Discovery of AZD4573, a Potent and Selective Inhibitor of CDK9 That Enables Short Duration of Target Engagement for the Treatment of Hematological Malignancies

Article abstract of DOI:10.1021/acs.jmedchem.0c01754

A CDK9 inhibitor having short target engagement would enable a reduction of Mcl-1 activity, resulting in apoptosis in cancer cells dependent on Mcl-1 for survival. We report the optimization of a series of amidopyridines (from compound 2), focusing on properties suitable for achieving short target engagement after intravenous administration. By increasing potency and human metabolic clearance, we identified compound 24, a potent and selective CDK9 inhibitor with suitable predicted human pharmacokinetic properties to deliver transient inhibition of CDK9. Furthermore, the solubility of 24 was considered adequate to allow i.v. formulation at the anticipated effective dose. Short-term treatment with compound 24 led to a rapid dose- and time-dependent decrease of pSer2-RNAP2 and Mcl-1, resulting in cell apoptosis in multiple hematological cancer cell lines. Intermittent dosing of compound 24 demonstrated efficacy in xenograft models derived from multiple hematological tumors. Compound 24 is currently in clinical trials for the treatment of hematological malignancies.

Full text of DOI:10.1021/acs.jmedchem.0c01754

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Drug Annotation
Discovery of AZD4573, a Potent and Selective Inhibitor of CDK9
That Enables Short Duration of Target Engagement for the
Treatment of Hematological Malignancies
Bernard Barlaam,* Robert Casella, Justin Cidado, Calum Cook, Chris De Savi, Allan Dishington,
Craig S. Donald, Lisa Drew, Andrew D. Ferguson, Douglas Ferguson, Steve Glossop, Tyler Grebe,
Chungang Gu, Sudhir Hande, Janet Hawkins, Alexander W. Hird, Jane Holmes, James Horstick,
Yun Jiang, Michelle L. Lamb, Thomas M. McGuire, Jane E. Moore, Nichole O’Connell, Andy Pike,
Kurt G. Pike, Theresa Proia, Bryan Roberts, Maryann San Martin, Ujjal Sarkar, Wenlin Shao,
Darren Stead, Neil Sumner, Kumar Thakur, Melissa M. Vasbinder, Jeffrey G. Varnes, Jianyan Wang,
Lei Wang, Dedong Wu, Liangwei Wu, Bin Yang, and Tieguang Yao
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ABSTRACT: A CDK9 inhibitor having short target engagement would enable a reduction of Mcl-1 activity, resulting in apoptosis
in cancer cells dependent on Mcl-1 for survival. We report the optimization of a series of amidopyridines (from compound 2),
focusing on properties suitable for achieving short target engagement after intravenous administration. By increasing potency and
human metabolic clearance, we identified compound 24, a potent and selective CDK9 inhibitor with suitable predicted human
pharmacokinetic properties to deliver transient inhibition of CDK9. Furthermore, the solubility of 24 was considered adequate to
allow i.v. formulation at the anticipated effective dose. Short-term treatment with compound 24 led to a rapid dose- and time-
dependent decrease of pSer2-RNAP2 and Mcl-1, resulting in cell apoptosis in multiple hematological cancer cell lines. Intermittent
dosing of compound 24 demonstrated efficacy in xenograft models derived from multiple hematological tumors. Compound 24 is
currently in clinical trials for the treatment of hematological malignancies.
neutralizes a subset of BH3-only proteins, such as Bim, Noxa and
Puma, preventing the activation of pro-apoptotic proteins.3,5
Furthermore, overexpression of Mcl-1 has been linked to
chemotherapy resistance and relapse in cancer patients.6−9
Given the pivotal role in tumor cell survival played by Mcl-1, it is
INTRODUCTION
■
Evasion of programmed cell death (apoptosis) is one of the
hallmarks of cancer.1 Apoptosis is a critical process for normal
development and tissue homeostasis and is therefore tightly
controlled by the balance of proteins that promote cell survival
and cell death. In many cancers, this balance is disrupted in favor
of cell survival, conferring a growth advantage. Myeloid cell
leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl2
family and a key survival factor in a wide range of human
cancers.2−4 Mcl-1 binds and sequesters pro-apoptotic proteins
Bak and Bax at the mitochondrial outer membrane, thereby
preventing activation of intrinsic apoptosis. Mcl-1 also binds and
Received: October 6, 2020
https://dx.doi.org/10.1021/acs.jmedchem.0c01754
© XXXX American Chemical Society
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Drug Annotation
unsurprising that depletion of Mcl-1 leads to rapid cell death in
cancer cells and tumor regression in several models of
hematological cancers.10,11 As a result, many medicinal
chemistry programs have attempted to develop Mcl-1 inhibitors.
However, Mcl-1 has been proven difficult to target pharmaco-
logically, due to the long, shallow hydrophobic protein−protein
interaction interface.12−15 Only very recently have small
molecule Mcl-1 inhibitors (e.g., AZD5991,16 AMG176,17
MIK66518) entered early clinical development.
An alternative approach would be to target Mcl-1 activity
indirectly by inhibiting the cyclin-dependent-kinase 9. Cyclin-
dependent-kinases (CDKs) are members of the serine/
threonine kinase family and are highly regulated by cyclins, a
family of regulatory subunits that bind to CDKs. The human
genome encodes for more than 20 CDKs and over 30 cyclins.19
CDKs are classified into two groups: CDKs involved in cell cycle
control,20 such as CDK1, 2, 4, and 6 and their associated cyclins
A, B, D, and E; and CDKs involved in transcription regulation/
RNA processing, such as CDK7, 8, and 9 and their associated
cyclins C, H and T.21 Dinaciclib, one of multiple nonselective
CDK small molecule inhibitors with potent CDK9 activity has
been shown to reduce expression of Mcl-1 and ultimately lead to
cancer cell death.22,23 The underlying hypothesis for this
observation was that transient inhibition of transcription driven
by CDK9 inhibition would result in the selective reduction of
protein levels for genes that have short-lived transcripts and
proteins, such as Mcl-1. However, because of the lack of
selectivity of dinaciclib, it was unclear whether the observed
effects are related to CDK9 inhibition alone.
Regulation of transcription is a complex process governed in
part through the activity of CDK9.24 Following successful
transcription initiation, RNA polymerase II (RNAP2) pauses
just downstream of the transcription start site, serving as a
checkpoint.25,26 To release RNAP2 from this pause, multiple
CDK9-mediated phosphorylation events are required, which
include phosphorylation of the serine 2 (Ser2) position in the 52
heptapeptide repeats (YSPTSPS) comprising the carboxyl-
terminal domain (CTD) of RNAP2.27 As an integral node of the
transcription regulatory network, CDK9 is the most studied
transcriptional CDK and has garnered considerable interest as a
potential target for cancer therapy,28,29 one working hypothesis
being that transient inhibition of transcription by CDK9
inhibition may provide a therapeutic opportunity to target
Mcl-1 activity indirectly.
Pan-CDK inhibitors, which have activity against CDK9 (e.g.,
1, also known as AZD5438,30 see Figure 1), have been known
for some time and have been previously evaluated in clinical
trials but with mixed outcomes, many of them showing complex
and challenging tolerability profiles.31 More selective CDK9
inhibitors have since emerged. We32 and others33 reported the
characterization of the in vitro and in vivo CDK9 pharmacology
using a preclinical tool 2 (AZ5576) identified as a reasonably
potent and highly selective CDK9 inhibitor from a series of
amidopyridines.34 In vitro treatment of the acute myeloid
leukemia (AML) cell line MV-4-11 with 2 resulted in a dose- and
time-dependent decrease in pSer2-RNAP2 and Mcl-1 ex-
pression with subsequent activation of caspase 3/7, leading to
rapid induction of cell death. This result was extended to
additional hematological cancer cell lines. In vivo, a similar
pharmacodynamic response was observed in MV-4-11 tumor
xenografts following a single oral dose of 2 (60 mg/kg), which
translated to significant antitumor efficacy after intermittent oral
administration.32 Other groups also have reported the
Figure 1. Structures of selected CDK9 inhibitors.
identification of more selective CDK9 inhibitors (e.g.,
LDC000067,35 LY285778536) and characterized the pharma-
cology of these compounds in vitro and/or in in vivo preclinical
hematological tumor models. However, at the start of this
project, no selective CDK9 inhibitor had entered clinical trials.28
More recently, two selective CDK9 inhibitors (BAY
114357237,38 and BAY 125115239) have been reported as
entering clinical trials.
On the basis of our understanding of the pharmacology of 2,
we set our objective to identify a highly potent and selective
CDK9 inhibitor having a predicted short half-life in humans (t1/2
< 2 h), and suitable physical properties (e.g., aqueous solubility)
for intravenous (i.v.) administration, to enable controlled
duration of target engagement in the clinic through modulation
of the infusion duration. This profile would allow maximal
flexibility in the dosing schedule to maximize the efficacy/
toxicity balance of such an agent. Compound 2 is a potent CDK9
inhibitor (CDK9 enzyme assay 5 mM [ATP], IC50 0.029 μM;
inhibition of pSer2-RNAP2 in MCF-7 cell line, IC50 0.13 μM;
induction of caspase in MV-4-11 cell line, EC50 0.22 μM) with
excellent kinase selectivity and a short half-life in rodents,
making it a suitable probe compound for characterizing short
target engagement of CDK9 in vitro and in vivo.32 However, its
low solubility (7.5 μM in 1× PBS buffer pH 7.4 (phosphate
buffered saline, phosphate final concentration of 0.01 M, pH
7.4); 24 μM in 0.1 M acetate buffer pH 4; both solubilities from
crystalline material) was seen as a significant limitation for i.v.
formulation, especially when considering the likely requirement
of a high therapeutic dose. Furthermore, it was thought that the
relatively low rate of metabolism, observed in human micro-
somes and hepatocytes in vitro, could result in the human t1/2
being too long to provide an optimal duration of target
engagement in the clinical setting. Details of the methodology
for predicting human pharmacokinetic parameters and clinical
dose are available in the section entitled “Protocol for human PK
predictions and clinical dose projections” in Supporting
Information. Predictions of human pharmacokinetic parameters
from in vitro scaled clearance and scaled volume from preclinical
species suggested a mean half-life of 4.3 h for compound 2 (Cl
2.3 mL/min/kg, Vss 0.88 L/kg, mean t1/2 4.3 h, see Table 1).
B
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Those two risks were considered significant, which drove our
decision not to progress 2 to the clinic.
Intermediate 40 was obtained by amide coupling of 39 with
carboxylic acid 38. Suzuki coupling of 40 with boronic ester 41,
followed by a two-step sequence of amine deprotection and
acetylation, gave compound 4. Alternatively, 40 was converted
to boronic ester 43. Suzuki coupling of 43 with the
corresponding halo-heterocycles followed by deprotection and
acetylation afforded compounds 20 and 22 (Scheme 2).
The synthesis of compounds 5, 8, 10, and 21 is described in
Scheme 3. Compounds 5 and 21 were synthesized from
compound 45. Suzuki coupling of 45 with boronic esters 41 and
49 gave chloro-pyridines 46 and 50 respectively. Palladium-
mediated coupling of 46 and 50 with amide 47 gave 48 and 51
respectively, which were converted to 5 and 21 after amine
deprotection and acetylation. Similarly starting from 52, Suzuki
coupling with the corresponding boronic ester (for 53a) or
Heck reaction with the corresponding imidazole (for 53b) gave
respectively 53a and 53b. The same three-step sequence of
palladium-mediated coupling with amide 47 followed by amine
deprotection and acetylation afforded compounds 8 and 10.
Compound 6 was made starting from 55 (Scheme 4).
Conversion of 2-fluoropyridine 55 to corresponding 2-amino-
pyridine 56, followed by Suzuki coupling with 41 gave
intermediate 57. Amide coupling of 57 with carboxylic acid
38, followed by amine deprotection and acetylation gave 6.
The route used for compounds 7, 9, 11−19. and 27−29
where the C-2 amido group was installed first, is described in
Scheme 5. 2-Amino-5-chloro-pyridine 59, obtained by chlori-
nation of compound 39, was coupled with carboxylic acid 38 to
give key intermediate 61 after an amine deprotection/
acetylation sequence. Suzuki coupling of 61 with the
corresponding boronic esters or Heck coupling of 61 with the
corresponding imidazoles respectively gave compounds 7, 19
and 9, 11. Compounds 12−18 and 27−29 were made starting
from 56 using a similar sequence (all using a Suzuki coupling,
except for 29 where a Heck reaction between 63 and the
corresponding imidazole was used).
Table 1. Pharmacokinetic Properties of Compound 2 in
Preclinical Species and Predicted Human Parameters
mouse
rat
58
77
1.3
0.22
dog
human
1.4
a
hepatocytes Clint (μL/min/106 cells)
52
40
1.1
0.26
5.9
7.5
0.62
1.0
b
c
plasma Cl (mL/min/kg)
2.3
0.88
b
c
Vss (L/kg)
c
t1/2 (h)
4.3
a
b
Intrinsic clearance measured from hepatocytes, Clint. For mouse
and Han Wistar rat studies (n ≥ 2), the compound was administered
respectively at a dose of 5.2 μmol/kg and 1.3 μmol/kg i.v. as a
solution formulation. For Beagle dogs (n = 2), the compound was
administered by intravenous infusion over 0.25 h at a dose of 5.2
μmol/kg as a solution in 15% captisol (w/v) solution adjusted to pH
c
4.0. Italicized text indicates human PK predictions (see Supporting
Information for methods).
Here we report further optimization of this amidopyridine
series as highly selective CDK9 inhibitors, with a focus on
potency, pharmacokinetic, and physicochemical properties
suitable for an intravenous agent with a short duration of target
engagement. This work led to the discovery of the clinical
candidate compound 24 (also known as AZD4573) for the
treatment of hematological malignancies.
RESULTS AND DISCUSSION
■
Synthetic Chemistry. The synthesis of compounds 3−34 is
outlined in Schemes 1−6 of the text and Schemes 7 and 8 of the
Supporting Information. In general terms, the C-4 headgroup on
the pyridine central core was introduced, in most cases, by a
Suzuki coupling reaction. Alternatively, the C-4 headgroup was
introduced by a Heck reaction. The C-2 amido group on the
pyridine central core was installed by a palladium-catalyzed
reaction between the 2-halopyridine and the corresponding
primary amide side chain. Alternatively, still starting from the 2-
halopyridine, the C-2 amido group on the pyridine central core
was installed in two steps: nucleophilic substitution of the 2-halo
substituent by ammonia, followed by amide coupling with the
corresponding carboxylic acid side chain. Depending on the
specific compound, the C-4 headgroup was introduced first on
the pyridine core, followed by the C-2 amido group, or vice
versa.
Compounds 23 and 24 were made according to Scheme 6.
Key boronic ester intermediate 69 in the synthesis of 23 and 24
was made as follows: intermediate 65 made by alkylation of
pyrazole was cyclized using the procedure developed by
Larsen40 to afford 66. Reduction of ketone 66 under Wolff−
Kishner conditions afforded previously described 67,41 which
was submitted to bromination conditions followed by
palladium-catalyzed borylation to afford 69. Compound 70,
which was obtained by Suzuki coupling of 45 with 69, was
subjected to palladium-catalyzed amidation with 47 to afford 71.
Final amine deprotection/acetylation sequence of 71 gave 23.
Suzuki coupling of 62 with 69, followed by a final amine
deprotection/acetylation sequence, gave 24.
Compound 3 was made as follows: Suzuki reaction of boronic
ester 35 with 36 gave 37, which was then coupled with
carboxylic acid 38. Amine deprotection and subsequent
acetylation afforded compound 3 (Scheme 1).
a
Scheme 1
a
Reagents: (a) 36, second generation XPhos precatalyst, K3PO4, dioxane−water, 110 °C; (b) 38, 1-chloro-N,N,2-trimethylprop-1-en-1-amine,
DCM, 0 °C to rt, then 37, pyridine, THF, rt; (c) HCl, dioxane−MeOH, rt then AcCl, DIPEA, DCM, rt.
C
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a
Scheme 2
a
Reagents: (a) 38, 1-chloro-N,N,2-trimethylprop-1-en-1-amine, DCM, 0 °C, then 39, pyridine, DCM, THF, 0 °C to rt; (b) 41, second generation
XPhos precatalyst, K3PO4, dioxane−water, 100 °C; (c) HCl, dioxane−DCM, rt; (d) Ac2O, NEt3, DCM, DMAP (optional), rt; (e) (BPin)2,
PdCl2(dppf), KOAc, dioxane, 90 °C; (f) Het-Br or Het-I, PdCl2(dppf), K3PO4, dioxane−water, 90 °C; (g) TFA, DCM, rt or HCl, dioxane−DCM,
rt.
The synthesis of compounds 25, 26, and 30−34 are described
in Scheme 7 and 8 (Supporting Information).
μM) while keeping high solubility. Previous work at
AstraZeneca had shown that halogen substitution, such as
fluorine or chlorine, at the C-5 position of the pyrimidine core of
compound 1 increased potency against some CDKs (e.g.,
CDK243). From 4, substitution of the 5-position of the pyridine
with a fluorine (compound 5) or a chlorine (compound 6)
significantly increased potency against CDK9. Compound 6 was
especially interesting in that it had similar potency against CDK9
and higher solubility when compared to 2. Therefore,
compound 6 was profiled further. Kinase selectivity of 6 was
evaluated at 1 μM in the “Eurofins kinase panel”. It showed
significant inhibition (>80% inhibition) on only 7 kinases out of
125 kinases: CDK9 (98%), GSK3α (101%), GSK3β (100%),
CDK1 (96%), CDK2 (93%), DYRK2 (87%), and CK1γ1
(87%). The selectivity among the CDK family was further
assessed in assays recapitulating the typical ATP concentration
in cells ([ATP] of 5 mM). The overall profile is summarized in
Table 3, supporting that 6 is a potent CDK9 inhibitor with >10-
fold selectivity against CDK1−7 and CDK12, except CDK3 (2-
fold).
The binding kinetics of 6 to CDK9 were determined by
surface plasmon resonance. Compound 6 showed a dissociation
rate constant (kd) of 3.64 × 10−3 s−1, which resulted in a
dissociation half-life (t1/2) of 6.3 min (see Table 4) and was
classified as “fast-off” binding kinetics. The reversibility of pSer2-
RNAP2 inhibition was also investigated in the MCF-7 cell line
by incubation of 6 followed by cell washout and measurement of
pSer2-RNAP2 inhibition at 30 min and 2 h after washout of the
inhibitor. Compound 6 showed reversible inhibition with an
increased IC50 at both time points following washout (IC50 > 3
μM at both time points vs IC50 0.11 μM without cell washout).
The reversibility of pSer2-RNAP2 inhibition (in line with the
“fast-off” binding kinetics of 6 to CDK9) was seen as a desirable
feature for a CDK9 inhibitor to deliver short target engagement.
The solubility of compound 6 was further assessed from a
crystalline batch: 94 mg/L in 1× PBS buffer pH 7.4 and 210 mg/
L in 0.1 M acetate buffer pH 4 (from crystalline Form B44). The
Discovery of Clinical Candidate 24. Docking of 2 into a
published structure of CDK9 in complex with cyclin T1 (PDB:
4BCF)42 provided some understanding of the key interactions
leading to the potency and selectivity achieved with this
compound. According to this model, compound 2 interacts with
the kinase hinge via a bidentate interaction through the pyridine
and the amidic NH with the backbone atoms of Cys106 (Figure
2). The cyclohexyl amide gives access to the solvent channel.
The 2-methoxy-4-fluoro phenyl motif is twisted with respect to
the pyridine ring (positioned 24° out of plane), bringing the
methoxy group in closer proximity to Ala153 and Leu156, which
follow the catalytic HRD-motif. From our understanding of the
SAR from published literature around 234 and our docking
model, our initial hypothesis was that the 4-fluoro-2-
methoxyphenyl at the C-4 position of the pyridine was a key
element for the potency and selectivity against CDK9.
Conversely, the lipophilic nature of this group was considered
as a key contributor to the poor solubility of 2. We decided to
investigate alternative groups at the C-4 position of the pyridine,
in particular, five-membered heterocycles which are present in
some CDK inhibitors (e.g., imidazole in 1) with the thought that
they might improve the physical properties of this series. Indeed,
compound 3, a hybrid of compounds 1 and 2, retained some
activity against CDK9. It is worth noting that compound 3 has a
similar ligand-lipophilicity efficiency (LLE) compared to 2, its
potency being reduced ca. 10-fold compared to 2, while its
lipophilicity is reduced by 1.1 units (see Table 3). In addition, 3
showed increased solubility compared to 2. With this
encouraging result in hand, we explored other five-membered
heterocycles (e.g., pyrazoles, imidazoles) and other [5,6]
bicyclic heterocycles from available boronic acids or esters.
