isotype is of paramount concern.
There is unequivocal clinical evidence that bIII-tubulin expression
is involved in resistance to taxoids and vinca alkaloids in multiple
tumor types including non-small cell lung,19–21 breast,22 and ovar-
ian cancers.23,24
identify and develop other classes of microtubule-targeted antican-
cer agents that might overcome the limitations of existing drugs.
Epothilones are a newer class of antimitotic agents and were
found to bind at the same site as the taxanes. Ixabepilone was
the first member from the epothilones family to be approved by
the USFDA for the treatment of metastatic breast cancer in October
2007.8,9
Though antimitotic agents have shown to be some of the most
successful agents against malignancies, resistance both intrinsic
and acquired, is of major concern, and results in treatment failures.
Multidrug or multiple drug resistance (MDR) is a major drawback
of cancer chemotherapy including the clinically used antimitotic
agents. These arise from intrinsic or acquired mechanisms of resis-
tance. A major mechanism of MDR occurs via an overexpression of
energy dependent (adenosine triphosphate; ATP), unidirectional
transmembrane efflux pumps. Ultimate failure of chemotherapy
with antimitotic drugs often results due to MDR.10 P-Glycoprotein
(Pgp) is a 170 kDa protein that belongs to the ATP-binding cassette
superfamily of transporters.11–13 A series of homologous proteins
termed multidrug-resistance-proteins (MRPs) have also been re-
ported.14,15 The first MRP termed MRP1 was identified in a drug
resistant lung cancer cell line that does not express Pgp.16 All these
transporters bind drugs within the cell and release them to the
extracellular space using ATP.16 Tumor cells preexposed to cyto-
toxic compounds often overexpress these pumps which allow the
cells to manifest resistance in the presence of the cytotoxic drug.
MDR affects cancer patients with leukemia as well as solid tumors.
Overexpression of Pgp has been reported in a number of tumor
types, particularly after the patient has received chemotherapy
indicating the clinical importance of Pgp in MDR.13 In addition, it
has also been reported that Pgp expression may be a prognostic
indicator in certain cancers and is associated with poor response
to chemotherapy.17 MRP1 overexpression is not consistently found
in tumors even though it is expressed in a high percentage of leu-
kemia and solid tumors; however, its prognostic relevance is not
Thus new agents that possess antimitotic and antitumor activ-
ities without substrate activity for Pgp are highly coveted and
would be useful antitumor agents as single agents or in combina-
tion with chemotherapeutic agents including antimitotic agents.
2. Design of antimitotic compounds
The initial series of compounds (1–5, Fig. 2) showed excellent to
moderate (10ꢀ8–10ꢀ5 M) tumor cell inhibitory activity in the NCI
60 tumor cell line panel.25 The debenzylated compounds showed
poor antitumor activity indicating the importance of the benzyl
moiety. In COMPARE analysis, the Pearson correlation coefficients
(PCC) of this series of compounds matched with microtubule tar-
geting agents (vinblastine, paclitaxel, rhizoxine, maystansine and
vincristine). These compounds were also compared in human tu-
mor cell lines (T-24 and MCF-7) with cell lines resistant to antican-
cer drugs due to overexpression of Pgp or MRP-1 (NCI/ADR, MCF-7/
VP). It was found that 1 and 4 exhibited potency against all the cell
lines (1: submicromolar inhibitor and 4: micromolar inhibitor).
Additionally, compounds 2–5 caused dose-dependent sensitization
of NCI/ADR cells to vinblastine indicating inhibition of Pgp. How-
ever, they were ineffective in blocking the actions of MRP1.

A. Gangjee et al. / Bioorg. Med. Chem. 19 (2011) 4355–4365
4357
from 2-acetylbutyrolactone 15 as described previously.25 Sono-
gashira coupling of either 16 or 17 with various substituted phe-
nylacetylenes (either commercially available or synthesized as
shown in Schemes 2 and 3) using catalytic amount of tetrakistri-
phenylphosphine palladium (0) {Pd(PPh3)4}, copper iodide (CuI)
and triethylamine afforded 19a–i in 75–95% yields. Subsequent
catalytic hydrogenation of 19a–i with 5% palladium-on-charcoal,
afforded 20a–i in 85–96% yield. Hydrolysis with 1 N NaOH afforded
analytically pure 6–14 in 70–80% yield. The structure of 6 was
unequivocally confirmed from its X-ray crystal structure; which
also proved the regiospecific iodination at the 5-position.26
Scheme 2 shows the synthesis of substituted acetylenes 18c, 18e
and 18f–i (other acetylenes were commercially available) as cou-
pling partners in the Sonogashira coupling reactions with 16 or 17
(Scheme 1). Substituted iodobenzenes 21c and 21e were reacted
with trimethylsilylacetylene by microwave irradiation at 120 °C
for 5 min using catalytic palladium on charcoal as a palladium-
source in the Sonogashira coupling reaction.27 Microwave irradia-
tion was necessary to force the reaction of these non-activated acet-
ylenes. Subsequent desilylation using tert-butylammonium fluoride
(t-BAF) lead to acetylenes 18c and 18e. Substituted bromobenzenes
21f–i did not react with trimethylsilylacetylene using the above
conditions. Hence Sonogashira coupling conditions developed by
Fu et al.28 for unactivated bromobenzenes were used. Thus, substi-
tuted bromobenzenes were reacted with trimethylsilylacetylene
using catalytic amount of dichlorobis(benzonitrile)palladium(II) as
a palladium source and tri-tert-butylphoshine (P(t-Bu)3), a bulky,
electron-rich ligand. The reaction proceeded smoothly at ambient
temperature with yields in the 90% range. A tetrafluoroborate salt
of this ligand (P(t-Bu)3).HBF4) worked equally efficiently for this
reaction. Subsequent desilylation lead to desired acetylenes 18f–i.
Substituted bromobenzenes, which were not available commer-
cially, were synthesized using literature methods.29–31 Thus, com-
pounds 21g and 21h were synthesized by regioselective bromin
ation (Scheme 3).29,30 While 21i was synthesized from 25 by a Dakin
reaction31 followed by methylation.
Me
N
1
2
R = 3',4',5'-triOMe
R = 4'-OMe
N
3 R = 3'-OMe
4
5
R = 2'-OMe
R = H
N
H2N
R
Ph
Figure 2. Lead compounds.
6
7
8
9
R = 3',5'-diOMe
R = 3',4'-diOMe
R = 2',4'-diOMe
R = 2'-chloro
Me
N
R
10 R = 2',6'-diOMe
N
H2N
N
11 R = 2',3',4'-triOMe
12
R = 2',3',4',5'-tetraOMe
Ph
13 R = 2',4',5'-triOMe
14 R = 2',3',5'-triOMe
Figure 3. Target compounds 6–14.
In an attempt to optimize the antitubulin, antitumor, and resis-
tance reversal activities of the parent compounds by variations of
the substitutions on the phenyl ring of the 5-phenylethyl side
chain, compounds 6–14 (Fig. 3) were designed and synthesized.
Since the most potent compounds of the parent series were 1
(3,4,5-trimethoxyphenylethyl side chain) and 4 (2-methoxyphe-
nylethyl side chain), various combinations of methoxy substitu-
tions were included on the phenyl ring of the 5-phenylethyl side
chain in order to probe for optimum cytotoxicity with resistance-
reversal activity.
3. Chemistry
The synthesis of the target compounds 6–14 were accomplished
as indicated in Scheme 1. Compounds 16 and 17 were obtained
Me
I
COMe
N
O
N
6 steps
(ref. 25)
R1
N
O
Ph
R1 = N(Piv)2
17 R1 = NHPiv
16
15
R
R
Me
N
Me
18a-i
b or c
N
a
16
17
or
R
N
N
N
R1
R1
N
Ph
Ph
20a-i
19a-i
Me
N
d
R
N
H2N
N
Ph
6- 14
Scheme 1. Synthesis of target compounds. Reagents and conditions: (a) 10 mol % Pd(PPh3)4, NEt3, 20 mol % CuI, dichloroethane, rt, 20–24 h; (b) 5% Pd/C, DCM/MeOH (5:1), H2
50 psi; (c) 5% Pd/C, DCM/MeOH (1:1), H2 50 psi; (d) 1 N NaOH, MeOH, 80 °C, 24 h.

4358
A. Gangjee et al. / Bioorg. Med. Chem. 19 (2011) 4355–4365
R
R
e or
f
R
18c
R = 2,4-diOMe
g
18e R = 2,6-diOMe
X
SiMe3
22c, 22e, 22f-i
18f R = 2,3,4-triOMe
18g
R = 2,3,4,5-tetraOMe
18h R = 2,4,5-triOMe
18i R = 2,3,5-triOMe
18c, 18e, 18f-i
21c, 21e, 21f-i
X = I for condition e
X = Br for condition f
Scheme 2. Synthesis of substituted aryl alkynes. Reagents and conditions: (e) trimethylsilylacetylene, 10% Pd/C (4 mol % Pd), 16 mol % PPh3, 16 mol % CuI, NEt3, CH3CN, M.W.
