5-(3-Bromophenyl)-7-(6-morpholin-4-ylpyridin-3-yl)pyrido[2,3-d] pyrimidin-4-ylamine: Structure-activity relationships of 7-substituted heteroaryl analogs as non-nucleoside adenosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2005.03.023

4-Amino-5,7-disubstituted pyridopyrimidines are potent, non-nucleoside inhibitors of adenosine kinase (AK). We recently identified a potent, orally efficacious analog, 4 containing a 7-pyridylmorpholine substituted ring system as the key structural element of this template. In this report, we disclose the pharmacologic effects of five- and six-membered heterocyclic ring replacements for the pyridine ring in 4. These replacements were found to have interesting effects on in vivo efficacy and genotoxicity as well as in vitro potency. We discovered that the nitrogen in the heterocyclic ring at C(7) is important for the modulation of mutagenic side effects (Ames assay).

Full text of DOI:10.1016/j.bmc.2005.03.023

Bioorganic & Medicinal Chemistry 13 (2005) 3705–3720
5-(3-Bromophenyl)-7-(6-morpholin-4-ylpyridin-3-yl)pyrido-
[2,3-d]pyrimidin-4-ylamine: structure–activity relationships of
7-substituted heteroaryl analogs as non-nucleoside
adenosine kinase inhibitors
Mark A. Matulenko,a,* Chih-Hung Lee,a Meiqun Jiang,a Robin R. Frey,a
Marlon D. Cowart,a Erol K. Bayburt,a Stanley DiDomenico, Jr.,a Gregory A. Gfesser,a
Arthur Gomtsyan,a Guo Zhu Zheng,a Jeffery A. McKie,a,ꢀ Andrew O. Stewart,a
Haixia Yu,a Kathy L. Kohlhaas,a Karen M. Alexander,a Steve McGaraughty,a
Carol T. Wismer,a Joseph Mikusa,a Kennan C. Marsh,b Ronald D. Snyder,c,ꢁ
Marilyn S. Diehl,c Elizabeth A. Kowaluk,a Michael F. Jarvisa
and Shripad S. Bhagwata,§
aNeuroscience Research, Global Pharmaceutical Research, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA
bExploratory Kinetics and Analysis, Global Pharmaceutical Research, Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, IL 60064, USA
cGenetic Toxicology, Global Pharmaceutical Research, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA
Received 30 January 2005; revised 10 March 2005; accepted 11 March 2005
Available online 9 April 2005
Abstract—4-Amino-5,7-disubstituted pyridopyrimidines are potent, non-nucleoside inhibitors of adenosine kinase (AK). We
recently identified a potent, orally efficacious analog, 4 containing a 7-pyridylmorpholine substituted ring system as the key struc-
tural element of this template. In this report, we disclose the pharmacologic effects of five- and six-membered heterocyclic ring
replacements for the pyridine ring in 4. These replacements were found to have interesting effects on in vivo efficacy and genotoxicity
as well as in vitro potency. We discovered that the nitrogen in the heterocyclic ring at C(7) is important for the modulation of muta-
genic side effects (Ames assay).
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
activation of ADO receptor (P1) subtypes.1,2 Activation
of ADO receptors produces a variety of homeostatic
inhibitory cellular events that contribute to anti-noci-
ceptive and anti-inflammatory actions in vivo.3 Thera-
peutic exploitation of the beneficial effects of ADO
receptor activation has focused on the development of
receptor selective agonists.4 Direct acting ADO A1
receptor agonists for CNS indications, however, have
suffered from cardiovascular side effects such as hypo-
tension and bradycardia.1
During cellular stress, local intracellular concentrations
of adenosine (ADO) increase, which is followed by the
active transport of ADO out of the cell and subsequent
Keywords: Adenosine kinase; Inhibitor; Pyridopyrimidine.
*
Corresponding author. Tel.: +1 847 938 2460; fax: +1 847 937
9195; e-mail: mark.a.matulenko@abbott.com
ꢀ Present address: Signal Research Division, 5555 Oberlin Drive, San
Diego, CA 92121.
ꢁ Present address: Schering-Plough, 144 Route 94, POB32, Lafayette,
NJ 07848.
An alternative approach to direct A1 receptor activation
is to modulate ADO concentration by inhibition of
known ADO metabolic pathways including the inhibi-
tion of adenosine kinase (AK) and adenosine deaminase
§ Present address: Ambit Biosciences, 4215 Sorrento Valley Blvd., San
Diego, CA 92121.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.03.023

3706
M. A. Matulenko et al. / Bioorg. Med. Chem. 13 (2005) 3705–3720
sic efficacy in vivo (ED50 = 30 lmol/kg, ip). Further
Br
optimization resulted in analog 3 with a morpholine resi-
due located in the 40-position. This analog potently
inhibited AK (IC50 = 1.7 nM (cytosolic); 112 nM (intact
cell)) and, in addition, showed improved thermal hyper-
algesia efficacy in vivo (ED50 = 8 lmol/kg, ip), but
lacked oral activity (data not shown). Incorporation of
a morpholine substituted pyridine ring at C(7)23,28,29
resulted in the discovery of ABT-702 (4) as the first,
non-nucleoside, inhibitor of AK containing a 4-amino-
5-bromophenyl-7-(4-aminosubstituted pyridyl)pyrido[2,
3-d]pyrimidine scaffold.24 ABT-702 is orally active to
reduce pain and inflammation in animal models.28,29
Expansion of the morpholine residue to include other
amino groups revealed a significant effect on oral bio-
availability30 as well as locomotor side effects.31 ABT-
702, however, was found to be clastogenic in an in vitro
Chinese Hamster micronucleus assay.32 In the current
report, we wish to report new data associated with
changes in the heteroaromatic ring at the C(7) position.
Our initial goal was to observe the influence of five- and
six-membered heterocyclic ring replacements at C(7) on
in vitro potency, in vivo efficacy and pharmacokinetic
characteristics. Unexpectedly, we observed an influence
on mutagenic side effects (Ames assay) depending on
the placement of nitrogen atoms in the heteroaromatic
ring.
NH2
5-aryl
substitution
N
N
NH2
N
N
5
N
N
7
N
NMe2
1
X
R
X=CH; R=NMe2 (2)
X=CH; R=morpholin-4-yl (3)
7-heteroaryl
substitution
X=N; R=morpholin-4-yl
ABT-702 (4)
Chart 1. Development of HTS hit 1 to ABT-702 (4).
(ADA). AK is a ubiquitous enzyme that is primarily
responsible for the intracellular phosphorylation of
ADO to AMP.5 Inhibition of AK, as compared to inhi-
bition of ADA, was shown to produce higher levels of
endogenous ADO release from nervous tissue and en-
hanced anti-convulsant and anti-nociceptive effects in
animal models.6
Previous strategies from our laboratories7 and others8–22
have focused on the identification of nucleoside-like
inhibitors. Recently, high-throughput screening of Ab-
bott proprietary compounds identified a pteridine lead
structure, 1 (Chart 1). Optimization23,24 of this structure
led to the conclusion that potent compounds of this
series contained a substituted aryl ring at C(5) on the
pyridopyrimidine ring core. The discovery of 223
represented one of the early, potent, non-nucleoside
inhibitors25–27 of AK (IC50 = 5 nM (cytosolic); 167 nM
(intact cell)), however, it displayed weak antihyperalge-
2. Results
2.1. Chemistry
The synthesis of 423 and other pyridopyrimidine AK
inhibitors are shown in Scheme 1. Condensation of 2-
(3-bromobenzylidene)malononitrile33,34 with ketone, 5
provided pyridine ring, 7.35 Completion of the pyrido-
pyrimidine ring core followed one of the two methods.
In the first method, the pyridine ring was refluxed with
Br
Br
O
3
4
9
Z1=Z2=Z3=Z4=CH
NH2
N
Z1=N; Z2=Z3=Z4=CH
Z2=N; Z1=Z3=Z4=CH
HET
NC
b
a
N
N
Z3
Z1
10 Z1=Z2=N; Z3=Z4=CH
11 Z1=Z4=N; Z2=Z3=CH
12 Z1=Z3=N; Z2=Z4=CH
N
Z4
O
H2N
N
HET
Z2
N
N
O
O
5 1-(6-morpholin-4-yl-pyridin-3-yl)ethanone
6 1-(2-morpholin-4-yl-thiazol-5-yl)-ethanone
7 (from 5)
8 (from 6)
c
Br
Br
R
14 2-morpholin-4-yl-1,3-thiazol-5-yl
15 5-morpholin-4-ylthien-2-yl
16 2-morpholin-4-yl-1,3-thiazo-4-yl
17 1-methyl-2-morpholin-4-yl-1H-imidazol-4-yl
18 2-morpholin-4-yl-1,3-oxazol-5-yl
NH2
N
NC
N
d
N
S
H2N
N
O
N
N
N
13
Scheme 1. Reagents and conditions: (a) 2-(3-bromobenzylidene)malononitrile, NH4OAc, DCE, reflux; (b) formamide, 180 ꢁC; (c) CH(OEt)3,
NH4SO4, reflux; NH3, EtOH, rt; (d) o-DCB, reflux.

M. A. Matulenko et al. / Bioorg. Med. Chem. 13 (2005) 3705–3720
3707
formamide to form the desired pyridopyrimidine ring
analogs 3, 4, and 9–12.36 Some analogs, however, did
not cyclize efficiently with formamide and were thus sub-
jected to a slightly longer, two-step sequence. First, ami-
dine, 13 was constructed by the treatment of 8 with
triethylorthoformate followed by reaction with ammo-
nia. The amidine was then heated in o-dichlorobenzene
to effect the ring closure and provide the desired pyrido-
pyrimidines, 14–18.37 Syntheses of all required ketones
of structure type 5 and 6 are delineated in Schemes 2
and 3.
proceeded from 5-bromo-2-chloropyrimidine41 through
a similar synthetic sequence developed for ketone 24.
Synthesis of pyrazine ketone, 30, was achieved from hy-
droxy acid 28 by treatment with thionyl chloride fol-
lowed by the addition of methyl magnesium malonate
and decarboxylation to afford the intermediate chloro
ketone, 29. Formation of 29 was followed by morphol-
ine displacement and proceeded without incident to give
30 in 29% overall yield.
The synthesis of all required five-membered ring hetero-
cyclic ketones are shown in Scheme 3. Thiazole 34 was
obtained by the addition of morpholine to 2-bromo-
thiazole followed by metallation with n-butyllithium
and trapping with acetic anhydride in 39% overall yield.
Thiophene ketone, 35, was available from commercial
bromide, 33 by reaction with morpholine at 145 ꢁC in
a sealed tube to provide the desired product in 68%
yield.
Ketone 21 was obtained from dibromide 19 (Scheme 2)
by a palladium coupling with trimethylsilyl acetylene
utilizing Sonogashiraꢂs protocol38 to give the desired
acetylenic intermediate. The acetylene was hydrolyzed
under acidic conditions to give bromo ketone 20, which
was followed by protection as the dioxolane. The result-
ing bromide was reacted with morpholine in a palladium
catalyzed coupling39,40 and subsequently reacted under
acidic conditions to give ketone, 21. Ketone 24 was
available from dichloropyridazine 22. Palladium-medi-
ated coupling with tributyl(1-ethoxyvinyl)tin followed
by acidic hydrolysis gave intermediate chloro ketone
(60% yield), which was displaced with morpholine to
provide ketone, 24. Synthesis of pyrimidine ketone, 27,
S
S
N
a
Br
N
O
N
95%
31
32
b
41%
O
O
O
O
Br
S
c
S
X
a,b
c,d
Br
N
O
N
N
N
N
N
68%
Br
Br
N
73%
51%
19
33
O
20
21
34 X=N
35 X=CH
O
O
O
O
Cl
Br
HO
e,f
g
O
N
O
N
N
N
22
Cl
Cl
d
e,f
N
23
Cl
N
24
N
N
N
60%
98%
Br
N
O
O
O
O
40%
O
O
19%
S
36
37
38
O
N
N
N
h,i
j
O
O
N
26
N
26
Cl
N
27
51%
68%
N
O
g,h
N
O
NH2
N
N
16%
Me
O
39
HO2C
N
N
N
N
N
40
m
k,l
OH
Cl
N
30
82%
35%
O
28
O
29
i
O
CHO
N
O
Scheme 2. Reagents and conditions: (a) TMSCCH, PdCl2(PPh3)2,
CuI, CH3CN, Et3N, rt, 1 h, 91%; (b) Hg(OAc)2, H2SO4, H2O, acetone,
reflux, 1 h, 80%; (c) HOCH2CH2OH, p-TsOH, PhH, reflux, 3 h, 86%;
(d) morpholine, Pd2(dba)3, BINAP, NaOt-Bu, PhMe, reflux, 6 h; 4 M
HCl, THF, 59%; (e) tributyl(1-ethoxyvinyl)tin, Pd2(dba)3, P(2-furyl)3,
DMF, 80 ꢁC, 4 h; (f) 1 N HCl, acetone, rt, 2 h; (g) morpholine, EtOH,
reflux, 4 h; (h) tributyl(1-ethoxyvinyl)tin, PdCl2(PPh3)2, DMF, 70 ꢁC,
2 h, 60%; (i) 1 N HCl, acetone, rt, 18 h; reflux, 1 h, 85%; (j)
morpholine, sealed tube, 90 ꢁC, 2 h, 68%; (k) SOCl2, DMF, reflux,
16 h; (l) MgCl2, CH2(CO2Me)2, Et3N, PhMe, rt to 160 ꢁC; (m)
morpholine, DMSO, rt, 3 h.
40%
N
Br
41
42
Scheme 3. Reagents and conditions: (a) morpholine, sealed tube,
100 ꢁC, 3 d; (b) n-BuLi, THF, ꢀ78 ꢁC; Ac2O, ꢀ60 ꢁC, 1 h; (c) morp-
holine, sealed tube 145 ꢁC; (d) Br2, hydroxylamine hydrochloride,
Na2CO3, H2O; (e) morpholine-4-carbothioic acid amide, EtOH, reflux,
2 h, 99%; (f) H2SO4, EtOH, NaNO2, 50 ꢁC, 2 h, 19%; (g) morpholine,
H2O, reflux, 33%; (h) NaH, MeI, THF, rt, 1.5 h, 49%; (i) morpholine-
4-carboxylic acid amide, acetone, rt, 1 h; reflux, 1 h.

