Synthesis of fluorinated and nonfluorinated tebufenpyrad analogues for the study of anti-angiogenesis MOA

Article abstract of DOI:10.1021/op500114v

In this contribution we report the synthesis of fluorinated and nonfluorinated tebufenpyrad analogues to explore potential druglike properties through the phenotypic screening as part of the Lilly Open Innovation Drug Discovery (OIDD) program.

Full text of DOI:10.1021/op500114v

Article
pubs.acs.org/OPRD
Synthesis of Fluorinated and Nonfluorinated Tebufenpyrad
Analogues for the Study of Anti-angiogenesis MOA
Raquel Roman,†,§ Antonio Navarro,∥,§ Dariusz Wodka,∥ Maria Alvim-Gaston,∥,⊥ Saba Husain,∥
́
Natalie Franklin,⊥ Antonio Simon-Fuentes,† and Santos Fustero*,†,‡
́
†Departamento de Química Organ
‡Laboratorio de Molec
∥Discovery Chemistry Research and Technologies, Open Innovation Drug Discovery, Lilly Research Laboratories, Eli Lilly and
́
ica, Universidad de Valencia, E-46100 Burjassot, Spain
icas, Centro de Investigacion Príncipe Felipe, E-46012 Valencia, Spain
́
ulas Organ
́
́
⊥
Company, Indianapolis, Indiana 46285, United States
S
* Supporting Information
ABSTRACT: In this contribution we report the synthesis of fluorinated and nonfluorinated tebufenpyrad analogues to explore
potential druglike properties through the phenotypic screening as part of the Lilly Open Innovation Drug Discovery (OIDD)
program.
of the measles virus RNA-dependent RNA polymerase
complex,8 and razaxaban bears a trifluoromethyl moiety at the
C3 position of the pyrazole ring. With these precedents in
mind, we decided to explore the potential druglike properties of
our tebufenpyrad analogues.
INTRODUCTION
■
Pyrazoles are well-known and important nitrogen-containing
five-membered heterocyclic compounds, found in a number of
small molecules1 that possess a wide range of agricultural and
pharmaceutical activities; some representative examples are the
acaricide tebufenpyrad,2 the selective COX-2 inhibitor
celecoxib,3 the phosphodiesterase inhibitor sildenafil,4 the
anorectic anti-obesity rimonabant,5 and the inhibitor of the
blood coagulation Factor Xa razaxaban (Figure 1).6 In previous
Through the Eli Lilly Open Innovation Drug Discovery
Program (OIDD), we submitted these analogues to be
evaluated for novelty, druglike characteristics and to access
proprietary, disease-relevant phenotypic assays (https://
openinnovation.lilly.com/dd/). Biological data from the
OIDD anti-angiogenesis effort, suggested that our analogues
could be active in the cord formation assay via mitochondrial
inhibition mechanisms. Mitochondrial dysfunction is a
common mechanism of drug-induced toxicity. Early identi-
fication of new chemical entities that perturb mitochondrial
function is of significant importance to avoid attrition in later
stages of drug development. One of the most informative ways
of assessing mitochondrial dysfunction is by measuring
mitochondrial oxygen consumption. In the pursuit of novel
anti-angiogenic molecules with a desired mechanism of action,
we designed new compounds, based on an in silico model, in
the attempt to eliminate the mitochondrial activity of these
series.
The structural study of these compounds allowed us to
identify four suggested domains to be modified (Figure 1): (i)
N-substituent pyrazole center core (R1); (ii) substituent at C3
(R2); (iii) substituent at C4 (R3), and (iv) amide backbone
(R4).
In this sense, we designed a number of fluorinated and
fluorine-free pyrazole derivatives, which were synthesized and
profiled in the OIDD Phenotypic modules available at the time
of the collaboration (Figure 2).9−12
Figure 1. Pyrazoles structurally related to the commercial acaricide
tebufenpyrad.
papers we reported the synthesis of a series of fluorinated
pyrazoles structurally related to the commercial acaricide
tebufenpyrad (Figure 1), some of them displaying important
acaricidal activities.7 Tebufenpyrad and razaxaban are pyrazole
5-carboxamide derivatives, whereas the carboxamide group is at
C3 in rimonabant. Moreover, 3-trifluoromethyl-1-methylpyr-
azole-5-carboxamides are known as non-nucleoside inhibitors
Special Issue: Fluorine Chemistry 14
Received: April 7, 2014
© XXXX American Chemical Society
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Figure 2. Phenotypic module flow scheme.
Table 1. Synthesis of 1,3,5-substituted pyrazole precursors
RESULTS AND DISCUSSION
■
1. Synthesis of 1,3,5-Substituted Pyrazole Precursors.
The pyrazole ring was obtained by condensation of
commercially or easily synthesized 1,3-diketones with the
corresponding monosubstituted hydrazines in the reaction
conditions previously described in the literature.1,7,13,14 The
reaction afforded mixtures of regioisomers in all cases, which
were properly separated by column chromatography (Table 1).
The ethoxycarbonyl group was used as a precursor of a variety
of fluorinated substituents at C3 in the pyrazole; however,
because of the carboxamide function at C5, two precursors
were employed, namely 2-furyl for the 3-fluorinated derivatives
and ethoxycarbonyl for the fluorine-free compounds.
2. Synthesis of 3-Fluoralkyl N-Methylpyrazole Deriv-
atives. 2.1. Tebufenpyrad Analogues. Tapping on our
research group experience in the synthesis of fluorinated
analogues of tebufenpyrad,7 we scaled up the synthesis of the
key intermediate aldehyde 8 to be able to make final
compounds with a variety of fluorinated substitutions (Scheme
1).
a
reagent
R
R1
R2
product
2:3
1a
1b
1c
1d
1e
1f
CH3
CF3
2-furyl
2-furyl
2-furyl
2-furyl
CO2Et
CO2Et
CO2Et
CO2Et
2a,3a
2b,3b
2c,3c
2d,3d
2e,3e
2f,3f
97:3 (98)
98:2 (99)
99:1 (98)
93:7 (98)
70:30 (65)
95:5 (87)
70:30 (83)
92:8 (49)
CH3
CF2CH3
CF2CF3
CH3
CH3
CO2Et
CH3
CH2CH3
cyclopropyl
3-pyridyl
CH2CH3
CH3
1g
1h
CH3
2g,3g
2h,3h
3-Pyridyl
a
In parentheses, overall yield.
