1.  Introduction {#sec1-molecules-22-01568}
===============

Pine wilt disease is a serious problem in the pine forests of several Asian and European countries \[[@B1-molecules-22-01568]\].  After pine wilt disease was first reported at Mt. Gumjung, Busan, this disease spread to several areas of the middle and southern parts of the Korean peninsula \[[@B2-molecules-22-01568]\], leading to 1.74 million dead pine trees in 2015 \[[@B3-molecules-22-01568]\].  Several kinds of methods have been developed for the control of pine wilt disease in Korea, and the Korean government has invested significant financial and human resources for several years to attempt to stop the spread of the disease \[[@B2-molecules-22-01568]\].  Felling and fumigation of dead trees with metham sodium is used to kill the larvae of *Monochamus alternatus* or *Monochamus saltuarius* and the pine wood nematode.  Aerial spraying with thiacloprid has been used to manage the adults of *M. alternatus* or *M. saltuarius*.  However, the application of aerial spraying has decreased annually because of concerns over environmental contamination and toxicity to non-target organisms.  Another control method is trunk injection of nematicides such as avamectin or emamectin benzoate, and this method is considered to be safer for the environment since the chemical agent is not widely dispersed.  Avamectin and emamectin benzoate have been widely used for several years in Korea.  Long and frequent usage of these nematicides increases the possibility of the emergence of resistance in the pine wood nematode, although there has been no research in this area as of yet. 

Concern for the occurrence of resistant strains increases the need for the development of new and safer types of pine wood nematode control agents.  Natural products derived from plants or microorganisms and their analogues provide good sources of lead compounds to develop new pest control agents.  For this purpose, nematicidal activities of plant-derived natural products and their analogues against the pine wood nematode have been investigated in several studies \[[@B4-molecules-22-01568],[@B5-molecules-22-01568],[@B6-molecules-22-01568],[@B7-molecules-22-01568],[@B8-molecules-22-01568],[@B9-molecules-22-01568]\]. 

In this study, we investigated the nematicidal activities of 98 3-acyltetramic acid analogues against the pine wood nematode, and developed their structure-activity relationships and physicochemical property--nematicidal activity relationships, in an attempt to find new alternatives for conventional nematicides. 

2.  Results and Discussion {#sec2-molecules-22-01568}
=========================

2.1.  Nematicidal Activities of 3-Acyltetramic Acids {#sec2dot1-molecules-22-01568}
---------------------------------------------------

The nematicidal activities of 3-acyltetramic acid analogues ([Figure 1](#molecules-22-01568-f001){ref-type="fig"} and [Figure 2](#molecules-22-01568-f002){ref-type="fig"}) and emamectin benzoate are shown in [Table 1](#molecules-22-01568-t001){ref-type="table"}.  Among the test compounds, **1c**, **1d**, **1h**, **2c**, **2f**, **2g**, and **2k** showed very strong nematicidal activities against the pine wood nematode, and mortalities for compounds **1c**, **2c**, **2f** and **2g** were 99.6%, 100%, 95.8% and 98.9% at a 10 μg/mL concentration, respectively, while compounds **1d**, **1h**, and **2k** showed slightly lower nematicidal activities (85.8%, 88.0% and 57.2%) at a 10 μg/mL concentration, respectively.  However, other 3-acyltetramic acid analogues showed less than 30% nematicidal activity.  The nematicidal activity of 3-acyltetramic acids with ≥50% mortality at 10 μg/mL was tested at lower concentrations.  Nematicidal activities of compounds **1c** and **2g** were 96.2% and 96.4% at a 5 μg/mL concentration, but their activities reduced to 15.0% and 6.3% at a 2.5 μg/mL concentration, respectively.  Compounds **1d** and **2f** showed 80.2% and 76.4% nematicidal activities against the pine wood nematode at a 5 μg/mL concentration, but showed less than 40% mortality at a 2.5 μg/mL concentration, respectively.  Other 3-acyltetaramic acids analogues showed \<60% mortality at a 5 μg/mL concentration.  This outcome is better than the nematicidal activities of emamectin benzoate, widely used for the prevention of pine wilt disease in Korea, which were 32.3%, 28.2% and 24.1% at 10, 5, and 2.5 μg/mL concentrations, respectively.  Analogues with less than 30% mortality are not shown in [Table 1](#molecules-22-01568-t001){ref-type="table"}.  Recently, finding novel lead compounds inspired by bioactive natural compounds has been demonstrated to be an effective strategy for the development of pesticides \[[@B10-molecules-22-01568]\].  Naturally occurring 3-acyltetramic acids are core structural skeletons with various biological activities such as magnesidin A \[[@B11-molecules-22-01568]\], reutericyclin \[[@B12-molecules-22-01568]\], integramycin \[[@B13-molecules-22-01568]\], the melophlins \[[@B14-molecules-22-01568]\] and the macrocidins \[[@B15-molecules-22-01568]\].  Although antibacterial activities of 3-acyltetramic acids have been reported in a previous study \[[@B16-molecules-22-01568]\], this is the first report of the nematicidal activities of 3-acyltetramic acid analogues against the pine wood nematode. 

