Scaffold Hopping and Optimization of Maleimide Based Porcupine Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.7b00662

Porcupine is an O-acyltransferase that regulates Wnt secretion. Inhibiting porcupine may block the Wnt pathway which is often dysregulated in various cancers. Consequently porcupine inhibitors are thought to be promising oncology therapeutics. A high throughput screen against porcupine revealed several potent hits that were confirmed to be Wnt pathway inhibitors in secondary assays. We developed a pharmacophore model and used the putative bioactive conformation of a xanthine inhibitor for scaffold hopping. The resulting maleimide scaffold was optimized to subnanomolar potency while retaining good physical druglike properties. A preclinical development candidate was selected for which extensive in vitro and in vivo profiling is reported.

Full text of DOI:10.1021/acs.jmedchem.7b00662

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Article
Scaffold hopping and Optimization of Maleimide based Porcupine Inhibitors
Soo Yei Ho, Jenefer Alam, Duraiswamy Athisayamani Jeyaraj, Weiling Wang, Grace Ruiting
Lin, Shi Hua Ang, Eldwin Sum Wai Tan, May Ann Lee, Zhiyuan Ke, Babita Madan, David
M Virshup, Li Jun Ding, Vithya Manoharan, Yun Shan Chew, Choon Bing Low, Vishal
Pendharkar, Kanda Sangthongpitag, Jeffrey Hill, Thomas H. Keller, and Anders Poulsen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00662 • Publication Date (Web): 03 Jul 2017
Downloaded from http://pubs.acs.org on July 4, 2017
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Scaffold hopping and Optimization of Maleimide
based Porcupine Inhibitors
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Soo Yei Ho,§* Jenefer Alam,§ Duraiswamy Athisayamani Jeyaraj,§ Weiling Wang,§ Grace
Ruiting Lin,§ Shi Hua Ang,§ Eldwin Sum Wai Tan,§ May Ann Lee,§ Zhiyuan Ke,§ Babita Madan,#
David M Virshup,# Li Jun Ding,§ Vithya Manoharan,§ Yun Shan Chew,§ Choon Bing Low,§
Vishal Pendharkar,§ Kanda Sangthongpitag,§ Jeffrey Hill,§ Thomas H. Keller§ and Anders
Poulsen§*
§Experimental Therapeutics Centre, 31 Biopolis Way, #03ꢀ01 Nanos, 138669 Singapore
#Duke&NUS Graduate Medical School Singapore, 8 College Road, Singapore 169857
ABSTRACT. Porcupine is an Oꢀacyltransferase that regulates Wnt secretion. Inhibiting
porcupine may block the Wnt pathway which is often dysregulated in various cancers.
Consequently porcupine inhibitors are thought to be promising oncology therapeutics. A high
throughput screen against porcupine revealed several potent hits that were confirmed to be Wnt
pathway inhibitors in secondary assays. We developed a pharmacophore model and used the
putative bioactive conformation of a xanthine inhibitor for scaffold hopping. The resulting
Maleimide scaffold was optimized to subꢀnanomolar potency while retaining good physical
druglike properties. A preꢀclinical development candidate was selected for which extensive in
vitro and in vivo profiling is reported.
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INTRODUCTION
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Wnt signaling is important in many cellular functions, ranging from tissue homeostasis,1,2 cell
polarization,3 cell differentiation4 and apoptosis.5 Aberrant Wnt signaling is found in a wide
variety of cancers6,7 as well as various diseases such as fibrosis,8 metabolic diseases9 and
disorders in endocrine function and bone metabolism.10 Hence various research groups have
been active in finding therapeutic solutions to target components of the Wnt pathway.11,12
In recent years, there has been an interest in Porcupine (PORCN) as a molecular target.13 Two
porcupine inhibitors, 1 (ETCꢀ159)14 (ClinicalTrials.gov NCT02521844), and 2 (LGK974)15
(ClinicalTrials.gov NCT01351103) are currently in Phase 1 clinical trials (Figure 1). PORCN, a
membrane bound Oꢀacyltransferase (MBOAT)16 enzyme residing in the endoplasmic reticulum,
is essential for the palmitoleation of the Wnt protein at the highly conserved serine residue 209.
Palmitoleation is critical for the extracellular secretion of Wnt and for its binding to the Frizzled
receptors.17 Thus small molecule inhibitors of PORCN should halt the secretion of all Wnt
ligands and therefore block Wnt signaling through both βꢀcatenin dependent and independent
pathways.
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Figure 1: Porcupine inhibitors in clinical development.
Our desire to find cancer therapeutics with new mechanisms of action prompted us to do a high
throughput screen (HTS) to find leads that block the Wnt pathway by inhibiting PORCN.18 We
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employed a cellular pathway screen for hit finding and lead optimization as no suitable
biochemical assays are available.16 A HEK293‐STF cell line expressing Wnt3A that contains a
luciferase reporter for β‐catenin mediated transcriptional activation was used. Hits that act
downstream of surface receptor activation by Wnt were identified with a second assay and
eliminated,18 as were cytotoxic compounds. Porcupine inhibition was confirmed for selected hits
using ωꢀalkynylꢀpalmitate and click chemistry19 as well as a PORCN overexpression assay.18 A
number of hits with diverse scaffolds were obtained and a xanthine20 and a phenoxyꢀacetamide
scaffold18 were found to be promising starting points and selected for lead optimization. In this
manuscript we describe the xanthine structure activity relationship and the use of a
pharmacophore model for the design of a novel scaffold as a followꢀup compound.
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CHEMISTRY
The anilines used for amide coupling described in this publication were either commercially
available or synthesized via Suzuki reaction using the respective boronic acid with the
corresponding aniline described in Schemes 1 and 2. Compound 3 was synthesized by amide
coupling using commercially available theophyllineꢀ7ꢀacetic acid with 4ꢀ(1,3ꢀthiazolꢀ2ꢀ
yl)aniline.
The Wnt pathway modulator 5 was synthesized via amide coupling from its commercially
available precursor, theophyllineꢀ7ꢀacetic acid and 4ꢀ(thiophenꢀ3ꢀyl)aniline, 4. Compounds 6 and
7 were also synthesized similarly using 2ꢀ(1Hꢀimidazolꢀ1ꢀyl)acetic acid and 2ꢀ(1Hꢀ
benzo[d]imidazolꢀ1ꢀyl)acetic acid respectively (Scheme 2).
