Combinatorial synthesis and biological evaluation of peptide-binding GPCR-targeted library

Article abstract of DOI:10.1016/j.bioorg.2009.04.001

As an effective strategy of the drug discovery for peptide-binding GPCRs based on the natural ligands, beta-turn peptidomimetic compound library with benzodiazepine skeleton was constructed using solid and solution phase parallel synthesis with four diffe

Full text of DOI:10.1016/j.bioorg.2009.04.001

Bioorganic Chemistry 37 (2009) 90–95
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Combinatorial synthesis and biological evaluation of peptide-binding
GPCR-targeted library
Ju Yeon Lee a, Isak Im a, Thomas R. Webb b, Douglas McGrath c, Mi-Ryoung Song a, Yong-Chul Kim a,
*
a Department of Life Science, Gwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea
b Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, MS 1000, 332 North Lauderdale, Memphis, TN 38105, USA
c Molecular Biology, RGo Bioscience, LLC, 10578 Science Center Dr., San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 February 2009
Available online 19 April 2009
As an effective strategy of the drug discovery for peptide-binding GPCRs based on the natural ligands,
beta-turn peptidomimetic compound library with benzodiazepine skeleton was constructed using solid
and solution phase parallel synthesis with four different scaffolds containing Phe, Lys, Ser and Glu,
respectively. The usefulness of 162 library compounds was evaluated by the cell based screening at mel-
anocortin 4 receptor in CHO-k1 cells, to find hit compounds showing agonistic effect at the receptor. The
screening of library afforded three hit compounds including the most effective analog, (S)-3-benzyl-7-(4-
fluorobenzyloxy)-4-(4-methoxyphenethyl)-4,5-dihydro-1H-benzo[e][1,4]diazepin-2(3H)-one, 13aiE, of
Keywords:
Benzodiazepine
Combinatorial synthesis
Peptidomimetics
Melanocortin 4 receptors
which EC50 was determined as 13 lM.
Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction
ical roles are different each other such as skin pigmentation, anti-
inflammatory effects, regulation of blood pressure, immune
modulation and food intake [7–11]. Human MC4R is found to be
expressed only in the brain and involved in the Leptin pathway; it
contributes to the controlling of human feeding behavior and energy
homeostasis [12, 13]. Over the last decade, the intensive efforts tar-
geting MC4R have led to the design of selective, peptidic, non-pep-
tidic molecule for the development of anti-obesity drugs [14–22].
Main strategy to identify small-molecule MC4R agonists is the pep-
tidomimetic approach based on piperazine, cyclohexane, and pyr-
rolidine core, which represent the important pharmacophores
from the His-Phe-Arg-Trp message sequence [23, 24].
G-protein coupled receptors (GPCRs) have been in the center of
pharmaceutical research as the most diverse and largest target
family showing important physiological relevance with various
diseases, participating in a lot of cell signal transduction processes.
Peptide-binding GPCRs belong to a small group GPCR family with
approximately 120 members which are activated by the binding
of intermediate size peptides consisting of typically 30–40 amino
acid residues [1]. Most of the peptide-binding GPCRs are related
with pathological events in several diseases and most of pharma-
ceutical companies have put a lot of efforts to modulate these
GPCRs for the management of corresponding diseases. Since the
lack of three-dimensional structures of GPCRs, an alternative strat-
egy toward drug discovery for peptide-binding GPCRs could be
peptidomimetic approaches based on the turn secondary structure
of peptide ligands. The turn conformation of natural peptide
ligands has been supported by structure–activity relationships of
native ligands [2], modified peptide fragments [3], cyclic peptide
analogues [4], and turn peptidomimetics [5].
In our recent study, we successfully developed two kinds of
b-turn-like template (Fig. 1) [25–27], adapting the outstanding
result of Garland and Dean [28] which are cluster analysis and
recombination of the observed patterns yielded a consensus posi-
tioning of the C
a atoms among the various b-turn types. The bio-
logical activity of the somatostatin and melanocortin mimic
analogues with 7’-alkoxy-2’,3-dihydro-1’H-spiro[imidazolidine-
4,4’-quinoline]-2,5-dione scaffold 1 were demonstrated, respec-
tively [25,27]. Also, we have developed a solid phase combinatorial
library synthesis of peptide-binding GPCR targeted library using
1,4-benzodiazepin-2-one based b-turn mimic scaffold with diverse
natural amino acids, and building blocks [26]. Several diverse
analogs of 1,4-benzodiazepines have been reported to show
activities toward diverse drug targets such as cholecystokinin
receptor (CCK), benzodiazepine receptor, neurokinin-1 receptor,
The melanocortin 4 receptor (MC4R), a member of melanocortin
receptor family [6] consisting of 332 amino acids, which is activated
uponbindingof melanocortin peptideandmediatessignaltransduc-
tion via G-protein coupled signaling system. In human, five receptor
subtypes which share high homology in primary amino acid
sequences have been identified (MC1R–MC5R), but their physiolog-
j
-secretase, and farnesyl transferase [29]. Herein, we report the
* Corresponding author. Fax: +82 62 970 2484.
E-mail address: yongchul@gist.ac.kr (Y.-C. Kim).
construction of expanded peptide-binding GPCR targeted library
0045-2068/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2009.04.001

J.Y. Lee et al. / Bioorganic Chemistry 37 (2009) 90–95
91
2.1.3. 2-[2-Amino-5-(2,2-dimethyl-propionyloxy)-benzylamino]-6-
tert-butoxycarbonylamino-hexanoic acid methyl ester (6b)
O
NR3
R2
O
NH
O
N
Under supplying hydrogen gas, 5a (8.47 g, 17.09 mmol) was dis-
solved in methanol (80 ml) and treated with solid 10% Pd/C (1.5 g).
