The discovery of fluazaindolizine: A new product for the control of plant parasitic nematodes

Article abstract of DOI:10.1016/j.bmcl.2017.02.029

Fluazaindolizine is a new highly effective and selective product for the control of plant parasitic nematodes. Specificity for nematodes coupled with absence of activity against the target sites of commercial nematicides suggests that fluazaindolizine has a novel mode of action. The discovery, structure-activity development and biological properties for this new class of nematicides are presented.

Full text of DOI:10.1016/j.bmcl.2017.02.029

Accepted Manuscript
The discovery of fluazaindolizine: A new product for the control of plant para-
sitic nematodes
George P. Lahm, Johan Desaeger, Ben K. Smith, Thomas F. Pahutski, Michel
A. Rivera, Tony Meloro, Roman Kucharczyk, Renee M. Lett, Anne Daly,
Brenton T. Smith, Daniel Cordova, Tim Thoden, John A. Wiles
PII:
S0960-894X(17)30161-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.02.029
BMCL 24701
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 January 2017
13 February 2017
14 February 2017
Please cite this article as: Lahm, G.P., Desaeger, J., Smith, B.K., Pahutski, T.F., Rivera, M.A., Meloro, T.,
Kucharczyk, R., Lett, R.M., Daly, A., Smith, B.T., Cordova, D., Thoden, T., Wiles, J.A., The discovery of
fluazaindolizine: A new product for the control of plant parasitic nematodes, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.02.029
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The discovery of fluazaindolizine: A new product for the control of
plant parasitic nematodes
George P. Lahm,a* Johan Desaeger,a Ben K. Smith,a Thomas F. Pahutski,a Michel A. Rivera,a
Tony Meloro,a Roman Kucharczyk,a Renee M. Lett,a Anne Daly,a Brenton T. Smith,a Daniel
Cordova,a Tim Thoden,b John A. Wilesc
aDuPont Crop Protection, Stine-Haskell Research Center, 1090 Elkton Rd., Newark, DE 19714, USA
bDuPont de Nemours (France) S.A.S., European Research Center, 68740 Nambsheim, France
cDuPont (U.K.) Limited, 4th Floor, Kings Court, London Road, Stevenage, SG1 2NG, United Kingdom
Abstract - Fluazaindolizine is a new highly effective and selective product for the control of plant parasitic
nematodes. Specificity for nematodes coupled with absence of activity against the target sites of commercial
nematicides suggests that fluazaindolizine has a novel mode of action. The discovery, structure-activity development
and biological properties for this new class of nematicides are presented.
The discovery and development of new nematicides that are highly effective against the target pest, work by new
modes of action, and meet societal demands of safety to humans and the environment are essential in the defense of
crops.
Soil dwelling nematodes are responsible for significant crop damage and yield loss in agricultural
production. Furthermore, many current nematicidal products are under regulatory pressure due to a range of
toxicological and environmental issues. Herein we describe the discovery of fluazaindolizine (1), a new product for
the control of plant parasitic nematodes.1
1
Figure 1. Chemical structure of fluazaindolizine.
Plant-parasitic nematodes are ubiquitous microscopic soil pests that feed on plant roots resulting in severe crop
losses. The damage often goes unnoticed due to the hidden nature of nematodes and the non-specific damage
symptoms, which can be confused with soil fertility, drought or other soil pest or pathogen problems. The most
recent nematode damage survey valued global crop losses at $100 billion annually.2 The increasing need for food
production and the growing pressure on agricultural land will likely cause crop losses to increase.
More than 4,000 species of plant-parasitic nematodes have been described but only a fraction of these cause
economic damage to crops.3 The most important nematode pest worldwide is the root-knot nematode (Meloidogyne
spp.), which is estimated to account for greater than 50% of all nematicide use and 5% of crop loss globally.4 Other
agriculturally important plant-parasitic nematodes include cyst nematodes (Globodera and Heterodera spp.), lesion
nematodes (Pratylenchus spp.), reniform nematodes (Rotylenchulus spp.) and sting nematodes (Belonolaimus spp.).
Soil fumigants account for about 50% of all commercial nematicides, with organophosphates and carbamates
comprising nearly all of the remaining nematicides.5 Many of these products are under regulatory pressure, and
certain fumigants and organophosphates have been banned. Despite increasing regulatory pressure and limited
choice, the global nematicide market continues to grow. Valued at $1 billion in 2011, the market is estimated to