3708
M. A. Matulenko et al. / Bioorg. Med. Chem. 13 (2005) 3705–3720
Table 1. In vitro characterization of AK inhibitors 3, 4, 9–12, 14–18 in
cytosolic and intact cell assays
Formation of thiazole 38 (regioisomer of ketone 34) was
achieved from 2,3-butanedione by treatment with bro-
mine followed by hydroxylamine hydrochloride to give
unstable oxime, 37 in 40% yield. Cyclization with mor-
pholine-4-carbothioic acid amide42 in refluxing EtOH
and deprotection provided 38 in 19% yield.43 Com-
pound 40 was synthesized from known oxazole 3944
by, first, displacement and rearrangement with morpho-
line in 33% yield followed by methylation to give the de-
sired imidazole ketone in 49% yield. Oxazole 42 was
available from a cyclization of morpholine-4-carboxylic
acid amide45 and known46 bromo ketone 41 in refluxing
acetone in 46% yield.
R1
Br
NH2
R2
N
N
Ar
O
N
N
a
Compound
Ar
R1
R1
R1
R1
R1
R1
AK inhibition (IC50)
Cytosolic
Intact cell
112 61
3
2.2. Biology
2
2
0.8
0.5
R2
R2
R2
R2
R2
Compounds were evaluated in vitro for their ability to
inhibit cytosolic AK (AKcyt) as well as ADO phosphor-
ylation in intact cells (AKcell).28 Analgesic activity was
measured utilizing the carrageenan-induced thermal
hyperalgesia model in rats (thermal hyperalgesia).29,47
Compounds were also evaluated for mutagenicity in
an Ames miniscreen assay using the procedure as de-
scribed earlier48 and clastogenicity in a V79 Chinese
Hamster lung cell micronucleus assay.32
4
50
8
N
N
9
14 30
75 25
30 10
79 28
37 25
N
10
11
12
3
6
2
2
2
4
N
N
3. Discussion
N
N
From earlier studies,23,30 we observed that compounds
similar to the phenyl morpholine analog, 3, and pyridyl
morpholine, 4, display similar in vitro potency but pyr-
idyl analogs, like 4, show superior thermal hyperalgesia
efficacy in vivo (4: ED50 = 0.6 lmol/kg, ip; ED50 = 5 l
mol/kg, po). Two conclusions were drawn from these
earlier studies. First, the pyridine ring in 4 is critical
for bioavailability and oral activity. Second, in vitro po-
tency was not greatly influenced by changes at the
40-amino position on the pyridine ring; a fact that is con-
trary to the observation at C(5).24,49
4
N
S
R2
R2
R1
14
15
12
127 73
105 14
N
S
R1
R1
R1
R2
R2
R2
86 18
55 19
N
16
17
18
81
2
S
N
94 30
785b
Extension (Table 1) to other six-membered heteroaro-
matic rings (9–12) at C(7) showed that all were equipo-
tent (AKcyt) with the phenyl analog, 3, and the pyridine
compound, 4 in inhibiting AK. Inhibition of AKcell
showed the same trend as in the purified enzyme assay,
however, the absolute activity values were about 10
times less potent. The factors contributing to the lack
of cell penetration for these AK inhibitors are not well
understood23,24,30,31,49 and does not seem to be confined
to the 5,7-substituted pyrido[2,3-d]pyrimidine scaf-
fold.25,50 To expand the scope of the 7-heteroaryl com-
pounds, a series of five-membered ring analogs were
completed and tested.
N
Me
R1
O
R2
19
3
159 10
N
a Mean values for inhibitors (IC50 in nM) calculated from at least three
determinations standard error of the mean (SEM). Inhibition of
AK and ADO phosphorylation assays as described by Jarvis et al.28
b n = 1.
active (AKcyt IC50 = 86 nM) than the phenyl analog, 3.
Additionally, it appears that if the nitrogen atom is in
a favorable position within the five-membered ring,
other heteroatoms are tolerated. For example, if the sul-
fur atom in 14 is replaced with oxygen, the activity of
the oxazole analog 18 (AKcyt IC50 = 19 nM) was re-
tained. Thus, contrary to the six-membered ring hetero-
cycles, in vitro potency in five-membered ring
heterocycles seems to be influenced by both the position
of the morpholine ring and the placement of the hetero-
atoms within the ring.
While changes in the spatial orientation of the morpho-
line ring relative to the 7-aryl substituent are tolerated to
some degree (18 vs 4), clearly, some five-membered het-
erocyclic analogs (15–17) show reduced potency (AKcyt).
Additionally, the bioisosteres 14 (to 4) and 15 (to 3)
exhibit an interesting trend depending on nitrogen
position. Thiazole 14 is nearly equipotent (AKcyt
IC50 = 12 nM) with pyridine 4 but thiophene 15 is less

M. A. Matulenko et al. / Bioorg. Med. Chem. 13 (2005) 3705–3720
3709
Table 2. In vivo characterization of AK inhibitors 3, 4, 9–12, 14–16,
and 18
Table 4. Genetic toxicology data for AK inhibitors 3, 4, 9–12, and 14
Compound
Ames bacterial
reversion assaya
In vitro
micronucleus
(MN) assayb
Compound Thermal hyperalgesiaa, ED50
Formalina, ED50
b
b
3
8
10
10
4
0.6
8
NAc
Ad
NA
A
9
8
30
3
ꢀ
ꢀ
ꢀ
ꢀ
nd
ꢀ
ꢀ
+
nde
+
nd
nd
ꢀ
10
11
12
14
15
16
18
3
4
ꢀ
+
5
>30
>30
ndc
nd
nd
nd
9
ꢀ
ꢀ
ꢀ
ꢀ
+
20
3
10
11
12
14
ꢀ
nd
ꢀ
ꢀ
ꢀ
ꢀ
>10
25
>10
ꢀ
nd
a Ames miniscreen was performed at 1–10,000 lg compound/plate as
described.48,60 Positive responses are defined as at least 2-fold
increases over background colony counts for TA-98 and TA-100 and
at least 3-fold increases over background for TA-1535 and TA-1538.
b V79 Chinese hamster lung cell micronucleus assay was performed as
described earlier.32 Positive micronucleus responses are defined as at
least 3-fold increase in percent micronucleated cells relative to solvent
controls.
a Values represent compounds administered by ip injection.
b ED50 values (in lmol/kg) reported for compounds for which a
complete dose-response relationship was obtained with at least six
animals (rats) at each dose, p < 0.05.
c nd = not determined.
c NA = with no S-9 mix activation.
The effect on AK inhibition in intact cells with these
five-membered ring heterocyclic analogs was also more
pronounced compared to the six-membered ring counter-
parts. The thiazole 14, imidazole 17 and oxazole 18
were approximately 10 times less potent in AKcell com-
pared to their activity in the AKcyt assay. The thiazole
regioisomer, 16, and thiophene 15 analogs, however,
were nearly equipotent in both assays. These results
suggest that the presence of the nitrogen atom is not
enough to explain the increased cell penetration and that
the position of the heteroatom may also be a requisite
for potent AKcell inhibition.
d A = with S-9 mix activation.
e nd = not determined.
mutation assay (Ames assay)48 after an S-9 mix51 activa-
tion. The introduction of a nitrogen atom and its posi-
tion in the aromatic ring were found to play a key role
in the Ames toxicity. The 30-pyridine analog, 4, displays
no mutagenic activity after S-9 mix activation. If, how-
ever, the nitrogen is moved to the 20-position (e.g., 9),
mutagenicity is observed. It appears that a nitrogen
atom in the 30 position is critical to avoid mutagenic
activity. Diazines 10 and 12 contain a 30 nitrogen and
display no mutagenic activity even after S-9 mix activa-
tion. The only five-membered ring heterocycle that was
evaluated in this Ames assay was the 30-pyridyl bioiso-
stere, thiazole 14. It, also, displayed no mutagenic side
effects. To further expand on this observation, other
amino substituted 7-aryl and heteroaryl pyrido pyrimi-
dines were tested for their mutagenicity in the Ames
assay (Table 5). Again, the same trend was observed
as in the morpholine case. Dimethylamino phenyl ana-
log, 2 and 20-pyridyl compound, 44 were both found
to be mutagenic; while the heteroaryl, dimethylamino
analogs, 43, 45, and 46 (all with a 30-nitrogen) were
non-mutagenic. Less electron rich nitrogen analogs,
such as 47–49 and 51 also follow this same general trend.
The effect of the five- and six-membered heterocycle
modifications on in vivo efficacy is shown in Table 2.
Earlier studies showed that the C(7) modification of
phenyl compound, 3 to the pyridine analog, 4, had a
favorable impact on in vivo efficacy in the thermal
hyperalgesia model when administered by intraperito-
neal (ip) injection. This could be attributed to the 2-fold
increase in AKcell going from 3 to 4. Further heterocy-
clic modifications do not seem to provide any observable
increase in efficacy. In fact, several of these analogs dis-
play decreased activity (15, 16, and 18) after ip adminis-
tration. We suspected that the pharmacokinetics
associated with each analog might be responsible for
the observed trends in the in vivo efficacy. Table 3 shows
the intravenous PK profile for analogs 4, 10, and 12. The
decreased in vivo activity of compounds 10 and 12
(compared to 4) could be attributed with their respective
plasma clearance values (Table 3).
In view of the need for S-9 activation in order to estab-
lish mutagenic side effects of some of these analogs,
metabolism studies were carried out on 30-pyridyl ana-
log, 4 as well as phenyl derivative, 2 in an attempt to
identify metabolites that could account for the observed
toxicity. Results from these studies revealed metabolism
primarily at the amino portion (morpholine or dimeth-
ylamino) of the molecule, which was shown to be non-
mutagenic (e.g., 48, Table 5). No additional metabolites
were observed with 2 that could account for the mutage-
nicity. At this time, it is not clear how the 30-nitrogen
influences the parent molecule to eliminate the mutage-
nicity. Some explanations could involve an effect on
the conformation of the 7-heteroaryl ring relative to
the pyridopyrimidine core through some chelation
Evaluation of these non-nucleoside AK inhibitors for
mutagenicity (Table 4) revealed a clear signal for the
7-phenyl derivatives, such as 3, in the bacterial reverse
Table 3. Pharmacokinetic dataa for AK inhibitors 4, 10, and 12
Compound
T1/2
Vb
CLp
4
0.9
1.2
1.2
1.0
3.0
3.0
0.8
1.7
1.7
10
12
a IV dose in rat at 5 lmol/kg. Units: T1/2 (h); Vb (L/kg); CLp (L/h kg).