120 °C, 5 min; (f) 3 mol % Pd(PhCN)2Cl2, 7 mol % P(t-Bu)3, 2 mol % CuI, HN(i-Pr)2, dioxane, rt 20 h; (g) t-BAF, THF, rt, 2 h.
and Pgp/MDR-reversal activities of compounds 1, 4 and 6–14 are
OMe
OMe
shown in Table 1. Compounds 6–14 exhibited two-digit micromo-
lar IC50 values against MCF-7 cells. Compound 13 with 2,4,5-trime-
thoxyphenyl side chain was the most potent compound with an
MeO
MeO
MeO
MeO
Br
h
IC50 of 15 lM against MCF-7 cells. Interestingly, all the compounds
OMe
OMe
were equipotent for the drug-resistant tumor cells than the paren-
tal MCF-7 cells. This suggests that these compounds were not sub-
strates for either Pgp or MRP1. In fact, several compounds were
somewhat more cytotoxic against NCI/ADR and/or MCF/VP cells
than that against MCF-7 cells (e.g., 6–10 and 12). For comparison,
paclitaxel, vinblastine, and vincristine inhibited MCF-7 cells at
nanomolar concentration. However, they exhibited diminished
inhibitory activity against drug-resistant NCI/ADR cells. Cisplatin
exhibited similar inhibitory activity in the micromolar range
against all the cell lines.
23
21g
OMe
OMe
MeO
MeO
i
Br
OMe
OMe
24
21h
Four (8, 10, 12 and 14) of the nine compounds inhibited Pgp
activity, making NCI/ADR cells more sensitive to vinblastine (indi-
cated as ‘Yes’ in the Pgp reversal column). Three (10, 11 and 12) of
the nine compounds inhibited MRP1, making MCF7/VP cells more
sensitive to vincristine (indicated as ‘Yes’ in MRP reversal column).
It is also possible that the increased sensitivity of vincristine in the
presence of 8, 10, 12 and 14 toward the resistant cell lines could be
due to a synergistic effect as well.
CHO
OH
OMe
j
k
MeO
Br
MeO
Br
MeO
Br
OH
25
OH
26
OMe
21i
Compound 14 was the most unusual compound and remarkably
afforded sensitization of resistant tumor cells where the resistance
was due to both Pgp and MRP1. To our knowledge, this is the only
known pyrrolo[2,3-d]pyrimidine analog that has antitumor activ-
ity of its own and restores sensitivity of antitumor agents to tumor
cells resistant to these agents due to both Pgp and MRP1. Though
14 has a modest antitumor activity it serves as a lead compound
for optimization of antitumor activity and ability to restore sensi-
tivity to antimitotic agents in tumor cells resistant to these agents
due to Pgp and/or MRP1. Such agents could be used alone or in
Scheme 3. Synthesis of substituted aryl bromides. Reagents and conditions: (h) Br2,
DCM, ꢀ10 °C, 1 h; (i) Br2, glacial HOAc, 0 °C, 1 h; (j) 1 N KOH, 3% H2O2, 30 min; (k)
dimethyl sulfate, acetone, rt, 14 h.
4. Results and discussion
The cytotoxicity, resistance factors [resistance factor = IC50 va-
lue against drug-resistant cancer cell line (NCI/ADR or MCF-7/
VP)/IC50 value against drug-sensitive cancer cell line (MCF-7)]
Table 1
Cytotoxicity data (IC50 values in lM), MDR reversal activities and effect on microtubule depolymerization of compounds 6–14
Compd #
MCF-7
Cytotoxicitya
Reversal
Microtubule depolymerization
NCI/ADR
(Pgp+/MRP1ꢀ)
0.25 0.12
2.7 0.4
Resistance
factorc
MCF-7/VP
(Pgp-/MRP1+)
Resistance
factorc
Pgp
MRP
(Pgp-/MRP1ꢀ)
1b
0.3 0.16
2.3 0.6
0.83
1.17
0.48
0.29
0.35
0.55
0.54
0.61
0.29
0.66
0.66
1080
>134
167
0.25 0.16
2.6 0.1
0.83
1.13
0.68
0.41
0.71
0.75
0.91
1.11
0.37
1
1.26
1.1
1.1
15.5
0.6
No
Yes
No
No
Yes
No
Yes
No
Yes
No
Yes
No
No
Yes
No
No
No
No
Yes
No
No
Yes
Yes
ND
No
No
No
No
Yes
Yes
Yes
No
No
No
Yes
Yes
NA
4b
6
7
8
9
10
11
12
13
25
17
17
20
22
18
24
15
6
6
4
4
4
3
6
3
12
5
6
5
17
7
6
2
2
4
4
6
5
7
3
2
12
15
20
20
9
11
12
11
7
5
5
5
4
10
4
15
14
50 11
33 11
2.7 0.5
>375
100
12.2 4.2
63 13
paclitaxel
vinblastine
vincristine
cisplatin
0.0025 0.0006
0.0028 0.0005
0.0006 0.0001
11.5 4.1
0.0028 0.0006
0.003 0.0006
0.0093 0.0014
7.0 2.9
1.1
a
b
c
Values are expressed as
From Ref. 25.
lM and represent the mean SEM for four experiments.
Resistance factor = IC50 value against drug-resistant cancer cell line (NCI/ADR or MCF-7/VP)/IC50 value against drug-sensitive cancer cell line (MCF-7).

A. Gangjee et al. / Bioorg. Med. Chem. 19 (2011) 4355–4365
4359
Table 2
Tumor cell inhibitory activity GI50 (lM) of compound 12 (NCI)
Leukemia
Colon cancer (ctd.)
Panel/cell line
Melanoma (ctd.)
Panel/cell line
Renal cancer (ctd.)
Panel/cell line
Panel/cell line
GI50
GI50
GI50
GI50
HL-60 (TB)
K-562
MOL T-4
RPMI-8226
SR
2.06
2.48
3.96
1.69
2.35
HCT-116
HCT-15
HT29
KM12
SW-620
3.68
3.2
3.6
3.33
3.19
SK-MEL-5
UACC-257
UACC-62
1.46
10.4
2.13
TK-10
UO-31
Prostate cancer
PC-3
DU-145
7.62
5.37
3.08
2.5
NSCLC
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
CNS cancer
SF-268
SF-295
SF-539
SNB-19
Ovarian cancer
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Breast cancer
MCF7
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
BT-549
T-47D
5.33
7.73
3.96
1.39
4.27
3.61
6.92
2.81
6.05
4.4
1.2
1.05
2.02
3.69
2.46
0.34
9.75
1.69
2.82
1.39
1.86
4.68
2.25
3.93
8.83
11.9
4.71
2.98
SNB-75
U251
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
Renal cancer
786-0
A498
ACHN
CAKI-1
SN12C
5.29
10.4
3.33
5.09
4.05
4.5
MDA-MB-468
2.25
9.36
2.5
2.8
5.89
6.86
combination with existing antimitotic agents against resistant tu-
mors. Additionally, compounds 10–12 caused microtubule depoly-
merization similar to 1 and vinca alkaloids.
actomyosin (A-6394) were obtained from Sigma Chemical
Company (St. Louis, MO). RPMI-1640 and -MEM culture media
and fetal bovine serum were from GibcoBRL (Grand Island, NY).
a
Compound 12, the 2,3,4,5-tetraOMe analog was also tested in
the NCI 6032 cell line panel. It inhibited the growth of most of
the cell lines at GI50 values that ranged from submicromolar to
two digit micromolar (Table 2). Compound 12 had a GI50 value of
0.34 lM against MDA-MD-435 breast cancer cell line. Additionally
it exhibited single digit micromolar GI50 values against almost 53
6.1.1. Assay of cytotoxicity and reversals of MDR
To test for reversal of Pgp-mediated MDR, NCI/ADR cells were
placed into 96-well tissue culture plates at approximately 15% con-
fluency and allowed to attach and recover for 24 h. The cells were
then treated with varying concentrations (as allowed by solubility)
of a test compound in the presence of 0 or 50 nM vinblastine for
48 h according to previously described procedures.34–40 After
48 h, cell survival was assayed using the sulforhodamine B (SRB)
binding assay.41 The percentage of cells killed is calculated as the
percentage decrease in SRB binding as compared with control cul-
tures and is taken from the mean of the absorbance measurements
of three equally treated wells. Reversal of MDR is indicated if the
compound enhances the toxicity of vinblastine toward the NCI/
ADR cells. The reversal index (Pgp Antagonism Score) is calculated
as the percentage of surviving NCI/ADR cells in the absence of vin-
blastine/the percentage of surviving NCI/ADR cells in the presence
of vinblastine. Control cultures included equivalent amounts of
ethanol (as the solvent control), which does not modulate the
growth or drug-sensitivity of these cells at the doses used in these
studies. To assess the toxicity of the compounds toward drug-sen-
sitive cells, the effects of the test modulators on the growth of
drug-sensitive MCF-7 cells were determined by the same methods.
To test for reversal of MRP1-mediated MDR, MCF-7/VP cells were
placed into 96-well tissue culture plates at approximately 15% con-
fluency and were allowed to attach and recover for 24 h. The cells
were then treated with varying concentrations (as allowed by sol-
ubility) of a test compound in the presence of 0 or 1 nM vincristine
for 48 h as above.
cell lines.
5. Conclusion
Nine pyrrolo[2,3-d]pyrimidine analogues are reported as antitu-
mor antimitotic agents. All the compounds were found to be one to
two-digit micromolar inhibitors of the proliferation of MCF-7 cells
in culture. Interestingly, all of these compounds (except 11 and 14)
were cytotoxic against sensitive and resistant MCF-7/VP (which
overexpresses MRP1) and NCI/ADR (which overexpresses pgp) cell
lines. Five (6, 8 ,10, 11, 12) out of the nine compounds were found
to restore sensitivity of vincristine or vinblastine to either Pgp or
MRP1 activity, respectively; while compound 14 restores sensitiv-
ity to both. This raises the possibility of further development of
these compounds as adjuvents to paclitaxel or such agents which
are not active against resistant cell lines. The advantage of this lies
in these compounds having their own antimicrotubule activity;
probably binding at a novel binding site on tubulin (other than that
of the paclitaxel domain, vinca domain or colchicine domain) and
also potentiating the activity of antimicrotubule agents used in
combination with these agents.