The synthesis started with a Knoevenagel condensation from
commercially available 1-(2-furyl) ethanone (130 g) and diethyl
oxalate to give diketone 1d with 70% yield and 99% purity after
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a
Scheme 1. Synthesis of the key aldehyde intermediate 8
a
Reaction conditions: Step 1: (CO2Et)2, t-BuOK, THF, DME, 3h; Step 2: CH3NHNH2, TFE, 1 h; Step 3: NaIO4/RuCl3·3H2O/EtOAc/H2O, 2 h;
Step 4: NaOCl, HOAc, addition 10−12 °C; Step 5: a) (COCl)2, DMF, AcCN, b) Et3N, DMAP, 4-tert-butylbenzylamine; Step 6: LiBH4, THF, 65
°C, 2 h; Step 7: (ClCO)2, DMSO, CH2Cl2, 1 h, −60 to 0 °C.
triturating the crude mixture with petroleum ether and ethyl
acetate (3:1). The formation of the pyrazole took place with
very high regioselectivity (96:4) and yield (92%), producing
167 g of the desired regioisomer. As the scale increased, there
was no change in the regiochemical outcome of the reaction or
the yield, as shown in Table 2.
step. Finally, the reduction to the aldehyde was carried out in
two steps. First, the ethyl ester was reduced to alcohol using
lithium borohydride and then was oxidized to the aldehyde
through a Swern oxidation with a 68% yield for both steps. A
total of 26 g of aldehyde intermediate 8 were produced in seven
steps with 18% overall yield (Scheme 1).
With this intermediate in hand, with or without chlorine
atom, we carried out the preparation of compounds 9 and 10
(Figure 3), already described previously, using the methodology
studied in our group.7 For the synthesis of 10b and 10c see
Supporting Information (SI).
Table 2. Effect of the scale in the regiochemistry of pyrazole
formation
a
entry
1d (g)
ratio (IPC) 2d/3d
yield (%)
b
1
2
3
0.5
36.6
95/5
94/6
−
90
c
c
195.0
96/4
90
a
b
c
IPC: In Process Control (LC−MS). Not isolated. Isolated yield.
The catalytic oxidation of the furan moiety was carried out in
cold temperatures by addition of small portions of sodium
periodate to control the exothermic reaction.15 Trituration of
the crude mixture with petroleum ether and ethyl acetate (3:1)
gave 78 g of pyrazole acid 4i in 52% yield.
Chlorination of the pyrazole using a mixture of sodium
hypochlorite and acetic acid furnished pure compound 5i with
76% yield after titration with MTBE. The excess of hazardous
chlorinating agent was reduced with an aqueous solution of
sodium pyrosulfite prior to workup. The amide was made by
activating the acid with thionyl chloride.16 The acid chloride
was not isolated, and it was treated with 1.1 equiv of 4-tert-
butylphenylmethanamine to give the corresponding amide with
quantitative yield, which was pure enough to use in the next
Figure 3. Fluorinated compounds prepared from the intermediate
scaling aldehyde.
2.2. Introducing Variability at the Carboxamide Group. In
order to fully explore the effect of substituent R2 (Scheme 2) in
the inhibition of cord formation in endothelial cells and move
away from mitochondrial inhibition activity, we used recursive
partitioning, a statistical method for multivariable analysis to
guide in the design of a variety of pyrazoles by keeping all the
substituents fixed except the substituent of the amide backbone,
for both series 4-chloro and 4-des-chloro. This model
considered HOMO/LUMO orbitals of the target molecules
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a
Scheme 2. Synthesis of the 4-chloro pyrazole amide derivatives 11−13
a
Reaction conditions: (i) NaIO4 (10 equiv), RuCl3·3H2O (5%), CH3CN/CCl4/H2O; rt (ii) NaOCl (13%), AcOH; −12 °C to rt. (iii) a) (ClCO)2,
Et3N, DMAP, CH2Cl2, DMF, rt. b) R3CH2NH2, rt. (iv) a) LiBH4, THF, reflux. b) MnO2, CH3CN, reflux. c) CH3OCH2PPh3, NaHMDS, THF, rt. d)
HCl 12 N, THF, rt. e) Deoxofluor, EtOH, CH2Cl2, rt for R1 = CH2CHF2, or f) NaBH4, MeOH, rt, followed by g) Deoxofluor, CH2Cl2, rt for R1 =
CH2CH2F.
as a surrogate to inhibit the mitochondria function,17 and
allowed us to select the substituents on the basis of the
predicted lack of mitochondrial disruption.
Thus, a variety of 4-chloro-N-methyl derivatives 11−13
(Scheme 2) bearing aliphatic and aromatic residues was
selected and prepared using two alternative pathways: one
consisted of the formation of the amide bond before
elaboration of the fluorinated chain (12 and 13, Scheme 2),
and the other used a more advanced intermediate with the
fluorinated chain fully installed, leaving the formation of the
amide for the last step (11, Scheme 2).
Two variants of this route have been developed using the same
stoichiometry of reagents: small excess of HATU (1.25 equiv)
and amine (1.25 equiv), and a base/solvent choice (DIEA/
NMP), that provided a medium compatible with the
purification step. Method 1 consisted of microwave irradiation
at 90 °C for 10 min, with 20 min preirradiation delay, to allow
active ester formation. In our hands, this preheating delay
resulted in better conversions and cleaner reactions. Upon
completion of the heating step, the reaction mixture was diluted
with methanol to quench the reactive intermediates and to
make the solution less viscous for liquid handling and injections
to RP purification system. In Method 2, the reaction mixtures
were agitated at room temperature for 12 h, and the post-
reaction treatment of the samples was the same as described for
Method 1.