After stirring for 3 h at room temperature, the reaction mixture
was filtered through a Celite bed and washed with methanol. The
methanol was evaporated, and the product was purified by silica
gel column chromatography and eluted with hexane/ethyl acetate
(3:1) to afford 6b (99.3% yield).
O
R2
R1
N
R3
R1O
N
R4
2
1
Fig. 1. The b-turn mimic scaffold based on the Garland–Dean triangles.
1H NMR (300 MHz, CDCl3) d (ppm) 6.78 (dd, J = 2.7 Hz, 8.5 Hz,
1H), 6.7 (d, J = 2.7 Hz, 1H), 6.62 (d, J = 8.4 Hz, 1H), 4.5 (s, 2H,
NH2), 3.64 (ABq, J = 10 Hz, 52.9 Hz, 2H), 3.69 (s, 3H), 3.24 (t,
J = 6.6 Hz, 1H), 3.05 (t, J = 5.7 Hz, 2H), 2.04 (s, 3H), 1.79–1.61 (m,
2H), 1.49 (s, 9H), 1.30 (s, 9H), 1.49–1.22 (m, 4H). MS (ESI) m/z:
466.19 ([M+H]+).
of 1,4-benzodiazepin-2-one skeleton for the discovery of new ago-
nists by the identification of hit compounds from a screening of the
combinatorial library targeting the human MC4R.
2. Materials and methods
2.1.4. 2,2-Dimethyl-propionic acid 3-(4-tert-butoxy carbonylamino-
butyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-7-yl ester (7b)
Compound 6a (6.11 g, 13.12 mmol) was dissolved in toluene
(40 ml), and 2 M AlMe3 in toluene (26 ml) was added drop wise
for 5 min at 0 °C and stirred for 10 min. The reaction temperature
was increased slowly by room temperature and stirred 1½ h. The
reaction was quenched with 45 ml of MeOH at 0 °C and stirred
more at room temperature. The mixture was partitioned between
saturated aqueous NaHCO3 and EtOAc. Before separating the or-
ganic layer, the mixture was filtered first. Then the biphasic filtrate
was separated and organic phase dried over, filtered, and concen-
trated. The product was purified by silica gel column chromatogra-
phy (CHCl3:MeOH = 25:1) to afford 7b (65.6% yield).
Thin-layer chromatography (TLC) was conducted on precoated
silica gel plates (Merck silica gel 60; F254, 0.25 mm) and visualized
with either short-wave UV light. Starting materials, reagents, and
solvents were obtained from Aldrich Chemical Co. (Milwaukee,
WI) and used as received unless otherwise noted. PL-FDMP resin
was purchased from Polymer Laboratories. Proton nuclear
magnetic resonance spectra were collected on a JEOL JNM-LA 300
WB instruments and were recorded in parts per million (ppm) d
values, relative to tetramethylsilane as the internal standard, and
spectra were taken in CDCl3. Mass spectroscopy was carried out
on VG BIOTECH platform. Parallel solid-phase synthesis was
performed on a MiniBlock from Mettler-Toledo Bohdan, Inc.
(Vernon Hills, IL). The SPE tube, SAX was purchased from Alltech
Associates (Lot No. 2312; Deerfield, IL). Parallel purification was
performed on Quad3, Parallel FLASH Purification System, Biotage,
Inc. (Charlottesvile, VA).
1H NMR (300 MHz, CDCl3) d (ppm) 7.72 (s, NH), 7.01–6.98 (m,
3H), 3.98 (ABq, J = 13.5 Hz, 68.4 Hz, 2H), 3.39 (t, J = 6.6 Hz, 1H),
3.09 (ABq, J = 13.5 Hz, 68.4 Hz, 2H), 1.91–1.76 (m, 2H), 1.87–1.75
(m, 2H), 1.59–1.54 (m, 2H), 1.42 (s, 9H), 1.35 (s, 9H). MS (ESI) m/
z: 434.17 ([M+H]+).
2.1. Representative synthetic procedure
2.2. General methods for solid-phase library synthesis
2.1.1. Preparation of 2,2-dimethyl-propionic acid 3-formyl-4-nitro-
phenyl ester (4)
Compounds 13a, 13b’, and 13c’ can be prepared as previously
described for 1,3,4,5-tetrahydrobenzo[e][1,4]diazepin-2-one [24].
First, to the 7a, 7b’, and 7c’ (12.9 mmol) in 1,2-dichloroethane
(100 ml) solution was added PL-FDMP resin (1.5 mmol/g,
12.15 mmol) and NaBH(OAc)3 (18.22 mmol) and the reaction mix-
ture was gently stirred for 12 h at room temperature. After the scaf-
folds disappeared on the TLC, the mixture was filtered and the resin
was washed with DMF (3 Â 30 ml), DCM (3 Â 30 ml), and MeOH
(3 Â 20 ml). The residue was dried in vacuo to a constant weight
(yield: 80.2%). The pivaloyl group of 9a, 9b, and 9c was hydrolyzed
to afford free phenolic OH group in 3% KOH solution of dioxane
and H2O (50 ml:50 ml) for 24 h at room temperature. The color of
resin changed to yellow-green. The resin was treated as same wash-
ing and drying procedure aforementioned. The syntheses were car-
ried out on the solid-phase in two set of 4 Â 6 reaction tubes in
MiniBlock. Resin-bound benzodiazeipne-2-one 10a, 10b, and 10c
(0.168 mmol) distributed in each reaction vessel suspended in 1:1
DMSO/NMP (4 ml) and followed by adding alkyl halide (0.84 mmol)
and DBU (0.84 mmol). Reaction mixture was shaken for 24 h at room
temperature. After washing the resin with DMF and DCM three
times, the reaction was repeated again, and the resin was washed
with THF additionally for next N-alkylation reaction. To the resin-
loaded O-alkylated benzodiazepine-2-one 11a, 11b, and 11c
(0.168 mmol) suspended in THF (3 ml) was added 1 M lithium-
tert-butoxide in THF, and the reaction tubes were shaken for 1 h.