6. Experimental
After 48 h, cell survival was assayed using the SRB binding as-
say. Reversal of MDR is indicated if the compound enhances the
toxicity of vincristine toward the MCF-7/VP cells. The reversal
index (MRP1 Antagonism Score) is calculated as the percentage
of surviving MCF-7/VP cells in the absence of vincristine/the per-
centage of surviving MCF-7/VP cells in the presence of vincristine.
6.1. General methods for biological evaluations: materials
MCF-7 breast carcinoma cells and NCI/ADR cells, an MDR line
that overexpresses Pgp, were obtained from the Division of Cancer
Treatment of the National Cancer Institute. MCF-7/VP cells that ex-
press MRP1 but not Pgp were provided by Drs. Schneider and Cow-
an.33 Human bladder carcinoma T24 cells were from the American
Type Culture Collection. Sulforhodamine B, antibodies against b-
tubulin (T-4026), proteinase K (P2308), RNase A (R-5503), and
6.1.2. Effect on tubulin assembly in vitro
Microtubule protein (MTP), comprising tubulin and MAPs, was
isolated from fresh bovine brain by three cycles of assembly and
disassembly, and pure tubulin was isolated by subsequent ion-ex-

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A. Gangjee et al. / Bioorg. Med. Chem. 19 (2011) 4355–4365
change chromatography, as described by Vallee.42 The effects of
compounds 6–14 on tubulin assembly were determined following
the procedures of Bai et al.43 Briefly, 0.25 mL samples containing
calculated from Ti = Tz. The LC50 (concentration of drug resulting in
a 50% reduction in the measured protein at the end of the drug
treatment as compared to that at the beginning) indicating a net
loss of cells following treatment is calculated from [(Ti ꢀ Tz)/
Tz] ꢂ 100 = ꢀ50. Values are calculated for each of these three
parameters if the level of activity is reached; however, if the effect
is not reached or is exceeded, the value for that parameter is ex-
pressed as greater or less than the maximum or minimum concen-
tration tested.
2.5 mg of tubulin/mL (67 lM tubulin) in 1 M glutamate, pH 6.6,
containing 4% DMSO were incubated with ethanol, compounds
6–14, paclitaxel, vinblastine or vincristine at 0 °C for 15 min. GTP
(0.5 mM) was then added to the samples followed by rapid warm-
ing to 37 °C, and the absorbance at 340 nm was continuously mon-
itored for approximately 60 min. Effect on microtubule assembly
was recorded as change in A340. A decreased A340 indicates
microtubule depolymerization.
6.2. General methods for synthesis
6.1.3. NCI-60 cancer cell line screening44
In these tests, the human tumor cell lines of the cancer screening
panel are grown in RPMI 1640 medium containing 5% fetal bovine
All evaporations were carried out in vacuo using a rotary evap-
orator. Analytical samples were dried in vacuo (0.2 mm Hg) in a
CHEM-DRY drying apparatus over P2O5 at 50–70 °C. Melting points
were determined on a MEL-TEMP II melting point apparatus with
FLUKE 51 K/J electronic thermometer and are uncorrected. Nuclear
magnetic resonance spectra for proton (1H NMR) were recorded on
a Bruker WH-300 (300 MHz) spectrometer. The chemical shift val-
ues are expressed in ppm (parts per million) relative to tetrameth-
ylsilane as an internal standard: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; br, broad singlet. The relative integrals of
peak areas agreed with those expected for the assigned structures.
Thin-layer chromatography (TLC) was performed on WHATMAN
UV254 silica gel plates with a fluorescent indicator, and the spots
were visualized under 254 and/or 365 nm illumination. Propor-
tions of solvents used for TLC are by volume. Column chromatogra-
phy was performed on a 230–400 mesh silica gel purchased from
Fisher Scietific. Elemental analyses were performed by Atlantic
Microlab, Inc., Norcross, GA. Element compositions are within
0.4% of the calculated values. All solvents and chemicals were pur-
chased from Aldrich Chemical Co. or Fisher Scientific and were
used as received.
serum and 2 mM
L-glutamine. For a typical screening experiment,
cells are inoculated into 96 well microtiter plates in 100
l
L at plating
densities ranging from 5000 to 40,000 cells/well depending on the
doubling time of individual cell lines. After cell inoculation, the
microtiter plates are incubated at 37 °C, 5% CO2, 95% air and 100%
relative humidity for 24 h prior to addition of experimental drugs.
After 24 h, two plates of each cell line are fixed in situ with TCA, to
represent a measurement of the cell population for each cell line
at the time of drug addition (Tz). Experimental drugs are solubilized
in dimethyl sulfoxide at 400-fold the desired final maximum test
concentration and stored frozen prior to use. At the time of drug
addition, an aliquot of frozen concentrate is thawed and diluted to
twice the desired final maximum test concentration with complete
medium containing 50
or 1/2 log serial dilutions are made to provide a total of five drug con-
centrations plus control. Aliquots of 100 l of these different drug
dilutions are added to the appropriate microtiter wells already con-
taining 100 l of medium, resulting in the required final drug con-
lg/mL gentamicin. Additional four, 10-fold
l
l
centrations. Following drug addition, the plates are incubated for
an additional 48 h at 37 °C, 5% CO2, 95% air, and 100% relative humid-
ity. For adherent cells, the assay is terminated by the addition of cold
6.2.1. General procedure for the synthesis of 19a–i
To a 50 mL round bottom flask equipped with a stir bar and cov-
ered with an aluminum foil was added 16 or 17 (0.5 mmol), substi-
tuted phenylacetylene (1.5 mmol), copper(I)iodide (20 mol %),
tetrakis(triphenylphosphine)palladium(0) (10 mol %) and flask
was sealed with a rubber septum. The flask was evacuated and
back filled with nitrogen (repeated three times). Anhydrous trieth-
ylamine (0.15 mL) and anhydrous dichloroethane (10 mL) were
then added with the help of a syringe. The yellow-brown solution
was stirred at room temperature under nitrogen atmosphere in
dark for 24 h (until disappearance of starting material; monitored
by TLC). At the end of the reaction, silica gel (1 g) was added and
solvents evaporated to make a silica gel plug which was loaded
on top of a silica gel column (3 cm ꢂ 15 cm) pre-treated with tri-
ethylamine/ethyl acetate/hexanes (1:1:25) and continued with a
gradient elution, collecting 10 mL fractions. For 19a, 19c, 19e and
19g–i (dipivalated compounds), the product eluted with triethyl-
amine/ethyl acetate/hexanes (1:1:15); while for 19b, 19d and 19f
(monopivalated compounds), the product eluted with triethyl-
amine/ethyl acetate/hexanes (1:1:10). The fractions containing
product spots (TLC) were pooled and evaporated under reduced
pressure. The residual solvents were further removed using high
vacuum oil pump.
TCA. Cells are fixed in situ by the gentle addition of 50 ll of cold 50%
(w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at
4 °C. The supernatant is discarded, and the plates are washed five
times with tap water and air dried. Sulforhodamine B (SRB) solution
(100 ll) at 0.4% (w/v) in 1% acetic acid is added to each well, and
plates are incubated for 10 min at room temperature. After staining,
unbound dye is removed by washing five times with 1% acetic acid
and the plates are air dried. Bound stain is subsequently solubilized
with 10 mM trizma base, and the absorbance is read on an auto-
mated plate reader at a wavelength of 515 nm. For suspension cells,
the methodology is the same except that the assay is terminated by
fixing settled cells at the bottom of the wells by gently adding 50 ll
of 80% TCA (final concentration, 16% TCA). Using the seven absor-
bance measurements [time zero, (Tz), control growth, (C), and test
growth in the presence of drug at the five concentration levels
(Ti)], the percentage growth is calculated at each of the drug concen-
trations levels. Percentage growth inhibition is calculated as:
½ðTi ꢀ TzÞ=ðC ꢀ TzÞꢁ ꢂ 100 for concentrations for which Ti > =
¼ Tz
½ðTi ꢀ TzÞ=Tzꢁ ꢂ 100 for concentrations for which Ti < Tz:
6.2.1.1. N-{7-Benzyl-4-methyl-5-[(3,5-dimethoxyphenyl)ethy-
nyl]-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-N-(2,2-dimethylpropano
yl)-2,2-dimethylpropanamide (19a). Compound 19a was syn-
thesized from 16 (266 mg, 0.5 mmol), 3,5-dimethoxyphenylacety-
lene 18a (243.3 mg, 1.5 mmol) using the general procedure
described above to afford after purification 272 mg (96%) as a col-
orless semisolid: TLC Rf 0.24 (ethyl acetate/triethylamine/hexanes,
1:1:6); 1H NMR (DMSO-d6): 1.18 (s, 18H, C(CH3)3), 2.89 (s, 3H, 4-
Three dose response parameters are calculated for each experi-
mental agent. Growth inhibition of 50% (GI50) is calculated from
[(Ti ꢀ Tz)/(C ꢀ Tz)] ꢂ 100 = 50, which is the drug concentration
resulting in a 50% reduction in the net protein increase (as mea-
sured by SRB staining) in control cells during the drug incubation.
