Structure-activity studies of a series of dipyrazolo[3,4-b:3′,4′-d]pyridin-3-ones binding to the immune regulatory protein B7.1

Article abstract of DOI:10.1016/S0968-0896(03)00183-4

The interaction of co-stimulatory molecules on T cells with B7 molecules on antigen presenting cells plays an important role in the activation of naive T cells. Consequently, agents that disrupt these interactions should have applications in treatment of transplant rejection as well as autoimmune diseases. To this end, specific small molecule inhibitors of human B7.1 were identified and characterized. Herein, we report the identification of potent small molecule inhibitors of the B7.1-CD28 interaction. In a high-throughput screen we identified several leads that prevented the interaction of B7.1 with CD28 with activities in the nanomolar to low micromolar range. One of these, the dihydrodipyrazolopyridinone 1, was subsequently shown to bind the V-like domain of human B7.1 at equimolar stoichiometry. With this as a starting point, we report here the synthesis and initial in vitro structure-activity relationships of a series of these compounds.

Full text of DOI:10.1016/S0968-0896(03)00183-4

Bioorganic & Medicinal Chemistry 11 (2003) 2991–3013
Structure–Activity Studies of a Series of
Dipyrazolo[3,4-b:30,40-d]pyridin-3-ones Binding
to the Immune Regulatory Protein B7.1
Neal J. Green,a,* Jason Xiang,a Jing Chen,a Lihren Chen,a Audrey M. Davies,a,y
Dave Erbe,b Steve Tama and James F. Tobinb
aDepartment of Chemical Sciences, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA
bDepartment of Metabolic Diseases, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA
Received 15 November 2002; accepted 26 February 2003
Abstract—The interaction of co-stimulatory molecules on T cells with B7 molecules on antigen presenting cells plays an important
role in the activation of naive T cells. Consequently, agents that disrupt these interactions should have applications in treatment of
transplant rejection as well as autoimmune diseases. To this end, specific small molecule inhibitors of human B7.1 were identified
and characterized. Herein, we report the identification of potent small molecule inhibitors of the B7.1–CD28 interaction. In a high-
throughput screen we identified several leads that prevented the interaction of B7.1 with CD28 with activities in the nanomolar to
low micromolar range. One of these, the dihydrodipyrazolopyridinone 1, was subsequently shown to bind the V-like domain of
human B7.1 at equimolar stoichiometry. With this as a starting point, we report here the synthesis and initial in vitro structure–
activity relationships of a series of these compounds.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
cialized contact area between T cell and APC
membranes known as the immunolgical synapse.8,9 For
CD28-dependent amplification, this involves bulk
recruitment of cell surface molecules and kinase-rich
rafts to the synapse, thereby increasing receptor phos-
phorylation and signaling.10,11 CTLA-4-dependent
attenuation involves recruitment of tyrosine phospha-
tases (such as SHP-2) to the synapse, resulting in
dephosphorylation.12,13 In experimental disease models,
altering these co-stimulatory signals has profound
effects on immunity. Blocking B7/CD28 interactions
with monoclonal antibodies or soluble receptors results
in immunosuppression and enhanced allograft survival,
while B7/CTLA-4 blockade results in enhanced anti-
tumor immune responses.14ꢀ16
Regulation of T cell responses plays a primary role in
determining the outcome of auto-immune disease, the
development of tumor immunity, and graft survival
following transplantation.1ꢀ4 These immune responses
are controlled by interaction of molecules on T cell and
antigen presenting cell (APC) surfaces. Activation of T
cells requires two signals, an antigen-specific signal
delivered through T cell antigen receptor (TcR), and a
second co-stimulatory signal. This co-stimulatory signal
dictates the outcome for T cells through the binding of
B7.1 and B7.2 expressed on antigen presenting cells to
CD28 and CTLA-4 on T cells.1,2 CD28 engagement by
B70s amplifies T cell receptor signaling and stimulates
production of cytokines required for T-cell prolifera-
tion. On the other hand, CTLA-4 engagement by B70s
down regulates the immune response.5ꢀ7 This amplifi-
cation or attenuation of TcR signaling occurs at a spe-
In recent clinical trials, co-stimulatory blockade has
shown considerable therapeutic potential for immu-
notherapy in both allograft transplantation and psor-
iasis.3,4 To date, co-stimulatory modifiers in the clinic
are either monoclonal antibodies or fusion proteins of
the CTLA4 receptor. Herein, we report the identifica-
tion of potent small molecule inhibitors of the B7.1–
CD28 interaction. In a high-throughput screen we
*Corresponding author. Tel.: +1-617-665-5604; fax: +1-617-665-
5685; e-mail: ngreen@wyeth.com
yCurrent address: AstraZeneca R&D Boston, 35 Gatehouse Drive,
Waltham, MA 02451, USA.
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00183-4

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N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
identified several leads that prevented the interaction of
B7.1 with CD28 with activities in the nanomolar to low
micromolar range. One of these, the dihydrodipyr-
azolopyridinone 1, was subsequently shown to bind the
V-like domain of human B7.1 at equimolar stoichio-
metry.17 With this as a starting point, we report here
the synthesis and initial in vitro structure–activity
relationships of a series of these compounds. Target
compounds were synthesized by several routes in
order to systematically modify peripheral substituents,
as well as the heterocyclic framework of 1. Of these
compounds, several tight-binding analogues were
identified and shown to block CD28 signaling in a
cell-based assay.
chemistry depicted in Scheme 1 was utilized to prepare
derivatives represented by the structure 1a. Inter-
mediates 2a–h were prepared by reaction of an aryl or
heteroaryl ester with sodiumhydride and ethyl acetate,
followed by methylation with (trimethylsilyl)diazo-
methane. Condensation with either ethyl 5-amino-1-
methylpyrazole-4-carboxylate or ethyl 2-aminothio-
phene-3-carboxylate using sodiumhydride in refluxing
dioxane gave the 4-hydroxy-1-methyl-pyrazolopyridine
or 4-hydroxy-thienylpyridine esters respectively.18 After
conversion of the hydroxypyridines to the chloropyr-
idines 3a–h and 4 with phosphorus oxychloride, anne-
lation of the pyrazolone ring was accomplished by
heating with an excess of the desired hydrazine in
refluxing ethanol, followed by cyclization with either
19
hot acetic acid or with sodiummethoxide.
The result-
ing products 6a–y (with the exception of 6n, 6r, 6s, 6v,
and 7a) were isolated in moderate yields (40–80%).21
Other analogues were prepared by the following con-
versions: Methylation on the remaining free nitrogen
atomof 1 was accomplished with NaH, MeOTf in
DMF to give 7a.20 Saponification of the nitrile group of
6p with sodiumhydroxide in hot ethylene glycol gave
the carboxylic acid 6s in 95% yield. Nitrile 6p was also
subjected to basic hydrolysis in the presence of hydro-
gen peroxide to afford the amide derivative 6t in a
respectable 40% yield. The nitro analogue, 6v was
reduced by hydrogen and catalytic palladiumon carbon
to give the amine 6w. Finally, reaction of 4a with alkyl-
hydrazines resulted in regioisomeric mixtures of addi-
tion products, with the product of the terminal nitrogen
atomaddition to the ester group predominating. To
avoid this and in order to prepare the N-benzyl-pyr-
Chemistry
In the structure–activity study reported here, we
attempted to make only single-point changes to the lead
compound 1 (Fig. 1), with a few exceptions. Modifications
to the N-aryl ring on the dihydropyrazole portion of 1 was
performed with chemistry that allowed the 6-methyl-4-(3-
[trifluoromethyl]phenyl) - dihydrodipyrazolopyridinone
core to remain constant, while variations on the 4-(3-
[trifluoromethyl]phenyl) group were performed utilizing
either the 2-substituted 40-chlorophenyl-, 40-fluoro-
phenyl-, or 30-fluorophenyl-6-methyl-dihydrodipyrazolo-
pyridinone core of 1a. In a similar fashion, analogues
with changes to the pyrazole ring (1c) contained either a
2-(40-chlorophenyl)-, 2-(40-fluorophenyl)-, or 2-(30-fluoro-
phenyl)- group in the ‘northern’ section, and a 4-[3-(tri-
fluoromethyl)phenyl]- ring in the ‘southern’ section. The
Figure 1.

N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
2993
Scheme 1. Synthesis of dihydrodipyrazolo- and dihydropyrazolo thienylpyridinone derivatives. Reagents: (a) EtOAc or MeOAc, NaH, rt to reflux,
60–95%; (b) TMSCHN2, 95%; (c) NaH, dioxane, reflux overnight 60–80%; (d) POCl3 reflux, 6 h, 75–85%; (e) arylhydrazine, ethanol, reflux, 50–
70%; (f) acetic acid, 100 ꢁC; (g) NaOMe, methanol, reflux, 40–80%; (h) anhydrous hydrazine, ethanol, reflux, 90%; (i) (1) (Boc)2O; (2) PS-PPh3,
PhCH2OH, DIAD, THF 0 ꢁC to rt; (3) TFA, 0 ꢁC to rt, 55%; (j) NaH, MeOTf, DMF, 90%; (k) NaOH, ethylene glycol, 130 ꢁC, 18 h, 95%; (l)
ethylene glycol, DMSO, 30% H2O2, K2CO3, H2O, 4 h, 40%; (m) EtOAc, MeOH, H2–Pd/C, 20 psi, 3 h, 67%.
azolone analogue 6o, the chloropyrazolopyridine 3a was
reacted with anhydrous hydrazine, which directly affor-
ded the unsubstituted dihydrodipyrazolopyridinone 6n.
Deprotonation of 6n with NaH in DMF and alkylation
with benzyl bromide gave 6o, after chromatographic
isolation.
in Scheme 1 to afford 10, which was cyclized in hot
acetic acid to give 11. Treatment of 11 with phosphorus
oxychloride gave the dichlorodipyrazolopyridine 12.
Compound 12 underwent a smooth regioselective
substitution at the 4-Cl position with 2,6-dimethylmor-
pholine to provide the 3-chloro-4-morpholinyl-
dipyrazolopyridine,24 which was converted to the
dihydrodipyrazolopyridinone 15e with sodiummethox-
ide followed by acidic hydrolysis. A few selected
4-aryl 2-(3-fluorophenyl)-6-methyl-dihydrodipyrazolo-
pyridinones were prepared via the triflate 13, which
underwent Suzuki coupling with several arylboronic
acids to give the corresponding aryl substituted chlor-
opyridines 14a–d.25,26 Compounds 14a–d were con-
verted to the desired analogues 15a–d according to
chemistry described in Scheme 1.
We also developed an alternative route for the synth-
esis of 4-substituted 2-(3-fluorophenyl)-6-methyl-dihy-
drodipyrazolopyridinones (Scheme 2). Condensation
of diethylmalonate and ethyl-5-amino-1-methylpyr-
azole-4-carboxylate with sodium methoxide in metha-
nol provided the hydroxypyridinone
8 in good
yield.22 Conversion 8 to the chloropyridinone 9 was
accomplished with POCl3 and BnEt3NCl in acetoni-
trile.23 The hydrazine moiety was added as described

2994
N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
Scheme 2. Representative synthesis of 4-substituted- (2-(3-fluorophenyl)-dihydrodipyrazolopyridinone derivatives. Reagents: (a) Na, ethanol, reflux
72 h, 82%; (b) POCl3, BnEt3NCl, CH3CN, 40 ꢁC to reflux, 3 h, 73%; (c) 3-fluorophenylhydrazine, ethanol, reflux, 12 h, 88%; (d) TfO2, 2,6-di-tert-
butyl-4-methylpyridine, CH2Cl2, 0 ꢁC, 18 h, 92%; (e) Pd(OAc)2, 2PPh3, K3PO4, BnEt3NCl, DME, H2O, Ar0B(OH)2, 2 h, 40–50%; (f) 3-fluoro-
phenylhydrazine, THF, reflux then acetic acid, 100 ꢁC, overnight, 40–55%; (g) acetic acid, 100 ꢁC, 6 h, 80%; (h) POCl3, reflux, 2 h, 75%; (i) 2,6-
dimethylmorpholine, DMF, 140 ꢁC, 4 h then NaOH, MeOH, THF, 16 h, 60% for two steps.
The monopyrazolopyridinone 24 was prepared via a five
step sequence shown in Scheme 3. Ethyl 2-chloropyr-
idine-3-carboxylate underwent a smooth Suzuki cou-
pling reaction with 3-(trifluoromethyl)phenylboronic
acid to give 22 in excellent yield. Ortholithiation and
subsequent lithium–halogen exchange was best accom-
plished with the corresponding carboxylic acid of 22
using s-BuLi and phenyltrimethylammonium tri-
bromide in THF.27 Alternative ortholithiation directing
groups resulted in dibromination products that could
not be separated fromeach other and/or characterized.
Following esterification of the carboxylic acid function
of 23, aromatic substitution with 3-fluorophenylhy-
drazine in refluxing ethanol resulted in concommitant
cyclization to give 24 directly.
For variations on the pyrazole ring of 1, the chemistry
in Scheme 4 was employed. Starting from either 25a or
25b and following the same chemistry as depicted in
Scheme 1, the 6-phenyl and 6-benzyl dihydrodipyr-
azolopyridinones 27 and 32 were obtained via the
phenyl and benzyl chloropyridine intermediates 26a and
26b, respectively. Hydrogenolysis of 32 afforded the
unsubstituted pyrazole analogue 29 in very good yield.
Attempted regioselective alkylation of the N-6 pyrazole
nitrogen atomover the N-3 dihydropyrazole atomof 29
with various alkyl halides gave a complicated mixture of
products. Alternatively, protection of the nitrogen atom
in the dihydropyrazole ring of 29 with diisopropylethyl-
amine gave the monobenzylated derivative 28a.
Alkylation via sodiumhydride in DMF with ethyl
Scheme 3. Synthesis of 2,4 substituted (monopyrazolo)pyridinones. Reagents: (a) 3-(trifluoromethyl)phenylboronic acid, Pd(PPh3)4, Na2CO3, H2O,
DME 85 ꢁC, 95%; (b) NaOH, H2O 84%; (c) s-BuLi, THF, PhMe3NBr3, 45%; (d) TMSCHN2 73%; (e) 3-F phenylhydrazine, ethanol, reflux 33%.

N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
2995
Scheme 4. Synthesis of 6-substituted dipyrazolopyridinones. Reagents: (a) 2NaH, dioxane, reflux overnight 60–80%; (b) POCl3 reflux, 6 h, 75–85%;
(c) 4-fluorophenylhydrazine, ethanol, reflux, 50–70%; (d) acetic acid, 100 ꢁC, 40–80%; (e) i-PrNEt2, BnBr, DMF 78%; (f) H2, Pd/C 80–92%; (g)
EtBr, or n-PrBr, NaH, DMF, 40–65%.
iodide or allyl bromide gave the ethyl and allyl deriva-
tives 28b and 28c, respectively. Hydrogenolysis then
afforded the ethyl-substituted pyrazole analogue 30 and
propyl-substituted pyrazole analogue 31.
were commercially available, the halogen-substituted
methyl hydrazinobenzoic acids and esters 34b–g were
obtained from commercially available halo-substituted
amino- or nitro-benzoates. Starting from the nitro-
benzoates 33e–g, the sequence included reduction of the
nitro group with tin chloride dihydrate, diazotization of
the isolated amine with sodium nitrite in aqueous
hydrochloric acid and finally in situ reduction, to afford
the hydrazines 34e–g as hydrochloride salts. The hydra-
zines 34b–d were prepared fromthe aminobenzoates
33b–d, starting fromthe diazotization step. The resulting
hydrazino salts were neutralized with aqueous NaOH.
This neutralization step was necessary to facilitate clean
addition to the chloropyrazolopyridine 3a. The addition
to 3a was accomplished by treating a warm ethylene
glycol solution of the hydrazines with sodium tert-but-
oxide, followed by slow introduction of 3a. By this
In parallel with characterizing the pharmacophore
binding requirements of 1 and analogues for in vitro
B7.1–CD28 inhibition, we also attempted to gain
increased potency through additional interactions
beyond the pyrazolopyridinone core. The extension of
functionality was facilitated through preparation of
carboxylic acid and amide derivatives (Scheme 5). These
derivatives were synthesized by addition of the desired
hydrazinobenzoic acids or esters to the chloro inter-
mediate 3a and in the case of amides by subsequent
acylation with a variety of amines. While 4-hydrazino-
benzoic acid (34h) and 3-hydrazinobenzoic acid (34a)
Scheme 5. General synthesis of 6-methyl-4-[3-(trifluoromethyl)phenyl]-dihydrodipyrazolopyridinyl-benzoates and benzamides. Reagents: (a) for
33b–d: (1) NaNO2, HCl; (2) SnCl2, HCl/H2O 30–50%; (a) for 33e–g: (1) SnCl2, HCl/H2O; (2) NaNO2, HCl; (3) SnCl2, HCl/H2O 30–50%; (b)
NaOtBu, ethylene glycol 100 ꢁC, 24 h, 50–60%; (c) NaH, DMF, 100 ꢁC, 70%; (d) for 36e,f: LiOH, THF, MeOH 99%; (e) EDCI, DMF, iPrNEt2,
heat 60–95%; (f) for 35a,b and c: MeOH, HCl 99%.