After removing THF solution, building blocks A–H (Table 1) in DMSO
(3 ml) was added to each reaction tube and shaken overnight. The
reaction mixture was removed by filtration, and the resins were
washed with DMF (4 ml), DCM (4 ml), and MeOH (4 ml) by three
Et3N(7.562 ml, 54.26 mmol) wasadded toa solutionof 3 (9.069 g,
54.26 mmol) in DCM (150 ml), followed by trimethyl acetic chloride
(7.34 ml, 59.66 mmol). Thereaction wasstirredat room temperature
for 30 min. The reaction mixture was quenched with saturated aque-
ous NH4Cl and extracted with chloroform. The organic phase was
separated and dried over Na2SO4, then evaporated under reduced
pressure. The residue was purified by flash silica gel column chroma-
tography (CHCl3:MeOH = 100:1) to afford 4 (99.5% yield).
1H NMR (300 MHz CDCl3): d (ppm) 10.44 (s, 1H), 8.10 (d,
J = 12 Hz, 1H), 7.25 (s, 1H), 7.07 (d, J = 12 Hz, 1H), 1.336 (s, 9H).
MS (ESI) m/z: 252.1 ([M+H]+).
2.1.2. 6-tert-Butoxycarbonylamino-2-[5-(2,2-dimethyl-
propionyloxy)-2-nitro-benzylamino]-hexanoic acid methyl ester (5b)
L-lysine(Boc) methyl ester hydrochloride salt (13.2 g,
44.57 mmol) was added to a solution of 4 (7 g, 27.86 mmol) in
DCE/DMF (70 ml/30 ml). After stirring for 30 min, NaBH(OAc)3
(5.51 g, 26 mmol) was added in the reaction solution and then stir-
red for 1hr and half at room temperature. The residue obtained
was extracted with chloroform and washed well with saturated
aqueous NaHCO3. The product was purified by silica gel column
chromatography by eluting it with hexane/ethyl acetate (5/1) to af-
ford 5b (62.8% yield).
1H NMR (300 MHz, CDCl3) d (ppm) 8.00 (d, J = 9 Hz, 1H), 7.38 (d,
J = 2.4 Hz, 1H), 7.11 (dd, J = 9 Hz, J = 2.4 Hz, 1H), 4.61 (bs, NH), 4.03
(ABq, J = 15 Hz, 117.9 Hz, 2H) 3.71 (s, 3H), 3.23 (m, 1H), 3.08 (m,
2H), 1.76–1.37 (m, 6H), 1.42 (s, 9H), 1.38 (s, 9H). MS (ESI) m/
z:496.12 ([M+H]+).

92
J.Y. Lee et al. / Bioorganic Chemistry 37 (2009) 90–95
Table 1
e–i (Table 1) (0.68 mmol) and shaken for 4 h. When TLC showed
Building blocks for the library synthesis.
that starting material had disappeared, the reaction was quenched
with aq. NH4Cl solution and extracted with CHCl3. All solvent and
remaining volatile residues were evaporated in parallel under
reduced pressure using a Genevac DD-4 system. The concentrated
crude products were dissolved in THF for the next alkylation.
Step (iii) of Scheme 3. To a solution of 11d (0.09 mmol) in 1 ml of
THF was added 1 M lithium-tert-butoxide in THF (0.27 mmol), sha-
ken for 1 h. The reaction mixture were treated with a solution of
blocks A–D (Table 1) (0.27 mmol) in 1 ml of DMSO and stirred at
room temperature for 9 h. The reaction was quenched with aq.
NH4Cl and extracted with CHCl3. Evaporation of the solvent and
purification via silica column chromatography afforded 13d in
ꢀ30–50% yield.
Building blocks for O-alkylation e-m (R2)
I
Br
I
I
O
e
g
f
h
I
F
I
Br
j
k
i
Br
m
Br
2.4. Representative spectral data: specific examples
l
2.4.1. (S)-3-benzyl-1-(2-fluorophenethyl)-7-(2-meth oxyethoxy)-4,5-
dihydro-1H-benzo[e][1,4]diazepin-2(3H)-one (13ahH)
Building blocks for N-alkylation A-H (R3)
1H NMR (300 MHz, CDCl3) d (ppm) 7.26–6.68 (m, 12H), 4.32–
4.27 (m, 1H), 4.09–4.06 (m, 2H), 3.85–3.74 (m, 4H), 3.63–3.57
(m, 1H), 3.49–3.44 (m, 1H), 3.45 (s, 3H), 3.20–3.14 (m, 1H), 3.12–
2.96 (m, 1H), 2.90–2.82 (m, 2H). MS (ESI) m/z: 449.20 ([M+H]+).
Br
OMs
F
A
B
2.4.2. (S)-3-(4-aminobutyl)-7-ethoxy-1-(4-fluorophen ethyl)-4,5-
dihydro-1H-benzo[e][1,4]diazepin-2(3H)-one (13beB)
Cl
1H NMR (300 MHz, CDCl3) d (ppm) 7.14–6.90 (m, 4H), 6.93–6.87
(m, 2H), 6.83 (d, J = 9H, 2H), 4.48–4.38 (m, 1H), 4.04 (q, J = 6.9 Hz,
2H), 3.72–3.64 (m, 2H), 3.67 (ABq, J = 11.7 Hz, 47.6 Hz, 2H), 3.17
(dd, J = 5.4 Hz, 7.8 Hz, 1H), 2.99–2.91 (m, 1H), 2.77–2.71 (m, 1H),
2.65 (t, J = 6.9 Hz, 2H), 1.87–1.82 (m, 1H), 1.52–1.45 (m, 1H), 1.43
(t, J = 6.9 Hz, 3H), 1.45–1.40 (m, 2H), 1.29–1.21 (m, 2H). MS (ESI)
m/z: 400.13 ([M+H]+).
