Pyrrolone Derivatives as Intracellular Allosteric Modulators for Chemokine Receptors: Selective and Dual-Targeting Inhibitors of CC Chemokine Receptors 1 and 2

Article abstract of DOI:10.1021/acs.jmedchem.8b00605

The recent crystal structures of CC chemokine receptors 2 and 9 (CCR2 and CCR9) have provided structural evidence for an allosteric, intracellular binding site. The high conservation of residues involved in this site suggests its presence in most chemokine receptors, including the close homologue CCR1. By using [³H]CCR2-RA-[R], a high-affinity, CCR2 intracellular ligand, we report an intracellular binding site in CCR1, where this radioligand also binds with high affinity. In addition, we report the synthesis and biological characterization of a series of pyrrolone derivatives for CCR1 and CCR2, which allowed us to identify several high-affinity intracellular ligands, including selective and potential multitarget antagonists. Evaluation of selected compounds in a functional [³⁵S]GTPγS assay revealed that they act as inverse agonists in CCR1, providing a new manner of pharmacological modulation. Thus, this intracellular binding site enables the design of selective and multitarget inhibitors as a novel therapeutic approach.

Full text of DOI:10.1021/acs.jmedchem.8b00605

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Article
Pyrrolone derivatives as intracellular allosteric
modulators for chemokine receptors: Selective and dual-
targeting inhibitors of CC Chemokine Receptors 1 and 2
Natalia Veneranda Ortiz Zacarías, Jacobus P. D. van Veldhoven, Laura Portner, Eric van Spronsen,
Salviana Ullo, Margo Veenhuizen, Wijnand J.C. van der Velden, Annelien J.M. Zweemer, Roy
M. Kreekel, Kenny Oenema, Eelke B. Lenselink, Laura H. Heitman, and Adriaan P. IJzerman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00605 • Publication Date (Web): 26 Sep 2018
Downloaded from http://pubs.acs.org on September 27, 2018
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Pyrrolone derivatives as intracellular allosteric
modulators for chemokine receptors: Selective and
dualꢀtargeting inhibitors of CC Chemokine
Receptors 1 and 2
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Natalia V. Ortiz Zacarías, Jacobus P.D. van Veldhoven, Laura Portner, Eric van Spronsen,
Salviana Ullo, Margo Veenhuizen, Wijnand J.C. van der Velden, Annelien J.M. Zweemer, Roy M.
Kreekel, Kenny Oenema, Eelke B. Lenselink, Laura H. Heitman, Adriaan P. IJzerman*
Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden
University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands
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ABSTRACT
The recent crystal structures of CC chemokine receptors 2 and 9 (CCR2 and CCR9) have
provided structural evidence for an allosteric, intracellular binding site. The high conservation of
residues involved in this site suggests its presence in most chemokine receptors, including the
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close homolog CCR1. By using [ H]CCR2ꢀRAꢀ[R], a highꢀaffinity, CCR2 intracellular ligand,
we report an intracellular binding site in CCR1, where this radioligand also binds with high
affinity. In addition, we report the synthesis and biological characterization of a series of
pyrrolone derivatives for CCR1 and CCR2, which allowed us to identify several highꢀaffinity
intracellular ligands, including selective and potential multiꢀtarget antagonists. Evaluation of
35
selected compounds in a functional [ S]GTPγS assay revealed that they act as inverse agonists
in CCR1, providing a new manner of pharmacological modulation. Thus, this intracellular
binding site enables the design of selective and multiꢀtarget inhibitors as a novel therapeutic
approach.
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INTRODUCTION
Chemokines are chemotactic cytokines that control the migration and positioning of immune
cells during physiological and pathological conditions by interacting with more than 20 different
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chemokine receptors. Chemokine receptors mainly belong to the class A of G proteinꢀcoupled
receptors (GPCRs), and can be divided into four different subtypes—namely C, CC, CXC and
CX3C—according to the pattern of specific cysteine residues in their major endogenous
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chemokines. To exert their function, chemokines bind at the extracellular side of their receptors
in a binding mechanism involving the Nꢀterminal domain, extracellular loops and the upper half
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, 4
of the transmembrane bundle.
After activation, most chemokine receptors signal through
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heterotrimeric G proteins, mainly G class, and βꢀarrestins. CC Chemokine receptors 1 (CCR1)
i/o
and 2 (CCR2) are two of the ten members of the CC subtype of chemokine receptors. CCR1 and
CCR2 are expressed in a variety of immune cells, such as monocytes, dendritic cells and T
helper typeꢀ1 (T 1) cells, from where they regulate diverse inflammatory and homeostatic
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functions. Multiple chemokines activate these two receptors, including CCL3, CCL5 and CCL8
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in the case of CCR1; and CCL2, CCL7 and CCL8 in the case of CCR2.
Dysregulation of CCR1, CCR2 and their ligands has been linked to several inflammatory and
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immune diseases,
which has resulted in many drug discovery efforts to develop small
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, 9
molecules that target these receptors. Several lines of evidence support a role for both CCR1
and CCR2 in the pathogenesis of diseases such as rheumatoid arthritis (RA) and multiple
sclerosis (MS): increased expression of both receptors and their ligands in disease models and
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patients;
protective effect of genetic knockout of CCR1 or CCR2 in disease models;
and
positive preclinical studies with chemokineꢀneutralizing monoclonal antibodies or smallꢀ
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molecule inhibitors of CCR1 or CCR2.
Yet, only few clinical studies have shown promising
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results,
while most of the drugs developed so far have failed in clinical trials due to lack of
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efficacy. In this regard, the development of multiꢀtarget drugs has been proposed as a strategy
to overcome the lack of efficacy. Multiꢀtarget drugs are designed to specifically act on more than
one drug target, which might be necessary in highly heterogeneous diseases, such as RA and MS,
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where more than one chemokine receptor is involved. The design of dualꢀantagonists has been
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previously undertaken for CCR1/CCR3,
CCR2/CCR5,
CCR5/CXCR4,
and
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CXCR1/CXCR2; however no CCR1/CCR2 dualꢀantagonists have so far been reported.
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Recently, the crystal structures of CCR2 and CCR9 have revealed a novel allosteric binding
site for small molecules in chemokine receptors. Both CCR2ꢀRAꢀ[R] in CCR2 and vercirnon in
CCR9 bind in a pocket located in the intracellular surface of the receptors, partially overlapping
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with the binding site for G proteins and βꢀarrestins.
These intracellular ligands can inhibit the
receptors in a nonꢀcompetitive and insurmountable manner with regard to chemokine binding, as
2
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demonstrated previously in CCR2. This might result in higher efficacy even in the presence of
a high local concentration of chemokines during a disease state. Together with the potential
advantages of allosteric modulators of chemokine receptors, this intracellular binding site seems
to be quite conserved among chemokine receptors, which suggests the presence of homologous
2
7
pockets in other receptors such as CCR1. This conservation might provide an opportunity for
the design of both selective and dualꢀtargeting inhibitors of CCR1 and CCR2, as a novel
approach to treat inflammatory and immune diseases.
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For CCR2, several compounds belonging to different scaffolds have already been reported to
bind to this intracellular binding site, including pyrrolone derivatives such as CCR2ꢀRAꢀ[R],
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sulfonamide derivatives and 2ꢀmercapto imidazoles.
When tested for selectivity, some of
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these compounds also displayed a moderate activity on CCR1,
suggesting that they might
also bind to CCR1. Thus, we selected the pyrrolone scaffold to explore a potential intracellular
binding site in CCR1. In our current study, we report the synthesis and the biological evaluation
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2, 33
of novel and previously patented pyrrolone derivatives
at both CCR1 and CCR2, in order to
determine their selectivity and structureꢀaffinity relationships (SAR) for both receptors. Finally,
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5
compounds were tested in a [ S]GTPγS binding assay, in order to determine their functional
effects in CCR1 and CCR2. Overall, our results provide evidence that CCR1 can also be targeted
with intracellular allosteric modulators, and that this binding site can be used for the design of
multiꢀtarget compounds.
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RESULTS AND DISCUSSION
Synthesis of pyrrolone derivatives
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The racemic pyrrolones (6ꢀ24, 26ꢀ46) depicted in Scheme 1 were synthesized via a one pot
three component condensation reaction, starting from the commercially available substituted
33
aldehydes 1aꢀl, anilines 2aꢀq and ethyl 2,4ꢀdioxoꢀbutanoates 3aꢀi in acetic acid (6ꢀ23, 26ꢀ46) or
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THF (24). The ethyl 2,4ꢀdioxoꢀbutanoates (3bꢀd, f, i), which were not commercially available,
were prepared by a Claisen condensation starting from the methyl ketones (4bꢀd,f,i) and diethyl
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oxalate 5. Pyrrolone 25 was prepared via a transesterification of 24 by the use of pꢀ
toluenesulfonic acid in 2ꢀpropanol.