Alkylation of 3,7ꢀdihydroꢀ1Hꢀpurineꢀ2,6ꢀdione with chloroacetic acid yielded the acid
intermediate 8. Compound 9 was obtained using amide coupling conditions. Compound 10 was
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also synthesized in a similar manner by alkylation, followed by hydrolysis and amide coupling
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(Scheme 3).
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Scheme 1. Synthesis of compound 3.a
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aReagents and conditions: (a) 4ꢀ(1,3ꢀthiazolꢀ2ꢀyl)aniline, HATU, Et3N, DCM.
Scheme 2. Synthesis of compounds 5, 6 and 7.a
aReagents and conditions: (a) 4ꢀbromoaniline, Pd(dppf)Cl2.DCM, K2CO3, 4:1 dioxaneꢀH2O, 100
oC. (b) respective acids, HATU, Et3N, DCM.
Scheme 3. Synthesis of compounds 9 and 10.a
aReagents and conditions: (a) ClCH2COOH, NaOH, water, 100oC. (b) 4ꢀ(thiophenꢀ3ꢀyl)aniline,
o
EDCl, HOBT, DMFꢀDMSO (1:1). (c) BrCH2COOEt, K2CO3, DMF, 80 C. (d) LiOH, MeOHꢀ
THFꢀH2O. (e) 4ꢀ(1,3ꢀthiazolꢀ2ꢀyl) aniline, HATU, Et3N, DCM.
Compound 12 was synthesized starting with 2ꢀchloropyridineꢀ3,4ꢀdiamine (Scheme 4).
Alkylation followed by ring closure with trimethylorthoformate yielded 4ꢀchloroꢀ3Hꢀ
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imidazol[4,5ꢀc]pyridine which then underwent hydrolysis to form intermediate 11. Alkylation of
intermediate 11 with 2ꢀchloroꢀNꢀ(4ꢀ(thiophenꢀ3ꢀyl)phenyl)acetamide followed by methylation
with methyl iodide yielded compound 12.
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Compound 14 was synthesized starting from 1Hꢀimidazoleꢀ5ꢀcarboxylic acid as described in
Scheme 5. Amidation was carried out on the acid chloride of 1Hꢀimidazoleꢀ5ꢀcarboxylic acid,
followed by alkylation and hydrolysis to yield acid intermediate 13. Compound 14 was obtained
via amide coupling as described previously.
Scheme 4. Synthesis of Compound 12.a
aReagents and conditions: (a) CH(OMe)3, 70 oC. (b) HCl in MeOH. (c) 2ꢀchloroꢀNꢀ(4ꢀ(thiophenꢀ
3ꢀyl)phenyl)acetamide, K2CO3, DMF. (d) MeI, K2CO3, DMF.
Scheme 5. Synthesis of Compound 14.a
aReagents and conditions: (a) Me2NH, SOCl2, DCM. (b) BrCH2COOEt, K2CO3, DMF, 70 oC. (c)
LiOH, MeOHꢀTHFꢀH2O. (d) 4ꢀ(thiophenꢀ3ꢀyl)aniline, HATU, Et3N, DCM.
The synthesis of 16 started with an acylation of 4ꢀ(thiazolꢀ2ꢀyl)aniline with
bromoacetylbromide, followed by Nꢀalkylation with methyl 2ꢀoxoꢀ1,2ꢀdihydroꢀ3ꢀpyridine
carboxylate to form acid intermediate 15. The acid then underwent amidation with methylamine
to yield the final compound 16 as shown in Scheme 6.
Compound 19 was synthesized starting from 3ꢀnitroꢀtriazine as shown in Scheme 7. 3ꢀnitroꢀ
triazine underwent alkylation with bromoethylacetate followed by hydrolysis to form the acid
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intermediate 17. Acid intermediate 17 was coupled with 4ꢀ(1,3ꢀthiazolꢀ2ꢀyl)aniline using HATU
as coupling reagent followed by nitro reduction using Fe in acidic conditions to yield
intermediate 18. Urea formation between 18 and 2,5ꢀdioxopyrrolidinꢀ1ꢀyl methylcarbamate
yielded the desired compound 19.
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Compound 21 was synthesized via three steps as shown in Scheme 8. 1ꢀmethyl maleimide
underwent monoꢀbromination to form 1ꢀmethylꢀ3ꢀbromo maleimide which was subsequently
treated with proline to yield acid intermediate 20. Compound 21 was obtained from coupling
acid 20 with 4ꢀ(1,3ꢀthiazolꢀ2ꢀyl)aniline using amide coupling reagents.
Scheme 6. Synthesis of Compound 16.a
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aReagents and conditions: (a) Bromoacetylbromide, Hunig’s base, DCM, ꢀ78 C to r.t. (b)
Methyl 2ꢀoxoꢀ1,2ꢀdihydroꢀ3ꢀpyridine carboxylate, NaH, DMF. (c) Methylamine, HATU, Et3N,
DMF.
Scheme 7. Synthesis of Compound 19.a
aReagents and conditions: (a) BrCH2COOEt, NaOEt in EtOH, reflux. (b) LiOH, MeOHꢀTHFꢀ
H2O. (c) 4ꢀ(1,3ꢀthiazolꢀ2ꢀyl)aniline, HATU, Et3N, DCM, r.t. (d) Fe, AcOH, EtOH, reflux. (e)
NaHCO3, EtOAc. (f) 2,5ꢀdioxopyrrolidinꢀ1ꢀyl methylcarbamate, pyridine, 70 oC
Scheme 8. Synthesis of compound 21.a
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aReagents and conditions: (a) Bromine, diethyl ether, 45oC. (b) Lꢀproline, Et3N, DMF. (c) 4ꢀ(1,3ꢀ
thiazolꢀ2ꢀyl)aniline, HATU, Et3N, DCM.
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The tricyclic compound 23 was synthesized from 1,3ꢀdimethyl,5,6ꢀdiamino uracil as described
in Scheme 9. 1,3ꢀdimethyl,5,6ꢀdiamino uracil underwent cyclization with dihydroꢀ2Hꢀpyranꢀ
2,6(3H)ꢀdione to form intermediate 22. Mono bromination was carried out at the carbon adjacent
to the carboxylic acid followed by intramolecular Nꢀalkylation to form the tricyclic precursor.
Lastly, compound 23 was obtained after amidation of the tricyclic precursor with 4ꢀ(1,3ꢀthiazolꢀ
2ꢀyl)aniline.
