Design, synthesis, and biological evaluation of novel catecholopyrimidine based PDE4 inhibitor for the treatment of atopic dermatitis

Article abstract of DOI:10.1016/j.ejmech.2017.12.069

Selective inhibition of phosphodiesterase (PDE) 4B favorably suppresses the synthesis of inflammatory cytokines and subsequently arrest the development of atopic dermatitis via modulating the intracellular cAMP levels. Considering the side effects of corticosteroids, selective PDE4 inhibition could constitute an effective alternative therapy for the treatment of atopic dermatitis (AD). In this study, a series of novel catechol based compounds bearing pyrimidine as the core have been synthesized and screened for the PDE4 inhibitory properties. The PDE4 selectivity of the active compounds over other PDEs has been investigated. Compound 23 bearing pyrimidine core functionalized with catechol, pyridine and trifluoromethyl groups can effectively inhibit the PDE4B with IC₅₀ value in nanomolar range (IC₅₀ = 15 ± 0.4 nM). Compound 23 exhibited seven fold higher selectivity towards PDE4B over PDE4D. Molecular Docking study confirmed its stronger affinity towards catalytic domain of PDE4B. In-vivo analysis confirmed that compound 23 effectively alleviated the symptoms of atopic dermatitis in DNCB–treated Balb/c mice by suppressing the synthesis of inflammatory mediators such as TNF-α and Ig-E. Taken together, this study suggested that compound 23 could be an effective PDE4 inhibitor for the potential treatment of AD.