2.2.  Structure-Activity Relationship {#sec2dot2-molecules-22-01568}
------------------------------------

Understanding the structure-activity relationship is very important for developing lead compounds for novel pesticides.  The chemical structures of compounds **1e**, **1f**, **1g**, and **1h** are very similar, but they differ in the identity of the acyl side chain, and among them, only compound **1h** showed strong nematidal activity against the pine wood nematode; compound **1h** has C~13~H~27~ at the C-acyl position while compounds **1e**, **1f** and **1g** have methyl, C~9~H~19~ and C~11~H~23~, respectively.  Compounds **2a**, **2b**, and **2c** also showed similar structure-activity relationships, again with the only structural difference being the acyl side chain length.  Compound **2c** with C~13~H~27~ at the R~4~ position showed strong nematicidal activity against the pine wood nematode, but compounds **2a** and **2b** with C~9~H~19~ and C~11~H~23~ on the side chain displayed very weak activity.  All of compounds **2h**, **2i**, **2j** and **2k** have the same N-group, but different acyl side chain lengths, and the nematicidal activity of **2k** with C~13~H~27~ on the side chain was much higher than that of compounds **2h**, **2i** and **2j** with C~6~H~13~, C~9~H~19~, and C~11~H~23~ as R~4~ substituents, respectively.  Compounds **1c** and **1d** have C~11~H~23~ and C~13~H~27~ on the side chain, and they all showed very strong nematicidal activities against the pine wood nematode.  Another 3-acyltetramic acid group, compounds **2d**, **2e**, **2f** and **2g,** showed a similar result, and the nematicidal activities of compounds **2f** and **2g** with C~11~H~23~ and C~13~H~27~ on the side chain were much better than those of compounds **2d** and **2e** with C~4~H~9~ and C~6~H~13~. These results clearly indicate that an optimal chain length is essential for nematicidal activity against the pine wood nematode, and that biological activity is strongly correlated to chemical structure; this is developed further below.  Seo et al.  \[[@B17-molecules-22-01568]\] investigated the structure-activity relationship of aliphatic compounds against the pine wood nematode, and also found that the chain length of the aliphatic compounds was very important for nematicidal activity; thus, among the alkanols and 2E-alkenols, the nematicidal activities of compounds with a C~9~--C~11~ chain length were much stronger than those of the other compounds with different chain lengths.  In the 2E-alkenals and alkanoic acids groups, compounds with C~8~, C~9~ and C~9~, C~10~ chain lengths, respectively, exhibited strong nematicidal activity compared to compounds with C~12~--C~14~ chain lengths. 

2.3.  Physiocochemical Property--Nematicidal Activity Relationships {#sec2dot3-molecules-22-01568}
------------------------------------------------------------------

