Design, synthesis and biological evaluation of renin inhibitors guided by simulated annealing of chemical potential simulations

Article abstract of DOI:10.1016/j.bmc.2017.05.032

We have applied simulated annealing of chemical potential (SACP) to a diverse set of ～150 very small molecules to provide insights into new interactions in the binding pocket of human renin, a historically difficult target for which to find low molecular

Full text of DOI:10.1016/j.bmc.2017.05.032

Accepted Manuscript
Design, synthesis and biological evaluation of renin inhibitors guided by simu-
lated annealing of chemical potential simulations
Ian S. Cloudsdale, John K. Dickson Jr, Thomas E. Barta, Brian S. Grella, Emilie
D. Smith, John L. Kulp III, Frank Guarnieri, John L. Kulp JR
PII:
S0968-0896(17)30568-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.032
BMC 13750
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 March 2017
10 May 2017
15 May 2017
Please cite this article as: Cloudsdale, I.S., Dickson, J.K. Jr, Barta, T.E., Grella, B.S., Smith, E.D., Kulp, J.L. III,
Guarnieri, F., Kulp, J.L. JR, Design, synthesis and biological evaluation of renin inhibitors guided by simulated
annealing of chemical potential simulations, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/
10.1016/j.bmc.2017.05.032
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Bioorganic & Medicinal Chemistry
Design, synthesis and biological evaluation of renin inhibitors guided by simulated
annealing of chemical potential simulations
Ian S. Cloudsdale,a* John K. Dickson, Jr,a Thomas E. Barta,a Brian S. Grella,a Emilie D. Smith,a John L.
Kulp III,a,b Frank Guarnieri,a,b and John L. Kulp JRa,b 
aBioLeap, Inc., 6350 Quadrangle Drive, Suite 110, Chapel Hill, North Carolina 27517, United States
bConifer Point Pharmaceuticals, 3805 Old Easton Road, Doylestown, PA 18902
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
We have applied Simulated Annealing of Chemical Potential (SACP) to a diverse set of ~150
very small molecules to provide insights into new interactions in the binding pocket of human
renin, a historically difficult target for which to find low MW inhibitors with good
bioavailability. In one of its many uses in drug discovery, SACP provides an efficient,
thermodynamically principled method of ranking chemotype replacements for scaffold hopping
and manipulating physicochemical characteristics for drug development. We introduce the use
of Constrained Fragment Analysis (CFA) to construct and analyze ligands composed of linking
those fragments with predicted high affinity. This technique addresses the issue of effectively
linking fragments together and provides a predictive mechanism to rank order prospective
inhibitors for synthesis. The application of these techniques to the identification of novel
inhibitors of human renin is described. Synthesis of a limited set of designed compounds
provided potent, low MW analogs (IC50s < 100 nM) with good oral bioavailability (F >20-58%).
Available online
Keywords:
Renin
Fragment-based simulations
Simulated Annealing of Chemical Potential
Hypertension
Physicochemical properties
2017 Elsevier Ltd. All rights reserved.
1. Introduction
(BP). Another method for therapeutic intervention of the RAAS
pathway would be directly target renin, the first and rate-limiting
Detailed investigations of the enormous costs and the
extensive amount of time involved in drug discovery have called
into question the benefits of early optimization efforts which
were aimed at creating potent, single-digit nanomolar
compounds.1-3 There is substantial evidence that early efforts to
drive hyper-potency typically lead to large and hydrophobic
compounds.4 Gleeson5 argues that such compounds inherently
possess a dichotomy between physicochemical parameters
associated with potency and those associated with desirable
ADME characteristics. Analysis by Wenlock6 indicated that in
the development pipeline, failure of oral drugs was associated
with the higher MW and LogP candidates. Another analysis by
Oprea7 added solubility to Wenlock’s analysis and confirmed that
drug-like molecules are more likely to be low MW (<425)
compounds.
step in the system. After more than 20 years of research in the
pharmaceutical industry, aliskiren, a sub-nanomolar compound
with less than 3% oral bioavailability10 in humans, was finally
approved as the only marketed renin inhibitor to date. Aliskiren
has a molecular weight of 551, which is a contributor to its poor
in vivo properties and one of the reasons for the continuing
research on better alternatives. Yokokawa11 has comprehensively
summarized the progress between 2009-2012 on investigations
into creating renin inhibitors with a better balance of potency to
physicochemical properties. While these efforts have produced
promising new molecules, there is still no marketed alternative to
aliskiren, which is a testament to how difficult it has been to
achieve both potency and desirable physicochemical properties
simultaneously.
The analyses of Gleeson,5 Wenlock6 and Oprea7 suggest that a
compound with MW<425 and a potency of around 50 nM
promises to have the right characteristics for drug development.
One approach adopted by the fragment-based drug design
(FBDD) community12, 13 has been to start with low affinity, small
molecular weight compounds. Multiple distinct fragments
predicted to bind in several adjacent pockets can be linked
together to form higher molecular weight, higher affinity
compounds. Ligand efficiency (LE),14 a metric used to describe
affinity per molecular unit, is very useful to compare different
A prominent and salient example of the tension between the
early drive for very high potency and the difficulty of developing
a real drug is the decades-long search for a renin inhibitor to treat
hypertension. The Renin−Angiotensin−Aldosterone System
(RAAS) plays a significant role in hypertension by regulating
9
blood pressure and extracellular fluid volume.8, It has been
shown that blockade of the RAAS, either with an angiotensin
converting enzyme inhibitor (ACEi) or with an angiotensin II
(AngII) AT1 receptor blocker (ARB) reduces blood pressure
———
 Corresponding authors. Tel.: (215) 589-6357; fax: (215) 489-4920; e-mails: jlkjr@pobox.com, isclou@gmail.com

Significant efforts over more than two decades by several
companies were invested in developing non-peptidomimetic
renin inhibitors such as the piperidines (2),16 piperazinones (3 and
4)17 and ureas (5) (Figure 1).18 We chose the 2IKO19 X-ray
protein structure as the input for SACP, because the
diaminopyrimidine scaffolds (6-8) reported by Pfizer19 had lower
pKa’s (~8.0) compared to the more basic amines inherent in
many renin inhibitors like 1 (pKa = 9.8) (Table 1). Maintaining
basicity closer to physiological pH was expected to provide
enhanced oral absorption.20 Our goals were three-fold: find
replacements for the diaminopyrimidine head group with similar
pKa values; find neutral fragments with equal or higher affinity
than the tetrahydroquinoline and dihydrobenzoxazinone moieties;
and, keep the MW and cLogP of the inhibitors low to enhance
the physicochemical characteristics for drug development.5-7 The
SACP technology was applied to the renin structure 2IKO using
150 fragments, resulting in the design of a small set of molecules
with MW<425 and cLogP<4 predicted to have 100 nM affinity or
better for renin. We were able to accomplish all the goals with a
minimum of effort. Oral bioavailability from 20-58% was
obtained with 341<MW<412 and 2.2<cLogP<3.8.
Figure 1. Examples of non-peptidomimetic renin inhibitors.
scaffolds during this process. Renin, however, presents a unique
challenge for fragment-based drug design, because many very
potent high molecular weight renin inhibitors have been reported.
Designing compounds that conform to the Gleeson-Wenlock-
Oprea constraints requires doing the opposite of standard FBDD,
i.e. instead of growing fragments to make larger molecules,
existing molecules must be shrunk by finding smaller fragments
that can replace the larger fragments without materially
degrading binding affinity. This task is quite difficult, because if
the molecular weight is to be reduced by more than 100 Daltons,
then multiple fragment replacements must be performed and thus
the design process must be exceptionally robust, since even one
incorrect fragment substitution could abolish all affinity. In silico
Simulated Annealing of Chemical Potential15 (SACP) is well
suited to this problem, because it can efficiently screen chemical
interactions in a specific protein environment and rank desirable
opportunities for scaffold hopping, plus it is not limited to the
solubility requirements of experimentally applied FBDD
approaches.
2. Materials and Methods
2.1. Computational methods
SACP is a way to determine the binding sites and the 3-
dimensional orientations of small molecular fragments on the
target protein, with binding affinity distinguished by excess
chemical potential (average free energy per molecule). A major
strength of the SACP technique is its ability to identify and
reliably rank non-obvious fragment substitution patterns and
chemotype replacements, including so-called scaffold hopping.
This enables optimizing the properties of a particular class of
molecules without appreciably degrading affinity or ligand
efficiency – a central goal of lead optimization. Because SACP
simulates how individual fragments separately interact with the
protein, it suffers the same limitation as experimental NMR and
X-ray fragment methods – it, by itself, provides no information
about whether or not two different fragments predicted to bind
with high affinity in adjacent sites on the protein can be
chemically linked. Previously we demonstrated that binding sites
can be identified using SACP to determine localized regions on a
protein that have high affinity for a diversity of fragments and a
low affinity for water (“hot-spots”).21 Once such a site is
determined, SACP can then be applied with a binding site biasing
option, including a detailed balance correction, to oversample a
critical locality on a protein in order to get more information, not
just on affinity, but also orientations of the fragment relative to
the protein and other fragments. Such biased sampling is
essential to discover poses in narrow clefts of the protein (“letter-
in-envelope”). Chemical potential () is a free energy metric, and
because the method is thermodynamically principled, one can
develop an understanding of the requirements of the ligand-
protein interaction. By comparing the individual fragments’
chemical potentials in binding sites, one can predict their relative
affinities and reliably prioritize which compounds to make.
Resources can be conserved by focusing on the higher affinity
fragments, thereby reducing the number of compounds to make
and test compared to conventional empirical SAR efforts.
Table 1. 2,4-Diaminopyrimidine Inhibitors
Cmpd
R1
X
Y
R2
R3
(CH2)3OMe
(CH2)3OMe
O
O
O
H,H
H,H
H,H
-
-
-
-
-
6a
6b
6c
6d
7a
7b
7c
(CH2)3OMe
CH2
CH2
-
-
-
(CH2)2NHCOMe
(CH2)3OMe
-
-
Me
Me
Me
Me
Me
(CH2)2NHCOMe
(CH2)3OMe
-
-
-
-
(CH2)2NHCOMe
(CH2)3CF3
-
-
-
-
Me
Me
7d
7e
To address the fundamental linkage problem, we have
generalized SACP to a process called constrained fragment
annealing (CFA).21 CFA grand canonical sampling goes one step
further than SACP by biasing the sampling of two (or more)
fragments simultaneously to oversample binding modes to the
protein that also place the fragments in correct binding

geometries22 for linkage. This distorts each fragment’s binding
mode to the protein from its optimal position and thus the
chemical potential of the individual fragment-protein interaction
is raised. The best candidates for linkage from CFA occur when
two adjacent fragments align with good bond lengths and angles
with the minimal distortion of the optimal binding chemical
potential of the individual fragments. This methodology of using
SACP followed by CFA is a theoretically grounded method to
first assess which individual fragments bind with the highest
affinity at a particular site on a protein and then to assess if two
distinct high affinity fragments at adjacent sites on the protein
should be linked. CFA allows one to answer the question of
where a full ligand, composed of two or more assembled
fragments identified by SACP, wants to interact and in what
orientation relative to the protein. The relative predicted affinity
(FE Pred) of a ligand is calculated by summation of the
fragments’ individual chemical potential values less the solvation
penalty (Gs)23 for moving the ligand from bulk solvent to the
interaction site.
2.2. Chemistry
The general strategy for construction of the aminopyridyl-
substituted bicylic heterocycles was to prepare the appropriately
substituted aminopyridines and bicyclic heterocycles separately
and to couple at or near the final step via a traditional Suzuki
Scheme 4. Synthesis of 2-Substituted Indole Intermediatesa
Scheme 1. Synthesis of 7-(2-Aminopyridyl)tetrahydroquinoline Analogsa
aReagents and conditions. (a) POCl3, DMF, DCE, 0 oC to rt; (b)
o
o
o
Ph3P+CH2YX-, BuLi, THF, -78 C or 0 C to rt; (c) I2, AgOTf, THF, -78 C;
(d) 3-methoxyprop-1-yne, Pd(Ph3P)2Cl2, CuI, Et3N, DMF.
aReagents and conditions. (a) NaOMe, MeOH; (b) Ph2CNH, Pd(OAc)2,
BINAP, Cs2CO3, toluene, 100 oC; (c) (i) Pd(PPh3)4, Cs2CO3, toluene/H2O, 100
oC; (ii) HCl, THF; (d) H2, Pd, MeOH; (e) Br2, AcOH (40b) or NBS, CH3CN
(40c-d); (f) Pd(PPh3)4, aq. Na2CO3 or Cs2CO3, dioxane, 80-100 oC.
Scheme 5. Synthesis of Indole 24a
Scheme 2. Synthesis of 7-(2-Aminopyridyl)- and 7-(2,4
Diaminopyrimidyl)indolesa
o
aReagents and conditions. (a) NaH, SEMCl, CH2Cl2, 0 C; (b) (i) NaBH4,
MeOH, THF, 0 oC; (ii) NaH, MeI, THF, 0 oC to RT; (iii) TBAF,
ethylenediamine, THF; (c) 47b, Pd(PPh3)4, Cs2CO3, dioxane/H2O, 100 oC.
Scheme 6. Synthesis of Indole Analogs 25-31a
aReagents and conditions. (a) 47a or 47b, Pd(PPh3)4, Na2CO3 or Cs2CO3,
aReagents and conditions. (a) 41a or 41b, Pd(PPh3)4, Na2CO3 or Cs2CO3,
dioxane/H2O, 100 oC; (b) H2, Pd/C, MeOH.
dioxane/H2O, 100 oC. (b) Pd(PPh3)4, Na2CO3, dioxane/H2O, 100 oC.
Scheme 3. Synthesis of Fluoroindole Analogsa
Scheme 7. Synthesis of 6-(2-Aminopyridyl)benzoxazinone Analogsa
aReagents and conditions. (a) Pd(PPh3)4, LiCl, CsOH.H2O, aq. dioxane,
100 oC; (b) Br(CH2)2NHCO2Me, NaH, 15-cr-5, DMF; (c) (i)
bis(pinacolato)diboron, Pd(dppf)Cl2, KOAc, dioxane, 85 oC; (ii) 41b,
Pd(dppf)Cl2, K3PO4.7H2O, toluene, 100 oC.
o
o
aReagents and conditions. (a) 1-propenylMgBr, THF, -78 C or 0 C to rt;
(b) 47b, Pd2(dba)3/X-Phos or Pd(PPh3)4, Cs2CO3, dioxane/H2O, 100 oC; (c)
bis(pinacolato)diboron, Pd(dppf)Cl2 or Ph(PPh3)4, KOAc, DMSO, 120 oC.

