Design, synthesis and biological activity of peptidomimetic analogs of insect allatostatins

Article abstract of DOI:10.1016/j.peptides.2010.10.016

Allatostatins (ASTs) comprise a family of insect neuropeptides isolated from cockroaches and found to inhibit the production of juvenile hormone (JH) by the corpora allata (CA). For this reason, the ASTs can be regarded as possible IGR candidates for pest control. Six peptidomimetic analogs according to the C-terminal pentapeptide of ASTs were prepared by solid-phase organic synthetic methods in an attempt to obtain new simple substitution agents. Assays of inhibition of JH biosynthesis in vitro by corpora allata from the cockroach Diploptera punctata showed that the activity of analog I (IC₅₀: 0.09 μM) was more active than that of the C-terminal pentapeptide (Tyr-Xaa-Phe-Gly-Leu-NH₂, IC₅₀: 0.13 μM) it mimicked and the activity of the analog II (IC₅₀: 0.13 μM) proved roughly equivalent to the C-terminal pentapeptide. The results indicate that a new simple mimicry for Tyr-Xaa-Phe-Gly has been discovered; analog I may be a novel compound candidate for potential IGRs. This study will be useful for the design of new AST analogs for insect management.

Full text of DOI:10.1016/j.peptides.2010.10.016

Peptides 32 (2011) 581–586
Contents lists available at ScienceDirect
Peptides
journal homepage: www.elsevier.com/locate/peptides
Design, synthesis and biological activity of peptidomimetic analogs of insect
allatostatins
Yong Xiea, Zhen Peng Kaia, Stephen S. Tobeb,∗, Xi Le Denga, Yun Linga, Xiao Qin Wua,
Juan Huanga, Li Zhanga, Xin Ling Yanga,∗
a Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, PR China
b Department of Cell and Systems Biology, University of Toronto, 25 Harbord St., Toronto, ON, Canada M5S 3G5
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 August 2010
Received in revised form 11 October 2010
Accepted 12 October 2010
Available online 20 October 2010
Allatostatins (ASTs) comprise a family of insect neuropeptides isolated from cockroaches and found to
inhibit the production of juvenile hormone (JH) by the corpora allata (CA). For this reason, the ASTs can
be regarded as possible IGR candidates for pest control. Six peptidomimetic analogs according to the
C-terminal pentapeptide of ASTs were prepared by solid-phase organic synthetic methods in an attempt
to obtain new simple substitution agents. Assays of inhibition of JH biosynthesis in vitro by corpora allata
from the cockroach Diploptera punctata showed that the activity of analog I (IC50: 0.09 ␮M) was more
active than that of the C-terminal pentapeptide (Tyr–Xaa–Phe–Gly–Leu–NH2, IC50: 0.13 ␮M) it mimicked
and the activity of the analog II (IC50: 0.13 ␮M) proved roughly equivalent to the C-terminal pentapeptide.
The results indicate that a new simple mimicry for Tyr–Xaa–Phe–Gly has been discovered; analog I may
be a novel compound candidate for potential IGRs. This study will be useful for the design of new AST
analogs for insect management.
Keywords:
Allatostatins
Peptide analogs
Peptidomimetics
Juvenile hormone
IGRs
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
non-amino acid replacement of specific amino acids that can inhibit
JH biosynthesis and display resistance to degradation. In particu-
The juvenile hormones (JH), produced by the corpora allata, play
a central role in both metamorphosis and reproduction in most
insect species [1,21]. Cockroach-type allatostatins (ASTs), a fam-
ily of insect neuropeptides isolated from the cockroach Diploptera
punctata [6,15,22,23], which inhibit the biosynthesis of JH by cor-
pora allata in vitro, were chosen as possible IGR candidates for pest
management [10,11]. However, native ASTs have some shortcom-
ings such as lack of effect in vivo, rapid degradation in vivo, and high
production cost [10]. Therefore, it is essential to discover new AST
analogs for pest control.