3710
M. A. Matulenko et al. / Bioorg. Med. Chem. 13 (2005) 3705–3720
Table 5. Genetic toxicology data for AK inhibitors 2 and 43–52
Br
NH2
N
N
N
Z3
Z2
R2
Z1
Z3
N
R1
c
Compound
R1
R2
Z1
Z2
Amesa
MNb
AK inhibition (IC50)
Cytosolic
Intact cell
2
Me
Me
Me
Me
Me
Me
Me
Me
Me
CHO
H
Me
Me
Me
Me
Me
CHO
CHO
CHO
H
CH
N
CH
CH
N
CH
CH
CH
CH
N
+
ꢀ
+
ꢀ
ꢀ
+
ꢀ
+
+
+
+
ꢀ
+
ꢀ
ꢀ
+
+
+
+
+
+
ꢀ
6
1
47
47
43
44
45
46
47
48
49
50
51
52
CH
N
3
73
48
N
1
N
CH
CH
CH
CH
CH
CH
CH
6
80
77
CH
N
CH
CH
CH
CH
CH
CH
2
2
68
CMe
CH
CH
CH
10
3
103
74
H
4
175
275
H
8
a Assay with S-9 mix activation.
b Assay without S-9 mix activation.
c Mean values for inhibitors (IC50 in nM) calculated from at least three determinations (standard error of the mean (SEM) 610%).
involving a nitrogen of the 7-heteroaryl substituent and
the 8-amino nitrogen of the pyridopyrimidine core with
some other ion present. The lack of genotoxicity could
also be the result of specific electronic factors attributed
to the electron deficient nature of the 30-substituted
heterocycle. Further expansion of this series will be
necessary in order to more fully understand these
observations.
genotoxicity. Placement of nitrogen in the 30 position
seems to eliminate mutagenic side effects although the
role of this substitution pattern and its effect on toxicity
is unclear. Clastogenetic effects did not seem to follow a
trend as observed in the Ames assay. The genotoxicity,
however, does not appear to be inherent to this series
or mechanism based.
As part of further toxicological evaluation, compounds
were also screened in an in vitro micronucleus (MN) as-
say.32 For the array of compounds in Tables 3 and 4,
clastogenicity was observed for both aryl and six-mem-
bered heteroaryl analogs, but not in all. The five-mem-
bered ring heterocycles, like 14, also do not seem to
offer an advantage in eliminating clastogenicity. The
conclusion from these studies is that there does not seem
to be a clear trend (as in the Ames assay) to predict clas-
togenic activity in this series. An important observation,
however, is that these clastogenic side effects do not ap-
pear related to the mechanism of AK inhibition.
5. Experimental
5.1. General procedures
General experimental procedures and techniques are re-
ported elsewhere.52
5.2. Chemical procedures
5.2.1. 1-[6-(4-Morpholinyl)-3-pyridinyl]ethanone (5). A
solution of MgCl2 (20.4 g, 0.214 mol) in toluene
(220 mL) at rt was treated with Et3N (101 mL,
0.722 mol) followed by dimethyl malonate (41.2 mL,
0.361 mol). After 1 h, 6-chloronicotinoyl chloride
(52.8 g, 0.300 mol) was added as a solution in toluene
(40 mL) and stirring continued for 20 min. The reaction
was quenched with concentrated HCl and H2O. The
aqueous layer was extracted with Et2O, the organic
phases combined, dried, and concentrated. The resulting
white solid was taken up in DMSO (350 mL) and H2O
(20 mL) and the reaction heated to 150 ꢁC for 2 h. The
mixture was cooled to rt, H2O added, and the solid fil-
tered and dried to give 40.2 g (78%) of 1-(6-chloro-3-
4. Conclusion
During the course of these studies, we were able to iden-
tify several 7-heteroaryl analogs that potently inhibited
AK. These new compounds had similar in vitro profiles,
however, some derivatives displayed lowered in vivo effi-
cacy (ip), compared to 4, to reduce nociception in an
animal model of inflammatory hyperalgesia. Initial
pharmacokinetic analysis seems to suggest that these
differences (of 10 and 12, for example) may be explained
by the increased clearance values in rat. Substitution of
the heterocyclic 7-substituent was found to influence
1
pyridinyl)ethone as a white solid. H NMR (300 MHz,
CDCl3) d 8.94 (dd, 1H, J = 2.5, 0.7), 8.20 (dd, 1H,
J = 8.4, 2.6), 7.45 (dd, 1H, J = 8.1, 0.7), 2.62 (s, 3H).

M. A. Matulenko et al. / Bioorg. Med. Chem. 13 (2005) 3705–3720
3711
13C NMR (75 MHz, CDCl3) d 195.3, 155.6, 150.1, 138.0,
131.1, 124.5, 26.6. MS (DCI/NH3) m/z 173 (M+NH4)+,
156 (M+H)+.
CDCl3) d 7.97 (d, 2H, J = 8.8), 7.73 (dd, 1H, J = 1.7,
1.7), 7.61 (m, 2H), 7.39 (dd, 1H, J = 7.8, 7.8), 7.10 (s,
1H), 6.96 (d, 2H, J = 9.2), 5.30 (br s, 2H), 3.88 (m,
4H), 3.28 (m, 4H).
A suspension of 1-(6-chloro-3-pyridinyl)ethone (40.0 g,
0.257 mol) in EtOH (300 mL) was treated with morpho-
line (47.1 mL, 0.540 mol) and the mixture heated to re-
flux for 4 h. Excess EtOH was removed under reduced
pressure and the residue taken up in H2O and extracted
with CH2Cl2. The organic layers were combined,
washed with 1 N HCl, dried, and concentrated to pro-
Following the procedure for 4 substituting 2-amino-4-
(3-bromophenyl)-6-(4-morpholin-4-ylphenyl)nicotino-
nitrile for 7 and purification by silica gel chromatogra-
phy (elution with 15% MeOH–CH2Cl2) to provide 3
1
(36%) as a yellow solid. H NMR (300 MHz, CDCl3)
d 8.73 (s, 1H), 8.26 (d, 2H, J = 9.2), 7.72 (ddd, 1H,
J = 7.1, 2.1, 2.1), 7.68 (m, 1H), 7.61 (s, 1H), 7.44 (m,
2H), 6.98 (d, 2H, J = 8.8), 3.88 (m, 4H), 3.31 (m, 4H).
13C NMR (75 MHz, CDCl3) d 161.9, 160.1, 159.8,
158.1, 152.6, 147.4, 140.6, 132.0, 131.3, 130.9, 128.8,
127.9, 127.1, 122.2, 119.4, 114.1, 104.4, 65.9, 47.2.
HRMS calcd for C23H2079BrN5O: 462.0929; observed:
462.0935. Anal. Calcd for C23H20BrN5O: C, 59.75; H,
4.36; N, 15.14. Found: C, 59.44; H, 4.21; N, 14.98.
1
vide 36.0 g (68%) of 5 as a light tan solid. H NMR
(300 MHz, CDCl3) d 8.75 (d, 1H, J = 2.6), 8.03 (dd,
1H, J = 9.2, 2.6), 6.61 (d, 1H, J = 9.2), 3.80 (m, 4H),
3.67 (m, 4H), 2.50 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 195.1, 160.4, 150.5, 137.1, 123.0, 105.3, 66.4, 44.8,
25.8. MS (DCI/NH3) m/z 207 (M+H)+.
5.2.2. 2-Amino-4-(3-bromophenyl)-6-[2-(4-morpholinyl)-
5-pyridyl]nicotinonitrile (7). A suspension of 5 (15.0 g,
72.7 mmol), 2-(3-bromobenzylidene)malononitrile33,34
(20.3 g, 87.1 mmol) and ammonium acetate (29.7 g,
385 mmol) in 1,2-dichloroethane (150 mL) was heated
to reflux for 10 h. The mixture was cooled, filtered,
washed with H2O, and dried to provide 20.3 g (64%)
of 7 as a yellow solid. This material was used without
further purification in the next step. 1H NMR
(300 MHz, DMSO-d6) d 8.94 (d, 1H, J = 2.0), 8.29
(dd, 1H, J = 9.1, 2.7), 7.86 (dd, 1H, J = 1.7, 1.7), 7.71
(m, 2H), 7.51 (dd, 1H, J = 7.8, 7.8), 7.27 (s, 1H), 6.94
(m, 3H), 3.71 (m, 4H), 3.59 (m, 4H). 13C NMR
(100 MHz, DMSO-d6) d 160.7, 159.4, 157.2, 152.7,
147.6, 139.3, 136.2, 132.2, 130.9, 130.7, 127.4, 122.2,
121.8, 117.0, 107.7, 106.1, 85.2, 65.9, 44.7. MS (DCI/
NH3) m/z 436/438 (M+H)+.
5.2.5. 1-(5-Bromo-2-pyridinyl)ethanone (20). A solution
of 2,5-dibromopyridine (20.0 g, 84.4 mmol) and trimeth-
ylsilyl acetylene (8.84 g, 90.0 mmol) in Et3N/CH3CN
(1:1, 150 mL) at rt was treated with dichlorobis(triphen-
ylphosphine)palladium(II)
(PdCl2(PPh3)2;
1.19 g,
1.70 mmol) and CuI (320 mg, 1.70 mmol). After 1 h,
the mixture was concentrated and the residue purified
by silica gel chromatography (elution with 50%
EtOAc–hexanes) to provide 19.5 g (91%) of 5-bromo-
2-[(trimethylsilyl)ethynyl]pyridine as a light orange
1
solid. H NMR (400 MHz, DMSO-d6) d 8.66 (dd, 1H,
J = 2.4, 0.6), 8.03 (dd, 1H, J = 8.3, 2.4), 7.48 (dd, 1H,
J = 8.2, 0.7), 0.23 (s, 9H). 13C NMR (100 MHz,
DMSO-d6) d 150.7, 140.4, 139.2, 128.6, 120.2, 103.2,
95.4, ꢀ0.6. MS (DCI/NH3) m/z 254/256 (M+H)+.
5.2.3. 5-(3-Bromophenyl)-7-[6-(4-morpholinyl)-3-pyridin-
yl]pyrido[2,3-d]pyrimidin-4-ylamine (4). A suspension of
7 (15.0 g, 34.4 mmol) in formamide (200 mL) was heated
to 180 ꢁC for 10 h. The reaction mixture was cooled,
H2O added, and filtered. The solid was purified by silica
gel chromatography (first column elution with 10%
MeOH–CH2Cl2; second column elution with 5% H2O–
CH3CN) to provide 6 g (38%) of 4 as a yellow solid.
1H NMR (300 MHz, DMSO-d6) d 9.08 (d, 1H,
J = 2.4), 8.53 (s, 1H), 8.47 (dd, 1H, J = 9.2, 2.4), 7.85
(m, 2H), 7.78 (m, 1H), 7.56 (m, 2H), 6.98 (d, 1H,
J = 9.1), 3.72 (m, 4H), 3.62 (m, 4H). 13C NMR
(100 MHz, DMSO-d6) d 161.9, 159.7, 159.6, 158.8,
158.1, 148.0, 147.6, 140.4, 136.5, 132.0, 131.3, 130.9,
127.9, 122.2, 122.1, 119.1, 106.3, 104.5, 65.9, 44.7.
HRMS calcd for C22H2079BrN6O: 463.0882; observed:
463.0888. Anal. Calcd for C22H19BrN6OÆ0.5 H2NC(O)H:
C, 55.62; H, 4.25; N, 18.74. Found: C, 55.43; H, 4.27; N,
18.76.
A solution of 5-bromo-2-[(trimethylsilyl)ethynyl]pyri-
dine (10.2 g, 40.1 mmol) in acetone/H2O (8:1, 150 mL)
at rt was treated with mercuric acetate (17.9 g,
42.0 mmol). After 15 min, the reaction was treated with
3 M H2SO4 (40 mL) and heated to reflux for 1 h. The
mixture was cooled, neutralized with 2 M NaOH, and
extracted with EtOAc. The organic layers were com-
bined, dried (Na2SO4), concentrated, and the residue
purified by silica gel chromatography (elution with
50% EtOAc–hexanes) to provide 5.90 g (80%) of 20 as
1
a light green solid. H NMR (400 MHz, DMSO-d6) d
8.86 (d, 1H, J = 1.9), 8.26 (dd, 1H, J = 8.6, 2.5), 7.88
(d, 1H, J = 8.6), 2.63 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) d 198.7, 151.8, 150.2, 140.4, 125.2, 123.2,
25.8. MS (DCI/NH3) m/z 200/202 (M+H)+.
5.2.6. 1-[5-(4-Morpholinyl)-2-pyridinyl]ethanone (21). A
solution of 20 (2.50 g, 13.6 mmol) and ethylene glycol
(3.72 g, 60.0 mmol) in benzene (100 mL) was treated
with a catalytic amount of p-toluenesulfonic acid, fitted
with a Dean–Stark condenser and the reaction heated to
reflux for 3 h. The mixture was cooled, washed with
aqueous NaHCO3, dried (MgSO4), and concentrated.
The residue was purified by silica gel chromatography
(elution with 50% EtOAc–hexanes) to give 2.47 g of 5-
bromo-2-(2-methyl-1,3-dioxolan-2-yl)pyridine (86%) as
5.2.4. 5-(3-Bromophenyl)-7-[4-(4-morpholinyl)phenyl]pyr-
ido[2,3-d]pyrimidin-4-ylamine (3). Following the proce-
dure for 7 substituting 1-[4-(4-morpholinyl)phenyl]-
ethanone (Aldrich) for 5 and purification by silica
gel chromatography (elution with 15% MeOH–CH2Cl2)
to give 2-amino-4-(3-bromophenyl)-6-(4-morpholin-4-
ylphenyl)nicotinonitrile (14%). 1H NMR (300 MHz,
1
a white solid. H NMR (400 MHz, DMSO-d6) d 8.69