OMs
C
D
OMs
O
OMs
O
O
E
F
2.4.3. (S)-1-(biphenyl-4-ylmethyl)-3-(hydroxylmeth yl)-7-propoxy-
4,5-dihydro-1H-benzo[e][1,4]diazepin-2(3H)-one (13cfD)
1H NMR (300 MHz, CDCl3) d (ppm) 7.58–7.49 (m, 5H), 7.46–7.40
(m, 2H), 7.37–7.28 (m, 2H), 7.21 (d, J = 9 Hz, 1H), 6.91 (dd,
J = 2.7 Hz, 9 Hz, 1H), 6.84 (d, J = 3H, 2H), 5.28 (d, J = 14.7 Hz, 1H),
4.90 (d, J = 14.7 Hz, 1H), 4.08 (dd, J = 3 Hz, 12 Hz, 1H), 3.715 (ABq,
J = 11.7 Hz, 34.5 Hz, 2H), 3.71–3.67 (m, 1H), 3.33 (t, J = 3.3 Hz,
1H), 1.82 (tq, J = 6.6 Hz, 7.2 Hz, 2H), 0.94 (t, J = 7.2 Hz, 3H). MS
(ESI) m/z: 417.01 ([M+H]+).
F
O
MsO
MsO
G
H
times. This alkylation was iterated one more time. All the reaction
resins were treated with 50% TFA/DCM (3 ml  2 times) for 2 h,
and the solution was collected by filtration and washed with DCM
by two times. The solution was evaporated in parallel manner using
Genevac DD-4 system, the concentrated products were dissolved in
chloroform and passed through the SAX resin to afford the free
amine form. Parallel evaporation of the eluents and purification by
a Quad3 parallel purification system with an appropriate mixture
of Chloroform/MeOH afforded final product 13a, 13b’, and 13c’.
2.4.4. (S)-7-(ethoxymethyl)-10-(4-methylphenethyl)-10,11a-dihydro-
1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-3,11(2H,5H)-dione (13dhC)
1H NMR (300 MHz, CDCl3) d (ppm) 7.25–7.16 (m, 5H), 7.09 (d,
J = 8.1 Hz, 1H), 6.94 (dd, J = 3 Hz, 8.1 Hz, 1H), 6.80 (d, J = 3 Hz,
1H), 4.75–4.58 (m, 2H), 4.06 (ABq, J = 13.8 Hz, 288 Hz, 2H), 4.04
(q, J = 6.9 Hz, 2H), 3.96 (t, J = 7.5 Hz, 2H), 3.74–3.67 (m, 1H),
2.88–2.79 (m, 2H), 2.71–2.61 (m, 1H), 2.59–2.47 (m, 1H), 2.44–
2.35 (m, 1H), 2.30 (s, 3H), 2.0–1.89 (m, 1H), 1.41 (t, J = 6.9 Hz,
3H). MS (ESI) m/z: 393.12 ([M+H]+).
2.3. Solution-phase library synthesis of (S)-7-R2-10-R3-10,11a-dihydro-
1H-benzo[e]pyrrolo[1,2-a][1,4]dia zepine-3,11(2H,5H)-diones
2.4.5. (S)-7-ethoxy-10-phenethyl-10,11a-dihydro-1H-
benzo[e]pyrrolo[1,2-a][1,4]diazepine-3,11(2H,5H)-dione (13deA)
1H NMR (300 MHz, CDCl3) d (ppm) 7.25–7.16 (m, 5H), 7.09 (d,
J = 8.1 Hz, 1H), 6.94 (dd, J = 3 Hz, 8.1 Hz, 1H), 6.80 (d, J = 3 Hz,
1H), 4.20 (m, 2H), 4.06 (ABq, J = 13.8 Hz, 288 Hz, 2H), 4.04 (q,
J = 6.9 Hz, 2H), 3.96 (t, J = 7.5 Hz, 1H), 2.85 (m, 2H), 2.21 (m, 2H),
1.41 (t, J = 6.9 Hz, 3H). MS (ESI) m/z: 365.21 ([M+H]+).
Step (i) of Scheme 3. Compound 8 (10.14 mmol) was dissolved
in 3% KOH MeOH/Water (8:2) solution and stirred at room temper-
ature for 40 min. Excess KOH was neutralized with 1 N aq. HCl
solution, and the resulting reaction mixtures were evaporated to
dryness. The residues were washed with pure water, extracted
with CHCl3, dried with anhydrous Na2SO4, and concentrated to
dryness to afford the free phenolic OH 10d in ꢀ92% yield.
2.4.6. (S)-10-phenethyl-7-propoxy-10,11a-dihydro-1H-
Step (ii) of Scheme 3. The solution of 10d (0.34 mmol) in DMF
(2 ml) was put in wells of 8-channel synthesizer and treated with
DBU (0.68 mmol). The mixture was treated with building blocks
benzo[e]pyrrolo[1,2-a][1,4]diazepine-3,11(2H,5H)-dione (13dfA)
1H NMR (300 MHz, CDCl3) d (ppm) 7.31–7.08 (m, 6H), 6.94 (dd,
J = 3 Hz, 4.3 Hz, 1H), 6.81 (d, J = 3 Hz, 1H), 4.35 (m, 2H), 4.07 (ABq,

J.Y. Lee et al. / Bioorganic Chemistry 37 (2009) 90–95
93
J = 13.5 Hz, 301 Hz, 2H), 3.99–3.90 (m, 3H), 2.88 (m, 2H), 2.41 (m,
2H), 2.32 (m, 2H), 1.82 (m, 2H), 1.04 (t, J = 7.5 Hz, 3H). MS (ESI)
m/z: 379.12 ([M+H]+).
