Pyrimidinyl Biphenylureas: Identification of New Lead Compounds as Allosteric Modulators of the Cannabinoid Receptor CB1

Article abstract of DOI:10.1021/acs.jmedchem.6b01448

The allosteric modulator 1-(4-chlorophenyl)-3-(3-(6-(pyrrolidin-1-yl)pyridin-2-yl)phenyl)urea (PSNCBAM-1, 2) bound the cannabinoid receptor 1 (CB₁) and antagonized G protein coupling. This compound demonstrated potent anorectic effects similar to the CB₁ antagonist rimonabant that once was marketed for the treatment of obesity, suggesting a new chemical entity for the discovery of antiobesity drugs. To increase structural diversity of this class of CB₁ ligands, we designed and synthesized two classes of novel analogues, in which the pyridine ring of 2 was replaced by a pyrimidine ring. These positively modulate the binding of the CB₁ orthosteric agonist CP55,940 while exhibiting an antagonism of G-protein coupling activity. Interestingly, compounds 7d and 8d demonstrated ERK1/2 phosphorylation mediated via β-arrestin unlike the orthosteric CP55,940 that does so in a G protein-dependent manner. These can serve as new lead compounds for the future development of CB₁ allosteric modulators that show biased agonism and potentially antiobesity behavior via a new mechanism.

Full text of DOI:10.1021/acs.jmedchem.6b01448

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Article
Pyrimidinyl Biphenylureas: Identification of New Lead Compounds
1
as Allosteric Modulators of the Cannabinoid Receptor CB
Leepakshi Khurana, Boqiao Fu, Anantha L. Duddupudi, Yu-
Hsien Liao, Sri Sujana Immadi, Debra A. Kendall, and Dai Lu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01448 • Publication Date (Web): 06 Jan 2017
Downloaded from http://pubs.acs.org on January 7, 2017
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Pyrimidinyl Biphenylureas: Identification of New
Lead Compounds as Allosteric Modulators of the
Cannabinoid Receptor CB1
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†
¥
¥
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Leepakshi Khurana , Bo-Qiao Fu , Anantha L. Duddupudi , Yu-Hsien Liao , Sri Sujana
¥
†
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Immadi , Debra A. Kendall *and Dai Lu *
†
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut, United
States
¥
Rangel College of Pharmacy, Health Science Center, Texas A&M University, Kingsville,
Texas, United States
*
coꢀcorresponding authors
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ABSTRACT
The
allosteric
modulator
1ꢀ(4ꢀchlorophenyl)ꢀ3ꢀ(3ꢀ(6ꢀ(pyrrolidinꢀ1ꢀyl)pyridinꢀ2ꢀ
yl)phenyl)urea (PSNCBAMꢀ1, 2) bound the cannabinoid receptor 1 (CB ) and antagonized G
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protein coupling. This compound demonstrated potent anorectic effects similar to the CB1
antagonist rimonabant that once was marketed for the treatment of obesity suggesting a new
chemical entity for the discovery of antiꢀobesity drugs. To increase structural diversity of this
class of CB ligands, we designed and synthesized two classes of novel analogs, in which the
1
pyridine ring of 2 was replaced by a pyrimidine ring. These positively modulate the binding of
the CB orthosteric agonist CP55,940, while exhibiting an antagonism of Gꢀprotein coupling
1
activity. Interestingly, compounds 7d and 8d demonstrated ERK1/2 phosphorylation mediated
via βꢀarrestin unlike the orthosteric CP55,940 that does so in a G proteinꢀdependent manner.
These can serve as new lead compounds for the future development of CB allosteric modulators
1
that show biased agonism and potentially antiꢀobesity behavior via a new mechanism.
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INTRODUCTION
The endocannabinoid system (ECS) consists of two wellꢀcharacterized receptor subtypes,
the cannabinoid receptor 1 (CB ) and the cannabinoid receptor 2 (CB ), the eicosanoid ligands
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synthesized in the body called endocannabinoids and several metabolic proteins (e.g. fatty acid
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amide hydrolase, monoacylglycerol lipase). Both the receptors are activated by ∆ ꢀ
tetrahydrocannabinol (THC), the psychoactive component of marijuana (Cannabis sativa) and
belong to class 1A rhodopsinꢀlike Gꢀprotein coupled receptors (GPCRs). Moreover, CB is one
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2
,3
of the most abundant GPCRs expressed in the central nervous system (CNS). Upon activation,
the CB receptor primarily couples to G protein, which causes downstream inhibition of
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i/o
adenylyl cyclase. The CB receptor can also activate inwardly rectifying and Aꢀtype outward
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4
,5
6,7
potassium channels and inhibit Nꢀtype and P/Q type of calcium channels. The CB receptor
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can also activate different members of mitogenꢀactivated protein kinases including p44/42 MAP
kinase, p38 kinase and JUNꢀterminal kinase through Gꢀprotein mediated pathways or pathways
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independent of Gꢀproteins via β−arrestins. Due to its complex signaling network, CB has been
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implicated in the pathology of many disorders and thus is a promising therapeutic target for
ameliorating diseases including nausea, obesity, neurodegenerative disorders, pain and substance
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abuse disorders.
However, despite extensive efforts to generate cannabinoidꢀbased
therapeutics, only a few medications from cannabinergic compounds are commercially available
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due to the extensive side effects associated with CB orthosteric ligands. Another CB1 ligand,
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SR141716A (rimonabant), which was initially developed as an antiꢀobesity drug, was withdrawn
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from the market due to its psychiatric side effects.
To overcome challenges posed by orthosteric ligands (ligands which compete with the
endogenous ligands for the same site), recent research in GPCRs including CB has shifted focus
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to allosteric modulators that bind to a topographically distinct site called the allosteric site.
Allosteric modulators offer many therapeutic advantages over their orthosteric counterparts.
First, due to less evolutionary pressure, allosteric binding pockets have aminoꢀacid sequences
which are not highly conserved like the orthosteric sites and thus are more specific for each
receptor subtype. Second, allosteric modulators have a ceiling effect due to their limited
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allosteric cooperativity and thus can be used to generate titrated pharmacological responses.
Third, allosteric modulators can be used to fineꢀtune endogenous signaling without affecting the
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spatial and temporal aspects of endogenous ligandꢀreceptor signaling.
Allosteric modulators, upon binding to a receptor, can induce an array of distinct
conformations that can be very different from those stabilized by orthosteric ligands and thus
have expanded the spectrum of biological conformations among the inactive and active states.