Earlier structure–activity studies have shown that the C-
terminal pentapeptide Tyr–Xaa–Phe–Gly–Leu–NH2 (Xaa = Ala, Asn,
Gly, Ser) is the minimum sequence capable of eliciting inhibi-
tion of JH production [13,18]. Hayes et al. [8] suggested that
the most important side chain of Dippu-AST 5 were Leu8 fol-
lowed by Phe6 and Tyr4 using the Ala scanning technique. Piulachs
et al. [14], Garside et al. [7] and Nachman et al. [12,13] also
synthesized Dippu-AST 5 or Dippu-AST 6 analogs by employing
lar, a pentapeptide analog (AST(b)␸2) containing the cyclopropyl
ring at the C-terminus and hydrocinnamic acid at the N-terminus
was synthesized by Nachman and co-workers (see ref. [12]), and
provided compelling evidence for the value of the peptidomimetic
approach in the development of compounds regulating physio-
logical processes such as JH production. In our previous studies
[10,11], we used the C-terminal pentapeptide region as the lead
compound and replaced Tyr/Phe residues in pentapeptide with
different aromatic acids, fatty acids or dicarboxylic acids. We sug-
gested that a potent AST analog should contain an aromatic group,
an appropriate length of linker and a Gly residue in the Y/FX
region.
In this study, we designed and synthesized a series of pep-
tidomimetic analogs only containing Leu, in an attempt to obtain
new simple substitution agents for novel IGRs candidates. As
shown in Fig. 1, the points of synthetic modification included (1)
replacement of Gly with succinic acid, (2) replacement of the Asp
side chain with succinic acid, 4-(1H-benzo[d][1,2,3]triazol-1-yl)-
4-oxobutanoic acid, or no substitution, (3) conservation of the
benzene ring and alteration of the backbone of Tyr and Phe. All
newly synthesized analogs were assayed for their ability to inhibit
JH biosynthesis in the cockroach in vitro; we describe here the syn-
thesis and biological activity.
∗
Corresponding authors. Tel.: +86 10 62732223 or +1 416 9783517.
E-mail addresses: stephen.tobe@utoronto.ca (S.S. Tobe),
yangxl@cau.edu.cn (X.L. Yang).
0196-9781/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2010.10.016

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Y. Xie et al. / Peptides 32 (2011) 581–586
Fig. 1. Design of peptidomimetic analogs. The key design features were the conservation of Leu5 and the benzene ring of Tyr1 and Phe3, the replacement of Gly4 with succinic
acid, and the modification of the backbone.
2. Experimental details
(2.2 equiv.) in absolute ethanol at room temperature over 20 min,
then heated at refluxed for 25 min, cooled and the solvent evapo-
ratedundervacuum. Theresiduewas dissolvedin MeOH and stirred
at 0 ◦C. Excess NaBH4 dissolved in methanol was added cautiously
over 40 min, then the mixture was stirred at room temperature
1.5 h. The solution was taken up in water and extracted with DCM
(2 ml × 30 ml). The organic extracts were dried with anhydrous
sodium sulfate, filtered and concentrated to give the target com-
pounds.
2.1. Synthesis
2.1.1. Material
Rink Amide-Am resin (0.68 mmol/g substitution), HOBt
(O-benzotriazole-N,N,Nꢀ,Nꢀ-tetramethyl-uronium-hexafluoro-
phosphate), HBTU (1-hydrozybenzotriazole anhydrate), DIEA,
TFA (trifluoroacetic acid), Dic (N,Nꢀ-diisopropylcarbodiimide)
and Fmoc–Leu–OH were purchased from GL Biochem (Shang-
hai) Ltd. HPLC grade DMF (N,N-dimethyl-formamide), DCM
(dichloromethane), acetonitrile were purchased from DIMA
Technology Inc. (USA), benzaldehyde, butane-1,4-diamine,
pentane-1,5-diamine, hexane-1,6-diamine, NaBH4, phenol,
thioanisole, 1H-benzotriazole and succinic anhydride, DPPA
(diphenylphosphorazidate) were purchased from Alfa Aesar, USA.