Full text of DOI:10.1016/j.ejmech.2017.12.069

Accepted Manuscript
Design, synthesis, and biological evaluation of novel catecholopyrimidine based
PDE4 inhibitor for the treatment of atopic dermatitis
Baskaran Purushothaman, Parthasarathy Arumugam, Goutam Kulsi, Joon Myong
Song
PII:
S0223-5234(17)31100-5
10.1016/j.ejmech.2017.12.069
EJMECH 10047
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 September 2017
Revised Date: 14 December 2017
Accepted Date: 18 December 2017
Please cite this article as: B. Purushothaman, P. Arumugam, G. Kulsi, J.M. Song, Design, synthesis,
and biological evaluation of novel catecholopyrimidine based PDE4 inhibitor for the treatment of atopic
dermatitis, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.069.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis, and biological evaluation of novel catecholopyrimidine based PDE4
inhibitor for the treatment of atopic dermatitis
Baskaran Purushothaman, Parthasarathy Arumugam, Goutam Kulsi, and Joon Myong Song*
College of Pharmacy, Seoul National University, Seoul 151-742, South Korea
*
Corresponding author: Prof. Joon Myong Song,
College of Pharmacy, Seoul National University,
Seoul 151-742, South Korea
Tel: +82 2 880 7841; Fax: + 82 2 871 2238
E-mail: jmsong@snu.ac.kr (Joon Myong Song)
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Design, synthesis, and biological evaluation of novel catecholopyrimidine based PDE4
inhibitor for the treatment of atopic dermatitis
Abstract
Selective inhibition of phosphodiesterase (PDE) 4B favorably suppresses the synthesis of
inflammatory cytokines and subsequently arrest the development of atopic dermatitis via
modulating the intracellular cAMP levels. Considering the side effects of corticosteroids,
selective PDE4 inhibition could constitute an effective alternative therapy for the treatment of
atopic dermatitis (AD). In this study, a series of novel catechol based compounds bearing
pyrimidine as the core have been synthesized and screened for the PDE4 inhibitory properties.
The PDE4 selectivity of the active compounds over other PDEs has been investigated.
Compound 23 bearing pyrimidine core functionalized with catechol, pyridine and
trifluoromethyl groups can effectively inhibit the PDE4B with IC50 value in nanomolar range
(
IC50 = 15±0.4 nM). Compound 23 exhibited seven fold higher selectivity towards PDE4B over
PDE4D. Molecular Docking study confirmed its stronger affinity towards catalytic domain of
PDE4B. In-vivo analysis confirmed that compound 23 effectively alleviated the symptoms of
atopic dermatitis in DNCB–treated Balb/C mice by suppressing the synthesis of inflammatory
mediators such as TNF-α, and Ig-E. Taken together, this study suggested that compound 23
could be an effective PDE4 inhibitor for the potential treatment of AD.
Keywords: atopic dermatitis; phosphodiesterase-4; catecholopyrimidine; dinitrochlorobenzene;
molecular docking.
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1
. Introduction
Atopic dermatitis (AD) is an inflammatory skin disease affecting young children, at a prevalence
rate of 10 – 25 % globally [1, 2]. Eczematous skin lesions, scaling, and unbearable itching
(pruritus) are the clinical features of AD [3]. The onset of AD involves complex interplay
between genetic, environmental, and immunological factors [4]. Sensitization of lymphocytes to
infiltrating allergens due to skin barrier dysfunction is considered to be the primary cause of the
disease. Sensitized T-helper lymphocyte cells, antigen-presenting cells, neutrophils, and mast
cells release a set of immunological mediators, which collectively determine the severity and
longevity of AD [5]. Several lines of evidence indicate that cyclic adenosine monophosphate
(cAMP) dependent protein kinase-A (PKA) negatively regulates the pro-inflammatory cytokine
synthesis in lymphocytes and macrophages [6, 7]. In addition, PKA has been reported to
suppress the tumor necrosis factor- α (TNF-α) secretion by inhibiting the expression of
inflammatory transcription factor Nuclear factor-kappaB (NF-κB) [8]. Previous studies reported
that elevated phosphodiesterase-4B (PDE4B) is responsible for the increased levels of pro-
inflammatory mediators in the leukocyte of AD patients [9]. It can thus be concluded that cAMP
level can negatively regulate inflammation in AD. Intracellular cAMP homeostasis is regulated
by fine-tuning between cAMP synthesis by adenylcyclase in response to extracellular signaling
and its degradation by phosphodiesterases (PDEs).
Phosphodiesterases are enzymes that hydrolyze cAMP and cyclic guanosine mono phosphate
(cGMP) into their inactive form of 5’-adenosine mono phosphate (5’AMP) and 5’- guanosine
mono phosphate (5’GMP), respectively [10]. PDEs are subdivided into 11 different groups based
on sequence similarity, biochemical properties, and inhibitor selectivity. Based on their affinity
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to cAMP or cGMP, PDEs are further classified into: (i) cAMP-specific (PDE4, 7, and 8); (ii)
cGMP- specific (PDE5, 6, and 9); and (iii) specific to both cAMP and cGMP (PDE1, 2, 3, 10,
and 11) [11, 12]. Among the cAMP-specific PDEs, PDE4 enzymes are significantly expressed in
inflammatory cells. Four isoforms of PDE4 have been discovered so far, i.e., PDE4A, B, C, and
D. Earlier, it has been reported that PDE4B is the predominant isoform of PDE4 expressed in
lymphocytes and macrophages [13]. It has been reported that the inhibition of PDE4D is
responsible for the emetic side effects [14]. Selective inhibition of the PDE4B may provide a
means to achieve efficacy while potentially mitigating the adverse effects such as nausea and
emesis. Thus, research efforts mainly focusing on the selective PDE4B inhibitors to minimize
the side effects [15-18]. Inhibition of PDE4B in lymphocytes and macrophages could effectively
elevate intracellular cAMP levels, which can suppress the expression of inflammatory cytokines
[19].
Antihistamines and corticosteroids are widely used to treat inflammatory skin diseases, but side
effects, such as skin-atrophy, present a serious issue [20, 21]. PDE4B inhibition has become an
attractive alternative therapy for inflammatory diseases such as COPD or asthma [22, 23]. It has
to be noted that overexpression of PDE4 has been reported in the activation of lymphocytes and
degranulation of mast cells [24-26]. Several oral PDE4 inhibitors are in clinical trials. For
example, the FDA recently approved roflumilast as a potential second generation PDE4B
inhibitor. However, several reports suggested that roflumilast causes nausea and emesis as its
major side effects due to abnormalities that resulted in the gastrointestinal tract (GIT) and the
central nervous system (CNS), respectively [27-30]. Apart from the antagonist role in the
regulation of pro-inflammatory mediators, the second messenger cAMP has been implicated in
the regulation of virtually every physiological process, including the opening of ion channels,
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muscle relaxation, cellular differentiation, neuro muscular interaction, carbohydrate and lipid
metabolism, and cell death mechanism [31-36]. Due to these active roles, it seems that side
effects are inevitable for the long-term administration of oral PDE4 inhibitors. Thus, a great deal
of clinical attention is required while administering oral PDE4 inhibitors. Conversely, topical
application of PDE4 inhibitors can be an optimal choice to treat AD, since it is expected to
exhibit less interference to cAMP-relevant metabolism. Recently, phase-2 clinical trials of
apremilast and LEO-29102, a PDE4 inhibitors, has been completed for AD [37, 38]. Nonetheless,
a safe and side-effect free non-steroidal anti-inflammatory PDE4 inhibitors is still needed for the
treatment of inflammatory disorders.
In order to develop a next generation novel PDE4 inhibitor, we rationally designed the
compounds by covalently combining the available ligands such as catechol (3,4-dialkoxyphenyl)
moiety with pyrimidine core into a single active molecule (Fig. 1). Design strategy and chemical
structures of different PDE4 inhibitors are shown in Fig.1. The functionalization of catechol
moiety to the pyrimidine core is one of the novel approaches for ligand based PDE4 drug
discovery. In addition to that, the catechol pyrimidine core is a potential bioisostere of adenosine
and also mimics the nucleotide part of the cAMP. A library of catechol functionalized
pyrimidine carboxamides has been synthesized in our laboratory which inhibits PDE4 enzyme in
the mid-to-low nanomolar range. Among all other synthesized compounds, compound 23
displayed most active compound (IC50 = 15±0.4 nM) through pharmacological evaluation.
Compound 23 possesses a pyrimidine core functionalized with catechol (3,4-dimethoxyphenyl)
scaffold, pyridinyl and trifluoromethyl (–CF ) groups. Pyrimidine derivatives, such as
3
pyrido[3’,2’:4,5]furo[3,2-d]pyrimidine and other heterocyclic compounds, have been extensively
described for their PDE4 inhibitory activity [39-41]. The phenyl pyrimidine, 5,5-dioxo-7,8-
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dihydro-6H-thiopyrano[3,2-d]pyrimidine, and pyridazino[4,5-b]indolizine derivatives also
reported as selective PDE4B inhibitors [16, 17, 42]. Many reports suggested that the -CF
3
functionalized compounds offers the characteristics of high electronegativity, enhanced
permeability to cell membrane, increased lipophilicity for a particular affinity to a target protein,
and minimized drug metabolism [43, 44]. These characteristics enhances the drug likeliness of
newly synthesized compounds. In the present study, the pharmacological profile of compound
2
3 was assessed by PDE4B enzyme assay and molecular docking with catalytic domain of
PDE4B. The anti-atopic profile was investigated by using DNCB-induced AD-like skin
inflammation in Balb-c mice. Our results show that compound 23 effectively inhibits PDE4B
activity, thereby alleviating AD-like symptoms in DNCB-induced Balb-c mice.
2
2
. Results and discussion
.1. Chemistry: Synthesis of small molecules
The synthesis of morpholine and catechol functionalized pyrimidine core compounds depicted in
Scheme 1 and 2. The condensation reaction of diethyl ethoxymethylenemalonate with different
substituted amidines, such as trifluoromethylacetamidine or S-methylisothiourea hemisulfate salt,
in the presence of sodium ethoxide as alkaline catalyst provided 2-(trifluoromethyl) or 2-
(methylthio) pyrimidinone (i). The chlorination of (trifluoromethyl) or 2-(methylthio)
pyrimidinone with POCl at 100° C yielded Ethyl 4-chloro-2-(methylthio) or Ethyl 4-chloro-2-
3
(
trifluoromethyl)pyrimidine-5-carboxylate (Scheme 1 and 2, (ii)). The nucleophilic substitution
SN2) reaction of chloropyrimidine (ii) with morpholine provided the corresponding
(
morpholinopyrimidine esters (Scheme 1, (iii)) in the presence of DIPEA in 2-propanol. These
morpholinopyrimidine ester were hydrolyzed to the corresponding acids (iv) by using aq.NaOH
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in ethanol. To this pyrimidine acid intermediates (iv) different aromatic amines were coupled by
acid-amine reaction to get morpholinopyrimidine carboxamides (v). The library of
catecholopyrimidine carboxamides 11-26 were synthesized from Ethyl 4-chloro-2-(methylthio)
or Ethyl 4-chloro-2-(trifluoromethyl)pyrimidine-5-carboxylate (ii). The classical Suzuki cross-
coupling reaction of chloropyrimidine intermediates (ii) with 3,4-dimethoxyphenylboronic acid
in the presence of tris(dibenzylideneacetone)dipalladium[0] and potassium fluoride under
thermal heating provided Ethyl 4-(3,4-dimethoxyphenyl)-2-(trifluoromethyl)pyrimidine-5-
carboxylate or Ethyl 4-(3,4-dimethoxyphenyl)-2-(methylthio)pyrimidine-5-carboxylate (Scheme
2
, (vi)) in good yields. The Suzuki cross-coupling reaction was optimized to get catechol
pyrimidine compounds [45]. The intermediate (vi) was produced in 74% yield by treating 1.5
equivalent of 3,4-dimethylphenylboronic acid. This was followed by the ester hydrolysis of
intermediates-(vi) with 1N NaOH in ethanol, providing the corresponding 4-(3,4-
dimethoxyphenyl)pyrimidine-5-carboxylic acids (Scheme 2, (vii)). Compounds 16 and 21 were
synthesized by oxidation of 13 and 20 through m-CPBA which act as oxidant. The
catecholopyrimidine compounds 11 and 18 were synthesized according to a previously reported
procedure.[46] Briefly, the acid derivatives 4-(3, 4-dimethoxyphenyl)-2-(methylthio or
trifluoromethyl) pyrimidine-5-carboxylic acid, (iv) or (vii) were activated using oxalylchloride to
afford pyrimidine acid chlorides such as 4-(3,4-dimethoxyphenyl)-2-(trifluoromethyl or
methylthio)pyrimidine-5-carbonyl chloride. This was followed by the reaction of p-nitrophenol,
obtaining pyrimidine nitrophenol ester compounds, (4-nitrophenyl 4-(3,4-dimethoxyphenyl)-2-
(trifluoromethyl or methylthio)pyrimidine-5-carboxylate) which reacted with 4-amino-3,5-
dichloropyridine in the presence of sodium hydride as base at 0°C to obtain final products 11 and
1
8. The synthetic scheme of compounds 11 and 18 were described in supporting information.
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A library consisting of morpholine (Scheme 1, (v)) and catecholopyrimidine (Scheme 2, (viii))
carboxamides were prepared by treating corresponding acid intermediates (iv, vii)) respectively,
with a variety of aromatic and aliphatic amines using solution-phase synthesis. The acid-amine
coupling reaction were performed by either acid-activating agent EDCI with Hunig’s base or the
activation of acid intermediates by vilsmeier reaction. In the vilsmeier reaction, addition of
oxalyl chloride and stoichiometric amount of DMF to the acid intermediate at 0⁰C formed
dimethylforminium chloride (DMFCl) by in-situ generation. Different substituted aromatic and
aliphatic amines were then reacted to these intermediates to form the final compounds. The
compounds’ novelty was checked, and they were not present in any accessible academic
database (e.g., PubChem, Scifinder). The structure and molecular weight of the compounds were
verified by NMR, IR, and HRMS (ESI) spectroscopy. We further characterized the active
compound 23 by 2D NMR (COSY, NOESY, HMBC, and HSQC) spectroscopy to assign all
protons and carbons of compound 23 (Supporting Information Figure S1-S6). In addition, we
have also confirmed the structure of compound 23 by X-ray crystal analysis (CCDC-1524592,
Fig. 2).
2
.2. In vitro evaluation of compounds: PDE4B enzyme assay
The library of morpholine and catechol functionalized pyrimidine compounds were tested for
dose-dependent PDE4B enzyme inhibition at different concentrations (1 nM – 5000 nM) using
rolipram as a control drug. IC50 values for the compounds were calculated based on the molar
concentration of PDE4B inhibitor required for 50% inhibition of the cAMP substrate into
5
’AMP. We have studied the structure activity relationship (SAR) of the pyrimidine nucleus
against PDE4B enzyme to get active compound. The different functional groups were substituted
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th
at 2, 4, and 5 -positions of pyrimidine nucleus and their PDE4B inhibitory potential was
evaluated. The main catechol (3,4-dimethoxyphenyl) scaffold was functionalized with different
th
th
substituents at the 4 - and 5 -positions of the -CF or -SCH functionalized pyrimidine nucleus
3
3
to determine the precise molecular feature required for PDE4 inhibition. Firstly, we have
th
introduced and fixed the morpholine ring to the 4 position of the pyrimidine nucleus (i.e.
th
morpholinopyrimidine). Then different aromatic amines were coupled at the 5 position of the
morpholinopyrimidine acid by EDCI coupling reaction to get morpholinopyrimidine
carboxamides and elucidated their PDE4 inhibitory activity. In particular, the acid-amine
coupling reaction of aromatic amine such as 3,4-dimethoxyphenyl (catechol) amine to the 2-
(trifluoromethyl) or 2-(methylthio)-4-morpholinopyrimidine-5-carboxylic acid, significantly
enhanced PDE4B inhibition activity (e.g., Compound 1). We introduced catechol moiety to the
th
4
position of pyrimidine nucleus to improve the inhibitory activity of the compounds against
PDE4B. Interestingly, the Suzuki coupling reaction of 3,4-dimethoxyphenylboronic acid at the
th
4
position of the pyrimidine nucleus obtained catecholopyrimidine intermediate (Scheme 2
(vii)). Substitution of aromatic amine, such as 4-aminopyridines, to the catecholopyrimidine
intermediate (vii) drastically increased PDE4B-inhibitory activity. Remarkably, the compounds
th
(
e.g., Compound 18, 23, and 25) bearing a catechol moiety to the 4 position of the pyrimidine
nucleus exhibited excellent PDE4 inhibitory ability, compared to that of catechol moiety
th
substituted at the 5 position. This was also observed in the results of 4-aminopyridine-
th
substituted at 5 position of pyrimidine acid intermediates (e.g., 7, 12, 18, and 23) (See Table 1).
th
However, coupling of aliphatic amines at the 5 position of 4-(3, 4-dimethoxyphenyl)-2-
(methylthio) pyrimidine-5-carboxylic acid decreased the PDE4B-inhibition activity (e.g. 15 and
1
7). The compound 23 showed fifty-five time’s higher activity than compound 15, this
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modification reveals that catechol moiety is ideal and required for PDE4B inhibition activity.
nd
Generally, the -CF
3
functionalization to the 2 position of the pyrimidine nucleus exhibited
better PDE4B activity compared with thiomethyl (-SCH ) substituted compounds. In conclusion,
3
catecholopyrimidine based small molecules 1, 5, 8, 18, 23, and 25 exhibited excellent PDE4B
inhibitory activity, with IC50 values in the nanomolar range. In particular, the IC50 values of
compound 1 and 23 are 31±7.17 nM and 15±0.4 nM, respectively. Table 1 shows the IC values
5
0
of synthesized small molecule inhibitors. Further to find whether the most active compounds 1,
, 8, 18 and 23 possess the selectivity against PDE4B, enzyme inhibition assay against PDE1A,
5
PDE3B, PDE11A, and all the isoforms of PDE4 were performed. Result depicted in Table-2
th
showed that catechol at the 4 position of pyrimidine nucleus have more preference for PDE4
enzyme over other PDEs (PDE1A, PDE3B and PDE11A). For example, the compound 23
selectively inhibited the PDE4B compared to the other PDEs and PDE4 iso-enzymes, and up to
several fold higher PDE4B inhibitory activity than rolipram. The dose dependent inhibition of
PDE4 isoenzymes and other PDEs by compound 23 are shown in Fig.3. Compound 23 showed
seven fold higher selectivity towards PDE4B compared to the PDE4D. However, the
th
compounds 1 and 8 bearing a 3,4-dimethoxyphenyl (catechol) group at the 5 position of
pyrimidine core showed low selectivity for PDE4B over PDE1A, PDE3B and PDE11A (Table 2).
2
.3. Molecular docking study
A molecular docking study was performed in order to obtain further insight into the binding
modes of the most active compound 23 into the cAMP binding site of PDE4A, B, C and D
enzymes. The reference compound rolipram, a well-known inhibitor of PDE4, was also docked
with PDE4B to compare with compound 23. (Fig.4). PDEs are mainly constituted of three
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domains: an N-terminal regulatory domain, a catalytic domain, and a C-terminal domain. The
most conserved catalytic domain usually consists of 17 α-helices [47-49] and forms a deep
pocket located at the junction of the three subdomains. It consists of a metal binding pocket (M
2
+
2+
pocket) containing bivalent (Zn , Mg ) metals which form a complex with phosphate moiety
of cAMP, and a (Q pocket) containing glutamine, which forms hydrogen bonds with the
nucleotide moiety of cAMP and a solvent pocket (S pocket). The crystal structure of the 3,4-
dialkoxyphenyl (catechol) functionalized compounds bound to PDE4B active site revealed that
the hydrophobic interactions with residues in the M pocket and Q pocket are very important for
PDE4 inhibition activity [50]. Molecular docking of compound 23 and reference compound
rolipram were performed on a binding model based on the PDE4B crystal structure (PDB: 1F0J)
by the CDOCKER protocol. The docking score of compound 23 and rolipram were -4.10 and -
3
.31, respectively. These computer simulated docking scores support the affinity and biological
activity of the compound 23. The 3D optimal conformation, schematic 2D diagram, and the
corresponding amino acids responsible for all of the major interactions of compound 23 and
rolipram in the PDE4B active site are shown in Fig. 4. From the docking study it was noted that
the amino acid residues are mostly responsible for binding with cAMP in PDE4B (Asp 392, Ile
4
10, Tyr 233, Cys 499) are present in both compound 23 as well as in rolipram. However,
compound 23 showed more binding interaction with other amino acid residues (Phe 414, Pro 396,
Leu 393 His 234, Thr 345) constituting strong affinity. From these interactions it is confirmed
that compound 23 is deeply embedded into the active site of PDE4B than rolipram. From the
docking result, it can be seen that the fluorine molecule (-CF , F ) within 3.23 Å of Gln 500
3
a
generates a strong conventional hydrogen bond, and another fluorine atom (-CF , F ) forms a
3
b
3
strong carbon hydrogen bond with His 234 within 3.71 Å. The –CF group in compound 23 itself
1
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exhibits an important hydrophobic interaction with His 234 and Phe 414 residue (Phe 414 is
3
highly conserved among PDE family). The -CF group has tremendous influence in medicinal
chemistry and responsible for very limited interaction in the reference compound rolipram
compared to compound 23. In halogen bonding, a fluorine atom is the donor can establish
conventional hydrogen bonding and other carbon hydrogen bonds, with the protein molecule
providing additional stability. The –CF group on an aryl system increases lipophilicity, whereas
3
fluorination of saturated alkyl groups results in decreased lipophilicity [51]. The -CF group also
3
polarizes the compound and increases the positive intermolecular interaction between the
3
receptor and fluorine atom [52]. The hydrogen bonds between fluorine atom of the –CF group
with Gln 500 and His 234 constitute important parameter to the PDE4 inhibition. This was also
in agreement with PDE4B enzyme assay. In contrast to the binding mode of rolipram, it was
observed that Asp 346 forms a strong hydrogen bond with catechol methoxy group with the
distance 3.22 Å, and Pro 396 forms another strong –hydrogen bond with pyridine nitrogen with
the distance 3.69 Å in compound 23. These seem to be the reason for the stability and
improvement of the biological activity and selectivity within the binding pocket of the target
protein PDE4B (Fig.4a and 4b). The hydrophobic interactions are also superior in compound 23
compared to the reference compound rolipram in the hydrophobic pocket. The other two
hydrogen bonds with Glu 304 and Asp 346 are also vital for the stabilization of the binding
mode. On the other hand, Leu 393, His 234, Phe 414, and Met 347 form strong hydrophobic
interactions with ligand atoms (compound 23), making the ligand more stable in the hydrophobic
pocket of the protein (Fig. 4a, 4b). The hydrophobic interactions constitute critically important
structural parameters, and exhibit a direct correlation with binding free energy in the
thermodynamics of the protein-ligand interaction. The interactions between the ligand and the
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hydrophobic side chains of the protein molecule contribute significantly to the binding free
energy in a receptor ligand association [53, 54]. The hydrophobic residues in the protein
molecule usually repel water and other polar groups, thereby increasing a net attraction of non-
polar groups of the ligand [55]. Hydrophobic amino acid residue, phenyl alanine, serves to affix
the ligand within the active site of PDE4B. In addition, the aromatic ring of phenyl alanine in
PDE4B may also participate in the stacking interaction with the aromatic moieties of the ligand.
The receptor surface model with aromatic overlay of compound 23 in Fig. S8 (Supporting
Information Fig.S8, c) also suggests that the compound is deeply embedded into the active site of
PDE4B.
In this study, we also successfully docked compound 23 with PDE4 A, C, and D enzymes with
respective PDB ID 2QYK, 2QYM, and 3G4K to validate the selectivity of our synthesized
compound 23 towards these enzymes with the docking score -8.85, -4.26, and -7.06. In all cases,
however, interactions of compound 23 with these PDE4 subtypes were limited. The 3D optimal
conformation of compound 23 in the active site of PDE4 A, C, and D and their comparative
study of all major interactions are shown in supplementary Fig.S7. From these computational
studies, we confirmed that the compound 23 is selective towards PDE4B. Thus, compound 23
could constitute a potential inhibitor of PDE4B with potent anti-inflammatory activity.
2
.4. X-ray crystal structure of compound 23
Crystallization of compound 23 was attempted using different solvent systems by changing the
°
°
polarity, boiling point, and temperature conditions (0 C - 30 C). Finally, single crystals of
compound 23 were obtained by slow evaporation of chloroform and hexane mixture in 1:1
°
volume ratio at 4 C. Compound 23 crystallized in monoclinic form with space group P2 /n. All
1
1
3