Following the synthetic sequence shown in Scheme 3, we
prepared 12 g of a key advanced intermediate of the fluorinated
Scheme 3. Large-scale synthesis of intermediate 14
For diamines, mono-Boc protected reagents were utilized,
and syntheses of the desired analogues were conducted as a
sequence of two steps (Route B): first, amide coupling with
Boc protected amine using either Method 1 or Method 2
described above, following deprotection by treatment with
hydrochloric acid. Two distinct protocols have been used for
this route: Method 3 (two-step), in which the Boc protected
intermediate 16 (see, e.g. 16b in Scheme 4) was isolated by RP
prior to deprotection; and Method 4 (one-pot), which consisted
of the coupling step and in situ deprotection. Both protocols
(Method 3 and Method 4) utilized RP purification of the final
product. Reaction conditions and yields of isolated products for
attempted reactions are shown in Table 3.
3. Synthesis of Fluorine-Free N-Substituted Pyrazoles
with R3 = 4-t-BuC6H4. In order to explore the effect of the
substituent at the C3 position, we prepared pyrazole precursors
2f and 2g (Table 1). As previously described,7 these
compounds can be chlorinated at C4 using N-chlorosuccini-
mide (NCS) under two different conditions: (1) by microwave
pyrazole derivative 14,7 which allowed us to access a variety of
target compounds by formation of the amide back-bond
through rapid parallel synthesis (RPS).
Simple amides 15 were prepared using HATU protocol
(Scheme 4), which has been modified to allow rapid synthesis
and purification of the products via reverse phase (RP)
chromatography without isolating crude materials (Route A).
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Scheme 4. RPS of amide derivatives 15
irradiation at 100 °C in CHCl3 (85% yield) or (2) by thermo
heating at 15 °C in AcOH (73% yield). The choice of the
method will depend on the nature of the starting material
(Scheme 5). The hydrolysis of the ethoxycarbonyl group was
then carried out at room temperature with a solution of LiOH
in THF:H2O (2:1) with almost quantitative yields (96−98%).
The activation of the acid could be done either with oxalyl
Table 3. Reaction conditions and yields of isolated products
15
yield
(%)
yield
(%)
cmpd route method
cmpd
route method
15a
15b
B
B
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
4
3
2
2
2
2
2
1
1
1
2
2
2
2
1
1
2
2
33
77
15s
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
2
2
2
2
2
2
1
1
2
2
2
2
2
2
1
1
−
66
65
53
a
15t
a
b
15c
21
15u
15v
a
b
15d
15e
15f
80
83
41
80
80
79
79
31
a
Scheme 5. Synthesis of fluorine-free N-methyl pyrazoles
15w
15x
55
62
96
84
a
15g
15h
15i
15y
15z
c
15a′
15b′
15c′
15d′
15e′
15f′
15g′
15h′
29
15j
70
82
75
80
63
81
82
81
b
15k
15l
25
59
33
53
47
68
81
73
15m
15n
15o
15p
15q
a,d
16b
a
15r
a
b
Compounds isolated as racemic mixtures. Low yield due to
c
byproducts formation. Low yield due to solubility problems during
RP purification. Boc-intermediate was isolated and characterized
a
Reaction conditions: (i) R1 = cyclopropyl: NCS, CHCl3, MW, 100
d
°C (85%); R1 = 3-pyridyl: NCS, HOAc, MW, 150 °C (73%). (ii) a)
LiOH, THF:H2O, rt (96−98%); b) R1 = cyclopropyl: (COCl)2,
CH2Cl2, rt; R1 = 3-pyridyl: SOCl2, 80 °C; c) 4-tBuC6H4CH2NH2
(75−80%, two steps).
following route B.
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chloride at room temperature or with thionyl chloride at 80 °C.
Finally, the addition of 4-tert-butylbenzylamine afforded the
final products 17 with good yields (Scheme 5).
An analogous treatment of precursor 2h led to the expected
pyrazole derivative with comparable yield (see SI). In addition,
two C4 chlorine-free tebufenpyrad analogues were prepared
from the pyrazole precursor 2e (Table 1 and Scheme 6). To
Table 4. Biological activity data of representative
compounds
a
IC50 (μM)
entry
cmpd
cord formation inhibition
oxygen consumption
1
9b
9c
9d
9e
9f
0.10
0.10
2.36
0.48
1.10
2.37
3.01
0.20
0.21
0.12
0.07
1.98
2.94
0.13
0.31
0.37
0.61
0.40
1.72
0.94
0.16
0.50
0.66
2
3
4
Scheme 6. Synthesis of C-4 chlorine-free N-methyl
a
5
pyrazoles
6
9g
b
7
10
8
10b
10c
9
b
b
10
11
12
13
14
12
−
−
b
b
13
15c′
15d′
20e
6.91
6.97
0.36
a
b
Biological data for additional compounds available in SI. No
biological data available.
inhibition of blood vessel growth in endothelial cells through
mitochondrial impairment. Therefore, even though some of the
compounds in the series are very potent anti-angiogenic agents
(9b, 9c, and 20e), we would anticipate drug-induced toxicity in
future in vivo studies due to mitochondrial dysfunction.
CONCLUSIONS
■
In summary, we have prepared a series of biologically active
fluorinated analogues of the commercial acaricide tebufenpyrad
through large-scale synthesis of the key intermediate aldehyde
8. With the objective of exploring the structure−activity
relationship as anti-angiogenic agents, we have changed the
structure of the substituents at the pyrazole ring: N-substituent
pyrazole center core, substituent at C3, substituent at C4 and
amide backbone by rapid parallel synthesis using the key
intermediate acid 14. These structural changes have allowed the
synthesis of a large library of fluorinated and fluorine-free
pyrazole derivatives.
