Synthesis of 5,7-disubstituted-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-amines as microtubule inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.12.029

Compounds 1-4 were previously reported as potent antimitotic and antitumor agents with Pgp modulatory effects. Compounds 5-18 have been synthesized in an attempt to optimize the various activities of 1-4. Compounds 5-10 explored the influence of methoxy substitutions on the 7-benzyl moiety in 1, while 11-18 investigated the influence of incorporation of a sulfur linker at C5 compared to 1-3. Compounds 5-10 demonstrated potent single-digit micromolar tumor cell cytotoxicity, Pgp modulation and microtubule inhibition. Compound 7 of this series was the most potent and showed GI₅₀ values in the nanomolar range against several human tumor cell lines in the standard NCI preclinical in vitro screen. Antitumor activity and Pgp modulatory effects were found to decrease for the 5-phenylthio compounds 11-14 compared to their 5-phenylethyl analogs 2-4 and the standard compound Taxol. Incorporation of methoxy substitutions on the 7-benzyl moiety improved antitumor activity for the 5-phenylthio compounds 16 and 17. Compounds 16 and 17 demonstrated single to two-digit micromolar inhibition of tumor cells.

Full text of DOI:10.1016/j.bmc.2012.12.029

Accepted Manuscript
Synthesis of 5,7-disubstituted-4-methyl-7H-pyrrolo[2, 3-d]pyrimidin-2-amines
as microtubule inhibitors
Aleem Gangjee, Sonali Kurup, Charles D. Smith
PII:
S0968-0896(12)01002-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2012.12.029
BMC 10495
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
17 September 2012
13 December 2012
22 December 2012
Please cite this article as: Gangjee, A., Kurup, S., Smith, C.D., Synthesis of 5,7-disubstituted-4-methyl-7H-
pyrrolo[2, 3-d]pyrimidin-2-amines as microtubule inhibitors, Bioorganic & Medicinal Chemistry (2013), doi: http://
dx.doi.org/10.1016/j.bmc.2012.12.029
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Synthesis of 5,7-disubstituted-4-methyl-7H-pyrrolo[2, 3-d]pyrimidin-2-amines as
microtubule inhibitors
Aleem Gangjee,•, † Sonali Kurup, †, § Charles D. Smith ‡
† Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences,
Duquesne University, Pittsburgh, PA 15282
‡ Department of Pharmaceutical Sciences, Medical University of South Carolina,
Charleston, SC 29425
§ Current address: Department of Biopharmaceutical Sciences, College of Pharmacy,
Roosevelt University, Schaumburg, IL 60173
* To whom correspondence should be addressed. Phone: 412-396-6070. Fax: 412-396-
5593. Email: gangjee@duq.edu
† Duquesne University
‡ Medical University of South Carolina
§ Roosevelt University
a
Abbreviations: MT, microtubule; MDR, multiple drug resistance; Pgp, P-glycoprotein;
MRP, multidrug-resistance proteins; TLC, thin layer chromatography; NMR, nuclear
magnetic resonance; HRMS, high resolution mass spectra.
1

Abstract
Compounds 1-4 were previously reported as potent antimitotic and antitumor agents with
Pgp modulatory effects. Compounds 5-18 have been synthesized in an attempt to
optimize the various activities of 1-4. Compounds 5-10 explored the influence of
methoxy substitutions on the 7-benzyl moiety in 1, while 11-18 investigated the influence
of incorporation of a sulfur linker at C5 compared to 1-3. Compounds 5-10 demonstrated
potent single-digit micromolar tumor cell cytotoxicity, Pgp modulation and microtubule
inhibition. Compound 7 of this series was the most potent and showed GI50 values in the
nanomolar range against several human tumor cell lines in the standard NCI preclinical in
vitro screen. Antitumor activity and Pgp modulatory effects were found to decrease for
the 5-phenylthio compounds 11-14 compared to their 5-phenylethyl analogs 2-4 and the
standard compound Taxol. Incorporation of methoxy substitutions on the 7-benzyl
moiety improved antitumor activity for the 5-phenylthio compounds 16 and 17.
Compounds 16 and 17 demonstrated single to two-digit micromolar inhibition of tumor
cells.
3

Keywords:
Pyrrolo[2,3-d]pyrimidines, Microtubule inhibitors, Microwave assisted organic synthesis
4

1. Introduction
The clinical prognosis of cancer remains relatively poor, suggesting a need for
new therapeutic strategies for the improvement of drugs in clinical use.1 Microtubules
(MTs) represent an excellent target and several inhibitors have a prominent role in cancer
chemotherapy. Microtubule inhibitors disrupt or suppress both microtubule structure and
normal functions by inhibition or promotion of microtubule assembly, resulting in cell
cycle arrest in the mitotic phase and induction of apoptosis.1,2 Microtubule inhibitors or
antimitotic agents have been classified on the basis of their mechanism of action and
effects on polymerization dynamics, and subsequently, on the basis of their binding site
on tubulin.3 MT-destabilizing agents inhibit MT polymerization at high concentrations
while MT-stabilizing agents promote microtubule assembly and prevent
depolymerization. Three main sites of binding have been identified for antimitotic agents
on microtubules or tubulin, namely the Vinca binding site, Taxol binding site and the
Colchicine binding site.4 The Vinca alkaloids bind to the β-subunit of soluble tubulin
dimers and induce a conformational change in tubulin which decreases tubulin self-
association.5 Taxol and its derivatives bind poorly to soluble tubulin, but with high
affinity to tubulin along the length of the microtubule. Taxol binding to the β-subunit
stabilizes the microtubule by inducing a conformational change in the tubulin that
increases its affinity for neighboring tubulin molecules. Ixabepilone, a recently approved
antimitotic agent for the treatment of metastatic breast cancer also binds to the Taxol
binding domain.6 Colchicine has been well studied for its antimitotic effects, however it
is not used clinically in cancer treatment due to its toxicity.7 It initially binds to soluble
5

tubulin, forming a tubulin–colchicine complexes that binds more tightly to their tubulin
neighbours than tubulin itself, so that the dissociation of tubulin is reduced.
Multidrug or multiple drug resistance (MDR) is a major drawback of the
clinically used antimitotic agents and has resulted in treatment failures.8 A major
mechanism of MDR occurs via the overexpression of the P-glycoprotein (Pgp) efflux
pump. Pgp is a membrane transport protein that has been associated with MDR. Pgp
binds drugs within the cell and releases them to the extracellular space using ATP,
thereby decreasing the intracellular drug concentration. Tumor cells pre-exposed to
cytotoxic compounds often overexpress these pumps, inducing resistance in the presence
of the cytotoxic drug. Due to the lack of detailed structural information on binding sites
for Pgp binders, the rational design of antimitotic agents with Pgp inhibitory effects has
been limited, although a few examples have also been reported. 9-11
Figure 1
Gangjee et. al.11 previously reported the 7-benzyl-4-methyl-5-substituted
phenylethyl-7H-pyrrolo-[2,3-d]pyrimidin-2-amines 1-4 (Figure 1) as antitumor agents
with submicromolar to micromolar tumor cell inhibition and Pgp inhibitory effects. The
antitumor effects were attributed to microtubule inhibition. Compounds 1-4 were
reported to bind to a novel/unknown site on microtubules that is distinct from the Taxol,
Vinca and Colchicine binding sites. Novel small molecules that bind to microtubules at
novel sites and are not substrates for drug efflux pumps (Pgp) would show significant
potential as therapeutic agents in cancer chemotherapy. Such agents could be used in
combination regimens with other antimitotic agents to afford a synergistic/additive effect,
overcome resistance and lower toxicity. Thus, it was of interest to further delineate the
6

