Discovery of novel 2,5-dioxoimidazolidine-based P2X7 receptor antagonists as constrained analogues of KN62

Article abstract of DOI:10.1021/jm500324g

Novel 2,5-dioxoimidazolidine-based conformationally constrained analogues of KN62 (1) were developed as P2X₇ receptor (P2X₇R) antagonists using a rigidification strategy of the tyrosine backbone of 1. SAR analysis of the 2,5-dioxoimidazolidine scaffold indicated that piperidine substitution at the N3 position and no substitution at N1 position were preferable. Further optimization of the substituents at the piperidine nitrogen and the spacer around the skeleton resulted in several superior antagonists to 1, including 1-adamantanecarbonyl analogue 21i (IC₅₀ = 23 nM in ethidium uptake assay; IC₅₀ = 14 nM in IL-1β ELISA assay) and (3-CF₃-4-Cl)benzoyl analogue (-)-21w (54 nM in ethidium uptake assay; 9 nM in IL-1β ELISA assay), which was more potent than the corresponding (+) isomer. Compound 21w displayed potent inhibitory activity in an ex vivo model of LTP-induced pain signaling in the spinal cord and significant anti-inflammatory activity in in vivo models of carrageenan-induced paw edema and type II collagen-induced joint arthritis.

Full text of DOI:10.1021/jm500324g

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Article
Discovery of Novel 2,5-Dioxoimidazolidine-based P2X7
Receptor Antagonists as Constrained Analogues of KN62
Jin-Hee Park, Ga Eun Lee, So-Deok Lee, Tran Thi Hien, Sujin Kim, Jin Won Yang, Joong-
Heui Cho, Hyojin Ko, Sung-Chul Lim, Yoon-Gyoon Kim, Keon Wook Kang, and Yong-Chul Kim
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500324g • Publication Date (Web): 18 Jan 2015
Downloaded from http://pubs.acs.org on February 15, 2015
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Discovery of Novel 2,5ꢀDioxoimidazolidineꢀbased P2X7
Receptor Antagonists as Constrained Analogues of KN62
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†1
¶1
†
‡
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JinꢀHee Park, GaꢀEun Lee, SoꢀDeok Lee, Tran Thi Hien, Sujin Kim, Jin Won Yang,
†
†
$
#
§*
JoongꢀHeui Cho, Hyojin Ko, SungꢀChul Lim, YoonꢀGyoon Kim, KeonꢀWook Kang,
†
*
YongꢀChul Kim
†
School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju
00ꢀ712, Republic of Korea
5
¶
Department of Pharmaceutical Industry, Korea Health Industry Development
Institute(KHIDI), Chungcheongbukꢀdo 363ꢀ700, Republic of Korea
§
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National
University, Seoul 151ꢀ742, Republic of Korea
$
‡
College of Pharmacy, Department of Pathology, College of Medicine, Chosun University,
Gwangju 501ꢀ759, Republic of Korea
#
College of Pharmacy, Dankook University, Cheonan 330ꢀ714, Republic of Korea.
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*To whom correspondence should be addressed. Phone: 82ꢀ62ꢀ970ꢀ2502. Fax: 82ꢀ62ꢀ970ꢀ
2484. Eꢀmail: kwkang@snu.ac.kr and yongchul@gist.ac.kr
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These authors contributed equally to this work.
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Abstract
Novel 2,5ꢀdioxoimidazolidineꢀbased conformationally constrained analogues of KN62 (1)
were developed as P2X receptor (P2X R) antagonists using a rigidification strategy of the
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tyrosine backbone of 1. SAR analysis of the 2,5ꢀdioxoimidazolidine scaffold indicated that
piperidine substitution at the N3 position and no substitution at N1 position were preferable.
Further optimization of the substituents at the piperidine nitrogen and the spacer around the
skeleton resulted in several superior antagonists to 1, including 1ꢀadamantanecarbonyl
analogue 21i (IC50 = 23 nM in ethidium uptake assay; IC50 = 14 nM in ILꢀ1β ELISA assay)
and (3ꢀCF ꢀ4ꢀCl)benzoyl analogue (ꢀ)ꢀ21w (54 nM in ethidium uptake assay; 9 nM in ILꢀ1β
3
ELISA assay), which was more potent than the corresponding (+) isomer. Compound 21w
displayed potent inhibitory activity in an ex vivo model of LTPꢀinduced pain signaling in the
spinal cord and significant antiꢀinflammatory activity in in vivo models of carrageenanꢀ
induced paw edema and type II collagenꢀinduced joint arthritis.
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Introduction
Purinergic P2X receptors, cationꢀselective ion channels gated by extracellular ATP, are
1, 2
divided into seven receptor subtypes, P2X
the crystal structure of the zP2X
1
ꢀP2X
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.
According to a recent report regarding
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receptor, these receptors exist as a trimeric subunit, with
each subunit consisting of two continuous transmembrane αꢀhelices, an intracellular terminus,
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and a large extracellular domain rich with disulfide bonds. In particular, the P2X
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receptor
4
(
P2X R), first identified from rat brain in 1996, is distinct compared with the other P2X
7
receptor subtypes because it has a considerably longer Cꢀterminus, with an additional 100ꢀ
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200 amino acids. Brief exposure of P2X
7
R to the agonist ATP facilitates a transient current
+
through the opening of cation channels, thereby triggering depolarization, homeostasis of Na
+
2+ 1ꢀ2, 5
and K , and the massive influx of Ca .
This process leads to activation of a diverse range
of signal transduction pathways related to inflammatory signals, including phospholipase A2,
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phospholipase D, mitogenꢀactivated protein kinase (MAPK), and nuclear κB factor.
Furthermore, repeated or sustained activation of the receptors of extracellular ATP induces
5
large and nonꢀspecific pore formation. The formation of an enlarged pore allows the
penetration of positively charged hydrophilic solutes, such as ethidium bromide and YOꢀProꢀ
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1, which has a molecular mass of up to 900 Da. Pannexinꢀ1, which acts as a plasma
membrane hemichannel, was recently reported to be explicitly involved in the formation of
8, 9
the large pore of P2X
P2X
and macrophages, and in brain glial cells, including microglia and astrocytes.
important role of P2X R in the production of ILꢀ1β in activated monocytes, macrophages,
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R.
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R is primarily expressed in hematopoieticꢀderived immune cells, including mast cells
6, 10
The most
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and microglia is part of a complex sequence of events. Initially, the activation of P2X R
+
triggers K efflux, leading to stimulation of the ILꢀ1βꢀconverting enzyme (caspaseꢀ1) in the
1
1ꢀ13
NLRP3/NALP3 inflammasome, which converts proꢀILꢀ1β to mature ILꢀ1β.
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Interleukinꢀ1β ( ILꢀ1β) is a cytokine protein belonging to the interleukinꢀ1 (ILꢀ1) family.
ILꢀ1, along with tumorꢀnecrosis factorꢀα (TNFꢀα) and ILꢀ6 is a major mediator of innate
immune reactions that play central roles in the development of pathological conditions
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1
1,14ꢀ15
leading to inflammation and chronic pain.
Therefore, the therapeutic regulation of
inflammation through the suppression or blockade of the proinflammatory activities of ILꢀ1
12, 16
has been widely studied using various methods.
For example, the recombinant form of
IL-1 receptor antagonist (ILꢀ1Ra), anakinra (Kineret, Amgen), has been used as a therapeutic
1
5, 17
agent in human patients with immuneꢀrelated disease.
as a crucial regulator of both ILꢀ1β maturation and externalization in immune cells,
antagonizing this receptor has been recognized as another promising strategy for blocking the
action of ILꢀ1. This potential has been confirmed by P2X R knockꢀout studies in which the
7
Because P2X R has been reported
8
, 14, 18
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absence of P2X
7
R in macrophage and microglia was found to impair ILꢀ1β secretion after
ATP stimulation. Furthermore, mice lacking P2X
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R have displayed suppressed nociceptive
19
sensitivity in preclinical pain models, especially those for arthritis and neuropathic pain. In
addition, the upregulated expression of P2X R has been observed in human dorsal root
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1
9
ganglia and injured nerves from patients with chronic neuropathic pain. Recently, the
activation of P2X
7
Rꢀinduced release of cathepsin B, a family of lysosomal proteases, in
20
macrophages independent from the mechanism of ILꢀ1 release was reported. Increased
levels of cathepsin B in the synovial fluid of RA patients assist in the degradation of
21
phagocytosed extracellular matrix protein, which is involved in bone and cartilage damage.
Therefore, it has been suggested that P2X R may be valuable as a therapeutic target to control
7
inflammation and chronic pain in patients with RA or neuropathic pain.
Several research groups and pharmaceutical companies are conducting researches on new
2
2ꢀ27
drugs targeting P2X
7
R.
7
KN62 (1) was one of the first generation antagonists of P2X R
2
8
discovered. Several attempts have been made to optimize analogues of 1, such as through
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the variation of substituents at the piperazine moiety (2), the replacement of the 5ꢀ
isoquinoline sulfonate with a substituted arylsulfonate at the paraꢀposition of the tyrosine
phenyl ring (3), and the preparation of ringꢀconstrained derivatives using 1,2,3,4ꢀ
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tetrahydroisoquinoline (TIC) (4).
