Synthesis and structure-activity relationship of trisubstituted thiazoles as Cdc7 kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.04.013

The Cell division cycle 7 (Cdc7) protein kinase is essential for DNA replication and maintenance of genome stability. We systematically explored thiazole-based compounds as inhibitors of Cdc7 kinase activity in cancer cells. Our studies resulted in the identification of a potent, selective Cdc7 inhibitor that decreased phosphorylation of the direct substrate MCM2 in vitro and in vivo, and inhibited DNA synthesis and cell viability in vitro.

Full text of DOI:10.1016/j.ejmech.2014.04.013

Accepted Manuscript
Synthesis and structure-activity relationship of trisubstituted thiazoles as Cdc7 kinase
inhibitors
Andreas Reichelt, Julie M. Bailis, Michael D. Bartberger, Guomin Yao, Hong Shu,
Matthew R. Kaller, John G. Allen, Margaret F. Weidner, Kathleen S. Keegan, Jennifer
H. Dao
PII:
S0223-5234(14)00332-8
10.1016/j.ejmech.2014.04.013
EJMECH 6884
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 January 2013
Revised Date: 1 April 2014
Accepted Date: 4 April 2014
Please cite this article as: A. Reichelt, J.M. Bailis, M.D. Bartberger, G. Yao, H. Shu, M.R. Kaller, J.G.
Allen, M.F. Weidner, K.S. Keegan, J.H. Dao, Synthesis and structure-activity relationship of trisubstituted
thiazoles as Cdc7 kinase inhibitors, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.04.013.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and structure-activity relationship of
trisubstituted thiazoles as Cdc7 kinase inhibitors
a,*
b
c
a
a
Andreas Reichelt , Julie M. Bailis , Michael D. Bartberger , Guomin Yao , Hong Shu ,
a
a
b
b
c
Matthew R. Kaller , John G. Allen , Margaret F. Weidner , Kathleen S. Keegan , Jennifer H. Dao
a
Departments of Medicinal Chemistry, Amgen, Inc., One Amgen Center Drive, Thousand Oaks,
b
California 91320. Oncology Research, Amgen, Inc., 1201 Amgen Court West, Seattle, Washington
c
9
8119. Molecular Structure and Characterization, Amgen, Inc., One Amgen Center Drive, Thousand
Oaks, California 91320.
ABSTRACT: The Cell division cycle 7 (Cdc7) protein kinase is essential for DNA replication and
maintenance of genome stability. We systematically explored thiazole-based compounds as inhibitors of
Cdc7 kinase activity in cancer cells. Our studies resulted in the identification of a potent, selective Cdc7
inhibitor that decreased phosphorylation of the direct substrate MCM2 in vitro and in vivo, and inhibited
DNA synthesis and cell viability in vitro.
KEYWORDS: Cdc7; MCM2; kinase inhibitor; DNA replication; thiazole
*
Corresponding Author. Phone: +1 (805) 313-5225. Fax: +1 (805) 480-3015. E-mail:
Andreas.Reichelt@amgen.com
1

ACCEPTED MANUSCRIPT
1
. Introduction
The complete, accurate replication of the DNA in each cell cycle is fundamental to maintaining
genomic stability. In cancer cells, oncogene activation or loss of tumor suppressor function can lead to
increased replication stress and deregulation of cell cycle controls. Proteins that control key transitions
in DNA replication and cell cycle progression therefore represent potential nodes for therapeutic
intervention in cancer.
The serine/threonine protein kinase Cdc7 is essential for the activation of individual origins of
replication and the initiation of DNA synthesis.[1,2] Cdc7 activation depends on the bipartite binding of
a subunit, Dbf4.[2] Cdc7/Dbf4 phosphorylates proteins associated with the replication origin, such as
the minichromosome maintenance (MCM) complex, to trigger origin firing.[2] In particular, direct
phosphorylation of the MCM2 subunit by Cdc7/Dbf4 promotes a conformational change in the MCM
complex that activates its helicase activity and initiates local DNA unwinding.[2] This allows the
association of other proteins required for DNA synthesis, such as DNA polymerase, to the exposed
regions of single-stranded DNA.[2]
Cdc7 plays a key role not only in activating origins to initiate synthesis, but also in regulating ongoing
replication. Cdc7 promotes the phosphorylation and activation of Chk1 and the replication
checkpoint,[3] which responds to blocks of replication fork progression or limiting amounts of
nucleosides by arresting cells in S phase. Checkpoint activation may downregulate Cdc7 kinase,
preventing additional replication origins from firing,[4] or Cdc7 may be required for recovery from
replication fork arrest.[5]
Given its essential function in cell proliferation, Cdc7 has been considered as a novel target for cancer
therapy.[6,7,8] Cdc7 inhibition with siRNA or small molecule inhibitors arrests normal cells at G1/S
after activation of a cell cycle checkpoint, but is lethal to cancer cells that cannot activate this
checkpoint due to defects in the DNA damage response pathway.[9,10,11,12,13] Expression levels of
Cdc7 and Dbf4 are increased in cancer cells, and amplification of Dbf4 has been identified in some
breast and ovarian cancer cell lines.[14,15,16] Although interfering with the DNA replication process is
2

ACCEPTED MANUSCRIPT
a proven strategy for cancer therapy, efficacy of current drugs that target this pathway is limited by
cytotoxicity to normal cells and inherent or acquired drug resistance. Compounds that inhibit Cdc7
kinase activity provide a potential advantage in that they target the initiation step of DNA replication,
rather than replication fork progression, and that toxicity may be decreased due to activation of a
cellular checkpoint response in normal cells. The interest in Cdc7 as a target for cancer therapy is
demonstrated by the report of several potent Cdc7 inhibitors by different research
groups[17,18,19,20,21,22,23,24] but clinical trials with Cdc7 inhibitors have not yet moved past Phase I
studies.
In our laboratories, high-throughput screening (HTS) efforts aimed at the discovery of inhibitors of
Cdc7 kinase activity led to the identification of compounds bearing a thiazole core. In particular,
thiazole 1 (Figure 1) was found to be a sub-micromolar inhibitor of Cdc7 kinase activity and showed no
detectable activity in our primary counter screen against the structural and functional homolog, cyclin-
dependent kinase CDK2. The synthetic approach to this compound allowed for the rapid synthesis of
analogues with variations at all substituents on the thiazole ring. Herein, we describe the synthesis and
structure-activity relationship (SAR) of compounds based on the thiazole scaffold of 1, eventually
leading to 74, a potent and selective Cdc7 inhibitor.
[Please, insert Figure 1 here]
2
. Chemistry
The thiazole cores of the compounds described in this paper were generally formed by reaction of
thioamides with diethyl bromomalonate (3) as demonstrated by the synthesis of compound 1 (Scheme
). The hydroxyl substituent in 1 was further elaborated by alkylation to form ether 4 or was
1
transformed into the corresponding triflate 5. Reaction of 5 with ammonia as well as primary or
secondary amines led to the 4-amino substituted thiazoles 6–10. Compound 13 that lacks a substituent at
C4 of the thiazole was synthesized by Suzuki coupling of commercially available ethyl 2-
3

ACCEPTED MANUSCRIPT
bromothiazole-5-carboxylate (12) with 4-pyridineboronic acid (11), while the synthesis of 4-methyl
thiazole 15 was achieved by reaction of pyridine-4-carbothioamide (2) with ethyl 2-chloro-3-
oxobutanoate (14). Suzuki-coupling of triflate 5 with a variety of boronic acids and esters gave
convenient access to compounds 16–24, which bear aromatic substituents at the 4-position of the
thiazole.
[Please, insert Scheme 1 here]
Amide 27 was synthesized analogously to its corresponding ester 1 by condensation of pyridine-4-
carbothiamide (2) with bromide 26 (Scheme 2). Saponification of the ester moiety in 1 delivered the
hydrochloride salt of acid 28, which was transformed into the corresponding acid chloride. Reaction of
the crude acid chloride with a variety of amines gave amides 29–31. Activation of acid 32, prepared by
saponification of ester 16, with HATU followed by treatment with ammonia, hydrazine, or
hydroxylamine hydrochloride delivered the primary amide 33, hydrazide 34, and hydroxamic acid 35,
respectively. Other amides bearing different functional groups at the 4-position of the thiazole were
prepared analogously.
[Please, insert Scheme 2 here]
The syntheses of thiazoles bearing heterocyclic carbonyl replacements at the 4-position are illustrated
in Scheme 3. Dehydration of amide 33 under Swern oxidation conditions[25] gave nitrile 38, which was
transformed into tetrazole 39 by reaction with sodium azide in the presence of zinc bromide.[26]
Oxadiazole 40 was obtained by reaction of hydrazide 34 with trimethyl orthoformate under acidic
conditions. Displacement of the triflate moiety in 5 with 1,2-phenylenediamine followed by cyclization
gave lactam 41.
4

ACCEPTED MANUSCRIPT
[Please, insert Scheme 3 here]
Our general strategy for the formation of the thiazole core was also applicable to the synthesis of
analogues of 1 with diverse substituents at the 2-position of the thiazole (Scheme 4). Thus, reaction of a
variety of thioamides 42 with bromides 3 or 52 gave easy access to compounds 43–51 and 53–54,
respectively. Thioamides that were not commercially available were synthesized from the corresponding
chlorides as shown in Scheme 4.
[Please, insert Scheme 4 here]
The complete reaction sequence to 2-(2-amino-4-pyrinidyl)thiazole-5-carboxamides bearing aromatic
or amino groups at the 4-position of the thiazole is outlined in Scheme 5. Transformation of 2-amino-4-
chloropyrimidine (55) into the corresponding iodide followed by palladium-mediated cyanation gave 2-
amino-4-cyanopyrimidine. Treatment with ammonium sulfite delivered thioamide 60, which was
cyclized to thiazole 46 following the standard protocol. The corresponding triflate was employed in
Suzuki-reactions or displaced with dimethylamine to give access to the 4-substituted thiazoles 63–70. A
two-step transformation of the esters to the primary amides then afforded analogues 71–78.
[Please, insert Scheme 5 here]
3
. Pharmacology
Cdc7/Dbf4 biochemical activity was assessed through a Protein A Amplified Luminescent Proximity
Homogenous Assay (AlphaScreen). Counter screens to determine the biochemical activity of related
protein kinases CDK2/Cyclin E, CDK1/Cyclin B, CDK4/Cyclin D1, Pim1, Pim2, CDK9/Cyclin T1,
CK2, and CK1 employed homogeneous time-resolved fluorescence (HTRF) assays. MCM2
phosphorylation was assessed by high content imaging in HCT-116 cells and with a Meso-Scale
5