The drug concentration resulting in total growth inhibition (TGI) is

A. Gangjee et al. / Bioorg. Med. Chem. 19 (2011) 4355–4365
4361
CH3), 3.78 (s, 6H, OCH3), 5.42 (s, 2H, CH2C6H5), 6.57–6.58 (d, 1H,
C6H3), 6.69 (s, 2H, C6H3), 7.21–7.32 (m, 5H, C6H5), 8.16 (s, 1H,
J = 8.7 Hz), 7.33–7.62 (m, 5H, C6H5), 7.88 (s, 1H, C6–H), 9.88 (br,
1H, NHPiv, exch); HRMS (EI): calcd for C30H32N4O4: 512.2424;
found: 512.2456.
C6–H); HRMS (EI): calcd for
C34H38N4O4: 566.2893; found:
566.2841.
6.2.1.7.
N-{7-Benzyl-4-methyl-5-[(2,3,4,5-tetramethoxyphe-
6.2.1.2. N-{7-Benzyl-4-methyl-5-[(3,4-dimethoxyphenyl)ethy-
nyl]-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-N-2,2-dimethylpropana-
mide (19b). Compound 19b was synthesized from 17 (225 mg,
0.5 mmol), 3,4-dimethoxyphenylacetylene 18b (243.3 mg,
1.5 mmol) using the general procedure described above to afford
after purification 237 mg (98%) as a lustrous off-white solid: TLC
Rf 0.21 (ethyl acetate/triethylamine/hexanes, 1:1:3); mp 158–
161 °C; 1H NMR (DMSO-d6): 1.26 (s, 9H, C(CH3)3), 2.87 (s, 3H, 4-
CH3), 3.80 (s, 6H, OCH3), 5.41 (s, 2H, CH2C6H5), 7.0–7.36 (m, 8H,
C6H3 and C6H5), 7.89 (s, 1H, C6–H), 9.92 (br, 1H, NHPiv, exch);
HRMS (EI): calcd for C29H30N4O3: 482.2318; found: 482.2303.
nyl)ethynyl]-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-N-(2,2-dimethyl
propanoyl)-2,2-dimethylpropanamide (19g). Compound 19g
was synthesized from 16 (191.5 mg, 0.36 mmol), 2,3,4,5-tetra-
methoxyphenylacetylene 18g (240 mg, 1.08 mmol) copper(I) io-
dide (13 mg, 0.072 mmol), tetrakis(triphenylphosphine)palladium
(0) (41 mg, 0.036 mmol), using the general procedure described
above to afford after purification 202 mg (89.5%) as a light brown
solid : TLC Rf 0.22 (ethyl acetate/triethylamine/hexanes, 1:1:6);
1H NMR (DMSO-d6): 1.18 (s, 18H, C(CH3)3), 2.96 (s, 3H, 4-CH3),
3.82–3.93 (t, 12H, OCH3), 5.40 (s, 2H, CH2C6H5), 6.71 (s, 1H, C6H),
7.21–7.34 (m, 5H, C6H5), 8.11 (s, 1H, C6–H); HRMS (EI): calcd for
C36H42N4O6: 626.3104; found: 626.3188.
6.2.1.3. N-{7-Benzyl-4-methyl-5-[(2,4-dimethoxyphenyl)ethy-
nyl]-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-N-(2,2-dimethylpropano
yl)-2,2-dimethylpropanamide (19c). Compound 19c was syn-
thesized from 16 (266 mg, 0.5 mmol), 2,4-dimethoxyphenylacety-
lene 18c (243.3 mg, 1.5 mmol) using the general procedure
described above to afford after purification 266 mg (94%) as a
off-white solid: TLC Rf 0.2 (ethyl acetate/triethylamine/hexanes,
1:1:6); 1H NMR (DMSO-d6): 1.15 (s, 18H, C(CH3)3), 2.89 (s, 3H, 4-
CH3), 3.79 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 5.38 (s, 2H, CH2C6H5),
6.62 (m, 1H, C6H3), 7.18–7.35 (m, 6H, C6H3 and C6H5), 8.04 (s, 1H,
6.2.1.8. N-{7-Benzyl-4-methyl-5-[(2,4,5-trimethoxyphenyl)ethy-
nyl]-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-N-(2,2-dimethylpropano
yl)-2,2-dimethylpropanamide (19h). Compound 19h was syn-
thesized from 16 (213 mg, 0.4 mmol), 2,4,5-trimethoxyphenylacet-
ylene 18h (230 mg, 1.2 mmol) copper(I)iodide (15 mg, 0.08 mmol),
tetrakis(triphenylphosphine)palladium(0) (45 mg, 0.04 mmol),
using the general procedure described above to afford after purifica-
tion 225 mg (94%) as a light brown solid: TLC Rf 0.19 (ethyl acetate/
triethylamine/hexanes, 1:1:6); mp 145–147 °C; 1H NMR (DMSO-
d6): 1.16–1.168 (d, 18H, C(CH3)3), 2.91 (s, 3H, 4-CH3), 3.71–3.72
(br, 3H, OCH3), 3.83–3.84 (br, 6H, OCH3), 5.39 (s, 2H, CH2C6H5),
6.73–6.74 (d, 1H, C6H2, J = 2.4 Hz), 6.98–6.99 (d, 1H, C6H2,
J = 2.4 Hz), 7.20–7.31 (m, 5H, C6H5), 8.05 (s, 1H, C6–H); HRMS (EI):
calcd for C35H40N4O5: 596.2998; found: 596.2959.
C6–H); HRMS (EI) calcd for
C34H38N4O4: 566.2893; found:
566.2921.
6.2.1.4. N-{7-Benzyl-4-methyl-5-[(2-chlorophenyl)ethynyl]-7H-
pyrrolo[2,3-d]pyrimidin-2-yl}-N-2,2-dimethylpropanamide
(19d). Compound 19d was synthesized from 17 (225 mg,
0.5 mmol), 2-chlorophenylacetylene 18d (205 mg, 1.5 mmol) using
the general procedure described above to afford after purification
218 mg (93%) as a white solid: TLC Rf 0.524 (ethyl acetate/triethyl-
amine/hexanes, 1:1:3); mp 134–136 °C; 1H NMR (DMSO-d6): 1.24
(s, 9H, C(CH3)3), 2.87 (s, 3H, 4-CH3), 5.40 (s, 2H, CH2C6H5), 7.30–
7.64 (m, 9H, C6H3 and C6H5), 8.01 (s, 1H, C6–H), 9.92 (br, 1H, NHPiv,
exch); Anal. Calcd for C27H25ClN4O: C, 70.96; H, 5.51; N, 12.26; Cl,
7.75. Found: C, 70.82; H, 5.52; N, 12.12; Cl, 7.85.
6.2.1.9. N-{7-Benzyl-4-methyl-5-[(2,3,5-trimethoxyphenyl)ethy-
nyl]-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-N-(2,2-dimethylpropa-
noyl)-2,2-dimethylpropanamide (19i). Compound 19i was
synthesized from 16 (186 mg, 0.35 mmol), 2,3,5-trimethoxyphe-
nylacetylene 18i (201 mg, 1.05 mmol) copper(I)iodide (13 mg,
0.07 mmol), tetrakis(triphenylphosphine)palladium(0) (39 mg,
0.035 mmol), using the general procedure described above to af-
ford after purification 200 mg (96%) as a light brown semisolid :
TLC Rf 0.2 (ethyl acetate/triethylamine/hexanes, 1:1:6); 1H NMR
(DMSO-d6): 1.15–1.19 (m, 18H, C(CH3)3), 2.94 (s, 3H, 4-CH3),
3.73–3.83 (m, 9H, OCH3), 5.43 (s, 2H, CH2C6H5), 6.59–7.32 (m,
7H, C6H2 and C6H5), 8.18 (s, 1H, C6–H); HRMS (EI): calcd for
6.2.1.5. N-{7-Benzyl-4-methyl-5-[(2,6-dimethoxyphenyl)ethy-
nyl]-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-N-(2,2-dimethylpropano
yl)-2,2-dimethylpropanamide (19e). Compound 19e was syn-
thesized from 16 (243 mg, 0.45 mmol), 2,6-dimethoxyphenylacet-
ylene 18e (230 mg, 1.35 mmol) using the general procedure
described above to afford after purification 250 mg (96.5%) as a
light green solid : TLC Rf 0.2 (ethyl acetate/triethylamine/hexanes,
1:1:6); mp 158–159.5 °C 1H NMR (DMSO-d6): 1.17–1.18 (s, 18H,
C(CH3)3), 2.91 (s, 3H, 4-CH3), 3.83 (s, 6H, OCH3), 5.40 (s, 2H,
CH2C6H5), 6.72–6.81 (m, 3H, C6H3), 7.18–7.35 (m, 5H, C6H5), 8.15
(s, 1H, C6–H); HRMS (EI): calcd for C34H38N4O4: 566.2893; found:
566.2871.
C35H40N4O5: 596.2998; found: 596.3001.
6.2.2. General procedure for the synthesis of compounds 20a,
20c, 20e, 20g–20i
To a Parr hydrogenation bottle was added 5% Pd/C followed by a
solution of 19a/19c/19e/19g–19i dissolved in dichloromethane
(20 mL) and methanol (5 mL). The mixture was hydrogenated at
50 psi at room temperature for 3.5 h. After filtration, the catalyst
was thoroughly washed with hot methanol–methylene chloride
mixture (1:1, 50 mL). The filtrate was concentrated in vacuo and
silica gel (1 g) was added to the residue. The solvent was evapo-
rated to afford a plug. The silica gel plug obtained was loaded onto
a silica gel column and eluted with 1:1:10 ethyl acetate/triethyl-
amine/hexanes. Fractions containing the product (TLC) were
pooled, and the solvent was evaporated to afford analytically pure
compound.