The SAR analysis illustrated that small structural modifica-
tions lead to significant changes in biological activity;
specifically the substituent on the amide backbone appears to
play a key role. Even though we made very potent compounds,
we could not separate the desired anti-angiogenic activity from
the undesired mitochondrial inhibition activity. Further
applications of the new compounds and biological studies are
underway in our laboratory.
a
Reaction conditions: (i) a) (COCl)2, CH2Cl2, DMF, rt. b) 4-t-
BuC6H4CH2NH2 (96%, two steps). (ii) 3-Bromopyridine, PdCl-
(C3H5)(dppb), KOAc, DMAC, 150 °C (32%). (iii) a) NBS, HOAc,
MW, 150 °C. b) Cyclopropylboronic acid, Pd(OAc)2, PPh3, Na2CO3,
toluene:H2O, MW, 140 °C (88%).
achieve this, we followed the two-step sequence of reactions
mentioned above, in which intermediate 2e was first converted
into its carboxamide derivative 18e with excellent yield (96%)
followed by the functionalization of the C4 position. Although
low yielding (32%), C−H activation of the C4 position in the
presence of catalytic amounts of PdCl(C3H5)(dppb) allowed us
to make and isolate the 4-(3-pyridyl) pyrazole 19e. As a higher-
yielding alternative, treatment of 18e with N-bromosuccini-
mide, followed by Suzuki coupling with cyclopropyl boronic
acid afforded the 4-(3-cyclopropyl) pyrazole derivative 20e in
88% yield (Scheme 6).
Finally, biological data were obtained for all the compounds
in the EDFC/ADSC coculture cord or tube formation assay.18
Data indicated that small structural modifications of the
molecules lead to significant changes in biological activity
(Table 4). The substituent on the amide backbone appears to
play a key role in the biological activity of the compounds;
however, it does not allow for the separation of the two in vitro
activities, i.e. cord formation inhibition and compound effect on
mitochondrial oxygen consumption.
EXPERIMENTAL SECTION
■
All reactions were carried out under nitrogen. The solvents
were purified prior to use. The reactions were monitored with
the aid of TLC on 0.25 mm precoated silica gel plates.
Visualization was carried out with UV light. Flash column
chromatography was performed on silica gel 60 (particle size
0.040−0.063 mm) with the solvents indicated in each case.
Melting points were measured with a Buchi B-540 apparatus.
̈
1H and 13C NMR spectra were recorded with 300 MHz Bruker
AC300 and 400 MHz Bruker Avance spectrometers. Chemical
shifts are given in ppm (δ) and are referenced to the residual
proton resonances of the solvents. Coupling constants (J) are
given in hertz (Hz). The letters m, s, d, t, and q represent
Notably, the most potent compounds in the cord formation
inhibition assay (9b, 9c, and 20e) also exhibit parallel potency
in the oxygen consumption assay (Figure 4). The lack of
selectivity seen may indicate a probable mechanism of
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Figure 4. Dual inhibitors. Vessel formation and mitochondrial function inhibitors.
multiplet, singlet, doublet, triplet, and quartet, respectively. The
letters br indicate that the signal is broad.
107.9, 123.5, 128.4, 132.6, 134.0, 146.7, 146.9, 148.9, 159.6
ppm. HRMS: calc for C12H13N3O2 (M+): 231.1008; found
231.1000.
Microwave reactions were carried out in a 0.5−2 mL vial
with a Biotage Initiator TM 2.0 microwave synthesizer. The
solutions were stirred before the irradiation was started, and the
absorbance of the solvent was set as “normal”. The reaction
time was initiated as soon the system reached the input
temperature, although it took approximately 2 min to reach it.
High-resolution mass spectra were recorded with a
VGmAutospec (VG Analytical, Micromass Instruments) by
Ethyl 1-Methyl-5-(3-pyridinyl)-1H-pyrazole-3-carbox-
ylate (3g). Flash chromatography (n-hexane/EtOAc, 2:1)
1
afforded 3g as a pale-yellow oil (55 mg; 58%). H NMR (300
MHz, CDCl3): δ 1.38 (t, J = 7.0 Hz, 3H; CH3), 3.95 (s, 3H;
CH3), 4.40 (q, J = 7.0 Hz, 2H; CH2), 6.89 (s, 1H; CH), 7.38−
7.43 (m, 1H; CH), 7.71−7.75 (m, 1H; CH), 8.66−8.69 (m,
2H; CH+CH) ppm; 13C NMR (75.5 MHz, CDCl3): δ 14.3,
38.3, 61.0, 109.4, 123.5, 125.7, 135.9, 141.5, 142.9, 149.2, 150.0,
161.9 ppm. HRMS: calc for C12H13N3O2 (M+): 231.1008;
found 231.1009.
Ethyl 3-Ethyl-1-(3-pyridinyl)-1H-pyrazole-5-carboxy-
late (2h). Flash chromatography (n-hexane/EtOAc, 3:1)
afforded 2h as a pale-yellow solid (31 mg; 30% two steps);
mp 48−50 °C. 1H NMR (300 MHz, CDCl3): δ 1.25 (t, J = 6.9
Hz, 3H; CH3), 1.30 (t, J = 7.5 Hz, 3H; CH3), 2.73 (q, J = 7.5
Hz, 2H; CH2), 4.24 (q, J = 7.2 Hz, 2H; CH2), 6.89 (s, 1H;
CH), 7.36−7.41 (m, 1H; CH), 7.76−7.80 (m, 1H; CH), 8.63,
(dd, 1J = 4.8 Hz, 2J = 1.5 Hz, 1H; CH), 8.68 (d, J = 2.4 Hz, 1H,
CH) ppm; 13C NMR (75.5 MHz, CDCl3): δ 13.5, 13.9, 21.3,
61.2, 111.5, 122.9, 133.2, 134.1, 137.0, 147.0, 149.1, 155.8,
159.0 ppm. HRMS: calc for C13H15N3O2 (M + 1): 246.1237;
found 246.1233.
̀
the Universitat de Valencia Mass Spectrometry Service.
Ethyl 3-cyclopropyl-1-methyl-1H-pyrazole-5-carboxy-
late (2f). Flash chromatography (n-hexane/EtOAc, 5:1)
afforded 2f as a colourless oil (EtOH: 259 mg; 57%, TFE:
1
3.5%). H NMR (300 MHz, CDCl3): δ 0.66−0.72 (m, 2H;
CHH+CHH), 0.88−0.93 (m, 2H; CHH+CHH), 1.35 (t, J =
7.2 Hz, 3H; CH3), 1.88−1.94 (m, 1H; CH), 4.08 (s, 3H; CH3),
4.30 (q, J = 7.2 Hz, 2H; CH2), 6.46 (s, 1H; CH) ppm; 13C
NMR (75.5 MHz, CDCl3): δ 7.8, 8.7, 14.2, 39.1, 60.8, 107.0,
132.8, 153.4, 159.8 ppm. HRMS: calc for C10H14N2O2 (M + 1):
195.1128; found 195.1126.