ACCEPTED MANUSCRIPT
Discovery (MSD) assay in tumor xenografts. Additional assays in HCT-116 cells were
bromodeoxyuridine (BrdU) incorporation measured using a colorometric ELISA assay, cell viability
assessed using a Cell Titer-glo assay, and DNA content evaluated by flow cytometry. See Experimental
protocols for details.
4
. Results and discussion
Intrigued by the arguments for Cdc7 as a potential novel target for cancer, we initiated a high-
throughput screen of a library of approximately 250,000 compounds for Cdc7 kinase inhibitors. In this
HTS campaign, we identified a high hit rate (1.1%) of Cdc7 inhibitors and we counter screened for
compounds with selectivity against CDK2, a closely related kinase that shares both structural and
functional homology with Cdc7.[1] Verified hits from the screen represented several, distinct chemical
series with remarkably low molecular weights and high binding efficiencies for inhibitors of kinase
activity. Prioritization of series based on drug-like properties led us to focus initially on a class of
compounds featuring a thiazole core.
Compound 1 was the most promising hit in a series of thiazoles that was obtained from the HTS
screen. In a biochemical assay for Cdc7 kinase activity (see Experimental protocols), the IC50 of 1 was
determined to be 0.323 µM, with no activity (IC >125 µM) detected in the counter screen assay
5
0
against CDK2. Mechanism of action studies indicated this compound to be ATP competitive
Supplementary Figure 1). Thiazole 1 appeared to be a very selective compound: in a panel of 100
(
kinases (KinomeSCAN, Ambit Bioscience, Inc.), compound 1 did not show any response with a percent
of control (POC) <50 at 1 µM (Supplementary Figure 2), and no agonist or antagonist activity was
detected in a panel of 13 receptors. The origin of this compound had been as an intermediate in a series
of potential CDK5 inhibitors;[27] its low molecular weight combined with its potency corresponds to a
binding efficiency of >0.5. It also exhibits other favorable characteristics including low intrinsic
clearance in human liver microsomes (HLM) and rat liver microsomes (RLM), (17 and <14 µl/min/mg,
–
6
respectively), good permeability and low efflux ratios (Papp ≥ 8.9 × 10 cm/s, ER = 1 for parental,
6

ACCEPTED MANUSCRIPT
MDR1- and rat mdr1-infected LLC-PK1 cells). The structure of 1 offered multiple points for
diversification and its efficient synthetic scheme allowed for the rapid synthesis of analogues.
An X-ray crystal structure, while not available during the course of our medicinal chemistry
campaign, has recently been reported for Cdc7 kinase and its activator Dbf4.[28] Although the
structure-activity relationships reported in this manuscript were carried out in a ligand-based fashion in
the absence of crystallographic information, the subsequent publication of the Cdc7/Dbf4 structure
made it possible to retrospectively analyze the binding of compound 1 and subsequent analogs in the
active site of Cdc7.
The predicted binding mode for the initial hit 1, arising from docking of the lowest-energy
conformation as determined by quantum mechanics into the ATP binding site, is depicted in Figure
2
.[29,30,31,32] The pyridine nitrogen of 1 is engaged in a hydrogen bond to the backbone NH of the
linker LEU137, whereas the ethyl ester functionality projects outward toward the exterior of the ATP
binding site, with an intramolecular hydrogen bond predicted between the thiazole hydroxyl group and
the carbonyl of the ethyl ester.
[Please, insert Figure 2 here]
Early SAR from our HTS campaign suggested that variations at the 4-position of the thiazole might
lead to compounds with improved affinity for Cdc7 but extensive SAR studies disproved this
hypothesis. Selected examples from a series of 2-(4-pyridyl)thiazole-5-carboxylates varying by the
substituent at C4 are shown in Table 1. Alkylation of the 4-hydroxy group was not tolerated, as methyl
ether 4 lost two orders of magnitude in its inhibitory activity against Cdc7 kinase compared with alcohol
1
. The precipitous loss of activity of ether 4 (17.2 ꢀM) upon O-alkylation of compound 1 can be
explained by disruption of the intramolecular H-bonding motif and likely resultant steric clash of the
alkoxyl and/or ester function with nearby residues LEU195, ASP96, or LYS90.
7

ACCEPTED MANUSCRIPT
Introduction of 4-amino substituents led to compounds with a wide range of potencies: the primary
amine 6 showed micromolar activity as did compound 7 that bears a methyl amino group at the 4-
position of the thiazole. Larger substituents at the amine as in 8 (NHBn) and in 9 (NHPh) were not
tolerated. The dimethylamino analogue 10 however, showed activities comparable with 1. Removal of
the substituent at the 4-position of the thiazole in 13 or replacement with a small alkyl group (e.g.
methyl in 15) led to compounds with micromolar activities. Thiazoles bearing unsubstituted aryl
moieties as C4-substituents (16 and 23) were 25–60-fold less potent than 1; however, this loss of
potency could be restored by introduction of substituents on the aromatic ring. In particular, compound
1
8 that bears an ortho-chloro phenyl group at the 4-position of the thiazole showed sub-micromolar
levels of activity. Substituents on the phenyl ring in addition to ortho-chloro were tolerated as well (19–
2). Large aromatic groups like the 2-naphthyl group in 24 were not tolerated at the 4-position of the
2
thiazole. None of the compounds showed any detectable inhibitory activity against CDK2. Overall, the
variations in the substituent on the 4-position of the thiazole ethyl esters depicted in Table 1 led to
compounds with activities at best as good as our starting point 1 and we focused our attention toward
the 5-substituent.
[Please, insert Table 1 here]
Exploration of the influence of substituents at C5 on the thiazole ring on the inhibitory activity against
Cdc7 revealed some promising trends (Table 2). Acids were generally found to be one to two orders of
magnitude more potent than their corresponding esters, as exemplified in the comparison of ester 16
(
IC50 = 20.5 µM) with acid 32 (IC50 = 0.172 µM). However, we noticed an interesting relationship
between the substituents at C4 and C5 in acid 28 as this compound, which was isolated as its
hydrochloride salt, exhibited an IC50 of 6.13 µM, 20-fold less potent than ester 1. Primary amides, on
the other hand, were uniformly found to be more potent: compounds 27 and 33, for example, showed
IC values of 0.115 µM and 0.101 µM, respectively. The activities of secondary and tertiary amides
5
0
8

ACCEPTED MANUSCRIPT
(e.g. 29–31) were in the micromolar range. Hydrazide 34, hydroxamic acid 35, and nitrile 38 as well as
the 5-heterocycle substituted thiazoles 39 and 40 showed improved activity compared to ethyl ester 16
but none of these compounds reached the activity level of primary amide 33. Only lactam 41 showed an
IC comparable with amides 27 and 33 but significantly reduced selectivity over CDK2 (11-fold vs.
5
0
>
300-fold).
[Please, insert Table 2 here]
Having established the primary amide as the optimal C5-substituent, several amides with different
substituents at the 4-position of the thiazole were synthesized. These compounds showed activities
around 0.1 µM, which in many cases equated to potency increases of two orders of magnitude compared
to their corresponding esters (Table 3). In order to test whether the biochemical potency would translate
into an effect on cells, phosphorylation of SER53 in MCM2, a Cdc7-dependent, Cdc7-specific
phosphorylation site,[33,34] was analyzed in the colon cancer cell line HCT-116 using a high content
imaging assay (see Experimental protocols). Potency in the Cdc7 biochemical assay did not result in
comparable potency in the cellular assay: compound 27, which bears a hydroxyl substituent at the 4-
position, did not show measurable activity in the cellular assay, whereas compounds 33, 36, and 37
inhibited MCM2 phosphorylation at micromolar concentrations, a greater than 100-fold shift from the
biochemical IC values. The shift in potency between the biochemical and cellular assays is consistent
5
0
with data on Cdc7 inhibitors from other chemical series (Reference 10, and data not shown), and may be
due in part to the low K (app) for ATP for Cdc7, which we determined as 0.4 µM (Supplementary
M
Figure 3).
[Please, insert Table 3 here]
9

