Design and synthesis of new bicyclic diketopiperazines as scaffolds for receptor probes of structurally diverse functionality

Article abstract of DOI:10.1039/b416349d

Diketopiperazines (DKPs) are a common motif in various biologically active natural products, and hence they may be useful scaffolds for the rational design of receptor probes and therapeutic agents. We constructed a new bicyclic scaffold that combines a D

Full text of DOI:10.1039/b416349d

View Article Online / Journal Homepage / Table of Contents for this issue
A R T I C L E
Design and synthesis of new bicyclic diketopiperazines as scaffolds
for receptor probes of structurally diverse functionality†
Pedro Besada,a Liaman Mamedova,a Craig J. Thomas,b Stefano Costanzia,b and
Kenneth A. Jacobson*a,b
a Molecular Recognition Section, Laboratory of Bioorganic Chemistry, NIDDK, National
Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892-0810,
USA. E-mail: kajacobs@helix.nih.gov; Fax: (301) 480-8422; Tel: (301) 496-9024
b Chemical Biology Core Facility, NIDDK, National Institutes of Health, Department of Health
and Human Services, Bethesda, MD 20892-0810, USA
Received (Pittsburgh, PA, USA) 26th October 2004, Accepted 31st March 2005
First published as an Advance Article on the web 21st April 2005
Diketopiperazines (DKPs) are a common motif in various biologically active natural products, and hence they may
be useful scaffolds for the rational design of receptor probes and therapeutic agents. We constructed a new bicyclic
scaffold that combines a DKP bridged with a 10-membered ring. In this way we obtained a three-dimensional
molecular skeleton, with several amendable sites that provide a starting point to design a new combinatorial library
having diverse substituent groups. Structural variation is based upon the flexibility of alkylation of the nitrogen
atoms of the DKP and on the side-chain olefin. We obtained a 10-membered secondary ring through a ring-closure
metathesis reaction using the second generation Grubbs catalyst. Rings containing both O-ethers and S-ethers were
compared. N-Alkyl or arylalkyl groups were introduced optionally at the two Na-atoms. This is a general scheme
that will allow us to test rings of varying sizes, linkages, and stereochemical parameters. The DKP derivatives were
tested for activity in astrocytoma cells expressing receptors coupled to phospholipase C. Inhibitory effects were
observed for signaling elicited by activation of human nucleotide P2Y receptors but not m3 muscarinic receptors.
Compound 20 selectively inhibited calcium mobilization (IC50 value of 486 16 nM) and phosphoinositide turnover
elicited by a selective P2Y1 receptor agonist, but this compound did not compete for binding of a radiolabeled
nucleotide-competitive receptor antagonist. Therefore, the new class of DKP derivatives shows utility as
pharmacological tools for P2Y receptors.
closure metathesis (RCM) reaction using the second generation
Introduction
Grubbs catalyst.16,17 This methodology provides a bicyclic DKP
The diketopiperazine (DKP) ring has been noted as a motif in
template with ether and olefinic groups present. This is a general
various biologically active natural products. Examples include
scheme that will allow us to test larger and smaller rings, vary
the commercial antibiotic bicyclomycin,1 the fungal metabolites
the ether linkage and manipulate stereochemical parameters.
TAN-14 A, C and E,2 and the thaxtomin A and B.3 DKPs may
be useful scaffolds for the rational design of receptor probes
and drugs. Owing to the presence of multiple H-bond acceptors
and donors and the side chains of the component amino acids,
The ether and olefinic functionality is desired for both chemical
advantages (i.e. the ability to derivatize at these positions) and
biological advantages (i.e. to provide additional recognition
elements for interaction with receptors and other biopolymers).
DKPs have multiple sites for structural elaboration of defined
Selected bicyclic DKP analogues were subjected to biological
stereochemistry. Others have explored the synthesis of DKPs
assays in a variety of receptor-based systems. Several of the
of varied structure,4–8 and combinatorial/polymer-supported
derivatives were found to inhibit the activity of extracellular
nucleotides through G protein-coupled P2Y receptors and
syntheses of DKPs have been reported.9–12
One of the limitations of conventional medicinal chemical
subsequent biochemical steps. Eight mammalian subtypes of
agents, as applied to the new domain of chemical genetics,13,14
such receptors (P2Y1,2,4,6,11–14) are currently sequence-defined.18–21
is the typical planarity found in most known pharmaceutical
The P2Y receptors are present in the cardiovascular, immune,
substances discovered through organic synthesis. We desired to
and nervous systems, as well as in other systems. They are
explore a three-dimensional molecular skeleton, which could
currently the focus of intense drug discovery efforts to design
be regarded as a starting point to design a new combinatorial
selective agonists and antagonists, which are lacking for most
library having diverse substituent groups. In the present study
of the subtypes. There are numerous therapeutic possibilities
we have built a new bicyclic scaffold that combines a DKP addi-
under exploration for P2Y receptor modulators. For example,
tionally bridged with a 10-membered ring. A similar approach
antagonists of the P2Y1 and P2Y12 receptors are of interest as
to synthesize a series of bridged DKPs, which were desired as
antithrombotic agents.22–32 Agonists of the P2Y2 receptor are
mimics of a peptide b-turn found in peptide hormones, was
of interest in the treatment of pulmonary diseases, including
introduced by Reitz and co-workers.15
cystic fibrosis. The P2Y6 receptor has recently been implicated
Construction of the DKP skeleton was achieved with amino
acid derivatives N-Boc-L-serine and L-allylglycine, thus provid-
in protection against apoptosis induced by TNFa.33
ing great flexibility for structural variation, including substitu-
tion at the nitrogen atoms of the DKP and on the side-chain
olefin. The 10-membered ring was obtained through a ring-
Results
Chemical synthesis
A new bicyclic scaffold that combines a DKP bridged with a
10-membered ring was prepared. This 10-membered secondary
ring was obtained through a RCM reaction using the second
† Electronic supplementary information (ESI) available: coordinates of
two representative conformations, and molecular model of compound 20
(in PDB format). See http://www.rsc.org/suppdata/ob/b4/b416349d/
2 0 1 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 0 1 6 – 2 0 2 5
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

View Article Online
generation Grubbs catalyst. The starting materials in this
synthesis (Schemes 1–3) were two amino acid derivatives of
defined stereochemistry, N-Boc-L-serine and L-allylglycine,
which provided the bicyclic DKP template with both ether
and olefinic groups present. In order to substitute the O-ether
moiety with an S-ether, L-cysteine 6 was used as the starting
material instead of N-Boc-L-serine 1.
The first step of the synthesis was the introduction of the allyl
group in the N-Boc-L-serine and L-cysteine using the methods
reported in the literature.34,35 Esterification of compound 2 with
TMSCHN2 followed by removal of the Boc group with TFA
afforded the amino ester 5. The intermediate 8 was obtained
after esterification of the amino acid 7 (Scheme 1).
The amino methyl ester 5 was subjected to reductive ami-
nation in the presence of 2,4-dimethoxybenzaldehyde or 2,4,6-
trimethoxybenzaldehyde to give compounds 13 and 14, respec-
tively. Following the same procedure, the thio derivatives 15
and 16 were obtained from 8 (Scheme 1). Our initial procedure,
in order to minimize racemization, involved mixing the amine,
NaBH(OAc)3 and the aldehyde simultaneously in CH2Cl2.15,36,37
The reactions carried at room temperature for 2 h afforded the
desired compounds but with racemization that was partial (70 :
30) for the dimethoxybenzyl derivatives 13 and 15 and total
(50 : 50) for the trimethoxybenzyl derivatives 14 and 16. An
alternative route consisted of the reductive amination followed
by esterification. The amino acid derivative 4 was subjected
to reductive amination using NaBH3CN in methanol at room
temperature for 24 h to obtain 9, which after esterification with
TMSCHN2 provided 13 with less than 5% of racemization.
Following the same procedure, 14–16 were obtained from 4 or 7
with a similar degree of racemization.
Fmoc-L-allylglycine (17) was coupled with the secondary
amine 13 using O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-
uronium hexafluorophosphate (HATU), 1-hydroxy-7-azabenzo-
triazole (HOAt) and N-ethylmorpholine (NEM) in a mixture
of CH2Cl2–DMF (9 : 1) to obtain the dipeptide 18 (Scheme 2).
RCM of 18 (1 mM) using the second generation Grubbs catalyst
in dichloromethane at room temperature provided 19. When the
first generation Grubbs catalyst was used with 18 no reaction
was observed. Finally, removal of the Fmoc group of 19 using
a mixture of piperidine–CH2Cl2 (1 : 4) was accompanied by in
situ formation of the DKP, and compound 20 was obtained in
high yield (93%).
Similar reaction sequences starting from compounds 14, 15
and 16, instead of 13, provided the bicyclic DKP templates 25,
33 and 36.
In order to introduce more diversity in the scaffold, the amide
in the DKP was alkylated with methyl and benzyl groups.38
Compounds 20 and 25 were treated with NaH and the resulting
anions reacted with methyl iodide and benzyl bromide to give
21, 22, 27 and 28.
The trimethoxybenzyl group in 25, 27 and 28 was removed
easily under acidic conditions; however, the normally acid-
labile dimethoxybenzyl group in 20 was stable under these
conditions.36
By this route the new bicyclic scaffold was obtained through
RCM followed by the formation of the DKP ring (Scheme 2).
The possibility was explored to obtain these derivatives through
Scheme 1 Preparation of protected Ser and Cys derivatives. Reagents and conditions: (i) NaH, allylbromide, DMF, rt, 94%; (ii) TMSCHN2,
Et2O–MeOH 1 : 1, rt, 78%; (iii) TFA, CH2Cl2, rt, 93% (5); (iv) EtONa, allylbromide, EtOH, rt, 76%; (v) TMSCHN2, Et2O–CH2Cl2–MeOH 1 : 5 : 5,
rt, 33% (13), 32% (14), 55% (15), 22% (16); (vi) 2,4-dimethoxybenzaldehyde or 2,4,6-trimethoxybenzaldehyde, NaBH3CN, MeOH, rt, 53% (9), 53%
(10), 41% (11), 41% (12); (vii) 2,4-dimethoxybenzaldehyde or 2,4,6-trimethoxybenzaldehyde, NaBH(OAc)3, CH2Cl2, rt, 85% (13), 82% (14), 74% (15),
58% (16), *partial racemization was obtained in this reaction.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 0 1 6 – 2 0 2 5
2 0 1 7

View Article Online
Scheme 2 Synthesis of bicyclic DKP derivatives. The RCM reaction preceded the closure of the DKP ring. Reagents and conditions: (i) HATU,
HOAt, NEM, CH2Cl2–DMF 9 : 1, rt, 53% (18), 40% (23), 37% (31), 41% (34); (ii) second generation Grubbs catalyst, CH2Cl2, rt, 60% (19), 57%
(24), 57% (32), 71% (35); (iii) piperidine–CH2Cl2 1 : 4, rt, 93% (20), 73% (25), 73% (33), 60% (36); (iv) CH2Cl2–TFA 2 : 1, rt, 96%; (v) MeI or BnBr,
NaH, THF, rt, 63% (21), 60% (22), 96% (27), 85% (28); (vi) CH2Cl2–TFA 2 : 1, rt, 63% (29), 89% (30).
a new route, by which the DKP ring was built first and
then the 10-membered ring was formed (Scheme 3). In this
case, compound 18 reacted with the mixture of piperidine–
CH2Cl2 (1 : 4) to obtain the DKP 37. The RCM reaction of
37 using first generation Grubbs catalyst was not satisfactory.