1H), 4.97 (ABq, J = 14.7 Hz, 119 Hz, 2H), 3.94 (m, 1H), 3.52 (ABq,
J = 11.7 Hz, 48.3 Hz, 2H), 3.17 (t, J = 5.7 Hz, 1H), 2.84 (t, J = 6.9 Hz,
2H), 1.85 (m, 2H), 1.64 (m, 2H), 1.38 (s, J = 2.1 Hz, 3H), 1.36 (s,
J = 2.1 Hz, 3H) 1.18 (m, 2H). MS (ESI) m/z: 458.16 ([M+H]+).
2.4.7. (S)-10-(4-fluorophenethyl)-7-propoxy-10,11a-dihydro-1H-
benzo[e]pyrrolo[1,2-a][1,4]diazepine-3,11(2H,5H)-dione (13dfB)
1H NMR (300 MHz, CDCl3) d (ppm) 7.31–7.08 (m, 5H), 6.94 (dd,
J = 3 Hz, 4.3 Hz, 1H), 6.81 (d, J = 3 Hz, 1H), 4.35 (m, 2H), 4.07 (ABq,
J = 13.5 Hz, 288 Hz, 2H), 3.99–3.90 (m, 3H), 2.88 (m, 2H), 2.41 (m,
2H), 2.32 (m, 2H), 1.82 (m, 2H), 1.04 (t, J = 7.5 Hz, 3H). MS (ESI)
m/z: 397.06 ([M+H]+).
2.5. Functional assay of the melanocortin 4 receptors
The MC4R agonist assay is a whole cell transcriptional assay
that detects the production of the b-lactamase enzyme induced
by agonist stimulation of the MC4 GPCR. The assay consists of an
engineered clonal Chinese Hamster Ovary (CHO-K1) cell line that
has been transfected with plasmids that allow for the expression
of the receptor and an adenylate cyclase responsive reporter
element (CRE) coupled to the coding sequence of the b-lactamase
enzyme. The transfected cells were plated at 5000 cells per well
2.4.8. (S)-10-(4-methylphenethyl)-7-propoxy-10,11a-dihydro-1H-
benzo[e]pyrrolo[1,2-a][1,4]diazepine-3,11(2H,5H)-dione (13dfC)
1H NMR (300 MHz, CDCl3) d (ppm) 7.31–7.08 (m, 5H), 6.94 (dd,
J = 3 Hz, 4.3 Hz, 1H), 6.81 (d, J = 3 Hz, 1H), 4.35 (m, 2H), 4.07 (ABq,
J = 13.5 Hz, 289 Hz, 2H), 3.99–3.90 (m, 3H), 2.88 (m, 2H), 2.41 (m,
2H), 2.32 (m, 2H), 2.30 (s, 3H), 1.82 (m, 2H), 1.04 (t, J = 7.5 Hz,
3H). MS (ESI) m/z: 393.14 ([M+H]+).
in a 96-well plate (black, clear bottom) in 50 ll per well of DMEM
supplemented with 10% fetal bovine serum. These cells were
allowed to adhere to the plate. The next day the media was
removed from the cells and replaced with serum-free DMEM med-
ia. After 12 h of serum starvation, media was aspirated, and then
2.4.9. (S)-7-(4-fluorobenzyloxy)-10-(4-fluorophen ethyl)-10,11a-dihy-
dro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-3,11(2H,5H)-dione (13diB)
1H NMR (300 MHz, CDCl3) d (ppm) 7.41–7.36 (m,2H), 7.25–6.86
(m, 8H), 5.01 (s,2H), 4.35 (m, 2H), 4.07 (ABq, J = 13.5 Hz, 289 Hz,
2H), 3.99–3.90 (m, 3H), 2.88 (m, 2H), 2.41 (m, 2H), 2.32 (m, 2H),
1.82 (m, 2H), 1.04 (t, J = 7.5 Hz, 3H). MS (ESI) m/z: 463.11 ([M+H]+).
25
uted to the each well at 10
Responses were compared to the effect of
as positive and negative control, respectively. After plates were
incubated for 4 h at 37 °C in a 5% CO2, 7 l of CCF2 dye stock pre-
pared per the manufacturer’s protocol was then added to each
well. Plates were then incubated for 1 h at room temperature,
and the fluorescence was determined on a BMG polarstar reader
with bottom reading optics.
l
l of DMEM was added. The library compounds were distrib-
M final concentration in DMEM media.
-MSH and only DMEM
l
a
l
2.4.10. (S)-7-isopropoxy-10-phenethyl-10,11a-dihydro-1H-
benzo[e]pyrrolo[1,2-a][1,4]diazepine-3,11(2H,5H)-dione (13dgA)
1H NMR (300 MHz, CDCl3) d (ppm) 7.27–7.10 (m, 6H), 6.94 (dd,
J = 3 Hz, J = 4.3 Hz, 1H), 6.81 (d, J = 3 Hz, 1H), 4.48 (m, 1H), 4.20 (m,
2H), 4.18 (ABq, J = 13.5 Hz, 315 Hz, 2H), 3.99 (t, J = 6 Hz, 1H), 2.88
(m, 2H), 2.44 (m, 2H), 2.31 (m, 2H), 1.38 (s, J = 2.1 Hz,3H), 1.36 (s,
J = 2.1 Hz, 3H). MS (ESI) m/z: 379.09 ([M+H]+).