3712
M. A. Matulenko et al. / Bioorg. Med. Chem. 13 (2005) 3705–3720
(dd, 1H, J = 2.2, 0.7), 8.06 (dd, 1H, J = 8.3, 2.5), 7.51
(dd, 1H, J = 8.3, 0.6), 4.03 (m, 2H), 3.85 (m, 2H), 1.63
(s, 3H). 13C NMR (100 MHz, DMSO-d6) d 159.3,
149.6, 139.3, 121.3, 119.6, 107.7, 64.6, 24.4. MS (DCI/
NH3) m/z 244/246 (M+H)+.
(Na2SO4) and concentrated. The residue was purified
by silica gel chromatography (elution with 20%
EtOAc–hexanes) to provide 4.71 g (60%) of 23 as a pale
yellow solid. 1H NMR (300 MHz, CDCl3) d 8.11 (d, 1H,
J = 8.8), 7.67 (d, 1H, J = 8.8), 2.88 (s, 3H). MS (DCI/
NH3) m/z 174 (M+NH4)+, 157 (M+H)+.
A solution of 5-bromo-2-(2-methyl-1,3-dioxolan-2-yl)-
pyridine (3.80 g, 17.9 mmol), morpholine (2.80 g,
32.0 mmol), sodium tert-butoxide (3.46 g, 36.0 mmol),
Pd2(dba)3 (275 mg, 0.300 mmol), and BINAP (373 mg,
0.599 mmol) in toluene (20 mL) was heated to reflux
for 6 h. The reaction mixture was cooled, poured into
aqueous NaHCO3 and extracted with CH2Cl2, and con-
centrated. The residue was taken up in THF (100 mL)
and treated with 4 M HCl (10 mL) and stirred for 2 h
at rt. The mixture was neutralized with 2 M NaOH
and extracted with CH2Cl2. The organic layers were
combined, concentrated, and the residue purified by sil-
5.2.9. 1-[6-(4-Morpholinyl)-3-pyridazinyl]ethanone (24).
Following the procedure for compound 5 substituting
23 for 1-(6-chloro-3-pyridinyl)ethone to give 24 in 98%
yield. 1H NMR (300 MHz, CDCl3) d 7.92 (d, 1H,
J = 9.6), 6.90 (d, 1H, J = 9.5), 3.86 (m, 4H), 3.80 (m,
4H), 2.78 (s, 3H). MS (ESI) m/z 208 (M+H)+.
5.2.10. 5-(3-Bromophenyl)-7-[6-(4-morpholinyl)-3-pyrid-
azinyl]pyrido[2,3-d]pyrimidin-4-ylamine (10). Following
the procedure for compound 7 substituting 24 for 5 to
afford 2-amino-4-(3-bromophenyl)-6-[6-(4-morpholin-
1
ica gel chromatography to provide 21 in 59% yield. H
yl)-3-pyridazinyl]nicotinonitrile (45%) as a brown
1
NMR (400 MHz, CDCl3) d 8.27 (d, 1H, J = 2.7), 7.94
(d, 1H, J = 8.9), 7.17 (dd, 1H, J = 8.9, 2.7), 3.87 (m,
4H), 3.34 (m, 4H), 2.64 (s, 3H). 13C NMR (100 MHz,
CDCl3) d 198.3, 148.4, 144.4, 134.8, 122.5, 119.6, 66.0,
46.7, 25.1. MS (DCI/NH3) m/z 207 (M+H)+.
powder. H NMR (300 MHz, CDCl3) d 8.26 (d, 1H,
J = 9.5), 8.10 (s, 1H), 7.82 (dd, 1H, J = 1.9, 1.9), 7.64
(m, 2H), 7.39 (dd, 1H, J = 7.7, 7.7), 6.98 (d, 1H,
J = 9.6), 5.33 (br s, 2H), 3.88 (m, 4H), 3.78 (m, 4H).
MS (DCI/NH3) m/z 437/439 (M+H)+.
5.2.7. 5-(3-Bromophenyl)-7-[5-(4-morpholinyl)-2-pyridin-
yl]pyrido[2,3-d]pyrimidin-4-ylamine (9). Following the
procedure of compound 7 substituting 21 for 5 provided
2-amino-4-(3-bromophenyl)-6-[5-(4-morpholinyl)-2-pyr-
idyl]nicotinonitrile in 71% yield. Material was used di-
rectly in the next reaction.
Following the procedure for 4 substituting 2-amino-4-
(3-bromophenyl)-6-[6-(4-morpholinyl)-3-pyridazinyl]-
nicotinonitrile for 7 to provide 10 (79%) as a brown
powder. 1H NMR (300 MHz, DMSO-d6) d 8.59 (s,
1H), 8.41 (d, 1H, J = 9.9), 8.26 (s, 1H), 7.85 (s, 1H),
7.82 (m, 1H), 7.53 (m, 3H), 3.75 (m, 4H), 3.73 (m,
4H). HRMS calcd for C21H18BrN7O: 464.0829; ob-
served: 464.0829. Portion of this sample converted to
the HCl salt for biological testing. Anal. Calcd for
C21H18BrN7OÆ2.0 HCl: C, 47.92; H, 3.77; N, 18.63.
Found: C, 47.77; H, 3.76; N, 18.97.
Following the procedure for compound 4 substituting
2-amino-4-(3-bromophenyl)-6-[5-(4-morpholinyl)-2-pyrid-
yl]nicotinonitrile for 5 and purification by silica gel chro-
matography (gradient elution with CH2Cl2 to 10%
MeOH–CH2Cl2) to provide 1.22 g (52%) of 9. This
material was converted into the HCl salt. 1H NMR
(400 MHz, CDCl3) d 10.09 (br d, 1H), 8.82 (s, 1H),
8.35 (d, 1H, J = 2.5), 8.30 (d, 1H, J = 8.9), 8.24 (s,
1H), 7.82 (s, 1H), 7.77 (d, 1H, J = 8.0), 7.56 (m, 3H),
7.34 (s, 1H), 3.73 (m, 4H), 3.38 (m, 4H). 13C NMR
(100 MHz, DMSO-d6) d 163.5, 158.5, 152.1, 150.0,
149.1, 148.1, 139.0, 137.8, 134.1, 133.1, 131.5, 131.5,
128.1, 124.1, 122.6, 122.2, 122.1, 104.1, 65.7, 46.3. MS
(DCI/NH3) m/z 207 (M+H)+. Anal. Calcd for
C22H19BrN6OÆ3 HCl: C, 46.14; H, 3.87; N, 14.67.
Found: C, 46.34; H, 4.19; N, 14.30.
5.2.11. 1-(2-Chloro-5-pyrimidinyl)ethanone (26). A solu-
tion of 2-chloro-5-bromopyrimidine41 (1.93 g, 10.0
mmol) and tributyl(1-ethoxyvinyl)tin (3.71 mL, 11.0
mmol) in DMF (20 mL) at rt was added PdCl2(PPh3)2
(140 mg) and the mixture heated to 70 ꢁC for 2 h. The
reaction was cooled and aqueous KF added and stirring
continued overnight. The mixture was poured into H2O
and extracted with Et2O. The organic phases were com-
bined, concentrated, and purified by silica gel chroma-
tography (elution with 50% hexane–CH2Cl2) to
provide 2-chloro-5-(1-ethoxyvinyl)pyrimidine in 60%
1
yield. H NMR (300 MHz, CDCl3) d 8.81 (s, 2H), 4.73
(d, 1H, J = 3.3), 4.40 (d, 1H, J = 3.3), 3.95 (q, 2H,
J = 6.9), 1.43 (t, 3H, J = 7.0).
5.2.8. 1-(6-Chloro-3-pyridazinyl)ethanone (23). A solu-
tion of ethoxyvinyltin (21.5 g, 59.6 mmol), tris(dibenzyl-
ideneacetone)dipalladium(0) (Pd2(dba)3, 670 mg, 0.73
mmol), tri-2-furylphosphine (670 mg, 2.85 mmol), and
3,6-dichloropyridazine (7.5 g, 50.3 mmol) was stirred in
DMF (60 mL) at 80 ꢁC for 4 h. Reaction mixture was
poured into 100 mL of aqueous KF and Et2O
(200 mL) and stirred for 30 min. The solids were filtered
through a pad of Celiteꢂ and the filtrate concentrated.
The residue was taken up in acetone (270 mL) and trea-
ted with 1 N HCl (50 mL) at rt. After 2 h, the mixture
was transferred to a separatory funnel and extracted
with Et2O (·3). The organic phases were combined,
washed with aqueous NaHCO3, H2O, brine; dried
A
solution of 2-chloro-5-(1-ethoxyvinyl)pyrimidine
(1.10 g, 5.96 mmol) in acetone (20 mL) was added 1 N
HCl (5 mL) and the mixture stirred at rt for 18 h then
heated to reflux for 1 h. The reaction was cooled, ex-
tracted with Et2O, dried, and concentrated to give 26
1
(85%) as a white solid. H NMR (300 MHz, CDCl3) d
9.11 (s, 2H), 2.66 (s, 3H).
5.2.12. 1-[2-(4-Morpholinyl)-5-pyrimidinyl]ethanone (27).
A mixture of ketone 26 and morpholine was combined
into a sealed tube and heated to 90 ꢁC for 2 h. The

M. A. Matulenko et al. / Bioorg. Med. Chem. 13 (2005) 3705–3720
3713
mixture was cooled, concentrated, and the residue puri-
fied by silica gel chromatography (elution with CH2Cl2)
to provide 27 in 68% yield. 1H NMR (300 MHz, CDCl3)
d 8.84 (s, 2H), 3.95 (m, 4H), 3.77 (m, 4H), 2.48 (s, 3H).
MS (DCI/NH3) m/z 208 (M+H)+.
(d, 1H, J = 1.5), 3.83 (m, 4H), 3.77 (m, 4H), 2.60 (s,
3H). MS (DCI/NH3) m/z 208 (M+H)+.
5.2.15. 5-(3-Bromophenyl)-7-[5-(4-morpholinyl)-2-pyrazin-
yl]pyrido[2,3-d]pyrimidin-4-ylamine (12). Following the
procedure for compound 7 substituting 30 for 5 pro-
vided 2-amino-4-(3-bromophenyl)-6-[5-(4-morpholinyl)-
2-pyrazinyl]nicotinonitrile in 61% yield. 1H NMR
(300 MHz, DMSO-d6) d 8.97 (d, 1H, J = 1.1), 8.39 (d,
1H, J = 1.4), 7.82 (dd, 1H, J = 2.0, 2.0), 7.75 (m, 1H),
7.63 (m, 1H), 7.52 (dd, 1H, J = 8.1, 8.1), 7.41 (s, 1H),
7.07 (br s, 2H), 3.72 (m, 4H), 3.68 (m, 4H). MS (DCI/
NH3) m/z 437/439 (M+H)+.
5.2.13. 5-(3-Bromophenyl)-7-[2-(4-morpholinyl)-5-pyri-
midinyl]pyrido[2,3-d]pyrimidin-4-ylamine (11). Following
the procedure for compound 7 substituting 27 for 5 to
provide 2-amino-4-(3-bromophenyl)-6-[2-(4-morpholin-
1
yl)-5-pyrimidinyl]nicotinonitrile in 38% yield. H NMR
(300 MHz, DMSO-d6) d 9.11 (s, 2H), 7.86 (m, 1H),
7.70 (m, 2H), 7.52 (dd, 1H, J = 7.8), 7.30 (s, 1H), 7.02
(br s, 2H), 3.82 (m, 4H), 3.70 (m, 4H). MS (DCI/NH3)
m/z 437/439 (M+H)+.
Following the procedure for compound 4 substituting 2-
amino-4-(3-bromophenyl)-6-[5-(4-morpholinyl)-2-pyr-
azinyl]nicotinonitrile for 7 and purification by silica gel
chromatography (elution with 10% MeOH–CH2Cl2)
provided 12. This material was converted to the HCl
salt. 1H NMR (300 MHz, DMSO-d6) d 9.19 (d, 1H,
J = 1.1), 8.56 (s, 1H), 8.42 (d, 1H, J = 1.1), 7.98 (s,
1H), 7.81 (m, 2H), 7.57 (m, 2H), 3.73 (m, 8H). MS
(DCI/NH3) m/z 464/466 (M+H)+. Anal. Calcd for
C21H18BrN7OÆ1.47 HCl: C, 48.70; H, 3.79; N, 18.93.
Found: C, 49.07; H, 3.82; N, 18.55.
Following the procedure for compound 4 substituting 2-
amino-4-(3-bromophenyl)-6-[2-(4-morpholinyl)-5-pyri-
midinyl]nicotinonitrile for 7 and purification by silica gel
chromatography (elution with 2% MeOH–CH2Cl2) to
provide 11 in 10% yield. This material was converted
to the HCl salt. 1H NMR (300 MHz, DMSO-d6) d
9.32 (s, 1H), 8.88 (s, 1H), 8.19 (s, 1H), 7.94 (m, 1H),
7.82 (m, 1H), 7.60 (m, 3H), 3.88 (m, 4H), 3.70 (m,
4H). 13C NMR (100 MHz, DMSO-d6/DCl) d 162.8,
160.6, 158.1, 157.6, 151.5, 149.5, 148.6, 137.0, 132.5,
131.3, 130.8, 127.8, 122.0, 121.0, 117.2, 102.7, 65.5,
43.6. MS (DCI/NH3) m/z 464/466 (M+H)+. Anal. Calcd
for C21H18BrN7OÆ1.2 HCl: C, 49.64; H, 3.81; N, 19.30.
Found: C, 49.69; H, 3.76; N, 18.90.
5.2.16. 1-[2-(4-Morpholinyl)-1,3-thiazol-5-yl]ethanone (34).
2-Bromothiazole (12.63 g, 77.00 mmol) in morpholine
(30.0 mL) was sealed into a tube and heated to 100 ꢁC
for 3 days. The mixture was cooled, partitioned between
H2O and CH2Cl2, the layers separated, and the organic
phase dried (Na2SO4) and concentrated to afford 12.5 g
(95%) of 2-morpholinothiazole53 as a brown oil. Mate-
5.2.14. 1-[5-(4-Morpholinyl)-2-pyrazinyl]ethanone (30). A
solution of 5-hydroxypyrazine-2-carboxylic acid (Lon-
za; 6.70 g, 47.8 mmol) in thionyl chloride (40 mL) was
treated with DMF (0.4 mL) and the reaction warmed
to reflux for 16 h. The volatiles were removed under re-
duced pressure and the residue was used directly in the
next reaction.
1
rial was used directly in the next reaction. H NMR
(400 MHz, DMSO-d6) d 7.19 (d, 1H, J = 3.7), 6.86 (d,
1H, J = 3.7), 3.71 (m, 4H), 3.37 (m, 4H). 13C NMR
(100 MHz, DMSO-d6) d 172.0, 139.7, 108.5, 65.7, 48.7.
MS (DCI/NH3) m/z 171 (M+H)+.
A
suspension of magnesium chloride (2.67 g,
A solution of 2-morpholinothiazole (7.20 g, 42.3 mmol)
in THF (80 mL) at ꢀ78 ꢁC was treated with n-BuLi
(2 M in hexanes, 23.5 mL). After 30 min, the reaction
mixture was transferred via cannula to a solution of ace-
tic anhydride (10 mL) in THF (50 mL) at ꢀ60 ꢁC and
stirred for 1 h. The slurry was then warmed to room
temperature for an additional 30 min, quenched with
saturated sodium bicarbonate, and extracted with
Et2O. The organic phase was dried (Na2SO4), concen-
trated, and purified by silica gel chromatography (elu-
tion with 50% CH2Cl2–EtOAc) to provide 3.70 g
(41%) of 34.54 1H NMR (400 MHz, DMSO-d6) d 8.09
(s, 1H), 3.71 (m, 4H), 3.53 (m, 4H), 2.39 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) d 188.7, 174.8, 149.2,
128.5, 65.3, 48.0, 25.7. MS (DCI/NH3) m/z 213 (M+H)+.
28.0 mmol), dimethyl malonate (7.70 g, 58.3 mmol),
and Et3N (11.72 g, 115.8 mmol) in toluene (70 mL)
was stirred for 1 h at rt. To this suspension was slowly
added the acid chloride in 50 mL of toluene and stirring
continued for 2 h. The reaction was quenched with
H2O, the acidity adjusted to pH 5 and the mixture ex-
tracted with CH2Cl2. The extracts were combined, con-
centrated, dissolved in DMSO (125 mL), H2O (3 mL),
and the mixture heated to 160 ꢁC for 90 min. The reac-
tion was cooled and portioned between H2O and
CH2Cl2. The organic phase was dried (Na2SO4), con-
centrated, and purified by silica gel chromatography
to provide 29 (35%), which was used directly in the next
reaction.
A solution of 29 (2.65 g, 16.9 mmol) in DMSO (30 mL)
was treated with morpholine (3.48 g, 39.9 mmol) and
stirred at rt for 3 h. The mixture was poured into H2O
(100 mL) and extracted with CH2Cl2. The organic layers
were combined, dried, concentrated, and the residue
purified by silica gel chromatography (elution with
50% hexanes–EtOAc) to give 2.87 g (82%) of 30. 1H
NMR (CDCl3, 300 MHz) d 8.79 (d, 1H, J = 1.5), 8.05
5.2.17. 2-Amino-4-(3-bromophenyl)-6-[2-(4-morpholinyl)-
1,3-thiazol-5-yl]nicotinonitrile (8). A slurry of 34 (4.91 g,
23.1 mmol) and ammonium acetate (9.75 g, 127 mmol)
in 1,2-dichloroethane (50 mL) was treated with 2-(3-bro-
mobenzylidene)malononitrile33,34 (10.78 g, 46.3 mmol)
and the mixture heated to reflux overnight. The solution
was cooled to room temperature, hexanes (50 mL)
added, and stirring continued for 3 h. The solid was

