Discovery of 6-(Fluoro-18F)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine ([18F]-MK-6240): A Positron Emission Tomography (PET) Imaging Agent for Quantification of Neurofibrillary Tangles (NFTs)

Article abstract of DOI:10.1021/acs.jmedchem.6b00166

Neurofibrillary tangles (NFTs) made up of aggregated tau protein have been identified as the pathologic hallmark of several neurodegenerative diseases including Alzheimer's disease. In vivo detection of NFTs using PET imaging represents a unique opportuni

Full text of DOI:10.1021/acs.jmedchem.6b00166

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Article
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Discovery of 6-(fluoro-F)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-
18
amine ([F]-MK-6240): A Positron Emission Tomography (PET)
Imaging Agent for Quantification of Neurofibrillary Tangles (NFTs)
Abbas M. Walji, Eric D. Hostetler, Harold Selnick, Zhizhen Zeng, Patricia Miller, Idriss Bennacef,
Cristian Salinas, Brett Connolly, Liza Gantert, Marie Holahan, Stacey O'Malley, Mona Purcell,
Kerry Riffel, Jing Li, Jaume Balsells, Julie A. OBrien, Stacey Melquist, Aileen Soriano,
Xiaoping Zhang, Aimie Ogawa, Serena Xu, Elizabeth M Joshi, Joseph Della Rocca, Fred
J. Hess, Joel Schachter, David Hesk, David Schenk, Arie Struyk, Kerim Babaoglu, Talakad
G Lohith, Yaode Wang, Kun Yang, Jianmin Fu, Jeffrey L. Evelhoch, and Paul J Coleman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00166 • Publication Date (Web): 18 Apr 2016
Downloaded from http://pubs.acs.org on April 19, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Published by American Chemical Society. Copyright © American Chemical Society.
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Discovery of 6-(fluoro-18F)-3-(1H-pyrrolo[2,3-
c]pyridin-1-yl)isoquinolin-5-amine ([18F]-MK-
6240): A Positron Emission Tomography (PET)
Imaging Agent for Quantification of
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Neurofibrillary Tangles (NFTs)
Abbas M. Walji*[a], Eric D. Hostetler[b], Harold Selnick[a], Zhizhen Zeng[b], Patricia Miller[b],
Idriss Bennacef[b], Cristian Salinas[b], Brett Connolly[b], Liza Gantert[b], Marie Holahan[b],
Stacey O’Malley[b], Mona Purcell[b], Kerry Riffel[b], Jing Li[c], Jaume Balsells[c], Julie A.
OBrien[d], Stacey Melquist[d], Aileen Soriano[e], Xiaoping Zhang[e], Aimie Ogawa[e], Serena
Xu[e], Elizabeth Joshi[f], Joseph Della Rocca[g], Fred J. Hess[h], Joel Schachter[h], David Hesk[i],
David Schenk[i], Arie Struyk[j], Kerim Babaoglu[k], Talakad G. Lohith[b], Yaode Wang[l], Kun
Yang[l], Jianmin Fu[l], Jeffrey L. Evelhoch[b], Paul J. Coleman[a].
[a]Discovery Chemistry, Merck & Co., Inc, West Point PA, USA; [b]Imaging, Merck & Co., Inc,
West Point PA, USA; [c]Process Chemistry, Merck & Co., Inc, West Point PA, USA;
[d]Pharmacology, Merck & Co., Inc, West Point NJ, USA; [e]Pharmacology, Merck & Co., Inc,
Kenilworth NJ, USA; [f]Drug Metabolism, Merck & Co., Inc, West Point PA, USA; [g]Discovery
Pharmaceutical Sciences, Merck & Co., Inc, West Point PA, USA; [h]Neuroscience, Merck &
[i]
Co., Inc, West Point PA, USA; Labelled Compound Synthesis, Merck & Co., Inc, West Point
[j]
PA, USA; Clinical Pharmacology, Merck & Co., Inc, West Point PA, USA; [k]Computational
Chemistry, Merck & Co., Inc, West Point PA, USA; [l]Pharmaron Beijing Co., Ltd, Beijing
100176, China;
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ABSTRACT
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Neurofibrillary tangles (NFTs) made up of aggregated tau protein have been identified as the
pathologic hallmark of several neurodegenerative diseases including Alzheimer’s disease. In
vivo detection of NFTs using PET imaging represents a unique opportunity to develop a
pharmacodynamic tool to accelerate discovery of new disease modifying therapeutics targeting
tau pathology. Herein we present the discovery of 6ꢀ(fluoroꢀ18F)ꢀ3ꢀ(1Hꢀpyrrolo[2,3ꢀc]pyridinꢀ1ꢀ
yl)isoquinolinꢀ5ꢀamine, 6 ([18F]ꢀMKꢀ6240), as a novel PET tracer for detecting NFTs. 6 exhibits
high specificity and selectivity for binding to NFTs, with suitable physicochemical properties
and in vivo pharmacokinetics.
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INTRODUCTION
The search for disease relevant biomarkers for neurodegenerative diseases such as
Alzheimer’s disease (AD), has received substantial attention within the scientific community
over the past decade.1ꢀ5 The main focus in recent clinical trials has been to use cognitive
endpoints to measure the effect of therapeutic agents. This requires studying a large number of
subjects over long time periods to detect robust changes in these standardized tests and has
resulted in an unsustainable financial burden for evaluation of novel disease modifying
therapeutics with limited benefits to patients. Therefore, there is an unmet need for sensitive
biomarkers that quantify early pathological changes and correlate closely to clinical phenotypes.1
To satisfy the key criteria for disease relevance the biomarker must be extant at early stages and
importantly, show good correlation with the progression of symptoms and signs of the disease.4
Once validated, clinical applications of the biomarker would go beyond patient selection since
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measuring pathological changes would provide definitive evidence of therapeutic responses and
more confidence for acceleration into larger patient populations.
