Parallel Synthesis of 1H-Pyrazolo[3,4-d]pyrimidines via Condensation of N-Pyrazolylamides and Nitriles

Article abstract of DOI:10.1021/acscombsci.7b00116

A novel parallel medicinal chemistry (PMC)-enabled synthesis of 1H-pyrazolo[3,4-d]pyrimidines employing condensation of easily accessible N-pyrazolylamides and nitriles has been developed. The presented studies describe singleton and library enablements that allowed rapid generation of molecular diversity to examine C4 and C6 vectors. This chemistry enabled access to challenging alkyl substituents, expanding the overall chemical space beyond that available via typical C(sp²)-C(sp²) coupling and S_NAr transformations. Furthermore, monomer group interconversions allowing the use of larger and more diverse amides and carboxylic acids as precursors to nitriles are discussed.

Full text of DOI:10.1021/acscombsci.7b00116

Technology Note
pubs.acs.org/acscombsci
Cite This: ACS Comb. Sci. 2017, 19, 675-680
Parallel Synthesis of 1H-Pyrazolo[3,4‑d]pyrimidines via Condensation
of N‑Pyrazolylamides and Nitriles
Akshay A. Shah,* Lois K. Chenard, Joseph W. Tucker, and Christopher J. Helal
Pfizer Worldwide Research and Development, Eastern Point Road, Groton, Connecticut 06340, United States
S
* Supporting Information
ABSTRACT: A novel parallel medicinal chemistry (PMC)-
enabled synthesis of 1H-pyrazolo[3,4-d]pyrimidines employ-
ing condensation of easily accessible N-pyrazolylamides and
nitriles has been developed. The presented studies describe
singleton and library enablements that allowed rapid
generation of molecular diversity to examine C4 and C6
vectors. This chemistry enabled access to challenging alkyl substituents, expanding the overall chemical space beyond that
available via typical C(sp2)−C(sp2) coupling and SNAr transformations. Furthermore, monomer group interconversions allowing
the use of larger and more diverse amides and carboxylic acids as precursors to nitriles are discussed.
KEYWORDS: parallel medicinal chemistry (PMC), pyrazolopyrimidine, monomer interconversion, condensation chemistry,
C(sp3) substitutions
itrogen-containing heterocycles are of significant im-
condensation of appropriately substituted 5-aminopyrazoles 4
Nportance for the design and development of pharmaco- and formamide surrogates4 and (2) condensation of pyrimidine
carbaldehyde derivatives 6 and hydrazine,5 as outlined in Figure
logically active molecules. Among many such heterocycles,
1B. These strategies are inherently more amenable to
installation of heteroatom substituents at C4 and C6, although
aryl and alkyl substituents can be introduced via metal-
mediated cross-coupling chemistry.6,7 Although significant
advances in C(sp2)−C(sp2) and C(sp2)−C(sp3) coupling
chemistry have been made, their scope is limited to
commercially available alkyl and aryl coupling reagents. A
survey of the literature indicated the need for the development
of a new synthetic method that can expand the scope to include
a wide range of alkyl substituents at C4 and C6 vectors of
pyrazolo[3,4-d]pyrimidines.
