Structure-based design and synthesis of covalent-reversible inhibitors to overcome drug resistance in EGFR

Article abstract of DOI:10.1016/j.bmc.2015.04.038

The clinical success of covalent kinase inhibitors in the treatment of EGFR-dependent non-small cell lung cancer (NSCLC) has rejuvenated the appreciation of reactive small molecules. Acquired drug resistance against first-line EGFR inhibitors remains the

Full text of DOI:10.1016/j.bmc.2015.04.038

Accepted Manuscript
Structure-based design and synthesis of covalent-reversible inhibitors to over-
come drug resistance in EGFR
Debjit Basu, André Richters, Daniel Rauh
PII:
S0968-0896(15)00344-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.04.038
BMC 12250
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 March 2015
12 April 2015
14 April 2015
Please cite this article as: Basu, D., Richters, A., Rauh, D., Structure-based design and synthesis of covalent-
reversible inhibitors to overcome drug resistance in EGFR, Bioorganic & Medicinal Chemistry (2015), doi: http://
dx.doi.org/10.1016/j.bmc.2015.04.038
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Structure-based design and synthesis of covalent-
reversible inhibitors to overcome drug resistance in
EGFR
Debjit Basu, André Richters, Daniel Rauh*
Department of Chemistry and Chemical Biology, Technische Universität Dortmund, Otto-
Hahn-Straße 6, 44227 Dortmund, Germany
* Corresponding author. Tel.: +49 231 755 7080
E-mail address: daniel.rauh@tu-dortmund.de (D. Rauh)
1

Abstract
The clinical success of covalent kinase inhibitors in the treatment of EGFR-dependent non-
small cell lung cancer (NSCLC) has rejuvenated the appreciation of reactive small molecules.
Acquired drug resistance against first-line EGFR inhibitors remains the major bottleneck in
NSCLC and is currently addressed by the application of fine-tuned covalent drugs. Here we
report the design, synthesis and biochemical evaluation of a novel class of EGFR inhibitors
with a covalent yet reversible warhead. A series of WZ4002 analogues, derived from
anilinopyrimidine and 3-substituted-2-cyanoacrylamide scaffolds, exhibit strong and selective
inhibitory activity against clinically relevant EGFRL858R and EGFRL858R/T790M
.
2

1. Introduction
The epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase
which plays pivotal roles in cell proliferation, survival, adhesion, migration and
differentiation through downstream signaling pathway activation.1 Dysregulation of EGFR
caused by overexpression or mutation is associated with the onset and development of various
cancers, including lung cancer, gastrointestinal malignancies, glioblastoma multiform and
thus EGFR represents an important drug target for the treatment of tumor diseases.2-4 In
particular, clinically acquired drug resistance against EGFR kinase inhibitors is a major
challenge in targeted cancer therapies for non-small cell lung cancer (NSCLC) treatment.5, 6
For instance, EGFRL858R mutant positive NSCLC patients receiving erlotinib or gefitinib as
first-line treatment suffer from a dramatic relapse which is caused by the acquired secondary
drug resistance mutation T790M.7-9 In this specific example, the oncogenic mutant form
L858R evoking increased catalytic activity of EGFR undergoes an additional mutation at the
gatekeeper position (T790M) of the kinase domain.10 The latter mutation increases the off-rate
of conventional reversible inhibitors rendering them as ineffective.11 Since the residence time
of an inhibitor toward its target is a relevant marker for its efficacy and potentially translates
into beneficial in vivo efficiency, the development of inhibitors with extended drug-target
residence time, amongst other strategies, is a powerful approach to tackling mutation
associated drug resistance.12 Second generation EGFR inhibitors (e.g., afatinib and
dacomitinib)13, are equipped with Michael acceptor systems that take advantage of the
14
covalent modification of a rare cysteine (Cys797) located at the lip of the ATP binding cleft
of EGFR.15, 16 Due to the covalent modification of Cys797, these irreversible inhibitors exhibit
a marginal off-rate and maximal drug-target residence time when compared to reversible
inhibitors. Such features are thought to be instrumental to overcome T790M associated drug
17, 18
resistance in NSCLC.10,
Despite promising in vitro results, second generation inhibitors
exhibit insufficient efficacy in patients at clinically achievable concentrations. Thus, on-target
3

toxicity represents the dose-limiting factor since in addition to the oncogenic mutant variants,
the wildtype form of EGFR is likewise addressed which results in severe side effects such as
skin rash and diarrhea.19-21 However, the third generation EGFR inhibitors, e.g. WZ4002, CO-
1686 (rociletinib) and AZD9291 combine the advantageous properties of covalent inhibition
with an improved selectivity profile since they spare the inhibition of wildtype EGFR. CO-
1686 and AZD9291 are at the leading edge of the treatment of acquired drugs resistance in
NSCLC. Both compounds exhibit beneficial properties in terms of residence times and
toxicity profiles.22-25 Notably, these compounds induce synergistic binding interactions with
the hydrophobic gatekeeper substitutions in T790M and, therefore, retain their inhibitory
efficiency towards clinically relevant EGFR mutants. However, solely few compounds
possessing such beneficial structural features are under clinical investigation. Since T790M
continues to be the predominant drug resistance mutation, there is an urgent medical need to
identify novel mutant selective molecules that potentially amalgamate the advantages of both
reversible and covalent inhibition yet devoid of the potential disadvantages associated with
both strategies.6, 9
Within the wide spectrum of kinase inhibitors, covalent-reversible inhibitors (CRIs) represent
an innovative class of promising inhibitors that can be optimized in terms of extended drug-
target residence times while retaining the advantages of non-covalent inhibition with respect
to limited off-target effects.26 Recent studies by Taunton and co-workers indicate potential
advantages of CRIs since the covalent protein modification is exclusively elicited and stable
within the properly formed binding site of the target protein. The covalent bond is formed
temporally and cleaved reversibly when the ligand binding site is disintegrated upon protein
unfolding or proteolysis. Michael acceptor elements of CRIs (Fig. 1C) are as a matter of
principle dually activated and thus lead to increased target affinity by covalent modification.
In combination with the reversible hinge binding properties, this class of inhibitors provides
prolonged pharmacodynamics over classical reversible inhibitors. Although CRIs are to date
4

exclusively used as tool compounds, they might very well translate into the development of
innovative therapeutics to overcome the challenges associated with conventional covalent
inhibition strategies, such as potential toxicity and off-target activities.26-28
Here, we set out to explore the concept of CRIs for the inhibition of clinically relevant EGFR
mutants while sparing wildtype EGFR. In this proof of concept study, we designed,
synthesized and biochemically characterized a focused library of CRIs based on the WZ4002
scaffold possessing the ability to address Cys797 in drug resistant mutants of EGFR with a
fine-tuned electron-deficient olefin.
2. Materials and methods
2.1. Molecular modeling
We utilized the co-crystal structure of WZ4002 in complex with EGFR (PDB-code: 3IKA) as
the template for the in silico design of covalent reversible analogues. We altered R1 and R2
and superposed the generated derivatives with the principal core of WZ4002. Extensions at R1
and R2 were introduced according to their spatial and electronic features to i) promote the
reversibility of binding (strong electron withdrawing groups at R1, CN and CF3) and ii) for
tuning the properties of the Michael acceptor (aliphatic and aromatic groups at R2). DS
Viewer Pro 6.0 (Accelrys) was used for defining structural features (e.g., aromaticity) of the
designed derivatives as well as performing energy minimization. The drafted compounds
were then superposed with the ligand WZ4002 using PyMOL (The PyMOL Molecular
Graphics System, Version 1.5.0.4 Schrödinger, LLC).
2.2. Chemistry
Unless otherwise noted, all reagents and solvents were purchased from Acros, Fluka, Sigma,
Aldrich, or Merck and used without further purification. Dry solvents were purchased as
1 13
anhydrous reagents from commercial suppliers. H and C NMR spectra were recorded on a
5

