Design, synthesis, and structure-activity relationship of 6-alkynylpyrimidines as potent adenosine kinase inhibitors

Article abstract of DOI:10.1021/jm020049a

Adenosine (ADO) is an extracellular signaling molecule within the central and peripheral nervous system. Its concentration is increased at sites of tissue injury and inflammation. One of the mechanisms by which antinociceptive and antiinflammatory effects of ADO can be enhanced consists of inhibition of adenosine kinase (AK), the primary metabolic enzyme for ADO. Novel nonnucleoside AK inhibitors based on 4-amino-6-alkynylpyrimidines were prepared, and the importance of the length of the linker at the 5-position for high affinity AK inhibition was demonstrated. Compounds with 2- and 3-atom linkers were the most potent AK inhibitors. Optimization of their physicochemical properties led to 31a and 37a that effectively reduced pain and inflammation in animal models.

Full text of DOI:10.1021/jm020049a

J . Med. Chem. 2002, 45, 3639-3648
3639
Design , Syn th esis, a n d Str u ctu r e-Activity Rela tion sh ip of 6-Alk yn ylp yr im id in es
a s P oten t Ad en osin e Kin a se In h ibitor s
Arthur Gomtsyan,* Stanley Didomenico, Chih-Hung Lee, Mark A. Matulenko, Ki Kim, Elizabeth A. Kowaluk,
Carol T. Wismer, J oe Mikusa, Haixia Yu, Kathy Kohlhaas, Michael F. J arvis, and Shripad S. Bhagwat†
Neuroscience Research, Global Pharmaceutical Products Division, Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, Illinois 60064
Received J anuary 31, 2002
Adenosine (ADO) is an extracellular signaling molecule within the central and peripheral
nervous system. Its concentration is increased at sites of tissue injury and inflammation. One
of the mechanisms by which antinociceptive and antiinflammatory effects of ADO can be
enhanced consists of inhibition of adenosine kinase (AK), the primary metabolic enzyme for
ADO. Novel nonnucleoside AK inhibitors based on 4-amino-6-alkynylpyrimidines were prepared,
and the importance of the length of the linker at the 5-position for high affinity AK inhibition
was demonstrated. Compounds with 2- and 3-atom linkers were the most potent AK inhibitors.
Optimization of their physicochemical properties led to 31a and 37a that effectively reduced
pain and inflammation in animal models.
In tr od u ction
Adenosine (ADO) is an endogenously released neuro-
modulator that functions as an extracellular signaling
molecule within the central nervous system (CNS) and
peripheral nervous system.1,2 Under adverse conditions
(e.g., pain, inflammation, tissue damage, etc.), the local
tissue levels of extracellular ADO are markedly in-
creased.3 Adenosine kinase (AK) is a key intracellular
enzyme regulating intra- and extracellular ADO con-
centrations. It rapidly phosphorylates ADO, maintain-
ing intracellular ADO concentrations at low levels.
Because ADO uptake is driven by its concentration
gradient, AK inhibition reduces the cellular uptake of
ADO,4 thus potentiating the local concentration of ADO
in the extracellular compartment. Consequently, AK
inhibition can enhance the endogeneous protective
action of ADO.2 The identification of compounds that
mimic or modulate the antinociceptive and antiinflam-
matory actions of ADO represents a potential approach
to the treatment of pain and inflammation.
Recently, our group reported the synthesis of the
novel nonnucleoside AK inhibitor 15 and described its
in vitro6 and in vivo7 properties. AK inhibitor 1 is a
member of the pyridopyrimidine class of compounds on
which we have performed several structure-activity
relationship (SAR) studies8,9 resulting in the develop-
ment of potent AK inhibitors with improved pharmaco-
F igu r e 1. Overlay of pyridopyrimidine 1 (green) and ethynyl-
kinetic and toxicological properties as compared to the
pyrimidine 2 (brown).
prototypic nucleoside AK inhibitors.10-13 Our efforts to
widen the structural diversity of AK inhibitors led to a
novel nonnucleoside class of AK inhibitors, 6-ethynyl-
pyrimidines, that were derived from the pyridopyrimi-
dine class by replacing a fused pyridine ring with an
superposition of the major fragments of the pyrido-
pyrimidine core of 1 and the ethynylpyrimidine core of
2. It also became apparent that the evaluation of
substituents at the 5-position could be an important
acetylenic unit at the 6-position of pyrimidine (Figure
direction for SAR studies since that position appeared
1). The overlay of compounds 1 and 2 showed good
to be the key point for filling a putative hydrophobic
pocket of the protein that in the case of pyridopyrimi-
dine substrate 1 is occupied by a 3-bromophenyl frag-
ment. The potential usefulness of such an approach to
SAR optimization of the 6-ethynylpyrimidines was
* To whom correspondence should be addressed. Tel: (847)935-4214.
Fax: (847)937-9195. E-mail: arthur.r.gomtsyan@abbott.com..
† Present address: Celgene, Signal Research Division, 5555 Oberlin
Drive, San Diego, CA 92130.
10.1021/jm020049a CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/09/2002

3640 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Gomtsyan et al.
Sch em e 1a
a
Reagents: (a) (i) Na, EtOH; (ii) RBr, EtOH, reflux, 16 h. (b) Na, EtOH, formamidine acetate, 0 °C, and then add 4, room temperature,
16 h. (c) POCl3, reflux, 14 h. (d) NaI, 45% HI, acetone, room temperature, 16 h. (e) NH3, EtOH, sealed tube, 100 °C, 16 h. (f) Compound
9, Pd(PPh3)2Cl2, CuI, MeCN-Et3N, room temperature, 1.5 h. (g) 3-Bromoaniline, 40-50 °C, 15 h. (h) NaH, MeI, THF, room temperature,
16 h.
indirectly confirmed by the previous successful utiliza-
tion of 2 as a core fragment in a nuclear magnetic
resonance (NMR)-based lead optimization of AK inhibi-
tors.14
of coupling of 6 with 9 followed by ammonolysis.
However, the coupling reaction turned out to be an
inefficient process; therefore, a longer three step pro-
cedure was more practical. For the anilino derivative
10f, diethyl chloromalonate 11 was chosen as the
starting material. It reacted with 3-bromoaniline, and
the resulting 12 was subjected to cyclization followed
by chlorination to obtain dichloro-pyrimidine 13. The
latter was N-methylated with MeI and further elabo-
rated to 10f.
Because there are not many commercially available
3-arylpropyl halides, Scheme 1 was not suited for the
synthesis of analogues of 10d with different substituents
in phenyl ring or heteroaromatic replacements. We
devised two synthetic routes (Scheme 2, routes A and
B) for the synthesis of such compounds. Both routes
utilized diethyl allylmalonate 15 as a starting material.
In route A, 15 was cross-coupled with 3-iodo-anisole to
give propenyl derivative 16, which was hydrogenated
to 17. The latter then was transformed to the target
compound 18a by a five step procedure shown in
Syn th esis
The preparation of benzyl-, phenethyl-, phenylpropyl-,
and phenylbutyl-substituted compounds 10a -e was
carried out in six steps from diethyl malonate 3 as
outlined in Scheme 1. Alkylation of 3 followed by cycli-
zation with formamidine gave dihydroxypyrimidine 5,
which was subjected to a three step sequence of chlo-
rination, iodination, and ammonolysis to afford the
4-amino-6-iodo-derivative 8. The latter underwent
Sonogashira coupling with 5-ethynylpyridine 9 (pre-
pared in three steps from 2,5-dibromopyridine by ami-
nation with morpholine, coupling with trimethylsilyl
acetylene and silyl group deprotection) to provide target
molecules 10a -e. The last three steps of this procedure,
iodination of 6 followed by ammonolysis and a coupling
reaction, can be replaced by a shorter two step sequence

6-Alkynylpyrimidines as Potent Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3641
Sch em e 2a
a
Reagents: Route A: (a) 4-Iodoanisole, n-Bu3N, Pd(OAc)2, MeCN, reflux, 4 h. (b) H2, 10% Pd/C, EtOAc, 15 h, 1 atm. (c) See Scheme
1, steps b-f. Route B: (a) Na, EtOH, formamidine acetate, 0 °C, and then add 15, room temperature, 16 h. (b) POCl3, reflux, 14 h. (c)
NH3, EtOH, sealed tube, 100 °C, 16 h. (d) 3-Br-pyridine, N,N-diisopropylethylamine, Pd(PPh3)4. tri-o-tolylphosphine, dioxane, reflux,
14 h. (e) H2, 4 atm, 10% Pd/C, EtOAc-MeOH, 17 h. (f) NaI, 40% HI, 70 °C, 10 min. (g) Compound 9, Pd(PPh3)2Cl2, CuI, MeCN-Et3N,
room temperature, 1.5 h.