6.2.1.6. N-{7-Benzyl-4-methyl-5-[(2,3,4-trimethoxyphenyl)ethy-
nyl]-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-N-2,2-dimethylpropana-
mide (19f). Compound 19f was synthesized from 17 (296 mg,
0.66 mmol), 2,3,4-trimethoxyacetylene 18f (384 mg, 2 mmol), cop-
per(I)iodide (25 mg, 0.13 mmol), tetrakis(triphenylphosphine)pal-
ladium(0) (76.2 mg, 0.066 mmol), using the general procedure
described above to afford after purification 329 mg (97%) as a
sticky brown semisolid : TLC Rf 0.27 (ethyl acetate/triethylamine/
hexanes, 1:1:3); 1H NMR (DMSO-d6): 1.25 (s, 9H, C(CH3)3), 2.87
(s, 3H, 4-CH3), 3.76–3.89 (m, 9H, OCH3), 5.39 (s, 2H, CH2C6H5),
6.82–6.85 (d, 1H, C6H2, J = 8.7 Hz), 7.17–7.20 (d, 1H, C6H2,
6.2.2.1. N-{7-Benzyl-4-methyl-5-[(3,5-dimethoxyphenyl)ethyl]-
7H-pyrrolo[2,3-d]pyrimidin-2-yl}-N-(2,2-dimethylpropanoyl)-
2,2-dimethylpropanamide (20a). Compound 20a was synthe-
sized from 19 (152 mg, 0.27 mmol) and 5% Pd/C (150 mg) using

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A. Gangjee et al. / Bioorg. Med. Chem. 19 (2011) 4355–4365
the general procedure described above to afford after purification
140 mg (91%) as a white solid: TLC Rf 0.238 (ethyl acetate/triethyl-
amine/hexanes, 1:1:6); 1H NMR (DMSO-d6): 1.10 (s, 18H, C(CH3)3),
2.71 (s, 3H, 4-CH3), 2.87 (t, 2H, –CH2–), 3.13 (t, 2H, –CH2–), 3.67 (s,
6H, OCH3), 5.33 (s, 2H, CH2C6H5), 6.31 (s, 1H, C6–H), 6.46–7.48 (m,
8H, C6H3 and C6H5); HRMS (EI): calcd for C34H42N4O4 570.3206;
found: 570.3235.
and C6H5); HRMS (EI): calcd for C35H44N4O5: 600.3311; found:
600.3285.
6.2.3. General procedure for the synthesis of compounds 20b,
20d, 20f
To a Parr hydrogenation bottle was added 5% Pd/C followed by
a solution of 19b/19d/19f dissolved in dichloromethane (10 mL)
and methanol (10 mL). The mixture was hydrogenated at 50 psi
at room temperature for 24 h. After filtration, the catalyst was
thoroughly washed with hot methanol–methylene chloride mix-
ture (1:1, 50 mL). The filtrate was concentrated in vacuo and sil-
ica gel (1 g) was added to the residue. The solvent was evaporated
to afford a plug. The silica gel plug obtained was loaded onto a
silica gel column and eluted with 1:1:6 ethyl acetate/triethyl-
amine/hexanes. Fractions containing the product (TLC) were
pooled, and the solvent was evaporated to afford analytically pure
compound.
6.2.2.2. N-{7-Benzyl-4-methyl-5-[(2,4-dimethoxyphenyl)ethyl]-
7H-pyrrolo[2,3-d]pyrimidin-2-yl}-N-(2,2-dimethylpropanoyl)-
2,2-dimethylpropanamide (20c). Compound 20c was synthe-
sized from 19c (150 mg, 0.265 mmol), 5% Pd/C (150 mg) using
the general procedure described above to afford after purification
140 mg (92%) as a white semisolid : TLC Rf 0.21 (ethyl acetate/tri-
ethylamine/hexanes, 1:1:6); 1H NMR (DMSO-d6): 1.14 (s, 18H,
C(CH3)3), 2.72 (s, 3H, 4-CH3), 2.75 (t, 2H, –CH2–), 2.95 (t, 2H, –
CH2–), 3.69 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 5.31 (s, 2H, CH2C6H5),
6.49 (s, 1H, C6–H), 6.99–7.39 (m, 8H, C6H3 and C6H5); HRMS (ESI):
calcd for C34H43N4O4 (M+H)+: 571.3284; found: 571.3311.
6.2.3.1. N-{7-Benzyl-4-methyl-5-[(3,4-dimethoxyphenyl)ethyl]-
7H-pyrrolo[2,3-d]pyrimidin-2-yl}-N-2,2-dimethylpropanamide
(20b). Compound 20b was synthesized from 19b (130 mg,
0.27 mmol), 5% Pd/C (130 mg) using the general procedure de-
scribed above to afford after purification 117 mg (89%) as a color-
less semisolid: TLC Rf 0.21 (ethyl acetate/triethylamine/hexanes,
1:1:3); 1H NMR (DMSO-d6): 1.22 (s, 9H, C(CH3)3), 2.68 (s, 3H, 4-
CH3), 2.86 (t, 2H, –CH2–), 3.04 (t, 2H, –CH2–), 3.66–3.69 (d, 6H,
OCH3), 5.31 (s, 2H, CH2C6H5), 6.75 (s, 1H, C6–H), 6.79–7.29 (m,
8H, C6H3 and C6H5), 9.68 (s, 1H, NHPiv, exch); HRMS (EI): calcd
for C29H34N4O3: 486.2631; found: 486.2667.
6.2.2.3. N-{7-Benzyl-4-methyl-5-[(2,6-dimethoxyphenyl)ethyl]-
7H-pyrrolo[2,3-d]pyrimidin-2-yl}-N-(2,2-dimethylpropanoyl)-
2,2-dimethylpropanamide (20e). Compound 20e was synthe-
sized from 19e (150 mg, 0.26 mmol) and 5% Pd/C (150 mg) using
the general procedure described above to afford after purification
139 mg (92%) as a white liquid : TLC Rf 0.22 (ethyl acetate/triethyl-
amine/hexanes, 1:1:6); 1H NMR (300 MHz) (DMSO-d6): 1.16 (s,
18H, C(CH3)3), 2.72 (s, 3H, 4-CH3), 2.79–2.83 (m, 4H, CH2CH2), (s,
6H, OCH3), 5.37 (s, 2H, CH2C6H5), 6.65–6.71 (m, 3H, C6H3), 7.12–
7.38 (m, 6H, C6H5 and C6–H); HRMS (ESI): calcd for C34H43N4O4
(M+H)+: 571.3284; found: 571.3286.
6.2.3.2.
N-{7-Benzyl-4-methyl-5-[(2-chlorophenyl)ethyl]-7H-
pyrrolo[2,3-d]pyrimidin-2-yl}-N-2,2-dimethylpropanamide
(20d). Compound 20d was synthesized from 19d (150 mg,
0.33 mmol), 5% Pd/C (150 mg) using the general procedure de-
scribed above to afford after purification 140 mg (92%) as a white
semisolid: TLC Rf 0.69 (ethyl acetate/triethylamine/hexanes,
1:1:3); 1H NMR (DMSO-d6): 1.22 (s, 9H, C(CH3)3), 2.69 (s, 3H, 4-
CH3), 3.04 (t, 4H, –CH2–CH2–), 5.31 (s, 2H, CH2C6H5), 7.21–7.30
(m, 10H, C6–H, C6H3 and C6H5), 9.71 (br, 1H, NHPiv, exch). HRMS
(EI): calcd for C27H30N4OCl: 416.2108; found: 416.2107.
6.2.2.4.
N-{7-Benzyl-4-methyl-5-[(2,3,4,5-tetramethoxyphe-
nyl)ethyl]-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-N-(2,2-dimethylpr
opanoyl)-2,2-dimethylpropanamide (20g). Compound 20g was
synthesized from 19g (150 mg, 0.24 mmol) and 5% Pd/C (150 mg)
using the general procedure described above to afford after purifi-
cation 136 mg (86%) as a colorless, sticky oil: TLC Rf 0.24 (ethyl ace-
tate/triethylamine/hexanes, 1:1:6); 1H NMR (DMSO-d6): 1.09 (s,
18H, C(CH3)3), 2.72 (s, 3H, 4-CH3), 2.86–2.87 (t, 2H, –CH2–), 3.02–
3.04 (t, 2H, –CH2–), 3.62–3.75 (m, 12H, OCH3), 5.31–5.33 (br, 2H,
CH2C6H5), 6.55 (s, 1H, C6–H), 7.07–7.43 (m, 6H, C6H1 and C6H5);
HRMS (EI): calcd for C36H46N4O6: 630.3417; found: 630.3403.