Bruker Avance DRX 400 or Bruker Avance DRX 500 spectrometer at 400 MHz or 500 MHz
1
and 101 MHz or 125 MHz respectively. H chemical shifts are reported in δ (ppm) as s
(singlet), d (doublet), dd (doublet of doublet), t (triplet), q (quartet), m (multiplet), and bs
(broad singlet) and are referenced to the residual solvent signal: CDCl3 (7.26), DMSO-d6
13
(2.50). Coupling constants (J) are expressed in hertz (Hz). C spectra are referenced to the
residual solvent signal: CDCl (77.0) or DMSO-d6 (39.0). All final compounds were purified
3
to >95% purity, as determined by high-performance liquid chromatography (HPLC). Purity
was measured using Agilent 1200 series HPLC systems with UV detection at 210 nm
(system: Agilent Eclipse XDB-C18 4.6 mm × 150 mm, 5 µM, 10-100% CH3CN in H2O, with
0.1% TFA, for 15 min at 1.0 mL/min). Analytical TLC was carried out on Merck 60 F245
aluminum-backed silica gel plates. Compounds were purified by column chromatography
using Baker silica gel (40-70 µm particle size). Preparative HPLC was conducted on a Varian
HPLC system (Pro Star 215) with a VP 250/21 nucleosil C18 PPN column from Macherey-
Nagel and monitored by UV at λ = 254 nm.
2.2.1. 2,5-Dichloro-4-(3-nitrophenoxy)pyrimidine (8)
Potassium carbonate (0.75 g, 5.45 mmol) and 2,4,5-trichloropyrimidine (0.50 g, 2.72 mmol)
were added to a solution of 3-nitrophenol (0.38 g, 2.72 mmol) in DMF (10 mL). The reaction
was heated to 60 °C and stirred for 2 h. Then it was cooled to ambient temperature and
filtered through celite. The filtrate was diluted with ethyl acetate (20 mL) and washed with
1M HCl (20 mL), water (10 mL) and brine (10 mL). The organic layer was dried over
anhydrous sodium sulfate and concentrated to afford 0.67 g of title compound 8 (2.34 mmol,
1
86%) as light yellow solid. The product was used without further purification. H NMR
(400 MHz, DMSO-d6) δ 8.89 (s, 1H), 8.30 (t, J = 1.9 Hz, 1H), 8.23 (d, J = 7.9 Hz, 1H), 7.89-
6

7.78 (m, 2H); 13C NMR (101 MHz, DMSO) δ 164.62, 159.28, 155.95, 151.57, 148.57,
131.27, 128.77, 121.56, 117.33, 116.88.
2.2.2. 1-(3-Methoxy-4-nitrophenyl)-4-methylpiperazine (10)
To a solution of 1-methyl-4-piperazine (1.56 mL, 11.7 mmol) in DMF (20 mL) was added
potassium carbonate (2.42 g, 17.5 mmol) and 4-fluoro-2-methoxy-1-nitrobenzene (2.0 g,
11.7 mmol). The resulting mixture was stirred at room temperature for 18 h. The reaction
mixture was then diluted with water (50 mL) and extracted with ethyl acetate (3 x 30 mL).
The organic extract was washed with brine (20 mL), dried over anhydrous sodium sulfate and
evaporated to dryness to obtain an oily crude product. The crude was purified by flash silica
gel chromatography using DCM/MeOH (97/3, v/v) as eluent to obtain 2.9 g of the desired
product 10 (11.5 mmol, 98%) as yellow oil.1H NMR (400 MHz, DMSO-d6) δ 7.87 (d,
J = 9.4 Hz, 1H), 6.58 (dd, J = 9.4, 2.4 Hz, 1H), 6.52 (d, J = 2.3 Hz, 1H), 3.90 (s, 3H), 3.49-
3.37 (m, 4H), 2.46-2.36 (m, 4H), 2.22 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 155.99,
155.48, 128.17, 128.02, 105.15, 96.93, 56.26, 54.34, 46.31, 45.62.
2.2.3. 2-Methoxy-4-(4-methylpiperazin-1-yl)aniline (11)
Nitro compound 10 (2.9 g, 11.5 mmol) was dissolved in a mixture of THF (12 mL) and
MeOH (8 mL) and cooled to 0 °C. Subsequently, zinc powder (3.7 g, 57.7 mmol), followed
by ammonium chloride (3.1 g, 57.7 mmol) were added and the reaction mixture was stirred at
room temperature for 18 h. The reaction mixture was then filtered over celite and the filtrate
was concentrated in vacuo. The crude was purified by flash silica gel chromatography using
DCM/MeOH (96/4, v/v) as eluent to obtain 1.9 g of the desired product 11 (8.59 mmol, 75%)
as dark brown oil.1H NMR (400 MHz, DMSO-d6) δ 6.52 (d, J = 8.3 Hz, 1H), 6.48 (d,
J = 2.1 Hz, 1H), 6.28 (dd, J = 8.3, 2.1 Hz, 1H), 4.08 (bs, 2H), 3.74 (s, 3H), 3.03-2.82 (m, 4H),
2.47-2.36 (m, 4H), 2.21 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 146.99, 143.29, 130.93,
114.20, 108.53, 101.86, 55.21, 54.96, 50.21, 45.81.
7

2.2.4. 5-Chloro-N-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)-4-(3-
nitrophenoxy)pyrimidin-2-amine (12)
To a solution of compound 8 (1.33 g, 4.66 mmol) and compound 11 (1.03 g, 4.66 mmol) in
anhydrous 1-butanol (20 mL), trifluoroacetic acid (0.36 mL, 4.66 mmol) was added. The
reaction mixture was heated to 100 °C and stirred for 18 h. Subsequently, it was cooled to
room temperature and saturated aqueous sodium bicarbonate solution was added drop wise
until basic pH was obtained. The volatiles were removed in vacuo and the obtained thick
slurry was dissolved in DCM (50 mL). The organic layer was washed with water (20 mL),
brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The
crude was purified by flash silica gel chromatography using DCM/MeOH (96:4, v/v) as eluent
1
to afford 1.42 g of the desired product 12 (3.02 mmol, 65%) as white solid. H NMR
(500 MHz, DMSO-d6) δ 8.37 (s, 1H), 8.27 (s, 1H), 8.18-8.14 (m, 2H), 7.77-7.71 (m, 2H), 7.09
(d, J = 6.8 Hz, 1H), 6.48 (d, J = 2.2 Hz, 1H), 6.14 (bs, 1H), 3.70 (s, 3H), 3.09-3.00 (m, 4H),
2.48-2.40 (m, 4H), 2.22 (s, 3H); 13C NMR (126 MHz, DMSO-d6) δ 163.12, 158.42, 158.15,
156.19, 152.10, 148.26, 147.39, 130.81, 128.85, 120.52, 119.86, 119.07, 117.71, 105.96,
103.59, 99.77, 55.33, 54.55, 48.39, 45.66.
2.2.5. 4-(3-Aminophenoxy)-5-chloro-N-(2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)pyrimidin-2-amine (13)
Nitro compound 12 (1.40 g, 2.9 mmol) was dissolved in a mixture of THF (12 mL) and
MeOH (8 mL) and cooled to 0 °C. Subsequently, zinc powder (0.97 g, 14.9 mmol), followed
by ammonium chloride (0.79 g, 14.9 mmol) were added and the reaction mixture was stirred
at room temperature for 18 h. The reaction mixture was then filtered over celite and the
filtrate was concentrated in vacuo. The crude was purified by flash silica gel chromatography
using DCM/MeOH (97/3, v/v) as eluent to obtain 1.19 g of the title compound 13 (2.7 mmol,
93%) as white solid.1H NMR (500 MHz, DMSO-d6) δ 8.35 (s, 1H), 8.05 (s, 1H), 7.42 (d,
8

J = 8.8 Hz, 1H), 7.12 (t, J = 8.0 Hz, 1H), 6.59 (d, J = 2.5 Hz, 1H), 6.54 (dd, J = 8.1, 1.3 Hz,
1H), 6.42 (t, J = 2.2 Hz, 1H), 6.37 (ddd, J = 8.0, 2.2, 0.7 Hz, 1H), 6.33 (d, J = 8.1 Hz, 1H),
5.32 (bs, 2H), 3.80 (s, 3H), 3.16-3.05 (m, 4H), 2.52-2.46 (m, 4H), 2.28 (s, 3H); 13C NMR
(126 MHz, DMSO-d6) δ 163.76, 158.30, 157.59, 153.02, 150.22, 148.24, 140.01, 136.07,
129.59, 122.87, 119.74, 111.17, 108.45, 106.65, 104.13, 99.89, 55.52, 54.56, 48.57, 45.63;
HRMS-ESI (m/z): [M+H]+ calcd. for C22H26O2N6Cl, 441.18003; found, 441.17968.
2.2.6. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)acetamide (14)
Compound 13 (0.10 g, 0.23 mmol) was dissolved in anhydrous DCM (4 mL) and cooled to
0 °C. Subsequently, acetic anhydride (27 µL, 0.27 mmol) and DIPEA (110 µL, 0.91 mmol)
were added and the resulting reaction mixture was stirred at room temperature for 4 h. The
reaction mixture was then diluted with DCM (10 mL) and washed with saturated aqueous
sodium bicarbonate solution (10 mL), saturated aqueous ammonium chloride solution
(10 mL) and water (10 mL). The organic layer was dried over anhydrous sodium sulfate and
concentrated in vacuo. The crude product was purified by flash silica gel chromatography
using DCM/MeOH (98/2, v/v) as eluent to obtain 0.031 g of compound 14 (0.064 mmol,
1
28%) as a yellow solid. H NMR (500 MHz, CDCl3) δ 8.22 (s, 1H), 7.60 (bs, 1H), 7.56 (d,
J = 7.8 Hz, 1H), 7.46-7.34 (m, 3H), 7.31 (s, 1H), 6.97 (d, J = 7.2 Hz, 1H), 6.45 (d, J = 2.2 Hz,
1H), 6.21 (bs, 1H), 3.82 (s, 3H), 3.16- 3.07 (m, 4H), 2.59- 2.62 (m, 4H), 2.38 (s, 3H), 2.17 (s,
3H); 13C NMR (126 MHz, CDCl3) δ 168.22, 157.83, 157.58, 140.49, 140.24, 139.22, 129.93,
121.96, 121.89, 119.40, 118.05, 116.98, 113.73, 113.70, 111.36, 108.14, 100.44, 55.76, 55.25,
50.06, 46.12, 24.86; HRMS-ESI (m/z): [M+H]+ calcd. for C24H28O3N6Cl, 483.19059; found,
483.19045.
2.2.7. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy) -phenyl)-2-cyanoacetamide (15a)
9