3714
M. A. Matulenko et al. / Bioorg. Med. Chem. 13 (2005) 3705–3720
collected by filtration, washed with methanol, and dried
to provide 4.57 g (45%) of 8 as an orange solid. 1H
NMR (300 MHz, DMSO-d6) d 8.14 (s, 1H), 7.81 (m,
1H), 7.72 (m, 1H), 7.63 (m, 1H), 7.50 (dd, 1H, J = 7.8,
7.8), 7.21 (s, 1H), 6.92 (br s, 2H), 3.73 (m, 4H), 3.49
(m, 4H). 13C NMR (100 MHz, DMSO-d6/DCl) d
170.3, 160.5, 153.1, 151.4, 138.8, 133.3, 132.8, 131.1,
131.0, 127.7, 125.8, 122.1, 116.7, 107.6, 86.1, 65.1,
49.2. MS (DCI/NH3) m/z 442/444 (M+H)+.
105.5, 83.2, 65.3, 49.6. MS (DCI/NH3) m/z 441/443
(M+H)+.
Following the procedure for compound 13 substituting
2-amino-4-(3-bromophenyl)-6-(5-morpholin-4-ylthiophen-
2-yl)nicotinonitrile for 8 provided N-[4-(3-bromophen-
yl)-3-cyano-6-(5-morpholin-4-ylthiophen-2-yl)pyridin-2-
yl]formamidine in 93% yield as an orange solid. MS
(DCI/NH3) m/z 468/470 (M+H)+.
5.2.18. N-[4-(3-Bromophenyl)-3-cyano-6-(2-morpholin-4-
yl-thiazol-5-yl)-pyridin-2-yl]-formamidine (13). A solu-
tion of 8 (1.50 g, 3.39 mmol) and triethylorthoformate
(34 mL) with a catalytic amount of ammonium sulfate
was heated to reflux for 6 h. The dark mixture was
cooled, ammonia in EtOH (2 M, 70 mL) added, and
the mixture stirred overnight. The solid product was
collected by filtration and dried to provide 1.17 g
(73%) of 13 as a yellow solid. MS (DCI/NH3) m/z 469/
471 (M+H)+.
Following the procedure for compound 14 substituting
N-[4-(3-bromophenyl)-3-cyano-6-(5-morpholin-4-ylthio-
phen-2-yl)pyridin-2-yl]formamidine for 13 provided 15
in 12% yield as an orange solid. mp: 265 ꢁC (dec); H
NMR (300 MHz, DMSO-d6) d 8.45 (s, 1H), 7.90 (d,
1H, J = 4.4), 7.77 (m, 2H), 7.69 (s, 1H), 7.52 (m, 2H),
6.29 (d, 1H, J = 4.4), 3.76 (m, 4H), 3.26 (m, 4H). 13C
1
NMR (100 MHz, DMSO-d6/DCl)
d 167.0, 163.2,
158.2, 152.0, 151.1, 147.4, 138.4, 135.7, 133.7, 132.3,
132.0, 128.6, 124.5, 123.3, 121.0, 107.4, 101.7, 66.0,
50.0. MS (ESI) m/z 468/470 (M+H)+. Anal. Calcd for
C21H18BrN5OS: C, 53.85; H, 3.87; N, 14.95. Found:
C, 53.59; H, 3.97; N, 14.62.
5.2.19. 5-(3-Bromophenyl)-7-[2-(4-morpholinyl)-1,3-thia-
zol-5-yl]pyrido[2,3-d]pyrimidin-4-ylamine (14). A solu-
tion of 13 (2.49 g, 5.30 mmol) in o-dichlorobenzene
(15 mL) was heated to reflux overnight. The reaction
mixture was cooled to room temperature, the solid col-
lected by filtration, and purified by silica gel chromato-
graphy (elution with 3% MeOH–CH2Cl2) to provide
1.06 g (43%) of 14 as a product as a yellow solid. mp:
5.2.22. 1-[2-(4-Morpholinyl)-1,3-thiazol-4-yl]ethanone (38).
2,3-Butanedione (30 mL, 355 mmol) at 0 ꢁC was treated
with 10 drops of bromine (caution: delayed exothermic
reaction). After 20 min, additional bromine (12.2 mL,
237 mmol) was added dropwise at such a rate as to
maintain reaction temperature between 20 and 30 ꢁC.
Vacuum distillation (10 mmHg; fractions between 37
and 48 ꢁC) provided 16.26 g (41%) of 1-bromo-2,3-
butanedione compound as a yellow oil. A solution of
1-bromo-2,3-butanedione (16.26 g, 98.55 mmol) in
H2O at 0 ꢁC was treated dropwise with a solution of
hydroxylamine hydrochloride (6.90 g, 99.29 mmol) and
sodium carbonate (4.20 g, 39.63 mmol) in H2O
(38 mL). After 1 h, the reaction was extracted with
CH2Cl2, concentrated to one fourth the original volume,
cooled, and filtered to provide 7.08 g (40%) of 37 as an
unstable (freezer) white solid.
1
>280 ꢁC. H NMR (400 MHz, DMSO-d6/DCl) d 9.86
(br s, 1H), 8.77 (s, 1H), 8.49 (s, 1H), 8.03 (s, 1H), 7.86
(s, 1H), 7.83 (m, 1H), 7.58 (m, 2H), 6.96 (br s, 1H),
3.77 (m, 4H), 3.60 (m, 4H). 13C NMR (100 MHz,
DMSO-d6/DCl) d 173.6, 162.8, 156.5, 151.5, 150.2,
147.5, 145.9, 137.7, 132.8, 131.4, 131.2, 128.0, 124.8,
122.4, 120.9, 102.0, 65.1, 48.0. MS (DCI/NH3) m/z
469/471 (M+H)+. Anal. Calcd for C20H17BrN6OS: C,
51.18; H, 3.65; N, 17.91. Found: C, 51.15; H, 3.76; N,
17.83.
5.2.20. 1-[5-(4-Morpholinyl)-2-thienyl]ethanone (35). A
solution
of
5-acetyl-2-bromothiophene
(8.01 g,
39.1 mmol) and morpholine (16 mL) was heated to
145 ꢁC overnight. The reaction mixture was cooled
and partitioned between H2O and CH2Cl2. The organic
phase was concentrated and purified by silica gel chro-
matography (elution with 30% hexane–EtOAc) to give
5.61 g (68%) of 35.54 1H NMR (300 MHz, CDCl3) d
7.47 (d, 1H, J = 4.4), 6.06 (d, 1H, J = 4.4), 3.84 (m,
4H), 3.28 (m, 4H), 2.46 (s, 3H). MS (DCI/NH3) m/z
212 (M+H)+.
A solution of known morpholine-4-carbothioic acid
amide42 (2.66 g, 18.2 mmol) and 1-bromobutane-2,3-
dione-3-oxime (3.29 g, 18.3 mmol) in EtOH (5.5 mL)
was heated to reflux for 2 h. The reaction mixture was
cooled to room temperature and the solid filtered, washed
with EtOH, and dried to provide 4.16 g (99%) of the de-
sired intermediate morpholine hydroxylamine com-
pound as pink solid. MS (DCI/NH3) m/z 228 (M+H)+.
A solution of 1-(2-morpholinothiazol-5-yl)ethanone-
oxime (6.00 g, 26.4 mmol) in H2O (250 mL), sulfuric
acid (27 mL), and EtOH (26 mL) at 50 ꢁC was
treated dropwise with sodium nitrite (1.97 g, 28.5 mmol)
as a solution in H2O (50 mL). After 2 h, the mixture was
cooled to 0 ꢁC, neutralized with ammonium hydroxide
and extracted with CH2Cl2. The organic phases were
concentrated and purified by silica gel chromatography
(elution with 10% EtOAc–CH2Cl2) to give 1.06 g
(19%) of 38 as a pink solid. 1H NMR (300 MHz,
DMSO-d6) d 7.77 (s, 1H), 3.72 (m, 4H), 3.41 (m, 4H),
2.44 (s, 3H). MS (DCI/NH3) m/z 213 (M+H)+.
5.2.21. 5-(3-Bromophenyl)-7-[5-(4-morpholinyl)-2-thienyl]-
pyrido[2,3-d]pyrimidin-4-ylamine (15). Following the
procedure for compound 8 substituting 35 for 34 pro-
vided
2-amino-4-(3-bromophenyl)-6-(5-morpholin-4-
1
ylthiophen-2-yl)nicotinonitrile in 38% yield. H NMR
(300 MHz, DMSO-d6) d 7.79 (m, 1H), 7.73 (m, 2H),
7.62 (m, 1H), 7.49 (dd, 1H, J = 7.8, 7.8), 7.12 (s,
1H), 6.80 (br s, 2H), 6.27 (d, 1H, J = 4.0), 3.75 (m,
4H), 3.19 (m, 4H). 13C NMR (100 MHz, DMSO-d6)
d 162.8, 160.6, 154.9, 151.8, 139.4, 132.1, 130.7 (over-
lapped), 129.5, 127.4, 127.2, 121.8, 117.3, 106.2,