The phthalimide compound 27 was obtained via a 7ꢀstep synthesis described in Scheme 10. 4ꢀ
nitro phthalimide underwent methylation followed by nitro reduction to form the aniline
intermediate 24. 24 was subjected to iodination and Suzuki reaction yielded intermediate 25. The
acid 26 was obtained via the oxidation of 25 and desired compound 27 was obtained via amide
coupling with 4ꢀ(1,3ꢀthiazolꢀ2ꢀyl)aniline.
Compounds 30ꢀ44 were synthesized similarly from 2,3ꢀpyridinedicarboxylic anhydride as
described in Scheme 11. The derivatives of the maleimide scaffold were prepared using the
corresponding amine, followed by hydrogenation to form the respective bicyclic intermediates
28 and 29. The aboveꢀmentioned intermediates then underwent Nꢀalkylation followed by
hydrolysis to form the respective acid intermediates and subsequently amide coupling with the
appropriate amines to yield the desired compounds.
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Scheme 9. Synthesis of compound 23.a
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aReagents and conditions: (a) dihydroꢀ2Hꢀpyranꢀ2,6(3H)ꢀdione, DMF. (b) 2M NaOH, reflux. (c)
Br2, PCl3, dichloroethane, reflux. (d) K2CO3, ACN, 70 oC. (e) 4ꢀ(1,3ꢀthiazolꢀ2ꢀyl)aniline, HATU,
Et3N, DCM.
Scheme 10. Synthesis of compound 27.a
aReagents and conditions: (a) NaH, MeI, DMF. (b) Pd/C, H2, MeOH. (c) aq. NaNO2, 6M HCl, 0
oC. followed by KI. (d) 2ꢀallylꢀ4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolane, CsF, Pd(PPh3)4, 1,4ꢀ
o
dioxane, 100 C. (e) RuCl3.3H2O, NaIO4, CCl4ꢀACNꢀH2O. (f) 4ꢀ(1,3ꢀthiazolꢀ2ꢀyl)aniline,
HATU, Et3N, DCM.
Scheme 11. Synthesis of compounds 30ꢀ44.a
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aReagents and conditions: (a) R1NH2, toluene, 130 C. (b) Pd/C, H2, MeOH. (c) BrCH2COOEt,
K2CO3, ACN, 70 oC. (d) LiOH, MeOHꢀTHFꢀH2O. (e) R2NH2, HATU, Et3N, DCM.
RESULTS AND DISCUSSION
Xanthine SAR. The xanthine development compound 1 was derived from HTS hit 3. During
the optimization it became very clear that both the biaryl substituent (green) and the amide linker
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(blue) are absolutely required for activity on the PORCN Oꢀacyltransferase.21 In fact all known
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porcupine inhibitors share these features, i.e. compare with compounds 2, 45 and 46 in Figure 2.
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N
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1 (
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IC50 = 1 nM
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ETC-159)
IC50 = 2.9 nM
(LGK974)
IC50 = 0.4 nM
IC50 = 60 nM
Figure 2. Porcupine inhibitors discussed in the literature. Compound 4518 and 4622 share
common features with 1 and 2. The red arrow depicts a ring hydrogen bond acceptor that is
discussed in the text.21
However the heterocyclic substituent attached to the 2ꢀacetamide carbon (black in Figure 2)
varies considerably between the different PORCN inhibitors. The SAR in Table 1 shows that for
compound 1 the xanthine is absolutely essential for activity. Removal of the uracil part from the
xanthine moiety in 6 resulted in loss of activity. The inactive benzimidazole analogue 7
demonstrated that at least one of the carbonyl hydrogen bond acceptors is essential for activity.
The inactive analogue 12 established that the carbonyl in the xanthine 2’ꢀposition (See Figure 2)
is essential for biological activity.
Table 1. Xanthine SAR
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Conformational
Energy (kJ/mol)b
Compound
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STF3a IC50 (µM)a
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0.0004
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<60% @ 100 µM
<60% @ 100 µM
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<60% @ 100 µM
<60% @ 100 µM
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a: data represent mean value of triplicate experiments. b: Energy difference between putative
bioactive conformation and global energy minima calculated by the OPLS3 force field. c: Global
energy minima is electrostatically collapsed and the force field energy is unlikely to be reliable.
All porcupine inhibitors have a ring hydrogen bond acceptor in the heterocyclic substituent
attached to the 2ꢀacetamide carbon (red arrow in Figure 2). Interestingly, when the putative
bioactive conformations of xanthine 1 and 2 were superimposed, it became apparent that the ring
hydrogen bond acceptor of 1 is about 2.8 Å from that of 2 (Figure 3). We therefore hypothesize
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that 1 binds to the porcupine protein through a water molecule signified by the purple dot in
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Figure 3.
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Figure 3. Superimposition of compound 1 (green carbon atoms) and 2 (grey carbon atoms) in
their putative bioactive conformations. It was hypothesized that in compounds like 1, the ring
acceptor hydrogen bonds to the porcupine protein through a water molecule (purple ball).
Scaffold Hopping. Following the promotion of 1 to preclinical development20 we started
working on a backꢀup compound. Since no further hit was available from HTS screening,
bioisosteric replacements for the xanthines were evaluated. The scaffolds were fitted to our
pharmacophore model21 and those with a compatible low energy conformation were synthesized
(Table 2).
Table 2. Proposed xanthine bioisosters.
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Conformational
Energy (kJ/mol)b
Compound
3
R1
R2
STF3a IC50 (µM)a
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0.024
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<60% @ 100 µM
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<60% @ 100 µM
<60% @ 100 µM
0.042
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a: data represent mean value of triplicate experiments. b: Energy difference between putative
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Figure 4. Putative bioactive conformations of some of the compounds in Table 2.
Figure 4 shows the putative bioactive conformations of some of these compounds. The
imidazoleꢀcarboxamide compound 14 may be considered a xanthine in which the 2’ꢀcarbonyl has
been removed and in agreement with our pharmacophore model this compound was found to be
inactive.21 In low energy conformations of the pyridoneꢀamide 16 the amide donor is forming an
intramolecular hydrogen bond with the pyridone carbonyl. The triazoleꢀurea compound 19 has
two low energy conformations with an internal hydrogen bond between the terminal hydrogen
bond donor of the urea and either of the triazole acceptor nitrogens. One of these conformations
may be considered equivalent to a xanthine in which one of the carbonyls has been deleted. All
of the compounds 14, 16, 19 and 21 (Table 2) were found to be inactive even though they fitted
the pharmacophore model with low energy conformations. It is likely that this is due to both
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carbonyls of the xanthine being important for porcupine inhibition. The tricyclic xanthine
compound 23 was our first successful attempt to make a rigid analogue of 3 and 5. We have
previously shown in a different scaffold that a proline ring can be used to rigidify the bioactive
conformation.18 Several of these tricyclic analogues of 23 were synthesized but the activities
were generally lower than the analogous xanthines, e.g compare compounds 3 and 23 (table 2)
for which there is a representative 2ꢀfold difference.