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Recently, a diverse series of P2X R antagonists with
enhanced drugꢀlike properties has been disclosed, including AstraZeneca’s adamantane
3
5
36,37
38
amide (5),
Pfizer’s 6ꢀazauracil (6),
Evotec’s pyridopyrimidine (7),
Abott’s
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cyanoguanidine (8), and GSK’s pyroglutamic acid amide (9) analogues. Smallꢀmolecule
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P2X R antagonists, such as AZD9056 (5), CEꢀ224,535 (6), and EVTꢀ401, have entered
clinical trials for the treatment of inflammatory diseases, including rheumatoid arthritis
25
(RA). Although 5 of AstraZeneca and 6 of Pfizer did not have sufficient efficacy in RA to
41, 42
proceed beyond Phase II clinical trials,
7
more novel P2X antagonists are in the drug
development pipeline, including EVTꢀ401, for which Phase I clinical trials were completed in
4
3
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2
009.
pyrazolodiazepine and 3,5ꢀdichloropyridine (11) derivatives as novel P2X
In this report, we describe the discovery of novel P2X R antagonists based on the design of
Our group has reported KNꢀ62 analogues
and protoberberine (10),
4
6
47
7
R antagonists.
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constrained structures of lowꢀenergy conformers of KNꢀ62 through the introduction of a 2,5ꢀ
dioxoimidazolidine scaffold as a rigidification strategy. We report extensive structureꢀactivity
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relationships between the designed analogues regarding their activity as P2X R antagonists
and in the suppression of the release of ILꢀ1β. In addition, we report the ex vivo antiꢀpain
signaling and the in vivo antiꢀinflammatory activity of 21w, one of the representative
compound.
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Results and Discussion
Design Strategy. Several research groups have conducted extensive SAR and
pharmacological studies of 1, which contains ꢀtyrosine. In these investigations, 1 (hP2X R;
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L
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pIC50 = 7.9) has demonstrated the potent and specific antagonism of ATPꢀinduced proꢀ
inflammatory ILꢀ1β release and the suppression of MAPK pathways, including p38 and
48ꢀ50
ERK.
Our strategy to design conformationally constrained derivatives of 1 was derived
from the computational analysis of lowꢀenergy conformers by optimizing the geometric
calculation in MOPAC using PM3 parameters in the CAChe program (BioMedCAChe
version 5.0, FUJITSU, Beaverton, Oregon, USA) (Figure 2). We calculated the distances
between the hydroxy group, carboxyl group, and the αꢀnitrogen of the tyrosine moiety in a
lowꢀenergy conformation of 1, which were the primary sites for modification in this analogꢀ
based derivatives (Figure 2A). Based on an analysis of the distance between these 3 points,
we chose (S)ꢀ5ꢀ(4ꢀhydroxybenzyl)imidazolidineꢀ2,4ꢀdione as a scaffold in order to restrict the
free rotation of the αꢀnitrogen and the carboxyl group of the Nꢀmethyl tyrosine moiety of 1
(Figure 2B). This scaffold was expected to facilitate the introduction of substituents at key
positions while maintaining the triangular geometries delineated by each pair of these 3
points of 1 (Figure 2C). Thus, we designed compound 19c, in which the same isoquinolinꢀ5ꢀ
sulfonyl group of 1 was retained at the R
1
position, the piperazine moiety of 1 was replaced
position, and an isoquinolinꢀ5ꢀyl methyl group
with a substituted piperidinyl group at the R
2
replaced the isoquinolinꢀ5ꢀsulfonyl group at the R position of 1. To support this basic idea,
3
the lowꢀenergy conformers of 1 and compound 19c were superimposed on one another. As
shown in Figure 2D, the overlap of the side chains was well matched in 3ꢀdimensional space,
suggesting that the 2,5ꢀdioxoimidazolidine derivatives might act as rigidified derivatives of 1.
As an initial trial of this strategy, we synthesized several derivatives through combined
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substitution of the isoquinolinꢀ5ꢀyl methyl group and the substituted piperidinyl group at the
N1 or N3 position and evaluated their P2X R antagonistic activity (Figure 2).
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Chemistry. The syntheses of the 2,5ꢀdioxoimidazolidine derivatives (15, 18ꢀ19, 21ꢀ24,
and 29) discussed in this paper are summarized in schemes 1ꢀ4. The 1ꢀsubsituted 4ꢀ((2,5ꢀ
dioxoimidazolidinꢀ4ꢀyl)methyl)phenylisoquinolineꢀ5ꢀsulfonates 15a,b were synthesized
starting from OꢀbenzylꢀLꢀtyrosine (12, Scheme 1). The reaction of 12 with potassium cyanate
yielded a urea intermediate that cyclized under acidic conditions at 90 °C; subsequent
51
deprotection of the benzyl group yielded the 5ꢀmembered imidazolidineꢀ2,4ꢀdione 13 .
Isoquinolineꢀ5ꢀsulfonate 14 was prepared by reaction of 4ꢀhydroxybenzyl compound 13 with
5ꢀisoquinolinyl sulfonyl chloride. The regioselective Nꢀalkylations to place substituents at the
N1 position were easily performed under weak basic conditions in high yield due to its
relatively acidic character compared to the amide NH at the N3 position, thus affording 15aꢀb.
Alkylation of the imide nitrogen at the N1 position occurred exclusively, as shown by the loss
1
of the imide proton signal in the H NMR.
Compounds 18aꢀe and 19aꢀc, with 4ꢀsubstituted piperidinyl, heteroꢀ or bulky aromatic
moieties at R and R , were obtained by a threeꢀ or fourꢀstep synthetic route (Scheme 2).
1
2
Compound 16 was prepared using sulfonylation with 5ꢀisoquinoline sulfonyl chloride
followed by Bocꢀdeprotection under acidic conditions. Compounds 17aꢀe were synthesized
via reductive amination using the appropriate aldehyde or ketone in the presence of sodium
.
triacetoxyborohydride Next, cyclization of 17aꢀe with potassium cyanate in acetic acid
afforded the imidazolidineꢀ2,4ꢀdione derivatives 18aꢀe in good yield. Finally, compounds
19aꢀc were obtained by alkylation of 18aꢀc using cesium bicarbonate.
Scheme 3 shows the preparation of 4ꢀsubstituted piperidinyl derivatives 20a, 21aꢀx, 24aꢀd,
4ꢀpiperidinyl methyl derivative 22a, and 4ꢀpiperidinyl ethyl derivatives 23aꢀb. After Bocꢀ
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deprotection of 18cꢀe with 10% TFA in dichloromethane, 4ꢀpiperidinyl amine 20aꢀc was
acylated or alkylated to attach various groups, including aliphatic acyl groups and substituted
benzoyl or benzyl groups, to yield 21aꢀx, 22a, and 23aꢀb. Furthermore, Nꢀalkylation of 21w
afforded compounds 24aꢀd.
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Scheme 4 shows the synthesis of the phenyl isoquinolineꢀ5ꢀsulfonate derivatives, which
uses the same procedures as Scheme 2 except for the first step. Compound 25 was
synthesized starting from (R)ꢀmethyl 2ꢀaminoꢀ2ꢀ(4ꢀhydroxyphenyl)acetate.
To determine the enantiomeric purity of the compounds, we selected compound 20a and
confirmed the structure of the corresponding Mosher amide by NMR analysis. Because
reductive amination and harsh chemical conditions were employed in subsequent steps, we
suspected that the synthesized derivatives may be racemized. The two singlet peaks for the
methoxy group in the Mosher amide showed a ratio of 5:7 (Figure S1). Therefore, the
synthesized 2,5ꢀdioxoimidazolidine derivatives were confirmed to be racemates. To enable
further studies, the two stereoisomers of compound (±)ꢀ21w were separated to afford (+)ꢀ21w
and (ꢀ)ꢀ21w using a chiral HPLC column (Figure S2).
SAR of 2,5ꢀDioxoimidazolidine Derivatives as P2X R Antagonists. To explore the
7
structureꢀactivity relationship (SAR) of the series of novel substituted 4ꢀ((2,5ꢀ
dioxoimidazolidinꢀ4ꢀyl)methyl)phenylisoquinolineꢀ5ꢀsulfonates, the compounds were
evaluated using an assay of ethidium ion accumulation stimulated by 2’(3’)ꢀOꢀ(4ꢀ
benzoylbenzoyl)ꢀATP (BzATP) in human HEK293 cells stably expressed with recombinant
human P2X R. A known P2X R antagonist, 1, was used as a positive control for comparison
7
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(Tables 1ꢀ4).
Compound 14, with no substitution on the imidazole skeleton, displayed no activity as a
P2X
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R antagonist. Therefore, we initially investigated the appropriate combinations of
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substituents at the R
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positions of the imidazolidineꢀ2,4ꢀdione scaffold to confirm our
rationale with respect to the design of conformationally constrained analogues of 1 (Table 1).
Compound 19c, which has isoquinolineꢀ5ꢀylmethyl and 1ꢀBocꢀ4ꢀpiperidinylmethyl groups at
0
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the R and R positions, respectively, was 6ꢀfold more potent against P2X R compared with
19a, the regioisomer of 19c. Thus, the substitution patterns of 19c may be preferred in order
to adopt the biologically active conformation of 1, although compound 19c displayed
approximately 12ꢀfold weaker activity than the parent compound 1. Replacement of the Boc
group of 19c with a benzyl group (19b), similar to 1, was not beneficial. Interestingly, when
we tested analogues with single substitutions at the N1 or N3 positions, R
clearly more favorable than R substitutions (15a vs. 18a and 15b vs. 18c) for hP2X
antagonistic activity. Notably, compound 18c, with a Boc piperidine group at the R position,
2
substitutions were
1
7
R
1
showed increased antagonistic activity with an IC50 in the submicromolar range and
demonstrated a 4ꢀfold increase in potency over compound 18a, which was substituted with an
isoquinoline group. Thus, the isoquinoline sulfonamide group of 1 does not appear to be
7
critical pharmacophore with our designed skeleton for P2X R antagonism. Compounds 18b
and 20b, with a benzyl substituent and no substitution, respectively, at the position of the Boc
group, showed 50% lower antagonistic activity and no activity, respectively. These results
suggest that the substitution on the piperidine nitrogen should be pivotal for P2X
7
R
antagonism.