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N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
method, analogues 35a–c, 35f, 36a, and 36e–f were
obtained directly. The ester analogues 35d (X=H,
Table 2. In vitro B7.1–CD28 binding inhibition for 4-substituted-2-
(halophenyl)-dipyrazolo(3,4b:3:40d)pyridinones
Y=CO2Me),
35e
(X=Cl,
Y=CO2Me),
36d
(Y=CO2Me, X=H) were prepared fromthe corre-
sponding acids 35a, 35b, and 36a, respectively by acid-
catalyzed esterification. The acid analogues 36b
(X=CO2H, Y=Cl) and 36c (X=CO2H, Y=F) were
prepared by saponication of the esters 36e and 36f,
respectively. Conversions to amide derivatives 35g–w
and 36g–z (see Tables 6 and 7) via the known water-
soluble carbodiimide protocol (EDCI).
Compd
R0
X
R
B7.1–CD28 inhibition
IC50 (nM)
1
6c
6p
6q
6r
4-Cl CH
3-CF3
3-CF3
3-CN
3-NO2
3-Br
3-CO2H
3-CONH2
3-Br
3-NO2
3-NH2
H
3-F
H
3,5-di-Cl
60ꢂ17
20ꢂ3
3-F
3-F
3-F
3-F
3-F
3-F
3-F
4-F
4-F
CH
CH
CH
CH
CH
CH
N
50ꢂ10
4ꢂ1
Structure–Activity Relationships
Compounds were first evaluated for inhibition of
human B7.1 binding to CD28 using a kinetic ELISA as
previously described.17 Briefly, this involved coating
wells with a CD28–Fc fusion protein and blocking
remaining sites with albumin. A preformed detection
complex of B7.1 coupled to alkaline phosphatase was
then incubated with inhibitors for 30 min. This mixture
was then added to the CD28–Fc coated wells, incubated
25 min and washed. Binding was then quantified using
the colourimetric substrate para-nitro phenyl phosphate
and measuring the optical density at 405 nm. Inhibition
constants (IC50’s) were then calculated by subtracting
background binding and comparing to uninhibited
(DMSO alone) controls. Data for the lead compound 1
or the structurally similar analogue 6c are included in
Tables 1–4 for the purposes of comparison.
22ꢂ3
6s
6t
72ꢂ13
1500ꢂ300
20ꢂ3
6u
6v
6w
6x
6y
6z
15a
15b
15c
15d
15e
CH
CH
50ꢂ13
400ꢂ100
1800ꢂ200
>3000
>3000
40ꢂ5
4-Cl CH
4-Cl CH
3-F
3-F
3-F
3-F
3-F
N
CH
3-Thienyl
3-OMe
3,4-Methylenedioxy
105ꢂ25
82ꢂ9
CH
CH
295ꢂ50
188ꢂ54
2,6-Dimethylmorpholine
steric, electronic and conformational requirements at
these two positions while maintaining a reasonable
molecular weight range and drug-like profile.30,31 For
the northern ring systemthe data indicated a general
trend that favored increased binding affinity with elec-
tron deficient meta- and para-substituted phenyl rings.
Substantially decreased affinity with ortho-substituted
phenyl rings and a tolerance for steric bulk in the para
position was seen when this ring substituent provided
hydrogen bond donor–acceptor properties (Table 1). In
Our first goal was to investigate the role of the two
peripheral aromatic rings on the dihydrodipyrazolopyr-
idinone core for inhibitory activity (see Fig. 1 for
‘northern’ and ‘southern’ rings). We sought to test
Table 1. In vitro B7.1–CD28 binding inhibition for 2-substituted-4-
(3-(trifluoromethyl)phenyl)-dipyrazolopyridinones
Table 3. In vitro B7.1–CD28 binding inhibition for (3,40d)-sub-
stituted-4-(3-(trifluoromethyl)phenyl)-pyrazolo(3,4b)pyridines
Compd
Ar=
B7.1–CD28 inhibition
IC50 (nM)a
Compd
3,4 d Group
B7.1–CD28 inhibition
IC50 (nM)
1
4-Chlorophenyl
4-Fluorophenyl
3-Fluorophenyl
60ꢂ17
50ꢂ3
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
20ꢂ3
1
60ꢂ17
3-Chloro-4-fluorophenyl
2-Fluorophenyl
2,4-Difluorophenyl
20-Carboxy-50-methyl-3-thienyl
2-Pyridizone
42ꢂ5
300ꢂ80
1000ꢂ250
>3000
70ꢂ12
900ꢂ250
150ꢂ25
25ꢂ3
1-tert-Butylphenyl
4-Aminosulfonylphenyl
4-Benzyloxyphenyl
4-Biphenyl
4-Hydroxymethylphenyl
H
Benzyl
6a
7a
200ꢂ70
>3000
39ꢂ9
33ꢂ7
>3000
>3000
aSee Experimental for assay protocol. Determinations were done in
triplicate. Dose–response curves were done at five concentrations.

N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
2997
Table 4. In vitro B7.1–CD28 binding inhibition for substituted-
fluorophenyl-(3-(trifluoromethyl)phenyl)-pyrazolopyridinones
resonance donating, IC50=82 nM), while the electron-
rich 3-amino analogue (6w) was only weakly active.
Two standouts fromthis trend were the electron defi-
cient 3-carboxamido analogue 6t which was only weakly
active (1500 nM), and the electronically neutral 3-car-
boxy analogue 6s (72 nM), which gave inhibitory activ-
ity comparable to 1. The disubstituted ring systems in
15a and 15d were weakly active and the 2,6-dimethyl-
morpholinyl derivative (15e) which contains a nonaro-
matic ring at the 4-position, was moderately active at
190 nM. The moderate binding affinity of (15e) is not easily
explained, by electrostatic contributions alone, but may
include the hydrogen bond acceptor properties of the
morpholine oxygen atom. Analogues lacking moderately
sized meta-substitution feature such as 6x, 6y and particu-
larly the 4-(3-pyridyl) analogue 6z, were weak inhibitors.
Compd
X
Y
Z
R
B7.1–CD28 inhibition
IC50 (nM)
24
6b
6a
30
27
32
31
29
7
3-F
3-F
4-F
4-F
3-F
4-F
4-F
4-F
4-F
—
N
N
N
N
N
N
N
CH
—
N
N
N
N
N
N
N
S
—
CH3
CH3
Ethyl
Phenyl
Benzyl
Propyl
H
1300ꢂ100
20ꢂ3
50ꢂ3
1500ꢂ60
750ꢂ90
70ꢂ20
Variations to the pyrazolone ring systemwere studied
(Table 3). As with the southern ring system, this portion
of the lead series also showed very tight SAR. A
requirement for hydrogen bond donation at the
pyrazolone binding site along with proper steric volume
and northern ring orientation appeared to play an
important role for binding. Methylation of the free
nitrogen atomon this ring led to inactivity ( 7a).
90ꢂ8
100ꢂ20
600ꢂ100
—
addition, it appeared that six-membered aromatic rings
where preferred to five-membered heteroaromatics.
Removal of the northern aromatic ring, or replacement
of the ring with alkyl groups completely abolished
activity (6n). The analogue with an unsubstituted phenyl
ring (6a, see Table 3) gave slightly diminished activity
compared to the lead compound 1. Electron-with-
drawing halogen groups in the 3- and/or 4-positions
gave compounds with similar inhibitory activity (6b, 6c,
and 6d), with the 3-fluorophenyl analogue 6c the most
active. Ortho-substituted ring systems such as 6e, and 6f,
were less active than 1, indicating a possible preference
for the ring to be nearly planar to the dihydrodipyr-
azolopyridinone core. Replacement of the 2-phenyl
moiety with a substituted thienyl ring as in 6g led to
significantly reduced activity. In contrast, six-membered
heteroaromatic ring systems led to compounds with
inhibitory activity comparable to 1 as in the 2-pyr-
idizone (6h) analogue. One notable trend was seen with
para-substituted phenyl ring analogues. The electron-
donating and bulky 4-tert-butyl group of 6i gave weak
activity; however, the isosteric but polar sulfonamide
group of 6j was only 2.5 times less active than 1. Other
groups in the para position such as the benzyloxy group
6k, the hydroxymethyl group 6m and the 4-biphenyl
ring system 6l, were comparable or more potent than 1.
Finally, increasing the freedomof rotation and distance
of the aromatic ring from the dihydrodipyrazolopyr-
idinone core, as in the benzyl analogue 6o, led to
reduced activity.
A preference for specific Van der Waals interactions at
the pyrazole binding site was noted when several N1
substituted pyrazole analogues and a thienyl analogue
were tested in the binding assay (Table 4). While the
unsubstituted pyrazole compound 29 was only half as
active as the lead 1, the thienyl ring analogue 7 was
considerably less active. The pyridine analogue 24 was
inactive, illustrating an essential role for a more fully
substituted pyridine nucleus. The ethyl-substituted
compound 30 and the phenyl analogue 27 were weak
inhibitors; however, the propyl and benzyl analogues
31 and 28 retained inhibitory activity comparable to
6a.
Crystallographic studies of protein–protein complexes
most frequently show that rather large surface area
contacts (>1600 angstroms2) exist between the two
interacting macromolecules.28 In order to inhibit con-
tacts of this magnitude with a small molecule, hydro-
phobic interactions over a large area may be necessary.
Toward that end, a further goal of this SAR study was
to facilitate improved binding with the B7.1 protein
beyond the dihydrodipyrazolopyridinone core. Due to a
greater tolerance of functionality in the ‘northern’
region, we decided to focus on this area and first tested
the incorporation of a hydrogen bonding group in the
formof a non-basic carboxylate at either the meta- or
para-positions (M or P, Table 5). Due to the tight
binding of ‘northern’ analogues containing halogen
substituents, we also studied the corresponding meta- or
para-carboxylates with vicinally substituted fluoro or
chloro groups with the possibility that these two groups
would operate in a synergistic manner for increased
inhibition. The presence of a larger chloro atombut not
a smaller fluoro atom adjacent to the carboxylate group
led to favorable interactions between the small molecule
and the protein. For example, the meta-acid analogue
In contrast to the ability of the northern-position to
accept a fair amount of functional group changes, large
changes fromthe 3-trifluoromethylphenyl group in the
southern aromatic ring were less well tolerated. In gen-
eral, electron-withdrawing groups in the meta position
of a six-membered aromatic ring led to potent inhibi-
0
tion; several analogues gave IC50 s ꢃ25 nM (Table 2)
with the electron-deficient nitro derivative 6q as the
most potent. This electrostatic trend was also seen with
the 3-methoxy analogue 15c (inductively withdrawing–

2998
N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
Table 5. In vitro B7.1–CD28 binding inhibition for 6-methyl-4-[3-
(trifluoromethyl)phenyl]-dihydrodipyrazolopyridinyl benzoates
Table 7. In vitro B7.1–CD28 binding inhibition for 6-methyl-4-[3-
(trifluoromethyl)phenyl]dihydrodipyrazolopyridinyl benzamide
R
R group position Compd
X
B7.1–CD28 inhibition
IC50 (nM)
Compd
Y
X
B7.1–CD28 inhibition
IC50 (nM)
M
P
35q
36p
H
H
120ꢂ75
79ꢂ15
35a
35b
35c
35d
35e
35f
36a
36b
36c
36d
36e
36f
CO2H
CO2H
CO2H
CO2Me
CO2Me
CO2Me
H
Cl
F
H
Cl
H
Cl
F
H
Cl
F
CO2H
CO2H
CO2H
CO2Me
CO2Me
CO2Me
48ꢂ10
7ꢂ3
70ꢂ14
8ꢂ4
M
M
35r
35s
H
H
180ꢂ30
120ꢂ20
98ꢂ9
300ꢂ100
190ꢂ40
16ꢂ2
P
M
M
36q
35t
35u
H
H
Cl
36ꢂ5
9ꢂ1
210ꢂ20
31ꢂ5
22ꢂ4
41ꢂ13
12ꢂ2
F
M
35v
H
55ꢂ9
Table 6. In vitro B7.1–CD28 binding inhibition for 6-methyl-4-[3-
(trifluoromethyl)phenyl]-dihydrodipyrazolopyridinyl benzamides
P
P
36r
36s
H
F
23ꢂ7
64ꢂ11
P
M
36t
35w
H
H
17ꢂ5
100ꢂ30
P
P
P
36u
36v
36w
H
H
H
110ꢂ20
94ꢂ32
44ꢂ15
R
R group position Compd
X
H
B7.1–CD28 inhibition
IC50 (nM)
P
36g
98ꢂ12
P
M
36h
35g
H
H
110ꢂ12
180ꢂ22
P
P
36x
36y
H
Cl
3ꢂ1
550ꢂ85
P
36i
H
500ꢂ95
trend of binding inhibition was seen for the para-acid
series of analogues 36b, 36a and 36c (Cl > >H>F).
The effect of an adjacent halogen atomon the methyl
ester series showed a non-linear trend. The unsub-
stituted meta-ester analogue 35d gave greater inhibition
than the 4-Cl analogue 35e and the 4-F analogue 35f,
while the 3-F para-ester 36f was a tighter binder than
either the 3-H or 3-Cl derivatives. It is not understood
whether the larger chloro atomfavorably effects the
rotational orientation of the carboxylate group com-
pared to the corresponding methyl ester, or if steric and/
or electrostatic forces are playing a larger role. Pre-
sumably the ester analogues would be metabolically
converted to the acids in vivo; nontheless, we used this
SAR information to guide us in preparing other analo-
gues of the northern ring section, particularly the amide
series.
P
P
M
M
36j
36k
35h
35i
H
F
H
F
13ꢂ4
310ꢂ60
54ꢂ24
2400ꢂ600
M
P
35k
36n
H
H
44ꢂ7
110ꢂ20
M
35n
H
270ꢂ44
M
M
35n
35o
H
Cl
20ꢂ4
44ꢂ4
M
35p
H
66ꢂ7
series 35a–c showed good activity, with the addition of a
para-chloro group (35b) improving activity 7-fold over
35a while the 4-fluoro-3-carboxy analogue 35c, was 10-
fold less active than the chloro derivative. The same
Two separate series of amides corresponding to the
meta-carboxylic acids 35a–c and para-carboxylic acid

N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
2999
36a–c were studied. For the ease of discussion, the
groups are further divided into amides derived from
alkyl or saturated heterocyclic amines (Table 6) and
amides prepared from benzylic or aromatic amines
(Table 7). Building on the SAR fromthe various 2-sub-
stituted dihydrodipyrazolopyridinones reviewed in
Table 1, we mainly focussed on incorporating function-
ality with the potential for hydrogen bonding, or groups
without large steric volume. The amides we prepared in
this study indicated that a cyclic ring systembeyond the
‘northern’ aromatic portion with small substituents
capable of accepting a hydrogen bond, or of making a
charge–charge interaction were beneficial for tighter
binding. The 3-hydroxy propyl amide 36g (Table 6)
gave moderate inhibition and the corresponding methyl
ether 36h was nearly as active, with a preference for this
group to be in the para-position. The hydroxy ethyl
chain was equally as active; however, longer chain deri-
vatives with hydroxy, methoxy, ester, or amide groups
were considerably less active (data not shown). Cyclic
rings with an ether or hydroxy group showed improved
binding over the acyclic analogues. The morpholine
derivatives 36j and 35h showed notable activity, with
the para-amides being tighter binders than the meta-
amides, however; here, the fluoro-substituted analogues
36k and 35i showed poor binding compared to the
fluoro methyl ester 36f. The pyrrolidine analogues 35k
and 36n gave equivalent inhibition with the meta-amides
again showing tighter binding. The l-proline methyl ester
35m was a weak inhibitor, however the l-proline acid 35n
at 20nM was almost 14-fold more active. The d-proline
isomer 35p showed weaker inhibition at 66nM indicating
only slight enantiospecificity for binding in this region.
While it was clear that these amide analogues did not
show improved binding over the carboxylates 35b, 35d, or
36f, a select group of aniline-based amides shown in Table
7 demonstrated comparable binding.
Table 8. In vitro B7.1–CD28 binding inhibition and cell-based inhi-
bition of selected 6-methyl-dihydrodipyrazolopyridinones
Compd X=
Y=
Z= B7.1–CD28 CD28–PIK3
inhibition association in
(nM)
Jurkat cells
inhibition @
50 mM (%)
6q
35b
H
Cl
F
CO2H
NO2
CF3
4ꢂ1
7ꢂ3
71
38
36x
35w
H
H
CF3
CF3
3ꢂ1
42
23
17ꢂ3
CD28 was evaluated by monitoring its association with
PI3-kinase. Briefly, CHO-B7.1 cells were pretreated
with compounds at a concentration of 50 mM for 30 min
on ice. Jurkat cells were added to these at a ratio of 8:1
for 12 min at 37 ꢁC. This reaction was stopped by addi-
tion of ice-cold NP-40 lysis buffer, cell debris was
removed by centrifugation, and the CD28 in the lysate
was immunoprecipitated using an antibody to CD28.
These immunoprecipitates were washed with lysis buffer
and resolved by electrophoresis. The CD28-associated
PI3-kinase was detected by western blot using an anti-
PI3-kinase antibody and quantified by densitometry.
Results were then expressed compared to uninhibited
controls. It should be noted that CHO-B7.1 and Jurkat
cells do not form an immunological synapse. As shown
in Table 8, these inhibitors were each able to inhibit
PI3-kinase association with CD28 between 23 and 71%,
but only at the relatively high concentration of 50 mM.
The SAR study of benzyl and aniline amides indicated
some apparent trends (Table 7). The secondary benzyl
analogues 35q and 36p were better inhibitors than the
N-methylated and bicyclic systems represented by 35r
and 35s, respectfully. The presence of hydroxy groups
on the aromatic ring of the benzyl analogues gave
improved binding over the unsubstituted derivatives as
was seen for 36q and 35t–v. For the aniline derivatives it
was clear that hydroxymethyl and hydroxyethyl groups
(36r–t and 35w) offered improved binding over the cor-
responding ester (36v), keto (36w) and methoxy (36u)
aniline amides, with the N-[3-(1-hydroxyethyl)phenyl]
derivative 36x giving an IC50 of 3 nM. As was the case
with the amides in Table 6, the few amides with chloro
or fluoro groups ortho to the benzamide carbonyl group
were significantly less active.
Conclusion
Identification of potent, orally active drugs to disrupt
co-stimulatory molecule interactions between T cells
and antigen presenting cells faces a number of thermo-
dynamic challenges.17 These derive fromthe need to
develop inhibitors with slow enough off rates to com-
pete effectively with the multiple interactions between
two cell surfaces, which result in high avidity interac-
tions. The molecular details of these interactions were
first revealed by the recently solved crystal structure of
B7.1 bound to CTLA4,29 in which CTLA4 and B7.1
pack in a strikingly periodic zipper-like arrangement.
Bivalent CTLA4 homodimers bridge bivalent B7.1
homodimers to form an unusually stable signaling
Some of the more potent compounds were selected for
further characterization using a cell-based assay of B7.1
stimulated signaling through CD28. The main objective
of this assay was to investigate how these compounds
would inhibit B7.1-CD28 signaling in an environment
of high avidity. In this assay, Chinese Hamster Ovary
cells expressing human B7.1 were used to stimulate the
Jurkat CD28+ human T cell line. Signaling through