Scheme 1. In the shorter route B, we prepared advanced
intermediate 4-amino-5-allyl-6-chloropyrimidine 21,
which was elaborated to the desired arylpropyl com-
pounds 18b-f by the sequence of cross-coupling with
aryl bromides or iodides, hydrogenation, iodination, and
Sonogashira coupling with 9. Overall yields of target
molecules in routes A and B were comparable. However,
the syntheses of 18 in route B required only four steps
from common intermediate 21, while a seven step
sequence needed to be applied in route A.
The preparation of 5-N-benzyl compounds 31 (Scheme
3) started from commercially available 5-amino-4,6-
dichloropyrimidine 26. Iodination followed by consecu-
tive N-benzylation and N-methylation gave an inter-
mediate 29 from which target molecules 31a -j were
obtained in two steps as illustrated in Scheme 1. It
should be noted that the addition of tetrabutylammo-
nium iodide helped to increase the yield of 27 and 28.
The synthesis of 5-aminomethyl derivatives 37
(Scheme 4) required 5-formyl-4.6-dichloropyrimidine 33,
which was synthesized from corresponding 4,6-dihydroxy-
pyrimidine 32 according to a literature procedure.15
Ammonolysis of 33 at 50-60 °C for 1 h afforded
monoamino compound 34, which was treated with the
appropriate secondary amine under reductive amination
conditions16 to provide 35. The synthesis of desired
37a -c was achieved by iodination of 35 followed by
cross-coupling reactions with pyridyl-acetylene 9 as
shown in Scheme 2.
Resu lts a n d Discu ssion
The synthesized 6-ethynylpyrimidines were tested for
their ability to inhibit AK enzyme activity and to inhibit
ADO phosphorylation in intact cells. The importance of
the latter assay stems from the fact that AK is an
intracellular target; therefore, the ability of AK inhibi-
tors to cross cell membranes is a physiologically relevant
property. The in vitro AK inhibition results are sum-
marized in Table 1 (the protocols used are described in
the Experimental section). 5-Unsubstituted pyrimidine
2, as it was expected, did not display high inhibitory
activity because of the absence of a lipophilic fragment
at the 5-position to fill a putative hydrophobic pocket
of the protein (Figure 1). Introduction of a 3-bromo-

3642 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Gomtsyan et al.
Sch em e 3a
a
Reagents: (a) NaI, 40% HI, room temperature, 3 h. (b) 95% NaH, THF, 0 °C, and then benzyl bromide, Bu4NI, room temperature, 2
h. (c) 95% NaH, THF, 0 °C, and then MeI, Bu4NI, room temperature, 14 h. (d) NH3, EtOH, sealed tube, 80 °C, 14 h. (e) Compound 9,
Pd(PPh3)2Cl2, CuI, MeCN-Et3N, room temperature, 1.5 h.
Sch em e 4a
a
Reagents: (a) POCl3, DMF, 1 h, 0 °C, and then add 32, stir 0.5 h at room temperature, and then reflux, 3 h. (b) NH3, toluene, 50-60
°C, 0.5 h, TLC monitoring. (c) N-Methylbenzylamine, AcOH, NaB (OAc)3H, CH2Cl2, room temperature, 15 h. (d) NaI, 40% HI, 70 °C, 10
min, and then Na2CO3 work-up. (e) Compound 9, Pd(PPh3)2Cl2, CuI, MeCN-Et3N, room temperature, 1.5 h.
benzyl group makes compound 10a closely resemble the
pyridopyrimidine counterpart 1. Because the activity of
10a was markedly higher as compared with the unsub-
stituted 2, we decided to further investigate the effect
of the substituent, especially the length of the chain
between the pyrimidine fragment of the core and the
aromatic group of the substituent. Thus, compounds
with 1-4 carbon atom linkers in the 5-position were
prepared to introduce the corresponding benzyl, phen-
ethyl, phenylpropyl, and phenylbutyl groups. 2-Phen-
ethyl and 3-phenylpropyl derivatives 10c,d exhibited
high AK potencies having IC50 values below 5 nM, which
were 5-10-fold better than those observed for benzyl
and 4-phenylbutyl analogues 10b,e. Therefore, ad-
ditional work was directed toward the synthesis of
target molecules with 2- and 3-atom linkers in the
5-position of the pyrimidine core. It should be noted that
early SAR by NMR studies14 have also indirectly proven
the relevance of such approximation.
The first stage of exploring the SAR of 3-atom-linked
compounds consisted of introducing arylpropyl groups
where an aryl group was represented by substituted
phenyl and pyridyl moieties and the linker was an all
carbon chain. The resulting compounds 18a -f exhibited
high but very similar inhibitory potencies at cytosolic
AK (IC50 values of 2.5-4 nM) and more variable activity
in the intact cell AK assay. Among the best in this latter
category were the unsubstituted phenylpropyl com-
pound 10d and the pyridylpropyl analogues 18b,f.
However, despite high inhibitory activities and, in some
instances, good cell-penetrating properties, these com-
pounds did not show potent activity in the formalin test
or in the carrageenan-induced thermal hyperalgesia
animal models (Table 2). One of the possible reasons

6-Alkynylpyrimidines as Potent Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3643
Ta ble 1. In Vitro Activity of AK Inhibitors
Ta ble 2. Activity (ip) of Selected AK Inhibitors in Animal Pain
Models
a
The data represent mean ( SEM; n ) 6-8 per dose group.
nt ) not tested. c na ) not active up to 100 µM/kg.
b
example, 31a ,f and the unsubstituted 31b, appeared to
be better inhibitors of cytosolic AK activity. From
several compounds in these series that were tested in
in vivo models, 31a produced significant reduction of
flinching in the formalin test with an ED50 value of 10-
12 µM/kg and blocked carrageenan-induced thermal
hyperalgesia with an ED50 value of 3-5 µM/kg. It should
be noted, however, that pharmacokinetic profiles of in
vivo active 31a and inactive 31f were very similar (t1/2
) 0.5 vs 0.6 h; Vâ ) 3.7 vs 3.4 L/kg; CLp ) 5.4 vs 4.5
L/hr‚kg; F ) 4 vs 5%). These data suggest that the
differential in vivo activity of 31a , as compared to 31f,
may be due to the relative ability of 31a to penetrate
into the CNS. Previous data with nucleoside-like AK
inhibitors indicated that the majority of the analgesic
effects in the inflammatory hyperalgesia model are
spinally mediated.17
a
The data represent mean ( SEM from at least three deter-
minations.
for such in vitro-in vivo discrepancy could have been a
poor water solubility of the synthesized compounds. To
test this assumption, we have synthesized 37a -c where
one of the carbon units in the linker was replaced with
the nitrogen atom. As a result, compound 37a showed
markedly increased in vivo activity with an ED50 value
of 5-7 µM/kg in the thermal hyperalgesia assay, a
model of inflammatory pain. Interestingly, this com-
pound was not active in the formalin model of persistent
pain, thus showing selectivity for inflammatory pain.
Encouraged by the fact that in vivo activity was
achievable in these series of compounds, we began to
explore the SAR of phenethyl-substituted molecules,
another class of compounds that we originally selected
based on their AK in vitro activity. However, instead of
focusing on an all carbon linker, we replaced one carbon
atom with nitrogen to prepare 31a -j that were anilino
analogues of phenethyl derivative 10c. The substituent
position in the phenyl ring of the N-benzyl moiety had
a clear effect on the in vitro potency of these compounds.
Unsubstituted or ortho- and meta-substituted com-
pounds showed increased AK inhibitory potencies as
compared with the para-substituted compounds. On the
other hand, compounds with the ortho substitution, for
Con clu sion
We have identified a novel nonnucleoside class of
potent AK inhibitors that contain a 4-amino-6-[6-(4-
morpholynyl)-3-pyridinylethynyl]pyrimidine core. The
substituent at the 5-position plays one of the most
important roles for AK potency as it is responsible for
the hydrophobic interactions with the protein. It was
shown that the length of the linker between the lipo-
philic fragment of the 5-substituent and the pyrimidine
core affects the AK inhibitory potency. Compounds with
2- and 3-atom linkers provided the highest AK inhibi-
tion. The critical step toward active compounds in vivo
was the introduction of a nitrogen unit in the linkers of
both types of series. Thus, compound 37a with a 3-atom
linker in the 5-position of the pyrimidine core exhibited
good efficacy (ED50 ) 5-7 µM/kg) in an animal model
of inflammatory pain. Additionally, compound 31a with
a 2-atom linker was very effective in both the inflam-
matory (ED50 ) 3-5 µM/kg) and the persistent chemical
pain (ED50 ) 10-12 µM/kg) models.