2.1.2.1. N,Nꢀ-dibenzyl-1,2-diaminoethane (1a). Yield: 76%; light yel-
low liquid; 1H NMR (CDCl3, 300 MHz): ıH (ppm) = 2.73 (4H, s,
CH2N), 3.78 (4H, s, CH2Ar), 7.23–7.35 (10H, m, ArH).
2.1.2.2. N,Nꢀ-dibenzyl-1,4-diaminobutpane (1b). Yield: 72%; light
yellow liquid; 1H NMR (CDCl3, 300 MHz): ıH (ppm) = 1.53–1.57
(4H, m, CH2), 2.71 (4H, d, J = 13.65 Hz, CH2N), 3.78 (4H, s, CH2Ar),
7.22–7.34 (10H, m, ArH).
2.1.2. General procedure for synthesis of compound 1
Compound 1 group was synthesized from diamines using reduc-
tion chemistry described by Ross et al. [16]. The diamine (1 equiv.)
was added dropwise to a stirred solution of the benzaldehyde
2.1.2.3. N,Nꢀ-dibenzyl-1,5-diaminopentane (1c). Yield: 72%; white
solid; mp 25–27 ◦C; 1H NMR (CDCl3, 300 MHz): ıH (ppm) =

Y. Xie et al. / Peptides 32 (2011) 581–586
583
1.31–1.39 (2H, m, CH2), 1.47–1.56 (4H, m, CH2), 2.63 (4H, t,
J = 7.18 Hz, CH2N), 3.78 (4H, s, CH2Ar), 7.22–7.35 (10H, m, ArH).
(1H, s, CONH2), 7.20–7.39 (10H, m, ArH), 7.43 (1H, s, CONH2), 7.96
(1H, d, J = 8.40 Hz, CONH), 11.91 (1H, s, COOH); ESI-MS: m/z 603.3
[M+Na]+ (calcd MNa+: 603.33).
2.1.2.4. N,Nꢀ-dibenzyl-1,6-diaminohexane (1d). Yield: 69%; light
yellow liquid; 1H NMR (CDCl3, 300 MHz): ıH (ppm) = 1.30–1.35 (4H,
m, CH2), 1.51 (4H, t, J = 6.66 Hz, CH2), 2.62 (4H, t, J = 7.17 Hz, CH2N),
3.78 (4H, s, CH2Ar), 7.24–7.33 (10H, m, ArH).
2.1.4.3. Analog V. Yield: 97%; 1H NMR (DMSO–d6, 300 MHz): ıH
(ppm) = 0.82 (3H, d, J = 6.24, CH3), 0.86 (3H, d, J = 6.39, CH3),
1.09–1.21 (2H, m, CH2), 1.36–1.49 (6H, m, CH2), 1.52–1.57 (1H, m,
CH), 2.41–2.59 (8H, m, CH2CO), 3.17–3.24 (4H, m, CH2N), 4.20–4.28
(1H, m, CH), 4.48–4.58 (4H, m, CH2Ar), 7.05 (1H, s, CONH2),
7.15–7.36 (10H, m, ArH), 7.37 (1H, s, CONH2), 7.98 (1H, d, J = 8.48 Hz,
CONH), 11.92 (1H, s, COOH); ESI-MS: m/z 617.4 [M+Na]+ (calcd
MNa+: 617.34).
2.1.3. General procedure for synthesis of compound 2
Compound 1 was added to a solution of succinic anhydride
(2.2 equiv.) in DCM (50 ml). The reaction mixture was stirred at
room temperature overnight, and then filtered to give the products.