Ethyl 5-Cyclopropyl-1-methyl-1H-pyrazole-3-carbox-
ylate (3f). Flash chromatography (n-hexane/EtOAc, 5:1)
afforded 3f as a colourless oil (EtOH: 80 mg; 18%, TFE: 160
mg, 83%). 1H NMR (300 MHz, CDCl3): δ 0.64−0.69 (m, 2H;
CHH+CHH), 0.96−1.02 (m, 2H; CHH+CHH), 1.35 (t, J =
7.0 Hz, 3H; CH3), 1.65−1.74 (m, 1H; CH), 3.95 (s, 3H; CH3),
4.35 (q, J = 7.0 Hz, 2H; CH2), 6.36 (s, 1H; CH) ppm; 13C
NMR (75.5 MHz, CDCl3): δ 5.9, 6.8, 14.3, 36.9, 60.6, 105.5,
141.9, 146.6, 162.5 ppm. HRMS: calc for C10H14N2O2 (M + 1):
195.1128; found 195.1125.
Ethyl 1-Methyl-3-(3-pyridinyl)-1H-pyrazole-5-carbox-
ylate (2g). Flash chromatography (n-hexane/EtOAc, 2:1)
afforded 2g as a pale-yellow solid (24 mg; 25%); mp 73−75 °C.
1H NMR (300 MHz, CDCl3): δ 1.39 (t, J = 7.5 Hz, 3H; CH3),
4.22 (s, 3H; CH3), 4.36 (q, J = 7.5 Hz, 2H; CH2), 7.15 (s, 1H;
CH), 7.29−7.34 (m, 1H; CH), 8.06−8.09 (m, 1H; CH), 8.54
(dd, 1J = 4.8 Hz, 2J = 1.5 Hz, 1H; CH), 9.00 (d, J = 1.5 Hz, 1H;
CH) ppm; 13C NMR (75.5 MHz, CDCl3): δ 14.2, 39.7, 61.2,
Ethyl 5-Ethyl-1-(3-pyridinyl)-1H-pyrazole-3-carboxy-
late (3h). Flash chromatography (n-hexane/EtOAc, 2:1)
afforded 3h as a pale-yellow solid (67 mg; 45%); mp 45−47
1
°C. H NMR (300 MHz, CDCl3): δ 1.21 (t, J = 7.5 Hz, 3H;
CH3), 1.35 (t, J = 6.9 Hz, 3H; CH3), 2.62 (q, J = 7.5 Hz, 2H;
CH2), 4.36 (q, J = 6.9 Hz, 2H; CH2), 6.75 (s, 1H; CH), 7.38−
7.42 (m, 1H; CH), 7.76−7.81 (m, 1H; CH), 8.63, (dd, 1J = 4.8
2
Hz, J = 1.2 Hz, 1H; CH), 8.69 (d, J = 1.8 Hz, 1H, CH) ppm;
13C NMR (75.5 MHz, CDCl3): δ 12.7, 14.2, 19.5, 60.9, 107.7,
123.6, 133.0, 134.1, 135.8, 144.8, 146.3, 147.2, 149.6, 162.2
ppm. HRMS: calc for C13H15N3O2 (M + 1): 246.1237; found
246.1232.
N-[4-(1,2,3-Thiadiazol-4-yl)benzyl]-4-chloro-3-(1,1-di-
fluoroethyl)-1-methyl-1H-pyrazole-5-carboxamide
G
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(11a). Flash chromatography (n-hexane/EtOAc, 2:1) afforded
11a as a white solid (40 mg, 38%). H NMR (300 MHz,
The resulting solution was heated in a Biotage Initiator
microwave synthesizer at 90 °C for 10 min (20 min incubation
delay, fixed heating time, absorption setting: very high, Method
A), or agitated at rt for 12 (Method B). The resulting solution
was then diluted with MeOH to 3 mL, stirred at rt for 30 min,
and used directly for high-pH reverse phase purification to
afford the corresponding 5-(1,1-difluoroethyl)-2-methyl-pyra-
zole-3-carboxamide.
Route B. A 5-mL microwave vial (Biotage) was charged with
5-(1,1-difluoroethyl)-2-methyl-pyrazole-3-carboxylic acid (76.1
mg, 0.400 mmol), HATU (190.1 mg, 1.25 equiv) and a
solution of corresponding carboxylate (1.25 equiv) for Method
C, or carbamate (1.25 equiv) for Method D, and DIEA (140
μL, 2 equiv) in NMP (2.5 mL). The resulting solution was
heated in a Biotage Initiator microwave synthesizer at 90 °C for
10 min (20 min incubation delay, fixed heating time, absorption
setting: very high). For Method D, 4 N HCl/dioxane (5 equiv)
was then added to the vial, and the vial was heated in a Biotage
Initiator microwave synthesizer at 120 °C for 10 min (fixed
heating time, absorption setting: very high).
High-pH Prep on LC/MS Prep Instrument for
Purification of Carboxamides 15. Mobile phase: Solvent
A - 10 mM ammonium bicarbonate pH 10/5% MeOH; Sovent
- acetonitrile. Phenomenex Gemini-NX 5 μm C18 110 Å, axial
75 cm × 30 cm column with 15 mm × 30 mm guard column.
Mobile phase at 50 °C via in-line heater. Reaction mixtures
after dilution with MeOH (final volume: 3 mL/inj) were used
directly for injections. Focused gradients used depended on
compound of interest elution time. Peak collection detection
via UV/MS combination thresholding.