In this communication, we report the development of a
general method for the synthesis of C4- and C6-substituted
pyrazolo[3,4-d]pyrimidines that allows the incorporation of a
range of alkyl, aryl, and heteroaromatic substituents by means
of a common synthetic technique. The methodology is well-
suited for the generation of libraries which is a valuable feature
in view of the growing interests in diversity-oriented library
generation and high-throughput screening technologies.8 Our
method employs condensation of easily accessible N-
pyrazolylamides 7 and readily available nitriles 8 to afford
pyrazolo[3,4-d]pyrimidine analogues 3 (Figure 1C). This
synthetic protocol is modified and optimized to facilitate the
generation of molecular libraries in parallel wherein nitrile
monomers can be reacted with chosen N-pyrazolylamide
templates. Furthermore, monomer group interconversion that
allows the transformation of amides and carboxylic acids into
pyrazolo[3,4-d]pyrimidines have emerged as one of the highly
sought chemical entities in pharmaceutical drug discovery.1
Their unique structural features that allow opportunities for
target engagement and rapid examination of structure−activity
relationship (SAR) have facilitated the development of a
number of small-molecule drug candidates. Specifically, much
attention has been dedicated to development of pyrazolo[3,4-
d]pyrimidine-based small-molecule kinase inhibitors.2 Recently,
the U.S. Food & Drug Administration approved the pyrazolo-
[3,4-d]pyrimidine-based inhibitor ibrutinib 1 (trade name
Imbruvica)3 for the treatment of mantle cell lymphoma and
chronic lymphocytic leukemia. A number of other derivatives
have shown promising preclinical activities and are under
investigation as inhibitors of kinase targets such as mTOR2a (2,
see Figure 1A), PI3K,2b P38 MAPK,2c GSK-3,2d CDK9,2e
Aurora kinase,2f Fyn,2g Src,2h Abl,2i and Sgk1.2j It is no surprise
that a growing number of synthetic resources have been
devoted to the development of new and efficient technologies
for the synthesis of pyrazolo[3,4-d]pyrimidines.
In connection to an ongoing drug discovery program, we
sought to develop pyrazolo[3,4-d]pyrimidine-based kinase
inhibitors. High-throughput fragment screening and molecular
modeling indicated the need for a comprehensive SAR study
with respect to C4 and C6 vectors as illustrated in general
structure 3 (Figure 1B). Our initial designs focused on
examination of C4- and C6-alkyl- and -aryl-substituted
pyrazolo[3,4-d]pyrimidines as a way to improve potency and
selectivity and to achieve drug-like absorption, distribution,
metabolism, and excretion (ADME) properties. A plethora of
methods exist for the synthesis of substituted pyrazolo[3,4-
d]pyrimidines that primarily employ two strategies: (1)
Received: July 26, 2017
Revised: October 1, 2017
Published: October 6, 2017
© 2017 American Chemical Society
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Technology Note
Scheme 1. (A) Early Attempt at Condensation of N-
Pyrazolylamides and Nitriles; (B) Working Mechanistic
Model
fonyl)-1H-pyrazol-3-yl)benzamide (7{1}) in ∼30% yield.
Suitable crystalline material was obtained from residual NMR
solvent (CDCl3), and an X-ray diffraction study provided an
unambiguous structural determination of 7{1}.13 Although
unexpected, the formation of 7{1} provided important
mechanistic clues to decipher events underlying the reaction.
These observed products indicated that 7{1} likely comes from
initial triflation of the protected pyrazole ring followed by
hydrolytic cleavage of the PMB group and that further
activation of 7{1} in the presence of additional Tf2O and 2-
ClPyr may effect the intended pyrimidine ring formation to
provide 3{1,1}. To verify this, we independently subjected 7{1}
to the same reaction conditions and noted full conversion to
the desired product, isolating 3{1,1} in 50% yield. Although this
result provided strong evidence for the reaction intermediacy of
7{1}, it did not preclude the possibility of the direct formation
of 3{1,1} from 11 as intended initially. On the basis of
mechanistic knowledge at the time, we attempted to increase
the conversion of 11 to 3{1,1} and minimize adduct 7{1}.
However, our efforts were met with limited success. A rational
experimental design suggested the use of more robust
protecting groups such as benzyl and tosyl instead of PMB,
but such a change may jeopardize the final electrophilic
aromatic substitution and elongate the reaction sequence by the
addition of a deprotection step. The mechanistic knowledge
proved to be key to our subsequent experimental designs in
that we proposed the deliberate formation and isolation of
interrupted adducts like 7{1}, which then could be smoothly
transformed into the desired pyrazolopyrimidine products as
exemplified above. Our proposed working mechanistic model is
outlined in Scheme 1B. The facile amide activation by triflic
anhydride provides imidoyl triflate11 12, which upon addition
of 2-chloropyridine leads to activated electrophile 13.8 At this
stage, the reversible addition of nitrile followed by electrophilic
aromatic substitution provides the desired cyclization to afford
pyrazolo[3,4-d]pyrimidine 3.