Compound 13 (0.58 g, 1.31 mmol) and cyanoacetic acid (0.17 g, 1.97 mmol) were dissolved
in anhydrous DCM (10 mL) and cooled to 0 °C. Subsequently, EDC.HCl (0.38 g, 1.97 mmol),
HOBt (0.27 g, 1.97 mmol) and DIPEA (0.68 mL, 3.95 mmol) were added. The reaction
mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with DCM
(20 mL) and washed with saturated aqueous sodium bicarbonate solution (10 mL), saturated
aqueous ammonium chloride solution (10 mL) and water (10 mL). The organic layer was
dried over anhydrous sodium sulfate and the volatiles were removed under reduced pressure.
The crude product was purified by flash silica gel chromatography to obtain 0.60 g of
1
compound 15a (1.18 mmol, 90%) as a yellow solid. H NMR (600 MHz, DMSO-d6) δ 10.53
(s, 1H), 8.34 (s, 1H), 8.10 (s, 1H), 7.49 (s, 1H), 7.47-7.37 (m, 2H), 7.23 (d, J = 8.5 Hz, 1H),
7.04-6.93 (m, 1H), 6.53 (d, J = 2.2 Hz, 1H), 6.20 (bs, 1H), 3.93 (s, 2H), 3.74 (s, 3H), 3.13-
3.01 (m, 4H), 2.52-2.44 (m, 4H), 2.26 (s, 3H); 13C NMR (151 MHz, DMSO-d6) δ 164.02,
163.62, 161.26, 158.31, 157.98, 155.53, 152.21, 139.54, 130.19, 129.95, 119.54, 117.28,
116.37, 115.72, 112.60, 106.44, 103.97, 99.89, 55.52, 54.47, 48.42, 45.50, 26.79; HRMS-ESI
(m/z): [M+H]+ calcd. for C25H27O3N7Cl, 508.18584; found, 508.18593.
General procedure I for the preparation of compounds 5a-5i: To a solution of an aldehyde of
choice in anhydrous ethanol was added intermediate 15a and piperidine. The reaction mixture
was stirred at 80 °C for 18 h and then allowed to cool to room temperature. Excess of solvent
was removed in vacuo and the remaining residue was dissolved in ethyl acetate (10 mL). The
organic layer was washed with water (10 mL), dried over anhydrous sodium sulfate and
evaporated to give the crude product as pale yellow oil. The crude was purified by flash silica
gel chromatography to yield the desired compounds.
2.2.8. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)-phenyl)-2-cyano-3-(4-fluorophenyl)acrylamide
(5a)
10

Compound 5a was prepared as described in general procedure I using 4-fluorobenzaldehyde
(0.018 g, 0.14 mmol), 15a (0.050 g, 0.10 mmol), piperidine (1.5 µL, 0.014 mmol) and
anhydrous ethanol (2 mL). The crude was purified by flash silica gel chromatography using
DCM/MeOH (98/2, v/v) as eluent to obtain 0.018 g of 5a (0.003 mmol, 30%) as yellow solid.
1H NMR (500 MHz, CDCl3) δ 8.36 (s, 1H), 8.23 (s, 1H), 8.08 (s, 1H), 8.00 (dd, J = 8.5,
5.3 Hz, 2H), 7.62 (s, 2H), 7.56 (d, J = 8.1 Hz, 1H), 7.47 (t, J = 8.1 Hz, 1H), 7.43 (bs, 1H),
7.20 (t, J = 8.5 Hz, 2H), 7.07 (d, J = 8.0 Hz, 1H), 6.44 (d, J = 1.9 Hz, 1H), 6.22 (bs, 1H), 3.80
(s, 3H), 3.15-3.07 (m, 4H), 2.62-2.52 (m, 4H), 2.34 (s, 3H); 13C NMR (126 MHz, CDCl3) δ
187.12, 166.48, 164.48, 158.09, 157.94, 157.60, 153.11, 152.77, 148.84, 147.26, 138.15,
133.52, 133.44, 130.03, 128.15, 121.79, 119.40, 117.58, 116.97, 116.79, 114.66, 108.02,
103.56, 100.31, 55.72, 55.19, 50.04, 46.16; HRMS-ESI (m/z): [M+H]+ calcd. for
C32H30O3N7ClF, 614.20772; found, 614.20877.
2.2.9. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)-phenyl)-2-cyano-3-(4-
(methylthio)phenyl)acrylamide (5b)
Compound 5b was prepared as described in general procedure I using 15a (0.050 g,
0.10 mmol), 4-(methylthio)benzaldehyde (0.022 g, 0.15 mmol), piperidine (1 µL, 0.01 mmol)
and anhydrous ethanol (2 mL). The crude product was purified by flash silica gel
chromatography using DCM/MeOH (98/2, v/v) as eluent to obtain 0.034 g of 5b
1
(0.053 mmol, 53%) as a pale-yellow solid. H NMR (500 MHz, CDCl3) δ 8.32 (s, 1H), 8.23
(s, 1H), 8.05 (s, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.62 (t, J = 1.9 Hz, 2H), 7.56 (d, J = 8.1 Hz,
1H), 7.46 (t, J = 8.1 Hz, 1H), 7.43 (s, 1H), 7.30 (d, J = 8.5 Hz, 2H), 7.06 (dd, J = 8.1, 1.5 Hz,
1H), 6.44 (d, J = 2.3 Hz, 1H), 6.23 (s, 1H), 3.81 (s, 3H), 3.21- 3.02 (m, 4H), 2.61- 2.58 (m,
4H), 2.54 (s, 3H), 2.35 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 164.78, 158.80, 158.20,
157.89, 153.67, 153.36, 149.14, 147.45, 141.45, 138.55, 131.61, 130.27, 128.19, 125.93,
11

122.10, 119.74, 119.49, 117.76, 117.68, 116.17, 114.89, 108.42, 102.21, 100.63, 55.98, 55.46,
50.24, 46.35, 15.07; HRMS-ESI (m/z): [M+H]+ calcd. for C33H33O3N7ClS, 642.20486; found,
642.20579.
2.2.10. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)-phenyl)-2-cyano-3-(4-nitrophenyl)acrylamide (5c)
Compound 5c was prepared as described in general procedure I using 15a (0.060 g,
0.12 mmol), 4-nitrobenzaldehyde (0.017 g, 0.12 mmol), piperidine (1.2 µL, 0.01 mmol) and
anhydrous ethanol (2 mL). The crude product was purified by flash silica gel chromatography
using DCM/MeOH (98.5/1.5, v/v) as eluent to obtain 0.065 g of 5c (0.10 mmol, 84%) as a
1
yellow solid. H NMR (400 MHz, DMSO-d6) δ 10.72 (s, 1H), 8.48-8.31 (m, 4H), 8.17 (d,
J = 8.1 Hz, 2H), 8.13 (s, 1H), 7.71-7.57 (m, 2H), 7.49 (t, J = 7.8 Hz, 1H), 7.25 (d, J = 8.4 Hz,
1H), 7.08 (d, J = 7.7 Hz, 1H), 6.53 (s, 1H), 6.22 (s, 1H), 3.73 (s, 3H), 3.12-2.98 (m, 4H), 2.50-
2.34 (s, 4H), 2.20 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 163.61, 160.03, 158.04, 152.15,
148.92, 148.74, 139.31, 137.87, 131.07, 129.95, 124.68, 124.32, 123.68, 119.52, 117.92,
117.65, 117.61, 115.44, 113.82, 110.90, 106.41, 103.88, 99.87, 55.54, 54.57, 48.54, 45.68;
HRMS-ESI (m/z): [M+H]+ calcd. for C32H30O5N8Cl, 641.20222; found, 641.20295.
2.2.11. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)-phenyl)-2-cyano-3-(3-nitrophenyl)acrylamide (5d)
Compound 5d was prepared as described in general procedure I using 15a (0.060 g,
0.12 mmol), 3-nitrobenzaldehyde (0.017 g, 0.12 mmol), piperidine (1.2 µL, 0.01 mmol) and
anhydrous ethanol (2 mL). The crude product was purified by flash silica gel chromatography
using DCM/MeOH (98.5/1.5, v/v) as eluent to obtain 0.064 g of 5d (0.1 mmol, 83%) as
yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 8.80 (s, 1H), 8.49-8.36 (m, 2H),
8.37-8.30 (m, 2H), 8.13 (s, 1H), 7.90 (t, J = 8.0 Hz, 1H), 7.71-7.58 (m, 2H), 7.49 (t,
J = 8.1 Hz, 1H), 7.25 (d, J = 8.7 Hz, 1H), 7.08 (d, J = 7.8 Hz, 1H), 6.53 (s, 1H), 6.20 (s, 1H),
12