When the second generation catalyst was used and the reac-
tion was performed in conditions of high dilution (0.2 mM),
20 (21%) was obtained with the dimer 38 (43%). However,
with higher concentrations (40 mM) only the dimer 38 was
obtained (79%).
MeSADP)-induced phospholipase C (PLC) was observed only
for 20 (45 10%), 33 (33 4%), and 36 (18 4%). At the
other P2Y receptor subtypes, substantial inhibition of PLC
activity (ca. 50% at 10 lM) was observed only for compounds
21 and 36 at the P2Y4 receptor and compound 20 at the P2Y6
receptors. Compound 20 and other DKPs (Fig. 1) inhibited
the mobilization of intracellular [Ca2+] in astrocytoma cells
stably expressing the human P2Y1 receptor, with concentration-
dependent reduction of the maximal effect. Compound 20 was
the most potent with complete inhibition of [Ca2+] mobilization
at 10 lM. Increasing concentrations of compound 20 progres-
sively attenuated the 2MeSADP effect (Fig. 2). Compound 20
was found to inhibit [Ca2+]i transients elicited by 2MeSADP with
an IC50 value of 486 16 nM. Control experiments in which PLC
was stimulated via an endogenous m3 muscarinic receptor (using
carbachol) in the control astrocytoma cells or via heterologously
expressed P2Y2,4,6 receptors (using the appropriate nucleotide)
demonstrated insignificant or weak inhibition by compound
20. Thus, the inhibitory effects of 20, albeit insurmountable,
Pharmacological activity
Compounds 20–22, 25–30, 33, and 36 were screened at a
10 lM concentration in an assay of phospholipase C in
1321N1 human astrocytoma cells stably expressing the human
P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors. The biological
characterization of the dimeric 38 will be reported elsewhere. In
P2Y1 receptor-expressing cells, inhibition of agonist (30 nM 2-
Scheme 3 Synthesis of bicyclic DKP derivatives. The closure of the DKP ring preceded the RCM reaction. DMB = 2,4-(MeO)2PhCH2. Reagents
and conditions: (i) piperidine–CH2Cl2 1 : 4, rt, 42%; (ii) 37 (0.2 mM), second generation Grubbs catalyst, CH2Cl2, rt, 21% (20), 43% (38); 37 (40 mM),
79% (38).
2 0 1 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 0 1 6 – 2 0 2 5

View Article Online
Discussion
These DKP scaffold molecules were intended for broad screen-
ing with no a priori bias for any particular protein target.
Among the targets thus far examined, inhibition was associated
exclusively with the P2Y receptors. These receptors are diverse
in the nature of their endogenous nucleotide ligands. Adenine
nucleotides are required for activation of the P2Y1, P2Y11, P2Y12,
and P2Y13 subtypes, and uracil nucleotides activate P2Y2, P2Y4,
and P2Y6 subtypes. The P2Y2 receptor is equipotently activated
by UTP and ATP. The most recently identified subtypes are
the P2Y13 receptor, which is present in human monocytes and
lymphocytes,42 and the P2Y14 receptor, which is activated by
UDP-glucose.43 P2Y receptor ligands are being investigated for
therapeutic applications in the cardiovascular, endocrine, and
other systems. Exploration of selective agonists and antagonists
modulators is most advanced at the P2Y1 and P2Y12 receptors,
but at most of the P2Y receptors selective pharmacological
probes are lacking.22–32 Compound 20 was shown to antagonize
signaling of the P2Y1 receptor selectively and with moderate
potency. At the P2Y1 receptor, the previously described high
affinity antagonists are nucleotide derivatives and therefore
highly charged, which is highly limiting in pharmacological
studies due to low bioavailability and stability.
Fig. 1 Effects of DKP derivatives (10 lM, structures in Scheme 2) on
concentration–response curves for intracellular Ca2+ changes induced by
2-MeSADP acting at P2Y1 receptors expressed in 1321N1 astrocytoma
cells. The cells were pre-treated for 20 min at room temperature with
antagonist before application of the agonist 2-MeSADP.
Therefore, owing to the ability to modify the chemical
functionality of this new class of DKP derivatives and the
already demonstrated biological activity, it shows promise as
an approach to designing new pharmacological tools. Further
study will be required to explore the mechanism of inhibition
of P2Y receptor-induced effects and to determine the overall
pharmacological selectivity of this class of compounds. It must
still be determined if manipulation of functional groups on the
DKP derivatives is able to vary the spectrum of interaction of
these scaffold molecules with signaling of specific P2Y receptor
subtypes, and among varied biochemical target proteins in
general. If the inhibition by the DKP is found to occur in direct
association with P2Y receptors, e.g. at an allosteric site, several
of the compounds already prepared, such as compound 20, will
provide insurmountable antagonists with receptor selectivity.
Fig. 2 Effect of compound 20 on the concentration–response curve of
the [Ca2+]i transient induced by 2MeSADP in P2Y1 receptors expressed
in 1321N1 astrocytoma cells. The cells were pre-treated for 20 min at
room temperature with different concentrations of compound 20 before
application of the agonist 2-MeSADP.
Experimental
were selective for signaling induced by P2Y1 receptor activation.
However, in a radioreceptor binding assay compound 20 at
10 lM failed to compete for the binding of a specific P2Y1
antagonist radioligand, [3H]MRS2279.39 Therefore, the binding
of the DKP was not occurring at the principal nucleotide binding
site of this receptor.
Chemical synthesis
Reagents and solvents were purchased from Sigma-Aldrich
(St. Louis, MO). Compounds 2 and 7 were synthesized as
reported.34,35
As an indication of the selectivity of these derivatives for the
P2Y receptor pathway, interactions with a limited number of
other receptors were examined (data not shown). Binding experi-
ments at the human A1 AR expressed in CHO cell membranes40
indicated that compound 20 at 10 lM displayed no significant
inhibition of radioligand binding. Similarly, this compound
failed to displace binding at the mouse TRH receptors, or
to either activate or antagonize functional effects of the m3
muscarinic receptor or the human calcium sensing receptor.
By means of a molecular dynamics simulation we carried out
a conformational analysis of compound 20. The DKP ring-
1H NMR spectra were obtained with a Varian Gemini 300
spectrometer using CDCl3, CD3OD or D2O as solvents. The
chemical shifts are expressed as ppm downfield from TMS. All
melting points were determined with a Thomas-Hoover appa-
ratus (A.H. Thomas Co.) and are uncorrected.
Purity of the compounds was checked using a Hewlett-
Packard 1100 HPLC equipped with a Luna 5 lm RP-C18(2)
analytical column (250 × 4.6 mm; Phenomenex, Torrance, CA).
System A: linear gradient solvent system: H2O–CH3CN from
80 : 20 to 0 : 100 in 20 min, then isocratic for 5 min; the flow rate
was 1 mL min−1. System B: linear gradient solvent system: 5 mM
TBAP–CH3CN from 80 : 20 to 20 : 80 in 20 min, then isocratic for
2 min; the flow rate was 1 mL min−1. Peaks were detected by UV
absorption with a diode array detector. All derivatives tested for
biological activity showed >98% purity in the HPLC systems.
TLC analysis was carried out on aluminium sheets precoated
with silica gel F254 (0.2 mm) from Aldrich. Low-resolution CI–
NH3 (chemical ionization) mass spectra were measured with a
Finnigan 4600 mass spectrometer, and low-resolution FAB (fast
atom bombardment) mass spectrometry was performed with a
JEOL SX102 spectrometer with 6 kV Xe atoms following des-
orption from a glycerol matrix or on LC/MS 1100 Agilent, 1100
MSD, with a Waters Atlantis C18 column. High-resolution mass
measurements were performed on a Micromass/Waters LCT
◦
◦
◦
∼
∼
∼
=
puckering coordinates (H −80 ; P −30 ; Q 40 ) indicated
=
=
2
a boat–screw/boat conformation.
Two different stable conformations were found for the 10-
membered ring: in the first conformation the ring bends toward
the dimethoxybenzyl group, while in the second one it bends
in the opposite direction. Two diametrically opposite stable
conformations were also found for the dimethoxybenzyl group.
The coordinates of two representative conformations, in PDB
format, are supplied as ESI.†
Consistent with the non-competitive nature of these ligands,
the stable conformations of compound 20 do not resemble
the bound conformation of the competitive P2Y1 antagonist
MRS2279, neither by steric nor electronic criteria.41
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 0 1 6 – 2 0 2 5
2 0 1 9

View Article Online
Premier Electrospray Time of Flight (TOF) mass spectometer
coupled with a Waters HPLC system.
1H-3), 3.54 (m, 1H, H-2). HRMS m/z found 326.1588 (M +
H)+. C16H24NO6 requires 326.1604.
(2S)-3-Allyloxy-2-tert-butoxycarbonylamino-propionic acid
methyl ester (3). To a solution of compound 2 (4.48 g,
18.3 mmol) in 130 mL of Et2O–MeOH (1 : 1) was added
TMSCHN2 (2 M in Et2O) dropwise until a yellow tint persisted.
The reaction mixture was stirred at room temperature for
30 min. The solvent was removed in vacuo, and the residue
was purified by silica gel column chromatography (petroleum
(2R)-3-Allylsulfanyl-2-(2,4-dimethoxybenzylamino)-propionic
acid (11). (Procedure A) Compound 11 (150 mg, 41%) as a white
solid was obtained from 7 (206 mg, 1.28 mmol), NaBH3CN
(100 mg, 1.51 mmol) and 2,4-dimethoxybenzaldehyde (197 mg,
1.16 mmol) in MeOH (40 mL). 1H NMR (CDCl3) d 7.30 (d, J =
8.7 Hz, 1H, Ar–H), 6.81 (br s, 1H, NH), 6.48 (m, 2H, Ar–H),
=
=
5.64 (m, 1H, CH CH2), 5.05 (m, 2H, CH CH2), 4.45 (d, J =
13.2 Hz, 1H, HCHAr), 4.18 (d, J = 13.2 Hz, 1H, HCHAr),
3.87 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.41 (m, 1H, H-2), 3.16
1
ether–ethyl acetate, 15 : 1) to afford 3 (3.71 g, 78%). H NMR
=
(CDCl3) d 5.83 (m, 1H, CH CH2), 5.38 (br d, J = 8.8 Hz, 1H,
=
NH), 5.21 (m, 2H, CH CH2), 4.43 (m, 1H, H-2), 3.98 (m, 2H,
=
=
(m, 1H, HCHCH CH2), 2.92 (m, 3H, HCHCH CH2, H-3).