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C-C bond lengths in the structure are almost similar (∼ 1.3 to 1.4 Å), except amide carbon (C16,
2
1
.50 Å) and –CF carbon (C5, 1.51 Å), and are typical sp hybridized carbon-carbon bonds.
3
Unfavorable H-H, N-H, and O-H interactions are not found within the molecule. The
aminopyridine ring is bifurcated by C1 and C16 in compound 23 through amide bond (C16, N17,
and O1), and orient near perpendicularly to the pyrimidine nucleus. The bond lengths and bond
angles of the fluorine atoms attached to carbon in the –CF
3
group are almost similar (F1, F2, F3
°
∼
1.3 Å and 107 ). The planes of catechol moiety attached to pyrimidine nucleus (i.e.,
°
°
catecholopyrimidine) were slightly twisted by 123 and 117 dihedral angle (C1-C7-C8 and N6-
C7-C8), which may be due to two methoxy groups attached in C11 and C10 position in the
catechol structure. The ORTEP representation of compound 23 is shown in Fig. 2, and the
solvent is omitted for clarity. The crystal data and structure refinement parameter for compound
2
2
2
3 are given in Table S1 in Supporting Information.
.5. In vivo evaluation of compound 23
.5.1. Compound 23 treatment alleviates AD-like symptoms in mice
Based on the IC values and the molecular docking analysis, compound 23 was chosen for in
5
0
vivo analysis. DNCB was used to induce AD-like symptoms on the dorsal skin of Balb/c mice, as
represented in the scheme (Fig. 5A). AD-like skin features were confirmed by evaluating the
dryness of the skin, erosion, erythema, and hemorrhage. AD symptoms was progressively
increased in response to the periodic doses of DNCB in Balb-c mice. The compound 23
treatment clearly reversed these DNCB-induced AD symptoms in experimental mice. (Fig. 5B).
To evaluate the degree of clinical response to the inhibitor treatment, dermatitis score was
calculated. Skin features of dermatitis, such as erythema/hemorrhage, edema, excoriation/erosion,
1
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and scaling/dryness were considered in the evaluation. Dermatitis scores were significantly very
high and progressive in DNCB treated mice as compared to the control group of mice (p<0.001).
However, topical application of compound 23 daily once to the AD-induced mice remarkably
relieved the mice from the AD like symptoms. The dermatitis score significantly reversed after
the second week of drug treatment compared to the DNCB-alone treated mice (Fig. 5C, 5D).
Itching is the major clinical outcome in AD. Scratching frequency was monitored to determine
the efficacy of the synthesized compounds against itching behavior in the experimental mice.
The observed results clearly indicated that the DNCB-induced scratching frequency was
significantly reduced in compound 23- (p<0.01) treated AD-induced experimental mice (Fig. 5E).
2
.5.2. Histological observation
Physical features of the skin of experimental mice were carefully examined. Marked hypertrophy
and edematous features were observed in the skin of the DNCB-treated mice. In contrast, skin
features of compound 23-treated atopic mice exhibited less hypertrophy and no signs of edema
compared to the DNCB-treated mice. Compound 23 alone treated mice exhibited normal skin
feature, no sign of allergy was observed. Furthermore, significantly increased dorsal skin
thickness was observed in DNCB-treated mice (1.19 mm, p<0.001) as compared to that of the
control group (0.45 mm). Treatment of compound 23 (0.71 mm, p<0.01) markedly reduced the
DNCB-induced dorsal skin thickness (Fig. 5E). Histopathological observation of tissue sections
obtained from the experimental mice of control, compound 23 alone treated mice shows normal
skin architecture. However, dorsal skin of the DNCB–treated experimental mice exhibited severe
epidermal hyperplasia, (acanthosis) hyperkeratosis, and dense infiltration of inflammatory cells
into both the epidermal and dermal region of the skin. Treatment of compound 23 significantly
1
5