6.2.3.3. N-{7-Benzyl-4-methyl-5-[(2,3,4-trimethoxyphenyl)eth
yl]-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-N-2,2-dimethylpropana-
mide (20f). Compound 20f was synthesized from 19 (150 mg,
0.29 mmol) and 5% Pd/C (150 mg) using the general procedure de-
scribed above to afford after purification 104 mg (69%) as a white
semisolid: TLC Rf 0.265 (ethyl acetate/triethylamine/hexanes,
1:1:3); 1H NMR (DMSO-d6): 1.23 (s, 9H, C(CH3)3), 2.72 (s, 3H, 4-
CH3), 2.80 (t, 2H, –CH2–), 2.97 (t, 2H, –CH2–), 3.72–3.74 (m, 9H,
OCH3), 5.32 (s, 2H, CH2C6H5), 6.69–7.31 (m, 9H, C6–H , C6H2,
C6H5), 9.71 (br, 1H, NHPiv, exch); HRMS (EI): calcd for
6.2.2.5. N-{7-Benzyl-4-methyl-5-[(2,4,5-trimethoxyphenyl)eth
yl]-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-N-(2,2-dimethylpropa-
noyl)-2,2-dimethylpropanamide (20h). Compound 20h was
synthesized from 19h (150 mg, 0.25 mmol) and 5% Pd/C (150 mg)
using the general procedure described above to afford after purifi-
cation 135 mg (89%) as a off-white semisolid : TLC Rf 0.35 (ethyl
acetate/triethylamine/hexanes, 1:1:3); 1H NMR (DMSO-d6): 1.14–
1.23 (m, 18H, C(CH3)3), 2.73 (s, 3H, 4-CH3), 2.79–3.02 (m, 4H, –
CH2–CH2–), 3.58–3.75 (t, 3H, OCH3), 5.32 (s, 2H, CH2C6H5), 6.63–
7.38 (m, 8H, C6–H, C6H2 and C6H5); HRMS (EI): calcd for
C30H36N4O4: 516.2737; found: 516.3350.
6.2.4. General procedure for the synthesis of compounds 6–14
To a round bottom flask were added 20a–i followed by metha-
nol (10 mL) and 1 N sodium hydroxide (2 mL). The reaction mix-
ture was heated at reflux for 24 h. The reaction was then cooled
and methanol evaporated under vacuum. The precipitated solid
obtained was filtered, washed with cold water and air-dried. This
solid was then recrystallized from a mixture of dichloromethane
and hexanes (1:2).
C35H44N4O5: 600.3311; found: 600.3333.
6.2.2.6. N-{7-Benzyl-4-methyl-5-[(2,3,5-trimethoxyphenyl)eth
yl]-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-N-(2,2-dimethylpropano
yl)-2,2-dimethylpropanamide (20i). Compound 20i was syn-
thesized from 19i (150 mg, 0.25 mmol) and 5% Pd/C (150 mg) using
the general procedure described above to afford after purification
135 mg (89%) as a white solid : TLC Rf 0.489 (ethyl acetate/trieth-
ylamine/hexanes, 1:1:3); mp 94.5–96 °C 1H NMR (DMSO-d6):
1.14–1.19 (d, 18H, C(CH3)3), 2.75 (s, 3H, 4-CH3), 2.87–2.88 (t, 2H,
–CH2), 3.03–3.04 (t, 2H, –CH2–), 3.58–3.76 (t, 9H, OCH3), 5.32 (s,
2H, CH2C6H5), 6.32–6.45 (m, 2H, C6H2), 7.10–7.44 (m, 6H, C6–H
6.2.4.1. 7-Benzyl-4-methyl-5-[2-(3,5-dimethoxyphenyl)ethyl]-
7Hpyrrolo[2,3-d]pyrimidin-2-amine (6). Compound 6 was syn-
thesized from 20a (70 mg, 0.12 mmol) using the general procedure
described above to afford 41 mg (83%) as a white crystalline solid:

A. Gangjee et al. / Bioorg. Med. Chem. 19 (2011) 4355–4365
4363
TLC Rf 0.45 (ethyl acetate/triethylamine/hexanes, 5:1:3); mp
144.2–145.8 °C; 1H NMR (DMSO-d6): 2.52 (s, 3H, 4-CH3), 2.82 (t,
2H, –CH2–), 2.94 (t, 2H, –CH2–), 3.70 (s, 6H, OCH3), 5.16 (s, 2H,
CH2C6H5), 6.05 (br, 2H, 2-NH2, exch), 6.30–6.31 (d, 1H, C6H3),
6.39–6.40 (d, 2H, C6H3), 6.78 (s, 1H, C6–H), 7.09–7.31 (m, 5H,
C6H5). Anal. Calcd for C24H26N4O2: C, 71.62; H, 6.51; N, 13.92.
Found: C, 71.28; H, 6.32; N, 13.92.
procedure described above to afford 34 mg (73.5%) as a lustrous
white crystals: TLC Rf 0.27 (ethyl acetate/triethylamine/hexanes,
5:1:3); mp 120.2–121.2 °C; 1H NMR (DMSO-d6): 2.54 (s, 3H, 4-
CH3), 2.78–2.79 (t, 2H, –CH2–), 2.80–2.81 (t, 2H, –CH2–), 3.66–
3.78 (m, 12H, OCH3), 5.17 (s, 2H, CH2C6H5), 6.07 (br, 2H, 2-NH2,
exch), 6.60 (s, 1H, C6H3), 6.81 (s, 1H, C6–H), 7.11–7.32 (m, 5H,
C6H5). Anal. Calcd for C26H30N4O4: C, 67.51; H, 6.54; N, 12.11.
Found: C, 67.14; H, 6.65; N, 11.99.
6.2.4.2. 7-Benzyl-4-methyl-5-[2-(3,4-dimethoxyphenyl)ethyl]-
7Hpyrrolo[2,3-d]pyrimidin-2-amine (7). Compound 7 was syn-
thesized from 20b (70 mg, 0.1145 mmol) using the general proce-
dure described above to afford 51 mg (87%) as a white fluffy solid:
TLC Rf 0.44 (ethyl acetate/triethylamine/hexanes, 5:1:3); mp
109.0–110.0 °C; 1H NMR (DMSO-d6): 2.51 (s, 3H, 4-CH3), 2.82 (t,
2H, –CH2–), 2.94 (t, 2H, –CH2–), 3.67–3.69 (d, 6H, OCH3), 5.15 (s,
2H, CH2C6H5), 6.12 (br, 2H, 2-NH2, exch), 6.72–7.29 (m, 9H, C6H3,
C6–H and C6H5). Anal. Calcd for C24H26N4O2ꢃ0.5H2O: C, 70.05; H,
6.61; N, 13.62. Found: C, 69.70; H, 6.61; N, 13.45.
6.2.4.8. 7-Benzyl-4-methyl-5-[2-(2,4,5-trimethoxyphenyl)ethyl]
-7Hpyrrolo[2,3-d]pyrimidin-2-amine (13). Compound 13 was
synthesized from 20g (80 mg, 0.133 mmol) using the general pro-
cedure described above to afford 51.5 mg (88%) as fluffy white
crystals: TLC Rf 0.30 (ethyl acetate/triethylamine/hexanes, 5:1:3);
mp 142–144 °C; 1H NMR (DMSO-d6): 2.52 (s, 3H, 4-CH3), 2.75–
2.76 (t, 2H, –CH2–), 2.82–2.85 (t, 2H, –CH2–), 3.60–3.74 (t, 9H,
OCH3), 5.15 (s, 2H, CH2C6H5), 6.05 (br, 2H, 2-NH2, exch), 6.63–
7.28 (m, 8H, C6H2, C6–H and C6H5). Anal. Calcd for: C25H28N4O3:
C, 69.42; H, 6.53; N, 12.95. Found: C, 69.31; H, 6.45; N, 12.81.
6.2.4.3. 7-Benzyl-4-methyl-5-[2-(2,4-dimethoxyphenyl)ethyl]-
7Hpyrrolo[2,3-d]pyrimidin-2-amine (8). Compound 8 was syn-
thesized from 20c (100 mg, 0.175 mmol) using the general
procedure described above to afford 61 mg (70.5%) as faint green
lustrous crystals: TLC Rf 0.49 (ethyl acetate/triethylamine/hexanes,
5:1:3); mp 132–133.3 °C; 1H NMR (DMSO-d6): 2.54 (s, 3H, 4-CH3),
2.74 (t, 2H, –CH2–), 2.76 (t, 2H, –CH2–), 3.72–3.74 (m, 6H, OCH3),
5.16 (s, 2H, CH2C6H5), 6.06 (br, 2H, 2-NH2, exch), 6.39–7.30 (m,
9H, C6H3, C6–H and C6H5). Anal. Calcd for C24H26N4O2: C, 71.62;
H, 6.51; N, 13.92. Found: C, 71.34; H, 6.49; N, 13.76.
6.2.4.9. 7-Benzyl-4-methyl-5-[2-(2,3,5-trimethoxyphenyl)ethyl]
-7Hpyrrolo[2,3-d]pyrimidin-2-amine (14). Compound 14 was
synthesized from 20h (80 mg, 0.133 mmol) using the general pro-
cedure described above to afford 45 mg (78%) as a white powder:
TLC Rf 0.33 (ethyl acetate/triethylamine/hexanes, 5:1:3); mp
111–111.6 °C; 1H NMR (DMSO-d6): 2.54 (s, 3H, 4-CH3), 2.80–2.90
(m, 4H, –CH2–CH2–), 3.60–3.76 (t, 9H, OCH3), 5.17 (s, 2H, CH2C6H5),
6.08 (br, 2H, 2-NH2, exch), 6.34–6.35 (d, 1H, C6H2, J = 2.7 Hz), 6.45–
6.45 (d, 1H, C6H2, J = 2.7 Hz), 6.82 (s, 1H, C6–H), 7.11–7.297 (m, 5H,
C6H5). Anal. Calcd for C25H28N4O3: C, 69.42; H, 6.53; N, 12.95.
Found: C, 69.47; H, 6.45; N, 12.84.