reaction. Which coupling partner in this penultimate reaction
contained the boronic ester acid depended on experimentation or
availability of starting material. The substituted 2-aminopyridines
utilized in this paper were commercially available or prepared as
shown in Scheme 1 through bromination of the requisite
pyridines. Synthesis of the tetrahydroquinoline derivatives 12-15
was accomplished by coupling of the known boronic acid ester
4224 with aminopyridines 41a-d.
compounds, a plot of FE Pred versus the pIC50 data shows an
excellent correlation (R2 = 0.82) (Figure 2). Although 2IKO
contains a weak ligand (8; IC50 = 4 M)19 that lacks a sidechain
in the S3 selectivity pocket (S3sp), the validation demonstrates
that SACP can predict relative affinity, even in regions not
occupied by an X-ray ligand. The crystal structure of 7a bound to
renin (2G1Y), with an alkyl ether occupying the S3sp site was
also available. Validation of the same set of data in the 2G1Y
structure gave a comparable correlation (R2 = 0.84)26 (Supporting
Information). Experience has shown that it is not uncommon to
observe three-fold variability in measured IC50 values for the
same compound based on day-to-day experimental variability in
assays. To this extent, we add error bars of pIC50 = 0.5 to the
graphs to represent that three-fold variance. If we can reliably
predict the affinity of the compounds within the variance of the
biological assay, the model can be used to predict the affinity of
the next compound to be made and, just as importantly, which
compounds would likely be less potent and need not be made.
Using a reliable predictive model also allows the chemist to
select compounds for synthesis that would maintain or enhance
those key physicochemical characteristics considered pertinent to
drug development.
Coupling of the simple unsubstituted or 3-methylindoles was
carried out using the commercially available indoleboronic acid
esters 43a-b to provide 16-20 as shown in Scheme 2. Preparation
of fluoro-substituted indoles was initiated using the Bartoli indole
synthesis25 to provide the 3-methylindoles 46a-c in one step
(Scheme 3). For the simple mono- and difluoroindole analogs,
Suzuki coupling was consistently unsuccessful or resulted in a
very poor yield of product when the boronic acid ester was
contained on the indole coupling partner. However, synthesis of
21-23 was accomplished when the 5-bromo-2-aminopyridines
were converted to their corresponding boronates 47a-b and
reacted with the requisite bromofluoroindoles 46a-c.
Elaboration of the 2-position of the indoles with substituents
extending into the S3sp region was performed by first
formylating or iodinating the indole ring, followed by further
extension via Wittig or Shonogashira chemistries as shown in
Scheme 4.
Our first goal was to replace the diaminopyrimidine group
present in literature compounds 6-8. This heterocycle is
protonated in its interaction with the aspartates at the catalytic
site of renin. The usual set of fragments used in SACP consists of
about 150 neutral fragments ranging from extremely small
fragments such as methane, water, methanol and acetamide to
moderately large fused heterocycles with a maximum MW of
170. The fragments are chosen to represent readily prepared
functional groups commonly found in drugs. Experience has
shown that the overlap of this small group of fragments readily
suggests alternative or more complicated moieties that are readily
analyzed by CFA. This set was supplemented by 15 protonated
fragments predicted to have pKa’s <8.5. A full list of the
fragments simulated can be found in the Supporting Information.
Fragments 9-11 were used for simulations. Two protonated
pyridine fragments, 9a and 10a, were compared to the pyrimidine
head group 11a (Figure 3). 9a and 10a showed predicted binding
affinity comparable to 11a in the simulations. Of interest was the
resulting replacement of the polar 4-amino substituent in 11 with
the neutral Me and OMe substituents. An ethane fragment with
good predicted affinity was found adjacent to the substituted
aminopyridines, and could be linked to the pyridine 6-position
such that the orientation of the Et group in 9b and 10b
corresponded exactly to the orientation of that in the pyrimidine
of 8 in the 2IKO X-ray structure. Subsequent CFA simulations
predicted improved affinity for the ethyl analogs 9b and 10b as
expected from filling of the lipophilic pocket between Val31 and
Val122 (Table 2).
For the synthesis of 24, it was found that protection of the
indole NH was necessary to complete elaboration of the 2-
position, as shown in Scheme 5. Following deprotection, 53 was
smoothly transformed into the final compound. Similarly,
coupling of the aminopyridine boronates 50a-e and 51 with the
appropriate substituted bromoindoles was readily accomplished,
followed by hydrogenation of the side chains to provide 25-31
(Scheme 6).
Preparation of the benzoxazinone derivatives 32-34 was
accomplished by coupling of the known boronates 54a-b18b with
the requisite bromoaminopyridines (Scheme 7). Synthesis of 35
required the assembly of the necessary boronate from 55.
3. Results and Discussion
To validate the technological fit of SACP to the design of
renin inhibitors, compounds 6a-d and 7a-d were deconstructed
into their corresponding fragments, and CFA was applied using
the 2IKO structure. By definition, a plot of the absolute free
energy of the binding interaction versus the log of the inhibition
constant (pKi) should be a straight line. Instead of pKi we use
pIC50, which is easier to obtain and is proportional to the pKi if
the assay conditions are kept the same. For the literature
Figure 3. Protonated amines interacting at the catalytic site.
Figure 2. Correlation plot of predicted relative Free Energies versus pIC50
for compounds 6a-d and 7a-d.

Table 2. Relative Interaction Values for Protonated Amines Compared to
11b.
For compound 15 we considered that the Ser79 hydroxyl could
either rotate or interact with the methoxy substituent of 15 via a
water-mediated H-bond, accounting for some of the affinity
observed. Molecular dynamics simulations of compounds 13 and
15 were performed to investigate the stability of the cavity
created by the flexible loop containing Ser79 and Thr80. Over a
10 ns simulation time course the cavity was maintained, no water
molecules penetrated this site, and the Ser79 hydroxyl did not
rotate. When comparing the affinity of 6c to 13 and 15, these
results suggest that the improved enthalpic interaction due to the
amino group is offset by the solvation penalty required by the
interaction. Here we show that free energy ranking coupled with
an efficient solvation penalty algorithm can discriminate the
contributions from various chemotypes and suggest non-obvious,
sometimes counterintuitive alternatives for synthesis.
Rel. H
Rel. H
proteina
(kcal/mol)
Ser79
Rel. FE
Pred
(kcal/mol) (kcal/mol)
Compound
Rel. Gs
+Thr80b
(kcal/mol)
10b
9b
-2.0
-1.6
0.0
-5.0
-4.5
0.0
1.7
-0.3
0.0
4.5
2.5
3.9
8.4
7.5
11b
10a
11a
9a
0.0
10.7
13.2
13.9
7.7
11.5
0.0
13.3
8.2
7.6
aEnthalpic interaction energy between fragment and entire protein. Enthalpic
b
interaction energy between fragment and Ser79+Thr80 only.
In the 2IKO structure the 4-amino group of 8 interacts via
hydrogen bonds to the O-atom on the side chain of Ser79 and O-
atom of the backbone carbonyl of Thr80. Medicinal chemists
involved in structure-based design emphasize the introduction of
polar functionalities in ligands for H-bond interactions with a
protein to increase the enthalpy of the interactions. In fact, in a
discussion of the X-ray structures of 7c-d and 8, the authors refer
to this H-bonded network as contributing significantly to the
binding enthalpy of the interactions.19 Here the SACP suggests
two fragment structures with considerably less polarity and less
or no ability to H-bond to these amino acids, yet with similar
predicted binding affinity. Although the pyrimidines 11a-b have
better enthalpic interactions than 9a-b or 10a-b with Ser79 and
Thr80, the difference in the enthalpic interactions is not as
pronounced when the whole protein is considered (Table 2,
column 4). More importantly, the solvation penalty (Table 2,
column 3) plays a larger role in determining the overall relative
affinity. Designed compounds 12-15 were synthesized and tested
and compared to the literature compound 6c (Table 3).
Gratifyingly, the potencies for replacement of the pyrimidine
head group by the pyridine analogs 13 and 15 were maintained.
The importance of the 6-Et group was clearly shown by the much
lower activity seen for compounds 12 and 14. We note that the 6-
ethylpyrimidine-2,4-diamine replacement by the 4-methyl or 4-
methoxy 6-ethylpyridin-2-amine was relatively straight forward,
and could have been discovered in a standard medicinal
chemistry program, but the computation allowed us to explore
many possibilities and only make and test the highest ranked
compounds.
Attention then focused on using the SACP of neutral fragments
to find replacements for the tetrahydroquinoline and
benzoxazinone moieties in compounds 6-7. Based on their
ranking and orientation relative to the 5-position of the ligand
heterocycle in the 2IKO model, two fragments were of greatest
interest - indole and benzimidazole. Indole was favored due to its
smaller solvation penalty in this site. The orientation of the
indole fragment when linked to the pyrimidine of the X-ray
structure was close to the global minimum of the assembled
compound 16. Interestingly, the indole NH interacts with the
protein forming an H-bond to the carbonyl of Gly223. In the
2G1R structure it is the amide NH of 7b that interacts with
Gly223, albeit in a different H-bonded configuration. A 2-
chlorotoluene fragment, although ranked considerably weaker
than indole, overlapped the benzo region of the indole fragment.
This prompted us to analyze the interactions of over 30
halogenated benzimidazoles, indoles, and 3-alkyllindoles. This
represents a common theme in design using such fragment data -
larger fragments are suggested by the relative superposition of
smaller overlapping fragments. This was seen here for multiple
fragment orientations and was previously described for a
methane fragment, highlighting the importance of the ethyl side
chain of the aminoheterocycles interacting at the catalytic site.
Using CFA, it is possible to analyze and rank such composite
fragments not present in the original dataset within a few hours.
The results of the analysis indicated that the fluoro-3-
methylindole fragment patterns were preferred.
Designed
compounds 17-23 were synthesized and tested. The improvement
in LE from 16 to 18, where the protonated heterocycle was the
only change, was encouraging. Also of note was the 36-fold
improvement in activity seen by judicious use of a single extra
heavy atom going from 17 to 19 and then to 20 as supported by
the simulation data. The differences observed in the fluoro-
substitution patterns 21-23 were within the error of the bioassay.
Table 3. Comparison of Heterocyclic Groups Interacting with the Catalytic
Aspartates.
The 2-position of the indole was ideally positioned to explore
S3sp. The fragments we found in our simulation set occupying
this pocket, alkanes (C1-C3), dimethyl ether, acetamides and
some simple 5-membered ring heterocyles, replicate those
previously reported in the literature. Analysis of alkyl
substitution off the 2-position of the indole indicated that this
6,5-bicyclic ring system was more tolerant of a wide range of
torsion angles compared to the 6,6-benzoxazinone and
tetrahydroquinoline ring systems (see Supporting Information).
The orientation of the indole allowed a linkage to an alkyl ether
fragment that resulted in ligand design 26, with a substitution
pattern containing one less rotatable bond for the extension into
S3sp than that found in the tetrahydroquinolines 6a-c and the
benzoxazinones 7a and 7c. The reduction in the overall entropy
of this side chain by removal of a rotatable bond was expected to
improve the affinity of the interaction as well. Further extension
Cmpd
X
R1
R2
IC50 (M)
Ligand
Efficiency
6c
N
CH
CH
CH
CH
-
Et
Me
Et
H
NH2
Me
0.512a,17
16.8
0.34
0.27
0.34
-
12
13
Me
0.491
14
15
OMe
OMe
-
>100
Et
-
0.500
0.33
0.30
1 (Aliskiren)
0.003b,27
aLit. IC50 = 0.691 µM, Kd = 0.535 µM. bLit. IC50 = 0.0006 µM.

of the side chain to a different alkyl ether fragment in an
alternative orientation of higher predicted affinity provided 30.
Upon testing 30 was found to be comparable in activity to 26,
illustrating the trade-off between affinity and entropy associated
with the longer side chain. The correlation plot for the indoles
containing the 2-amino-6-ethyl-4-methylpyridyl moiety is shown
in Figure 4. The predicted binding pose of the most potent indole
derivative 27 illustrates the interactions with Gly223 and the S3sp
pocket compared to the 2IKO and 2G1R X-ray ligands (Figures 5
and 6).
Unlike the alkyl ether substitutions, linkage from the indole to
an acetamide or a heterocyclic fragment could not be achieved
without imposing a large conformational penalty upon binding.
This was predicted by CFA and exemplified by the much poorer
affinity observed for 31. Using predictive ranking of the
substituents, it was possible to drive the potency to 59 nM by
synthesizing only 13 compounds (Table 4) while maintaining LE.
Table 4. SAR of the indoles that were designed, synthesized, and tested.
IC50
Cmpd
X
N
Y
G
R1
R2 R3 R4
LE
(M)
29.2 0.32
124 0.30
NH2 Et
H
H
H
H
H
H
H
F
H
H
H
H
H
H
F
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
CH Me Me
CH Me Et
CH Me Me
CH Me Et
CH Me Et
CH Me Et
CH Me Et
CH Me Et
CH Me Me
CH Me Et
CH Me Et
CH Me Et
CH Me Et
CH Me Et
CH Me Et
H
H
H
H
H
10.2 0.36
8.53 0.36
3.37 0.37
4.52 0.35
8.26 0.33
7.76 0.32
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
Figure 4. Correlation plot of versus predicted relative Free Energies versus
pIC50 for compounds 17-30 where G=Et.
H
H
H
F
H
F
CH2OMe
(CH2)2OMe
(CH2)2OMe
(CH2)2OMe
(CH2)2OMe
(CH2)2OMe
(CH2)3OMe
(CH2)2CONHMe
H
H
H
F
H
H
H
H
F
3.9
0.32
1.33 0.35
0.23 0.38
0.06 0.39
0.36 0.35
0.11 0.36
0.22 0.36
1.93 0.29
H
F
F
H
H
H
H
Table 5. Comparison of benzoxazinones that were designed, synthesized, and
tested.
Figure 5. Predicted binding pose of the fragments comprising 27 (cyan,
yellow, blue) compared to 8 (green) in 2IKO showing the interaction of the
indole NH with Gly223, and the ether penetrating S3sp.
Cmpd
X
N
R1
Et
R2
R3
IC50
(M)
LE
7b
32
33
34
35
NH2
Me
(CH2)2NHCOMe
(CH2)2NHCOMe
(CH2)2NHCOMe
(CH2)3OMe
0.147 0.33
0.069 0.35
0.045 0.33
0.267 0.31
0.121 0.30
CH Et
CH Et OMe
CH Et
CH Et
Me
Me
(CH2)2NHCO2Me
Having established the substituted pyridine as a replacement
for the pyrimidine in the tetrahydroquinoline scaffold, and
considering its activity in the indole series, we examined this
head group replacement in the potent benzoxazinone Pfizer
scaffold. The 6-ethyl-2-aminopyridine groups improved affinity
2- to 3-fold,28 with a modest improvement in LE (Table 5, 7b vs
32 and 34). The SACP data suggested that an appropriately
Figure 6. Predicted binding pose of 27 (cyan, yellow, blue) compared to 7d
(light green) in 2G1R showing the two different NH interactions with
Gly223.