ACCEPTED MANUSCRIPT
reversed the DNCB-induced alterations. H&E stained sections of DNCB-induced mice showed
thickened epidermis compared to the control groups. However, reduced epidermal thickness was
observed in compound 23-treated AD-induced mice, as compared to the DNCB-alone treated
mice (Fig. 6A).
2
.5.3. Compound 23 treatment suppress the inflammatory mediators
Serum IgE level is directly co-related to the severity of AD. IgE is crucially involved in the
activation of mast cell and subsequently the secretion of pruritus factor such as histamine and
prostaglandins. Moreover, activated mast cell releases chemokines and cytokines such as IL-4,
IL-13, and TNF-α which collectively promote the mobilization and sensitization of Th2 cells [56,
5
7]. The observed result in the present study show that serum IgE level in the DNCB-treated
mice were significantly increased (p<0.001), as compared to the normal and drug control mice.
However, the DNCB-induced immunoglobulin-E (Ig-E) synthesis was significantly reduced in
the compound 23 (p<0.001) treated mice (Fig. 6B). Reduced levels of serum IgE can be co-
related to the reduced scratching behavior among the compound 23 treated AD affected mice.
Similarly pro-inflammatory mediator, TNF-α level was also measured in the tissue extracts of
experimental mice. The observed result show that compound 23 treatment significantly reduced
the DNCB–induced TNF-α levels in the experimental mice (Fig. 6C). Suppression of TNF-α is
highly beneficial in the treatment AD since the TNF-α is a well-known activator of pro-
inflammatory transcription factor NF-kB in lymphocytes.
3
. Conclusion
1
6