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N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
complex, which is likely similar to the interactions
between CD28 and B7.1 on cells. Because of this, it is
not surprising that the compounds identified here,
although potent inhibitors of B7.1 binding to CD28 in
in vitro cell-free assays, are only weak inhibitors of
CD28 signaling in cells.
blocked with 1% BSA in TBS for 1 h at 22 ꢁC, then
washed 3ꢄ in TBS prior to assay. The detection com-
plex was formed as follows: B7.1–Fc-biotin, prepared
using NHS-LC-biotin (Pierce #21335) according to the
manufacturers instructions (4.1 mol biotin/mol Fc), was
added at 0.8 mg/mL to streptavidin–alkaline phospha-
tase (Caltag SA1008) at 1:1000 in TBS. Inhibitors or
DMSO (1% final) were added to this complex and
incubated 30 min at 22 ꢁC. Detection complex
(ꢂinhibitors) was then added to the CD28 coated wells
for 25 min at 22 ꢁC, washed 5ꢄ with TBS, developed
with the colorimetric substrate pNPP (Pierce #34045) in
diethanolamine/MgCl2 buffer (pH 9.5) and read at
405 nm.
Although many of the analogues reported here were
capable of blocking the activation of CD28 associated
PI3 kinase in Jurkat cells following stimulation with
CHO-B7.1 cells, high compound concentrations were
required to obtain significant levels of inhibition. This
requirement for high inhibitor concentrations likely
reflects the differences in dissociation rates of the small
molecules from B7.1 compared to that of CD28. Since
small molecules dissociate much faster from B7.1 than
does CD28, therefore a high small molecule concen-
tration is required to maintain a high percentage of
occupancy of B7.1. As noted above, this difference is
dissociation rates is further amplified by the fact that
under physiological conditions the B7.1/CD28 interac-
tion occurs in a specialized cell surface environment,
known as the immunological synapse, where the strong
multivalent complexes form. In fact, the small molecules
presented here are incapable of preventing the adhesion
of cells expressing CD28 to those expressing B7.1 due to
the strength of these multivalent complexes.17 Thus, it is
likely that the development of suitable antagonists for
such interactions will involve the identification of potent
compounds with dissociation rates slow enough to
effectively compete with the stable complexes formed
between immune system cells.32
Cell-based assay for B7.1 inhibitors (co-immunoprecipi-
tation of CD28 and PI3-kinase). CHO-B7.1 cells were
pretreated with B7.1 compounds (final concentration
50 mM) at 25 ꢁC for 10 min, followed by a 30-min incu-
bation at 4 ꢁC. The cells were then mixed with Jurkat
cells (Human T cell line, ATCC) and incubated for an
additional 12 min at 37 ꢁC. The ratio of CHO-B7.1 cells
to Jurkat Cells in the mixture was 1:8. The reaction was
stopped by addition of ice-cold NP-40 lysis buffer and
the cells were incubated on ice for 30 min. The cell deb-
ris was removed by centrifugation (6 min at 14,000 rpm)
and the CD28 in the lysate was immunoprecipitated
using anti-CD28 (Clone 3-D10, GI) at 4 ꢁC for 4 h. The
immunoprecipitate was washed 4 ꢄ with NP-40 lysis
buffer and resolved by SDS–PAGE. The CD28-asso-
ciated PI3-kinase was detected by western blot using
anti-PI3-kinase (UBI) and quantified by densitometry.
To develop analogues with slower off rates will likely
involve the use of co-crystals of these current com-
pounds with B7.1 to guide further improvements.
Importantly, previous studies had mapped the binding
of the dihydrodipyrazolopyridinone 1 lead compound
to a site on the GFCC0C00 face of the N-terminal V-set
domain of human B7.1, very near to the site of its
interactions with counter receptors.17 This small mole-
cule binding site is not present in the homologous
human B7.2, or even mouse B7.1, thus explaining the
exquisite specificity of the compounds described here.
Occupancy of this site by inhibitors blocked B7.1 bind-
ing not only to CD28, but also to CTLA4, although at
much higher concentrations of inhibitors.17 Therefore,
this site on B7.1 may represent a rare ‘hot spot’ for
small molecule antagonist design. To this end, the SAR
reported here represent an important initial insight into
modifications of this lead structure resulting in analo-
gues with improved potency. Drug candidates from
these analogues my then provide a potent means of
inhibiting B7.1 mediated co-stimulation, resulting in a
novel approach to immune modulation.
Chemistry
1H NMR spectra were determined on a Bruker instru-
ment at 300 MHz unless otherwise stated. Chemical
shifts (d) are expressed in parts per million relative to
the internal standard tetramethylsilane. Coupling con-
stants (J) are given in Hz. Electrospray and chemical
ionization (NH3) mass spectra were recorded on a
Hewlett-Packard 1100 series MSD spectrometer. LC
spectra were recorded on a Hewlett-Packard 1100 series
ALS with a diode array detector at 254 nm. Chromato-
graphic purifications were performed either by flash
chromatography using Baker 40 mm silica gel or by
BiotageTM silica gel cartridges. Melting points were deter-
mined in open capillary tubes and are uncorrected. Com-
pound purity was assessed via the results of two diverse
HPLC systems; Method A: MeOH–H2O (both containing
0.1% HCO2H and 0.1% iPrOH). Gradient: 5% MeOH to
100% MeOH, over 7min. Method B: CH3CN–H2O (both
containing 0.1% HCO2H and 0.1% iPrOH). Gradient:
5% CH3CN to 100% CH3CN, over 7 min.
Methyl 3-methoxy-3-(5-bromo-3-pyridinyl)-2-propenoate
(2e). To a solution of methyl 5-bromonicotinate (15.0 g,
69.4 mmol) in ethyl acetate (500 mL) was added NaH
(60% in mineral oil, 2.4 g) and the mixture was gently
heated at 40 ꢁC until a mild exotherm occurred. After
the solvent ceased fromrefluxing, additional NaH was
added (2.27 g, total of 139 mmol) and the mixture
heated at reflux temperature for 16 h. After allowing the
Experimental
Biology
CD28/B7.1 ELISA. Wells were coated with 300 ng
CD28-Fc in carbonate buffer (pH 9.4) overnight at 4 ꢁC,

N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
3001
solution to cool to room temperature methylene chlo-
ride (300 mL) and water (350 mL) were added. The
organic phase was washed with brine, dried over sodium
sulfate and concentrated. To this oil was added aceto-
nitrile (240 mL) and methanol (60 mL) followed by a
solution of TMSCHN2 in hexanes (70 mL, 2 M,
139 mmol). This solution was stirred for 36 h at which
time aq HCl (5%, 50 mL) was added. After nitrogen
evolution ceased, the organic layer was separated,
washed with brine, dried over sodiumsulfate and con-
centrated. The residue was chromatographed through a
plug of silica gel (4:1, hexane/EtOAc) to give 2e (10.1 g,
53%) as a light-brown solid. 1H NMR (CDCl3) d 8.73 (s,
2H), 8.04 (s, 1H), 5.62 (s, 1H), 3.96 (s, 3H), 3.79 (s, 3H).
layer was washed with brine, dried over sodiumsulfate
and concentrated. The off-white solid was triturated
with hexanes to give 9 g of a white solid. 1H NMR
(CDCl3) d 8.77 (s, 3H), 8.07 (s, 1H), 5.62 (s, 1H), 3.89 (s,
3H), 3.78 (s, 3H). This solid was dissolved in phospho-
rus oxychloride (150 mL) and heated to reflux tempera-
ture for 2 h. The reaction mixture was concentrated,
dissolved in EtOAc (200 mL), cooled to 0 ꢁC and neu-
tralized with aqueous sodiumcarbonate. The organic
layer was washed with brine, dried over sodiumsulfate
and concentrated. Chromatography on silica gel (3:1
hexane/EtOAc) gave 3e (7.43 g, 65% for two steps) as a
1
white powder. H NMR (CDCl3) d 8.78 (m, 3H), 8.17
(s, 1H), 4.18 (s, 3H), 3.81 (s, 3H); MS (ES+) m/z 381.3,
383.3, 385.3 (M+H)+1
.
Methyl 3-methoxy-3-(3-cyanophenyl)-2-propenoate (2b).
1
Yield 8.98 g (64%). H NMR (CDCl3) d 7.59 (s, 1H),
7.55 (d, J=5.6 Hz, 1H), 7.43 (m, 1H), 7.33 (d,
Ethyl 4-chloro-6-(3-cyanophenyl)-1-methyl-5-pyrazolo-
pyridinecarboxylate (3b). 95% yield as a white powder.
1H NMR (CDCl3) d 8.21 (s, 1H), 8.04 (s, 1H), 7.91 (m,
1H), 7.79 (d, J=7.6 Hz, 1H), 7.62 (dd, J=7.83, 8.10 Hz,
1H), 4.27 (s, 3H), 380 (s, 3H). MS (ES+) m/z
J=4.6 Hz, 1H), 4.9 (s, 1H), 3.95 (s, 3H), 3.78 (s, 3H).
MS (ES+) m/z 218 (M+H)+1
.
Methyl 3-methoxy-3-(3-nitrophenyl)-2-propenoate (2c).
1
327(M+H)+1
.
Yield 8.47 g (75%). H NMR (CDCl3) d 8.61 (s, 1H),
8.38 (d, J=8.6 Hz, 1H), 8.13 (d, J=8.87 Hz, 1H), 7.60
Ethyl 4-chloro-6-(3-nitrophenyl)-1-methyl-5-pyrazolopyr-
1
(dd, J=8.82, 8.30 Hz, 1H), 5.81 (s, 1H), 3.83 (s, 3H),
3.79 (s, 3H). MS (ES+) m/z 238 (M+H)+1
idinecarboxylate (3c). H NMR (CDCl3) d 8.62 (s, 1H),
8.35 (d, J=7.4 Hz, 1H), 8.21 (s, 1H), 8.04 (d,
.
J=7.69 Hz, 1H), 7.68 (dd, J=7.42, 7.63 Hz, 1H), 4.23
(s, 3H), 3.84 (s, 3H). MS (ES+) m/z 347 (M+H)+1
.
Ethyl 3-methoxy-3-(3-bromophenyl)-2-propenoate (2d).
Using ethyl acetate afforded 15.55 g (92%). The crude
material was used in the next step and characterized as 3d.
Ethyl 4-chloro-6-(3-bromophenyl)-1-methyl-5-pyrazolo-
pyridinecarboxylate (3d). Chromatography on silica gel
(5:1 hexane/EtOAc) gave 3d (12.21 g, 57%) as a white
Ethyl 3-methoxy-3-(3-trifluoromethylphenyl)-2-propeno-
ate (2a). Using ethyl acetate afforded 65.5 g (78%). The
crude material was used in the next step.
1
solid. H NMR (CDCl3) d 8.09 (s, 1H), 7.76 (s, 1H),
7.51 (m, 2H), 7.24 (m, 1H), 4.31 (q, J=7.5 Hz, 2H), 4.18
(s, 3H), 1.26 (t, J=7.5 Hz, 3H).
Ethyl 3-methoxy-3-phenyl-2-propenoate (2f). Prepared
according to the procedure for 2e from commercially
available ethyl benzoylacetate. Yield 2.67 g, 100%. MS
(ES+) m/z 207.3 (M+H)+.
Ethyl
4-chloro-6-(3-trifluoromethylphenyl)-1-methyl-5-
pyrazolo pyridine carboxylate (3a). Chromatography on
silica gel (3:1 hexane/EtOAc) gave 65 g of a white solid.
1H NMR (CDCl3) d 8.41 (s, 1H), 8.06 (d, J=0.9 Hz,
1H), 7.91 (dd, J=7.1, 0.85 Hz, 1H), 7.76 (d, J=7.1 Hz,
1H), 7.68 (ddd, J=8.2, 2.2 Hz, 1H) 4.31 (q, J=7.5 Hz,
2H), overlapping with 4.26 (s, 3H), 1.36 (t, J=7.5 Hz,
3H).
Ethyl 3-methoxy-3-(3-fluorophenyl)-2-propenoate (2g).
Prepared according to the procedure for 2e fromcom-
mercially available ethyl 3-fluorobenzoylacetate. Yield
2.86 g, 43%. MS (ES+) m/z 225.2 (M+H)+.
Methyl 3-methoxy-3-(3-pyridinyl)-2-propenoate (2h).
Prepared according to the procedure for 2e fromcom-
mercially available methyl nicotinoylacetate. Yield
82%. 1H NMR (CDCl3) d 8.82 (s, 1H), 8.64 (d,
J=4.2 Hz, 1H), 7.74 (d, J=6.3 Hz, 1H), 7.38 (m, 1H),
5.59 (s, 1H), 3.88 (s, 3H), 3.73 (s, 3H); MS (ES+) m/z
194 (90%, M+H+).
Ethyl 4-chloro-6-phenyl-1-methyl-5-pyrazolopyridine car-
boxylate (3f). Yield 1.4 g, 60% 1H NMR (CDCl3) d
8.19 (s, 1H), 7.69 (m, 2H), 7.49 (m, 3H), 4.22 (s, 3H),
overlapping 4.21 (q, 2H), 1.09 (t, J=7.2 Hz, 3H).
Ethyl 4-chloro-6-(3-fluorophenyl)-1-methyl-5-pyrazolo-
1
pyridine carboxylate (3g). Yield 1.59 g, 73% H NMR
(CDCl3) d 8.19 (s, 1H), 7.46 (m, 3H), 7.19 (m, 1H), 4.22
(s, 3H), 3.78 (s, 3H).
Ethyl 4-chloro-6-(5-bromo-3-pyridinyl)-1-methyl-5-pyra-
zolopyridinecarboxylate (3e). To a solution of ethyl 5-
amino-1-methyl-4-pyrazole
carboxylate
(5.91 g,
Ethyl 4-chloro-6-(3-pyridinyl)-1-methyl-5-pyrazolopyri-
dine carboxylate (3h). Yield 44%. 1H NMR (CDCl3)
8.73 (d, J=4.94 Hz, 1H), 8.33 (s, 1H), 8.18 (s, 1H), 7.59
(m, 2H), 4.23 (q, J=3.8 Hz, 2H), 1.11 (t, J=3.9 Hz,
35 mmol) in THF (350 mL) was added NaH (60% in
mineral oil, 4.1 g, 122 mmol). After the mixture was
stirred for 30 min, 2e (10 g, 35 mmol) was added and the
mixture was heated at reflux temperature for 36 h. The
resulting homogeneous solution was cooled to 0 ꢁC and
carefully acidified to pH 5 with aq HCl. The mixture
was extracted with EtOAc (3 ꢄ 100 mL). The organic
3H). MS (ES+) m/z 317 (M+H)+1
.
Ethyl 4-chloro-6-[3-(trifluoromethyl)phenyl)-1-phenyl-5-
1
pyrazolopyridine carboxylate (26a). Yield 1.9 g, 25% H

3002
N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
NMR (CDCl3) d 8.39 (s, 1H), 8.24 (d, J=6.1 Hz, 2H),
7.96 (s, 1H), 7.92 (d, J=6.1 Hz, 1H), 7.75 (d, J=6.1 Hz,
1H), 7.62 (m, 1H), 7.48 (m, 2H), 7.39 (m, 1H), 4.23 (q,
J=7.2 Hz, 2H), 1.15 (t, J=7.2 Hz, 3H). MS (ES+) m/z
1H), 7.5-7.92 (m, 3H), 8.03 (s, 1H), 8.08 (s, 1H), 8.28 (br
s, 1H), 8.34 (s, 1H). MS m/z 460 (MꢀH).
2-(2-Fluorophenyl)-6-methyl-4-[3-(trifluoromethyl)phenyl]
-1,6-dihydrodipyrazolo[3,4-b:30,40 -d]pyridin-3(2H)-one
(6e). Prepared from2-fluorophenylhydrazine and 3a
and obtained in 71% yield, mp 275.9–278.5 ꢁC. 1H
NMR (DMSO-d6) d 8.56 (s, 1H), 8.49 (d, J=8.71 Hz,
1H), 8.13 (s, 1H), 7.83 (d, J=8.42 Hz, 1H), 7.73 (m,
2H), 7.48 (m, 1H), 7.34 (m, 2H), 4.19 (s, 3H). MS
(ES+) m/z 428 (M+H)+1. HPLC Method A: room
temperature, 3.70 min, 98%. HPLC Method B: room
temperature, 4.54 min, 98%.
446.4 (M+H)+1
.
Methyl 1-benzyl-4-chloro-6-[3-(trifluoromethyl)phenyl)-
5-pyrazolopyridine carboxylate (26b). Prepared from
methyl
5-amino-1-benzyl-1H-pyrazole-4-carboxylate
and 2a according to the procedure for 3a. Purified by
flash chromatography on silica gel (10:85:5 EtOAc/hex-
ane/CHCl3) in 72% yield (two steps).
2-(4-Chlorophenyl)-6-methyl-4-[3-(trifluoromethyl)phenyl]-
1,6-dihydro-dipyrazolo[3,4-b:30,40-d]pyridin-3(2H)-one (1).
A solution of 3a (0.38 g, 1 mmol) and 4-chloro-
phenylhydrazine (3 equiv) in anhydrous ethanol (10 mL)
was stirred at reflux for 4 h. An additional 3 equiv of the
hydrazine was added and the mixture was stirred for an
additional 16 h. After evaporating to dryness, the resi-
due was chromatographed over silica gel (heptane/
EtOAc, 3:1) to give 357 mg (74%) of 4a as a light-yellow
solid. This material was stirred in AcOH (30 mL) at
reflux temperature for 6 h. The AcOH was removed in
vacuo, the resulting solid was dissolved in EtOAc and
washed with saturated aqueous NaHCO3. The organic
layer was dried (Na2SO4), filtered and evaporated to
give an orange solid that was recrystallized fromEtOH/
EtOAc/heptane to give 250 mg (78%) as a pink crystal-
6-Methyl-4-[3-(trifluoromethyl)phenyl]-2-[4-(trifluorome-
thyl)-2-pyrimidinyl]-1,6-dihydrodipyrazolo[3,4-b:30,40-d]pyr-
idin-3(2H)-one (6f). Prepared from4-trifluoromethyl-2-
pyrimidinylhydrazine and 3a and isolated in 56% yield,
mp 265.8–269 C. H NMR (DMSO-d6) d 9.21 (s, 1H),
8.25 (m, 3H), 7.88 (d, J=8.01 Hz, 1H), 7.81 (d,
J=7.79 Hz, 1H), 7.80 (m, 1H), 4.11 (s, 3H). MS (ES+)
m/z 480 (M+H)+1. HPLC Method A: roomtempera-
ture, 4.75 min, 98%. HPLC Method Example B: room
temperature, 4.77 min, 98%.
ꢁ
1
5-Methyl-3-(6-methyl-3-oxo-4-[3-(trifluoromethyl)phenyl]-
3,6-dihydrodipyrazolo[3,4-b:3,4-d]pyridin-2(1H)-yl)-2-thio-
phenecarboxylic acid (6g). Prepared from2-carboxy-5-
methyl-3-thiophenohydrazine and 3a and isolated in
95% yield, mp 275 ꢁC (dec.). H NMR (DMSO-d6) d
1
1
line solid. H NMR (acetone-d6) d 8.55 (d, J=6.7 Hz,
1H), 8.48 (d, J=8.2 Hz, 1H), 8.32 (d, J=8.2 Hz, 2H),
7.91 (s, 1H), 7.67 (dd, J=7.1, 0.85 Hz, 1H), 7.59 (ddd,
J=8.2, 2.2 Hz, 1H), 7.38 (d, J=8.2 Hz, 1H), 4.07 (s,
3H). MS (ES+) m/z 444.1 (M+H)+1 HPLC Method
A: room temperature, 4.06 min, 100%. HPLC Method
B: room temperature, 4.27 min, 100%.
8.47 (s, 1H), 8.33 (m1H), 8.25 (s, 1H), 7.89 (m, 1H),
7.67 (m, 2H), 4.15 (s, 3H), 2.22 (s, 3H). MS (ES+) m/z
472 (MꢀH)+1. HPLC Method A: roomtemperature,
4.43 min, 99.0%. HPLC Method B: room temperature,
4.04 min, 99.0%.
6-Methyl-2-(6-oxo-1,6-dihydro-3-pyridazinyl)-4-[3-(trifluoro-
methyl)phenyl]-1,6-dihydrodipyrazolo[3,4-b:3,4-d]pyridin-
3(2H)-one (6h). From 3a (1.53 g, 4 mmol) and 3-chloro-
6-hydrazinopyridazine (0.87 g, 6 mmol). Yield: 0.956 g,
4-Fluorophenyl)-6-methyl-4-[3-(trifluoromethyl)phenyl]-
1,6-dihydrodipyrazolo[3,4-b:30,40-d]pyridin-3(2H)-one (6b).
Prepared from4-fluorophenylhydrazine and 3a and
obtained in 79% yield, mp 275 ꢁC (dec.). 1H NMR
(DMSO-d6) d 8.38 (s, 1H), 8.31 (d, J=7.49 Hz, 1H),
8.20 (s, 1H), 7.91 (m, 3H), 7.76 (dd, J=7.75, 8.73 Hz,
1H), 7.35 (dd, J=7.71, 8.76 Hz, 2H), 4.12 (s, 3H). MS
(ES+) m/z 428.2 (M+H)+1. HPLC Method A: room
temperature, 3.93 min, 97.52%. HPLC Method B: room
temperature, 4.71 min, 97.32%.
1
48% H NMR (THF-d8) d 9.8 (s, 1H), 8.15 (s, 1H),
7.79–7.55 (m, 4H), 7.38 (d, J=0.026 Hz, 1H), 7.07 (d,
J=0.026 Hz, 1H), 3.93 (s, 3H), 3.82 (m, 2H), 0.83 (m,
3H). MS (ES+) m/z 492.2 (M+H)+1. To a solution of
this intermediate (0.95 g, 1.9 mmol) in DMF (5 mL) was
added NaH (0.16 g, 4.8 mmol). The solution was heated
at 70 ꢁC for 24 h, poured into ice water and the pH
adjusted to 3. The mixture was filtered and the solid
washed with cold EtOAc to give 0.8 g of a white pow-
2-(3-Fluorophenyl)-6-methyl-4-[3-(trifluoromethyl)phenyl]-
1,6-dihydrodipyrazolo[3,4-b:30,40-d]pyridin-3(2H)-one (6c).
Prepared from3-fluorophenylhydrazine and 3a and
obtained in 82% yield, mp 276 ꢁC (dec.). 1H NMR
(DMSO-d6) d 8.49 (s, 1H), 8.41 (d, J=8.75 Hz, 1H),
8.18 (s, 1H), 7.90 (m, 1H), 7.81 (d, J=8.43 Hz, 1H), 7.73
(d, J=8.42, 8.71 Hz, 1H), 7.50 (m, 1H), 7.01 (m, 1H),
4.15 (s, 3H). MS (ES+) m/z 428 (M+H)+1. HPLC
Method A: room temperature, 4.79 min, 98%. HPLC
Method B: room temperature, 4.77 min, 98%.
1
der. H NMR (THF-d8) d 8.59 (d, J=0.032 Hz, 1H),
8.44 (s, 1H), 8.35 (d, J=0.022 Hz, 1H), 8.06 (s, 1H),
7.80 (m, 1H), 7.72 (m, 1H), 7.29 (m, 1H), 4.48 (q,
J=0.024 Hz, H), 4.02 (s, 3H), 1.41 (t, J=0.047 Hz,
J=0.024, 3H). MS (ES+) m/z 478.3 (M+H)+1. To a
solution of this material (0.1 g, 0.2 mmol) in HOAc
(2 mL) and water (0.2 mL) was added KI (1 mg) The
solution was heated at 100 ꢁC for 5 days and poured
into ice water. The mixture was filtered and the solid
washed with cold EtOAc to give 6h (0.51 g, 55%) as a
white powder. 1H NMR (DMSO-d6) d 8.49 (d,
J=10.2 Hz, 1H), 8.33 (s, 1H), 8.28–8.23 (m, 2H), 7.88
(d, J=7.8 Hz, 1H), 7.75 (m, 1H), 7.09 (d, J=10.2 Hz,
2-(3-Chloro-4-fluorophenyl)-6-methyl-4-[3-(trifluorome-
thyl)phenyl]-1,6-dihydrodipyrazolo[3,4-b:30,40 -d]-pyridin-
3(2H)-one (6d). Yield: 0.031 g (74%) of a gold-brown
1
solid. H NMR (acetone-d6) d 4.05 (s, 3H), 7.32 (m,
1H), 4.10 (s, 3H). MS (ES+) m/z 428.1 (M+H)+1
.