HRMS m/z found 312.1265 (M + H)+. C15H22NO4S requires
312.1270. Mp 150–151 ◦C.
=
CH2CH CH2), 3.85 (m, 1H, 1H-3), 3.77 (s, 3H, OCH3), 3.65 (m,
1H, 1H-3), 1.46 [s, 9H, C(CH3)3]; MS (FAB) m/z 260 (M + H)+.
(2S)-3-Allyloxy-2-amino-propionic acid (4). To a solution of
compound 2 (242 mg, 0.99 mmol) in CH2Cl2 (15 mL) was
added TFA (1 mL). The reaction mixture was stirred at room
temperature for 2 h. The solvent was removed in vacuo to obtain
compound 4 as a crude solid (520 mg), which then was used
directly in the next step without further purification. 1H NMR
=
=
(D2O) d 5.96 (m, 1H, CH CH2), 5.33 (m, 2H, CH CH2), 4.11
(m, 2H, CH2CH CH2), 3.93 (m, 3H, H-2, H-3).
=
(2S)-3-Allyloxy-2-amino-propionic acid methyl ester (5). To
a solution of compound 3 (1.02 g, 3.93 mmol) in CH2Cl2 (60 mL)
was added TFA (10 mL). The reaction mixture was stirred at
room temperature for 30 min, and then was neutralized with
the addition of saturated NaHCO3 (225 mL). The product was
extracted with CH2Cl2, dried over Na2SO4, and evaporated to
1
obtain compound 5 (585 mg, 93%). H NMR (CDCl3) d 5.87
=
=
(m, 1H, CH CH2), 5.22 (m, 2H, CH CH2), 4.01 (m, 2H,
CH2CH CH2), 3.75 (s, 3H, OCH3), 3.68 (m, 3H, H-2, H-3).
=
(2R)-3-Allylsulfanyl-2-amino-propionic acid methyl ester (8).
To a suspension of compound 7 (1.21 g, 7.49 mmol) in 55 mL
of Et2O–MeOH–CH2Cl2 (1:5:5), was added TMSCHN2 (2 M
in Et2O) dropwise until a yellow tint persisted. The reaction
mixture was stirred at room temperature for 2 h. The solvent was
removed in vacuo to obtain compound 8 as a crude solid (1.46 g),
which then was used directly in the next step without further
1
=
purification. H NMR (CDCl3) d 5.78 (m, 1H, CH CH2), 5.12
=
(m, 2H, CH CH2), 3.75 (s, 3H, OCH3), 3.64 (m, 1H, H-2), 3.15
=
(m, 2H, CH2CH CH2), 2.88 (m, 1H, 1H-3), 2.71 (m, 1H, 1H-3);
MS (CI) m/z 176 (M + H)+.
(2S)-3-Allyloxy-2-(2,4-dimethoxybenzylamino)-propionic acid
(9). (Procedure A) To a solution containing the crude solid
4 (260 mg) in MeOH (20 mL) was added NaBH3CN
(38 mg, 0.58 mmol) and 2,4-dimethoxybenzaldehyde (75 mg,
0.45 mmol). The reaction mixture was stirred at room temper-
ature for 24 h. Water (3 mL) was added, and the product was
extracted with CH2Cl2 and dried over Na2SO4. After removal of
the solvent, the residue was purified by column chromatography
on silica gel (CH2Cl2–MeOH, 92 : 8) to give 9 (77 mg, 53% from
1
2) as a white solid. H NMR (CDCl3) d 7.25 (d, J = 8.7 Hz,
=
1H, Ar–H), 6.47 (m, 2H, Ar–H), 5.83 (m, 1H, CH CH2), 5.19
(m, 2H, CH CH2), 4.76 (br s, 1H, NH), 4.39 (d, J = 13.0 Hz,
=
1H, HCHAr), 4.24 (d, J = 13.0 Hz, 1H, HCHAr), 4.01–3.69
=
(m, 4H, CH2CH CH2, H-3), 3.85 (s, 3H, OCH3), 3.80 (s, 3H,
OCH3), 3.56 (m, 1H, H-2). HRMS m/z found 296.1501 (M +
H)+. C15H22NO5 requires 296.1498. Mp 135–136 ◦C.
(2S)-3-Allyloxy-2-(2,4,6-trimethoxybenzylamino)-propionic
acid (10). (Procedure A) Compound 10 (84 mg, 53% from
2) was obtained from a solution of crude solid 4 (260 mg),
NaBH3CN (38 mg, 0.58 mmol) and 2,4,6-trimethoxybenz-
1
aldehyde (89 mg, 0.45 mmol) in MeOH (20 mL). H NMR
=
(CDCl3) d 6.11 (s, 2H, Ar–H), 5.79 (m, 1H, CH CH2), 5.54
(br s, 1H, NH), 5.17 (m, 2H, CH CH2), 4.49 (d, J = 13.2 Hz,
=
1H, HCHAr), 4.40 (d, J = 13.2 Hz, 1H, HCHAr), 3.98–3.74
=
(m, 3H, CH2CH CH2, 1H-3), 3.82 (s, 9H, OCH3), 3.68 (m, 1H,
2 0 2 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 0 1 6 – 2 0 2 5

View Article Online
0.42 mmol) and TMSCHN2 (2 M in Et2O) in Et2O–MeOH (1 : 1)
(10 mL). The product was purified by column chromatography
on silica gel (petroleum ether–ethyl acetate, 4 : 1). H NMR
added second generation Grubbs catalyst (35 mg, 0.04 mmol).
The reaction mixture was stirred at room temperature for 48 h.
The solvent was removed in vacuo, and the residue was purified
by column chromatography on silica gel (petroleum ether–ethyl
1
(CDCl3) d 7.12 (d, J = 8.7 Hz, 1H, Ar–H), 6.42 (m, 2H, Ar–H),
1
=
=
5.73 (m, 1H, CH CH2), 5.06 (m, 2H, CH CH2), 3.81 (s, 3H,
OCH3), 3.79 (s, 3H, OCH3), 3.76 (m, 2H, CH2Ar), 3.69 (s, 3H,
acetate, 2 : 1) to afford 19 (40 mg, 60%). H NMR (CDCl3)
d 7.78 (d, J = 7.2 Hz, 2H, Ar–H), 7.63 (d, J = 7.2 Hz, 2H,
Ar–H), 7.41 (t, J = 7.2 Hz, 2H, Ar–H), 7.32 (t, J = 7.1 Hz, 2H,
Ar–H), 7.08 (d, J = 8.1 Hz, 1H, Ar–H), 6.40 (m, 2H, Ar–H),
6.07 (d, J = 7.5 Hz, 1H, NH), 5.83 (m, 2H, H-8, H-9), 5.25 (m,
1H, H-6), 4.94 (d, J = 15.0 Hz, 1H, HCHAr), 4.51–4.34 (m,
3H, CHCH2OCO, 1H-10), 4.29–4.08 (m, 3H, CHCH2OCO,
H-2), 3.90 (m, 2H, HCHAr, 1H-10), 3.79 (s, 3H, OCH3), 3.75
(m, 1H, H-3), 3.69 (s, 3H, OCH3), 3.43 (s, 3H, OCH3), 2.68 (m,
1H, 1H-7), 2.53 (m, 1H, 1H-7); MS (FAB) m/z 601 (M + H)+.
(1S,8S)-11-(2,4-Dimethoxybenzyl)-3-oxa-9,11-diazabicyclo-
[6.2.2]dodec-5-ene-10,12-dione (20). (Procedure F) Compound
19 (24 mg, 0.04 mmol) was stirred at room temperature in 5 mL
of CH2Cl2–piperidine (4 : 1) for 2 h. After the solvent was
removed, the resulting residue was purified by silica gel column
chromatography (CH2Cl2–MeOH, 96 : 4) to obtain 20 (13 mg,
93%) as a white solid. 1H NMR (CDCl3) d 7.24 (m, 1H, Ar–H),
6.45 (m, 2H, Ar–H), 5.93 (br s, 1H, NH), 5.62 (m, 2H, H-5,
H-6), 4.98 (d, J = 14.3 Hz, 1H, HCHAr), 4.25 (m, 3H, H-4,
H-8), 4.14 (d, J = 14.3 Hz, 1H, HCHAr), 3.89 (m, 3H, H-1,
H-2), 3.79 (s, 6H, OCH3), 3.11 (m, 1H, 1H-7), 2.83 (m, 1H,
1H-7); 13C NMR (CDCl3) d 169.5, 168.1, 161.0, 158.9, 132.0,
131.4, 125.9, 116.4, 104.9, 98.6, 73.2, 71.5, 61.5, 55.6, 53.8,
41.6, 34.0; HRMS m/z found 347.1591 (M + H)+. C18H23N2O5
requires 347.1607; HPLC (System A) 8.1 min (99%), (System
B) 9.1 min (99%). Mp 155–156 ◦C.
=
OCH3), 3.41 (m, 1H, H-2), 3.05 (m, 2H, CH2CH CH2), 2.74
(m, 2H, H-3); MS (CI) m/z 326 (M + H)+.
(2R)-3-Allylsulfanyl-2-(2,4,6-trimethoxybenzylamino)-propionic
acid methyl ester (16). (Procedure B) Compound 16 (697 mg,
58% from 7) was obtained from a solution containing crude
solid 8 (731 mg), NaBH(OAc)3 (988 mg, 4.43 mmol) and
2,4,6-trimethoxybenzaldehyde (682 mg, 3.41 mmol) in CH2Cl2
(65 mL). The product was purified by column chromatography
on silica gel (petroleum ether–ethyl acetate, 2 : 1). (Procedure
C) Compound 16 (35 mg, 22%) was obtained from 12 (154 mg,
0.45 mmol) and TMSCHN2 (2M in Et2O) in Et2O–MeOH (1 : 1)
(12 mL). The product was purified by column chromatography
1
on silica gel (petroleum ether–ethyl acetate, 4 : 1). H NMR
=
(CDCl3) d 6.10 (s, 2H, Ar–H), 5.72 (m, 1H, CH CH2), 5.05 (m,
2H, CH CH2), 3.81 (m, 2H, CH2Ar), 3.80 (s, 3H, OCH3), 3.79
=
(s, 6H, OCH3), 3.65 (s, 3H, OCH3), 3.37 (m, 1H, H-2), 3.03 (m,
=
2H, CH2CH CH2), 2.72 (m, 2H, H-3), 2.42 (br s, 1H, NH);
MS (CI) m/z 356 (M + H)+.