ACCEPTED MANUSCRIPT
In conclusion, the catechol functionalized pyrimidine amides were designed and synthesized as
topical PDE4 inhibitors for the treatment of atopic dermatitis. Among these compounds, only
th
th
catechol group at the 4 and 5 position of pyrimidine nucleus exhibited the most significant
PDE4 inhibitory activities confirmed by PDE4 enzyme assay (e.g., 1, 5, 8, 12, 18, 23 and 25).
From PDE selectivity assay, it was observed that the compound 23 selectively inhibited the
PDE4B over other PDEs. The molecular docking study showed that the compound 23 enhances
the probability of binding interactions at the catalytic domain of the PDE4B. In addition,
trifluoromethyl group at the compound 23 provides strong halogen bond interactions between the
ligand and PDE4 enzyme, which results low IC50 value in nano molar range (23, 15±0.4 nM)
against PDE4B. Moreover, in vivo animal study demonstrated that compound 23 suppressed the
inflammatory mediators such as IgE and TNF-α in DNCB-induced Balb-c mice. Further
development is in progress for PDE4 inhibitors with higher activity and selectivity in our
laboratory.
4
. Experimental section
4
.1. Chemistry: Materials and Methods
All the reagents and solvents were purchased from sigma Aldrich, TCI or Alfa Aesar, and used
without further purification. The column chromatography was carried out in silica gel (Merck,
silica gel 60, particle size 0.063−0.200 mm, 70−230 mesh ASTM) with mentioned commercial
grade solvent as mobile phase. The progress of all reactions was monitored by thin layer
2
chromatography, which was performed on 2.0 × 4.0 cm aluminum sheets precoated with silica
gel 60 (HF-254, Merck) to a thickness of 0.25 mm. The developed chromatograms were viewed
1
7

ACCEPTED MANUSCRIPT
under ultraviolet light (254 and 365 nm). All the compounds including intermediates were
1
13
detected with 254nm UV light. H and C NMR spectra were recorded in deuterated chloroform
or dimethyl sulfoxide (Cambridge Isotope Labs), containing 0.05% v/v TMS as an internal
standard, using Bruker AVANCE500 and AVANCE400 spectrometer. Chemical shifts are
reported in values (ppm) relative to internal TMS, and J values are reported in Hz. The
abbreviations for the peak multiplicities are as follows: s (singlet), d (doublet), dd (doublet of
-1
doublets), t (triplet), q (quartet), m (multiplet), and br (broad). IR spectrum (cm ) was taken on
FT/IR-4200 JASCO (ATR) spectrometer. Mass spectroscopy data was obtained with an Agilent
6
530 Q-TOF LC-MS spectrometer. Single-crystal X-ray diffraction data were collected an
Agilent Technologies SuperNova diffractometer. The solid compounds melting point was
recorded in Bernard Electrothermal digital melting point apparatus (Model: IA9100, Cole-
Parmer, UK). Elemental analysis was performed in Flash1112, Flash2000 Elemental Analyzer
(Thermo Fisher Scientific, Germany).
4
.1.1. General procedure for the synthesis of compounds 1-6, 8-10, 13, 14, 16, 22, 24, 25 and 26
To a solution of morpholino or catecholopyrimidine carboxylic acid ((iv) or (vii)) (1 mmol) in
anhydrous DMF (5 ml) were added N-(3-dimethylaminopropyl)-N-ethylcarbodiimide
hydrochloride (EDCI.HCl) (1.2 mmol), Hunig’s base (2.5 mmol) and hydroxybenzotriazole
hydrate (HOBt) (1.2 mmol). The reaction mixture stirred for 30 min to dissolve and activate the
acid starting material at 0°C then to room temperature for 1 h. Then amine (1 mmol) was slowly
added and stirred the reaction mixture for overnight at room temperature under N atmosphere.
2
The completion of the reaction was monitored by TLC. Then the reaction mixture was slowly
poured into DI water and extracted with dichloromethane (DCM) (2×50 ml). The organic layer
1
8

ACCEPTED MANUSCRIPT
was washed with sat.NaHCO
3
and then dried over anhydrous sodium sulfate. The crude product
was purified by column chromatography using silica gel as stationary phase.
.1.2. General procedure for the synthesis of compounds 7, 11, and 18
4
The synthesis of compounds 7, 11, and 18 is required two-step process. Step-1: To a solution of
pyrimidine carboxylic acid (1 mmol) in dry DCM were added catalytic amount of DMF and
followed by oxalyl chloride (2.5 mmol) at 0⁰C. The reaction mixture was stirred at 0⁰C for 10
minutes then slowly changed to room temperature and stirred for 1 h. The progression of the
reaction was monitored by TLC. After completion of the reaction, the oxalyl chloride distil off
using vacuo completely. To this reaction mixture, dry THF was added and cooled to 0⁰C. Then
followed by triethylamine (2.5 mmol) and p-nitrophenol (1 mmol) was added. The reaction
mixture slowly changed to room temperature and stirring continued for 5 hours. The completion
of the reaction was monitored by TLC. Then the reaction mixture was poured slowly into ice-
cold DI water. The yellow color solid was formed. Then filtered, washed with water and hexane,
dried by suction pump. The 4-nitrophenyl pyrimidine carboxylate intermediate product was used
for next step without analysis. Step-2: To a solution of 4-amino-3,5-dichloropyridine (1.2 mmol)
in anhydrous DMF (5 ml) was added 60% NaH (2 mmol) at 0⁰C. The reaction mixture was
stirred for 30 minutes at 0⁰C. Then 4-nitrophenyl-5-pyrimidine carboxylate intermediate (1
mmol) was added slowly. The reaction mixture turn into room temperature and stirred for 1.5 h.
The reaction mixture slowly poured into DI water (100 ml) and extracted with DCM (2x100 ml).
The combined organic layer was dried over anhydrous Na SO . Then the crude product was
2
4
purified by silica gel column chromatography.
.1.3. General procedure for the synthesis of compounds 12, 15, 17, 19-21 and 23
4
1
9

ACCEPTED MANUSCRIPT
To a solution of morpholino or catecholopyrimidine carboxylic acid ((iv) or (vii), 1 mmol) in 5
ml of dry DCM were added oxalyl chloride (2 mmol) and anhydrous DMF (catalytic amount) at
0
⁰C. The reaction mixture was stirred at 0⁰C for 30 min then to room temperature for 1 h. The
reaction mixture was concentrated under N atmosphere in vacuo to give crude pyrimidine-5-
2
carbonyl chloride as a pale yellow solid. This compound was used for next reaction without
further purification. To this crude material 5 ml of dry DCM and triethylamine (2.5 mmol) was
added, followed by different substituted amines (1 mmol) at 0 ⁰C. The reaction mixture was
stirred at 0 ⁰C for 15 minutes, then slowly to room temperature for 1 h. The completion of the
reaction was monitored by TLC. The reaction mixture was concentrated in vacuo. The resulting
residue was purified by column chromatography using silica gel as a stationary phase.
4
4
.1.4. Reaction conditions and spectroscopic characterization of compounds 1-26
.1.4.1. N-(3,4-dimethoxyphenyl)-2-(methylthio)-4-morpholinopyrimidine-5-carboxamide (1)
Reagents
morpholinopyrimidine-5-carboxylic acid (250 mg, 0.98 mmol),
-aminoveratrole (150 mg, 0.98 mmol). Room temperature.
Column purification: methanol: dichloromethane (0.5:9.5).
Appearance: off-white Solid. Yield: 350 mg, 87%. Mp 170-172 °C; IR: 3262, 1639, 1519, 1399,
and
conditions:
2-(methylthio)-4-
4
-1 1
1
247, 1165, 1126 cm . H NMR (400 MHz, DMSO-d ) δ 10.33 (s, 1H), 8.21 (s, 1H), 7.35 (d, J
6
=
2.4 Hz, 1H), 7.20 (dd, J = 8.7, 2.4 Hz, 1H), 6.92 (d, J = 8.7 Hz, 1H), 3.73 (d, J = 2.5 Hz, 6H),
1
3
3
.63 (dd, J = 5.5, 3.4 Hz, 4H), 3.55 (dd, J = 5.4, 3.5 Hz, 4H), 2.48 (s, 3H). C NMR (500 MHz,
CDCl ) δ 173.31, 163.81, 160.93, 157.86, 149.30, 146.24, 131.25, 111.75, 111.46, 111.34,
04.50, 66.55, 56.15, 56.01, 48.64, 14.18. HRMS (ESI+): Calcd for C18 S (M+1)
3
1
22 4 4
H N O
2
0