Among the compounds from this initial exploration, compound
4 caught our attention, having slightly improved potency against
CDK9 (CDK9 enzyme assay 5 mM [ATP], IC50: 0.23 μM;
CDK9 cellular assay using pSer2-RNAP2 end-point, IC50: 0.52
D
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a
Scheme 3
a
Reagents: (a) Het-BPin, second generation XPhos precatalyst, K3PO4, dioxane−water, 90 °C; (b) 47, Pd(PPh3)4, xantphos, Cs2CO3, dioxane, 120
°C; (c) TFA, DCM, rt; (d) Ac2O, NEt3, DMAP (optional), DCM, rt; (e) Het-BPin, Pd(PPh3)4, Cs2CO3, dioxane−water, 100 °C (for 53a); (f)
1,2-dimethylimidazole, Pd(OAc)2, PPh3, Cs2CO3, dioxane, 120 °C (for 53b).
a
Scheme 4
a
Reagents: (a) conc. aq. ammonia, NMP, 100 °C (microwave); (b) 41, PdCl2(dppf), Cs2CO3, dioxane−water, 95 °C; (c) 38, 1-chloro-N,N,2-
trimethylprop-1-en-1-amine, DCM, 0 °C, then 57, pyridine, DCM, 0 °C to rt; (d) HCl, dioxane−DCM−MeOH, 0 °C; (e) AcCl, pyridine, DCM,
rt, 0 °C.
pharmacokinetics of compound 6 were subsequently evaluated
in multiple species to facilitate prediction of its human
pharmacokinetics. In mouse, rat, and dog, compound 6 showed
a moderate volume of distribution typical of its neutral ionic
state.45 Clearance was high in rats and moderate in dogs with the
observed plasma clearance values in both species being
accurately predicted by scaling the intrinsic clearance derived
from isolated hepatocyte incubations. Overall, this resulted in
short half-lives of less than 1 h in all three species. Compound 6
was, however, metabolized much more slowly in human
microsomes and hepatocytes, resulting a predicted human
clearance of <2.8 mL/min/kg. The predicted human
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a
Scheme 5
a
Reagents: (a) NCS, DMF, −78 °C to rt; (b) 38, 1-chloro-N,N,2-trimethylprop-1-en-1-amine, DCM, 0 °C, then 57, pyridine, DCM, THF, 0 °C to
rt; (c) HCl, dioxane-MeOH, 0 °C; (d) Ac2O, DMAP, NEt3, DCM, rt; (e) Het-BPin, second-generation XPhos precatalyst, K3PO4, dioxane−water,
rt (for 7 and 19) or Het-H, Pd(OAc)2, PPh3, NEt3, Cs2CO3, dioxane, 100 °C (for 9 and 11); (f) 38, 1-chloro-N,N,2-trimethylprop-1-en-1-amine,
DCM, 0 °C, then 56, pyridine, rt; (g) HCl, dioxane-DCM, rt; (h) Ac2O, DMAP (optional), NEt3, DCM, rt; (i) Het-BPin, second generation
XPhos precatalyst, K3PO4, dioxane−water, 60 °C (for 12 from 63) or Het-B(OH)2, PdCl2(dppf), Ba(OH)2, dioxane−water, 75 °C (for 14 from
63) or Pd(OAc)2, KOAc, DMA, 150 °C (microwave) (for 29 from 63); (j) Het-BPin, second generation XPhos precatalyst, K2HPO4 or K3PO4,
dioxane-water, rt to 50−80 °C (for 13, 15−18, 27−28 from 62); (k) chiral purification (for 27−28 only).
pharmacokinetic parameters, when integrated into a PBPK
model, resulted in a predicted t1/2 in humans of >2.8 h (see
Table 5), which was longer than we were seeking to achieve.
Despite concerns that the human pharmacokinetic properties
of compound 6 might not be optimal for a short target
engagement CDK9 inhibitor, compound 6 was a promising lead.
Another hypothetical concern with the series was the potential
for pharmacologically active metabolites with longer human
half-lives that would result in more sustained target engagement.
Therefore, metabolite identification studies were conducted
with compound 6 in HLM (and human hepatocytes) resulting
in the identification of three oxidative metabolites: 6-M1, 6-M2,
and 6-M3. 6-M1 was identified as the major metabolite. Open
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a
Scheme 6
a
Reagents: (a) BrCH2C(Me)2CO2Et, Cs2CO3, DMA, 80 °C; aq. NaOH, MeOH, rt; (b) n-BuLi, 2-MeTHF, −78 °C to rt; (c) hydrazine hydrate,
2,2′-oxidiethanol, 180 °C, then NaOH, 150 °C; (d) NBS, DCM, rt; (e) Pd(P(Cy)3)2Cl2, (BPin)2, KOAc, PdCl2(dppf), DMA, 85 °C; (f) 69,
second generation XPhos precatalyst, K2HPO4, dioxane−water, 80 °C; (g) 47, Pd(PPh3)4, xantphos, Cs2CO3, dioxane, 120 °C; (h) HCl, dioxane−
DCM−MeOH, rt; (i) Ac2O, pyridine or NEt3, DMAP (optional), DCM, rt ; (j) 69, second generation XPhos precatalyst, K2HPO4, dioxane-water,
50 °C.
hepatocytes, and 6-M2 was present only as a trace metabolite
in HLM. Therefore, it was considered unlikely that either would
have sufficient exposure to significantly contribute to CDK9
inhibition in the clinic.
Having established the potential for five-membered and [5,6]-
bicyclic systems at the C-4 position of the pyridine to balance
CDK9 potency with aqueous solubility, and the potential to
further improve potency through C-5 substitution of the
pyridine, we decided to further mine this SAR by again varying
the C-4 position. From the initial exploration of the C-4
substituent, the pyrazolopiperidine headgroup (compound 6)
combined acceptable CDK9 potency and high solubility. 1,5-
Dimethyl-3-pyrazole 7 was significantly less potent against
CDK9, whereas pyrazolopyridine 8 kept comparable potency to
6, but was much less soluble. Initial compounds comparing an
imidazole group to the pyrazole group (e.g., imidazopiperidine 9
vs pyrazolopiperidine 6, 1,2-dimethyl-5-imidazole 10 vs 1,5-
dimethyl-3-pyrazole 7, and imidazopyridine 11 vs pyrazolopyr-
idine 8) showed reduced CDK9 potency by about 10-fold, but
provided some evidence of increased solubility (probably
related to the lower lipophilicity). Finally, triazolopiperidine
12 gave much reduced potency (ca. 100-fold) against CDK9 vs
pyrazolopiperidine 6.
Figure 2. Compound 2 (green) docked into the ATP binding site of a
publicly available crystal structure of CDK9−cyclinT1 (PDB: 4BCF),
with CDK9 shown as a blue ribbon and CyclinT1 as a gray surface.
chain primary alcohol 6-M2 was detected as a trace metabolite,
and 6-M3 as a minor carboxylic acid metabolite (see Figure 3).
The structures of 6-M1 and 6-M2 were subsequently
confirmed by chemical synthesis of authentic standards (see
Supporting Information). Pharmacological evaluation of 6-M1
and 6-M2 showed that they were significantly less potent (6.5-
fold for 6-M1 and >18-fold for 6-M2 in the pSer2-RNAP2
cellular assay) than compound 6 (see Table 6). In addition, 6-
M1 also showed a high rate of metabolism in human
We then explored modifications of the piperidine portion of
the pyrazolopiperidine headgroup in compound 6. Pyrrolidine
13 showed slightly reduced potency (ca. 5-fold) when compared
to piperidine 6, but surprisingly, imidazopyrrolidine 14 was
nearly equipotent compared vs pyrazolopyrrolidine 13 (potency
reduced by only ca. 2-fold). Azepane 15 showed comparable
potency (CDK9 5 mM [ATP], IC50: 0.023 μM; pSer2-RNAP2,
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Drug Annotation
Table 2. Structure, CDK9 Enzyme and Cell Inhibitory Potencies, and Other Properties of Compounds 2−34
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Drug Annotation
Table 2. continued
a
b
Geometric means of at least two IC50 determinations per compound. Measured using shake-flask methodology with a buffer/octanol volume
c
d
ratio of 100:1. Solubility from phosphate buffer (pH: 7.4); sample from dried DMSO solution. Intrinsic clearance measured from fresh rat
e
hepatocytes and cryopreserved human hepatocytes, Clint; μL·min−1·106 cells−1. Human liver microsome intrinsic clearance (HLM Clint); μL·
min−1·mg−1.
Table 3. Selectivity of Compounds 6 and 23 against Kinases
within the CDK Family
Table 4. Binding Kinetics of 6, 23, and 24 to CDK9
Determined by Surface Plasmon Resonance
a
a
compound 6 IC50 (μM)
compound 23 IC50 (μM)
compound compound
kinase
[ATP] 5 mM
[ATP] 5 mM
compound 6
23
24
CDK9
CDK1
CDK2
CDK3
CDK4
CDK5
CDK6
CDK7
CDK12
0.039
0.44
0.61
0.084
2.1
4.1
2.5
5.9
>30
0.004
0.095
0.066
0.020
0.334
2.78
0.532
0.548
9.85
equilibrium dissociation constant
D (nM)
0.295
0.123
0.0894
K
association rate constant ka
8.93
3640
6.3
4.62
569
20.3
7.95
711
16.2
(μM−1·s−1
)
dissociation rate constant kd (10−6
·
s−1
)
dissociation half-life t1/2 (min)
observed with them, we were concerned by the potential higher
volume of distribution associated with this ion class and the
consequence on the pharmacokinetic half-life. We then looked
at adding substituents on the piperidine in an attempt to further
interact with the P-loop. Indeed, gem-dimethylpiperidine 20
showed significantly increased potency (about 10−20-fold,
CDK9 5 mM [ATP], IC50: 0.008 μM; pSer2-RNAP2, IC50:
0.048 μM) versus the unsubstituted piperidine 4. In addition, 20
was highly soluble and showed an increased rate of metabolism
in HLM and human hepatocytes. Introduction of a 5-fluoro on
the pyridine led to a further increase in potency (compound 21:
CDK9 5 mM [ATP], IC50: < 0.003 μM; pSer2-RNAP2, IC50:
a
[ATP] 5 mM: concentration of ATP used in the kinase assay 5 mM.
IC50: 0.092 μM) to piperidine 6. In addition, 15 exhibited an
increased rate of metabolism in HLM and human hepatocytes,
although its solubility was slightly lower. Introduction of
heteroatoms to the piperidine ring of compound 6 (e.g.,
compounds 16−19) showed reduced potency against CDK9
(range of 3−30-fold) compared to compound 6. It is noteworthy
that only a very small number of basic compounds (e.g.,
compound 17) were investigated. Despite the good solubility
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Table 5. Pharmacokinetic Properties of Compound 6 in
Preclinical Species and Predicted Human Parameters
Table 6. CDK9 Enzymatic and Cellular Potencies and in
Vitro Intrinsic Clearance of 6 and Its Metabolites 6-M1 and 6-
M2 in HLM and Human Hepatocytes
mouse
rat
46
dog
human
<1.2
a
hepatocytes Clint (μL/min/106 cells)
27
14
CDK9 enz. [ATP] 5 mM pSer2 cell IC50
HLM
b
Hu. heps
a
a
c
Cpd
IC50 (μM)
(μM)
Cllint
Cllint
b
c
plasma Cl (mL/min/kg)
65
78
30
<2.8
b
c
6
0.039
0.060
0.085
0.11
0.72
>2
17
18
15
<1.2
33
2.9
Vss (L/kg)
0.65
0.45
0.75
0.25
0.90
0.50
0.68
>2.8
c
6-M1
6-M2
t1/2 (h)
a
b
Intrinsic clearance measured from hepatocytes, Clint. For mouse
a
Geometric means of at least two IC50 determinations per compound.
and Han Wistar rat studies (n ≥ 2), the compound was administered
respectively at a dose of 4.8 μmol/kg and 1.2 μmol/kg i.v. as a
solution formulation. For Beagle dogs (n = 2), the compound was
administered by intravenous infusion over 0.25 h at a dose of 5.2
μmol/kg as a solution in 5% DMSO and made to volume with 0.9%
b
Human liver microsome intrinsic clearance (HLM Clint); μL·min−1·
c
mg−1. Intrinsic clearance measured from human hepatocytes, Clint;
μL·min−1·106 cells−1.
c
sodium chloride. Italicized text indicated human PK predictions (see
(data not shown), but we were keen to explore the subtleties of
the SAR in this position, following the extensive optimization in
other areas of the molecule. The different stereoisomers of the 3-
acetamido-cyclohexane-1-carboxamide substituent were eval-
uated (compounds 30−32 vs 24), confirming the initial
observation on related compounds that the (R,R)-diaster-
eoisomer was the most potent. Finally, further modification of
this region (e.g., compounds 33 and 34) did not improve activity
versus the 3-acetamido-cyclohexane-1-carboxamide group
(compound 24).
The cocrystallographic structure of 24 bound to CDK9 in
complex with cyclin T1 confirmed the ligand binding mode
(Figure 4). The cyclin is bound as expected near the αC-helix of
CDK9, with no direct interaction with the ATP binding site or
the ligand. Consistent with the predicted binding mode of 2
(Figure 2), Cys106 makes two hydrogen-bonds with the pyridyl
amide core, and the cyclohexyl ring of solvent channel group is
orthogonal to this motif. The pyrazole−pyridine biaryl system is
twisted out of plane by 37 deg, such that one of the gem-
dimethyl substituents of 24 fills a small pocket formed near
Asp167 of the activation loop, in close proximity to the aliphatic
portion Cβ of its side chain, the Cα of Asn154 and the side chain
of Ala166.
Supporting Information for methods).
0.010 μM) while showing an increased rate of metabolism in
HLM and human hepatocytes with only a modest reduction in
solubility. Similarly, substitution of the pyrrolidine with gem-
dimethyl (compounds 22−24) was very fruitful. Compound 22
was significantly more potent than 4. Again, introduction of a 5-
fluoro or a 5-chloro on the pyridine further improved potency
(respectively compound 23: CDK9 5 mM [ATP], IC50: 0.004
μM; pSer2-RNAP2, IC50: 0.025 μM and 24: CDK9 5 mM
[ATP], IC50 < 0.004 μM; pSer2-RNAP2, IC50: 0.0134 μM),
while introduction of a 5-cyano (compound 25) or a 5-methyl
(compound 26) was less favorable. In addition, 23 and 24 were
highly soluble (550 and 150 μM, solubility at pH 7.4 phosphate
buffer, data obtained from dried DMSO sample method) and
showed a higher rate of metabolism in HLM and human
hepatocytes than compound 22. Gem-dimethyl substitution
provided more CDK9 potency than a single methyl substituent
(see compound 24 vs compounds 27 and 28). The
imidazopyrrolidine analogue of 24 (compound 29) also
displayed high potency against CDK9 (CDK9 5 mM [ATP],
IC50 0.011 μM; pSer2-RNAP2, IC50: 0.036 μM) and showed
high solubility and a similar rate of metabolism in HLM to
compound 24.
Selected compounds (e.g., compounds 23 and 24) were
further profiled. Kinase selectivity of 23 was evaluated at 1 μM in
the “Eurofins kinase panel”. It showed significant inhibition
(>80% inhibition) on 8 kinases out of 125 kinases in the
The C-2 position of the pyridine had been previously
optimized, following a library on a less optimized scaffold
Figure 3. Metabolites of 6 after incubation in HLM. Metabolites shown were considered to have the potential to retain pharmacological activity and do
not represent a complete metabolic scheme.
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Drug Annotation
Table 7. Selectivity of Compound 24 against Selected Kinases
a
kinase
IC50 (μM) [ATP] 5 mM
CDK9
CDK1
CDK2
CDK3
CDK4
CDK5
CDK6
CDK7
CDK12
CK1γ1
CK1γ2
DYRK2
GSK3α
GSK3β
JNK1
<0.004
0.117
0.052
0.023
0.499
1.27
0.363
1.37
8.07
5.52
6.66
8.62
0.187
0.247
2.80
Figure 4. Crystal structure of 24 bound to the complex of CDK9 (blue)
and cyclin T1 (gray) (PDB: 6Z45). The ligand carbon atoms are shown
in green.
a
[ATP] 5 mM: concentration of ATP used in the kinase assay 5 mM.
7.4 and 57 mg/L in 0.1 M acetate buffer pH 4 for compound 23
(from crystalline Form B44); 17 mg/L in 1× PBS buffer pH 7.4
and 50 mg/L in 0.1 M acetate buffer pH 4 for compound 24
(from crystalline Form A44). In addition, evaluation of common
pharmaceutical excipients suitable for i.v. clinical formulation
demonstrated further solubility enhancement. As a result,
development of a suitable clinical formulation for i.v.
administration was seen as a highly viable option, especially
given the anticipated lower therapeutic dose resulting from the
increased CDK9 potency of compound 24 compared to
compounds 2 and 6.
The pharmacokinetic properties were evaluated in mouse, rat,
and dog for compound 23 and in mouse, rat, dog, and
cynomolgus monkey for compound 24: in all species evaluated,
compounds 23 and 24 showed a moderate volume of
distribution, as anticipated from their neutral ionic state.
Clearance was high in all preclinical species, with the observed
plasma clearance values being accurately predicted by scaling the
intrinsic clearance derived from isolated hepatocyte incubations.
Overall, the high clearance combined with the low/medium
volume of distribution resulted in short half-lives (below 1 h) in
all species. Scaling of in vitro intrinsic clearances values from
human hepatocytes resulted in similar predicted human
clearance values for both compounds, which, when combined
with the predicted human volume of distribution, resulted in a
predicted human t1/2 value of 1.5 h for compound 23 (predicted
human PK parameters: Cl 4.8 mL/min/kg, Vss 0.61 L/kg, mean
“Eurofins kinase panel”: CDK9 (100%), GSK3α (101%),
GSK3β (101%), CDK1 (100%), CDK2 (100%), CDK7
(92%), CK1γ1 (91%), and DYRK2 (89%). The selectivity of
23 among the CDK family was further assessed in assays
recapitulating the typical ATP concentration in cells ([ATP] of
5 mM), supporting that 23 is a potent CDK9 inhibitor with at
least 16-fold selectivity against CDK1-7 and CDK12, except
CDK3 (5-fold); see Table 2. Kinase selectivity of 24 was
evaluated at 1 μM in the “Eurofins kinase panel” and at 0.1 μM in
the “Thermofisher kinase panel”. It showed significant inhibition
(>80% inhibition) on 8 kinases outside the CDK family out of
125 kinases in the “Eurofins kinase panel”: GSK3β (104%),
DYRK2 (100%), GSK3α (100%), CK1γ1 (94%), Jnk1 (90%),
MAP2K7 (85%), INSR (83%), and MAP3K9 (82%). In the
“Thermofisher kinase panel”, only 7 kinases outside the CDK
family out of 362 kinases tested were significantly inhibited
(>80% inhibition): GSK3β (100%), GSK3α (96%), DYRK2
(85%), MAP4K4 (85%), Jnk1 (84%), ERK7 (83%), and
DYRK1A (80%). The selectivity among the CDK family and
other kinase hits was further assessed in assays recapitulating the
typical ATP concentration in cells ([ATP] of 5 mM). The
overall profile is summarized in Table 7, supporting that 24 is a
potent CDK9 inhibitor with >10-fold selectivity against CDK1-
7 and CDK12, except CDK3 (>5.8-fold) and >47-fold
selectivity for other non-CDK kinases. This selectivity profile
among the CDK family was confirmed in cells, showing that 24
exhibited >25-fold cellular selectivity for CDK9 over CDK1,
CDK2, CDK4, CDK6, and CDK7 upon short-term treatment.46
The binding kinetics of 23 and 24 to CDK9 was determined
by surface plasmon resonance (Table 4). Both compounds
showed a short dissociation half-life: 20.3 min for compound 23
and 16.2 min for compound 24. The reversibility of pSer2-
RNAP2 inhibition in the MCF-7 cell line was investigated by
incubation of 23 and 24 followed by cell washout and
measurement of pSer2-RNAP2 inhibition at 30 min and 2 h
after wash-out. Compounds 23 and 24 showed reversibility,
where inhibition was very significantly reduced at 30 min (IC50
reduced by 19-fold for 23 and 32-fold for 24) and at 2 h (IC50 > 3
μM), which was expected from the “fast-off” binding kinetics of
23 and 24 to CDK9 measured by surface plasmon resonance.
Thermodynamic solubilities of 23 and 24 were further
assessed from crystalline batches: 30 mg/L in 1× PBS buffer pH
t
1/2 1.5 h, see Table 8) and 1.6 h for compound 24 (predicted
human PK parameters: Cl 5.3 mL/min/kg, Vss 0.73 L/kg, mean
t
1/2 1.6 h, see Table 9) respectively
Metabolite identification studies were performed in HLM
(and human hepatocytes) for compounds 23 and 24. Both
compounds showed analogous metabolic pathways to those
observed with compound 6, leading to the formation of 23-M1,
23-M2, and 23-M3 from compound 23 and 24-M1, 24-M2, and
24-M3 from compound 24 (see Figure 5), the M1 metabolite
being the most abundant. The structures of 24-M1 and 24-M2
were subsequently confirmed by chemical synthesis of authentic
standards. From their biological activity and their properties
(Table 10), the risk that the metabolites formed from 24 would
contribute to significant CDK9 activity in the clinical setting was
viewed as low. The detailed metabolism of 24 in vitro and in vivo
will be the subject of a separate publication in due course.