orientated amide would have a higher affinity in the S3sp pocket
than an ether. The amides 32 and 33 and the carbamate 35 do
indeed show higher affinity than the ether analog 34 because, in
contrast to the indole analog 31, they can adopt a low energy
conformation when linked to the benzoxazinone scaffold.
system using either tetramethylsilane (TMS) or the appropriate
solvent reference as an internal standard. Column
chromatography was carried out using 230-400 mesh silica gel.
Preparative HPLC was performed on a Gilson GX-281 system
using conditions specified in the experimental descriptions.
Analytical LC/MS was performed on an Agilent 1200
purification system coupled to an Agilent 6110 quadrupole mass
spectrometer (column: Halo 2.7 m C18 50 x 4.6 mm; injection
volume: 0.8 L; flow rate: 1.8 mL/min; wavelength: 214 nm and
254 nm; elution: linear gradient of 5:95 CH3CN:water (0.05%
v/v formic acid) to 95:5 CH3CN:water (0.05% v/v formic acid)
over 1 min, with a hold for 1 min). All compounds were isolated
as amorphous solids unless noted otherwise. The purity of all
compounds was determined to be >95% by HPLC analysis.
Experiments were performed at BioDuro, LLC.
The three most potent compounds, 27, 32 and 33, were chosen
for pharmacokinetic studies in rats. Data is shown in Table 6. All
three compounds achieved maximum concentrations upon oral
dosing that were many times greater than their corresponding in
vitro renin IC50 values, and they demonstrated reasonable half-
lives and good oral bioavailability. In Table 7, we compare key
measurements of ligand optimization (potency, MW, and cLogP)
for the synthesized compounds with those presented in the
literature for compound 7b. Since the values obtained for the
standards used were three to seven times higher than reported
elsewhere, the LE values were normalized to 7b (Table 7,
column 7). Aliskiren 1, 7c and 7d were not as efficient as 7b at
interacting with the protein, and their bioavailability was poor,
ranging from 2-12%. Manipulation of the side chain of 7b to give
7e improved bioavailability at the expense of LE. In addition
cLogP increased significantly to >5. In contrast, compounds 27,
32 and 33 maintained or improved LE relative to 7b, exhibited a
lower cLogP range (2.2-3.8) and resulted in oral bioavailabilities
from 20-58%.
5.2. 5-(1-(3-Methoxypropyl)-1,2,3,4-tetrahydroquinolin-7-yl)-
4,6-dimethylpyridin-2-amine (12).
A mixture of 42 (180 mg, 0.54 mmol), 2-amino-5-bromo-4,6-
dimethylpyridine (109 mg, 0.54 mmol),
tetrakis(triphenylphosphine)palladium(0) (31 mg, 0.03 mmol),
and aqueous Na2CO3 solution (2 N, 0.7 mL) in dioxane (5 mL)
was stirred at 80 oC overnight under argon. The mixture was
cooled to room temperature, filtered, and the filtrate was
concentrated under vacuum. The crude residue was purified by
silica gel column chromatography, eluting with 20:1 petroleum
ether: EtOAc, to give 88 mg of impure product. The resulting
material was purified by preparative HPLC (column:
4. Conclusion
These results demonstrate that the SACP and CFA techniques
can be utilized as effective medicinal chemistry design tools to
rapidly search for, and rank novel chemotype and substituent
replacements. The data generated frequently suggests non-
obvious ligands and substitutions. With a predictive tool in hand,
only a limited number of compounds need to be assessed for
affinity. The combination of SACP+CFA has broad applicability
to all aspects of discovery and optimization provided structural
information is available. It is complementary to experimental
FBDD approaches, allowing one to assess alternative chemotype
replacements that might be too insoluble to be addressed early in
the experimental phase or a priori to rank the effectiveness of
adding functionality. Applying these techniques, we were able to
achieve our initial goals of lower MW and cLogP with new
fragments, and to efficiently identify novel inhibitors of human
renin with increased LE and comparable or improved
bioavailability relative to literature comparators.
Phenomenex Gemini 5 m C18 150 × 21.2 mm; injection
volume: 4 mL; flow rate: 20 mL/min; wavelength: 214 nm and
254 nm; elution: linear gradient of 30:70 CH3CN:water (0.1%
v/v TFA) to 50:50 CH3CN:water (0.1% v/v TFA) over 10 min) to
1
give 12 (53 mg, 30%) as the TFA salt as a yellow oil. H NMR
(300 MHz, CDCl3) δ 14.72 (br s, 2H), 7.17 (d, 1H, J = 6.0 Hz),
6.84 (s, 1H), 6.69 (d, 1H, J = 6.0 Hz), 6.52 (s, 1H), 5.32 (br s,
5H), 3.47-3.44 (m, 6H), 3.31 (s, 3H), 2.92 (m, 2H), 2.28 (s, 3H),
2.13-2.08 (m, 5H), 1.94 (m, 2H). LCMS 326 [M + H]+.
5.3. 6-Ethyl-5-(1-(3-methoxypropyl)-1,2,3,4-tetrahydroquinolin-
7-yl)-4-methylpyridin-2-amine (13).
A mixture of 42 (200 mg, 0.6 mmol), 2-amino-5-bromo-6-
ethyl-4-methylpyridine
(130
mg,
0.6
mmol),
tetrakis(triphenylphosphine)palladium(0) (35 mg, 0.03 mmol),
and aqueous Na2CO3 solution (2 N, 0.7 mL) in dioxane (5 mL)
o
was stirred at 80 C overnight under argon. The mixture was
5. Experimental
cooled to room temperature, filtered, and the filtrate was
concentrated under vacuum. The crude residue was purified by
preparative HPLC (column: Phenomenex Gemini 5 m C18 150
× 21.2 mm; injection volume: 4 mL; flow rate: 20 mL/min;
wavelength: 214 nm and 254 nm; elution: linear gradient of
25:75 CH3CN:water (0.1% v/v TFA) to 50:50 CH3CN:water
(0.1% v/v TFA) over 10 min) to give 13 (12 mg, 4%) as the TFA
5.1. General methods.
All solvents and reagents were obtained from commercially
available sources and used without further purification.
Reactions were carried out under nitrogen atmosphere unless
otherwise stated. NMR was performed on a Varian Oxford
Table 6. Rat Pharmacokinetic Data.
Cmpd
t1/2a (h)
2.2
Vdssa (L/kg)
Cla (mL/kg min)
%Fb
20
58
39
AUC b (ng.h/mL)
Cmax b (ng/mL)
10
3.4
5.4
68
66
56
473
1810
1493
87
529
331
27
1.0
32
2.0
33
a1 mg/kg iv dose. b10 mg/kg po dose. Compounds dosed as their HCl salts; vehicle = D5W.
Table 7. Comparison of Some Key Metrics
Cmpd
Measured IC50
Literature IC50
MW
cLogP
LE
Relative LE cf. 7b
%F (rat)
(µM)
0.003
0.147
-
-
-
(µM)
0.0006
0.020
0.007
0.001
0.141
0.048
-
551
385
483
496
521
521
341
396
412
3.5
2.8
4.3
3.3
5.0
5.0
3.8
3.6
2.2
0.30a/0.32b
0.33a/0.37b
0.32
0.9
1
2.4
ndc
0.4
8
12
74
20
58
39
1
7b
7c
0.86
0.91
0.68
0.73
1.18
1.06
1.05
7d
0.34
0.25
0.27
0.39
0.35
0.33
rac-7e
(S)-7e
27
32
33
-
0.059
0.069
0.045
-
-
aBased on measured IC50. bBased on literature IC50. cNot determined.

salt as a yellow oil. 1H NMR (300 MHz, CD3OD) δ 8.45 (s, 1H),
6.92 (d, 1H, J = 0.6 Hz), 6.56 (s, 1H), 6.32 (s, 1H), 6.23 (d, 1H, J
= 0.9 Hz), 3.28-3.27 (m, 2H), 3.26-3.24 (m, 3H), 2.81-2.76 (m,
2H), 2.45-2.41 (m, 2H), 2.01 (s, 3H), 1.95-1.93 (m, 2H), 1.78-
1.76 (m, 2H), 1.09 (t, 3H, J = 7.5 Hz). LCMS 340 [M + H]+.
- 7.149 (m, 2H), 6.61 - 6.52 (m, 2H), 6.16 (d, 1H, J = 2.1 Hz),
6.02 (dd, 1H, J1 = 17.4 Hz, J2 = 2.1 Hz), 5.31 (dd, 1H, J1 = 10.6
Hz, J2 = 1.5 Hz), 3.68 (s, 3H). LCMS 315.1 [M + 1]+.
A
solution of the vinylpyridine (460 mg, 1.46 mmol) in 1M
HCl/THF (10 mL) was stirred overnight at room temperature.
The solvent was evaporated under reduced pressure to give the
hydrochloride salt of 39 (160 mg, 73%) as a white solid. LCMS
151.2 [M + 1]+.
5.4. 4-Methoxy-5-(1-(3-methoxypropyl)-1,2,3,4-
tetrahydroquinolin-7-yl)pyridin-2-amine (14).
(41c). A mixture of 2-amino-4-methoxypyridine (2.4 g, 19
mol) and N-bromosuccinimide (3.1 g, 17 mmol) was dissolved in
acetic acid (6 mL) and stirred at room temperature for 1 h. The
reaction mixture was concentrated under vacuum, basified with
saturated aqueous K2CO3 and extracted with EtOAc. The
combined organics were dried, concentrated under vacuum, and
purified by silica gel column chromatography, eluting with 50:1
CH2Cl2:MeOH, to give 41c (1.85 g, 47%). LCMS 203 [M+H]+.
(41d). A suspension of 39 (160 mg, 1.06 mmol) and 12%
palladium on carbon (20 mg) in MeOH (10 mL) was
hydrogenated under 1 atm of hydrogen (balloon) with stirring
overnight at room temperature. The reaction mixture was
filtered, and the filtrate was concentrated under vacuum. The
resulting residue was diluted with CH2Cl2 (30 mL) and washed
with saturated aqueous NaHCO3 (30 mL). The organic phase
was washed with brine (20 mL), dried over Na2SO4 and
concentrated under vacuum. The crude product was purified by
silica gel column chromatography, eluting with a gradient of
200:1 to 50:1 CH2Cl2:MeOH, to give 40d (120 mg, 74%) as a
yellow oil. LCMS [M+1]+ 153.1. To a solution of 40d (120 mg,
1.06 mmol) in CH3CN (10 mL) was added N-bromosuccinimide
(126 mg, 0.71 mmol), and the reaction mixture was stirred in the
dark at room temperature for 2 h, then diluted with water (30 mL)
and extracted with ethyl acetate (3 x 30 mL). The combined
organic phases were washed with brine (50 mL), dried over
Na2SO4 and concentrated under vacuum to give 41d (145 mg,
A mixture of 42 (100 mg, 0.3 mmol), 41c (61 mg, 0.3 mmol),
tetrakis(triphenylphosphine)palladium(0) (18 mg, 0.015 mmol),
and aqueous Na2CO3 solution (2 N, 0.3 mL) in dioxane (5 mL)
o
was stirred at 80 C overnight under argon. The mixture was
cooled to room temperature, filtered, and the filtrate was
concentrated under vacuum. The crude residue was purified by
preparative HPLC (column: Phenomenex Gemini 5 m C18 150
× 21.2 mm; injection volume: 4 mL; flow rate: 20 mL/min;
wavelength: 214 nm and 254 nm; elution: linear gradient of
25:75 CH3CN:water (0.1% v/v TFA) to 50:50 CH3CN:water
(0.1% v/v TFA) over 10 min) to give 14 (25 mg, 19%) as the
TFA salt as a white solid. 1H NMR (300 MHz, CD3OD) δ 7.65
(s, 1H), 6.98 (d, 1H, J = 9.0 Hz), 6.77 (s, 1H), 6.64 (d, 1H, J =
9.0 Hz), 3.97 (s, 3H), 3.48-3.34 (m, 6H), 3.32 (s, 3H), 2.81-2.77
(m, 2H), 2.03-1.95 (m, 2H), 1.92-1.83 (m, 2H). LCMS 328
[M+H]+.
1
80%) as a yellow solid. H NMR (300 MHz, DMSO-d6) δ 5.97
(br s, 2H), 5.94 (s, 1H), 3.76 (s, 3H), 2.63 (q, 2H, J = 8.0 Hz),
1.13 (t, 3H, J = 8.0 Hz). LCMS 231.0, 233.0 [M + 1]+.
A mixture of 41d (120 mg, 0.52 mmol), 42 (172 mg, 0.52
mmol), tetrakis(triphenylphosphine)palladium(0) (120 mg, 0.11
mmol) and Cs2CO3 (338 mg, 10.4 mmol) in dioxane (10 mL) and
water (2.5 mL) was stirred at 100 oC overnight under argon. The
mixture was cooled to room temperature, filtered, and the filtrate
was concentrated under vacuum. The crude residue was purified
by preparative HPLC (column: Phenomenex Gemini 5 m C18
150 × 21.2 mm; injection volume: 4 mL; flow rate: 20 mL/min;
wavelength: 214 nm and 254 nm; elution: linear gradient of
20:80 CH3CN:water (0.1% v/v TFA) to 55:45 CH3CN:water
(0.1% v/v TFA) over 10 min) to give 15 (50 mg, 27%) as the
TFA salt as a brown solid. 1H NMR (300 MHz, CDCl3) δ 13.58
(br s, 1H), 11.50 (br s, 1H), 7.50 (br s, 1H), 7.06 (d, 1H, J = 7.8
Hz), 6.79 (s, 1H), 6.65 (d, 1H, J = 7.7 Hz), 6.10 (s, 1H), 3.72 (s,
3H), 3.45 - 3.31 (m, 6H), 3.22 (s, 3H), 2.83 (t, 2H, J = 6.5 Hz),
2.45 (q, 2H, J = 7.5 Hz), 2.07 - 1.99 (m, 2H), 1.89 - 1.82 (m, 2H),
1.08 (t, 3H, J = 7.5 Hz). LCMS 356.3 [M + 1]+.
5.5. 6-Ethyl-4-methoxy-5-(1-(3-methoxypropyl)-1,2,3,4-
tetrahydroquinolin-7-yl)pyridin-2-amine (15).
(37). A solution of sodium methoxide in MeOH (33 mL, 1M,
33 mmol) was added to a suspension of 2,4,6-trichloropyridine
(36) (6.0 g, 33 mmol) in MeOH (82 mL) and stirred for 5 h at
room temperature. The reaction mixture was poured into water
(200 mL). The solid was removed by filtration, and the filtrate
was concentrated under vacuum to give 37 (4.8 g, 80%) as a
1
white solid. H NMR (300 MHz, DMSO-d6) δ 7.19 (s, 2H), 3.89
(s, 3H). LCMS 178.0, 180.1 [M + 1]+.
(38). A mixture of 37 (2.2 g, 12.5 mmol), benzophenone
imine (2.7 g, 15.0 mmol), palladium (II) acetate (280 mg, 1.2
mmol), 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (777 mg, 1.2
mmol) and Cs2CO3 (8.1 g, 25.0 mmol) in toluene (30 mL) was
stirred at 100 oC overnight under argon. The mixture was cooled
to room temperature and filtered. The filtrate was concentrated
under vacuum and the resulting residue was purified by silica gel
column chromatography, eluting with a gradient of 50:1 to 10:1
petroleum ether:EtOAc, to give 38 (900 mg, 20%) as a brown oil.
LCMS 323.1 [M + 1]+.
5.6. 6-Ethyl-5-(1H-indol-7-yl)pyrimidine-2,4-diamine (16).
(44). A mixture of 2,4-diamino-6-ethylpyrimidine (543 mg,
3.93 mmol), N-iodosuccinimide (884 mg, 3.93 mmol) in
methanol (10 mL) was stirred at room temperature for 30 min, at
which time analysis by TLC showed the starting material was
completely consumed. The resulting mixture was concentrated
and purified by silica gel chromatography, eluting with 50:50
petroleum ether:EtOAc, to give 44 (400 mg, 40 %). 1H NMR
(300 MHz, DMSO-d6) δ 6.29 (br s, 2H), 6.03 (br s, 2H), 2.57 (q,
2H, J = 7.5 Hz), 1.09 (t, 3H, J = 7.5 Hz). LCMS 265.1 [M + H]+.
(39). A mixture of 38 (900 mg, 2.8 mmol), 4,4,5,5-
tetramethyl-2-vinyl-1,3,2-dioxaborolane (650 mg, 4.2 mmol),
tetrakis(triphenylphosphine)palladium(0) (650 mg, 0.56 mmol)
and Cs2CO3 (1.8 g, 5.6 mmol) in toluene/water (4:1, 20 mL) was
o
A mixture of 44 (200 mg, 0.758 mmol), indole-7-boronic acid
stirred at 100 C overnight under argon. The reaction mixture
pinacol
ester
(184.1
mg,
0.758
mmol),
was cooled to room temperature and filtered. The filtrate was
concentrated under vacuum and the resulting residue was purified
by silica gel column chromatography, eluting with a gradient of
200:1 to 100:1 CH2Cl2:MeOH, to give the desired vinylpyridine
tetrakis(triphenylphosphine)palladium(0) (87.5 mg, 0.076 mmol),
and Na2CO3 (201 mg, 1.89 mmol) in dioxane/water (4:1, 10 mL)
was stirred at 100 oC overnight under argon. The reaction
mixture was cooled to room temperature and concentrated under
vacuum. The resulting residue was purified by preparative
1
(460 mg, 52%) as a brown oil. H NMR (300 MHz, DMSO-d6) δ
7.68 -7.66 (m, 2H), 7.57 - 7.44 (m, 3H), 7.31 - 7.29 (m, 3H), 7.17