3. Results and discussion
The synthesis of benzodiazepine library was summarized in
Schemes 1 and 2. Hydroxyl group of 5-hydroxy-2-nitro-benzalde-
hyde (3) was first protected with trimethylacetyl chloride in
CH2Cl2. Four amino acid esters, phenylalanine methyl ester a,
lysine methyl ester b, serine methyl ester c, and glutamate methyl
ester d, were used to form secondary amine unit of the benzodiaz-
epine skeleton with reductive alkylation reactions using NaB-
H(OAc)3. For the cyclization, the nitro group of compound 5a–d
was reduced to amine in a condition of catalytic hydrogenation
and subsequent intramolecular cyclization with AlMe3 in toluene
gave the skeleton of tetrahydro-1,4-benzodiazepin-2-one 7a–c
and 8 as the key scaffold for solid and solution phase library
synthesis (Scheme 1).
For the efficient and fast library synthesis, Phe 7a, Lys 7b’,
and Ser-scaffold 7c’ were loaded on the 4-formyl-3,5-dimethoxy-
phenoxy (PL-FDMP) resin by reductive alkylation with high yield
(>80%). To incorporate R2 building blocks at the phenolic oxygen,
the pivaloyl group of 9a–c was first hydrolyzed in 3% KOH in
dioxane/H2O (1:1) and resulting 10a–c were treated with 9 alkyl
halides (Table 1) in the presence of DBU as a base in the mixture
of DMSO/NMP (1:1), respectively (Scheme 2). Each resin-bound
O-substituted benzodiazepine-2-one 9a–c were treated with
1 M lithium-tert-butoxide in THF, and after wash, subsequently
reacted with 8 alkyl halide as the R3 building blocks (Table 1)
in DMSO. To cleave the final product out of each resin, 50%
TFA/ CH2Cl2 (3 ml) was treated for 2 h, and the filtrate was col-
lected, evaporated, passed through SAX resin to remove TFA in a
parallel fashion. In the case of glutamate scaffold 8, the t-butyl
protective group was cleaved by AlMe3 during cyclization pro-
cess, forming pyrrolidinone structure. Thus, parallel solution
phase synthesis was applied using eight channel parallel synthe-
sizers for the introduction of limited number of R2 and R3 groups
to afford 24 compounds (Scheme 3). After each step of parallel
2.4.11. (S)-3-(4-aminobutyl)-7-ethoxy-1-phenethyl-4,5-dihydro-1H-
benzo[e][1,4]diazepin-2(3H)-one (13beA)
1H NMR (300 MHz, CDCl3) d (ppm) 7.48–7.14 (m, 5H), 7.03 (d,
J = 4.5 Hz, 1H), 6.88 (dd, J = 1.5 Hz, 4.5 Hz, 1H), 6.81 (d, J = 1.5H,
1H), 4.05 (q, J = 6.9 Hz, 2H), 3.72 (m, 2H), 3.67 (ABq, J = 11.7 Hz,
41.7 Hz, 2H), 3.17 (t, J = 5.7 Hz, 1H), 2.77 (m, 2H), 2.65 (t,
J = 6.9 Hz, 2H), 1.85 (m, 2H), 1.55–1.21 (m, 4H), 1.43 (t, J = 6.6 Hz,
3H), 1.31 (m, 2H). MS (MALDI), m/z: 380.2 ([M+H]À).
2.4.12. (S)-3-(4-aminobutyl)-1-(4-fluorophenethyl)-7-isopropoxy-
4,5-dihydro-1H-benzo[e][1,4]diazepin-2(3H)-one (13bgB)
1H NMR (300 MHz, CDCl3) d (ppm) 7.20–6.81 (m, 7H), 4.48 (m,
1H), 3.72 (m, 2H), 3.67 (ABq, J = 11.7 Hz, 41.6 Hz, 2H), 3.17 (t,
J = 5.7 Hz, 1H), 2.77 (m, 2H), 2.65 (t, J = 6.9 Hz, 2H), 1.85 (m, 2H),
1.38 (s, J = 2.1 Hz,3H), 1.36 (s, J = 2.1 Hz, 3H). MS (ESI) m/z:
414.22 ([M+H]+).
2.4.13. (S)-3-(4-aminobutyl)-7-(hexyloxy)-1-phenethyl-4,5-dihydro-
1H-benzo[e][1,4]diazepin-2(3H)-one (13bkA)
1H NMR (300 MHz, CDCl3) d (ppm) 7.36–7.14 (m, 5H), 7.03 (d,
J = 8.4 Hz, 1H), 6.88 (dd, J = 3 Hz, 8.4 Hz, 1H), 6.80 (m, J = 3H, 1H),
3.96 (t, J = 6.3 Hz, 2H), 3.72 (m, 2H),3.69 (ABq, J = 11.4 Hz,
43.5 Hz, 2H), 3.17 (t, J = 5.7 Hz, 1H), 2.77 (m, 2H), 2.65 (t,
J = 6.9 Hz, 2H), 1.9 4–1.75 (m, 4H), 1.49–1.14 (m, 10H), 0.91 (t,
J = 6.6 Hz, 3H). MS (ESI) m/z: 438.12 ([M+H]+).
2.4.14. (S)-3-(4-aminobutyl)-1-(biphenyl-4-ylmethyl)-7-isopropoxy-
4,5-dihydro-1H-benzo[e][1,4]diazepin-2(3H)-one (13bgD)
1H NMR (300 MHz, CDCl3) d (ppm) 7.53–7.23 (m, 9H), 7.07 (d,
J = 8.4 Hz, 1H), 6.81 (dd, J = 2.7 Hz, 8.4 Hz, 1H), 6.69 (d, J = 2.7 Hz,

94
J.Y. Lee et al. / Bioorganic Chemistry 37 (2009) 90–95
O
R1
OMe
O
H
O
H
N
H
a
b
NO2
NO2
NO2
HO
Piv O
O
4
Piv
3
5a,5b,5c,5d
R1 = Bn(a), 4-Boc-amino butyl(b),
t-butoxy methyl(c), t-butyl propionate(d)
c
R1
O
O
H
H
N
R1
N
N
OMe
NH2
O
d
O
NH
NH
Piv
Piv
Piv
O
O
O
6a,6b,6c,6d
7a,7b,7c
8
Scheme 1. Preparation of four tetrahydro-1,4-benzodiazepine-2-one scaffolds. (a) trimethylacetyl chloride, TEA, DCM, 30 min RT. (b) Amino acid ester, NaBH(OAc)3, DCE/
DMF, 2 h, RT. (c) Pd/C, H2, MeOH, 3 h, RT. (d) AlMe3, toluene, 1 h, 0–25 °C.