Several classes of allosteric modulators of CB have been identified (Figure 1), which include 5ꢀ
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chloroꢀ3ꢀethylꢀNꢀ(4ꢀ(piperidinꢀ1ꢀyl)phenethyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (1, ORG27569), 1ꢀ
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(
4ꢀchlorophenyl)ꢀ3ꢀ(3ꢀ(6ꢀ(pyrrolidinꢀ1ꢀyl)pyridinꢀ2ꢀyl)phenyl)urea (2, PSNCBAMꢀ1), 3ꢀ(4ꢀ
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chlorophenyl)ꢀ5ꢀ(8ꢀmethylꢀ3ꢀpꢀtolylꢀ8ꢀazabicyclo[3.2.1]octanꢀ2ꢀyl)isoxazole (3, RTIꢀ371), the
endogenous ligand (5S,6R,9E,11Z,13E,15S)ꢀ5,6,15ꢀtrihydroxyicosaꢀ9,11,13ꢀtrienoic acid (4,
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lipoxin A4)
and 6ꢀmethylꢀ3ꢀ(2ꢀnitroꢀ1ꢀ(thiophenꢀ2ꢀyl)ethyl)ꢀ2ꢀphenylꢀ1Hꢀindole (5,
2
0
ZCZ011).
These ligands are positive allosteric modulators (PAMs) of orthosteric agonist
binding at the CB receptor. A family of peptide endocannabinoids (Pepcans) represented by
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pepcanꢀ12 (6) has recently been reported to exhibit negative allosteric modulation (NAM) of the
CB receptor at the level of regulating orthosteric agonist binding as well as the signaling
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2
1
functions of CB receptor. Since allosteric modulators can induce distinct conformations than
1
those induced by orthosteric ligands, they offer potential to generate pharmacological responses
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that may be difficult to achieve with orthosteric ligands alone. Furthermore, the different
receptor states induced by allosteric modulators can be biased for coupling to different G
proteins or βꢀarrestin likely due to the different conformations they induce. This can impact
some intracellular signaling pathways as is seen with some allosteric modulators including 1 for
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the CB receptor and consequently offer promising avenues to develop subtypeꢀspecific and
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pathwayꢀspecific therapeutics.
Cl
O
N
A
B
Cl
N
C
N
NH
O
N
N
H
H
N
H
1 (ORG27569)
2 (PSNCBAM-1)
N
N
O
HO OH
Cl
COOH
OH
3 (RTI-371)
4 (Lipoxin A4)
NO2
S
Primary sequence:
RVDPVNFKLLSH
6 (Pepcan-12)
N
H
5
(ZCZ011)
Figure 1. Structures of representative allosteric modulators of the CB receptor.
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Some in vivo pharmacological assessments have indicated that these allosteric modulators can
generate therapeutic effects relevant to various clinical therapies. For instance, 1 was
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demonstrated to be effective in inhibiting reinstatement of drugꢀseeking behavior and 4 was
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found to reduce βꢀamyloidꢀprovoked neurotoxicity. The positive CB allosteric modulator 5
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was shown to generate antinociceptive effects without psychoactive effects typically found with
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orthosteric ligands of the CB receptor. Interestingly, 2 was found capable of reducing food
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intake and body weight in an acute feeding study. Thus, this scaffold holds promise for the
development of antiꢀobesity drugs by targeting CB allosteric sites. A recent SAR study of 2 has
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revealed that two structural variations can preserve its activity. These include the replacement of
the chloro group on the ring A with either a fluoro (F) or a cyano (CN) group and the
replacement of the pyrrolidine ring with a N,Nꢀdimethyl amino group. These analogues
exhibited a doseꢀdependent reduction of Emax values with the agonist CP55,940 in a calcium
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mobilization assay, as expected for CB G protein NAMs. In order to increase the structural
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diversity from this scaffold, we designed novel analogs with the hypothesis that a pyrimidinyl
ring can be employed to replace the pyridine ring of 2. This hypothesis was supported by two
considerations. First, many allosteric modulators of various GPCRs possess cyclic moieties that
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contain multiple nitrogen atoms. The pyrimidine moiety has been found in a number of
allosteric modulators of different GPCRs such as for the glucagonꢀlike peptide 1 (GLPꢀ1)
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receptor, the metabotropic glutamate receptor 5 (mGluR5), and the γꢀaminobutyric acidB
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(
GABA ) receptor
among others. Second, in comparison with pyridine, the pyrimidine
B
structure offers more synthetic versatility. The πꢀelectron density is decreased to an even greater
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extent than in pyridine.
Therefore, nucleophilic aromatic substitution is facilitated.
Synthetically, the electronꢀdeficient nature of pyrimidine accounts for better opportunities when
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nucleophilic aromatic substitution chemistry is employed in functionalization of the electronꢀ
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deficient heteroaromatic ring. Based on the above features, we synthesized and assessed novel
analogs 7aꢀ7h and 8aꢀ8t whose structures are shown in Figure 2.
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O
O
X
X
N
R
N
R
N
N
H
N
N
H
H
H
N
N
7
8
7
7
a. X = p-Cl, R = N-pyrrolidinyl
b. X = p-Br, R = N-pyrrolidinyl
7c. X = p-F, R = N-pyrrolidinyl
7d. X = p-CN, R = N-pyrrolidinyl
e. X = p-F, R = N,N-dimethyl
f. X = p-CN, R = N,N-dimethyl
g. X = p-I, R = N,N-dimethyl
h. X = m-F, R = N,N-dimethyl
8a. X = p-Cl, R = N-pyrrolidinyl
8b. X = p-Br, R = N-pyrrolidinyl
8c. X = p-F, R = N-pyrrolidinyl
8d. X = p-CN, R = N-pyrrolidinyl
8e. X = p-F, R = N,N-dimethyl
8f. X = p-CN, R = N,N-dimethyl
8g. X = p-I, R = N,N-dimethyl
8h. X = m-F, R = N,N-dimethyl
8l. X = p-COOH, R = N-pyrrolidinyl
m. X = p-OCH3, R = N-pyrrolidinyl
n. X = p-OH, R = N-pyrrolidinyl
o. X = m-CN, R = N-pyrrolidinyl
p. X = p-CN, R = N-azetidinyl
q. X = p-CN, R = N-piperidinyl
8r. X = p-CN, R = N(Et)
8s. X = p-CN, R = CH CH NCH
8
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7
7
7
7
8
2
8
8
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i. X = p-CF , R = N-pyrrolidinyl
j. X = p-CH C(O), R = N-pyrrolidinyl
k. X = p-EtOC(O), R = N-pyrrolidinyl
3
3
2
3
3 8t. X = p-CN, R = N-cyclopropyl amino
Figure 2. The designed and synthesized pyrimidinyl biphenyl ureas.