(2S)-(9H-Fluoren-9-ylmethoxycarbonylamino)-pent-4-enoic
acid (17). A solution of Fmoc-Cl (900 mg, 3.38 mmol)
in dioxane (5 mL) was added, at 0 ◦C, to a suspension of
L-allylglycine (299 mg, 2.60 mmol) and NaHCO3 (655 mg,
7.79 mmol) in 16.8 mL H2O–dioxane (1.4 : 1). The mixture
was stirred for 15 min at 0 ◦C and then at room temperature
for 4.5 h. The pH was adjusted to approximately 9 with the
addition of solid NaHCO3 and the mixture was diluted with
H2O (120 mL) and washed with Et2O. The aqueous layer was
acidified to pH = 3 with an aqueous HCl solution (6 M). The
product was extracted with EtOAc, dried over Na2SO4 and
the solvent was removed under reduced pressure. The resulting
residue was purified by column chromatography on silica gel
(CH2Cl2–MeOH, 97 : 3) to afford 17 (615 mg, 70%). 1H NMR
(CDCl3) d 7.75 (d, J = 7.5 Hz, 2H, Ar–H), 7.57 (d, J = 6.9 Hz,
2H, Ar–H), 7.39 (t, J = 7.4 Hz, 2H, Ar–H), 7.29 (t, J = 7.4 Hz,
2H, Ar–H), 5.71 (m, 1H, H-4), 5.34 (br d, J = 7.8 Hz, 1H, NH),
5.17 (m, 2H, H-5), 4.47 (m, 1H, H-2), 4.39 (d, J = 6.9 Hz, 2H,
CHCH2), 4.21 (t, J = 6.9 Hz, 1H, CHCH2), 2.58 (m, 2H, H-3);
MS (FAB) m/z 338 (M + H)+.
(1S,8S)-11-(2,4-Dimethoxybenzyl)-9-methyl-3-oxa-9,11-diaza-
bicyclo[6.2.2]dodec-5-ene-10,12-dione (21). (Procedure G) A
solution of compound 20 (10 mg, 0.03 mmol) in THF (1 mL)
was added, at 0 ◦C, to a suspension of NaH (3.5 mg, 0.09 mmol,
60% dispersion in mineral oil) in THF (1 mL). After stirring at
room temperature for 30 min, the reaction mixture was cooled
to 0 ◦C and CH3I (18 lL, 0.29 mmol) was added. The reaction
mixture was stirred at room temperature for 5 h, followed by
quenching with H2O. The product was extracted with CH2Cl2,
dried over Na2SO4, and the solvent was removed under reduced
pressure. The resulting residue was purified by preparative
thin-layer chromatography (CH2Cl2–MeOH, 96 : 4) to obtain
1
21 (6.6 mg, 63%). H NMR (CDCl3) d 7.25 (m, 1H, Ar–H),
6.44 (m, 2H, Ar–H), 5.67 (m, 1H, H-6), 5.55 (m, 1H, H-5),
4.95 (d, J = 14.4 Hz, 1H, HCHAr), 4.21 (m, 2H, H-4), 4.16 (d,
J = 14.4 Hz, 1H, HCHAr), 4.15 (m, 1H, H-8), 3.90 (m, 3H,
H-1, H-2), 3.79 (s, 6H, OCH3), 3.07 (m, 1H, 1H-7), 2.91 (s, 3H,
NCH3), 2.88 (m, 1H, 1H-7); 13C NMR (CDCl3) d 168.1, 167.3,
161.0, 158.9, 132.1, 130.8, 125.7, 116.5, 104.9, 98.6, 72.8, 71.4,
61.7, 61.1, 55.6, 41.3, 33.9, 32.9; HRMS m/z found 377.1777
(M + H)+. C19H25N2O5 requires 361.1763; HPLC (System A)
9.1 min (98%), (System B) 10.4 min (98%).
(2S,2ꢀS)-3-Allyloxy-2-{(2,4-dimethoxybenzyl)-[2ꢀ-(9H-fluoren-
9-ylmethoxycarbonylamino)-pent-4ꢀ-enoyl]-amino}-propionic acid
methyl ester (18). (Procedure D) A solution containing
compound 17 (638 mg, 1.89 mmol), HATU (742 mg,
1.89 mmol), HOAt (3.79 mL, 1.89 mmol, solution 0.5–0.7 M in
DMF) and NEM (0.49 mL, 3.79 mmol) in CH2Cl2 (34.2 mL)
was added to a solution of compound 13 (117 mg, 0.38 mmol)
in 15.5 mL CH2Cl2–DMF (9 : 1). The reaction mixture was
stirred at room temperature overnight. The solvent was removed
in vacuo, and the residue was purified by silica gel column
chromatography (petroleum ether–ethyl acetate, 2 : 1) to afford
18 (126 mg, 53%). 1H NMR (CDCl3) d 7.77 (d, J = 7.5 Hz, 2H,
Ar–H), 7.61 (d, J = 7.2 Hz, 2H, Ar–H), 7.40 (t, J = 7.4 Hz, 2H,
Ar–H), 7.31 (t, J = 7.4 Hz, 2H, Ar–H), 7.17 (d, J = 7.8 Hz,
(1S,8S)-9-Benzyl-11-(2,4-dimethoxybenzyl)-3-oxa-9,11-diaza-
bicyclo[6.2.2]dodec-5-ene-10,12-dione (22). (Procedure G)
Compound 22 (7.5 mg, 60%) was obtained as a white solid
from 20 (10 mg, 0.03 mmol), NaH (4.2 mg, 0.10 mmol, 60%
dispersion in mineral oil) and BnBr (54 lL, 0.45 mmol) in
THF (2 mL) after for 8 h stirring at room temperature. The
product was purified by preparative thin-layer chromatography
=
1H, Ar–H), 6.43 (m, 2H, Ar–H), 5.74 (m, 2H, CH CH2),
5.14 (m, 4H, CH CH2), 4.80 (m, 1H, HCHAr), 4.53–4.02 (m,
=
1
(CH2Cl2–MeOH, 97 : 3). H NMR (CDCl3) d 7.29 (m, 3H,
=
6H, HCHAr, CHCH2OCO, CHCH2OCO, CHCH2CH CH2,
Ar–H), 7.19 (m, 3H, Ar–H), 6.44 (m, 2H, Ar–H), 5.68 (m, 1H,
H-6), 5.55 (m, 1H, H-5), 5.29 (d, J = 14.7 Hz, 1H, HCHPh),
4.96 (d, J = 14.7 Hz, 1H, HCHAr), 4.20 (m, 2H, H-4), 4.16
(m, 2H, H-8, HCHAr), 3.99 (m, 2H, H-1, 1H-2), 3.80 (s, 3H,
OCH3), 3.79 (m, 2H, 1H-2, HCHPh), 3.77 (s, 3H, OCH3), 2.89
(m, 2H, H-7); 13C NMR (CDCl3) d 167.9, 167.4, 161.1, 158.9,
135.7, 131.8, 130.5, 129.1, 128.4, 128.1, 126.3, 116.4, 104.9,
98.7, 72.8, 71.5, 61.7, 57.8, 55.6 (2C), 48.7, 41.7, 32.9; HRMS
=
H-2), 3.88 (m, 2H, OCH2CH CH2), 3.84–3.55 (m, 2H, H-3),
3.79 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 3.59 (s, 3H, OCH3),
2.48 (m, 2H, CH2CH CH2); MS (FAB) m/z 629 (M + H) .
+
=
(3S,6S)-4-(2,4-Dimethoxybenzyl)-6-(9H-fluoren-9-ylmethoxy-
carbonylamino)-5-oxo-3,4,5,6,7,10-hexahydro-2H-[1,4]oxazecine-
3-carboxylic acid methyl ester (19). (Procedure E) To a solution
of compound 18 (70 mg, 0.11 mmol) in CH2Cl2 (500 mL) was
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 0 1 6 – 2 0 2 5
2 0 2 1

View Article Online
m/z found 437.2095 (M + H)+. C25H29N2O5 requires 437.2076;
HPLC (System A) 13.4 min (98%), (System B) 16.2 min (98%).
Mp 129–130 ◦C.
7 h of stirring at room temperature. The product was purified
by preparative thin-layer chromatography (CH2Cl2–MeOH,
96 : 4). H NMR (CDCl3) d 6.08 (s, 2H, Ar–H), 5.68 (m, 1H,
1
H-6), 5.57 (m, 1H, H-5), 5.23 (d, J = 13.8 Hz, 1H, HCHAr),
4.24 (m, 2H, H-4), 4.15 (m, 1H, H-8), 4.10 (d, J = 13.8 Hz,
1H, HCHAr), 3.96 (m, 1H, 1H-2), 3.80 (s, 3H, OCH3), 3.78
(m, 1H, 1H-2), 3.77 (s, 6H, OCH3), 3.69 (m, 1H, H-1), 3.08
(m, 1H, 1H-7), 2.89 (s, 3H, NCH3), 2.86 (m, 1H, 1H-7); 13C
NMR (CDCl3) d 167.7, 167.6, 161.8, 160.3, 130.7, 126.0, 103.2,
90.7, 72.5, 71.2, 61.1, 60.0, 55.9, 55.6, 35.2, 33.8, 32.9; HRMS
m/z found 391.1867 (M + H)+. C20H27N2O6 requires 391.1869;
HPLC (System A) 9.1 min (98%), (System B) 10.6 min (98%).
Mp 158–159 ◦C.
(2S,2ꢀS)-3-Allyloxy-2-[[2ꢀ-(9H-fluoren-9-ylmethoxycarbonyl-
amino)-pent-4ꢀ-enoyl]-(2,4,6-trimethoxybenzyl)-amino]-propionic
acid methyl ester (23). (Procedure D) Compound 23 (278 mg,
40%) was obtained from 14 (357 mg, 1.05 mmol) in 45 mL
CH2Cl2–DMF (9 : 1), and 17 (1.77 g, 5.26 mmol), HATU
(2.06 g, 5.26 mmol), HOAt (10.5 mL, 5.26 mmol, solution
0.5–0.7 M in DMF) and NEM (1.35 mL, 10.5 mmol), in
1
CH2Cl2 (99 mL) and DMF (0.47 mL). H NMR (CDCl3) d
7.76 (d, J = 7.5 Hz, 2H, Ar–H), 7.61 (d, J = 7.5 Hz, 2H,
Ar–H), 7.40 (t, J = 7.2 Hz, 2H, Ar–H), 7.30 (t, J = 7.2 Hz,
=
(1S,8S)-9-Benzyl-11-(2,4,6-trimethoxybenzyl)-3-oxa-9,11-
diazabicyclo[6.2.2]dodec-5-ene-10,12-dione (28). (Procedure G)
Compound 28 (10.6 mg, 85%) was obtained from 25 (10 mg,
0.03 mmol), NaH (4 mg, 0.1 mmol, 60% dispersion in mineral
oil) and BnBr (64 lL, 0.53 mmol) in THF (2 mL) after 24 h
of stirring at room temperature. The product was purified by
preparative thin-layer chromatography (CH2Cl2–MeOH, 97 : 3).