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Our first successful design of a xanthine bioisostere was the phtalimide 27. Both the carbonyls
of the phthalimide superimpose well on those of the xanthine. Several of the phthalimides were
synthesized and were generally more potent than the analogous xanthines but also less soluble,
e.g. the solubility of 27 and 30 is 0.42 and 1.26 µg/ml, respectively. We hypothesized that the
limited solubility may be due to stacking interactions of the flat aromatic groups in the solid
state. The fused piperidineꢀmaleimide compound 30 was an attempt to reduce the sp2 character
of the phthalimide. Although the piperidineꢀmaleimide compounds were found to be less potent
than the phthalimides, they were more soluble and approximately equipotent with the xanthines.
The physical properties of 30 were satisfactory (MW = 382.4 Dalton, cLogP = 2.2, PSA =
105Å2 and 4 rotatable bonds) and the scaffold seemed to be novel, which is why it was chosen
for lead optimization.
Lead Optimization. In vitro profiling of 30 showed a reasonable profile for a lead with
Cytochrome P450 2D6 and 3A4 inhibition of >20 and 5.2 µM, respectively. Mice and human
liver microsome (MLM and HLM) clearance was moderate at 65 and 24 µl/min/mg, while
aqueous solubility at pH 7.4 and 4.0 was poor at 1.3 and 1.1 µg/ml. We first turned our attention
to improving the metabolic stability and solubility of the compounds. One of the strategies
pursued was to lower the lipophilicity of the compounds by introducing polar functionalities in
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the form of aromatic nitrogens. Table 3 shows selected compounds from a nitrogen scan around
the biphenyl of compound 31. Nitrogen was tolerated in most positions although the activity
decreased approximately 6ꢀfold for compounds 32 and 34 with nitrogen in the ortho and para
position of the terminal ring. In compound 35 with a nitrogen ortho to the amide group, the
activity increased 10ꢀfold compared to the biphenyl 31 but metabolic stability and solubility
deteriorated. Introduction of a nitrogen meta to the amide as in 36 resulted in a metabolically
very stable compound but solubility was poor. Unfortunately the introduction of a single nitrogen
did not increase solubility compared to the biphenyl. A double nitrogen scan around the biphenyl
was subsequently performed and selected compounds with in vitro profiling data are also shown
Table 3. Introduction of two or more nitrogen in the biphenyl rings gives rise to improved
metabolic stability with modest improvements in solubility as demonstrated in compounds 37
and 39.
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Table 3. Introduction of nitrogen atoms in the biaryl group.
MLMb/HLMc
CL'int
(µl/min/mg)
STF3a IC50
(µM)a
Sol. pH 7.4
(µg/ml)d
Compound
R
0.015
0.012
0.067
65/24
25/22
38/28
1.26
2.10
N.A.
30
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0.008
0.072
0.001
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N.A.
N.A.
0.24
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67/175
0.006
0.011
0.001
0.029
13/7
17/11
36/48
12/7
0.75
13.04
3.34
9.5
36
37
38
39
adata represent mean value of triplicate experiments; mouse liver microsomes; human liver
b
c
microsomes; dthermodynamic solubility at pH 7.4.
Selection of the preclinical candidate. We next synthesized a number of compounds with a
combination of small substituents on the maleimide nitrogen and the biaryl system. Table 4
summarized the in vitro pharmacokinetic data for compounds 39ꢀ44. Although in isolation the
introduction of aromatic nitrogens and small substituents did not improve solubility and
metabolic stability (data not shown), the combination produced compounds with improved
properties. Addition of a third nitrogen at the meta position of the terminal aromatic ring in
compounds 39ꢀ44 increased solubility without compromising metabolism. Since compounds 39ꢀ
44 overall had an acceptable profile the in vivo pharmacokinetics were investigated at a dose of
5mg/kg in female ICR mice by oral gavage (Table 5). Generally, the cyclopropyl analogues had
a better in vivo pharmacokinetic profile compared with the respective methyl analogues. There
was a good correlation between MLM CL’int and in vivo clearance with an R2 of 0.71. While the
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halfꢀlife was relatively constant around 2 hours, the Tmax varied between 10 min and 2 hours.
The Cmax was 750 to 4000 ng/mL where longer Tmax values resulted in a lower Cmax. An
excellent correlation (R2) of 0.84 was observed between Cmax and area under curve which
varied from 800 to 15500. Compound 40 and 42 had the best absorption and exposures among
the compounds investigated in vivo with Cmax greater than 3000 ng/ml and area under the curve
greater that 10000 ng*h/ml. The biological activity of compounds 40 and 42 were comparable
but compound 40 had a more desirable solubility compared to compound 42. In addition, the free
plasma fraction of 40 was three times higher than that of 42. Consequently we chose compound
40 for efficacy studies in a mouse xenograft model and more extensive in vivo and in vitro
profiling.
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Table 4. In vitro pharmacokinetics for compounds 39ꢀ44.
Plasma
Protein
STF3aa
CYP
MLMc /
Sol. pH
Compound
R1
R2 IC50 3A4/2D6b HLMd CL'int
(µM) IC50(µM) (µl/min/mg)
7.4/4e(µg/ml) Binding
(%)
Me 0.029
cPr 0.012
6.2/>20
9.3/9.3
12/7
9.5/13.9
60/141
95.00
97.40
39
40
24/18
Me 0.009
cPr 0.011
7.1/>30
16/8.5
21/13
16/25
41/82
98.26
99.16
41
42
14.9/13.8
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Me 0.029 >30/>30
8/8
35.5/>194
11.1/>41
96.87
98.10
43
44
cPr 0.012
8/>30
15/23
8
9
c
adata represent mean value of triplicate experiments; bCytochrome P450 enzyme; mouse liver
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d
e
microsomes; human liver microsomes; thermodynamic solubility at pH 7.4 and pH 4
respectively.
Table 5. In vivo PK for best compounds at a dose of 5 mg/kg PO in mice. See table 4 for
structures.