ACCEPTED MANUSCRIPT
In analogy to general kinase inhibitors, we envisioned the 2-substituents on the thiazole to interact
with the hinge region of the enzyme, a hypothesis that was retrospectively supported by the proposed
binding of compound 1 in the active site of Cdc7. Hence, we expected variations at this position to have
a pronounced effect on the inhibitory activity against Cdc7 kinase and focused our SAR studies towards
the 2-position of the thiazole (Table 4). Indeed, compound 43, which bears a 3-pyridyl moiety, lost 2
orders of magnitude in potency compared to 1, and the corresponding 2-pyridyl analogue 44 did not
show any inhibitory activity against Cdc7. We rationalize this decrease in activity of pyridyl
regioisomers 43 and 44 by the loss of the key intermolecular hydrogen bonding contact between the
pyridine nitrogen of 1 and the backbone NH of the linker LEU137 of Cdc7 (Figure 2). The importance
of the ring-nitrogen para to the connection to the thiazole ring was further demonstrated by the high
micromolar activities of compounds 48–51. An alkyl substituent at the 2-position of the 4-pyridyl
moiety in 45 led to 20-fold loss in potency, whereas an amino substituent at this position was tolerated
(53 was equipotent to 16). When we explored other heterocycles at the 2-position of the thiazole our
efforts were rewarded by the discovery of 46. This compound features a 2-amino-4-pyrimidyl moiety
and showed an 8-fold improvement in potency over our starting point 1. Interestingly, its regioisomer 54
did not show any inhibitory activity against Cdc7. Again, a pyrazine ring in 47, which lacks the ring-
nitrogen para to the connection to the thiazole ring, was poorly tolerated.
[Please, insert Table 4 here]
So far, our SAR studies had established the 2-amino-4-pyrimidyl moiety as the optimal substituent at
the 2-position of the thiazole and we had discovered that thiazole-5-carboxamides were exquisitely
potent. Keeping these groups constant, we reevaluated the influence of substituents at the 4-position on
the inhibitory activity against Cdc7 kinase as well as the cellular activity against MCM2
phosphorylation in HCT-116 cells. The compounds depicted in Table 5 showed cellular activities <10
µM, and sub-micromolar potency was observed for compounds 74 and 77 equating to shifts in potency
1
0

ACCEPTED MANUSCRIPT
between the biochemical and cellular Cdc7 kinase assays of >30-fold. To further characterize these
compounds, we tested their stability in microsomes in vitro, using HLM and RLM assays (see
Experimental protocols). All compounds tested were stable in HLM and RLM assays, with clearance of
<
100 µl/min/mg for all but one (Table 5).
[Please, insert Table 5 here]
Compounds 74 and 77 were potent in the biochemical and cellular assays and displayed low (<100
µl/min/mg) clearance in both HLM and RLM assays. We also tested these compounds in a mouse liver
microsome (MLM) assay (see Experimental protocols), because we intended to further test the best
compound in vivo using a mouse model. The MLM assay resulted in intrinsic clearance of 57 µl/min/mg
for 74, and 155 µl/min/mg for 77, showing a similar trend with the results of the RLM assay.
Additional profiling of these two compounds against a panel of 100 protein kinases (KinomeSCAN,
Ambit Bioscience, Inc.) revealed similar overall selectivity, with a POC < 35 at 1 µM for 12/100 and
1
0/100 kinases for 74 and 77, respectively (Supplementary Figure 2). As the KinomeSCAN used
binding competition assays to assess kinase inhibition, and Cdc7 kinase was not included in that panel,
we tested these and select other compounds in enzymatic assays for kinases of particular interest (Table
6
).
Compounds 74 and 77 were not active against the cell cycle kinases CDK1 or CDK4, but did inhibit
CDK9 (IC values of 0.75 µM and 0.73 µM, respectively), a kinase which primarily functions in
5
0
cellular transcription through regulation of RNA polymerase II.[35] Cdc7/CDK9 inhibitors reported by
Nerviano Medical Sciences have been shown to be efficacious in tumor models, in part because
inhibition of CDK9 downregulates transcripts of anti-apoptotic proteins such as Mcl-1.[11,36] We also
tested for activity of our Cdc7 inhibitors against protein kinases from the casein kinase family, which
are structurally related to Cdc7 and have also been considered as anticancer targets.[37,38] Compounds
7
4 and 77 did not inhibit CK2, but did show activity against CK1 kinases. Compound 74 inhibited
1
1

ACCEPTED MANUSCRIPT
CK1α with an IC50 of 0.69 µM, CK1γ with an IC50 of 0.76 µM, and CK1δ with an IC50 of 0.084 µM.
Compound 77 inhibited CK1α with an IC of 0.55 µM, CK1γ with an IC of 1.9 µM, and CK1δ with
5
0
50
an IC of 0.104 µM. We did not detect any inhibition of Pim1, another structurally related kinase, by
5
0
compounds 74 and 77 in enzymatic assays. Compound 74 also did not show any agonist or antagonist
activity in a panel of 13 receptors.
[Please, insert Table 6 here]
We further characterized compounds 74 and 77 in different cellular assays (Table 7 and Figure 3).
The compounds inhibited DNA synthesis in HCT-116 cells, with IC values of 2.6 µM and 6.9 µM, for
5
0
7
4 and 77 respectively, in a colorometric bromodeoxyuridine (BrdU) assay (Figure 3A). Compounds 74
and 77 also inhibited cell proliferation, resulting in IC values of 4.2 µM and 4.6 µM in a Cell Titer-
5
0
Glo assay, which measures cellular ATP metabolism (Figure 3B). The IC values for the BrdU and cell
5
0
viability assays were similar to each other, but were 5- to 9-fold less potent than the IC values for the
5
0
proximal assay, MCM2 phosphorylation. This suggests that inhibition of MCM2 close to the IC90 is
required for maximal inhibition of cell proliferation (pMCM2 IC90 = 6.37 µM for 74). Flow cytometry
for DNA content of HCT-116 cells treated with compound 74 revealed an accumulation of cells in the
S/G2/M phases of the cell cycle (Figure 3C), suggesting delayed progression through S phase following
Cdc7 inhibition. Caspases 3 and 7 were not activated at the time points analyzed (Table 7), raising the
possibility that the cells did not undergo apoptosis, although an increase in sub-G1 DNA content was
observed at higher compound concentrations or at later time points (Figure 3 and data not shown).
Imaging analysis of HCT-116 cells treated with compound 74, as well as with Cdc7 inhibitors from
other chemical series, suggested that loss of cell viability occurred following aberrant mitosis
(Supplementary Figure 5).
[Please, insert Table 7 here]
1
2

ACCEPTED MANUSCRIPT
[Please, insert Figure 3 here]
Mechanism of action studies demonstrated that 74, like the initial thiazole 1 and other compounds
from this series, showed ATP-competitive inhibition of Cdc7 (Supplementary Figure 4). Its predicted
binding mode as determined in a manner analogous to that of compound 1 is shown in Figure 4. The
binding orientation of compound 74 displays a syn orientation of the sulfur atom of the thiazole with
respect to the proximal nitrogen atom of the adjacent pyrimidine ring, predicted from our own quantum
mechanical calculations as well as previous studies,[39,40] which demonstrate the syn preference of
aromatic sulfur atoms to lone-pair donor atoms when oriented in a 2,2'-arrangement. This syn S-N
arrangement, favored over the anti S-N conformer of 74 by greater than 4 kcal/mol, necessitates an
outward orientation of the pendant 2,4-dichloroaryl group toward the solvent front. The 2,4-
dichlorophenyl substituent may potentially engage in an electrostatic or van der Waals interaction with
the sidechain of HIS139 (depending on protonation state) and/or the backbone carbonyl oxygen of
SER181. [41] The aminopyridine moiety interacts with the linker residue in a manner typical of this
functionality, with the ring nitrogen atom of 74 interacting with the NH of linker LEU137, augmented
by an H-bonding contact between the pendant NH and backbone C=O of PRO135 in the rear of the
2
binding cavity. The carbonyl and NH groups of the primary amide moiety of 74 are predicted to
2
interact with the salt-bridged LYS90 and ASP196, respectively.
[Please, insert Figure 4 here]
Based on its potency, selectivity, and microsomal clearance in vitro (Tables 5–7), we prioritized
compound 74 for in vivo studies. Pharmacokinetic (PK) studies in rat following intravenous (iv)
administration of 74 resulted in a high clearance of 3890 ml/kg/h, and a high volume of distribution of
4
430 ml/kg (Table 8). Following oral administration, the bioavailability of 74 was low (6.6%), as was
1
3

ACCEPTED MANUSCRIPT
the Cmax (13 ng/ml), although the terminal half-life was 3.73 h. Mouse plasma protein binding assays
showed a free fraction (fu) of 9.0% for compound 74.
[Please, insert Table 8 here]
Despite suboptimal PK, for proof of principle we investigated whether compound 74 inhibits Cdc7
kinase activity in vivo. Because of its low oral bioavailability, NCR/nude mice bearing HCT-116 tumor
xenografts were administered compound 74 as a single iv dose at 50 mg/kg or 150 mg/kg, and blood
plasma and tumor samples were collected at 1, 3, 6, and 16 h post dose for
pharmacokinetic/pharmacodynamic (PK/PD) analysis (Figure 5). For the PD readout, we used a meso-
scale discovery (MSD) assay to detect MCM2 serine 53 phosphorylation in tumor lysates (see
Experimental protocols). The 50 mg/kg dose of 74 was well tolerated and resulted in partial inhibition
of MCM2 phosphorylation in tumors. MCM2 phosphorylation was inhibited by 39% at the 3 h time
point, but showed less of an effect at later time points, with 20% inhibition of the signal at the 6 h time
point, and near control levels of MCM2 phosphorylation at the 16 h time point. The 50 mg/kg dose
resulted in an unbound plasma concentration of >1 µM at 1 h, and decreased thereafter. The 150 mg/kg
dose of compound 74 also inhibited MCM2 phosphorylation in tumors, with 30% inhibition at 1 h, 38%
inhibition at 3 h, and 31% inhibition at 6 h following dosing. The 150 mg/kg dose resulted in unbound
plasma levels of nearly 5 µM at the 1 h time point, and remained above 1 µM through 6 h post-dose.
However, this high dose was not well tolerated: even at 6 h, mice appeared moribund and consequently,
we did not test a 16 h time point. For both doses tested, inhibition of MCM2 phosphorylation occurred
after the maximal plasma concentrations were observed, consistent with the idea that cells must pass
through S phase for Cdc7 kinase activity and MCM2 phosphorylation to be inhibited. Compound 74
therefore demonstrated that a potent, selective inhibitor of Cdc7 kinase activity can decrease MCM2
phosphorylation in vitro as well as in vivo.
1
4