structure-activity relationship (SAR) for 1-4. Compounds 5-18 were designed as analogs
of 1-4 to optimize antitumor potential and Pgp modulatory effects.
Removal of the 7-benzyl moiety in 1 was previously reported to decrease activity
by 100 to 10,000-fold against tumor cell lines, indicating the importance of the N7-
benzyl moiety on the inhibitory potency of 1.11 Compounds 5-10 (Figure 1) were
synthesized to determine the effects of variation in the substitutions on the 7-benzyl
moiety of 1 on antitumor activities and Pgp modulation. Methoxy substitutions were
incorporated at varied positions of the 7-benzyl moiety while maintaining the 3,4,5-
triOMephenylethyl substitution at C5. Compounds 11-14 (Figure 1) were synthesized as
5-thio analogs of 2-4 to determine the impact of variation in the C5-phenylethyl linker on
antitumor activity and Pgp modulation. The sulfur atom incorporated at C5 was
anticipated to mimic the two-atom bridge of 2-4 due to a larger atomic size of the sulfur
atom compared to a carbon or nitrogen atom. This modification would allow a side chain
phenyl distance somewhere between a one- and two-carbon-atom bridge and also cause a
decrease in the C-S-C angle (98°) compared to a C-C-C angle (109°), consequently
altering the orientation of the C5 phenyl ring. Compounds 11-14 incorporated a small set
of electron donating and withdrawing groups on the C5-thiophenyl moiety while
maintaining the 7-benzyl substitution. Additionally, compounds 15-18 were synthesized
to determine the influence on biological activity of varied substitutions on the 7-benzyl
moiety in combination with a 5-thiophenyl moiety.
2. Chemistry
7

Gangjee et. al.11 synthesized 1-4 in a ten-step sequence starting from 2-
acetylbutyrolactone. A convergent synthesis of the proposed target compounds 5-10
could be envisioned to proceed via removal of the 7-benzyl followed by alkylation at N7
with the appropriately substituted benzyl chlorides. Debenzylation and alkylation at N7
proceeded in poor yields, hence 5-10 were synthesized following a modification of the
previously reported procedures as shown in Schemes 1 and 2.
Scheme 1
Reaction of 19a-f12 with pivaloyl chloride in the presence of triethylamine and DMAP for
12 h afforded a mixture of the dipivaloylated 22a-d and/or monopivaloylated compounds
23a-f (Scheme 1). Decreasing the electron density of the pyrrole ring promotes
regioselective halogenation at C5. Pivaloylation of the 2-amino group in 20a-d12 and
21a-f directs the iodination regiospecifically to C5 yielding compounds 22a-f. Based on
the success of reported microwave assisted Sonogashira reactions,13,14 the Sonogashira
reaction of 22a-f with 3,4,5-trimethoxyphenyl acetylene was attempted using
dichloroethane as solvent, triethylamine as base, Pd(PPh3)4 as catalyst and CuI as an
o
additive under microwave irradiation at 100 C for 10 min. Compounds 23a-f were
synthesized in 71-80% yields. Microwave irradiation significantly shortened the duration
of reaction (10 min at 100 oC vs 12 h at room temperature). Reduction with hydrogen and
5% palladium-on-charcoal in a mixture of methanol and methylene chloride, followed by
removal of the pivaloyl protecting group on reflux with 1 N NaOH and methanol
afforded target compounds 5-10 in 55-66 % yield.
Scheme 2
8

The synthesis of target compounds 11-18 was envisioned to proceed via
substitution of the 5-iodo in N-[5-iodo-7-benzylsubstituted-4-methyl-7H-pyrrolo[2,3-
d]pyrimidin-2-yl]-2,2-dimethyl propanamide, 24a,11 24b, 22d and 24c with the
appropriate thiol (Scheme 2). Among heterocyclic halides, there are few reports on C-S
bond formation via copper mediated cross coupling.15-18 We elected to utilize a
microwave assisted, copper catalyzed reaction under ligand-free conditions for the
synthesis of target compounds 11-18. It was anticipated that microwave irradiation and
the appropriate choice of solvent would lower reaction duration. Attempts were made to
couple 24a with the appropriate thiol (1.2 eq) using stoichiometric amount of CuI (1.3
eq), K2CO3 as base (1.5 eq) and DMF as the solvent under time and temperature
o
variations. Optimal results were obtained at 100 C for 4 h. Compounds 25a-h were
synthesized in 68-81% yields. The phenyl thiols were utilized in slight excess (1.2 eq)
due to the formation of the corresponding phenyl disulfides as a byproduct in this
reaction. This is the first report on microwave assisted, copper-catalyzed reaction of 5-
iodo substituted pyrrolo[2,3-d]pyrimidines with substituted phenyl thiols at a lower
reaction temperature of 100 oC under ligand free conditions. Higher reaction temperatures
resulted in lower yields and increased disulfide formation. The microwave assisted
reaction of 24a with the appropriate thiol, K2CO3 in DMF, in the absence of CuI from 60-
o
150 C was unsuccessful. No reaction occurred and the starting material was recovered,
confirming that the reaction proceeds via a copper-catalyzed, Ullman-type coupling.
Removal of the pivaloyl protecting group in 25a-h was achieved on reflux with 1 N
NaOH to afford target compounds 11-18 in 50-65 % yield.
9

3. Biological Evaluation and Discussion
Table 1
Target compounds 5-18 were evaluated for cytotoxicity toward JC mammary
adenocarcinoma cells and for their impact on Pgp activity according to the reported
protocols (Table 1).19 JC mouse adenocarcinoma cells express high levels of Pgp and can
be used for the evaluation of Pgp substrate activity and/or inhibition. The lowest
concentration of the target compounds displaying a maximal increase in the intracellular
accumulation of [3H] Taxol, a known substrate of Pgp was determined. A known
microtubule inhibitor and antitumor agent, Taxol was included for comparison.
Compounds 5-10 with varied N7-benzyl substitutions demonstrated IC50 values
ranging from 0.6 M to 3 M in the cytotoxicity assay. Compound 7 was the most potent
compound in this series being only 4-fold less potent than the standard compound Taxol,
while compounds 5 and 10 were 6.6-fold less potent than Taxol. Among the mono-OMe
benzyl substituted compounds, the 4-OMe was slightly better than the 2- and 3-OMe
substituted compounds. In the di- and tri-OMe benzyl substituted compounds, the
presence of a 4-OMe slightly improved potency, with the 3,4-diOMe and 3,4,5-triOMe
both being better than 3,5-diOMe substituted compound, although less potent than 7. In
the drug accumulation assay for determination of Pgp activity, all of the compounds with
the exception of 10, demonstrated a significant increase in the intracellular accumulation
of [3H] Taxol at values close to their IC50 ranging from 0.2 M to 2 M. Compound 7
demonstrated an increase in the intracellular accumulation of [3H] Taxol at the lowest
concentration for this series of compounds, followed by compounds 6, 8 and 9 suggesting
Pgp modulatory effects for compound 7 >6 >8 >9.
10

The 5-thiosubstituted compounds 11-14 demonstrated micromolar cytotoxicity in
JC cells. Of compounds 11-14, compound 11 demonstrated IC50 values (cytotoxicity) at
the lowest concentration, and was approximately 2-fold more cytotoxic in JC cells than
12-14. Against Pgp, compound 12 demonstrated an increase in the intracellular
accumulation of [3H] Taxol below its IC50, while 11, 13 and 14 did not show any increase
in the accumulation of [3H] Taxol at the evaluated concentrations. Based on the
cytotoxicity and Pgp modulation for 12, a combination of the 5-(2-methoxy)phenylthio
substitution was attempted with a variety of 7-benzyl substitutions for compounds 15-17.
Antitumor activity improved for the 4-OMe and 3,4,5-triOMe benzyl substitutions in 16
and 17 respectively. Compounds 16 and 17 demonstrated single-digit micromolar
cytotoxicity (IC50 values) in JC cells and were approximately 4- fold better than 11. Pgp
modulation however, was abolished with substitution at the 7-benzyl moiety. A
combination of the 3,4,5-triOMe phenylthio substitution at C5 with the 3,4,5-triOMe
benzyl substitution at N7 was attempted in compound 18 in an effort to improve
antitumor activity. Compound 18 showed a dramatic decrease in antitumor activity
compared to its phenylethyl analog, 10.
Figure 2
Based on the promising results for JC cell inhibition and Pgp modulation,
compound 7 was evaluated specifically for its effect on microtubules at varied doses
(Figure 2). Compound 7 produced dose-dependent microtubule depolymerization,
confirming inhibition of microtubule polymerization as the mechanism for tumor cell
inhibition.
Table 2
11