Figure 1. (A) Examples of pyrazolo[3,4-d]pyrimidine-based kinase
inhibitors. (B) Prior strategies for the synthesis of pyrazolo[3,4-
d]pyrimidines. (C) Current strategy for the synthesis of C4- and C6-
substituted pyrazolo[3,4-d]pyrimidines.
nitriles in traditional batch and parallel synthetic workflows is
described. The synthetic enablement and substrate scope are
described in detail below.
In 2006, Movassaghi and Hill reported the one-step synthesis
of pyrimidines and quinazolines via condensation of N-vinyl or
N-aryl amides with nitriles.9,10 This transformation leverages
the unique reactivity of electrophilically activated amides,11,12 in
the presence of 2-chloropyridine (2-ClPyr) and trifluorome-
thanesulfonic anhydride (Tf2O), to trap weakly nucleophilic
nitriles. We sought to build upon this discovery to effect the
formation of pyrazolo[3,4-d]pyrimidines via activation of N-
pyrazolylamides toward nucleophilic addition and subsequent
electrophilic aromatic substitution with nitriles. Preliminary
studies focused on understanding the feasibility of our
mechanistic framework to guide the synthesis of the desired
pyrazolo[3,4-d]pyrimidines. Commercially available 1-p-me-
thoxybenzyl-1H-pyrazol-5-amine (9) served as an entry point
for the introduction of 1H-pyrazole. The choice of p-
methoxybenzyl (PMB) as a masking agent was thought to
provide favorable electronic activation of the pyrazole motif, as
required for electrophilic aromatic substitution, and easily allow
subsequent removal under mild reaction conditions. Amine 9
was readily converted to test substrate 11 (Scheme 1A).
Preliminary experiments showed that when benzamide
derivative 11 was reacted with cyclopropyl carbonitrile (8{1})
in the presence of Tf2O and 2-ClPyr at −10 to 23 °C, only a
∼10% yield of the desired cyclization was observed, leading to
pyrazolo[3,4-d]pyrimidine 3{1,1} lacking the benzyl protecting
group. Surprisingly, we also isolated N-(1-(trifluoromethylsul-
With the mechanistic model laid out, we then optimized the
reaction conditions and examined the generality of the method,
initially via the use of standard batch-style laboratory
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techniques. N-(1-(Trifluoromethylsulfonyl)-1H-pyrazol-3-yl)-
amide derivatives 7{1}, 7{2}, and 7{3} (shown in Table 1)
Table 2. Pyrazolopyrimidine Synthesis via a Two-Step
Parallel Method
a b
,
a
Table 1. Pyrazolopyrimidine Synthesis
a
General reaction procedure: To amide 7 (1 equiv) in 1,2-DCE were
added Tf2O (2 equiv) and 2-ClPyr (2.1−2.5 equiv) followed by nitrile
8 (3 equiv) at 0 °C, and the mixture was stirred for 1−2 h at 55 °C.
The reaction mixtures were then subjected to aqueous workup and
b
purified via flash column chromatography. NOESY studies for
a
For the reaction procedure, see the Supporting Information.
selected compounds noted correlations between the N−H and C3−H
b
Reactions were run on a 0.1 mmol scale. Isolated amounts over
two steps after final purification are reported.
hydrogens and may suggest a majority of the tautomeric population in
c
CHCl3-d. Isolated yields upon purification are reported.