CHEMISTRY
The syntheses of the target compounds 7 and 8 were achieved through the routes
illustrated by Scheme 1 and 2, respectively. To obtain compounds 7aꢀ7h, the synthesis of
intermediate 3ꢀ(2ꢀchloropyrimidinꢀ4ꢀyl)aniline 12 was critical. Coupling of the commercially
available 2,4ꢀdichloropyrimidine 9 with (3ꢀnitrophenyl)boronic acid 10 under typical Suzuki
reaction conditions exclusively gave the 2ꢀchloroꢀ4ꢀ(3ꢀnitrophenyl)pyrimidine 11. We found that
this Suzuki coupling reaction only took place at the 4ꢀchloro position of the starting material 9,
whilst its 2ꢀchloro group was spared. The structure of the Suzuki coupling product 11 was
confirmed by NOESY signals between the protons Hꢀ8 and Hꢀ12 with proton Hꢀ5 of 11 (see
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supporting information Figure S3 for 2D H NMR of 11). Upon reduction of 11 catalyzed with
stannous chloride dihydrate, the desired intermediate 3ꢀ(2ꢀchloropyrimidinꢀ4ꢀyl)aniline 12 was
obtained in an acceptable yield (40%). Reaction of the pyrimidinyl aniline 12 with properly
substituted isocyanate (13aꢀ13f) yielded the key intermediates 14aꢀ14f. Thereafter, amination of
the pyrimidinyl ring of individual 14 with either pyrrolidine or dimethyl amine in THF generated
the final compounds 7aꢀ7h.
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a
Scheme 1. Synthesis of Pyrimidinyl Biphenyl Ureas 7aꢀ7h
a
.
2
Reagents and conditions: (i) Na CO , Pd(PPh ) , DME, H O; 90 °C, 7 h, 61%; (ii) SnCl 2H O,
2
3
3 4
2
2
DCM/MeOH (1:1), 0 °C followed by reflux at 60 °C, 4 h, 40%; (iii) DCM, 0 °C to rt, 2 h, 40ꢀ
8
6%; (iv) pyrrolidine (for 7aꢀ7d) or dimethyl amine (for 7eꢀ7h), THF, reflux, 2ꢀ4 h, 35ꢀ81%.
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a
Scheme 2. Synthesis of Pyrimidinyl Biphenyl Ureas 8aꢀ8t
a
Reagents and conditions: (i) amine 15 (15a: pyrrolidine; 15b: NH(CH ) ; 15c: MeNHEt; 15d:
3
2
NH(Et) ; 15e: piperidine; 15f: cyclopropylamine; 15g: azetidine), THF, rt, 2 h, 46%ꢀ80%; (ii)
2
Na CO , Pd(PPh ) , 1,4ꢀdioxane, H O (4:1), 110 °C, 10 h, 46ꢀ75%; (iii) 10% Pd/C, H , EtOAc or
2
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ethanol, rt, 4 h, 38ꢀ97%; (iv) DCM, 0 °Cꢀrt, 2 h, 25ꢀ86%; (v) BBr , DCM, 0 °Cꢀrt; (vi) LiOH,
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THF/H O, rt.
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Since the Suzuki coupling reaction between 9 and 10 could not lead to the intermediate
that can provide the desired amine 19, we took the route illustrated in Scheme 2 and aminated the
2,4ꢀdichloropyrimidine 9 first. This yielded two regioisomers 16 and 17. The 4ꢀamino
pyrimidine 16 was obtained as the major product whilst the 2ꢀamino pyrimidine 17 was the
minor product. The structures of individual isomers (i.e. 16aꢀ16g and 17aꢀ17g) were either
1
verified by comparison with the NMR data reported in the literature or determined by 2D H
NMR (see supporting information). Coupling the 4ꢀamino pyrimidine 16aꢀ16g, respectively,
with (3ꢀnitrophenyl)boronic acid 10 under Suzuki reaction conditions yielded the desired 2ꢀ(3ꢀ
nitrophenyl)pyrimidinꢀ4ꢀamines 18aꢀ18g in acceptable yields (46%ꢀ75%). Hydrogenation of
18aꢀ18g produced the 2ꢀ(3ꢀaminophenyl)ꢀpyrimidinꢀ4ꢀamines 19aꢀ19g in good yield (87ꢀ97%).
Coupling the selected isocyanates 13gꢀ13k with amines 19aꢀ19g yielded the final compounds
8aꢀ8k, 8m and 8oꢀt. The carboxylic acid 8l was further derived from 8k through hydrolysis, and
8n was obtained from demethylation of 8m.
RESULTS AND DISCUSSION
The synthesized analogs were evaluated for allosteric modulation of CB , using the
1
orthosteric agonist CP55,940, and two key parameters were determined: K the equilibrium
B,
dissociation constant of the allosteric modulator which reflects the affinity of the allosteric
modulator for the receptor and α, the binding cooperativity factor which defines the magnitude
and direction of the allosteric effect on the binding of the orthosteric ligand, when both occupy
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the receptor. Modulators with an α>1 promote orthosteric ligand binding and are classified as
positive allosteric modulators (PAMs) whereas modulators with α<1 inhibit orthosteric ligand
binding and are classified as negative allosteric modulators (NAMs). Compounds with no
allosteric activity on orthosteric ligand binding have an α = 1.
10
11
12
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To evaluate the scaffolds 7 and 8, we first compared 7a and 8a with the lead compound
2
. The results, shown in Tables 1 and 2 (entry 2 and 10), suggested that replacing the pyridine
ring of 2 with a pyrimidine ring reduced the binding affinity relative to the parent compound
while the modulation on orthosteric ligand binding was enhanced (i.e. the α values are
increased). This promoted our further investigation of the two new scaffolds by optimizing the
substituents. We synthesized and assessed compounds 7bꢀ7h and their counterpart compounds
8bꢀ8h. The results shown in Table 1 (entry 3ꢀ9) and Table 2 (entry 11ꢀ17) revealed that the
scaffolds 7 and 8 do not show significant difference in binding affinity (K ) and binding
B
cooperativity factor (α) when compounds with the same substitutions on the two different
scaffolds are compared. However, the binding affinity is significantly influenced by the
substitution on the phenyl ring A and the amino substituent on the pyrimidine ring C. We found
that a paraꢀcyano group on the phenyl ring A and a pyrrolidinyl group on the pyrimidine ring C
provided a compound (i.e. 8d) that showed activity comparable to the lead compound 2. It also
appears that a dimethyl amino substituent on the pyrimidinyl ring C is suboptimal than a
pyrrolidinyl group within each series of compounds from scaffolds 7 and 8.
A cyano group is a strong electronꢀwithdrawing group (EWG). To investigate if other
substituents on the phenyl ring A can increase the binding affinity and cooperativity, we
synthesized and assessed the compounds 8iꢀ8n (Table 2, entry 18ꢀ23), which bear either an EWG
such as CF (8i), acetyl (8j), ethoxyacyl (8k) and COOH (8l) or an electronꢀdonating group
3
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(
EDG) such as OMe (8m) and OH (8n). However, none of them surpassed the cyanoꢀsubstituted
compound 8d. Introducing EDGs on the phenyl ring A (i.e. compound 8m and 8n) significantly
reduced the binding affinity to the allosteric site. Some of the EWGs are able to retain some
binding affinity to the allosteric site (i.e. 8i and 8k). These suggested that variation of the
electron density of the phenyl ring A alone is unable to enhance the binding affinity. It seems
that the electronꢀwithdrawing cyano group is not only to polarize the adjacent electron density on
the ring A, but is also likely involved in the molecular recognition process. The multiple
0
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2
biological functions of a cyano group have been well recognized. Its functions include serving
as carbonyl and halogen bioisosteres, a hydroxyl and carboxyl surrogate, an inducer of nonꢀ
specific dipole interactions with amino acids and metal ions, and a functionality able to replace a
32
conserved water molecule from the binding domain. Expulsion of a conserved water molecule
from the binding domain by a cyano group has been indicated to provide additional entropic
3
3,34
improvement of binding affinity.