HPLC (column: Phenomenex Gemini 5 m C18 150 × 21.2 mm;
injection volume: 3 mL; flow rate: 20 mL/min; wavelength: 214
nm and 254 nm; elution: linear gradient of 25:75 CH3CN:water
(0.1% v/v TFA) to 45:55 CH3CN:water (0.1% v/v TFA) over 10
min) to give 16 (17 mg, 6%) as the TFA salt. 1H NMR (300
MHz, DMSO-d6) δ 11.06 (br s, 1H), 8.05 (br s, 1H), 7.63 (d, 1H,
J = 8.7 Hz), 7.34 (t, 1H, J = 2.7 Hz), 7.10 (t, 1H, J = 7.5 Hz),
6.94 (d, 1H, J = 7.5 Hz), 6.56 (br s, 1H), 6.52 - 6.50 (m, 1H),
2.20 - 2.02 (m, 2H), 0.94 (t, 3H, J = 7.5 Hz). LCMS 254.1 [M +
H]+.
(0.1% v/v TFA) over 10 min) to give 19 (20 mg, 30%) as the
TFA salt as a white solid. 1H NMR (300 MHz, CD3OD) δ 10.04
(br s, 1H), 7.58 (d, 1H, J = 9.0 Hz), 7.13 (t, 1H, J = 7.2 Hz), 6.99
(s, 1H), 6.89 (d, 1H, J = 9.0 Hz), 6.81 (s, 1H), 2.33 (s, 3H), 2.14
(s, 3H), 2.01 (s, 3H). LC-MS 252.2 [M + H]+.
5.10. 6-Ethyl-4-methyl-5-(3-methyl-1H-indol-7-yl)pyridin-2-
amine (20).
To a mixture of 3-methylindole-7-boronic acid pinacol ester
(43b) (prepared from 7-bromo-3-methylindole according to
Koshino, N. et al. Nitrogen-Containing Aromatic Compounds
and Metal Complexes. PCT Application WO 2011/052805 A1, 5
May 2011) (70 mg, 0.27 mmol), 41b (70 mg, 0.327 mmol), and
Cs2CO3 (176 mg, 0.54 mmol) in dioxane/water (3:1, 4 mL) was
added tetrakis(triphenylphosphine)palladium(0) (31 mg, 0.027
mmol) quickly under argon. The reaction mixture was stirred at
5.7. 5-(1H-Indol-7-yl)-4,6-dimethylpyridin-2-amine (17).
A mixture of indole-7-boronic acid pinacol ester (43a) (243
mg,
1
mmol),
41a
(200
mg,
1
mmol),
tetrakis(triphenylphosphine)palladium(0) (58 mg, 0.05 mmol),
and aqueous Na2CO3 solution (2M, 1.25 mL) in dioxane (5 mL)
o
o
100 C under argon overnight and cooled to room temperature.
was stirred at 100 C for 14 h under argon. The mixture was
Water (10 mL) was added and the resulting mixture was
extracted with EtOAc (3 x 5 mL). The combined organic phases
were dried over Na2SO4 and concentrated under vacuum. The
crude residue was purified by preparative HPLC (column:
Phenomenex Gemini 5 m C18 150 × 21.2 mm; injection
volume: 4 mL; flow rate: 20 mL/min; wavelength: 214 nm and
254 nm; elution: linear gradient of 20:80 CH3CN:water (0.1%
v/v TFA) to 70:30 CH3CN:water (0.1% v/v TFA) over 10 min) to
give 20 (15 mg, 21%) as the TFA salt as a white solid. 1H NMR
(300 MHz, CD3OD) δ 10.08 (br s, 1H), 7.58 (d, 1H, J = 9.0 Hz),
7.13 - 7.10 (m, 1H), 6.98 (d, 1H, J = 1.2 Hz), 6.89 (dd, 1H, J1 =
7.2 Hz，J2 = 1.2 Hz), 6.81 (d, 1H, J = 0.9 Hz), 2.53-2.35 (m,
2H), 2.33 (d, 3H, J = 0.6 Hz), 1.97 (d, 3H, J = 1.2 Hz), 1.05 (t,
3H, J = 7.5 Hz). LCMS 266.2 [M + H]+.
cooled to room temperature, poured into ice water (20 mL) and
extracted with EtOAc (3 x 10 mL). The combined organic
phases were washed with brine, dried over Na2SO4 and
concentrated under vacuum. The crude product was purified by
preparative TLC (20:1 CH2Cl2:MeOH), then recrystallized (5:1
hexane:EtOAc, 6 mL) to give 17 (60 mg, 25%) as a white solid.
1H NMR (300 MHz, CDCl3) δ 8.45 (br s, 1H), 7.65 (d, 1H, J =
9.0 Hz), 7.20 - 7.14 (m, 2H), 6.93 (d, 1H, J = 9.0 Hz), 6.61 – 5.97
(m, 1H), 6.35 (s, 1H), 4.47 (br s, 2H), 2.08 (s, 3H), 1.92 (s, 3H).
LCMS 238.1 [M + H]+.
5.8. 6-Ethyl-5-(1H-indol-7-yl)-4-methylpyridin-2-amine (18).
A mixture of indole-7-boronic acid pinacol ester (43a) (203
mg,
0.8
mmol),
41b
(150
mg,
0.7
mmol),
tetrakis(triphenylphosphine)palladium(0) (40 mg, 0.03 mmol),
5.11. 6-Ethyl-5-(4-fluoro-3-methyl-1H-indol-7-yl)-4-
methylpyridin-2-amine (21).
and aqueous Na2CO3 solution (2M, 0.9 mL) in dioxane (10 mL)
o
was stirred at 100 C under argon overnight. The mixture was
(46a). To a suspension of 1-bromo-4-fluoro-2-nitrobenzene
(45a) (2.0 g, 9.12 mmol) in THF (40 mL) at -78 oC was added 1-
propenylmagnesium bromide (60 mL, 1M in THF, 60 mmol).
The reaction mixture was warmed to room temperature, stirred
for 2 h, and quenched with saturated aqueous NH4Cl. The
mixture was extracted with EtOAc (3 x 50 mL), and the
combined organic layers were washed with brine (50 mL), dried
over Na2SO4, and concentrated under vacuum to afford 46a (1.1
g, 55%) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ
11.21 (br s, 1H), 7.32 - 6.96 (m, 2H), 6.68 (dd, 1H, J1 = 12.0 Hz,
J2 = 9.0 Hz), 2.35 (s, 3H). GCMS 226, 228 [M]+.
cooled to room temperature, filtered, and concentrated under
vacuum. The crude residue was purified by preparative HPLC
(column: Phenomenex Gemini 5 m C18 150 × 21.2 mm;
injection volume: 4 mL; flow rate: 20 mL/min; wavelength: 214
nm and 254 nm; elution: linear gradient of 10:90 CH3CN:water
(0.1% v/v TFA) to 50:50 CH3CN:water (0.1% v/v TFA) over 10
min) to give 18 (65 mg, 26%) as the TFA salt as a pale yellow
solid. 1H NMR (300 MHz, CD3OD) δ 8.49 (s, 1H), 7.62 (d, 1H,
J = 7.5 Hz), 7.20 (d, 1H, J = 3.6 Hz), 7.11 (t, 1H, J = 7.5 Hz),
6.89 (d, 1H, J = 7.2 Hz), 6.72 (s, 1H), 6.52 (d, 1H, J = 3.0 Hz),
2.51-2.34 (m, 2H), 1.95 (s, 3H), 1.03 (t, 3H, J = 1.5 Hz). LCMS
252 [M + H]+.
(47b). A mixture of 41b (1.4 g, 6.5 mmol), 4,4,4',4',5,5,5',5'-
octamethyl-2,2'-bi(1,3,2-dioxaborolane) (2.5 g, 9.8 mmol),
tetrakis(triphenylphosphine)palladium(0) (2.3 g, 1.9 mmol) and
potassium acetate (1.28 g, 13 mmol) in DMSO (15 mL) was
stirred at 120 oC overnight under argon. The mixture was cooled
to room temperature and poured into water (30 mL). The aqueous
phase was extracted with EtOAc (3 x 100 mL), and the combined
organic phases were washed with water (50 mL) and brine (50
mL), dried over Na2SO4, concentrated under vacuum and purified
by silica gel column chromatography, eluting with 100:1 CH2Cl2:
methanol, to give 47b (0.4 g, 24%) as a brown oil. LC-MS 263
[M + H]+.
5.9. 4,6-Dimethyl-5-(3-methyl-1H-indol-7-yl)pyridin-2-amine
(19).
To a solution of 43b (prepared from 7-bromo-3-methylindole
according to Koshino, N. et al. (Sumitomo Chemical Co.).
Nitrogen-Containing Aromatic Compounds and Metal
Complexes. PCT Application WO 2011/052805 A1, 5 May
2011) (70 mg, 0.27 mmol), 41a (65 mg, 0.327 mmol), and
Cs2CO3 (176 mg, 0.54 mmol) in 3:1 dioxane:water (4 mL) was
addded tetrakis(triphenylphosphine)palladium(0) (31 mg, 0.027
o
mmol) quickly under argon. The mixture was heated at 100 C
under argon overnight. Water (10 mL) was added and the
resulting mixture was extracted with EtOAc (3 x 5 mL). The
combined organic phases were dried over Na2SO4 and
concentrated under vacuum. The product residue was purified by
preparative HPLC (column: Phenomenex Gemini 5 m C18 150
× 21.2 mm; injection volume: 4 mL; flow rate: 20 mL/min;
wavelength: 214 nm and 254 nm; elution: linear gradient of
25:75 CH3CN:water (0.1% v/v TFA) to 60:40 CH3CN:water
A mixture of 46a (150 mg, 0.66 mmol), 47b (150 mg, 0.79
mmol), tris(dibenylideneacetone)dipalladium(0) (60 mg, 0.066
mmol),
2-Dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl
(X-Phos) (40 mg, 0.066 mmol) and Cs2CO3 (430 mg, 1.32 mmol)
o
in dioxane/water (4:1, 15 mL) was stirred at 100 C overnight
under argon. The mixture was cooled to room temperature,
filtered, and concentrated under vacuum. The crude product was
purified by silica gel column chromatography, eluting with a