ACCEPTED MANUSCRIPT
[Please, insert Figure 5 here]
5
. Conclusions
Cdc7 presents a challenge as a target for drug discovery, because the presence of a kinase insert
domain adjacent to the active site, together with dependence on binding of the Dbf4 subunit for kinase
activity, are not straightforward for building homology models [1], and a crystal structure of the protein
had not been revealed until very recently [28]. Our primary HTS screen identified a high hit rate of
Cdc7 inhibitors that were remarkable, relative to previously described protein kinase inhibitors, for their
low molecular weight and high binding efficiency. We focused on compounds bearing a thiazole
moiety, systematically exploring SAR by building off the core of compound 1. Rapid synthesis and
characterization of analogs identified amide 74 as the most potent, selective Cdc7 inhibitor from our
SAR studies with on-target activity demonstrated in vitro and in vivo.
Our studies demonstrated a large shift in potency between enzymatic activity and activity in cells.
Additionally, we found that plasma exposure levels of compound 74 over the cellular IC for 3–6 h
5
0
were not sufficient to cause 50% inhibition of MCM2 phosphorylation in tumors in vivo. Together,
these observations suggest that in vivo inhibition of Cdc7 with small molecules may require constant
inhibition of the target at levels much greater than the cellular IC .
5
0
Comparison of Cdc7 inhibitors from distinct chemical series, including thiazole-based compounds,
will facilitate better understanding of on-target activity and the requirements for target coverage for
efficacy.
6
6
. Experimental protocols
.1. Chemistry
All chemicals were used as received from the commercial sources. Reactions were performed without
significant exclusion of moisture or air. Reaction temperatures describe the temperature of the bath
surrounding the reaction vessel. Reactions under microwave irradiation were performed in a Biotage®
1
5

ACCEPTED MANUSCRIPT
Initiator microwave reactor in sealed vessels. Column chromatography was conducted under medium
pressure either on silica gel (Merck® Grade 9385, pore size 60 Å, 230–400 mesh) or on prepacked
silica gel cartridges (Biotage®). Analytical HPLC and mass spectroscopy were conducted using a
reverse-phase Agilent 1100 Series HPLC-mass spectrometer. Purities for final compounds were
measured using UV detection at 215 nm and 254 nm as >95%. NMR spectra were acquired as solutions
in the indicated solvents on Bruker Avance DRX400 or Bruker Avance DPX300 spectrometers at 400
and 300 MHz field strengths, respectively. Chemical shifts are reported in parts per million (ppm, δ),
downfield from tetramethylsilane (TMS, δ = 0.00 ppm), and are referenced to the residual solvent.
Coupling constants are reported in Hertz (Hz). Spectral splitting patterns are designated as s: singlet, d:
doublet, t: triplet, q: quartet, quin: quintet, m: multiplet, br.: broad.
The general reaction sequence is demonstrated by the synthesis of lead compound 74 as depicted in
Scheme 5:
6.1.1. 2-Amino-4-iodopyrimidine (55-iodide). 2-Amino-4-chloropyrimidine (55) (13.0 g, 100 mmol)
was added to a 57 wt.% aqueous solution of hydriodic acid (115 ml, 1.00 mol) at 0 °C and the mixture
was stirred at room temperature for 3 h. The mixture was cooled to 0 °C and the resulting precipitate
2 3
was removed by filtration and taken up in cold 5N aqueous Na CO (200 ml). The mixture was
extracted with EtOAc (3 × 500 ml) and the combined organic layers were concentrated under reduced
1
pressure to deliver 2-amino-4-iodopyrimidine (21.1 g, 95.0 mmol, 95% yield) as a white solid. H NMR
(
400 MHz, DMSO-d ) δ ppm 7.78 (d, J = 5.0 Hz, 1 H), 7.02 (br. s, 2 H), 7.00 (d, J = 5.0 Hz, 1 H). MS
6
(ESI, pos. ion) m/z: 222.1 (M+1).
6.1.2. 2-Aminopyrimidine-4-carbonitrile (57). A flask containing a solution of 55-iodide (4.09 g, 18.5
mmol) in NMP (75 ml) was purged with argon for 5 minutes. Zinc cyanide (2.28 g, 19.4 mmol) and
tetrakis(triphenylphosphine)palladium (0) (1.71 g, 1.48 mmol) were added and the mixture was stirred
at 80 °C for 2 h. The mixture was cooled to room temperature, EtOAc (200 ml) and 30% aqueous
4
NH OH (200 ml) was added, and stirring was continued for 1 h. The layers were separated, the aqueous
layer was extracted with EtOAc (4 × 200 ml), and the combined organic layers were concentrated under
1
6

ACCEPTED MANUSCRIPT
reduced pressure. Et
2
O (30 ml) was added and the precipitate was collected by filtration and washed
with Et O to deliver 2-aminopyrimidine-4-carbonitrile (1.66 g, 13.8 mmol, 75% yield) as a white solid.
2
1
H NMR (400 MHz, DMSO-d ) δ ppm 8.51 (d, J = 4.7 Hz, 1 H), 7.31 (br. s, 2 H), 7.08 (d, J = 4.7 Hz, 1
6
H). MS (ESI, pos. ion) m/z: 121.1 (M+1).
6
.1.3 2-Aminopyrimidine-4-carbothioamide (60). 40-48 wt.% aqueous ammonium sulfide (8.27 ml,
8.5 mmol) was added to a mixture of 57 (5.3 g, 44.1 mmol), triethylamine (6.14 ml, 44.1 mmol), and
pyridine (28.8 ml, 353 mmol) and the resulting suspension was stirred at 52 °C for 2 h. The mixture was
concentrated under reduced pressure and H O was added to the residue. The mixture was chilled to 0 ºC
4
2
and the resulting precipitate was removed by filtration to deliver 2-aminopyrimidine-4-carbothioamide
1
(
5.5 g, 35.7 mmol, 80% yield) as a yellow solid. H NMR (400 MHz, DMSO-d ) δ ppm 10.23 (br. s, 1
6
H), 9.58 (br. s, 1 H), 8.42 (d, J = 4.9 Hz, 1 H), 7.35 (d, J = 4.9 Hz, 1 H), 6.80 (br. s, 2 H). MS (ESI, pos.
ion) m/z: 155.1 (M+1).
6
.1.4. Ethyl 2-(2-aminopyrimidin-4-yl)-4-hydroxythiazole-5-carboxylate (46). A mixture of 60 (5.4 g,
5.0 mmol), pyridine (11.4 ml, 140 mmol), and diethyl bromomalonate (3) (8.84 ml, 52.5 mmol) in
EtOH (15 ml) was heated under reflux for 16 h. The mixture was concentrated under reduced pressure
and H O was added to the residue. The resulting precipitate was removed by filtration and washed with
3
2
Et O to deliver ethyl 2-(2-aminopyrimidin-4-yl)-4-hydroxythiazole-5-carboxylate (5.30 g, 19.9 mmol,
2
1
5
7% yield) as a tan solid. H NMR (400 MHz, DMSO-d ) δ ppm 12.27 (s, 1 H), 8.46 (d, J = 4.9 Hz, 1
6
H), 7.11 (d, J = 4.9 Hz, 1 H), 7.01 (s, 2 H), 4.23 (q, J = 7.1 Hz, 2 H), 3.35 (br. s, 3 H), 1.27 (t, J = 7.1
Hz, 3 H). MS (ESI, pos. ion) m/z: 267.0 (M+1).
6.1.5. Ethyl 2-(2-aminopyrimidin-4-yl)-4-(trifluoromethylsulfonyloxy)thiazole-5-carboxylate (46-
triflate). Trifluoromethanesulfonic anhydride (7.95 g, 28.2 mmol) was added to a mixture of 46 (5.0 g,
1
2
8.8 mmol) and pyridine (4.46 g, 56.3 mmol) in CH
2 2
Cl (80 ml) at 0 ºC and the mixture was stirred for
h at 0 °C. H O (200 ml) was added, the layers were separated, and the organic layer was washed with
2
H O (4 × 200 ml). The organic layer was dried (Na SO ) and concentrated under reduced pressure. The
2
2
4
crude product was purified by a flash chromatography (silica gel, 0% to 40% EtOAc/hexanes) to deliver
1
7