ACCEPTED MANUSCRIPT
3
5
4
91.1395; found 391.1401. Anal.Calcd for C18
5.40; H, 5.66; N, 14.36.
22 4 4
H N O S: C, 55.37; H, 5.68; N, 14.35; found: C,
.1.4.2.
N-(3,4-dimethoxyphenethyl)-4-morpholino-2-(trifluoromethyl)pyrimidine-5-
carboxamide (2)
Reagents and conditions: 4-morpholino-2-(trifluoromethyl)
pyrimidine-5-carboxylic acid (100 mg, 0.36 mmol) and 3,4-
dimethoxyphenethylamine (60 µl, 0.36 mmol). Room
temperature, 12 h. Column purification: hexane:
ethylacetate (7:3). Yield: 40 mg, 50%. Mp 183-185 °C; IR: 3279, 2931, 1632, 1254, 1144, 808
-1 1
cm . H NMR (400 MHz, DMSO-d ) δ 8.76 (t, J = 5.4 Hz, 1H), 8.26 (s, 1H), 6.91 – 6.83 (m,
6
2
H), 6.75 (dd, J = 8.1, 2.0 Hz, 1H), 3.74 (s, 2H), 3.71 (s, 3H), 3.57 (dd, J = 5.7, 3.8 Hz, 4H), 3.48
13
(
dt, J = 6.2, 3.4 Hz, 6H), 2.75 (t, J = 7.0 Hz, 2H). C NMR (500 MHz, CDCl ) δ 165.95, 159.56,
3
1
6
56.69, 156.30, 156.01, 149.34, 148.10, 130.34, 120.66, 120.25, 118.05, 116.03, 111.66, 111.44,
6.32, 55.95, 47.14, 40.98, 34.87. HRMS (ESI+): Calcd for C20 (M+1) 441.1705;
23 3 4 4
H F N O
found 441.1712. Anal.Calcd for C H F N O : C, 54.54; H, 5.26; N, 12.72, found: C, 54.58; H,
2
0
23
3
4
4
5
4
.36; N, 12.74.
.1.4.3. N-(2-(1H-indol-3-yl)ethyl)-2-(methylthio)-4-morpholinopyrimidine-5-carboxamide (3)
Reagents and conditions: 2-(methylthio)-4-
morpholinopyrimidine-5-carboxylic acid (100 mg, 0.39 mmol)
and tryptamine (63 mg, 0.39 mmol), RT. Column purification:
methanol: dichloromethane (1:9). Yield: 75 mg, 60%. Mp
-1 1
1
66-168 °C; IR: 3719, 2970, 2885, 2353, 1634, 1355, 1203, 742 cm . H NMR (500 MHz,
CDCl ) δ 8.19 (s, 1H), 8.12 (s, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.20 (ddd,
3
2
1

ACCEPTED MANUSCRIPT
J = 8.1, 7.0, 1.2 Hz, 1H), 7.14 – 7.07 (m, 1H), 7.02 (d, J = 2.3 Hz, 1H), 6.29 (t, J = 5.8 Hz, 1H),
3
.75 (q, J = 6.2 Hz, 2H), 3.53 – 3.47 (m, 4H), 3.37 – 3.31 (m, 4H), 3.05 (t, J = 6.5 Hz, 2H), 2.46
1
3
(
s, 2H). C NMR (500 MHz, CDCl ) δ 172.57, 166.51, 160.16, 157.01, 136.45, 127.09, 122.45,
3
1
22.22, 119.70, 118.57, 112.51, 111.40, 111.16, 66.26, 47.77, 39.91, 24.96, 22.64, 14.09. HRMS
(
ESI+): Calcd for C H N O S (M+1) 398.1606; found 398.1654. Anal.Calcd for C H N O S:
2
0
23
5
2
20 23
5
2
C, 60.43; H, 5.83; N, 17.62, found: C, 60.48; H, 5.92; N, 17.64.
.1.4.4. N-(4-fluorophenyl)-2-(methylthio)-4-morpholinopyrimidine-5-carboxamide (4)
Reagents and conditions: 2-(methylthio)-4-
4
morpholinopyrimidine-5-carboxylic acid (100 mg, 0.39 mmol)
and 4-fluoroaniline (37 µl, 0.39 mmol). Room temperature, 12
h. Column Purification: hexane: ethyl acetate (5:5). Yield: 90
-1 1
mg, 95%. Mp 190-192 °C; IR: 3293, 2911, 1640, 1520, 1209, 972, 826 cm . H NMR (500 MHz,
CDCl ) δ 8.46 (s, 1H), 8.42 (s, 1H), 7.58 (dd, J = 8.9, 4.7 Hz, 2H), 7.10 – 7.01 (m, 2H), 3.75 (t,
3
1
3
4
1
H), 3.55 (t, 4H), 2.50 (s, 3H). C NMR (500 MHz, CDCl
3
) δ 173.40, 164.09, 160.79, 160.57,
58.62, 157.79, 133.65, 121.37, 121.31, 116.08, 115.90, 111.45, 66.51, 48.56, 14.18. HRMS
(
ESI+): Calcd for C H FN O S (M+1) 348.1117; found 348.1113. Anal.Calcd for
16 17 4 2
C H FN O S: C, 55.16; H, 4.92; N, 16.08, found: C, 55.24; H, 5.0; N, 16.12.
16
17
4
2
4
.1.4.5. N-(3,4-dimethoxyphenethyl)-2-(methylthio)-4-morpholinopyrimidine-5-carboxamide (5)
Reagents and conditions: 2-(methylthio)-4-
morpholinopyrimidine-5-carboxylic acid (100 mg, 0.39
mmol) and 3,4-dimethoxyphenylethylamine (66 µl, 0.39
mmol). Room temperature, 12 h. Column Purification:
hexane: ethyl acetate (4:6). Yield: 100 mg, 90%. Mp 152-154 °C; IR: 3727, 3272, 2304, 1628,
2
2

ACCEPTED MANUSCRIPT
-
1 1
1
7
3
525, 1247, 968, 791 cm . H NMR (500 MHz, CDCl
3
) δ 8.21 (d, J = 1.2 Hz, 1H), 6.80 (d, J =
.9 Hz, 1H), 6.73 (d, J = 9.3 Hz, 2H), 6.25 (t, J = 5.8 Hz, 1H), 3.84 (d, J = 1.2 Hz, 6H), 3.69 –
1
3
.60 (m, 6H), 3.43 (t, J = 4.7 Hz, 4H), 2.84 (q, J = 6.9, 5.6 Hz, 2H), 2.47 (d, J = 1.2 Hz, 3H). C
NMR (500 MHz, CDCl3) δ 172.77, 166.52, 160.25, 157.16, 149.23, 147.95, 130.75, 120.63,
11.77, 111.45, 111.11, 66.35, 55.96, 55.92, 47.91, 40.90, 34.99, 14.10. HRMS (ESI+): Calcd
for C H N O S (M+1) 419.1698; found: 419.1704. Anal. Calcd for C H N O S: C, 57.40; H,
1
2
0
26
4
4
20 26
4
4
6
4
.26; N, 13.39, found: C, 57.44; H, 6.28; N, 13.40.
.1.4.6. N-(2,4-dimethoxybenzyl)-2-(trifluoromethyl)-4-morpholinopyrimidine-5-carboxamide
(6)
Reagents
and
conditions:
4-morpholino-2-
(trifluoromethyl)pyrimidine-5-carboxylic acid (70 mg, 0.25
mmol) and 2,4-Dimethoxybenzylamine (38 µl, 0.25 mmol).
Room temperature, 14 h. Column purification: hexane:
ethylacetate (5:5). Yield: 60 mg, 60%. Mp 163-165 °C; IR: 3727, 3298, 2943, 1624, 1537, 1167,
-1 1
9
70, 799 cm . H NMR (500 MHz, CDCl ) δ 8.36 (s, 1H), 6.51 – 6.40 (m, 3H), 4.48 (d, J = 5.9
3
Hz, 2H), 3.80 (d, J = 16.3 Hz, 6H), 3.58 (dd, J = 5.8, 3.6 Hz, 4H), 3.51 (dd, J = 5.6, 3.8 Hz, 4H).
1
3
C NMR (500 MHz, CDCl ) δ 165.46, 161.05, 159.57, 158.55, 156.55, 156.19, 155.91, 131.23,
3
1
20.29, 118.10, 117.44, 116.49, 104.00, 98.77, 77.20, 66.30, 55.50, 55.46, 47.08, 39.93. HRMS
(
ESI+): Calcd for C H F N O (M+1) 427.1548; found 427.1599. Anal.Calcd for
19 21 3 4 4
C
19 21 3 4 4
H F N O : C, 53.52; H, 4.96; N, 13.14, found: C, 53.58; H, 4.94; N, 13.12.
4
.1.4.7. N-(3,5-dichloropyridin-4-yl)-2-(methylthio)-4-morpholinopyrimidine-5-carboxamide (7)
2
3