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Characterization of [ H]-CCR2-RA-[R] binding on CCR1 and CCR2
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3
[
H]ꢀCCR2ꢀRAꢀ[R] is the (R)ꢀisomer of [ H]ꢀCCR2ꢀRA, a highꢀaffinity radioligand previously
2
6
characterized in our group for CCR2. To avoid a possible effect of the lowerꢀaffinity isomer,
3
we used the tritiumꢀlabeled (R)ꢀisomer in the present study. As expected, [ H]ꢀCCR2ꢀRAꢀ[R]
binds with highꢀaffinity to osteosarcoma (U2OS) cells stably expressing CCR2b (U2OSꢀCCR2)
as shown by saturation experiments (K of 6.3 nM and B of 2.6 pmol/mg, Supplementary Fig
D
max
3
1
and Supplementary Table 1). Kinetic characterization showed that [ H]ꢀCCR2ꢀRAꢀ[R]
associates and dissociates in a biphasic manner (Supplementary Table 1), consistent with the
3
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3
previously reported [ H]ꢀCCR2ꢀRA kinetics. We had reported that [ H]ꢀCCR2ꢀRA binds with
2
8
low affinity to CCR5 (K of 100 nM), suggesting that CCR2ꢀRAꢀ[R] is a nonꢀselective
D
antagonist that can bind several chemokine receptors. In this regard, CCR1 is a close homolog of
CCR2, with 61% amino acid similarity and 47% identity; furthermore, this amino acid similarity
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is > 90% when only considering the amino acid residues involved in the intracellular binding site
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of CCR2ꢀRAꢀ[R] in CCR2 (Supplementary Fig 2). This prompted us to investigate the binding
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of [ H]ꢀCCR2ꢀRAꢀ[R] in membrane preparations from U2OS cells stably expressing CCR1
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(
U2OSꢀCCR1). [ H]ꢀCCR2ꢀRAꢀ[R] homologous displacement assays on U2OSꢀCCR1 yielded a
K of 13.5 nM and a B
of 6.1 pmol/mg (Fig 1a, Supplementary Table 1), suggesting the
D
max
presence of an intracellular site in CCR1 and making it a suitable tool to study such binding
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pocket. Binding of [ H]ꢀCCR2ꢀRAꢀ[R] to U2OSꢀCCR1 was also assessed in kinetic experiments
3
at 25 ºC. These experiments showed that [ H]ꢀCCR2ꢀRAꢀ[R] associates and dissociates in a
biphasic manner, similar to our findings in CCR2, but the association and dissociation rates were
significantly higher in CCR1 than in CCR2 (Supplementary Fig 1 and Supplementary Table 1).
3
Overall, these findings allowed us to set up a [ H]ꢀCCR2ꢀRAꢀ[R] competitive displacement
assay on both U2OSꢀCCR1 and U2OSꢀCCR2, to determine the binding affinity (K ) of unlabeled
i
compounds. Using this assay, we first determined the ability of known ligands to displace this
26, 28
radioligand from CCR1, i.e. the CCR2 intracellular ligands SDꢀ24 and JNJꢀ27141491,
and
3
5
the CCR1 orthosteric antagonist BX471 (Fig 1b). SDꢀ24 and JNJꢀ27141491 fully displaced
3
[
H]ꢀCCR2ꢀRAꢀ[R] from CCR1 in a concentrationꢀdependent manner, indicating that these
compounds bind at the same binding site as CCR2ꢀRAꢀ[R]. SDꢀ24 displaced the radioligand with
3
a pK of 7.45 ± 0.05 (K = 36 nM), while JNJꢀ27141491 displaced [ H]ꢀCCR2ꢀRAꢀ[R] with a pK
i
i
i
30, 31
of 6.9 ± 0.06 (K = 138 nM), consistent with previously reported activities in CCR1.
To rule
i
out that these compounds bind at the orthosteric binding site of CCR1, we also investigated the
3
effect of BX471 in [ H]ꢀCCR2ꢀRAꢀ[R] binding. As expected, BX471 was not able to displace
3
the radioligand (Fig 1b); on the contrary, BX471 significantly enhanced the binding of [ H]ꢀ
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CCR2ꢀRAꢀ[R] by approximately 20% (116 ± 2% in the presence of 10 µM BX471), in a similar
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manner as previously reported with CCR2 orthosteric antagonists.
This allosteric
enhancement is consistent with two different binding sites in CCR1: the orthosteric binding site
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where BX471 binds and an intracellular pocket for CCR2ꢀRAꢀ[R], SDꢀ24 and JNJꢀ27141491.
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This [ H]ꢀCCR2ꢀRAꢀ[R] assay was also used to determine the affinity of the synthesized
pyrrolone derivatives. All pyrrolone derivatives 6 – 46 were first tested at a single concentration
of 1 µM in both U2OSꢀCCR1 and U2OSꢀCCR2 (Tables 1 – 3). Compounds which displaced
3
more than 50% of [ H]ꢀCCR2ꢀRAꢀ[R] binding were further evaluated in this assay using at least
six different concentrations of unlabeled compound in order to determine their binding affinity
for the corresponding receptor subtypes (Fig 1c,d and Tables 1 – 3). Finally, we selected four
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compounds (39, 41, 43 and 45) to be tested in a functional [ S]GTPγS binding assay (Fig 3).
The potency (pIC ) of these compounds was determined in the presence of an EC
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concentration of CCL3 (8 nM) or CCL2 (20 nM) in U2OSꢀCCR1 or U2OSꢀCCR2 membranes,
respectively.
Docking of CCR2-RA-[R] in CCR1 and CCR2
In order to better understand the binding mode of CCR2ꢀRAꢀ[R] in both human CCR1 and
CCR2b, we docked this compound into models of both receptors (Fig 2). In the case of CCR2,
5
x62
6x32
homology modeling was used to model the CCR2 residues between Ser226
and Lys240
,
which correspond to the M2 muscarinic acetylcholine receptor sequence in the CCR2b crystal
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structure (PDB ID:5T1A). These residues were modelled because this region is in close
proximity to the CCR2ꢀRAꢀ[R] binding site. As expected from the sequence alignment
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(
Supplementary Fig 2), CCR2ꢀRAꢀ[R] was predicted to bind to CCR1 in an overlapping binding
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site as the one reported in the crystal structure of CCR2 , in a solventꢀexposed intracellular
pocket found between the intracellular ends of transmembrane segments 1 – 3, 6, 7 and helix 8
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(Fig 2). The vinylogous carboxylic acid functionality makes similar interactions in CCR1 as in
CCR2: the hydroxyl and the two carbonyl groups are involved in hydrogenꢀbond interactions
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x50
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8x50
with the side chain of Arg131 , and the backbone of Arg307
and Phe308
(Fig 2). A
similar hydrophobic subpocket is also observed around the cyclohexyl moiety, which interacts
6x33
6x36
6x37
6x40
6x36
with Ala , Val/Leu , Ile
and Ile . Interestingly, Val244
in CCR2 is replaced by the
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x36
3x50
bigger Leu240
in CCR1, which pushes the ligand down against Arg131 , resulting in a
slightly different binding orientation of CCR2ꢀRAꢀ[R] in this receptor (Fig 2). In addition, the
8x49
8x49
exchange of Lys311
in CCR2 by Arg307
in CCR1 might also contribute to the
stabilization of this slightly altered binding pose. This difference in orientation could result in
CCR1 selectivity, as this orientation seems to open up the subpockets in the proximity of the
cyclohexyl and the acetyl group of CCR2ꢀRAꢀ[R] in CCR1, allowing the introduction of bigger
and more lipophilic substituents at these positions.
Structure-Affinity Relationships (SAR)
1
Modifications replacing the cyclohexyl group (R , Table 1)
29, 32, 33, 36
Several pyrrolone derivatives have been previously evaluated at CCR2,
resulting in
2
9
the identification of CCR2ꢀRAꢀ[R] as a hit compound for further development, but
characterization of these compounds in CCR1 is mostly missing. Compound 6, previously
3
6
reported and characterized in CCR2 by Zou et al. (2007), was selected as our starting point for
the analysis of SAR in both CCR1 and CCR2. In our assay, compound 6 showed an affinity of
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1 nM for CCR2, and a slightly higher affinity of 56 nM for CCR1 (Table 1). To note, the
1
25
binding affinities reported previously for these pyrrolone derivatives were obtained with a Iꢀ
2
9, 36
3
CCL2 binding assay,
resulting in lower affinities compared with our [ H]ꢀCCR2ꢀRAꢀ[R]
2
6
0
1
2
3
4
5
6
7
8
9
0
1
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0
binding assay, as previously observed in our group. For our SAR study, we first examined
1
different C5 substituents of the pyrrolone core (R ), as shown in Table 1. In line with previous
29
studies, we found that increasing the size of the cycloalkyl group from cyclohexyl (6) to
cycloheptyl (7) or cyclooctyl (8) resulted in a decrease in binding affinity for CCR2; however,
the affinity for CCR1 was retained, indicating that bulkier groups are better tolerated in CCR1
than in CCR2, and providing an avenue for selectivity on CCR1 over CCR2. Previous studies
29
showed that decreasing the size of the cycloalkyl group was also detrimental for CCR2, so we
decided not to explore smaller ring sizes.