Next, we examined substitution on the 4ꢀpiperidinyl moiety (R
the length of the carbon linker between the piperidinyl group and the N3 position of the 2,5ꢀ
dioxoimidazolidine (Table 2). Regarding the effects of substitutions at the R position of the
3
position) and the effects of
3
7
4ꢀpiperidinyl moiety, P2X R antagonistic activity increased in the following order: benzylꢀ <
benzoylꢀ < Bocꢀ. Therefore, we expected that acyl substitution at the 4ꢀpiperidinyl moiety
would be preferred over aryl alkyl substitutions for antagonistic activity (21a vs. 21b, 18b vs.
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22a, and 23a vs. 23b). Furthermore, aliphatic hydrophobic groups may be preferred instead
of aromatic hydrophobic groups at the R position of the 4ꢀpiperidinyl moiety (18e vs. 21aꢀb;
3
18c vs. 18b, 22a; 18d vs. 23aꢀb). Regarding the optimum length of the alkyl spacer between
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the 4ꢀpiperidinyl moiety and the N3 position (from n = 2 to n = 0), analogues directly
substituted with the 4ꢀpiperidinyl group (n = 0) at the N3 position (18e, 21a, 21b) afforded
7
the best P2X R antagonistic activity. Thus, we chose the 2,5ꢀdioxoimidazolidine skeleton as a
new lead structure for further optimization of P2X R antagonism.
7
Because compound 20a exhibited no antagonistic activity at P2X R up to 10 ꢀM, various
7
3
aliphatic acyl groups (R groups) were substituted on the 4ꢀpiperidinyl moiety of compound
20a (Table 3). Of this series of acyclic aliphatic acyl derivatives, compounds with branched
alkyl groups (21d, 21e, 21g) were relatively more potent than those with linear alkyl groups
(21c, 21f). Therefore, we introduced monoꢀ or polycycloalkylcarbonyl groups (21hꢀo) at the
R
3
position and obtained compounds with dramatically increased potencies in the low
nanomolar range. For example, the substitution of a cyclohexanoyl (cꢀC 11CO) group (21h)
at the R position increased the antagonistic activity of P2X R more than 30ꢀfold (IC = 133
6
H
3
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50
nM) compared with the compounds with acyclic alkyl groups. Moreover, substitutions with
polycycloalkylcarbonyl groups further increased the antagonist activities, with IC50 values in
the range of 20ꢀ100 nM. In particular, a 1ꢀadamantylcarbonyl group (21i) at the R
3
position
resulted in the most potent antagonist for P2X
7
R (IC50 = 23 nM), which was approximately 5ꢀ
fold more potent than reference compound 1.
Additional polycycloalkylcarbonyl derivatives (21kꢀo) were tested to optimize the R
group. Except for 21l, which has a 3ꢀbromoꢀadamantylcarbonyl group at the R position, all
other derivatives, including those with 3,5ꢀdimethyladamantylcarbonyl (21m), 1ꢀ
adamantylmethylcarbonyl (21k), noradamantylcarbonyl (21n), and
3
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norboranylmethylcarbonyl (21o) groups, exhibited a 2ꢀ to 3ꢀfold decrease in antagonistic
activity compared to compound 21i, suggesting that the hydrophobic pocket of the receptor
should be tightly regulated for optimum antagonistic activity of the polycycloalkyl carbonyl
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derivatives. The importance of acyl substitutions at the R
by testing compound 21j, which exhibited dramatically decreased antagonistic activity for
P2X R.
3
position was confirmed once again
7
Most of the compounds with micromolar and high submicromolar IC50 values in the
ethidium accumulation assay (e.g., 15aꢀb, 18aꢀd, 19aꢀc, 21aꢀg, 21j, 21p, 22a, and 23aꢀb)
exhibited a relatively low inhibitory effect on the release of ILꢀ1β, ranging from 30 to 68%
inhibition at 1 ꢀM. However, compounds with low nanomolar and subnanomolar activities in
the ethidium accumulation assay exhibited more than 70% inhibition of ILꢀ1β release.
Furthermore, compounds such as 21k, 21l, 21n, 21o, and 21w, which showed greater than 95%
inhibition values against the release of ILꢀ1β by BzATP at 1 ꢀM, were further evaluated by
obtaining full concentrationꢀresponse curves to compare the IC50 values. As a result, the
inhibitory effect of the above 2,5ꢀdioxoimidazolidine derivatives on the release of ILꢀ1β
exhibited patterns similar to those observed in the ethidium accumulation assay.
In the experiments for inhibitory activity against BzATPꢀinduced ILꢀ1β release in LPSꢀ
primed THPꢀ1 cells, all of the polycycloalkylacylꢀsubstituted analogues in Table 3 displayed
twoꢀdigit nanomolar IC values. The most potent P2X R antagonists in this series, 21i and
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2
1l, exhibited parallel inhibitory activity against BzATPꢀinduced ILꢀ1β release in LPSꢀ
primed THPꢀ1 cells, with IC50 values of 14 and 27 nM, respectively. Interestingly, 21k and
1o, which are both weaker P2X R antagonists, also displayed potent inhibitory activity in
2
7
the ILꢀ1β ELISA assay, with IC50 values of 15 and 29 nM, respectively.
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Next, we investigated the effect of substituents on the benzoyl group at the R position, as
shown in Table 4. Overall, metaꢀ and/or paraꢀsubstitutions with halides were more favorable
than substitutions with electronꢀdonating groups or orthoꢀsubstitution with halides. Thus,
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compounds with monoꢀ or diꢀhalogen substituted benzoyl groups at the R
3
position (21qꢀx)
exhibited IC50 values in the 10 to 10 M range, whereas 21p, which has a 3,4ꢀ
dimethoxybenzoyl group at the R position, exhibited significantly decreased antagonistic
activity, with an IC50 value of 3.76 ꢀM. In the SAR analysis of halogen substitutions on the
benzoyl moiety, the profile of P2X R antagonistic activity with chlorination displayed the
ꢀ8
ꢀ7
3
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following pattern: orthoꢀ (21q) < metaꢀ (21r) < paraꢀ (21s, IC50 = 133 nM). In the case of
fluorine substitution, the paraꢀfluorobenzoyl analogue, 21t, was 5ꢀfold less potent than the
corresponding chlorinated compound, 21s, suggesting that the size of the substituent on the
benzoyl moiety has a significant effect on P2X R antagonism. Additionally, we investigated
7
3
disubstituted (ortho, meta, para) benzoyl derivatives (21uꢀx) at the R position. Among these
derivatives, 3,4ꢀdisubstitution (21uꢀw) resulted in synergistically enhanced antagonist
activities, with IC50 values of 217, 73, and 70 nM, respectively, compared with the
corresponding derivatives monoꢀsubstituted at the para position (21s, 21t). The antagonistic
activity decreased by more than 3ꢀfold with 2ꢀfluoroꢀ4ꢀchloro substitution (21x). Overall, the
3
SAR of the substituents on the benzoyl moiety at the R position suggests that the electronic
effects, relative size, and positions of the substitutions may play important roles in P2X
7
R
antagonistic activity.
In addition, the inhibitory activities of monoꢀ and diꢀhalogen substituted benzoyl groups at
the R position against ILꢀ1β release through the activation of P2X R were well correlated
with the results of an ethidium accumulation assay (Table 4). In particular, the 3,4ꢀdichloro
and 3ꢀCF ꢀ4ꢀchloro derivatives 21v and 21w, respectively, exhibited the best ILꢀ1β inhibitory
activities, with IC values in the range of 50ꢀ80 nM.
3
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Although substitution at the R position was not appropriate for antagonism, as shown in
1
1
Table 1, we examined the effect of alkyl substitution at the R position. As shown in Table 5,
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the size of the substituent at the R position of the derivatives (24aꢀd) influenced the
1
antagonistic activity. Compound 24a, which has a methyl substituent, was well tolerated,
with an IC50 of 542 nM, whereas compounds 24bꢀd displayed gradually reduced antagonistic
activities as the substituents increased in size.
For the derivatives with an aromatic ring directly linked to a 2,5ꢀdioxoimidazolidine
skeleton, such as compound 29aꢀc, P2X
combination of the best substituents at the R
spacer between the aromatic ring and the 2,5ꢀdioxoimidazolidine skeleton, as is the case with
7
R antagonism was completely absent despite the
3
position (Table 6). This result suggests that the
2
1i, 21k, and 21w, is critical for antagonistic activity.