gradient of 200:1 to 50:1 CH2Cl2:MeOH, to give 21 (50 mg,
20%) as a brown solid. 1H NMR (300 MHz, CDCl3) δ 7.83 (brs,
1H), 6.79 - 6.76 (m, 3H), 6.32 (s, 1H), 4.46 (s, 2H), 2.47 (s, 3H),
2.39 - 2.23 (m, 2H), 1.86 (s, 3H), 1.00 (t, 3H, J = 7.5 Hz). LCMS
284.1 [M + H]+.
1H), 6.84 (s, 1H), 2.38-2.51 (m, 5H), 2.01 (s, 3H), 1.09 (t, 3H, J=
8.0 Hz). LCMS 302 [M + H] +.
5.14. 6-Ethyl-5-(2-(methoxymethyl)-3-methyl-1H-indol-7-yl)-4-
methylpyridin-2-amine (24).
(52). To a suspension of sodium hydride (160 mg, 54 wt. %
in oil, 3.6 mmol) in dry CH2Cl2 (30 mL) at 0 C under nitrogen
was added 48d (237 mg, 1 mmol). After warming to room
5.12. 6-Ethyl-5-(5-fluoro-3-methyl-1H-indol-7-yl)-4-
methylpyridin-2-amine (22).
o
(46b). To a solution of 2-bromo-4-fluoro-1-nitrobenzene
(45b) (13.7 g, 62.5 mmol) in THF (20 mL) at -60 C was added
temperature
over
1
h,
a
solution
of
2-
o
(trimethylsilyl)ethoxymethyl chloride (200 mg, 1.2 mmol) in dry
CH2Cl2 (2mL) was added. The mixture was stirred for 1 h at
room temperature. Water (15mL) was added and the mixture
was extracted with CH2Cl2 (3 x 10 mL). The combined organic
layers were dried, concentrated under vacuum, and purified by
silica gel column chromatography, eluting with 20:1 petroleum
ether: EtOAc, to give 52 (368 mg, 100%) as a brown oil. 1H
NMR (300 MHz, DMSO) δ 10.19 (s, 1H), 7.82 (d, 1H, J = 6.0
Hz), 7.67 (d, 1H, J = 6.0 Hz), 7.14-7.09 (m, 1H), 6.26 (s, 2H),
3.43-3.32 (m, 2H), 2.61 (s, 3H), 0.76-0.71 (m, 2H), 0.14 (s, 9H).
LCMS 392 [M + Na]+. The reaction was repeated on a similar
scale (1.14 mmol of 48d) to provide an additional 410 mg of 52.
1-propenylmagnesium bromide (500 mL, 0.5M in THF, 250
mmol) dropwise. The reaction mixture was stirred at -60 oC for 1
h, quenched with saturated aqueous NH4Cl (100 mL) and
extracted with EtOAc (2 x 500 mL). The combined organic
layers were dried over Na2SO4 and concentrated under vacuum.
The crude residue was purified by silica gel column
chromatography, eluting with 50:1 petroleum ether: EtOAc, to
give 46b (5.6 g, 39%) as a light brown oil. 1H NMR (300 MHz,
CDCl3) δ 8.02 (s, 1H), 7.17-7.12 (m, 2H), 7.06 (s, 1H), 2.27 (d,
3H, J = 1.6 Hz). GC-MS 227 [M]+.
A mixture of 46b (80 mg, 0.35 mmol), 47b (113 mg, 0.42
mmol), tetrakis(triphenylphosphine)palladium(0) (40 mg, 0.035
mmol), and Cs2CO3 (228 mg, 0.7 mmol) in dioxane (10 mL) and
(53). To a solution of 52 (740 mg, 2.0 mmol) dissolved in
THF/MeOH (1:1, 10 mL) was added sodium borohydride (90
0
o
water (2 mL) was stirred at 100 C overnight under argon, then
mg, 2.4 mmol) in portions at 0 C. The reaction mixture was
cooled to room temperature. Water (10 mL) was added and the
mixture was extracted with EtOAc (3 x 5 mL). The combined
organic layers were dried over Na2SO4 and concentrated under
vacuum. The crude product was purified by preparative HPLC
(column: Phenomenex Gemini 5 m C18 150 × 21.2 mm;
injection volume: 4 mL; flow rate: 20 mL/min; wavelength: 214
nm and 254 nm; elution: linear gradient of 60:40 CH3CN:water
(0.05% v/v NH4OH) to 90:10 CH3CN:water (0.05% v/v NH4OH)
over 10 min) to give 22 (33 mg, 33%) as a light brown solid. 1H
NMR (400 MHz, CD3OD) δ 7.17 (dd, 1H, J = 10.0 Hz, J = 4.0
Hz), 6.99 (s, 1H), 6.65 (dd, 1H, J = 8.0 Hz, J = 4.0 Hz), 6.45 (s,
1H), 2.42-2.21 (m, 5H), 1.86 (s, 3H), 0.99 (t, 3H, J = 8.0 Hz).
LCMS 284.1 [M + H]+.
stirred for 1 h at 0 oC and purified by silica gel column
chromatography, eluting with 8:1 petroleum ether:EtOAc, to give
1
the desired alcohol (560 mg, 70%) as a pale yellow oil. H NMR
(300 MHz, CDCl3) δ 7.50 (dd, 1H, J = 7.8 Hz, 0.9 Hz), 7.41 (dd,
1H, J = 7.6 Hz, 0.8 Hz), 7.01-6.96 (m, 1H), 7.14-7.09 (m, 1H),
6.12 (s, 2H), 4.80 (s, 2H), 3.66-3.60 (m, 2H), 2.34 (s, 3H), 0.92-
0.86 (m, 2H), -0.05 (s, 9H). LCMS 332 [M + H]+.
To a suspension of sodium hydride (78 mg, 54 wt. % in oil,
1.76 mmol) in dry THF (10 mL) cooled to 0 C under nitrogen
was added the alcohol prepared above (500 mg, 1.35 mmol).
After stirring for 1 h at 0 oC, methyl iodide (0.7 mL, 10.8 mmol)
was added. The reaction mixture was stirred at room temperature
overnight. Water was added and the mixture was extracted with
o
5.13. 5-(4,5-Difluoro-3-methyl-1H-indol-7-yl)-6-ethyl-4-
CH2Cl2.
The combined organic layers were dried and
methylpyridin-2-amine (23).
concentrated under vacuum. The crude product was purified by
silica gel column chromatography, eluting with 10:1 petroleum
ether:EtOAc to give the desired SEM-protected methyl ether
(510 mg, 98%) as a yellow oil. 1H NMR (300 MHz, CDCl3) δ
7.46 (dd, 1H, J = 7.8 Hz, 1.1 Hz), 7.39 (dd, 1H, J = 7.7 Hz, 1.1
Hz), 6.97-6.92 (m, 1H), 5.95 (s, 2H), 4.66 (s, 2H), 3.58-3.53 (m,
2H), 3.33 (s, 3H), 2.31 (s, 3H), 0.91-0.85 (m, 2H), -0.06 (s, 9H).
(46c). To a mixture of 2-bromo-4,5-difluoronitrobenzene
(45c) (238 mg, 1.0 mmol) in THF (10 mL) at 0 oC under nitrogen
was added 1-propenylmagnesium bromide (20 mL, 0.5M in THF,
10 mmol) dropwise. The reaction mixture was stirred at room
temperature overnight, poured into ice water (20 mL), and
extracted with EtOAc (3 x10 mL). The combined organic phases
were washed with brine, dried over anhydrous Na2SO4, and
concentrated under vacuum. The crude product was purified by
silica gel column chromatography to afford 46c (85 mg, 35%).
1H NMR (300 MHz, CDCl3) δ 8.00 (br s, 1H), 7.12-7.17 (m, 1H),
2.41 (s, 3H).
A mixture of the SEM-protected methyl ether prepared above
(250 mg, 0.65 mmol), ethylenediamine (118 mg, 1.95 mmol) and
tetrabutylammonium fluoride (3.9 mL, 1M in THF, 3.9 mmol) in
THF (1 mL) was stirred at 70 oC under nitrogen for 4 h. The
reaction mixture was cooled to room temperature, concentrated
under vacuum, and purified by preparative TLC to give 53 (143
mg, 87%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 8.23
(s, 1H), 7.48 (d, 1H, J = 9.0 Hz), 7.34-7.31 (m, 1H, J = 9 Hz),
7.01-6.96 (m, 1H), 4.62 (s, 2H), 3.40 (s, 3H), 2.29 (s, 3H).
A mixture of 46c (110 mg, 0.45 mmol), 47b (128 mg, 0.49
mmol), tetrakis(triphenylphosphine)palladium(0) (140 mg, 0.12
mmol), and Cs2CO3 (398 mg, 1.22 mmol) in water (2 mL) and
o
dioxane (10 mL) was stirred at 100 C overnight under argon.
A mixture of 53 (188 mg, 0.74 mmol), 47b (195 mg, 0.74
mmol), tetrakis(triphenylphosphine)palladium(0) (43 mg, 0.04
mmol) and Cs2CO3 (604 mg, 1.86 mmol) in dioxane (5 mL) and
The reaction mixture was poured into ice water (15 mL) and
extracted with EtOAc (3 x 5 mL). The combined organic phases
were washed with brine, dried over anhydrous Na2SO4 and
concentrated under vacuum. The crude product was purified by
preparative HPLC (column: Phenomenex Gemini 5 m C18 150
× 21.2 mm; injection volume: 4 mL; flow rate: 20 mL/min;
wavelength: 214 nm and 254 nm; elution: linear gradient of
30:70 CH3CN:water (0.1% v/v TFA) to 50:50 CH3CN:water
(0.1% v/v TFA) over 10 min) to give 23 (11 mg, 8%) as the TFA
salt. 1H NMR (400 MHz, CD3OD) δ 7.04 (s, 1H), 6.87-6.92 (m,
o
water (1 mL) was stirred at 100 C for overnight under argon.
The reaction mixture was cooled to room temperature and
filtered. The filtrate was concentrated under vacuum and purified
by preparative HPLC (column: Phenomenex Gemini 5 m C18
150 × 21.2 mm; injection volume: 4 mL; flow rate: 20 mL/min;
wavelength: 214 nm and 254 nm; elution: linear gradient of
35:65 CH3CN:water (0.05% v/v NH4OH) to 55:45 CH3CN:water