1
CDCl3): δ 2.03 (t, JHF = 17.7 Hz, 3H; CH3), 4.19 (s, 3H, CH3),
4.71 (d, J = 5.6 Hz, 2H; CH2), 7.15 (t, J = 5.6 Hz, 1H; NH),
7.49 (d, J = 8.4 Hz, 2H; 2CH), 8.05 (d, J = 8.4 Hz, 2CH), 8.65
(s, 1H; SCH) ppm; 13C NMR (75.5 MHz, CDCl3): δ 23.3 (t,
1
2JCF = 26.6 Hz), 41.4, 43.3, 107.2, 118.6 (t, JCF = 234.3 Hz),
127.9, 128.3, 130.1, 130.3, 132.7, 138.5, 143.3, 157.9, 162.3
ppm; 19F NMR (282.4 MHz, CDCl3): δ −87.4 (q, J = 17.7 Hz,
2F; CF2) ppm. HRMS: calc for C16H14ClF2N5OS (M + 1):
420.0468; found 420.0470.
4-Chloro-3-(1,1-difluoroethyl)-N-(3-fluoro-2-trifluoro-
methylbenzyl)-1-methyl-1H-pyrazole-5-carboxamide
(11b). Flash chromatography (n-hexane/EtOAc, 5:1) afforded
1
11b as a white solid (106 mg, 85%); mp: 100−102 °C. H
NMR (300 MHz, CDCl3): δ 2.02 (t, J = 18.6 Hz, 3H; CH3),
4.16 (s, 3H; CH3), 4.80−4.82 (m, 2H; CH2), 7.13−7.19 (m,
1H; CH), 7.26 (br, 1H; NH), 7.40 (d, J= 7.8 Hz, 1H; CH),
7.53 (m, 1H; CH) ppm; 13C NMR (75.5 MHz, CDCl3): δ 23.3
(t, 2JCF = 26.6 Hz), 41.0 (q, 5JCF = 3.4 Hz), 107.4, 116.9 (d, 2JCF
1
= 22.8), 118.6 (t, JCF = 234.5 Hz), 111.6−125.4 (CF3), 126.5
(d, 1JCF = 3.4 Hz), 132.3, 133.6 (d, 4JCF = 9.8 Hz), 137.7, 143.4
2
1
(t, JCF = 32.7), 157.9, 160.5 (d, JCF = 258.1) ppm; 19F NMR
1
2
(282.4 MHz, CDCl3): δ −111.8 (qdd, JFF = 26.1 Hz, JFH
=
3
11.0 Hz, JHF = 5.1 Hz, 1F; CF), −87.5 (q, JFH = 18.6 Hz, 2F;
CF2), −55.0 (d, JFF = 26.1 Hz, 3F; CF3) ppm. HRMS: calc for
C15H12ClF6N3O (M + 1): 400.0646; found 400.0650.
N-[4-(1,2,3-Thiadiazol-4-yl)benzyl]-4-chloro-3-(2-fluo-
roethyl)-1-methyl-1H-pyrazole-5-carboxamide (12a).
Flash chromatography (n-hexane/EtOAc, 2:1) afforded 12a
as a white solid (80.3 mg, 70% two steps); mp: 165−167 °C.
1H NMR (300 MHz, CD3COCD3): δ 2.99 (dt, 1JHF = 22.5 Hz,
2J = 6.6 HZ, 2H; CH2), 4.02 (s, 3H; CH3), 4.69 (dt, 1JHF = 47.1
3-(1,1-Difluoroethyl)-1-methyl-N-[2-(methylamino)ethyl]-
1H-pyrazole-5-carboxamide (15a). Purification with high-pH
1
RP afforded 15a as a colourless oil (32.5 mg, 33%). H NMR
(400 MHz, DMSO) δ 8.48 (br, 1H; NH), 7.08 (s, 1H; CH),
4.02 (s, 3H; CH3), 3.28 (t, J = 6.5 Hz, 2H; CH2), 2.60 (t, J =
6.5 Hz, 2H; CH2), 2.28 (s, 3H; CH3), 1.97 (t, JHF = 18.8 Hz,
3H; CH3) ppm; 13C NMR (125.8 MHz, DMSO) δ 23.5 (t, 2JCF
2
Hz, J = 6.6 Hz, 2H; CH2F), 4.73 (d, J = 6.3 Hz, 2H; CH2),
7.60 (d, J = 8.4 Hz, 2H; 2CH), 8.07 (br, 1H; NH), 8.15 (d, J =
8.4 Hz, 2H; 2CH), 9.35 (s, 1H; SCH) ppm; 13C NMR (75.5
MHz, CD3COCD3): δ 26.8 (d, 2JCF = 13.3 Hz), 39.2, 42.6, 81.5
1
= 27.0 Hz), 35.8, 38.6, 50.4, 104.3, 119.2 (t, JCF = 231.7 Hz),
1
2
136.7, 146.0 (t, JCF = 33.4 Hz), 158.7 ppm; 19F NMR (376.2
(d, JCF = 100.6 Hz), 107.9, 127.4, 128.3, 130.1, 131.4, 133.1,
3
140.2, 143.5 (d, JCF = 4.4 Hz), 158.4, 162.3 ppm; 19F NMR
MHz, DMSO) δ −82.3 (q, JFH = 19.1 Hz; 2F; CF2). HRMS:
calc for C10H16F2N4O (M+): 246.1306; found 246.1292.
3-(1,1-Difluoroethyl)-1-methyl-N-(3-pyrrolidinyl)-1H-pyra-
zole-5-carboxamide (15b). Purification with high-pH RP
1
2
(282.4 MHz, CD3COCD3): δ −218.3 (tt, JFH = 47.1 Hz, JFH
= 22.4 Hz, 1F; CH2F) ppm. HRMS: calc for C16H15ClFN5OS
(M + 1): 380.0743; found 380.0732.