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Within the field of ADꢀrelevant pharmacodynamic biomarkers, earlier research efforts were
directed towards measuring human βꢀamyloid deposition in vivo, which culminated in the
discoveries of several βꢀamyloid positron emission tomography (PET) imaging agents.6ꢀ14 Based
on this work βꢀamyloid PET imaging is recognized as a viable imaging platform for the
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detection of βꢀamyloid pathology.11 However, the recognition that deposited βꢀamyloid does not
correlate to disease progression has rekindled the search for a more relevant biomarker.15 In
contrast to amyloid plaques, neurofibrillary tangles (NFTs) comprised of aggregated tau proteins
have shown to better correlate with cognitive decline based on evaluation of Alzheimer’s disease
autopsy cases.16ꢀ20 Importantly, the density and spread of NFTs within different brain regions
has been successfully used to stage disease severity with higher consistency.21ꢀ23 These data
strongly supports the quantification of tau pathology as a disease relevant biomarker for inꢀlife
diagnosis of AD, more appropriate staging of disease states, and measuring the effect of diseaseꢀ
modifying therapeutics targeting tau pathology.
More recent research efforts have been directed towards extending the success of PET
imaging for the in vivo quantification of NFTs.24ꢀ30 The challenges of discovering PET imaging
agents for NFTs have been recognized and are subjects of recent reviews.24, 26, 28 Several
discovery efforts have translated into the selection of novel small molecule clinical candidates
with high binding affinity to NFTs and selectivity over βꢀamyloid. From these efforts 1 ([18F]ꢀ
AVꢀ145131ꢀ34 a.k.a. [18F]ꢀT80733, 34) and 2 ([18F]ꢀT808)35 were identified as promising early
agents, and were followed by the discoveries of 3 ([18F]ꢀTHKꢀ5117),36ꢀ38 4 ([18F]ꢀTHKꢀ5351),39
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5 ([11C]ꢀPBB3),40, 41 which have been evaluated in early clinical studies (Scheme 1). Currently, 1
has been most extensively studied in AD patients. Although 1 shows excellent selectivity over
βꢀamyloid and has the requisite properties for optimal cell permeability and brain penetration,
the signal to background ratio in early AD patients may be smaller than desired.33 Furthermore,
recent reports have suggested 1 shows widespread, highꢀaffinity offꢀtarget binding.42ꢀ44 We
believe this will have a significant impact on accurately measuring NFT burden in early disease
states and confound interpretation of changes in the longitudinal quantification of NFTs. These
limitations are attributed to subꢀoptimal affinity for NFTs and lower selectivity over other brain
targets, both of which can be addressed with further optimization of the imaging agent.
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Development of an optimal NFT PET tracer as a pharmacodynamic tool for preclinical and
clincial research represents a strategic advantage in the development of diseaseꢀmodifying
therapeutics targeting tau pathology. With the objective of identifying a more sensitive and
selective NFT PET tracer we initiated an internal PET tracer discovery program. Herein, we
describe our research efforts that culminated in the discovery of 6, an optimized PET imaging
agent exhibiting superior in vitro characteristics for NFT binding and clean offꢀtarget profile
with suitable physicochemical properties and pharmacokinetics in rhesus monkeys.
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Scheme 1. Chemical structures of NFT PET tracers: 1, 2, 3, 4, 5, 6 and βꢀamyloid PET tracer 7.
RESULTS and DISCUSSION
The key elements for the identification of an NFT PET tracer were based on achieving high
affinity for NFTs and exquisite levels of selectivity over binding to βꢀamyloid. These were
driven by iterative design cycles to establish the SAR with a strong emphasis on improving
physicochemical properties to reduce nonꢀspecific binding.45 This was achieved using predictive
tools such as lipophilic ligand efficiency (LLE) and measuring the lipophilicity using the shake
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flask method (sfLogD).46,
To further prioritize compound selection for optimal brain
penetration we used Pfizer’s CNSꢀMPO tool and measured Pꢀgp efflux and permeability.48
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Central to the routine clinical application of a PET tracer for NFTs is the limitation around the
use of an [11C] tracer due to the short halfꢀlife (~20 min) of carbonꢀ11 requiring an onꢀsite
cyclotron. Therefore, the chemistry strategy focused on establishing the molecular connectivity,
starting from the minimal pharmacophore, which allowed the incorporation of the 18F isotope
without compromising the desired properties. Finally, the PET candidate needed to demonstrate
no white matter binding in nonhuman primates and no evidence of brainꢀpenetrant metabolites.
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Our discovery efforts began with assessing the most suitable screening strategy to identify
novel chemical matter for lead optimization. First we initiated a competitive binding assay in in
vitro assembled tau filaments using radiolabeled Thioflavin T as the reference radioligand.
However, we quickly recognized a similar discrepancy to that reported by Kolb and coworkers,34
in correlating the observed binding affinity to human AD brain tissue. Based on this data and
our previous experience in the discovery of 7 ([18F]ꢀMKꢀ3328),14, 49 a novel βꢀamyloid PET
ligand, we developed a complimentary competition binding assay for NFTs in human AD cortex
homogenates to characterize newly designed compounds. Although the tau filament based assay
could not be used directly to drive our discovery efforts, we were able to screen the Merck
compound collection and prioritized a novel scaffold for identification of a new reference
radioligand and lead optimization using the AD cortical homogenate assays.
From our lead identification efforts we selected azaꢀindole 8, which was synthesized using
copper catalyzed CꢀN bond formation from commercially available starting materials (8a & 8b)
(Scheme 1). After rhodium mediated tritium labelling, [3H]8 was evaluated as a radioligand in
human AD brain homogenates.50 [3H]8 has a Kd of 0.3 nM for binding to NFTs in human AD
brain homogenates with low levels of nonspecific binding, making it a highly suitable
radioligand for competition binding assays. Furthermore, it exhibited high selectivity (Ki >10
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ꢀM) for binding to βꢀamyloid using [3H]7 as the reference radioligand in human AD brain
homogenates. Based on the in vitro specificity, selectivity, and the excellent physicochemical
properties we dedicated further efforts to investigate this series.
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Scheme 2. Chemical synthesis of 8 and [3H]8.
A thorough SAR analysis of the structural elements revealed that the azaꢀindole core with a
2,4ꢀsubstituted pyridine, represented by compound 9 (Ki = 8.8 nM) (Scheme 3), was the minimal
pharmacophore required for high binding affinity to NFTs within this series. Unfortunately,
introduction of fluorine atoms within this minimal pharmacophore on positions enabled for direct
radiochemical introduction of fluorineꢀ18 resulted in a >10 fold loss in affinity (Scheme 3). This
was the case when either heterocyclic ring was fluorinated, suggesting a specific electronic
contribution to NFT binding from the basic nitrogen atoms. Therefore we shifted our lead
optimization efforts to compound 8 and functionalized the isoquinoline with fluorine atoms on
the 6 and 8 position (compound 10 & 11), which are also activated for nucleophilic aromatic
substitution (SNAr).