1H NMR (CDCl3) d 7.28 (m, 3H, Ar–H), 7.16 (m, 2H, Ar–H),
6.10 (s, 2H, Ar–H), 5.70 (m, 1H, H-6), 5.60 (m, 1H, H-5), 5.33
(d, J = 15.3 Hz, 1H, HCHPh), 5.20 (d, J = 14.0 Hz, 1H,
HCHAr), 4.27 (m, 2H, H-4), 4.15 (m, 1H, H-8), 4.13 (d, J =
14.0 Hz, 1H, HCHAr), 3.89 (m, 2H, H-2), 3.82 (s, 3H, OCH3),
3.75 (s, 6H, OCH3), 3.73 (m, 1H, H-1), 3.70 (d, J = 15.3 Hz, 1H,
HCHPh), 2.98 (m, 1H, 1H-7), 2.86 (m, 1H, 1H-7); 13C NMR
(CDCl3) d 167.9, 167.7, 161.8, 160.3, 135.8, 130.6, 128.9, 128.1,
128.0, 126.5, 103.1, 90.7, 72.4, 71.1, 59.9, 57.7, 55.9, 55.6, 48.4,
35.3, 32.8; HRMS m/z found 467.2208 (M + H)+. C26H31N2O6
requires 467.2182; HPLC (System A) 13.3 min (98%), (System
B) 16.3 min (98%).
2H, Ar–H), 6.10 (s, 2H, Ar–H), 5.84 (m, 2H, CH CH2), 5.15
(m, 4H, CH CH2), 4.61 (m, 2H, CH2Ar), 4.49–4.07 (m, 5H,
=
=
CHCH2OCO, CHCH2OCO, CHCH2CH CH2, H-2), 3.98 (m,
2H, OCH2CH CH2), 3.81 (s, 3H, OCH3), 3.79 (s, 3H, OCH3),
3.78 (s, 3H, OCH3), 3.70 (m, 2H, H-3), 3.50 (s, 3H, OCH3), 2.58
=
=
=
(m, 1H, HCHCH CH2), 2.44 (m, 1H, HCHCH CH2); MS
(API-ES) m/z 681 (M + Na)+.
(3S,6S)-6-(9H-Fluoren-9-ylmethoxycarbonylamino)-5-oxo-4-
(2,4,6-trimethoxybenzyl)-3,4,5,6,7,10-hexahydro-2H-[1,4]oxa-
zecine-3-carboxylic acid methyl ester (24). (Procedure E)
Compound 24 (5.5 mg, 57%) was obtained from 23 (10 mg,
0.015 mmol) and second generation Grubbs catalyst (4 mg,
0.005 mmol) in CH2Cl2 (60 mL). The product was purified by
preparative thin-layer chromatography (petroleum ether–ethyl
1
acetate, 2 : 1). H NMR (CDCl3) d 7.78 (d, J = 7.5 Hz, 2H,
Ar–H), 7.63 (d, J = 7.5 Hz, 2H, Ar–H), 7.41 (t, J = 7.4 Hz,
2H, Ar–H), 7.32 (t, J = 7.4 Hz, 2H, Ar–H), 6.08 (m, 3H, Ar–H,
NH), 5.83 (m, 2H, H-8, H-9), 5.33 (m, 1H, H-6), 4.78–4.37
(m, 5H, CH2Ar, CHCH2OCO, 1H-10), 4.29–4.08 (m, 3H,
CHCH2OCO, H-2), 3.92 (m, 2H, H-3, 1H-10), 3.80 (s, 3H,
OCH3), 3.75 (s, 3H, OCH3), 3.74 (s, 3H, OCH3), 3.40 (s, 3H,
OCH3), 2.70 (m, 1H, 1H-7), 2.49 (m, 1H, 1H-7); MS (API-ES)
m/z 653 (M + Na)+.
(1S,8S)-9-Methyl-3-oxa-9,11-diazabicyclo[6.2.2]dodec-5-ene-
10,12-dione (29). (Procedure H) Compound 29 (1.7 mg, 63%)
was obtained from 27 (5 mg, 0.01 mmol) and TFA (0.35 mL) in
CH2Cl2 (0.7 mL). 1H NMR (CDCl3) d 6.12 (br s, 1H, NH), 5.62
(m, 2H, H-5, H-6), 4.32 (m, 2H, H-4), 4.07 (m, 2H, 1H-2, H-8),
3.96 (m, 1H, H-1), 3.73 (m, 1H, 1H-2), 3.14 (m, 1H, 1H-7), 2.95
(s, 3H, NCH3), 2.88 (m, 1H, 1H-7); HRMS m/z found 211.1086
(M + H)+. C10H15N2O3 requires 211.1083.
(1S,8S)-11-(2,4,6-Trimethoxybenzyl)-3-oxa-9,11-diazabicyclo-
[6.2.2]dodec-5-ene-10,12-dione (25). (Procedure F) Compound
25 (35 mg, 73%) was obtained as a white solid from 24 (80 mg,
0.13 mmol) and 15 mL of CH2Cl2–piperidine (4 : 1). 1H NMR
(CDCl3) d 6.17 (br s, 1H, NH), 6.09 (s, 2H, Ar–H), 5.63 (m, 2H,
H-5, H-6), 5.26 (d, J = 14.1 Hz, 1H, HCHAr), 4.29 (m, 2H,
H-4), 4.24 (m, 1H, H-8), 4.10 (d, J = 14.1 Hz, 1H, HCHAr),
4.04 (m, 1H, 1H-2), 3.81 (s, 3H, OCH3), 3.77 (s, 6H, OCH3),
3.74 (m, 1H, 1H-2), 3.64 (m, 1H, H-1), 3.11 (m, 1H, 1H-7),
2.81 (m, 1H, 1H-7); 13C NMR (CDCl3) d 169.9, 167.7, 161.8,
160.3, 131.5, 126.0, 103.2, 90.7, 73.0, 71.4, 59.7, 55.9, 55.6, 53.8,
35.5, 34.1; HRMS m/z found 377.1705 (M + H)+. C19H25N2O6
requires 377.1713; HPLC (System A) 8.3 min (99%), (System
B) 9.3 min (99%). Mp 123–124 ◦C.
(1S,8S)-9-Benzyl-3-oxa-9,11-diazabicyclo[6.2.2]dodec-5-ene-
10,12-dione (30). (Procedure H) Compound 30 (2.7 mg, 89%)
was obtained from 28 (5 mg, 0.01 mmol) and TFA (0.35 mL)
in CH2Cl2 (0.7 mL). The product was purified by preparative
1
thin-layer chromatography (CH2Cl2–MeOH, 96 : 4). H NMR
(CDCl3) d 7.31 (m, 3H, Ar–H), 7.25 (m, 2H, Ar–H), 6.22 (br s,
1H, NH), 5.64 (m, 2H, H-5, H-6), 5.38 (d, J = 15.0 Hz, 1H,
HCHPh), 4.35 (m, 2H, H-4), 4.11 (m, 2H, 1H-2, H-8), 4.04 (m,
1H, H-1), 3.73 (d, J = 15.0 Hz, 1H, HCHPh), 3.70 (m, 1H,
1H-2), 3.01 (m, 1H, 1H-7), 2.86 (m, 1H, 1H-7); HRMS m/z
found 287.1391 (M + H)+. C16H19N2O3 requires 287.1396.
(1S,8S)-3-Oxa-9,11-diazabicyclo[6.2.2]dodec-5-ene-10,12-
dione (26). (Procedure H) To a solution of compound 25
(10 mg, 0.03 mmol) in CH2Cl2 (1.5 mL) was added TFA
(0.75 mL). After stirring at room temperature for 7 h, the
solvent was removed under reduced pressure. The residue was
purified by preparative thin-layer chromatography (CH2Cl2–
(2R,2ꢀS)-3-Allylsulfanyl-2-{(2,4-dimethoxybenzyl)-[2ꢀ -(9H-
fluoren-9 - ylmethoxycarbonylamino ) - pent - 4ꢀ - enoyl ] - amino } -
propionic acid methyl ester (31). (Procedure D) Compound
31 (107 mg, 37%) was obtained from 15 (146 mg, 0.45 mmol)
in 20 mL CH2Cl2–DMF (9 : 1), and 17 (755 mg, 2.24 mmol),
HATU (878 mg, 2.24 mmol), HOAt (4.48 mL, 2.24 mmol,
solution 0.5–0.7 M in DMF) and NEM (0.58 mL, 4.48 mmol)
in CH2Cl2 (40.3 mL). The product was purified by column
chromatography on silica gel (petroleum ether–ethyl acetate, 3 :
1). 1H NMR (CDCl3) d 7.77 (d, J = 7.2 Hz, 2H, Ar–H), 7.61 (d,
J = 7.2 Hz, 2H, Ar–H), 7.40 (t, J = 7.4 Hz, 2H, Ar–H), 7.31
(t, J = 7.4 Hz, 2H, Ar–H), 7.16 (d, J = 8.7 Hz, 1H, Ar–H),
1
MeOH, 94 : 6) to afford 26 (5 mg, 96%) as a white solid. H
NMR (CD3OD) d 5.59 (m, 2H, H-5, H-6), 4.33 (m, 2H, H-4),
4.06 (m, 1H, H-8), 3.95 (m, 1H, 1H-2), 3.77 (m, 2H, H-1, 1H-2),
3.04 (m, 1H, 1H-7), 2.77 (m, 1H, 1H-7); 13C NMR (CD3OD) d
173.0, 171.1, 132.5, 126.2, 76.0, 74.9, 59.1, 54.4, 35.0; HRMS
m/z found 197.0932 (M + H)+. C9H13N2O3 requires 197.0926.
(1S,8S)-9-Methyl-11-(2,4,6-trimethoxybenzyl)-3-oxa-9,11-
diazabicyclo[6.2.2]dodec-5-ene-10,12-dione (27). (Procedure
G) Compound 27 (10 mg, 96%) was obtained from 25 (10 mg,
0.03 mmol), NaH (3.5 mg, 0.09 mmol, 60% dispersion in
mineral oil) and CH3I (18 lL, 0.29 mmol) in THF (2 mL) after
=
6.43 (m, 2H, Ar–H), 5.75 (m, 2H, CH CH2), 5.11 (m, 4H,
=
CH CH2), 4.78 (d, J = 15.6 Hz, 1H, HCHAr), 4.49–4.18 (m,
=
5H, HCHAr, CHCH2OCO, CHCH2OCO, CHCH2CH CH2),
4.07 (m, 1H, H-2), 3.78 (s, 3H, OCH3), 3.76 (s, 3H, OCH3),
=
3.55 (s, 3H, OCH3), 3.10 (m, 3H, SCH2CH CH2, 1H-3), 2.94
2 0 2 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 0 1 6 – 2 0 2 5

View Article Online
=
(m, 1H, 1H-3), 2.61 (m, 1H, HCHCH CH2), 2.46 (m, 1H,
HCHCH CH2); MS (API-ES) m/z 667 (M + Na) .