Compound Cmax
Tmax T1/2(h) CL
AUC0ꢀinf
(ng/mL) (h)
(L/h/kg) (ng*h/mL)
759
4031
845
2.00 1.32
0.50 1.48
2.00 2.00
0.50 1.80
0.17 2.20
0.50 2.10
1.01
0.39
0.80
0.30
5.80
4.00
4948
12663
6465
15509
859
39
40
41
42
43
44
3292
1051
1189
1244
Profiling of preclinical candidate. To confirm that compound 40 is a porcupine inhibitor, we
examined whether PORCN overexpression could blunt its effect on the Wnt/βꢀcatenin reporter.
HT1080 cells were treated with either a control plasmid or a murine PORCN containing plasmid
in combination with a Wnt/βꢀcatenin reporter plasmid and Wnt3a. Treatment of the wildꢀtype
cells with 40 inhibited luciferase activity in a dose dependent manner, but in cells overexpressing
porcupine the luciferase activity was inhibited to a much lesser extent (Figure 5).
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Figure 5. HT1080 cells were transfected with empty vector (EV) or mPORCNꢀD expression
plasmids followed by treatment with 40 at indicated concentrations. Luciferase activity was
measured after 24 hours. Error bars represent SD.
In vivo PK for an intravenous dose of 1 mg/kg of 40 in mice was obtained and the oral
absolute bioavailability was calculated to be 100%. Oral PK in rats was established with a dose
of 10 mg/kg. T1/2 in rat were 1.6 hours, Cmax was 17273 ng/ml with an exposure of 81171
ng*h/ml. An intravenous dose of 2 mg/kg allowed us to calculate the oral bioavailability in rats
to be 81%. The plasma concentration profile for both oral and intravenous administration is
shown in figure 6 for mice (left) and rats (right).
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Figure 6. Plasma concentration profile of Compound 40 in mice (left) and rat (right).
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In vivo efficacy of 40 was established in a Wntꢀdriven tumor model. MMTVꢀWnt1 tumor
fragments were implanted in the mammary fat pads of Balb/c nude mice. After 17 days, mice
bearing MMTVꢀWnt1 tumors were selected and divided into 5 groups. These were treated with
either vehicle or 1, 3 or 10 mg/kg of compound 40 once daily by oral gavage for 14 days. The
last group was treated with 3 mg/kg of our reference compound 2 which have previously been
shown to be efficacious in this model.23 Compound 40 was found to be equipotent with 2
inhibiting tumor growth in a doseꢀdependent manner as shown in figure 7. Tumor growth
inhibition for 40 ranged between 24% at 1 mg/kg to 97% at 10 mg/kg and found to be statistical
significant with a p value < 0.0001 for 2 doses (3 mg/kg and 10 mg/kg). No significant
bodyweight loss was observed in this model (See supplementary material).
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Figure 7. Inhibition of growth of MMTVꢀWnt1 tumors for 40 and reference compound 2 (Figure
1). Tumor growth inhibition for 40 was 24% at 1 mg/kg, 72% at 3 mg/kg, and 97% at 10 mg/kg.
Tumor growth inhibition compared with vehicle was statistically significant for the two highest
doses with a p value of <0.0001.
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Compound 40 was then tested in enzyme and receptor panels. No significant inhibition was
found at 10 µM in Eurofins 87 receptor panel. In a cytochrome panel with 8 isoforms Cyp3A4,
Cyp2D6 and Cyp1A2 were inhibited with IC50 values between 8.4 and 19.4 µM while no
inhibition of the other isoforms were found. This is in contrast to the Cyp3A4 assay used during
lead optimization where 40 had an IC50 of 2.1 ꢁM. In the KINOMEscan panel with 456 wild type
and mutated kinases, compound 40 was found to inhibit MEK5 and RIOK2 with Kd’s of 1.3 and
7.4 µM. No other kinases were significantly inhibited at 10 µM which resulted in the S1
selectivity score of 0. 40 was also found to be AMES negative while it had marginal hERG
inhibition in a patch clamp assay with an IC50 value of 48µM. A summary of the data for 40 is
provided in the supporting materials.
CONCLUSION
Inhibition of the Wnt pathway is an attractive approach for developing new therapeutics for the
treatment of various types of fibrosis and cancers. However the pathway is very complex with
19 human Wnt ligands that act on 10 Frizzled (Fzd) receptors. Our strategy was to find a point of
intervention that blocks signaling through all Frizzled receptors. This is accomplished through
inhibition of porcupine, an enzyme that palmitoleates Wnt ligands, thereby blocking Wnt
excretion.
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We designed a novel porcupine inhibitor scaffold using a previously developed pharmacophore
model19 and scaffold hopping. The new piperidineꢀmaleimide scaffold had reasonable physical
properties except for limited solubility. Medicinal chemistry optimization led to compounds with
nanomolar activity in our cell based assay for porcupine inhibition. The solubility problem was
solved by a combination of the addition of small substituents that may disrupt solid state
interactions and substitution of aromatic carbon atoms for nitrogen.
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Our inhibitors have good drugꢀlike properties and good in vitro PK which generally translated
into good oral PK in mice and high oral bioavailability. We selected a preꢀclinical development
candidate, compound 40 that was extensively profiled in cytochrome and receptor panels. This
compound has high efficacy in a Wntꢀdriven murine tumor model.
EXPERIMENTAL
General. All reagents were purchased from commercial sources and used as received. Reaction
progress was monitored by TLC using Merck silica gel 60 F254 coated glass plates with UV
detection at 254 nM. Shimadzu LCꢀ20AD and LCMSꢀ2020 was used for LC−MS analysis with a
Phenomenex Kinetex 2.6 ꢁm, 50 × 2.10 mm column. Proton nuclear magnetic resonance (1H
NMR) spectra were obtained using a Bruker Ultrashield 400 PLUS/R system, operating at 400
MHz. All resonance bands were referenced to tetramethylsilane (internal standard). Splitting
patterns are indicated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad peak. The
compounds’ purities were ≥95% determined by a VARIAN ProStar HPLC instrument.
Compound Synthesis.
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General Synthetic procedure for amide coupling. A stirred solution of 2ꢀ(6ꢀmethylꢀ5,7ꢀdioxoꢀ
2,3,4,5,6,7ꢀhexahydroꢀ1Hꢀpyrrolo[3,4ꢀb]pyridinꢀ1ꢀyl)acetic acid (0.21 mmol.) in DCM (5 ml)
was treated with HATU (0.252 mmol), TEA (0.42 mmol) and the corresponding amine (0.21
mmol) and stirred at r.t for 16 h. Upon reaction completion, water was added to the reaction
mixture and extracted with DCM. The combined organics was washed with brine and dried over
Na2SO4 and concentrated in vacuo. The crude compound was purified by column
chromatography to afford the desired product.