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Scheme 3. (A) Identification of minimal pharmacophore for NFT affinity. (B) SAR
exploration of fluorinated analogs of compound 8.
Chemical syntheses of compound 10 & 11 (Schemes 4 & 5) were initiated from the
appropriately substituted indanones which were reacted with isoamylnitrite to form 2ꢀ
(oximino)indanꢀ1ꢀones 10b & 11b. Ring expansion to the 1,3ꢀdichloroisoquinolines was
achieved after reaction with PCl5 in POCl3 followed by treatment with anhydrous HCl.
Regioselective reduction of the chlorine atom in the 3 position with red phosphorous provided
the key coupling partners for palladium catalyzed CꢀN bond formation to give the final products
10 & 11. Both fluorinated isoquinoline isomers exhibited similar high affinity binding to NFTs
using [3H]7, and high selectivity to βꢀamyloid using [3H]7 in competition binding assays using
human AD brain homogenates (Table 1). Compound 10 was prioritized for further in vitro and
in vivo studies based on higher affinity for NFTs than 10 and more robust radiochemistry
starting from the corresponding 6ꢀsubstituted nitro precursor (Scheme 6) to yield [18F]10. [3H]10
was also prepared directly from 10 using the direct tritiation method.50
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Table 1. Physicochemical properties, NFT binding affinity and βꢀamyloid selectivity of
selected compounds 1-6. (nd = not detemined)
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Scheme 4: Chemical Synthesis of compound 10
Scheme 5: Chemical Synthesis of compound 11
Scheme 6: Radiochemical Synthesis of compound [18F]10
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In NFTꢀrich AD brain tissue homogenates, saturation binding studies show [3H]10 has a
high affinity for NFTs (Kd = 1.2 nM) (Table 2) which translates to a large predicted in vivo
signal based on the high Bmax / Kd ratio (~121).51 However, the lipophilicity of compound 10
was higher than desired as measured using the shake flask method (sfLogD = 3.6). To further
investigate the potential impact of the high lipophilicity we performed in vivo studies in rhesus
monkeys. Although these monkeys do not exhibit tau pathology, the PET studies were designed
to inform on the intrinsic physicochemical and pharmacokinetic properties of the molecule.
[18F]10 distributes rapidly across the bloodꢀbrain barrier (BBB) followed by rapid clearance
(Figure 2, panel A). However, the distribution is not homogeneous across all brain regions with
significant retention in white matter (Figure 2, panels A and C). This data was very informative
as it unveiled a significant liability which would reduce the in vivo sensitivity of compound 10,
due to higher background signal directly attributed to suboptimal physicochemical properties.
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With the goal of reducing nonꢀspecific binding of compound 10, we designed several
modifications aimed at modulating the polar surface area to decrease overall lipophilicity. Based
on predicted properties the most promising modifications included: (a) addition of a nitrogen
within the isoquinoline converting to a naphthyridine ring (compound 12) and (b) introduction of
an amine on the isoquinoline ring system (compound 6). Compound 12 was prepared in 7 steps
(Scheme 7) starting from direct nucleophilic aromatic substitution of azaꢀindole 8a with methyl
5ꢀbromoꢀ2ꢀchloroisonicotinate 12a under basic conditions to give the hydrolyzed isonicotinic
acid 12b. The carboxylic acid was subjected to Curtius rearrangement in the presence of tꢀ
BuOH and deprotected to give the triꢀsubstituted pyridine 12d. Heck coupling with ethyl
acrylate followed by intramolecular lactamization and phosphorus oxychloride mediated
chlorination gave the chlorinated napthyridine 12g. Introduction of flouride was achieved under
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standard conditions using potassium flouride to give fluoroꢀnaphthyridine 12. Synthesis of
compound 6 (Scheme 8) was initiated from direct nitration of previously synthesized
isoquoniline 10d. Reduction of the nitro group using iron in acetic acid followed by protection
of the aniline nitrogen gave the key substrate (6d) for coupling with azaꢀindole 8a. Palladium
catalyzed CꢀN bond formation with tꢀBuXPhos followed by deprotection with trifluoroacetic
acid gave the fluoroꢀaniline 6.
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Scheme 7: Chemical synthesis of compound 12.
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Scheme 8: Chemical synthesis of compound 6.
The strategic introduction of nitrogen atoms resulted in reduction of lipophilicity and
predicted overall improvement in the physicochemical properties for both compounds 5 & 6, as
calculated using the CNSꢀMPO (>5.5) scores and measured shake flask log D (2.9ꢀ3.3) (Table
1). Fluoroꢀnaphthyridine 5 presented the potential for improved 18Fꢀradiochemistry via direct
nucleophilic displacement starting from the activated 2ꢀnitroꢀnaphthyridine. However, the
fluoroꢀnaphthyridine 5 exhibited a significant loss in affinity for binding to NFT compared to the
fluoroꢀisoquinoline 3 (>200 fold loss in NFT affinity, Table 1). Interestingly, fluoroꢀaniline 6
maintained a similar in vitro profile to 3 in competition binding assays with [3H]1 for binding to
NFT and against [3H]ꢀ7 for selectivity against βꢀamyloid. Compound 6 was not a substrate for
human or rat Pꢀglycoprotein (Pꢀgp) mediated efflux (BA:AB ~1.3 @ 0.1 ꢀM) in the Pꢀgp
expressing LLCꢀPK1 cell line, and showed high passive permeability in control LLCꢀPK1 cells
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(Papp > 29 x 10ꢀ6cm•sꢀ1). Evaluation of 6 in a counterꢀscreen panel of 118 receptor and enzyme
targets revealed no ancillary activities with IC50 values > 1 ꢀM. Based on this data tritium
labeled [3H]6 was prepared for further characterization in in vitro studies.