3.74 (s, 3H), 3.13-2.99 (m, 4H), 2.50-2.38 (m, 4H), 2.20 (s, 3H); 13C NMR (101 MHz,
DMSO-d6) δ 163.65, 160.06, 158.31, 158.09, 152.14, 148.82, 148.43, 148.10, 139.33, 135.80,
133.32, 131.03, 129.95, 126.54, 124.36, 119.54, 117.92, 117.59, 115.47, 113.83, 113.70,
109.87, 109.83, 106.40, 103.97, 99.85, 55.54, 54.56, 48.52, 45.65; HRMS-ESI (m/z): [M+H]+
calcd. for C32H30O5N8Cl, 641.20222; found, 641.20323.
2.2.12. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)-phenyl)-2-cyano-3-(4-
(dimethylamino)phenyl)acrylamide (5e)
Compound 5e was prepared as described in general procedure I using 15a (0.050 g,
0.10 mmol), 4-(dimethylamino)benzaldehyde (0.022 g, 0.15 mmol), piperidine (1.5 µL,
0.01 mmol) and anhydrous ethanol (2 mL). The precipitate was filtered and washed with cold
1
ethanol (10 mL) to obtain 0.045 g of 5e (0.07 mmol, 70%) as bright yellow solid. H NMR
(500 MHz, CDCl3) δ 8.23 (d, J = 2.4 Hz, 2H), 7.99 (s, 1H), 7.91 (d, J = 9.0 Hz, 2H), 7.64 (s,
2H), 7.54 (d, J = 8.2 Hz, 1H), 7.43 (t, J = 8.1 Hz, 2H), 7.02 (dd, J = 8.1, 1.4 Hz, 1H), 6.70 (d,
J = 9.0 Hz, 2H), 6.44 (d, J = 2.2 Hz, 1H), 6.24 (s, 1H), 3.81 (s, 3H), 3.11- 3.08 (m, 10H),
2.61-2.49 (m, 4H), 2.32 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 164.58, 160.21, 157.86,
157.69, 153.72, 153.67, 153.11, 148.87, 139.82, 138.88, 133.98, 129.89, 121.75, 119.68,
119.57, 119.04, 118.62, 117.25, 114.38, 111.66, 108.14, 103.87, 100.27, 95.05, 55.75, 55.28,
50.10, 46.23, 40.15; HRMS-ESI (m/z): [M+H]+ calcd. for C34H36O3N8Cl, 639.25934; found,
639.26022.
2.2.13. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)-phenyl)-2-cyano-3-(4-cyanophenyl)acrylamide (5f)
Compound 5f was prepared as described in general procedure I using 15a (0.060 g,
0.12 mmol), 4-cyanobenzaldehyde (0.015 g, 0.12 mmol), piperidine (2.0 µL, 0.02 mmol) and
anhydrous ethanol (2 mL). The crude product was purified by flash silica gel chromatography
13

using DCM/MeOH (98.5/1.5, v/v) as eluent to obtain 0.064 g of 5f (0.1 mmol, 86%) as an
1
off-white solid. H NMR (400 MHz, DMSO-d6) δ 10.71 (s, 1H), 8.37 (s, 2H), 8.22-7.98 (m,
5H), 7.75-7.55 (m, 2H), 7.49 (t, J = 7.7 Hz, 1H), 7.26 (d, J = 8.2 Hz, 1H), 7.08 (d, J = 7.6 Hz,
1H), 6.54 (s, 1H), 6.21 (s, 1H), 3.74 (s, 3H), 3.13-2.98 (m, 4H), 2.41-2.37 (m, 4H), 2.21 (s,
3H); 13C NMR (101 MHz, DMSO-d6) δ 163.69, 160.06, 158.29, 158.10, 152.19, 151.09,
149.22, 148.47, 139.32, 136.14, 133.11, 130.45, 129.95, 119.53, 118.23, 117.95, 117.62,
115.48, 114.04, 113.84, 110.33, 106.42, 103.97, 100.14, 99.87, 55.55, 54.57, 48.54, 45.68;
HRMS-ESI (m/z): [M+H]+ calcd. for C33H30O3N8Cl, 621.21239; found, 621.21303.
2.2.14. (E)-N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-2-cyano-3-(pyridin-4-yl)acrylamide (5g)
Compound 5g was prepared as described in general procedure I using 15a (0.060 g,
0.12 mmol), 4-pyridinecarboxaldehyde (16 µL, 0.17 mmol), piperidine (1.2 µL, 0.01 mmol)
and anhydrous ethanol (2 mL). The crude product was purified by flash silica gel
chromatography using DCM/MeOH (96/4, v/v) as eluent to obtain 0.058 g of 5g
1
(0.097 mmol, 81%) as pale yellow solid. H NMR (500 MHz, DMSO-d6) δ 10.74 (s, 1H),
8.84 (d, J = 5.3 Hz, 2H), 8.37 (s, 1H), 8.33 (s, 1H), 8.11 (s, 1H), 7.83 (d, J = 5.1 Hz, 2H),
7.70-7.61 (m, 2H), 7.50 (t, J = 8.1 Hz, 1H), 7.28 (d, J = 8.6 Hz, 1H), 7.08 (d, J = 7.9 Hz, 1H),
6.54 (s, 1H), 6.22 (bs, 1H), 3.75 (s, 3H), 3.15-3.00 (m, 4H), 2.51-2.39 (m, 4H), 2.24 (s, 3H);
13C NMR (126 MHz, DMSO-d6) δ 164.52, 160.61, 158.91, 152.97, 151.66, 149.51, 140.08,
139.75, 130.73, 123.77, 123.44, 122.93, 120.46, 118.79, 118.43, 115.90, 114.69, 112.68,
110.58, 107.32, 104.89, 104.78, 100.78, 56.43, 55.35, 49.33, 46.39; [M+H]+ calcd. for
C31H30O3N8Cl, 597.21239; found, 597.21285.
2.2.15. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)-phenyl)-2-cyano-3-(furan-3-yl)acrylamide (5h)
14

Compound 5h was prepared as described in general procedure I using 15a (0.048 g,
0.09 mmol), 3-furaldehyde (15 µL, 0.19 mmol), piperidine (1.0 µL, 0.01 mmol) and
anhydrous ethanol (2 mL). The crude product was purified by flash silica gel chromatography
using DCM/MeOH (98/2, v/v) to obtain 0.048 g of 5h (0.08 mmol, 89%) as brown solid.
1H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 8.26 (s, 1H), 8.08 (s, 1H), 7.96 (s, 1H), 7.70-7.61
(m, 2H), 7.61-7.53 (m, 2H), 7.49 (t, J = 8.1 Hz, 1H) 7.45 (bs, 1H), 7.25 (s, 1H), 7.09 (d,
J = 8.0 Hz, 1H), 6.47 (d, J = 2.3 Hz, 1H), 6.24 (bs, 1H), 3.84 (s, 3H), 3.19-3.09 (m, 4H), 2.66-
2.55 (m, 4H), 2.38 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 163.64, 160.43, 158.08,
152.07, 150.86, 146.27, 144.77, 142.49, 139.53, 137.59, 130.09, 129.81, 124.53, 120.43,
119.58, 117.61, 116.32, 115.62, 113.85, 107.54, 106.40, 105.02, 103.98, 99.91, 55.50, 54.42,
48.32, 45.46; [M+H]+ calcd. for C30H29O4N7Cl, 586.19641; found, 586.19564.
2.2.16. N-(3-((5-chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)-phenyl)-2-cyano-3-(1H-imidazol-5-yl)acrylamide
(5i)
Compound 5i was prepared as described in general procedure I using 15a (0.050 g,
0.10 mmol), 4-formylimidazole (0.014 g, 0.15 mmol), piperidine (1.0 µL, 0.01 mmol) and
anhydrous ethanol (2 mL). The crude product was purified by flash silica gel chromatography
using DCM/MeOH (97/3, v/v) to obtain 0.044 g of 5i (0.075 mmol, 75% yield) as brown
1
solid. H NMR (400 MHz, DMSO-d6) δ 12.84 (bs, 1H), 10.31 (s, 1H), 8.35 (s, 1H), 8.18 (s,
1H), 8.14 (s, 1H), 8.05 (s, 1H), 7.99 (s, 1H), 7.65 (d, J = 6.7 Hz, 2H), 7.43 (t, J = 8.4 Hz, 1H),
7.25 (d, J = 8.8 Hz, 1H), 7.01 (d, J = 7.4 Hz, 1H), 6.52 (s, 1H), 6.20 (s, 1H), 3.73 (s, 3H),
3.09-2.99 (m, 4H), 2.45-2.36 (m, 4H), 2.19 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ
164.48, 162.07, 159.20, 158.89, 152.85, 140.61, 139.41, 139.38, 130.56, 120.31, 118.38,
118.11, 117.89, 117.48, 116.69, 114.63, 112.80, 111.37, 107.17, 104.73, 101.75, 100.68,
15