1
N-[4-(1,2,3-Thiadiazol-4-yl)benzyl]-4-chloro-3-(2,2-di-
fluoroethyl)-1-methyl-1H-pyrazole-5-carboxamide
(13a). Flash chromatography (n-hexane/EtOAc, 1:1) afforded
13a as a white solid (82.9 mg, 73% two steps); mp: 178−180
°C. 1H NMR (300 MHz, CD3COCD3): δ 3.20 (td, 1JHF = 16.8
Hz, 2J = 4.5 Hz, 2H; CH2), 4.04 (s, 3H; CH3), 4.73 (d, J = 6.0
afforded 15b as a colourless oil (66.6 mg, 77%). H NMR
(400 MHz, DMSO) δ 8.46 (d, J = 7.0 Hz, 1H; NH), 7.12 (s,
1H; CH), 4.25 (m, 1H; CH), 4.06 (s, 3H; CH3), 2.96−2.84 (m,
1
2
2H; CH2), 2.73 (m, 1H; CH), 2.64 (dd, J = 11.4 Hz, J = 4.4
Hz, 2H; CH2), 1.92 (t, JHF = 18.9 Hz, 3H; CH3), 1.97 (m, 1H;
CH), 1.63 (m, 1H; CH) ppm; 13C NMR (125.8 MHz, DMSO)
δ 23.5 (t, 2JCF = 27.0 Hz), 32.5, 45.3, 50.3, 52.6, 104.5, 119.2 (t,
1
2
Hz, 2H; CH2), 6.21 (tt, JHF = 56.4 Hz, J = 4.7 Hz, 1H;
CHF2), 7.60 (d, J = 8.7 Hz, 2H; 2CH), 8.15 (d, J = 8.7 Hz, 2H;
2CH), 8.15 (br, 1H; NH), 9.35 (s, 1H; SCH) ppm; 13C NMR
2
1JCF = 231.7 Hz), 136.6, 146.0 (t, JCF = 33.8 Hz), 158.3 ppm;
19F NMR (376.2 MHz, DMSO) δ −82.3 (q, JFH = 19.1 Hz; 2F;
CF2). HRMS: calc for for C11H17F2N4O (M + H)+: 259.1365;
found 259.1388.
2
(75.5 MHz, CD3COCD3): δ 30.0 (t, JCF = 14.1 Hz), 39.3,
1
42.6, 108.7, 115.6 (t, JCF = 143.8 Hz), 127.4, 128.3, 130.1,
131.4, 139.9, 140.2, 158.3, 163.2 ppm; 19F NMR (282.4 MHz,
N-(4-tert-Butylbenzyl)-4-chloro-3-cyclopropyl-1-methyl-
1H-pyrazole-5-carboxamide (17f). Flash chromatography (n-
hexane/EtOAc, 6:1) afforded 17f as a colourless oil (63.5 mg,
1
2
CD3COCD3): δ −116.5 (dt, JFH = 56.2 Hz, JFH = 16.7 Hz,
2F; CHF2) ppm. HRMS: calc for C16H14ClF2N5OS (M + 1):
398.0648; found 398.0639.
1
82% two steps). H NMR (300 MHz, CDCl3): δ 0.87−0.94
Synthesis and Purification of Carboxamides 15. Route
A. A 5-mL vial was charged with 5-(1,1-difluoroethyl)-2-methyl-
pyrazole-3-carboxylic acid (76.1 mg, 0.400 mmol), HATU
(190.1 mg, 1.25 equiv) and a solution of corresponding amine
(1.25 equiv) and DIEA (140 μL, 2 equiv) in NMP (2.5 mL).
(m, 4H; 4CH), 1.32 (s, 9H; 3CH3), 1.80−1.95 (m, 1H; CH);
4.09 (s, 3H; CH3), 4.61 (d, J = 5.7 Hz, 2H; CH2), 7.03 (br, 1H;
NH), 7.29 (d, J = 8.4 Hz, 2CH), 7.39 (d, J = 8.4 Hz, 2H; 2CH)
ppm; 13C NMR (75.5 MHz, CDCl3): δ 6.5, 6.9, 31.7, 34.5,
40.6, 43.1, 108.3, 125.7, 127.3, 130.9, 134.4, 149.1, 150.6, 158.4
H
dx.doi.org/10.1021/op500114v | Org. Process Res. Dev. XXXX, XXX, XXX−XXX

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ppm. HRMS: calc for C19H24ClN3O (M + 1): 346.1681; found
346.1681.
completion of the reaction, as indicated by TLC, the solvent
was removed, and the residue was purified by flash
chromatography to afford the corresponding 4-bromopyrazole
derivative, which was dissolved in aqueous toluene (toluene/
H2O, 15:1) (1.4 mL), and cyclopropylboronic acid (14.5 mg,
0.17 mmol), Pd(OAc)2 (3 mg, 0.013 mmol), PPh3 (15.7 mg,
0.06 mmol) and potassium carbonate (63 mg, 0.45 mmol) were
added. The reaction mixture was heated under microwave
irradiation at 140 °C. The resulting solution was filtered, and
the solvent was evaporated under reduced pressure. The
residue was purified with silica gel column chromatography.
N-(4-tert-Butylbenzyl)-4-cyclopropyl-3-ethyl-1-methyl-1H-
pyrazole-5-carboxamide (20e). Flash chromatography (n-
hexane/EtOAc, 3:1) afforded 20e as a white solid (35 mg,
N-(4-tert-Butylbenzyl)-4-chloro-1-methyl-3-(3-pyridinyl)-
1H-pyrazole-5-carboxamide (17g). Flash chromatography (n-
hexane/EtOAc, 2:1) afforded 17g as a colourless oil (103.5 mg,
1
80% two steps). H NMR (300 MHz, CDCl3): δ 1.32 (s, 9H;
3CH3), 4.24 (s, 3H; CH3), 4.64 (d, J = 5.7 Hz, 2H; CH2), 7.09
(br, 1H; NH), 7.30 (d, J = 8.4 Hz, 2H; 2CH), 7.33−7.38 (m,
1
2
1H; CH), 8.09−8.13 (m, 1H; CH), 8.60 (dd, J = 4.8 Hz, J =
1.8 Hz, 1H; CH), 9.07 (d, J = 1.5 Hz, 1H; CH) ppm; 13C NMR
(75.5 MHz, CDCl3): δ 31.2, 34.5, 41.2, 43.3, 107.2, 123.3,
125.8, 127.0, 127.4, 132.7, 134.1, 134.7, 143.4, 148.4, 149.4,
150.8, 158.1 ppm. HRMS: calc for C21H23ClN4O (M + 1):
383.1633; found 383.1634.