(0.05% v/v NH4OH) over 10 min) to give 24 (33 mg, 14%) as a
white solid. 1H NMR (400 MHz, DMSO) δ 10.44 (s, 1H), 7.42
(d, 1H, J = 6.0 Hz), 7.04-7.00 (m, 1H), 6.78 (d, 1H, J = 6.0 Hz),
6.26 (s, 1H), 5.73 (s, 2H), 4.43 (s, 2H), 3.19 (s, 3H), 2.26 (s, 3H),
2.23-2.07 (m, 2H), 1.73 (s, 3H), 0.93-0.89 (m, 3H). LCMS 310
[M +H]+.
mmol), and aqueous Na2CO3 solution (0.51 mL, 2M) in dioxane
(5 mL) was stirred at 100 oC overnight under argon. The reaction
mixture was cooled to room temperature, poured into ice water
(15 mL), and extracted with EtOAc (3 x 5 mL). The combined
organic phases were washed with brine, dried over Na2SO4, and
concentrated in vacuo. The crude product was purified by
preparative TLC, eluting with 5:1 petroleum ether:EtOH, to give
the desired (E)- and (Z)-5-(2-(2-methoxyvinyl)-3-methyl-1H-
indol-7-yl)-4,6-dimethylpyridin-2-amine (80 mg, 77%). LCMS
308.0 [M + H]+.
5.15. 5-(2-(2-Methoxyethyl)-3-methyl-1H-indol-7-yl)-4,6-
dimethylpyridin-2-amine (25).
(48d). Phosphorus oxychloride (523 mg, 2.50 mmol) was
o
added dropwise into DMF (456 mg, 6.25 mmol) at 0 C under
A mixture of the olefins prepared above (80 mg, 0.26 mmol)
and a catalytic amount of 10% palladium on carbon in MeOH (5
mL) was stirred at room temperature under an atmosphere of
hydrogen (balloon) for 2 h. The reaction mixture was filtered
and the filtrate was concentrated under vacuum. The crude
product was purified by preparative HPLC (column:
Phenomenex Gemini 5 m C18 150 × 21.2 mm; injection
volume: 4 mL; flow rate: 20 mL/min; wavelength: 214 nm and
254 nm; elution: linear gradient of 30:70 CH3CN:water (0.1%
v/v TFA) to 38:62 CH3CN:water (0.1% v/v TFA) over 10 min) to
give 25 (4 mg, 3%) as the TFA salt. 1H NMR (400 MHz,
CD3OD) δ 9.91 (br s, 1H), 7.53 (d, 1H, J = 8.0 Hz), 7.12 (t, 1H, J
= 8.0 Hz), 6.86 (d, 2H, J = 8.0 Hz), 3.61 (t, 2H, J = 8.0 Hz), 3.32
(s, 3H), 2.98 (t, 2H, J = 8.0 Hz), 2.29 (s, 3H), 2.17 (s, 3H), 2.07
(s, 3H). LCMS 310.2 [M + H]+.
nitrogen, stirred at 0 oC for 1 h, and treated with a solution of 46d
(523 mg, 2.5 mmol) in 1,2-dichloroethane (10 mL). The
resulting mixture was heated at reflux for 4 h, and cooled to room
temperature. Saturated aqueous sodium acetate (20 mL) was
added and the mixture was heated at reflux for an additional 4 h.
The reaction mixture was cooled to room temperature, diluted
with water (20 mL), and extracted with CH2Cl2 (3 x 50 mL). The
combined organic phases were washed with brine, dried over
anhydrous Na2SO4 and concentrated under vacuum. The crude
product was purified by silica gel column chromatography,
eluting with a gradient of 30:1 to 20:1 petroleum ether:EtOAc, to
give 48d (279 mg, 47%) as a brown oil. 1H NMR (300 MHz,
CDCl3) δ 10.06 (s, 1H), 8.83 (br s, 1H), 7.65 (d, 1H, J = 9.0 Hz),
7.54 (dd, 1H, J1 = 9.0 Hz, J2 = 3.0 Hz), 7.05 (t, 1H, J = 9.0 Hz),
2.64 (s, 3H). LCMS 237.9, 239.9 [M + H]+.
5.16. 6-Ethyl-5-(2-(2-methoxyethyl)-3-methyl-1H-indol-7-yl)-4-
methylpyridin-2-amine (26).
(50d).
To
a
suspension
of
(methoxymethyl)triphenylphosphonium chloride (515 mg, 1.5
mmol) in dry THF (5 mL) was added n-butyllithium (0.75 mL,
2.2M in hexane, 1.65 mmol) at 0 oC under nitrogen. The mixture
A mixture of 50d (90 mg, 0.34 mmol), 47b (134 mg, 0.40
mmol), tetrakis(triphenylphosphine)palladium(0) (40 mg, 0.034
mmol), and aqueous Na2CO3 solution (0.51 mL, 2M) in dioxane
o
was stirred at 0 C for 1 h and a solution of 48d (237 mg, 1
o
mmol) in THF (5 mL) was added quickly. The reaction mixture
was stirred at room temperature overnight and saturated aqueous
NH4Cl (20 mL) was added. The mixture was extracted with
EtOAc (3 x 10 mL). The combined organic phases were washed
with brine, dried over Na2SO4, and concentrated under vacuum.
The crude product was purified by silica gel column
chromatography, eluting with a gradient of 100:0 to 5:95
petroleum ether:EtOAc, to give 50d (125 mg, 47%) as a white
solid. LCMS 266.0, 268.1 [M + H] +. A small sample of 50d
(10 mg, mixture of isomers) was purified by preparative TLC,
eluting with 20:1 petroleum ether:EtOAc, to afford the Z isomer
exclusively: 1H NMR (300 MHz, CDCl3) δ 9.26 (br s, 1H), 7.46
(d, 1H, J = 7.8 Hz), 7.38-7.31 (m, 1H), 6.98 (t, 1H, J = 7.8 Hz),
6.22 (d, 1H, J = 9.0 Hz), 5.52 (d, 1H, J = 9.0 Hz), 3.88 (s, 3H),
2.31 (s, 3H).
(5 mL) ) was stirred at 100 C overnight under argon. The
reaction mixture was cooled to room temperature, poured into ice
water (15 mL), and extracted with EtOAc (3 x 5 mL). The
combined organic phases were washed with brine, dried over
Na2SO4, and concentrated under vacuum. The crude product was
purified by preparative TLC, eluting with 5:1 petroleum
ether:EtOH, to give the desired (E)- and (Z)-6-ethyl-5-(2-(2-
methoxyvinyl)-3-methyl-1H-indol-7-yl)-4-methylpyridin-2-
amine (90 mg, 82%). LCMS 322.1 [M + H]+.
A mixture of the olefins prepared above (90 mg, 0.28 mmol)
and a catalytic amount of 10% palladium on carbon in MeOH (15
mL) was stirred at room temperature under an atmosphere of
hydrogen (balloon) for 2 h. The reaction mixture was filtered
and the filtrate was concentrated under vacuum. The crude
product was purified by preparative HPLC (column:
Phenomenex Gemini 5 m C18 150 × 21.2 mm; injection
volume: 4 mL; flow rate: 20 mL/min; wavelength: 214 nm and
254 nm; elution: linear gradient of 30:70 CH3CN:water (0.1%
v/v TFA) to 50:50 CH3CN:water (0.1% v/v TFA) over 10 min) to
give 26 (5 mg, 4%) as the TFA salt as a yellow oil. 1H NMR
(400 MHz, CD3OD) δ 9.93 (br s, 1H), 7.53 (d, 1H, J = 8.0 Hz),
7.12 (t, 1H, J = 8.0 Hz), 6.86 (d, 2H, J = 8.0 Hz), 3.60 (t, 2H, J =
8.0 Hz), 3.35 - 3.30 (m, 3H), 2.97 (t, 2H, J = 6.0 Hz), 2.54 - 2.40
(m, 2H), 2.29 (s, 3H), 2.02 (s, 3H), 1.09 (t, 3H, J = 8.0 Hz).
LCMS 324.3 [M + H]+.
(47a).
To a mixture of 41a (200 mg, 1 mmol),
4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2- dioxaborolane) (508
mg, 2 mmol) and potassium acetate (196 mg, 2 mmol) in DMSO
(3
mL)
was
added
[1,1’-bis-
(diphenylphosphino)ferrocene]dichloropalladium(II) (220 mg,
0.3 mmol) quickly under argon. The mixture was stirred at 120
oC under overnight. The reaction mixture was cooled to room
temperature, poured into water (20 mL), and extracted with
EtOAc (3 x 10 mL). The combined organic phases were washed
with brine (2 x 10 mL), dried over Na2SO4 and concentrated
under vaccum. The product residue was purified by silica gel
column chromatography, eluting with 15:1 petroleum ether:
EtOAc, to give 47a (41 mg, 16%) as a brown oil. 1H NMR (300
MHz, CDCl3) δ 6.13 (s, 1H), 4.60 (br s, 2H), 2.50 (s, 3H), 2.31
(s, 3H), 1.36 (s, 12H). LC-MS 249.3 [M + H] +. A second batch
using 180 mg of 41a gave an additional 102 mg (45%) of 47a.
5.17. 6-Ethyl-5-(4-fluoro-2-(2-methoxyethyl)-3-methyl-1H-indol-
7-yl)-4-methylpyridin-2-amine (27).
(48a). Phosphorus oxychloride (1.01 g, 6.6 mmol) was added
o
to DMF (482 mg, 6.6 mmol) at 0 C, followed by a solution of
46a (500 mg, 2.2 mmol) in 1,2-dichloroethane (8 mL). The
reaction mixture was heated at reflux for 5 h, followed by the
addition of saturated aqueous sodium acetate solution (10 mL),
and the reaction was continued at reflux for an additional 1 h.
A mixture of 50d (90 mg, 0.34 mmol), 47a (127 mg, 0.40
mmol), tetrakis(triphenylphosphine)palladium(0) (40 mg, 0.034

The reaction mixture was cooled to room temperature and
extracted with CH2Cl2 (3 x 10 mL). The combined organic layers
were dried over Na2SO4 and concentrated under vacuum. The
crude product was purified by silica gel column chromatography,
eluting with 10:1 petroleum ether: EtOAc, to give 48a (477 mg,
65%) as a yellow solid. H NMR (300 MHz, CDCl3) δ 10.03 (s,
1H), 8.83 (br s, 1H), 7.42-7.38 (m, 1H), 6.72-6.66 (m, 1H), 2.75
(d, 3H, J = 4.0 Hz).
column chromatography, eluting with 50:1 petroleum ether:
EtOAc, to give the desired indole pinacol ester (212 mg, 36%) as
a brown oil. LCMS: 332.2 [M + H]+.
To a solution of the indole boronic ester prepared above (212
mg, 0.64 mmol), 41b (164 mg, 0.77 mmol) and Cs2CO3 (417 mg,
1.28 mmol) in dioxane (10 mL) and water (2 mL) was added
tetrakis(triphenylphosphine)palladium(0) (74 mg, 0.064 mmol)
1
o
under argon. The mixture was stirred at 100 C under argon
(50a).
To
a
solution
of
overnight, and cooled to room temperature. Water (50 mL) was
added and the mixture was extracted with EtOAc (3 x 20 mL).
The combined organic layers were dried over Na2SO4 and
concentrated under vacuum. The crude product was purified by
silica gel column chromatography, eluting with 50:1 petroleum
ether: EtOAc, to give (E)- and (Z)-6-ethyl-5-(4-fluoro-2-(2-
methoxyvinyl)-3-methyl-1H-indol-7-yl)-4-methylpyridin-2-
amine (200 mg, 92%) as a brown oil. LCMS 340.0 [M + H]+.
(methoxymethyl)triphenylphosphonium chloride (349 mg, 1.02
mmol) in THF (5 mL) was added n-BuLi (0.36 mL, 0.892 mmol,
2.5 M in n-hexane) at 0 C. The mixture was stirred at 0 oC for 1
o
h. The crude (methoxymethylene)triphenylphosphorane product
solution was used in the next step without any purification.
A solution of 48a (65 mg, 0.255 mmol) in THF (3 mL) was
added to the crude (methoxymethylene)triphenylphosphorane
solution above, and the reaction mixture was stirred at room
temperature overnight. Water (5 mL) was added and the mixture
was extracted with EtOAc (3 x 3 mL). The organic layer was
dried over Na2SO4 and concentrated under vacuum. The
resulting residue was purified by preparative TLC (10:1
petroleum ether: EtOAc) to give 50a (26 mg, 36%) as a light
brown oil. 1H NMR (300 MHz, CDCl3) δ 9.26 (br s, 1H), 7.13-
7.09 (m, 1H), 6.62-6.56 (m, 1H), 6.24 (d, 1H, J = 8.8 Hz), 5.46
(d, 1H, J = 8.8 Hz), 3.90 (s, 3H), 2.39 (s, 3H).
To a solution of the olefin mixture prepared above (200 mg,
0.59 mmol) in MeOH (5 mL) was added 10% palladium on
carbon (400 mg) and the mixture was stirred under an
atmosphere of hydrogen (balloon) at room temperature overnight.
The reaction mixture was filtered and the filtrate was
concentrated under vacuum. The crude product was purified by
preparative HPLC (column: Phenomenex Gemini 5 m C18 150
× 21.2 mm; injection volume: 4 mL; flow rate: 20 mL/min;
wavelength: 214 nm and 254 nm; elution: linear gradient of
70:30 CH3CN:water (0.05% v/v NH4OH) to 90:10 CH3CN:water
(0.05% v/v NH4OH) over 10 min) to give the desired product as
the free amine. The free amine was dissolved in MeOH, treated
with concentrated HCl, and lyophilized to give 27 (37 mg, 19%)
To a solution of 50a (150 mg, 0.53 mmol), 47b (171 mg, 0.64
mmol) and Cs2CO3 (346 mg, 1.06 mmol) in dioxane (10 mL) and
water
tetrakis(triphenylphosphine)palladium(0) (61 mg, 0.053 mmol)
under argon. The mixture was stirred at 100 C under argon
overnight, then cooled to room temperature. Water (10 mL) was
added and the mixture was extracted with EtOAc (3 x 5 mL).
The combined organic layers were dried over Na2SO4 and
concentrated under vacuum. The crude product was purified by
preparative TLC, eluting with 10:1 CH2Cl2:MeOH, to give the
desired (E)- and (Z)-6-ethyl-5-(4-fluoro-2-(2-methoxyvinyl)-3-
methyl-1H-indol-7-yl)-4-methylpyridin-2-amine (58 mg, 32%) as
a light brown oil. LCMS 340.1 [M + H]+.
(2
mL)
was
added
1
as the HCl salt as a white solid. H NMR (400 MHz, CD3OD) δ
o
6.83 (s, 1H), 6.78-6.75 (m, 2H), 3.58 (t, 2H, J = 8.0 Hz), 3.33 (s,
3H), 2.94 (t, 2H, J = 8.0 Hz), 2.51-2.40 (m, 5H), 2.01 (s, 3H),
1.09 (t, 3H, J = 8.0 Hz). LCMS 342.1 [M + H]+.
5.18. 6-Ethyl-5-(5-fluoro-2-(2-methoxyethyl)-3-methyl-1H-indol-
7-yl)-4-methylpyridin-2-amine (28).
(48b). Phosphorus oxychloride (0.6 mL, 6.6 mmol) was
added to DMF (0.5 mL, 6.6 mmol) at 0 oC, followed by a
solution of 46b (500 mg, 2.2 mmol) in 1,2-dichloroethane (8
mL). The cooling bath was removed, and the reaction mixture
was heated at reflux overnight, followed by cooling to room
temperature. Sodium hydroxide solution was added until a pH 8-
9 was reached. The reaction mixture was extracted with CH2Cl2
(3 x 20 mL), and the combined organic layers were dried over
Na2SO4 and concentrated under vacuum. The crude product was
purified by silica gel column chromatography, eluting with 10:1
petroleum ether: EtOAc, to give 48b (70 mg, 13%) as a yellow
To a solution of the olefin mixture above (58 mg, 0.17 mmol)
in MeOH (3 mL) was added 10% palladium on carbon (58 mg),
and the mixture was stirred under an atmosphere of hydrogen
(balloon) at room temperature overnight, filtered and
concentrated under vacuum. The crude product was purified by
preparative HPLC (column: Phenomenex Gemini 5 m C18 150
× 21.2 mm; injection volume: 4 mL; flow rate: 20 mL/min;
wavelength: 214 nm and 254 nm; elution: linear gradient of
70:30 CH3CN:water (0.05% v/v NH4OH) to 75:25 CH3CN:water
(0.05% v/v NH4OH) over 10 min) to give 27 (32 mg, 55%) as a
white solid. 1H NMR (400 MHz, CD3OD) δ 6.68-6.66 (m, 2H),
6.44 (s, 1H), 3.56 (t, 2H, J = 8.0 Hz), 3.31 (s, 3H), 2.91 (t, 2H, J
= 8.0 Hz), 2.39-2.24 (m, 5H), 1.85 (s, 3H), 0.98 (t, 2H, J = 8.0
Hz). LCMS 342.1 [M + H]+.
1
solid. H NMR (300 MHz, CDCl3) δ 10.05 (s, 1H), 8.78 (s, 1H),
7.38-7.29 (m, 2H), 2.59 (s, 3H).
(50b). To a solution of 48b (400 mg, 1.57 mmol) in THF (5
mL)
was
added
a
solution
of
(methoxymethylene)triphenylphosphorane in THF (5.49 mmol)
(prepared as for 50a), and the reaction mixture was stirred at
room temperature overnight. Water (10 mL) was added and the
mixture was extracted with EtOAc (3 x 5 mL). The combined
organic layers were dried over Na2SO4 and concentrated under
vacuum. The crude product was purified by preparative TLC,
eluting with 10:1 petroleum ether: EtOAc, to give 50b (220 mg,
Alternative synthesis of 27. To a solution of 50a (500 mg,
1.767 mmol), bis(pinacolato)boron (897 mg, 3.534 mmol) and
potassium acetate (346 mg, 3.543 mmol) in DMSO (10 mL) was
added
[1,1’-bis-
(diphenylphosphino)ferrocene]dichloropalladium(II) (130 mg,
1
0.177 mmol) under argon. The reaction mixture was heated to
50%) as a light brown oil. H NMR (300 MHz, CDCl3) δ 9.16 (s,
o
120 C overnight under argon, and cooled to room temperature.
1H), 7.07-7.04 (m, 2H), 6.24 (d, 1H, J = 6.9 Hz), 5.48 (d, 1H, J =
6.6 Hz), 3.90 (s, 3H), 2.21 (d, 3H, J = 1.8 Hz).
Water (100 mL) was added and the mixture was extracted with
EtOAc (3 x 20 mL). The combined organic layers were washed
with brine (2 x 20 mL), dried over Na2SO4 and concentrated
under vacuum. The crude product was purified by silica gel
To a solution of 50b (200 mg, 0.71 mmol), 47b (227 mg, 0.85
mmol) and Cs2CO3 (461 mg, 1.41 mmol) in dioxane (10 mL) and
water
(2
mL)
was
added