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Table 8. Pharmacokinetic Properties of Compound 23 in
Preclinical Species and Predicted Human Parameters
Table 10. CDK9 Enzymatic and Cellular Potencies and in
Vitro Intrinsic Clearance of 24 and Its Metabolites 24-M1
and 24-M2 in HLM and Human Hepatocytes
mouse
rat
81
104
0.80
0.18
dog
human
2.6
4.8
0.61
1.5
a
hepatocytes Clint (μL/min/106 cells)
75
70
0.49
0.16
23
53
1.2
CDK9 enz. [ATP] 5 mM pSer2 cell IC50
HLM
b
Hu. heps
a
a
c
Cpd
24
24-
IC50 (μM)
(μM)
Cllint
Cllint
b
c
plasma Cl (mL/min/kg)
b
c
<0.004
0.020
0.0134
0.17
50
14
5.5
48
Vss (L/kg)
c
t
1/2 (h)
0.43
M1
24-
M2
a
b
Intrinsic clearance measured from hepatocytes, Clint. For mouse
0.139
>2.3
24
<1
and Han Wistar rat studies (n ≥ 2), the compound was administered
respectively at a dose of 2.4 μmol/kg and 1.2 μmol/kg i.v. as a
solution formulation. For Beagle dogs (n = 2), the compound was
administered by intravenous infusion over 0.25 h at a dose of 0.24
μmol/kg as a solution in 10/90 DMSO/10% captisol (w/v) at the
a
Geometric means of at least two IC50 determinations per compound.
b
Human liver microsome intrinsic clearance (HLM Clint); μL·min−1·
c
mg−1. Intrinsic clearance measured from human hepatocytes, Clint;
μL·min−1·106 cells−1.
c
concentration of 0.2 mg/mL. Italicized text indicates human PK
predictions (see Supporting Information for methods).
The activity of compound 24 was recently reported in
multiple AML cell lines in vitro and multiple AML xenograft
models in vivo.46 Consistent with the expected mode of action of
a CDK9 inhibitor, compound 24 showed inhibition of pSer2-
RNAP2, reduction of Mcl-1 protein and induction of caspase 3/
7 activation in the acute myeloid leukemia cell lines MV-4-1146
and Nomo-1 (see Figure 6) in a dose- and time-dependent
manner.
In the MV-4-11 xenograft mouse model, compound 24
showed a dose-dependent cell death driven antitumor efficacy
when dosed intermittently (BID 2 h apart by i.p. administration,
2 days on 5 days off) at 5 mg/kg (97% tumor growth inhibition,
measurement taken 33 days after commencement of dosing)
and 15 mg/kg (100% complete tumor regression that was
sustained out to more than 125 days). This antitumor response
was driven by cell death as evidenced by induction of caspase 3/
7. A concomitant reduction in pSer2-RNAP2 and Mcl-1
demonstrated that this effect is indeed mediated by inhibition
of CDK9.46 Here we report the in vivo activity of compound 24
in the Nomo-1 AML xenograft model. Compound 24 resulted in
a similar extent of cell death driven antitumor efficacy using the
same doses and schedules, with the 5 mg/kg BID schedule
resulting in 65% tumor growth inhibition, and the 15 mg/kg BID
Table 9. Pharmacokinetic Properties of Compound 24 in
Preclinical Species and Predicted Human Parameters
mouse
98
rat
73
dog monkey human
hepatocytes Clint (μL/min/106
11
32
5.5
a
cells)
b
c
plasma Cl (mL/min/kg)
55
0.45
0.18
71
0.68
0.18
16
19
5.3
b
c
Vss (L/kg)
0.67
0.66
1.3
0.89
0.73
1.6
c
t1/2 (h)
a
b
Intrinsic clearance measured from hepatocytes, Clint. For mouse
and Han Wistar rat studies (n ≥ 2), the compound was administered
respectively at a dose of 2.3 μmol/kg and 1.2 μmol/kg i.v. as a
solution formulation. Male Beagle dogs (n = 2) were administered
0.23 μmol/kg by intravenous infusion over 0.25 h formulated as a
solution in 10% dimethyl sulfoxide (DMSO)/90% sterile water for
injection (adjusted to pH 7−9). For cynomolgus monkeys (n = 2),
the compound was administered by intravenous infusion over 0.25 h
at a dose of 0.46 μmol/kg as a solution in 2% dimethylacetamide
(DMA)/30% polyethylene glycol 400 (PEG400)/68%, 1% (w/v)
c
Tween 80 in sterile water for injection. Italicized text indicates
human PK predictions (see Supporting Information for methods).
Figure 5. Metabolite identification of 23 and 24 after incubation in HLM. Metabolites shown were considered to have the potential to retain
pharmacological activity and do not represent a complete metabolic scheme.
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Figure 8. Dose-dependent reduction of pSer2-RNAP2 in the acute
myeloid leukemia Nomo-1 xenograft model by i.p. administration of
compound 24, two doses 2 h apart by i.p. administration.
sensitive AML cell lines screened with compound 24,46 MV-4-
11 was selected as a representative AML cell line for the
purposes of predicting the efficacious dose in clinical AML. The
quantitative PKPD/antitumor efficacy model, derived from
mouse MV-4-11 xenograft study data (described previously46)
was translated to the clinical AML setting (by adjusting system
parameter values and replacing the mouse PK model for
compound 24 with a predicted human PBPK model; see
Supporting Information) and used to predict clinical efficacy.
Clinical i.v. dose was optimized such that each 2 h i.v. infusion
was predicted to result in a 60% reduction in the leukemic cell
burden. This extent of antitumor efficacy (per dosing occasion)
was similar to what had been achieved in mouse MV-4-11
xenograft models when dosing compound 24 and was predicted
to be sufficient to drive a sustained progressive reduction in
leukemic cell burden in AML patients when utilizing a 2 days-
on/12 days-off dosing schedule (assuming a 10 day doubling
time). The predicted clinical dose of compound 24 that would
be required to achieve this extent of antitumor efficacy was 44
mg. The predicted time-course of free concentration in plasma
for a typical patient, at this dose, is shown in Figure 9.
Figure 6. Inhibition of pSer2-RNAP2, depletion of Mcl-1 protein, and
induction of caspase 3/7 activation after a 6-h incubation of compound
24 (dose response) in the acute myeloid leukemia Nomo-1 cell line.
Top panel: Western blot; bottom panel: quantification.
schedule resulting in 65% tumor volume regression (assessed 12
days after the commencement of dosing), Figure 7. The extent
and duration of reduction of pSer2-RNAP2 in the Nomo-1
xenograft tumors (Figure 8) were also similar to that observed in
MV-4-11 tumors at both dose levels.
As the half-maximal effective concentration (EC50) for
caspase activation in MV-4-11 cells closely aligns with other
Preclinical in vivo toxicological evaluation of compound 24
supported dosing to patients, and 24 (AZD4573) is currently in
phase 1 clinical trials in patients with relapsed or refractory
Figure 7. Dose-dependent cell death driven antitumor efficacy in the
acute myeloid leukemia Nomo-1 xenograft model of compound 24 by
i.p. administration, twice daily 2 h apart, 2 days on 5 days off.
Figure 9. Simulated human pharmacokinetic profile of compound 24
following a 44 mg dose administered by a 2 h continuous i.v. infusion.
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A = 0.1% formic acid in water (for acid work) or 0.1% ammonia in water
(for base work) B = acetonitrile. For acid analysis, the column used was
Waters Acquity HSS T3 1.8 μm 2.1 × 50 mm, and for base analysis the
column used was Waters Acquity BEH 1.7 μm 2.1 × 50 mm.
Alternatively, UPLC was carried out using a Waters UPLC fitted with a
Waters SQ mass spectrometer (column temp 30, UV = 210−400 nm,
mass spec = ESI with positive/negative switching) at a flow rate of 1
mL/min using a solvent gradient of 2−98% B over 1.5 min (total
runtime with equilibration back to starting conditions: 2 min), where A
= 0.1% formic acid in water and B = 0.1% formic acid in acetonitrile (for
acid work) or A = 0.1% ammonium hydroxide in water and B =
acetonitrile (for base work). For acid analysis, the column used was a
Waters Acquity HSS T3 1.8 μm 2.1 × 30 mm, and for base analysis the
column used was a Waters Acquity BEH C18 1.7 μm 2.1 × 30 mm;
LCMS was carried out using a Waters Alliance HT (2795) fitted with a
Waters ZQ ESCi mass spectrometer and a Phenomenex Gemini − NX
(5 μm × 2.1 mm) column at a flow rate of 1.1 mL/min 95% A to 95% B
over 4 min with a 0.5 min hold. The modifier was kept at a constant 5%
C (50:50 acetonitrile/water 0.1% formic acid) or D (50:50 acetonitrile/
water 0.1% ammonium hydroxide depending on whether it was an
acidic or basic method. Intermediates were not fully purified, but their
structure and purity were assessed by NMR, HPLC, or UPLC and mass
techniques (unless stated otherwise) and are consistent with the
proposed structures.
hematological malignancies (ClinicalTrials.gov Identifier:
NCT03263637).
CONCLUSION
■
Here we report the optimization of a series of pyrimidine amides
as potent and selective CDK9 inhibitors having suitable
pharmacokinetic and physicochemical properties for an intra-
venous agent with short duration of target engagement. Starting
from 2, a highly selective inhibitor of CDK9 but with physical
and pharmacokinetic properties that appeared to be unsuitable
for the desired profile, we identified compound 24 (also known
as AZD4573). Compound 24 is a potent inhibitor of CDK9
(IC50 of <0.004 μM) with fast-off binding kinetics (t1/2 16 min)
and high selectivity versus other kinases, including other CDK
family members. Compound 24 exhibited a short pharmacoki-
netic half-life in multiple preclinical species (less than 1 h in rat,
dog, and monkey) ,and PBPK modeling predicted a short half-
life in humans. While the predictions of human PK may never be
completely accurate due to species differences, the predictions
for compounds relative to one another are useful in providing a
rank order in order to select the most appropriate clinical
candidate. Compound 24 also exhibited suitable solubility for
intravenous administration. Short-term treatment with com-
pound 24 led to a rapid dose- and time-dependent decrease in
pSer2-RNAP2 and Mcl-1 in cells, resulting in activation of
caspase 3/7 and cell apoptosis in a broad range of hematological
cancer cell lines. In vivo efficacy was demonstrated in xenograft
models derived from multiple hematological tumors (e.g.,
regression at 15 mg/kg, i.p administration, BID 2 days on 5
days off, in acute myeloid lymphoma MV-4-11 and Nomo-1
xenografts), with evidence of a decrease of pharmacologically
relevant biomarkers (e.g., pSer2-RNAP2). Compound 24 is
currently in phase 1 clinical trials for the treatment of
hematological malignancies.
All experimental activities involving animals were carried out in
accordance with AstraZeneca animal welfare protocols, which are
consistent with the American Chemical Society Publications rules and
ethical guidelines.
(1S,3R)-3-Acetamido-N-[4-(1-isopropyl-2-methyl-1H-imidazol-5-
yl)-2-pyridinyl]cyclohexanecarboxamide (3). A mixture of 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (35, 0.650 g, 2.95
mmol), potassium phosphate (0.941 g, 4.43 mmol), and 5-bromo-1-
isopropyl-2-methyl-1H-imidazole (36, 0.30 g, 1.5 mmol) in water (8
mL) and 1,4-dioxane (24 mL) was degassed using a stream of nitrogen
for 20 min. Then, chloro(2-dicyclohexylphosphino-2′,4′,6′-triisoprop-
yl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (0.12 g,
0.15 mmol) was added to the mixture, and the reaction was heated at
110 °C for 3 h. After concentration under reduced pressure, the
resulting residue was suspended in MeOH (10 mL) and DCM (30
mL), and the mixture was filtered and concentrated under reduced
pressure to give crude 4-(1-isopropyl-2-methyl-1H-imidazol-5-yl)-
pyridin-2-amine (37, 137 mg, 42%), which was used without
purification. MS-ESI m/z 217 [MH+].
1-Chloro-N,N,2-trimethylprop-1-en-1-amine (0.232 g, 1.73 mmol)
was added to (1S,3R)-3-((tert-butoxycarbonyl)amino)-
cyclohexanecarboxylic acid (38, 0.337 g, 1.39 mmol) in DCM (8
mL) at 0 °C. The solution was stirred at room temperature for 30 min.
A solution of 37 (0.25 g, 1.2 mmol) and pyridine (0.14 mL, 1.7 mmol)
in THF (8 mL) was then added. The mixture was stirred at room
temperature for 18 h. The mixture was then partitioned between EtOAc
(50 mL) and water (50 mL), and the layers were separated. The organic
layer was concentrated under reduced pressure. The resulting residue
was purified by flash silica chromatography, eluting with 10−20%
MeOH in DCM to afford tert-butyl ((1R,3S)-3-((4-(1-isopropyl-2-
methyl-1H-imidazol-5-yl)pyridin-2-yl)carbamoyl)cyclohexyl)-
carbamate (62 mg, 12%). MS-ESI m/z 442 [MH+].
EXPERIMENTAL SECTION
■
General Methods. All experiments were carried out at ambient
temperature under an inert atmosphere. Evaporations were carried out
by rotary evaporation or utilizing Genevac equipment or a Biotage v10
evaporator in vacuo, and workup procedures were carried out after the
removal of residual solids by filtration. Flash chromatography
purifications were performed on an automated Teledyne Isco
CombiFlash Rf or Teledyne Isco CombiFlash Companion using
prepacked RediSep Rf Gold Silica Columns (20−40 μm, spherical
particles), GraceResolv Cartridges (Davisil silica), or Silicycle
cartridges (40−63 μm). Ion exchange purification was generally
performed using an SCX-2 (Biotage) cartridge. Preparative chromatog-
raphy was performed on a Gilson prep HPLC instrument with UV
collection or on a Waters AutoPurification HPLC-MS instrument with
MS- and UV- triggered collection. The purities of the compounds for
biological testing were assessed by NMR and mass spectral techniques
following liquid chromatography (LCMS or UPLC) and are consistent
with the proposed structures characterized; purity was at least 95%.
Proton NMR were determined using a Bruker Avance 500 (500 MHz)
or Bruker Avance 400 (400 MHz) instrument. Measurements were
taken at ambient temperature unless otherwise specified; the following
abbreviations have been used: s, singlet; d, doublet; t, triplet; q, quartet;
m, multiplet; dd, doublet of doublets; ddd, doublet of doublet of
doublet; dq, double of quartets; dt, doublet of triplets; tt, triplet of
triplets; p, pentet; br, broad signal. UPLC was carried out using a
Waters UPLC fitted with Waters SQ mass spectrometer (column temp
40, UV = 220−300 nm, mass spec = ESI with positive/negative
switching) at a flow rate of 1 mL/min using a solvent system of 97% A +
3% B to 3% A to 97% B over 1.50 min (total run time with equilibration
back to starting conditions back to starting conditions 1.70 min), where
Hydrochloric acid in dioxane (4 M; 0.49 mL, 14.0 mmol) was added
to a solution of tert-butyl ((1R,3S)-3-((4-(1-isopropyl-2-methyl-1H-
imidazol-5-yl)pyridin-2-yl)carbamoyl)cyclohexyl)carbamate (0.062 g,
0.14 mmol) in MeOH (2 mL). The reaction was stirred at room
temperature for 18 h. After concentration under reduced pressure, the
resulting residue was diluted in DCM (5 mL) and DIPEA (0.098 mL,
0.56 mmol). Acetyl chloride (0.020 mL, 0.28 mmol) was then added.
The reaction was stirred at room temperature for 10 min. The reaction
was then concentrated under reduced pressure, and the resulting
residue was purified by preparative HPLC (column: Xbridge Phenyl 19
mm × 150 mm 5 μm), eluting with 25−45% acetonitrile in water
containing 0.2% NH4OH (pH 10). Product fractions were concen-
1
trated under reduced pressure to give 3 (26 mg, 48%) as a solid. H
NMR (400 MHz, DMSO-d6): 1.01−1.17 (1H, m), 1.21−1.39 (3H, m),
N
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Drug Annotation
1.44 (6H, d, J = 7.0 Hz), 1.73−1.82 (6H, m), 1.89 (1H, br d, J = 11.5
Hz), 2.47 (3H, s), 2.58−2.70 (1 H, m), 3.52−3.61 (1H, m), 4.50 (1H,
td, J = 7.0, 14.1 Hz), 6.89 (1H, s), 7.05 (1H, dd, J = 1.4, 5.1 Hz), 7.77
(1H, d, J = 8.0 Hz), 8.10 (1H, s), 8.34 (1H, d, J = 5.0 Hz) 10.53 (1 H, s);
MS-ESI m/z 384 [MH+]. HRMS-ESI: m/z found 384.2390 [MH+],
C22H29ClN5O2 requires 384.2394.
(1S,3R)-3-Acetamido-N-(5-fluoro-4-(4,5,6,7-tetrahydropyrazolo-
[1,5-a]pyridin-3-yl)pyridin-2-yl)cyclohexanecarboxamide (5).
Chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)-
[2-(2′-amino-1,1′-biphenyl)]palladium(II) (0.092 g, 0.12 mmol) was
added to a degassed mixture of 41 (0.347 g, 1.40 mmol), 2-chloro-5-
fluoro-4-iodopyridine 45 (0.30 g, 1.2 mmol) and potassium phosphate,
tribasic (0.61 g, 3.5 mmol) in 1,4-dioxane (10 mL), and water (2 mL).
The mixture was degassed and stirred at 90 °C for 2 h under nitrogen.
The reaction mixture was concentrated under reduced pressure, and the
resulting residue was taken up in water (20 mL). The mixture was
extracted with DCM (3 × 20 mL). The combined organic layers were
dried over MgSO4, filtered, and concentrated under reduced pressure.
The resulting residue was purified by flash silica chromatography,
elution gradient 0−60% EtOAc in heptane. Product fractions were
concentrated under reduced pressure to afford 3-(2-chloro-5-
fluoropyridin-4-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine 46 (200
mg, 68%) as a yellow gum. MS-ESI m/z 252 [MH+].
(1S,3R)-3-Acetamido-N-(4-(4,5,6,7-tetrahydropyrazolo[1,5-a]-
pyridin-3-yl)pyridin-2-yl)cyclohexanecarboxamide (4). 1-Chloro-
N,N,2-trimethylprop-1-en-1-amine (0.57 mL, 4.33 mmol) was added
to a solution of 38 (1.01 g, 4.16 mmol) in DCM (40 mL) at 0 °C. After
1.5 h, a mixture of 4-bromopyridin-2-amine 39 (0.60 g, 3.5 mmol) and
pyridine (1.1 mL, 14 mmol) in DCM (33 mL) was added via cannula.
The resulting yellow mixture was allowed to warm to room temperature
and was stirred under these conditions for 72 h. The now white mixture
was filtered and rinsed with a cold DCM wash, and the white precipitate
was dried under a vacuum at 70 °C for 30 min to afford tert-butyl
((1R,3S)-3-((4-bromopyridin-2-yl)carbamoyl)cyclohexyl)carbamate
40 (1.38 g, 100%) as a white solid. 1H NMR (300 MHz, DMSO-d6):
0.99−1.35 (4H, m) 1.38 (9H, s) 1.68−1.80 (3H, m) 1.88 (1H, d)
2.53−2.64 (1H, m) 3.15−3.35 (1H, m) 6.76 (1H, d) 7.34 (1H, dd)
8.21 (1H, d) 8.33 (1H, d) 10.63 (1H, s). MS-ESI m/z 398, 400 [MH+].
Chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-
biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (9.88 mg, 0.01
mmol) was added in one portion to a degassed mixture of 40 (100 mg,
0.25 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyridine 41 (93 mg, 0.38 mmol), potassium
phosphate (160 mg, 0.75 mmol), 1,4-dioxane (2 mL), and water (0.2
mL) at 21 °C under nitrogen. The resulting mixture was stirred at 100
°C for 16 h. The reaction mixture was diluted with EtOAc (10 mL) and
washed with saturated NaHCO3 (10 mL). The aqueous layer was
extracted with EtOAc (2 × 10 mL), and the combined organic layers
were dried over Na2SO4, filtered, and concentrated under reduced
pressure to afford crude product. The crude product was purified by
flash silica chromatography, elution gradient 20−80% EtOAc in
heptane. Pure fractions were evaporated to dryness to afford tert-
butyl ((1R,3S)-3-((4-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-yl)-
pyridin-2-yl)carbamoyl)cyclohexyl)carbamate (42, 70 mg, 63%) as a
white powder. 1H NMR (400 MHz, DMSO-d6): 1.12 (1H, d), 1.21−
1.33 (3H, m), 1.39 (9H, s), 1.76 (3H, s), 1.82−1.95 (3H, m), 2.00 (2H,
d), 2.59 (1H, s), 2.97 (2H, t), 3.89 (1H, s), 4.12 (2H, t), 6.75 (1H, s),
7.19 (1H, dd), 7.85 (1H, s), 8.22 (2H, d), 10.32 (1H, s). MS-ESI m/z
440 [MH+].
Hydrochloric acid in dioxane (4 M; 0.20 mL, 0.80 mmol) was added
dropwise to 42 (70 mg, 0.16 mmol) in DCM (2 mL) at 21 °C under
nitrogen. The resulting mixture was stirred at 21 °C for 16 h. MeOH (1
mL) was added, and the mixture was purified directly by ion exchange
chromatography, using an SCX-2 column. The desired product was
eluted using 1 M NH3 in MeOH, and product fractions were
concentrated under reduced pressure to afford (1S,3R)-3-amino-N-
(4-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-yl)pyridin-2-yl)-
cyclohexanecarboxamide (47 mg, 87%) as a white crystalline solid. 1H
NMR (400 MHz, CD3OD): 0.95−1.08 (1H, m), 1.2−1.4 (3H, m),
1.76−1.91 (5H, m), 1.99 (3H, dtt), 2.44 (1H, ddd), 2.61 (1H, tt), 2.95
(2H, t), 3.25 (1H, s), 4.07 (2H, t), 7.11 (1H, dd), 7.74 (1H, s), 8.07−
8.16 (2H, m), NH2 peak not observed. MS-ESI m/z 340 [MH+].