Compound 7 was further evaluated in the NCI 60 tumor cell line panel.20
Compound 7 inhibited most of the cell lines with GI50 values that ranged from two-digit
nanomolar to submicromolar concentrations (Table 2). In addition, compound 7 also
demonstrated submicromolar GI50 values toward resistant tumor cell lines NCI/ADR-
RES (202 nM), HCT-15 colon (242 nM) and renal cancer cell lines, TK-10 (686 nM) and
UO-31 (397 nM).
4. Conclusions
We synthesized fourteen novel microtubule inhibitors 5-18 to determine the effect of the
substitution pattern of the 5,7-disubstituted pyrrolo[2,3-d]pyrimidines on antitumor
activity and Pgp modulation. Compounds 5-10 and 16 demonstrated single digit
micromolar effects in JC tumor cells. The ethyl linker was found to be preferred over the
thio linker at C5 for antitumor activity. Methoxy substitutions on the N7-benzyl ting
improved antitumor activity over the unsubstituted benzyl moiety for both the C5-ethyl
and C5-thio linked compounds. Compounds 5-9 induced significant Pgp modulation as
demonstrated in the in vitro drug accumulation assay. The ethyl linker was also found to
be preferred over the thio linker at C5 for Pgp modulation. Although substitutions at the
N7-benzyl improved antitumor activity over the unsubstituted benzyl moiety for both the
C5-ethyl and C5-thio linked compounds, Pgp modulation was not observed for C5-thio
linked compounds with substitutions at the N7-benzyl moiety. Our results indicate that
the potency of antitumor activity and Pgp modulation does indeed vary with different
substitutions and that an optimal combination of the substitutions in the 7-benzyl moiety,
the C5-linker and the C5-phenyl moiety is essential for inhibition of microtubules, tumor
12

cells and Pgp. Compound 7 emerged as the most viable candidate for future evaluation
with its potent microtubule and Pgp modulatory effects and remarkable nanomolar
cytotoxic effects in the NCI 60 cell line panel.
5. Experimental Section
5.1 Synthesis All evaporations were carried out in vacuo with a rotary evaporator. Thin-
layer chromatography (TLC) was performed on silica gel plates (Whatman 250 M PE
SilG/UV) with fluorescent indicator. Spots were visualized by UV (254 and 366 nm)
illumination. Column chromatography was performed using Merck silica gel (200-400
mesh). Proton nuclear magnetic resonance (1H) spectra were recorded on a Bruker WH-
300 (300 MHz) or Bruker WH-400 (400 MHz) spectrometer. The chemical shift values
were expressed in ppm (parts per million) relative to tetramethylsilane as internal
standard; s = singlet, d = double, t = triplet, q = quartet, m = multiplet, br = broad singlet.
The relative integrals of peak areas agreed with those expected for the assigned
structures. Melting points were determined on a Mel-Temp II melting point apparatus
with FLUKE 51 K/J electronic thermometer and were uncorrected. Analytical samples
were dried in vacuo (0.2mm Hg) in a CHEM DRY drying apparatus over P2O5.
Elemental analysis was performed by Altlantic Microlabs, Norcross, GA, USA. Element
compositions are within ±0.4% of the calculated values. Fractional moles of water or
organic solvents frequently found in some analytical samples could not be prevented
despite 24–48 h of drying in vacuo and were confirmed where possible by their presence
1
in the H NMR spectra. High-resolution mass spectra (HRMS), using Electron Impact
13

(EI), were recorded on a VG Autospec (Fisons Instruments) micromass (EBE Geometry)
double-focusing mass spectrometer. All solvents and chemicals were purchased from
Aldrich Chemical Co. and Fisher Scientific and were used as received.
5.1.1 N-(2-methoxybenzyl)-N-(2,2-dimethylpropanoyl)-7H-pyrrolo[2,3-d]pyrimidin-
2-yl)-2,2-dimethylpropanamide (20a) and N-[7-(2-methoxybenzyl)-4-methyl-7H-
pyrrolo[2,3-d]pyrimidin-2-yl]-2,2-dimethylpropanamide (21a)
To a 100 mL round-bottom flask, under nitrogen, was added 19a (0.8 g, 2.43 mmol),
PivCl (12 mL), DMAP (100 mg, 0.5 mmol), NEt3 (15 ml) and dichloroethane (55 mL).
o
The mixture was stirred at 50-55 C for 12 h. The reaction mixture was diluted with
CH2Cl2 (120 mL) and washed sequentially with H2O (30 mL) and brine (30 mL). The
organic layer was separated and dried over Na2SO4, filtered, and dried in vacuo to afford
brown oil. The crude product was purified by flash chromatography on silica gel
(gradient, 1:1:15 EtOAc/NEt3/hexane to 1:1:5 EtOAc/NEt3/hexane) and washed with
diethyl ether or methanol to afford 0.8 g (62 %) of 20a as an off-white solid (Rf, 1H NMR
as that reported)12 and 0.39 g (39 %) of 21a as a yellow solid; TLC Rf 0.54
o
1
(EtOAc/NEt3/Hexane, 5:1:3); mp 119-120 C; H NMR (DMSO-d6):  1.21 (s, 9 H,
C(CH3)3), 2.61 (s, 3 H, CH3), 3.86 (s, 3 H, OCH3), 5.33 (s, 2 H, CH2), 6.65 (d, 1 H, CH),
6.71 (d, 2 H, C6H4), 6.82 (t, 1 H, C6H4), 7.02 (d, 2 H, C6H4), 7.25 (t, 2 H, C6H4), 7.36 (d,
1 H, CH), 9.70 (s, 1 H, NH); HRMS (EI) Calcd for C20H24N4O2 m/z = 352.1899, found
m/z = 352.1888.
14

5.1.2 N-(3-methoxybenzyl)-N-(2,2-dimethylpropanoyl)-7H-pyrrolo[2,3-d]pyrimidin-
2-yl)-2,2-dimethylpropanamide (20b) and N-[7-(3-methoxybenzyl)-4-methyl-7H-
pyrrolo[2,3-d]pyrimidin-2-yl]-2,2-dimethylpropanamide (21b)
Compounds 20b and 21b were synthesized as described for 20a and 21a with 19b. The
crude mixture was purified by flash chromatography on silica gel to afford 0.36 g of 20b
1
12
(25%) as a yellow solid (Rf, H NMR as that reported) and 21b as a yellow semisolid
1
(0.9 g, 70 %); TLC Rf 0.55 (EtOAc/NEt3/Hexane, 5:1:3); H NMR (DMSO-d6):  1.22
(s, 9 H, C(CH3)3), 2.59 (s, 3 H, CH3), 3.70 (s, 3 H, OCH3), 5.34 (s, 2 H, CH2), 6.63 (d, 1
H, CH), 6.81-7.20 (m, 4 H, C6H4), 7.46 (d, 1 H, CH), 9.95 (s, 1 H, NH). HRMS (EI)
Calcd for C20H24N4O2 m/z = 352.1899, found m/z = 352.1909.
5.1.3 N-(4-methoxybenzyl)-N-(2,2-dimethylpropanoyl)-7H-pyrrolo[2,3-d]pyrimidin-
2-yl)-2,2-dimethylpropanamide (20c) and N-[7-(4-methoxybenzyl)-4-methyl-7H-
pyrrolo[2,3-d]pyrimidin-2-yl]-2,2-dimethylpropanamide (21c)
Compounds 20c and 21c were synthesized as described for 20a and 21a with 19c. The
crude mixture was purified by flash chromatography on silica gel to afford 20c (25%) as
1
a yellow semisolid (Rf, H NMR as that reported) 12 and 21c as a yellow semisolid (65
1
%); TLC Rf 0.54 (EtOAc/NEt3/Hexane, 5:1:3); H NMR (DMSO-d6):  1.24 (s, 3 H,
C(CH3)3), 2.60 (s, 3 H, CH3), 3.69 (s, 3 H, OCH3), 5.29 (s, 2 H, CH2), 6.66 (d, 1 H, CH),
6.84 (d, 2 H, C6H4), 7.28 (d, 2 H, C6H4), 7.42 (s, 1 H, CH), 9.81 (s, 1 H, NH).
5.1.4
N-[7-(3,4-Dimethoxybenzyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl]-2,2-
dimethylpropanamide (21d)
15