(1R,8S)-11-(2,4,6-Trimethoxybenzyl)-3-thia-9,11-diazabicyclo-
[6.2.2]dodec-5-ene-10,12-dione (36). (Procedure F) Compound
36 (9 mg, 60%) was obtained from 35 (25 mg, 0.04 mmol) and
3.75 mL of CH2Cl2–piperidine (4 : 1). The product was purified
by preparative thin-layer chromatography (CH2Cl2–MeOH,
+
=
(3R,6S)-4-(2,4-Dimethoxybenzyl)-6-(9H-fluoren-9-ylmethoxy-
carbonylamino)-5-oxo-3,4,5,6,7,10-hexahydro-2H-[1,4]thiazecine-
3-carboxylic acid methyl ester (32). (Procedure E) Compound
32 (52 mg, 57%) was obtained from 31 (95 mg, 0.15 mmol)
and second generation Grubbs catalyst (46 mg, 0.05 mmol)
in CH2Cl2 (148 mL). The product was purified by column
chromatography on silica gel (petroleum ether–ethyl acetate,
1
96 : 4). H NMR (CDCl3) d 6.09 (s, 3H, Ar–H, NH), 5.71 (m,
2H, H-5, H-6), 5.39 (d, J = 13.8 Hz, 1H, HCHAr), 4.41 (d, J =
13.8 Hz, 1H, HCHAr), 4.25 (m, 1H, H-8), 4.07 (m, 1H, H-1),
3.80 (s, 3H, OCH3), 3.78 (s, 6H, OCH3), 3.45 (m, 1H, 1H-4),
3.26–3.01 (m, 4H, H-2, 1H-4, 1H-7), 2.54 (m, 1H, 1H-7); 13C
NMR (CDCl3) d 169.6, 166.4, 161.8, 160.6, 130.7, 127.5, 103.2,
90.7, 60.1, 56.0, 55.6, 55.3, 55.2, 38.2, 32.9, 30.6, 29.5; HRMS
m/z found 393.1495 (M + H)+. C19H25N2O5S requires 393.1484;
HPLC (System A) 9.7 min (99%), (System B) 11.3 min (99%).
1
2.5 : 1). H NMR (CDCl3) d 7.77 (d, J = 7.5 Hz, 2H, Ar–H),
7.63 (d, J = 7.5 Hz, 2H, Ar–H), 7.41 (t, J = 7.4 Hz, 2H, Ar–H),
7.32 (t, J = 7.5 Hz, 2H, Ar–H), 7.12 (d, J = 7.5 Hz, 1H, Ar–H),
6.41 (m, 2H, Ar–H), 6.15 (br d, J = 6.3 Hz, 1H, NH), 5.67
(m, 2H, H-8, H-9), 5.30 (m, 1H, H-6), 5.05 (d, J = 14.4 Hz,
1H, HCHAr), 4.43 (m, 2H, CHCH2OCO), 4.29–4.07 (m, 2H,
CHCH2OCO, HCHAr), 3.79 (s, 3H, OCH3), 3.76–3.63 (m, 1H,
H-3), 3.71 (s, 3H, OCH3), 3.52–3.28 (m, 2H, H-10), 3.37 (s, 3H,
OCH3), 3.02–2.72 (m, 3H, H-2, 1H-7), 2.45 (m, 1H, 1H-7); MS
(API-ES) m/z 639 (M + Na)+.
(3S,6S)-3-Allyl-6-allyloxymethyl-1-(2,4-dimethoxybenzyl)-
piperazine-2,5-dione (37). (Procedure F) Compound 37 (52 mg,
42%) was obtained from 18 (206 mg, 0.33 mmol) and 10 mL of
CH2Cl2–piperidine (4 : 1). The product was purified by column
chromatography on silica gel (petroleum ether–ethyl acetate, 1 :
2). 1H NMR (CDCl3) d 7.24 (d, J = 8.2 Hz, 1H, Ar–H), 6.45 (m,
(1R,8S)-11-(2,4-Dimethoxybenzyl)-3-thia-9,11-diazabicyclo-
[6.2.2]dodec-5-ene-10,12-dione (33). (Procedure F) Compound
33 (19 mg, 73%) was obtained from 32 (45 mg, 0.07 mmol) and
5 mL of CH2Cl2–piperidine (4 : 1). The product was purified
by preparative thin-layer chromatography (CH2Cl2–MeOH,
=
2H, Ar–H), 6.19 (br s, 1H, NH), 5.82 (m, 2H, CH CH2), 5.22
(m, 4H, CH CH2), 5.10 (d, J = 14.7 Hz, 1H, HCHAr), 4.12
=
(d, J = 14.7 Hz, 1H, HCHAr), 4.01 (m, 1H, H-6), 3.98 (m, 2H,
=
OCH2CH CH2), 3.93 (m, 1H, H-3), 3.82 (m, 2H, CHCH2O),
=
3.79 (2s, 6H, OCH3), 2.90 (m, 1H, HCHCH CH2), 2.60 (m,
1
96 : 4). H NMR (CDCl3) d 7.26 (d, J = 8.4 Hz, 1H, Ar–H),
1H, HCHCH CH2); 13C NMR (CDCl3) d 166.9, 166.4, 161.0,
=
6.45 (m, 3H, Ar–H, NH), 5.71 (m, 2H, H-5, H-6), 5.21 (d, J =
14.6 Hz, 1H, HCHAr), 4.33 (m, 1H, H-1), 4.28 (d, J = 14.6 Hz,
1H, HCHAr), 4.24 (m, 1H, H-2), 3.79 (s, 6H, OCH3), 3.45 (m,
1H, 1H-4), 3.25–3.02 (m, 4H, H-2, 1H-4, 1H-7), 2.55 (m, 1H,
1H-7); 13C NMR (CDCl3) d 169.6, 167.0, 161.1, 159.1, 132.5,
130.7, 127.4, 116.2, 104.7, 98.7, 60.1, 56.9, 55.6, 55.2, 45.4, 32.4,
30.4, 29.3; HRMS m/z found 363.1371 (M + H)+. C18H23N2O4S
requires 363.1379; HPLC (System A) 9.8 min (99%), (System
B) 11.4 min (99%).
158.8, 134.1, 133.7, 131.9, 120.1, 118.2, 116.3, 104.8, 98.6, 72.6,
67.8, 60.1, 55.6, 55.4, 41.7, 40.6; MS (FAB) m/z 375 (M + H)+.
(1S,8S,11S,18S)-21,23-Bis-(2,4-dimethoxybenzyl)-3,13-dioxa-
9,19,21,23-tetraazatricyclo[16.2.2.28,11]tetracosa-5,15-diene-10,
20,22,24-tetraone (38). (Procedure E) To
a solution of
compound 37 (15 mg, 0.04 mmol) in CH2Cl2 (1 mL) was added
second generation Grubbs catalyst (5 mg, 0.006 mmol). The
reaction mixture was stirred at room temperature for 16 h. The
solvent was removed in vacuo, and the residue was purified by
preparative thin-layer chromatography (CHCl3–MeOH, 97 : 3)
(2R,2ꢀS)-3-Allylsulfanyl-2-[[2ꢀ -(9H-fluoren-9-ylmethoxycar-
bonylamino)-pent-4ꢀ -enoyl]-(2,4,6-trimethoxybenzyl)-amino]-
propionic acid methyl ester (34). (Procedure D) Compound
34 (124 mg, 41%) was obtained from 16 (159 mg, 0.45 mmol)
in 20 mL CH2Cl2–DMF (9 : 1), and 17 (755 mg, 2.24 mmol),
HATU (878 mg, 2.24 mmol), HOAt (4.48 mL, 2.24 mmol, solu-
tion 0.5–0.7 M in DMF) and NEM (0.58 mL, 4.48 mmol)
in CH2Cl2 (40.3 mL). The product was purified by column
chromatography on silica gel (petroleum ether–ethyl acetate,
1
to afford 38 (11 mg, 79%). H NMR (CDCl3) d 9.18 (m, 2H,
NH), 7.24 (d, J = 8.3 Hz, 2H, Ar–H), 6.45 (m, 4H, Ar–H),
5.65 (m, 4H, H-5, H-6, H-15, H-16), 5.18 (d, J = 14.5 Hz,
2H, HCHAr), 4.33 (m, 2H, 1H-4, 1H-14), 4.25 (m, 2H, 1H-2,
1H-12), 4.11 (d, J = 14.5 Hz, 2H, HCHAr), 3.99 (m, 2H, H-8,
H-18), 3.93 (m, 2H, H-1, H-11), 3.79 (s, 6H, OCH3), 3.78 (s,
6H, OCH3), 3.76 (m, 2H, 1H-2, 1H-12), 3.67 (m, 2H, 1H-4,
1H-14), 2.90 (m, 2H, 1H-7, 1H-17), 2.68 (m, 2H, 1H-7, 1H-17);
13C NMR (CDCl3) d 169.5, 166.3, 161.0, 158.8, 131.8, 130.6,
127.9, 116.2, 104.9, 98.6, 72.5, 69.8, 60.5, 55.6, 55.3, 40.8, 40.0;
HRMS m/z found 693.3149 (M + H)+. C36H45N4O10 requires
693.3136.
1
2.5 : 1). H NMR (CDCl3) d 7.76 (d, J = 7.5 Hz, 2H, Ar–H),
7.62 (m, 2H, Ar–H), 7.40 (t, J = 7.2 Hz, 2H, Ar–H), 7.30 (m,
=
2H, Ar–H), 6.10 (s, 2H, Ar–H), 5.82 (m, 2H, CH CH2), 5.13
=
(m, 4H, CH CH2), 4.72–4.18 (m, 5H, CH2Ar, CHCH2OCO,
CHCH2OCO), 3.89–3.66 (m, 2H, CHCH2CH CH2, H-2), 3.81
(s, 3H, OCH3), 3.78 (s, 6H, OCH3), 3.47 (s, 3H, OCH3), 3.15 (m,
=
=
3H, SCH2CH CH2, 1H-3), 2.98 (m, 1H, 1H-3), 2.65 (m, 1H,
HCHCH CH2), 2.48 (m, 1H, HCHCH CH2); MS (API-ES)
Pharmacological analyses
=
=
Cell culture and membrane preparation. Human 1321N1
astrocytoma cells transfected individually with the hP2Y1,2,4,6,11
receptors26,33,40 were grown at 37 ◦C in a humidified incubator
with 5% CO2–95% air in Dulbecco’s modified Eagle’s medium
(JRH Biosciences, Inc.) supplemented with 10% fetal bovine
serum (FBS), 100 Units mL−1 penicillin, 100 lg mL−1 strepto-
mycin and 2 mM L-glutamine. The cells were grown to ca. 60%
confluence for the experiments.