2.1.3.1. Compound 2a. Yield: 95%; 1H NMR (DMSO–d6, 300 MHz):
ıH (ppm) = 2.42–2.56 (8H, m, CH2CO), 3.30–3.41 (4H, m, CH2N),
4.46–4.58 (4H, m, CH2Ar), 7.13–7.38 (10H, m, ArH), 11.98 (2H, s,
COOH).
2.1.4.4. Analog VI. Yield: 95%; 1H NMR (DMSO–d6, 300 MHz): ıH
(ppm) = 0.82–0.88 (6H, m, CH3), 1.17–1.20 (4H, m, CH2), 1.38–1.52
(6H, m, CH2), 1.52–1.58 (1H, m, CH), 2.40–2.60 (8H, m, CH2CO),
3.17–3.24 (4H, m, CH2N), 4.21–4.32 (1H, m, CH), 4.49–4.59 (4H, m,
CH2Ar), 7.07 (1H, s, CONH2), 7.18–7.40 (10H, m, ArH), 7.41 (1H, s,
CONH2), 7.98 (1H, d, J = 8.45 Hz, CONH), 11.92 (1H, s, COOH); ESI-
MS: m/z 609.4 [M+H]+ (calcd MH+: 608.36).
2.1.3.2. Compound 2b. Yield: 96%; 1H NMR (DMSO–d6, 300 MHz):
ıH (ppm) = 1.45–1.51 (4H, m, CH2), 2.41–2.59 (8H, m, CH2CO), 3.20
(4H, s, CH2N), 4.47 (2H, s, CH2Ar), 4.55 (2H, s, CH2Ar), 7.19–7.36
(10H, m, ArH), 11.90 (2H, s, COOH).
2.1.5. Synthesis of analog I
DPPA (1 equiv.) was added to a stirred solution of analog III
(1 equiv.), 1H-benzotriazole (1 equiv.) and triethylamine in abso-
lute DMF at 0 ◦C for 6 h, then heated to room temperature for 20 h,
the solvent evaporated under vacuum, the residue dissolved in
diethyl ether and filtered to give the products [9,17]. The crude
compounds were purified on a C18 reversed-phase preparative col-
umn with a flow rate of 10 ml/min using acetonitrile/water (50:50).
UV detection was at 215 nm. Yield: 70%; 1H NMR (DMSO–d6,
300 MHz): ıH (ppm) = 0.81–0.88 (6H, m, CH3), 1.40–1.50 (2H, m,
CH2), 1.55–1.59 (1H, m, CH), 2.42–2.56 (8H, m, CH2CO), 3.30–3.42
(4H, m, CH2N), 4.18–4.23 (1H, m, CH), 4.49–4.59 (4H, m, CH2Ar),
7.09 (1H, s, CONH2), 7.17–7.40 (14H, m, ArH), 7.41 (1H, s, CONH2),
8.02 (1H, d, J = 8.46, CONH); ESI-MS: m/z 654.3 [M+H]+ (calcd MH+:
654.33).
2.1.3.3. Compound 2c. Yield: 93%; 1H NMR (DMSO–d6, 300 MHz):
ıH (ppm) = 1.09–1.23 (2H, m, CH2), 1.38–1.49 (4H, m, CH2),
2.41–2.61 (8H, m, CH2CO), 3.17–3.25 (4H, s, CH2N), 4.49 (2H, s,
CH2Ar), 4.56 (2H, s, CH2Ar), 7.17–7.44 (10H, m, ArH), 11.72 (2H,
s, COOH).
2.1.3.4. Compound 2d. Yield: 91%; 1H NMR (DMSO–d6, 300 MHz):
ıH (ppm) = 1.17–1.19 (4H, m, CH2), 1.39–1.47 (4H, m, CH2),
2.39–2.28 (8H, m, CH2CO), 3.19 (4H, t, J = 7.06 Hz, CH2N), 4.50 (2H,
s, CH2Ar), 4.56 (2H, s, CH2Ar), 7.18–7.38 (10H, m, ArH), 11.90 (2H,
s, COOH).