7
Effect of the Enantiomers (+)ꢀ21w and (−)ꢀ21w on the P2X R Antagonism. A series
of 2,5ꢀdioxoimidazolidine derivatives were synthesized and tested as racemic mixtures. To
investigate the stereochemical preference of 2,5ꢀdioxoimidazolidine derivatives for P2X R
7
antagonism, the chiral separation of a representative compound, 21w, was conducted using a
chiral HPLC column and afforded two enantiomers, (+)ꢀ21w and (ꢀ)ꢀ21w, which were
subsequently tested in two biological assay systems. The (ꢀ) enantiomer of 21w exhibited 1.4ꢀ
and 5.5ꢀfold higher inhibitory effects than racemic 21w, whereas the (+) enantiomer of 21w
was 2.4ꢀ and 2ꢀfold less active in the ethidium accumulation assay and ILꢀ1β release assay,
respectively (Table 7). Thus, the absolute stereochemistry of 2,5ꢀdioxoimidazolidine
7
derivatives is important for P2X R antagonistic activity. To determine the absolute
configuration of an enantiomer of compound 21w by XRD (Xꢀray diffraction), we attempted
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to obtain crystals of each enantiomer of 21w as well as derivatives of 21w such as Nꢀ
alkylated compounds at R position with various groups (e.g. benzyl group, methyl group and
1
chiral auxiliary group) or Nꢀmethylated compounds at 5ꢀisoquinoline group. Unfortunately,
we had failed all the trials due to the poor crystal formation. All schemes, solubility
information and HPLC data of derivatives were shown in supporting information (Synthesis
S2 and Figure S5).
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Selectivity Evaluation of 21w at mP2X and hP2X receptors using a TEVC Assay.
1
3
From these derivatives, a representative P2X
7
R antagonist, compound 21w, was further
1 3
evaluated for its selectivity against the mP2X and hP2X receptors; the activities of 21w
were measured in twoꢀelectrode voltage clamping (TEVC) experiments on Xenopus oocytes
with overexpression of mP2X or hP2X receptors by microinjection of the corresponding
1
3
capped ribonucleic acids (cRNA). As a result, 10 ꢁM 21w exhibited only a 5−15% reduction
of the 2 ꢁM ATPꢀinduced ion current of either the P2X or P2X receptor, suggesting that the
compound was highly selective for P2X R vs. P2X R and P2X R. Detailed data are provided
1
3
7
1
3
in the Supporting Information (Figure S3).
7
Effect of Compound 21w at Rodent P2X Receptor. We further assessed whether 21w
inhibits rodent P2X receptor using J774.A1, a murine monocyte/macrophage cell line. ILꢀ1β
7
secretion was 170 fold increased by ATP stimulus in LPSꢀprimed J774.A1 cells. Although
relative potency is obviously lower than human THPꢀ1 monocytes, compound 21w
concentrationꢀdependently suppressed ILꢀ1β secretion in J774.A1 cells, and the IC50 value
was determined as 11.8 ꢀM (Figure 3). 5 was used as a reference compound, which was
7
known as a low affinity P2X receptor antagonist. However, 5 did not inhibit ATPꢀstimulated
ILꢀ1β release up to 60 ꢀM in the rodent macrophage cells.
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Ex Vivo Antiꢀpain Signaling Activity of Compound 21w. P2X R is abundantly
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expressed in microglia and has been proposed to play a pivotal role in the crossꢀtalk between
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microglia and neurons. Microglial P2X R and its downstream signaling pathways
53
contribute to the induction of spinal longꢀterm potentiation (LTP). The antiꢀpain signaling
effects of the 2,5ꢀdioxoimidazolidine derivative, with pregabalin as a positive control, were
54
assessed in the neuropathic rat dorsal horn. Pregabalin is an anticonvulsant drug used to
3
treat neuropathic pain and partial seizures. Compound 21w, which contains a (3ꢀCF ꢀ4ꢀ
Cl)benzoyl moiety, was selected for animal experiments due to its solubility and high potency
in both assay systems (IC in EtBr uptake: 70 nM; IC in ILꢀ1β release: 50 nM) compared
50
50
with other derivatives.
Compound 21w reduced ex vivo painꢀrelated LTP signaling by 76% at 20 µM, whereas
pregabalin exhibited only a 31% reduction at the same concentration, suggesting that 2,5ꢀ
dioxoimidazolidine derivatives may be potential therapeutic agents for the treatment of
neuropathic pain.
In Vivo Antiꢀinflammatory Activity of Compound 21w. P2X R may play a major role
7
5
5
in the regulation of processes related to inflammation, as demonstrated in in vivo
1
9, 55
inflammatory animal models.
The antiꢀinflammatory effect of compound 21w, the potent
and selective hP2X R antagonist, was evaluated in carrageenanꢀinduced paw edema in SD
7
rats. The paw edema volume was increased after carrageenan injection, and the maximal
increase occurred at 6 h (Figure 4A). The paw edema volume of the 21wꢀtreated group was
significantly decreased compared to the control. Rats intraperitoneally injected with 10 mg/kg
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21w also exhibited significant suppression of hind paw swelling 3ꢀ6 h after carrageenan
injection (Figure 4B).
We then assessed the antiꢀarthritic effect of 21w in CIIꢀinduced arthritis in DBA/1J mice.
Histopathological RA grades were estimated by the severity of inflammation, fibrosis,
damage to the articular cartilage and bone, and ankylosis in the ankle joints. Increased
histological signs of damage were observed in the CIIꢀinduced arthritis group. However,
these features were ameliorated by 21w treatment (Figure 5A). Injection of 21w (50 mg/kg)
greatly suppressed the histological damage and decreased the cumulative arthritis injury
scores compared with the vehicleꢀtreated CIIꢀRA group (Table 8). To verify whether 21w
inhibits the production of proinflammatory mediators, we further determined the COXꢀ2 and
iNOS protein levels in ankle tissues. Compound 21w (25 and 50 mg/kg) reduced the protein
expression of COXꢀ2 and iNOS in the RA tissues (Figure 5B). These results suggest that the
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novel P2X R antagonists could be utilized in antiꢀinflammatory treatment.
7
In Vivo PK Study of Compound 21w. Because we determined in vivo antiꢀarthritis
effects of compound 21w by using mouse and rat models, we checked pharmacokinetics
profile of compound 21w in rats (Table 9). The absolute bioavailability of compound 21w
after intraperitoneal injection was 86.0%. The plasma concentrations of compound 21w
declined with mean terminal halfꢀlife of 77.2 min after intravenous injection. After
intraperitoneal injection of compound 21w (10 mg/kg) in rats, maximal plasma concentration
of compound 21w (Cmax) was 400 ± 200 ng/ml at 1.5 hr. Considering in vitro IC50 value of
compound 21w [50 nM in ILꢀ1β release assay (THPꢀ1 human monocytes) and 11.8 ꢀM
(J774,A1 mouse monocytes), respectively], the plasma concentration may not be enough to
completely inhibit P2X
7
R in the animal study. We speculate that lower plasma concentration
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than the in vitro IC50 value (11.8 ꢀM) in mouse ILꢀ1β assay might be efficaciously enough
7
for the blocking of P2X R in arthritic tissues, considering that the analysis of in vitro ILꢀ1β
release was performed in supraphysiological concentration of ATP (5 mM), and compound
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21w is a ATPꢀcompetitive antagonist of P2X R (Figure S4).
Conclusions
In conclusion, this report describes the identification of a 2,5ꢀdioxoimidazolidine class of
novel potent P2X R antagonists designed from conformationally constrained analogues of 1
KN62). The results of the SAR studies for P2X R antagonism are summarized as follows: (1)
7
(
7
substitution at the N1 position of 2,5ꢀdioxoimidazolidine is unfavored, (2) acyl substitution at
the N3 position of the piperidine moiety is essential (Table 1), (3) a short chain length
between the 2,5ꢀdioxoimidazolidine and piperidine moieties is crucial for achieving high
potency (18c, 18d, and 18e), and (4) hydrophobic and bulky aliphatic groups are preferred as
acyl substituents on the piperidine. Compounds containing an adamantyl group (21i, 21k, 21l,
and 21m) exhibited superior antagonistic effects compared to other derivatives. A
representative optimized compound, 21w, exhibited not only lowꢀnanomolar IC50 values in
P2X R antagonism and the inhibition of ILꢀ1β release but also significant in vivo or ex vivo
7
efficacy in animal models of inflammation, pain, and rheumatoid arthritis.
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Experimental Section
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13
Chemistry. H and C NMR spectra were determined with a JEOL JNMꢀLA 300WB
spectrometer at 300 MHz or JEOL JNMꢀECX 400P spectrometer at 400 MHz, and spectra
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were taken in CDCl
3 6 3 4
, DMSOꢀd or CD ODꢀd . Unless otherwise noted, chemical shifts (δ)
are expressed as ppm downfield from internal tetramethylsilane. Optical rotations were
25
obtained on a ATAGO APꢀ100 polarimeter. Specific rotations ([α]
D
) are reported in deg/dm,
and the concentration (c) is given as g/100mL in the specified solvent. Mass spectroscopy
was carried out on electrospray and highꢀresolution mass spectra (m/z) were recorded on a
FAB. Highꢀresolution mass analysis was performed at Korea Basic Science Institute (Daegu).
The purity of all final products was determined by HPLC (at least 95% purity unless
otherwise noted, Table S1). The determination of purity was performed on a Shimadzu SCLꢀ
1
0A VP HPLC system using a Shimadzu Shimꢀpack C18 analytical column (250 mm x 4.6
mm, 5 mm, 100 Å) in linear gradient solvent systems. Solvent system was
O:CH CN=60:40 to 10:90 over 30 min at a flow rate = 1 mL/min. Peaks were detected at
H
2
3
254 nm. Synthesized starting materials were prepared according to Supporting Information.
The other starting materials were purchased from Aldrich or Fluka. 5 (AZD9056) was
56
synthesized according to the report from AstraZeneca.