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2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-(thiazol-2-
yl)phenyl)acetamide (3). The titled compound was obtained as a white solid (yield, 39 %) with
the general amide coupling procedure using 2ꢀ(1,3ꢀdimethylꢀ2,6ꢀdioxoꢀ1,2,3,6ꢀtetrahydroꢀ7Hꢀ
1
purinꢀ7ꢀyl)acetic acid and 4ꢀ(thiazolꢀ2ꢀyl)aniline . H NMR (400 MHz; DMSOꢀd6) δ 10.67 (s,
1H), 8.08 (s, 1H), 7.92 (m, 2H), 7.88 (d, J = 3.2 Hz, 1H), 7.72 (m, 2H), 7.70 (s, 1H), 5.24 (s,
2H), 3.47 (s, 3H), 3.20 (s, 3H); MS (ESI) m/z 397.1 [C18H16N6O3S + H]+.
4-(thiophen-3-yl) aniline (4). To a stirred solution of 4ꢀbromoaniline (5.75 mmol) in 1,4ꢀ
dioxane (40 ml) and water (8 ml) was added thiophenꢀ3ꢀylboronic acid (8.62 mmol) and K2CO3
(11.49 mmol) at r.t and passed nitrogen gas through reaction mixture for 15 minutes.
Pd(dppf)Cl2.DCM (0.287 mmol) was added to the reaction mixture and degassed for another 15
o
min. The reaction mixture heated to 100 C for 4 h. After completion, the reaction mixture was
cooled to r.t, added water and extracted with ethyl acetate. The combined organic layers were
washed with brine solution, dried over anhydrous Na2SO4, filtered and dried under vacuum to
afford the crude product. The crude product was purified by column chromatography to afford 4ꢀ
1
(thiophenꢀ3ꢀyl) aniline as a pale yellow solid (yield, 48 %). H NMR (400 MHz; CDCl3) δ 7.41
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(d, J = 8.4 Hz, 2H), 7.33 (d, J = 3.1 Hz, 1H), 7.32 (d, J = 0.9 Hz, 1H), 7.31 (s, 1H), 6.71 (d, J =
8.4 Hz, 2H), 3.7 (br s, 2H); MS (ESI) m/z 176.0 [C10H9NS + H]+.
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2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-(thiophen-3-yl)phenyl)
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acetamide (5). The titled compound was obtained as a white solid (yield, 68 %) with the general
1
amide coupling procedure using theophyllineꢀ7ꢀacetic acid and 4ꢀ(thiophenꢀ3ꢀyl) aniline, 4. H
NMR (400 MHz; DMSOꢀd6) δ 10.43 (s, 1H), 8.07 (s, 1H), 7.78 (br s, 1H), 7.68 (m, 2H), 7.61 (m,
3H), 7.52 (m, 1H), 5.22 (s, 2 H), 3.47 (s, 3H), 3,21 (s, 3H); MS (ESI) m/z 396.0 [C19H17N5O3S +
H]+.
2-(1H-imidazol-1-yl)-N-(4-(thiophen-3-yl)phenyl)acetamide (6). The titled compound was
obtained as a white solid (yield, 42 %) with the general amide coupling procedure using 2ꢀ(1Hꢀ
imidazolꢀ1ꢀyl)acetic acid (0.5 mmol) and 4ꢀ(thiophenꢀ3ꢀyl) aniline (0.5 mmol), 4. 1H NMR (400
MHz; DMSOꢀd6) δ 10.51 (s, 1H), 8.60 (s, 1H), 7.80 (s, 1H), 7.69 (m, 2H), 7.61 (m, 3H), 7.54 (m,
2H), 7.42 (s, 1H), 5.11 (s, 2H); MS (ESI) m/z 396.0 [C15H13N3OS + H]+.
2-(1H-benzo[d]imidazol-1-yl)-N-(4-(thiophen-3-yl)phenyl)acetamide
(7).
The
titled
compound was obtained as a white solid (yield, 39 %) with the general amide coupling
procedure using 2ꢀ(1Hꢀbenzo[d]imidazolꢀ1ꢀyl)acetic acid and 4ꢀ(thiophenꢀ3ꢀyl) aniline, 4.1H
NMR (400 MHz, DMSOꢀd6) δ 10.53 (s, 1H), 8.23 (s, 1H), 7.80 (d, J = 2.0 Hz, 1H), 7.70ꢀ7.60
(m, 6H), 7.53 (t, J = 8.4 Hz, 2H), 7.27ꢀ7.19 (m, 2H), 5.19 (s, 2H); MS (ESI) m/z 334.3
[C19H15N3OS +H]+.
2-(2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetic acid (8). 3,7ꢀdihydroꢀ1Hꢀpurineꢀ2,6ꢀdione
(6.57 mmol) in water (4.2 ml) at r.t was added 2M NaOH solution (10.4 ml) and the resulting
solution was stirred for 30 min and chloroacetic acid (6.57 mmol) was added. The resulting
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reaction mixture was refluxed for 5 h before cooling to r.t and stirred overnight. The precipitated
solid was removed by filtration and water was acidified with concentrated HCl. The solid
collected by filtration, washed with hot EtOH to afford the titled compound as white solid (yield,
23 %). 1H NMR (400 MHz; DMSOꢀd6) δ 13.35 (br s, 1H), 11.59 (s, 1H), 10.88 (br s, 1H), 7.91(s,
1H), 5.0 (s, 2H); MS (ESI) m/z 211.1 [C7H6N4O4 + H]+.
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2-(2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-(thiophen-3-yl)phenyl)acetamide (9). A
stirred solution of 8 (0.476 mmol) in DMF (5 ml) and DMSO (5 ml) was added EDCI (0.57
mmol), HOBT (0.57 mmol) and stirred at r.t for 15 min followed by the addition of 4 (0.476
mmol). The reaction mixture was stirred at r.t for 16 h. After completion, the reaction mixture
was concentrated under vaccum, water was added to the reaction mixture the precipitated solid
1
was collected by filtration to give the titled compound as an offꢀwhite solid (yield, 40 %). H
NMR (400 MHz; DMSOꢀd6) δ 11.60 (s, 1H), 10.88 (br s, 1H), 10.43 (br s, 1H), 7.95 (s, 1H),
7.79 (d, J = 2.0 Hz, 1H), 7.68 (d, J = 8.8 Hz, 2H), 7.61ꢀ7.59 (m, 3H), 7.53 (d, J = 4.8 Hz, 1H),
5.14 (s, 2H); MS (ESI) m/z 366.10 [C17H13N5O3S + H]+.