Additionally, we found that the position of the cyano
substitution impacts the allosteric activity drastically. When the cyano group on the ring A of 8
was shifted from the paraꢀposition to the metaꢀposition, the binding affinity to the allosteric site
and the allosteric modulation of orthosteric ligand binding were nearly abolished (i.e. 8d vs 8o,
Table 2).
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Table 1. Allosteric parameters of analogs derived from scaffold 7 (7aꢀ7h)
a
b
Entry
1
Compd
R1
R2
K (nM)
α
B
5
4.3
21.9ꢀ134.6)
26.9
2
pꢀCl
NꢀPyrrolidinyl
7.3
11.6
16.8
3.5
(
(
2
2
3
4
5
6
7
8
9
7a
7b
7c
7d
7e
7f
pꢀCl
pꢀBr
pꢀF
NꢀPyrrolidinyl
NꢀPyrrolidinyl
NꢀPyrrolidinyl
NꢀPyrrolidinyl
N(CH3)2
61.8ꢀ832.2)
394.9
79.3ꢀ1964)
(
1
95.6
(
113.8ꢀ336.1)
167.8
pꢀCN
pꢀF
10.5
7.3
(57.1ꢀ492.1)
116
506.6ꢀ2459)
16.6
1
(
4
pꢀCN
pꢀI
N(CH3)2
6.1
(308.1ꢀ563.2)
965.3
7g
7h
N(CH3)2
2.7
(
629.0ꢀ1481)
67.4
9
mꢀF
N(CH3)2
2.5
(
540.3ꢀ1722)
a
b
K : equilibrium dissociation constant of a potential allosteric ligand. α: binding cooperativity
B
factor for the tested allosteric modulator. The two allosteric parameters were tested using
3
[ H]CP55,940 as the orthosteric ligand.
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1
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Allosteric parameters of analogs derived from scaffold 8 with variation of substituents
on the phenyl ring A (8aꢀ8o)
a
b
Entry
1
Compd
R1
R2
K (nM)
α
B
54.3
2
pꢀCl
NꢀPyrrolidinyl
7.3
18.1
6.1
4.9
4.6
5.2
4.5
2.5
2.0
3.8
2.5
1.6
(21.9ꢀ134.6)
64.4
61.9ꢀ1129)
01.9
(32.8ꢀ316.9)
222.9
2
10
8a
8b
8c
8d
8e
8f
pꢀCl
pꢀBr
pꢀF
NꢀPyrrolidinyl
NꢀPyrrolidinyl
NꢀPyrrolidinyl
NꢀPyrrolidinyl
N(CH3)2
(
1
1
1
1
2
(
154.2ꢀ322.1)
4
9.1
13
pꢀCN
pꢀF
(
27.9ꢀ86.3)
1221
14
(849.6ꢀ1755)
13.3
216.2ꢀ454.2)
97.1
(480.7ꢀ2068)
69.8
173.6ꢀ2584)
44.2
3
15
pꢀCN
pꢀI
N(CH3)2
(
9
1
1
1
1
6
7
8
9
8g
8h
8i
N(CH3)2
6
mꢀF
N(CH3)2
(
1
pꢀCF3
NꢀPyrrolidinyl
(
85.8ꢀ242.4)
2108
8j
pꢀCH C(O) NꢀPyrrolidinyl
3
(
1299ꢀ3422)
4
63.0
20
8k
pꢀEtOC(O) NꢀPyrrolidinyl
(
51.8ꢀ4132)
c
c
2
2
1
2
8l
pꢀCOOH
pꢀOCH3
NꢀPyrrolidinyl
NꢀPyrrolidinyl
NB
NB
8m
1826
3.8
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8
9
(
991.7ꢀ3364)
c
c
2
2
3
4
8n
8o
pꢀOH
mꢀCN
NꢀPyrrolidinyl
NꢀPyrrolidinyl
NB
NB
1980
1.5
(388.1ꢀ10100)
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a
b
K : equilibrium dissociation constant of a potential allosteric ligand. α: binding cooperativity
B
factor for the tested allosteric modulator. The two allosteric parameters were tested using
3
c
[
[
H]CP55,940 as the orthosteric ligand. NB: no detectable binding or modulation of
3
H]CP55,940 with the receptor using up to 32 µM of test compound.
Following the identification of 8d, we investigated if other amino substituents on the
pyrimidine ring can enhance activity (Table 3). We reduced the ring size to a 4ꢀmembered
azetidinyl ring (8p). This led to a decline in K . When the ring size was increased to a 6ꢀ
B
membered piperidinyl ring (8q), it showed a K comparable to 8d with slightly reduced binding
B
cooperativity factor (α). This promoted our synthesis of analogs with acyclic amino substituents
of the pyrimidinyl ring (i.e. 8rꢀ8t). The results (entry 27ꢀ29, Table 3) indicated that only the N,Nꢀ
diethyl amino group (8r) preserve the activity of 8d. The results from 8r to 8t suggested that a
dialkyl amino group is preferred as the substituent on the pyrimidinyl ring C and each of the
alkyl substitutions should be greater than methyl.
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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2
3
4
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2
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9
0
Table 3. Allosteric parameters of analogs derived from scaffold 8 (8pꢀ8t)
a
b
entry
Compd
R
K (nM)
B
α
49.1
(27.9ꢀ86.3)
1
2
3
5
8d
4.6
4.5
3.2
211.0
8p
8q
8r
(
100.7ꢀ442.0)
6
6.4
26
27
(
24.9ꢀ176.4)
6
9.6
4.8
(
20.1ꢀ240.3)
2
06.9
110.3ꢀ388.1)
13.3
(216.2ꢀ454.2)
2
8
8s
8f
4.5
4.4
(
3
7
9
7.7
2
9
8t
1.7
(
13.4ꢀ711.0)
a
b
K : equilibrium dissociation constant of a potential allosteric ligand. α: binding cooperativity
B
factor for the tested allosteric modulator. The two allosteric parameters were tested using
3
[
H]CP55,940 as the orthosteric ligand.