2.1.4. General procedure for synthesis of analog III–VI
Leu with resin (compound 3) was synthesized from Rink Amide-
AM resin (1 equiv.) using the standard Fmoc/tBu chemistry and
HBTU/HOBt protocol [2–4]. Fmoc–Leu–OH (3 equiv.) was activated
with HBTU (3 equiv.), HOBt (3 equiv.) and DIEA (6 equiv.) in DMF for
5 min, and couplings were run for 4 h. Removal of the N-terminal
Fmoc group from the residues was accomplished with 20% piperi-
dine in DMF for 20 min. Compound 2 (8 equiv.) was coupled to the
Leu with resin with Dic (4 equiv.) in DMF for 4 h at room tem-
perature [5]. The product was cleaved from the resin with TFA
containing 2.5% phenol, 2.5% thioanisole and 5% water for 2 h at
room temperature. The resin suspension was filtered and volatile
reagents were removed in vacuo on a rotary evaporator at 40 ◦C.
Phenol and thioanisole were removed by a diethyl ether extrac-
tion. The crude compounds were purified on a C18 reversed-phase
preparation column with a flow rate of 10 ml/min using acetoni-
trile/water (50:50) at room temperature. UV detection was at
215 nm.
2.1.6. Synthesis of analog II
Compound 4 was synthesized from compound 3 using the
symmetrical anhydride method. Succinic anhydride (4 equiv.) was
added to compound 3 (1 equiv.) in DMF for 12 h at room tem-
perature. Compound 4 was activated with HATU (3 equiv.), HOAt
(3 equiv.) and DIEA (8 equiv.) in DMF for 5 min, then compound 1a
was added, and coupling was run for 4 h. The product was cleaved
from the resin with TFA containing 2.5% phenol, 2.5% thioanisole
and 5% water for 2 h at room temperature. The crude compounds
were purified on a C18 reversed-phase preparative column with a
flow rate of 10 ml/min using acetonitrile/water (50:50). UV detec-
tion was at 215 nm. Yield: 91%; 1H NMR (DMSO–d6, 300 MHz): ıH
(ppm) = 0.81–0.90 (6H, m, CH3), 1.42–1.50 (2H, m, CH2), 1.55–1.58
(1H, m, CH), 2.43–2.54 (4H, m, CH2CO), 3.30–3.41 (4H, m, CH2N),
3.77 (1H, s, NH), 4.19–4.22 (1H, m, CH), 4.49–4.58 (4H, m, CH2Ar),
6.89 (1H, s, CONH2), 7.18 (1H, s, CONH2), 7.19–7.38 (10H, m, ArH),
7.83 (1H, d, J = 7.99 Hz, CONH); ESI-MS: m/z 453.3 [M+H]+ (calcd
MH+: 453.28).
2.1.4.1. Analog III. Yield: 95%; 1H NMR (DMSO–d6, 300 MHz): ıH
(ppm) = 0.82–0.89 (6H, m, CH3), 1.41–1.50 (2H, m, CH2), 1.55–1.59
(1H, m, CH), 2.43–2.56 (8H, m, CH2CO), 3.31–3.43 (4H, m, CH2N),
4.18–4.23 (1H, m, CH), 4.49–4.59 (4H, m, CH2Ar), 7.09 (1H, s,
CONH2), 7.17–7.38 (10H, m, ArH), 7.41 (1H, s, CONH2), 8.01 (1H, d,
J = 8.46 Hz, CONH), 11.96 (1H, s, COOH); ESI-MS: m/z 553.3 [M+H]+
(calcd MNa+: 552.29).
2.2. Bioassays in vitro
All radiochemical assays for JH biosynthesis were performed
using individual CA from day 7 mated females. Compounds were
dissolved in medium 199 for assay as described previously [19,20].
Compounds were used in the bioassay on the same day of sample
preparation. The solutions were discarded at the end of each day.