tetrakis(triphenylphosphine)palladium(0) (82 mg, 0.071 mmol)
under argon. The reaction mixture was stirred at 100 C under
mmol), and Cs2CO3 (362 mg, 1.11 mmol) in water (1 mL) and
dioxane (5 mL) was stirred at 100 oC overnight under argon. The
reaction mixture was cooled to room temperature, poured into ice
water (15 mL), and extracted with EtOAc (3 x 5 mL). The
combined organic layers were washed with brine, dried over
Na2SO4, and concentrated under vacuum. The crude product was
purified by preparative TLC, eluting with 5:1 petroleum ether:
EtOH, to give the desired (E)- and (Z)-5-(4,5-difluoro-2-(2-
methoxyvinyl)-3-methyl-1H-indol-7-yl)-6-ethyl-4-
o
argon overnight and cooled to room temperature. Water (10 mL)
was added and the mixture was extracted with EtOAc (2 x 10
mL). The combined organic layers were dried over Na2SO4 and
concentrated under vacuum. The crude product was purified by
preparative TLC, eluting with 16:1 CH2Cl2:MeOH, to give the
desired (E)- and (Z)-6-ethyl-5-(5-fluoro-2-(2-methoxyvinyl)-3-
methyl-1H-indol-7-yl)-4-methylpyridin-2-amine (70 mg, 29%) as
a brown oil. LCMS: 340.1 [M + H]+.
methylpyridin-2-amine (78 mg, 59%). LCMS 358 [M + H]+.
To a solution of the olefin mixture above (70 mg, 0.21 mmol)
in MeOH (3 mL) was added 10% palladium on carbon (140 mg)
and the mixture was stirred under an atmosphere of hydrogen
(balloon) at room temperature overnight. The catalyst was
filtered and the filtrate was concentrated under vacuum. The
crude product was purified by preparative HPLC (column:
Phenomenex Gemini 5 m C18 150 × 21.2 mm; injection
volume: 4 mL; flow rate: 20 mL/min; wavelength: 214 nm and
254 nm; elution: linear gradient of 70:30 CH3CN:water (0.05%
v/v NH4OH) to 99:1 CH3CN:water (0.05% v/v NH4OH) over 10
min) to give 28 (15 mg, 21%) as a white solid. 1H NMR (400
MHz, CD3OD) δ 7.08 (dd, 1H, J = 10.0 Hz, J = 4.0 Hz), 6.57 (dd,
1H, J = 10.0 Hz, J = 4.0 Hz), 6.45 (s, 1H), 3.57 (t, 2H, J = 8.0
Hz), 3.30 (s, 3H), 2.93 (t, 2H, J = 8.0 Hz), 2.42-2.23 (m, 5H),
1.87 (s, 3H), 1.00 (t, 3H, J = 8.0 Hz). LCMS 342.1 [M + H]+.
A mixture of the olefin prepared above (78 mg, 0.22 mmol)
and 10% palladium on carbon (30 mg) in MeOH (15 mL) was
stirred at room temperature under an atmosphere of hydrogen
(balloon) for 2 h. The reaction mixture was filtered and the
filtrate was concentrated under vacuum. The crude product was
purified by preparative HPLC (column: Phenomenex Gemini 5
m C18 150 × 21.2 mm; injection volume: 4 mL; flow rate: 20
mL/min; wavelength: 214 nm and 254 nm; elution: linear
gradient of 30:70 CH3CN:water (0.1% v/v Et3N) to 50:50
CH3CN:water (0.1% v/v Et3N) over 10 min) to give 29 (12 mg,
15%). 1H NMR (400 MHz, CD3OD) δ 6.63-6.67 (m, 1H), 6.45
(s, 1H), 3.56 (t, 2H, J = 8.0 Hz), 3.31 (s, 3H), 2.91 (t, 2H, J = 8.0
Hz), 2.22-2.29 (m, 5H), 1.87 (s, 3H), 1.00 (t, 3H, J = 8.0 Hz).
LCMS 324.3 [M + H]+.
5.20. 6-Ethyl-5-(2-(3-methoxypropyl)-3-methyl-1H-indol-7-yl)-4-
methylpyridin-2-amine (30).
5.19. 5-(4,5-Difluoro-2-(2-methoxyethyl)-3-methyl-1H-indol-7-
yl)-6-ethyl-4-methylpyridin-2-amine (29).
(49). Iodine (279 mg, 1.1 mmol) was added in one portion to
a solution of 46d (209 mg, 1 mmol) in dry THF (10 mL) at -78
oC. After 3 min, silver trifluoromethanesulfonate (283 mg, 1.1
mmol) was added in one portion. The reaction mixture was
(48c). Phosphorus oxychloride (1.84 g, 12 mmol) was added
dropwise into DMF (876 mg, 12 mmol) at 0 oC under an
atmosphere of nitrogen. The reaction mixture was stirred at 0 oC
for 1 h, followed by the addition of a solution of 46c (984 mg, 4
mmol) in 1,2-dichloroethane (20 mL). The reaction mixture was
heated at reflux for 4 h, cooled to room temperature, and treated
with saturated aqueous sodium acetate (20 mL). The reaction
mixture was heated at reflux for an additional 4 h, cooled to room
temperature, and water (20 mL) was added. The mixture was
extracted with CH2Cl2 (3 x 50 mL). The combined organic layers
were washed with brine, dried over Na2SO4, and concentrated
under vacuum. The crude product was purified by silica gel
column chromatography, eluting with a gradient of 30:1 to 20:1
petroleum ether:EtOAc, to give 48c (234 mg, 21%). 1H NMR
(300 MHz, CDCl3) δ 10.03 (br s, 1H), 8.80 (br s, 1H), 7.36-7.42
(m, 1H), 2.75 (s, 3H).
o
stirred at -78 C for 3 h and NaHCO3 (185 mg, 2.2 mmol) was
added. The reaction mixture was allowed to warm to room
temperature. After 30 min, the mixture was filtered through
Celite and rinsed with EtOAc. To the filtrate was added a
mixture of saturated aqueous sodium thiosulfate solution and
saturated aqueous NaHCO3 solution (1:1, 30 mL). The organic
layer was separated and dried, and then concentrated and purified
to give 49 (200 mg, 60%) as a brown oil. 1H NMR (300 MHz,
CDCl3) δ 8.08 (s, 1H), 7.43 (d, 1H, J = 9.0 Hz), 7.28 (s, 1H), 7.00
- 6.97 (m, 1H), 7.27 (s, 3H). LCMS 335 [M + H]+.
(51). A mixture of 49 (200 mg, 0.6 mmol), copper (I) iodide
(23 mg, 0.12 mmol) and bis(triphenylphosphine)palladium(II)
dichloride (42 mg, 0.06 mmol) in DMF/triethylamine (1:1, 2 mL)
under argon was heated to 50 oC, and then 3-methoxyprop-1-yne
(0.05 mL, 0.5 mmol) was added. The mixture was stirred
overnight at 50 oC under argon and cooled to room temperature.
The mixture was treated with 1% aqueous HCl and extracted
with EtOAc. The combined organic layers were dried and
purified to give 51 (71 mg, 43%) as a pale yellow solid. 1H
NMR (300 MHz, DMSO) δ 11.55 (s, 1H), 7.50 (d, 1H, J = 6.0
Hz,), 7.36-7.34 (m, 1H), 6.98-6.93 (m, 1H), 4.42 (s, 2H), 3.37 (s,
3H), 2.29 (s, 3H). LCMS 278 [M + H]+.
(50c).
To
a
suspension
of
(methoxymethyl)triphenylphosphonium chloride (879 mg, 2.56
mmol) in THF (5 mL) was added n-butyllithium (1.16 mL, 2.2M
o
in hexane, 2.56 mmol) at 0 C under nitrogen. The reaction
mixture was stirred at 0 oC for 1 h, and a solution of 48c (234 mg,
1 mmol) in THF (5 mL) was added quickly. The reaction
mixture was allowed to stir at room temperature overnight,
followed by the addition of saturated aqueous NH4Cl (20 mL).
The mixture was extracted with EtOAc (3 x 10 mL). The
combined organic layers were washed with brine, dried over
anhydrous Na2SO4 and concentrated under vacuum. The crude
product was purified by silica gel column chromatography,
eluting with a gradient of 100:0 to 5:95 petroleum ether:EtOAc,
to give 50c (64 mg, 21%) as a white solid. 1H NMR (300 MHz,
CDCl3) δ 9.18 (brs, 1H), 7.05-7.10 (m, 1H), 6.26 (d, 1H, J = 9.0
Hz), 5.45 (d, 1H, J = 9.0 Hz), 3.90 (s, 3H), 2.37 (s, 3H). The
reaction was repeated with 95 mg of 48c to provide an additional
27 mg (25%) of 50c.
A mixture of 51 (111 mg, 0.4 mmol), 47b (126 mg, 0.48
mmol), tetrakis(triphenylphosphine)palladium(0) (23 mg, 0.02
mmol) and Cs2CO3 (326 mg, 1.0 mmol) in dioxane (5 mL) and
o
water (1 mL) was stirred at 100 C overnight under argon. The
reaction mixture was cooled to room temperature and filtered.
The filtrate was concentrated under vacuum and purified by
preparative HPLC (column: Phenomenex Gemini 5 m C18 150
× 21.2 mm; injection volume: 4 mL; flow rate: 20 mL/min;
wavelength: 214 nm and 254 nm; elution: linear gradient of
25:75 CH3CN:water (0.1% v/v TFA) to 50:50 CH3CN:water
(0.5% v/v NH4OH) over 10 min) to give the desired 6-ethyl-5-(2-
A mixture of 50c (91 mg, 0.37 mmol), 47b (117 mg, 0.44
mmol), tetrakis(triphenylphosphine)palladium(0) (118 mg, 0.11