Compound 21d was synthesized as described for 21a with 19d and was obtained as a
yellow foam; TLC Rf 0.52 (EtOAc/NEt3/Hexane, 5:1:3); 1H NMR (DMSO-d6):  1.23 (s,
9 H, C(CH3)3), 2.58 (s, 3 H, CH3), 3.68 (s, 3 H, OCH3), 3.71 (s, 3 H, OCH3), 5.26 (s, 2 H,
CH2), 6.58 (d, 1 H, CH), 6.82-7.19 (m, 3 H, C6H3), 7.45 (d, 1 H, CH), 9.76 (s, 1 H, NH);
HRMS (EI) [M]+: Calcd for C21H26N4O3 m/z = 382.2004, found m/z = 382.1998.
5.1.5
N-[7-(3,5-Dimethoxybenzyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl]-2,2-
dimethylpropanamide (21e)
Compound 21e was synthesized as described for 21a with 19e and was obtained as a light
1
brown semisolid (0.82 g, 68 %); TLC Rf 0.53 (EtOAc/NEt3/Hexane, 5:1:3); H NMR
(DMSO-d6):  1.22 (s, 9 H, C(CH3)3), 2.60 (s, 3 H, CH3), 3.68 (s, 6 H, (OCH3)2), 5.28 (s,
2 H, CH2), 6.37 (s, 1 H, C6H3), 6.48 (s, 2 H, C6H3), 6.62 (d, 1 H, CH), 7.47 (d, 1 H, CH),
9.79 (s, 1 H, NH); HRMS (EI) [M]+: Calcd for C21H26N4O3 m/z = 382.2004, found m/z =
382.2003.
5.1.6
N-(3,4,5-Trimethoxybenzyl)-N-(2,2-dimethylpropanoyl)-7H-pyrrolo[2,3-
(20d) and 5.1.20 N-[7-(3,4,5-
d]pyrimidin-2-yl)-2,2-dimethylpropanamide
Trimethoxybenzyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl]-2,2
dimethylpropanamide (21f)
Compounds 20d and 21f were synthesized as described for 20a and 21a with 19f. The
crude mixture was purified by flash chromatography on silica gel to afford 20d (62%) as
a yellow solid (Rf, 1H NMR as that reported) 12 and 21f as a yellow foam (26 %); TLC Rf
1
0.50 (EtOAc/NEt3/Hexane, 5:1:3); H NMR (DMSO-d6):  1.20 (s, 9 H, C(CH3)3), 2.57
16

(s, 3 H, CH3), 3.55 (s, 3 H, OCH3), 3.71 (s, 6 H, (OCH3)2), 5.23 (s, 2 H, CH2), 6.57 (d, 1
H, CH), 6.85 (s, 1 H, C6H2), 7.52 (d, 1 H, CH), 9.79 (s, 1 H, NH); HRMS (EI) [M]+:
Calcd for C22H28N4O4 m/z = 412.2110, found m/z = 412.2122.
5.1.7
N-[5-Iodo-7-(2-methoxybenzyl)-N-(2,2-dimethylpropanoyl)-7H-pyrrolo[2,3-
d]pyrimidin-2-yl)-2,2-dimethylpropanamide (22a)
To a 100 mL round-bottom flask, protected from light, under nitrogen, was added
compound 20a (350 mg, 0.7 mmol), N-iodo succinimide (NIS) (350 mg, 1.5 mmol) and
dry DMF (10 mL). The mixture was stirred at room temperature for 12 h. The reaction
mixture was dried in vacuo, purified by flash chromatography on silica gel (isocratic,
1:1:15 EtOAc/NEt3/hexane) and washed with diethyl ether or methanol to afford 360 mg
(82%) of 22a as a white solid (Rf, 1H NMR as that reported). 12
5.1.8
N-[5-Iodo-7-(3,4,5-Trimethoxybenzyl)-N-(2,2-dimethylpropanoyl)-7H-
pyrrolo[2,3-d]pyrimidin-2-yl)-2,2-dimethylpropanamide (22b)
Compound 22b was synthesized as described for 22a with 20d(1 g, 2.29 mmol) using the
general procedure described above to afford a yellow solid (0.92 g, 78%) (Rf, 1H NMR as
that reported) 12
5.1.9
N-[5-Iodo-7-(3-methoxybenzyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl]-
2,2-dimethylpropanamide (22c)
Compound 22c was synthesized as described for 22a with 20b and was obtained as a
1
yellow foam (0.6 g, 60 %); TLC Rf 0.64 (EtOAc/NEt3/Hexane, 5:1:3); H NMR (DMSO-
17

d6):  1.23 (s, 9 H, C(CH3)3), 2.79 (s, 3 H, CH3), 3.71 (s, 3 H, OCH3), 5.29 (s, 2 H, CH2),
6.82-7.20 (m, 4 H, C6H4), 7.73 (s, 1 H, CH), 9.89 (s, 1 H, NH); HRMS (ESI) [M + H]+:
Calcd for C20H24N4O2I m/z = 479.0944, found m/z = 479.0938.
5.1.10 N-[5-Iodo-7-(4-methoxybenzyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl]-
2,2-dimethylpropanamide (22d)
Compound 22d was synthesized was synthesized as described for 22a with 21c and was
obtained as a yellow foam (820 mg, 86 %); TLC Rf 0.63 (EtOAc/NEt3/Hexane, 5:1:3); 1H
NMR (DMSO-d6):  1.24 (s, 9 H, C(CH3)3), 2.80 (s, 3 H, CH3), 3.70 (s, 3 H, OCH3), 5.26
(s, 2 H, CH2), 6.88 (s, 2 H, C6H4), 7.33 (d, 2 H, C6H4), 7.77 (s, 1 H, CH), 9.86 (s, 1 H,
NH); HRMS (EI): Calcd for C20H23N4O2I m/z = 478.0865, found m/z = 478.0856.
5.1.11 N-[5-Iodo-7-(3,4-dimethoxybenzyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-
yl]-2,2-dimethylpropanamide (22e)
Compound 22e was synthesized as described for 22a with 21d and was obtained as a
1
yellow semisolid (0.42 g, 70 %); TLC Rf 0.61 (EtOAc/NEt3/Hexane, 5:1:3); H NMR
(DMSO-d6):  1.24 (s, 9 H, C(CH3)3), 2.78 (s, 3 H, CH3), 3.67 (s, 3 H, OCH3), 3.71 (s, 3
H, OCH3), 5.23 (s, 2 H, CH2), 6.85-7.25 (m, 3 H, C6H3), 7.74 (s, 1 H, CH), 9.89 (s, 1 H,
NH); HRMS (EI) : Calcd for C21H25IN4O3 m/z = 508.0971, found m/z = 508.0973.
5.1.12 N-[5-Iodo-7-(3,5-dimethoxybenzyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-
yl]-2,2-dimethylpropanamide (22f)
18