ACCEPTED MANUSCRIPT
ethyl 2-(2-aminopyrimidin-4-yl)-4-(trifluoromethylsulfonyloxy)thiazole-5-carboxylate (4.3 g, 10.8
1
mmol, 58% yield). H NMR (400 MHz, DMSO-d ) δ ppm 8.54 (d, J = 4.9 Hz, 1 H), 7.17 (s, 2 H), 7.14
6
(d, J = 4.9 Hz, 1 H), 4.39 (q, J = 7.1 Hz, 2 H), 1.34 (t, J = 7.1 Hz, 3 H). MS (ESI, pos. ion) m/z: 398.9
(M+1).
6.1.6. Ethyl 2-(2-aminopyrimidin-4-yl)-4-(2,4-dichlorophenyl)thiazole-5-carboxylate (66). A mixture
of 46-triflate (485 mg, 1.22 mmol), 2,4-dichlorophenylboronic acid (279 mg, 1.46 mmol), trans-
dichlorobis(triphenyl-phosphine)palladium (ii) (85.0 mg, 0.122 mmol), and sodium carbonate
monohydrate (453 mg, 3.65 mmol) in 1,4-dioxane/H
mixture was partitioned between H O (10 ml) and CH
aqueous layer was extracted with CH Cl (2 × 15 ml). The combined organic layers were dried (MgSO )
2
O (1:1, 2.4 ml) was stirred at 60 °C for 1.5 h. The
2
2
Cl (15 ml), the layers were separated, and the
2
2
2
4
and concentrated under reduced pressure. The resulting brown oil was absorbed onto silica gel and
purified by flash chromatography (silica gel, 20% to 60% EtOAc/hexanes) to deliver ethyl 2-(2-
aminopyrimidin-4-yl)-4-(2,4-dichlorophenyl)thiazole-5-carboxylate (259 mg, 0.655 mmol, 54% yield)
1
as a yellow solid. H NMR (400 MHz, DMSO-d ) δ ppm 8.47 (d, J = 4.9 Hz, 1 H), 7.78 (d, J = 2.0 Hz, 1
6
H), 7.59 (d, J = 8.2 Hz, 1 H), 7.55 (dd, J = 8.2, 2.0 Hz, 1 H), 7.22 (d, J = 4.9 Hz, 1 H), 7.06 (s, 2 H),
4
.18 (q, J = 7.2 Hz, 2 H), 1.14 (t, J = 7.2 Hz, 3 H). MS (ESI, pos. ion) m/z: 395.0 (M+1).
.1.7. 2-(2-Aminopyrimidin-4-yl)-4-(2,4-dichlorophenyl)thiazole-5-carboxylic acid (66-acid). Lithium
6
hydroxide monohydrate (78.0 mg, 1.86 mmol) was added to a solution of 66 (245 mg, 0.620 mmol) in
THF/H O (1:1, 1.2 ml) and the mixture was stirred at room temperature for 2 h and under reflux for 12
2
h. The THF was removed under reduced pressure and the mixture was diluted with H O (10 ml). The
2
precipitate was collected by filtration and titurated in boiling MeOH to deliver 2-(2-aminopyrimidin-4-
yl)-4-(2,4-dichlorophenyl)thiazole-5-carboxylic acid (180 mg, 0.490 mmol, 79% yield) as an off-white
1
solid. H NMR (400 MHz, DMSO-d
6
) δ ppm 13.65 (br. s, 1 H), 8.45 (d, J = 5.0 Hz, 1 H), 7.76 (d, J =
1
2
.9 Hz, 1 H), 7.57 (d, J = 8.3 Hz, 1 H), 7.53 (dd, J = 8.3, 1.9 Hz, 1 H), 7.21 (d, J = 5.0 Hz, 1 H), 7.03 (s,
H). MS (ESI, pos. ion) m/z: 367.0 (M+1).
1
8

ACCEPTED MANUSCRIPT
6.1.8. 2-(2-Aminopyrimidin-4-yl)-4-(2,4-dichlorophenyl)thiazole-5-carboxamide (74). HATU (211
mg, 0.556 mmol) was added to a mixture of 66-acid (170 mg, 0.463 mmol) and diisopropylethylamine
242 µl, 1.39 mmol) in DMF (1 ml) and the mixture was stirred at room temperature for 30 min. A 0.5M
(
solution of ammonia in 1,4-dioxane (2.78 ml, 1.39 mmol) was added and the mixture was stirred at
room temperature for 2 h. The mixture was diluted with MeOH/CH Cl , absorbed onto silica gel, and
2
2
purified by flash chromatography (silica gel, 5% to 10% MeOH/CH Cl ). The resulting yellow solid
2
2
was further purified by reversed phase HPLC (Phenomenex Gemini-NX 10µ C18 110Å, 100 × 50 mm,
0% to 95% H O/MeCN, 0.1% TFA). The product containing fractions were combined, basified with
solid NaHCO , and extracted with CH Cl (3 × 20 ml). The combined organic layers were dried
MgSO ) and concentrated under reduced pressure to deliver 2-(2-aminopyrimidin-4-yl)-4-(2,4-
1
2
3
2
2
(
4
1
dichlorophenyl)thiazole-5-carboxamide (120 mg, 0.328 mmol, 71% yield) as a light yellow solid. H
NMR (400 MHz, DMSO-d ) δ ppm 8.44 (d, J = 5.0 Hz, 1 H), 7.73 (d, J = 1.8 Hz, 1 H), 7.67 (br. s, 2 H),
6
7
.56 (d, J = 8.2 Hz, 1 H), 7.53 (dd, J = 8.2, 1.8 Hz, 1 H), 7.21 (d, J = 5.0 Hz, 1 H), 7.01 (br. s, 2 H). MS
(ESI, pos. ion) m/z: 365.9 (M+1).
6.1.9. Ethyl 4-hydroxy-2-(4-pyridyl)-thiazole-5-carboxylate (1). Prepared in 44% yield analogously to
1
4
6 using 2 instead of 60. H NMR (400 MHz, DMSO-d
6
): δ 12.35 (br. s, 1 H), 8.75 (dd, J = 4.5, 1.5 Hz,
2
H), 7.87 (dd, J = 4.5, 1.5 Hz, 2 H), 4.26 (q, J = 7.0 Hz, 2 H), 1.28 (t, J = 7.0 Hz, 3 H). MS (ESI, pos.
ion) m/z: 251 (M+1).
.1.10. Ethyl 4-methoxy-2-(pyridin-4-yl)thiazole-5-carboxylate (4). Iodomethane (267 µl, 4.30 mmol)
6
was added to a mixture of 1 (717 mg, 2.86 mmol) and potassium carbonate (792 mg, 5.73 mmol) in
DMF (5 ml) and the mixture was heated in a sealed vial at 80 °C for 18 h. Another 1.5 equiv.
iodomethane (267 µl, 4.30 mmol) was added and the mixture was heated in a sealed vial at 80 °C for 6
h. The mixture was cooled to room temperature and partitioned between H
2
O (30 ml) and CH
Cl
2
Cl
2
(20
ml). The layers were separated, and the aqueous layer was extracted with 10% MeOH/CH
2
2
(3 × 30
ml). The combined organic layers were dried (MgSO ) and concentrated under reduced pressure. The
4
crude product was absorbed onto silica gel and purified by flash chromatography (silica gel, 20% to
1
9

ACCEPTED MANUSCRIPT
6
0% EtOAc/hexanes) to deliver ethyl 4-methoxy-2-(pyridin-4-yl)thiazole-5-carboxylate (199 mg, 0.753
1
mmol, 26% yield) as an orange solid. H NMR (300 MHz, CDCl ) δ ppm 8.75 (dd, J = 4.5, 1.5 Hz, 2
3
H), 7.81 (dd, J = 4.5, 1.5 Hz, 2 H), 4.36 (q, J = 7.2 Hz, 2 H), 4.25 (s, 3 H), 1.38 (t, J = 7.2 Hz, 3 H). MS
(ESI, pos. ion) m/z: 265.1 (M+1).
6.1.11. Ethyl 2-(pyridin-4-yl)-4-(trifluoromethylsulfonyloxy)thiazole-5-carboxylate (5). Prepared in
1
8
4
9% yield analogously to 46-triflate using 1 instead of 46. H NMR (CDCl , 400 MHz): δ 8.80 (dd, J =
3
.5, 1.5 Hz, 2 H), 7.78 (dd, J = 4.5, 1.5 Hz, 2 H), 4.46 (q, J = 7.1 Hz, 2 H), 1.43 (t, J = 7.1 Hz, 3 H). MS
(
ESI, pos. ion) m/z: 383.0 (M+1).
.1.12. Ethyl 4-amino-2-(pyridin-4-yl)thiazole-5-carboxylate (6). A mixture of 5 (200 mg, 0.523
mmol) and a 0.5 M solution of ammonia in 1,4-dioxane (3.14 ml, 1.57 mmol) was heated in a Biotage
6
®
reactor at 120 °C for 1 h and at 160 °C for 1 h and for 3 h. The mixture was cooled to room temperature
and concentrated under reduced pressure. The residue was partitioned between saturated aqueous
NaHCO (20 ml) and EtOAc (20 ml) and the aqueous layer was extracted with EtOAc (2 × 20 ml). The
3
combined organic layers were washed with brine (40 ml), dried (Na SO ), and concentrated under
2
4
reduced pressure. The crude product was purified by flash chromatography (silica gel, 70%
EtOAc/hexanes) to deliver ethyl 4-amino-2-(pyridin-4-yl)thiazole-5-carboxylate (16 mg, 0.064 mmol,
1
1
2% yield) as a light yellow solid. H NMR (400 MHz, CDCl ) δ ppm 8.73 (d, J = 4.5 Hz, 2 H), 7.77 (d,
3
J = 5.7 Hz, 2 H), 5.92 (br. s, 2 H), 4.35 (q, J = 7.0 Hz, 2 H), 1.39 (t, J = 7.0 Hz, 3 H). MS (ESI, pos. ion)
m/z: 250.1 (M+1).
6
.1.13. Ethyl 4-(methylamino)-2-(pyridin-4-yl)thiazole-5-carboxylate (7). A 2M solution of
methylamine in THF (392 µl, 0.785 mmol) was added to a solution of 5 (300 mg, 0.785 mmol) in THF
5 ml) and the mixture was stirred at 75 °C for 1.5 h. The mixture was filtered and H O (10 ml) was
added to the filtrate whereupon a suspension formed slowly. The solid was collected by filtration and
(
2
washed with H
2
O to deliver ethyl 4-(methylamino)-2-(pyridin-4-yl)thiazole-5-carboxylate (29 mg,
1
0
.110 mmol, 14% yield) as a yellow solid. H NMR (400 MHz, CDCl ) δ ppm 8.73 (d, J = 5.1 Hz, 2 H),
3
2
0