(L)-5ꢀ(4ꢀHydroxybenzyl)imidazolidineꢀ2,4ꢀdione (13). OꢀBenzylꢀ(L)ꢀtyrosine (12) (400
mg, 1.47 mmol) and potassium cyanate (299 mg, 3.69 mmol) were added to a solution of
water (15 mL) and pyridine (15 mL). The mixture was heated to 90 °C for 3 h, cooled to
room temperature, and pyridine was washed out with chloroform (4 x 30 mL). Glacial acetic
acid (15 mL) and 6 N HCl (15 mL) were added to the aqueous phase and the solution was
refluxed for 1.5 h. The solution was kept at 120 °C for 1 h 30 min. The precipitate product
51
was filtered, and washed with cold water and dried under reduced pressure to give 13 (164
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mg, 54.1% yield): H NMR (DMSOꢀd
6
, 300 MHz) δ 10.36 (1H, s), 9.23 (1H, s), 7.85 (1H, s),
6
4
.93 (2H, d, J = 8.8 Hz), 6.61 (2H, d, J = 8.8 Hz), 4.21 (1H, t, J = 4.8 Hz), 2.76 (2H, d, J =
+
.4 Hz); MS (ESI): [M] = 207.1.
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8
9
0
(
L)-4ꢀ((2,5ꢀDioxoimidazolidinꢀ4ꢀyl)methyl)phenyl isoquinolineꢀ5ꢀsulfonate (14). To a
suspension of NaH (87.3 mg of 60% oil suspension, 2.18 mmol) in dried THF (15 mL) was
.
added 13 (150 mg, 0.73 mmol). After 10 min, the isoquinolineꢀ5ꢀsulfonyl chloride HCl (480
mg, 1.82 mmol) in dried DCM was added. This mixture was stirred for 5 h at room
temperature, and then concentrated under vacuum. The residue was taken up in solution of
4
saturated aq. NH Cl and extracted with ethyl acetate. The combined extracts were dried over
sodium sulfate, filtered, and evaporated. The residue was purified by silica gel column
1
chromatography (CHCl :MeOH = 60:1) to give 14 (232 mg, 80.2% yield): H NMR (DMSOꢀ
3
6
ꢀd , 400 MHz) δ 10.41(1H, br s), 9.56 (1H, s), 8.82 (1H, d, J = 6.0 Hz), 8.59 (1H, d, J = 8.4
Hz), 8.37 (1H, d, J = 6.0 Hz), 8.27 (1H, d, J = 8 Hz), 7.81ꢀ7.77 (2H, m), 7.08 (2H, d, J = 8.4
Hz), 6.80 (2H, d, J = 8.4 Hz), 4.24 (1H, t, J = 4.8 Hz), 2.86 (1H, dd, J = 14.4, 4.8 Hz), 2.80
+
(
1H, dd, J = 14.4, 5.2 Hz); HRMS (FAB) [M + H] (C19
H
16
N
3
O
5
S): calcd. 398.0811, found
3
98.0814; Purity 97.3%.
L)-4ꢀ((1ꢀ(Isoquinolinꢀ5ꢀylmethyl)ꢀ2,5ꢀdioxoimidazolidinꢀ4ꢀyl)methyl)phenyl
isoquinolineꢀ5ꢀsulfonate (15a). To a solution of 14 (95 mg, 0.24 mmol) in dried DMF (10
mL) in the presence of CsHCO (185 mg, 0.96 mmol) was added isoquinolineꢀ5ꢀylmethyl
(
3
methanesulfonate (114 mg, 0.48 mmol). The mixture was stirred overnight at 50 °C, and then
concentrated under vacuum. The residue was taken up in solution of saturated aq. NH Cl and
4
extracted with ethyl acetate. The combined extracts were dried over sodium sulfate, filtered,
and evaporated. The residue was purified by silica gel column chromatography
1
(CHCl
3
:MeOH = 75:1) to give 15a (56.3 mg, 43.7% yield): H NMR (CDCl
3
, 400 MHz)
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δ 9.41 (1H, s), 9.23 (1H, s), 8.83 (1H, d, J = 6.0 Hz), 8.56ꢀ8.52 (2H, m), 8.29 (1H, d, J = 8.0
Hz), 8.23 (1H, d, J = 7.2 Hz), 8.06 (1H, d, J = 6.0 Hz), 7.92 (1H, d, J = 8.0 Hz), 7.65 (1H, t, J
=
8.0 Hz), 7.59 (1H, d, J = 7.2 Hz), 7.52 (1H, t, J = 7.6 Hz), 6.95 (2H, d, J = 8.8 Hz), 6.65
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(2H, d, J = 8.4 Hz), 5.50 (1H, s), 5.00 (1H, d, J = 14.8 Hz), 4.95 (1H, d, J = 14.8 Hz), 4.23
13
(1H, dd, J = 7.6, 4.0 Hz), 3.11 (1H, dd, J = 14.4, 4.0 Hz), 2.83 (1H, dd, J = 14.4, 7.6 Hz); C
NMR (CDCl
3
) δ 199.2, 177.6, 175.9, 170.8, 164.6, 152.4, 150.4, 149.8, 144.4, 144.1, 139.2,
+
1
38.0, 135.2, 133.6, 129.2, 126.7, 122.2, 58.4, 29.9, 25.5; HRMS (FAB) [M + H]
S): calcd. 539.1389, found 539.1393; Purity 95.3%.
L)-tertꢀButyl 4ꢀ((4ꢀ(4ꢀ(isoquinolinꢀ5ꢀylsulfonyloxy)benzyl)ꢀ2,5ꢀdioxoimidazolidinꢀ1ꢀ
23 4 5
(C29H N O
(
yl)methyl)piperidineꢀ1ꢀcarboxylate (15b). It was synthesized from 14 following the
1
procedure described for 15a: Yield 80.6%; H NMR (CDCl
3
, 400 MHz) δ 9.42 (1H, s), 8.82
(1H, d, J = 6.0 Hz), 8.54 (1H, d, J = 6.0 Hz), 8.31ꢀ8.28 (2H, m), 8.30 (1H, d, J = 8.0 Hz),
.68 (1H, t, J = 8.0 Hz), 7.07 (2H, d, J = 8.4 Hz), 6.86 (2H, d, J = 8.4 Hz), 5.25 (NH, s), 4.20
1H, dd, J = 8.0, 4.0 Hz), 4.04ꢀ4.02 (2H, m), 3.32 (1H, dd, J = 13.2, 7.2 Hz), 3.27 (1H, dd, J
13.2, 7.2 Hz), 3.17 (1H, dd, J = 14.0, 4.0 Hz), 2.85 (1H, dd, J = 14.0, 8.0 Hz), 2.58ꢀ2.53
7
(
=
13
(2H, m), 1.76ꢀ1.68 (1H, m), 1.43 (9H, s), 1.35ꢀ1.24 (2H, m), 1.10ꢀ1.00 (2H, m); C NMR
(CDCl
3
) δ 195.6, 176.72, 174.0, 172.4, 166.6, 163.3, 149.8, 149.3, 145.3, 144.3, 143.7, 138.0,
+
1
33.6, 127.5, 80.0, 52.9, 35.4, 32.0, 27.0, 24.3, 17.8, 16.3; HRMS (FAB) [M + H]
S): calcd. 595.2226, found 595.2223; Purity 96.9%.
L)-Methyl 2ꢀaminoꢀ3ꢀ(4ꢀ(isoquinolinꢀ5ꢀylsulfonyloxy)phenyl)propanoate
30 35 4 7
(C H N O
(
(16).
Compound 16 was obtained through twoꢀstep process. First, to a suspension of NaH (406 mg
of 60% oil suspension, 10.2 mmol) in dried THF (50 mL) was added Bocꢀ(L)ꢀtyrosine methyl
.
ester (1 g, 3.39 mmol). After 10 min, the isoquinolinesulfonyl chloride HCl (2.24 g, 8.47
mmol) dissolved in dried DCM was added. This mixture was stirred for 5 h at room
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temperature, and then concentrated under vacuum. The residue was taken up in solution of
saturated aq. NH Cl and extracted with ethyl acetate. The combined extracts were dried over
4
sodium sulfate, filtered, and evaporated. The residue was purified by silica gel column
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chromatography (CHCl :MeOH = 80:1). Next, to a 0.08 M solution of protected amide
product (1.5 g, 3.08 mmol) in dichloromethane at 0 °C was added TFA (8 mL), and the
resulting mixture was allowed to stir for 3 h at room temperature. The crude reaction was
concentrated under vacuum with azeotropic removal of TFA with hexane, and the residue
3
was diluted with saturated aq. NaHCO (50 mL). The aqueous mixture was extracted with
dichloromethane (3 x 50 mL), and the combined organic extracts were dried over sodium
sulfate and concentrated under vacuum. The residue was purified by silica gel column
3
chromatography (CHCl :MeOH = 60:1) to give 16 (977 mg, 62.7% twoꢀstep overall yield):
1
H NMR (CDCl , 400 MHz) δ 9.41 (1H, s), 8.81 (1H, d, J = 6.0 Hz), 8.54 (1H, d, J = 6.0 Hz),
3
8.29ꢀ8.27 (2H, m), 7.66 (1H, t, J = 7.6 Hz), 7.04 (2H, d, J = 8.8 Hz), 6.81 (2H, d, J = 8.8 Hz),
3.65 (3H, s), 3.64 (1H, dd, J = 7.6, 5.2 Hz), 2.98 (1H, dd, J = 13.6, 5.2 Hz), 2.79 (1H, dd, J =
+
1
3.6, 7.6 Hz); MS (ESI): [M] = 387.0.