100.64, 56.35, 55.43, 49.40, 46.59; [M+H]+ calcd. for C30H29O4N7Cl, 586.20764; found,
586.20699.
General procedure II for the preparation of compounds 5j-5m: In a sealed tube an aldehyde
of choice and piperidine were added to a solution of 15a in acetic acid. The solution was
heated to 80 °C for 0.5 h, cooled to ambient temperature and then poured into ice-water. The
aqueous layer was extracted with DCM (3 x 10 mL) and the combined extract was further
washed with saturated aqueous sodium bicarbonate solution (10 mL) and water (10 mL). The
organic layer was dried over anhydrous sodium sulfate and concentrated under reduced
pressure. The crude product was purified by flash silica gel chromatography to obtain the
desired products as a mixture of cis/trans isomers.
2.2.17. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-2-cyano-3-cyclopropylacrylamide (5j)
Compound 5j was prepared as described in general procedure II using 15a (0.050 g,
0.10 mmol), cyclopropanecarbaldehyde (7.3 µL, 0.10 mmol), piperidine (1 µL, 0.01 mmol)
and acetic acid (1 mL). The crude product was purified by flash silica gel chromatography
using DCM/MeOH (98/2, v/v) as eluent to obtain 0.046 g of 5j (0.082 mmol, 82%) as a
mixture of cis/trans isomers. Solely the spectroscopic data of the major isomer are reported.
1H NMR (500 MHz, CDCl3) δ 8.23 (s, 1H), 7.83 (s, 1H), 7.62 (bs, 1H), 7.56 (s, 1H), 7.51 (d,
J = 7.7 Hz, 1H), 7.44 (t, J = 7.8 Hz, 2H), 7.14 (d, J = 11.5 Hz, 1H), 7.04 (d, J = 7.6 Hz, 1H),
6.44 (s, 1H), 6.21 (bs, 1H), 3.81 (s, 3H), 3.-3.14 (m, 4H), 2.77-2.65 (m, 4H), 2.44 (s, 3H),
2.14-2.04 (m, 1H), 1.32-1.34 (m, 2H), 1.06-0.98 (m, 2H); 13C NMR (126 MHz, CDCl3) δ
167.74, 164.50, 157.87, 157.57, 153.12, 148.88, 147.82, 146.86, 138.28, 129.98, 122.19,
119.43, 119.14, 117.58, 115.89, 114.65, 108.44, 106.77, 105.73, 100.55, 55.78, 54.96, 49.72,
45.75, 29.79, 16.42, 11.77; [M+H]+ calcd. for C29H31O3N7Cl, 560.21714; found, 560.21751.
16

2.2.18. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-2-cyano-4-methylpent-2-enamide (5k)
Compound 5k was prepared as described in general procedure II using 15a (0.060 g,
0.12 mmol), isobutyraldehyde (21 µL, 0.24 mmol), piperidine (1 µL, 0.01 mmol) and acetic
acid (1 mL). The crude product was purified by flash silica gel chromatography using
DCM/MeOH (98/2, v/v) to obtain 0.032 g of 5k (0.057 mmol, 48%) as a mixture of cis/trans
1
isomers. Solely the spectroscopic data of the major isomer are reported. H NMR (500 MHz,
CDCl3) δ 8.24 (s, 1H), 7.81 (s, 1H), 7.61 (d, J = 10.5 Hz, 2H), 7.55 (s, 1H), 7.52 (d,
J = 8.5 Hz, 1H), 7.45 (t, J = 8.1 Hz, 1H), 7.42 (bs, 1H), 7.06 (d, J = 9.1 Hz, 1H), 6.46 (d,
J = 2.1 Hz, 1H), 6.21 (bs, 1H), 3.82 (s, 3H), 3.13-3.11 (m, 4H), 3.00 (qd, J = 13.3, 6.6 Hz,
1H), 2.64-2.53 (m, 4H), 2.36 (s, 3H), 1.19 (d, J = 6.6 Hz, 6H); 13C NMR (126 MHz, CDCl3) δ
168.31, 164.51, 157.95, 157.64, 157.46, 153.13, 147.21, 138.09, 135.85, 130.03, 121.87,
119.41, 118.19, 118.17, 117.59, 114.97, 114.65, 108.53, 108.10, 100.35, 55.75, 55.23, 50.03,
46.13, 29.84, 21.58; [M+H]+ calcd. for C29H33O3N7Cl, 562.23279; found, 562.23313.
2.2.19. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-2-cyano-4,4-dimethylpent-2-enamide (5l)
Compound 5l was prepared as described in general procedure II using 15a (0.078 g,
0.15 mmol), trimethylacetalaldehyde (34 µL, 0.30 mmol), piperidine (1.5 µL, 0.01 mmol) and
acetic acid (1 mL). The crude product was purified by flash silica gel chromatography using
DCM/MeOH (98/2, v/v) as eluent to obtain 0.062 g of 5l (0.11 mmol, 73%) as a mixture of
cis/trans isomers. Solely the spectroscopic data of the major isomer are only reported.
1H NMR (500 MHz, CDCl3) δ 8.23 (s, 1H), 8.00 (s, 1H), 7.79 (s, 1H), 7.57 (s, 1H), 7.51 (d,
J = 8.4 Hz, 1H), 7.45 (t, J = 8.1 Hz, 2H), 7.26 (s, 1H), 7.06 (dd, J = 8.0, 1.2 Hz, 1H), 6.45 (d,
J = 2.3 Hz, 1H), 6.21 (s, 1H), 3.82 (d, J = 3.8 Hz, 3H), 3.17-3.07 (m, 4H), 2.55-2.65 (m, 4H),
2.36 (s, 3H), 1.33 (s, 9H); 13C NMR (126 MHz, CDCl3) δ 170.90, 164.53, 158.03, 157.90,
17

157.62, 153.12, 148.88, 147.23, 138.19, 129.97, 121.85, 119.32, 117.61, 117.10, 115.99,
114.68, 108.12, 106.46, 100.35, 99.87, 55.75, 55.18, 49.99, 46.07, 35.35, 29.06; [M+H]+
calcd. for C30H35O3N7Cl, 576.24844; found, 576.24998.
2.2.20. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-2-cyano-3-cyclopentylacrylamide (5m)
Compound 5m was prepared as described in general procedure II using 15a (0.05 g,
0.10 mmol), cyclopentanecarbaldehyde (21 µL, 0.20 mmol), piperidine (1 µL, 0.01 mmol)
and acetic acid (1 mL). The crude product was purified by flash silica gel chromatography
using DCM/MeOH (98/2, v/v) as eluent to obtain 0.032 g of 5m (0.054 mmol, 54%) as a
mixture of cis/trans isomers. Solely the spectroscopic data of the major isomer are reported.
1H NMR (500 MHz, CDCl3) δ 8.23 (s, 1H), 7.86 (s, 1H), 7.68 (d, J = 10.6 Hz, 2H), 7.55 (s,
1H), 7.51 (d, J = 7.8 Hz, 1H), 7.47-7.36 (m, 2H), 7.05 (d, J = 7.8 Hz, 1H), 6.44 (s, 1H), 6.21
(bs, 1H), 3.81 (s, 3H), 3.21-3.03 (m, 5H), 2.69-2.53 (m, 4H), 2.37 (s, 3H), 2.06-1.92 (m, 2H),
1.89-1.63 (m, 4H), 1.57-1.41 (m, 2H); 13C NMR (126 MHz, CDCl3) δ 167.18, 164.50, 157.97,
157.64, 153.19, 138.07, 132.74, 130.00, 125.01, 121.88, 119.31, 117.58, 115.23, 114.65,
114.58, 108.79, 108.15, 102.95, 102.83, 100.37, 55.76, 55.21, 49.99, 45.91, 42.96, 33.19,
25.96; [M+H]+ calcd. for C31H35O3N7Cl, 588.24844; found, 588.24740.
General procedure III for the preparation of compounds 17a-c: To a stirred solution of an
aldehyde of choice and trifluoropropionic acid in dry THF was added TiCl4 at room
temperature. The reaction mixture was stirred for 0.5 h, followed by drop wise addition of
Et3N. After being stirred for additional 24 h, the reaction was quenched by the addition of
water (20 mL), and the obtained mixture was extracted with DCM (3 x 30 mL). The
combined organic layers were dried over anhydrous sodium sulfate and concentrated under
reduced pressure. The crude product was purified by flash silica gel chromatography to obtain
the desired products as a mixture of cis/trans isomers.
18