Substitution of the cycloalkyl group by a phenyl group (9) led to a great loss of CCR2 affinity
(39% displacement at 1 µM), consistent with previously reported values showing a decreased
36
affinity for an almost similar pair of compounds. Yet this substitution only led to a 3ꢀfold
decrease in CCR1 affinity (K of 162 nM), thus showing much higher selectivity for CCR1.
i
Next, we explored the effect of N-aryl modifications in both affinity and selectivity (compounds
1
1
0 – 17), specifically the effect of para and meta substituents. In general, N-aryl groups on the R
position resulted in increased selectivity towards CCR1, as most compounds did not displace
3
more than 36% [ H]ꢀCCR2ꢀRAꢀ[R] binding in CCR2 at a concentration of 1 µM. Only
compounds 12 and 13, with halogen substitutions in para position (Cl and Br, respectively)
regained CCR2 affinity (12, 207 nM; 13, 214 nM). Furthermore, paraꢀsubstituted derivatives
displayed significantly higher affinities compared with their metaꢀsubstituted analogues.
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9
In the case of CCR1, introduction of a paraꢀmethyl moiety (10) resulted in a slight decrease in
affinity compared with the unsubstituted 9; in contrast, the metaꢀsubstituted analogue (14)
showed less than 50% displacement at 1 µM. Introduction of an electronꢀdonating substituent
1
1
1
1
1
1
1
1
1
1
2
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2
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6
0
1
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9
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(methoxy, 11 and 15) was not well tolerated in any position, as it led to an approximately 3ꢀfold
decrease in affinity when placed in para position (11, 541 nM) and a near complete loss of
affinity when placed in meta position (15, 28% displacement at 1 µM). Halogen substituents in
para position were also more favored in the case of CCR1, yielding higher affinities compared
1
with the unsubstituted 9 and regardless of the halogen used (67 nM for R = 4ꢀCl phenyl (12), p
1
< 0.0001 to 9; 87 nM for R = 4ꢀBr phenyl (13); p = 0.0002 to 9). However, selectivity for CCR1
was notably reduced considering that these compounds displayed binding affinities of around
2
00 nM in CCR2. Although moving the halogens to the meta position (16 and 17) decreased the
affinities more than 2ꢀfold compared with their para analogues, selectivity for CCR1 was
3
restored as these compounds showed less than 20% displacement of [ H]ꢀCCR2ꢀRAꢀ[R] binding
in CCR2. Together, the results for compounds 6 to 17 indicate that in CCR1 aliphatic groups
yield higher affinities, while aromatic groups yield lower affinities but improved selectivity over
CCR2.
3
Modifications to the acetyl group (R , Table 1)
Previous modifications to the vinylogous carboxylic acid functionality in CCR2 showed
29, 36
detrimental effects in binding affinity.
Indeed, mutagenesis and structural studies have
8
x48
shown crucial interactions of the hydroxyl and the two carbonyl groups with Glu310
,
8
x49
8x50
Lys311
and Phe312
(residues according to structureꢀbased BallesterosꢀWeinstein
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6
37
24, 28
numbering ) in CCR2.
Sequence alignment of CCR1 and CCR2 (Supplementary Fig 2) and
our docking study (Fig 2) suggest similar interactions in CCR1, as only position 8.49 differs
(arginine in CCR1 and lysine in CCR2). Therefore, we decided to keep the vinylogous
3
0
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0
carboxylic acid moiety and explore different modifications to the acetyl group at the R position
(
Table 1). A gradual increase in the length of the alkyl chain from a methyl group (6) to a butyl
3
group (18 – 20) resulted in a ~2ꢀfold increase in CCR1 affinity (30 nM for R = ethyl (18), p =
3
3
0
.0004 against 6; 29 nM for R = propyl (19), p = 0.0002 against 6; and 31 nM for R = butyl
(
20), p = 0.0010 against 6). In contrast, for CCR2 we observed a similar or a slight decrease in
affinity. Introduction of a bulkier isopropyl group led to a decrease in affinity in both receptors,
with a more drastic effect in CCR2 affinity. Replacing the isopropyl group with cyclopropyl (22)
or tertꢀbutyl (23) restored the affinity in CCR2 to values similar to compound 20 (22, 160 nM;
2
3, 158 nM); in CCR1, these modifications further improved the binding affinity to
approximately 20 nM, yielding compounds with the highest affinity and selectivity observed in
1
3
these series of R and R modifications (22, 19 nM; 23, 22 nM). These results suggest a larger
hydrophobic subpocket in CCR1, able to accommodate larger and branched alkyl chains.
We also explored the effect of adding heteroatoms—oxygen in this case—between the
carbonyl and an ethyl or isopropyl group (24 and 25, respectively). Overall, this led to a drastic
drop in affinity for both receptors. This detrimental effect was most pronounced in compound 25,
3
which displaced less than 40% of [ H]ꢀCCR2ꢀRAꢀ[R] binding in CCR1 and less than 10% in
CCR2. The transformation of the ketone into an ester might decrease the electron density on the
carbonyl oxygen, as well as the acidity of the adjacent protons, thus weakening or disrupting key
8
x49
24, 28
8x49
hydrogen bonding interactions with Lys
in CCR2
or Arg
in CCR1. The need of an
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acidic function for intracellular antagonists has also been reported in a study with Nꢀ
benzylindoleꢀ2ꢀcarboxylic acids, where the authors found a correlation between higher acidity
3
8
3
and higher CCR2 affinity. Finally, replacing the methyl group in R with a phenyl group (26)
3
1
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0
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9
0
had no effect on CCR1 affinity, while it only displaced 37% of [ H]ꢀCCR2ꢀRAꢀ[R] binding in
3
CCR2. Altogether these findings indicate that bigger, more lipophilic groups in R are better
tolerated in CCR1, while in CCR2 methyl is preferred.
2
Modifications to the phenyl ring (R , Table 2)
2
In addition, we explored different Nꢀaryl modifications in the phenyl ring (R , Table 2),
starting with modifications in para position. Removing the methyl group in 6 yielded compound
2
7, with an unsubstituted phenyl group, which displaced less than 50% of the radioligand in both
receptors. Increasing the size of the alkyl group from methyl (6) to ethyl (28) caused a 3ꢀfold
decrease in CCR1 affinity, while the affinity in CCR2 was maintained (28, 168 nM in CCR1
versus 66 nM in CCR2). Adding an electronꢀdonating methoxy group was unfavorable for both
receptors, as affinities dropped to 260 nM in CCR1 and 217 nM in CCR2. In contrast, an
electronꢀwithdrawing substituent (trifluoromethyl, 32) restored the affinity to 92 nM in CCR2,
similar to our starting compound 6, and to 144 nM in CCR1. The substitution of the paraꢀmethyl
group with halogens yielded derivatives with improved binding affinities in both receptors (30
and 31), but no gain in selectivity. Substitution with a chlorine (30) or bromine atom (31) led to a
4
.5ꢀfold increase in CCR2 affinity compared with 6, with K values around 20 nM regardless of
i
the halogen. In the case of CCR1, the bromine atom (31) led to a 2ꢀfold increase compared with
6 (31, 24 nM), while the smaller chlorine atom did not affect the affinity much (30, 39 nM).
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29
Although not synthesized in our study, Dasse et al. (2007), showed that the paraꢀfluoro
analogue performed worse in CCR2 than other paraꢀhalogen derivatives. In this regard, from
fluoro to chloro there is an important increase in polarity (σ), lipophilicity (π) and size, whereas
3
9, 40
0
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from chloro to bromo only lipophilicity and size increase.
Taken together, these results
suggest that lipophilicity and size of the halogen might be more important in CCR1 than in
CCR2, while electronegativity or polarity could play a bigger role in CCR2.