Acetic anhydride (0.016 mL, 0.17 mmol) was added to (1S,3R)-3-
amino-N-(4-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-yl)pyridin-2-
yl)cyclohexanecarboxamide (47 mg, 0.14 mmol) and triethylamine
(0.023 mL, 0.17 mmol) in DCM (1 mL) at 21 °C under nitrogen. The
resulting solution was stirred under these conditions for 60 h. The
reaction mixture was loaded directly onto silica and purified by flash
silica chromatography, elution gradient 1−10% MeOH in DCM.
Product fractions were concentrated under reduced pressure to afford 4
(43 mg, 81%) as a white solid. 1H NMR (400 MHz, CDCl3): 1.07−1.23
(1H, m), 1.37−1.53 (3H, m), 1.87−2.03 (8H, m), 2.03−2.11 (2H, m),
2.25 (1H, d), 2.39−2.51 (1H, m), 3.06 (2H, t), 3.88 (1H, dtq), 4.20
(2H, t), 5.40 (1H, d), 7.10 (1H, dd), 7.80 (1H, s), 8.17 (1H, dd), 8.32
(1H, s), 8.35 (1H, s). MS-ESI m/z 382 [MH+]. HRMS-ESI: m/z found
382.2248 [MH+], C21H28N5O2 requires 382.2238.
Tetrakis(triphenylphosphine)palladium(0) (0.092 g, 0.080 mmol)
was added to a mixture of 46 (0.20 g, 0.79 mmol), tert-butyl ((1R,3S)-3-
carbamoylcyclohexyl)carbamate 47 (0.231 g, 0.95 mmol, see
Supporting Information), 9,9-dimethyl-4,5-bis(diphenylphosphino)-
xanthene (0.092 g, 0.16 mmol), and cesium carbonate (0.777 g, 2.38
mmol) in 1,4-dioxane (6 mL). The mixture was degassed (vacuum) and
backfilled with nitrogen, and the resulting suspension was stirred at 120
°C for 2 h in the microwave reactor. The reaction mixture was
partitioned between water (20 mL) and DCM (40 mL) and separated
using a phase separation cartridge. The organic layer was adsorbed onto
silica and purified by flash silica chromatography, elution gradient 0−
60% EtOAc in heptane. Product fractions were concentrated under
reduced pressure to afford tert-butyl ((1R,3S)-3-((5-fluoro-4-(4,5,6,7-
tetrahydropyrazolo[1,5-a]pyridin-3-yl)pyridin-2-yl)carbamoyl)-
cyclohexyl)carbamate 48 (136 mg). This material was used directly in
the next step without further purification. MS-ESI m/z 458 [MH+].
Trifluoroacetic acid (0.17 mL, 2.2 mmol) was added to 48 (0.10 g,
0.22 mmol) in DCM (5 mL). The resulting solution was stirred at room
temperature for 1 h. The reaction mixture was purified by ion exchange
chromatography using an SCX-2 column. The desired product was
eluted from the column using 1 M NH3 in MeOH, and product
fractions were concentrated under reduced pressure to afford (1S,3R)-
3-amino-N-(5-fluoro-4-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-
yl)pyridin-2-yl)cyclohexanecarboxamide as a yellow gum. This material
was used directly in the next step without further purification. MS-ESI
m/z 358 [MH+]. Acetic anhydride (0.032 mL, 0.34 mmol) was added
to (1S,3R)-3-amino-N-(5-fluoro-4-(4,5,6,7-tetrahydropyrazolo[1,5-a]-
pyridin-3-yl)pyridin-2-yl)cyclohexanecarboxamide (0.10 g, 0.28
mmol), triethylamine (0.12 mL, 0.84 mmol), and N,N-dimethylpyr-
idin-4-amine (2 mg, 0.01 mmol) in DCM (5 mL) at room temperature
under air. The resulting solution was stirred at room temperature. for 2
h. The reaction mixture was quenched with saturated aqueous
ammonium chloride (20 mL) and extracted with DCM (2 × 20 mL).
The combined organic layers were dried over MgSO4, filtered, and
concentrated under reduced pressure. The resulting residue was
purified by preparative HPLC (Waters XBridge Prep C18 OBD
column, 5 μ silica, 30 mm diameter, 100 mm length), using decreasingly
polar mixtures of water (containing 1% NH3) and MeCN as eluents.
Fractions containing the desired compound were concentrated under
reduced pressure to afford 5 (0.047 g, 42%) as a gum. 1H NMR (500
MHz, DMSO-d6): 1.03−1.15 (1H, m), 1.23−1.37 (3H, m), 1.74−1.82
(6H, m), 1.83−1.94 (3H, m), 2.00−2.08 (2H, m), 2.56−2.68 (1H, m),
2.91 (2H, t), 3.58−3.61 (1H, m), 4.15 (2H, t), 7.73−7.78 (2H, m), 8.26
(1H, d), 8.30 (1H, d), 10.48 (1H, s). MS-ESI m/z 400 [MH+]. HRMS-
ESI: m/z found 400.2144 [MH+], C21H27FN5O2 requires 400.2143.
(1S,3R)-3-Acetamido-N-(5-chloro-4-(4,5,6,7-tetrahydropyrazolo-
[1,5-a]pyridin-3-yl)pyridin-2-yl)cyclohexanecarboxamide (6). The
reaction was split into four separate sealed microwave reaction vessels,
each containing 5-chloro-2-fluoro-4-iodopyridine 55 (750 mg, 2.95
mmol), concentrated aqueous ammonium hydroxide (8.4 mL), and
NMP (7.5 mL). The reaction vessels were each heated at 100 °C for 17
h. The combined batches were then diluted with water (50 mL) and
extracted with EtOAc (3 × 120 mL). The combined organic layers were
O
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Journal of Medicinal Chemistry
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Drug Annotation
dried over MgSO4, filtered, and concentrated under reduced pressure to
afford a pale-yellow oil. The oil was loaded onto a 20 g SCX-2 column
and eluted sequentially with DCM, MeOH, and 1% NH3 in MeOH.
Basic fractions were concentrated to provide 5-chloro-4-iodopyridin-2-
amine 56 (2.9 g, 99%) as a colorless solid. 1H NMR (400 MHz, DMSO-
d6): 6.21 (2H, s), 7.05 (1H, s), 7.93 (1H, s). MS-ESI m/z 255 [MH+].
Cesium carbonate (13.4 g, 41.2 mmol) and PdCl2(dppf)·CH2Cl2
(0.94 g, 1.2 mmol) were added sequentially to a degassed mixture of 56
(4.19 g, 16.5 mmol), 41 (5.72 g, 23.1 mmol), 1,4-dioxane (141 mL),
and water (23.5 mL). The resulting red mixture was warmed to 95 °C
and became clear. With vigorous stirring, some precipitate formed
which gradually redissolved. After 4 h, another 800 mg of 41 were
added; after another 40 min, the reaction was cooled to room
temperature and stirred under these conditions for 18 h. The mixture
was then diluted with ethyl acetate, and the layers were separated. The
organic layer was washed with saturated aqueous sodium chloride, dried
over sodium sulfate, filtered, and concentrated under reduced pressure.
The resulting oil was purified by flash silica chromatography, elution
gradient 0−10% methanol in ethyl acetate. Product fractions were
combined, concentrated under reduced pressure, and the resulting
residue was stirred vigorously in 1:1 DCM: hexane for 20 min. The
mixture was then diluted with hexane and filtered with a hexane wash.
The resulting solid was dried under vacuum to afford 5-chloro-4-
(4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-yl)pyridin-2-amine 57
g). This solid was repurified by flash silica (plug) chromatography,
elution gradient 0−10% MeOH in ethyl acetate, to afford a white foam
solid. The solid was treated with 20 mL of acetonitrile, and the resulting
mixture was warmed to reflux conditions before being allowed to cool
to rt. Additional acetonitrile (∼5 mL) was added, and the process was
repeated until all solid dissolved. The resulting faint yellow solution was
cooled to rt, and a precipitate formed. After 1 h the precipitate was
filtered and washed with acetonitrile before being dried under vacuum
at 65 °C for 1 h. The solid was cooled to rt to afford 6 (1.76 g, > 98%
e.e.).
6: 1H NMR (300 MHz, DMSO-d6): 0.97−1.17 (1H, m), 1.20−1.38
(3H, m), 1.68−1.94 (9H, m), 1.96−2.07 (2H, m), 2.54−2.68 (1H, m),
2.80 (2H, t), 3.46−3.65 (1H, m), 4.14 (2H, t), 7.73 (1H, d), 7.76 (1H,
s), 8.14 (1H, s), 8.38 (1H, s), 10.57 (1H, s). 13C NMR (126 MHz,
DMSO-d6): 20.13, 22.89, 22.96, 23.22, 24.49, 28.69, 32.38, 35.64,
43.91, 47.39, 48.22, 113.46, 114.37, 123.66, 138.38, 138.62, 141.16,
148.10, 151.40, 168.56, 174.98. MS-ESI m/z 416 [MH+]. HRMS-ESI:
m/z found 416.1851 [MH+], C21H27ClN5O2 requires 416.1848.
Analytical SFC: flow: 1 mL/min, column: Chiralpak IA, 5 μ, 4.6 ×
150 mm, eluent: 40% methanol in CO2 containing 0.1% dimethylethyl-
amine), tR: (6, 1.42 min), ((1R,3S), 2.42 min). [α]D25 °C: + 70.2° in
MeOH.
(1S,3R)-3-Acetamido-N-(4-bromo-5-chloropyridin-2-yl)-
cyclohexanecarboxamide (7). N-Chloro-succinimide (3.70 g, 27.7
mmol) dissolved in DMF (20 mL) was added dropwise to 4-
bromopyridin-2-amine 39 (4.40 g, 25.4 mmol) in DMF (50 mL) at −78
°C over a period of 30 min under nitrogen. The resulting suspension
was then allowed to warm to room temperature. After stirring under
these conditions for 24 h, the reaction mixture was diluted with Et2O
(50 mL) and washed sequentially with aqueous NaOH (1 M; 2 × 50
mL), water (50 mL), and saturated aqueous sodium chloride (25 mL).
The organic layer was dried over MgSO4, filtered, and concentrated
under reduced pressure. The resulting residue was purified by flash
silica chromatography, elution gradient 0−25% EtOAc in DCM.
Product fractions were concentrated under reduced pressure to afford
4-bromo-5-chloropyridin-2-amine 59 (2.30 g, 43.7%) as a cream-
colored solid. 1H NMR (400 MHz, DMSO-d6): 6.35 (2H, s), 6.82 (1H,
s), 8.01 (1H, s). MS-ESI m/z 207, 209 [MH+].
1
(2.79 g, 68%) as light orange-beige needles. H NMR (300 MHz,
DMSO-d6): 1.74−1.88 (2H, m), 1.96−2.06 (2H, m), 2.76 (2H, t), 4.12
(2H, t), 6.03 (2H, br. s), 6.43 (1H, s), 7.63 (1H, s), 7.94 (1H, s). MS-
ESI m/z 249 [MH+].
1-Chloro-N,N,2-trimethylprop-1-en-1-amine (1.1 mL, 8.4 mmol)
was added to a solution of 38 (2.01 g, 8.24 mmol) in DCM (50 mL) at 0
°C. The reaction was maintained under these conditions for 100 min.
During this time, 58 (1.64 g, 6.59 mmol), pyridine (2.1 mL, 26 mmol),
and DCM (20 mL) were combined in a separate flask. The resulting
mixture was warmed gently (∼40 °C) until all solids dissolved. The
resulting solution was then cooled to 0 °C, whereupon a homogeneous
light-yellow mixture formed. This mixture was added via cannula
rapidly to the previously prepared solution of 38 and 1-chloro-N,N,2-
trimethylprop-1-en-1-amine, resulting in a darker yellow solution. The
reaction was allowed to warm to room temperature overnight and was
then evaporated to dryness to afford tert-butyl ((1R,3S)-3-((5-chloro-
4-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-yl)pyridin-2-yl)-
carbamoyl)cyclohexyl)carbamate 58 as a gray mixture (3.12 g). This
crude material was taken on to the next step without further
purification. MS-ESI m/z 474 [MH+].
Hydrochloric acid in dioxane (4 M; 10 mL, 40 mmol) was added to a
mixture of crude 58 (3.12 g, 6.59 mmol) in DCM (5 mL) and methanol
(5 mL) at 0 °C. The mixture became an amber solution. After 1 h, the
amber solution was concentrated under reduced pressure, and the
resulting residue was dried under a vacuum to afford (1S,3R)-3-amino-
N-(5-chloro-4-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-yl)pyridin-
2-yl)cyclohexanecarboxamide as a beige/gray foam solid. This material
was carried on to the next step without further purification. MS-ESI m/z
374 [MH+]. Acetyl chloride (1.0 mL, 14.5 mmol) was added dropwise
to a mixture of (1S,3R)-3-amino-N-(5-chloro-4-(4,5,6,7-
tetrahydropyrazolo[1,5-a]pyridin-3-yl)pyridin-2-yl)-
cyclohexanecarboxamide (2.46 g, 6.59 mmol) and pyridine (6.4 mL, 79
mmol) in DCM (58 mL) at 0 °C. After 45 min, the mixture was washed
with saturated aqueous sodium hydrogen carbonate and saturated
aqueous sodium chloride before being dried over sodium sulfate,
filtered, and concentrated under reduced pressure. The resulting dark
amber oil was purified by flash silica chromatography, elution gradient
0−10% methanol in DCM. Product fractions were concentrated under
reduced pressure to afford 6 (2.6 g, 93% yield over three steps, 96% e.e.)
as a light beige foam solid. This material was further purified by
preparative SFC conditions (Chiralpak IA column, 5 μm, 30 mm
diameter, 250 mm length, 40 °C column temperature, 100 bar outlet
pressure, 120 mL/min flow rate), eluting with 40% methanol
containing 0.1% dimethylethylamine in CO2, to afford faster eluting 6
and the slower eluting (1R,3S) enantiomer. Product fractions for 6 were
concentrated under reduced pressure to afford an amber-pink solid (2.3
A solution of 38 (1.50 g, 6.15 mmol) dissolved in DCM (20 mL) at 0
°C was treated with 1-chloro-N,N,2-trimethylprop-1-en-1-amine
(0.976 mL, 7.38 mmol). The mixture was stirred at room temperature
for 1.5 h before 59 (1.02 g, 4.92 mmol) and pyridine (0.59 mL, 7.4
mmol) were added sequentially. The resulting solution was stirred at
room temperature for 16 h. The reaction mixture was diluted with
DCM (25 mL) and washed sequentially with water (2 × 25 mL) and
saturated aqueous sodium chloride (25 mL). The organic layer was
dried over MgSO4, filtered, and concentrated under reduced pressure.
The resulting crude product was purified by ion exchange
chromatography, using an SCX-2 column. The desired product was
eluted from the column using methanol to afford tert-butyl ((1R,3S)-3-
((4-bromo-5-chloropyridin-2-yl)carbamoyl)cyclohexyl)carbamate 60
1
(2.34 g, quantitative) as a white solid. H NMR (400 MHz, DMSO-
d6): 1.12 (1H, dd), 1.22−1.32 (3H, m), 1.38 (9H, s), 1.72 (3H, dd),
1.83−1.94 (2H, m), 2.11 (1H, dt), 8.48 (1H, s), 8.50 (1H, s), 10.77
(1H, s), one proton not observed. MS-ESI m/z 430, 432 [M-H−].
Hydrochloric acid in dioxane (4 M; 5.9 mL, 24 mmol) was added to
60 (1.20 g, 2.77 mmol) in MeOH (7.0 mL) under air. The resulting
solution was stirred at ambient temperature for 16 h. The reaction
mixture was concentrated under reduced pressure to afford crude
(1S,3R)-3-amino-N-(4-bromo-5-chloropyridin-2-yl)-
cyclohexanecarboxamide dihydrochloride as a white solid. This solid
was taken up in DCM (8.4 mL), and 4-dimethylaminopyridine (0.014
g, 0.11 mmol) and triethylamine (1.0 mL, 7.1 mmol) were added
sequentially. Then acetic anhydride (0.26 mL, 2.7 mmol) was added
dropwise. The resulting solution was stirred at room temperature for 18
h before being quenched with saturated aqueous NH4Cl (50 mL) and
extracted with DCM (2 × 50 mL). The combined organic layers were
dried over MgSO4, filtered, and concentrated under reduced pressure to
afford (1S,3R)-3-acetamido-N-(4-bromo-5-chloropyridin-2-yl)-
cyclohexanecarboxamide 61 (0.96 g, 95%) as a white solid. The
P
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Drug Annotation
product was used in the next step without further purification. 1H NMR
(400 MHz, DMSO-d6): 1.23−1.41 (4H, m), 1.67−1.85 (4H, m), 2.39
(3H, tt), 2.75−2.92 (1H, m), 3.53 (1H, dtd), 7.59−7.83 (1H, m), 8.50
(2H, dd), 10.80 (1H, d). MS-ESI m/z 374, 376 [MH+].
TFA (2.0 mL, 26 mmol) was added to 54a (0.12 g, 0.26 mmol) in
DCM (10 mL). The resulting mixture was stirred at room temperature
for 16 h and then concentrated under reduced pressure. The resulting
crude yellow gum (100 mg) was dissolved in DCM (2 mL), and then
TEA (0.11 mL, 0.81 mmol) and acetic anhydride (0.051 mL, 0.54
mmol) were added sequentially. The resulting mixture was stirred at
room temperature for 1 h and was then concentrated under reduced
pressure. The crude product was purified by preparative HPLC (Waters
XBridge Prep C18 OBD column, 5 μ silica, 19 mm diameter, 150 mm
length), using decreasingly polar mixtures of water (containing 0.1%
NH4HCO3) and MeCN as eluents. Fractions containing the desired
compound were concentrated under reduced pressure to afford impure
8 (0.050 g, 45%) as a white solid. This material was repurified by
preparative HPLC (Waters XBridge Prep C18 OBD column, 5 μ silica,
19 mm diameter, 150 mm length), using decreasingly polar mixtures of
water (containing 0.05% TFA) and MeCN as eluents. Product fractions
were concentrated under reduced pressure to afford 8 (0.030 g, 27%) as
61 (0.10 g, 0.27 mmol) was added to a mixture of 1,5-dimethyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.065 g,
0.29 mmol), chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-
1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (0.021 g,
0.03 mmol), and potassium phosphate (0.170 g, 0.80 mmol) in 1,4-
dioxane (2.3 mL) and water (0.23 mL). The reaction mixture was then
degassed with a stream of nitrogen for 5 min. The resulting mixture was
stirred at room temperature for 16 h and subsequently purified by ion
exchange chromatography, using an SCX-2 column and eluting first
with methanol and then with 1 M NH3 in methanol. Product fractions
were concentrated under reduced pressure to afford a yellow oil. This
oil was further purified by preparative HPLC (Waters XBridge Prep
C18 OBD column, 5 μ silica, 50 mm diameter, 100 mm length), using
decreasingly polar mixtures of water (containing 1% NH3) and MeCN
as eluents. Fractions containing the desired compound were
concentrated under reduced pressure to afford 7 (0.011 g, 11%) as a
colorless solid. 1H NMR (500 MHz, DMSO-d6): 0.91−1.17 (1H, m),
1.19−1.4 (3H, m), 1.78 (6H, s), 1.89 (1H, d, J = 12.2 Hz), 2.28 (3H, s),
2.54−2.69 (1H, m), 3.45−3.65 (1H, m), 3.82 (3H, s), 7.60 (1H, s),
7.75 (1H, d, J = 7.9 Hz), 8.08 (1H, s), 8.41(1H, s), 10.60 (1H, s). MS-
ESI m/z 390 [MH+]. HRMS-ESI: m/z found 390.1704 [MH+],
C19H25ClN5O2 requires 390.1691.
1
a white solid. H NMR (300 MHz, DMSO-d6): 1.02−1.12 (1H, m),
1.19−1.40 (3H, m), 1.74−1.80 (6H, m), 1.85−1.95 (1H, m), 2.59−
2.65 (1H, m), 3.52−3.58 (1H, m), 7.07 (1H, dt, J = 1.3, 6.9 Hz), 7.47
(1H, ddd, J = 1.1, 6.8, 9.0 Hz), 7.73−7.84 (2H, m), 8.35 (1H, s), 8.44
(1H, s), 8.47 (1H, s), 8.84 (1H, td, J = 1.1, 7.0 Hz), 10.68 (1H, s).
HRMS-ESI: m/z found 412.1537 [MH+], C21H23ClN5O2 requires
412.1535.
(1S,3R)-3-Acetamido-N-(5-chloro-4-(5,6,7,8-tetrahydroimidazo-
[1,2-a]pyridin-3-yl)pyridin-2-yl)cyclohexanecarboxamide (9). 61
(0.20 g, 0.53 mmol), 5,6,7,8-tetrahydroimidazo[1,2-a]pyridine (47
mg, 0.38 mmol), cesium carbonate (0.14 g, 0.42 mmol), triethylamine
(0.11 mL, 0.76 mmol), triphenylphosphine (0.02 g, 0.06 mmol) and
diacetoxypalladium (6.85 mg, 0.030 mmol) were suspended in 1,4-
dioxane (5 mL) and sealed in a microwave tube. The reaction was
heated to 100 °C for 16 h in a microwave reactor and then cooled to
room temperature. The reaction was concentrated under reduced
pressure, and the resulting residue was purified by ion exchange
chromatography using an SCX-2 column. The desired product was
eluted from the column using 1 M NH3 in MeOH, and product
fractions were concentrated under reduced pressure. The resulting
residue was further purified by preparative HPLC (Waters XBridge
Prep C18 OBD column, 5 μ silica, 50 mm diameter, 100 mm length),
using decreasingly polar mixtures of water (containing 1% NH3) and
MeCN as eluents. Fractions containing the desired compound were
concentrated under reduced pressure to afford 9 (0.069 g, 44%) as a
(1S,3R)-3-Acetamido-N-(5-chloro-4-(pyrazolo[1,5-a]pyridin-3-
yl)pyridin-2-yl)cyclohexanecarboxamide (8). Pd(PPh3)4 (0.21 g, 0.18
mmol) was added to 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)pyrazolo[1,5-a]pyridine (0.490 g, 2.01 mmol), 2,5-dichloro-4-
iodopyridine (52, 0.500 g, 1.83 mmol), and Cs2CO3 (1.78 g, 5.48
mmol) in dioxane (2 mL) and water (0.2 mL) under nitrogen. The
resulting mixture was stirred at 100 °C for 1 h. The reaction mixture was
then concentrated under reduced pressure. The resulting mixture was
diluted with EtOAc (100 mL) and washed sequentially with water (100
mL) and saturated aqueous sodium chloride (100 mL). The organic
layer was dried over sodium sulfate, filtered, and concentrated under
reduced pressure. The resulting residue was purified by flash silica
chromatography, elution gradient 0−25% EtOAc in petroleum ether.