(3-methoxyprop-1-yn-1-yl)-3-methyl-1H-indol-7-yl)-4-
methylpyridin-2-amine (26 mg, 20%) as a white solid. 1H NMR
(300 MHz, CD3OD) δ 7.59 (d, 1H, J = 9.0 Hz), 7.12-7.07 (m,
1H), 6.88 (d, 1H, J = 9.0 Hz), 6.43 (s, 1H), 4.37 (s, 2H), 3.43 (s,
3H), 2.38 (s, 3H), 2.35-2.19 (m, 2H), 1.84 (s, 3H), 0.99 - 0.94 (m,
3H). LCMS 334 [M +H] +. The reaction was repeated on the
same scale to provide an additional 25 mg of 6-ethyl-5-(2-(3-
methoxyprop-1-yn-1-yl)-3-methyl-1H-indol-7-yl)-4-
mL) was stirred at room temperature under an atmosphere of
hydrogen (balloon) for 2 h. The reaction mixture was filtered
and concentrated under vacuum. The crude product was purified
by preparative HPLC (column: Phenomenex Gemini 5 m C18
150 × 21.2 mm; injection volume: 4 mL; flow rate: 20 mL/min;
wavelength: 214 nm and 254 nm; elution: linear gradient of
30:70 CH3CN:water (0.1% v/v TFA) to 33:67 CH3CN:water
(0.1% v/v TFA) over 10 min) to give 31 (6 mg, 6%) as the TFA
salt. 1H NMR (400 MHz, CD3OD) δ 7.52 (d, 1H, J = 8.0 Hz),
7.12 (t, 1H, J = 8.0 Hz), 6.86 (d, 2H, J = 8.0 Hz), 3.00 - 2.98 (m,
2H), 2.68 (s, 3H), 2.49 - 2.42 (m, 2H), 2.28 (s, 3H), 2.01 (s, 3H),
1.08 (t, 3H, J = 8.0 Hz). LCMS 351.2 [M + H]+.
methylpyridin-2-amine.
A mixture of the alkyne prepared above (35 mg, 0.1 mmol)
and 10% palladium on carbon (10 mg) in MeOH (2 mL) was
stirred for 4 h at room temperature under an atmosphere of
hydrogen (balloon). The reaction mixture was filtered and the
filtrate was concentrated under vacuum. The crude product was
purified by preparative HPLC (column: Phenomenex Gemini 5
m C18 150 × 21.2 mm; injection volume: 4 mL; flow rate: 20
mL/min; wavelength: 214 nm and 254 nm; elution: linear
gradient of 25:75 CH3CN:water (0.1% v/v TFA) to 50:50
CH3CN:water (0.5% v/v NH4OH) over 10 min) to give 30 (7 mg,
17%) as a white solid. 1H NMR (400 MHz, DMSO) δ 10.16 (s,
1H), 7.32 (d, 1H, J = 4.0 Hz), 6.99-6.95 (m, 1H), 6.68 (d, 1H, J =
8.0 Hz), 6.25 (s, 1H), 5.71 (s, 2H), 3.27-3.24 (m, 2H), 3.20 (s,
3H), 2.67-2.63 (m, 2H), 2.23-2.10 (m, 5H), 1.79-1.73 (m, 5H),
0.93-0.89 (m, 3H). LCMS 338 [M +H]+.
5.22. N-(2-(6-(6-Amino-2-ethyl-4-methylpyridin-3-yl)-2,2-
dimethyl-3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-
yl)ethyl)acetamide (32).
A mixture of 54b (448 mg, 1.15 mmol), 41b (248 mg, 1.15
mmol), tetrakis(triphenylphosphine)palladium(0) (133 mg, 0.115
mmol), cesium hydroxide monohydrate (579 mg, 3.45 mmol) and
lithium chloride (146 mg, 3.45 mmol) in a mixture of dioxane
o
(18 mL) and water (3 mL) was stirred at 100 C for 12 h under
argon. The reaction mixture was cooled to room temperature and
concentrated under vacuum to give 1.1 g of a crude product
mixture. The crude residue was purified by preparative HPLC
(column: Phenomenex Gemini 5 m C18 150 × 21.2 mm;
injection volume: 5 mL; flow rate: 20 mL/min; wavelength: 214
nm and 254 nm; elution: linear gradient of 30:70 CH3CN:water
(0.1% v/v TFA) to 70:30 CH3CN:water (0.1% v/v TFA) over 10
min) to give the desired product as the free base. The free base
was dissolved in concentrated HCl (10 mL) and lyophilized to
5.21. 3-(7-(6-Amino-2-ethyl-4-methylpyridin-3-yl)-3-methyl-1H-
indol-2-yl)-N-methylpropanamide (31).
(50e).
To
a
suspension of (2-(methylamino)-2-
oxoethyl)triphenylphosphonium bromide (828 mg, 2.0 mmol)
(prepared from heating 2-bromo-N-methylacetamide (1 eq) and
triphenylphosphine (1 eq) in toluene at reflux overnight, followed
by cooling and filtration to give the desired solid salt) in dry THF
1
give 32 (120 mg, 26%) as the HCl salt as a white solid. H NMR
(400 MHz, CD3OD) δ 7.28 (s, 1 H), 7.13 (d, 1H, J = 8.1 Hz),
6.90 (dd, 1H, J = 1.8, 8.1 Hz), 6.77 (s, 1H), 4.04 (t, 2H, J = 7.2
Hz), 3.34 (t, 2H, J = 7.2 Hz), 2.58 (q, 2H, J = 7.5 Hz), 2.15 (s,
3H), 1.90 (s, 3H), 1.52 (s, 6H), 1.18 (t, 3H, J = 7.5 Hz). LCMS
397 [M + H]+.
o
(5 mL) at -78 C under nitrogen was added n-butyllithium (0.91
mL, 2.2M in hexane, 2.0 mmol). The reaction mixture was
allowed to warm to room temperature and stir for 1 h, followed
by the rapid addition of a solution of 48d (237 mg, 1 mmol) in
THF (5 mL). The reaction mixture was stirred at room
temperature overnight, quenched with saturated aqueous NH4Cl
(10 mL), and extracted with EtOAc (3 x 5 mL). The combined
organic phases were washed with brine, dried over Na2SO4, and
concentrated under vacuum. The crude product was purified by
silica gel column chromatography, eluting with a gradient of 15:1
to 5:1 petroleum ether:EtOAc, to give 50e (93 mg, 32%) as a
mixture of isomers. A small sample of 50e (10 mg, mixture of
isomers) was purified by preparative TLC, eluting with 3:1
petroleum ether:EtOAc, to afford the E isomer exclusively: 1H
NMR (300 MHz, CDCl3) δ 12.96 (br s, 1H), 7.49 (d, 1H, J = 9.0
Hz), 7.38 (d, 1H, J = 9.0 Hz), 6.93 (t, 1H, J = 9.0 Hz), 6.85 (d,
1H, J = 15.0 Hz), 5.75 (br s, 1H), 6.64 (d, 1H, J = 15.0 Hz), 2.98
(d, 1H, J = 6.0 Hz), 2.40 (s, 3H).
5.23. N-(2-(6-(6-Amino-2-ethyl-4-methoxypyridin-3-yl)-2,2-
dimethyl-3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-
yl)ethyl)acetamide (33).
A mixture of 54b (224 mg, 0.58 mmol), 41d (134 mg, 0.58
mmol), tetrakis(triphenylphosphine)palladium(0) (67 mg, 0.058
mmol), cesium hydroxide monohydrate (292 mg, 1.74 mmol) and
lithium chloride (74 mg, 1.74 mmol) in dioxane (12 mL) and
o
water (1.5 mL) was stirred at 100 C for 12 h under argon. The
reaction mixture was cooled to room temperature and
concentrated under vacuum to give 80 mg of a crude product
mixture. The crude residue was purified by preparative HPLC
(column: Phenomenex Gemini 5 m C18 150 × 21.2 mm;
injection volume: 5 mL; flow rate: 20 mL/min; wavelength: 214
nm and 254 nm; elution: linear gradient of 30:70 CH3CN:water
(0.1% v/v TFA) to 40:60 CH3CN:water (0.1% v/v TFA) over 10
min) to give the desired product as the free base. The free base
was dissolved in 2N aqueous HCl (2 mL) and lyophilized to give
33 (40 mg, 17%) as the HCl salt as a white solid. 1H NMR (400
MHz, CD3OD) δ 7.24 (s, 1 H), 7.04 (d, 1H, J = 8.1 Hz), 6.94 (dd,
1H, J = 1.8, 8.1 Hz), 6.37 (s, 1H), 4.02 (t, 2H, J = 7.2 Hz), 3.89
(s, 3H), 3.38 (m, 2H), 2.59 (q, 2H, J = 7.5 Hz), 1.88 (s, 3H), 1.52
(s, 6H), 1.20 (t, 3H, J = 7.5 Hz). LCMS 413 [M + H]+.
A mixture of 50e (93 mg, 0.32 mmol), 47b (125 mg, 0.38
mmol), tetrakis(triphenylphosphine)palladium(0) (37 mg, 0.03
mmol), and Cs2CO3 (311 mg, 0.95 mmol) in dioxane (5 mL) and
o
water (1 mL) was stirred at 100 C overnight under argon. The
reaction mixture was cooled to room temperature, poured into ice
water (10 mL), and extracted with EtOAc (3 x 5 mL). The
combined organic phases were washed with brine, dried over
Na2SO4, and concentrated under vacuum. The crude product was
purified by preparative TLC, eluting with 3:1 petroleum
ether:EtOH, to give the desired (E)- and (Z)-3-(7-(6-amino-2-
ethyl-4-methylpyridin-3-yl)-3-methyl-1H-indol-2-yl)-N-
5.24. 6-(6-Amino-2-ethyl-4-methylpyridin-3-yl)-4-(3-
methoxypropyl)-2,2-dimethyl-2H-benzo[b][1,4]oxazin-3(4H)-one
(34).
methylacrylamide (73 mg, 66%). LCMS 349.2 [M + H]+.
A mixture of 54a (140 mg, 0.37 mmol), 41b (66.7 mg, 0.31
mmol), tetrakis(triphenylphosphine)palladium(0) (36 mg, 0.03
mmol), cesium hydroxide monohydrate (156 mg, 0.93 mmol) and
A mixture of the olefins prepared above (73 mg, 0.21 mmol)
and a catalytic amount of 10% palladium on carbon in MeOH (5

lithium chloride (39.4 mg, 0.93 mmol) in dioxane (10 mL) and
water (2 mL) was stirred at 100 C for 12 h under argon. The
instructions by BPS Bioscience (San Diego, CA). Final
concentration of DMSO was 1% in all assays. Fluorescence
intensity was measured using a Tecan Infinite M1000 microplate
reader. Data analysis and curve fitting was performed using
GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). All
IC50 assays included a concentration response for commercially
obtained aliskiren hydrochloride (standard) included on the same
assay plate.
o
reaction mixture was cooled to room temperature and
concentrated under vacuum to give 100 mg of a crude product
mixture. The crude residue was purified by preparative HPLC
(column: Phenomenex Gemini 5 m C18 150 × 21.2 mm;
injection volume: 5 mL; flow rate: 20 mL/min; wavelength: 214
nm and 254 nm; elution: linear gradient of 25:75 CH3CN:water
(0.1% v/v TFA) to 45:55 CH3CN:water (0.1% v/v TFA) over 10
min) to give 34 (30 mg, 25%) as the TFA salt as a yellow oil. 1H
NMR (400 MHz, CD3OD) δ 7.12 (s, 1 H), 7.10 (d, 1H, J = 8.4
Hz), 6.89 (dd, 1H, J1 = 8.4 Hz, J2 = 2.0 Hz), 6.80 (s, 1H), 4.04 (t,
2H, J = 5.1 Hz), 3.33 (t, 2H, J = 1.2 Hz), 3.32 (s, 3H), 2.57 (q,
2H, J = 8.4 Hz), 2.13 (s, 3H), 1.88 (t, 2H, J = 2.8 Hz), 1.51 (s,
6H), 1.19 (t, 3H, J = 7.2 Hz). LCMS 384 [M + H]+.
5.27. Rat In Vivo Pharmacokinetic Studies.
All studies were performed by BioDuro, LLC under IACUC-
approved protocols using 8 week-old jugular-cannulated Sprague
Dawley rats weighing between 250 and 350 g.
Single
intravenous bolus doses were administered via jugular cannula,
and single oral doses were administered via gavage. Blood
samples were collected for analysis at 2 min (iv only), 5 min, 15
min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h after dosing. Plasma
concentrations were determined by LC/MS/MS after
precipitation with acetonitrile. Test compounds (as their HCl
salts) were formulated in an aqueous 5% glucose solution (D5W)
at a concentration of 1 mg/mL, and dosed at 1 mg/kg body
weight for the intravenous study arms and 10 mg/kg body weight
for the oral study arms. Data are reported as the mean (n = 3).
5.25. Methyl (2-(6-(6-amino-2-ethyl-4-methylpyridin-3-yl)-2,2-
dimethyl-3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-
yl)ethyl)carbamate (35).
(56). A mixture of 55 (prepared according to Powell, et al.18b
)
(200 mg, 0.78 mmol) and sodium hydride (37.5 mg, 60 wt %,
o
0.94 mmol) in DMF (10 mL) was stirred at 0 C for 15 min,
followed by the addition of 15-crown-5 (0.1 mL) and methyl (2-
bromoethyl)carbamate (171 mg, 0.94 mmol). The reaction
mixture was warmed to room temperature, stirred for 12 h,
poured into water (50 mL), and extracted with EtOAc (2 x 100
mL). The combined organic layers were dried over anhydrous
Na2SO4 and concentrated under vacuum. The resulting residue
was purified by silica gel column chromatography, eluting with
2:1 petroleum ether: EtOAc, to give 56 (0.22 g, 79%) as a white
solid. LCMS 357, 359 [M + H]+.
Acknowledgments
Part of the research reported in this publication was supported by
National Institute of General Medical Sciences (NIGMS) of the
National Institutes of Health under award number
R43GM109549. We thank Qiang Wang for assisting with the
SACP computations.
A mixture of 56 (0.2 g, 0.56 mmol), bis(pinacolato)boron
Supplementary Material
(0.17
g,
0.67
mmol),
[1,1’-bis-
(diphenylphosphino)ferrocene]dichloropalladium(II) (46 mg,
0.056 mmol) and potassium acetate (165 mg, 1.68 mmol) in
dioxane (10 mL) was stirred under argon at 85 C for 12 h. The
List of fragments used in SACP; correlation plot of the validation
set in 2G1Y; torsion angle analysis of representative
heterocycles.
o
resulting mixture was cooled to room temperature and
concentrated under vacuum. The resulting residue was purified
by silica gel column chromatography, eluting with 2:1 petroleum
ether: EtOAc to give the desired boronic ester (0.18 g, 80%) as a
white solid. LCMS 405 [M + H] +. A mixture of the boronic
ester (100 mg, 0.25 mmol), 41b (54 mg, 0.25 mmol), [1,1’-bis-
(diphenylphosphino)ferrocene]dichloropalladium(II) (20 mg,
0.025 mmol), and potassium phosphate tribasic heptahydrate
(253 mg, 0.75 mmol) in toluene (10 mL) was stirred at 100 oC for
12 h under argon. The reaction mixture was cooled to room
temperature and concentrated under vacuum to give 280 mg of a
crude product mixture. The crude residue was purified by
preparative HPLC (column: Phenomenex Gemini 5 m C18 150
× 21.2 mm; injection volume: 5 mL; flow rate: 20 mL/min;
wavelength: 214 nm and 254 nm; elution: linear gradient of
30:70 CH3CN:water (0.1% v/v TFA) to 80:20 CH3CN:water
(0.1% v/v TFA) over 10 min) to give 35 (35 mg, 34%) as a TFA
salt as a white solid. 1H NMR (400 MHz, CD3OD) δ 7.15 (s, 1
H), 7.08 (d, 1H, J = 8.4 Hz), 6.89 (dd, 1H, J = 2.0, 8.4 Hz), 6.79
(s, 1H), 4.02 (t, 2H, J = 2.8 Hz), 3.54 (s, 3H), 3.31 (t, 2H, J = 1.6
Hz), 2.54 (q, 2H, J = 7.6 Hz), 2.12 (s, 3H), 1.50 (s, 6H), 1.18 (t,
3H, J = 7.6 Hz). LCMS 413 [M + H]+.
Abbreviations
SACP, simulated annealing of chemical potential; CFA,
constrained fragment analysis; FE Pred, predicted relative free
energy; Gs, relative solvation change; D5W, 5% dextrose
(glucose) in water; Vdss, steady-state volume of distribution; Cl,
clearance; Cmax, maximum concentration.
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