3644 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Gomtsyan et al.
2-Mor p h olin yl-5-eth yn ylp yr id in e (9). A solution of 2,5-
dibromopyridine (2.10 g, 8.86 mmol) in morpholine (4 mL) was
heated to 90 °C for 15 h. The mixture was concentrated, and
the residue was chromatographed (EtOAc-hexane, 1:1) to
provide 5-bromo-2-morpholinylpyridine (1.93 g, 89%). MS
(DCI/NH3): m/e 243/245 (M + H)+. To a slurry of this compond
in piperidine (5 mL) was added trimethylsilyl acetylene (4.03
g, 41.0 mmol), Pd(PPh3)2Cl2 (0.20 g, 0.29 mmol), and copper(I)
iodide (0.15 g, 0.79 mmol), and the mixture was heated in the
sealed tube at 90 °C for 3 days. The reaction mixture was
concentrated, and the residue was chromatographed (EtOAc-
hexane, 1:3) to give 2-(4-morpholinyl)-5-pyridinylethynyltri-
methylsilane (1.43 g, 69%). MS (DCI/NH3): m/e 261 (M + H)+.
Desilylation of this product was carried out in methanol with
1 M aqueous K2CO3. After 2 h, the solvent was removed in
vacuo, the residue was partitioned between water and dichloro-
methane, and the organic phase was dried (Na2SO4) and
concentrated to obtain the desired product 9 (1.42 g, 95%) that
was used in the next step without further purification. 1H
NMR (CDCl3): δ 8.32 (d, J ) 1.5 Hz, 1H), 7.58 (dd, J ) 11
and 1.5 Hz, 1H), 6.55 (d, J ) 11 Hz, 1H), 3.80 (m, 4H), 3.57
(m, 4H), 3.07 (s, 1H). MS (DCI/NH3): m/e 189 (M + H)+.
4-Am in o-5-(3-br om oben zyl)-6-[6-(4-m or ph olin yl)-3-pyr i-
d in yleth yn yl]p yr im id in e (10a ). A solution of 4-amino-6-
iodo-5-(3-bromobenzyl)pyrimidine 8a (0.38 g, 0.97 mmol) in
MeCN (8 mL) and triethylamine (6 mL) at ambient temper-
ature was treated with 2-morpholinyl-5-ethynylpyrimidine 9
(0.2 g, 1.07 mmol), Pd(PPh3)2Cl2 (0.034 g, 0.046 mmol), and
copper(I) iodide (0.009 g, 0.06 mmol) for 1.5 h, filtered, and
crystallized from ethanol to give 0.34 g (80%) of the desired
product 10a ; mp 229-231 °C. 1H NMR (CDCl3): δ 8.51 (s, 1H),
8.38 (d, J ) 2 Hz, 1H), 7.60 (dd, J ) 7.5 and 2 Hz, 1H), 7.15-
7.43 (m, 4H), 6.60 (d, J ) 7.5 Hz, 1H), 4.82 (s, 2H), 4.17 (s,
2H), 3.80 (t, J ) 4.5 Hz, 4H), 3.58 (t, J ) 4.5 Hz, 4H). MS
(DCI/NH3): m/e 450 (M + H)+. Anal. Calcd. (C22H20BrN5O‚
0.25H2O): C, H, N.
Exp er im en ta l Section
1H NMR spectra were obtained at 300 and 400 MHz using
tetramethylsilane as internal standard. The mass spectra
(electron spray ionization (ESI) and dissolvable chemical
ionization (DCI)) and high-resolution mass spectra were
recorded on a Finnigin-4000 instrument. Elemental combus-
tion analyses were within +0.4% of theoretical values and
obtained from Robertson Microlit Laboratories. Chromato-
graphic separations were performed on silica gel 60 (230-400
mesh).
4-Am in o-6-[6-(4-m or ph olin yl)-3-pyr idin yleth yn yl]pyr im -
id in e (2). This compound was synthesized from 4,6-dichloro-
pyrimidine in three steps by the procedure described below
for the synthesis of 10a from 6a (yield 61%). 1H NMR (DMSO-
d6): δ 8.38 (d, J ) 2.0 Hz, 1H), 8.32 (s, 1H), 7.72 (dd, J ) 2.0
and 7.5 Hz, 1H), 7.00 (broad s, 2H), 6.87 (d, J ) 7.5 Hz, 1H),
6.53 (s, 1H), 3.70 (m, 4H), 3.58 (m, 4H). MS (DCI/NH3): m/e
282 (M + H)+. Anal. Calcd. (C15H15N5O‚0.1H2O): C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
10a -e: Dieth yl (3-Br om oben zyl)m a lon a te (4a ). A solution
of sodium (0.88 g, 38 mmol) in ethanol (50 mL) was treated
with diethyl malonate 3 (6.40 g, 40 mmol) dropwise over 15
min. A solution of 3-bromobenzyl bromide (10.0 g, 40 mmol)
in ethanol (40 mL) was added dropwise over 1 h, and the
heterogeneous solution was refluxed for 16 h. The solvent was
removed in vacuo, and the residue was diluted in EtOAc,
washed with aqueous NH4Cl and water, and concentrated. The
residue was chromatographed (EtOAc-hexane, 5:95) to pro-
1
vide 11.0 g (88%) of the title compound. H NMR (CDCl3): δ
7.36 (m, 2H), 7.15 (m, 2H), 4.19 (q, J ) 7.5 Hz, 4H), 3.60 (t, J
) 9.0 Hz, 1H), 3.19 (d, J ) 9.0 Hz, 2H), 1.21 (t, J ) 7.5 Hz,
6H). MS (DCI/NH3): m/z 329 (M + H)+.
4,6-Dih yd r oxy-5-(3-br om oben zyl)p yr im id in e (5a ). A
solution of sodium ethoxide in ethanol (prepared from 3.87 g,
168.4 mmol of sodium, and 100 mL of ethanol) was treated at
0 °C with formamidine acetate (5.5 g, 53 mmol), the resulting
mixture was stirred for 20 min, and then, the solution of
diethyl (3-bromobenzyl)malonate 4a (15.84 g, 48.1 mmol) in
ethanol (10 mL) was added. The mixture was stirred at
ambient temperature for 16 h, the solvent was removed at
reduced pressure, water was added, and the pH was adjusted
to 6-7 with 3 N HCl. The heterogeneous mixture was stirred
for 15 min, and the pale yellow precipitate was filtered. The
solid was suspended in EtOAc, refluxed for 30 min, filtered,
4-Am in o-5-ben zyl-6-[6-(4-m or ph olin yl)-3-pyr idin yleth yn -
yl]p yr im id in e (10b). Compound 10b was synthesized using
benzyl bromide, and the process was described for the syn-
1
thesis of 10a (yield 77%). H NMR (DMSO-d6): δ 8.28 (d, J )
2.0 Hz, 1H), 8.22 (s, 1H), 7.63 (dd, J ) 7.5 and 2.0 Hz, 1H),
7.32-7.12 (m, 5H), 6.95 (broad s, 2H), 6.87 (d, J ) 7.5 Hz,
1H), 4.09 (s, 2H), 3.70 (t, J ) 4.5 Hz, 4H), 3.58 (t, J ) 4.5 Hz,
4H). MS (DCI/NH3): m/e 372 (M + H)+. Anal. Calcd.
(C22H21N5O‚0.4H2O): C, H, N.
1
and dried to give 7.41 g (55%) of the title compound. H NMR
4-Am in o-5-(2-ph en eth yl)-6-[6-(4-m or ph olin yl)-3-pyr idin -
yleth yn yl]p yr im id in e (10c). Compound 10c was synthesized
using (2-bromoethyl)benzene and the process described for the
synthesis of 10a (yield 83%). 1H NMR (CDCl3): δ 8.47 (s, 1H),
8.38 (d, J ) 2.0 Hz, 1H), 7.68 (dd, J ) 7.5 and 2 Hz, 1H), 7.18-
7.37 (m, 5H), 6.62 (d, J ) 7.5 Hz, 1H), 4.80 (s, 2H), 4.17 (s,
2H), 3.82(t, J ) 4.5 Hz, 4H), 3.60 (t, J ) 4.5 Hz, 4H), 3.00 (t,
J ) 3.0 Hz, 4H). MS (DCI/NH3): m/e 386 (M + H)+. Anal.