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Evaluation of [3H]6 in saturation binding studies in NFTꢀrich AD cortex homogenates
confirmed the high affinity saturable binding to NFTs (Kd = 0.42 nM) with consistent high Bmax
/
Kd ratio (~211) to that observed in studies with [3H]10 (Table 2). To evaluate the selectivity of 6
for binding to NFTs, we used 2 as the blocking agent to represent a structurally distinct and
selective NFT ligand. We observed a similar Bmax / Kd ratio (~238) to that obtained with selfꢀ
block (Table 2), suggesting minimal to no offꢀtarget binding. A lack of binding was also
confirmed in nonꢀAD cortex homogenates under similar conditions (data not shown).
A
comparative study using 2 to define nonꢀdisplaceable binding, revealed that compound [3H]6 had
at least 2 fold higher Bmax / Kd ratio compared to [3H]1. The higher Bmax / Kd ratio suggests
higher sensitivity for compound 6 for binding to NFTs which is proportional to the in vivo signal
from a PET imaging agent.51 Autoradiographic imaging studies with human AD brain slices
were used to qualitatively validate the binding characteristics of 6. [3H]6 showed a strong signal
with high amount of displaceable binding in the grey matter (region of high NFT load) by either
selfꢀblock (Figure 1B) or block with 2 (Figure 1C), and the observed binding pattern matched
closely with immunostaining for NFTs using PHF6 (Figure 1D). Based on this data compound 6
exhibited superior in vitro specificity and selectivity characteristics, and was prioritized for
further studies in vivo.
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Table 2. Bmax and Kd values for [3H]10, [3H]6 and [3H]1 binding in NFTꢀrich AD brain tissues.
Data are determined using same pool of brain homogenates.
Figure 1(A-E). Results from autoradiography studies with [3H]6 and immunohistochemistry in
the frontal cortex (FC) of an NFTꢀrich AD brain donor. A. [3H]6 total binding GM/WM ratio =
5.4; B. [3H]6 + 50 nM (6) GM/WM ratio = 1.2; C. [3H]6 + 500 nM 2 GM/WM ratio = 1.1; D.
PHF6 staining for phosphoꢀtau. E. 6E10 IHC staining for amyloid; GM = grey matter, WM =
white matter;
To enable in vivo evaluation of its physicochemical and pharmacokinetic properties in rhesus
monkey PET studies, [18F]6 was synthesized from the 5ꢀdiBocꢀ6ꢀnitro precursor 6e in 2 synthetic
steps (Scheme 9). Baseline PET studies showed [18F]6 has favorable pharmacokinetic
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properties, with high distribution into the brain followed by rapid clearance (Figure 2, panel B).
Gratifyingly, the effect of improved physicochemical properties was also evident in vivo as no
white matter retention was observed with homogenous distribution across all brain regions since
rhesus monkey do not exhibit tau pathology (Figure 2, panel D). In vitro metabolism studies in
monkey liver microsomes predicted low levels of oxidative defluorination which manifested as
minimal skull uptake in vivo. In contrast, similar studies in human liver microsomes showed
lower levels of oxidative defluorination (data not shown). To ensure minimal impact on
quantifying changes in NFT signal the extent of defluorination will need to be monitored in early
clinical validation studies.
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Boc
N
H2N 18F
Boc
N
NO2
N
K[18F]
N
N
kryptofix, DMF
then 5% TFA
N
N
6e
[
18F]6
18F]-MK-6240
[
Scheme 9: Radiochemical Synthesis of compound [18F]6
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Figure 2: Regional timeꢀactivity curves and summed PET image (40ꢀ80 min) from a rhesus
monkey overlaid on an MR image in saggital plane. Panels A and C are from [18F]10 and panels
B and D are from [18F]6. Radioactivity concentration is expressed as standardized uptake value
(SUV) that is normalized to injected dose and body weight.
To further evaluate the imaging characteristics of [18F]6 we designed an in vivo PET
blocking study in rhesus monkey. In this experiment a high dose of 6 was administered prior to
tracer level dosage of [18F]6, and the resulting PET data was compared to a baseline PET study
with [18F]6. As shown in figure 3, the level of tracer retention did not change between the
baseline and selfꢀblock studies, indicating 6 has no high affinity offꢀtarget binding to CNS
targets that could interfere with detection NFTs in human. Based on these favorable preclinical
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data, [18F]6 was selected to move into early clinical studies and designated as [18F]ꢀMKꢀ6240.
Extensive preclinical in vitro and in vivo studies with 6 have been completed and will be
communicated shortly. Further exploratory in vitro studies will be required to determine the
utility and binding characteristics of 6 to NFTs in tissue from nonꢀAD tauopathies.
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Figure 3. Comparison of summed PET images (90ꢀ120 min, sagittal view) overlaid on an MR
image from PET studies with [18F]6 at baseline (left image) and after 1 mpk 6 (right image) in
rhesus monkey; scale is in SUV.
CONCLUSIONS
In summary, we have identified a novel imaging agent for detecting tau pathology, 6, with
exquisite in vitro binding affinity to NFTs, high selectivity to βꢀamyloid, and excellent
physicochemical properties for brain penetration and cellular permeability. 6 exhibits no offꢀ
target binding with suitable in vivo pharmacokinetics and merits clinical evaluation as a PET
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ligand for the quantification of NFTs in AD patients. Clinical studies to evaluate [18F]6 as an
NFT PET tracer in Alzherimer’s disease patients are currently underway and will be reported in
due course.
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EXPERIMENTAL SECTION
General Methods
All solvents used were commercially available “anhydrous” grade, and reagents were used
without purification unless otherwise noted. Nonꢀaqueous reactions were carried out in ovenꢀ or
heatgunꢀ dried glassware under a N2(g) atmosphere. Magnetic stirring was used to agitate the
reactions and they were monitored for completion by either TLC (silica gel 60, Merck) or LC–
MS. A Smith Creator microwave from Personal Chemistry was used for microwave heating, and
a CombiFlash system using RediSep cartridges by Teledyne Isco was used for silica gel
chromatography with fraction collection at 254 nm. Reversedꢀphase HPLC purification was
carried out on a Waters HPLC (XBridge Prep C18 5 ꢀm 19_150 mm column) using a gradient of
0.1% TFA in H2O/CH3CN with sample collection triggered by photodiode array detection. The
reported yields are for isolated compounds of ≥95% purity which was determined by HPLC and
1HꢀNMR. HPLC was carried out with a Waters 2690 Separations Module equipped with a YMC
Pro 50x3 mm i.d. C18 column interfaced with a Waters Micromass ZMD spectrometer using a
1
gradient of 0.05% TFA in H2O/CH3CN with UV detection at 215 and 254 nm. H spectra were
recorded on a Varian INOVA 400 or 500 MHz spectrometer, and all chemical shifts are
referenced to an internal standard of tetramethylsilane or the CDCl3 solvent peak, respectively.