were synthesized via a two-step protocol: coupling of 9 with
appropriate carboxylic acids followed by treatment with triflic
anhydride and 2-chloropyridine (see the Supporting Informa-
tion for detailed reaction procedures and spectral data). The
pyrimidine ring formation was optimized with respect to
temperature, solvent, and reagent stoichiometry. We replaced
dichloromethane with safer and environmentally friendlier 1,2-
dichloroethane. Heating the reaction mixture to 55 °C provided
complete conversions and greater yields. The reagent
stoichiometry was optimized at 2 equiv of Tf2O and 2.5
equiv of 2-ClPyr with respect to 7. Table 1 illustrates the
synthesis of representative pyrazolo[3,4-d]pyrimidines 3{1−
3,1−2} that incorporates sp3 hydrocarbons and heteroalkyls at
C4 and alkyl and aryl substituents at C6. Pyrazolo[3,4-
d]pyrimidines 3{1−3,1−2} were isolated in moderate yields.
The ease of reaction setup, fast in situ reaction monitoring
techniques, and good conversions of the transformations
involved suggested the suitability of these reactions in parallel
mode. To demonstrate this, a two-step library was created using
N-(1-(trifluoromethylsulfonyl)-1H-pyrazol-3-yl)amide 7{2} as
a template and 11 structurally diverse nitriles according to a
protocol identical to the standard procedure developed
previously. The results are summarized in Table 2. The two-
step protocol comprising initial pyrimidine ring formation and
subsequent tert-butyldiphenylsilyl (TBDPS) ether deprotection
was executed without purification of the intermediate products,
and the final products were separated using a mass-triggered
semiautomated HPLC purification system.14 The goal of the
parallel medicinal chemistry (PMC) was to access more
compounds in quick fashion and in sufficient quantities to
facilitate biological testing15 as opposed to maximizing chemical
yields. Hence, the success was measured in terms of the overall
efficiency expressed as the “success rate” and not in terms of
individual chemical yields. The data presented in Table 2
demonstrated a success rate of 64%, defined as the number of
final test compounds isolated divided by the number of
monomers submitted (see the Supporting Information for
complete list of monomers submitted). A library of seven test
compounds was quickly generated, allowing SAR studies with
respect to the C4 vector. The examples in Table 2 illustrate the
range of alkyls (Table 2, examples 3{2,1}, 3{2,3}, 3{2,4},
3{2,6}, and 3{2,8}) and aryls (Table 2, examples 3{2,5} and
3{2,7}) that can be introduced at the C4 vector. Comparison
with existing literature methods underscored the unique and
diverse sp3 chemical space that can be accessed via our
method.16
One of the issues often encountered in a PMC-enabled
synthesis is the limited size of the available monomer sets. The
larger the available monomer set is, the greater is the chemical
space accessible via the method. To extend and diversify the
accessible chemical space, we sought to expand the method-
ology to include amide and carboxylic acid monomer sets. This
strategy would not only increase the accessible chemical space
but also allow the use of relatively abundant and inexpensive
alternative feedstock materials at both exploratory and
preparative scales. In view of these considerations, we
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developed protocols that could use amide as well as carboxylic
acid monomer sets to access C4- and C6-substituted pyrazolo-
[3,4-d]pyrimidines. Importantly, we were able to integrate
monomer group interconversions with the key ring formation
while avoiding intermediate purification steps. Table 3 outlines
Scheme 2. (A) Use of a Primary Amide for Diversity
Building at the C4 Vector; (B) Use of a Carboxylic Acid for
Diversity Building at the C6 Vector (Cy = Cyclohexyl)
Table 3. Pyrazolopyrimidine Synthesis Utilizing Primary
a b
,
Amide Monomers in a Parallel Workflow
Scheme 2B successfully demonstrated the use of a larger and
more diverse nitrile surrogate in the form of carboxylic acid,
which is of significant value.