ACCEPTED MANUSCRIPT
Reagents
and
conditions:
2-(methylthio)-4-
morpholinopyrimidine-5-carboxylic acid (100 mg, 0.39 mmol)
and 4-amino-2,6-dichloropyridine (0.39 mmol). 0⁰C for 30 min
then room temperature for 1hr. Column purification: methanol:
dichloromethane (1:9). Appearance: white solid. Yield: 70 mg, 60%. Mp 152-154 °C; IR: 3727,
1
3
204, 2352, 1655, 1558, 1390, 1176, 972, 810. H NMR (500 MHz, CDCl ) δ 8.58 (s, 1H), 8.57
3
1
3
(
s, 2H), 7.94 (s, 1H), 3.79 – 3.73 (m, 4H), 3.64 (t, J = 4.7 Hz, 4H), 2.53 (s, 3H). C NMR (500
MHz, CDCl ) δ 174.60, 163.61, 160.73, 158.30, 148.48, 138.98, 129.24, 109.06, 66.45, 48.55,
3
1
4.24. HRMS (ESI+) Calcd for C H C N O S (M+1), 401.0294; found 401.0299. Anal.Calcd
15 15 l2 5 2
2 5 2
for C15H15Cl N O S: C, 45.01; H, 3.78; N, 17.50, found: C, 45.05; H, 3.80; N, 17.52.
4
.1.4.8. 2-(trifluoromethyl)-N-(3,4-dimethoxyphenyl)-4-morpholinopyrimidine-5-carboxamide
(8)
Reagents
and
conditions:
4-morpholino-2-
(trifluoromethyl)pyrimidine-5-carboxylic acid (80 mg, 0.28
mmol) and 4-Aminoveratraole (44.23 mg, 0.28 mmol). Room
temperature, 14 hr. Column purification: hexane: ethylacetate
-1 1
(
4:6). Yield: 90%, 80 mg. Mp 214-216 °C; IR: 3245, 1647, 1519, 1339, 974, 800 cm . H NMR
(
500 MHz, CDCl ) δ 8.55 (s, 1H), 7.92 (s, 1H), 7.39 (d, J = 2.5 Hz, 1H), 6.98 (dd, J = 8.6, 2.5 Hz,
3
1
4
1
H), 6.85 (d, J = 8.6 Hz, 1H), 3.88 (d, J = 12.0 Hz, 6H), 3.79 – 3.73 (m, 4H), 3.74 – 3.68 (m,
1
3
H). C NMR (500 MHz, CDCl ) δ 163.33, 159.92, 157.24, 156.46, 156.17, 149.36, 146.74,
3
30.56, 120.25, 116.55, 111.73, 111.44, 104.59, 66.43, 56.16, 56.04, 47.61. HRMS (ESI+):
Calcd for C18
19 3 4 4 19 3 4
H F N O (M+1) 413.1548; found 413.1542. Anal.Calcd: C18H F N O4: C, 52.43;
H, 4.64; N, 13.59, found: C, 52.45; H, 4.67; N, 13.60.
2
4

ACCEPTED MANUSCRIPT
4
.1.4.9. N-(3,4-dimethoxyphenethyl)-4-(4-(2-hydroxyethyl)piperazin-1-yl)-2-
(
methylthio)pyrimidine-5-carboxamide (9)
Reagents and conditions: 4-(4-(2-hydroxyethyl) piperazin-1-yl)-2-
methylthio) pyrimidine-5-carboxylic acid (60 mg, 0.20 mmol) and
,4-dimethoxyphenethylamine (36.5 mg, 0.20 mmol). Room
(
3
temperature, 14h. Column purification: methanol: dichloromethane
(2:8). Yield: Appearance: pale brown solid. Yield: 50 mg, 70%. Mp 108-110 °C; IR: 3264, 2926,
-
1 1
1
7
634, 1524, 1393, 1241, 962 cm . H NMR (400 MHz, DMSO-d ) δ 8.48 (t, J = 5.6 Hz, 1H),
6
.93 (s, 1H), 6.90 – 6.81 (m, 2H), 6.73 (dd, J = 8.1, 2.0 Hz, 1H), 4.43 (t, J = 5.4 Hz, 1H), 3.72 (d,
J = 8.6 Hz, 6H), 3.51 (t, J = 5.9 Hz, 2H), 3.41 (t, J = 5.0 Hz, 5H), 2.73 (t, J = 7.0 Hz, 2H), 2.44 (s,
3
4
5
4
H), 2.43 (s, 3H), 2.37 (d, J = 6.1 Hz, 6H). HRMS (ESI+): Calcd for C H N O S (M+1)
22 31 5 4
62.2130; found: 462.2160. Anal.Calcd for C H N O S: C, 57.25; H, 6.77; N, 15.17, found: C,
2
2
31
5
4
7.28; H, 6.80; N, 15.18.
.1.4.10.
4-(4-(2-hydroxyethyl)piperzin-1-yl)-N-(3,4-dimethoxyphenyl)-2-
(
methylthio)pyrimidine-5-carboxamide (10)
Reagents and conditions: 4-(4-(2-hydroxyethyl) piperazin-1-yl)-
-(methylthio) pyrimidine-5-carboxylic acid (60 mg, 0.20 mmol)
and 4-aminoveratrole (30 mg, 0.22 mmol). Room temperature,
4hr. column purification: methanol: dichloromethane (2:8).
Appearance: brown solid. Yield: 40 mg, 45%. Mp 109-111 °C;
2
1
-1 1
IR: 3316, 2946, 1737, 1652, 1508, 1357, 1208, 1132, 791 cm . H NMR (500 MHz, CDCl ) δ
3
8
8
.49 (s, 1H), 8.32 (s, 1H), 7.47 (d, J = 2.5 Hz, 1H), 6.93 (dd, J = 8.5, 2.5 Hz, 1H), 6.82 (d, J =
.7 Hz, 1H), 3.88 (d, J = 17.2 Hz, 6H), 3.62 (t, J = 5.3 Hz, 2H), 3.58 (t, J = 4.9 Hz, 4H), 2.60 (t,
2
5