Calcd. (C23H23N5O): C, H, N.
4-Am in o-5-(3-ph en ylpr opyl)-6-[6-(4-m or ph olin yl)-3-pyr i-
d in yleth yn yl]p yr im id in e (10d ). Compound 10d was syn-
thesized using 1-bromo-3-phenylpropane and the process
described for the synthesis of 10a (yield 76%). 1H NMR
(CDCl3): δ 8.42 (s, 1H), 8.39 (d, J ) 2.0 Hz, 1H), 7.59 (dd, J )
7.5 and 2.0 Hz, 1H), 7.32-7.18 (m, 5H), 6.60 (d, J ) 7.5 Hz,
1H), 4.78 (s, 2H), 3.82 (t, J ) 4.5 Hz, 4H), 3.60 (t, J ) 4.5 Hz,
4H), 2.75 (m, 4H), 1.95 (m, 2H). MS (DCI/NH3): m/e 400 (M +
H)+. Anal. Calcd. (C24H25N5O): C, H, N.
(DMSO-d6): δ 7.98 (s, 1H), 7.33 (m, 2H), 7.20 (m, 2H), 3.58 (s,
2H). MS (DCI/NH3): m/e 281 (M + H)+.
4,6-Dich lor o-5-(3-br om oben zyl)p yr im id in e (6a ). A solu-
tion of 4,6-dihydroxy-5-(3-bromobenzyl)pyrimidine 5a (2.8 g,
10 mmol) in POCl3 (20 mL) was refluxed for 16 h. After the
excess of POCl3 was removed at reduced pressure, the residue
was dissolved in CH2Cl2, washed twice with water, and dried
over MgSO4. The solvent was removed, and the residue was
precipitated from ethanol to give 2.46 g (77%) of the product.
1H NMR (CDCl3): δ 8.70 (s, 1H), 7.38 (m, 2H), 7.15 (m, 2H),
4.24 (s, 2H). MS (DCI/NH3): m/e 318 (M + H)+.
4,6-Diiod o-5-(3-br om oben zyl)p yr im id in e (7a ). A solu-
tion 4,6-dichloro-5-(3-bromobenzyl)pyrimidine 6a (4.93 g, 15.5
mmol) in acetone (75 mL) was treated with NaI (11.6 g, 77
mmol) and HI (45% solution, 15 mL) at ambient temperature
for 16 h. The resulting mixture was poured into a beaker with
ice water, filtered, and air-dried to give 6.17 g (80%) of the
1
desired product. H NMR (CDCl3): δ 8.35 (s, 1H), 7.40-7.08
(m, 4H), 4.40 (s, 2H). MS (DCI/NH3): m/e 501 (M + H)+.
4-Am in o-6-iod o-5-(3-br om oben zyl)p yr im id in e (8a ). A
solution of diiodo derivative 7a (1.0 g, 2 mmol) in ethanol (3
mL) was treated with ammonia, and the solution was heated
in the sealed tube at 100 °C for 16 h. After the mixture was
cooled to ambient temperature and concentrated, the residue
was precipitated from ethanol to give 0.52 g (48%) of the
desired product. 1H NMR (CDCl3): δ 8.18 (s, 1H), 7.41 (d, J )
8.0 Hz, 1H), 7.35 (s, 1H), 7.23 (m, 1H), 7.10 (d, J ) 8.0 Hz,
1H), 4.82 (broad s, 2H), 4.03 (s, 2H). MS (DCI/NH3): m/e 390
(M + H)+.
4-Am in o-5-(4-p h en ylbu tyl)-6-[6-(4-m or p h olin yl)-3-p yr i-
d in yleth yn yl]p yr im id in e (10e). Compound 10e was syn-
thesized using 1-bromo-3-phenylbutane18 and the process
described for the synthesis of 10a (yield 75%). 1H NMR
(CDCl3): δ 8.41 (s, 1H), 8.39 (d, J ) 2.0 Hz, 1H), 7.62 (dd, J )
7.5 and 2.0 Hz, 1H), 7.31-7.16 (m, 5H), 6.59 (d, J ) 7.5 Hz,
1H), 4.90 (s, 2H), 3.82 (t, J ) 4.5 Hz, 4H), 3.60 (t, J ) 4.5 Hz,
4H), 2.78-2.55 (m, 4H), 1.94-1.60 (m, 4H). MS (DCI/NH3):
m/e 414 (M + H)+. Anal. Calcd. (C25H27N5O): C, H, N.
Dieth yl 2-(3-Br om oa n ilin o)m a lon a te (12). A solution of
3-bromoaniline (150 g, 872 mmol) was treated with diethyl

6-Alkynylpyrimidines as Potent Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3645
chloromalonate 11 (69.5 g, 291 mmol) for 15 h at 40-50 °C.
Diethyl ether was added, and 3-bromoaniline hydrobromide
was filtered off. The filtrate was washed twice with water, 1
N HCl, and water. The organic phase was dried (Na2SO4),
decolorized with charcoal, concentrated, and crystallized from
hexane to obtain the desired product 12 (73.9 g, 77%); mp 83-
1.71 (m, 2H). MS (DCI/NH3): m/e 430 (M + H)+. Anal. Calcd.
(C25H27N5O2): C, H, N.
4-Am in o-5-a llyl-6-ch lor op yr im id in e (21). Compound 21
was prepared by ammonolysis of 5-allyl-4,6-dichloropyrimi-
1
dine19 20 as described for the synthesis of 8a (yield 88%). H
NMR (CDCl3): δ 8.27 (s, 1H), 5.71 (m, 1H), 5.22-5.08 (m, 4H),
1
3.40 (m, 2H). MS (DCI/NH3): m/e 170 (M + H)+.
84 °C. H NMR (CDCl3): δ 7.03 (t, J ) 8.0 Hz, 1H), 6.90 (m,
1H), 6.81 (t, J ) 2.5 Hz, 1H), 6.58 (m, 1H), 4.90 (broad s, 1H),
4.68 (s, 1H), 4.26 (q, J ) 7.5 Hz, 4H), 1.30 (t, J ) 7.5 Hz, 6H).
MS (DCI/NH3): m/e 330 (M + H)+.
4,6-Dih ydr oxy-5-(3-br om oan ilin o)pyr im idin e. This com-
pound was prepared using the procedure described for the
synthesis of 5a (yield 63%). 1H NMR (DMSO-d6): δ 7.91 (s,
1H), 7.00-6.90 (m, 2H), 6.65 (m, 1H), 6.59 (d, J ) 4.0 Hz, 1H),
6.50 (m, 1H). MS (DCI/NH3): m/e 282 (M + H)+.
4-Am in o-5-(3-p yr id in -3-ylp r op -2-en yl)-6-ch lor op yr im i-
d in e (22b). A solution of 4-amino-5-allyl-6-chloropyrimidine
21 (5.0 g, 29.6 mmol) in dioxane (50 mL) was treated with
diisopropylethylamine (5 mL), 3-bromopyridine (4.9 g, 31.0
mmol), tri-o-tolylphosphine (0.31 g), and Pd(PPh3)4 (0.8 g), and
the mixture was refluxed for 14 h. After it was cooled to
ambient temperature, the mixture was concentrated, and the
residue was diluted in EtOAc and washed with water. The
organic layer was dried (MgSO4) and concentrated, and the
residue was chromatographed (CH2Cl2-MeOH, 95:5) to obtain
the desired product 22b (1.25 g, 17%). 1H NMR (DMSO-d6): δ
8.55(d, J ) 2.5 Hz, 1H), 8.40 (d, J ) 4.0 Hz, 1H), 8.11 (s, 1H),
7.81 (m, 1H), 7.31 (dd, J ) 4.0 and 7.5 Hz, 1H), 7.16 (broad
sd, 2H), 6.50-6.32 (m, 2H), 3.50 (d, J ) 5.0 Hz, 2H). MS (DCI/
NH3): m/e 247 (M + H)+.
4,6-Dich lor o-5-(3-br om oa n ilin o)p yr im id in e (13). A so-
lution of dihydroxy intermediate (2.2 g, 7.8 mmol), POCl3 (8
mL), and dimethylformamide (DMF, 3 mL) was refluxed for
15 h. The resulting viscous mixture was poured into a beaker
with ice, the pH was adjusted between 6 and 7 with concen-
trated NH4OH, extracted twice with Et2O, dried (Na2SO4), and
concentrated, and the residue was chromatographed (EtOAc-
hexane, 1:9) to obtain the desired compound 13 (0.94 g, 38%).