Product fractions were concentrated under reduced pressure to afford
3-(2,5-dichloropyridin-4-yl)pyrazolo[1,5-a]pyridine (53a, 0.310 g,
1
64%) as a white solid. H NMR (300 MHz, DMSO-d6): 7.09 (1H,
1
dt, J = 1.3, 6.9 Hz), 7.46 (1H, ddd, J = 1.1, 6.8, 9.0 Hz), 7.74 (1H, s),
7.80 (1H, td, J = 1.2, 9.0 Hz), 8.44 (1H, s), 8.60 (1H, s), 8.84 (1H, td, J
= 1.1, 7.0 Hz). MS-ESI m/z 264 [MH+].
yellow gum. H NMR (500 MHz, DMSO-d6): 0.95−1.16 (1H, m),
1.19−1.39 (3H, m), 1.78 (3H, s), 1.83−1.97 (2H, m), 2.55−2.68 (1H,
m), 2.84 (2H, s), 3.18 (2H, dd), 3.31 (3H, s), 3.57 (1H, dt), 3.83 (2H,
s), 4.08 (1H, q), 7.13 (1H, s), 7.75 (1H, d), 8.16 (1H, s), 8.47 (1H, s),
10.70 (1H, s). MS-ESI m/z 416 [MH+]. HRMS-ESI: m/z found
416.1851 [MH+], C21H27ClN5O2 requires 416.1848.
Pd(PPh3)4 (0.02 g, 0.02 mmol) was added to a mixture of 53a (0.054
g, 0.21 mmol), 47 (0.05 g, 0.21 mmol), 4,5-bis(diphenylphosphino)-
9,9-dimethylxanthene (0.024 g, 0.04 mmol), and Cs2CO3 (0.202 g, 0.62
mmol) in dioxane (2 mL) under nitrogen. The resulting mixture was
stirred at 120 °C for 2 h. In a separate flask, Pd(PPh3)4 (0.05 g, 0.04
mmol) was added to a mixture of 53a (0.109 g, 0.41 mmol), 47 (0.10 g,
0.41 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (0.048
g, 0.08 mmol), and Cs2CO3 (0.403 g, 1.24 mmol) in dioxane (2 mL)
under nitrogen. The resulting mixture was stirred at 120 °C for 2 h.
Both reactions were then combined and filtered through a plug of silica
with ethyl acetate wash. The filtrate was concentrated under reduced
pressure, and the resulting residue was redissolved in EtOAc (100 mL)
before being washed sequentially with water (100 mL) and saturated
aqueous sodium chloride (100 mL). The organic layer was dried over
sodium sulfate, filtered, and concentrated under reduced pressure. The
resulting residue was purified by preparative TLC (eluting with 50%
EtOAc in petroleum ether), to afford tert-butyl ((1R,3S)-3-((5-chloro-
4-(pyrazolo[1,5-a]pyridin-3-yl)pyridin-2-yl)carbamoyl)cyclohexyl)-
(1S,3R)-3-Acetamido-N-(5-chloro-4-(1,2-dimethyl-1H-imidazol-
5-yl)pyridin-2-yl)cyclohexanecarboxamide (10). Pd(OAc)2 (0.041 g,
0.18 mmol) was added to 1,2-dimethyl-1H-imidazole (0.175 g, 1.83
mmol), 52 (0.5 g, 1.83 mmol), Cs2CO3 (1.78 g, 5.48 mmol), and PPh3
(0.048 g, 0.18 mmol) in dioxane (10 mL) under nitrogen. The resulting
mixture was stirred at 120 °C for 16 h and then filtered with an ethyl
acetate wash. The filtrate was concentrated under reduced pressure and
redissolved in EtOAc (50 mL). This mixture was washed sequentially
with water (2 × 50 mL) and saturated aqueous sodium chloride (50
mL). The organic layer was dried over sodium sulfate, filtered, and
concentrated under reduced pressure. The resulting residue was
purified by flash silica chromatography, elution gradient 0−30% EtOAc
in petroleum ether. Product fractions were concentrated under reduced
pressure, and the resulting oil was crystallized from hexane to afford 2,5-
dichloro-4-(1,2-dimethyl-1H-imidazol-5-yl)pyridine (53b, 0.170 g,
38.5%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6): 3.17 (3H,
s), 3.48 (3H, s), 7.16 (1H, s), 7.65 (1H, s), 8.64 (1H, s). MS-ESI m/z
242 [MH+].
1
carbamate (54a, 0.13 g, 44%) as a white solid. H NMR (400 MHz,
DMSO-d6): 1.06−1.13 (1H, m), 1.26−1.30 (3H, m), 1.39 (9H, s),
1.73−1.80 (3H, m), 1.86−1.94 (1H, m), 2.56−2.63 (1H, m), 3.17−
3.32 (1H, m), 6.82 (1H, d, J = 8.2 Hz), 7.09 (1H, t, J = 6.8 Hz), 7.45−
7.54 (1H, m), 7.80 (1H, d, J = 9.0 Hz), 8.37 (1H, s), 8.45 (1H, s), 8.48
(1H, s), 8.85 (1H, d, J = 6.9 Hz), 10.67 (1H, s). MS-ESI m/z 470
[MH+].
Pd(PPh3)4 (0.05 g, 0.04 mmol) and 4,5-bis(diphenylphosphino)-
9,9-dimethylxanthene (0.05 g, 0.08 mmol) were added to a mixture of
53b (0.20 g, 0.41 mmol), 47 (0.10 g, 0.41 mmol), and Cs2CO3 (0.403 g,
1.24 mmol) in dioxane (3 mL) under nitrogen. The resulting mixture
Q
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Drug Annotation
was stirred at 120 °C for 30 min. The reaction was then filtered with an
ethyl acetate wash, and the filtrate was concentrated under reduced
pressure. The resulting residue was purified by preparative TLC (100%
EtOAc), to afford tert-butyl (1R,3S)-3-((5-chloro-4-(1,2-dimethyl-1H-
imidazol-5-yl)pyridin-2-yl)carbamoyl)cyclohexyl)carbamate (54b,
3.62 (2H, m), 4.44 (1H, s), 7.80 (1H, s), 8.19 (1H, s), 8.81 (1H, s). MS-
ESI m/z 478 [M − H−].
62 (1 g, 2.08 mmol) was suspended in DCM (15 mL) at ambient
temperature. Hydrochloric acid in dioxane (4 M; 2.61 mL, 10.4 mmol)
was added, and the resulting mixture stirred for 16 h. The reaction
mixture was then loaded onto a 50 g SCX-2 column and eluted
sequentially with DCM, MeOH, and 1% NH3 in MeOH. Basic fractions
were concentrated under reduced pressure to afford crude 3-amino-N-
(5-chloro-4-iodopyridin-2-yl)cyclohexanecarboxamide as a colorless
amorphous solid (782 mg). This solid was dissolved in triethylamine
(0.632 mL, 4.53 mmol) in DCM (10 mL) at ambient temperature.
Then acetic anhydride (0.214 mL, 2.27 mmol) was added dropwise.
The reaction mixture was stirred for 5 days under these conditions
before being filtered with a DCM wash to provide (1S,3R)-3-
acetamido-N-(5-chloro-4-iodopyridin-2-yl)cyclohexanecarboxamide
63 (480 mg, 55%) as a colorless solid. The filtrate was concentrated
under reduced pressure, and the resulting residue was purified by flash
silica chromatography, elution gradient 20 to 60% EtOAc in heptane.
Product fractions were concentrated under reduced pressure to afford
additional 63 (193 mg, 22%) as a colorless crystalline solid (combined
1
0.140 g, 76%) as a pale yellow solid. H NMR (400 MHz, DMSO-
d6): 1.08−1.12 (1H, m), 1.22−1.35 (3H, m), 1.38 (9H, s), 1.73−1.77
(3H, m), 1.85−1.93 (1H, m), 2.38 (3H, s), 2.57−2.61 (1H, m), 3.23−
3.31 (1H, m), 3.44 (3H, s), 6.77−6.83 (1H, m), 7.01 (1H, s), 8.12 (1H,
s), 8.48 (1H, s), 10.72 (1H, s). MS-ESI m/z 448 [MH+].
TFA (1 mL, 13 mmol) was added to 54b (0.14 g, 0.31 mmol) in
DCM (5 mL). The resulting mixture was stirred at room temperature
for 1 h and then concentrated under reduced pressure to afford a yellow
gum (110 mg). This gum was taken up in DCM (5 mL), and TEA (0.13
mL, 0.95 mmol) was added. Then acetic anhydride (0.032 g, 0.32
mmol) was added dropwise. The resulting mixture was stirred at rt for 1
h and was then concentrated under reduced pressure. The resulting
residue was purified by preparative HPLC (Waters XBridge Prep C18
OBD column, 5 μ silica, 19 mm diameter, 150 mm length), using
decreasingly polar mixtures of water (containing 0.1% NH4HCO3) and
MeCN as eluents. Product fractions were concentrated under reduced
pressure to afford 10 (40 mg) as a white solid. This solid was repurified
by chiral reverse phase HPLC (Chiralpak IA column, 5 μ silica, 20 mm
diameter, 250 mm length; flow rate 20 mL/min), eluting with isocratic
30% IPA in hexanes, to afford 10 (0.030 g, 25%) as a white solid. 1H
NMR (400 MHz, DMSO-d6): 1.01−1.14 (1H, m), 1.19−1.38 (3H, m),
1.69−1.84 (6H, m), 1.85−1.92 (1H, m), 2.38 (3H, s), 2.60−2.64 (1H,
m), 3.44 (3H, s), 3.51−3.63 (1H, m), 7.01 (1H, s), 7.78 (1H, d, J = 7.9
Hz), 8.13 (1H, s), 8.48 (1H, s), 10.74 (1H, s). HRMS-ESI: m/z found
390.1701 [MH+], C19H25ClN5O2 requires 390.1691.
(1S,3R)-3-Acetamido-N-(5-chloro-4-(imidazo[1,2-a]pyridin-3-yl)-
pyridin-2-yl)cyclohexanecarboxamide (11). Imidazo[1,2-a]pyridine
(0.045 mL, 0.45 mmol), 61 (0.235 g, 0.63 mmol), cesium carbonate
(0.161 g, 0.49 mmol), triethylamine (0.125 mL, 0.90 mmol),
triphenylphosphine (0.019 g, 0.07 mmol), and diacetoxypalladium
(8.05 mg, 0.04 mmol) were suspended in 1,4-dioxane (5 mL) in a
microwave tube. The reaction was heated at 100 °C for 2 h in a
microwave reactor and then cooled to rt. The reaction was then
resubjected to microwave conditions (100 °C) for another 4 h and
cooled. The reaction mixture was diluted with DCM (20 mL) and
washed with water (3 × 25 mL). The organic layer was dried over
sodium sulfate, filtered, and concentrated under reduced pressure. The
resulting residue was purified by flash silica chromatography, elution
gradient 0−2% MeOH in EtOAc. Product fractions were concentrated
under reduced pressure to afford (0.078 g, 42%) as a colorless solid. 1H
NMR (400 MHz, CDCl3): 1.08−1.28 (1H, m), 1.32−1.58 (3H, m),
1.87−1.97 (3H, m), 1.98 (3H, s), 2.26 (1H, d, J = 12.3 Hz), 2.54 (1H,
ddd, J = 3.4, 8.2, 11.6 Hz), 3.88 (1H, dtd, J = 4.1, 7.9, 11.7 Hz), 5.69
(1H, d, J = 8.1 Hz), 6.91 (1H, td, J = 1.1, 6.9 Hz), 7.29 (1H, ddd, J = 1.2,
6.8, 9.1 Hz), 7.72 (1H, dt, J = 1.0, 9.1 Hz), 7.95 (1H, s), 8.15 (1H, dt, J =
1.1, 6.9 Hz), 8.38 (1H, s), 8.43 (1H, s), 8.70 (1H, s). MS-ESI m/z 412
[MH+]. HRMS-ESI: m/z found 412.1545 [MH+], C21H23ClN5O2
requires 412.1535.
1
yield: 77%). H NMR (400 MHz, DMSO-d6): 1.01−1.17 (1H, m),
1.18−1.39 (3H, m), 1.68−1.84 (2H, m), 1.78 (3H, s), 1.89 (1H, m),
2.51 (2H, m), 3.48−3.65 (1H, m), 7.74 (1H, d), 8.38 (1H, s), 8.71 (1H,
s), 10.66 (1H, s). MS-ESI m/z 422 [MH+].
Crude 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4,5,6,7-tetra-
hydro-[1,2,3]triazolo[1,5-a]pyridine (approximately 0.3 mL solution;
see Supporting Information) was added to a mixture of 63 (10 mg, 0.02
mmol), Cs2CO3 (15 mg, 0.05 mmol), and chloro(2-dicyclohexylphos-
phino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-
biphenyl)]palladium(II) (1.9 mg, 2.4 μmol) in 1,4-dioxane (2 mL) and
water (0.5 mL) under nitrogen. The resulting mixture was warmed to
60 °C and maintained under these conditions for 45 min. This reaction
was then allowed to cool to room temperature. In a separate flask the
remaining suspension mixture containing crude 3-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-4,5,6,7-tetrahydro-[1,2,3]triazolo[1,5-a]-
pyridine (approximately 2.0 mL) was added to a mixture of 63 (70 mg,
0.17 mmol), Cs2CO3 (325 mg, 1.00 mmol) and chloro(2-
dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-
amino-1,1′-biphenyl)]palladium(II) (26 mg, 0.03 mmol) in 1,4-
dioxane (16 mL) and water (4 mL) under nitrogen. The resulting
mixture was stirred at 60 °C for 45 min. This reaction was then allowed
to cool to room temperature. Both cooled reaction mixtures were
combined and then diluted with saturated aqueous sodium chloride
(100 mL). The resulting mixture was extracted with EtOAc (3 × 100
mL), and the combined organic layers were dried over Na2SO4, filtered,
and concentrated under reduced pressure. The resulting residue was
purified by flash silica chromatography, using an elution gradient of 0 to
100% EtOAc in petroleum ether followed by an elution gradient of 0 to
20% MeOH in EtOAc. Pure fractions were concentrated under reduced
pressure. The resulting residue was further purified by preparative
HPLC (XBridge Prep C18 OBD column, 5 μ silica, 19 mm diameter,
150 mm length), using decreasingly polar mixtures of water (containing
0.8% NH4HCO3) and MeCN as eluents. Fractions containing the
desired compound were concentrated under reduced pressure to afford
(1S,3R)-3-Acetamido-N-(5-chloro-4-(4,5,6,7-tetrahydro-[1,2,3]-
triazolo[1,5-a]pyridin-3-yl)pyridin-2-yl)cyclohexanecarboxamide
(12). 1-Chloro-N,N,2-trimethylpropenylamine (1.149 mL, 8.68 mmol)
was added to a stirred solution of 38 (1.41 g, 5.79 mmol) in DCM (25
mL) cooled in an ice bath under a nitrogen atmosphere. The resulting
mixture was stirred at ambient temperature for 1 h. 56 (1.47 g, 5.79
mmol) and pyridine (0.70 mL, 8.7 mmol) were added, and the resulting
mixture was stirred at ambient temperature for 16 h. The reaction was
quenched by the addition of saturated aqueous NH4Cl (50 mL). The
resulting mixture was extracted with DCM (3 × 75 mL), and the
combined organic layers were dried over MgSO4, filtered, and
concentrated under reduced pressure. The resulting pale-yellow solid
was slurried with Et2O (10 mL) and filtered to yield tert-butyl ((1R,3S)-
3-((5-chloro-4-iodopyridin-2-yl)carbamoyl)cyclohexyl)carbamate 62
1
12 (20 mg, 25%) as a white solid. H NMR (DMSO-d6, 400 MHz):
1.00−1.14 (1H, m), 1.19−1.37 (3H, m), 1.68−1.81 (6H, m), 1.81−
1.92 (3H, m), 1.99−2.10 (2H, m), 2.56−2.70 (1H, m), 2.82 (2H, t),
3.51−3.63 (1H, m), 4.42 (2H, t), 7.80 (1H, d), 8.26 (1H, s), 8.48 (1H,
s), 10.73 (1H, s). MS-ESI m/z 417 [MH+]. HRMS-ESI: m/z found
417.1795 [MH+], C20H26ClN6O2 requires 417.1800.
(1S,3R)-3-Acetamido-N-(5-chloro-4-(5,6-dihydro-4H-pyrrolo[1,2-
b]pyrazol-3-yl)pyridin-2-yl)cyclohexanecarboxamide (13). A mix-
ture of 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydro-
4H-pyrrolo[1,2-b]pyrazole (0.14 g, 0.58 mmol; see Supporting
Information), 62 (0.18 g, 0.38 mmol), chloro(2-dicyclohexylphosphi-
no-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]-
palladium(II) (0.03 g, 0.04 mmol), potassium phosphate, dibasic
(0.200 g, 1.15 mmol), 1,4-dioxane (4 mL), and water (0.8 mL) was
stirred at 21 °C for 18 h. The mixture was then heated at 40 °C for 17 h,
and then at 50 °C for another 2 h. The mixture was diluted with EtOAc
1
(1.79 g, 3.73 mmol, 64%) as a cream-colored solid. H NMR (400
MHz, CDCl3): 1.04−1.18 (1H, m), 1.24−1.41 (2H, m), 1.44 (9H, s),
1.92 (2H, dq), 2.00 (1H, d), 2.28 (1H, d), 2.31−2.41 (1H, m), 3.27−
R
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Drug Annotation
(30 mL) and then washed with water (10 mL). The organic layer was
concentrated under reduced pressure, and the resulting crude product
was purified by flash silica chromatography, elution gradient 0 to 70%
EtOAc in heptane. Product fractions were concentrated under reduced
pressure to afford tert-butyl ((1R,3S)-3-((5-chloro-4-(5,6-dihydro-4H-
pyrrolo[1,2-b]pyrazol-3-yl)pyridin-2-yl)carbamoyl)cyclohexyl)-
purified by ion exchange chromatography using an SCX-2 column,
eluting with 7 N ammonia in methanol. Product fractions were
concentrated under reduced pressure to afford crude (1S,3R)-3-amino-
N-(5-chloro-4-(5,6,7,8-tetrahydro-4H-pyrazolo[1,5-a]azepin-3-yl)-
pyridin-2-yl)cyclohexanecarboxamide (550 mg) as a solid. A portion of
this solid (450 mg, 1.16 mmol) was dissolved in DCM (10 mL) and
triethylamine (0.34 mL, 2.4 mmol). Acetic anhydride (0.13 mL, 1.4
mmol) was added. The reaction mixture was stirred at rt for 4 h. Silica
was added, and the mixture was concentrated under reduced pressure.
The resulting residue was purified by flash silica chromatography,
eluting with 0.5% methanol in ethyl acetate, to give 15 (260 mg, 52%) as
1
carbamate 64a (0.119 g, 67%) as a white solid. H NMR (400 MHz,
CDCl3): 1.04−1.17 (1H, m), 1.34−1.41 (2H, m), 1.44 (9H, s), 1.89−
2.03 (4H, m), 2.29 (1H, d), 2.33−2.44 (1H, m), 2.69 (2H, p), 3.14−
3.21 (2H, m), 3.45−3.59 (1H, m), 4.17−4.24 (2H, m), 4.44 (1H, s),
7.93 (1H, s), 8.15 (1H, s), 8.23 (1H, s), 8.33 (1H, s). MS-ESI m/z 460
[MH+].
1
a white solid. H NMR (400 MHz, DMSO-d6): 1.05−1.12 (1H, m),
To a solution of 64a (0.12 g, 0.26 mmol) dissolved in DCM (3 mL)
was added hydrochloric acid in dioxane (4 M; 1.29 mL, 5.17 mmol).
The mixture was stirred at rt for 30 min before being concentrated
under reduced pressure. The resulting residue was dissolved in DCM (2
mL) and treated with triethylamine (0.079 mL, 0.57 mmol) followed by
acetic anhydride (0.029 mL, 0.31 mmol). The reaction mixture was
stirred at rt for 0.5 h and then washed with water. The organic layer was
concentrated under reduced pressure and purified by flash silica
chromatography, elution gradient 0−10% MeOH in DCM. Product
fractions were concentrated under reduced pressure to afford 13 (0.075
g, 72%) as a white solid. 1H NMR (400 MHz, CDCl3): 1.09−1.22 (1H,
m), 1.38−1.58 (2H, m), 1.88−2.03 (6H, m), 2.26 (1H, d), 2.43−2.56
(1H, m), 2.69 (2H, p), 3.14−3.21 (2H, m), 3.49 (1H, s), 3.87 (1H, dtt),
4.21 (2H, t), 5.59 (1H, d), 8.14 (1H, s), 8.22 (1H, s), 8.33 (1H, s), 8.43
(1H, s). MS-ESI m/z 402 [MH+]. HRMS-ESI: m/z found 402.1681
[MH+], C20H25ClN5O2 requires 402.1691.