ACCEPTED MANUSCRIPT
7
.82 (d, J = 5.9 Hz, 2 H), 6.72 (br. s, 1 H), 4.32 (q, J = 7.1 Hz, 2 H), 3.24 (d, J = 5.1 Hz, 3 H), 1.37 (t, J
=
7.1 Hz, 3 H). MS (ESI, pos. ion) m/z: 264.0 (M+1).
.1.14. Ethyl 4-(benzylamino)-2-(pyridin-4-yl)thiazole-5-carboxylate (8). Prepared in 41% yield
6
analogously to 7 using benzylamine instead of 2M methylamine in THF. The compound was
1
additionally purified by tituration with MeOH. H NMR (400 MHz, DMSO-d ) δ ppm 8.73 (d, J = 5.7
6
Hz, 2 H), 7.87 (d, J = 5.7 Hz, 2 H), 7.61 (t, J = 6.3 Hz, 1 H), 7.38–7.43 (m, 2 H), 7.33 (t, J = 7.5 Hz, 2
H), 7.20–7.27 (m, 1 H), 4.77 (d, J = 6.3 Hz, 2 H), 4.27 (q, J = 7.0 Hz, 2 H), 1.28 (t, J = 7.0 Hz, 3 H). MS
(ESI, pos. ion) m/z: 340.0 (M+1).
6.1.15. Ethyl 4-(phenylamino)-2-(pyridin-4-yl)thiazole-5-carboxylate (9). Prepared in 45 yield
analogously to 7 using aniline instead of 2M methylamine in THF. The reaction took 3 d at 75 °C for
1
completion. H NMR (400 MHz, DMSO-d ) δ ppm 9.14 (s, 1 H), 8.78 (d, J = 6.1 Hz, 2 H), 7.97 (d, J =
6
6
7
.1 Hz, 2 H), 7.69 (d, J = 8.0 Hz, 2 H), 7.37 (t, J = 8.0 Hz, 2 H), 7.05 (t, J = 7.3 Hz, 1 H), 4.35 (q, J =
.1 Hz, 2 H), 1.33 (t, J = 7.1 Hz, 3 H). MS (ESI, pos. ion) m/z: 326.0 (M+1).
6.1.16. Ethyl 4-(dimethylamino)-2-(pyridin-4-yl)thiazole-5-carboxylate (10). Prepared in 93% yield
1
analogously to 7 using dimethylamine instead of 2M methylamine in THF. H NMR (400 MHz,
DMSO-d
H), 3.17 (s, 6 H), 1.27 (t, J = 7.0 Hz, 3 H). MS (ESI, pos. ion) m/z: 278.1 (M+1).
.1.17. Ethyl 2-(pyridin-4-yl)thiazole-5-carboxylate (13). A mixture of 4-pyridylboronic acid (11) (3
6
) δ ppm 8.73 (dd, J = 4.5, 1.4 Hz, 2 H), 7.88 (dd, J = 4.5, 1.4 Hz, 2 H), 4.21 (q, J = 7.0 Hz, 2
6
g, 24.4 mmol), ethyl 2-bromothiazole-5-carboxylate (12) (5.76 g, 24.4 mmol), sodium carbonate (7.76
g, 73.2 mmol) in DME/H O (3:1, 100 ml) was purged with argon for 10 min. trans-
2
Dichlorobis(triphenylphosphine)palladium (ii) (1.37 g, 1.95 mmol) was added and flushing with argon
was continued for 5 min. The mixture was stirred at 80 °C for 3 d, concentrated under reduced pressure,
2
and partitioned between H O (100 ml) and EtOAc (100 ml). Celite was added, the layers were
separated, and the aqueous layer was extracted with EtOAc (2 × 100 ml). The combined organic layers
were washed with brine (150 ml), dried (Na SO ), and concentrated under reduced pressure. The solid
2
4
was dissolved in MeOH, and the solution was dried (Na SO ) and concentrated under reduced pressure.
2
4
2
1

ACCEPTED MANUSCRIPT
The crude product was purified by flash chromatography (silica gel, 100% EtOAc) to deliver ethyl 2-
1
(
pyridin-4-yl)thiazole-5-carboxylate (25 mg, 0.107 mmol, 0.4% yield) as a tan solid. H NMR (400
MHz, CDCl ) δ ppm 8.77 (d, J = 6.1 Hz, 2 H), 8.50 (s, 1 H), 7.85 (d, J = 6.1 Hz, 2 H), 4.43 (q, J = 7.2
3
Hz, 2 H) 1.43 (t, J = 7.2 Hz, 2 H). MS (ESI, pos. ion) m/z: 235.0 (M+1).
6
.1.18. Ethyl 4-methyl-2-(pyridin-4-yl)thiazole-5-carboxylate (15). A mixture of pyridine-4-
carbothioamide (2) (479 mg, 3.47 mmol) and ethyl 2-chloroacetoacetate (14) (575 µl, 4.16 mmol) in
EtOH (5 ml) was heated under reflux for 30 h. The mixture was diluted with CH Cl (5 ml), absorbed
onto silica gel, and purified by flash chromatography (silica gel, 2% to 8% MeOH/CH Cl ) to deliver
2
2
2
2
1
ethyl 4-methyl-2-(pyridin-4-yl)thiazole-5-carboxylate (628 mg, 2.53 mmol, 73% yield) as a tan solid. H
NMR (300 MHz, CDCl ) δ ppm 8.74 (d, J = 5.3 Hz, 2 H), 7.82 (dd, J = 4.5, 1.3 Hz, 2 H), 4.38 (q, J =
3
7
.1 Hz, 2 H), 2.81 (s, 3 H), 1.41 (t, J = 7.1 Hz, 3 H). MS (ESI, pos. ion) m/z: 249.1 (M+1).
6.1.19. Ethyl 4-phenyl-2-(pyridin-4-yl)thiazole-5-carboxylate (16). Prepared in 66% yield analogously
1
to 66 using 5 and phenylboronic acid instead of 46-triflate and 2,4-dichlorophenylboronic acid. H
NMR (300 MHz, CDCl ) δ ppm 8.76 (dd, J = 4.6, 1.5 Hz, 2 H), 7.91 (dd, J = 4.6, 1.5 Hz, 2 H), 7.84–
3
7
.80 (m, 2 H), 7.40–7.53 (m, 3 H), 4.33 (q, J = 7.1 Hz, 2 H), 1.32 (t, J = 7.1 Hz, 3 H). MS (ESI, pos.
ion) m/z: 311.0 (M+1).
.1.20. Ethyl 4-(2-hydroxyphenyl)-2-(pyridin-4-yl)thiazole-5-carboxylate (17). Prepared in 54% yield
6
analogously to 66 using 5 and 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol instead of 46-
1
triflate and 2,4-dichlorophenylboronic acid. Reaction at 40 °C for 2 h. H NMR (300 MHz, DMSO-d )
6
δ ppm 8.44 (d, J = 4.8 Hz, 1 H), 8.31 (t, J = 5.0 Hz, 1 H), 7.72 (s, 1 H), 7.46–7.62 (m, 2 H), 7.21 (d, J =
5
2
.0 Hz, 1 H), 6.98 (s, 2 H), 3.16 (d, J = 7.0 Hz, 2 H), 1.01 (t, J = 7.0 Hz, 3 H). MS (ESI, pos. ion) m/z:
81.0 (M+1–EtOH, M+1 of lactone).
6.1.21. Ethyl 4-(2-chlorophenyl)-2-(pyridin-4-yl)thiazole-5-carboxylate (18). Prepared in 56% yield
analogously to 66 using 5 and 2-chlorophenylboronic acid instead of 46-triflate and 2,4-
1
dichlorophenylboronic acid. H NMR (400 MHz, DMSO-d ) δ ppm 8.76 (d, J = 6.2 Hz, 2 H), 7.98 (d, J
6
2
2

ACCEPTED MANUSCRIPT
=
6.2 Hz, 2 H), 7.41–7.64 (m, 4 H), 4.17 (q, J = 7.5 Hz, 2 H), 1.11 (t, J = 7.5 Hz, 3 H). MS (ESI, pos.
ion) m/z: 345.0 (M+1).
.1.22. Ethyl 4-(2-chloro-4-fluorophenyl)-2-(pyridin-4-yl)thiazole-5-carboxylate (19). Prepared in
6
5
2
5% yield analogously to 66 using 5 and 2-chloro-4-fluorophenylboronic acid instead of 46-triflate and
1
,4-dichlorophenylboronic acid. H NMR (400 MHz, DMSO-d ) δ ppm 8.76 (d, J = 6.2 Hz, 2 H), 7.98
6
(
d, J = 6.2 Hz, 2 H), 7.53–7.69 (m, 2 H), 7.35 (td, J = 8.8, 2.6 Hz, 1 H), 4.18 (q, J = 7.4 Hz, 2 H), 1.13
t, J = 7. Hz, 3 H). MS (ESI, pos. ion) m/z: 363.0 (M+1).
(
6.1.23. Ethyl 4-(2,3-dichlorophenyl)-2-(pyridin-4-yl)thiazole-5-carboxylate (20). Prepared in 80%
yield analogously to 66 using 5 and 2,3-dichlorophenylboronic acid instead of 46-triflate and 2,4-
1
dichlorophenylboronic acid. H NMR (400 MHz, DMSO-d ) δ ppm 8.78 (dd, J = 4.7, 1.2 Hz, 2 H), 8.00
6
(dd, J = 4.7, 1.6 Hz, 2 H), 7.80 (dd, J = 7.8, 1.7 Hz, 1 H), 7.56 (dd, J = 7.8, 1.7 Hz, 1 H), 7.50 (t, J = 7.8
Hz, 1 H), 4.20 (q, J = 7.1 Hz, 2 H), 1.12 (t, J = 7.1 Hz, 3 H). MS (ESI, pos. ion) m/z: 379.0 (M+1).
6.1.24. Ethyl 4-(2,4-dichlorophenyl)-2-(pyridin-4-yl)thiazole-5-carboxylate (21). Prepared in 74%
1
yield analogously to 66 using 5 instead of 46-triflate. H NMR (300 MHz, CDCl ) δ ppm 8.76 (d, J =
3
5
.0 Hz, 2 H), 7.86 (dd, J = 4.4, 1.2 Hz, 2 H), 7.53 (d, J = 1.7 Hz, 1 H), 7.42 (d, J = 8.2 Hz, 1 H), 7.37
(dd, J = 8.2, 1.7 Hz, 1 H), 4.27 (q, J = 7.1 Hz, 2 H), 1.24 (t, J = 7.1 Hz, 3 H). MS (ESI, pos. ion) m/z:
3
6
79.0 (M+1).
.1.25. Ethyl 4-(2-chloro-4-methoxyphenyl)-2-(pyridin-4-yl)thiazole-5-carboxylate (22). Prepared in
6
0% yield analogously to 66 using 5 and 2-chloro-4-methoxyphenylboronic acid instead of 46-triflate
1
and 2,4-dichlorophenylboronic acid. H NMR (400 MHz, DMSO-d ) δ ppm 8.77 (dd, J = 4.7, 1.4 Hz, 2
6
H), 7.99 (dd, J = 4.7, 1.4 Hz, 2 H), 7.49 (d, J = 8.5 Hz, 1 H), 7.18 (d, J = 2.5 Hz, 1 H), 7.04 (dd, J = 8.5,
2
3
.5 Hz, 1 H), 4.21 (q, J = 7.1 Hz, 2 H), 3.86 (s, 3 H), 1.16 (t, J = 7.1 Hz, 3 H). MS (ESI, pos. ion) m/z:
75.8 (M+1).
6.1.26. Ethyl 4-(pyridin-3-yl)-2-(pyridin-4-yl)thiazole-5-carboxylate (23). Prepared in 22% yield
analogously to 66 using 5 and 3-pyridylboronic acid instead of 46-triflate and 2,4-
1
dichlorophenylboronic acid. H NMR (300 MHz, CDCl ) δ ppm 9.06 (s, 1 H), 8.78 (d, J = 5.6 Hz, 2 H),
3
2
3