1H NMR (CDCl3): δ 8.58 (s, 1H), 7.13 (m, 2H), 6.92 (m, 1H),
6.65 (m, 1H), 5.93 (broad s, 1H). MS (DCI/NH3): m/e 318 (M
+ H)+.
4,6-Dich lor o-5-[(3-br om op h en yl)m eth yla m in o]p yr im i-
d in e (14). A solution of 13 (1.1 g, 3.5 mmol) in tetrahydrofuran
(THF, 10 mL) was treated with NaH (0.095 g, 4.15 mmol) at
ambient temperature. The mixture was stirred for 10 min and
then treated with MeI (5.0 g, 35 mmol). After 15 h, the mixture
was diluted with EtOAc, washed with aqueous NH4Cl and
water. The organic phase was concentrated to give the desired
product 14 (1.05 g, 90%). 1H NMR (CDCl3): δ 8.78 (s, 1H),
7.15-6.95 (m, 2H), 6.70 (t, J ) 1.5 Hz, 1H), 6.37 (m, 1H), 3.21
(s, 3H). MS (DCI/NH3): m/e 332 (M + H)+.
4-Am in o-5-(3-p yr id in -3-ylp r op yl)-6-ch lor op yr im id in e
(23b). A mixture of 22b (0.7 g, 2.84 mmol), Pt/C (5%, 0.35 g)
in EtOAc (25 mL), and MeOH (25 mL) was hydrogenated at 4
atm for 17 h. The resulting mixture was filtered through Celite
and concentrated to obtain the desired product 23b (0.42 g,
60%). 1H NMR (DMSO-d6): δ 8.44 (s, 1H), 8.40 (d, J ) 4.5 Hz,
1H), 8.05 (s, 1H), 7.14 (d, J ) 7.5 Hz, 1H), 7.30 (m, 1H), 7.20
(broad s, 2H), 2.69 (t, J ) 8.0 Hz, 2H), 2.60 (t, J ) 8.0 Hz,
2H), 1.72 (m, 2H). MS (DCI/NH3): m/e 249 (M + H)+.
4-Am in o-5-(3-p yr id in -3-ylp r op yl)-6-iod op yr im id in e
(24b). A solution of 23b (0.19 g, 0.76 mmol) in 40% HI (4
mL) was treated with NaI (0.57 g, 3.8 mmol) and stirred for
10 min at 70 °C. The precipitate was filtered, taken up in
aqueous NaHCO3, extracted with EtOAc, dried (MgSO4), and
concentrated to give the desired product 24b (0.17 g, 67%).
1H NMR (DMSO-d6): δ 8.43 (d, 2.0 Hz, 1H), 8.39 (dd, J ) 2.0
and 4.5 Hz, 1H), 8.04 (s, 1H), 7.64 (m, 1H), 7.30 (m, 1H), 7.18
(broad s, 2H), 2.70 (t, J ) 8.0 Hz, 2H), 2.58 (t, J ) 8.0 Hz,
2H), 1.72 (m, 2H). MS (DCI/NH3): m/e 341 (M + H)+.
4-Am in o-5-[3-(3-pyr idin yl)pr opyl)-6-[6-(4-m or ph olin yl)-
3-p yr id in yleth yn yl]p yr im id in e (18b). Compound 18b was
prepared from 24b and 2-morpholinyl-5-ethynylpyridine 9
according to the procedure described for the synthesis of 10a
4-Am in o-5-[(3-br om oph en yl)m eth ylam in o]-6-[6-(4-m or -
p h olin yl)-3-p yr id in yleth yn yl]p yr im id in e (10f). Compound
10f was prepared from 14 following the procedures described
1
for the synthesis of 10a (yield 78%). H NMR (DMSO-d6): δ
8.28 (s, 1H), 7.98 (d, J ) 2 Hz, 1H), 7.36 (dd, J ) 7.5 and 2
Hz, 1H), 7.18-7.00 (m, 3H), 6.88-6.78 (m, 3H), 6.69 (m, 1H),
6.53 (d, J ) 2 Hz, 1H), 3.65 (t, J ) 4.5 Hz, 4H), 3.55 (t, J ) 4.5
Hz, 4H), 3.12 (s, 3H). MS (DCI/NH3): m/e 465 (M + H)+. Anal.
Calcd. (C22H22BrN6O‚0.5H2O): C, H, N.
1
(yield 29%). H NMR (DMSO-d6): δ 8.45-8.35 (m, 2H), 8.21
Dieth yl 2-[3-(4-Meth oxyp h en yl)p r op -2-en yl]m a lon a te
(16). To a solution of diethyl allylmalonate 15 (2.0 g, 10 mmol)
in MeCN (10 mL) were added 4-iodoanisole (2.33 g, 10 mmol),
n-tributylamine (2.4 mL), and palladium acetate (0.04 g). After
it was refluxed for 4 h, the resulting mixture was cooled,
concentrated, diluted with Et2O, and washed with 1 N HCl,
water, and brine. The organic phase was dried (MgSO4) and
concentrated, and the residue was chromatographed (EtOAc-
(d, J ) 2.0 Hz, 1H), 8.18 (s, 1H), 7.61 (m, 1H), 7.51 (dd, J )
7.5 and 2.0 Hz, 1H), 7.25 (dd, J ) 8 and 4.5 Hz, 1H), 6.90
(broad s, 2H), 6.88 (d, J ) 7.5 Hz, 1H), 3.70 (t, J ) 4.5 Hz,
4H), 3.58 (t, J ) 4.5 Hz, 4H), 2.78-2.67 (m, 4H), 1.89-1.72
(m, 2H). MS (DCI/NH3): m/e 401 (M + H)+. Anal. Calcd.
(C23H24N6O‚1.22H2O): C, H, N.
4-Am in o-5-[3-(3-br om op h en yl)p r op yl)-6-[6-(4-m or p h o-
lin yl)-3-pyr idin yleth yn yl]pyr im idin e (18c). Compound 18c
was prepared from the 1-bromo-3-iodobenzene and using the
synthetic sequence described for the synthesis of 18b (yield
42%). 1H NMR (DMSO-d6): δ 8.41 (s, 1H), 8.32 (d, J ) 2.0 Hz,
1H), 7.55 (dd, J ) 7.5 and 2.0 Hz, 1H), 7.39-7.29 (m, 2H),
7.15-7.08 (m, 2H), 6.60 (d, J ) 7.5 Hz, 1H), 4.88 (s, 2H), 3.81
(t, J ) 4.5 Hz, 4H), 3.61 (t, J ) 4.5 Hz, 4H), 2.79-2.66 (m,
4H), 2.01-1.87 (m, 2H). MS (APCI): m/e 478/480 (M + H)+.
Anal. Calcd. (C24H24BrN5O): C, H, N.
4-Am in o-5-[3-(4-br om op h en yl)p r op yl)-6-[6-(4-m or p h o-
lin yl)-3-p yr id in yleth yn yl]p yr im id in e (18d ). Compound
18d was prepared from the 1-bromo-4-iodobenzene and using
the synthetic sequence described for the synthesis of 18b (yield
40%). 1H NMR (DMSO-d6): δ 8.40 (s, 1H), 8.33 (d, J ) 2.0 Hz,
1H), 7.46 (dd, J ) 7.5 and 2.0 Hz, 1H), 7.41-7.35 (m, 2H),
7.11-7.04 (m, 2H), 6.61 (d, J ) 7.5 Hz, 1H), 4.81 (s, 2H), 3.81
(t, J ) 4.5 Hz, 4H), 3.60 (t, J ) 4.5 Hz, 4H), 2.77-2.66 (m,
4H), 2.01-1.89 (m, 2H). MS (DCI/NH3): m/e 478/480 (M + H)+.
Anal. Calcd. (C24H24BrN5O): C, H, N.
1
hexane, 2:3) to give the desired product 16 (1.76 g, 58%). H
NMR (CDCl3): δ 7.35-7.22 (m, 2H), 6.90-6.80 (m, 2H), 6.42
(d, J ) 15.0 Hz, 1H), 6.02 (m, 1H), 4.26-4.12 (m, 4H), 3.70 (s,
3H), 3.47 (t, J ) 6.0 Hz, 1H), 2.79 (m, 2H), 1.25 (t, J ) 7.5 Hz,
6H). MS (DCI/NH3): m/e 307 (M + H)+.
Dieth yl 2-[3-(4-Meth oxyp h en yl)p r op yl]m a lon a te (17).