M. A. Matulenko et al. / Bioorg. Med. Chem. 13 (2005) 3705–3720
3715
5.2.23. 5-(3-Bromophenyl)-7-[2-(4-morpholinyl)-1,3-thia-
zol-4-yl]pyrido[2,3-d]pyrimidin-4-ylamine (16). Following
the procedure for compound 8 substituting 38 for 34 pro-
yl-1H-imidazol-4-yl)nicotinonitrile for 8 provided N-[4-
(3-bromo-phenyl)-3-cyano-6-(1-methyl-2-morpholin-4-
yl-1H-imidazol-4-yl)-pyridin-2-yl]-formamidine. This mat-
erial was used directly in the next reaction.
vided
ylthiazol-4-yl)nicotinonitrile in 74% yield as yellow
2-amino-4-(3-bromophenyl)-6-(2-morpholin-4-
1
solid. H NMR (300 MHz, DMSO-d6) d 7.80 (m, 1H),
A
solution of N-[4-(3-bromophenyl)-3-cyano-6-(1-
7.75 (m, 1H), 7.61 (m, 1H), 7.55 (s, 1H), 7.52 (dd, 1H,
J = 7.8, 7.8), 7.24 (s, 1H), 7.01 (br s, 2H), 3.72 (m, 4H),
3.43 (m, 4H). MS (DCI/NH3) m/z 442/444 (M+H)+.
methyl-2-morpholin-4-yl-1H-imidazol-4-yl)pyridin-2-yl]-
formamidine in 30% sodium methoxide in MeOH
(5 equiv) was heated to reflux. After 2 h, the reaction
was cooled filtered and the solid purified by silica gel
chromatography (gradient CH2Cl2; 2% MeOH–CH2Cl2;
4% MeOH–CH2Cl2) to provide 17 (48%) as a pale yel-
Following the procedure for compound 13 substituting
2-amino-4-(3-bromophenyl)-6-(2-morpholin-4-ylthiazol-
4-yl)nicotinonitrile for 8 provided N-[4-(3-bromophen-
yl)-3-cyano-6-(2-morpholin-4-ylthiazol-4-yl)pyridin-2-yl]-
1
low solid. H NMR (300 MHz, DMSO-d6) d 8.50 (s,
1H), 7.87 (s, 1H), 7.79 (m, 2H), 7.63 (s, 1H), 7.54 (m,
1
formamidine in 83% yield as a yellow solid. H NMR
2H), 3.73 (m, 4H), 3.60 (s, 3H), 3.04 (m, 4H). 13C
(300 MHz, DMSO-d6) d 8.77 (m, 1H), 8.22 (m, 1H),
7.76 (m, 4H), 7.60 (m, 1H), 7.52 (m, 2H), 3.72 (m, 4H),
3.46 (m, 4H). MS (DCI/NH3) m/z 469/471 (M+H)+.
NMR (100 MHz, DMSO-d6/DCl) d 164.0, 152.6,
152.5, 150.4, 150.1, 150.0, 137.5, 133.9, 132.1 (over-
lapped), 128.8, 125.9, 125.6, 123.1, 122.8, 104.5, 66.2,
49.5, 36.1. MS (DCI/NH3) m/z 466/468 (M+H)+. Anal.
Calcd for C21H20BrN7OÆ0.7 H2O: C, 52.66; H, 4.50;
N, 20.47. Found: C, 52.38; H, 4.46; N, 20.30.
Following the procedure for compound 14 substituting
N-[4-(3-bromophenyl)-3-cyano-6-(2-morpholin-4-ylthia-
zol-4-yl)pyridin-2-yl]formamidine for 13 provided 16 in
40% yield as a tan solid. mp: 278–282 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d 8.55 (s, 1H), 7.86 (s, 1H), 7.81
(m, 3H), 7.56 (m, 2H), 3.72 (m, 4H), 3.47 (m, 4H). MS
(DCI/NH3) m/z 469/471 (M+H)+. Anal. Calcd for
C20H17BrN6OSÆ0.6 H2O: C, 50.03; H, 3.82; N, 17.50.
Found: C, 49.77; H, 3.92; N, 17.28.
5.2.26. 1-[2-(4-Morpholinyl)-1,3-oxazol-5-yl]ethanone (42).
A solution of 2-bromo-3-oxobutyraldehyde46 (6.00 g,
36.4 mmol) and morpholine-4-carboxylic acid amide45
(9.01 g, 69.2 mmol) in acetone (40 mL) was stirred at
rt for 1 h then heated to reflux for 1 h. The solvent re-
moved under reduced pressure and the residue purified
by silica gel chromatography (gradient elution with
10% EtOAc–CH2Cl2 to EtOAc) to provide 2.85 g
5.2.24. 1-[1-Methyl-2-(4-morpholinyl)-1H-imidazol-4-yl]-
ethanone (40). A solution of 5-acetyl-2-aminooxaz-
ole44 (7.00 g, 55.5 mmol) and morpholine (20 mL) in
H2O (14 mL) was heated to reflux overnight. The reac-
tion was cooled to room temperature, concentrated
and triturated with EtOAc, filtered, and dried to provide
3.52 g (33%) of the desired compound. A slurry of so-
dium hydride (60% in oil, 590 mg, 14.7 mmol) and
methyl iodide (0.86 mL, 13.7 mmol) in THF (20 mL) at
room temperature was added a solution of 1-[2-(4-mor-
pholinyl)-1H-imidazol-4-yl]ethanone (2.44 g, 12.5 mmol)
in DMF (13 mL) and stirring continued for 1.5 h. Reac-
tion quenched with EtOH, H2O added and extracted
with CH2Cl2. The organic phases were combined, dried
(Na2SO4), concentrated, and the residue purified by sil-
ica gel chromatography (elution with 25% CH2Cl2–
EtOAc) to provide 1.27 g (49%) of 40. 1H NMR
(300 MHz, DMSO-d6) d 7.75 (s, 1H), 3.72 (m, 4H),
3.53 (s, 3H), 3.01 (m, 4H), 2.30 (s, 3H). MS (DCI/
NH3) m/z 210 (M+H)+.
1
(40%) of the desired compound as a yellow solid. H
NMR (300 MHz, DMSO-d6) d 7.99 (s, 1H), 3.68 (m,
4H), 3.53 (m, 4H), 2.26 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) d 182.1, 163.0, 144.7, 139.8, 65.5, 45.2,
25.2. MS (DCI/NH3) m/z 197 (M+H)+.
5.2.27. 5-(3-Bromophenyl)-7-[2-(4-morpholinyl)-1,3-oxa-
zol-5-yl]pyrido[2,3-d]pyrimidin-4-ylamine (18). Following
the procedure for compound 8 substituting 42 for 34
provided 2-amino-4-(3-bromophenyl)-6-(2-morpholin-
4-yloxazol-5-yl)nicotinonitrile in 59% yield as a yellow
1
solid. H NMR (300 MHz, DMSO-d6) d 7.79 (dd, 1H,
J = 2.1, 2.1), 7.74 (m, 1H), 7.62 (s, 1H), 7.61 (m, 1H),
7.50 (dd, 1H, J = 7.8, 7.8), 6.97 (br s, 2H), 6.93 (s,
1H), 3.70 (m, 4H), 3.53 (m, 4H). MS (DCI/NH3) m/z
426/428 (M+H)+.
Following the procedure for compound 13 substituting
2-amino-4-(3-bromophenyl)-6-(2-morpholin-4-yloxazol-
5-yl)nicotinonitrile for 8 provided N-[4-(3-bromophen-
yl)-3-cyano-6-(2-morpholin-4-yloxazol-5-yl)pyridin-2-yl]-
5.2.25. 5-(3-Bromophenyl)-7-[1-methyl-2-(4-morpholinyl)-
1H-imidazol-4-yl]pyrido[2,3-d]pyrimidin-4-ylamine (17).
Following the procedure for compound 8 substituting
40 for 34 provided 2-amino-4-(3-bromophenyl)-6-(1-
methyl-2-morpholin-4-yl-1H-imidazol-4-yl)nicotinonit-
rile in 75% yield as an orange solid. 1H NMR (300 MHz,
DMSO-d6) d 7.76 (m, 1H), 7.73 (m, 1H), 7.59 (m, 1H),
7.56 (s, 1H), 7.53 (m, 1H), 7.06 (s, 1H), 6.79 (br s,
2H), 3.72 (m, 4H), 3.55 (s, 3H), 3.02 (m, 4H). MS
(DCI/NH3) m/z 439/441 (M+H)+.
1
formamidine in 86% yield as a yellow solid. H NMR
(300 MHz, DMSO-d6) d 8.65 (m, 1H), 8.20 (m, 1H),
7.81 (m, 1H), 7.74 (m, 3H), 7.64 (m, 1H), 7.51 (dd,
1H, J = 8.1, 8.1), 7.23 (s, 1H), 3.70 (m, 4H), 3.54 (m,
4H). MS (DCI/NH3) m/z 453/455 (M+H)+.
Following the procedure for compound 14 substituting
N-[4-(3-bromophenyl)-3-cyano-6-(2-morpholin-4-yl-
oxazol-5-yl)pyridin-2-yl]formamidine for 13 provided 18
in 31% yield as a yellow solid. 1H NMR (300 MHz,
DMSO-d6) d 8.50 (s, 1H), 7.95 (s, 1H), 7.80 (m, 3H),
7.54 (m, 4H), 3.72 (m, 4H), 3.57 (m, 4H). 13C NMR
Following the procedure for compound 13 substituting
2-amino-4-(3-bromophenyl)-6-(1-methyl-2-morpholin-4-