Rates of JH release were determined using the modified in vitro
radiochemical assay [19,20]. This assay measures the incorporation
2.1.4.2. Analog IV. Yield: 94%; 1H NMR (DMSO–d6, 300 MHz): ıH
(ppm) = 0.81–0.87 (6H, m, CH3), 1.39–1.52 (6H, m, CH2), 1.53–1.61
(1H, m, CH), 2.41–2.57 (8H, m, CH2CO), 3.20–3.26 (4H, m, CH2N),
4.21–4.33 (1H, m, CH), 4.51 (2H, s, CH2Ar), 4.57 (2H, s, CH2Ar), 7.10

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Y. Xie et al. / Peptides 32 (2011) 581–586
Scheme 1. Synthesis of analogs I–VI. Reagents: (a) benzaldehyde, CH3CH2OH, room temperature, 2 h, then NaBH4, 0 ◦C, 1.5 h; (b) succinic anhydride, DMF, room temperature,
12 h; (c) 20% piperidine in DMF, room temperature, 20 min; (d) Fmoc–Leu–OH, HBTU, HOBt, DIEA, room temperature, 4 h; (e) compound 2a–2d, Dic, room temperature, 4 h;
(f) TFA, 2.5% phenol, 2.5% thioanisole, 5% water, room temperature, 2 h; (g) 1H-benzotriazole, DPPA, 0 ◦C, 6 h, then room temperature, 20 h; (h) compound 1a, HATU, HOAt,
DIEA, room temperature, 4 h.
Tyr–Xaa–Phe–Gly. The key design feature was that the Leu5 and the
benzene ring of Tyr1 and Phe3 were conserved, Gly4 was replaced
with succinic acid, and the backbone was modified (Fig. 1). In the
first, the Tyr–Xaa–Phe–Gly was replaced with compound 2a, 2b, 2c
and 2d, containing the benzene ring and a carboxylic acid group
(Scheme 1). In the second, the last two analogs were designed to
replace the carboxylic acid group of 2a. In analog I, the group was
replaced by amidation, and in analog II, the group was removed by
elimination (Scheme 1).
of radiolabeled methionine into JH III in the final step of biosynthe-
sis by CA maintained in vitro. CA were incubated for 3 h in 100 ␮L
of medium 199 (GIBCO, 1.3 mM Ca2+, 2% Ficoll, methionine-free)
containing L [14C-S-methyl] methionine (40 ␮M, specific radioac-
tivity 1.48–2.03 GBq/mmol (Amersham)). Samples were extracted
and JH release determined. Each data point on the dose–response
figure represents replicate incubations of 10–27 experimental CA
compared to control CA (i.e. no analog added).
The analogs were synthesized using solid-phase organic syn-
thesis employing the Fmoc protecting group strategy. Leu with
resin was synthesized from rink amide-am resin using HBTU/HOBt
protocol. For avoiding the generation of OBt esters, analogs III–VI
containing a carboxylic acid group were synthesized using Dic as
the coupling reagent instead of HBTU/HOBt [5]. In step g (Scheme 1),
we planned the acylation between 1H-benzotriazole and analog
III by exploring several coupling reagents, and we found that the
coupling could not be accomplished by HBTU/HOBt, HATU/HOAt
or Dic. So analog I was synthesized using azide method. In step
h (Scheme 1), analog II was synthesized using HATU/HOAt for
enhancing the coupling yields and to shorten coupling times.
3. Results and discussion
3.1. Design and synthesis of analogs
Based on previous studies [11], the most important amino acids
for the ability of the C-terminal pentapeptide to inhibit JH biosyn-
thesis were Gly4 and Leu5. Tyr1 and Xaa2 could be replaced by an
aromatic group and an appropriate length of linker. Phe3 could be
replaced with an Aic residue [13]. We discovered that Gly4 could be
substituted by a linear group, such as succinic acid (unpublished).