The 3-acyltetramate library comprises a series of substituted systems, varying in ring and nitrogen groups, as shown in [Figure 1](#molecules-22-01568-f001){ref-type="fig"} and [Figure 2](#molecules-22-01568-f002){ref-type="fig"}.  Of interest is that only a small subset of the examined compounds showed nematicidal activity against the pine wood nematode *B. xylophilus* at a 10 μg/mL concentration, and those compounds which were active (namely **2c**, **1c**, **2g**, **2f**, **1h**, **1d** and **2k**) were very similar in structure and possessed similar physicochemical properties ([Table 2](#molecules-22-01568-t002){ref-type="table"}); thus, all active compounds had C~13~H~27~ or C~11~H~23~ acyl substituents in two closely related structural groups with the common physicochemical properties of a polar surface area (PSA) 57.6A ^2^, rel-PSA (relative polar surface area) 7.8--8.6% and ClogP 5.1--5.9 and a PSA 75--84A^2^, rel-PSA 11.1--11.6% and ClogP 4.7--5.1, respectively.  This is a comparatively narrow range of parameters, at least in comparison with the full library, which has a much wider range of physicochemical properties (−0.43 \< ClogD~7.4~ (calculated distribution coefficient at pH 7.4) \< 5.3; −0.43 \< ClogP \< 6.1; 57 \< PSA \< 117A^2^; 7.8 \< rel-PSA \< 27.6% and 257 \< MSA (molecular surface area) \< 800A^3^).  Of interest is that analysis of the physicochemical properties of acyltetramates in relation to their antibacterial activity has also been studied, and maximal activity also occurs for a narrow band of physicochemical descriptors, consistent with similar structures giving similar activities \[[@B16-molecules-22-01568],[@B18-molecules-22-01568]\]. 

3.  Materials and Methods {#sec3-molecules-22-01568}
========================

3.1.  Chemicals {#sec3dot1-molecules-22-01568}
--------------

The chemical structures of 98 3-acyltetramic acid analogues are shown in [Figure 1](#molecules-22-01568-f001){ref-type="fig"} and [Figure 2](#molecules-22-01568-f002){ref-type="fig"}.  Their synthesis and characterization have been previously documented \[[@B16-molecules-22-01568],[@B19-molecules-22-01568]\].  Emamectin benzoate (purity \> 98%) was used as a positive control, and supplied form Syngenta Korea (Seoul, Korea). 

3.2.  Collection of the Pine Wood Nematode {#sec3dot2-molecules-22-01568}
-----------------------------------------

Pine wood nematode *B. xylophilus* was supplied from National Institute of Forest Science, Seoul, Korea.  We reared pine wood nematode on a lawn of *Botrytis cinerea* cultured on potato dextrose agar medium (PDA) in the dark at 28 °C.  We extracted pine wood nematode by using Baermann funnel method \[[@B20-molecules-22-01568]\] one day before bioassay. 

3.3.  Nematicidal Activity Test {#sec3dot3-molecules-22-01568}
------------------------------

To test the nematicidal activity of 3-acyltetramic acid analogues, test analogues were dissolved in ethanol (Daejung Chemical, Siheun, Gyeonggi-do, Korea) at a concentration of 1 mg/mL. 3-acyltetramic acid solutions (1 µL) were applied to the wells of a 96-well plate (JET Bio-Filtration co., Ltd, Guangzhou, China).  Numbers of pine wood nematode in each well were about 50--150 nematodes (mixture of juvenile and adult nematodes, male:female:juvenile ≈ 1:1:2) in 99 µL of water.  The total volume of the solution in each well was 100 µL, and the concentration of the test 3-acyltetramic acid analogues was 10 μg/mL. Ethanol (1 µL) and emamectin benzoate (Syngenta Korea, Seoul, Korea) were used as negative and positive control, respectively.  In four adjacent wells (i.e., in a column) on the plate, pine wood nematodes were treated with 3-acyltetramic acid and a set of other 3-acyltetramic acid was placed in the wells of every next column.  All experiments were replicated 4 times.  We applied 3-acyltetramic acids randomly.  The treated 96-well plates were stored in the dark at 25 ± 1 °C and 60% relative humidity.  Mortality of pine wood nematode was determined after 48 h of treatment under microscope (65×).  Nematodes were considered as dead if their bodies were motionless and straightened. 

3.4.  Statistical Analysis {#sec3dot4-molecules-22-01568}
-------------------------

The percentages of mortality of pine wood nematode were transformed to arcsine square-root values prior to analysis of variance (ANOVA).  Treatment mean values were compared and separated using Scheffe's test.  Statistical analyses were performed using IBM SPSS Statistics 23.0 (2015).  Mean (±SE) values of untransformed data have been reported. 

4.  Conclusions {#sec4-molecules-22-01568}
==============

In this study, the nematicidal activities of 98 3-acyltetramic acids analogues against the pine wood nematode were evaluated.  Among test compounds, compounds **1c**, **1d**, **1h**, **2c**, **2f**, **2g**, and **2k** showed very strong nematicidal activity.  The structure-activity relationship study indicated that the chain length at the C-acyl position is essential for high nematicidal activity against the pine wood nematode.  Further studies including the safety of active 3-acyltetramic acid analogues to human and non-target organisms, their formulations, and their modes of action are necessary to develop the practical use of 3-acyltetramic acid analogues as novel pine wood nematode control agents. 