2.2.21. 3-(4-Fluorophenyl)-2-(trifluoromethyl)acrylic acid (17a)
Compound 17a was prepared as described in general procedure III using 4-
flurobenzaldehyde (0.21 g, 1.75 mmol), trifluoropropionic acid (0.15 g, 1.17 mmol), dry THF
(2 mL), TiCl4 (1.0 M in DCM, 1.7 mL, 1.75 mmol) and Et3N (0.82 mL, 5.85 mmol). The
crude product was purified by flash silica gel chromatography using DCM/MeOH (98/2, v/v)
as eluent to obtain 0.25 g of 17a (1.07 mmol, 91%) as a mixture of cis/trans isomers. Solely
1
the spectroscopic data of the single isomer are reported. H NMR (500 MHz, DMSO-d6) δ
8.12 (s, 1H), 7.56-7.45 (m, 2H), 7.28 (t, J = 8.8 Hz, 2H); 13C NMR (126 MHz, DMSO-d6) δ
163.87, 161.89, 146.41, 131.66 (JC-F = 6.5 Hz), 128.97, 123.25, 121.07, 115.42
(JC-F = 21.9 Hz).
2.2.22. 3-(4-(Dimethylamino)phenyl)-2-(trifluoromethyl)acrylic acid (17b)
Compound 17b was prepared as described in general procedure III using 4-
dimethylaminobenzaldehyde (0.24 g, 1.75 mmol), trifluoropropionic acid (0.15 g,
1.17 mmol), TiCl4 (1.0 M in DCM, 1.7 mL, 1.75 mmol), Et3N (0.82 mL, 5.85 mmol) and dry
THF (3 mL). The crude product was purified by flash silica gel chromatography using
DCM/MeOH (98/2, v/v) as eluent to obtain 0.19 g of 17b (0.73 mmol, 62%) as a mixture of
1
cis/trans isomers. Solely the spectroscopic data of the single isomer are reported. H NMR
(500 MHz, DMSO-d6) δ 7.93 (s, 1H), 7.42 (d, J = 8.7 Hz, 2H), 7.25 (bs, 1H), 6.75 (d, J = 8.7
Hz, 2H), 3.01 (s, 6H); 13C NMR (101 MHz, DMSO-d6) δ 189.83, 167.18, 151.26, 150.61,
132.06, 131.29, 120.63, 111.36, 39.73.
2.2.23. 3-(4-Methoxyphenyl)-2-(trifluoromethyl)acrylic acid (17c)
Compound 17c was prepared as described in general procedure III using 4-
methoxybenzaldehyde (0.24 g, 1.75 mmol), trifluoropropionic acid (0.15 g, 1.17 mmol), TiCl4
(1.0 M in DCM, 1.7 mL, 1.75 mmol), Et3N (0.82 mL, 5.85 mmol) and dry THF (3 mL). The
crude product was purified by flash silica gel chromatography using DCM/MeOH (98/2, v/v)
19

as eluent to obtain 0.20 g of 17c (0.08 mmol, 68%) as a mixture of cis/trans isomers. Solely
1
the spectroscopic data of the single isomer are reported. H NMR (500 MHz, DMSO-d6) δ
8.04 (s, 1H), 7.45 (d, J = 8.7 Hz, 2H), 7.01 (d, J = 8.7 Hz, 2H), 3.80 (s, 3H); 13C NMR
(126 MHz, DMSO-d6) δ 164.35, 161.13, 147.27, 132.03 (JC-F = 2.3 Hz), 131.51, 124.40,
114.10, 113.98, 55.36.
General procedure IV for the preparation of compounds 5n-p: Intermediate 13 and an
acrylic acid of choice (17a-c) were dissolved in anhydrous DMF and cooled to 0 °C.
Subsequently, EDC.HCl, HOBt and DIPEA were added and the resulting reaction mixture
was stirred at room temperature for 18 h. The reaction mixture was diluted with DCM
(20 mL) and washed with saturated aqueous sodium bicarbonate solution (10 mL), saturated
aqueous ammonium chloride solution (10 mL) and water (10 mL). The organic layer was
dried over anhydrous sodium sulfate and the solvent was removed in vacuo. The crude was
purified by flash silica gel chromatography to obtain the desired compounds.
2.2.24. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)-phenyl)-3-(4-fluorophenyl)-2-
(trifluoromethyl)acrylamide (5n)
Compound 5n was prepared as described in general procedure IV using 13 (0.020 g,
0.045 mmol), 17a (0.016 g, 0.068 mmol), anhydrous DMF (2 mL), EDC.HCl (0.013 g,
0.068 mmol), HOBt (0.008 g, 0.054 mmol) and DIPEA (23 µL, 0.136 mmol). The crude was
purified by flash silica gel chromatography using DCM/MeOH (98/2, v/v) as eluent to obtain
0.006 g of 5n (0.009 mmol, 20%) as yellow solid. 1H NMR (500 MHz, CDCl3) δ 8.21 (s, 1H),
7.48-7.38 (m, 3H), 7.36-7.23 (m, 5H), 7.08-6.94 (m, 4H), 6.48 (t, J = 2.8 Hz, 1H), 6.17 (bs,
1H), 3.85 (s, 3H), 3.14-3.10 (m, 4H), 2.65 – 2.54 (m, 4H), 2.36 (s, 3H); HRMS-ESI (m/z):
[M+H]+ calcd. for C32H30O3N6ClF4, 657.19986; found, 657.20108.
20

2.2.25. N-(3-((5-chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-(4-(dimethylamino)phenyl)-2-
(trifluoromethyl)acrylamide (5o)
Compound 5o was prepared as described in general procedure IV using 13 (0.020 g,
0.045 mmol), 17b (0.017 g, 0.068 mmol), EDC.HCl (0.013 g, 0.068 mmol), HOBt (0.008 g,
0.054 mmol), DIPEA (23 µL, 0.136 mmol) and anhydrous DMF (2 mL). The crude was
purified by flash silica gel chromatography using DCM/MeOH (98/2, v/v) as eluent to obtain
0.005 g of 5o as yellow solid (0.007 mmol, 16%).1H NMR (500 MHz, CDCl3) δ 8.20 (d,
J = 3.9 Hz, 1H), 7.61-7.33 (m, 5H), 7.32-7.29 (m, 1H), 7.16 (d, J = 8.3 Hz, 1H), 7.10 (d,
J = 8.1 Hz, 1H), 6.96 (t, J = 6.4 Hz, 2H), 6.61 (d, J = 8.3 Hz, 1H), 6.56 (d, J = 8.3 Hz, 1H),
6.47 (s, 1H), 6.21 (bs, 1H), 3.84 (s, 3H), 3.22-3.14 (m, 4H), 2.91 (s, J = 3.3 Hz, 6H), 2.74-
2.63 (m, 4H), 2.42 (s, 3H); HRMS-ESI (m/z): [M+H]+ calcd. for C34H36O3N7ClF3, 682.25148;
found, 682.25264.
2.2.26. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)-phenyl)-3-(4-methoxyphenyl)-2-
(trifluoromethyl)acrylamide (5p)
Compound 5p was prepared as described in general procedure IV using 13 (0.060 g,
0.13 mmol), 17b (0.067 g, 0.27 mmol), EDC.HCl (0.039 g, 0.20 mmol), HOBt (0.022 g,
0.16 mmol), DIPEA (70 µL, 0.41 mmol) and anhydrous DMF (3 mL). The crude was purified
by flash silica gel chromatography using DCM/MeOH (98/2, v/v) as eluent to obtain 0.021 g
1
of 5p (0.031 mmol, 24%) as yellow solid. H NMR (500 MHz, CDCl3) δ 8.20 (d, J = 2.8 Hz,
1H), 7.59-7.34 (m, 5H), 7.26-7.23 (m, 2H), 7.18 (d, J = 8.0 Hz, 1H), 7.08-6.92 (m, 2H), 6.84
(d, J = 7.8 Hz, 1H), 6.79 (d, J = 8.1 Hz, 1H), 6.48 (s, 1H), 6.19 (bs, 1H), 3.85 (s, 3H), 3.75 (s,
3H), 3.20-3.07 (m, 4H), 2.66-2.54 (m, 4H), 2.38 (s, 3H).; HRMS-ESI (m/z): [M+H]+ calcd.
for C33H33O4N6ClF3, 669.21984; found, 669.22087.
21