Moving the substituents from the para to the meta position resulted in poor affinities for both
receptors, compared with their paraꢀsubstituted analogues. In CCR1, the metaꢀmethyl (33) and
metaꢀchlorine (35) groups led to a 9ꢀfold and 13ꢀfold decrease in affinity, respectively; in CCR2,
the affinities decreased 3ꢀfold and 13ꢀfold after the same substitutions. The addition of a
trifluoromethyl group in meta position (36) also led to a 3ꢀfold decrease in CCR2 affinity
3
compared with its paraꢀsubstituted analogue 32. In CCR1 36 only displaced 25% of [ H]ꢀCCR2ꢀ
RAꢀ[R] binding at a concentration of 1 µM, displaying the highest selectivity towards CCR2 in
these series of modifications. Also detrimental was the addition of a fluorine group in meta
3
position (34), which led to less than 50% displacement of [ H]ꢀCCR2ꢀRAꢀ[R] binding in both
receptors. Overall, substituents in para position were more favored in both receptors, especially
halogen substituents, yet none of the compounds displayed selectivity towards CCR1. Similarly
2
9
as reported by Dasse et al. (2007), attempts to introduce different substituents in the ortho
position were unsuccessful, thus we continued to explore different combinations of phenyl
substituents.
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As part of our SAR analysis we synthesized compound 38 (also referred as CCR2ꢀRA), which
3
corresponds to the racemic mixture of the radioligand [ H]ꢀCCR2ꢀRAꢀ[R] used in this study.
This compound displayed an affinity of 15 nM in CCR1 and 10 nM in CCR2, similar to the KD
values obtained in homologous displacement or saturation assays (Supplementary Table 1).
Replacing the paraꢀchloro group in 38 with a methyl moiety (37), while keeping the orthoꢀ
fluorine group, led to an expected decrease in affinity for both receptors, as compound 6 with a
methyl group in para position performed worse than 30 with a chlorine atom in the same
position. When the para substituent was replaced with a bromine atom (39), the affinity was
restored to 11 nM in CCR1 and 6 nM in CCR2. Subsequent combinations of meta and para
substituents (40 – 42) generated compounds with decreased CCR1 affinities compared with 38,
as expected from the data on the monoꢀsubstituted meta analogues. Compound 41 displayed a
slightly higher selectivity for CCR2 (8 nM in CCR2 versus 31 nM in CCR1). Overall,
disubstituted derivatives performed better than the monoꢀsubstituted compounds in both
receptors; however, no clear trend in selectivity was observed in these series.
1
1
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2
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4
4
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5
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5
6
0
1
2
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0
In an attempt to improve both affinity and selectivity for CCR1, we decided to combine some
1
2
3
of the best features observed at R , R and R positions: a disubstituted phenyl ring with an
2
orthoꢀfluoro and paraꢀbromo moieties for R , in order to retain the high affinity of 39; a
3
cyclopropyl group or an unsubstituted phenyl ring at R (22 and 26) to gain selectivity; and a
1
metaꢀbromo phenyl ring at R (17) to further improve selectivity for CCR1. These combinations
resulted in four final compounds shown in Table 3 (43 – 46). To maintain a high affinity for
2
CCR1, we kept the 2ꢀfluoroꢀ4ꢀbromophenyl group at R constant and we combined it with
1
3
3
different R and R substituents. The combination with a cyclopropyl group at R position (43)
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6
led to the highest CCR1 affinity in our study (K of 5 nM), but selectivity over CCR2 was
i
3
reduced compared with 22 (3ꢀfold versus 8ꢀfold). Replacing the cyclopropyl group at R by a
phenyl group (44) decreased the affinity for CCR1 by more than 5ꢀfold compared with 43.
0
1
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Compound 43, somewhat unexpectedly, bound to CCR2 with an affinity of 66 nM, more than
1
1
5ꢀfold better than 26. Replacing the cyclohexyl group at R (43) by a 3ꢀbromoꢀphenyl group
(45) resulted in an improved selectivity over CCR2, as this compound did not displace more than
3
5
0% of [ H]ꢀCCR2ꢀRAꢀ[R] binding at 1 µM, whereas it showed an affinity of 50 nM in CCR1.
3
Finally, replacing the cyclopropyl with a methyl group at R (46) maintained the affinity for
CCR1 and restored the affinity for CCR2 (65 nM in CCR1 and 216 nM in CCR2), with a
concomitant loss of selectivity.
Functional characterization of selected compounds
Following the SAR analysis, four compounds (39, 41, 43 and 45) were selected for further
characterization in a G proteinꢀdependent functional assay, in order to assess their inhibitory
potencies (pIC ) in both CCR1 and CCR2. The four compounds were selected based on their
5
0
affinity and selectivity profile: compounds 43 and 39, with the highest affinity for either CCR1
or CCR2 respectively; compound 41, with higher selectivity towards CCR2; and compound 45,
with higher selectivity towards CCR1. As functional assay we used a previously reported
35
[
S]GTPγS binding assay on U2OSꢀCCR2 membranes, which had been applied in the functional
2
6
characterization of several allosteric and orthosteric CCR2 ligands. Similarly as reported by
26
35
Zweemer et al. (2013), CCL2 stimulated [ S]GTPγS binding in a concentrationꢀdependent
manner, displaying a potency of 5 nM in CCR2 (pEC = 8.3 ± 0.09, Fig 3a). Using the same
5
0
assay conditions, we characterized the G protein activation of CCL3 in U2OSꢀCCR1
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membranes. In this assay, CCL3 induced [ S]GTPγS binding in CCR1 with a higher potency
than CCL2 in CCR2 (1.3 nM, pEC = 8.9 ± 0.06), and with a higher maximum effect (Emax) (Fig
5
0
4
1
3a). It should be noted that the potency of CCL3 in our study is lower than previously reported,
which might be related to the differences in cell line and/or assay conditions.
1
1
1
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For the antagonist assays, we used a submaximal EC concentration of CCL3 (8 nM) and
80
CCL2 (20 nM) in CCR1 or CCR2, respectively, in order to evoke 80% stimulation of
35
[
S]GTPγS binding. Although all compounds were able to inhibit CCL3ꢀ or CCL2ꢀinduced G
protein activation, their potencies (IC ) ranged between 30 nM to 8 µM (Table 4 and Fig 3b,c).
5
0
In CCR2, the potency of the compounds increased in the same order observed for affinity (Fig
c, 45 < 43 < 41 < 39). In CCR1 39 displayed the highest potency (590 nM), followed by 43
950 nM), contrary to their binding affinity (Fig 3b, 43 > 39). In addition, the moderate
3
(
selectivity observed in the binding assays was lost in this functional assay: except for 45, all
compounds were more potent inhibitors of CCR2 than CCR1, as their potencies were 3ꢀfold (43),
35
1
9ꢀfold (39) or 48ꢀfold (41) lower in CCR1. Upon comparison of potencies in the [ S]GTPγS
3
assay and the affinities in the [ H]ꢀCCR2ꢀRAꢀ[R] binding assay, we observed that all compounds
displayed between 5 to 10ꢀfold difference between assays in CCR2 (Tables 2 – 4), in agreement
26
with previous characterization of CCR2ꢀRAꢀ[R] on this receptor. In contrast, all compounds
displayed at least a 50ꢀfold difference between assays when tested on CCR1. Such lack of
correlation between apparent potencies and binding affinities in CCR1 might be dependent on
the assay conditions used, G protein concentrations, or the chemokine used in this study; thus,
further studies are warranted to fully characterize these ligands for their selectivity.
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6
In CCR1, all compounds behaved as inverse agonists, as they all significantly decreased the
basal activity of CCR1 at the highest concentration tested (Supplementary Fig 3a). In this regard,
it was previously demonstrated that CCR1 exhibits constitutive activity leading to ligandꢀ
4
2
0
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0
independent G proteinꢀactivation, βꢀarrestin recruitment and receptor internalization, which
points to the development of inverse agonists as a potential therapeutic option for inflammatory
3
5
42
diseases. Yet, only BXꢀ471 has been reported to act as inverse agonist in CCR1. This
prompted us to further characterize these compounds as inverse agonists in CCR1, by measuring
their inhibitory potency in absence of the agonist CCL3 (Supplementary Fig 3b and
Supplementary Table 2). Compounds 39 and 41 were more potent inverse agonists than
antagonists, displaying a 3ꢀfold and almost 10ꢀfold higher potency, respectively, as inverse
agonists. As such, their potencies as inverse agonists were more comparable to their binding
affinities (Table 2 and Supplementary Table 2). In contrast, 43 and 45 showed similar potencies
when measured in the absence or presence of CCL3, and thus, displayed more than 130ꢀfold
difference between functional and binding assays (Table 2 and Supplementary Table 2).