The allosteric binding parameters of the compounds suggest using pyrimidine in lieu of
the pyridine ring led to two compounds (i.e. 8d and 8r) showing similar binding affinity (K ) to
B
an allosteric site as the lead compound 2. Their allosteric effects on orthosteric agonist binding
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(
i.e. the α values) are comparable to 2. To further investigate the value of these novel allosteric
3
5
modulators, compounds 7a and 8a, 7d and 8d, and 8r were compared with 2 via [ S]GTPγS
binding activity indicative of G protein coupling. The results are shown in Figure 3. All of these
tested compounds showed a concentrationꢀdependent inhibition of CP55,940ꢀinduced GTPγS
binding. In this functional assay, compounds 7a and 8a are weaker than 2 in reducing the Gꢀ
protein coupling to the CB1 receptor. In contrast, compounds 7d (D, Figure 3) and 8d (E, Figure
10
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3
) showed a potency comparable to 2 in reducing the Gꢀprotein coupling. Notably, although 7d
has a weaker binding affinity for the allosteric site than 8d and 8r, it displayed a reduction in G
protein coupling comparable to 8d and 2 in the functional assay. This was indicated by their
35
relative reduction in G protein coupling at the 0.3 ꢀM testing concentration in the [ S]GTPγS
binding assay. In each assay in Figure 3, cells not transfected and transfected with the CB1
receptor treated with the inverse agonist SR141716A are also given. As expected, the later
3
5
reduces the [ S]GTPγS activity below the basal (constitutive activity in the absence of ligand)
level of coupling activity. In combination with the allosteric binding parameters, we conclude
that compounds 7d and 8d are viable CB allosteric modulators showing a binding profile and
1
functional activity similar to 2.
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6
Figure 3. CP55,940ꢀinduced GTPγS levels in the presence and absence of varying
concentrations of test allosteric modulators. HEK293 cell membranes expressing the CB1
receptor were tested for GTPγS levels in the absence and presence of compounds (A) 2, (B) 7a,
0
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(C) 7d, (D) 8a, (E) 8d, (F) 8r at the indicated concentrations in the presence of CP55,940 (1
3
5
µM). The basal levels of [ S]GTPγS binding was also measured in the absence of any
orthosteric and allosteric ligand (Basal CB ), and its inhibition was tested by treatment with
1
SR141716A (1 µM). Data are presented as a percentage of the GTPγS levels in the presence of
CP55,940 (1µM). Each data point represents the mean ± SE (error bars) of at least three
independent experiments performed in duplicate. NonꢀCB ꢀmediated GTPγS levels were
1
3
5
measured by obtaining [ S]GTPγS binding to membrane preparations transfected with vector
PCDNA3.1 not incorporating the gene for CB (No CB ).
1
1
Since the compounds tested antagonize the CP55,940ꢀinduced G protein coupling, we
determined if signaling via βꢀarrestin, instead of G protein, was utilized. For this purpose, we
used ERK1/2 phosphorylation as a good earmark of cellular signaling that can exhibit both Gꢀ
3
5,36
protein dependent and Gꢀprotein independent phosphorylation.
Consistent with previous
observations, ERK1/2 phosphorylation induced by treatment with the orthosteric agonist
CP55,940 alone for 5 min was substantially reduced by G proteinꢀsensitive pertussis toxin
(PTX). However, knockdown of βꢀarrestin 1 or 2 did not impact ERK1/2 phosphorylation by
CP55940, collectively indicating phosphorylation by this compound alone is G ꢀmediated (Figure
i
9
4). However, treatment with the allosteric modulators 7d and 8d alone demonstrated ERK1/2
phosphorylation was insensitive to treatment by PTX, indicating that phosphorylation of ERK1/2
induced by these allosteric modulators is not G ꢀmediated. This is consistent with the results
i
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showing inhibition of Gꢀprotein coupling shown for these allosteric modulators (Figure 3 above).
Interestingly, knockdown of βꢀarrestinꢀ1 with siRNA attenuated ERK1/2 phosphorylation by the
37, 38
modulators 7d and 8d. Compound 2 exhibited a similar response shown for comparison.
10
11
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These results, and the enhancement in binding of the orthosteric agonist CP55,940, suggest that
these compounds are PAMs and promote an active state of the receptor that may be biased to
signal via βꢀarrestinꢀ1 (at least for ERK1/2 phosphorylation) and not G protein.
Figure 4. Impact of compound 7d and 8d on ERK1/2 phosphorylation. HEK293 cells expressing
the CB receptor were untreated (DMSO vehicle only) or treated with CP55,940 (0.5 µM) or 2
1
(10ꢀM), 7d (10 ꢀM), 8d (10 ꢀM) for 5min. Control siRNA, or βꢀarrestin 1 siRNA, or βꢀarrestin
2
siRNA or PTX (5 ng/mL) were used to treat the cells as indicated. Protein from cell lysates
were separated on SDSꢀPAGE and analyzed by Western blots probed with phosphꢀERK1/2 (pꢀ
ERK1/2). The total level of ERK1/2 detected is shown for comparison. Noted that the two bands
correspond to the predominant isoforms, p42 (ERK2) and p44 (ERK1), for ERK1/2.
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CONCLUSIONS
The recent identification of CB allosteric modulators indicated a new approach for
1
developing safer therapeutics based on regulating the pharmacologically important CB receptor.
1
0
1
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Unlike CB orthosteric ligands, which typically present undesired psychotropic and psychiatric
1
side effects, allosteric modulators hold promise as therapeutics with lesser side effects. This is
because allosteric modulation of the receptor can be more finely regulated. Furthermore, some
allosteric modulators may utilize biased signaling pathways such that signal transduction is more
pathwayꢀspecific. In contrast to CB orthosteric ligands, the structural diversity of CB allosteric
1
1
modulators is fairly limited. This work identified novel lead compounds from the biphenyl ureas
that possess a pyrimidine ring in their structures. The new compounds bearing a pyrimidine
moiety showed biological activities comparable to the wellꢀestablished CB allosteric modulator
1
2
that has a pyridine moiety. The two new scaffolds (i.e. 7 and 8) offered broader opportunity for
structural optimization due to the chemical nature of pyrimidine ring. Collectively, the newly
identified pyrimidinyl biphenyl urea analogs provide new chances for the development of novel
and potent CB allosteric modulators. Additionally, the bias exhibited by these allosteric
1
modulators for βꢀarrestin mediated signaling can confer selective fineꢀtuning of CB ꢀimpacted
1
3
9
pathways. In many other GPCRs, βꢀarrestin biased ligands have been shown to have
therapeutically relevant benefits. Carvedilol ((±)ꢀ[3ꢀ(9Hꢀcarbazolꢀ4ꢀyloxy)ꢀ2ꢀhydroxypropyl][2ꢀ
(2ꢀmethoxyphenoxy)ethyl]amine), for example, is a βꢀarrestin biased ligand for both β AR and
1
β2AR which acts as an antagonist for Gꢀprotein mediated signaling and promotes βꢀarrestin
mediated signaling that promotes epidermal growth factor transactivation to confer
4
0
cardioprotection. Thus, biased CB allosteric modulators offer unique opportunities to develop
1
therapeutics.