All temperatures are degrees Celsius unless otherwise noted. Mass spectra (MS) were measured
by electrospray ionꢀmass spectroscopy (ESI).
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Chemical synthesis of final compounds 6, 8, 9, 10, 11, 12:
5
6
7
6-Fluoro-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine (6):
8
9
Step 1. 3-Chloro-6-fluoro-5-nitroisoquinoline (6a):
To
a
solution of 3ꢀchloroꢀ6ꢀ
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fluoroisoquinoline (10d) (2.0 g, 11.0 mmol) in concentrated sulfuric acid (30 mL) was added
potassium nitrate (1.2 g, 11.6 mmol) at 0 °C. The resulting mixture was stirred for 3 hours at
ambient temperature then poured onto ice/water (200 g). A filtration was performed and the filter
cake was washed with water (2 x 10 mL), purified by silica gel column chromatography, eluted
with 10~20% ethyl acetate in petroleum ether to afford 6a as a lightꢀyellow solid (1.0 g, 40%):
1H NMR (300 MHz, DMSOꢀd6) δ 9.43 (s, 1H), 8.70ꢀ8.65 (m, 1H), 8.07 (s, 1H), 8.00ꢀ7.93 (m,
1H); LC/MS: [(M +1)]+ = 227.0.
Step 2. 3-Chloro-6-fluoroisoquinolin-5-amine (6b):
To a solution of 3ꢀchloroꢀ6ꢀfluoroꢀ5ꢀ
nitroisoquinoline (6a) (1.0 g, 4.41 mmol) in acetic acid (30 mL) was added iron powder (1.24 g,
22.07 mmol). The resulting mixture was stirred for 16 hours at ambient temperature, then filtered
through Celite and the filtrate was concentrated under reduced pressure. The residue was taken
up with water (50 mL, saturated with sodium bicarbonate) and the resulting mixture was
extracted with ethyl acetate (4 x 100 mL). The combined organic layers was dried over
anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure
and the residue was purified by silica gel column chromatography, eluted with 1% methanol in
dichloromethane to afford 6b as a yellow solid (0.75 g, 86%): 1H NMR (300 MHz, DMSOꢀd6) δ
9.06 (s, 1H), 8.26 (s, 1H), 7.51ꢀ7.44 (m, 1H), 7.40ꢀ7.35 (m, 1H), 6.01 (br s, 2H); LC/MS: [(M
+1)]+ = 197.1.
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Step
3.
tert-Butyl
N-[(tert-butoxy)carbonyl]-N-(3-chloro-6-fluoroisoquinolin-5-
5
6
7
8
yl)carbamate (6c): To a solution of 3ꢀchloroꢀ6ꢀfluoroisoquinolinꢀ5ꢀamine (0.35 g, 1.78 mmol)
in dichloromethane (60 mL) were added diꢀtertꢀbutyl dicarbonate (0.97 g, 4.45 mmol),
triethylamine (0.36 g, 3.56 mmol) and N,Nꢀdimethylpyridinꢀ4ꢀamine (0.02 g, 0.18 mmol). The
resulting mixture was stirred for 12 hours at ambient temperature. After that, the reaction was
quenched with water (100 mL) and extracted with ethyl acetate (3 x 100 mL). The combined
organic layers was washed with brine (30 mL) and dried over anhydrous sodium sulfate. After
filtration, the filtrate was concentrated under reduced pressure and the residue was purified by
silica gel column chromatography, eluted with 1~2% methanol in dichloromethane to afford 6c
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as a yellow solid (0.45 g, 62%): H NMR (300 MHz, CDCl3) δ 9.07 (s, 1H), 8.01ꢀ7.98 (m, 1H),
7.67 (s, 1H), 7.45ꢀ7.40 (m, 1H), 1.37 (s, 18H); LC/MS: [(M +1)]+ = 197.0.
Step 4. tert-Butyl 6-fluoro-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-ylcarbamate
(6d): To a solution of 1Hꢀpyrrolo[2,3ꢀc]pyridine (8a) (0.20 g, 1.70 mmol) in tetrahydrofuran (80
mL) were added tertꢀbutyl Nꢀ[(tertꢀbutoxy)carbonyl]ꢀNꢀ(3ꢀchloroꢀ6ꢀfluoroisoquinolinꢀ5ꢀ
yl)carbamate (6c) (0.45 g, 1.13 mmol), sodium 2ꢀmethylpropanꢀ2ꢀolate (0.22 g, 2.27 mmol) and
tꢀBuXPhos Palladium (II) biphenylꢀ2ꢀamine mesylate (0.09 g, 0.11 mmol) at ambient
temperature. The resulting mixture was stirred for 2 hours at 50 °C under nitrogen atmosphere.
After cooling down to ambient temperature, the resulting mixture was diluted with water (100
mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers was washed
with brine (100 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was
concentrated under reduced pressure and the residue was purified by silica gel column
chromatography, eluted with 1~2% methanol in dichloromethane to afford 6d as a yellow solid
(0.35 g, 79%): 1H NMR (300 MHz, CDCl3) δ 9.68 (s, 1H), 9.22 (s, 1H), 8.37 (d, J = 4.8 Hz, 1H),
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8.07 (d, J = 3.3 Hz, 1H), 7.99ꢀ7.93 (m, 2H), 7.63 (d, J = 5.7 Hz, 1H), 7.44ꢀ7.26 (m, 1H), 6.80 (d,
J = 3.3 Hz, 1H), 6.50 (br s, 1H), 1.56 (s, 9H); LC/MS: [(M +1)]+ = 379.2.