A mixture of 16 (1.63 g, 5.3 mmol) and 10% Pd/C (0.17 g) in
EtOAc (25 mL) was hydrogenated at 1 atm for 15 h. The
resulting mixture was filtered through Celite and concentrated
to give the desired product 17 (1.47 g, 92%). 1H NMR
(CDCl3): δ 7.09 (m, 2H), 6.82 (m, 2H), 4.18 (q, J ) 7.5 Hz,
4H), 3.79 (s, 3H), 3.33 (t, J ) 9.0 Hz, 1H), 2.57 (t, J ) 9.0 Hz,
2H), 1.91 (m, 2H), 1.60 (m, 2H), 1.25 (t, J ) 7.5 Hz, 6H). MS
(DCI/NH3): m/e 309 (M + H)+.
4-Am in o-5-[3-(4-m eth oxyph en yl)pr opyl]-6-[-(4-m or ph o-
lin yl)-3-p yr id in ylet h yn yl]p yr im id in e (18a ). Compound
18a was prepared from 17 using a synthetic sequence de-
scribed for the synthesis of 10a (yield 54%). 1H NMR (DMSO-
d6): δ 8.19 (d, J ) 2.0 Hz, 1H), 8.16 (s, 1H), 7.49 (dd, J ) 7.5
and 2.0 Hz, 1H), 7.10 (d, J ) 7.5 Hz, 2H), 6.93-6.75 (m, 5H),
3.73-3.67 (m, 7H), 3.57 (t, J ) 4.5 Hz, 4H), 2.73-2.60 (m, 4H),
4-Am in o-5-[3-(2-ch lor op h en yl)p r op yl)-6-[6-(4-m or p h o-
lin yl)-3-pyr idin yleth yn yl]pyr im idin e (18e). Compound 18e

3646 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Gomtsyan et al.
was prepared from the 1-chloro-2-iodobenzene and using the
synthetic sequence described for the synthesis of 18b (yield
56%). 1H NMR (DMSO-d6): δ 8.24 (d, J ) 2.0 Hz, 1H), 8.18 (s,
1H), 7.53 (dd, J ) 8.0 and 2.0 Hz, 1H), 7.36 (m, 2H), 7.24 (m,
2H), 6.95 (s, 2H), 6.88 (d, J ) 9.0 Hz, 1H), 3.70 (m, 4H), 3.56
(m, 4H), 2.83 (m, 2H), 2.74 (m, 2H), 1.77 (m, 2H). MS (ESI):
m/e 434 (M + H)+. Anal. Calcd. (C24H24ClN5O‚0.2H2O): C, H, N.
4-Am in o-5-[3-(4-pyr idin yl)pr opyl)-6-[6-(4-m or ph olin yl)-
3-p yr id in yleth yn yl]p yr im id in e (18f). Compound 18f was
prepared from the 4-bromopyridine and using the synthetic
sequence described for the synthesis of 18b (yield 46%). 1H
NMR (DMSO-d6): δ 8.40 (broad s, 2H), 8.26 (d, J ) 2.0 Hz,
1H), 8.19 (s, 1H), 7.53 (dd, J ) 7.5 and 2.0 Hz, 1H), 7.22 (d, J
) 4.5 Hz, 2H), 6.95 (broad s, 2H), 6.89 (d, J ) 7.5 Hz, 1H),
3.70 (t, J ) 4.5 Hz, 4H), 3.55 (t, J ) 4.5 Hz, 4H), 2.79-2.65
(m, 4H), 1.87-1.71 (m, 2H). MS (DCI/NH3): m/e 401 (M + H)+.
Anal. Calcd. (C23H24N6O‚2.0H2O): C, H, N.
using the synthetic sequence described for the synthesis of 31a
(yield 28%). 1H NMR (CDCl3): δ 8.39 (d, J ) 1.5 Hz, 1H), 8.05
(s, 1H), 7.72 (dd, J ) 7.5 and 2.0 Hz, 1H), 7.60 (s, 1H), 7.41-
7.30 (m, 2H), 7.23 (t, J ) 7.5 Hz, 1H), 6.95 (broad s, 2H), 6.91
(d, J ) 7.5 Hz, 1H), 4.30 (s, 2H), 3.72 (t, J ) 4.5 Hz, 4H), 3.58
(t, J ) 4.5 Hz, 4H), 2.75 (s, 3H). MS (DCI/NH3): m/e 479/481
(M + H)+. Anal. Calcd. (C23H23BrN6O‚0.3H2O): C, H, N.
4-Am in o-5-[(4-br om oben zyl)m eth yla m in o]-6-[6-(4-m or -
p h olin yl)-3-p yr id in ylet h yn yl]p yr im id in e (31d ). Com-
pound 31d was prepared from the 4-bromobenzyl bromide and
using the synthetic sequence described for the synthesis of 31a
(yield 70%). 1H NMR (CDCl3): δ 8.39 (d, J ) 1.5 Hz, 1H), 8.08
(s, 1H), 7.73 (dd, J ) 7.5 and 2.0 Hz, 1H), 7.45 (d, J ) 7.0 Hz,
2H), 7.31 (d, J ) 7.0 Hz, 2H), 7.05-6.90 (m, 3H), 4.28 (s, 2H),
3.71 (t, J ) 4.5 Hz, 4H), 3.59 (t, J ) 4.5 Hz, 4H), 2.72 (s, 3H).
MS (DCI/NH3): m/e 479/481 (M + H)+. Anal. Calcd. (C23H23
BrN6O‚2.2H2O‚0.2EtOH): C, H, N.
-
4,6-Diiodo-5-am in opyr im idin e (27). A mixture of 5-amino-
4,6-dichloropyrimidine 26 (4.0 g, 24.2 mmol), sodium iodide
(18.0 g, 120 mmol), and 40% HI (60 mL) was stirred for 3 h at
ambient temperature. After it was filtered, the precipitate was
washed with aqueous NaHCO3 and dried to give the desired
product (8.0 g, 94%). MS (DCI/NH3): m/e 348 (M + H)+.
4,6-Diiod o-5-(2-ch lor oben zyla m in o)p yr im id in e (28a ).
A solution of 27 (3.5 g, 10 mmol) in THF (100 mL) was treated
with 95% NaH (0.29 g, 12 mmol) at 0 °C, then the mixture
was warmed to ambient temperature, and 2-chlorobenzyl
bromide (1.6 mL, 12 mmol) and tetrabutylammonium iodide
(4.4 g, 12 mmol) were added. The mixture was stirred for 2 h,
concentrated, and chromatographed (EtOAc-hexane, 1:4) to
give the desired product 28a (3.5 g, 74%). 1H NMR (DMSO-
d6): δ 8.10 (s, 1H), 7.49-7.20 (m, 4H), 4.52 (m, 2H), 4.12 (t, J
) 7.5 Hz, 1H). MS (DCI/NH3): m/e 472 (M + H)+.
4,6-Diiodo-5-[N-m eth yl-N-(2-ch lor oben zylam in o)]pyr im -
id in e (29a ). A solution of 28a (4.0 g, 8.5 mmol) in THF (80
mL) was treated with 95% NaH (0.25 g, 10.2 mmol) at 0 °C,
then the mixture was warmed to ambient temperature, and
methyl iodide (0.8 mL, 12.8 mmol) and tetrabutylammonium
iodide (3.1 g, 8.5 mmol) were added. The mixture was stirred
for 14 h, concentrated, and chromatographed (EtOAc-hexane,
5:95) to give desired product 29a (2.8 g, 68%). 1H NMR
(CDCl3): δ 8.21 (s, 1H), 7.56 (m, 1H), 7.38 (m, 1H), 7.25 (m, 2H),
4.47 (s, 2H), 2.91 (s, 3H). MS (DCI/NH3): m/e 486 (M + H)+.
4-Am in o-5-[(2-ch lor oben zyl)m eth ylam in o]-6-iodopyr im -
id in e (30a ). A solution of diiodo derivative 29a (0.49 g, 1.0
mmol) in ethanol (3 mL) was treated with ammonia, and the
solution was heated in the sealed tube at 80 °C for 14 h. After
the mixture was cooled to ambient temperature and concen-
trated, the residue was precipitated from ethanol to give 30a
(0.2 g, 51%). 1H NMR (DMSO-d6): δ 7.80 (s, 1H), 7.56 (m, 1H),
7.42 (m, 1H), 7.30 (m, 2H), 6.92 broad s, 2H), 4.43-4.15 (m,
2H), 2.75 (s, 3H). MS (DCI/NH3): m/e 375 (M + H)+.