For membrane preparation, human astrocytoma cells express-
ing human P2Y1 receptors were grown to approximately 80%
confluence and then harvested. The cells were homogenized
and suspended and then centrifuged at 100 g for 5 min at
room temperature. The pellet was resuspended in 50 mM
tris(hydroxymethyl)aminomethane (Tris) hydrochloride buffer
(pH 7.4). The suspension was homogenized with a Polytron
homogenizer (Brinkmann) for 10 s and was then recentrifuged
at 20 000 g for 20 min at 4 ◦C. The resultant pellets were
resuspended in Tris buffer (pH 7.4), and the suspension was
m/z 697 (M + Na)+.
(3R,6S)-6-(9H-Fluoren-9-ylmethoxycarbonylamino)-5-oxo-4-
(2,4,6-trimethoxybenzyl)-3,4,5,6,7,10-hexahydro-2H-[1,4]thiaze-
cine-3-carboxylic acid methyl ester (35). (Procedure E)
Compound 35 (30 mg, 71%) was obtained from 34 (44 mg,
0.07 mmol) and second generation Grubbs catalyst (17 mg,
0.02 mmol) in CH2Cl2 (65 mL). The product was purified by
preparative thin-layer chromatography (petroleum ether–ethyl
1
acetate, 2 : 1). H NMR (CDCl3) d 7.77 (d, J = 7.2 Hz, 2H,
Ar–H), 7.63 (m, 2H, Ar–H), 7.40 (t, J = 7.4 Hz, 2H, Ar–H),
7.31 (t, J = 7.2 Hz, 2H, Ar–H), 6.16 (d, J = 6.9 Hz, 1H, NH),
6.09 (m, 2H, Ar–H), 5.65 (m, 2H, H-8, H-9), 5.36 (m, 1H, H-6),
4.70 (m, 2H, CH2Ar), 4.43 (m, 2H, CHCH2OCO), 4.24 (t, J =
7.2 Hz, 1H, CHCH2OCO), 3.84–3.62 (m, 1H, H-3), 3.80 (s, 3H,
OCH3), 3.77 (s, 6H, OCH3), 3.52–3.27 (m, 2H, H-10), 3.36 (s,
3H, OCH3), 3.05–2.65 (m, 3H, H-2, 1H-7), 2.43 (m, 1H, 1H-7);
MS (API-ES) m/z 669 (M + Na)+.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 0 1 6 – 2 0 2 5
2 0 2 3

View Article Online
stored at −80 ◦C until the binding experiments. The protein
Data analysis
concentration was measured with the Bradford assay.44
IC50 values obtained in radioligand binding assays and in assays
of inhibition of agonist-stimulated inositol phosphate accumu-
lation were calculated by a four-parameter logistic equation
and the GraphPad software package (GraphPad, San Diego,
CA). All concentration–effect curves were repeated in at least
three separate experiments, carried out in duplicate or triplicate
with different membrane preparations or different 1321N1 cell
cultures.
Storage of test substances. Agonists were dissolved as stock
solutions in Tris buffer (pH 7.4), and the DKP derivatives
were stored as frozen stock solutions in DMSO (5 mM) at
−20 ◦C. Prior to use the DMSO solutions were warmed briefly
to 50–60 ◦C.
Determination of inositol phosphates. The quantity of in-
ositol phosphates was measured essentially as reported.25,26,33,41
The P2Y1,2,4,6,11-1321N1 cells were grown to confluence in 6-well
plates in the presence of myo-[3H]inositol (2 lCi mL−1) for 24 h.
Cells were then treated for 30 min at 37 ◦C with antagonists
or buffer in the presence of 20 mM LiCl, followed by another
30 min of incubation at 37 ◦C with the appropriate agonist.
Agonists used were: P2Y1, 2-MeSADP; hP2Y2, UTP; hP2Y4,
UTP; hP2Y6, UDP; hP2Y11, ATP. The reaction was terminated
upon aspiration of the medium and addition of cold formic
acid (20 mM). After 30 min, supernatants were neutralized
with NH4OH, and applied to Bio-Rad Dowex AG 1-X8 anion
exchange columns. All of the columns were then washed with
water followed by a 60 mM sodium formate solution containing
5 mM sodium tetraborate. Total inositol phosphates were eluted
with 1 M ammonium formate containing 0.1 M formic acid, and
radioactivity values were measured using a liquid scintillation
counter.
Molecular modeling
Molecular modeling was carried out with the module Discover3
of InsightII (Accelrys, Inc.) on Silicon Graphics Origin 200
computer, using the CFF91 forcefield.45
An NVT (constant-volume/constant-temperature) molecular
dynamics simulation was carried out for 500 ns at 298 K, with
a time step of 1 fs. Conformations were sampled at regular
intervals of 100 ns and energy minimized employing the BFGS
Newton method, until an RMS gradient lower than 0.00001 kcal
−1
˚
A
mol−1
was reached.
For a description of the puckering of the DKP ring we used
the coordinates H (phase angle of symmetrical interconversion),
P2 (phase angle of pseudorotation), and Q (total puckering
amplitude) as defined by Haasnoot,46 utilizing the final formulae
that we recently reported.40
Acknowledgements
Radioligand binding assay. P2Y1 receptor binding experi-
ments were performed as previously described.39 Briefly, mem-
branes (40 lg protein) from astrocytoma cells stably expressing
human P2Y1 receptors were incubated with [3H]MRS2279 (8
nM) for 30 min at 4 ◦C in a total assay volume of 200 lL. For
adenosine A1 receptor binding, an agonist radioligand [3H]R-
PIA (2.0 nM) was incubated with membranes (40 lg protein
per tube) from CHO cells stably expressing human adenosine
Dr Pedro Besada thanks the Cystic Fibrosis Foundation for
financial support. Mass spectral measurements were carried out
by Dr Victor Livengood and NMR by Wesley White (NIDDK).
We thank Heng T. Duong and Brian Harris (NIDDK) for
proofreading the manuscript, and Dr Zhan-Guo Gao, Lanh
Bloodworth, Dr Jianxin Hu, and Dr Xinping Lu (NIDDK) for
biological assays.
◦
A1 receptors for 60 min at 25 C.40 Radiolabeled ligand
References
concentrations used in all assays approximated the Kd values
of the receptor. Binding reactions were terminated by filtration
through Whatman GF/B glass-fiber filters under reduced pres-
sure with an MT-24 cell harvester (Brandel), and radioactivity
was determined with a 1414 liquid scintillation counter (Wallac,
Win Spectral, Perkin Elmer Life Sciences).
1 R. M. Williams and C. A. Durham, Bicyclomycin: synthetic,
mechanistic, and biological studies, Chem Rev., 1988, 88, 511–540.
2 Y. Funabashi, T. Horiguchi, S. Iinuma, S. Tanida and S. Harada,
TAN-1496 A, C and E, diketopiperazine antibiotics with inhibit
activity against mammalian DNA topoisomerase I, J. Antibiot., 1994,
47, 1202–18.
3 J. Moyroud, J. Gelin, A. Chene and J. Mortier, Synthese d’analogues
structuraux de Thaxtomines, phytotoxines responsables de la gale de
la pomme de terre, Tetrahedron, 1996, 52, 8525–8534.
Calcium mobilization assay
4 A. K. Szardenings and T. S. Burkoth, A simple procedure for the
solid phase synthesis of diketopiperazine and diketomorpholine
derivatives, Tetrahedron, 1997, 53, 6573–6593.
5 J. Alsina, K. J. Jensen, F. Albericio and G. Barany, Solid-phase
synthesis with tris(alkoxy)benzyl backbone amide linkage (BAL),
Chem. Eur. J., 1999, 5, 2787–2795.
6 E. Perrotta, M. Altamura, T. Barani, S. Bindi, D. Giannotti, N. J. S.
Harmat, R. Nannicini and C. A. Maggi, 2,6-Diketopiperazines from
amino acids, from solution-phase to solid-phase organic synthesis,
J. Comb. Chem., 2001, 3, 453–460.
7 C. J. Dinsmore and D. C. Beshore, Recent advances in the synthesis
of diketopiperazines, Tetrahedron, 2002, 58, 3297–3312.
8 T. Guo, G. Dong, D. Fitzpatrick, P. Geng, K. K. Ho, C. H. Jibilian,
S. G. Kultgen, R. Liu, E. McDonald, K. W. Saionz, K. J. Valenzano,
D. Xie, A. E. P. Adang, N. C. R. van Straten, M. L. Webb, Discovery
of potent biaryl diketopiperazine FSH receptor agonists: rapid lead
optimization through parallel synthesis Poster of the 228th ACS
National Meeting, Philadelphia, PA, 2004.
9 A. K. Szardenings, D. Harris, S. Lam, L. Shi, D. Tien, Y. Wang, D. V.
Patel, M. Navre and D. A. Campbell, Rational design and combi-
natorial evaluation of enzyme inhibitor scaffolds: identification of
novel inhibitors of matrix metalloproteinases, J. Med. Chem., 1998,
41, 2194–2200.
Human astrocytoma cells stably expressing human P2Y1 re-
ceptors were cultured in Dulbecco’s modified Eagle’s medium
(DMEM, JRH Biosciences, Inc., Lenexa, KS, USA) and F12 (1 :
1) supplemented with 10% fetal bovine serum, 100 units peni-
cillin mL−1, 100 lg streptomycin mL−1, 2 lmol glutamine mL−1,
and 500 lg geneticin mL−1. For the assay, cells were grown
overnight in 100 lL of media in 96-well flat-bottom plates at
37 ◦C at 5% CO2 or until approximately 60–80% confluency. The
calcium assay kit (Molecular Devices, Sunnyvale, CA) was used
as directed without washing of the cells, and with probenecid
added to the loading dye at a final concentration of 2.5 mM
to increase dye retention. Cells were loaded with 50 lL of
dye with probenecid to each well and incubated for 45 min
at room temperature. The compound plate was prepared using
dilutions of various compounds in Hank’s buffer. For antagonist
studies, both agonist and antagonist were added to the sample
plate. Samples were run in duplicate using a Molecular Devices
Flexstation I at room temperature. Cell fluorescence (excitation
= 485 nm, emission = 525 nm) was monitored following
exposure to the compound. Increases in intracellular calcium
are reported as the maximum fluorescence value after exposure
minus the basal fluorescence value before exposure.
10 A. K. Szardenings, V. Antonenko, D. A. Campbell, N. DeFrancisco,
S. Ida, L. Shi, N. Sharkov, D. Tien, Y. Wang and M. Navre,
Identification of highly selective inhibitors of collagenase-1 from
2 0 2 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 0 1 6 – 2 0 2 5

View Article Online
combinatorial libraries of diketopiperazines, J. Med. Chem., 1999,
42, 1348–1357.
11 A. Bianco, C. P. Sonksen, P. Roepstorff and J.-P. Briand, Solid-
phase synthesis and structural characterization of highly substituted
hydroxyproline-based 2,5-diketopiperazines, J. Org. Chem., 2000, 65,
2179–2187.