7
8
9
Step 5. 6-Fluoro-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine (6): A solution of
tertꢀbutyl 6ꢀfluoroꢀ3ꢀ(1Hꢀpyrrolo[2,3ꢀc]pyridinꢀ1ꢀyl)isoquinolinꢀ5ꢀylcarbamate (6d) (0.35 g, 0.93
mmol) in dichloromethane (50 mL) was treated with trifluoroacetic acid (3.16 g, 27.70 mmol)
for 12 hours at ambient temperature. The resulting mixture was concentrated under reduced
pressure and the residue was dissolved into dichloromethane (50 mL), washed with saturated
aqueous solution of sodium bicarbonate (50 mL). The organic layer was dried over anhydrous
sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure and the
residue was purified by silica gel column chromatography, eluted with 1ꢀ2% methanol in
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dichloromethane to afford 6 as a yellow solid (0.24 g, 90%): H NMR (300 MHz, DMSOꢀd6) δ
9.70 (s, 1H), 9.27 (s, 1H), 8.40 (s, 1H), 8.35ꢀ8.27 (m, 2H), 7.68ꢀ7.66 (m, 1H), 7.45ꢀ7.43 (m, 2H),
6.88 (d, J = 3.3 Hz, 1H), 6.03 (br s, 2H); LC/MS: [(M +1)]+ = 279.1.
3-(1H-Pyrrolo[2,3-c]pyridin-1-yl)isoquinoline (8):
To
a
stirred solution of 1Hꢀ
pyrrolo[2,3ꢀc]pyridine (8a) (0.20 g, 1.69 mmol) in dimethyl sulfoxide (30 mL) were added 3ꢀ
bromoisoquinoline (8b) (0.38 g, 1.86 mmol), dimethylglycine (0.07 g, 0.68 mmol), cesium
carbonate (2.20 g, 6.77 mmol) and copper (I) iodide (0.19 g, 1.01 mmol). The resulting mixture
was stirred for 16 hours at 120 °C under a nitrogen atmosphere. After cooling down to ambient
temperature, the reaction was quenched by water (200 mL) and extracted with ethyl acetate (3 x
50 mL). The combined organic layers was washed with brine (3 x 100 mL), dried over
anhydrous sodium sulfate and filtered. The filtration was concentrated under reduced pressure
and the residue was purified by silica gel column chromatography, eluted with 1~1.5% methanol
1
in dichloromethane to afford 8 as a brown solid (0.38 g, 92%): H NMR (400 MHz, DMSO-d6)
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δ 9.69 (br s, 1H), 9.42 (s, 1H), 8.35 (d, J = 3.2 Hz, 1H), 8.27 (s, 2 H), 8.20 (d, J = 8.4 Hz, 1H),
8.06 (d, J = 8.0 Hz, 1H), 7.84 (t, J = 8.0 Hz, 1H), 7.68ꢀ7.65 (m, 2H), 6.87 (d, J = 3.6 Hz, 1H);
LC/MS: [(M +1)]+ = 246.1.
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1-(5-Methylpyridin-2-yl)-1H-pyrrolo[2,3-c]pyridine (9): To a stirred solution of 1Hꢀ
pyrrolo[2,3ꢀc]pyridine (8a) (0.10 g, 0.84 mmol) in dimethyl sulfoxide (20 mL) were added 2ꢀ
bromoꢀ5ꢀmethylpyridine (9a) (0.19 g, 1.10 mmol), dimethylglycine (0.035 g, 0.34 mmol),
cesium carbonate (1.10 g, 3.39 mmol) and copper (I) iodide (0.097 g, 0.51 mmol). The resulting
mixture was stirred for 16 hours at 120 °C under a nitrogen atmosphere. After cooling down to
ambient temperature, the reaction was quenched by water (30 mL) and extracted with ethyl
acetate (3 x 30 mL). The combined organic layers was washed with brine (100 mL), dried over
anhydrous sodium sulfate and filtered. The filtration was concentrated under reduced pressure
and the residue was purified by silica gel column chromatography, eluted with 1~4% methanol
1
in dichloromethane to afford 9 as a light yellow solid (51 mg, 29%): H NMR (300 MHz,
DMSO-d6) δ 9.68 (s, 1H), 8.54 (s, 1H), 8.27ꢀ8.26 (m, 2H), 7.85 (d, J = 8.7 Hz, 1H), 7.77 (d, J =
8.4 Hz, 1H), 7.64 (d, J = 5.1 Hz, 1H), 6.81 (d, J = 3.0 Hz, 1H), 2.36 (s, 3H); LC/MS: [(M +1)]+ =
210.1.
6-Fluoro-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinoline (10):
Step 1. (E)-5-fluoro-2-(hydroxyimino)-2,3-dihydroinden-1-one (10b):
To a stirred
solution of 5ꢀfluoroꢀ2,3ꢀdihydroindenꢀ1ꢀone (10a) (22 g, 147 mmol) in diethyl ether (500 mL)
was added a saturated solution of dry hydrochloride gas in methanol (22 mL) followed by the
addition of isopentyl nitrite (26 g, 220 mmol) at 0 °C. After stirred for 2 hours at ambient
temperature, solids were collected by filtration and washed with diethyl ether (200 mL) to afford
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10b as a yellow solid (21 g, 79%): H NMR (400 MHz, DMSO-d6) δ 12.67 (s, 1H), 7.85ꢀ7.81
5
6
(m, 1H), 7.46ꢀ7.43 (m, 1H), 7.31ꢀ7.28 (m, 1H), 3.79 (s, 2H); LC/MS: [(M +1)]+ = 180.0.
7
8
9
Step 2. 1,3-Dichloro-6-fluoroisoquinoline (10c): To a stirred solution of (E)ꢀ5ꢀfluoroꢀ2ꢀ
(hydroxyimino)ꢀ2,3ꢀdihydroindenꢀ1ꢀone (10b) (25 g, 140 mmol) in phosphorus oxytrichloride
(400 mL, saturated with hydrochloride) was added pentachlorophosphorane (73 g, 349 mmol).
The resulting mixture was stirred for 16 hours at 60 °C. After cooling down to ambient
temperature, the resulting mixture was concentrated under reduced pressure and the residue was
triturated with water (500 mL) and filtered. The filter cake was washed with water (200 mL) and
dried in a vacuum oven to afford 10c as a yellow solid (28 g, 93%): 1H NMR (400 MHz, CDCl3)
δ 8.42ꢀ8.35 (m, 1H), 7.68 (s, 1H), 7.47ꢀ7.41 (m, 2H); LC/MS: [(M +1)]+ = 216.0.