3
Interestingly, both compounds 43 and 45 have a cyclopropyl in the R position while 39 and 41
have a methyl group (Tables 2 and 3), which suggests that this larger group might be responsible
for the difference in their efficacy and functional profile. Moreover, most compounds displayed
pseudoꢀHill slopes of less than unity in CCR1, when tested in the presence or absence of CCL3
(Table 4 and Supplementary Table 2), indicative of a more complex mechanism of inhibition,
4
3
combining negative allosteric modulation and inverse agonism. Of note, the basal levels of
35
constitutive activity in the [ S]GTPγS assay are very dependent on the assay conditions used,
such as GDP concentrations. Yet, at a singleꢀconcentration (100 ꢁM) tested, all compounds
consistently decreased the basal activity in CCR1 after varying GDP concentrations. For
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instance, compound 41 decreased basal activity by 22% (1 ꢁM GDP), 26% (10 ꢁM GDP) and
2
5% (20 ꢁM GDP) (data not shown). To the best of our knowledge, these compounds represent
the first intracellular ligands with demonstrated inverse agonism in CCR1. Both 45 and 43
decreased the basal activity of CCR2 to a similar or smaller level than in CCR1 (45, maximal
decrease of 58%; 43, maximal decrease of 27%), indicative of inverse agonism (Supplementary
Fig 3a). However, no constitutive activity has been reported for CCR2, with only one
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constitutively active mutant (CAM) described so far. In fact, Gilliland et al. (2013) showed that
CCR2 was not able to induce ligandꢀindependent cell migration or to constitutively associate
4
2
with βꢀarrestin, pointing to a lack of constitutive activity. Moreover, several classes of
orthosteric and allosteric CCR2 ligands did not show evidence of inverse agonism when
35
26
previously tested in a similar [ S]GTPγS binding assay. Thus, the inverse agonism observed in
this study might be the consequence of the expression level, ligand concentration and/or assay
conditions employed, so further research is warranted to investigate ligandꢀindependent signaling
in CCR2.
CONCLUSIONS
3
In this study we have characterized [ H]ꢀCCR2ꢀRAꢀ[R], a highꢀaffinity intracellular antagonist
26
previously described for CCR2, in both CCR1 and CCR2, which allowed us to conclude that
this radioligand binds to CCR1 with a similar highꢀaffinity. By characterizing this radioligand in
CCR1, we have provided evidence that CCR1 possesses an intracellular binding site that can be
used for the design of nonꢀcompetitive compounds. In addition, this intracellular radioligand
allowed us to explore the SAR of a series of pyrrolone derivatives in both CCR1 and CCR2.
Although some of these derivatives had been previously described for CCR2, their
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characterization in CCR1 had not been reported. With the SAR analysis we learned that
1
3
introduction of bulkier and more lipophilic groups at R and R positions was better tolerated in
CCR1, allowing us to obtain better selectivity for this receptor. The high conservation between
the intracellular pockets of CCR1 and CCR2 prevented us from finding high selectivity in these
series of compounds, but allowed us to find several potential dualꢀtarget antagonists. Finally,
characterization of four selected compounds in a functional assay allowed us to determine their
functional effects as antagonists in CCR2 and inverse agonists in the constitutivelyꢀactive CCR1,
which opens up a novel avenue to modulate these receptors in inflammatory diseases. In
addition, this highlyꢀconserved binding site might allow the design of both selective and multiꢀ
target inhibitors for chemokine receptors, beyond CCR1 and CCR2.
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EXPERIMENTAL SECTION
Chemistry. General methods.
All solvents and reagents were purchased from commercial sources and were of analytical grade.
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Demineralized water is simply referred to as H O, as was used in all cases unless stated
2
1
otherwise (i.e. brine). H NMR spectra were recorded on a Bruker AV 400 liquid spectrometer
1
(
H NMR, 400 MHz) at ambient temperature. Chemical shifts are reported in parts per million
(ppm), are designated by δ and are downfield to the internal standard tetramethylsilane (TMS) in
CDCl . Couplingꢀconstants are reported in Hz and are designated as J. As a representative
3
1
1
example of the obtained H NMR spectra, supplementary Fig 4 shows the H NMR spectrum of
compound 43. Analytical purity of the final compounds was determined by high pressure liquid
chromatography (HPLC) with a Phenomenex Gemini 3 × C18 110A column (50 × 4.6 mm,
3
ꢁm), measuring UV absorbance at 254 nm. Sample preparation and HPLC method was –
unless stated otherwise – as follows: 0.3–0.8 mg of compound was dissolved in 1 mL of a 1:1:1
mixture of CH CN/H O/tBuOH and eluted from the column within 15 min, with a three
3
2
component system of H O/CH CN/1% TFA in H O, decreasing polarity of the solvent mixture in
2
3
2
time from 80/10/10 to 0/90/10. All compounds showed a single peak at the designated retention
time and are at least 95% pure. Liquid chromatography–mass spectrometry (LC–MS) analyses
were performed using Thermo Finnigan Surveyor – LCQ Advantage Max LC–MS system and a
Gemini C18 Phenomenex column (50 × 4.6 mm, 3 ꢁm). The elution method was set up as
follows: 1–4 min isocratic system of H O/CH CN/1% TFA in H O, 80:10:10, from the 4th min, a
2
3
2
gradient was applied from 80:10:10 to 0:90:10 within 9 min, followed by 1 min of equilibration
at 0:90:10 and 1 min at 80:10:10. Thinꢀlayer chromatography (TLC) was routinely performed to
monitor the progress of reactions, using aluminum coated Merck silica gel F254 plates.
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Purification by column chromatography was achieved by use of Grace Davison Davisil silica
column material (LC60A 30–200 micron). Yields and reaction conditions were not optimized.
4
5, 46
Additionally, all compounds were screened using FAFꢀDrugs4
in order to detect potential
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PanꢀAssay Interference Compounds (PAINS). None of the compounds was identified as PAINS
47
after application of three different filters based on Baell et al.
33
General procedure for the synthesis of compounds 6ꢀ23, 26ꢀ46.
The respective aldehyde 1aꢀl (1.0 eq.), aniline 2aꢀq (1.0 eq.) and ethyl 2,4ꢀdioxoꢀbutanoate
analogue 3aꢀi (1.0 eq.) were dissolved in acetic acid (2.5 mL/mmol) and heated at 95⁰C for 2ꢀ4 h
under a nitrogen atmosphere. Upon completion of the reaction (TLC 1/7 EtOAct/Pet ether) acetic
acid was removed under reduced pressure, the residue was triturated with Et O and stirred for 30
2
minutes after which the pure product was collected by filtration.
3
3
4ꢀAcetylꢀ5ꢀcyclohexylꢀ3ꢀhydroxyꢀ1ꢀ(4ꢀmethylphenyl)ꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone (6).
Started from cyclohexane carboxaldehyde 1a (243 µL, 2.00 mmol, 1.00 eq.), 4ꢀmethylaniline
(214 mg, 2.00 mmol, 1.00 eq.) and ethyl 2,4ꢀdioxopentanoate 3a (251 µL, 2.00 mmol, 1.00 eq.)
1
in 5 mL of acetic acid. Yield: 287 mg, 46%, white solid. H NMR (400 MHz, DMSO): δ 7.38 (d,
J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 4.99 (d, J = 1.2 Hz, 1H), 2.43 (s, 3H), 2.32 (s, 3H),
1
.83 (t, J = 11.2 Hz, 1H), 1.65ꢀ1.56 (m, 1H), 1.52ꢀ1.27 (m, 4H), 0.53 (qd, J = 12.4 , 2.8 Hz, 1H)
+
ppm. MS [ESI+H] : 313.93.
3
2
4ꢀAcetylꢀ5ꢀcycloheptylꢀ3ꢀhydroxyꢀ1ꢀ(4ꢀmethylphenyl)ꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone (7).
48
Started from cycloheptylcarboxaldehyde 1b (375 mg, 3.00 mmol, 1.00 eq.), 4ꢀmethylaniline 2b
(321 mg, 3.00 mmol, 1.00 eq.) and ethyl 2,4ꢀdioxopentanoate 3a (377 µL, 3.00 mmol, 1.00 eq.)
in 7.5 mL of acetic acid. Purified by recrystallization from a mixture of EtOAc and Pet. Ether.
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1
Yield: 102 mg, 13%, offꢀwhite solid. H NMR (400 MHz, CDCl ): δ 7.26ꢀ7.22 (m, 4H), 4.95 (d,
3
J = 1.6 Hz, 1H), 2.54 (s, 3H), 2.38 (s, 3H) ppm, 2.09ꢀ2.03 (m, 1H), 1.61ꢀ1.47 (m, 4H), 1.46ꢀ1.32
+
(m, 4H), 1.31ꢀ1.12 (m, 4H), 0.80 (qd, J = 10.8, 3.2 Hz, 1H) ppm. MS: [ESI+H] : 328.13.