(1S,3R)-3-Acetamido-N-(5-chloro-4-(6,7-dihydro-5H-pyrrolo[1,2-
a]imidazol-3-yl)pyridin-2-yl)cyclohexanecarboxamide (14). 63 (130
mg, 0.31 mmol), (6,7-dihydro-5H-pyrrolo[1,2-a]imidazol-3-yl)-
boronic acid hydrochloride (145 mg, 0.77 mmol; see Supporting
Information), barium hydroxide (211 mg, 1.23 mmol) ,and
PdCl2(dppf) (22 mg, 0.030 mmol) were suspended in dioxane (2
mL) and water (0.4 mL) and sealed into a microwave tube. The
reaction was heated to 75 °C in a microwave reactor and maintained
under these conditions for 2 h before being cooled to rt. The reaction
mixture was filtered with a methanol wash, and the filtrate was then
concentrated under reduced pressure. The resulting residue was
purified by preparative HPLC (Waters XBridge Prep C18 OBD
column, 5 μm silica, 19 mm diameter, 100 mm length), using
decreasingly polar mixtures of water (containing 1% NH3) and MeCN
as eluents. Fractions containing the desired compound were evaporated
to dryness to afford 14 (21 mg, 17%) as a solid. 1H NMR (500 MHz,
DMSO-d6): 1.09 (1H, d), 1.30 (3H, q), 1.76−1.80 (5H, m), 1.91 (1H,
d), 2.57−2.73 (4H, m), 2.85 (2H, t), 3.58 (1H, dd), 4.16 (2H, t), 7.55
(1H, s), 7.75 (1H, d), 8.35 (1H, s), 8.42 (1H, s), 10.67 (1H, s). MS-ESI
m/z 402 [MH+]. HRMS-ESI: m/z found 402.1707 [MH+],
C20H25ClN5O2 requires 402.1691.
(1S,3R)-3-Acetamido-N-(5-chloro-4-(5,6,7,8-tetrahydro-4H-
pyrazolo[1,5-a]azepin-3-yl)pyridin-2-yl)cyclohexanecarboxamide
(15). Chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-
biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (0.16 g, 0.21
mmol) was added to a degassed mixture of 3-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-5,6,7,8-tetrahydro-4H-pyrazolo[1,5-a]-
azepine (0.656 g, 2.50 mmol; see Supporting Information), 62 (1.00 g,
2.08 mmol) and potassium phosphate, tribasic (1.09 g, 6.25 mmol) in
1,4-dioxane (20 mL) and water (2 mL). The mixture was again
degassed and was stirred at 85 °C for 24 h under nitrogen. The reaction
mixture was allowed to cool, and silica added. This new mixture was
concentrated under reduced pressure, and the resulting residue was
purified by flash silica chromatography, eluting with 50% ethyl acetate
in heptane to give tert-butyl ((1R,3S)-3-((5-chloro-4-(5,6,7,8-tetrahy-
dro-4H-pyrazolo[1,5-a]azepin-3-yl)pyridin-2-yl)carbamoyl)-
cyclohexyl)carbamate (64b, 0.70 g, 69%) as a solid. This material was
carried on to the next step without further purification. MS-ESI m/z
488 [MH+].
1.17−1.37 (3H, m), 1.57−1.66 (2H, m), 1.69−1.95 (11H, m), 2.56−
2.65 (1H, m), 2.70−2.77 (2H, m), 3.50−3.61 (1H, m), 4.21−4.45 (2H,
m), 7.48 (1H, s), 7.73 (1H, d), 8.05 (1H, s), 8.40 (1H, s), 10.58 (1H, s).
MS-ESI m/z 430 [MH+]. HRMS-ESI: m/z found 430.2007 [MH+],
C22H29ClN5O2 requires 430.2004.
(1S,3R)-3-Acetamido-N-(5-chloro-4-(6,7-dihydro-4H-pyrazolo-
[5,1-c][1,4]oxazin-3-yl)pyridin-2-yl)cyclohexanecarboxamide (16).
A mixture of 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-6,7-dihy-
dro-4H-pyrazolo[5,1-c][1,4]oxazine (0.094 g, 0.38 mmol; see Support-
ing Information), 62 (0.120 g, 0.25 mmol), chloro(2-dicyclohex-
ylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-
biphenyl)]palladium(II) (0.020 g, 0.03 mmol) and potassium
phosphate dibasic (0.131 g, 0.75 mmol) in 1,4-dioxane (4 mL), and
water (0.8 mL) was stirred at 50 °C for 1 h. The mixture was then
diluted with EtOAc (30 mL). The resulting mixture was washed with
water (10 mL), and the organic layer was concentrated under reduced
pressure. The resulting residue was purified by flash silica
chromatography, elution gradient 0 to 70% EtOAc in heptane. Product
fractions were concentrated under reduced pressure to afford tert-butyl
((1R,3S)-3-((5-chloro-4-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-
3-yl)pyridin-2-yl)carbamoyl)cyclohexyl)carbamate 64c (0.081 g,
1
68%). H NMR (400 MHz, DMSO-d6): 1.22−1.35 (4H, m), 1.38
(9H, s), 1.75 (3H, s), 1.90 (1H, d), 2.54−2.63 (1H, m), 4.12−4.26
(4H, m), 4.90 (2H, s), 5.75 (1H, s), 6.76 (1H, d), 7.89 (1H, s), 8.01
(1H, s), 8.39 (1H, s), 10.58 (1H, s). MS-ESI m/z 476 [MH+].
To a mixture of 64c (0.072 g, 0.15 mmol) suspended in DCM (3
mL) at room temperature was added HCl in dioxane (4 M; 0.756 mL,
3.03 mmol). The mixture became a solution which was stirred at room
temperature for 30 min. The reaction was concentrated under reduced
pressure to yield a solid. This solid was dissolved in DCM (2 mL), and
the resulting solution was treated sequentially with triethylamine (0.047
mL, 0.33 mmol) and acetic anhydride (0.017 mL, 0.18 mmol) at room
temperature. The reaction mixture was stirred at room temperature for
30 min and then concentrated under reduced pressure. The resulting
residue was purified by flash silica chromatography, elution gradient 50
to 100% EtOAc in heptane, then 0 to 10% MeOH in DCM. Product
fractions were concentrated under reduced pressure to afford 16 (0.056
g, 88%) as a white solid. 1H NMR (400 MHz, DMSO-d6): 1.08 (1H, d),
1.29 (4H, q), 1.78 (1H, s), 1.91 (3H, s), 2.61 (2H, s), 3.57 (1H, dt),
4.08−4.27 (4H, m), 4.89 (2H, s), 7.74 (1H, d), 7.88 (1H, s), 8.01 (1H,
s), 8.39 (1H, s), 10.59 (1H, s), 11.90 (1H, s). MS-ESI m/z 418 [MH+].
HRMS-ESI: m/z found 418.1640 [MH+], C20H25ClN5O3 requires
418.1640.
(1S,3R)-3-Acetamido-N-(5-chloro-4-(5-methyl-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrazin-3-yl)pyridin-2-yl)-
cyclohexanecarboxamide (17). A mixture of 62 (0.200 g, 0.42 mmol),
5-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrazine (0.197 g, 0.750 mmol; see Support-
ing Information), chloro(2-dicyclohexylphosphino-2′,4′,6′-triisoprop-
yl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (0.033 g,
0.040 mmol), potassium phosphate dibasic (0.218 g, 1.25 mmol), 1,4-
dioxane (4 mL), and water (0.8 mL) was stirred at 45 °C for 18 h. More
chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)-
[2-(2′-amino-1,1′-biphenyl)]palladium(II) (0.033 g, 0.04 mmol) was
added, and the reaction temperature was raised to 60 °C for 1 h. The
reaction mixture was then cooled and passed through an SCX-2
column. The desired product was eluted from the column using 1 M
NH3 in MeOH, and product fractions were concentrated under
TFA (2 mL) was added to a stirred solution of 64b (700 mg, 1.43
mmol) in DCM (10 mL). The reaction was stirred at rt for 24 h, the
volatiles removed under a vacuum, and the resulting residue was
S
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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
reduced pressure. The resulting residue was purified by flash silica
chromatography, elution gradient 0 to 100% EtOAc in heptane, then
0−10% MeOH in DCM. Product fractions were concentrated under
reduced pressure to afford tert-butyl ((1R,3S)-3-((5-chloro-4-(5-
methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-3-yl)pyridin-2-yl)-
carbamoyl)cyclohexyl)carbamate 64d (0.054 g, 27%). 1H NMR (400
MHz, CDCl3, 30 °C): 1.04−1.19 (1H, m), 1.44 (12H, s), 1.87−2.02
(3H, m), 2.29 (1H, d), 2.33−2.46 (1H, m), 2.53 (3H, s), 2.95−3.00
(2H, m), 3.50 (1H, s), 3.76 (2H, s), 4.28 (2H, t), 4.52 (1H, s), 7.85
(1H, s), 8.12 (2H, s), 8.26 (1H, s). MS-ESI m/z 489 [MH+].
To a solution of 64d (0.042 g, 0.090 mmol) dissolved in DCM (2
mL) was added HCl in dioxane (4 M; 0.429 mL, 1.72 mmol). The
mixture was stirred at room temperature for 2 h before being
concentrated under reduced pressure to afford a solid (33 mg). This
solid was dissolved in DCM (2 mL) and triethylamine (0.026 mL, 0.19
mmol). Then acetic anhydride (9.6 μL, 0.10 mmol) was added. The
mixture was stirred at room temperature for 30 min and then
concentrated under reduced pressure. The resulting residue was
purified by flash silica chromatography, elution gradient 0 to 100%
EtOAc in (10% MeOH in DCM). Product fractions were concentrated
under reduced pressure to afford 17 (0.027 g, 74%, 94% purity by
HPLC) as a colorless dry film. 1H NMR (400 MHz, CDCl3): 1.09−1.24
(1H, m), 1.41−1.56 (3H, m), 1.87−2.04 (6H, m), 2.25 (1H, d), 2.51
(4H, s), 2.9−2.98 (2H, m), 3.73 (2H, s), 3.87 (1H, dtd), 4.26 (2H, t),
5.60 (1H, d), 7.84 (1H, s), 8.11 (1H, s), 8.25 (1H, d), 8.30 (1H, s). MS-
ESI m/z 431 [MH+]. HRMS-ESI: m/z found 431.1955 [MH+],
C21H28ClN6O2 requires 431.1957.
2.20 (3H, dd), 2.39−2.5 (1H, m), 3.39−3.45 (2H, m), 4.16 (2H, t),
4.81 (1H, s), 5.49 (1H, d), 7.79 (1H, s), 8.14 (1H, s), 8.19 (1H, s), 8.30
(1H, s). MS-ESI m/z 417 [MH+]. HRMS-ESI: m/z found 417.1796
[MH+], C20H26ClN6O2 requires 417.1800.
(1S,3R)-3-Acetamido-N-(5-chloro-4-(6,7-dihydro-5H-pyrazolo-
[5,1-b][1,3]oxazin-3-yl)pyridin-2-yl)cyclohexanecarboxamide (19).
61 (0.100 g, 0.27 mmol) was added in one portion to a degassed
mixture of 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-6,7-dihy-
dro-5H-pyrazolo[5,1-b][1,3]oxazine (0.067 g, 0.27 mmol; see
Supporting Information), chloro(2-dicyclohexylphosphino-2′,4′,6′-
triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II)
(0.021 g, 0.03 mmol), potassium phosphate (0.170 g, 0.80 mmol), 1,4-
dioxane (2.3 mL) and water (0.45 mL) at room temperature. The
resulting mixture was stirred at room temperature for 16 h and then
concentrated under reduced pressure before being purified by ion
exchange chromatography using an SCX-2 cartridge. The desired
product was eluted from the column using 1 M NH3 in MeOH, and
product fractions were concentrated under reduced pressure. The
resulting residue was purified by preparative HPLC (Waters XBridge
Prep C18 OBD column, 5 μ silica, 30 mm diameter, 100 mm length)
using decreasingly polar mixtures of water (containing 1% NH3) and
MeCN as eluents. Fractions containing the desired compound were
concentrated under reduced pressure to afford 19 (0.017 g, 16%) as a
white gum. 1H NMR (500 MHz, DMSO-d6): 1.21−1.32 (1H, m), 1.4−
1.53 (3H, m), 1.95 (1H, s), 1.96 (3H, s), 2.06 (1H, d), 2.39−2.48 (2H,
m), 2.7−2.85 (3H, m), 3.74 (1H, dt), 4.34 (2H, t), 4.53−4.68 (2H, m),
7.93 (1H, d), 8.05 (1H, s), 8.48 (1H, d), 8.58 (1H, s), 10.62 (1H, s).
MS-ESI m/z 418 [MH+]. HRMS-ESI: m/z found 418.1639 [MH+],
C20H25ClN5O3 requires 418.1640.
(1S,3R)-3-Acetamido-N-(5-chloro-4-(4,5,6,7-tetrahydropyrazolo-
[1,5-a]pyrimidin-3-yl)pyridin-2-yl)cyclohexanecarboxamide (18).
Crude 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrimidine (see Supporting Information)
was added to 62 (0.160 g, 0.33 mmol), chloro(2-dicyclohexylphosphi-
no-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]-
palladium(II) (0.026 g, 0.03 mmol) and potassium phosphate tribasic
(0.175 g, 1.00 mmol) in 1,4-dioxane (4 mL) and water (0.8 mL) at 50
°C. The resulting mixture was stirred at 50 °C for 2 h and then at 80 °C
for 16 h. The reaction mixture was concentrated under reduced
pressure, and the resulting residue was redissolved in DCM (20 mL)
and washed with water (20 mL). The organic layer was dried over
MgSO4, filtered, and concentrated under reduced pressure. The
resulting residue was purified by flash silica chromatography, elution
gradient 0 to 30% EtOAc in heptane. Product fractions were
concentrated under reduced pressure to afford tert-butyl ((1R,3S)-3-
((5-chloro-4-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-3-yl)-
pyridin-2-yl)carbamoyl)cyclohexyl)carbamate 64e (0.082 g, 52%) as a
yellow gum. 1H NMR (400 MHz, CDCl3): 1.44 (12H, s), 1.82−2.46
(8H, m), 3.27−3.36 (3H, m), 4.12 (3H, t), 5.33 (1H, d), 7.80 (1H, s),
8.13 (1H, s), 8.19 (1H, s), 8.20 (1H, s). MS-ESI m/z 475 [MH+].
64e (0.086 g, 0.18 mmol) and HCl in dioxane (4 M; 0.362 mL, 1.45
mmol) were dissolved in methanol (2 mL) at room temperature under
air. The resulting solution was stirred at room temperature for 3 h
before being concentrated under reduced pressure. The resulting
material (66 mg) was dissolved in triethylamine (0.081 mL, 0.58 mmol)
in DCM (1 mL) at room temperature under nitrogen. Then 4-
dimethylaminopyridine (1.14 mg, 9.3 μmol) was added followed by
dropwise addition of acetic anhydride (0.021 mL, 0.22 mmol). The
resulting solution was stirred at room temperature for 2 h before being
quenched with saturated aqueous NH4Cl (10 mL). The resulting
mixture was extracted with DCM (2 × 10 mL). The combined organic
layers were dried over MgSO4, filtered, and concentrated under reduced
pressure. The resulting white solid was purified by flash silica
chromatography, elution gradient 0 to 100% EtOAc in heptane.
Fractions were concentrated under reduced pressure to afford semipure
product, which was further purified by preparative HPLC (Waters
XBridge Prep C18 OBD column, 5 μm silica, 30 mm diameter, 100 mm
length) using decreasingly polar mixtures of water (containing 1%
NH3) and MeCN as eluents. Fractions containing the desired
compound were concentrated under reduced pressure to afford 18
(1S, 3R)-3-Acetamido-N-(4-(5, 5-dimethyl-4, 5, 6, 7-
tetrahydropyrazolo[1,5-a]pyridin-3-yl)pyridin-2-yl)-
cyclohexanecarboxamide (20). 40 (1.50 g, 3.77 mmol), potassium
acetate (1.11 g, 11.3 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi-
(1,3,2-dioxaborolane) (1.44 g, 5.65 mmol), and PdCl2(dppf) (0.276 g,
0.380 mmol) were charged to a flask. 1,4-Dioxane (30 mL) was added,
and the mixture was heated at 90 °C under nitrogen for 3 h. The mixture
was allowed to cool, and the solids were removed by filtration. Ethyl
acetate (100 mL) and water (50 mL) were added, and the layers were
separated. The aqueous layer was extracted with EtOAc (2 × 50 mL),
and the combined organic layers were dried over Na2SO4 and
concentrated under reduced pressure to afford tert-butyl ((1R,3S)-3-
((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)-
carbamoyl)cyclohexyl)carbamate 43 (2.76 g) as a dark brown oil. This
oil was used directly in the next step without further purification. MS-
ESI m/z 446 [MH+].
Dichloro[1,1′-bis(di-t-butylphosphino)ferrocene]palladium(II) (44
mg, 0.070 mmol) was added to a degassed solution of 43 (300 mg, 0.67
mmol), 3-iodo-5,5-dimethyl-4,5,6,7-tetrahydropyrazolo[1,5-a]-
pyridine (242 mg, 0.88 mmol; see Supporting Information) and
potassium phosphate, tribasic (429 mg, 2.02 mmol) in 1,4-dioxane (10
mL), and water (1 mL). The resulting mixture was stirred at 90 °C for
18 h. The crude reaction was cooled and purified by ion exchange
chromatography using an SCX-2 column. The desired product was
eluted from the column using 1 M NH3 in MeOH, and product
fractions were concentrated under reduced pressure to afford a brown
oil. This oil was purified by flash silica chromatography, elution gradient
0−100% EtOAc in heptane. Product fractions were concentrated under
reduced pressure to afford tert-butyl ((1R,3S)-3-((4-(5,5-dimethyl-
4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-yl)pyridin-2-yl)-
1
carbamoyl)cyclohexyl)carbamate (44a, 170 mg, 54%) as a solid. H
NMR (400 MHz, DMSO-d6): 1.03 (6H, s), 1.04−1.15 (1H, m), 1.21−
1.41 (12H, m), 1.72−1.81 (3H, m), 1.83−1.92 (3H, m), 2.53−2.62
(1H, m), 2.65−2.69 (2H, m), 3.30 (2H, m), 4.16 (2H, t), 6.76 (1H, br
d), 7.76 (1H, d), 8.19 (1H, d), 8.29 (1H, d), 10.43 (1H, s). MS-ESI m/z
468 [MH+].
Trifluoroacetic acid (1 mL) was added to 44a (170 mg, 0.36 mmol)
in DCM (10 mL). The resulting mixture was stirred at rt for 6 h. The
reaction was then concentrated under reduced pressure, and the
resulting residue was subjected to ion exchange chromatography using
an SCX-2 column. The desired product was eluted from the column
1
(9.8 mg, 13%, 94% purity by HPLC). H NMR (400 MHz, CDCl3):
1.13 (1H, dd), 1.31−1.52 (4H, m), 1.87−1.95 (2H, m), 1.96 (4H, s),
T
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Journal of Medicinal Chemistry
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Drug Annotation
using 2 M NH3 in MeOH. Product fractions were concentrated under
reduced pressure, and the resulting residue was purified by flash silica
chromatography, eluting with 7% (1% ammonia in methanol) in DCM
to afford (1S,3R)-3-amino-N-(4-(5,5-dimethyl-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyridin-3-yl)pyridin-2-yl)-
cyclohexanecarboxamide (70 mg, 52%) as a solid. MS-ESI m/z 368
[MH+].
eluted from the column with 7 N NH3 in MeOH. Product fractions
were concentrated under reduced pressure to afford (1S,3R)-3-amino-
N-(4-(5,5-dimethyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-yl)-5-
fluoropyridin-2-yl)cyclohexanecarboxamide (0.87 g, 100%) as a solid.
1H NMR (400 MHz, CDCl3): 1.01−1.12 (7H, m), 1.31−1.49 (3H, m),
1.83−1.99 (5H, m), 2.14 (1H, d), 2.35 (1H, td), 2.66−2.85 (3H, m),
4.23 (2H, t), 7.85 (1H, d), 7.99−8.18 (2H, m), 8.29 (1H, d). NH2
signal not observed. MS-ESI m/z 386 [MH+].