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Step 3. 3-Chloro-6-fluoroisoquinoline (10d):
To a stirred solution of 1,3ꢀdichloroꢀ6ꢀ
fluoroisoquinoline (10c) (28 g, 130 mmol) in acetic acid (500 mL) and hydriodic acid (250 mL,
55% w/w aqueous solution) was added red phosphorus (10 g, 324 mmol) at ambient temperature.
The resulting mixture was stirred for 4 hours at 110 °C. After cooling down to ambient
temperature, the resulting mixture was concentrated under reduced pressure. The residue was
taken up by ethyl acetate (500 mL) and water (500 mL). The organic layer was separated out and
the aqueous layer was extracted with ethyl acetate (2 x 500 mL). The combined organic layers
was washed with brine (300 mL) and dried over anhydrous sodium sulfate. After filtration, the
filtrate was concentrated under reduced pressure and the residue was purified by silica gel
column chromatography, eluted with 0~10% ethyl acetate in petroleum ether to afford 10d as a
yellow solid (20 g, 85%): 1H NMR (300 MHz, CDCl3) δ 9.04 (s, 1H), 8.06ꢀ7.97 (m, 1H), 7.68 (s,
1H), 7.39ꢀ7.35 (m, 2H); LC/MS: [(M +1)]+ = 182.1.
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Step 4. 6-Fluoro-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinoline (10): To a stirred solution
of 1Hꢀpyrrolo[2,3ꢀc]pyridine (8a) (1.14 g, 9.66 mmol) and 3ꢀchloroꢀ6ꢀfluoroisoquinoline (10d)
(1.17 g, 6.44 mmol) in tetrahydrofuran (60 mL) was added sodium 2ꢀmethylpropanꢀ2ꢀolate (1.24
g, 12.89 mmol) and tꢀBuXPhos palladium(II) biphenylꢀ2ꢀamine mesylate (0.26 g, 0.32 mmol) at
ambient temperature. The resulting mixture was stirred for 4 hours at 50 °C under a nitrogen
atmosphere. After cooling down to ambient temperature, the resulting mixture was concentrated
under reduced pressure and the residue was purified by silica gel column chromatography, eluted
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with 0~1% methanol in dichloromethane to afford 10 as an offꢀwhite solid (1.50 g, 88%): H
NMR (400 MHz, DMSO-d6) δ 9.69 (s, 1H), 9.43 (s, 1H), 8.35ꢀ8.28 (m, 4H), 7.85ꢀ7.82 (m, 1H),
7.68 (d, J = 5.2 Hz, 1H), 7.61ꢀ7.56 (m, 1H), 6.89 (d, J = 3.9 Hz, 1H); LC/MS: [(M +1)]+ = 264.2.
8-Fluoro-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinoline (11):
Step 1. (E)-7Ffluoro-2-(hydroxyimino)-2,3-dihydro-1H-inden-1-one (11b): To a stirred
solution of 7ꢀfluoroꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀone (11a) (2.0 g, 13.3 mmol) in diethyl ether (100
mL) was added a saturated solution of dry hydrochloride gas in methanol (10 mL) followed by
the addition of isopentyl nitrite (2.3 g, 19.9 mmol) at 0 °C. After additional 1 hour, solids were
collected by filtration and washed with diethyl ether (200 mL) to afford 11b as a brown solid
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(1.5 g, 60%): H NMR (300 MHz, DMSO-d6) δ 12.75 (br s, 1H), 7.78ꢀ7.72 (m, 1H), 7.45ꢀ7.43
(m, 1H), 7.27ꢀ7.21 (m, 1H), 3.79 (s, 2H); LC/MS: [(M +1)]+ = 180.2.
Step 2. 1,3-Dichloro-8-fluoroisoquinoline (11c): To a stirred solution of (E)ꢀ7ꢀfluoroꢀ2ꢀ
(hydroxyimino)ꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀone (11b) (1.50 g, 8.37 mmol) in phosphorus
oxytrichloride (20 mL, saturated with hydrochloride gas) was added pentachlorophosphorane
(2.09 g, 10.05 mmol). The resulting mixture was stirred for 6 hours at 60 °C. After cooling down
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to ambient temperature, the resulting mixture was concentrated under reduced pressure and the
residue was triturated with water (100 mL) and filtered. The filter cake was washed with water
(100 mL) and dried in a vacuum oven to afford 11c as a brown solid (1.0 g, 53%): 1H NMR (300
MHz, DMSO-d6) δ 8.12 (s, 1H), 7.91ꢀ7.88 (m, 2H), 7.63ꢀ7.58 (m, 1H).
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Step 3. 3-Chloro-8-fluoroisoquinoline (11d):
To a stirred solution of 1,3ꢀdichloroꢀ8ꢀ
fluoroisoquinoline (11c) (1.0 g, 4.63 mmol) in acetic acid (30 mL) and hydriodic acid (15 mL,
55% w/w aqueous solution) was added red phosphorus (0.36 g, 11.57 mmol) at ambient
temperature. The resulting mixture was stirred for 3 hours at 120 °C. After cooling down to
ambient temperature, the resulting mixture was concentrated under reduced pressure. The residue
was taken up by ethyl acetate (100 mL) and water (100 mL). The organic layer was separated out
and the aqueous layer was extracted with ethyl acetate (2 x 100 mL). The combined organic
layers was washed with brine (100 mL) and dried over anhydrous sodium sulfate. After filtration,
the filtrate was concentrated under reduced pressure to afford 3ꢀchloroꢀ8ꢀfluoroisoquinoline as a
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yellow solid (0.70 g, 79%): H NMR (400 MHz, DMSO-d6) δ 9.45 (s, 1H), 8.25 (s, 1H), 7.89ꢀ
7.81 (m, 2H), 7.55ꢀ7.51 (m, 1H); LC/MS: [(M +1)]+ = 182.2.