ACCEPTED MANUSCRIPT
8
.69 (d, J = 4.9 Hz, 1 H), 8.19 (d, J = 7.8 Hz, 1 H), 7.88 (d, J = 5.6 Hz, 2 H), 7.42 (dd, J = 7.8, 4.9 Hz, 1
H), 4.35 (q, J = 7.1 Hz, 2 H), 1.33 (t, J = 7.1 Hz, 3 H). MS (ESI, pos. ion) m/z: 312.0 (M+1).
.1.27. Ethyl 4-(naphthalen-2-yl)-2-(pyridin-4-yl)thiazole-5-carboxylate (24). Prepared in 59% yield
6
analogously to 66 using 5 and 2-naphthylboronic acid instead of 46-triflate and 2,4-
1
dichlorophenylboronic acid. H NMR (300 MHz, CDCl ) δ ppm 9.06 (s, 1 H), 8.78 (d, J = 5.6 Hz, 2 H),
3
8
.69 (d, J = 4.8 Hz, 1 H), 8.19 (d, J = 7.8 Hz, 1 H), 7.88 (d, J = 5.6 Hz, 2 H), 7.42 (dd, J = 7.8, 4.8 Hz, 1
H), 4.35 (q, J = 7.0 Hz, 2 H) 1.33 (t, J = 7.0 Hz, 3 H). MS (ESI, pos. ion) m/z: 361.1 (M+1).
.1.28. Methyl 3-amino-2-bromo-3-oxopropanoate (26). Bromine (0.875 ml, 17.1 mmol) was added
6
portionwise to a solution of methyl 3-amino-3-oxopropanoate (25) (2 g, 17.1 mmol) in glacial acetic
acid (30 ml) and the mixture was stirred at room temperature until it became colorless. The mixture was
partitioned between EtOAc (100 ml) and H O (100 ml), the layers were separated, and the organic layer
2
was washed with H O (3 × 100 ml), saturated aqueous NaHCO (2 × 100 ml), and brine (100 ml). The
2
3
organic layer was dried (Na SO ) and concentrated under reduced pressure to deliver methyl 3-amino-2-
2
4
bromo-3-oxopropanoate (1.50 g, 7.65 mmol, 45% yield) as a yellow oil, which used without further
purification.
6.1.29. 4-Hydroxy-2-(pyridin-4-yl)thiazole-5-carboxamide (27). A mixture of 26 (1.50 g, 7.65 mmol),
pyridine-4-carbothioamide (2) (1.00 g, 7.24 mmol) and pyridine (1.75 ml, 21.7 mmol) in toluene (40
ml) was heated under reflux for 16 h. The mixture was cooled to room temperature, the solvent was
decanted and the resulting solid was titurated with hexane. H O was added, the solid was collected by
2
filtration and titurated with MeOH to deliver 4-hydroxy-2-(pyridin-4-yl)thiazole-5-carboxamide (50 mg,
1
0
.226 mmol, 3% yield) as a tan solid. H NMR (400 MHz, DMSO-d ) δ ppm 13.04 (br. s, 1 H), 8.74 (d,
6
J = 5.9 Hz, 2 H), 7.83 (d, J = 5.9 Hz, 2 H), 7.68 (br. s, 1 H), 7.24 (br. s, 1 H). MS (ESI, pos. ion) m/z:
22.1 (M+1).
.1.30. 4-Hydroxy-2-(pyridin-4-yl)thiazole-5-carboxylic acid hydrochloride (28). A mixture of 1 (700
2
6
mg, 2.80 mmol) and lithium hydroxide monohydrate (1.2 g, 50 mmol) in MeOH/THF/H O (1:1:1, 30
2
ml) was stirred at room temperature for 1 h and at 70 °C for 2 h. The volatile solvents were removed
2
4

ACCEPTED MANUSCRIPT
under reduced pressure, H
2
O (10 ml) was added, and the solution was acidified by the dropwise addition
of 10% aqueous HCl. The resulting solid was removed by filtration and titurated with MeOH to deliver
4
-hydroxy-2-(pyridin-4-yl)thiazole-5-carboxylic acid hydrochloride (450 mg, 1.74 mmol, 62% yield) as
1
a tan solid. H NMR (400 MHz, DMSO-d ) δ ppm 10.89 (s, 1 H), 8.67 (d, J = 6.1 Hz, 2 H), 7.79 (d, J =
6
6
.1 Hz, 2 H), 6.47 (s, 1 H). MS (ESI, pos. ion) m/z: 222.9 (M+1).
.1.31. 4-Hydroxy-2-(pyridin-4-yl)thiazole-5-carbonyl chloride hydrochloride (28-acid chloride). A
M solution of oxalyl chloride in CH Cl (3.30 ml, 6.60 mmol) was added dropwise to a suspension of
8 (853 mg, 3.30 mmol) in CH Cl (7 ml). N,N-dimethylformamide (50 µl, 3.30 mmol) was added and
6
2
2
2
2
2
2
the mixture was stirred at room temperature for 1 h. The mixture was concentrated under reduced
pressure to deliver a yellow solid, which was used without further purification.
6
.1.32. 4-Hydroxy-N-methyl-2-(pyridin-4-yl)thiazole-5-carboxamide (29). A 2M solution of
methylamine in THF (0.92 ml, 1.840 mmol) was added to a solution of 28-acid chloride (170 mg,
.613 mmol) in DMF (1.5 ml) and the mixture was stirred at room temperature for 2 h. The mixture was
filtered and purified by reversed phase HPLC (Phenomenex Gemini-NX 10µ C18 110Å, 100 × 50 mm,
0% to 95% H O/MeCN, 0.1% TFA). The product containing fractions were combined, basified with
solid NaHCO , and extracted with CH Cl (3 × 20 ml). The combined organic layers were dried
MgSO ) and concentrated under reduced pressure. The resulting solid was taken up in 2N NH in
0
1
2
3
2
2
(
4
3
MeOH/CH Cl , absorbed onto silica gel, and further purified by flash chromatography (silica gel, 2% to
2
2
1
0
5% MeOH/CH Cl ) to deliver 4-hydroxy-N-methyl-2-(pyridin-4-yl)thiazole-5-carboxamide (11 mg,
2 2
1
.047 mmol, 8% yield) as an orange solid. H NMR (300 MHz, CD OD) δ ppm 8.62 (d, J = 4.0 Hz, 1
3
H), 7.88 (d, J = 5.7 Hz, 2 H), 2.92 (s, 3 H). MS (ESI, pos. ion) m/z: 236.0 (M+1).
6.1.33. N-Benzyl-4-hydroxy-2-(pyridin-4-yl)thiazole-5-carboxamide (30). Prepared in 13% yield
1
analogously to 29 using benzylamine instead of 2M methylamine in THF. H NMR (300 MHz, CD
3
OD)
δ ppm 8.66 (dd, J = 4.8, 1.1 Hz, 2 H), 7.93 (dd, J = 4.8, 1.3 Hz, 1 H), 7.30–7.42 (m, 2 H) 7.21–7.30 (m,
H), 4.58 (s, 2 H). MS (ESI, pos. ion) m/z: 312.0 (M+1).
1
2
5