2-((1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)amino)-N-(4-(thiazol-2-
yl)phenyl)acetamide (10). To a solution of 2,4ꢀpyrimidinedione, 5ꢀaminoꢀ1,3ꢀdimethyl (0.645
mmol) in DMF (3.2 ml) was added methyl bromoacetate (0.838 mmol) and K2CO3 (0.968
o
mmol). The reaction was allowed to stir at 80 C for 16 h. Upon completion of reaction, the
reaction mixture was dried in vacuo. Water was added and the aqueous was extracted with
CHCl3. The combined organics was washed with brine, dried over anhydrous Na2SO4 and dried
in vacuo to afford the crude product methyl (1,3ꢀdimethylꢀ2,4ꢀdioxoꢀ1,2,3,4ꢀ
tetrahydropyrimidinꢀ5ꢀyl)glycinate which is carried on to the next step. MS (ESI) m/z 228.2
[C9H13N3O4 + H]+.
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To a stirred solution of methyl methyl (1,3ꢀdimethylꢀ2,4ꢀdioxoꢀ1,2,3,4ꢀtetrahydropyrimidinꢀ5ꢀ
yl)glycinate (0.484 mmol) in THF (5 ml), MeOH (1 ml) and water (1 ml) was added LiOH.H2O
(0.968 mmol) and stirred at r.t for 5 h. Upon completion of reaction, the reaction mixture was
concentrated. The residue was dissolved in water, washed with EtOAc, acidified with aq.KHSO4
and the product was extracted with 10 % MeOH/CHCl3. The combined organics was dried over
Na2SO4 and concentrated to give (1,3ꢀdimethylꢀ2,4ꢀdioxoꢀ1,2,3,4ꢀtetrahydropyrimidinꢀ5ꢀ
yl)glycine as white solid. MS (ESI): m/z 212.2 [C8H11N3O4 – H]+.
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The titled compound was obtained as a white solid (yield, 52 %) with the general amide
coupling procedure using (1,3ꢀdimethylꢀ2,4ꢀdioxoꢀ1,2,3,4ꢀtetrahydropyrimidinꢀ5ꢀyl)glycine
1
(0.484 mmol) and 4ꢀ(1,3ꢀthiazolꢀ2ꢀyl) aniline (0,484 mmol). H NMR (400 MHz; DMSOꢀd6) δ
10.18 (s, 1H), 7.88 (m, 3H), 7.73 (m, 3H), 6.71 (s, 1H), 4.95 (t, J = 6.8 Hz, 1H), 3.77 (d, J = 6.4
Hz, 2H), 3.26 (s, 3H), 3.23 (s, 3H); MS (ESI) m/z 372.4 [C17H17N5O3S + H]+.
3H-imidazo[4,5-c]pyridin-4(5H)-one (11). 2ꢀchloropyridineꢀ3,4ꢀdiamine (3.496 mmol) in
o
trimethylorthoformate (10 ml) was heated at 70 C for 16 h. After completion, the reaction
mixture was cooled to r.t, and the reaction mixture was concentrated under vacuum. The residue
was purified by column chromatography to give 4ꢀchloroꢀ3Hꢀimidazo[4,5ꢀc]pyridine as a white
solid (yield, 58 %). 1H NMR (400 MHz; DMSOꢀd6) δ 13.20 (br s, 1H), 8.47 (s, 1H), 8.11 (d, J =
5.6 Hz, 1H), 7.62 (d, J = 5.2 Hz, 1H); MS (ESI) m/z 154.1 [C6H4ClN3 + H]+.
4ꢀchloroꢀ3Hꢀimidazo[4,5ꢀc]pyridine (1.633 mmol) in methanolic HCl (20 ml) was stirred at r.t
for 24 h. Upon completion, the reaction mixture was concentrated under vacuum. The crude
residue was washed with diethyl ether to give the titled compound as a light brown solid (yield,
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95 %). 1H NMR (400 MHz; DMSOꢀd6) δ 11.88 (br s, 1H), 9.11 (s, 1H), 7.39ꢀ7.37 (m, 1H), 6.66
3
4
5
6
(d, J = 7.2 Hz, 1H); MS (ESI) m/z 136.0 [C6H5N3O + H]+.
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2-(5-methyl-4-oxo-4,5-dihydro-3H-imidazo[4,5-c]pyridin-3-yl)-N-(4-(thiophen-3-
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yl)phenyl)acetamide (12). To a solution of 3Hꢀimidazo[4,5ꢀc]pyridinꢀ4(5H)ꢀone, 11 (1.555
mmol) in DMF (5 ml) was added K2CO3 (1.711 mmol) and 2ꢀchloroꢀNꢀ(4ꢀ(thiophenꢀ3ꢀ
yl)phenyl)acetamide (1.555 mmol) and stirred at r.t for 16 h. Upon reaction completion, the
reaction mixture was poured into water and precipitated solid was collected by filtration, washed
with excess water and diethyl ether before drying under vacuum to afford 2ꢀ(4ꢀoxoꢀ4,5ꢀdihydroꢀ
3Hꢀimidazo[4,5ꢀc]pyridinꢀ3ꢀyl)ꢀNꢀ(4ꢀ(thiophenꢀ3ꢀyl)phenyl)acetamide as a light brown solid
1
(yield, 55 %). H NMR (400 MHz; DMSOꢀd6) δ 11.27 (s, 1H), 10.45 (s, 1H), 8.14 (s, 1H), 7.79
(s, 1H), 7.69ꢀ7.61 (m, 5H), 7.53 (d, J = 4.8 Hz, 1H), 7.09 (t, J = 6.0 Hz, 1H), 6.58 (d, J = 6.8 Hz,
1H), 5.36 (s, 2H); MS (ESI) m/z 351.1 [C18H14N4O2S +H]+.