Compound 22f was synthesized as described for 22a with 21e and was obtained as a
1
yellow semisolid (0.74 g, 75 %); TLC Rf 0.62 (EtOAc/NEt3/Hexane, 5:1:3); H NMR
(DMSO-d6):  1.23 (s, 9 H, C(CH3)3), 2.81 (s, 3 H, CH3), 3.69 (s, 6 H, (OCH3)2), 5.24 (s,
2 H, CH2), 6.38 (s, 1 H, C6H3), 6.58 (s, 2 H, C6H3), 7.76 (s, 1 H, CH), 9.91 (s, 1 H, CH);
HRMS (ESI) [M + H]+: Calcd for C21H26IN4O3 m/z = 509.1041, found m/z = 509.1050.
5.1.13
N-[7-(2-Methoxybenzyl)-4-methyl-5-(3,4,5-trimethoxyphenylethynyl)-7H-
pyrrolo[2,3-d]pyrimidin-2-yl]-N-(2,2-dimethylpropanoyl)-2,2-dimethylpropanamide
(23a)
To a 5 mL microwave reaction vial was added 22a (500 mg, 0.80 mmol), 2-
methoxyphenyl acetylene (2.26 eq), CuI (0.16 eq), Pd(Ph3)4 (0.16 eq), NEt3 (2 mL) and
dry dichloroethane (3 mL). The mixture was evacuated and back-filled with nitrogen
(three cycles). The reaction mixture was irradiated in a microwave apparatus at 100 °C
for 10 min. After the reaction mixture was cooled to ambient temperature, the mixture
was filtered. The filtrate was dried in vacuo and the crude product was purified by flash
chromatography on silica gel (gradient, 1:1:15 EtOAc/NEt3/hexane to 1:1:5
EtOAc/NEt3/hexane) and washed with diethyl ether or methanol to afford 415 mg (75%)
1
of 23a as a yellow semisolid; TLC Rf 0.52 (EtOAc/NEt3/Hexane, 5:1:10); H NMR
(DMSO-d6):  1.15 (s, 18 H, C(CH3)6), 2.89 (s, 3 H, CH3), 3.69 (s, 3 H, OCH3), 3.81 (s, 9
H, (OCH3)3), 5.35 (s, 2 H, CH2), 6.84 (s, 2 H, C6H4), 6.92-7.29 (m, 4 H, C6H4), 7.92 (s, 1
H, CH); Anal.(C36H42N4O6 • 0.22 H2O): C, 68.53; H, 6.78; N, 8.88. Found C, 68.57; H,
6.71; N, 8.59.
19

5.1.14 N-[7-(3,4,5-Trimethoxybenzyl)-4-methyl-5-(3,4,5-trimethoxyphenylethynyl)-
7H-pyrrolo[2,3-d]pyrimidin-2-yl]-N-(2,2-dimethylpropanoyl)-2,2-
dimethylpropanamide (23b)
Compound 23b was synthesized as described for 23a with 22b and was obtained as a
1
yellow semisolid (73 %); TLC Rf 0.48 (5:1:10 EtOAc/NEt3); H NMR (DMSO-d6): 
1.18 (s, 18 H, C(CH3)6), 2.89 (s, 3 H, CH3), 3.60 (s, 3 H, OCH3), 3.68 (s, 3 H, OCH3),
3.70 (s, 6 H, (OCH3)2), 3.80 (s, 6 H, (OCH3)2), 5.28 (s, 2 H, CH2), 6.62 (s, 2 H, C6H2),
6.82 (s, 2 H, C6H2), 8.13 (s, 1 H, CH); HRMS (ESI) [M + Na]+: Calcd for C38H46N4O8Na
m/z = 709.3213, found m/z = 709.3157.
5.1.15
N-7-(3-methoxybenzyl)-5-[(3,4,5-trimethoxyphenyl)ethynyl]-4-methyl-7H-
pyrrolo[2,3-d]pyrimidin-2-yl]-2,2-dimethylpropanamide (23c)
Compound 23c was synthesized as described for 23a with 22c and was obtained as a
1
yellow semisolid (273 mg, 80 %); TLC Rf 0.52 (EtOAc/NEt3/Hexane, 5:1:3); H NMR
(DMSO-d6):  1.22 (s, 9 H, C(CH3)3), 2.85 (s, 3 H, CH3), 3.69 (s, 3 H, OCH3), 3.79 (s, 6
H, (OCH3)2), 3.86 (s, 3 H, OCH3), 5.35 (s, 2 H, CH2), 6.83 (s, 2 H, C6H2), 6.83 (m, 2 H,
C6H4), 7.06 (d, 1 H, C6H4), 7.29 (t, 1 H, C6H4), 7.75 (s, 1 H, CH), 9.88 (s, 1 H, NH);
Anal.(C31H34N4O5 • 0.3 CH3OH): C, 68.12; H, 6.41; N, 10..16. Found C, 68.20; H, 6.29;
N, 9.83.
5.1.16
N-7-(4-methoxybenzyl)-5-[(3,4,5-trimethoxyphenyl)ethynyl]-4-methyl-7H-
pyrrolo[2,3-d]pyrimidin-2-yl]-2,2-dimethylpropanamide (23d)
20

Compound 23d was synthesized as described for 23a with 22d and was obtained as a
1
yellow foam (250 mg, 74 %); TLC Rf 0.52 (EtOAc/NEt3/Hexane, 5:1:3); H NMR
(DMSO-d6):  1.25 (s, 3 H, C(CH3)3), 2.84 (s, 3 H, CH3), 3.68 (s, 3 H, OCH3), 3.71 (s, 3
H, OCH3), 3.80 (s, 6 H, (OCH3)2), 5.30 (s, 2 H, CH2), 6.81 (s, 2 H, C6H2), 6.88 (d, 2 H,
C6H4), 7.35 (d, 2 H, C6H4), 7.87 (s, 1 H, CH), 9.93 (s, 1 H, NH).
5.1.17 N-7-(3,4-Dimethoxybenzyl)-5-[(3,4,5-trimethoxyphenyl)ethynyl]-4-methyl-7H-
pyrrolo[2,3-d]pyrimidin-2-yl]-2,2-dimethylpropanamide (23e)
Compound 23e was synthesized as described for 23a with 22e and was obtained as a
1
yellow semisolid (240 mg, 71 %); TLC Rf 0.50 (EtOAc/NEt3/Hexane, 5:1:3); H NMR
(DMSO-d6):  1.27 (s, 9 H, C(CH3)3), 2.55 (s, 3 H, CH3), 3.67 (s, 3 H, OCH3), 3.79 (s, 6
H, (OCH3)2), 3.68-3.70 (s, 6 H, (OCH3)2), 5.28 (s, 2 H, CH2), 6.81 (s, 2 H, CH2), 6.89 (d,
2 H, C6H3), 6.96 (s, 1 H, C6H3), 7.26 (d, 1 H, C6H3), 7.91 (s, 1 H, CH), 9.92 (s, 1 H, CH).
5.1.18 N-7-(3,5-Dimethoxybenzyl)-5-[(3,4,5-trimethoxyphenyl)ethynyl]-4-methyl-7H-
pyrrolo[2,3-d]pyrimidin-2-yl]-2,2-dimethylpropanamide (23f)
Compound 23f was synthesized as described for 23a with 22f and was obtained as a
1
yellow semisolid (246 mg, 73 %); TLC Rf 0.48 (EtOAc/NEt3/Hexane, 5:1:3); H NMR
(DMSO-d6):  1.23 (s, 9 H, C(CH3)3), 2.84 (s, 3 H, CH3), 3.69 (s, 3 H, OCH3), 3.70 (s, 6
H, (OCH3)2), 3.79 (s, 6 H, (OCH3)2), 5.27 (s, 2 H, CH2), 6.39 (s, 1 H, C6H3), 6.58 (s, 2 H,
C6H3), 6.81 (s, 2 H, C6H2), 7.94 (s, 1 H, CH), 9.93 (s, 1 H, NH); HRMS (ESI) [M + H]+:
Calcd for C32H37N4O6 m/z = 573.2713, found m/z = 573.2697.
21