2.2.27. (E)-3-Cyclopropylacrylic acid (20)
Malonic acid (0.29 g, 2.85 mmol), cyclopropanecarbaldehyde (0.10 g, 1.42 mmol) and
piperidine (0.14 mL, 1.42 mmol) were dissolved with pyridine (1 mL) and stirred at 80 °C for
2 h. After cooling to ambient temperature the reaction mixture was stirred for further 18 h.
The mixture was poured into 2 M hydrochloric acid (10 mL) and then extracted with DCM
(30 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated under
reduced pressure and the remaining residue was purified by flash silica gel chromatography
using ethyl acetate/petroleum ether (1/1, v/v) as eluent to obtain 0.067 g of 20 as off-white
1
solid (0.597 mmol, 42%). H NMR (400 MHz, DMSO-d6) δ 11.96 (bs, 1H), 6.30 (dd,
J = 15.3, 10.2 Hz, 1H), 5.83 (d, J = 15.4 Hz, 1H), 1.72-1.36 (m, 1H), 0.89 (d, J = 5.6 Hz, 2H),
0.63-0.61 (m, 2H); 13C NMR (101 MHz, DMSO-d6) δ 167.15, 154.02, 118.72, 14.12, 8.36.
2.2.28. (E)-N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy) -phenyl)-3-cyclopropylacrylamide (21)
Intermediate 13 (0.124 g, 0.28 mmol) and acrylic acid 20 (0.031 g, 0.28 mmol) were
dissolved in anhydrous DCM (4 mL) and cooled to 0 °C. Subsequently, EDC.HCl (0.080 g,
0.41 mmol), HOBt (0.044 g, 0.33 mmol) and DIPEA (0.14 mL, 0.83 mmol) were added and
the resulting mixture was stirred for 18 h at room temperature. The reaction mixture was
diluted with DCM (20 mL) and washed with saturated aqueous sodium bicarbonate solution
(10 mL), saturated aqueous ammonium chloride solution (10 mL) and water (10 mL). The
organic layer was dried over anhydrous sodium sulfate and concentrated under reduced
pressure. The crude product was purified by flash silica gel column chromatography with
DCM/MeOH (98/2, v/v) to obtain 0.053 g of the desired product 21 (0.099 mmol, 35%) as
1
white solid. H NMR (400 MHz, DMSO-d6) δ 10.09 (s, 1H), 8.34 (s, 1H), 8.10 (s, 1H), 7.61
(s, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.38 (t, J = 8.1 Hz, 1H), 7.25 (d, J = 8.6 Hz, 1H), 6.91 (d,
J = 7.1 Hz, 1H), 6.52 (s, 1H), 6.32 (dd, J = 15.0, 10.0 Hz, 1H), 6.18 (d, J = 15.3 Hz, 2H), 3.73
22

(s, 3H), 3.11-2.97 (m, 4H), 2.50-2.37 (m, 4H), 2.23 (s, 3H), 1.70-1.56 (m, 1H), 0.91 (d,
J = 5.5 Hz, 2H), 0.67-0.57 (m, 2H); 13C NMR (101 MHz, DMSO-d6) δ 163.68, 158.27,
158.00, 152.11, 150.08, 148.42, 148.34, 140.70, 129.76, 121.36, 119.55, 116.28, 116.22,
112.66, 112.57, 108.16, 106.39, 103.98, 99.84, 55.52, 54.59, 48.54, 45.70, 14.12, 8.27;
HRMS-ESI (m/z): [M+H]+ calcd. for C28H32O3N6Cl, 535.22189; found, 535.22118.
2.2.29. 2-Cyano-3-cyclopropylacrylic acid (23)
To a solution of cyclopropanecarbaldehyde (0.36 g, 5.25 mmol) in dry toluene (10 mL) was
added cyanoacetic acid (0.44 g, 5.25 mmol), piperidine (1.0 mL, 10.5 mmol) and glacial
acetic acid (1.2 mL, 20.9 mmol). The reaction mixture was stirred at 80 °C for 2 h and was
then cooled to room temperature. Excess solvent was removed in vacuo and the remaining
residue was dissolved in ethyl acetate (20 mL). The organic layer was washed with aqueous
1 M HCl solution (10 mL), water (10 mL), dried over anhydrous sodium sulfate and
evaporated to obtain the crude oily product. Purification by silica gel flash chromatography
using DCM/MeOH (98/2, v/v) as eluent yielded 0.35 g of the desired product 23 as white
1
solid (2.55 mmol, 49%). H NMR (500 MHz, DMSO-d6) δ 6.82 (d, J = 11.3 Hz, 1H), 3.96
(bs, 1H), 1.81 (dd, J = 7.7, 3.7 Hz, 1H), 1.20- 1.04 (m, 2H), 0.86- 0.75 (m, 2H); 13C NMR
(126 MHz, DMSO-d6) δ 164.38, 163.44, 117.29, 111.10, 14.74, 9.76.
2.2.30. 2-Cyano-3-cyclopropylpropanoic acid (24)
Pd/C (0.05 g, 10 wt. %) was suspended in anhydrous MeOH (4 mL) and to that a solution of
acrylic acid 23 (0.25 g, 1.82 mmol) in MeOH (2 mL) was added. The atmosphere of the
reaction mixture was exchanged for H2 using a balloon equipped with a needle adapter and
the suspension was stirred at room temperature for 5 h. The crude reaction mixture was
diluted with ethyl acetate (10 mL) and filtered through celite. The filtrate was concentrated in
vacuo to afford 0.25 g compound 24 (1.80 mmol, 99%) as colorless oil which was used
1
without further purification. H NMR (400 MHz, DMSO-d6) δ 3.75 (bs, 1H), 3.46-3.28 (m,
23

1H), 1.87-1.54 (m, 2H), 1.46-1.17 (m, 2H), 0.85-0.68 (m, 1H), 0.47-0.33 (m, 1H), 0.19-0.07
(m, 1H); 13C NMR (101 MHz, DMSO-d6) δ 170.05, 121.66, 35.66, 9.82, 5.32, 4.84.
2.2.30. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)pyrimidin-4-yl)oxy)-phenyl)-2-cyano-3-cyclopropylpropanamide (25)
Intermediate 13 (0.080 g, 0.18 mmol) and acrylic acid 24 (0.038 g, 0.27 mmol) were
dissolved in anhydrous DMF (2 mL) and cooled to 0 °C. Subsequently, EDC.HCl (0.070 g,
0.36 mmol), HOBt (0.030 g, 0.21 mmol) and DIPEA (0.12 mL, 0.72 mmol) were added and
the resulting mixture was stirred for 18 h at room temperature. The reaction mixture was
diluted with DCM (20 mL) and washed with aqueous sodium bicarbonate solution (10 mL),
saturated aqueous ammonium chloride solution (10 mL) and water (10 mL). The organic layer
was dried over anhydrous sodium sulfate and the solvent was removed in vacuo. The crude
product was purified by flash silica gel chromatography using DCM/MeOH (97/3, v/v) as
eluent to obtain 0.054 g of the desired product 25 (0.096 mmol, 53%) as white solid. 1H NMR
(500 MHz, DMSO-d6) δ 10.56 (s, 1H), 8.35 (s, 1H), 8.08 (s, 1H), 7.52-7.50 (m, 1H), 7.49-
7.40 (s, 2H), 7.23 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 7.9 Hz, 1H), 6.53 (d, J = 2.3 Hz, 1H), 6.18
(bs, 1H), 3.99-3.87 (m, 1H), 3.74 (s, 3H), 3.11-3.03 (m, 4H), 2.51-2.43 (m, 4H), 2.25 (s, 3H),
1.95-1.77 (m, 2H), 1.37-1.29 (m, 1H), 0.91-0.81 (m, 2H), 0.52-0.51 (m, 1H), 0.24-0.13 (m,
1H); 13C NMR (126 MHz, DMSO-d6) δ 164.13, 164.03, 163.62, 157.98, 152.21, 148.24,
139.37, 129.94, 119.52, 118.22, 118.16, 117.44, 116.62, 112.88, 109.89, 106.37, 103.87,
99.85, 55.51, 54.52, 48.47, 45.57, 34.75, 13.59, 8.41, 4.45, 3.91; ESI-MS (m/z) calcd. for
C28H32O3N6Cl, 562.0; found 562.2.
2.3. Activity-based assay for IC50 determination.
IC50 determinations for EGFRWT and its mutants (Invitrogen, Lot# 279551C for EGFRL858R and
Invitrogen, Lot# 350247C for EGFRL858R/T790M) were performed with the HTRF KinEASE-TK
24

assay from Cisbio according to the manufacturer’s instructions. An artificial substrate peptide
was phosphorylated by EGFR. After completion of the reaction, an anti-phosphotyrosine
antibody labeled with europium cryptate and streptavidin labeled with the fluorophore XL665
were added. FRET between europium cryptate and XL665 was measured to quantify the
phosphorylation of the substrate peptide. ATP concentrations were set at their respective Km-
values (2 µM for EGFRWT, 9 µM for EGFRL858R and 4 µM for EGFRL858R/T790M) while a substrate
concentration of 500 nM, 225 nM and 200 nM, respectively, was used. Kinase and inhibitor
were preincubated for 30 min before the reaction was started by addition of ATP and substrate
peptide. A Tecan infinite M1000 plate reader was used to measure the fluorescence of the
samples at 620 nm (Eu-labeled antibody) and 665 nm (XL665 labeled streptavidin) 60 µs
after excitation at 317 nm. The quotient of both intensities for reactions made with eight
different inhibitor concentrations was fit to a Hill four-parameter equation to determine IC50
values. Each reaction was performed in duplicate, and at least three independent
determinations of each IC were made.
50
2.4. Protein expression and purification.
2.4.1. cSrc
Protein expression and purification of mutant cSrc (T338M, S345C) was performed as
described elsewhere utilizing a construct that includes kinase domain residues 251–533.29-31
2.4.2. Construct Design of EGFRWT
.
DNA encoding residues comprising the kinase domain of human EGFR (uniprot entry
P00533, residues 702-1016) were synthesized (GeneArt, life technologies) including an N-
terminal His -tag, eGFP and a recognition site for HRV 3C protease (LEVLFQGP). The
6
construct was cloned into pIEX/Bac3 expression vector (Merck Millipore), using NcoI and
Bsu36I restriction sites, for deployment in BacMagic expression system (Merck Millipore).
25