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EXPERIMENTAL SECTION
Compounds. The compound 2 was purchased from Tocris Bioscience. All other tested
compounds were synthesized in our synthetic laboratory at Texas A&M University.
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Synthesis. All chemical reagents and solvents were purchased from SigmaꢀAldrich Chemical
Co. unless specified otherwise and used without further purification. All anhydrous reactions
were performed under a static argon atmosphere in dried glassware using anhydrous solvents.
Organic phases in work up were dried over anhydrous Na SO , and removed by evaporation
2
4
under reduced pressure. The crude compounds were purified by a Combiflash R chromatography
f
system (Teledyne Technologies, Inc., Thousand Oaks, CA) unless specified otherwise. Purities
of the intermediates were established by several analytical methods including thinꢀlayer
1
chromatography (TLC), melting point, H NMR and mass spectrometry. Analytical thinꢀlayer
®
chromatography (TLC) was run on preꢀcoated silica gel TLC aluminum plates (Whatman ,
UV , layer thickness 250 ꢁm), and the chromatograms were visualized under ultraviolet (UV)
254
light. Melting points were determined on a capillary Electrothermal melting point apparatus and
1
are uncorrected. H NMR spectra of some intermediates and all final compounds were recorded
1
on a Bruker Avance DPXꢀ300 spectrometer operating at 300 MHz. The 2D H NMR data of
some intermediates were recorded on a Bruker Ascendꢀ600 spectrometer operating at 600 MHz.
All NMR spectra were recorded using CDCl or DMSOꢀd as solvent unless otherwise specified.
3
6
The chemical shifts are reported in ppm (parts per million) relative to tetramethylsilane (TMS) as
an internal standard. Multiplicities are indicated as brs (broad singlet), s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplets), dt (doublet of triplets), ddd (doublet of doublet of doublets)
and coupling constants (J) are reported in hertz (Hz). Low and high resolution mass spectra were
obtained at either the Laboratory for Biological Mass Spectrometry of Texas A&M University
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for APCI ionization method or at the School of Chemical Sciences, University of Illinois at
Urbana−Champaign for ESI ionization method. The purities of biologically tested compounds
were analyzed either by combustion elemental analysis performed in Roberson Microlit
laboratories (Madison, NJ) or by the combination of HRMS and HPLC. The results of
combustion elemental analysis indicated by the symbols of the elements were within ±0.4% of
the calculated values for the proposed formula. The purity of each tested compound was
determined to be greater than 95% either by combustion elemental analyses (C, H, N) or by
HPLC with two different mobile phase systems.
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General Procedure A: Preparation of biphenyl ureas (14aꢀ14f and 8aꢀ8t). To the solution of
0.5 mmol of amine 12 (for synthesis of 14aꢀ14f) or amine 19 (for synthesis of 8aꢀ8t) in
anhydrous dichloromethane (5ꢀ8 mL) was added the selected isocyanate 13 (0.75ꢀ0.95 mmol,
1.2ꢀ1.9 equiv) at 0 °C. The reaction mixture was stirred at 0 °C for 10 min and then at room
temperature for 2 h. The reaction was monitored by TLC (30% acetone in hexane or 50ꢀ70%
ethyl acetate in hexane). After completion of the reaction, if white suspension was formed, it was
filtered. The filtered solid was washed with dichloromethane (2 mL) and diethyl ether (5 mL)
successively, and then dried in vacuum oven. If no solid was precipitated out upon the
completion of the reaction, the reaction mixture was condensed in vacuo. The resultant crude
compound was purified by Combiflash chromatography (0 to 50ꢀ80% ethyl acetate in hexane) to
obtain the pyrimidinyl biphenyl urea compounds 14aꢀ14f and 8aꢀ8t.
General Procedure B: Preparation of pyrimidinyl biphenyl ureas 7aꢀ7d. To the solution of
biphenyl urea 14 (0.1 mmol) in anhydrous tetrahydrofuran (2ꢀ10 mL), pyrrolidine (7ꢀ13 equiv)
was added dropwise at room temperature. The reaction mixture was refluxed for 2ꢀ4 h. The
progress of reaction was monitored by TLC (40 % acetone in hexane). Upon completion of the
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reaction, the mixture was cooled to room temperature and condensed under reduced pressure.
The residue was purified by Combiflash chromatography to obtain the target compounds 7aꢀ7d.
General Procedure C: Preparation of pyrimidinyl biphenyl ureas 7eꢀ7h. To the solution of
biphenyl urea 14 (0.3 mmol, 1.0 equiv) in anhydrous tetrahydrofuran (2ꢀ10 mL) in a pressure
tube was added dimethylamine (2ꢀ10 equiv, 2.0 M in methanol) at room temperature. The
reaction mixture was refluxed for 2ꢀ4 h. The progress of the reaction was monitored by TLC
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(
50% ethyl acetate in hexane). Upon completion of the reaction, it was cooled to room
temperature and condensed under reduced pressure. The residue was partitioned between water
15 mL) and ethyl acetate (3 x 10 mL). The organic layer was separated and was washed with
water, brine and dried over anhydrous Na SO . Filtration and removal of the solvent under
(
2
4
reduced pressure provided the crude compound, which was purified by Combiflash
chromatography (0ꢀ50% ethyl acetate in hexane) to obtain the desired compounds 7eꢀ7h.
1ꢀ(4ꢀChlorophenyl)ꢀ3ꢀ(3ꢀ(2ꢀ(pyrrolidineꢀ1ꢀyl)pyrimidineꢀ4ꢀyl)phenyl)urea
(7a).
The
compound 7a was prepared from the biphenyl urea 14a (28 mg, 0.078 mmol) and pyrrolidine (76
mg, 1.06 mmol) in anhydrous THF (2 mL) according to the general procedure B. The crude
product was purified by Combiflash chromatography (0ꢀ50% ethyl acetate in hexane) to provide
1
product 7a (14 mg, 46%) as a white solid; mp 171ꢀ174 °C. H NMR (300 MHz, DMSOꢀd ): δ
6
8.87 (d, J = 3.9 Hz, 2H), 8.41 (d, J = 4.8 Hz, 1H), 8.18 (s, 1H), 7.73ꢀ7.64 (m, 2H), 7.52ꢀ7.32 (m,
+
5
H), 7.07 (d, J = 5.1 Hz, 1H), 3.58 (s, 4H), 1.96 (s, 4H). MS (ESI): m/z = 394.1 [M+H] , 396.1
+
[
M+H+2] . Anal. Calcd for (C H N OCl): C, 64.04; H, 5.12; N, 17.78. Found: C, 63.89; H,
2
1
20
5
5.37; N, 17.42.
1ꢀ(4ꢀBromophenyl)ꢀ3ꢀ(3ꢀ(2ꢀ(pyrrolidineꢀ1ꢀyl)pyrimidineꢀ4ꢀyl)phenyl)urea
(7b).