4-Am in o-5-[(4-ch lor oben zyl)m eth yla m in o]-6-[6-(4-m or -
p h olin yl)-3-p yr id in ylet h yn yl]p yr im id in e (31e). Com-
pound 31e was prepared from the 4-chlorobenzyl bromide and
using the synthetic sequence described for the synthesis of 31a
(yield 51%). 1H NMR (CDCl3): δ 8.38 (d, J ) 1.5 Hz, 1H), 8.05
(s, 1H), 7.73 (dd, J ) 7.5 and 2.0 Hz, 1H), 7.40-7.29 (m, 4H),
6.91 (broad s, 2H), 6.91 (d, J ) 7.5 Hz, 1H), 4.29 (s, 2H), 3.70
(t, J ) 4.5 Hz, 4H), 3.58 (t, J ) 4.5 Hz, 4H), 2.73 (s, 3H). MS
(DCI/NH3): m/e 435 (M + H)+. Anal. Calcd. (C23H23ClN6O‚
0.1H2O): C, H, N.
4-Am in o-5-[(2-m eth ylben zyl)m eth ylam in o]-6-[6-(4-m or -
p h olin yl)-3-p yr id in yleth yn yl]p yr im id in e (31f). Compound
31f was prepared from the 2-methylbenzyl bromide and using
the synthetic sequence described for the synthesis of 31a (yield
40%). 1H NMR (CDCl3): δ 8.37 (d, J ) 1.5 Hz, 1H), 8.08 (s,
1H), 7.71 (dd, J ) 7.5 and 2.0 Hz, 1H), 7.34 (m, 1H), 7.15-
7.07 (m, 3H), 6.92 (d, J ) 7.5 Hz, 1H), 6.78 (broad s, 2H), 4.31
(s, 2H), 3.70 (t, J ) 4.5 Hz, 4H), 3.57 (t, J ) 4.5 Hz, 4H), 2.71
(s, 3H), 2.27 (s, 3H). MS (DCI/NH3): m/e 415 (M + H)+. Anal.
Calcd. (C24H26N6O‚0.2H2O): C, H, N.
4-Am in o-5-[(3-ch lor oben zyl)m eth yla m in o]-6-[6-(4-m or -
p h olin yl)-3-p yr id in ylet h yn yl]p yr im id in e (31g). Com-
pound 31g was prepared from the 3-chlorobenzyl bromide and
using the synthetic sequence described for the synthesis of 31a
(yield 67%). 1H NMR (CDCl3): δ 8.35 (d, J ) 1.5 Hz, 1H), 8.12
(s, 1H), 7.70 (overlapped d, 1H), 7.70 (dd, J ) 7.5 and 2.0 Hz,
1H), 7.46 (d, J ) 7.0 Hz, 2H), 7.32 (dd, J ) 8.5 and 7.0 Hz,
1H), 6.92 (d, J ) 7.5 Hz, 1H), 4.50 (s, 2H), 3.70 (t, J ) 4.5 Hz,
4H), 3.59 (t, J ) 4.5 Hz, 4H), 2.79 (s, 3H). MS (DCI/NH3): m/e
435 (M + H)+. Anal. Calcd. (C23H23ClN6O‚0.7H2O): C, H, N.
4-Am in o-5-[m eth yl(2-tr iflu or om eth ylben zyl)a m in o]-6-
[6-(4-m or p h olin yl)-3-p yr id in yleth yn yl]p yr im id in e (31h ).
Compound 31h was prepared from the 2-trifluorobenzyl
bromide and using the synthetic sequence described for the
1
synthesis of 31a (yield 68%). H NMR (CDCl3): δ 8.38 (d, J )
1.5 Hz, 1H), 8.14 (s, 1H), 7.97 (d, J ) 6.0 Hz, 1H), 7.75-7.63
(m, 3H), 7.48 (t, J ) 6.0 Hz, 1H), 6.92 (d, J ) 7.5 Hz, 1H),
4.53 (s, 2H), 3.70 (t, J ) 4.5 Hz, 4H), 3.59 (t, J ) 4.5 Hz, 4H),
2.70 (s, 3H). MS (DCI/NH3): m/e 469 (M + H)+. Anal. Calcd.
(C24H23F3N6O‚0.25H2O): C, H, N.
4-Am in o-5-[(2-ch lor oben zyl)m eth yla m in o]-6-[6-(4-m or -
p h olin yl)-3-p yr id in ylet h yn yl]p yr im id in e (31a ). Com-
pound 31a was prepared from 30a and 2-morpholinyl-5-
ethynylpyridine 9 as described for the synthesis of 10a (60%).
1H NMR (CDCl3): δ 8.38 (d, J ) 2.0 Hz, 1H), 8.10 (s, 1H),
7.70 (dd, J ) 7.5 and 2.0 Hz, 1H), 7.56 (m, 1H), 7.38 (m, 1H),
7.30-7.21 (m, 2H), 6.90 (d, J ) 7.5 Hz, 1H), 4.43 (s, 2H), 3.70
(t, J ) 4.5 Hz, 4H), 3.58 (t, J ) 4.5 Hz, 4H), 2.77 (s, 3H). MS
(DCI/NH3): m/e 435 (M + H)+. Anal. Calcd. (C23H23ClN6O‚
0.6H2O): C, H, N.
4-Am in o-5-(ben zylm eth yla m in o)-6-[6-(4-m or p h olin yl)-
3-p yr id in yleth yn yl]p yr im id in e (31b). Compound 31b was
prepared from the benzyl bromide and using the synthetic
sequence described for the synthesis of 31a (yield 72%). 1H
NMR (CDCl3): δ 8.38 (d, J ) 1.5 Hz, 1H), 8.03 (s, 1H), 7.73
(dd, J ) 7.5 and 2.0 Hz, 1H), 7.40-7.18 (m, 5H), 6.95 (d, J )
7.5 Hz, 1H), 6.87 (broad s, 2H), 4.30 (s, 2H), 3.70 (t, J ) 4.5
Hz, 4H), 3.58 (t, J ) 4.5 Hz, 4H), 2.75 (s, 3H). MS (DCI/NH3):
m/e 401 (M + H)+. Anal. Calcd. (C23H24N6O‚0.4H2O): C, H, N.
4-Am in o-5-[(3-br om oben zyl)m eth yla m in o]-6-[6-(4-m or -
p h olin yl)-3-p yr id in ylet h yn yl]p yr im id in e (31c). Com-
pound 31c was prepared from the 3-bromobenzyl bromide and
4-Am in o-5-[(3,4-d ich lor oben zyl)m eth yla m in o]-6-[6-(4-
m or p h olin yl)-3-p yr id in yleth yn yl]p yr im id in e (31i). Com-
pound 31i was prepared from the 3,4-dichlorobenzyl bromide
and using the synthetic sequence described for the synthesis
of 31a (yield 56%). 1H NMR (CDCl3): δ 8.49 (d, J ) 2.0 Hz,
1H), 8.08 (s, 1H), 7.72 (dd, J ) 7.5 and 2.0 Hz, 1H), 7.67 (d, J
) 1.0 Hz, 1H), 7.53 (d, J ) 6.0 Hz, 1H), 7.30 (dd, J ) 6.0 and
1.0 Hz, 1H), 7.00 (broad s, 2H), 6.92 (d, J ) 7.5 Hz, 1H), 4.29
(s, 2H), 3.70 (t, J ) 4.5 Hz, 4H), 3.58 (t, J ) 4.5 Hz, 4H), 2.73
(s, 3H). MS (DCI/NH3): m/e 469 (M + H)+. Anal. Calcd. (C23H22
Cl2N6O‚0.25H2O): C, H, N.
-
4-Am in o-5-[m et h yl(2-n a p h t h ylm et h yl)a m in o]-6-[6-(4-
m or p h olin yl)-3-p yr id in yleth yn yl]p yr im id in e (31j). Com-
pound 31j was prepared from the 2-(bromomethyl)naphthalene
and using the synthetic sequence described for the synthesis
of 31a (yield 47%). 1H NMR (CDCl3): δ 8.40 (d, J ) 2.0 Hz,
1H), 8.04 (s, 1H), 7.86-7.78 (m, 4H), 7.71 (dd, J ) 7.5 and 2.0

6-Alkynylpyrimidines as Potent Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3647
Hz, 1H), 7.57 (d, J ) 8.5 Hz, 1H), 7.50-7.41 (m, 2H), 6.94
(broad s, 2H), 6.90 (d, J ) 7.5 Hz, 1H), 4.49 (s, 2H), 3.70 (t, J
) 4.5 Hz, 4H), 3.58 (t, J ) 4.5 Hz, 4H), 2.80 (s, 3H). MS (DCI/
NH3): m/e 451 (M + H)+. Anal. Calcd. (C27H26N6O‚0.2H2O):
C, H, N.