6.2.4.4. 7-Benzyl-4-methyl-5-[2-(2-chlorophenyl)ethyl]-7Hpyr-
rolo[2,3-d]pyrimidin-2-amine (9). Compound 9 was synthe-
sized from 20d (65 mg, 0.14 mmol) using the general procedure
described above to afford 44 mg (90%) as yellow needles: TLC Rf
0.46 (ethyl acetate/triethylamine/hexanes, 5:1:3); mp 144.5–
146.5 °C; 1H NMR (DMSO-d6): 1H NMR (DMSO-d6): 2.54 (s, 3H, 4-
CH3), 2.98 (br, 4H, –CH2–CH2–), 5.16 (s, 2H, CH2C6H5), 6.07 (br,
2H, 2-NH2, exch), 6.76 (s, 1H, C6–H), 7.08–7.39 (m, 8H, C6H4 and
C6H5). Anal. Calcd for C22H21N4Cl: C, 70.11; H, 5.62; N, 14.87; Cl,
9.41. Found: C, 70.40; H, 5.72; N, 14.97; Cl, 9.16.
6.2.5. General procedure for the synthesis of compounds 22c
and 22e
To a 5 mL microwave reaction tube (Biotage Inc.) equipped with
a stir bar was added 21c or 21e (1 mmol), 10% Pd/C (0.04 mmol),
triphenylphosphine (0.16 mmol), copper (I) iodide (0.16 mmol),
trimethylsilylacetylene (1.05 mmol), triethylamine (3 mL) and ace-
tonitrile (2 mL). The microwave tube was sealed with a crimp-cap
and subjected to microwave heating in a microwave reactor (Initi-
ator, Biotage Inc.) set at 120 °C with ‘fixed hold time’ for 5 min. At
the end of the reaction, the grey-black suspension was passed
through Celite pad, washing thoroughly with hot ethyl acetate
(20 mL). The filtrate concentrated in reduced pressure and silica
gel (1 g) was added. Further evaporation lead to a silica gel plug
which was loaded on top of a silica gel column (2 cm ꢂ 20 cm)
and eluted with 5% ethyl acetate in hexanes. Fractions correspond-
ing to the product spot were evaporated under reduced pressure to
obtain an analytically pure product.
6.2.4.5. 7-Benzyl-4-methyl-5-[2-(2,6-dimethoxyphenyl)ethyl]-7
Hpyrrolo[2,3-d]pyrimidin-2-amine (10). Compound 10 was
synthesized from 20e (135 mg, 0.236 mmol) using the general pro-
cedure described above to afford 85 mg (89%) as white fluffy crys-
tals: TLC Rf 0.45 (ethyl acetate/triethylamine/hexanes, 5:1:3); mp
179.1–180.6 °C; 1H NMR (DMSO-d6): 2.56 (s, 3H, 4-CH3), 2.76 (t,
2H, –CH2–), 2.81 (t, 2H, –CH2–), 3.72 (m, 6H, OCH3), 5.17 (s, 2H,
CH2C6H5), 6.06 (br, 2H, 2-NH2, exch), 6.60–7.15 (m, 9H, C6H3, C6–
H and C6H5). Anal. Calcd for C24H26N4O2: C, 71.62; H, 6.51; N,
13.92. Found: C, 71.45; H, 6.50; N, 13.83.
6.2.5.1. (2,4-Dimethoxyphenylethynyl)trimethylsilane (22c).
Compound 22c was synthesized from 21c (264 mg, 1.0 mmol) using
the general procedure described above to afford 170 mg (73%) as a
off-white solid: TLC Rf 0.63 (ethyl acetate/hexanes, 1:3); mp 46.1–
47.2 °C (lit.45 mp 45.0–47.0 °C); 1H NMR (CDCl3): 0.26 (s, 9H,
Si(CH3)3), 3.95–3.99 (m, 6H, OCH3), 6.43 (d, 2H, C6H3), 7.42 (d, 1H,
C6H3); HRMS (EI): calcd for C13H18O2Si: 234.1076; found: 234.1067.
This compound matched in all aspects (1H NMR) with that reported
in the literature.45
6.2.4.6. 7-Benzyl-4-methyl-5-[2-(2,3,4-trimethoxyphenyl)ethyl]
-7Hpyrrolo[2,3-d]pyrimidin-2-amine (11). Compound 11 was
synthesized from 20f (82 mg, 0.16 mmol) using the general proce-
dure described above to afford 60 mg (87%) as a lustrous off-white
solid: TLC Rf 0.47 (ethyl acetate/triethylamine/hexanes, 5:1:3); mp
115–116.5 °C; 1H NMR (DMSO-d6): 2.55 (s, 3H, 4-CH3), 2.76–2.84
(m, 4H, –CH2–CH2–), 3.72–3.74 (m, 9H, OCH3), 5.16 (s, 2H,
CH2C6H5), 6.07 (br, 2H, 2-NH2, exch), 6.67–7.29 (m, 8H, C6H2, C6–
H and C6H5). Anal. Calcd for: C25H28N4O3: C, 69.42; H, 6.53; N,
12.95. Found: C, 69.25; H, 6.58; N, 12.90.
6.2.5.2. (2,6-Dimethoxyphenylethynyl)trimethylsilane (22e).
Compound 22e was synthesized from 21e (264 mg, 1.0 mmol)
using the general procedure described above to afford 176 mg
(75%) as a off-white solid: TLC Rf 0.63 (ethyl acetate/hexanes,
1:3); mp 105–106 °C; 1H NMR (CDCl3): 0.17 (s, 9H, Si(CH3)3),
3.75 (s, 6H, OCH3), 6.37–6.40 (d, 2H, C6H3), 7.15 (d, 1H, C6H3);
6.2.4.7. 7-Benzyl-4-methyl-5-[2-(2,3,4,5-tetramethoxyphenyl)
ethyl]-7Hpyrrolo[2,3-d]pyrimidin-2-amine
(12). Compound
12 was synthesized from 20f (63 mg, 0.1 mmol) using the general

4364
A. Gangjee et al. / Bioorg. Med. Chem. 19 (2011) 4355–4365
HRMS (EI): calcd for C13H18O2Si: 234.1076; found: 234.1106. The
1H NMR spectrum of this compound matched with that reported
in the literature.46
light to dark brown. The solution was stirred for 2 h at room tem-
perature under nitrogen. At the end of the reaction, silica gel
(500 mg) was added to the reaction mixture and solvent evapo-
rated in reduced pressure to get a silica gel plug, which was loaded
on top of a silica gel column (2 cm ꢂ 5 cm) and eluted with 5%
ethyl acetate in hexanes. Fractions corresponding to the product
spots were evaporated under reduced pressure to get substituted
aryl alkynes.
6.2.6. General procedure for the synthesis of compounds 22f–i
Aryl bromide (1.00 mmol; in case it is solid), Pd(PhCN)2Cl2
(11.5 mg, 0.030 mmol) and CuI (3.8 mg, 0.020 mmol) are added
to a dry, 10-mL round bottom flask, which is then sparged with ar-
gon and charged with dioxane (1.0 mL; Aldrich Sure/Seal anhy-
drous/99.8%). P(t-Bu)3 (70
0.07 mmol; made from 1gm P(t-Bu)3 and 4.95 mL anhydrous diox-
ane; preserved under argon), HN(i-Pr)2 (170 L, 1.20 mol; freshly
lL of a 1 M solution in dioxane;
6.2.7.1. 1-Ethynyl-2,4-dimethoxybenzene (18c). Compound
18c was synthesized from 22c (540 mg, 2.3 mmol) and tert-butyl-
ammonium fluoride solution (1.15 mL, 1.15 mmol) using the gen-
eral procedure described above to afford 240 mg (70%) as an
yellow-brown oil: TLC Rf 0.48 (ethyl acetate/hexanes, 1:3); 1H
NMR (CDCl3): 3.25 (s, 1H, C2H), 3.79–3.94 (m, 6H, OCH3), 6.47
(d, 2H, C6H3), 7.41 (d, 1H, C6H3). The 1H NMR spectrum of this
compound matched with that reported in the literature.45
l
distilled over CaH2), aryl bromide (1.00 mmol, in case it is liquid),
and the trimethylsilylacetylene (1.20 mmol) are added via syringe
to the stirred reaction mixture. After the aryl bromide has been
consumed, the reaction mixture is diluted with EtOAc (5 mL), fil-
tered through a small pad of Celite (with EtOAc rinsings), concen-
trated in reduced pressure and silica gel (1 g) was added. Further
evaporation lead to a silica gel plug which was loaded on top of
a silica gel column (2 cm ꢂ 20 cm) and eluted with 5% ethyl acetate
in hexanes. Fractions corresponding to the product spot were evap-
orated under reduced pressure to obtain analytically pure product.
6.2.7.2. 1-Ethynyl-2,6-dimethoxybenzene (18e). Compound
18e was synthesized from 22e (150 mg, 0.64 mmol) and tert-butyl-
ammonium fluoride solution (0.3 mL, 0.3 mmol) using the general
procedure described above to afford 99 mg (90%) as off-white so-
lid: TLC Rf 0.46 (ethyl acetate/hexanes, 1:3); 1H NMR (CDCl3):
3.35 (s, 1H, C2H), 3.90 (s, 6H, OCH3), 6.53–6.56 (d, 3H, C6H3); HRMS
(EI): calcd for C10H10O2 162.0680; found 162.0681. The 1H NMR
spectrum of this compound matched with that reported in the
literature.46
6.2.6.1.
(2,3,4-Trimethoxyphenylethynyl)trimethylsilane
(22f). Compound 22f was synthesized from 21f (247.1 mg,
1.0 mmol) using the general procedure described above to afford
218 mg (82.5%) as an yellow oil: TLC Rf 0.47 (ethyl acetate/hexanes,
1:3); 1H NMR (CDCl3): 0.25 (s, 9H, Si(CH3)3), 3.85 (s, 6H, OCH3),
3.97 (s, 3H, OCH3), 6.57–6.60 (d, 1H, C6H2, J = 8.7 Hz), 7.12–7.14
(d, 1H, C6H2, J = 8.7 Hz); HRMS (EI): calcd for C14H20O3Si:
264.1181; found: 264.1235.