(±)-Methyl
2ꢀ(isoquinolinꢀ5ꢀylmethylamino)ꢀ3ꢀ(4ꢀ(isoquinolinꢀ5ꢀ
ylsulfonyloxy)phenyl)propanoate (17a). A solution of 16 (550 mg, 1.42 mmol) and
isoquinolineꢀ5ꢀcarboxaldehyde (224 mg, 1.42 mmol) in dried DCE (30 mL) was treated with
sodium triacetoxyborohydride (754 mg, 3.56 mmol). After 4 h, the resulting mixture was
treated with 10% aq. potassium hydroxide (40 mL). The organic phase was extracted with
dichloromethane (3 x 20 mL), and combined organic phases were dried over sodium sulfate
and concentrated under vacuum. The residue was purified by silica gel column
1
chromatography (CHCl
3
:MeOH = 40:1) to give 17a (602 mg, 80.2% yield): H NMR (CDCl
3
,
400 MHz) δ 9.40 (1H, s), 9.23 (1H, s), 8.81 (1H, d, J =6.4 Hz), 8.55 (1H, d, J = 6.4 Hz), 8.47
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(1H, d, J =6.0 Hz), 8.28ꢀ8.25 (2H, m), 7.88 (1H, d, J = 7.6 Hz), 7.77 (1H, d, J = 6.0Hz), 7.63
(1H, t, J = 7.6 Hz), 7.52ꢀ7.25 (2H, m), 7.00 (2H, d, J = 8.0 Hz), 6.77 (2H, d, J = 8.4 Hz), 4.19
(1H, d, J = 13.2 Hz), 3.95 (1H, d, J = 13.2 Hz), 3.65 (3H, s), 3.50 (1H, dd, J = 7.6, 6.0 Hz),
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+
2
.91 (1H, dd, J = 14.0, 6.0 Hz), 2.83 (1H, dd, J = 14.0, 7.6 Hz); MS (ESI): [M] = 528.0.
(±)-
methyl
2ꢀ((1ꢀbenzylpiperidinꢀ4ꢀyl)methylamino)ꢀ3ꢀ(4ꢀ(isoquinolinꢀ5ꢀ
ylsulfonyloxy)phenyl)propanoate (17b). It was synthesized from 16 and 1ꢀ
1
benzylpiperidineꢀ4ꢀcarbaldehyde following the procedure described for 17a: Yield 62.7%; H
NMR (CDCl3, 400 MHz) δ 9.39 (1H, s), 8.81 (1H, d, J = 6.4 Hz), 8.55 (1H, d, J = 6.4 Hz),
8
.25ꢀ8.21 (2H, m), 7.61 (1H, t, J = 7.6 Hz), 7.32ꢀ7.27 (5H, m), 7.01 (2H, d, J = 8.8 Hz), 6.77
(
2H, d, J = 8.4 Hz), 3.57 (3H, s), 3.46 (2H, s), 3.34 (1H, t, J = 6.8 Hz), 2.84ꢀ2.79 (4H, m),
2
.43 (1H, dd, J = 11.6, 6.8 Hz), 2.24 (1H, dd, J = 11.6, 6.8 Hz), 1.90ꢀ1.10 (7H, m); MS (ESI):
+
[
M] = 574.0.
(
±)-tertꢀButyl 4ꢀ((3ꢀ(4ꢀ(isoquinolinꢀ5ꢀylsulfonyloxy)phenyl)ꢀ1ꢀmethoxyꢀ1ꢀoxopropanꢀ
2
ꢀylamino)methyl)piperidineꢀ1ꢀcarboxylate (17c). It was synthesized from 16 and tertꢀ
butyl 4ꢀformylpiperidineꢀ1ꢀcarboxylate following the procedure described for 17a: Yield
1
65.7%; H NMR (CDCl
3
, 400 MHz) δ 9.42 (1H, s), 8.81 (1H, d, J = 6.0 Hz), 8.55 (1H, d, J =
6
.0 Hz), 8.29ꢀ8.26 (2H, m), 7.66 (1H, t, J = 7.6 Hz), 7.02 (2H, d, J = 8.8 Hz), 6.79 (2H, d, J =
.8 Hz), 4.04 (2H, m), 3.58 (3H, s), 3.42 (1H, t, J = 6.8 Hz), 2.83 (2H, m), 2.62 (2H, m), 2.44
8
(
1H, dd, J = 11.2, 6.8 Hz), 2.24 (1H, dd, J = 11.2, 6.8 Hz), 1.75ꢀ1.08 (3H, m), 1.43 (9H, s),
+
0.99 (2H, m); MS (ESI): [M] = 584.2.
(±)-tertꢀButyl
4ꢀ(2ꢀ(3ꢀ(4ꢀ(isoquinolinꢀ5ꢀylsulfonyloxy)phenyl)ꢀ1ꢀmethoxyꢀ1ꢀ
oxopropanꢀ2ꢀylamino)ethyl)piperidineꢀ1ꢀcarboxylate (17d). It was synthesized from 16
and tertꢀbutyl 4ꢀ(2ꢀoxoethyl)piperidineꢀ1ꢀcarboxylate following the procedure described for
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1
1
3
7a: Yield 66.7%; H NMR (CDCl , 400 MHz) δ 9.42 (1H, s), 8.81 (1H, d, J = 6.0 Hz), 8.54
(
1H, d, J = 6.0 Hz), 8.29ꢀ8.27 (2H, m), 7.66 (1H, t, J = 7.6 Hz), 7.02 (2H, d, J = 8.4 Hz), 6.79
2H, d, J = 8.4 Hz), 4.09ꢀ3.94 (2H, m), 3.57 (3H, s), 3.40ꢀ3.37 (1H, m), 2.83ꢀ2.81 (2H, m),
(
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
6
7
8
9
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2
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5
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8
9
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2
3
4
5
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8
9
0
2.83ꢀ2.81 (2H, m), 2.59ꢀ2.55 (3H, m), 2.42ꢀ2.41 (2H, m), 1.42 (9H, s), 1.39ꢀ1.30 (3H, m),
+
1.07 (2H, q, J = 11.6 Hz); MS (ESI): [M] = 584.0.
(
±)-tertꢀButyl 4ꢀ(3ꢀ(4ꢀ(isoquinolinꢀ5ꢀylsulfonyloxy)phenyl)ꢀ1ꢀmethoxyꢀ1ꢀoxopropanꢀ2ꢀ
ylamino)piperidineꢀ1ꢀcarboxylate (17e). It was synthesized from 16 and tertꢀbutyl 4ꢀ
1
oxopiperidineꢀ1ꢀcarboxylate following the procedure described for 17a: Yield 74.5%; H
NMR (CDCl , 400 MHz) δ 9.41 (1H, s), 8.82 (1H, d, J = 6.4 Hz), 8.56 (1H, d, J =6.0 Hz),
3
8
3
.29ꢀ8.24 (2H, m), 7.66 (1H, t, J = 7.6 Hz), 7.01 (2H, d, J = 8.4 Hz), 6.77 (2H, d, J = 8.4 Hz),
.85 (2H, m), 3.59 (3H, s), 3.47 (2H, t, J = 7.2 Hz), 2.82 (4H, m), 2.47 (1H, m), 1.59 (4H, m),
+
1.43 (9H, s); MS (ESI): [M] = 569.9.
(
±)-4ꢀ((3ꢀ(Isoquinolinꢀ5ꢀylmethyl)ꢀ2,5ꢀdioxoimidazolidinꢀ4ꢀyl)methyl)phenyl
isoquinolineꢀ5ꢀsulfonate (18a). To a solution of 17a (300 mg, 0.57 mmol) in acetic acid (15
mL) was added potassium cyanate (231 mg, 2.84 mmol), and the resulting mixture was
allowed to stir for 5 h at room temperature. The crude reaction was concentrated under
vacuum with azeotropic removal of acetic acid with toluene, and the residue was diluted with
saturated aq. NaHCO
3
(20 mL). The aqueous mixture was extracted with ethyl acetate (3 x 20
mL) and the combined organic extracts were dried over sodium sulfate and concentrated
under vacuum. The residue was purified by silica gel column chromatography (CHCl :MeOH
3
1
=
35:1) to give 18a (234 mg, 76.3% yield): H NMR (CDCl3, 400 MHz) δ 9.42 (1H, s), 9.29
(
1H, s), 8.81 (1H, d, J = 6.0 Hz), 8.58 (1H, d, J = 6.0 Hz), 8.53 (1H, d, J = 6.0 Hz), 8.30ꢀ8.27
2H, m), 8.00 (1H, d, J = 8.4 Hz), 7.81 (1H, d, J = 6.0 Hz), 7.67 (1H, t, J = 8.0 Hz), 7.56 (1H,
(
t, J = 8.0 Hz), 7.36 (1H, d, J = 7.2 Hz), 6.96 (2H, d, J = 8.8 Hz), 6.80 (2H, d, J = 8.4 Hz),
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5.48 (1H, d, J = 14.8 Hz), 4.37 (1H, d, J = 15.2 Hz), 3.78 (1H, t, J = 4.8 Hz), 3.05 (2H, d, J =
13
4
.8 Hz); C NMR (CDCl
3
) δ 195.8, 175.3, 172.3, 166.6, 162.9, 160.9, 149.8, 148.4, 147.8,
1
45.3, 144.3, 143.5, 142.08, 141.6, 139.0, 138.0, 133.6, 127.6, 125.9, 56.4, 33.6, 23.8;
S): calcd. 539.1389, found 539.1391; Purity 99.3%.