ACCEPTED MANUSCRIPT
6.1.34. 4-Hydroxy-N,N-dimethyl-2-(pyridin-4-yl)thiazole-5-carboxamide (31). Prepared in 10% yield
1
analogously to 29 using 2M dimethylamine in THF instead of 2M methylamine in THF. H NMR (300
MHz, CD OD) δ ppm 8.66 (d, J = 5.9 Hz, 2 H), 7.93 (d, J = 5.9 Hz, 2 H), 3.21 (s, 6 H). MS (ESI, pos.
3
ion) m/z: 250.1 (M+1).
6.1.35. 4-Phenyl-2-(pyridin-4-yl)thiazole-5-carboxylic acid (32). Prepared in 82% yield analogously
1
to 66-acid using 16 instead of 66. H NMR (300 MHz, DMSO-d ) δ ppm 13.66 (br. s, 1 H), 8.77 (d, J =
6
5
.2 Hz, 2 H), 7.98 (d, J = 5.2 Hz, 2 H), 7.81 (dd, J = 5.6, 2.2 Hz, 2 H), 7.30–7.60 (m, 3 H). MS (ESI,
pos. ion) m/z: 283.1 (M+1).
6.1.36. 4-Phenyl-2-(pyridin-4-yl)thiazole-5-carboxamide (33). Prepared in 77% yield analogously to
1
7
4 using 32 instead of 66-acid. H NMR (300 MHz, DMSO-d ) δ ppm 8.76 (d, J = 5.3 Hz, 2 H) 7.97–
6
7
.82 (m, 6 H), 7.38–7.56 (m, 3 H). MS (ESI, pos. ion) m/z: 282.0 (M+1).
6.1.37. 4-Phenyl-2-(pyridin-4-yl)thiazole-5-carbohydrazide (34). Prepared in 25% yield analogously
1
to 74 using 32 and hydrazine hydrate instead of 66-acid and 0.5M ammonia in 1,4-dioxane. H NMR
(
400 MHz, DMSO-d ) δ ppm 9.89 (br. s, 1 H), 8.77 (dd, J = 4.5, 1.8 Hz, 2 H), 7.98 (dd, J = 4.5, 1.6 Hz,
6
2
H), 7.85 (dt, J = 6.7, 1.6 Hz, 2 H), 7.40–7.53 (m, 3 H), 4.65 (d, J = 2.9 Hz, 2 H). MS (ESI, pos. ion)
m/z: 297.1 (M+1).
.1.38. N-Hydroxy-4-phenyl-2-(pyridin-4-yl)thiazole-5-carboxamide (35). Prepared in 11% yield
6
analogously to 74 using 32 and hydroxylamine hydrochloride instead of 66-acid and 0.5M ammonia in
1
1
,4-dioxane. H NMR (400 MHz, DMSO-d ) δ ppm 11.34 (br. s, 1 H), 9.48 (br. s, 1 H), 8.76 (d, J = 5.9
6
Hz, 2 H), 7.97 (d, J = 5.9 Hz, 2 H), 7.85 (d, J = 7.0 Hz, 2 H), 7.39–7.58 (m, 3 H). MS (ESI, pos. ion)
m/z: 298.1 (M+1).
6.1.39. 4-Methoxy-2-(pyridin-4-yl)thiazole-5-carboxylic acid (4-acid). Prepared in 85% yield
1
analogously to 66-acid using 4 instead of 66. H NMR (300 MHz, DMSO-d
6
) δ ppm 13.07 (br. s, 1 H),
8
.75 (d, J = 3.9 Hz, 2 H), 7.91 (d, J = 3.9 Hz, 2 H), 4.13 (s, 3 H). MS (ESI, pos. ion) m/z: 237.1 (M+1).
6.1.40. 4-Methoxy-2-(pyridin-4-yl)thiazole-5-carboxamide (36). Prepared in 87% yield analogously to
1
7
4 using 4-acid instead of 66-acid. H NMR (300 MHz, DMSO-d ) δ ppm 8.74 (d, J = 5.7 Hz, 2 H),
6
2
6

ACCEPTED MANUSCRIPT
7
.89 (d, J = 5.7 Hz, 2 H), 7.77 (br. s, 1 H), 7.11 (br. s, 1 H), 4.17 (s, 3 H). MS (ESI, pos. ion) m/z: 236.0
(
M+1).
.1.41. 4-(2,4-Dichlorophenyl)-2-(pyridin-4-yl)thiazole-5-carboxylic acid (21-acid). Prepared in 88%
6
1
yield analogously to 66-acid using 21 instead of 66. H NMR (300 MHz, DMSO-d ) δ ppm 13.73 (br. s,
6
1
H), 8.77 (d, J = 5.5 Hz, 2 H), 7.97 (d, J = 5.5 Hz, 2 H), 7.78 (d, J = 1.6 Hz, 1 H), 7.60 (d, J = 8.2 Hz, 1
H), 7.55 (dd, J = 8.2, 1.6 Hz, 1 H). MS (ESI, pos. ion) m/z: 350.9 (M+1).
6.1.42. 4-(2,4-Dichlorophenyl)-2-(pyridin-4-yl)thiazole-5-carboxamide (37). Prepared in 40% yield
1
analogously to 74 using 21-acid instead of 66-acid. H NMR (300 MHz, DMSO-d
6
) δ ppm 8.75 (d, J =
5
.5 Hz, 2 H), 7.92 (d, J = 5.5 Hz, 2 H), 7.75 (s, 1 H), 7.72–7.52 (m, 4 H). MS (ESI, pos. ion) m/z: 350.0
(M+1).
6.1.43. 4-Phenyl-2-(pyridin-4-yl)thiazole-5-carbonitrile (38). A solution of oxalyl chloride (124 µl,
1
.39 mmol) in CH Cl (1 ml) was added to a mixture of 33 (261 mg, 0.928 mmol) and dimethyl
2
2
sulfoxide (132 µl, 1.86 mmol) in CH Cl (3 ml) at –78 °C and the mixture was stirred at –78 °C for 15
2
2
min. Triethylamine (387 µl, 2.78 mmol) was added dropwise and the mixture was stirred at –78 °C for
5 min. H O (10 ml) was added and the mixture was stirred at room temperature for 10 min. It was
diluted with H O (30 ml) and extracted with CH Cl (2 × 50 ml). The combined organic layers were
dried (Na SO ) and concentrated under reduced pressure. The resulting yellow solid was titurated with
1
2
2
2
2
2
4
EtOAc to deliver 4-phenyl-2-(pyridin-4-yl)thiazole-5-carboxamide (170 mg, 0.646 mmol, 70% yield) as
1
a yellow solid. H NMR (400 MHz, DMSO-d ) δ ppm 8.84 (dd, J = 4.5, 1.8 Hz, 2 H), 8.15 (dt, J = 6.5,
6
1
.8 Hz, 2 H), 8.06 (dd, J = 4.5, 1.8 Hz, 2 H), 7.54–7.71 (m, 2 H). MS (ESI, pos. ion) m/z: 264.0 (M+1).
.1.44. 4-Phenyl-2-(pyridin-4-yl)-5-(2H-tetrazol-5-yl)thiazole (39). A mixture of 38 (133 mg, 0.505
mmol), sodium azide (36.1 mg, 0.556 mmol) and zinc bromide (114 mg, 0.505 mmol) in H O (2 ml)
6
2
was heated in a Biotage® microwave reactor at 140 °C for 20 min and at 155 °C for 30 min. The
suspension was filtered and the solid was taken up in 0.25 N aqueous NaOH (10 ml). The resulting
suspension was stirred for 30 min and filtered. The filtrate was brought to pH = 6.5 by the addition of
1
0% aqueous HCl and the resulting solid was removed by filtration of deliver 4-phenyl-2-(pyridin-4-yl)-
2
7

ACCEPTED MANUSCRIPT
1
5
-(2H-tetrazol-5-yl)thiazole (42 mg, 0.137 mmol, 27% yield) as a light yellow solid. H NMR (400
MHz, CD OD) δ ppm 8.79 (dd, J = 4.6, 1.4 Hz, 2 H), 8.03 (dd, J = 4.6, 1.6 Hz, 2 H), 7.73 (dd, J = 6.6,
3
2
.8 Hz, 2 H), 7.43–7.49 (m, 3 H). MS (ESI, pos. ion) m/z: 307.1 (M+1).
.1.45. 2-(4-Phenyl-2-(pyridin-4-yl)thiazol-5-yl)-1,3,4-oxadiazole (40). A mixture of 34 (260 mg,
6
0
0
.877 mmol), trimethyl orthoformate (144 µl, 1.32 mmol), and 4-methylbenzenesulfonic acid (2.12 mg,
.012 mmol) in THF (3 ml) was heated under reflux for 2.5 h. Another 1.5 mol% 4-
methylbenzenesulfonic acid (2.12 mg, 0.012 mmol) was added and the mixture was heated under reflux
for 1 h. The mixture was concentrated under reduced pressure and partitioned between saturated
aqueous NaHCO
extracted with EtOAc (2 × 50 ml). The combined organic layers were washed with brine (50 ml), dried
Na SO ), and concentrated under reduced pressure. The crude product was taken up in DMSO, filtered,
3
(50 ml) and EtOAc (50 ml). The layers were separated and the aqueous layer was
(
2
4
and purified by reversed phase HPLC (Phenomenex Gemini-NX 10µ C18 110Å, 100 × 50 mm, 10% to
5% H O/MeCN, 0.1% TFA). The product containing fractions were combined, basified with solid
9
2
NaHCO , and extracted with CH Cl . The combined organic layers were dried (Na SO ) and
3
2
2
2
4
concentrated under reduced pressure to deliver 2-(4-phenyl-2-(pyridin-4-yl)thiazol-5-yl)-1,3,4-
1
oxadiazole (11 mg, 0.036 mmol, 4% yield) as a yellow solid. H NMR (400 MHz, CD
3
OD) δ ppm 8.97
(
s, 1 H), 8.74 (d, J = 5.7 Hz, 2 H), 8.09 (d, J = 6.0 Hz, 2 H), 7.78 (dd, J = 6.7, 3.0 Hz, 2 H), 7.47–7.54
m, 3 H). MS (ESI, pos. ion) m/z: 307.1 (M+1).
.1.46. Ethyl 4-[(2-aminophenyl)amino]-2-(4-pyridyl)-1,3-thiazole-5-carboxylate (5-amide). A
(
6
solution of 5 (2.01 g, 5.26 mmol) and 1,2-phenylenediamine (1.71 g, 15.8 mmol) in dioxane (10.5 ml)
was heated under reflux for 48 h. The solvent was removed under reduced pressure and the resulting
3 2 2
residue was purified by flash chromatography (silica gel, 1% to 2% 2M NH in MeOH/CH Cl ) to
deliver ethyl 4-[(2-aminophenyl)amino]-2-(4-pyridyl)-1,3-thiazole-5-carboxylate (1.29 g, 3.79 mmol,
1
7
2% yield) as a bright-yellow solid. H NMR (400 MHz, DMSO-d
6
) δ ppm 8.95 (dd, J = 4.5, 1.7 Hz, 2
H), 8.79 (s, 1 H), 8.08 (dd, J = 4.5, 1.7 Hz, 2 H), 7.38 (dd, J = 7.9, 1.1 Hz, 1 H), 7.03–7.12 (m, 2 H),
2
8