6.2.7.3. 1-Ethynyl-2,3,4,5-tetramethoxybenzene (18g). Com-
pound 18g was synthesized from 22g (150 mg, 0.64 mmol) and
tert-butylammonium fluoride solution (0.3 mL, 0.3 mmol) using
the general procedure described above to afford 115 mg (84%) as
an orange-red oil: TLC Rf 0.47 (ethyl acetate/hexanes, 1:3); 1H
NMR (CDCl3): 3.25 (s, 1H, C2H), 3.83–3.94 (m, 12H, OCH3), 6.82
(s, 1H, C6H); HRMS (EI): calcd for C12H14O4: 222.0892; found:
222.0861.
6.2.6.2. (2,3,4,5-Tetramethoxyphenylethynyl)trimethylsilane
(22g). Compound 22g was synthesized from 21g (247.1 mg,
1.0 mmol) using the general procedure described above to afford
218 mg (82.5%) as an orange oil: TLC Rf 0.576 (ethyl acetate/hex-
anes, 1:3); 1H NMR (CDCl3): 0.27 (s, 9H, Si(CH3)3), 3.83–3.94 (m,
12H, OCH3), 6.27 (s, 1H, C6H); HRMS (EI): calcd for C15H22O4Si:
294.1287; found: 294.1298.
6.2.7.4. 1-Ethynyl-2,4,5-trimethoxybenzene (18h). Compound
18h was synthesized from 22h (300 mg, 1.135 mmol) and tert-
butylammonium fluoride solution (0.6 mL, 0.6 mmol) using the
general procedure described above to afford 170 mg (78.5%) as a
white solid: TLC Rf 0.239 (ethyl acetate/hexanes, 1:3); mp 113.8–
114.2 °C; 1H NMR (CDCl3): 3.14 (s, 1H, C2H), 3.72–3.81 (m, 9H,
OCH3), 6.38–6.39 (d, 1H, C6H2, J = 4.2 Hz); 6.84–6.85 (d, 1H, C6H2,
J = 4.2 Hz); HRMS (EI): calcd for C11H12O3: 192.0786; found:
192.0786.
6.2.6.3.
(2,4,5-Trimethoxyphenylethynyl)trimethylsilane
(22h). Compound 22h was synthesized from 21h (247 mg,
1.0 mmol) using the general procedure described above to afford
185 mg (70%) as an yellow-orange oil: TLC Rf 0.475 (ethyl ace-
tate/hexanes, 1:3); 1H NMR (CDCl3): 0.26 (s, 9H, Si(CH3)3), 3.84–
3.89 (m, 9H, OCH3), 6.57 (s, 1H, C6H2), 7.04 (s, 1H, C6H2); HRMS
(EI): calcd for C14H20O3Si: 264.1181; found: 264.1153.
6.2.7.5. 1-Ethynyl-2,3,5-trimethoxybenzene (18i). Compound
18i was synthesized from 22i (519 mg, 1.96 mmol) and tert-butyl-
ammonium fluoride solution (1 mL, 1.0 mmol) using the general
procedure described above to afford 315 mg (83%) as an colorless
oil: TLC Rf 0.43 (ethyl acetate/hexanes, 1:3); 1H NMR (CDCl3):
3.25 (s, 1H, C2H), 3.76–3.86 (m, 9H, OCH3), 6.51–6.53 (m, 2H,
C6H2); HRMS (EI): calcd for C11H12O3: 192.0786; found: 192.0759.
6.2.6.4.
(2,3,5-Trimethoxyphenylethynyl)trimethylsilane
(22i). Compound 22i was synthesized from 21i (618 mg,
2.5 mmol), Pd(PhCN)2Cl2 (28.75 mg, 0.075 mmol), CuI (9.5 mg,
0.050 mmol), P(t-Bu)3 (175
HN(i-Pr)2 (425 L, 1.20 mol), and trimethylsilylacetylene (390
3 mmol) using the general procedure described above to afford
519.5 mg (84%) as a colorless oil: TLC Rf 0.56 (ethyl acetate/hex-
anes, 1:3); 1H NMR (CDCl3): 0.27 (s, 9H, Si(CH3)3), 3.77–3.88 (m,
9H, OCH3), 6.44–6.64 (m, 2H, C6H2); HRMS (EI): calcd for
lL of a 1 M solution; 0.175 mmol),
l
lL,
6.2.8. Synthesis of 1-bromo-2,3,4,5-tetramethoxybenzene (21g)
1,2,3,4-Tetramethoxybenzene 23 (80 mg, 0.40 mmol) wag dis-
solved in dichloromethane (0.5 ml) and cooled down to ꢀ10 °C.
Bromine (32 mg, 0.80 mmol) in methylene chloride (0.2 ml) was
added dropwise within 1 min while the mixture was stirred. Reac-
tion was continued for 1 h at ꢀ10 °C. At the end of the reaction, sil-
ica gel (500 mg) was added and solvent evaporated under reduced
pressure to obtain a plug. The product was purified by column
chromatography using 10% EtOAc in hexanes to obtain 89.2 mg
(80%) of 21g, The compound solidified on refrigeration, mp 36.6–
C14H20O3Si: 264.1181; found: 264.1199.
6.2.7. General procedure for the synthesis of compounds 18c,
18e and 18f–i
To a 25 mL round bottom flask was added 22c/22e/22f–i
(1 mmol) followed by the addition of 5 mL anhydrous tetrahydro-
furan. To this solution was added 1 M tert-butylammonium fluo-
ride solution in THF (0.5 mmol). The colorless solution becomes

A. Gangjee et al. / Bioorg. Med. Chem. 19 (2011) 4355–4365
4365
38.2 °C (lit.29 36.0–38.0 °C); 1H NMR (CDCl3): 3.8–3.9 (m, 12H,
References and notes
OCH3), 6.8 (s, 1H, C6H1). HRMS (EI): calcd for C10H13O4Br:
275.9997; found: 275.9945.
1. Gangjee, A.; Namjoshi, O. A.; Keller, S. N.; Smith, C. D. Abstract of Papers, 233rd
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6.2.9. Synthesis of 1-bromo-2,4,5-trimethoxybenzene (21h)
Bromine (79 mg, 1 mmol) in glacial acetic acid (1 mL) was
added dropwise within 1 min to a vigorously stirred solution of
1,2,4-trimethoxybenzene 24 (168 mg, 1 mmol) in glacial acetic
acid (1 mL) at 0 °C. The reaction was completed in 10 min as mon-
itored by TLC. To the resulting mixture was added water (25 mL)
and was extracted with methylene chloride (10 mL ꢂ 2). The or-
ganic layer was washed with satd Na2CO3 (20 mL), water (10 mL)
and brine (10 mL). Organic extracts were dried over anhyd MgSO4.
Silica gel (500 mg) was added and the solvent was evaporated un-
der reduced pressure to afford a silica plug which was loaded on a
silica column (2 cm ꢂ 10 cm) and eluted with 10% EtOAc in hex-
anes. Fractions containing the product spot (TLC) were pooled
and evaporated under reduced pressure to afford 21h (156 mg,
63%) as off-white lustrous solid, TLC Rf 0.2 (ethyl acetate/hexanes,
1:5); mp 51.6–52.2 °C (lit.30 mp 54.0–55.5 °C); 1H NMR (CDCl3):
3.92–4.0 (m, 9H, OCH3), 6.6 (s, 1H, C6H2), 7.1 (s, 1H, C6H2).
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6.2.10. Synthesis of 1-bromo-2,3,5-trimethoxybenzene (21i)
To a mixture of 5-Bromovanillin 25 (2.3 g, 10 mmol) in 1 N KOH
(10.5 mL) was added 3% aqueous hydrogen peroxide solution
(22.5 mL). The dark purple solution thus obtained was stirred at
40 °C for 30 min and then cooled to room temperature, acidified
with dil HCl and extracted with ether (100 mL ꢂ 2). Organic extracts
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as a buff colored solid; mp 141.5 (lit.31 mp 141 °C). 1H NMR (CDCl3):
3.86 (s, 3H, OCH3), 4.62 (br, 1H, OH, exch.), 5.48 (br, 1H, OH, exch.),
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anhydrous K2CO3 (1.93 g, 14 mmol) and acetone (20 mL; freshly
distilled over CaH2), followed by dimethyl sulphate (1.33 mL,
14 mmol) under nitrogen. The reaction was continued at room tem-
perature for 14 h and then quenched by the addition of cold water
(20 mL) and stirred for additional 2 h. Acetone was then evaporated
under reduced pressure and aqueous layer was extracted with
EtOAc (25 mL ꢂ 3). Organic extracts washed with water and dried
over anhyd sodium sulfate. Silica gel (2 g) was added and the sol-
vent evaporated to afford a plug which was loaded on a silica col-
umn (3 cm ꢂ 15 cm) and eluted with 5% EtOAc in hexanes.
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orated under reduced pressure to afford 21i (799 mg, 92%) as a col-
orless oil which solidified on refrigeration, mp 36–38 °C (lit31 mp
37–38 °C); 1H NMR (CDCl3): 3.76–3.83 (t, 9H, OCH3), 6.43–6.44 (d,
1H, C6H2, J = 2.7 Hz), 6.63 (d, 1H, C6H2, J = 2.7 Hz).
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Acknowledgements
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This work was supported, in part, by a Grant from the National
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Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.05.030.