The
compound 7b was prepared from the biphenyl urea 14b (40 mg, 0.1 mmol) and pyrrolidine (83
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2
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mg, 1.2 mmol) according to the general procedure B. The product was purified by Combiflash
chromatography (0-50% ethyl acetate in hexane) to provide product 7b (19 mg, 47%) as a white
1
solid; mp 180ꢀ183 °C. HNMR (300 MHz, DMSOꢀd ): δ 8.87 (d, J = 3.6 Hz, 2H), 8.40 (d, J =
6
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5.1 Hz, 1H), 8.17 (s, 1H), 7.72 (d, J = 7.5 Hz, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.45ꢀ7.38 (m, 5H),
+
7.07 (d, J = 4.8 Hz, 1H), 3.57 (s, 4H), 1.96 (s, 4H). MS (ESI): m/z = 438.1 [M+H] ,
+
4
5
1
40.1[M+H+2] . Anal. Calcd for (C H N OBr·H O): C, 56.39; H, 4.73; N, 15.66. Found: C,
2
1
20
5
2
6.46; H, 4.35; N, 15.50.
ꢀ(4ꢀFluorophenyl)ꢀ3ꢀ(3ꢀ(2ꢀ(pyrrolidineꢀ1ꢀyl)pyrimidineꢀ4ꢀyl)phenyl)urea
(7c).
The
compound 7c was prepared from the biphenyl urea 14c (0.12 g, 0.35 mmol) and pyrrolidine
(0.17 g, 2.45 mmol) in anhydrous THF (12 mL) according to the general procedure B. The crude
product was isolated by Combiflash chromatography (0ꢀ50% ethyl acetate in hexane) to yield 7c
1
(
98 mg, 74%) as a white solid; mp 206ꢀ208 °C. H NMR (300 MHz, DMSOꢀd ): δ 8.84 (s, 1H),
6
8.75 (s, 1H), 8.40 (d, J = 5.1 Hz, 1H), 8.17 (s, 1H), 7.71ꢀ7.64 (m, 2H), 7.50ꢀ7.38 (m, 3H), 7.16ꢀ
+
7
.06 (m, 3H), 3.57 (s, 4H), 1.96 (s, 4H). MS (ESI): m/z = 378.2 [M+H] . Anal. Calcd for
(C H FN O·¼H O): C, 66.04; H, 5.41; N, 18.34. Found: C, 66.16; H, 5.34; N, 18.12.
2
1
20
5
2
1ꢀ(4ꢀCyanophenyl)ꢀ3ꢀ(3ꢀ(2ꢀ(pyrrolidineꢀ1ꢀyl)pyrimidineꢀ4ꢀyl)phenyl)urea
(7d).
The
compound 7d was prepared from the biphenyl urea 14d (30 mg, 0.085 mmol) and pyrrolidine
(84 mg, 1.18 mmol) in anhydrous THF (2 mL) according to the general procedure B. The crude
product was purified by Combiflash chromatography (0ꢀ50% ethyl acetate in hexane) to obtain
°
1
product 7d (17 mg, 51%) as a white solid; mp 153ꢀ156 C. H NMR (300 MHz, DMSOꢀd ): δ
6
9.25 (s, 1H), 9.03 (s, 1H), 8.41 (d, J = 5.1 Hz, 1H), 8.19 (s, 1H), 7.76ꢀ7.64 (m, 6H), 7.43 (t, J =
+
7
.9 Hz, 1H), 7.08 (d, J = 5.1 Hz, 1H), 3.58 (s, 4H), 1.96 (s, 4H). MS (ESI): m/z = 385.2 [M+H] .
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Anal. Calcd for (C H N O·H O): C, 65.66; H, 5.51; N, 20.88. Found: C, 65.51; H, 5.26; N,
2
2
20
6
2
2
1
7
0.76.
ꢀ(3ꢀ(2ꢀ(Dimethylamino)pyrimidinꢀ4ꢀyl)phenyl)ꢀ3ꢀ(4ꢀfluorophenyl)urea (7e). The compound
e was prepared from the biphenyl urea 14c (0.1 g, 0.3 mmol) and dimethyl amine (1 mL, 2.0
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mmol, 2.0 M in MeOH) in anhydrous THF (10 mL) according to the general procedure C. The
crude product was purified by Combiflash chromatography (0ꢀ50% ethyl acetate in hexane) to
1
obtain product 7e (80 mg, 78%) as a white solid; mp 182ꢀ184 °C. H NMR (300 MHz, DMSOꢀ
d6): δ 8.85 (s, 1H), 8.76 (s, 1H), 8.42 (d, J = 5.1 Hz, 1H), 8.19 (t, J = 1.8 Hz, 1H), 7.72ꢀ7.69 (m,
1H), 7.66ꢀ7.63 (m, 1H), 7.50ꢀ7.38 (m, 3H), 7.16ꢀ7.07 (m, 3H), 3.21 (s, 6H). MS (ESI): m/z =
+
.
352.1 [M+H] Anal. Calcd for (C H FN O): C, 64.95; H, 5.16; N, 19.93. Found: C, 64.85; H,
19 18
5
5.26; N, 19.79.
1ꢀ(4ꢀCyanophenyl)ꢀ3ꢀ(3ꢀ(2ꢀ(dimethylamino)pyrimidinꢀ4ꢀyl)phenyl)urea (7f). The compound
7f was prepared from the biphenyl urea 14d (0.10 g, 0.286 mmol) and dimethylamine (0.275 mL,
.55 mmol, 2.0 M in MeOH) in anhydrous THF (10 mL) according to the general procedure C.
0
The crude product was purified by Combiflash chromatography (0ꢀ50% ethyl acetate in hexane)
1
to provide 7f (0.069 g, 67.6%) as a white solid; mp 132ꢀ134 °C. H NMR (300 MHz, DMSOꢀd ):
6
δ 9.27 (s, 1H), 9.05 (s, 1H), 8.42 (d, J = 5.1 Hz, 1H), 8.22 (t, J = 1.8 Hz, 1H), 7.77ꢀ 7.73 (m, 3H),
7.67ꢀ7.63 (m, 3H), 7.43 (t, J = 7.8 Hz, 1H), 7.09 (d, J = 5.1 Hz, 1H), 3.21 (s, 6H). MS (ESI): m/z
+
=
359.2 [M+H] . Anal. Calcd for (C H N O·½H O): C, 65.38; H, 5.21; N, 22.87. Found: C,
2
0
18
6
2
65.75; H, 4.99; N, 22.58.
1ꢀ(3ꢀ(2ꢀ(Dimethylamino)pyrimidinꢀ4ꢀyl)phenyl)ꢀ3ꢀ(4ꢀiodophenyl)urea (7g). The compound
7g was prepared from the biphenyl urea 14e (0.10 g, 0.22 mmol) and dimethylamine (1.2 mL,
2.4 mmol, 2.0 M in MeOH) in anhydrous THF (15 mL) according to the general procedure C.