ACCEPTED MANUSCRIPT
1
3
J = 4.8 Hz, 4H), 2.56 (t, J = 5.3 Hz, 2H), 2.51 (s, 3H). C NMR (500 MHz, CDCl
3
) δ 173.26,
63.82, 161.05, 158.02, 149.25, 146.17, 131.28, 111.78, 111.44, 111.38, 104.55, 59.32, 57.74,
6.14, 55.99, 52.63, 48.41, 14.19. HRMS (ESI+): Calcd for C H N O S (M+1) 434.1972;
1
5
20
27
5
4
found 434.1977. Anal.Calcd for C H N O S: C, 55.41; H, 6.28; N, 16.15, found: C, 55.44; H,
2
0
27
5
4
6
4
.30; N, 16.17.
.1.4.11. N-(3,5-dichloropyridin-4-yl)-4-(3,4-dimethoxyphenyl)-2-(methiothio)pyrimidine-5-
carboxamide (11)
Reagents
and
conditions:
4-(3,4-dimethoxyphenyl)-2-
(methylthio)pyrimidine-5-carboxylic acid (80 mg, 0.26 mmol) and
4
-amino-3,5-dichloropyridine (43 mg, 0.26 mmol). Column
purification: methanol: dichloromethane (3:7). Appearance: white
solid. Yield: 80 mg, 90%. Mp 261-263 °C; IR: 3194, 1665, 1552, 1398, 1266, 1209, 1028, 807
-1
1
cm . H NMR (500 MHz, CDCl
3
) δ 8.91 (s, 1H), 8.49 (s, 2H), 7.39 (dd, J = 8.3, 2.1 Hz, 1H),
7
3
1
.34 (d, J = 2.1 Hz, 1H), 7.18 (s, 1H), 6.94 (d, J = 8.3 Hz, 1H), 3.91 (d, J = 3.2 Hz, 6H), 2.64 (s,
1
3
H). C NMR (500 MHz, CDCl ) δ 175.08, 163.17, 158.48, 151.83, 149.46, 148.39, 138.71,
3
28.78, 128.29, 123.11, 121.24, 111.92, 111.26, 56.10, 14.35. HRMS (ESI+): Calcd for
C H C N O S (M+1) 451.0320; found 451.0325. Anal.Calcd for C H C N O S: C, 50.56; H,
19
16 l2
4
3
19 16 l2
4
3
3
4
.57; N, 12.41, found: C, 50.60; H, 3.59; N, 12.43.
.1.4.12. 4-(3,4-dimethoxyphenyl)-2-(methylthio)-N-(pyridin-4-yl)pyrimidine-5-carboxamide
(12)
Reagents
and
conditions:
4-(3,4-dimethoxyphenyl)-2-
(methylthio)pyrimidine-5-carboxylic acid (70 mg, 0.22 mmol) and
2
6

ACCEPTED MANUSCRIPT
4
-aminopyridine (21 mg, 0.22 mmol). 0⁰C-room temperature. Column purification: methanol:
dichloromethane (1:9). Appearance: pale yellow solid. Yield: 65 mg, 75%. Mp 173-175 °C; IR:
1
3
8
1
1
2
322, 3194, 1662, 1550, 1264, 1196, 1022, 792. H NMR (400 MHz, DMSO-d ) δ 10.96 (s, 1H),
6
.80 (s, 1H), 8.50 – 8.43 (m, 2H), 7.60 – 7.53 (m, 2H), 7.45 – 7.34 (m, 2H), 7.06 (d, J = 8.5 Hz,
1
3
6
H), 3.78 (s, 3H), 3.67 (s, 3H), 2.63 (s, 3H). C NMR (500 MHz, DMSO-d ) δ 174.67, 166.02,
62.14, 158.27, 151.78, 150.60, 149.17, 144.11, 127.90, 122.84, 113.42, 111.37, 55.65, 33.94,
5.08, 13.90. HRMS (ESI+): Calcd for C H N O S (M+1) 383.1133; found 383.1128.
1
9
18
4
3
Anal.Calcd for C H N O S: C, 59.67; H, 4.74; N, 14.65, found: C, 59.70; H, 4.76; N, 14.66.
1
9
18
4
3
4
.1.4.13.
H-pyran-4-yl)pyrimidine-5-carboxamide (13)
and conditions: 4-(3,4-dimethoxyphenyl)-2-
methylthio)pyrimidine-5-carboxylic acid (70 mg, 0.22 mmol) and
4-(3,4-dimethoxyphenyl)-2-(methylthio)-N-(tetrahydro-
2
Reagents
(
4
-aminotetrahydropyran (28 µl, 0.27 mmol). Room temperature, 12
h. Column purification: hexane: ethylacetate (4:6). Yield: 85 mg, 90%. Mp 193-195 °C; IR: 3275,
-
1 1
2
950, 1632, 1541, 1215, 1019, 791 cm . H NMR (500 MHz, CDCl
m, 2H), 6.92 (d, J = 8.1 Hz, 1H), 4.04 (ddp, J = 11.4, 8.2, 4.2 Hz, 1H), 3.92 (d, J = 10.0 Hz, 6H),
.83 (dt, J = 11.9, 3.4 Hz, 2H), 3.41 (td, J = 11.6, 2.1 Hz, 2H), 2.60 (s, 3H), 1.76 (ddd, J = 12.5,
3
) δ 8.72 (s, 1H), 7.34 – 7.27
(
3
4
1
3
1
3
.4, 2.2 Hz, 2H), 1.27 – 1.16 (m, 2H). C NMR (500 MHz, CDCl3) δ 173.74, 165.81, 162.19,
57.87, 151.49, 149.13, 128.67, 122.98, 122.52, 111.93, 110.87, 66.45, 56.10, 56.07, 46.24,
2.50, 14.26. HRMS (ESI+): Calcd for C H N O S (M+1) 390.1485; found 390.1491.
1
9
23
3
4
Anal.Calcd for C H N O S: C, 58.59; H, 5.95; N, 10.79, found: C, 58.61; H, 5.98; N, 10.81.
1
9
23
3
4
2
7

ACCEPTED MANUSCRIPT
4
5
.1.4.14. 2-(trifluoromethyl)-N-(tetrahydro-2H-pyran-4-yl)-4-(3,4-dimethoxyphenyl)pyrimidine-
-carboxamide (14)
Reagents
and
conditions:
2-(trifluoromethyl)-4-(3,
4-
dimethoxyphenyl) pyrimidine-5-carboxylic acid (50 mg, 0.15 mmol)
and 4-aminotetrahydropyran (16µl, 0.15mmol). Room temperature,
2
4hr. Column purification: methanol: dichloromethane (0.5:9.5).
-1 1
Yield: 50 mg, 60%. Mp 200-202 °C; IR: 3250, 2949, 2844, 1641, 1555, 1139, 815, 729 cm . H
NMR (500 MHz, DMSO-d ) δ 9.01 (s, 1H), 8.76 (d, J = 7.5 Hz, 1H), 7.52 – 7.42 (m, 2H), 7.14
6
(d, J = 8.5 Hz, 1H), 3.95 (s, 1H), 3.84 (s, 3H), 3.79 (s, 4H), 3.76 (t, J = 3.7 Hz, 1H), 3.38 (td, J =
1
3
1
1.2, 2.2 Hz, 2H), 1.78 – 1.71 (m, 2H), 1.37 (qd, J = 10.9, 4.1 Hz, 2H). C NMR (500 MHz,
DMSO-d ) δ 164.53, 162.24, 157.59, 151.71, 148.55, 130.83, 127.27, 122.58, 111.85, 111.63,
6
6
5.43, 55.72, 55.54, 45.51, 31.79. HRMS (ESI+): Calcd for C H F N O (M+1) 412.1439;
19 20 3 3 4
20 3 3 4
found 412.1432. Anal.Calcd for C19H F N O : C, 55.47; H, 4.90; N, 10.21, found: C, 55.51; H,
4
4
.92; N, 10.23.
.1.4.15. N, N-diethyl-2-(trifluoromethyl)-4-(3, 4-dimethoxyphenyl) pyrimidine-5-carboxamide
15)
(
Reagents and conditions: 2-(trifluoromethyl)-4-(3, 4-dimethoxyphenyl)
pyrimidine-5-carboxylic acid (80 mg, 0.24 mmol) and Diethylamine
(25
µl, 0.24 mmol). Room temperature, 24 h. Column purification:
methanol: dichloromethane (2:8). Appearance: White solid. Yield: 40
-1
mg, 45%. Mp 162-164 °C; IR: 3307, 2938, 2828, 1649, 1561, 1510, 1358, 1209, 1138, 972 cm .
1
H NMR (500 MHz, CDCl ) δ 8.74 (s, 1H), 7.54 (d, J = 7.4 Hz, 2H), 6.94 – 6.88 (m, 1H), 3.93
3
(d, J = 1.5 Hz, 6H), 3.68 (dt, J = 13.7, 6.8 Hz, 1H), 3.35 (dq, J = 13.9, 7.0 Hz, 1H), 2.88 (p, J =
2
8