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Agro-Bio Industry Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant number 316030-3) to IK Park.  MGM gratefully acknowledges funding from the Oxford Innovation Fund. 

**Sample Availability:** Samples of the compounds are not available from the authors. 

H.-R.L., M.G.M. and I.K.P. conceived and designed the experiments; H.-R.L., S.-C.L., S.-M.S., J.-E.L. and C.-S.J. performed the experiments; Y.-C.J. synthesized 3-acyltetramic acid analogues; H.R.L., M.G.M. and I.-K.P. analyzed the data; H.R.L., M.G.M. and I.-K.P. wrote the paper. 

The authors declare no conflict of interest. 

![ Chemical structures of monocyclic 3-acyltetramic acid analogues.](molecules-22-01568-g001){#molecules-22-01568-f001}

![ Chemical structures of 3-acyltetramic acid analogues.](molecules-22-01568-g002){#molecules-22-01568-f002}

molecules-22-01568-t001_Table 1

###### 

Nematicidal activities of 3-acyltetramic acid analogues against *B. xylophilus.* 

  -----------------------------------------------------------------------------------------------------
  Compounds ^1^        Mortality (%, Mean ± SE, *n* = 4)                          
  -------------------- ----------------------------------- ---------------------- ---------------------
  **1c**               99.6 ± 0.7a ^3^                     96.2 ± 0.9a            15.0 ± 3.0bc

  **1d**               85.8 ± 3.5a                         80.2 ± 6.8ab           35.5 ± 3.2a

  **1h**               88.0 ± 7.3a                         0.7 ± 1.3e             − ^4^

  **2c**               100a                                66.8 ± 8.1bc           6.3 ± 0.5cd

  **2f**               95.8 ± 1.1a                         76.4 ± 3.1b            10.8 ± 2.8c

  **2g**               98.9 ± 1.9a                         96.4 ± 1.8a            6.3 ± 3.6cd

  **2k**               57.2 ± 18.8b                        50.6 ± 10.1c           13.1 ± 3.9c

  Emamectin benzoate   32.3 ± 2.7c                         28.2 ± 3.9d            24.1 ± 4.3b

  Control              0d                                  0e                     0d

                       F~8,\ 27~ = 80.208\                 F~8,\ 27~ = 156.542\   F~7,\ 24~ = 41.067\
                       *p* \< 0.0001                       *p* \< 0.0001          *p* \< 0.0001
  -----------------------------------------------------------------------------------------------------

Compounds ^1^ with \>30% mortality at 10 μg/mL are shown.  ^2^ μg/mL. ^3^ Means within a column followed by the same letters are not significantly different (Scheffe's test).  ^4^ Not tested. 

molecules-22-01568-t002_Table 2

###### 

Physicochemical properties of active 3-acyltetramic acid analogues. 

  Compounds   Mw ^1^   MSA(A ^3^) ^2^   PSA(A ^2^) ^3^   rel-PSA (%) ^4^   ClogP ^5^   ClogD~7.4~ ^6^   H-D ^7^/H-A ^8^   RB ^9^
  ----------- -------- ---------------- ---------------- ----------------- ----------- ---------------- ----------------- --------
  **1c**      366      674              57.6             8.55              5.12        4.51             1/3               15
  **1d**      394      735              57.6             7.84              5.92        5.30             1/3               17
  **1h**      410      721              83.9             11.6              5.13        4.26             1/4               14
  **2c**      380      673              74.7             11.1              4.69        3.91             1/4               14
  **2f**      366      674              57.6             8.55              5.12        4.51             1/3               15
  **2g**      394      735              57.6             7.84              5.92        5.30             1/3               17
  **2k**      378      642              74.7             11.6              4.77        3.92             1/4               13

Mw ^1^: Molecular weight.  MSA ^2^: Molecular surface area.  PSA ^3^: Polar surface area.  rel-PSA ^4^: Relative polar surface area (%) = (PSA/MSA) × 100.  ClogP ^5^: Calculated partition coefficient.  ClogD~7.4~ ^6^: Calculated distribution coefficient at pH 7.4.  H-D ^7^: Hydrogen bond donor count.  H-A ^8^: Hydrogen bond acceptor count.  RB ^9^: Rotatable bond count.