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J.-L. Huang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 927–930
Scheme 1.
Scheme 2.
2-cyclohexen-1-one 3 (IC50=30 nM).8 Further SAR
studies suggested that a C-2 substitution on 3 has a
large effect on inhibition potency, while a C-5 substitu-
tion has minimal effect.9
The IC50 values for compounds 7 and 10 are 0.04 and
10 mM, respectively.16 This result shows that compound
10 is 250-fold less potent than compound 7. Apparently,
the presence of an amide functionality at the C-3 posi-
tion of 10 is highly detrimental to the inhibition
potency. This observation is consistent with our pre-
vious SAR studies9 and might be attributed to the
resulting conformational-constrained structure that
prevents tight binding with the enzyme active site. Hav-
ing established that compound 7 is an effective in vitro
4-HPPD inhibitor, we turned our attention to investi-
gating the possible metabolic fate of compound 7 in
vivo. When compound 7 was dissolved in D2O at room
temperature for 24 h, more than 50% of 7 was hydro-
lyzed back to compound 1, presumably due to the
intrinsic electrostatic repulsion between the 2-acyl oxy-
gen atom and the two 1,3-diketone oxygens.16 Further
basic hydrolysis of the ester functional group at the C-5
position gave the active GA20 3b-hydroxylase inhibitor
2. Thus, it is reasonable to assume that compound 7 will
be nonenzymatically hydrolyzed or enzymatically
degradated in vivo to give 1 and subsequently into its
corresponding acid 2, as indicated in Scheme 4.
In this paper, we report our attempt to design and syn-
thesize a potent inhibitor for 4-HPPD as a potential
herbicide which would block the biosynthesis of
plastoquinones and tocopherols in plants, and when
metabolized in vivo could become a potent GA20 3b-
hydroxylase inhibitor and act as a plant growth
regulator by interfering with gibberellin biosynthesis.
The molecules designed and synthesized as potential
sequential inhibitors are compounds 7 and 10, as depic-
ted in Scheme 3. The approach began with base-cata-
lyzed cyclization of keto ester 4 via intramolecular
Dieckmann condensation to afford enol 5,10 which upon
treatment with cyclopropanecarbonyl chloride under
basic conditions gave rise to enol ester 6. Preparation of
1 was accomplished by the cyanide-catalyzed iso-
merization11 of enol ester 6 using triethylamine as a base
in methylene chloride. Further esterification of 1 with
cyclopropanecarbonyl chloride under basic conditions
yielded 2,3,5-trisubstituted cyclohexane-1,3-dione deri-
vative 7, quantitatively. When treated with oxalyl
chloride at room temperature, enol 1 was converted to
the corresponding chloride 8.12 Amination of 8 with
liquid ammonia in methylene chloride at room tem-
perature gave the expected enamine 9.13 Final N-acyla-
tion of 9 with cyclopropanecarbonyl chloride as
described previously afforded amide 10.
Although further investigations of compound 7 as a
sequential inhibitor for 4-HPPD and GA20 3b-
hydroxylase in vivo are needed, the results presented
here demonstrate that compound 7 is a strong 4-HPPD
inhibitor and it provides a good example of how to
develop biologically active molecules with multi-func-
tional purposes. Conceptually, successive inhibition of
two enzymes in plants would offer the advantage of
using a lower amount of the active ingredients for the
same effect, as compared to selective inhibition of a
single enzyme. Furthermore, it is less likely that the
treated plants would develop resistance to the applied
herbicides or PGRs.
In summary, we have designed and synthesized a potent
4-hydroxyphenylpyruvate dioxygenase inhibitor 7 with
IC50 of 40 nM by functionalizing the 2-, 3-, and 5-posi-
tions of cyclohexane-1,3-dione. After metabolism, we
expect compound 7 will have the potential to serve as a
potent plant growth regulator by inhibiting the activity
of a second enzyme, GA20 3b-hydroxylase.
The synthesized compounds were evaluated in vitro for
inhibition activity against 4-HPPD purified from pig
liver14 by the spectrophotometric enol-borate method.15
Huang, Jian-Lin