3716
M. A. Matulenko et al. / Bioorg. Med. Chem. 13 (2005) 3705–3720
(100 MHz, DMSO-d6/DCl) d 163.7, 161.4, 152.5, 151.4,
150.8, 149.4, 142.8, 137.8, 133.7, 132.5, 132.1, 131.9,
128.5, 123.1, 121.2, 103.4, 65.5, 45.9. MS (DCI/NH3)
m/z 453/455 (M+H)+. Anal. Calcd for C20H17Br-
N6O2Æ0.3 H2O: C, 52.37; H, 3.87; N, 18.37. Found: C,
52.01; H, 3.69; N, 18.26.
7.57 (ddd, 1H, J = 7.8, 1.6, 1.6), 7.54 (m, 1H), 6.75
(dd, 1H, J = 8.9, 0.7), 3.13 (s, 6H). 13C NMR
(100 MHz, DMSO-d6) d 161.9, 159.7, 159.6, 159.1,
158.1, 148.2, 147.4, 140.5, 136.1, 132.0, 131.3, 130.9,
127.9, 122.2, 120.5, 118.9, 105.4, 104.3, 37.6. MS
(DCI/NH3) m/z 421/423 (M+H)+. Anal. Calcd for
C20H17BrN6Æ3.0 HClÆ2.0 H2O: C, 42.39; H, 4.27; N,
14.83. Found: C, 42.06; H, 4.44; N, 15.15.
5.2.28. 5-(3-Bromophenyl)-7-(4-dimethylaminophenyl)-
pyrido[2,3-d]pyrimidin-4-ylamine (2). Following the
procedure for
7
substituting 1-[4-(dimethylamino)-
5.2.30. 5-(3-Bromophenyl)-7-(5-dimethylaminopyridin-2-
yl)pyrido[2,3-d]pyrimidin-4-ylamine (44). To a solution
of 3-dimethylaminopyridine55 (3.53 g, 28.9 mmol) in
CH2Cl2 (100 mL) at 0 ꢁC was added NBS (5.34 g,
30.0 mmol) and the reaction stirred for 3 h. The mixture
was washed with saturated NaHCO3, dried (MgSO4),
and concentrated. The residue was purified by silica
gel chromatography (elution with 20% EtOAc–hexanes)
to provide 2.67 g (46%) of (5-bromopyridin-2-yl)di-
phenyl]ethanone for 5 and purification by silica gel
chromatography (elution with 15% MeOH–CH2Cl2)
gave 2-amino-4-(3-bromophenyl)-6-(4-dimethylamino-
phenyl)nicotinonitrile (54%) as a yellow solid. 1H
NMR (300 MHz, DMSO-d6) d 8.03 (d, 2H, J = 8.9),
7.84 (dd, 1H, J = 1.1, 1.1), 7.73 (m, 1H), 7.65 (m, 1H),
7.51 (dd, 1H, J = 8.1, 8.1), 7.18 (s, 1H), 6.85 (br s,
2H), 6.77 (d, 2H, J = 9.2), 2.99 (s, 6H). 13C NMR
(100 MHz, DMSO-d6) d 160.6, 159.0, 152.3, 151.6,
139.6, 132.0, 130.9, 130.7, 128.4, 127.4, 124.3, 121.8,
117.2, 111.4, 107.6, 84.2, 39.7. MS (DCI/NH3) m/z
393/395 (M+H)+.
1
methylamine. H NMR (300 MHz, CDCl3) d 7.83 (d,
1H, J = 3.3), 7.25 (d, 1H, J = 8.8), 6.86 (dd, 1H,
J = 8.8, 3.3), 2.96 (s, 6H). MS (DCI/NH3) m/z 201/203
(M+H)+.
Following the procedure for 4 substituting 2-amino-4-
(3-bromophenyl)-6-(4-dimethylaminophenyl)nicotino-
nitrile for 7 and purification by silica gel chromatogra-
phy (elution with 15% MeOH–CH2Cl2) to provide 2
To a solution of (5-bromopyridin-2-yl)dimethylamine
(2.67 g, 13.4 mmol) in CH3CN (20 mL) and Et3N
(20 mL) was added PdCl2(PPh3)2 (175 mg, 0.25 mmol),
CuI (48 mg, 0.25 mmol) and trimethylsilyl acetylene
(2.46 g, 25.0 mmol), and the mixture stirred at rt for
48 h. The mixture was diluted with a mixture of hexanes
and EtOAc and filtered through Celiteꢂ. The filtrate was
concentrated and crude dimethyl(6-trimethylsilanylethyn-
ylpyridin-3-yl)amine used directly in the next reaction.
1
(52%) as a yellow solid. H NMR (500 MHz, DMSO-
d6) d 8.51 (s, 1H), 8.20 (d, 2H, J = 9.0), 7.82 (m, 1H),
7.78 (m, 1H), 7.75 (s, 1H), 7.55 (m, 2H), 6.81 (d, 2H,
J = 9.1), 3.02 (s, 6H). 13C NMR (125 MHz, DMSO-d6)
d 161.9, 160.5, 159.9, 158.1, 152.0, 147.2, 140.7, 132.1,
131.3, 131.0, 128.9, 127.9, 124.2, 122.2, 119.2, 111.7,
104.1, 39.3. MS (DCI/NH3) m/z 420/422 (M+H)+. Anal.
Calcd for C21H18BrN5Æ1.5 H2O: C, 56.39; H, 4.73; N,
15.66. Found: C, 56.24; H, 4.33; N, 15.47.
A solution of dimethyl(6-trimethylsilanylethynylpyridin-
3-yl)amine in acetone (25 mL) and H2O (1 mL) was
added 8 M H2SO4 (4 mL) followed by Hg(OTf)2
(6.40 g, 15.0 mmol) and the reaction heated to reflux
for 4 h cooled to rt and stirring continued overnight.
The mixture was diluted with EtOAc and H2O, filtered
through Celiteꢂ and the filtrated partitioned, and ex-
tracted with EtOAc then CH2Cl2. The organic phases
were combined, dried (MgSO4), and concentrated. The
residue was purified by silica gel chromatography (elu-
tion with 25% EtOAc–hexanes) to give 960 mg of 1-(5-
5.2.29. 5-(3-Bromophenyl)-7-(6-dimethylaminopyridin-3-
yl)pyrido[2,3-d]pyrimidin-4-ylamine (43). Following the
procedure for compound 5 substituting dimethylamine
for morpholine gave 1-[6-(dimethylamino)-3-pyridinyl]-
1
ethone as a yellow solid in 81% yield. H NMR (300
MHz, DMSO-d6) d 8.72 (d, 1H, J = 2.2), 7.96 (dd, 1 H
J = 9.2, 2.6), 6.68 (dd, 1H, J = 9.1, 0.7), 3.12 (s, 6H),
2.44 (s, 3H). MS (DCI/NH3) m/z 165 (M+H)+.
1
dimethylaminopyridin-2-yl)ethone. H NMR (300 MHz,
CDCl3) d 8.10 (d, 1H, J = 3.1), 7.96 (d, 1H, J = 8.8),
6.95 (dd, 1H, J = 8.8, 3.3), 3.10 (s, 6H), 2.63 (s, 3H).
MS (DCI/NH3) m/z 165 (M+H)+.
Following the procedure for compound 7 substituting 1-
[6-(dimethylamino)-3-pyridinyl]ethone for 5 with the
following modified work up. After the reaction was
judged complete by TLC, the solvent was removed
under reduced pressure, the residue taken up in
MeOH–CH2Cl2, filtered, and dried to provide 2-amino-
4-(3-bromophenyl)-6-[2-dimethylamino-5-pyridyl]nicot-
inonitrile (28%). MS (DCI/NH3) m/z 394/396
(M+H)+.
Following the procedure for compound 7 substituting
1-(5-dimethylaminopyridin-2-yl)ethone for 5 to provide
6-amino-4-(3-bromophenyl)-50-dimethylamino-[2,20]bipyr-
1
idyl-5-carbonitrile (88%). H NMR (300 MHz, DMSO-
d6) d 8.19 (s, 1H), 8.17 (d, 1H, J = 5.8), 7.81 (dd, 1H,
J = 1.7, 1.7), 7.74 (m, 1H), 7.63 (m, 1H), 7.54 (s, 1H),
7.51 (dd, 1H, J = 8.2, 8.2), 7.20 (dd, 1H, J = 9.2, 3.1),
6.97 (br s, 2H), 3.03 (s, 6H). MS (DCI/NH3) m/z 394/
396 (M+H)+.
Following the procedure for compound 4 substituting 2-
amino-4-(3-bromophenyl)-6-[2-dimethylamino-5-pyridyl]-
nicotinonitrile for 7 to provide compound 43 in 73%
1
yield. This material was converted to the HCl salt. H
Following the procedure for compound 4 substituting
6-amino-4-(3-bromophenyl)-50-dimethylamino-[2,20]bipyr-
idyl-5-carbonitrile for 7 to provide compound 44 in 25%
NMR (400 MHz, DMSO-d6) d 9.04 (dd, 1H, J = 2.4,
0.7), 8.52 (s, 1H), 8.40 (dd, 1H, J = 9.0, 2.4), 7.84 (m,
1H), 7.80 (s, 1H), 7.78 (ddd, 1H, J = 7.5, 2.1, 2.1),
1
yield. This material was converted to the HCl salt. H

M. A. Matulenko et al. / Bioorg. Med. Chem. 13 (2005) 3705–3720
3717
NMR (300 MHz, DMSO-d6) d 8.54 (s, 1H), 8.40 (d, 1H,
J = 8.8), 8.21 (d, 1H, J = 2.9), 8.12 (s, 1H), 7.79 (m, 2H),
7.57 (m, 2H), 7.28 (dd, 1H, J = 9.2, 3.3), 3.07 (s, 6H).
MS (DCI/NH3) m/z 421/423 (M+H)+.
Following the procedure for compound 7 substituting
1-(2-dimethylaminopyrimidin-5-yl)ethanone for 5 to pro-
vide 2-amino-4-(3-bromophenyl)-6-(2-dimethylamino-
pyrimidin-5-yl)nicotinonitrile in 27% yield. 1H NMR
(300 MHz, DMSO-d6) d 9.07 (s, 2H), 7.86 (dd, 1H,
J = 1.7, 1.7), 7.74 (br d, 1H, J = 8.5), 7.66 (br d, 1H,
J = 7.8), 7.51 (dd, 1H, J = 7.8, 7.8), 7.28 (s, 1H), 7.03
(s, 2H), 3.20 (s, 6H).
5.2.31. 5-(3-Bromophenyl)-7-(6-dimethylaminopyridazin-
3-yl)pyrido[2,3-d]pyrimidin-4-ylamine (45). Following
the procedure for compound 5 substituting 23 for
1-(6-chloro-3-pyridinyl)ethone
and
dimethylamine
hydrochloride for morpholine to give to provide 1-[6-
(dimethylamino)-3-pyridazinyl]ethanone in 77% yield
Following the procedure for 4 substituting 2-amino-4-
(3-bromophenyl)-6-(2-dimethylaminopyrimidin-5-yl)-
1
1
nicotinonitrile for 7 to give 46 in 50% yield. H NMR
as yellow crystals. H NMR (300 MHz, CDCl3) d 7.88
(d, 1H, J = 9.5), 6.80 (d, 1H, J = 9.5), 3.28 (s, 6H),
(300 MHz, DMSO-d6) d 9.93 (br s, 1H), 9.29 (s, 2H),
8.87 (s, 1H), 8.16 (s, 1H), 7.94 (dd, 1H, J = 1.7, 1.7),
7.82 (ddd, 1H, J = 7.8, 1.7, 1.7), 7.62 (ddd, 1H,
J = 7.8, 1.7, 1.7), 7.57 (dd, 1H, J = 7.8, 7.8), 7.32 (br s,
1H), 3.26 (s, 6H). 13C NMR (125 MHz, DMSO-d6) d
163.2, 161.6, 158.9, 157.8, 151.8, 150.0, 148.9, 137.5,
132.9, 131.7, 131.1, 128.2, 122.4, 121.2, 116.7, 103.0,
36.9.
2.77 (s, 3H). MS (DCI/NH3) m/z 166 (M+H)+.
Following the procedure for compound 7 substituting 1-
[6-(dimethylamino)-3-pyridazinyl]ethanone for 5 to pro-
vide 2-amino-4-(3-bromophenyl)-6-[6-(dimethylamino)-
3-pyridazinyl]nicotinonitrile in 44% yield as an orange
solid. 1H NMR (300 MHz, CDCl3) d 8.20 (d, 1H,
J = 9.8), 8.12 (s, 1H), 7.83 (dd, 1H, J = 2.0), 7.64 (m,
2H), 7.37 (dd, 1H, J = 7.8, 7.8), 6.88 (d, 1H, J = 9.5),
3.27 (s, 6H). MS (DCI/NH3) m/z 395/397 (M+H)+.
5.2.33. N-{4-[4-Amino-5-(3-bromophenyl)pyrido[2,3-d]-
pyrimidin-7-yl]phenyl}-N-methylformamide (47). To a
solution of 40-fluoroacetophenone (10.0 g, 72.4 mmol)
in DMSO (20 mL) was added methylamine (40% in
H2O, 65 mL), the reaction flask fitted with a reflux con-
denser and the mixture heated to 90 ꢁC for 18 h. The
reaction was cooled, H2O added, the solid filtered, dried,
and purified by chromatography (gradient elution: 0–
60% EtOAc–hexanes) to provide 6.12 g (57%) of 1-(4-
methylaminophenyl)ethanone58 as a light yellow solid.
1H NMR (300 MHz, DMSO-d6) d 7.72 (AA0BB0, 2H,
J = 8.8), 6.59 (m, 1H), 6.54 (AA0BB0, 2H, J = 8.8),
2.73 (d, 3H, J = 5.1), 2.39 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) d 195.1, 153.8, 130.5, 124.8,
110.5, 29.2, 25.8. MS (DCI/NH3) m/z 150 (M+H)+.
Following the procedure for 4 substituting 2-amino-4-
(3-bromophenyl)-6-[6-(dimethylamino)-3-pyridazinyl]-
nicotinonitrile for 7 to give 45 in 74% yield as a tan
powder. This material was converted to the HCl salt.
1
mp: >230 ꢁC; H NMR (300 MHz, DMSO-d6) d 8.76
(s, 1H), 8.33 (d, 1H, J = 4.4), 8.18 (m, 1H), 7.77 (m,
1H), 7.70 (m, 1H), 7.53 (m, 1H), 7.45 (dd, 1H, J = 7.7,
7.7), 7.28 (m, 1H), 3.12 (s, 6H). 13C NMR (125 MHz,
DMSO-d6) d 163.7, 158.1, 156.9, 152.4, 150.3, 149.5,
146.0, 137.7, 133.3, 131.6, 131.5, 128.1, 127.6, 122.6,
121.9, 116.4, 105.0, 38.7. HRMS calcd for
C19H1679BrN7: 422.0729; observed: 422.0726. Anal.
Calcd for C19H16BrN7Æ1.6 HCl: C, 47.48; H, 3.69; N,
20.40. Found: C, 47.41; H, 3.72; N, 20.57.
Following the procedure for compound 7 substituting 1-
(4-methylaminophenyl)ethanone for 5 to provide 2-amino-
4-(3-bromophenyl)-6-(4-methylaminophenyl)nicotinoni-
trile (39%) as a yellow solid. 1H NMR (300 MHz,
DMSO-d6) d 7.96 (AA0BB0, 2H, J = 8.8), 7.83 (dd,
1H, J = 1.7, 1.7), 7.72 (ddd, 1H, J = 7.8, 1.7, 1.0), 7.64
(ddd, 1H, J = 8.1, 1.7, 1.7), 7.50 (dd, 1H, J = 8.1, 8.1),
7.13 (s, 1H), 6.81 (br s, 2H), 6.59 (AA0BB0, 2H,
J = 8.8), 6.24 (q, 1H, J = 5.1), 2.74 (d, 3H, J = 5.1).
13C NMR (75 MHz, DMSO-d6) d 160.6, 159.2, 152.3,
151.8, 139.6, 132.0, 130.74, 130.66, 128.6, 127.4, 124.0,
121.8, 117.3, 111.1, 107.4, 83.9, 29.3. MS (ESI) m/z
378/380 (M+H)+.
5.2.32. 5-(3-Bromophenyl)-7-(2-dimethylaminopyrimidin-
5-yl)pyrido[2,3-d]pyrimidin-4-ylamine (46). To a solution
of 5-bromo-2-(dimethylamino)pyrimidine56 (8.11 g,
40.3 mmol; from 2-dimethylaminopyrimidine57) in
DMF (80 mL) at rt was added tributyl(1-ethoxyvi-
nyl)stannane (15.0 mL, 44.4 mmol) followed by Pd-
(PPh3)Cl2 (560 mg, 0.80 mmol) and the mixture heated
to 90 ꢁC for 2 h. The reaction was quenched with aque-
ous KF and extracted with Et2O. The organic layers
were combined, dried, and concentrated to provide
[5-(1-ethoxyvinyl)pyrimidin-2-yl]dimethylamine.
The
resulting vinyl ether was used without further
purification.
Following the procedure for 4 substituting 2-amino-4-
(3-bromophenyl)-6-(4-methylaminophenyl)nicotinonit-
1
rile for 7 to give 47 in 51% yield. H NMR (300 MHz,
To a solution of [5-(1-ethoxyvinyl)pyrimidin-2-yl]di-
methylamine in acetone (80 mL) was added 1 N HCl
(20 mL) and the reaction stirred at rt overnight. The
reaction was concentrated and the residue partitioned
between H2O and Et2O. The organic layers were com-
bined, concentrated, and purified by column chromat-
ography (100% CH2Cl2) to provide 3.86 g (58% yield)
of 1-(2-dimethylaminopyrimidin-5-yl)ethanone. 1H
NMR (300 MHz, CDCl3) d 8.84 (s, 2H), 3.28 (s, 6H),
2.47 (s, 3H).
CDCl3) d 10.03 (br s, 1H), 8.92 (s, 1H), 8.83 (s, 1H),
8.46 (AA0BB0, 2H, J = 8.8), 8.27 (s, 1H), 7.98 (m, 1H),
7.83 (m, 1H), 7.59 (m, 4H), 7.46 (br s, 1H), 3.30 (s,
3H). MS (DCI/NH3) m/z 435/437 (M+H)+.
5.2.34. N-{5-[4-Amino-5-(3-bromophenyl)pyrido[2,3-d]-
pyrimidin-7-yl]pyridin-2-yl}-N-methylformamide
(48).
To a solution of 6-chloro-3-acetylpyridine23 in DMSO
(5 M) was added methylamine (40% in H2O; 5 eq) the