5.1.19
7-(2-Methoxybenzyl)-5-[(3,4,5-trimethoxyphenyl)ethyl]-4-methyl-7H-
pyrrolo[2,3-d]pyrimidin-2-amine (5)
To a Parr hydrogenation bottle was added 23a (200 mg, 0.36 mmol), 5% Pd/C (1:3 by
weight) 4 mL MeOH and 10 mL CH2Cl2. The mixture was shaken under 50 p.s.i.
hydrogen for 24 h. The reaction mixture was filtered, dried in vacuo. To the resulting off-
white semisolid (160 mg, 0.23 mmol) was added aqueous 1 N NaOH (4 mL) and MeOH
(20 mL). The reaction mixture was refluxed for 12 h, and concentrated. The slurry
obtained was dissolved in water (5 mL) and extracted with CH2Cl2 (30 mL). The organic
layer was dried over Na2SO4 and concentrated to afford a colourless oil. The crude
product was purified by flash chromatography on silica gel (gradient, 5:1:10
EtOAc/NEt3/hexane to 5:1:3 EtOAc/NEt3/hexane) and washed with diethyl ether or
methanol to afford 78 mg (66%) of 5 as a white solid; TLC Rf 0.26 (EtOAc/NEt3/Hexane,
o
1
5:1:3); mp 147-148 C; H NMR (DMSO-d6):  2.53 (s, 3 H, CH3), 2.82 (t, 2 H, CH2),
2.99 (t, 2 H, CH2), 3.60 (s, 3 H, OCH3), 3.71 (s, 6 H, (OCH3)2), 3.83 (s, 3 H, OCH3), 5.12
(s, 2 H, CH2), 6.06 (s, 2 H, NH2), 6.45 (d, 1 H, C6H4), 6.48 (s, 2 H, C6H2), 6.74 (s, 1 H,
CH), 6.79 (t, 1 H, C6H4), 6.99 (d, 1 H, C6H4), 7.23 (t, 1 H, C6H4); Anal.(C26H30N4O4): C,
66.90; H, 6.57; N, 12.00. Found C, 66.97; H, 6.56; N, 11.81.
5.1.20
7-(3-Methoxybenzyl)-5-[(3,4,5-trimethoxyphenyl)ethyl]-4-methyl-7H-
pyrrolo[2,3-d]pyrimidin-2-amine (6)
Compound 6 was synthesized as described for 5 with 23c and was obtained as a white
1
solid (70 mg, 61 %); TLC Rf 0.27 (EtOAc/NEt3/Hexane, 5:1:3); mp 144-146 oC; H
NMR (DMSO-d6):  2.48 (s, 3 H, CH3), 2.80 (t, 2 H, CH2), 2.96 (t, 2 H, CH2), 3.59 (s, 3
22

H, OCH3), 3.68 (s, 9 H, (OCH3)3), 5.12 (s, 2 H, CH2), 6.08 (s, 2 H, NH2), 6.64 (s, 2 H,
CH2), 6.73-6.74 (m, 3 H, C6H4), 6.80 (s, 1 H, CH), 7.18 (t, 1 H, C6H4); HRMS (ESI) [M
+ H]+: Calcd for C26H31N4O4 m/z = 463.2345, found m/z = 463.2325.
5.1.21
7-(4-Methoxybenzyl)-5-[(3,4,5-trimethoxyphenyl)ethyl]-4-methyl-7H-
pyrrolo[2,3-d]pyrimidin-2-amine (7)
Compound 7 was synthesized as described for 5 with 23d and was obtained as a white
solid (60 mg, 57 %); TLC Rf 0.26 (EtOAc/NEt3/Hexane, 5:1:3); mp 137-138 oC; 1H NMR
(DMSO-d6):  2.50 (s, 3 H, CH3), 2.78 (t, 2 H, CH2), 2.94 (t, 2 H, CH2), 3.58 (s, 3 H,
OCH3), 3.69 (s, 9 H, (OCH3)3), 5.06 (s, 2 H, CH2), 6.06 (s, 2 H, NH2), 6.52 (s, 2 H, CH2),
6.74 (s, 1 H, CH), 6.83 (d, 2 H, C6H4), 7.09 (d, 2 H, C6H4); HRMS (ESI) [M + H]+: Calcd
for C26H31N4O4 m/z = 463.2345, found m/z = 463.2331.
5.1.22
7-(3,4-Dimethoxybenzyl)-5-[(3,4,5-trimethoxyphenyl)ethyl]-4-methyl-7H-
pyrrolo[2,3-d]pyrimidin-2-amine (8)
Compound 8 was synthesized as described for 5 with 23e and was obtained as a light
o
yellow solid (65 mg, 55 %); TLC Rf 0.24 (EtOAc/NEt3/Hexane, 5:1:3); mp 135-137 C;
1H NMR (DMSO-d6):  2.51 (s, 3 H, CH3), 2.79 (t, 2 H, CH2), 2.94 (t, 2 H, CH2), 3.59 (s,
3 H, OCH3), 3.69 (s, 6 H, (OCH3)2), 3.69 (s, 6 H, (OCH3)2), 5.06 (s, 2 H, CH2), 6.08 (s, 2
H, NH2), 6.51 (s, 2 H, C6H3), 6.57 (d, 2 H, C6H3), 6.76 (s, 1 H, CH), 6.84 (s, 1 H, CH),
6.96 (s, 1 H, CH). HRMS (ESI) [M + H]+: Calcd for C27H33N4O5 m/z = 493.2451, found
m/z = 493.2430.
23

5.1.23
7-(3,5-Dimethoxybenzyl)-5-[(3,4,5-trimethoxyphenyl)ethyl]-4-methyl-7H-
pyrrolo[2,3-d]pyrimidin-2-amine (9)
Compound 9 was synthesized as described for 5 with 23f and was obtained as a light
o
yellow solid (70 mg, 57 %); TLC Rf 0.22 (EtOAc/NEt3/Hexane, 5:1:3); mp 133-135 C;
1H NMR (DMSO-d6):  2.49 (s, 3 H, CH3), 2.80 (t, 2 H, CH2), 2.95 (t, 2 H, CH2), 3.60 (s,
3 H, OCH3), 3.67 (s, 6 H, (OCH3)2), 3.71 (s, 6 H, (OCH3)2), 5.09 (s, 2 H, CH2), 6.09 (s, 2
H, NH2), 6.29 (s, 2 H, C6H3), 6.38 (s, 1 H, C6H3), 6.54 (s, 2 H, C6H2), 6.82 (s, 1 H, CH);
HRMS (ESI) [M + H]+: Calcd for C27H33N4O5 m/z = 493.2451, found m/z = 493.2433.
5.1.24 7-(3,4,5-Trimethoxybenzyl)-5-[(3,4,5-trimethoxyphenyl)ethyl]-4-methyl-7H-
pyrrolo[2,3-d]pyrimidin-2-amine (10)
Compound 10 was synthesized as described for 5 with 23b and was obtained as a white
solid (78 mg, 66%); TLC Rf 0.20 (EtOAc/NEt3/Hexane, 5:1:3); mp 136-137 oC; 1H NMR
(DMSO-d6):  2.52 (s, 3 H, CH3), 2.81 (t, 2 H, CH2), 2.94 (t, 2 H, CH2), 3.61 (s, 6 H,
OCH3), 3.69-3.71 (s, 12 H, (OCH3)4), 5.07 (s, 2 H, CH2), 6.12 (s, 2 H, NH2), 6.54 (s, 2 H,
C6H2), 6.60 (s, 1 H, C6H2), 6.84(s, 1 H, CH); Anal.(C38H50N4O8 · 0.38 (C2H5)2O): Calcd
C, 66.01; H, 7.54; N, 7.78. Found C, 66.32; H, 7.29; N, 7.51.
5.1.25 N-[5-Iodo-7-(2-methoxybenzyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl]-
2,2-dimethylpropanamide (24b)
Compound 24b was synthesized as described for 22a with 21a and was obtained as a
yellow foam (0.86 g, 66 %); TLC Rf 0.65 (EtOAc/NEt3/Hexane, 5:1:3); 1H NMR
(DMSO-d6):  1.22 (s, 9 H, C(CH3)3), 2.82 (s, 3 H, CH3), 3.86 (s, 3 H, OCH3), 5.32 (s, 2
24