N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
3003
HPLC Method A: room temperature, 4.51 min, 100%.
HPLC Method B: room temperature, 3.91 min, 100%.
3H), 7.55–7.66 (m, 2H), 7.72 (d, J=7.84 Hz, 1H), 8.06
(s, 1H), 8.31 (d, J=7.88 Hz, 1H), 8.37 (br s, 1H). MS m/
z 514 (MꢀH). HPLC Method A: roomtemperature,
4.46 min, 100%. HPLC Method Example B: room tem-
perature, 4.59 min, 100%.
2-(4-tert-Butylphenyl)-6-methyl-4-[3-(trifluoromethyl)phe-
nyl]-1,6-dihydrodipyrazolo[3,4-b:30,40-d]pyridin-3(2H)-one
(6i). Prepared from4- tert-butylphenylhydrazine and 3a
and obtained in 83% yield, mp 326 ꢁC (dec.). H NMR
2-[4-(Hydroxymethyl)phenyl]-6-methyl-4-[3-(trifluorome-
thyl)phenyl]-1,6-dihydrodipyrazolo[3,4 -b:3,4-d]pyridin-
3(2H)-one (6m). To a solution of 36a (2.0 g, 4.4 mmol)
in THF (40 mL) at room temperature, was added a
solution of BH3 in THF (22 mL, 1 M) at a rate of 50 mL/
min. A precipitate began to form, the reaction was stir-
red overnight at roomtemperature, at which time it
became a homogeneous red solution. The reaction was
quenched with MeOH/H2O (1:1, 30 mL) and the
organic layer was removed under reduced pressure. The
residue was redissolved in EtOAc, washed with satu-
rated aqueous NaHCO3, water and brine, dried and
concentrated. Flash chromatography EtOAc/CH2Cl2;
9:1) gave 1.01 g (55%) of 6m as a white crystalline solid,
1
(DMSO-d6) d 8.40 (s, 1H), 8.32 (d, J=8.25 Hz, 1H),
8.12 (s, 1H), 7.87 (d, J=8.1 Hz 1, 1H), 7.75 (m, 3H),
7.53 (d, J=8.71 Hz, 2H), 4.15 (s, 3H), 1.33 (s, 9H). MS
(ES+) m/z 466 (M+H)+1. HPLC Method A: room
temperature, 5.04 min, 100%. HPLC Method B: room
temperature, 5.03 min, 100%.
4-(6-Methyl-3-oxo-4-[3-(trifluoromethyl)phenyl]-3,6-dihy-
drodipyrazolo[3,4-b:3,4-d]pyridin-2(1H)-yl)benzenesulfo-
namide (6j). This compound was obtained in 56%
yield, mp 272–274 ꢁC. 1H NMR (acetone-d6) d 8.53–8.45
(m, 3H), 8.01 (s, 1H), 7.88–7.83 (m, 3H), 7.76–7.73 (m,
2H), 4.16 (s, 3H). MS (ES+) m/z 489 (M+H)+1
.
mp 97.2–100.1 ꢁC. H NMR (DMSO-d6) d 8.51 (s, 1H),
1
HPLC Method A: room temperature, 3.40 min, 100%.
HPLC Method B: room temperature, 4.49 min, 100%.
8.42 (d, J=7.6 Hz, 1H), 8.07 (d, J=8.7 Hz, 2H), 7.93 (s,
1H), 7.69 (d, J=7.7 Hz, 1H), 7.60 (t, J=7.7 Hz, 1H),
7.34 (d, J=8.7 Hz, 2H), 4.56 (s, 2H), 4.05 (s, 3H). MS
(ES+) m/z 440 (M+H)+1. HPLC Method A: room
temperature, 3.37 min, 98.1%. HPLC Method B: room
temperature, 4.49 min, 98.8%.
2-(1,10-Biphenyl-4-yl)-6-methyl-4-[3-(trifluoromethyl)phe-
nyl]-1,6-dihydrodipyrazolo-[3,4-b:30,40-d]pyridin-3(2H)-one
(6k). To a dry, single-necked, round-bottomed flask,
was added biphenyl-4-yl-hydrazine HCl salt (574 mg,
2.6 mmol) and ethylene glycol (6.5 mL), followed by the
addition of sodium-tert-butoxide (260 mg, 2.7 mmol).
This heterogenous mixture was then heated to 70 ꢁC and
allowed to stir for 1 h. As the mixture cooled down to
6-Methyl-4-[3-(trifluoromethyl)phenyl]-1,6-dihydrodipyr-
azolo[3,4-b:3,4-d]pyridin-3(2H)-one (6n). A solution of
chloride (767 mg, 2.0 mmol) and hydrazine mono-
hydrate (0.29 mL, 300 mg, 6.0 mmol) in 20 mL of etha-
nol was refluxed for 4 h. After cooling to room
temperature, the precipitate was collected by filtration
and then washed with water and ethyl acetate. The
light-tan solid was dried in vacuumoven overnight and
474 mg (71%) of pure product was obtained, mp 281–
roomtemperature
3a (500 mg, 1.3 mmol) was added.
The reaction mixture was set to heat at 105 ꢁC for 16 h.
After heating, the reaction mixture was allowed to cool
to roomtemperature and quenched with dilute HCl
(0.6 N) and partitioned with EtOAc. The layers were
separated and the organic layer was dried over Na2SO4,
filtered, and concentrated in vacuo. The crude residue
obtained was purified by flash chromatography on silica
gel using 7:2:1 toluene/EtOAc/hexanes to give the
hydrazine derivative. A portion of this purified material
was used directly in the next step: To a solution of this
intermediate (80 mg, 0.15 mmol) in EtOH (1 mL), was
added NaH (60% in oil; 13 mg, 0.32 mmol). After 1 h at
roomtemperature, the reaction was quenched with
dilute HCl (0.5 N) and partitioned with EtOAc. The
organic layer was washed with saturated NaCl solution
and dried over Na2SO4, filtered, and concentrated in
vacuo. The title compound was obtained in 0.037 g
(51%) as a gold-orange solid, mp 321.3–324 ꢁC. 1H
NMR (acetone-d6) d 3.74 (br s, 3H), 6.95 (br m, 1H),
7.07 (m, 2H), 7.15–7.54 (br m, 6H), 7.54–7.93 (br m,
3H), 7.93–8.2 (m, 2H). MS m/z 484 (MꢀH). HPLC
Method A: room temperature, 4.40 min, 100%. HPLC
Method B: room temperature, 3.09 min, 100%.
283.2 ꢁC. H NMR (DMSO) d 12.81 (br s, 1H), 11.30
1
(br s, 1H), 8.29 (s, 1H), 8.27 (d, J=8.23 Hz, 1H), 8.12 (s,
1H), 7.88 (d, J=8.11 Hz, 1H), 7.76 (dd, J=8.22,
8.11 Hz, 1H), 4.14 (s, 3H); MS (ES+) m/z 334 (100%,
M+H+). HPLC Method A: roomtepmerature,
4.32 min, 99.0%. HPLC Method B: room temperature,
3.15 min, 99.0%.
2-Benzyl-6-methyl-4-(3-trifluoromethyl-phenyl)-1,6-dihy-
drodipyrazolo[3,4-b:3,4-d]pyridin-3(2H)-one (6o). To a
solution of 6-methyl-4-(3-trifluoromethyl-phenyl)-1,6-
dihydro-2H-1,2,5,6,7-pentaaza-as-indacen-3-one (256mg,
0.768 mmol) in a mixed solvent of dioxane and H2O
(2:1, v/v, 6 mL) at 0 ꢁC were added Na2CO3 (89 mg,
0.84 mmol) and BOC2O (184 mg, 0.843 mmol) and the
reaction mixture was allowed to warm to room tem-
perature. After 18 h, an aqueous workup provided 6-
methyl-3-oxo-4-(3-trifluoromethyl-phenyl)-3,6-dihydro-
2H-1,2,5,6,7-pentaaza-as-indacene-1-carboxylic
acid
2-[4-(Benzyloxy)phenyl]-6-methyl-4-[3-(trifluoromethyl)-
phenyl]-1,6-dihydrodipyrazolo[3,4-b:30,40-d]-pyridin-3(2H)-
one (6l). Prepared according to the procedure for 6l in
45% yield after flash chromatography (1:1, hexanes/
EtOAꢁc), followed by trituration fromhexanes, mp 221–
tert-butyl ester in 95% yield (318 mg). To 6-methyl-3-
oxo - 4 - (3 - trifluoromethyl - phenyl) - 3,6 - dihydro - 2H-
1,2,5,6,7-pentaaza-as-indacene-1-carboxylic acid tert-
butyl ester (0.10 mmol) in THF (1.5 mL) at 0 ꢁC were
added PS-triphenylphosphine (120 mg, 1.61 mmol/g,
0.19 mmol, Argonaut), benzyl alcohol (0.13 mmol), and
DIAD (0.16 mmol). The reaction mixture was allowed
1
223.4 C. H NMR (acetone-d6) d 4.05 (s, 3H), 5.04 (s,
2H), 6.8 (m, 1H), 7.15–7.32 (m, 4H), 7.35–7.44 (br m,

3004
N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
to warmto roomtemperature and stirred for 4 h before
an aqueous workup. Silica gel chromatography yielded
the desired 2-benzyl-6-methyl-3-oxo-4-(3-trifluoro-
methyl-phenyl)-3,6-dihydro-2H-1,2,5,6,7-pentaaza-as-
indacene-1-carboxylic acid tert-butyl ester which was
subjected to TFA treatment to give the title compound.
1H NMR (CDCl3) d 9.35 (s, 1H), 8.40 (s, 1H), 8.31 (d,
J=7.5 Hz, 1H), 7.89 (s, 1H), 7.67 ( d, J=7.5 Hz, 1H),
7.54 (t, J=7.7 Hz, 1H), 7.35–7.20 (m, 5H), 4.98 (s, 2H),
21.0 Hz, 2H), 7.65 (t, J=7.69 Hz, 1H), 7.51 (m, 1H),
7.07 (m, 1H), 4.11 (s, 3H). MS (ES+) m/z 404
(M+H)+1. HPLC Method A: 3.14 min, 100%. HPLC
Method B: 4.28 min, 100%.
3-[2-(3-Fluorophenyl)-6-methyl-3-oxo-1,2,3,6-tetrahydro-
dipyrazolo[3,4-b:30,40-d]pyridin-4-yl]benzamide (6t). To a
solution of 6p (217 mg, 0.56 mmol) in DMSO (25 mL)
and ethylene glycol (5 mL) was added 30% H2O2
(10 mL) followed by potassium carbonate (34 mg) in
H2O (4 mL). After stirring for 4 h, the pH of the reac-
tion mixture was adjusted to 5 and extracted with
EtOAc (50 mL). The organic layer was dried and con-
centrated to afford 6t (92 mg, 40%), mp 332–335 ꢁC. 1H
NMR (D2O, 600 MHz) d 8.40 (s, 1H), 8.23 (d, 10.3,
1H), 8.07 (m, 2H), 7.78 (m, 2H), 7.67 (dd, J=10.1,
7.8 Hz, 1H), 7.51 (m, 1H), 7.09 (m, 1H), 4.07 (s, 3H).
MS (ES+) m/z 403 (M+H)+1. HPLC Method A: room
temperature 4.04 min, 100%. HPLC Method B: room
temperature 2.84 min, 100%.
4.15 (s, 3H). MS (ES+) m/z 424 (M+H)+1
.
3-[2-(3-Fluorophenyl)-6-methyl-3-oxo-1,2,3,6-tetrahydro-
dipyrazolo[3,4-b:3,4-d]pyridin-4-yl]benzonitrile (6p). Pre-
1
pared according to the procedure described for 1. H
NMR (CDCl3) d 8.42 (s, 1H), 8.31 (d, J=7.8 Hz, 1H),
8.24 (s, 1H), 8.03 (J=7.6 Hz, 1H), 7.82 (m, 2H), 7.80
(m, 1H), 7.55 (m, 1H), 7.11 (m, 1H), 4.13 (s, 3H). MS
(ES+) m/z 385 (M+H)+1
.
2-(3-Fluorophenyl)-6-methyl-4-(3-nitrophenyl)-1,6-dihy-
drodipyrazolo[3,4-b:3,4-d]pyridin-3(2H)-one (6q). Pre-
pared according to the procedure of 1 to give 4c. The
crude material was used without further purification in
4-(5-Bromo-3-pyridinyl)-2-(3-fluorophenyl)-6-methyl-1,6-
dihydrodipyrazolo[3,4-b:3,4-d]pyridin-3(2H)-one (6u). To
a solution of 3e (1.3 g, 3.28 mmol) in ethanol (30 mL)
was added 3-fluorophenylhydrazine (1.03 g, 8.2 mmol).
The reaction was heated at reflux temperature for 6 h
then concentrated. Chromatography on silica gel (hex-
ane/EtOAc, 1:1) gave 1.1 g of 4e (69%) as a white foam.
1H NMR (CDCl3) d 10.1 (s, 1H) 8.73 (s, 1H), 8.67 (s,
1H), 8.27 (s, 1H), 8.16 (d, J=0.035 Hz, 1H), 7.22 (m,
1H), 6.66 (m, 3H), 6.28 (s, 1H), 4.08 (s, 3H), 4.04 (s,
3H); MS (ES+) m/z 471.3, 473.3 (M+H)+1. To a
solution of this foam(1.0 g) in DMF (20 mL) was added
NaH (0.15 g). The solution was heated at 90 ꢁC for 12 h,
poured into ice water and the pH adjusted to 4. The
mixture was filtered and the solid washed with cold
EtOAc to give 6u (0.8 g, 55% for two steps) as an off-
1
the next step. Yield 63%. H NMR (CDCl3) d 8.42 (s,
1H), 8.28 (s, 1H), 7.88 (d, J=6.3 Hz, 2H), 7.62 (m, 1H),
7.24 (d, J=6.55 Hz, 2H), 6.97 (d, J=8.9 Hz, 2H), 4.08
(s, 3H), 3.46 (s, 3H). Cyclization in refluxing HOAc and
recrystallization (EtOAc, EtOH) afforded 150 mg (85%)
of 6q as a off-white powder, mp 313 ꢁC (dec.). 1H NMR
(D2O, 600 MHz) d 8.73 (s, 1H), 8.42 (d, J=7.32 Hz,
1H), 8.25 (d, J=7.66 Hz, 1H), 8.20 (s, 1H), 7.79 (m,
1H), 7.64 (m, 2H), 7.58 (m, 1H), 7.11 (m, 1H), 4.12 (s,
3H). HPLC Method A: room temperature, 4.61 min,
100%. HPLC Method B: room temperature, 3.73 min,
100%.
2-(3-Fluorophenyl)-6-methyl-4-[3-bromophenyl]-1,6-dihy-
drodipyrazolo[3,4-b:30,40-d]pyridin-3(2H)-one (6r). Pre-
1
white foam. H NMR (300 M, THF-d8) d 9.24 (br s,
pared from3-bropmhenylhydrazine and
3a.
1H), 8.78 (br s, 1H), 8.72 (s, 1H), 8.20 (s, 1H), 7.83 (d,
1H), 7.81 (d, 1H), 7.55 (m, 1H), 6.99 (m, 1H), 4.08 (s,
3H); MS (ES+) m/z 441.2, 439.2 (M+H)+1. HPLC
Method A: room temperature 5.13 min, 100%. HPLC
Method B: room temperature 3.59 min, 100%.
Recrystallization fromEtOAc/EtOH provided 2.5 g
(74%) of 6r as a tan crystalline solid. 1H NMR (DMSO-
d6) d 8.23 (s, 1H), 8.21 (d, J=1.6 Hz, 1H), 8.03 (d,
J=7.8 Hz, 1H), 7.95 (d, J=7.8 Hz, 1H), 7.87 (d,
J=11.5 Hz, 1H), 7.79 (dd, J=7.9, 0.85 Hz, 1H), 7.60 (d,
J=7.4 Hz, 1H), 7.53 (d, J=7.4 Hz, 1H), 7.08 (ddd,
J=8.3, 2.1 Hz, 1H), 4.21 (s, 3H); HPLC Method A:
room temperature, 3.70 min, 98%; HPLC Method
Example B: room temperature, 4.54 min, 98%.
2-(4-Fluorophenyl)-6-methyl-4-(3-nitrophenyl)-1,6-dihy-
drodipyrazolo[3,4-b:3,4-d]pyridin-3(2H)-one (6v). Pre-
pared from4-chlorophenylhydrazine and 3c in 25%
overall yield, mp 317–319.3 ꢁC; H NMR (DMSO-d6)
1
8.86 (s, 1H), 8.48 (d, 7.83, 1H), 8.39 (d, 7.97, 1H), 8.24
(s, 1H), 7.85 (m, 2H), 7.81 (m, 1H), 7.40 (m, 2H), 4.18
(s, 3H). HPLC Method A: room temperature 4.51 min,
99.1%. HPLC Method B: room temperature 3.61 min,
99.2%.
3-[2-(3-Fluorophenyl)-6-methyl-3-oxo-1,2,3,6-tetrahydro-
dipyrazolo[3,4-b:3,4-d]pyridin-4-yl]benzoic acid (6s). To a
solution of 6p (332 mg, 0.86 mmol) in ethylene glycol
(50 mL) was added with aqueous NaOH (4.3 mL, 1 N,
4.32 mmol). The resulted yellow solution was heated to
130 ꢁC for 20 h. The mixture was acidified with 1 N HCl
solution and partitioned between H2O and EtOAc.
Organic phase was neutralized by aqueous NaHCO3
solution. The organic layer was removed and the aqu-
eous phase was reacidified to pH 1. The resulting pre-
cipitate was collected by filtration to afford 6s (331 mg,
95%) as a white solid, mp 352–354 ꢁC. 1H NMR
(DMSO-d6) d 8.49 (s, 1H), 8.28 (s, 1H), 8.17 (dd,
J=7.82, 20.3 Hz, 1H), 8.06 (m, 1H), 7.85 (dd, J=11.5,
4-(3-Aminophenyl)-2-(4-fluorophenyl)-6-methyl-1,6-dihy-
drodipyrazolo[3,4-b:3,4-d]pyridin-3(2H)-one (6w). To a
solution of 6v (102 mg, 0.25 mmol) in EtOAc (5 mL) and
MeOH (10 mL) in a Parr bottle, Pd/C catalyst (27 mg)
was added under nitrogen and the mixture was hydro-
genated at 20 psi for 3 h. Filtration and silica gel chro-
matography (EtOAc) gave 6w in 67% yield, mp 204–
207 ꢁC. 1H NMR (DMSO-d6) 8.15 (s, 1H), 8.04 (m, 2H),
7.30 (m, 2H), 7.27 (dd, J=7.97, 8.65, 1H), 7.07 (m, 2H),