±)-4ꢀ((3ꢀ((1ꢀBenzylpiperidinꢀ4ꢀyl)methyl)ꢀ2,5ꢀdioxoimidazolidinꢀ4ꢀyl)methyl)phenyl
10
11
12
13
14
15
16
17
18
19
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56
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58
59
60
23 4 5
HRMS (FAB) [M + H]+ (C29H N O
(
isoquinolineꢀ5ꢀsulfonate (18b). It was synthesized from 17e following the procedure
1
described for 18a: Yield 74.7%; H NMR (CDCl
3
, 400 MHz) δ 9.39 (1H, s), 8.79 (1H, d, J =
6.0 Hz), 8.52 (1H, d, J = 5.6 Hz), 8.27 (1H, d, J = 8.4 Hz), 8.24 (1H, d, J = 7.2 Hz), 7.65 (1H,
t, J = 8.0 Hz), 7.32ꢀ7.22 (5H, m), 6.98 (2H, d, J = 8.4 Hz), 6.78 (2H, d, J = 8.8 Hz), 4.20 (1H,
t, J = 4.4 Hz), 3.57ꢀ3.50 (3H, m), 3.06 (2H, d, J = 4.4 Hz), 2.88 (2H, d, J = 10.4 Hz), 2.73
13
(1H, dd, J = 13.6, 6.0 Hz), 1.93ꢀ1.91 (4H, m), 1.50ꢀ1.48 (2H, m), 1.27ꢀ1.24 (2H, m);
C
NMR (CDCl ) δ 195.9, 175.1, 172.3, 166.6, 163.0, 153.1, 149.7, 149.5, 147.8, 145.3, 144.1,
3
1
1
9
43.5, 142.2, 141.6, 141.0, 139.6, 138.0, 133.5, 127.6, 59.5, 57.7, 46.8, 38.3, 23.8, 23.2, 18.1,
7.7; HRMS (FAB) [M + H]+ (C32
5.1%.
33 4 5
H N O S): calcd. 585.2172, found 585.2174; Purity
(±)-tertꢀButyl 4ꢀ((5ꢀ(4ꢀ(isoquinolinꢀ5ꢀylsulfonyloxy)benzyl)ꢀ2,4ꢀdioxoimidazolidinꢀ1ꢀ
yl)methyl)piperidineꢀ1ꢀcarboxylate (18c). It was synthesized from 17c following the
1
procedure described for 18a: Yield 82.1%; H NMR (CDCl
3
, 400 MHz) δ 9.41 (1H, s), 8.80
(1H, d, J = 6.0 Hz), 8.53 (1H, d, J = 6.8 Hz), 8.29 (1H, d, J = 8.4 Hz), 8.26 (1H, d, J = 7.6
Hz), 7.66 (1H, t, J = 8.0 Hz), 7.04 (5H, d, J = 8.4 Hz), 6.80 (2H, d, J = 8.4 Hz), 4.20ꢀ4.18 (2H,
m), 4.09ꢀ4.06 (2H, m), 3.56 (1H, dd, J = 14.4, 9.2 Hz), 3.08ꢀ3.06 (2H, d, J = 4.8 Hz), 2.75
(1H, dd, J = 14.0, 5.6 Hz), 2.64ꢀ2.59 (2H, m), 1.88ꢀ1.64 (2H, m), 1.42 (9H, s), 1.13 (2H, qd, J
13
= 12.4, 4.4 Hz); C NMR (CDCl ) δ 172.1, 155.7, 154.6, 153.2, 148.6, 145.7, 135.2, 135.0,
3
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133.6, 131.6, 130.7, 130.1, 128.6, 125.8, 122.2, 117.5, 79.6, 61.4, 45.9, 34.7, 34.0, 29.4, 28.4;
HRMS (FAB) [M + H]+ (C H N O S): calcd. 595.2226, found 595.2223; Purity 97.0%.
3
0
35
4
7
(±)-tertꢀButyl 4ꢀ(2ꢀ(5ꢀ(4ꢀ(isoquinolinꢀ5ꢀylsulfonyloxy)benzyl)ꢀ2,4ꢀdioxoimidazolidinꢀ1ꢀ
0
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9
0
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0
yl)ethyl)piperidineꢀ1ꢀcarboxylate (18d). It was synthesized from 17d following the
1
procedure described for 18a: Yield 75.8%; H NMR (CDCl
3
, 400 MHz) δ 9.41 (1H, s), 8.80
(1H, d, J = 6.0 Hz), 8.52 (1H, d, J = 6.0 Hz), 8.29 (1H, d, J = 8.4 Hz), 8.26 (1H, d, J = 7.6
Hz), 7.67 (2H, t, J = 7.6 Hz), 7.00 (2H, d, J = 8.4 Hz), 6.80 (2H, d, J = 8.4 Hz), 4.20ꢀ4.17 (1H,
m), 4.13ꢀ4.01 (2H, m), 3.74ꢀ3.69 (1H, m) 3.06ꢀ3.04 (2H, m), 2.90ꢀ2.83 (1H, m), 2.66 ꢀ2.58
1
3
(
2H, m), 1.66ꢀ1.54 (4H, m), 1.43 (9H, s), 1.26 (1H, t, J = 7.2 Hz), 1.12ꢀ0.98 (2H, m); C
NMR (CDCl ) δ 195.9, 175.0, 174.2, 172.3, 166.5, 163.0, 146.8, 149.5, 147.9, 145.3, 144.1,
43.6, 141.6, 138.0, 133.5, 127.6, 80.0, 56.4, 28.4, 23.4, 23.0, 22.6, 20.8, 20.2, 16.3; HRMS
(FAB) [M + H]+ (C31 S): calcd. 609.2383, found 609.2381; Purity 99.4%.
±)-tertꢀButyl 4ꢀ(5ꢀ(4ꢀ(isoquinolinꢀ5ꢀylsulfonyloxy)benzyl)ꢀ2,4ꢀdioxoimidazolidinꢀ1ꢀ
3
1
37 4 7
H N O
(
yl)piperidineꢀ1ꢀcarboxylate (18e). It was synthesized from 17b following the procedure
1
described for 18a: Yield 80.9%; H NMR (CDCl
3
, 400 MHz) δ 9.41 (1H, s), 8.80 (1H, d, J =
6.0 Hz), 8.51 (1H, d, J =6.0 Hz), 8.29 (1H, d, J = 8.4 Hz), 8.26 (1H, d, J = 7.6 Hz), 7.66 (1H,
t, J = 7.6 Hz), 7.00 (2H, d, J = 8.4 Hz), 6.79 (2H, d, J = 8.4 Hz), 4.19 (1H, t, J = 4.4 Hz), 3.74
(1H, m), 3.05 (2H, m), 2.89 (1H, m), 2.62 (2H, m), 1.43 (9H, s), 1.26 (2H, t, J = 7.2 Hz), 1.10
1
3
(
2H, m); C NMR (CDCl
43.8, 141.7, 137.9, 132.9, 127.7, 80.1, 55.2, 46.9, 45.5, 30.0, 20.0, 16.3; HRMS (FAB) [M +
S): calcd. 581.2070, found 581.2072; Purity 97.0%.
4ꢀ((3ꢀ(isoquinolinꢀ5ꢀylmethyl)ꢀ4ꢀ(4ꢀ(isoquinolinꢀ5ꢀ
3
) δ 199.7, 174.1, 172.4, 165.9, 163.1, 152.0, 149.6, 149.4, 145.4,
1
33 4 7
H]+ (C29H N O
(±)-tertꢀButyl
ylsulfonyloxy)benzyl)ꢀ2,5ꢀdioxoimidazolidinꢀ1ꢀyl)methyl)piperidineꢀ1ꢀcarboxylate (19a).
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To a solution of 18a (100 mg, 0.19 mmol) in dried DMF in the presence of CsHCO (103 mg,
0
.74 mmol) was added 1ꢀbocꢀ4ꢀpiperidinylmethyl methanesulfonate (109 mg, 0.37 mmol).
The mixture was stirred overnight at 50 °C, and then concentrated under vacuum. The residue
was taken up in solution of saturated aq. NH Cl and extracted with ethyl acetate. The
combined extracts were dried over sodium sulfate, filtered, and evaporated. The residue was
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purified by silica gel column chromatography (CHCl :MeOH = 75:1) to give 19a (96.6 mg,
1
7
0.7% yield): H NMR (CDCl
3
, 400 MHz) δ 9.43 (1H, s), 9.29 (1H, s), 8.83 (1H, d, J = 6.0
Hz), 8.55 (2H, t, J = 6.4 Hz), 8.31ꢀ8.28 (2H, m), 8.00 (1H, d, J = 8.4 Hz), 7.80 (1H, d, J = 6.0
Hz), 7.67 (1H, t, J = 8.0 Hz), 7.56 (1H, t, J = 8.0 Hz), 7.37 (1H, d, J = 6.8 Hz), 6.96 (2H, d, J
=
8.8 Hz), 6.82 (2H, d, J = 8.4 Hz), 5.57 (1H, d, J = 15.2 Hz), 4.41 (1H, d, J = 15.2 Hz), 3.98ꢀ
3
.96 (2H, m), 3.68 (1H, t, J = 4.8 Hz), 3.25 (1H, dd, J = 14.0, 7.6 Hz), 3.20 (1H, dd, J = 14.0,
.6 Hz), 3.08 (1H, dd, J = 14.8, 4.8 Hz), 3.03 (1H, dd, J = 14.8, 4.8 Hz), 2.55 (2H, q, J = 13.2
7
1
3
Hz), 1.44 (9H, s), 1.30ꢀ0.91 (3H, m); C NMR (CDCl
3
) δ 195.1, 176.1, 174.0, 172.5, 166.7,
63.3, 161.1, 149.8, 149.2, 148.3, 147.6, 145.4, 144.6, 143.8, 143.3, 142.1, 139.0, 137.9,
33.4, 127.5, 125.8, 80.1, 54.5, 35.9, 34.0, 24.2, 23.4, 17.4, 16.3; HRMS (FAB) [M + H]+
1
1
(
C H N O S): calcd. 736.2805, found 736.2807; Purity 98.1%.