Acetic anhydride (0.022 mL, 0.23 mmol) was added to a stirred
solution of (1S,3R)-3-amino-N-(4-(5,5-dimethyl-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyridin-3-yl)pyridin-2-yl)-
cyclohexanecarboxamide (70 mg, 0.19 mmol), triethylamine (0.056
mL, 0.40 mmol), and N,N-dimethylpyridin-4-amine (1.2 mg, 9.5 μmol)
in DCM (10 mL). The reaction mixture was stirred at rt for 4 h, and the
crude reaction was purified by ion exchange chromatography using an
SCX-2 column. The desired product was eluted from the column using
1 M NH3 in MeOH and product fractions were concentrated under
reduced pressure. The resulting residue was further purified by
preparative HPLC (Waters XBridge Prep C18 OBD column, 5 μ silica,
30 mm diameter, 100 mm length), using decreasingly polar mixtures of
water (containing 1% NH3) and MeCN as eluents. Fractions containing
the desired compound were concentrated under reduced pressure to
Acetic anhydride (0.088 mL, 0.93 mmol) was added to a stirred
solution of (1S,3R)-3-amino-N-(4-(5,5-dimethyl-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyridin-3-yl)-5-fluoropyridin-2-yl)-
cyclohexanecarboxamide (300 mg, 0.78 mmol), triethylamine (0.23
mL, 1.6 mmol) and DCM (10 mL). The reaction mixture was stirred at
ambient temperature for 4 h. Silica was added, and the volatiles were
removed by concentration under reduced pressure. The resulting
residue was purified by flash silica chromatography, eluting with 0.5%
methanol in ethyl acetate, to afford 21. 1H NMR (400 MHz, DMSO-
d6): 1.03 (6H, s), 1.02−1.14 (1H, m), 1.24−1.38 (3H, m), 1.72−1.81
(6H, m), 1.86−1.91 (3H, m), 2.55−2.64 (1H, m), 2.69 (2H, s), 3.52−
3.64 (1H, m), 4.16 (2H, t), 7.64−7.81 (2H, m), 8.19 (1H, d), 8.29 (1H,
d), 10.45 (1H, s). MS-ESI m/z 428 [MH+]. 13C NMR (126 MHz,
DMSO-d6): 22.75, 24.03, 27.03, 27.06, 28.29, 28.49, 31.91, 34.37,
35.12, 36.14, 43.38, 44.81, 46.93, 109.90, 112.12, 129.97, 135.67,
137.69, 138.80, 148.78, 152.23, 168.07, 174.12. HRMS-ESI: m/z found
428.2459 [MH+], C23H31FN5O2 requires 428.2456.
(1S,3R)-3-Acetamido-N-(4-(5,5-dimethyl-5,6-dihydro-4H-pyrrolo-
[1,2-b]pyrazol-3-yl)pyridin-2-yl)cyclohexanecarboxamide (22).
Dichloro[1,1′-bis(di-t-butylphosphino)ferrocene]palladium(II) (45.5
mg, 0.07 mmol) was added to a degassed solution of 43 (518 mg, 0.70
mmol), 3-bromo-5,5-dimethyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole
(68, 150 mg, 0.70 mmol; see preparation as an intermediate in the
synthesis of 23), and potassium phosphate tribasic (444 mg, 2.09
mmol) in 1,4-dioxane (5 mL) and water (0.5 mL). The resulting
mixture was stirred at 90 °C for 18 h and then purified by ion exchange
chromatography using an SCX-2 column. The desired product was
eluted from the column using 1 M NH3 in MeOH, and pure fractions
were concentrated under reduced pressure to afford crude product as a
brown oil. This oil was purified by flash silica chromatography, elution
gradient 0−100% EtOAc in heptane. Product fractions were
concentrated under reduced pressure to afford tert-butyl ((1R,3S)-3-
((4-(5,5-dimethyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)pyridin-
2-yl)carbamoyl)cyclohexyl)carbamate 44b (100 mg, 31%) as a white
solid. MS-ESI m/z 454 [MH+].
44b (93 mg, 0.21 mmol) was dissolved in HCl in dioxane (4 M;
0.436 mL, 1.74 mmol) and MeOH (5 mL), and the reaction mixture
was stirred at room temperature for 18 h. The reaction mixture was then
purified by ion exchange chromatography using an SCX-2 column. The
desired product was eluted from the column using 1 M NH3 in MeOH,
and product fractions were concentrated under reduced pressure. The
resulting residue was further purified by flash silica chromatography,
elution gradient 0−10% (7 N ammonia in methanol) in DCM. Product
fractions were concentrated under reduced pressure to afford (1S,3R)-
3-amino-N-(4-(5,5-dimethyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
yl)pyridin-2-yl)cyclohexanecarboxamide (68 mg, 94%) as a white solid.
MS-ESI m/z 354 [MH+]. To a stirred solution of (1S,3R)-3-amino-N-
(4-(5,5-dimethyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)pyridin-
2-yl)cyclohexanecarboxamide (65 mg, 0.18 mmol), triethylamine
(0.054 mL, 0.39 mmol) and N,N-dimethylpyridin-4-amine (1.12 mg,
9.2 μmol) in DCM (5 mL) was added acetic anhydride (0.021 mL, 0.22
mmol). The reaction mixture was stirred at room temperature for 1 h
and then purified by ion exchange chromatography using an SCX-2
column. The desired product was eluted from the column using 1 M
NH3 in MeOH, and product fractions were concentrated under
reduced pressure to afford 22 (60.0 mg, 82.0%) as a colorless oil which
was crystallized from an ether/heptane mix to afford a white solid. 1H
NMR (400 MHz, DMSO-d6): 1.10 (1H, t), 1.29 (9H, s), 1.79 (6H, s),
1.85−1.94 (1H, m), 2.57−2.66 (1H, m), 2.93 (2H, s), 3.58 (1H, dt),
3.90 (2H, s), 7.21 (1H, dd), 7.74 (1H, d), 7.96 (1H, s), 8.18−8.24 (2H,
m), 10.32 (1H, s). MS-ESI m/z 396 [MH+]. HRMS-ESI: m/z found
396.2393 [MH+], C22H30N5O2 requires 396.2394.
1
afford 20 (44 mg, 56%) as a solid. H NMR (500 MHz, DMSO-d6):
1.04 (6H, s), 1.06−1.15 (1H, m), 1.32−1.38 (3H, m), 1.66−1.82 (6H,
m), 1.83−1.94 (3H, m), 2.58−2.64 (1H, m), 2.78 (2H, s), 3.54−3.62
(1H, m), 4.14 (2H, t), 7.16 (1H, dd), 7.76 (1H, d), 7.84 (1H, s), 8.18
(1H, s), 8.24 (1H, d), 10.34 (1H, s). MS-ESI m/z 410 [MH+]. HRMS-
ESI: m/z found 410.2547 [MH+], C23H32N5O2 requires 410.2551.
(1S, 3R)-3-Acetamido-N-(4-(5, 5-dimethyl-4, 5, 6, 7-
tetrahydropyrazolo[1,5-a]pyridin-3-yl)-5-fluoropyridin-2-yl)-
cyclohexanecarboxamide (21). 45 (1.55 g, 6.03 mmol), 5,5-dimethyl-
3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyridine (49, 2.0 g, 7.2 mmol; see Support-
ing Information), chloro(2-dicyclohexylphosphino-2′,4′,6′-triisoprop-
yl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (0.48 g,
0.60 mmol) and potassium phosphate, dibasic (3.15 g, 18.1 mmol)
were dissolved in degassed dioxane (20 mL) and water (1 mL) at 21 °C.
The mixture was stirred at 90 °C for 24 h and then allowed to cool to
room temperature. The mixture was diluted with EtOAc (30 mL),
washed with water (10 mL), and the organic layer was concentrated
under reduced pressure. The resulting residue was purified by flash
silica chromatography, elution gradient 0−50% EtOAc in heptane.
Product fractions were concentrated under reduced pressure to afford
3-(2-chloro-5-fluoropyridin-4-yl)-5,5-dimethyl-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyridine 50 (1.3 g, 77%) as a white solid. 1H
NMR (400 MHz, CDCl3): 1.10 (6H, s), 1.89 (2H, m), 2.68 (2H, s),
4.26 (2H, t), 7.27 (1H, d), 7.80 (1H, d), 8.23 (1H, d). MS-ESI m/z 280
[MH+].
Tetrakis(triphenylphosphine)palladium(0) (0.496 g, 0.43 mmol)
was added to 50 (1.2 g, 4.29 mmol), 47 (1.04 g, 4.29 mmol), 9,9-
dimethyl-4,5-bis(diphenylphosphino)xanthene (0.496 g, 0.86 mmol),
and cesium carbonate (4.19 g, 12.9 mmol) in 1,4-dioxane (10 mL). The
resulting mixture was degassed for 5 min under nitrogen and then
subjected to microwave conditions (120 °C; 17 h). The reaction
mixture was diluted with water (20 mL) and ethyl acetate (100 mL)
before being filtered. The layers were separated, and the organic layer
was adsorbed onto silica and purified by flash silica chromatography,
eluting with isocratic 50% EtOAc in heptane. Product fractions were
concentrated under reduced pressure to afford tert-butyl ((1R,3S)-3-
((4-(5,5-dimethyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-yl)-5-
fluoropyridin-2-yl)carbamoyl)cyclohexyl)carbamate 51 (1.1 g, 53%) as
a white solid. 1H NMR (400 MHz, DMSO-d6): 1.03 (6H, s), 1.02−1.14
(1H, m), 1.20−1.35 (3H, m), 1.39 (9H, s), 1.70−1.79 (3H, br m),
1.82−1.92 (3H, m), 2.54−2.63 (1H, m), 2.68 (2H, s), 4.16 (2H, t),
6.76 (1H, br d), 7.76 (1H, d), 8.19 (1H, d), 8.29 (1H, d), 10.43 (1H, s).
1H multiplet under water peak. MS-ESI m/z 486 [MH+].
TFA (2 mL) was added to a solution of 51 (1.1 g, 2.27 mmol) in
DCM (20 mL). The resulting mixture was stirred at ambient
temperature for 24 h, and then the reaction was concentrated under
reduced pressure. The resulting residue was purified by ion exchange
chromatography using an SCX-2 column. The desired product was
U
https://dx.doi.org/10.1021/acs.jmedchem.0c01754
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
(1S,3R)-3-Acetamido-N-(4-(5,5-dimethyl-5,6-dihydro-4H-pyrrolo-
[1,2-b]pyrazol-3-yl)-5-fluoropyridin-2-yl)cyclohexanecarboxamide
(23). 1H-Pyrazole (20 g, 293.78 mmol), ethyl 3-bromo-2,2-
dimethylpropanoate (61.4 g, 293.78 mmol), and cesium carbonate
(144 g, 440.68 mmol) in DMA (200 mL) were stirred at 80 °C for 16 h.
The mixture was then poured into water (400 mL) and extracted with
ethyl acetate (150 mL). The organic layer was concentrated under
reduced pressure to give a colorless oil. This oil was purified by flash
silica chromatography, elution gradient 10 to 40% ethyl acetate in
heptane). Product fractions were concentrated under reduced pressure
to afford ethyl 2,2-dimethyl-3-(1H-pyrazol-1-yl)propanoate (46.0 g,
80.0%), as a colorless oil. 1H NMR (400 MHz, CDCl3): 0.97 (6H, s),
1.02 (3H, t), 3.93 (2H, q), 4.10 (2H, s), 6.00 (1H, t), 7.16 (1H, d), 7.26
(1H, d). MS-ESI m/z 197 [MH+].
Aqueous sodium hydroxide (5 M; 94 mL, 46 mmol) was added
portion-wise to a stirred solution of ethyl 2,2-dimethyl-3-(1H-pyrazol-
1-yl)propanoate (46 g, 234 mmol) dissolved in methanol (250 mL) at
room temperature. The mixture was allowed to exotherm to 37 °C
during addition. The resulting solution was stirred under these
conditions for 30 min and then cooled to room temperature before
being concentrated under reduced pressure to 1/3 volume. This new
solution was acidified to ∼pH 3 with concentrated aqueous HCl. A
colorless oil separated from the mixture. The flask was swirled in an ice
bath, and a colorless solid crystallized. The mixture was allowed to stand
overnight at room temperature. The solid was isolated by filtration and
dried under reduced pressure to afford 2,2-dimethyl-3-(1H-pyrazol-1-
yl)propanoic acid 65 (30.0 g, 76%) as a colorless crystalline solid. 1H
NMR (400 MHz, DMSO-d6): 1.05 (6H, s), 4.23 (2H, s), 6.21 (1H, t),
7.35−7.44 (1H, m), 7.54−7.67 (1H, m), 12.41 (1H, br s). MS-ESI m/z
169 [MH+].
(0.720 g, 3.35 mmol), and potassium acetate (1.15 g, 11.7 mmol) in
DMA (7 mL). The resulting suspension was degassed and stirred at 85
°C for 5 h. Dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium
DCM adduct (0.273 g, 0.33 mmol) was then added to the reaction
mixture, and stirring was continued under these conditions for 18 h
before the reaction mixture was cooled to rt. The reaction mixture was
diluted with EtOAc (20 mL) and washed sequentially with water (2 ×
15 mL), and saturated aqueous sodium chloride (15 mL). The organic
layer was dried over MgSO4, filtered, and concentrated under reduced
pressure. The resulting crude product was purified by flash silica
chromatography, elution gradient 0−50% EtOAc in heptane. Product
fractions were concentrated under reduced pressure to afford 5,5-
dimethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydro-
4H-pyrrolo[1,2-b]pyrazole 69 (0.458 g, 52%) as a cream-colored solid.
1H NMR (400 MHz, CDCl3): 1.24 (6H, s), 1.27 (12H, s), 2.79 (2H, s),
3.87 (2H, s), and 7.76 (1H, s).
45 (1.00 g, 3.88 mmol), 69 (1.53 g, 5.83 mmol), chloro(2-
dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-
amino-1,1′-biphenyl)]palladium(II) (0.31 g, 0.39 mmol), and dibasic
potassium phosphate (2.03 g, 11.65 mmol) were dissolved in degassed
dioxane (10 mL) and water (2 mL) at 21 °C. The reaction mixture was
stirred at 80 °C for 3 h, and then the mixture was cooled, diluted with
EtOAc (30 mL), and washed with water (10 mL). The organic layer was
concentrated under reduced pressure. The resulting residue was
purified by flash silica chromatography, elution gradient 0−50% EtOAc
in heptane. Product fractions were concentrated under reduced
pressure to afford 3-(2-chloro-5-fluoropyridin-4-yl)-5,5-dimethyl-5,6-
dihydro-4H-pyrrolo[1,2-b]pyrazole 70 (1.00 g, 97%) as a white solid.
1H NMR (500 MHz, CDCl3): 1.36 (6H, s), 2.95 (2H, d), 3.97 (2H, s),
7.31 (1H, d), 7.94 (1H, d), 8.20 (1H, d). MS-ESI m/z 266 [MH+].
Tetrakis(triphenylphosphine)palladium(0) (0.13 g, 0.12 mmol) and
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (0.13 g, 0.23
mmol) were added together in one portion to a degassed mixture of
47 (0.670 g, 2.76 mmol), 70 (0.61 g, 2.3 mmol), cesium carbonate
(1.88 g, 5.76 mmol), and 1,4-dioxane (26 mL). The mixture was rapidly
heated to reflux. After 20 h, the reaction was cooled, diluted with 50%
saturated aqueous sodium chloride, and extracted with ethyl acetate
(2×). The combined organic layers were dried over sodium sulfate,
filtered, and concentrated under reduced pressure to afford crude tert-
butyl ((1R,3S)-3-((4-(5,5-dimethyl-5,6-dihydro-4H-pyrrolo[1,2-b]-
pyrazol-3-yl)-5-fluoropyridin-2-yl)carbamoyl)cyclohexyl)carbamate
71 as a light-yellow solid. Hydrochloric acid in dioxane (4 M; 10 mL, 40
mmol) and DCM (5 mL) was added, resulting in a clear orange solution
that rapidly became cloudy and yellow. Methanol (∼3 mL) was titrated
into the reaction until the mixture became mostly clear. After 15 min,
the orange mixture was concentrated under reduced pressure to afford
an orange solid. Pyridine (3.7 mL, 46 mmol) was added to this solid
along with DCM (19 mL). A slight exotherm was noted, and the
reaction was immersed in a water bath. Then acetic anhydride (0.43
mL, 4.6 mmol) was added dropwise. After another 10 min, another 200
μL of acetic anhydride was added. After another 30 min, another 600 μL
of anhydride and 6 mL of pyridine were added. The reaction was
maintained under these conditions for another 45 min and was then
poured into saturated aqueous sodium bicarbonate and ethyl acetate.
The layers were separated, and the aqueous layer was extracted with
ethyl acetate. The combined organic layers were dried over sodium
sulfate, filtered, and concentrated under reduced pressure. The
resulting orange residue was purified by flash silica chromatography,
elution gradient 50 to 100% EtOAc in hexane followed by 0−20%
methanol in ethyl acetate, and product fractions were concentrated
under reduced pressure to afford 23 (0.89 g, 94%) as a faint yellow foam
solid. 1H NMR (DMSO-d6): 1.00−1.16 (1H, m), 1.22−1.40 (9H, m),
1.74−1.81 (6H, m), 1.83−1.94 (1H, m), 2.55−2.68 (1H, m), 2.93 (2H,
s), 3.49−3.65 (1H, m), 3.94 (s, 2H), 7.75 (1H, d), 7.88 (1H, d), 8.28
(1H, d), 8.30 (1H, d), 10.46 (1H, s). 13C NMR (126 MHz, DMSO-d6):
22.75, 24.03, 27.55, 28.25, 31.90, 35.21, 42.99, 43.38, 46.94, 60.23,
107.64, 109.96, 129.59, 135.86, 141.88, 144.44, 148.96, 151.91, 168.07,
174.18. HRMS-ESI: m/z found 414.2302 [MH+], C22H29FN5O2
requires 414.2300.
n-BuLi in hexane (9.03 mL, 24.4 mmol) was added dropwise to 65
(2.0 g, 12 mmol) in 2-methyl tetrahydrofuran (40 mL) at −78 °C over a
period of 20 min under nitrogen. The resulting suspension was stirred at
−78 °C for 15 min, and then the reaction was stirred at approximately
−45 °C for 1 h. The mixture was allowed to warm to 15 °C before the
reaction was quenched slowly onto ice-cold saturated ammonium
chloride (100 mL). The reaction mixture was diluted with EtOAc (100
mL), and the ammonium chloride layer was separated and extracted
with EtOAc (50 mL). The combined organic layers were washed with
saturated aqueous sodium chloride (50 mL), dried over MgSO4,
filtered, and concentrated under reduced pressure to afford 5,5-
dimethyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-4-one 66 (0.97 g,
1
54%) as a pale-yellow oil which crystallized on standing. H NMR
(400 MHz, DMSO-d6): 1.29 (6H, s), 4.36 (2H, s), 6.77 (1H, d), 7.89
(1H, d). MS-ESI m/z 151 [MH+].
Hydrazine hydrate (4.13 mL, 85.2 mmol) was added to a stirred
solution of 66 (2.56 g, 17.1 mmol) dissolved in 2,2′-oxydiethanol (48.5
mL, 511 mmol). The resulting solution was stirred at 180 °C for 1 h.
Potassium hydroxide (3.35 mL, 59.7 mmol) was carefully added to the
mixture, and the resulting suspension was stirred at 150 °C for 2 h. After
being cooled to room temperature, the reaction mixture was diluted
with water (50 mL), and the pH was adjusted to 4.5 with aqueous HCl
(2 N). Following extraction with Et2O (5 × 50 mL), the combined
organic layers were washed with water (2 × 20 mL) and then saturated
aqueous sodium chloride (20 mL). The organic layers were dried over
MgSO4, filtered, and concentrated under reduced pressure to give 5,5-
dimethyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole 67 (0.922 g, 40%) as
a clear yellow oil. 1H NMR (400 MHz, CDCl3): 1.21 (6H, s), 2.61 (2H,
s), 3.80 (2H, s), 5.82−5.93 (1H, m), 7.41 (1H, d).
N-Bromosuccinimide (1.17 g, 6.55 mmol) was added to a stirred
solution of 67 (892 mg, 6.55 mmol) dissolved in DCM (10 mL) at 23
°C. The resulting mixture was stirred at 23 °C for 16 h before being
diluted with DCM (20 mL) and washed sequentially with water (2 × 20
mL) and saturated aqueous sodium chloride (20 mL). The organic
layer was dried over MgSO4, filtered, and concentrated under reduced
pressure to afford 3-bromo-5,5-dimethyl-5,6-dihydro-4H-pyrrolo[1,2-
1
b]pyrazole 68 (1.39 g, 99%) as a yellow oil. H NMR (400 MHz,
CDCl3): 1.23 (6H, s), 2.58 (2H, s), 3.83 (2H, s), and 7.35 (1H, s).
Pd(P(Cy)3)2Cl2 (0.25 g, 0.33 mmol) was added to 4,4,4′,4′,5,5,5′,5′-
octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.70 g, 6.69 mmol), 68
V
https://dx.doi.org/10.1021/acs.jmedchem.0c01754
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
(1S,3R)-3-Acetamido-N-(5-chloro-4-(5,5-dimethyl-5,6-dihydro-
4 H - p y r r o l o [ 1 , 2 - b ] p y r a z o l - 3 - y l ) p y r i d i n - 2 - y l ) -
cyclohexanecarboxamide (24). A mixture of 69 (433 mg, 0.83 mmol),
62 (360 mg, 0.75 mmol), chloro(2-dicyclohexylphosphino-2′,4′,6′-
triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II)
(59 mg, 0.08 mmol), and potassium phosphate dibasic (392 mg, 2.25
mmol) in 1,4-dioxane (4 mL) and water (0.8 mL) was stirred at 50 °C
for 5 h. The reaction mixture was cooled to room temperature and then
purified by ion exchange chromatography using an SCX-2 column. The
desired product was eluted from the column using 1 M NH3 in MeOH,
and product fractions were concentrated under reduced pressure. The
resulting residue was purified by flash silica chromatography, elution
gradient 0 to 100% EtOAc in heptane. Product fractions were
concentrated under reduced pressure to afford tert-butyl ((1R,3S)-3-
((5-chloro-4-(5,5-dimethyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
yl)pyridin-2-yl)carbamoyl)cyclohexyl)carbamate 72 (188 mg, 51%) as
8.00 (1H, s), 8.27 (1H, s), 8.31−8.38 (1H, m), 10.52 (1H, s). MS-ESI
m/z 475 [MH+].