N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
3005
6.78 (d, J=7.83 Hz, 1H), 4.08 (s, 3H). HPLC Method
A: room temperature 3.64 min, 96.9%. HPLC Method
B: room temperature 2.46 min, 100%.
resulting solid was washed with cyclohexane (3 ꢄ
200 mL) and dried in a vacuum oven for 24 h to give
7.87 g (73%). H NMR (CDCl3): 8.15 (s, 1H), 4.59 (q,
2H, J=7.3 Hz), 4.07 (s, 3H), 1.51 (t, 3H, J=7.3 Hz).
1
4-(3-Fluorophenyl)-2-phenyl-6-methyl-1,6-dihydrodipyra-
zolo[3,4-b:3,4-d]pyridin-3(2H)-one
(6x).
Prepared
Ethyl-4-chloro-1-methyl-6-trifluoromethanesulfonyloxy-
1H-pyrazolo[3,4-b]pyridine-5-carboxylate (13). To a
according to the procedure for 1 from 2f. 1H NMR
(MeOH-d4) d 8.08 (s, 1H), 7.88 (m, 4H), 7.41 (m, 5H),
5.41 (s, 1H), 4.02 (s, 3H). MS (ES+) m/z 376.4
(M+H)+1. HPLC Method A: roomtepmerature
4.72 min, 100%. HPLC Method B: room temperature
3.85 min, 100%.
solution of the chloropyridone (2.56 g, 10 mmol) in
methylene chloride (50 mL) and 2,6-di-tert-butyl-4-
methylpyridine (3.07 g, 15 mmol) was cooled to ꢀ78 ꢁC
and trifluoromethanesulfonyl anhydride (3.30 mL,
18 mmol) in methylene chloride (20 mL) was added
dropwise. After the addition the suspension was stirred
at 0 ꢁC for 4 h, diluted with ethyl acetate (40 mL),
washed with saturated aqueous sodiumbicarbonate (3
ꢄ 30 mL), water, 10% aqueous hydrochloric acid (3 ꢄ
30 mL), water and then brine. The organic layer was
dried over sodiumsulfate and concentrated to give
4-(3-Fluorophenyl)-2-(4-chlorophenyl)-6-methyl-1,6-dihy-
drodipyrazolo[3,4-b:3,4-d]pyridin-3(2H)-one (6y). Pre-
pared according to the procedure for 1 from 2g. 1H
NMR (300 M, MeOH-d4) d 8.39 (s, 1H), 8.01 (d,
J=0.032 Hz, 2H), 7.92 (m, 1H), 7.71 (m, 2H), 7.62 (d,
J=0.032 Hz, 2H), 7.43 (m, 1H), 4.4 (s, 3H). MS (ES-)
m/z 394.3 (MꢀH)ꢀ1. HPLC Method A: roomtempera-
ture, 4.68 min, 100%. HPLC Method B: room tem-
perature, 4.67 min, 100%.
1
3.56 g (92%) of a tan oil that solidified in a freezer. H
NMR (CDCl3): 8.15 (s, 1H), 4.48 (q, 2H, J=7.3 Hz),
4.05 (s, 3H), 1.41 (t, 3H, J=7.3 Hz); MS (ES+) m/z:
456, 457 (M+H)+1
.
4-(3-Fluorophenyl)-6-methyl-4-(3-pyridinyl)-1,6-dihydro-
dipyrazolo[3,4-b:3,4-d]pyridin-3(2H)-one (6z). Prepared
from4-chlorophenylhydrazine and 3h in 25% overall
yield. 1H NMR (CDCl3): 8.88 (s, 1H), 8.75 (d,
J=5.4 Hz, 1H), 8.38 (d, J=7.7 Hz, 1H), 8.32 (s, 1H),
7.84 (m, 1H), 7.63 (m, 1H), 6.65 (d, J=6.5 Hz, 1H), 6.61
(m, 2H), 4.08 (s, 3H), 4.02 (q, J=5.72 Hz, 2H), 0.95 (t,
4-(3,5-Dichlorophenyl)-2-(3-fluorophenyl)-6-methyl-1,6-
dihydrodipyrazolo[3,4-b:30,40-d]pyridin-3(2H)-one (15a).
To a solution of 13 (387 mg, 1 mmol) in THF (4 mL)
was added tetrakistriphenylphosphine palladium
(115 mg, 10 mol%). After stirring for 20 min at room
temperature, 3,5-dichlorobenzeneboronic acid (285 mg,
1.5 mmol) was added, followed by 2 N K2CO3 (0.7 mL)
J=5.9 Hz, 3H). MS (CI) m/z=407 (100%, M+H)+1
.
and
benzyltriethylammonium
chloride
(319 mg,
HPLC Method A: room temperature 3.88 min, 100%.
HPLC Method B: room temperature 2.52 min, 100%.
1.4 mmol). The mixture was heated at reflux tempera-
ture for 4 h, allowed to cool to roomtemperature and
diluted with EtOAc (20 mL). The organic layer was
washed with water and brine then dried over sodium
sulfate and concentrated. The crude residue was chro-
matographed (hexane/EtOAc, 5:1) to give 127 mg (33%)
of 14a as a white solid. To this solid (120 mg,
0.31 mmol) in DMF (3 mL) was added 3-fluoro-
phenylhydrazine (99 mg, 0.78 mmol). The reaction was
heated at 100 ꢁC for 10 h and then allowed to cool to
roomtemperature. The reaction was diluted with
EtOAc (10 mL) and water (5 mL) and the resulting pre-
cipitate collected by filtration to afford 80 mg of an off-
white solid. To a suspension of this intermediate (65 mg,
0.13 mmol) in DMF (1 mL) was added NaH (14 mg,
0.41 mmol). The homogeneous solution was stirred at
100 ꢁC for 3 h, allowed to cool to roomtemperature,
diluted with EtOAc and collected by filtration to give
Ethyl-4-hydroxy-1-methyl-6-oxo-6,7-dihydro-1H-pyra-
zolo[3,4-b]pyridine-5-carboxylate (8). Sodiumemtal
(12.7 g, 0.55 mol) was dissolved ethanol (200 mL) at
roomtemperature. To this solution was added the ethyl
aminopyrazole (25 g, 0.148 mmol) and the mixture was
allowed to stir for 30 min. A solution of diethylmalonate
(80 mL, 0.52 mol) in ethanol (75 mL) was added drop-
wise over a period of 30 min during which time the
reaction was heated to reflux temperature for 56 h. After
allowing the thick mixture to cool to room temperature
water (500 mL) was added with stirring. The aqueous
phase was washed with ethyl acetate (3 ꢄ 250 mL), then
acidified with hydrochloric acid to pH 2. The resulting
white solid was filtered, washed with water then ethanol
then ethyl acetate and finally toluene. The cake was
ꢁ
1
dried in a vacuumoven at 40 C overnight to give 28.7 g
1
44 mg (22% for three steps) of 15a as a pink solid. H
(82%). H NMR (DMSO-d6): 7.35 (s, 1H), 3.77 (q, 2H,
J=7.3 Hz), 3.31 (s, 3H), 0.81 (t, 3H, J=7.3 Hz).
NMR (DMSO) d 8.29 (s, 1H), 7.88 (s, 3H), 7.79 (m,
2H), 7.66 (m, 1H), 7.09 (s, 1H), 4.08 (s, 3H); MS (ES+)
m/z: 430, 429 (M+H)+1. HPLC Method A: roomtem-
perature, 4.47 min, 100%. HPLC Method B: room
temperature, 3.47 min, 100%.
Ethyl-4-chloro-1-methyl-6-oxo-6,7-dihydro-1H-pyrazolo
[3,4-b]pyridine-5-carboxylate (9). To a solution of 8
(10 g, 42.2 mmol) in acetonitrile (165 mL) was added
benzyltriethylammonium chloride (40.4 g, 169 mmol),
followed by phosphorus oxychloride (17.6 mL,
190 mmol). The mixture was heated at 40 ꢁC for 30 min
and at reflux temperature for 2.5 h. After the mixture
cooled to roomtepmerature, water was added
(300 mL—CAUTION EXOTHERMIC!!). This mixture
was allowed to stir overnight and then filtered. The
2-(3-Fluorophenyl)-6-methyl-4-thien-3-yl-1,6-dihydrodi-
pyrazolo[3,4-b:30,40-d]pyridin-3(2H)-one (15b). Prepared
according to the same reaction sequence as 15a to give
35 mg (25% yield) of the title compound as an off-white
solid. 1H NMR (acetone-d6) d 8.89 (s, 1H), 8.08 (s, 1H),
8.01 (m, 1H), 7.76 (m, 2H), 7.45 (m, 2H), 6.94 (m, 1H),
4.03 (s, 3H); MS (ES+) m/z: 434 (M+H)+1
.

3006
N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
4-(1,3-Benzodioxol-5-yl)-2-(3-fluorophenyl)-6-methyl-1,6-
dihydrodipyrazolo[3,4-b:30,40-d]pyridin-3(2H)-one (15d).
Prepared according to the same reaction sequence as
15a to give 41 mg (39% yield) of the title compound as
an off-white solid. HPLC Method A: roomtemperature,
4.46 min, 100%. HPLC Method B: room temperature,
3.54 min, 100%.
methyl-2,6-dihydrodipyrazolo[3,4-b:30,40-d]-pyridine as
a yellow solid, mp 194.5–197 ꢁC. H NMR (CDCl3) d
1
1.21 (d, J=6.27 Hz, 6H), [minor isomer 1.26 (d, J=6.47
Hz, 6H)], 2.74 (m, 2H), [minor isomer 3.07 (m, 2H)],
[minor isomer 3.57 (d, J=12.73 Hz, 2H)], 3.74 (d,
J=12.62 Hz, 2H), 3.9 (m, 2H), 4.00 (s, 3H), [minor iso-
mer 4.26 (m, 2H)], 7.13–7.22 (m, 1H), 7.33–7.49 (m,
3H), 8.02 (s, 1H). MS m/z: 415 (M+H).
2-(3-Fluorophenyl)-4-(3-methoxyphenyl)-6-methyl-1,6-di-
(15c).
hydrodipyrazolo[3,4-b:30,40-d]pyridin-3(2H)-one
Prepared according to the same reaction sequence as
To a solution of 3-chloro-4-(2,6-dimethyl-4-morpholi-
nyl)-2-(3-flurophenyl)-6-methyl-2,6-dihydrodipyrazolo-
15a to give 39 mg (34% yield) of the title compound as
1
[3,4-b:30,40-d]-pyridine
(48 mg,
0.116 mmol)
and
an off-white solid. H NMR (DMSO) d 8.31 (s, 1H),
3:1 MeOH/THF (1.6 mL), was added 4 N NaOH
(0.4 mL). The reaction mixture was heated at reflux
temperature for 16 h. Upon cooling to room tempera-
ture, the mixture was quenched with 3 N HCl and par-
titioned with EtOAc. The organic layer was dried over
Na2SO4, filtered, and concentrated in vacuo to obtain
40 mg (87%) of the title compound as a beige powder,
mp 204.7–207 ꢁC. 1H NMR (acetone-d6) d 1.06 (d,
J=6.26 Hz, 6H), [minor isomer 1.14 (m, 6H)], 2.5 (dd,
J=12.74 Hz, 10.38, 2H), [minor isomer 3.46 (m, 2H)],
3.7 (m, 2H), 3.81 (s, 3H), [minor isomer 4.05 (m, 2H)],
4.43 (d, J=12.53 Hz, 2H), 6.86 (m, 1H), 7.37 (m, 1H),
7.59 (m, 1H), 7.71 (m, 1H), 7.78 (s, 1H). MS m/z: 395
(MꢀH). HPLC Method A: room temperature 3.59 min,
100%. HPLC Method Example B: 4.68 min, 100%.
8.00 (m, 1H), 7.91 (m, 1H), 7.75–7.40 (m, 4H), 7.01 (m,
1H), 6.95 (m, 1H), 4.21 (s, 3H), 3.96 (s, 3H). HPLC
Method A: room temperature, 4.23 min, 98.2%. HPLC
Method B: room temperature, 3.51 min, 100%.
2-(3-Fluorophenyl)-6-methyl-1,2,5,6-tetrahydrodipyrazo-
lopyridin-3,4-dione (11). To a suspension of the chloro-
pyridone (524 mg, 2.05 mmol) in toluene (15 mL) was
added the hydrazine (651 mg, 5.13 mmol) and a catalytic
amount of 2,6-di-tert-butyl-4-methylpyridine. The mix-
ture was heated at reflux temperature for 36 h slowly
becoming homogeneous then becoming a thick slurry.
The mixture was filtered and washed with cold toluene
to give 624 mg (88%) of a white powder. To 550 mg of
this product in 35 mL of THF was added NaH (187 mg,
60% in oil, 5.58 mmol). The mixture was heated for 24 h
at reflux temperature, cooled and carefully quenched
with 10% hydrochloric acid, diluted with ethyl acetate
(30 mL), filtered, then washed with water and ethyl ace-
tate to give 391 mg (83%, 73% for both steps) of a white
powder. 1H NMR (DMSO) d 7.91 (s, 1H), 7.69 (m, 3H),
7.01 (m, 1H), 3.98 (s, 3H).
2-(4-Chlorophenyl)-1,6-dimethyl-4-[3-(trifluoromethyl)phe-
nyl]-1,6-dihydrodipyrazolo[3,4-b:30,40-d]pyridin-3(2H)-one
(7a). A solution of 46284 (49 mg, 0.12 mmol) in dry
DMF ( 5 mL) was added with NaH (12 mg, 60%,
0.3 mmol) under argon at room temperature. The sus-
pension was stirred for 30 min and then followed by the
dropwise addition of methyl triflate. The mixture was
stirred at room temperature for a further 30 min, and
diluted with ethyl acetate. The organic layer was washed
with H2O (3ꢄ), brine, and dried over MgO4. The crude
residue obtained was purified by flash chromatography
on silica gel using 1:1 EtOAc/hexane as the eluant to
give 7a as a yellow solid (41 mg, 82%), mp 203.9–
204.4 ꢁC. 1H NMR (CDCl3) d 8.34 (s, 1H), 8.29 (d,
J=8.65 Hz, 1H), 8.22 (s, 1H), 7.75 (d, J=8.43 Hz, 1H),
7.63 (dd, J=8.62, 8.43 Hz, 1H), 7.50 (m, 4H), 4.30 (s,
3-Chloro-4-chloro-2-(3-fluorophenyl)-6-methyl-2,6-dihy-
drodipyrazolo[3,4-b:30,40-d]-pyridine (12). A mixture of
11 (1.5 g, 5.02 mmol) and phosphorous oxychloride
(20 mL) was heated at reflux temperature for 5 h. The
phosphorous oxychloride was removed in vacuo the
crude residue was treated with ice water and the result-
ing precipitate was neutralized to pH 7 with aqueous
Na2CO3. The slurry was extracted with EtOAc and the
organic layer was dried over Na2SO4, filtered, and con-
centrated in vacuo to afford 1.2 g (75%) of the title
3H), 3.61 (s, 3H). MS (ES+) m/z 458 (M+H)+1
.
HPLC Method A: room temperature, 4.44 min, 100%.
HPLC Method B: room temperature, 4.82 min, 100%.
1
compound as a light-yellow solid. H NMR (DMSO) d
4.1 (s, 3H), 7.54 (br m, 1H), 7.63–7.82 (m, 3H), 8.32 (s,
1H). MS m/z: 336 (M+H).
7-Methyl-2-phenyl-5-[3-(trifluoromethyl)phenyl]-1,7-dihy-
dro-4H-pyrazolo[40,30:5,6]pyrido[4,3-d]pyrimidin-4-one
(19). Benzamidine hydrochloride salt (5.0 g, 32 mmol)
was dissolved in CH2Cl2 (100 mL) and H2O (100 mL)
and neutralized by adding NaOH (12.8 mL, 2.5 M,
32 mmol). After stirring at room temperature for 2 h,
organic layer was separated, washed with brine and
dried with MgO4. Benzamidine was obtained as white
solid (3.01 g, 78%) and used for the following reaction.
Starting material chloride (0.43 g, 1.12 mmol) was dis-
solved in 20 mL of ethanol and added with benzamidine
(0.404 g, 3.36 mmol). The mixture was heat to reflux for
2 days and then cool to roomtemperature. After
removing ethanol in vacuo, the residue was partitioned
between EtOAc and H2O. Regular workup and chro-
4-(2,6-Dimethyl-4-morpholinyl)-2-(3-fluorophenyl)-6-
methyl-1,6-dihydrodipyrazolo[3,4-b:30,40-d]pyridin-3(2H)-
one (15e). A mixture of 12 (100 mg, 0.315 mmol) and
2,6-di-methylmorpholine (0.097 mL, 0.787 mmol) in
DMF (0.4 mL) was heated at 140 ꢁC for 4 h. The reac-
tion mixture was cooled to room temperature, quenched
with water, and the pH was adjusted to ꢅ3 with 3 N
HCl. The aqueous mixture was extracted with EtOAc
and the organic layer was dried over Na2SO4, filtered,
and concentrated in vacuo. The crude material was
purified by flash chromatography on silica gel using
0.5% MeOH/CHCl3 to afford 90 mg (69%) of 3-chloro-
4-(2,6-dimethyl-4-morpholinyl)-2-(3-flurophenyl)-6-