3718
M. A. Matulenko et al. / Bioorg. Med. Chem. 13 (2005) 3705–3720
reaction flask sealed and the mixture heated to 90 ꢁC for
18 h. The reaction was cooled, H2O added, the solid fil-
tered, and dried to provide 1-(6-methylaminopyridin-3-
aminophenyl)nicotinonitrile (36%). 1H NMR (300
MHz, CDCl3) d 7.91 (dd, 1H, J = 8.5, 2.0), 7.84 (m,
1H), 7.74 (dd, 1H, J = 1.7, 1.7), 7.64 (m, 1H), 7.59
(m, 1H), 7.40 (dd, 1H, J = 8.1, 8.1), 7.07 (s, 1H), 6.67
(d, 1H, J = 8.5), 2.98 (s, 3H), 2.21 (s, 3H).
1
yl)ethanone in 67% yield. H NMR (300 MHz, CDCl3)
d 8.71 (d, 1H, J = 2.4), 8.02 (dd, 1H, J = 9.2, 2.4), 6.40
(d, 1H, J = 9.2), 3.30 (m, 1H), 3.00 (d, 3H, J = 5.1),
2.50 (s, 3H).
Following the procedure for 4 substituting 2-amino-4-
(3-bromophenyl)-6-(3-methyl-4-methylaminophenyl)-
nicotinonitrile for 7 to give 48 (54%). 1H NMR
(300 MHz, CDCl3) d 8.79 (s, 1H), 8.32 (m, 1H), 8.21
(s, 1H), 8.11 (dd, 1H, J = 8.5, 2.2), 7.75 (ddd, 1H,
J = 7.4, 1.8, 1.8), 7.68 (m, 2H), 7.47 (m, 2H), 7.26 (d,
1H, J = 8.5), 5.31 (br s, 2H), 3.25 (s, 3H), 2.38 (s, 3H).
To formic-pivalic anhydride59 (2 eq) at 0 ꢁC was added
1-(6-methylaminopyridin-3-yl)ethanone (1 eq) as a solu-
tion in CH2Cl2 (1 M), the reaction warmed to rt and
stirred for 5 h. The reaction was washed with aqueous
sodium hydroxide, dried (Na2SO4), and concentrated
to provide N-(5-acetylpyridin-2-yl)-N-methylformamide
(70%). 1H NMR (300 MHz, CDCl3) d 9.63 (s, 1H),
8.94 (d, 1H, J = 2.2), 8.30 (dd, 1H, J = 8.8, 2.2), 7.08
(d, 1H, J = 8.5), 3.38 (s, 3H), 2.63 (s, 3H).
5.2.36. 5-(3-Bromophenyl)-7-(4-methylaminophenyl)pyr-
ido[2,3-d]pyrimidin-4-ylamine (50). A solution of 47 in
concentrated HCl was stirred overnight at rt. The reac-
tion mixture was lyophilized to provide 50 in quantita-
1
Following the procedure for compound 7 substituting
N-(5-acetylpyridin-2-yl)-N-methylformamide for 5 to
provide N-[6-amino-4-(3-bromophenyl)-5-cyano[2,30]bi-
pyridinyl-60-yl]-N-methylformamide. Material was used
directly in the next reaction.
tive yield. H NMR (300 MHz, DMSO-d6) d 9.75 (br
s, 1H), 8.82 (s, 1H), 8.21 (AA0BB0, 2H, J = 8.8), 8.00
(s, 1H), 7.91 (s, 1H), 7.80 (m, 1H), 7.57 (m, 2H), 7.06
(br s, 1H), 6.69 (AA0BB0, 2H, J = 8.8), 2.80 (s, 3H).
5.2.37. N-{4-[4-Amino-5-(3-bromophenyl)pyrido[2,3-d]-
pyrimidin-7-yl]phenyl}formamide (51) and 7-(4-aminophe-
nyl)-5-(3-bromophenyl)pyrido[2,3-d]pyrimidin-4-ylamine
(52). Following the procedure for compound 7 sub-
stituting 1-(4-aminophenyl)ethanone (Aldrich) for 5
to provide 2-amino-6-(4-aminophenyl)-4-(3-bromophen-
Following the procedure for 4 substituting N-[6-amino-
4-(3-bromophenyl)-5-cyano[2,30]bipyridinyl-60-yl]-N-meth-
ylformamide for 7 to give 48 in 31% yield. 1H NMR (300
MHz, DMSO-d6) d 9.47 (s, 1H), 9.31 (d, 1H, J = 2.2),
8.76 (dd, 1H, J = 8.8, 2.2), 8.59 (s, 1H), 8.03 (s, 1H),
7.90 (dd, 1H, J = 1.8, 1.8), 7.80 (ddd, 1H, J = 7.4, 1.8,
1.8), 7.57 (m, 3H), 3.32 (s, 3H).
1
yl)nicotinonitrile (44%). H NMR (300 MHz, DMSO-
d6) d 7.89 (AA0BB0, 2H, J = 8.5), 7.83 (dd, 1H, J = 1.7,
1.7), 7.72 (br d, 1H, J = 8.1), 7.64 (br d, 1H, J = 7.8),
7.50 (dd, 1H, J = 7.8, 7.8), 7.11 (s, 1H), 6.79 (br s,
2H), 6.61 (AA0BB0, 2H, J = 8.8), 5.65 (br s, 2H).
5.2.35. N-4-[4-Amino-5-(3-bromophenyl)pyrido[2,3-d]pyr-
imidin-7-yl]-2-methylphenyl-N-methylformamide (49). A
solution of 2-fluorotoluene (10.0 g, 90.8 mmol) and ace-
tyl chloride (6.64 mL, 93.4 mmol) in dry CH2Cl2
(50 mL) was treated with AlCl3 (15.3 g, 115 mmol), por-
tionwise, and the reaction stirred at rt for 24 h. To com-
plete reaction, the mixture was heated to reflux in the
last 2 h. The reaction mixture was cooled, quenched
with H2O and extracted with CH2Cl2. Organic layers
combined, concentrated, and the residue purified by sil-
ica gel chromatography (elution with 100% hexanes then
100% CH2Cl2) to provide 1-(4-fluoro-3-methylphen-
yl)ethanone (82%). 1H NMR (500 MHz, CDCl3) d
8.03 (d, 1H, J = 7.5), 8.00 (m, 1H), 7.27 (dd, 1H,
J = 8.7), 2.78 (s, 3H), 2.53 (s, 3H).
Following the procedure for 4 substituting 2-amino-6-
(4-aminophenyl)-4-(3-bromophenyl)nicotinonitrile for 7
to give 51 and 52 as a separable mixture. 51: H NMR
1
(300 MHz, DMSO-d6) d 10.49 (s, 1H), 8.56 (s, 1H),
8.34 (m, 1H), 8.32 (AA0BB0, 2H, J = 8.8), 7.88 (m,
2H), 7.80 (m, 1H), 7.76 (AA0BB0, 2H, J = 8.8), 7.56
1
(m, 2H). 52 HCl salt: H NMR (300 MHz, DMSO-d6)
d 8.82 (s, 1H), 8.16 (AA0BB0, 2H, J = 8.8), 7.99 (s,
1H), 7.91 (dd, 1H, J = 1.5, 1.5), 7.80 (ddd, 1H, J = 7.7,
1.5, 1.5), 7.60 (ddd, 1H, J = 8.1, 1.1, 1.1), 7.56 (m,
1H), 6.74 (AA0BB0, 2H, J = 8.8).
5.3. Biological procedures
To a solution of 1-(4-fluoro-3-methylphenyl)ethanone
(4.59 g, 30.2 mmol) in DMSO (7 mL) was added methyl-
amine (40% in H2O, 7 mL), the reaction sealed in a tube
and heated to 96 ꢁC for 18 h. The mixture was cooled,
poured into H2O, extracted with CH2Cl2, the organic
phases combined, and concentrated. The residue was
purified by silica gel chromatography (elution with 80%
CH2Cl2–hexanes) to provide 1-(3-methyl-4-methylamino-
Assays for AK inhibition and ADO phosphorylation
in intact cells (IMR-32) cells were conducted using
radiochemical methods as described by Jarvis et al.28
Inflammatory hyperalgesia was assessed in male Spra-
gue–Dawley rats (200-400 g, Charles River, Wilming-
ton, MA) 2 h after an injection of 100 lL of a 1%
solution of k-carrageenan (Sigma Chemical Co., St.
Louis, MO) into the plantar surface of the right hind
paw.29 A hyperalgesic response to thermal stimulation
was determined using a commercially available paw
thermal stimulator (UARDG, Department of Anesthe-
siology, University of California, San Diego, La Jolla,
CA) modeled after that described by Hargreaves et al.47
All animal handling and experimental protocols were
1
phenyl)ethanone (31%). H NMR (300 MHz, CDCl3) d
7.80 (dd, 1H, J = 8.1, 1.7), 7.71 (m, 1H), 6.56 (d, 1H,
J = 8.5), 2.97 (s, 3H), 2.51 (s, 3H), 2.16 (s, 3H).
Following the procedure for compound 7 substituting 1-
(3-methyl-4-methylaminophenyl)ethanone for 5 to pro-
vide 2-amino-4-(3-bromophenyl)-6-(3-methyl-4-methyl-

M. A. Matulenko et al. / Bioorg. Med. Chem. 13 (2005) 3705–3720
3719
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5.4. Genetic toxicology
Assays for bacterial reverse mutation (Ames assay)
using a Salmonella strain (TA-98) with and without
S-9 mix metabolic activation were conducted as previ-
ously described.48 A positive response was defined as
at least a two-fold increase the number of mutant colo-
nies in the presence of test compound (2000 lg/well) as
compared to vehicle controls. Additional assays were
conducted to assess clastogenic and aneugenic potential
using a Chinese hamster lung V79 cell micronucleus
assay.32 In this assay, a positive response was defined
as at least a three-fold increase in the percent of micro-
nucleated cells in the presence of test compound
(5000 lg/well) rela- tive to vehicle controls and/or a clear
concentration dependent response.
Acknowledgements
25. Gomtsyan, A.; Didomenico, S.; Lee, C.-H.; Matulenko,
M. A.; Kim, K.; Kowaluk, E. A.; Wismer, C. T.; Mikusa,
J.; Yu, H.; Kohlhaas, K.; Jarvis, M. F.; Bhagwat, S. S. J.
Med. Chem. 2002, 45, 3639.
The authors would like to thank Abbott Structural
Chemistry group for helpful NMR and mass spec
characterizations.
26. Gomtsyan, A.; Lee, C.-H. Curr. Pharm. Design 2004, 10,
1093.
27. Bauser, M.; Delapierre, G.; Hauswald, M.; Flessner, T.;
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