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The crude product was purified by Combiflash chromatography (0ꢀ50% ethyl acetate in hexane)
1
to provide 7g (35 mg, 35%) as a white solid; mp 230ꢀ232 °C. H NMR (300 MHz, DMSOꢀd ): δ
6
8.89 (s, 1H), 8.85 (s, 1H), 8.42 (d, J = 5.1 Hz, 1H), 8.19 (s, 1H), 7.73ꢀ7.59 (m, 4H), 7.44ꢀ7.31
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8
9
0
+
(
m, 3H), 7.08 (d, J = 5.1 Hz, 1H), 3.21 (s, 6H). MS (ESI): m/z = 460.1 [M+H] . Anal. Calcd for
(C H IN O): C, 49.69; H, 3.95; N, 15.25. Found: C, 50.01; H, 4.02; N, 14.81.
1
9
18
5
1ꢀ(3ꢀ(2ꢀ(Dimethylamino)pyrimidinꢀ4ꢀyl)phenyl)ꢀ3ꢀ(3ꢀfluorophenyl)urea (7h). The compound
7h was prepared from the biphenyl urea 14f (0.12 g, 0.35 mmol) and dimethylamine (1.22 mL,
2.44 mmol, 2.0 M in MeOH) in anhydrous THF (15 mL) according to the general procedure C.
The crude compound was purified by Combiflash chromatography (0ꢀ50% ethyl acetate in
1
hexane) to provide 7h (0.10 g, 81%) as a white solid; mp 195ꢀ198 °C. H NMR (300 MHz,
DMSOꢀd ): δ 8.98 (s, 1H), 8.94 (s, 1H), 8.42 (d, J = 5.1 Hz, 1H), 8.20 (s, 1H), 7.74ꢀ7.64 (m,
6
2
3
5
H), 7.53ꢀ7.28 (m, 3H), 7.16ꢀ7.08 (m, 2H), 6.83ꢀ6.76 (m, 1H), 3.21 (s, 6H). MS (ESI): m/z =
+
52.2 [M+H] . Anal. Calcd for (C H FN O): C, 64.95; H, 5.16; N, 19.93. Found: C, 64.91; H,
1
9
18
5
.27; N, 19.72.
1ꢀ(4ꢀChlorophenyl)ꢀ3ꢀ(3ꢀ(4ꢀ(pyrrolidinꢀ1ꢀyl)pyrimidinꢀ2ꢀyl)ꢀphenyl)urea (8a). The title
compound 8a was synthesized from 3ꢀ(4ꢀ(pyrrolidineꢀ1ꢀyl)pyrimidinꢀ2ꢀyl)aniline 19a (0.07 g,
0
.29 mmol) and 4ꢀchlorophenyl isocyanate 13a (0.044 g, 0.29 mmol) in anhydrous CH Cl (5
2 2
mL) according to the general procedure A. The crude compound was purified by Combiflash
chromatography (0ꢀ50% ethyl acetate in hexane) to yield the product 8a (0.085 g, 74.5%) as a
1
white solid; mp 192ꢀ195 °C. H NMR (300 MHz, DMSOꢀd ): δ 8.88 (s, 1H), 8.83 (s,1H), 8.38
6
(s, 1H), 8.26 (d, J = 6.3 Hz, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 8.1 Hz, 1H), 7.50 (d, J =
8.7 Hz, 2H), 7.40ꢀ7.32 (m, 3H), 6.44 (d, J = 6.0 Hz, 1H), 3.61 (brs, 2H), 3.33 (brs, 2H), 1.98 (s,
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+
+
4
H). MS (ESI): m/z = 394.2 [M+H] , 396.1 [M+H+2] . Anal. Calcd for (C H N OCl·⅓H O):
21 20 5 2
C, 63.08; H, 5.21; N, 17.51. Found: C, 63.20; H, 5.38; N, 17.15.
1
ꢀ(4ꢀBromophenyl)ꢀ3ꢀ(3ꢀ(4ꢀ(pyrrolidinꢀ1ꢀyl)pyrimidinꢀ2ꢀyl)ꢀphenyl)urea (8b). The title
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18
19
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compound 8b was synthesized from 3ꢀ(4ꢀ(pyrrolidineꢀ1ꢀyl)pyrimidinꢀ2ꢀyl)aniline 19a (0.07 g,
0.3 mmol) and 4ꢀbromo phenyl isocyanate 13b (0.086 g, 0.43 mmol) in anhydrous CH Cl (5
2
2
mL) according to the general procedure A. The crude compound was purified by Combiflash
chromatography (0ꢀ50% ethyl acetate in hexane) to yield product 8b (0.105 g, 82.6%) as a white
1
solid; mp 223ꢀ225 °C. H NMR (300 MHz, DMSOꢀd ): δ 8.85 (s, 1H), 8.79 (s,1H), 8.39 (s, 1H),
6
8.25 (d, J = 6.0 Hz, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 8.1 Hz, 1H), 7.46 (s, 4H), 7.36 (t,
J = 7.8 Hz, 1H), 6.42 (d, J = 6.0 Hz, 1H), 3.62 (brs, 2H), 3.39 (brs, 2H), 1.98 (s, 4H). MS (ESI):
+
+
m/z = 438.1 [M+H] , 440.1 [M+H+2] . Anal. Calcd for (C H N OBr): C, 57.54; H, 4.60; N,
2
1
20
5
15.98. Found: C, 57.73; H, 4.53; N, 15.78.
1ꢀ(4ꢀFluorophenyl)ꢀ3ꢀ(3ꢀ(4ꢀ(pyrrolidinꢀ1ꢀyl)pyrimidinꢀ2ꢀyl)ꢀphenyl)urea (8c). The title
compound 8c was synthesized from 3ꢀ(4ꢀ(pyrrolidineꢀ1ꢀyl)pyrimidinꢀ2ꢀyl)aniline 19a (0.075 g,
0
.31 mmol) and 4ꢀfluorophenyl isocyanate 13c (0.051 g, 0.37 mmol) in anhydrous CH Cl (5
2 2
mL) according to the general procedure A. The white suspension was filtered. The solid was
washed with dichloromethane (3 mL), then dried in vacuum oven to yield the compound 8c
1
(0.079 g, 67 %) as a white solid; mp 185ꢀ187 °C. H NMR (300 MHz, DMSOꢀd ): δ 8.80 (s,
6
1H), 8.68 (s, 1H), 8.39 (s, 1H), 8.26 (d, J = 6.0 Hz, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 8.1
Hz, 1H), 7.50ꢀ7.46 (m, 2H), 7.36 (t, J = 7.8 Hz, 1H), 7.13 (t, J = 9.0 Hz, 2H), 6.42 (d, J = 6.0 Hz,
+
1H), 3.64 (brs, 2H), 3.39 (brs, 2H), 1.99 (s, 4H). HRMS (ESI) m/z for C H FN O [M+H] :
21
21
5
Calcd 378.1730; Found: 378.1727.
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