AK In h ibition Assa y. Routine AK inhibition assays were
carried out at 23 °C in a volume of 100 µL. The reaction
mixture contained 64 mM Tris-HCl (pH 7.5), 0.2 mM MgCl2,
1mM ATP, 0.2 µM [U-14C]ADO or [2-3H]ADO, and appropriate
volumes of rat brain cytosol as a source of AK.7 The reaction
was incubated for 15 min and terminated by aliquoting 40 µL
of the reaction mixture into DE-81 anion exchange filter disks.
The filter disks were air-dried, washed for 15 min in 2 mM
ammonium formate, rinsed with water, methanol, and acetone,
and dried under an atmosphere of nitrogen. Bound radioactiv-
ity was determined by standard scintillation spectrometry.
In ta ct Cell ADO P h osp h or yla tion Assa y. Routine ADO
phosphorylation assays in intact cells were conducted by using
IMR-32 human neuroblastoma cells (American Type Culture
Collection, Gaithersburg, MD). Appropriate concentrations of
test compounds (10-11 to 10-4 M) were added to each well, and
the cells were incubated for 10 min. The reaction was initiated
by the addition of 50 µL of 2 µM [U-14C]ADO. After a 20 min
incubation, the assay buffer was rapidly aspirated, and the
cells were quickly frozen by the addition of liquid nitrogen.
The plates were allowed to thaw at room temperature for 20
min, and a 50 µL aliquot of the supernatant was placed onto
DE-81 filter disks. The filter disks were then processed as
described above for the AK enzyme assay.
4,6-Dich lor o-5-for m ylp yr im id in e (33). A mixture of DMF
(64 mL) and POCl3 (200 mL) at 0 °C was stirred for 1 h, treated
with 4,6-dihydroxypyrimidine 32 (50.0 g, 446 mmol), and
stirred for 0.5 h at ambient temperature, and the heteroge-
neous mixture was refluxed for 3 h. The volatiles were removed
at reduced pressure, and the residue was poured into ice water
and extracted six times with diethyl ether. The organic phase
was washed with aqueous NaHCO3 and water, dried over
Na2SO4, concentrated, and crystallized (EtOAc-petroleum
ether) to give the desired product 33 (43.5 g, 55%); mp 67-69
1
°C (literature15 69-70 °C). H NMR (CDCl3): δ 10.45 (s, 1H),
8.90 (s, 1H). MS (DCI/NH3): m/e 177 (M + H)+.
4-Am in o-6-ch lor o-5-for m ylp yr im id in e (34). Ammonia
was bubbled through a solution of 33 (5.0 g, 28.4 mmol) in
toluene (250 mL) for 3-4 min, and the solution was heated to
60 °C. After 0.5 h, more ammonia gas was bubbled and stirring
was continued for 0.5 h at 50-60 °C. The reaction was
monitored by thin-layer chromatography (TLC) to avoid
formation of 4,6-diamino-5-formylpyrimidine. The mixture was
cooled to ambient temperature and concentrated, and the
residue was chromatographed (EtOAc-hexane, 3:7) to give the
desired compound 34 (3.0 g, 67%). 1H NMR (DMSO-d6): δ
10.25 (s, 1H), 8.72 (broad s, 1H), 8.58 (broad s, 1H), 8.40 (s,
1H). MS (DCI/NH3): m/e 158 (M + H)+.
4-Am in o-5-ben zylm eth yla m in om eth yl-6-ch lor op yr im i-
d in e (35a ). A solution of 34 (0.5 g, 3.18 mmol) in CH2Cl2 (10
mL) at ambient temperature was treated with N-methyl-
benzylamine (0.4 mL, 3.18 mmol), acetic acid (0.2 mL, 3.18
mmol), and sodium triacetoxyborohydride (1.0 g, 4.77 mmol).
After 15 h, the mixture was concentrated and the residue was
chromatographed (EtOAc-hexane, 35:65) to give the desired
product 35a (0.38 g, 46%). 1H NMR (DMSO-d6): δ 8.12 (s, 1H),
7.30 (m, 7H), 3.60 (s, 2H), 3.50 (s, 2H), 2.06 (s, 3H). MS (DCI/
NH3): m/e 263 (M + H)+.
In Vivo Eva lu a tion of AK In h ibitor s. Nociceptive paw
flinching in rats (Formalin test) was assessed 30 min follow-
ing an intraplantar injection of 5% formalin (50 µL) into the
right hindpaw. The antihyperalgesic effects of AK inhibitors
were assessed in the carrageenan-induced thermal hyper-
algesia pain model.20 Compounds were administered intra-
peritoneally 30 min before injection of 100 µL of 1% solution
of λ-carrageenan. The hyperalgesia to thermal stimulation was
determined 2 h after carrageenan injection.
Ack n ow led gm en t. We thank Dr. Tom Pagano,
Adam Huffman, David Whittern, and J an Waters for
excellent NMR support.
Refer en ces
4-Am in o-5-b en zylm et h yla m in om et h yl-6-iod op yr im i-
d in e (36a ). A solution of 35a (0.2 g, 0.76 mmol) in 40% HI (4
mL) was treated with NaI (0.57 g, 3.8 mmol) and stirred for
10 min at 70 °C. The precipitate was filtered, taken up in
aqueous NaHCO3, and extracted with EtOAc. The organic
phase was dried over Na2SO4 and evaporated to give the
desired product 36a (0.18 g, 67%). 1H NMR (DMSO-d6): δ 7.92
(s, 1H), 7.30 (m, 7H), 3.60 (s, 2H), 3.52 (s, 2H), 2.08 (s, 3H).
MS (DCI/NH3): m/e 355 (M + H)+.
4-Am in o-5-(b en zylm et h yla m in om et h yl)-6-[6-(4-m or -
p h olin yl)-3-p yr id in ylet h yn yl]p yr im id in e (37a ). Com-
pound 37a was prepared by using 36a and 2-morpholinyl-5-
ethynylpyridine 9 as described for the synthesis of 10a (yield
62%). 1H NMR (DMSO-d6): δ 8.35 (d, J ) 2.0 Hz, 1H), 8.28 (s,
1H), 7.68 (dd, J ) 7.5 and 2.0 Hz, 1H), 7.33-7.20 (m, 5H),
7.10 (broad s, 2H), 6.90 (d, J ) 7.5 Hz, 1H), 3.72 (m, 6H), 3.58
(m, 6H), 2.05 (s, 3H). MS (DCI/NH3): m/e 415 (M + H)+. Anal.
Calcd. (C24H26N6O‚0.2H2O): C, H, N.
4-Am in o-5-(1,2,3,4-tetr a h yd r oisoqu in olin -2-ylm eth yl)-
6-[6-(4-m or ph olin yl)-3-pyr idin yleth yn yl]pyr im idin e (37b).
Compound 37b was synthesized according to a scheme de-
scribed for the synthesis of 37a (yield 46%). 1H NMR (DMSO-
d6): δ 8.32 (d, J ) 2.0 Hz, 1H), 8.29 (s, 1H), 7.67 (dd, J ) 7.5
and 2.0 Hz, 1H), 7.13-7.03 (m, 4H), 6.87 (d, J ) 7.5 Hz, 1H),
3.83 (s, 2H), 3.70-3.51 (m, 10H), 2.88-2.70 (m, 4H). MS (DCI/
NH3): m/e 427 (M + H)+. Anal. Calcd. (C25H26N6O): C, H, N.
4-Am in o-5-[(2-ch lor oben zyl)m eth yla m in om eth yl)-6-[6-
(4-m or p h olin yl)-3-p yr id in ylet h yn yl]p yr im id in e (37c).
Compound 37c was synthesized according to a scheme de-
scribed for the synthesis of 37a (yield 61%). 1H NMR (DMSO-
d6): δ 8.40 (d, J ) 2.0 Hz, 1H), 8.25 (s, 1H), 7.73 (dd, J ) 7.5
and 2.0 Hz, 1H), 7.52 (m, 2H), 7.35 (m, 2H), 7.08 (broad s,
2H), 6.90 (d, J ) 7.5 Hz, 1H), 3.78 (s, 2H), 3.75-3.64 (m, 6H),
3.59 (t, J ) 4.5 Hz, 4H), 2.09 (s, 3H). MS (DCI/NH3): m/e 449
(M + H)+. Anal. Calcd. (C24H25ClN6O‚0.4H2O): C, H, N.
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