12 A. Bianco, J. Furrer, D. Limal, G. Guichard, K. Elbayed, J. Raya, M.
Piotto and J.-P. Briand, Multistep synthesis of 2,5-diketopiperazines
on different solid supports monitored by high resolution magic angle
spinning NMR spectroscopy, J. Comb. Chem., 2000, 2, 681–690.
13 A. N. Koehler, A. F. Shamji and S. L. Schreiber, Discovery of an
inhibitor of a transcription factor using small molecule microarrays
and diversity-oriented synthesis, J. Am. Chem. Soc, 2003, 125, 8420–
8421.
14 K. Shokat and M. Velleca, Novel chemical genetic approaches to
the discovery of signal transduction inhibitors, Drug Discov. Today,
2002, 7, 872–879.
15 C. J. Creighton, G. C. Leo, Y. Du and A. B. Reitz, Design,
synthesis, and conformational analysis of eight-membered cyclic
peptidomimetics prepared using ring closing metathesis, Bioorg.
Med. Chem., 2004, 12, 4375–4385.
16 M. Scholl, S. Ding, C. W. Lee and R. H. Grubbs, Synthesis and
activity of a new generation of ruthenium-basedolefin metathesis cat-
alysts coordinated with 1,3-dimesityl-4,5-dihydroimidazol-2-ylidene
ligands, Org. Lett., 1999, 1, 953–956.
tagogues acting through P2Y-Receptors, J. Med. Chem., 1999, 42,
3636–3646.
30 P. Raboisson, A. Baurand, J. P. Cazenave, C. Gachet, M. Retat, B.
Spiess and J. J. Bourguignon, Novel antagonists acting at the P2Y1
purinergic receptor: synthesis and conformational analysis using
potentiometric and nuclear magnetic resonance titration techniques,
J. Med. Chem, 2002, 45, 962–972.
31 P. Raboisson, A. Baurand, J. P. Cazenave, C. Gachet, D. Schultz,
B. Spiess and J. J. Bourguignon, A general approach toward the
synthesis of C-nucleoside pyrazolo[1,5-a]-1,3,5-triazines and their
3ꢀ,5ꢀ-bisphosphate C-nucleotide analogues as the first reported in vivo
stable P2Y1-receptor antagonists, J. Org. Chem, 2002, 67, 8063–8071.
32 A. H. Ingall, J. Dixon, A. Bailey, M. E. Coombs, D. Cox, J. I.
McInally, S. F. Hunt, N. D. Kindon, B. J. Theobald, P. A. Willis,
R. G. Humphries, P. Leff, J. A. Clegg, J. A. Smith and W. Tomlinson,
Antagonists of the platelet P2T receptor: a novel approach to
antithrombotic therapy, J. Med. Chem, 1999, 42, 213–220.
33 S. G. Kim, K. A. Soltysiak, Z. G. Gao, T. S. Chang, E. Chung
and K. A. Jacobson, Tumor necrosis factor a-induced apoptosis
in astrocytes is prevented by the activation of P2Y6, but not P2Y4
nucleotide receptors, Biochem. Pharmacol., 2003, 65, 923–931.
34 M. P. Glenn, L. K. Pattenden, R. C. Reid, D. P. Tyssen, J. D. A.
Tyndall, C. J. Birch and D. P. Fairlie, b-Strand mimicking macrocyclic
amino acids: templates for protease inhibitors with antiviral activity,
J. Med. Chem., 2002, 45, 371–381.
17 S. J. Connon and S. Blechert, Recents developments in olefin cross-
metathesis, Angew. Chem., Int. Ed., 2003, 42, 1900–1923.
18 I. von Ku¨gelgen and A. Wetter, Molecular pharmacology of P2Y-
receptors, Naunyn Schmiedebergs Arch. Pharmacol., 2000, 362, 310–
323.
19 B. F. King and A. Townsend-Nicholson, Recombinant P2Y recep-
tors: the UCL experience, J. Auton. Nerv. Syst, 2000, 81, 164–170.
20 G. Hollopeter, H. M. Jantzen, D. Vincent, G. Li, L. England, V.
Ramakrishnan, R. B. Yang, P. Nurden, A. Nurden, D. Julius and
P. B. Conley, Identification of the platelet ADP receptor targeted by
antithrombotic drugs, Nature, 2001, 409, 202–207.
21 D. Communi, N. Suarez Gonzalez, M. Detheux, S. Bre´zillon, V.
Lannoy, M. Parmentier and J.-M. Boeynaems, Identification of a
novel human ADP receptor goupled to Gi, J. Biol. Chem., 2001, 276,
41 479–41 485.
22 K. A. Jacobson, Purine and pyrimidine nucleotide (P2) receptors,
Ann. Rep. Med. Chem., 2002, 37, 75–84.
35 I. Koch and M. Keusgen, Diastereoselective synthesis of alliin by an
asymmetric sulfur oxidation, Pharmazie, 1998, 53, 668–671.
36 C. G. Boojamra, K. M. Burow, L. A. Thompson and J. A. Ellman,
Solid-phase synthesis of 1,4-benzodiazepine-2,5-diones. Library
preparation and demonstration of synthesis generality, J. Org. Chem.,
1997, 62, 1240–1256.
37 K. J. Jensen, J. Alsina, M. F. Songster, J. Vagner, F. Albericio and
G. Barany, Backbone amide linker (BAL) strategy for solid-phase
synthesis of C-terminal-modified and cyclic peptides, J. Am. Chem.
Soc., 1998, 120, 5441–5452.
38 M. Fresno, J. Alsina, M. Royo, G. Barany and F. Albericio, Solid-
phase synthesis of diketopiperazine, useful scaffolds for combinato-
rial chemistry, Tetrahedron Lett., 1998, 39, 2639–2642.
39 G. L. Waldo, J. Corbitt, J. L. Boyer, G. Ravi, H. S. Kim, X. D. Ji,
J. Lacy, K. A. Jacobson and T. K. Harden, Quantitation of the
P2Y1 receptor with a high affinity radiolabeled antagonist, Mol.
Pharmacol., 2002, 62, 1249–1257.
23 E. Nandanan, E. Camaioni, S. Y. Jang, Y.-C. Kim, G. Cristalli, P.
Herdewijn, J. A. Secrist, K. N. Tiwari, A. Mohanram, T. K. Harden,
J. L. Boyer and K. A. Jacobson, Structure–activity relationships of
bisphosphate nucleotide derivatives as P2Y1 receptor antagonists and
partial agonists, J. Med. Chem., 1999, 42, 1625–1638.
24 E. Nandanan, S. Y. Jang, S. Moro, H. Kim, M. A. Siddiqi, P. Russ,
V. E. Marquez, R. Busson, P. Herdewijn, T. K. Harden, J. L. Boyer
and K. A. Jacobson, Synthesis, biological activity, and molecular
modeling of ribose-modified adenosine bisphosphate analogues as
P2Y1 receptor ligands, J. Med. Chem., 2000, 43, 829–842.
25 H. S. Kim, D. Barak, T. K. Harden, J. L. Boyer and K. A. Jacob-
son, Acyclic and cyclopropyl analogues of adenosine bisphosphate
antagonists of the P2Y1 receptor: structure activity relationships and
receptor docking, J. Med. Chem., 2001, 44, 3092–3108.
26 J. Boyer, M. Adams, R. G. Ravi, K. A. Jacobson and T. K. Harden,
2-Chloro-N6-methyl-(N)-methanocarba-2ꢀ-deoxyadenosine-3ꢀ,5ꢀ-
bisphosphate is a selective high affinity P2Y1 receptor antagonist,
Br. J. Pharmacol, 2002, 135, 2004–2010.
27 B. Fischer, J. L. Boyer, C. H. V. Hoyle, A. U. Ziganshin, A. L.
Brizzolara, G. E. Knight, J. Zimmet, G. Burnstock, T. K. Harden and
K. A. Jacobson, Identification of potent, selective P2Y-purinoceptor
agonists: structure–activity relationships for 2-thioether derivatives
of adenosine 5ꢀ-triphosphate, J. Med. Chem., 1993, 36, 3937–3946.
28 J. L. Boyer, S. Siddiqi, B. Fischer, T. Romera-Avila, K. A. Jacobson
and T. K. Harden, Identification of potent P2Y purinoceptor agonists
that are derivatives of adenosine 5ꢀ-monophosphate, Br. J. Pharma-
col., 1996, 118, 1959–1964.
40 M. Ohno, S. Costanzi, H. S. Kim, V. Kempeneers, K. Vastmans, P.
Herdewijn, S. Maddileti, Z. G. Gao, T. K. Harden and K. A. Jacob-
son, Nucleotide analogues containing 2-oxa-bicyclo[2.2.1]heptane
and L-a-threofuranosyl ring rystems: interactions with P2Y recep-
tors, Bioorg. Med. Chem., 2004, 12, 5619–5630.
41 S. Costanzi, L. Mamedova, Z.-G. Gao and K. A. Jacobson,
Architecture of P2Y nucleotide receptors: structural comparison
based on sequence analysis, mutagenesis, and homology modeling,
J. Med. Chem., 2004, 47, 5393–5404.
42 L. Wang, S. E. W. Jacobsen, A. Bengtsson and D. Erlinge, P2 receptor
mRNA expression profiles in human lymphocytes, monocytes and
CD34+ stem and progenitor cells, BMC Immunol., 2004, 5, 16.
43 J. K. Chambers, L. E. Macdonald, H. M. Sarau, R. S. Ames, K.
Freeman, J. J. Foley, Y. Zhu, M. M. McLaughlin, P. Murdock, L.
McMillan, J. Trill, A. Swift, N. Aiyar, P. Taylor, L. Vawter, S. Naheed,
P. Szekeres, G. Hervieu, C. Scott, J. M. Watson, A. J. Murphy, E.
Duzic, C. Klein, D. J. Bergsma, S. Wilson and G. P. Livi, A G protein-
coupled receptor for UDP-glucose, J. Biol. Chem., 2000, 275, 10 767–
10 771.
44 M. M. Bradford, A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of protein
dye binding, Anal. Biochem., 1976, 76, 248–254.
45 J. R. Maple, M. J. Hwang, T. P. Stockfisch, U. Dinur, M. Waldman,
C. S. Ewig and A. T. Hagler, Derivation of Class II force fields. 1.
Methodology and quantum force field for the alkyl functional group
and alkane molecules, J. Comput. Chem., 1994, 15, 162–182.
46 C. A. G. Haasnoot, Conformational analysis of six-membered rings
in solution: ring-puckering coordinates derived from vicinal NMR
proton–proton coupling constants, J. Am. Chem. Soc., 1993, 115,
1460–1468.
29 B. Fischer, A. Chulkin, J. L. Boyer, K. T. Harden, F. P. Gendron,
A. R. Beaudoin, J. Chapal, D. Hillaire-Buys and P. Petit, 2-Thioether
5ꢀ-O-(1-thiotriphosphate)adenosine derivatives as new insulin secre-
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 0 1 6 – 2 0 2 5
2 0 2 5