R1
R1
R1
b
N
N
N
c
a
O
O
O
NH
NH
O
NH
R2
Piv
O
O
HO
10a, 10b, 10c
O
9a, 9b, 9c
11a, 11b, 11c
R1 = Bn(a), 4-Boc-amino butyl(b),
t-butoxy methyl(c)
d
R1
H
R1
N
N
O
R3
e
N
N
R3
R2
R2
O
O
13a, 13b', 13c'
12a, 12b, 12c
R1 = Bn(a), 4-Boc-amino butyl(b'),
t-butoxy methyl(c')
Scheme 2. Solid phase synthetic route of tetrahydro-1,4-benzodiazepine-2-one library. (a) PL-FDMP resin, 7a (7b or 7c), NaBH(OAc)3, DCE, 12 h, RT. (b) 5% KOH dioxane/H2O
(1:1) 24 h, RT. (c) R2–X, DBU, NMP/DMSO, 48 h, RT. (d) i, LiOtBu in THF, 1 h, RT. ii, R3–X in DMSO 24 h, RT. (e) TFA, DCM.
O
O
O
O
N
N
N
N
a
b
c
O
O
O
O
NH
NH
N
NH
R3
R2
R2
Piv
O
HO
O
O
10d
11d
13d
8
Scheme 3. Solution phase synthetic route of tetrahydro-1,4-benzodiazepine-2-one library. (a) 5% KOH dioxane/H2O, 40 min (1:1) (b) R2–X, DBU, NMP/DMSO, 4 h, RT (c) i,
LiOtBu in THF; ii, R3–X in DMSO, 10 h, RT.
procedure including extraction, washing and evaporation, all
products were purified by Quad3TM parallel purification system
with chloroform/methanol elution condition to give 162 final
library compounds. The purified products were characterized
randomly by 1H NMR and ESI mass.
All the compounds described here were evaluated for cellular
activity using a whole cell transcriptional assay that detects the
production of the beta lactamase enzyme induced by agonist stim-
ulation of the MC4 G-protein coupled receptor using a publishing
procedure [25]. In the assay, every compound was tested in three

J.Y. Lee et al. / Bioorganic Chemistry 37 (2009) 90–95
95
Table 2
Biological evaluation of the benzodiazepine derivatives.
R1
H
N
O
N
R3
R2
O
13
Compounds
R1
R2
R3
% Activity @ 10
lM
EC50
(l
M)
13beC
13bfD
13ahH
13alC
13afC
13amG
13aiE
4-Amino-butyl
4-Amino-butyl
Ethyl
Propyl
4-Methyl-phenethyl
4-Phenyl-benzyl
27
23
27
53
75
26
88
n.d.a
n.d.
n.d.
20.0
17.0
n.d.
Bn
Bn
Bn
Bn
Bn
2-Methoxy-ethyl
2-Methyl-naphtalene
propyl
3-Phenyl-benzyl
4-Fluoro-benzyl
2-Fluoro-phenethyl
4-Methyl-phenethyl
4-Methyl-phenethyl
2-Methoxy-benzyl
4-Methoxy-phenethyl
13.0
a
n.d., not determined.
[4] V.J. Hruby, D. Lu, S.D. Sharma, A.L. Castrucci, R.A. Kesterson, F.A. Al-Obeidi, M.E.
Hadley, R.D. Cone, J. Med. Chem. 38 (1995) 3435–3461.
[5] F. Mutulis, J. Kreicberga, S. Yahorava, I. Mutule, B.-J. Larisa, R. Muceniece, S.
Acena, S. Veiksina, R. Petrovska, J.E.S. Wikberg, Bioorg. Med. Chem. 15 (2007)
5787–5810.
times. Only the compounds showing over 50% activation at 10 lM
concentration were determined in EC50. The results are listed in Ta-
ble 2. Some of these compounds showed micromolar activity
including compounds 13alC (20.0
lM), 13afC (17.0 lM), and
[6] I. Gantz, H. Miwa, Y. Konda, Y. Shimoto, T. Tashiro, S.J. Watson, J. DelValle, T.
Yamada, J. Biol. Chem. 268 (1993) 15147–15179.
13aiE (13.0 M), for MC4R. The benzyl group at R1 position showed
l
better agonistic activity than others groups. Relatively small
groups are more tolerated at the R2 position, as shown by the com-
parison between compounds 13afC and 13ahC (2-methyl-naphta-
lene vs. propyl) and 13alC and 13aiE (2-methyl-naphtalene vs. 4-
fluoro-benzyl). A 4-methoxy-phenethyl group at R3 position dis-
played improved agonistic activity than 4-methyl-phenethyl
group. The results of screening and conformational analysis sup-
ported that the rationale of designing b-turn mimic non-peptide
small molecules using tetrahydro-1,4-benzodiazepin-2-one scaf-
fold could be valid.
[7] V. Chhajlani, J.E.S. Wikberg, FEBS 309 (1992) 417–420.
[8] V. Chhajlani, R. Muceniece, J.E.S. Wikberg, Biochem. Biophys. Res. Commun.
195 (1993) 866–873.
[9] L. Roselli-Rehfuss, K.G. Mountjoy, L.S. Robbins, M.T. Mortrud, M.J. Low, J.B.