N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
3007
matography afford the desired product (0.208 g, 44%)
as a white solid with startꢁing material (0.129 g, 30%)
Method A: room temperature, 3.40 min, 100%. HPLC
method B: room temperature, 4.63 min, 100%.
1
recovered, mp 344.3–346.1 C. H NMR (DMSO-d6) d
8.58 (s, 1H), 8.34 (d, J=7.01 Hz, 2H), 7.90 (m, 2H),
7.81 (d, J=6.83 Hz, 1H), 7.65 (m, 4H), 4.11 (s, 3H). MS
(ES+) m/z 421 (M+H)+1. HPLC Method A: room
temperature 4.14 min, 100%; HPLC Method B:
4.89 min, 100%.
2-(3-Fluorophenyl)-6-phenyl-4-[3-(trifluoromethyl)phenyl]-
1,6-dihydrodipyrazolo[3,4-b:3,4-d]pyridin-3(2H)-one (27).
Prepared according to the procedure of 1 from3-fluoro-
phenyl hydrazine and 26a (0.03 g, 21% overall) as a
1
white foam. H NMR (300 M, THF-d8) d 11.45 (s, 1H),
8.61 (s, 1H), 8.41 (m, 3H), 7.80 (m, 3H), 7.64 (m, 1H),
7.50 (m, 4H), 7.35 (m, 1H), 6.99 (m, 1H) MS (ES-) m/z
488.2 (MꢀH)ꢀ1. HPLC Method A: roomtemperature
5.15 min, 98.2%. HPLC method B: room temperature
4.71 min, 100%.
Ethyl 2-[3-(trifluoromethyl)phenyl]nicotinate (22). In a
flask flushed with nitrogen was placed ethyl 2-chloro-
nicotinate (1.24 g, 6.67 mmol), DME (20 mL), and tet-
rakistriphenylphosphine palladium (385 mg, 0.33 mmol,
0.05 equiv). Potassium carbonate (1.84 g, 13.34 mmol,
2 equiv),
in
water
(4 mL)
and
m-tri-
6-Benzyl-2-(4-fluorophenyl)-4-[3-(trifluoromethyl)phenyl]-
1,6-dihydrodipyrazolo[3,4-b:3,4-d]pyridin-3(2H)-one (32).
Prepared according to the procedure of 1 from4-fluoro-
phenyl hydrazine and 26b. Cyclization and flash chro-
matography on silica gel (25:60:15 to 40:40:20 EtOAc/
hexane/CH2Cl2) gave 32 as a brownish foamin 82%
yield. 1H NMR (CDCl3+acetone-d6) d 8.36 (s, 3H),
8.33 (s, 3H), 8.14 (s, 1H), 7.84–7.80 (m, 2H), 7.70 (d,
J=8.2 Hz, 1H), 7.62 (m, 1H), 7.36 (d, J=7.2 Hz, 2H),
7.29–7.21 (m, 3H), 7.13 (t, J=8.8 Hz, 2H). MS (ES+)
m/z 504.2 (M+H)+1. HPLC Method A: roomtem-
perature 5.00 min, 97.2%. HPLC method B: room tem-
perature 4.47 min, 100%.
fluoromethylphenylboronic acid (1.65 g, 8.67 mmol,
1.3 equiv) in 4 mL of DME were added sequentially, and
the mixture was heated to reflux temperature for 20 h.
Evaporation of the solvent left a residue, which was
purified by chromatography on silica gel to give 1.67 g
1
of desired coupling product 22 in 85% yield. H NMR
(CDCl3) d 8.82 (d, J=6.9 Hz, 1H), 8.25 (d, J=8.5 Hz,
1H), 7.83 (s, 1H), 7.75 (m, 2H), 7.6 (dd, J=6.5 Hz,
J=8.3 Hz, 1H), 7.4 (dd, J=6.6 Hz, J=6.5 Hz, 1H), 4.20
(q, J=6.3 Hz, 2H), 1.1 (t, J=6.2 Hz, 3H). MS (CI) m/
z=296 (M+H+).
4-Bromo-2-[3-(trifluoromethyl)phenyl]nicotinic acid (23).
To a solution of 22 (1.38 g, 4.71 mmol) dissolved in
DMSO (15 mL) and methanol (15 mL) was added
with a 2 N NaOH solution (14 mL, 28 mmol, 6 equiv).
The mixture was stirred at room temperature for 3 h.
The mixture was then diluted with ethyl acetate,
neutralized with 2 N HCl, and washed with water
(3ꢄ), brine, and dried. Concentration in vacuo gave
1.18 g of 2-[3-(trifluoromethyl)phenyl]nicotinic acid in
95% yield. 1H NMR (CDCl3) d 8.82 (d, J=4.2 Hz,
1H), 8.27 (d, J=6.3 Hz, 1H), 7.78 (s, 1H), 7.54 (m,
4H). MS (CI) m/z=266 (MꢀH+). This intermediate
was dissolved in dry THF (20 mL) and TMEDA
(0.31 mL, 2.1 equiv). The solution was cooled to ꢀ78 ꢁC
and added to a solution of 2.5 M n-BuLi (0.84 mL,
2.1 mmol, 2.1 equiv) dropwise via cannula. After the
addition was complete, the resulting yellow solution
was stirred for 1 h. A solution of trimethylpheny-
lammonium tribromide (789 mg, 2.1 mmol, 2.1 equiv) in
THF (8 mL) was added slowly and the mixture was
kept at this temperature for 0.5 h then slowly warm to
room temperature. Regular workup and column chro-
matography provided 155 mg of product 23 in 45%
2-(4-Fluorophenyl)-4-[3-(trifluoromethyl)phenyl]-1,6-dihy-
drodipyrazolo[3,4-b:3,4-d]pyridin-3(2H)-one (29). To a
suspension of 32 (964 mg, 1.91 mmol) in methanol
(96 mL) were added 4 M HCl (20 mL, in 1,4-dioxane)
and Pd/C (10%, 650 mg). The reaction mixture was
stirred at roomtemperature under 1 atmH
for 18 h
2
before it was filtered through Celite and rinsed with
methanol. The volatiles were evaporated and the residue
was redistributed in EtOAc and water. After adjustment
of the pH to 8, the layers were separated and the aqu-
eous was further extracted with EtOAc. The combined
organic layers were dried over K2CO3 and evaporated
to yield the title compound in quantitative yield as an
orange solid. MS (ES+) m/z 414.2 (M+H)+1. HPLC
Method A: room temperature, 4.44 min, 100%. HPLC
method B: room temperature, 3.33 min, 100%.
1-Benzyl-2-(4-fluorophenyl)-4-[3-(trifluoromethyl)phenyl]-
1,6 - dihydrodipyrazolo[3,4 - b:30,40 - d]pyridin - 3(2H) - one
(28a). To a solution of 29 (720 mg, 1.74 mmol) in DMF
(20 mL) at 0 ꢁC were added iPr2NEt (606 mL, 3.48 mmol)
and BnBr (228 mL, 1.91 mmol). The reaction mixture
was maintained at 4 ꢁC for 16 h before partitioning into
EtOAc/water. Extractive workup followed by column
chromatography on silica gel (30–40% EtOAc in hex-
anes) gave the title compound in 78% yield.
1
yield. H NMR (MeOH) d 8.31 (d, J=4.1 Hz, 1H), 8.0
(s, 1H), 7.94 (m, 1H), 7.7 (m, 4H). MS (CI) m/z=345
(MꢀH+).
2-(3-Fluorophenyl)-4-[3-(trifluoromethyl)phenyl]-1,2-dihy-
dro-3H-pyrazolo[4,3-c]pyridin-3-one (24). Treatment of
23 with TMS-diazomethane, addition of 3-fluorophenyl
hydrazine and cyclization in refluxing ethanol gave
compound 24 in 39% yield, mp 185–187 C. H NMR
(CDCl3) d 8.80 (d, J=6.10 Hz, 1H), 8.47 (s, 1H), 8.39
(d, J=7.75 Hz, 1H), 7.96 (d, J=7.85 Hz, 1H), 7.81 (dd,
J=7.75, 7.76 Hz, 1H), 7.71 (dd, J=8.96, 7.28 Hz, 2H),
7.60 (m, 1H), 7.54 (m, 1H), 7.33 (m, 1H). HPLC
6-Ethyl-2-(4-fluorophenyl)-4-[3-(trifluoromethyl)phenyl)-
1,2-dihydro-3H-pyrazolo[4,3-c]pyridin-3-one (30). To
28a in methanol (1 mL) was added NaH (0.70 mmol) at
0 ꢁC and the reaction mixture was stirred at the same
temperature for 20 min before iodoethane (0.89 mmol)
was introduced. The reaction mixture was allowed to
warmto roomtemperature and stirring was continued
for 4.5 days before aqueous workup. Silica gel chroma-
ꢁ
1

3008
N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
tography gave 28b in 38% yield along with 32% of its
N-1 regioisomer. 28b was subjected to hydrogenolysis
using Pd(OH)2 in methanol to give the title compound
in 41% yield after silica gel chromatography (70%
5 ꢁC for an additional 1 h and filtered. The crude mate-
rial was dissolved in hot water (250 mL), filtered twice
and the crystals collected upon cooling to give 9.6 g,
71% of 2-chloro-5-hydrazinobenzoic acid hydrochloride
(34b). 1H NMR (DMSO-d6) d 8.49 (s, 1H), 7.46 (d,
J=6.3 Hz, 1H), 7.35 (s, 1H), 7.06 (d, J=6.3 Hz, 1H).
EtOAc in hexanes). MS (ES+) m/z 442.3 (M+H)+1
.
2-(4-Fluorophenyl)-6-propyl-4-[3-(trifluoromethyl)phenyl]-
1,6-dihydrodipyrazolo[3,4-b:3,4-d]pyridin-3(2H)-one (31).
The title compound was prepared according to the pro-
cedure for 30 except that allyl bromide was used in the
alkylation step to give 28c. Hydogenolysis afforded 31
in 43% yield (for two steps). MS (ES+) m/z 456.4
Starting from4-amino-2-chlorobenzoic acid
33c, 2-
chloro-4-hydrazinobenzoic acid 34c was prepared in the
same manner. Yield: 7.62 g, 57%. A solution of 2-
chloro-5-hydrazino-benzoic acid-HCl 34b (2.32 g,
10.4 mmol) and ethylene glycol (15 mL) was treated with
sodium-tert-butoxide (2.0 g, 20.8 mmol) in portions
maintaining the temperature <35 ꢁC. Following the
addition this solution was inversely added to a hot
(120 ꢁC) solution of 3a (2.0 g, 5.2 mmol) and ethylene
glycol (12 mL). The reaction mixture was stirred at
125 ꢁC for 15 h. After allowing the mixture to cool to
room temperature, more sodium-tert-butoxide (0.76 mg,
7.9 mmol) was added and heating was resumed (125 ꢁC)
for an additional 2–3 h. The reaction mixture was
allowed to cool to roomtemperature and quenched with
dilute HCl (0.6 N) and partitioned with EtOAc. The
layers were separated and the organic layer was dried
over Na2SO4, filtered, and concentrated in vacuo. The
crude residue obtained was subjected to a hot tritura-
tion in EtOAc/MeOH (1.25:1) for 1 h at reflux. When
cooled to roomtemperature, the slurry was filtered and
washed with a 4:1 EtOAc/MeOH solution to afford
1.2 g (42%) of the title compound as a tan solid, mp
335.6–338 ꢁC. 1H NMR [acetone-d6 (5% v/v DMSO-d6)]
d 3.74 (s, 3H), 7.21 (d, J=8.82 Hz, 1H), 7.34 (t, J=7.81
Hz, 1H), 7.44 (d, J=7.78 Hz, 1H), 7.82 (br s, 2H), 7.96
(d, J=7.72 Hz, 1H), 8.02 (br s, 2H). MS m/z: 488
(M+H). HPLC Method A: room temperature 4.69 min,
100%. HPLC method B: room temperature 3.64 min,
100%.
(M+H)+1
.
2-(4-Fluorophenyl)-4-[3-(trifluoromethyl)phenyl]-1,2-dihy-
dro-3H-pyrazolo[3,4-d]thieno[2,3-b]pyridin-3-one (7).
The title compound was prepared according to the pro-
cedure for 6a except that 2-amino-thiophene-3-car-
boxylic acid methyl ester was used in the condensation
1
with 2a. H NMR (CDCl3) d 8.03 (d, J=7.14 Hz, 1H),
7.98–7.93 (m, 2H), 7.87 (s, 1H), 7.59 (d, J=8.0 Hz, 1H),
7.49 (d, J=8.0 Hz, 1H), 7.14 (d, J=5.2 Hz, 1H), 6.82
(app t, J=9.1 Hz, 2H).
3-(6-Methyl-3-oxo-4-[3-(trifluoromethyl)phenyl]-3,6-dihy-
drodipyrazolo[3,4-b:3,4-d]pyridin-2(1H)-yl)benzoic acid
(35a). To a solution of 3-hydrazino-benzoic acid 34a
(7.14 g, 0.047 mol) and ethylene glycol (160 mL) was
added sodium-tert-butoxide (4.5 g, 0.047 mol). The mix-
ture was stirred at 75 ꢁC for 1 h. Upon cooling to room
temperature 3a (6 g, 15.6 mmol) was added. Heating was
resumed and increased to 100 ꢁC where the reaction
mixture was allowed to stir for an additional 16 h. After
16 h, additional sodium tert-butoxide (2.5 g, 0.026 mol)
was added. The mixture was allowed to cool to room
temperature and quenched with water (200 mL) fol-
lowed by acidification using 3 N HCl to pH ꢅ3. The
organic layer was dried over Na2SO4, filtered, and con-
centrated in vacuo. The crude residue was subjected to a
hot trituration (65 ꢁC for 1 h) in EtOAc/MeOH (1.5:1)
with stirring at roomtemperature for 3 h. The slurry
was filtered and washed with cold EtOAc to afford 5.8 g
(82%) of the title compound as a tan powder, mp 289–
291 ꢁC. 1H NMR [acetone-d6 (5% v/v DMSO-d6)] d 4.05
(s, 3H), 7.51 (t, J=7.97 Hz, 1H), 7.63 (dd, J=7.83 Hz,
7.55, 1H), 7.75 (m, 2H), 8.11 (s, 1H), 8.2 (d, J=7.97 Hz,
1H), 8.3 (d, J=7.83 Hz, 1H), 8.36 (s, 1H), 8.44 (s, 1H).
MS (ES+) m/z: 454 (M+H)+1. HPLC Method A:
room temperature, 3.52 min, 100%. HPLC method B:
room temperature, 3.94 min, 100%.
2-Fluoro-5-[6-methyl-3-oxo-4-[3-(trifluoromethyl)phenyl]-
3,6-dihydrodipyrazolo[3,4-b:30,40-d]pyridin-2(1H)-yl]ben-
zoic acid (35c). Prepared in 92% yield according to the
procedure above from 34d and 3a, m p>375 ꢁC. 1H
NMR (methanol-d4) 8.25 (s, 1H), 8.15 (m, 3H), 7.82 (m,
3H), 7.19 (m, 1H), 4.13 (br s, 3H). MS (ES+) m/z 472.1
(M+H)+1. HPLC Method A: roomtepmerature
4.56 min, 100%. HPLC method B: room temperature
3.50 min, 100%.
Methyl 3-(6-methyl-3-oxo-4-[3-(trifluoromethyl)phenyl]-
3,6-dihydrodipyrazolo[3,4-b:3,4-d]pyridin-2(1H)-yl)benzo-
ate (35d). To a dry, single-necked, round-bottomed
flask was added 35a, (300 mg, 0.662 mmol) followed by
MeOH (2 mL) and HCl (4 M solution in dioxane,
0.7 mL). The resulting suspension was heated to reflux
for 10 h. The reaction mixture was quenched with H2O
and partitioned with EtOAc and saturated NaCl solu-
tion. The organic layer was dried over Na2SO4, filtered,
and concentrated. The crude residue obtained was tri-
turated fromdiethyl ether followed by filtration to
afford 205 mg (66%) of the title compound as a bright
2-Chloro-5-(6-methyl-3-oxo-4-[3-(trifluoromethyl)phenyl]-
3,6-dihydrodipyra-zolo[3,4-b:3,4-d]pyridin-2(1H)-yl)ben-
zoic acid (35b). To solution of 5-amino-2-chlorobenzoic
acid 33b (10.3 g, 60 mmol) in hydrochloric acid 40 mL at
0 ꢁC was added NaNO2 (4.8 g, 70 mmol) in water
(20 mL) at a rate that maintained the temperature below
10 ꢁC. After stirring for 30 min, a solution of tin chlo-
ride dihydrate (33.9 g, 150 mmol) in water (10 mL) and
HCl (40 mL) was added at a rate that maintained the
temperature below 10 ꢁC. Stirring of this viscous mix-
ture was maintained by periodically adding additional
water and HCl as necessary. The mixture was stirred at
orange solid, mp 275–277 ꢁC. H NMR (DMSO-d6) d
1
3.91 (s, 3H), 4.13 (s, 3H), 7.6–8.08 (m, 4H), 8.15–8.42
(m, 4H), 8.51 (s, 1H). MS (ES+) m/z: 468.2 (M+H)+1