3
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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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
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2
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4
5
6
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9
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2
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8
9
0
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2
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7
8
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4
ꢀAcetylꢀ5ꢀcyclooctylꢀ3ꢀhydroxyꢀ1ꢀ(4ꢀmethylphenyl)ꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone (8).
Started from cyclooctylcarboxaldehyde 1c (648 mL, 4.42 mmol, 1.00 eq.), 4ꢀmethylaniline 2b
474 mg, 4.42 mmol, 1.00 eq.) and ethyl 2,4ꢀdioxopentanoate 3a (554 µL, 4.42 mmol, 1.00 eq.)
(
in 10 mL of acetic acid. Purified by column chromatography using as eluent 1/6 EtOAc/Pet
1
ether. Yield: 118 mg, 8%, white solid. H NMR (400 MHz, CDCl ): δ 7.26ꢀ7.21 (m, 4H), 4.90
3
(
d, J = 1.6 Hz, 1H), 2.53 (s, 3H), 2.37 (s, 3H), 2.22ꢀ2.14 (m, 1H), 1.62ꢀ1.52 (m, 1H), 1.50ꢀ1.15
+
(m, 13H) 0.89ꢀ0.78 (m, 1H) ppm. MS: [ESI+H] : 342.20.
32
4
ꢀAcetylꢀ3ꢀhydroxyꢀ1ꢀ(4ꢀmethylphenyl)ꢀ5ꢀphenylꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone
(9).
Started from benzaldehyde 1d (449 mL, 4.42 mmol, 1.00 eq.), 4ꢀmethylaniline 2b (474 mg, 4.42
mmol, 1.00 eq.) and ethyl 2,4ꢀdioxopentanoate 3a (554 µL, 4.42 mmol, 1.00 eq.) in 10 mL of
1
acetic acid. Yield: 867 mg, 64%, offꢀwhite solid. H NMR (400 MHz, CDCl ): δ 7.28ꢀ7.24 (m,
3
5
H), 7.22 (d, J = 6.0 Hz, 2H), 7.07 (d, J = 8.0 Hz, 2H), 5.75 (s, 1H), 2.49 (s, 3H), 2.16 (s, 3H)
+
ppm. MS [ESI+H] : 308.00.
4
ꢀAcetylꢀ3ꢀhydroxyꢀ5ꢀ(4ꢀmethylphenyl)ꢀ1ꢀ(4ꢀmethylphenyl)ꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone
(
10). Started from 4ꢀmethylbenzaldehyde 1e (521 mL, 4.42 mmol, 1.00 eq.), 4ꢀmethylaniline 2b
(
474 mg, 4.42 mmol, 1.00 eq.) and ethyl 2,4ꢀdioxopentanoate 3a (554 µL, 4.42 mmol, 1.00 eq.)
in 10 mL of acetic acid. Purified by recrystallization from acetone/hexanes. Yield: 257 mg, 18%
1
yellowish solid. H NMR (400 MHz, DMSOꢀd ): δ 7.42 (d, J = 8.4 Hz, 2H), 7.12ꢀ7.04 (m, 4H),
6
+
6
.98 (d, J = 8.0 Hz, 2H), 5.94 (s, 1H), 2.30 (s, 3H), 2.19 (s, 3H), 2.16 (s,3H) ppm. MS [ESI+H] :
322.00.
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ꢀAcetylꢀ3ꢀhydroxyꢀ5ꢀ(4ꢀmethoxyphenyl)ꢀ 1ꢀ(4ꢀmethylphenyl)ꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀ
4
9
one (11). Started from 4ꢀmethoxybenzaldehyde 1f (527 mL, 4.42 mmol, 1.00 eq.), 4ꢀ
methylaniline 2b (474 mg, 4.42 mmol, 1.00 eq.) and ethyl 2,4ꢀdioxopentanoate 3a (554 µL, 4.42
mmol, 1.00 eq.) in 10 mL of acetic acid. The desired product was obtained by column
0
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8
9
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1
2
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4
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7
8
9
0
1
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4
5
6
7
8
9
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8
9
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4
5
6
7
8
9
0
chromatography using a gradient of 1/6 EtOAc/Pet Ether to 1/3 EtOAc/Pet Ether, yielding 34
1
mg, 2% as an offꢀwhite solid. H NMR (400 MHz, DMSOꢀd ): δ 7.42 (d, J = 8.4 Hz, 2H), 7.12
6
(
d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H), 6.73 (d, J = 8.8 Hz, 2H) 5.93 (s, 1H), 3.64 (s, 3H),
+
2
.30 (s, 3H), 2.20 (s, 3H) ppm. MS [ESI+H] : 337.80.
4
ꢀAcetylꢀ5ꢀ(4ꢀchlorophenyl)ꢀ3ꢀhydroxyꢀ1ꢀ(4ꢀmethylphenyl)ꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone
3
2
(12). Started from 4ꢀchlorobenzaldehyde 1g (621 mg, 4.42 mmol, 1.00 eq.), 4ꢀmethylaniline 2b
474 mg, 4.42 mmol, 1.00 eq.) and ethyl 2,4ꢀdioxopentanoate 3a (554 µL, 4.42 mmol, 1.00 eq.)
(
in 10 mL of acetic acid. The desired product was obtained by column chromatography using 1/6
1
EtOAc/Pet ether as eluent, yielding 96 mg, 6% as a white solid. H NMR (400 MHz, DMSOꢀ
d6): δ 7.43 (d, J = 8.4 Hz, 2H), 7.30ꢀ7.18 (m, 4H), 7.08 (d, J = 8.4 Hz, 2H), 5.98 (s, 1H), 2.30 (s,
+
3
H), 2.20 (s, 3H) ppm. MS [ESI+H] : 342.00.
4
ꢀAcetylꢀ5ꢀ(4ꢀbromophenyl)ꢀ3ꢀhydroxyꢀ1ꢀ(4ꢀmethylphenyl)ꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone
3
2
(
13). Started from 4ꢀbromobenzaldehyde 1h (818 mg, 4.42 mmol, 1.00 eq.), 4ꢀmethylaniline 2b
(
474 mg, 4.42 mmol, 1.00 eq.) and ethyl 2,4ꢀdioxopentanoate 3a (554 µL, 4.42 mmol, 1.00 eq.)
1
in 10 mL of acetic acid. Yield: 1.23 g, 72%, yellowish solid. H NMR (400 MHz, CDCl ): δ 7.39
3
(d, J = 8.8 Hz, 2H), 7.25 (d, J = 8.8 Hz, 2H), 7.11ꢀ7.08 (m, 4H), 5.73 (s, 1H), 2.27 (s, 3H), 2.23
+
(
s, 3H) ppm. MS [ESI+H] : 387.93.
4
ꢀAcetylꢀ3ꢀhydroxyꢀ5ꢀ(3ꢀmethylphenyl)ꢀ1ꢀ(4ꢀmethylphenyl)ꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone
(14). Started from 3ꢀmethylbenzaldehyde 1i (600 mg, 5.00 mmol, 1.00 eq.), 4ꢀmethylaniline 2b
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(
536 mg, 5.00 mmol, 1.00 eq.) and ethyl 2,4ꢀdioxopentanoate 3a (627 µL, 5.00 mmol, 1.00 eq.)
1
in 12 mL of acetic acid. Yield: 560 mg, 35%, white solid. H NMR (400 MHz, DMSOꢀ d ): δ
6
7.43 (d, J = 8.4 Hz, 2H), 7.11ꢀ7.05 (m, 3H), 7.02 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 7.2 Hz, 1H),
+
1
1
1
1
1
1
1
1
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
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
5
.94 (s, 1H), 2.31 (s, 3H), 2.19 (s, 3H), 2.18 (s, 3H) ppm. MS [ESI+H] : 321.93
4
ꢀAcetylꢀ3ꢀhydroxyꢀ5ꢀ(3ꢀmethoxyphenyl)ꢀ1ꢀ(4ꢀmethylphenyl)ꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀ
one (15). Started from 3ꢀmethoxylbenzaldehyde 1j (681 mg, 5.00 mmol, 1.00 eq.), 4ꢀ
methylaniline 2b (536 mg, 5.00 mmol, 1.00 eq.) and ethyl 2,4ꢀdioxopentanoate 3a (627 µL, 5.00
1
mmol, 1.00 eq.) in 12 mL of acetic acid. Yield: 1.27 g, 75%, white solid. H NMR (400 MHz,
DMSOꢀ d ): δ 7.44 (d, J = 8.4 Hz, 2H), 7.12 ꢀ 7.07 (m, 3H), 6.79 (s, 1H), 6.75 (d, J = 0.8 Hz,
6
1H), 6.69 (dd, J = 8.0, 2.2 Hz, 1H), 5.97 (s, 1H), 3.65 (s, 3H), 2.32 (s, 3H), 2.20 (s, 3H) ppm. MS
+
[
ESI+H] : 337.39.
