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terminus region on JH inhibitory activity and to explore the
structure-activity relationship as well as to highlight the structural
requirements of AST analogs for further discovering new analogs
with higher activity and simpler structure, we designed two series
of ASTs analogs by modifying the N-terminus region of lead B1 via
replacing 1H-benzotriazole with aniline, heterocyclic amines or
other substitutes (the design strategy is shown in Fig.1). The target
compounds were prepared via a simpler synthetic route in the
compound 2 was directly reacted with 4-aminobenzotrifluoride
followed by cleavage and purification, and we successfully
obtained the target compound II12. Comparing to lead B1, the
route of II12 was shortened from nine steps with twice purification
to eight steps with once purification. Moreover, the dosage of HPLC
grade solvent used in target compounds preparation was reduced
by half.
Natural ASTs are susceptible to inactivation in vivo as a
consequence of hydrolysis by hemolymph and poor penetration
through the insects. The primarily hydrolytic site of the core
pentapeptide between the residues Y/F-X and the other hydrolytic
site such as G-L was also detected [6]. Analogs with modification
and replacement of amino acids at the hydrolysis not only showed
resistance to peptidases but also retained their bioactivities. In our
previous study [11], analog B1, a new mimic for Y/FXFGL in which Y
was replaced with 4-(1H-benzo[d][1,2,3]triazol-1-yl)-4-oxobuta-
noic acid, showed good inhibition activity without hydrolytic site.
To find new analogs with higher bioactivity and lower cost, further
optimization on B1 was carried out in present work. As shown in
Fig. 1, the first step of the strategy was to replace the N-terminus
1H-benzotriazole with aniline and heterocyclic amines (Series I).
The bioassay result indicated that analog I1 with N-terminus
containing benzene showed the best activity. We subsequently
selected I1 as the second lead, and a series of analogs (Series II)
with different substituents on benzene at the N-terminus were
designed and synthesized to explore the substituent effect on the
benzene ring.
According to the results shown in Table 1, most of the target
analogs showed obvious ability to inhibit JH biosynthesis by the
corpora allata (CA) of D. punctata in vitro at 1 Â10À5 mol/L. Analogs
showing inhibition rate more than 70% were chosen to determine
their IC50 values. The results indicated that these analogs had
different potencies owing to vary modification at N-terminus.
Series I (I1–I5), in which aniline and heterocyclic amines mimicked
the 1H-benzotriazole region, showed lower activity than B1.
However, analog I1 showed the best activity compared to other
analogs of series I and therefore was chosen to be the second lead
for further optimization. Fortunately, we found that the activities
of series II were higher than that of series I. Fig. 2 clearly exhibited
that different positions of substituents on the benzene ring at N-
terminus can lead to different activities. In particular, substituents
in the phenyl para-position were beneficial for activity compared
with meta- and ortho- substituents (p-F (II6) > m-F (II5) > o-F
(II4)). Meanwhile, in the same para-position on the phenyl ring, the
present work and their structures were confirmed by 1H NMR, 13
C
NMR and HRMS. Their inhibition of JH biosynthesis against
Diploptera punctata was evaluated in vitro and the preliminary
structure-activity relationship was analyzed. In addition, 3D-QSAR
studies were performed to explore the vital factors for designing
new IGRs candidate.
The synthesis of analog B1 (1) and analog II12 (2) was
illustrated in Scheme S1 (Supporting information). The rest
analogs were obtained using the same synthetic route (2). Leu
with resin was synthesized from Rink amide-AM resin (1 equiv.)
using the standard Fmoc/tBu chemistry and HBTU/HOBt protocol
[12,13]. 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-terminus Fmoc group
from the residues was accomplished with 20% piperidine in DMF
for 20 min. Compound 2 was obtained from compound 1 (8 equiv.)
which was coupled to the Leu with resin with Dic (4 equiv.) in DMF
for 4 h at room temperature [14]. The product with the resin was
prepared by the acyl reaction of compound 2 with 4-amino-
benzotrifluoride in DMF for 8 h, and then the product was cleaved
from the resin with TFA containing 5% water for 2.5 h at room
temperature. The crude compounds were purified on a C18
reversed-phase preparation column with a flow rate of 10 mL/min
using acetonitrile/water (50:50) at room temperature. UV detec-
tion was at 215 nm. The physical and identification data (melting
point, 1H NMR and HRMS of all target compounds and 13C NMR of
typical analogs I2 and II12) are given in the Supporting
information.
The method involves the acyl reaction and solid phase peptide
synthesis. Using the reported method [11], the lead B1 was
obtained totally in nine steps and twice purification. The key
intermediate compound 2 was cleaved with TFA and then the
solution was purified on a C18 reversed-phase preparative column
and freeze-dried in vacuum. The crude target compound B1 was
obtained by repeating above purification steps. In the present work
with purpose to simplify the route and reduce the cost, the
Fig. 1. Design strategy of the N-terminus modified B1 analogs.
Please cite this article in press as: M. Wang, et al., Exploring the N-terminus region: Synthesis, bioactivity and 3D-QSAR of allatostatin analogs
Wang, Meizi