64f (1.11 g, 2.34 mmol) was dissolved in DCM (20 mL).
Trifluoroacetic acid (1.8 mL, 23 mmol) was added, and the reaction
mixture was stirred at rt for 18 h. The reaction was purified by ion
exchange chromatography using an SCX-2 column. The desired
product was eluted from the column using 1 M NH3 in MeOH, and
product fractions were concentrated under reduced pressure to afford
(1S,3R)-3-amino-N-(5-chloro-4-(5-methyl-5,6-dihydro-4H-pyrrolo-
[1,2-b]pyrazol-3-yl)pyridin-2-yl)cyclohexanecarboxamide (0.68 g,
77%) as a white solid. MS-ESI m/z 374 [MH+].
Acetic anhydride (0.20 mL, 2.2 mmol) was added to a stirred
solution of (1S,3R)-3-amino-N-(5-chloro-4-(5-methyl-5,6-dihydro-
4H-pyrrolo[1,2-b]pyrazol-3-yl)pyridin-2-yl)cyclohexanecarboxamide
(670 mg, 1.79 mmol), triethylamine (0.52 mL, 3.8 mmol) and N,N-
dimethylpyridin-4-amine (11 mg, 0.09 mmol) in DCM (10 mL). The
reaction mixture was stirred at rt for 18 h. The mixture was purified by
ion exchange chromatography using an SCX-2 column, and the desired
product was eluted from the column using 1 M NH3 in MeOH. Product
fractions were concentrated under reduced pressure. The resulting
residue was purified by flash silica chromatography, using an elution
gradient of 0 to 100% EtOAc in heptane followed by isocratic 10%
MeOH in EtOAc. Product fractions were concentrated under reduced
pressure to afford (1S,3R)-3-acetamido-N-(5-chloro-4-(5-methyl-5,6-
dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)pyridin-2-yl)-
1
a white solid. H NMR (400 MHz, DMSO-d6): 1.25 (12H, d), 1.37
(7H, s), 1.74 (3H, s), 1.87 (1H, d), 2.52−2.62 (1H, m), 2.88 (2H, s),
3.18−3.29 (1H, m), 3.93 (2H, s), 6.80 (1H, d), 7.99 (1H, s), 8.24 (1H,
s), 8.32−8.35 (1H, m), 10.56 (1H, s). MS-ESI m/z 488 [MH+].
72 (186 mg, 0.380 mmol) was dissolved in HCl in dioxane (4 M;
0.81 mL, 3.2 mmol) and MeOH (5 mL), and the resulting solution was
stirred at room temperature for 18 h. The reaction mixture was then
purified by ion exchange chromatography using an SCX-2 column. The
desired product was eluted from the column using 1 M NH3 in MeOH,
and product fractions were concentrated under reduced pressure to
afford (1S,3R)-3-amino-N-(5-chloro-4-(5,5-dimethyl-5,6-dihydro-4H-
pyrrolo[1,2-b]pyrazol-3-yl)pyridin-2-yl)cyclohexanecarboxamide (114
mg, 77%) as a white solid. This material was used directly in the next
step. MS-ESI m/z 388 [MH+]. This solid was dissolved in DCM (10
mL) and triethylamine (0.084 mL, 0.60 mmol) and N,N-dimethylpyr-
idin-4-amine (1.7 mg, 0.010 mmol) were added. Then acetic anhydride
(0.032 mL, 0.34 mmol) was added dropwise. The reaction mixture was
stirred at room temperature for 4 h and then purified by ion exchange
chromatography using an SCX-2 column. The desired product was
eluted using 1 M NH3 in MeOH, and product fractions were
concentrated under reduced pressure. The resulting residue was
purified by flash silica chromatography, elution gradient 0 to 100%
EtOAc in heptane. Product fractions were concentrated under reduced
1
cyclohexanecarboxamide (693 mg, 93%) as a white solid. H NMR
(400 MHz, DMSO-d6): 1.05−1.11 (1H, m), 1.23 (3H, d), 1.27−1.38
(3H, m), 1.72−1.81 (6H, m), 1.89 (1H, br d), 2.52−2.63 (1H, m), 2.67
(1H, dd), 3.13−3.19 (1H, m), 3.20−3.28 (1H, m), 3.50−3.63 (1H, m),
3.76 (1H, dd), 4.27−4.37 (1H, m), 7.75 (1H, d), 8.00 (1H, s), 8.27
(1H, s), 8.35 (1H, s), 10.55 (1H, s). MS-ESI m/z 416 [MH+].
(1S,3R)-3-Acetamido-N-(5-chloro-4-(5-methyl-5,6-dihydro-4H-
pyrrolo[1,2-b]pyrazol-3-yl)pyridin-2-yl)cyclohexanecarboxamide (670
mg, 1.79 mmol) was resolved by preparative HPLC (Chiral
Technologies IA column, 20 μm silica, 100 mm diameter, 250 mm
length), using a 70/15/15 mixture of heptane/EtOH/MeOH as eluents
and a flow rate of 450 mL/min, fractions containing the desired
compounds were concentrated under reduced pressure to give the
faster eluting isomer 1 of (1S,3R)-3-acetamido-N-(5-chloro-4-(5-
methyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)pyridin-2-yl)-
cyclohexanecarboxamide (27, 356 mg, 48%, 99.3% e.e.) and the slower
eluting isomer 2 of (1S,3R)-3-acetamido-N-(5-chloro-4-(5-methyl-5,6-
dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)pyridin-2-yl)-
cyclohexanecarboxamide (28, 348 mg, 47%, 97.5% e.e.).
1
pressure to afford 24 (86 mg, 70%) as a white solid. H NMR (400
MHz, DMSO-d6): 1.09 (1H, d), 1.28 (9H, s), 1.78 (6H, s), 1.90 (1H,
d), 2.62 (1H, s), 2.89 (2H, s), 3.57 (1H, dt), 3.95 (2H, s), 7.73 (1H, d),
7.99 (1H, s), 8.25 (1H, s), 8.33−8.36 (1H, m), 10.53 (1H, s). 13C NMR
(126 MHz, DMSO-d6): 23.23, 24.50, 27.95, 28.72, 32.37, 35.60, 43.63,
43.94, 47.40, 60.74, 111.50, 112.56, 122.30, 140.86, 142.35, 145.18,
148.50, 151.56, 168.56, 174.99. MS-ESI m/z 430 [MH+]. HRMS-ESI:
m/z found 430.2003 [MH+], C22H29ClN5O2 requires 430.2004.
Isomer 1 and Isomer 2 of (1S,3R)-3-acetamido-N-(5-chloro-4-(5-
methyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)pyridin-2-yl)-
cyclohexanecarboxamide (27 and 28). Chloro(2-dicyclohexylphos-
phino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-
biphenyl)]palladium(II) (0.25 g, 0.31 mmol) was added to a degassed
mixture of 5-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole (1.03 g, 3.75 mmol; see
Supporting Information), 62 (1.50 g, 3.13 mmol), and dibasic
potassium phosphate (1.63 g, 9.38 mmol) in 1,4-dioxane (15 mL)
and water (3 mL). The resulting mixture was degassed and then stirred
at 90 °C for 18 h under nitrogen. The reaction mixture was allowed to
cool to rt, diluted with EtOAc (100 mL) and washed sequentially with
water (100 mL) and saturated aqueous sodium chloride (50 mL). The
organic extract was dried over MgSO4, filtered, and concentrated under
reduced pressure. The resulting residue was purified by flash silica
chromatography, elution gradient 0−70% EtOAc in heptane. Product
fractions were concentrated under reduced pressure to afford tert-butyl
((1R,3S)-3-((5-chloro-4-(5-methyl-5,6-dihydro-4H-pyrrolo[1,2-b]-
pyrazol-3-yl)pyridin-2-yl)carbamoyl)cyclohexyl)carbamate (64f, 1.0 g,
27: 1H NMR (400 MHz, DMSO-d6): 1.05−1.15 (1H, m), 1.25 (3H,
d), 1.26−1.39 (3H, m), 1.72−1.83 (6H, m), 1.90 (1H, br d), 2.55−2.62
(1H, m), 2.67 (1H, dd), 3.11−3.19 (1H, m), 3.21−3.28 (1H, m),
3.51−3.62 (1H, m), 3.76 (1H, dd), 4.27−4.37 (1H, m), 7.75 (1H, d
br), 8.00 (1H, s), 8.27 (1H, s), 8.35 (1H, s), 10.55 (1H, s). MS-ESI m/z
416 [MH+]. HRMS-ESI: m/z found 416.1860 [MH+], C21H27ClN5O2
requires 416.1848.
28: 1H NMR (400 MHz, DMSO-d6): 1.08−1.13 (1H, m), 1.24 (3H,
d), 1.25−1.36 (3H, m), 1.62−1.85 (6H, m), 1.91 (1H, br d), 2.52−2.61
(1H, m), 2.67 (1H, dd), 3.13−3.19 (1H, m), 3.21−3.29 (1H, m),
3.51−3.60 (1H, m), 3.76 (1H, dd), 4.32 (1H, dd), 7.74 (1H, d), 7.98
(1H, s), 8.28 (1H, s), 8.34 (1H, s), 10.54 (1H, s). MS-ESI m/z 416
[MH+]. HRMS-ESI: m/z found 416.1849 [MH+], C21H27ClN5O2
requires 416.1848.
Analytical HPLC: flow: 2 mL/min, column: Chiral Technologies IA,
5 μ, 4.6 × 250 mm, eluent: 1:1 ethanol/methanol in heptane), tR: (27,
7.9 min), (28, 9.3 min).
(1S,3R)-3-Acetamido-N-(5-chloro-4-(6,6-dimethyl-6,7-dihydro-
5 H - p y r r o l o [ 1 , 2 - a ] i m i d a z o l - 3 - y l ) p y r i d i n - 2 - y l ) -
cyclohexanecarboxamide (29). 63 (800 mg, 1.90 mmol), 6,6-
dimethyl-6,7-dihydro-5H-pyrrolo[1,2-a]imidazole (825 mg, 5.69
mmol; see Supporting Information), palladium acetate (171 mg, 0.76
mmol), and potassium acetate (372 mg, 3.79 mmol) were suspended in
DMA (15 mL) and sealed into a microwave tube. The tube was
degassed and purged with nitrogen (3×). The reaction was then
subjected to microwave conditions (150 °C, 16 h) and cooled to rt. The
1
69%) as a yellow foam. H NMR (400 MHz, DMSO): 1.08 (2H, s),
1.22−1.3 (6H, m), 1.38 (9H, s), 1.76 (3H, s), 1.90 (1H, d), 2.53−2.75
(2H, m), 3.2−3.3 (2H, m), 3.76 (1H, dd), 4.32 (1H, dd), 6.76 (1H, d),
W
https://dx.doi.org/10.1021/acs.jmedchem.0c01754
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
reaction mixture was purified by preparative HPLC (Waters XBridge
Prep C18 OBD column, 5 μ silica, 30 mm diameter, 100 mm length),
using decreasingly polar mixtures of water (containing 1% NH3) and
MeCN as eluents. Fractions containing the desired compound were
concentrated under reduced pressure. The resulting light brown solid
was recrystallized using EtOAc/heptane and dried under a vacuum to
give 29 (180 mg, 22%) as a white solid. The filtrate was concentrated
under reduced pressure to provide a second batch of 29 (118 mg, 14%).
1H NMR (500 MHz, DMSO-d6): 1.03−1.16 (1H, m), 1.19−1.41 (9H,
m), 1.72−1.81 (6H, m), 1.91 (1H, br. d), 2.57−2.68 (1H, m), 2.71
(2H, s), 3.50−3.62 (1H, m), 3.91 (2H, s), 7.51 (1H, s), 7.75 (1H, d),
8.28 (1H, s), 8.42 (1H, s), 10.66 (1H, s). 13C NMR (126 MHz, DMSO-
d6): 22.74, 24.01, 27.31, 28.24, 31.86, 35.05, 38.10, 43.14, 43.47, 46.91,
58.27, 111.18, 121.37, 123.87, 134.70, 136.99, 148.20, 151.12, 155.97,
168.09, 174.68. MS-ESI m/z 430 [MH+]. HRMS-ESI: m/z found
430.2006 [MH+], C22H25ClN5O2 requires 430.2004.
Steve Glossop − Oncology R&D, AstraZeneca, Cambridge CB4
0WG, United Kingdom
Tyler Grebe − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States
Chungang Gu − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States
Sudhir Hande − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States
Janet Hawkins − Oncology R&D, AstraZeneca, Cambridge
CB4 0WG, United Kingdom
Alexander W. Hird − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States
Jane Holmes − Oncology R&D, AstraZeneca, Cambridge CB4
0WG, United Kingdom
James Horstick − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States
Yun Jiang − Pharmaron Beijing Co., Ltd., Beijing 100176, P. R.
China
Michelle L. Lamb − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States; orcid.org/0000-
0002-3005-8065
Thomas M. McGuire − Oncology R&D, AstraZeneca,
Cambridge CB4 0WG, United Kingdom
Jane E. Moore − Oncology R&D, AstraZeneca, Cambridge CB4
0WG, United Kingdom
Nichole O’Connell − Discovery Sciences, AstraZeneca, Boston,
Massachusetts 02451, United States
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01754.
■
sı
Experimental procedures for the synthesis and character-
ization of intermediates and metabolites 6-M1, 6-M2, 24-
M1, and 24-M2; crystal structure of compound 24 bound
to CDK9 in complex with cyclin T1; ligand docking
protocol; procedures for determination of DMPK proper-
ties; protocol for human PK predictions and clinical dose
projections; biological testing protocol, data, and
associated errors (PDF)
Andy Pike − Oncology R&D, AstraZeneca, Cambridge CB4
0WG, United Kingdom
Molecular formula strings (CSV)
Kurt G. Pike − Oncology R&D, AstraZeneca, Cambridge CB4
0WG, United Kingdom; orcid.org/0000-0002-9731-7500
Theresa Proia − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States
Bryan Roberts − Oncology R&D, AstraZeneca, Cambridge CB4
0WG, United Kingdom
Accession Codes
The coordinates of the crystal structure of compound 24 bound
to CDK9 in complex with cyclin T1 have been deposited under
the following code 6Z45. Authors will release the atomic
coordinates and experimental data upon article publication.
Maryann San Martin − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States
Ujjal Sarkar − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States
Wenlin Shao − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States
Darren Stead − Oncology R&D, AstraZeneca, Cambridge CB4
0WG, United Kingdom
Neil Sumner − Oncology R&D, AstraZeneca, Cambridge CB4
0WG, United Kingdom
Kumar Thakur − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States
Melissa M. Vasbinder − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States
Jeffrey G. Varnes − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States
Jianyan Wang − Advanced Drug Delivery, Pharmaceutical
Sciences, R&D, AstraZeneca, Boston, Massachusetts 02451,
United States
Lei Wang − Pharmaron Beijing Co., Ltd., Beijing 100176, P. R.
China
Dedong Wu − Advanced Drug Delivery, Pharmaceutical
Sciences, R&D, AstraZeneca, Boston, Massachusetts 02451,
United States
Liangwei Wu − Pharmaron Beijing Co., Ltd., Beijing 100176, P.
R. China
AUTHOR INFORMATION
Corresponding Author
Bernard Barlaam − Oncology R&D, AstraZeneca, Cambridge
CB4 0WG, United Kingdom; orcid.org/0000-0002-0904-
7556; Phone: +44(0)1625 237335;
■
Email: bernard.barlaam2@astrazeneca.com
Authors
Robert Casella − Advanced Drug Delivery, Pharmaceutical
Sciences, R&D, AstraZeneca, Boston, Massachusetts 02451,
United States
Justin Cidado − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States
Calum Cook − Oncology R&D, AstraZeneca, Macclesfield
SK10 2NA, United Kingdom
Chris De Savi − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States
Allan Dishington − Oncology R&D, AstraZeneca, Cambridge
CB4 0WG, United Kingdom
Craig S. Donald − Oncology R&D, AstraZeneca, Cambridge
CB4 0WG, United Kingdom
Lisa Drew − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States
Andrew D. Ferguson − Discovery Sciences, AstraZeneca,
Boston, Massachusetts 02451, United States; orcid.org/
0000-0002-1018-9631
Douglas Ferguson − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States
X
https://dx.doi.org/10.1021/acs.jmedchem.0c01754
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
Mc Henry, K. T.; Pinchback, R. M.; Ligon, A. H.; Cho, Y. J.; Haery, L.;
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M. S.; Demichelis, F.; Rubin, M. A.; Janne, P. A.; Daly, M. J.; Nucera, C.;
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Ladanyi, M.; Letai, A.; Garraway, L. A.; Loda, M.; Beer, D. G.; True, L.
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Bin Yang − Oncology R&D, AstraZeneca, Boston,
Massachusetts 02451, United States; orcid.org/0000-
0002-1499-5569
Tieguang Yao − Pharmaron Beijing Co., Ltd., Beijing 100176,
P. R. China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01754
Author Contributions
The manuscript was written through contributions of all
authors. All authors were employees of AstraZeneca at the
time of the described work and have given approval to the final
version of the manuscript.
(6) Wenzel, S. S.; Grau, M.; Mavis, C.; Hailfinger, S.; Wolf, A.; Madle,
Notes
̈
H.; Deeb, G.; Dorken, B.; Thome, M.; Lenz, P.; Dirnhofer, S.;
The authors declare no competing financial interest.
Hernandez-Ilizaliturri, F. J.; Tzankov, A.; Lenz, G. MCL1 is deregulated
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ACKNOWLEDGMENTS
■
̀
(7) Wuilleme-Toumi, S.; Robillard, N.; Gomez, P.; Moreau, P.; Le
The authors would like to acknowledge Matt Peters for assessing
the safety risks of this project, Anne Marie Mazzola and Scott
Boiko for in vitro and in vivo biology data generation, Tim Ikeda
for his input into assay design and data generation, and past and
present members of the CDK9 project.
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NONSTANDARD ABBREVIATIONS AND
ACRONYMS
■
second-generation XPhos precatalyst, chloro(2-dicyclohexyl-
phosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-
1,1′-biphenyl)]palladium(II); 2-MeTHF, 2-methyl tetrahydro-
furan; AML, acute myeloid leukemia; Bak, Bcl2 homologous
antagonist/killer; Bax, Bcl2-associated X protein; BCL2, B-cell
lymphoma 2; BID, bis in die (twice a day); BPin, boronic pinacol
ester; CDK, cyclin dependent kinase; CDT, carboxyl-terminal
domain; CSNK1G1, casein kinase 1 gamma 1; DCM,
dichloromethane; DIPEA, N-ethyl-N-isopropylpropan-2-
amine; DYRK, dual specificity tyrosine phosphorylation
regulated kinase; ERK7, extracellular signal-regulated kinase 7;
GSK, glycogen synthase kinase; HLM, human liver microsome;
INSR, insulin receptor kinase; i.p., intraperitoneal; i.v., intra-
venous; Jnk1, c-Jun N-terminal kinase 1; MAP2K7, dual
specificity mitogen-activated protein kinase kinase 7;
MAP3K9, mitogen-activated protein kinase kinase kinase 9;
MAP4K4, mitogen-activated protein kinase kinase kinase kinase
4; Mcl-1, myeloid cell leukemia 1; MeCN, acetonitrile; NBS, N-
bromosuccinimide; NCS, N-chlorosuccinimide; NMP, N-
methylpyrrolidone; PBPK, physiologically based pharmacoki-
netic; PdCl2(dppf), [1,1′-bis(diphenylphosphino)ferrocene]-
dichloropalladium(II); Pd(P(Cy)3)2Cl2, dichlorobis-
(tricyclohexylphosphine)palladium(II); Puma, p53 upregulated
modulator of apoptosis; RNAP2, RNA polymerase II; rt, room
temperature; SFC, supercritical fluid chromatography; TEA,
triethylamine; Vss, volume of distribution at steady state;
xantphos, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene.
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critical survival factor for multiple myeloma. Blood 2002, 99, 1885−
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Moujalled, D. M.; Bruno, A.; Csekei, M.; Paczal, A.; Szabo, Z. B.; Sipos,
S.; Radics, G.; Proszenyak, A.; Balint, B.; Ondi, L.; Blasko, G.;
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Smith, J.; Pedder, C.; Graham, C.; Studeny, A.; Lysiak-Auvity, G.;
Girard, A.-M.; Grave, F.; Segal, D.; Riffkin, C. D.; Pomilio, G.;
Galbraith, L. C. A.; Aubrey, B. J.; Brennan, M. S.; Herold, M. J.; Chang,
C.; Guasconi, G.; Cauquil, N.; Melchiore, F.; Guigal-Stephan, N.;
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https://dx.doi.org/10.1021/acs.jmedchem.0c01754
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
Sekhar Reddy Chilamakuri, C.; D’Santos, C. S.; Shao, W.; Saeh, J. C.;
Koch, R.; Weinstock, D. M.; Zinda, M.; Fawell, S. E.; Drew, L.
AZD4573 is a highly selective CDK9 inhibitor that suppresses Mcl-1
and induces apoptosis in hematological cancer cells. Clin. Cancer Res.
2020, 26, 922−934.
AA
https://dx.doi.org/10.1021/acs.jmedchem.0c01754
J. Med. Chem. XXXX, XXX, XXX−XXX