4
ꢀAcetylꢀ5ꢀ(3ꢀchlorophenyl)ꢀ3ꢀhydroxyꢀ1ꢀ(4ꢀmethylphenyl)ꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone
(16). Started from 3ꢀchlorobenzaldehyde 1k (703 mg, 5.00 mmol, 1.00 eq.), 4ꢀmethylaniline 2b
(536 mg, 5.00 mmol, 1.00 eq.) and ethyl 2,4ꢀdioxopentanoate 3a (627 µL, 5.00 mmol, 1.00 eq.)
1
in 12 mL of acetic acid. Yield: 619 mg, 36%, light yellow solid. H NMR (400 MHz, CDCl ): δ
3
7
.28 (d, J = 8.4 Hz, 2H), 7.21ꢀ7.18 (m, 3H), 7.17ꢀ7.13 (m, 1H), 7.10 (d, J = 8.4 Hz, 2H), 5.75 (s,
+
1
H), 2.29 (s, 3H), 2.27 (s, 3H) ppm. MS [ESI+H] : 341.80
4
ꢀAcetylꢀ5ꢀ(3ꢀbromophenyl)ꢀ3ꢀhydroxyꢀ1ꢀ(4ꢀmethylphenyl)ꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone
(17). Started from 3ꢀbromobenzaldehyde 1l (925 mg, 5.00 mmol, 1.00 eq.), 4ꢀmethylaniline 2b
(536 mg, 5.00 mmol, 1.00 eq.) and ethyl 2,4ꢀdioxopentanoate 3a (627 µL, 5.00 mmol, 1.00 eq.)
1
in 12 mL of acetic acid. Yield: 993 mg, 51%, brown solid. H NMR (400 MHz, DMSOꢀd ): δ
6
7
.48 (t, J = 1.6 Hz, 1H), 7.44 (d, J = 8.4 Hz, 2H), 7.34ꢀ7.30 (m, 1H), 7.20 (dt, J = 8.0, 1.6 Hz,
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H), 7.14 (t, J = 7.6 Hz, 1H), 7.10 (d, J = 8.4 Hz, 2H), 6.02 (s, 1H), 2.33 (s, 3H), 2.20 (s, 3H)
+
ppm. MS [ESI+H] : 386.67
5ꢀCyclohexylꢀ3ꢀhydroxyꢀ4ꢀpropionylꢀ1ꢀ(4ꢀmetylphenyl)ꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone
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(18). Started from cyclohexane carboxaldehyde 1a (129 mg, 1.15 mmol, 1.00 eq.), 4ꢀ
50
methylaniline 2b (123 mg, 1.15 mmol, 1.00 eq.) and ethyl 2,4ꢀdioxohexanoate 3b (198 mg,
1
1
.15 mmol, 1.00 eq.) in 3 mL of acetic acid. Yield: 65 mg, 19%, white solid. H NMR (400
MHz, CDCl ): δ 7.31ꢀ7.23 (m, 4H), 4.96 (s,1H), 2.95ꢀ2.82 (m, 2H), 2.38 (s, 3H), 1.90 (t, J = 10.8
3
Hz, 1H), 1.66ꢀ1.54 (m, 4H), 1.43ꢀ1.41 (m, 1H), 1.17 (t, J = 7.2 Hz, 3H), 1.09ꢀ1.03 (m, 3H), 0.98ꢀ
+
0
.86 (m, 1H), 0.71ꢀ0.61 (m, 1H) ppm. MS [ESI+H] : 328.13.
4ꢀButyrylꢀ5ꢀcyclohexylꢀ3ꢀhydroxyꢀ1ꢀ(4ꢀmethylphenyl)ꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone
32
(19). Started from cyclohexane carboxaldehyde 1a (605 µL, 5.00 mmol, 1.00 eq.), 4ꢀ
3
4
methylaniline 2b (536 mg, 5.00 mmol, 1.00 eq.) and ethyl 2,4ꢀdioxoheptanoate 3c (198 mg,
1
1.15 mmol, 1.00 eq.) in 12 mL of acetic acid. Yield: 669 mg (39%) as a white solid. H NMR
(400 MHz, DMSO): δ 7.39 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 5.02 (s, 1H), 2.89ꢀ2.70
(m, 2H), 2.32 (s, 3H), 1.84ꢀ1.78 (m, 1H), 1.61ꢀ1.32 (m, 7H), 0.97ꢀ0.80 (m, 6H), 0.80ꢀ0.73 (m,
+
1
H), 0.57ꢀ0.48 (m, 1H) ppm. MS [ESI+H] : 341.87.
4
ꢀPentanoylꢀ5ꢀcyclohexylꢀ3ꢀhydroxyꢀ1ꢀ(4ꢀmethylphenyl)ꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone
(20). Started from cyclohexane carboxaldehyde 1a (266 mg, 2.37 mmol, 1.00 eq.), 4ꢀ
3
4
methylaniline 2b (253 mg, 2.37 mmol, 1.00 eq.) and ethyl 2,4ꢀdioxooctanoate 3d (475 mg, 2.37
1
mmol, 1.00 eq.) in 5 mL of acetic acid. Yield: 237 mg, 28%, white solid. H NMR (400 MHz,
DMSO): δ 12.02 (br s 1H), 7.39 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 7.6 Hz, 2H), 5.02 (s, 1H), 2.92ꢀ
2
.73 (m, 2H), 2.32 (s, 3H), 1.85ꢀ1.75 (m, 1H), 1.58ꢀ1.28 (m, 9H), 0.91ꢀ0.65 (m, 7H) 0.57ꢀ0.50
+
(m,1H) ppm. MS [ESI+H] : 356.00.
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ꢀCyclohexylꢀ3ꢀhydroxyꢀ4ꢀisobutyrylꢀ1ꢀ(4ꢀmethylphenyl)ꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone
(
21). Started from cyclohexane carboxaldehyde 1a (535 µL, 4.42 mmol, 1.00 eq.), 4ꢀ
methylaniline 2b (474 mg, 4.42 mmol, 1.00 eq.) and ethyl 2,4ꢀdioxoꢀ5ꢀmethylhexanoate 3e (823
1
1
1
1
1
1
1
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1
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0
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0
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7
8
9
0
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6
7
8
9
0
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
mg, 4.42 mmol, 1.00 eq.) in 10 mL of acetic acid. Yield: 255 mg, 17%, white solid. H NMR
(
400 MHz, DMSOꢀd ): δ 12.07 (br s, 1H), 7.40 (d, J = 7.6 Hz, 2H), 7.24 (d, J = 7.6 Hz, 2H),
6
5
1
0
.03 (d, J = 1.6 Hz, 1H), 3.44ꢀ3.41 (m, 1H), 2.32 (s, 3H), 1.80ꢀ1.70 (m, 1H), 1.62ꢀ1.59 (m, 1H),
.46ꢀ1.37 (m, 4H), 1.09 (d, J = 6.8 Hz, 3H), 1.02 (d, J = 6.8 Hz, 3H), 0.97ꢀ0.77 (m, 4H), 0.59ꢀ
+
.53 (m, 1H) ppm. MS [ESI+H] : 342.13.
5
ꢀCyclohexylꢀ4ꢀ(cyclopropanecarbonyl)ꢀ3ꢀhydroxyꢀ1ꢀ(4ꢀmethylphenyl)ꢀ1,5ꢀdihydroꢀ2Hꢀ
pyrrolꢀ2ꢀone (22). Started from cyclohexane carboxaldehyde 1a (605 µL, 5.00 mmol, 1.00 eq.),
51
4
ꢀmethylaniline 2b (550 µL, 5.00 mmol, 1.00 eq.) and ethyl 4ꢀcyclopropylꢀ2,4ꢀdioxobutanoate
1
3
f (920 mg, 5.00 mmol, 1.00 eq.) in 10 mL of AcOH. Yield: 60 mg, 4%, white solid. H NMR
(400 MHz, MeOD): δ 7.34 (d, J = 8.4 Hz, 2H), 7.27 (d, J = 7.6 Hz, 2H), 5.01 (d, J = 2.0 Hz, 1H),
3
.01ꢀ2.95 (m, 1H), 2.38 (s, 3H), 1.88 (t, J = 10.4 Hz, 1H), 1.72ꢀ1.64 (m, 1H), 1.60ꢀ1.48 (m, 3H),
+
1
.41 (d, J = 11.2 Hz, 1H), 1.04ꢀ0.86 (m, 8H), 0.72ꢀ0.62 (m, 1H) ppm. MS [ESI+Na] : 363.10.