H, CH2), 6.84-7.30 (m, 4 H, C6H4), 7.57 (s, 1 H, CH), 9.82 (s, 1 H, NH);
Anal.(C20H23IN4O2 · 1.02 (C4H8)O): Calcd C, 52.41; H, 5.69; N, 10.14; I, 22.97. Found
C, 52.81; H, 5.78; N, 10.10; I, 23.25.
5.1.26 N-[5-Iodo-7-(3,4,5-Trimethoxybenzyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-
2-yl]-2,2-dimethylpropanamide (24c)
Compound 24c was synthesized as described for 22a with 21f and was obtained as a
yellow solid (0.73 g, 73 %); TLC Rf 0.59 (EtOAc/NEt3/Hexane, 5:1:3); 1H NMR
(DMSO-d6):  1.23 (s, 9 H, C(CH3)3), 2.79 (s, 3 H, CH3), 3.57 (s, 3 H, OCH3), 3.75 (s, 6
H, (OCH3)2), 5.20 (s, 2 H, CH2), 6.99 (s, 2 H, C6H2), 7.82 (s, 1 H, CH), 9.95 (s, 1 H,
NH).
5.1.27 N-[7-benzyl-5-(4-phenyl)sulfanyl-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl]-
2,2-dimethylpropanamide (25a)
To a 5 mL microwave reaction vial was added 24a (300 mg, 0.48 mmol), phenylthiol (1.2
eq), CuI (1.3 eq), K2CO3 (1.5 eq) and 4 mL of dry DMF. The mixture was evacuated and
back-filled with nitrogen (three cycles). The reaction mixture was irradiated in a
microwave apparatus at 100 °C for 4 h. After the reaction mixture was cooled to ambient
temperature, the mixture was filtered. The filtrate was dried in vacuo and the crude
product was purified by flash chromatography on silica gel (gradient, 1:8 to 1:2, EtOAc:
hexane) and washed with diethyl ether/methanol to afford 230 mg (78%) of 25a as a
1
yellow semisolid; Rf 0.50 (5:1:3, EtOAc/NEt3/Hexane); H NMR (DMSO-d6):  1.23 (s,
25

9 H, C(CH3)3), 2.54 (s, 3 H, CH3), 5.44 (s, 2 H, CH2), 7.04-7.36 (m, 10 H, C6H5 and
C6H5), 7.95 (s, 1 H, CH), 9.93 (s, 1 H, NH).
5.1.28
N-[7-benzyl-5-(2-methoxyphenyl)sulfanyl-4-methyl-7H-pyrrolo[2,3-
d]pyrimidin-2-yl]-2,2-dimethylpropanamide (25b)
Compound 25b was synthesized as described for 25a with 2-methoxyphenylthiol and was
obtained as a white foam (76%); Rf 0.45 (5:1:3, EtOAc/NEt3/Hexane); 1H NMR (DMSO-
d6):  1.20 (s, 9 H, C(CH3)3), 2.39 (s, 3 H, 4-CH3), 3.83 (s, 3 H, OCH3), 5.4 (s, 2 H, CH2),
6.42-7.33 (m, 9 H, C6H5 and C6H4), 7.84 (s, 1 H, CH), 9.85 (s, 1 H, NH). HRMS(ESI)
[M+H]+ m/z Calcd for (C26H29N4O2S) 461.2011 Found 461.1987.
5.1.29
N-[7-benzyl-5-(4-methoxyphenyl)sulfanyl-4-methyl-7H-pyrrolo[2,3-
d]pyrimidin-2-yl]-2,2-dimethylpropanamide (25c)
Compound 25c was synthesized as described for 25a with 4-methoxyphenylthiol and was
1
obtained as a yellow foam (78%); Rf 0.48 (5:1:3, EtOAc/NEt3/Hexane); H NMR
(DMSO-d6):  1.22 (s, 9 H, C(CH3)3), 2.58 (s, 3 H, CH3), 3.68 (s, 3 H, OCH3), 5.41 (s, 2
H, CH2), 6.86 (d, 2 H, C6H4), 7.02 (d, 2 H, C6H4), 7.22-7.34 (m, 5 H, C6H5), 7.89 (s, 1 H,
CH), 9.87 (s, 1 H, NH). HRMS (ESI) [M + H]+: m/z Calcd for (C26H29N4O2S) 461.2011
Found 461.1982.
5.1.30
N-[7-benzyl-5-(4-chlorophenyl)sulfanyl-4-methyl-7H-pyrrolo[2,3-
d]pyrimidin-2-yl]-2,2-dimethylpropanamide (25d)
26

Compound 25d was synthesized as described for 25a with 4-chlorophenylthiol and was
1
obtained as a yellow semisolid (83%); Rf 0.53 (5:1:3, EtOAc/NEt3/Hexane); H NMR
(DMSO-d6):  1.23 (s, 9 H, C(CH3)3), 2.54 (s, 3 H, CH3), 5.32 (s, 2 H, CH2), 7.04-7.73
(m, 9 H, C6H5 and C6H4), 7.98 (s, 1 H, CH), 9.92 (s, 1 H, NH). HRMS (ESI) [M + H]+:
m/z Calcd for (C25H26N4O2SCl) 465.1481 Found 465.1516.
5.1.31 N-(2-Methoxybenzyl)-4-methyl-5-[(2-methoxyphenyl)sulfanyl]-7H-
pyrrolo[2,3-d]pyrimidin-2-yl]-2,2-dimethylpropanamide (25e)
Compound 25e was synthesized as described for 25a with 24 and was obtained as a white
foam (77%); TLC Rf 0.47 (5:1:3, EtOAc/NEt3/Hexane); 1H NMR (DMSO-d6):  1.20 (s,
3 H, C(CH3)3), 2.52 (s, 3 H, CH3), 3.83 (s, 3 H, OCH3), 3.85 (s, 3 H, OCH3), 5.37 (s, 2
H, CH2), 6.47 (d, 1 H, C6H4), 6.84-7.27 (m, 7 H, C6H4 and C6H4), 7.69 (s, 1 H, CH), 9.82
(s, 1 H, NH); Anal.(C27H30N4O3S • 0.533 (C2H5)2O): C, 66.00; H, 6.71; N, 10.56; S, 6.04.
Found C, 65.95; H, 6.48; N, 10.30; S, 6.23.
5.1.32 N-(4-Methoxybenzyl)-4-methyl-5-[(2-methoxyphenyl)sulfanyl]-7H-
pyrrolo[2,3-d]pyrimidin-2-yl]-2,2-dimethylpropanamide (25)
Compound 25 was synthesized as described for 25a with 22d and was obtained as a
yellow semisolid (72%); TLC Rf 0.46 (5:1:3, EtOAc/NEt3/Hexane); 1H NMR (DMSO-
d6):  1.27 (s, 3 H, C(CH3)3), 2.50 (s, 3 H, CH3), 3.73 (s, 3 H, OCH3), 3.89 (s, 3 H,
OCH3), 5.37 (s, 2 H, CH2), 6.48 (d, 1 H, C6H4), 6.77 (t, 1 H, C6H4), 6.89 (d, 2 H, C6H4),
6.92 (d, 1 H, C6H4), 7.03 (t, 1 H, C6H4), 7.38 (d, 2 H, C6H4), 7.87 (s, 1 H, CH), 9.95 (s, 1
27