4
0
42
5
7
(±)-4ꢀ((3ꢀ((1ꢀBenzylpiperidinꢀ4ꢀyl)methyl)ꢀ1ꢀ(isoquinolinꢀ5ꢀylmethyl)ꢀ2,5ꢀ
dioxoimidazolidinꢀ4ꢀyl)methyl)phenyl isoquinolineꢀ5ꢀsulfonate (19b). It was synthesized
from 18e and isoquinolinꢀ5ꢀylmethyl methanesulfonate following the procedure described for
1
1
9a: Yield 59.1%; H NMR (CDCl
3
, 400 MHz) δ 10.32 (NH, s), 9.41 (1H, s), 9.20 (1H, s),
8.85 (1H, d, J = 6.0 Hz), 8.50 (2H, d, J = 6.0 Hz), 8.28 (1H, d, J = 8.4 Hz), 8.16 (1H, d, J =
.2 Hz), 7.97 (1H, d, J = 6.0 Hz), 7.87 (1H, d, J = 7.6 Hz), 7.62 (1H, t, J = 8.0 Hz), 7.52 (2H,
7
m), 7.44 (5H, m), 6.71 (2H, d, J = 8.0 Hz), 6.35 (2H, d, J = 8.4 Hz), 4.83 (2H, qd, J = 14.8
Hz), 4.20 (1H, t, J = 4.0 Hz), 3.69 (4H, m), 3.01 (4H, m), 1.97 (2H, m), 1.76 (4H, m), 1.25
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(
2H, m); C NMR (CDCl
3
) δ 195.9, 192.2, 185.9, 177.8, 176.4, 174.8, 172.5, 163.3, 153.6,
1
1
51.7, 149.6, 149.2, 146.8, 145.7, 144.9, 144.0, 143.4, 142.2, 141.1, 138.9, 137.9, 137.2,
36.4, 132.9, 129.2, 60.6, 57.4, 50.6, 48.0, 46.5, 42.0, 37.8, 34.1; HRMS (FAB) [M + H]+
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(C42
H
40
N
5
O
5
S): calcd. 726.2750, found 726.2753; Purity 97.4%.
(
±)-tertꢀButyl 4ꢀ((3ꢀ(isoquinolinꢀ5ꢀylmethyl)ꢀ5ꢀ(4ꢀ(isoquinolinꢀ5ꢀ
ylsulfonyloxy)benzyl)ꢀ2,4ꢀdioxoimidazolidinꢀ1ꢀyl)methyl)piperidineꢀ1ꢀcarboxylate (19c).
It was synthesized from 18c and isoquinolinꢀ5ꢀylmethyl methanesulfonate following the
1
procedure described for 19a: Yield 68.3%; H NMR (CDCl
3
, 400 MHz) δ 9.42 (1H, s), 9.02
(
1H, s), 8.87 (1H, d, J = 6.0 Hz), 8.52 (2H, d, J = 6.0 Hz), 8.27 (1H, d, J = 8.4 Hz), 8.16 (1H,
d, J = 7.6 Hz), 7.99 (1H, d, J = 5.6 Hz), 7.87 (1H, d, J = 7.6 Hz), 7.62 (1H, t, J = 8.0 Hz),
.44 (2H, m), 6.72 (2H, d, J = 8.4 Hz), 6.36 (2H, d, J = 8.4 Hz), 4.91 (2H, qd, J = 14.8 Hz),
4.15 (2H, m), 3.63 (1H, m), 3.00 (2H, m), 2.81 (1H, d, J = 5.6 Hz), 2.62 (2H, m), 1.70 (4H,
7
13
m), 1.43 (9H, s), 1.15 (2H, qd, J = 12.4 Hz); C NMR (CDCl ) δ 195.0, 175.7, 174.0, 172.4,
3
1
1
72.1, 166.1, 163.2, 160.1, 149.6, 149.2, 148.0, 146.7, 145.8, 145.2, 143.9, 143.6, 143.3,
41.6, 141.2, 138.9, 137.9, 132.9, 127.6, 126.3, 80.3, 55.8, 38.5, 29.7, 24.4, 22.7, 18.0, 17.6,
16.2; HRMS (FAB) [M + H]+ (C40
5.9%.
±)-4ꢀ((2,5ꢀDioxoꢀ3ꢀ(piperidinꢀ4ꢀyl)imidazolidinꢀ4ꢀyl)methyl)phenyl isoquinolineꢀ5ꢀ
42 5 7
H N O S): calcd. 736.2805, found 736.2803; Purity
9
(
sulfonate (20a). To a 0.08 M solution of 18e (1g, 1.72 mmol) in dichloromethane at 0 °C was
added TFA (5 mL), and the resulting mixture was allowed to stir for 1 h at 0 °C. The crude
reaction was concentrated under vacuum with azeotropic removal of TFA with hexane, and
3
the residue was diluted with saturated aq. NaHCO (50 mL). The aqueous mixture was
extracted with dichloromethane (3 x 50 mL), and the combined organic extracts were dried
over sodium sulfate and concentrated under vacuum. The residue was purified by flash
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column chromatography (silica gel, CHCl
3
:MeOH:ammonia = 95:4.5:0.5) to give 20a (705
1
mg, 85.2% yield): H NMR (DMSOꢀd
6
, 400 MHz) δ 9.56 (1H, s), 8.82 (1H, d, J = 6.0 Hz),
8.59 (1H, d, J = 8.0 Hz), 8.36 (1H, d, J = 6.0 Hz), 8.25 (1H, d, J = 7.6 Hz), 7.80 (1H, t, J =
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8.0 Hz), 7.08 (2H, d, J = 7.6 Hz), 6.79 (2H, d, J = 7.2 Hz), 4.34ꢀ4.32 (1H, m), 3.13 (1H, s),
3.07ꢀ3.00 (1H, m), 2.98 (2H, dd, J = 15.2, 4.8 Hz), 2.91ꢀ2.81 (2H, m), 2.26ꢀ2.14 (2H, m),
13
1
.86ꢀ1.77 (1H, q, J = 12.0 Hz), 1.59ꢀ1.55 (2H, m), 1.37ꢀ1.34 (1H, m); C NMR (CDCl
3
)
δ 173.6, 156.3, 153.9, 147.6, 145.9, 136.2, 135.3, 131.3, 130.6, 128.7, 128.4, 126.5, 121.4,
116.5, 72.3, 60.2, 29.0, 22.1, 19.4; HRMS (FAB) [M + H]+ (C24
found 481.1543; Purity 95.9%.
25 4 5
H N O S): calcd. 481.1546,
(±)-4ꢀ((2,5ꢀDioxoꢀ3ꢀ(piperidinꢀ4ꢀylmethyl)imidazolidinꢀ4ꢀyl)methyl)phenyl
isoquinolineꢀ5ꢀsulfonate (20b). It was synthesized from 18c following the procedure
1
described for 20a: Yield 83.7%; H NMR (DMSOꢀd
6
, 400 MHz) δ 9.56 (1H, s), 8.82 (1H, d,
J = 6.0 Hz), 8.60 (1H, d, J = 8.0 Hz), 8.35 (1H, d, J = 6.0 Hz), 8.26 (1H, d, J = 7.6 Hz), 7.81
1H, t, J = 8.0 Hz), 7.06 (2H, d, J = 7.6 Hz), 6.79 (1H, d, J = 7.2Hz), 4.19ꢀ4.16 (1H, m), 3.56ꢀ
(
3.49 (1H, m), 3.07ꢀ3.00 (3H, m), 2.74 (1H, dd, J = 14.8, 5.2 Hz), 2.55ꢀ2.48 (2H, m), 1.97ꢀ
+
1.94 (1H, m), 1.53ꢀ1.38 (4H, m), 1.18ꢀ1.07 (2H, m); MS (ESI): [M] = 494.7.
(±)-4ꢀ((2,5ꢀDioxoꢀ3ꢀ(2ꢀ(piperidinꢀ4ꢀyl)ethyl)imidazolidinꢀ4ꢀyl)methyl)phenyl
isoquinolineꢀ5ꢀsulfonate (20c). It was synthesized from 18d following the procedure
1
described for 20a: Yield 82.5%; H NMR (DMSOꢀd
6
, 400 MHz) δ 9.41 (1H, s), 8.80 (1H, d,
J = 6.0 Hz), 8.52 (1H, d, J = 6.0 Hz), 8.29 (1H, d, J = 8.4 Hz), 8.26 (1H, d, J = 7.1 Hz), 7.67
2H, t, J = 7.6 Hz), 7.00 (2H, d, J = 8.4 Hz), 6.80 (2H, d, J = 8.4 Hz), 4.195(1H, t, J = 4.4 Hz),
(
4.13 (2H, m), 3.74 (1H, m) 3.05 (2H, s), 2.90 (1H, m), 2.62 (2H, m), 1.66 (4H, m), 1.43 (9H,
+
s), 1.31 (1H, t, J = 7.2 Hz), 1.10 (2H, m); MS (ESI): [M] = 508.8.
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