In summary, it appears that the Leu5 was the most important
residue for activity. Therefore, we designed a new mimicry for

Y. Xie et al. / Peptides 32 (2011) 581–586
585
100
80
IC50= 0.09 µM
100
50
0
IC50=0.13 µM
60
40
20
-8
-7
-6
-5
-4
0
-9
-8
-7
-6
-5
-4
-50
log [pentapeptide] M
log [ analog I ] M
Fig. 2. Effect of pentapeptide and analog I on JH release by CA from day 7 mated female Diploptera in vitro as a function of dose. Each point represents mean SEM. N = 10–20.
Table 1
phase organic chemistry, and their biological activities assessed.
Compared with the lead compound (C-terminal pentapeptide), the
Inhibition of JH release by corpora allata of Diploptera punctata.
Analog
IC50
Maximal responsea
analog I (IC50: 0.09 ␮M) showed excellent ability to inhibit JH pro-
duction in vitro. Analog II (IC50: 0.13 ␮M) proved roughly equivalent
to the C-terminal pentapeptide. Our results suggest that a novel
mimicry for Tyr–Xaa–Phe–Gly has been discovered and the analog
I family may be potential compounds for IGR development.
C-terminal pentapeptide
I
II
III
IV
V
0.13 ␮M
0.09 ␮M
0.13 ␮M
9.82 ␮M
–
72.3%
90.2%
85.0%
62.7%
19.8%
92.1%
86.8%
1.15 ␮M
2.63 ␮M
Acknowledgements
VI
IC50 values determined from dose–response curves whose data points represent
means of more than eight replicates.
We are grateful for financial support from the China 973 Pro-
gram (2010CB126104), the NSFC (20972185, 20672138), and the
Natural Sciences and Engineering Research Council of Canada.
a
Maximal response represents the inhibition of JH release from CA at 10−5 M.
3.2. Effects of analogs on JH biosynthesis in vitro
References
The analogs (I–VI) were evaluated for their ability to inhibit
biosynthesis of JH by the corpora allata (CA) of the cockroach D.
punctata, maintained in vitro, by measuring the incorporation of
radiolabeled methionine. The rates of JH biosynthesis with the
analogs were compared to those in the presence of the C-terminal
pentapeptide alone (control) (Table 1). Analog I (IC50: 0.09 ␮M)
showed higher activity than that of the C-terminal pentapeptide
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(IC50: 0.13 ␮M) it mimicked and the activity of analog II (IC50
:
0.13 ␮M) proved roughly equivalent to the C-terminal pentapep-
tide. Comparison of the dose response curve of analog I on JH release
with the C-terminal pentapeptide (Fig. 2), revealed that analog I and
this pentapeptide showed similar activity at high concentrations
(10−6 M and 10−5 M). In particular, analog I showed better results
when the concentration was decreased to below 10−7 M. This result
demonstrates that analog I had a significant effect in vitro when
compared to the pentapeptide.
Analog III (IC50: 9.82 ␮M) and IV containing a hydrophilic car-
boxyl group, showed low potencies and only 62.7% and 19.8%
inhibition of JH release by CA at 10−5 M. Analog V and VI, synthe-
sized from 1,5-diaminepentane, and 1,6-diaminehexane proved to
have allatostatin-like activity, resulting in inhibition of JH biosyn-
thesis with an IC50 of 1.15 ␮M and 2.63 ␮M; they showed 92.1%
and 86.8% of inhibition of JH release at 10−5 M. From a compari-
son of results from analogs III–VI, it may be seen that differences in
activity may be attributable to differences in length of the diamine,
and this result is consistent with the HQASR model [11]. Comparing
analogs III, IV, V, VI to I and II, it appears that the carboxyl group
had a negative impact on activity.
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4. Conclusion
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In the present study, to obtain new simple substitution agents
of the active pentapeptide core of the FGLa allatostatins, a series
of novel peptidomimetic analogs were designed by substitution of
the Tyr–Xaa–Phe–Gly with peptidomimetics, synthesized by solid

586
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