1
88%); mp: 60−62 °C. H NMR (300 MHz, CDCl3): δ 0.52−
N-(4-tert-Butylbenzyl)-4-chloro-3-ethyl-1-(3-pyridinyl)-1H-
pyrazole-5-carboxamide (17h). Flash chromatography (n-
hexane/EtOAc, 2:1) afforded 17h as a white solid (110 mg,
75% two steps); mp: 80−82 °C. 1H NMR (300 MHz, CDCl3):
δ 1.19 (t, J = 7.5 Hz, 3H; CH3), 1.21 (s, 9H; 3CH3), 2.61 (q, J
= 7.5 Hz, 2H; CH2), 4.45 (d, J = 5.7 Hz, 2H; CH2), 6.81 (t, J =
4.8 Hz, 1H; NH), 7.15 (d, J = 8.4 Hz, 2H; 2CH), 7.27 (d, J =
8.4 Hz, 2H; 2CH), 7.65−7.69 (m, 1H; CH), 8.48 (dd, 1J = 4.8
Hz, 2J = 1.5 Hz, 1H; CH), 8.55 (d, J = 2.4 Hz, 1H; CH) ppm;
13C NMR (75.5 MHz, CDCl3): δ 12.4, 19.4, 31.3, 34.5, 43.4,
110.6, 123.1, 125.7, 127.5, 132.4, 132.9, 134.0, 137.0, 146.0,
148.9, 150.8, 152.5, 157.5 ppm. HRMS: calc for C22H25ClN4O
(M + 1): 397.1790; found 397.1787.
Synthesis of 19e by Cross-Coupling Reaction with 3-
Bromopyridine. Starting of compound 2e, carboxamide 18e
was obtained following the methodology described above.
Preparation of the PdCl(C3H5)(dppb) Catalyst. An oven-
dried, 25-mL Schlenk tube equipped with a magnetic stirring
bar under a nitrogen atmosphere was charged with [Pd(C3H5)-
Cl]2 (128 mg, 0.36 mmol) and dppb (312 mg, 0.72 mmol).
Anhydrous dichloromethane (7 mL) was added, and then the
solution was stirred at room temperature for 20 min. The
solvent was removed in vacuum. The yellow powder was used
without purification.
0.57 (m, 2H, 2CH), 0.80−0.86 (m, 2H; 2CH), 1.22 (t, J = 7.5
Hz, 3H; CH3), 1.31 (s, 9H; 3CH3), 2.65 (q, J = 7.5 Hz, 2H;
CH2), 4.08 (s, 3H; CH3), 4.59 (d, J = 5.8 Hz, 2H; CH2), 6.98
(br, 1H; NH), 7.29 (d, J = 8.5 Hz, 2H; 2CH), 7.38 (d, J = 8.5
Hz, 2H; 2CH) ppm; 13C NMR (75.5 MHz, CDCl3): δ 5.1, 7.2,
13−7, 20.1, 31.3, 34.5, 39.4, 43.3, 118.5, 125.7, 127.7, 134.4,
134.6, 150.7, 152.6, 160.3 ppm. HRMS: calc for C21H30N3O
(M + 1): 340.2383; found 340.2389.
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed experimental procedures and complete character-
ization data including 1H, 13C, and 19F NMR spectra for all new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-Mail: santos.fustero@uv.es
■
Author Contributions
§R.R. and A.N. contributed equally to this paper.
Notes
Procedure for Direct Arylation Reactions. 3-Bromopyridine
(0.4 mmol), pyrazol derivative 18e (0.33 mmol), KOAc (0.66
mmol), and PdCl(C3H5)(dppb) (0.003 mmol) were dissolved
in DMAc (1.3 mL) under nitrogen atmosphere. The reaction
mixture was stirred at 150 °C for 16 h. Then, the solvent was
evaporated, and the product was purified by silica gel column
chromatography.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Dr. Fat
́
ima Iradier, Dr. Ivan
́
Collado, and Dr.
Marta Pineiro-Nunez for the organization and logistics behind
̃
̃
the collaboration; Dr. Daniel J. Sall and Dr. Mark T. Uhlik for
their valuable help with the generation of the biological data.
We also thank Robert D. Boyer and Christopher T. Reutter for
the analytical and spectral support. We also would like to
acknowledge the OIDD team (Eli Lilly & Company) for their
partnership. Support for research carried out at University of
Valencia was provided in part by Eli Lilly and Company. We
N-(4-tert-Butylbenzyl)-3-ethyl-1-methyl-4-(3-pyridinyl)-
1H-pyrazole-5-carboxamide (19e). Flash chromatography (n-
hexane/EtOAc, 1:1) afforded 19e as a white solid (40 mg, 32%
1
yield, 40% conv.); mp: 138−140 °C. H NMR (300 MHz,
CDCl3): δ 1.2 (t, J = 7.5 Hz, 3H; CH3), 1.31 (s, 9H; 3CH3),
2.53 (q, J = 7.5 Hz, 2H; CH2), 4.13 (s, 3H; CH3), 4.35 (d, J =
5.7 Hz, 2H; CH2), 5.72 (t, J = 5.5 Hz, 1H; NH), 6.94 (d, J = 8.4
Hz, 2H; 2CH), 7.16−7.20 (m, 1H; CH), 7.27 (d, J = 8.3 Hz,
2H; 2CH), 7.49−7.53 (m, 1H; CH), 8.50−8.52 (m, 2H; 2CH)
ppm; 13C NMR (75.5 MHz, CDCl3): δ 13.9, 19.6, 31.2, 34.4,
38.9, 43.3, 116.4, 123.5, 125.6, 127.3, 128.5, 133.9, 134.1, 137.4,
148.7, 150.3, 150.6, 150.9, 159.8 ppm. HRMS: calc for
C23H28N4O (M + 1): 377.2336; found 377.2328.
́
thank the Spanish Ministerio de Ciencia e Innovacion
(CTQ2010-19774-C02-01) and the Generalitat Valenciana
(PROMETEO/2010/061) for their financial support.
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