5
ꢀCyclohexylꢀ3ꢀhydroxyꢀ1ꢀ(4ꢀmethylphenyl)ꢀ4ꢀpivaloylꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone
3
2
(
23). Started from cyclohexane carboxaldehyde 1a (121 µL, 1.00 mmol, 1.00 eq.), 4ꢀ
methylaniline 2b (107 mg, 1.00 mmol, 1.00 eq.) and ethyl 5,5ꢀdimethylꢀ2,4ꢀdioxohexanoate 3g
1
(
175 µL, 1.00 mmol, 1.00 eq.) in 3 mL of acetic acid. Yield: 20 mg, 6%, white solid. H NMR
(400 MHz, DMSOꢀd ): δ 7.41 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H), 5.11 (d, J = 2.2 Hz,
6
1
0
H), 2.32 (s, 3H), 1.63ꢀ1.58 (m, 2H), 1.52ꢀ1.46 (m, 3H), 1.31ꢀ1.28 (m, 1H) 1.25 (s, 9H) 1.01ꢀ
+
.69 (m, 4H), 0.69ꢀ0.59 (m, 1H) ppm. MS [ESI+H] : 356.13.
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Ethyl
2ꢀcyclohexylꢀ4ꢀhydroxyꢀ5ꢀoxoꢀ1ꢀ(4ꢀmethylphenyl)ꢀ2,5ꢀdihydroꢀ1Hꢀpyrroleꢀ3ꢀ
2
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carboxylate (24). Sodium 1,4ꢀdiethoxyꢀ1,4ꢀdioxobutꢀ2ꢀenꢀ2ꢀolate (1.25 g, 6.00 mmol) was
dissolved in 25 ml H O and 25 ml Et O was added. Acidified to pH 2 with 6M HCl (aq.) and was
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2
0
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2
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extracted with Et O from the aqueous phase, dried over MgSO4 and concentrated in vacuo
2
52
yielding 1.05 g, 4.97 mmol, 83% diethyl 2ꢀoxosuccinate as a yellow oil.
Diethyl 2ꢀoxosuccinate 3h (1.05 g, 4.97 mmol, 1.12 eq.) was added to a mixture of
cyclohexane carboxaldehyde 1a (534 µL, 4.42 mmol, 1.00 eq.) and 4ꢀmethylaniline 2b (474 mg,
4
.42 mmol, 1.00 eq.) in 10 mL of dry THF and stirred at room temperature overnight. The
reaction mixture was concentrated in vacuo, Et O was added and the white precipitate was
2
1
collected by filtration. Yield: 400 mg, 26%, white solid. H NMR (400 MHz, CDCl ): δ 11.24 (s,
3
1
2
H), 7.40 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 5.00 (d, J = 1.8 Hz, 1H), 4.32ꢀ4.13 (m,
H), 2.32 (s, 3H), 1.85ꢀ1.76 (m, 1H), 1.66ꢀ1.59 (m, 1H), 1.56ꢀ1.44 (m, 3H), 1.32 (d, J = 12.0 Hz,
+
1H), 1.26 (t, J = 7.2 Hz, 3H), 1.06ꢀ0.75 (m, 4H), 0.63ꢀ0.53 (m, 1H) ppm. MS [ESI+H] : 344.07.
Isopropyl 2ꢀcyclohexylꢀ4ꢀhydroxyꢀ5ꢀoxoꢀ1ꢀ(4ꢀmethylphenyl)ꢀ2,5ꢀdihydroꢀ1Hꢀpyrroleꢀ3ꢀ
carboxylate (25). Ester 22 (343 mg, 1.00 mmol, 1.00 eq.) and pꢀtoluenesulfonic acid (172 mg,
1
.00 mmol, 1.00 eq.) were dissolved in 10 mL of 2ꢀpropanol and the reaction mixture was
refluxed for 48 hours. The solvent was removed under reduced pressure, the crude product was
dissolved in 50 ml EtOAc and washed 3x with H O, dried over MgSO filtered and concentrated
2
4,
under reduced pressure. The crude product was purified by column chromatography (4/1
1
EtOAc/Pet ether) and yielded 150 mg, 9.5%, brownish solid. H NMR (400 MHz, CDCl ): δ
3
9
.33 (s, 1H), 7.30 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H), 5.31ꢀ5.23 (m, 1H), 4.79 (d, J =
.0 Hz, 1H), 2.37 (s, 3H), 1.88 (td, J = 9.6, 2.8 Hz, 1H), 1.73ꢀ1.66 (m, 1H), 1.63ꢀ1.50 (m, 3H),
2
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.38 (d, J = 6.8 Hz, 6H), 1.29ꢀ1.19 (m, 1H), 1.13ꢀ0.90 (m, 4H), 0.74ꢀ0.64 (m, 1H) ppm. MS
[
ESI+H]+: 357.93
4ꢀBenzoylꢀ5ꢀcyclohexylꢀ3ꢀhydroxyꢀ1ꢀ(4ꢀmethylphenyl)ꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone (26).
1
1
1
1
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1
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1
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0
Started from cyclohexane carboxaldehyde 1a (484 µL, 4.00 mmol, 1.00 eq.), 4ꢀmethylaniline 2b
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(428 mg, 4.00 mmol, 1.00 eq.) and ethyl 2,4ꢀdioxoꢀ4ꢀphenylbutanoate 3i (880 mg, 4.00 mmol,
1
1
7
.00 eq.) in 10 mL of AcOH. Yield: 53 mg, 4%, white solid. H NMR (400 MHz, DMSOꢀ d ): δ
6
.85 (d, J = 7.6 Hz, 2H), 7.69ꢀ7.62 (m, 1H), 7.58ꢀ7.52 (m, 2H), 7.49 (d, J = 8.0 Hz, 2H), 7.29 (d,
J = 8.4 Hz, 2H), 5.32 (s, 1H), 2.35 (s, 3H), 1.73ꢀ1.62 (m, 2H), 1.58ꢀ1.51 (m, 2H), 1.49ꢀ1.37 (m,
+
2
H), 1.05ꢀ0.94 (m, 1H), 0.88ꢀ0.74 (m, 4H) ppm. MS [ESI+H] : 375.93.
3
2
4ꢀAcetylꢀ5ꢀcyclohexylꢀ3ꢀhydroxyꢀ1ꢀphenylꢀ1,5ꢀ dihydroꢀ2Hꢀpyrrolꢀ2ꢀone (27). Started
from cyclohexane carboxaldehyde 1a (534 µL, 4.42 mmol, 1.00 eq.), aniline 2a (400 µL, 4.42
mmol, 1.00 eq.) and ethyl 2,4ꢀdioxopentanoate 3a (554 µL, 4.42 mmol, 1.00 eq.) in 10 mL of
1
AcOH. Yield: 1.00 g, 76%, yellow solid. H NMR (400 MHz, CDCl ): δ 7.47ꢀ7.44 (m, 4H),
3
7
1
3
.32ꢀ7.28 (m, 1H), 4.99 (d, J = 2.0 Hz, 1H), 2.54 (s, 3H), 1.97ꢀ1.90 (m, 1H), 1.69ꢀ1.66 (m, 1H),
.59ꢀ1.48 (m, 3H), 1.45ꢀ1.41 (m, 1H), 1.10ꢀ0.99 (m, 3H), 0.92ꢀ0,86 (m, 1H), 0.63 (qd, J = 9.2,
+
.6 Hz, 1H) ppm. MS [ESI+H] : 300.07
32
4
ꢀAcetylꢀ5ꢀcyclohexylꢀ1ꢀ(4ꢀethylphenyl)ꢀ3ꢀhydroxyꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone (28).
Started from cyclohexane carboxaldehyde 1a (534 µL, 4.42 mmol, 1.00 eq.), 4ꢀethylaniline 2c
(553 µL, 4.42 mmol, 1.00 eq.) and ethyl 2,4ꢀdioxopentanoate 3a (554 µL, 4.42 mmol, 1.00 eq.)
1
in 10 mL of AcOH. Yield: 134 mg, 9%, lightꢀyellow solid. H NMR (400 MHz, CDCl ): δ 7.32
3
(d, J = 8.4 Hz, 2H), 7.25 (d, J = 6.4 Hz, 2H), 4.94 (s, 1H), 2.68 (q, J =7.6 Hz, 2H) 2.54 (s, 3H),
1
.95ꢀ1.90 (m, 1H), 1.68ꢀ1.66 (m, 1H), 1.67ꢀ1.51 (m, 3H), 1.43ꢀ1.41 (m, 1H), 1.26 (t, J = 7.6 Hz,
+
3H), 1.10ꢀ0.98 (m, 4H), 0.90ꢀ0.87 (m, 1H), 0.69ꢀ0.60 (m, 1H) ppm. MS [ESI+H] : 328.13
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