Transfection, virus generation and amplification was carried out in Spodoptera frugiperda cell
line Sf21 following the BacMagic protocol.
2.4.3 EGFRWT protein expression and purification.
The protein was expressed in Sf9 cells using the BacMagic system. Following protein
expression the cells were harvested (3000 x g, 10 min), resuspended in buffer A (50 mM
TRIS-HCl, 500 mM NaCl, 5% glycerol, 1 mM DTT, pH 8) and homogenized by french press.
The lysate was cleared by centrifugation at 40.000 x g for 1 h and loaded on a column packed
with Ni-NTA Superflow resin (Qiagen). The elution was done with a gradient of buffer B
(buffer A + 500 mM imidazole) from 0-250 mM imidazole. HRV 3C cleavage was carried out
by dialysis against buffer C (20 mM TRIS-HCl, 150 mM NaCl, 2% glycerol, 1 mM DTT,
1 mM EDTA pH 7.5) overnight at 8 °C. For improved cleavage, 0.01%-0.1% (v/v) Triton X-
100 were added to the sample. Subsequently, the NaCl concentration of the preparation was
adjusted to 50-80 mM by dialysis or dilution and the protein solution was loaded on a HiTrap-
Q-HP column (GE Healthcare). The protein was eluted with buffer D (buffer C + 1 M NaCl)
with a gradient of 0-250 mM NaCl. For the final purification step the fractions containing
EGFR were combined, concentrated and applied to a HiLoad 16/600 superdex 75 pg column
(GE Healthcare) in buffer E (25 mM TRIS, 250 mM NaCl, 1 mM DTT, 10% Glycerol, pH 8).
The purified protein was concentrated to 1.74 mg/mL and stored at -80 °C until further use.
2.5. Mass spectrometry experiments.
We used the drug-resistant mutant variant of cSrc (T338M, S345C) for mass spectrometry
experiments as a model system for EGFRT790M. We incubated 52 µM of protein with 100 µM of
inhibitor in buffer (25 mM TRIS, 250 mM NaCl, 10% glycerol, 1 mM TCEP, pH 8) on ice for
1 h. We analyzed the aliquots by mass spectrometry using a Finnigan LCQ Advantage Max
mass spectrometer from Thermo. Deconvolution and visualization of the generated data was
performed using MagTran (freeware).
26

3. Results and discussion
3.1. Structure-based design of covalent reversible inhibitors (CRIs)
The basic idea of our CRI design approach was aimed for the amalgamation of the superior
inhibitory properties of the highly EGFRT790M mutant selective WZ4002 scaffold and the
potential benefits of covalent-reversible electrophiles. Thus, we designed a series of CRIs by
modifying the Michael acceptor of WZ4002 (Fig. 1C), in a way that allows the electron
deficient electrophile to reach out for Cys797 of EGFR without disrupting the overall binding
mode of the parent inhibitor (Fig. 1D). Designed covalent-reversible WZ4002-analogues (5)
equipped with various tuned electrophiles were modeled into the ATP binding pocket of
EGFRT790M (PDB-code: 3IKA) according to the binding mode of the parent inhibitor. Similar
to WZ4002, we expected the CRIs to bind to the active conformation of the kinase, with both
the C-helix and the DFG-motif in inward conformation resembling the active state of the
kinase domain. The aniline pyrimidine core forms bidentate hydrogen bond interactions with
the backbone amide of hinge residue Met793 and the chlorine substituent on the pyrimidine
core directs towards the gatekeeper.23 Our optimization efforts were directed towards the
extension of the WZ4002 core at the β-carbon of the Michael acceptor with various aromatic
(5a-i) and aliphatic (5j-n) substituents (Table 1). We speculated that small groups would have
potential advantages over bulkier substituents due to potential steric repulsions with protein
surface around Arg841. We contemplated that the introduction of polar motifs (e.g., pyridyl)
may lead to beneficial charged interactions with the side chain of Asn800 of EGFR.32 We
predicted that an additional interaction in this area would further strengthen the ligand’s
affinity towards the gatekeeper mutant.
3.2. Synthesis of a focused CRI-library
27

We synthesized a focused library of EGFR inhibitors (Table 1) utilizing WZ4002 as lead
structure by utilizing a generic route to obtain the common intermediate 13 (Scheme 1).11
Subsequently, we altered the Michael-acceptor by the introduction of various covalent-
reversible warheads and further derivatized the β-carbon of the resulting 2-substituted
acrylamide with suitable aliphatic and aromatic groups to explore the associated structure-
activity relationships (SARs). Initially, 3-nitrophenol was regio-selectively attached at the 4-
position of 2,4,5-trichloropyrimidine to obtain aryl ether 8 (Scheme 1). In parallel, 5-fluoro-2-
nitroanisole was decorated with N-methylpiperizine at the 5-position. Following reduction of
the nitro group led to compound 11, which was coupled under acidic condition with aryl ether
8 to obtain the core intermediate 12. The nitro group of 12 was reduced to obtain the
corresponding amine 13, which was coupled with cyanoacetic acid and 3,3,3-
trifluoropropanoic acid to the corresponding amide using standard coupling conditions (EDC,
HOBt, DIPEA) to obtain key intermediates 15a and 15b respectively. Next, we performed an
aldol condensation of compound 15a with various commercially available aldehydes to
generate our designed CRIs. The aromatic aldehydes underwent efficient aldol condensation
with compound 15a using piperidine as base in refluxing ethanol to afford the desired
products 5a-i predominantly as trans-olefins.33 The exclusive E-configuration was
exemplarily assigned for CRI 5g by NMR experiments (data not shown), which clearly
illustrated the NOE cross peak between the amide proton of the cyano acrylamide and the
olefin proton (Scheme 1). Equivalent signals were not observed for pyridyl and amide protons
thus confirming E-configuration for our designed CRIs. Only in few cases a small amount of
cis-olefin was observed as detected by LC-MS and NMR (data not shown). Tool compound
14 was prepared by treating compound 13 with acetic anhydride and DIPEA.
Although aldol condensations using aromatic aldehydes were successful, analogous reactions
using aliphatic aldehydes did not result in the desired products. Constitutive studies revealed
28

that strong dehydrating reaction conditions are required for the complete formation of the
olefin from the respective intermediate. Reaction conditions using various bases such as
piperidine, DBU, Et3N and KH2PO4 were applied, but did not translate into the formation of
the desired reaction products (Table 2). However, the use of aqueous ammonia or ammonium
acetate led to the formation of traces of the aldol products. Ultimately, conditions utilizing
catalytic amounts of piperdine and excess of acetic acid in refluxing benzene afforded the
desired condensation product in moderate yield (65%) after heating overnight at 80 °C.
Further optimizations led to the execution of the aldol condensations in neat acetic acid as
solvent with an equivalent amount of piperidine, 15a and the aliphatic aldehyde of choice to
afford the desired condensation products 5j-m in good yields (90%).
Having successfully synthesized the 3-substituted-2-cyanoacrylamides, we aimed for the
synthesis of a novel class of CRIs disclosing a trifluoromethyl- instead of cyano-functionality
as electron withdrawing group. The aldol condensation of aromatic aldehydes with compound
15b to produce the desired products by applying the previously used method was likewise
unsuccessful. Hence, 5n-p were synthesized in a two-step process from amine precursor 13
(Scheme 2). Initially, the 3-aryl-2-trifluoromethylacrylic acids were prepared by aldol
condensation of appropriate aromatic aldehydes with 3,3,3-trifluoropropanoic acid using TiCl4
in DCM as solvent. Subsequently, the 3-aryl-2-trifluoromethylacrylic acids were coupled to
the amine 13 using EDCI and HOBt in DMF to obtain the desired products 5n-p in good
yields (80-90%).
Tool compound 21 lacking a substituent at the α-carbon was likewise prepared in two steps.
Therefore the condensation product 20 of malonic acid and cyclopropane carboxaldehyde was
coupled to amine 13 by utilizing EDC/HOBt coupling conditions (Scheme 3). The trans-
geometry of compound 20 was confirmed by the coupling constant of the olefin protons in
1H NMR. Similarly, cyanoacetic acid was condensed with cyclopropane carboxaldehyde to
29