H, NH); HRMS (ESI) [M + H]+: Calcd for C27H31N4O3S m/z = 491.2117, found m/z =
491.2091.
5.1.33
N-(2-Methoxybenzyl)-4-methyl-5-[(3,4,5-trimethoxyphenyl)sulfanyl]-7H-
pyrrolo[2,3-d]pyrimidin-2-yl]-2,2-dimethylpropanamide (25g)
Compound 25g was synthesized as described for 25a with 24c and 3,4,5-
trimethoxyphenyl thiol and was obtained as a yellow semisolid (72%); TLC Rf 0.47
1
(5:1:3 Hexane/ TEA /EtOAc); H NMR (DMSO-d6):  1.21 (s, 3 H, C(CH3)3), 2.63 (s, 3
H, CH3), 3.59 (s, 6 H, (OCH3)2), 3.86 (s, 3 H, OCH3), 5.41 (s, 2 H, CH2), 6.37 (s, 2 H,
C6H2), 6.77-6.81 (m, 2 H, C6H4), 7.04 (d, 1 H, C6H4), 7.28 (t, 1 H, C6H4), 7.80 (s, 1 H,
CH), 9.85 (s, 1 H, NH); HRMS (ESI) [M + H]+: Calcd for C29H35N4O5S m/z = 551.2328,
found m/z = 551.2304.
5.1.34 N-(3,4,5-Trimethoxybenzyl)-4-methyl-5-[(3,4,5-trimethoxyphenyl)sulfanyl]-
7H-pyrrolo[2,3-d]pyrimidin-2-yl]-2,2-dimethylpropanamide (25h)
Compound 25h was synthesized as described for 25a with 24c and was obtained as a
yellow semisolid (76%); TLC Rf 0.41 (5:1:3 EtOAc/NEt3); 1H NMR (DMSO-d6):  1.23
(s, 3 H, C(CH3)3), 2.65 (s, 3 H, CH3), 3.53 (s, 6 H, (OCH3)2), 3.60 (s, 3 H, OCH3), 3.68
(s, 6 H, (OCH3)2), 3.74 (s, 6 H, (OCH3)2), 5.33 (s, 2 H, CH2), 6.30 (s, 2 H, C6H2), 6.61
(s, 2 H, C6H4), 6.97 (s, 1 H, CH), 8.19 (s, 1 H, NH).
5.1.35 7-Benzyl-5-phenylsulfanyl-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine (11)
28

To a 50 mL round-bottom flask was added 25a (200 mg, 0.33 mmol), aqueous 1 N NaOH
(4 mL) and MeOH (20 mL). The reaction mixture was refluxed for 12 h, and
concentrated. The slurry obtained was dissolved in water (5 mL) and extracted with
CH2Cl2 (30 mL). The organic layer was dried over Na2SO4 and concentrated to afford a
colourless oil. The crude product was purified by flash chromatography on silica gel
(gradient, 5:1:10 EtOAc/NEt3/hexane to 5:1:3 EtOAc/NEt3/hexane) and washed with
diethyl ether to afford 11 (56%) as a white solid; TLC Rf
0.30 (5:1:3,
o
1
EtOAc/NEt3/Hexane); mp 142-143 C; H NMR (DMSO-d6)  2.37 (s, 3 H, CH3), 5.30
(s, 2 H, CH2), 5.71 (s, 2 H, NH2), 7.04-7.34 (m, 10 H, C6H5 and C6H5), 7.54 (s, 1 H, CH);
Anal. [C20H18N4S· 0.2 (C2H5)2O]: C, 69.16; H, 5.35; N, 15.64; S, 9.16. Found C, 69.22;
H, 5.45; N, 15.75; S, 9.01.
5.1.36
7-Benzyl-5-[(2-methoxyphenyl)sulfanyl]-4-methyl-7H-pyrrolo[2,3-
d]pyrimidin-2-amine (12)
Compound 12 was synthesized as described for 11 with 25b and was obtained as a white
1
solid (58%); Rf 0.26 (5:1:3, EtOAc/NEt3/Hexane); mp 164–165 °C; H NMR (DMSO-
d6)  2.31 (s, 3 H, CH3), 3.84 (s, 3 H, OCH3), 5.29 (s, 2 H, CH2), 6.35 (s, 2 H, NH2), 6.35
(d, 1 H, C6H4), 6.54 (t, 1 H, C6H4), 6.78 (d, 1 H, C6H4), 6.99 (d, 1 H, C6H4), 7.08 (t, 1 H,
C6H4), 7.20-7.33 (m, 6 H, C6H5), 7.46 (s, 1 H, CH); HRMS (ESI) [M + H]+: m/z Calcd
for (C21H21N4OS) 377.1436 Found 377.1399.
5.1.37
7-Benzyl-5-[(4-methoxyphenyl)sulfanyl]-4-methyl-7H-pyrrolo[2,3-
d]pyrimidin-2-amine (13)
29

Compound 13 was synthesized as described for 11 with 25c and was obtained as a white
o
1
solid (52%); TLC Rf 0.28 (5:1:3 EtOAc/NEt3/Hexane); mp 158-160 C; H NMR
(DMSO-d6)  2.43 (s, 3 H, CH3), 3.56 (s, 3 H, OCH3), 3.57 (s, 6 H, (OCH3))2, 5.30 (s, 2
H, CH2), 6.34 (s, 2 H, NH2), 6.32 (s, 2 H, C6H2), 7.17-7.30 (m, 5 H, C6H5), 7.53 (s, 1 H,
CH); HRMS (ESI) [M + H]+: m/z Calcd for (C21H21N4OS) 377.1436 Found 377.1454.
5.1.38 7-Benzyl-5-[(4-chlorophenyl)sulfanyl]-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-
2-amine (14)
Compound 14 was synthesized as described for 11 with 25d and was obtained as a
1
yellow solid (51%); TLC Rf 0.31 (5:1:3, EtOAc/NEt3/Hexane); mp 134-135 oC; H
NMR (DMSO-d6)  2.31 (s, 3 H, CH3), 5.28 (s, 2 H, CH2), 6.32 (s, 2 H, NH2), 6.87 (d, 2
H, C6H4), 7.02-7.33 (m, 7 H, C6H5 and C6H4), 7.51 (s, 1 H, CH); HRMS (ESI) [M + H]+:
m/z Calcd for (C20H18N4SCl) 381.0941 Found 381.0916.
5.1.39 7-(2-Methoxybenzyl)-5-[(2-methoxyphenyl)sulfanyl]-4-methyl-7H-
pyrrolo[2,3-d]pyrimidin-2-amine (15)
Compound 15 was synthesized as described for 11 with 25e and was obtained as a white
solid (105 mg, 53%); TLC Rf 0.20 (5:1:3, EtOAc/NEt3/Hexane); mp 171-172 oC; 1H
NMR (DMSO-d6):  2.33 (s, 3 H, CH3), 3.84 (s, 3 H, OCH3), 3.86 (s, 3 H, OCH3), 5.24
(s, 2 H, CH2), 6.34 (s, 2 H, NH2), 6.55-7.27 (m, 8 H, C6H4 and C6H4), 7.35 (s, 1 H, CH);
Anal.(C22H22N4O2S): C, 65.00; H, 5.45; N, 13.78; S, 7.88. Found C, 64.78; H, 5.37; N,
13.68; S, 7.86.
30