Step 4. 8-Fluoro-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinoline (11): To a stirred solution
of 1Hꢀpyrrolo[2,3ꢀc]pyridine (8a) (98.0 mg, 0.8 mmol) and 3ꢀchloroꢀ8ꢀfluoroisoquinoline (11d)
(100.0 mg, 0.6 mmol) in tetrahydrofuran (20 mL) was added sodium 2ꢀmethylpropanꢀ2ꢀolate
(106.0 mg, 1.1 mmol) and tꢀBuXPhos palladium(II) biphenylꢀ2ꢀamine mesylate (43.7 mg, 0.06
mmol) at ambient temperature. The resulting mixture was stirred for 12 hours at 50 °C under a
nitrogen atmosphere. After cooling down to ambient temperature, the resulting mixture was
diluted with water (30 mL) and extracted with ethyl acetate (3 x 30 mL). The combined organic
layers was washed with brine (30 mL) and dried over anhydrous sodium sulfate. After filtration,
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the filtrate was concentrated under reduced pressure and the residue was purified by silica gel
column chromatography, eluted with 1~2% methanol in dichloromethane to afford 11 as an offꢀ
white solid (54.1 mg, 37%): 1H NMR (300 MHz, DMSO-d6) δ 9.74 (s, 1H), 9.56 (s, 1H), 8.39 (s,
2H), 8.30ꢀ8.27 (m, 1H), 7.93ꢀ7.83 (m, 2H), 7.69ꢀ7.68 (m, 1H), 7.47ꢀ7.43 (m, 1H), 6.90 (d, J =
3.3 Hz, 1H); LC/MS: [(M +1)]+ = 264.2.
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2-Fluoro-7-(1H-pyrrolo[2,3-c]pyridin-1-yl)-1,6-naphthyridine (12)
Step1. 5-Bromo-2-(1H-pyrrolo[2,3-c]pyridin-1-yl)isonicotinic acid (12b): To a stirred
solution of 1Hꢀpyrrolo[2,3ꢀc]pyridine (8a) (5.6 g, 47.9 mmol) in N,Nꢀdimethylformamide (250
mL) were added cesium carbonate (46.8 g, 144.0 mmol) and methyl 5ꢀbromoꢀ2ꢀ
chloroisonicotinate (12a) (12.0 g, 47.9 mmol). The resulting mixture was stirred for 16 hours at
80 °C. After cooling down to ambient temperature, solvent was evaporated out under reduced
pressure and the residue was triturated with dichloromethane (500 mL). After filtration, the filter
cake was collected, suspended in water (500 mL) and acidified by diluted hydrochloric acid (300
mL, 1N). A filtration was performed and the filter cake was collected and dried in the air to
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afford 12b as a light yellow solid (10 g, 66%): H NMR (300 MHz, DMSO-d6) δ 14.70 (br s,
1H), 9.89 (s, 1H), 8.97ꢀ8.94 (m, 2H), 8.52 (d, J = 6.0 Hz, 1H), 8.28 (s, 1H), 8.23 (d, J = 6.3 Hz,
1H), 7.24 (d, J = 3.3 Hz, 1H); LC/MS: [(M +1)]+ = 318.0, 320.0.
Step2. tert-Butyl (5-bromo-2-(1H-pyrrolo[2,3-c]pyridin-1-yl)pyridin-4-ylcarbamate (12c):
To a stirred solution of 5ꢀbromoꢀ2ꢀ(1Hꢀpyrrolo[2,3ꢀc]pyridinꢀ1ꢀyl)isonicotinic acid (12b) (10.0
g, 31.4 mmol) and triethylamine (4.8 g, 47.2 mmol) in tertꢀbutyl alcohol (250 mL) was added
diphenyl phosphorazidate (26.0 g, 94.0 mmol) at ambient temperature. The resulting mixture was
refluxed for 4 hours under a nitrogen atmosphere. After cooling down to ambient temperature,
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the resulting mixture was concentrated under reduced pressure and the residue was purified by
silica gel column chromatography, eluted with 2~10% methanol in dichloromethane to afford
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12c as a light yellow solid (3.0 g, 25%): H NMR (300 MHz, DMSOꢀd6) δ 9.56 (br s, 1H), 8.95
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(s, 1H), 8.69 (s, 1H), 8.29 (d, J = 5.4 Hz, 1H), 8.20 (s, 1H), 8.18 (d, J = 3.3 Hz, 1H), 7.69 (d, J =
5.1 Hz, 1H), 6.87 (d, J = 3.3 Hz, 1H), 1.53 (s, 9H).
Step 3. 5-Bromo-2-(1H-pyrrolo[2,3-c]pyridin-1-yl)pyridin-4-amine (12d): A solution of
tertꢀbutyl (5ꢀbromoꢀ2ꢀ(1Hꢀpyrrolo[2,3ꢀc]pyridinꢀ1ꢀyl)pyridinꢀ4ꢀyl)carbamate (12c) (0.70 g, 1.79
mmol) in dichloromethane (32 mL) was treated with trifluoroacetic acid (4 mL) at ambient
temperature for 3 hours. The resulting solution was concentrated under reduced pressure and the
residue was dissolved into dichloromethane (50 mL), washed with saturated aqueous solution of
sodium bicarbonate (2 x 50 mL). The organic layer was dried over anhydrous sodium sulfate and
filtered. The filtrate was concentrated under reduced pressure and the residue was purified by
silica gel column chromatography, eluted with 1~10% methanol in dichloromethane to afford
12d as a light yellow solid (0.50 g, 91%): 1H NMR (300 MHz, DMSOꢀd6) δ 9.46 (br s, 1H), 8.29
(s, 1H), 8.25 (d, J = 5.4 Hz, 1H), 8.04 (d, J = 3.3 Hz, 1H), 7.64 (d, J = 5.4 Hz, 1H), 7.04 (s, 1H),
6.78 (d, J = 3.3 Hz, 1H), 6.63 (br s, 2H); LC/MS: [(M +1)]+ = 289.0, 291.0.
Step4. (E)-Ethyl 3-(4-amino-6-(1H-pyrrolo[2,3-c]pyridin-1-yl)pyridin-3-yl)acrylate (12e):
To a solution of 5ꢀbromoꢀ2ꢀ(1Hꢀpyrrolo[2,3ꢀc]pyridinꢀ1ꢀyl)pyridinꢀ4ꢀamine (12d) (1.0 g, 3.46
mmol) in N,Nꢀdimethylformamide (50 mL) were added ethyl acrylate (1.38 g, 13.83 mmol),
triethylamine (1.40 g, 13.83 mmol), palladium (II) acetate (0.078 g, 0.35 mmol) and triꢀoꢀ
tolylphosphine (0.21 g, 0.69 mmol) at ambient temperature under nitrogen atmosphere. The
resulting solution was stirred for 16 hours at 120 oC. After cooling down to ambient temperature,
the resulting mixture was concentrated under reduced pressure and the residue was purified by
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