N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
3009
HPLC Method A: room temperature, 3.99 min, 100%.
HPLC Method Example B: room temperature,
4.79 min, 100%.
HPLC Method A: room temperature 3.53 min, 97.2%;
HPLC Method Example B: room temperature
4.92 min, 100%.
Methyl-2-chloro-5-(6-methyl-3-oxo-4-[3-(trifluorom-
ethyl)phenyl]-3,6-dihydrodipyrazolo[3,4-b:30,40-d]-pyridin-
2(1H)-yl)benzoate (35e). Prepared by the method
described for 35d starting from 35b. Yield: 65 mg
2-Chloro-4-(6-methyl-3-oxo-4-[3-(trifluoromethyl)phenyl]
-3,6-dihydrodipyrazolo[3,4-b:3,4-d]pyridin-2(1H)-yl)ben-
zoic acid (36b). To a solution of THF (4 mL), MeOH
(1 mL), and 1 N LiOH (2.57 mL) was added 36e
(129 mg). The mixture was stirred overnight and acid-
ified with aqueous HCl to pH 4. The aqueous layer was
extracted with EtOAc, dried and concentrated to give a
white foamthat was recrystallized fromhot EtOAc to
give the title compound as a white solid (121 mg, 97%).
1H NMR (DMSO-d6) d 8.55 (d, J=1.6 Hz, 1H), 8.39–
8.30 (m, 3H), 7.94 (s, 1H), 7.88 (d, J=8.7 Hz, 1H), 7.77
(d, J=7.7 Hz, 1H), 7.68 (m, 1H), 3.97 (s, 3H).
(63%) of an orange-brown solid, mp 211–214 ꢁC. H
1
NMR (acetone-d6): 3.75 (s, 3H), 3.89 (s, 3H), 7.29 (d,
J=8.86 Hz, 1H), 7.45 (m, 1H), 7.55 (m, 1H), 7.77 (s,
1H), 8.39 (d, J=7.79 Hz, 1H), 8.44 (s, 1H), 8.46 (s,
1H), 8.70 (s, 1H). MS m/z 502 (M+H). HPLC
Method A: 4.13 min, 100%. HPLC Method Example
B: 4.87 min, 97.29%.
Methyl
2-chloro-4-(6-methyl-3-oxo-4-[3-(trifluorome-
thyl)phenyl] - 3,6 - dihydrodipyrazolo[3,4 - b:3,4 - d]pyridin-
2(1H)-yl)benzoate (36e). To a solution of methyl 2-
chloro-4-nitrobenzoate (5 g, 23 mmol) in EtOAc
(100 mL) was added a tin chloride dihydrate (17.6 g,
78 mmol). After stirring overnight at room temperature
a solution of 1 N NaOH was added to bring the pH 9.
The aqueous solution was extracted with EtOAc (5 ꢄ
100 mL), washed with water, brine and concentrated to
give methyl 4-amino-2-chlorobenzoate as a white solid
2.74 g, 64%. This ester was then treated according to the
procedure for the preparation of 2-chloro-5-hydrazino-
benzoic acid hydrochloride (the internal temperature of
these reactions were maintained at ꢀ5 ꢁC to prevent
possible ester hydrolysis) to give methyl 2-chloro-4-
hydrazinobenzoate 34e as a white crystalline solid
Methyl 4-(6-methyl-3-oxo-4-[3-(trifluoromethyl)phenyl]-
3,6-dihydrodipyrazolo[3,4-b:3,4-d]pyridin-2(1H)-yl)benzo-
ate (36d). Obtained in 57% yield according to the pro-
cedure for 35d starting from 36a: mp 292–294.2 ꢁC. H
1
NMR (DMSO-d6) d 8.31 (m, 4H), 8.01 (s, 1H), 7.94 (d,
J=8.8 Hz, 2H), 7.63 (d, J=7.7 Hz, 1H), 7.55 (t, J=7.7
Hz, 1H), 3.94 (s, 3H), 3.68 (s, 3H); MS (ES+) m/z 468.1
(M+H)+1. HPLC Method A: roomtemperature,
4.07 min, 97.4%; HPLC Method Example B: room
temperature, 4.85 min, 100%.
Methyl-2-fluoro-4-[6-methyl-3-oxo-4-[3-(trifluoromethyl)-
phenyl]-3,6-dihydrodipyrazolo[3,4-b:30,40-d]pyridin-2(1H)-
yl]benzoate (36f). Isolated in 95% yield according to
the procedure for 36e, mp 260–262.4 ꢁC. 1H NMR
(CDCl3) 8.16 (s, 1H), 8.08 (d, J=7.40 Hz, 1H), 7.97 (s,
1H), 7.75 (dd, J=8.23 Hz, 8.32, 1H), 7.51 (m, 4H), 4.14
1
(2.35 g, 50%). H NMR (DMSO-d6) 8.01 (s, 1H), 7.68
(dd, J=8.7 Hz, 1H), 6.86 (d, J=1.9 Hz, 1H), 6.69 (dd,
J=8.7 Hz, 1.9, 1H) 5.02 (br s, 2H) 3.74 (s, 3H).
(s, 3H), 3.81 (s, 3H). MS (ES+) m/z 486.2 (M+H)+1
.
HPLC Method A: room temperature 4.04 min, 100%.
HPLC Method B: 4.86 min, 100%.
Starting frommethyl 2-chloro-5-nitrobenzoate
33f,
methyl 2-chloro-5-hydrazinobenzoate 34f was prepared
in the same manner. Yield: 7.4 g, 80% 1H NMR
(DMSO-d6) d 8.43 (br s, 1H) 8.01 (s, 1H), 7.52 (dd,
J=2.6 Hz, 1H), 7.26 (d, J=8.7 Hz, 1H), 7.22 (dd,
J=8.7 Hz, 2.6, 1H), 3.86 (s, 3H). 2.92 (br s, 2H).
2-Fluoro-4-[6-methyl-3-oxo-4-[3-(trifluoromethyl)phenyl]-
3,6-dihydrodipyrazolo[3,4-b:30,40-d]pyridin-2(1H)-yl]ben-
zoic acid (36c). Obtained in 86% yield from 36f accord-
ing to the procedure for 36b, mp 235 ꢁC (dec.). 1H NMR
(methanol-d4) 8.27 (s, 1H), 8.21 (d, J=7.64 Hz, 1H),
8.14 (s, 1H), 7.97 (m, 2H), 7.85 (m, 2H), 7.68 (dd,
J=7.73, 7.77 Hz, 1H), 4.12 (s, 3H). MS (ES+) m/z
472.1 (M+H)+1. HPLC Method A: roomtemperature
4.63 min, 100%. HPLC method B: room temperature
3.56 min, 100%.
Methyl-2-fluoro-5-[6-methyl-3-oxo-4-[3-(trifluoromethyl)-
phenyl]-3,6-dihydrodipyrazolo[3,4-b:30,40-d]pyridin-2(1H)-
yl]benzoate (35f). Methyl 2-fluoro-4-hydrazinobenzoate
34g was prepared frommethyl 2-fluoro-4-nitrobenzoate
33g as described in the procedure for 36e. The title
compound was prepared in 53% yield following the
procedure above, mp 263–265.8 ꢁC. H NMR (acetone-
1
General procedure for the preparation of dihyrodipyr-
azolopyridine carboxamides
d6) d 8.42 (s, 1H), 8.35 (m, 4H), 7.55 (m, 2H), 7.48 (m,
2H), 3.82 (s, 3H), 1.95 (s, 3H). MS (ES+) m/z 486.2
(M+H)+1. HPLC Method A: roomtemperature,
3.95 min, 100%; HPLC Method B: room temperature,
4.73 min, 96.3%.
To a dry, single-necked, round-bottomed flask were
added the appropriate dihydrodipyrazolo pyridine ben-
zoic acid and DMF (0.2 M) followed by the subsequent
addition of the desired amine (3 equiv), EDC HCl salt
(2 equiv), and diisopropylethylamine (1.8 equiv). After
the additions were complete, the solution was allowed
to stir at room temperature for 16 h. The reaction mix-
ture was diluted with ethyl acetate and partitioned with
dilute acid (0.5 N HCl). After the layers were separated,
the organic layer was washed with saturated aqueous
NaCl, dried over Na2SO4, filtered, and concentrated on
4-(6-Methyl-3-oxo-4-[3-(trifluoromethyl)phenyl]-3,6-dihy-
drodipyrazolo[3,4-b:3,4-d]pyridin-2(1H)-yl)benzoic acid
(36a). Obtained in 69% yield according to the proce-
dure for 35a starting from4-hydrazinobenzoic acid 34h,
mp 315–318.6 ꢁC; H NMR (DMSO) d 8.25 (m, 3H),
1
8.07 (br s, 4H), 7.94 (d, J=7.5 Hz, 1H), 7.78 (t, J=7.5
Hz, 1H), 4.15 (s, 3H); MS (ES+) m/z 454.1 (M+H)+1

3010
N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
a rotary evaporator. The crude residue obtained was
further purified by flash chromatography where needed.
yield, mp 219 ꢁC (dec.). 1H NMR (CDCl3) 8.18 (s,
1H), 8.07 (d, J=6.38 Hz, 2H), 7.55 (br s, 1H), 7.42
(m, 3H), 6.89 (m, 1H), 4.14 (s, 3H), 3.65 (m, 4H),
3.45 (m, 2H), 3.14 (m, 2H). MS (ES+) m/z 541
(M+H)+1. HPLC Method A: roomtepmerature
4.59 min, 100%. HPLC Method B: room temperature
4.57 min, 100%.
N-(3-Hydroxypropyl)-4-[6-methyl-3-oxo-4-[3-(trifluoro-
methyl)phenyl]-3,6-dihydrodipyrazolo[3,4-b:30,40 -d]pyri-
din-2(1H)-yl]benzamide (36g). Obtained in 42% yield,
mp 193 ꢁC (dec.). H NMR (DMSO-d6) d 8.21 (s, 1H),
1
8.16 (d, J=7.69 Hz, 1H), 8.05 (m, 3H), 7.84 (d, J=8.79
Hz, 2H), 7.69 (d, J=8.35 Hz, 1H), 7.61 (dd, J=7.69,
8.73 Hz, 1H), 4.87 (s, 1H), 4.05 (s, 3H), 3.61 (t, J=6.32
Hz, 2H), 3.44 (t, J=6.80 Hz, 2H), 3.26 (br s, 1H), 1.79
(m, 2H). MS (ES+) m/z 511 (M+H)+1. HPLC Method
A: room temperature 3.31 min, 100%. HPLC Method
B: room temperature 4.88 min, 98.9%.
6-Methyl-2-[3-(4-morpholinylcarbonyl)phenyl]-4-[3-(tri-
fluoromethyl)phenyl] - 1,6 - dihydrodipyrazolo - [3,4 - b:3,4 -
d]pyridin-3(2H)-one (35h). Obtained in 66% yield
(0.113 g) of a yellow solid after flash chromatography
(80:20%, EtOAc/EtOH), mp 234.8 C (dec.). H NMR
(CD3OD): 3.33–3.77 (br m, 8H), 4.08 (s, 3H), 7.24 (d,
J=7.28 Hz, 1H), 7.52 (dd, J=7.69 Hz, 7.42, 1H), 7.64
(dd, J=7.89 Hz, 8.1, 1H), 7.76 (d, J=7.69 Hz, 1H),
7.94, (br s, 2H), 8.12 (s, 1H), 8.22 (d, J=7.55 Hz, 1H),
8.28 (s, 1H). MS m/z: 525 (M+H). HPLC Method A:
room temperature 4.41 min, 100%. HPLC method B:
room temperature 3.44 min, 100%.
ꢁ
1
N-(3-Methoxypropyl)-4-(6-methyl-3-oxo-4-[3-(trifluoro-
methyl)phenyl]-3,6-dihydrodipyrazolo[3,4-b:3,4-d]pyridin-
2(1H)-yl)benzamide (36h). Isolated in 85% yield as yel-
ꢁ
1
low solid. mp: 160 C (dec). H NMR (CDCl3) 8.24 (d,
J=9.2 Hz, 1H), 8.2 (s, 1H), 7.61 (d, J=7.96 Hz, 2H),
7.49 (m, 1H), 7.38 (d, J=7.82 Hz, 2H), 7.35 (m, 1H),
4.19 (s, 3H), 3.44 (m, 4H), 3.28 (s, 3H), 1.77 (m, 2H).
MS (ES+) m/z 525 (M+H)+1. HPLC Method A: room
temperature 3.48 min, 96%. HPLC Method B: room
temperature 4.33 min, 99%.
6-Methyl-2-[3-(1-pyrrolidinylcarbonyl)phenyl]-4-[3-(tri-
fluoromethyl)phenyl] - 1,6 - dihydrodipyrazolo[3,4 - b:3,4 -
d]pyridin-3(2H)-one (35k). Yield: 0.06 g (36%) of a light
yellow solid after flash chromatography (95:5%,
CH2Cl2/MeOH), mp 255–258 ꢁC. 1H NMR (CDCl3):
1.9 (m, 2H), 2.0 (m, 2H), 3.36 (m, 2H), 3.7 (m, 2H),
4.26 (s, 3H), 6.76 (d, J=7.28 Hz, 1H), 7.12 (dd,
J=7.89 Hz, 7.83, 1H), 7.33 (d, J=8.38 Hz, 1H), 7.6
(dd, J=7.76, 7.69 Hz, 1H), 7.72 (d, J=7.83 Hz, 1H),
7.8 (s, 1H), 8.24 (d, J=7.55 Hz, 1H), 8.3 (s, 1H), 8.41
(s, 1H). MS m/z: 507 (M+H). HPLC Method A: room
temperature 4.03 min, 100%. HPLC method B: room
temperature 3.65 min, 100%.
N-(3-Methoxypropyl)-3-(6-methyl-3-oxo-4-[3-(trifluoro-
methyl)phenyl]-3,6-dihydrodipyrazolo[3,4-b:3,4-d]pyridin-
2(1H)-yl)benzamide (35g). Yield: 0.025 g (14%) of an
orange-brown solid after flash chromatography (95:5%,
CH2Cl2/MeOH), mp 180.9–183.7 ꢁC. 1H NMR
(CDCl3): 3.36 (s, 3H), 3.4–3.57 (br m, 4H), 4.24 (s, 3H),
7.15 (br s, 2H), 7.32 (br s, 1H), 7.57 (m, 1H), 7.61–7.75
(br m, 2H), 7.84 (s, 1H), 8.16–8.27 (m, 2H), 8.33 (s, 1H).
MS m/z: 523 (MꢀH). HPLC Method A: roomtem-
perature 3.53 min, 100%. HPLC Method B: room tem-
perature 4.84 min, 100%.
6-Methyl-2-[4-(1-pyrrolidinylcarbonyl)phenyl]-4-[3-(tri-
fluoromethyl)phenyl]-1,6-dihydrodipyrazolo[3,4-b:3,4-d]
pyridin-3(2H)-one (36n). Isolated in 78% yield as yel-
1
6-Methyl-2-{4-[(4-methyl-1-piperazinyl)carbonyl]phenyl}-4
-[3-(trifluoromethyl)phenyl]-1,6-dihydrodipyrazolo[3,4-b:
3,4-d]pyridin-3(2H)-one (36i). Obtained in 70% yield,
low solid. H NMR (DMSO-d6) 8.30 (s, 1H), 8.19 (d,
8.3 Hz, 1H), 8.01 (d, 7.9 Hz, 2H), 7.89 (s, 1H), 7.66 (d,
7.88 Hz, 1H), 7.60 (m, 1H), 7.36 (d, 8.21 Hz, 2H), 3.94
(s, 3H), 3.60 (m, 4H), 2.58 (m, 4H); MS (CI) m/z=507
(95%, M+H+). HPLC Method A: roomtemperature
2.77 min, 97%; HPLC Method B: room temperature
3.87 min, 98%.
ꢁ
1
mp 191–193.5 C. H NMR (CDCl3) 8.32 (s, 1H), 8.29
(s, 1H), 8.25 (d, J=7.97 Hz, 1H), 8.04 (d, J=8.51 Hz,
2H), 7.89 (d, J=7.83 Hz, 1H), 7.75 (dd, J=7.97 Hz,
8.41, 1H), 7.60 (d, J=8.51 Hz, 2H), 4.10 (s, 3H), 3.4 (m,
4H), 3.18 (m, 2H), 2.77 (br s, 3H), 2.43 (m, 2H). MS
(ES+) m/z 536 (M+H)+1. HPLC Method A: room
temperature 2.69 min, 100%. HPLC Method B: room
temperature 3.80 min, 100%.
Methyl-(2R)-1-[3-(6-methyl-3-oxo-4-[3-(trifluoromethyl)-
phenyl]-3,6-dihydrodipyrazolo[3,4-b:3,4-d]pyri-din-2(1H)-
yl)benzoyl]-2-pyrrolidinecarboxylate
(35m).
Yield:
0.114 g (61%) of a gold-orange solid fromflash chro-
matography (95:5%, CH2Cl2/MeOH), mp 180 ꢁC (dec.).
1H NMR (CDCl3): 1.86–2.16 (m, 3H), 2.39 (m, 1H),
3.56–3.72 (m, 2H), 3.9 (s, 3H), 4.26 (s, 3H), 4.7 (dd,
J=8.17 Hz, 4.81, 1H), 6.92 (d, J=7.42 Hz, 1H), 7.16
(dd, J=7.97 Hz, 8.1, 1H), 7.3 (br s, 1H), 7.59 (dd, J=7.69,
7.83 Hz, 1H), 7.72 (d, J=7.97 Hz, 1H), 7.86 (s, 1H), 8.24
(d, J=7.83 Hz, 1H), 8.32 (s, 2H). MS m/z: 565 (M+H).
HPLC Method A: room temperature 3.71min, 100%.
HPLC Method B: room temperature 4.63min, 100%.
6-Methyl-2-[4-(4-morpholinylcarbonyl)phenyl]-4-[3-(tri-
fluoromethyl)phenyl]-1,6-dihydrodipyrazolo[3,4-b:3,4-d]
pyridin-3(2H)-one (36j). Obtained in 35% yield, mp
286.8–290.1 ꢁC. 1H NMR (DMSO-d6) 8.49–8.32 (m,
4H), 7.94 (s, 1H), 7.80 (d, J=7.9 Hz, 1H), 7.62 (t,
J=7.9 Hz, 1H), 7.41 (d, J=8.8 Hz, 2H), 3.96 (s, 3H),
3.62–3.35 (m, 8H). MS (ES+) m/z 523 (M+H)+1
.
HPLC Method A: room temperature 3.41 min, 100%.
HPLC Method B: room temperature 4.52 min, 100%.
2-[3-Fluoro-4-(morpholin-4-ylcarbonyl)phenyl]-6-methyl-
4-[3-(trifluoromethyl)phenyl]-1,6-dihydrodipyrazolo[3,4-
b:30,40-d]pyridin-3(2H)-one (36k). Obtained in 26%
(2R)-1-[3-(6-Methyl-3-oxo-4-[3-(trifluoromethyl)phenyl]-
3,6-dihydrodipyrazolo[3,4-b:3,4-d]pyridin-2(1H)-yl)ben-
zoyl]-2-pyrrolidinecarboxylic acid (35n). Yield: 0.06 g