Tatro, M.L. Entwistle, R.B. Simerly, R.D. Cone, Proc. Natl. Acad. Sci. USA 90
(1993) 8856–8860.
[10] I. Gantz, H. Miwa, Y. Konda, Y. Shimoto, T. Tashiro, S.J. Watson, J.D. Valle, T.
Yamada, J. Biol. Chem. 268 (1993) 15174–15179.
[11] K.G. Mountjoy, L.S. Robbins, M.T. Mortrud, R.D. Cone, Science 257 (1992)
1248–1251.
[12] G.S. Barsh, I.S. Farooqi, S. O’Rahilly, Nature 404 (2000) 644–651.
[13] M.W. Schwartz, S.C. Woods, D.P. Jr., R.J. Seeley, D.G. Baskin, Nature 404 (2000)
661–671.
[14] C.G. Joseph, K.R. Wilson, M.S. Wood, N.B. Sorenson, D.V. Phan, Z. Xiang, R.M.
Witek, C. Haskell-Luevano, J. Med. Chem. 51 (2008) 1423–1431.
[15] W. Jiang, J.A. Tran, F.C. Tucci, B.A. Fleck, S.R. Hoare, S. Markison, J. Wen, C.W.
Chen, D. Markison, J. Wen, C.W. Chem, D. Marinkovic, M. Arellano, A.C. Foster,
C. Chen, Bioorg. Med. Chem. Lett. 17 (2007) 6546–6552.
[16] R.K. Bakshi, Q. Hong, R. Tang, R.N. Kalyani, T. MacNeil, D.H. Weinberg, L.H.T.
Van der Ploeg, A.A. Patchett, R.P. Nargund, Bioorg. Med. Chem. Lett. 16 (2006)
1130–1133.
[17] C. Fotsch, N. Han, P. Arasasingham, Y. Bo, M. Carmouch, N. Chen, J. Davis, M.H.
Goldberg, C. Hale, F.-Y. Hsiegh, M.G. Kelly, Q. Liu, M.H. Norman, D.M. Smith, M.
Stec, N. Tamayo, N. Xi, S. Xu, A.W. Bannon, J.W. Baumagartner, Bioorg. Med.
Chem. Lett. 15 (2005) 1623–1627.
[18] L.N. Koikov, F.H. Ebetino, J.C. Hayes, D. Cross-Doersen, J.J. Knittel, Bioorg. Med.
Chem. Lett. 14 (2004) 4839–4842.
[19] R. Ruel, T.F. Herpin, L. Iben, G. Luo, A. Martel, H. Mason, G. Mattson, B. Poirier,
E.H. Ruediger, D. Shi, C. Thibault, G. Yu, L.A. Zimanyi, G.S. Poindexter, J.E.
Macor, Bioorg. Med. Chem. Lett. 13 (2003) 4341–4344.
4. Conclusions
A 162-member compound library was synthesized from tetra-
hydro-1,4-benzodizepin-2-one by an efficient solid and solution
phase synthetic route with three major points of diversity. We
present verification of our designing rationale, b-turn mimic
approach, with a screening of peptide-binding GPCR targeted li-
brary of tetrahydro-1,4-benzodizepin-2-one derivatives at MC4R.
Based on the results in this study, progress on the increase of ago-
nist potency by the introduction of key amino acid moiety of
melanocyte stimulating hormone ( -MSH) and selectivity against
subtype of MCR family is presently underway.
a-
a
[20] J.R. Holder, Z. Xian, R.M. Bauzo, C. Haskell-Luevano, J. Med. Chem. 45 (2002)
5736–5744.
[21] C. Haskell-Luevano, J.R. Holder, E.K. Monck, R.M. Bauzo, J. Med. Chem. 44
(2001) 2247–2252.
Acknowledgment
[22] C. Haskell-Luevano, S. Hendrata, C. North, T.K. Sawyer, M.E. Hadley, V.J. Hruby,
C. Dickinson, I. Gantz, J. Med. Chem. 40 (1997) 2133–2139.
[23] M.A. Bednarek, T.M. Fong, Expert Opin. Ther. Pat. (2004) 14, 327–336.
[24] R.S. Boyce, D.M. Duhl, Curr. Opin. Invest. Drugs (2004) 5, 1063–1071.
[25] D. Chianelli, Y.-C. Kim, D. Lvovskiy, T.R. Webb, Bioorg. Med. Chem. 11 (2003)
5059–5068.
This study was supported by a Grant from the institute of Med-
ical System Engineering (iMSE) in the GIST, Korea.
References
[26] I. Im, T.R. Webb, Y.D. Gong, J.I. Kim, Y.C. Kim, J. Comb. Chem. 6 (2004) 207–
213.
[27] T.R. Webb, L. Jiang, S. Sviridov, R.E. Venegas, A.V. Vlaskina, D. McGrath,
[1] J.D.A. Tyndall, B. Pfeiffer, G. Abbenante, D.P. Fairlie, Chem. Rev. 105 (2005)
793–826.
[2] C.H. –Luevano, T.K. Sawyer, S.H. Hendrata, C. North, L. Panahinia, M. Stum, D.J.
Staples, A.L. Castrucci, M.E. Handley, V.J. Hruby, Peptides 17 (1996) 995–1002.
[3] T.K. Sawyer, P.J. Sanfilippo, V.J. Hruby, M.H. Engel, C.B. Heward, J.B. Burnett,
M.E. Handley, Proc. Natl. Acad. Sci. USA 77 (1980) 5754–5758.
J. Tucker, J. Wang, A. Deschenes, R. Li, J. Comb. Chem.
710.
[28] S.L. Garland, P.M. Dean, J. Comput.-Aid. Mol. Des. 13 (1999) 469–483.
[29] D.A. Horton, G.T. Bourne, M.L. Smythe, Chem. Rev. (2003) 893–930.
9 (2007) 704–