To assess the full potential of our methodology in terms of
building diversity, we carried out an analysis to determine the
size of the virtual library utilizing the various viable starting
materials as determined via our functional group interconver-
sion strategy. The virtual library is defined as the total accessible
chemical diversity based on available and usable monomers
present in Pfizer’s corporate chemical store. The analysis was
performed using proprietary software that allowed identification
of usable monomers via application of a number of filters such
as material availability (>25 mg), range of molecular weight,
reactivity and compatibility of functional groups, etc. This
analysis enumerated 2461 usable nitriles, 2896 usable amides,
and 11 965 usable carboxylic acids, thus representing a large
virtual library and significant promise for our methodology to
drive SAR around the C4 and C6 vectors of pyrazolo[3,4-
d]pyrimidines in our efforts to identify compounds of
pharmacological value.
In conclusion, we have developed a novel PMC-enabled
methodology for the preparation of pyrazolo[3,4-d]pyrimidines
via condensation of easily accessible N-pyrazolylamides and
nitriles. The power of this method lies in the ability to access
challenging chemical space, including that with greater C(sp3)
character, and was demonstrated effectively in both batch and
library modes. Further enablements have established protocols
that use abundant and inexpensive amide and carboxylic acid
monomers, allowing rapid access to a range of substituents. The
mechanistic underpinnings suggest promise for syntheses of
other related heteroaromatics that are of value to drug
discovery programs. Efforts are currently underway to define
this scope and will be reported in due course.
a
For the reaction procedure, see the Supporting Information.
b
Reactions were run on a 0.1 mmol scale. Isolated amounts after
purification are reported.
a two-step library that used primary amide monomers 15 and
involved initial conversion to nitriles17 followed by condensa-
tion with triflated pyrazolylamide template 7{4}. As displayed
in Table 3, a success rate of 60% (see the Supporting
Information for a complete list of monomers submitted) was
achieved, and the test compounds were isolated in moderate to
good yields. As illustrated in Table 3, the C4 substituents
installed in earlier examples shown in Table 2 (compare
examples 3{2,1}, 3{2,3}, 3{2,4}, and 3{2,5} with examples
3{4,1}, 3{4,3}, 3{4,4}, and 3{4,5}, respectively) were now
accessed from amides. Such monomer group interconversion
adds tremendous value by providing choices in feedstock
selection. We also note the ability to easily introduce all of the
substituted carbon substituents at C4, as exemplified in 3{1,11}
(Scheme 2A). The ease of introduction of such quaternary
carbon substituents opens the door for the design of
challenging analogues that were virtually inaccessible before.
Further enablements led to the application of carboxylic acid
monomers to generate diversity at the C4 vector of
pyrazolopyrimidines. Scheme 2B describes the reaction scheme
that employed cyclohexanecarboxylic acid (16) as the C4
diversity introduction element leading to product 3{4,12}. The
protocol involved the formation of amide intermediate 15{12},
which facilitated the smooth conversion to the corresponding
nitrile under the same reaction conditions as for the pyrimidine
ring formation. Attempts to transfer the protocol in Scheme 2B
to the library platform met with limited success, indicating
complexity associated with multicomponent and multistep
procedures. Despite PMC limitations, the strategy shown in
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acscombs-
ci.7b00116.
■
S
Complete description of reactions, characterization data
(1H and 13C NMR, IR, and HRMS) of all new
1
compounds, and copies of H and 13C NMR spectra
(PDF)
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ACS Combinatorial Science
Technology Note
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AUTHOR INFORMATION
Corresponding Author
*E-mail: akshayshah515@gmail.com.
ORCID
Akshay A. Shah: 0000-0001-9451-0869
Christopher J. Helal: 0000-0002-9091-2091
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
■
Funding
The work was supported by Pfizer Inc.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Drs. Omar K. Ahmad, Daniel Smaltz, and
Christopher amEnde for helpful discussions, Mr. Patrick
Mullins for purification assistance, Mr. Matthew Teague and
Mr. Victor Soliman for high-resolution mass spectrometric
analysis, Mr. Ivan Samardjiev and Dr. Brian Samas for X-ray
crystallographic assistance, and Drs. Anabella Villalobos and
Patrick Verhoest for support.
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