N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
3011
(>99%) of a gold-orange solid, mp 199–202 ꢁC. 1H
NMR (DMSO-d6): 1.95–2.24 (br m, 3H), 2.5 (br s,
1H), 3.62–3.84 (br m, 2H), 4.29 (s, 3H), 4.63 (br s,
1H), 7.6 (d, J=7.69 Hz, 1H), 7.8 (m, 1H), 7.96 (m,
1H), 8.1 (d, J=7.69 Hz, 1H), 8.16 (d, J=8.52 Hz,
1H), 8.25 (s, 1H), 8.39–8.49 (m, 3H). MS m/z: 551
(M+H). HPLC Method A: roomtepmerature
4.53 min, 100%. HPLC method B: room temperature
3.38 min, 100%.
MS m/z: 557 (M+H). HPLC Method A: roomtem-
perature 4.07 min, 100%. HPLC Method B: room tem-
perature 4.87 min, 100%.
2-[3-(3,4-Dihydro-2(1H)-isoquinolinylcarbonyl)phenyl]-6-
methyl-4-[3-(trifluoromethyl)phenyl]-1,6-dihydrodipyra-
zolo[3,4-b:3,4-d]pyridin-3(2H)-one (35s). Yield: 0.154 g
(82%) of a yellow solid after flash chromatography
(80:20%, EtOAc/EtOH), mp 234 ꢁC (dec.). 1H NMR
(cis/trans rotamer mixture) (DMSO-d6): 2.9 (br s, 2H),
3.62/3.88 (cis/trans 0.6:1, br s, 2H), 4.06 (s, 3H), 4.61/4.8
(cis/trans 1.7:1, br s, 2H), 6.99–7.34 (m, 5H), (dd,
J=7.97, 7.69 Hz, 1H), 7.72 (dd, J=7.89, 7.97 Hz, 1H),
7.84 (d, J=7.55 Hz, 1H), 8.08 (s, 1H), 8.12–8.3 (m, 2H),
8.35 (d, J=8.1 Hz, 1H), 8.42 (br s, 1H). MS m/z: 569
(M+H). HPLC Method A: room temperature 4.16 min,
100%. HPLC Method B: room temperature 4.17 min,
100%.
1-{2-Chloro-5-[6-methyl-3-oxo-4-[3-(trifluoromethyl)phe-
nyl]-3,6-dihydrodipyrazolo[3,4-b:30,40-d]pyridin-2(1H)-yl]-
benzoyl}-D-proline (35o). Yield: 0.07 g (92%) of a gold-
orange solid, mp 214–218 ꢁC. 1H NMR (acetone-d6):
1.84–2.08 (m, 3H), 2.26 (m, 1H), 3.28 (m, 2H), 4.05 (s,
3H), 4.51 (m, 1H), 7.44 (d, J=8.88 Hz, 1H), 7.61 (m,
1H), 7.72 (br d, J=7.38 Hz, 2H), 7.96 (br s, 1H), 8.08 (s,
1H), 8.27 (br d, J=7.40 Hz, 1H), 8.33 (br s, 1H). MS m/
z: 583 (MꢀH).
N-(3,4-Dihydroxybenzyl)-4-(6-methyl-3-oxo-4-[3-(trifluor-
omethyl)phenyl]-3,6-dihydrodipyrazolo[3,4-b:3,4-d]pyridin-
2(1H)-yl)benzamide (36q). Isolated in 64% yield as light
yellow solid, mp: 206–209 ꢁC. 1H NMR (DMSO-d6)
8.35 (s, 1H), 8.29 (d, J=8.4 Hz, 1H), 8.20 (s, 1H), 8.02
(m, 4H), 7.78 (m, 1H), 7.75 (dd, J=6.8, 7.5 Hz, 1H),
6.72 (s, 1H), 6.63 (d, J=7.8 Hz, 2H), 4.63 (s, 2H), 4.21
(s, 3H). MS (ES+) m/z 575 (M+H)+1. HPLC Method
A: room temperature 3.38 min, 100%. HPLC Method
B: room temperature 4.55 min, 99%.
1-{3-[6-Methyl-3-oxo-4-[3-(trifluoromethyl)phenyl]-3,6-di-
hydrodipyrazolo[3,4-b:30,40-d]pyridin-2(1H)-yl]benzoyl}-L-
proline (35p). Yield: 0.136 g (50%) of a gold-orange
solid, mp 168–171 ꢁC. 1H NMR (DMSO-d6): 1.92 (br m,
3H), 2.25 (br s, 1H), 3.52 (br m, 2H), 4.05 (s, 3H), 4.50
(br m, 1H), 7.32 (br m, 1H), 7.44 (br m, 1H), 7.61 (br d,
J=7.57 Hz, 1H), 7.71 (br d, J=6.39 Hz, 1H), 7.94 (br s,
2H), 8.09 (s, 1H), 8.30 (d, J=6.98 Hz, 1H), 8.37 (s, 1H).
MS m/z: 551 (M+H). HPLC Method A: roomtem-
perature 3.39 min, 100%. HPLC Method B: room tem-
perature 4.56 min, 100%.
N-(3,4-Dihydroxybenzyl)-3-(6-methyl-3-oxo-4-[3-(trifluor-
omethyl)phenyl]-3,6-dihydrodipyrazolo[3,4-b:3,4-d]pyridin-
2(1H)-yl)benzamide (35t). Yield: 0.045 g (24%) of a
gold-orange solid after flash chromatography (90:10%,
CH2Cl2/MeOH), mp 177.7–179.8 ꢁC. 1H NMR (ace-
tone-d6): 4.11 (s, 3H), 4.44 (d, J=5.49 Hz, 2H), 6.7 (m,
2H), 6.88 (s, 1H), 7.46 (m, 1H), 7.6–7.8 (m, 3H), 8.09 (s,
1H), 8.27 (d, J=7.69 Hz, 1H), 8.38–8.52 (m, 3H). MS
m/z: 575 (M+H). HPLC Method A: roomtemperature
4.49 min, 100%. HPLC method B: room temperature
3.39 min, 100%.
N-Benzyl-3-(6-methyl-3-oxo-4-[3-(trifluoromethyl)phenyl]-
3,6-dihydrodipyrazolo[3,4-b:3,4-d]-pyridin-2(1H)-yl)benza-
mide (35q). This compound was prepared by the general
procedure for amides described above from the appro-
priate starting materials. Yield: 0.049 g (27%) of a yel-
low solid after flash chromatography (95:5%, EtOAc/
EtOH). MS m/z: 543 (M+H). HPLC Method A: room
temperature 4.85 min, 100%. HPLC method B: room
temperature 3.94 min, 100%.
N-Benzyl-4-(6-methyl-3-oxo-4-[3-(trifluoromethyl)phenyl]-
3,6-dihydrodipyrazolo[3,4-b:3,4-d]pyridin-2(1H)-yl)benza-
mide (36p). Obtained in 45% yield, mp 440 ꢁC (dec.).
1H NMR (DMSO-d6) 9.05 (m, 1H), 8.35–8.29 (m, 2H),
8.25 (s, 1H), 8.09–8.01 (m, 3H), 7.89 (d, J=7.9 Hz, 1H),
7.80 (t, J=7.9 Hz, 1H), 7.34–7.24 (m, 5H), 4.50 (d,
J=5.8 Hz, 2H), 4.11 (s, 3H). MS (ES+) m/z 543
(M+H)+1. HPLC Method A: roomtepmerature
3.92 min, 97.9%. HPLC Method B: room temperature
4.79 min, 98.0%.
2-Chloro-N-(3,4-dihydroxybenzyl)-5-(6-methyl-3-oxo-4-[3-
(trifluoromethyl)phenyl]-3,6-dihydrodipyrazolo[3,4-b:3,4-
d]pyridin-2(1H)-yl)benzamide (35u). Yield: 0.030 g (8%)
of a gold-orange solid after flash chromatography
(90:10%, CH2Cl2/MeOH), mp 212–215 ꢁC. 1H NMR
(acetone-d6) d 3.97 (s, 3H), 4.31 (d, J=5.78 Hz, 2H),
6.61 (br s, 2H), 6.86 (s, 1H), 7.27 (d, J=8.41 Hz, 1H),
7.55 (m, 1H), 7.66 (d, J=8.71 Hz, 1H), 7.83 (br m, 1H),
8.05 (d, J=5.31 Hz, 3H), 8.28 (d, J=7.44 Hz, 1H), 8.35
(s, 1H). MS m/z 607 (MꢀH). HPLC Method A: room
temperature, 4.48 min, 100%. HPLC method B: room
temperature, 3.45 min, 100%.
N-Benzyl-N-methyl-3-(6-methyl-3-oxo-4-[3-(trifluorome-
thyl)phenyl]-3,6-dihydrodipyrazolo[3,4-b:3,4-d]-pyridin-
2(1H)-yl)benzamide (35r). Yield: 0.076 g (41%) of a
beige solid after flash chromatography (95:5%, CH2Cl2/
N-(4-Hydroxy-3-methoxybenzyl)-3-(6-methyl-3-oxo-4-[3-
(trifluoromethyl)phenyl]-3,6-dihydrodipyrazolo[3,4-b:3,4-
d]pyridin-2(1H)-yl)benzamide (35v). Yield: 0.087 g
(45%) of a gold-orange solid after flash chromato-
graphy (96:4%, CH2Cl2/MeOH), mp 172–176 ꢁC. 1H
NMR (acetone-d6) d 3.8 (s, 3H), 4.17 (s, 3H), 4.52 (d,
J=6.04 Hz, 2H), 6.76 (d, J=7.97 Hz, 1H), 6.83 (d,
J=7.97 Hz, 1H), 7.02 (s, 1H), 7.54 (m, 1H), 7.69–7.88
MeOH), mp 163.7–165.6 ꢁC. H NMR (cis/trans rota-
1
mer mixture) (CDCl3): 2.87/3.1 (cis/trans 0.85:1), s, 3H),
4.24 (s, 3H), 4.44/4.81 (cis/trans 1.12:1, s, 2H), 6.78 (m,
1H), 7.02–7.21 (m, 2H), 7.26–7.44 (m, 5H), 7.58 (m,
1H), 7.72 (d, J=7.55 Hz, 1H), 7.8/7.89 (cis/trans 1:1, s,
1H), 8.22 (d, J=7.97 Hz, 1H), 8.29 (s, 1H), 8.34 (s, 1H).

3012
N. J. Green et al. / Bioorg. Med. Chem. 11 (2003) 2991–3013
(m, 3H), 8.18 (s, 1H), 8.28 (m, 1H), 8.36–8.5 (m, 3H).
MS m/z 589 (M+H). HPLC Method A: roomtem-
perature, 4.59 min, 100%. HPLC method B: room tem-
perature, 3.57 min, 100%.
Methyl-3-{[4-(6-methyl-3-oxo-4-[3-(trifluoromethyl)phenyl]
-3,6-dihydrodipyrazolo[3,4-b:3,4-d]pyridin-2(1H)-yl)ben-
zoyl]amino}benzoate (36v). Obtained in 27% yield, mp
220.1–222.4 ꢁC. H NMR (DMSO-d6) d 10.34 (s, 1H),
1
8.42 (s, 1H), 8.32 (d, J=8.2 Hz, 2H), 8.28–8.23 (m, 2H),
8.08 (m, 3H), 7.78 (d, J=8.2 Hz, 2H), 7.65 (t, J=7.4 Hz,
1H), 7.43 (t, J=8.2 Hz, 1H), 4.09 (s, 3H), 3.8 (s, 3H).
MS (ES+) m/z 587 (M+H)+1. HPLC Method A: room
temperature, 4.05 min, 100%. HPLC Method B: room
temperature, 4.87 min, 100%.
N-[4-(2-Hydroxyethyl)phenyl]-4-(6-methyl-3-oxo-4-[3-
(trifluoromethyl)phenyl]-3,6-dihydrodipyrazolo[3,4-b:3,4-
d]pyridin-2(1H)-yl)benzamide (36r). Obtained in 100%
yield, mp 270–274 ꢁC. H NMR (DMSO-d6) d 10.17 (s,
1
1H), 8.34–8.25 (m, 3H), 8.10 (br s, 3H), 7.92 (d,
J=7.6 Hz, 1H), 7.78 (t, J=7.6 Hz, 1H), 7.67 (d,
J=8.4 Hz, 2H), 7.20 (d, J=8.4 Hz, 2H), 4.12 (s, 3H),
3.60 (t, J=7.1 Hz, 2H), 2.71 (t, J=7.1 Hz, 2H). MS
(ES+) m/z 573 (M+H)+1. HPLC Method A: room
temperature, 3.58 min, 100%. HPLC Method B: room
temperature, 4.66 min, 100%.
N-(3-Acetylphenyl)-4-(6-methyl-3-oxo-4-[3-(trifluorome-
thyl)phenyl] - 3,6 - dihydrodipyrazolo[3,4 - b:3,4 - d]pyridin -
2(1H)-yl)benzamide (36w). Obtained in 15% yield, mp
300 ꢁC (dec.). H NMR (DMSO-d6: MeOH/d4, 1: 1) d
1
8.37–8.00 (m, 7H), 7.90 (d, J=7.7 Hz, 2H), 7.82 (br s,
1H), 7.67 (d, J=7.7 Hz, 2H), 7.44 (t, J=8.2 Hz, 1H),
4.08 (br s, 3H), 2.54 (s, 3H). MS (ES+) m/z 571
(M+H)+1. HPLC Method A: roomtemperature,
3.91 min, 100%. HPLC Method B: room temperature,
4.80 min, 100%
2-Fluoro-N-[4-(2-hydroxyethyl)phenyl]-4-[6-methyl-3-oxo-
4 - [3 - (trifluoromethyl)phenyl] - 3,6 - dihydrodipyrazolo[3,4 -
b:30,40-d]pyridin-2(1H)-yl]benzamide (36s). Obtained in
44.4% yield, mp 198–201.2 ꢁC. H NMR (DMSO-d6) d
1
10.25 (s, 1H), 8.29 (s, 1H), 8.24 (d, J=7.82 Hz, 1H),
8.19 (s, 1H), 8.02 (d, J=8.32 Hz, 1H), 7.95 (d,
J=7.77 Hz, 1H), 7.84 (d, J=8.20 Hz, 1H), 7.82 (m, 2H),
7.79 (d, J=8.31 Hz, 2H), 7.63 (d, J=8.28 Hz, 2H), 4.10
(s, 3H), 3.61 (t, J=6.25 Hz, 2H), 2.73 (t, J=6.35 Hz,
2H). MS (ES+) m/z 591 (M+H)+1. HPLC Method A:
room temperature, 3.69 min, 100%. HPLC Method B:
room temperature, 4.70 min, 100%.
N-[3-(1-Hydroxyethyl)phenyl]-4-(6-methyl-3-oxo-4-[3-(tri-
fluoromethyl)phenyl] - 3,6 - dihydrodipyrazolo[3,4 - b:3,4 -
d]pyridin-2(1H)-yl)benzamide (36x). Obtained in 68%
yield, mp 251–254 ꢁC. 1H NMR (CDCl3) d 10.21 (s,
1H), 8.32 (m, 3H), 8.20 (s, 1H), 8.17 (m, 4H), 7.86 (m,
1H), 7.68 (m, 2H), 7.27 (t, J=7.83 Hz, 1H), 7.08 (d,
J=7.52 Hz, 1H), 4.75 (m, 1H), 4.11 (s, 3H), 1.39 (d, 6.2,
3H). MS (ES+) m/z 573 (M+H)+1. HPLC Method A:
room temperature, 3.66 min, 100%. HPLC Method B:
room temperature, 4.71 min, 100%.
N-[3-(Hydroxymethyl)phenyl]-4-(6-methyl-3-oxo-4-[3-
(trifluoromethyl)phenyl]-3,6-dihydrodipyrazolo[3,4-b:3,4-
d]pyridin-2(1H)-yl)benzamide (36t). Obtained in 22%
yield, mp 440 ꢁC (dec.). H NMR (DMSO-d6) d 8.16–
2-Chloro-N-[3-(1-hydroxyethyl)phenyl]-4-[6-methyl-3-oxo-
4-[3-(trifluoromethyl)phenyl]-3,6-dihydrodipyrazolo[3,4-
b:30,40-d]pyridin-2(1H)-yl]benzamide (36y). Obtained in
1
8.11 (m, 4H), 8.01 (s, 1H), 7.92 (d, J=8.8 Hz, 2H), 7.67–
7.51 (m, 4H), 7.24 (t, J=7.8 Hz, 1H), 7.07 (d, J=7.5 Hz,
1H), 4.53 (s, 2H), 4.00 (s, 3H). MS (ES+) m/z 559
(M+H)+1. HPLC Method A: roomtemperature,
3.63 min, 100%. HPLC Method B: room temperature,
4.66 min, 100%.
50% yield, mp 326 ꢁC (dec.). H NMR (MeOH-d4) d
1
8.30 (s, 1H), 8.22 (d, J=7.6 Hz, 1H), 8.07 (s, 2H), 7.97–
7.89 (m, 1H), 7.79–7.75 (m, 5H), 7.33 (t, J=7.8 Hz, 1H),
7.18 (d, J=7.6 Hz, 1H), 4.09 (s, 3H), 1.46 (d, J=6.4 Hz,
3H). MS (ES+) m/z 608 (M+H)+1. HPLC Method A:
room temperature, 4.00 min, 100%. HPLC Method B:
room temperature, 4.81 min, 100%.
N-[3-(Hydroxymethyl)phenyl]-3-(6-methyl-3-oxo-4-[3-
(trifluoromethyl)phenyl]-3,6-dihydrodipyra-zolo[3,4-b:3,4-
d]pyridin-2(1H)-yl)benzamide (35w). This compound
was prepared by the general procedure for amides
described above fromthe appropriate starting materials.
Yield: 0.154 g (82%) of yellow solid after flash chroma-
tography (80:20%, EtOAc/EtOH). MS m/z 569
(M+H). HPLC Method A: roomtepmerature,
4.67 min, 97.5%. HPLC method B: room temperature,
3.58 min, 97.1%.
Acknowledgements
The authors gratefully acknowledge Dr. Gary Stack for
the original synthesis of 1, Drs. Melissa Lin and Frank
Koehn for structural elucidation of 12 and 15e, and Drs.
Andre Asselin, Wei Li and Mrs. Diane DeVincentis for
scale-up of 9. The authors also give special thanks to Dr.
John Knopf for support of this project.
N-[3-(Methoxymethyl)phenyl]-4-(6-methyl-3-oxo-4-[3-
(trifluoromethyl)phenyl]-3,6-dihydrodipyrazolo[3,4-b:3,4-
d]pyridin-2(1H)-yl)benzamide (36u). Obtained in 57%
References and Notes
yield, mp 210 ꢁC (dec.). H NMR (DMSO-d6) d 8.33
1
(m, 2H), 8.24 (s, 1H), 8.09 (m, 4H), 7.92 (m, 1H), 7.73
(m, 2H), 7.71 (d, J=7.32 Hz, 1H), 7.31 (dd, J=6.85,
7.42 Hz, 1H), 7.02 (d, J=6.83 Hz, 1H), 4.40 (s, 2H),
4.10 (s, 3H), 3.29 (s, 3H). MS (ES+) m/z 573
(M+H)+1. HPLC Method A: roomtemperature,
3.99 min, 96%. HPLC Method B: room temperature,
4.84 min, 100%.
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