To a solution of 2ꢀ(4ꢀoxoꢀ4,5ꢀdihydroꢀ3Hꢀimidazo[4,5ꢀc]pyridinꢀ3ꢀyl)ꢀNꢀ(4ꢀ(thiophenꢀ3ꢀ
yl)phenyl)acetamide(1.287 mmol) in DMF (5 mL) at r.t was added K2CO3 (1.711 mmol) and
stirred for 30 min. Methyl iodide (0.571 mmol) was added at r.t and stirred for 3 h. Upon
completion of reaction, the reaction mixture was poured into water and precipitated solid was
collected by filtration, washed with excess water and diethyl ether then dried under vacuum. The
solid compound was further purified by column chromatography to afford the titled compound as
1
an offꢀwhite solid (yield, 29 %). H NMR (400 MHz; DMSOꢀd6) δ 10.45 (s, 1H), 8.14 (s, 1H),
7.79 (d, J = 1.6 Hz, 1H), 7.69ꢀ7.67 (m, 2H), 7.62ꢀ7.61 (m, 3H), 7.53 (d, J = 5.2 Hz, 1H), 7.42 (d,
J = 7.2 Hz, 1H), 6.62 (d, J = 7.6 Hz, 1H), 5.36 (s, 2H), 3.47(s, 3H), 3.31(s, 3H); MS (ESI) m/z
365.1 [C19H16N4O2S + H]+.
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2-(5-(dimethylcarbamoyl)-1H-imidazol-1-yl)acetic acid (13). A stirred solution of 1Hꢀ
imidazoleꢀ5ꢀcarboxylic acid (4.464 mmol) in SOCl2 (8 ml) was heated to reflux for 5 h, before
cooling to r.t. The reaction mixture was added to toluene and the solid was collected by filtration
and dried to give the corresponding acid chloride. The acid chloride in DCM (20 ml) was added
dimethylamine in THF (2 ml, 2M solution) and stirred at r.t for 16 h. Upon completion of
reaction, the reaction mixture was concentrated and washed with diethyl ether to give N,Nꢀ
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dimethylꢀ1Hꢀimidazoleꢀ5ꢀcarboxamide as a yellow solid (yield, 80 %).
[C6H9N3O + H]+.
MS (ESI) m/z 140.6
To a solution of N,Nꢀdimethylꢀ1Hꢀimidazoleꢀ5ꢀcarboxamide(3.597 mmol) in DMF (10 ml)
o
o
was added K2CO3 (4.316 mmol) at r.t and heated to 100 C for 1 h and cooled to 70 C;
o
bromoethylacetate (5.395 mmol) was added to reaction mixture and stirred at 70 C for 16 h.
After completion of reaction, the reaction mixture was cooled to r.t and diluted with water and
extracted with EtOAc. The combined organics were dried over Na2SO4 and concentrated under
vacuum. The crude compound was purified by column chromatography to give ethyl 2ꢀ(5ꢀ
1
(dimethylcarbamoyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)acetate as a yellow solid (yield, 25 %). H NMR (400
MHz; CDCl3) δ 7.55 (s, 1H), 7.46 (s, 1H), 4.69 (s, 2H), 4.26 (q, J = 7.2 Hz 2H), 3.48 (s, 3H),
3.08 (s, 3H), 1.30 (t, J = 7.2 Hz, 3H); MS (ESI) m/z 225.8 [C10H15N3O3 + H]+.
To a stirred solution of 2ꢀ(5ꢀ(dimethylcarbamoyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)acetate (0.888 mmol) in
THF (10 ml), MeOH (10 ml) and water (5 ml) was added LiOH.H2O (1.333 mmol) and stirred at
r.t for 2 h. The reaction mixture was concentrated, the residue was dissolved in water, washed
with EtOAc and acidified with aq.KHSO4; product was extracted with EtOAc. The combined
organics was washed with brine, dried over Na2SO4 and concentrated in vacuo to provide the
titled compound as an offꢀwhite solid (yield, 80 %). 1H NMR (400 MHz, DMSOꢀd6) δ 13.20 (br
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s, 1H), 7.63 (s, 1H), 7.59 (s, 2H), 3.40 (s, 3H), 2.92 (s, 3H); MS (ESI) m/z 198.2 [C8H11N3O3 +
5
6
H]+.
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N,N-dimethyl-1-(2-oxo-2-(4-(thiophen-3-yl)phenylamino)ethyl)-1H-imidazole-5-carboxamide
(14). The titled compound was obtained as a white solid (yield, 60 %) with the general amide
coupling procedure using 2ꢀ(5ꢀ(dimethylcarbamoyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)acetic acid (0.71 mmol)
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and 4ꢀ(thiophenꢀ3ꢀyl) aniline, 4 (0.71 mmol). H NMR (400 MHz, DMSOꢀd6) δ 10.39 (s, 1H),
7.80 (s, 1H), 7.70ꢀ7.61 (m, 7H), 7.53 (s, 1H), 4.97 (s, 2H), 3.42 (s, 3H), 2.94 (s, 3H); MS (ESI)
m/z 355.5 [C18H18N4O2S+ H]+.
2-oxo-1-(2-oxo-2-((4-(thiazol-2-yl)phenyl)amino)ethyl)-1,2-dihydropyridine-3-carboxylic
acid (15). To a solution of 4ꢀ(1,3ꢀthiazolꢀ2ꢀyl) aniline (1.6 mmol) and Hunig’s base (1.6 mmol)
in anhydrous DCM (8 ml) at ꢀ78 °C was added bromoacetyl bromide (1.6 mmol) dropwise. The
reaction mixture was stirred at ꢀ78 °C for 1 h, warmed to r.t and stirred further for 1 h. Upon
completion of reaction, the reaction mixture was diluted with ethyl acetate, washed with H2O and
brine, dried over Na2SO4 and concentrated in vacuo. The crude was purified by column
chromatography to afford 2ꢀbromoꢀNꢀ(4ꢀ(thiazolꢀ2ꢀyl)phenyl)acetamide as a yellow solid (yield,
1
34%). H NMR (400 MHz; MeOD) δ 7.91ꢀ 7.93 (m, 2H), 7.83 (d, J = 3.2 Hz, 1H), 7.70 – 7.73
(m, 2H), 7.56 (d, J = 3.2Hz, 1H), 3.99 (s, 2H); MS (ESI) m/z 298.2 [C11H9BrN2OS + H]+.
Methyl 2ꢀoxoꢀ1,2ꢀdihydroꢀ3ꢀpyridine carboxylate (0.5 mmol) and 2ꢀbromoꢀNꢀ(4ꢀ(thiazolꢀ2ꢀ
yl)phenyl)acetamide (0.544 mmol) were taken up in anhydrous DMF (10 ml) and cooled to 0 °C
using an iceꢀbath. NaH (2 mmol) was added carefully into the reaction. The mixture was left to
warm up to r.t under N2 for 4 h. Upon completion of reaction, the reaction mixture was diluted
with DCM and quenched with 1N HCl. The reaction mixture was dried with Na2SO4, filtered and
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