increase to $1.4 billion by 2020.5 Recently there has been a resurgence in nematicide discovery across the crop
protection industry, and several new nematicides including fluensulfone and fluopyram have been introduced.6-8 The
renewed interest in nematicide discovery is long overdue and offers hope for growers that are currently struggling to
manage nematode problems. The challenge is to align the needs of the grower with the demands of regulators and
society. An ideal nematicide would 1) offer highly effective plant parasitic nematode control, without negatively
impacting beneficial soil organisms, beneficial arthropods or pollinators, 2) have good soil root zone movement, but
low leaching potential, 3) provide effective residual plant parasitic nematode control without persisting in soil for
unacceptably long durations and 4) provide an appropriate level of plant root protection, by either contact or
systemic activity, without resulting in residues in crop produce.
High throughput screening of our internal compound libraries against root-knot nematode (RKN) identified several
active N-phenylsulfonylimidazopyridine-2-carboxamides, such as 2 and 3 (Figure 2). Testing at lower rates showed
3 to be highly effective on the root-knot nematode, however, some associated plant phytotoxicity was observed. The
resulting optimization program focused on the dual objective of maximizing nematicidal activity with a
corresponding reduction or elimination of the plant effects.
2
3
Figure 2. N-phenylsulfonylimidazopyridine-2-carboxamide lead compounds.
The compounds of Tables 1-3 were prepared as outlined in Scheme 1. The imidazopyridine-2-carboxylic acid 7 was
prepared by the reaction of 2-amino-3,5-disubstituted pyridines with ethyl bromopyruvate to afford the ethyl esters
of formula 6, which were subsequently hydrolyzed to the acids 7. The acids were then coupled with a series of
phenylsulfonamides 8, typically using EDC as the coupling reagent, to afford the N-phenylsulfonylimidazopyridine-
2-carboxamides 9 in yields generally ranging from 40-60%.9
a
b, c
4
5
6
d
7
8
9
Scheme 1. (a) DME, 0oC to RT or heat (b) aq. NaOH, EtOH (c) aq. HCl (d) EDC, DMAP, DCM:tBuOH (1:1).

Nematicidal activity is summarized in Tables 1-3. Control of RKN through contact and/or systemic means was
evaluated using standard laboratory procedures. Efficacy was determined by the amount of root gall formation
observed in a 7-day evaluation when compared to an untreated control.10 Lack of gall formation was indicative of
100% nematode control, whereas gall formation equivalent to that found in the untreated control was indicative of
0% control. Both EC50 and EC90 values are reported in Tables 1-3. Compounds showing a wider range between the
EC50 and EC90 tended to have a flatter dose response with generally higher use rates required for economic control.
Compounds D01 through D07 of Table 1 compare a series of mono-substituted aryl sulfonamides containing a
common left side, i.e. the 8-chloro-6-trifluoromethyl-imidazopyridine-2-carboxamide group. Lead compound D01
demonstrated excellent efficacy in our RKN assay and was in fact one of the more potent analogs we evaluated
(EC50 = 3.9 ppm). However, significant plant effects were observed which precluded further advancement. The
corresponding 2-Me analog, D02, also demonstrated strong nematicidal activity but with similar phytotoxicity.
While the meta-substituted derivatives D03-D05 were less active as nematicides (EC50 = 16.2-57.3 ppm), several
analogs demonstrated significant safety to plants including the 3-OMe (D04) and 3-CF3 (D05) derivatives.
Compounds D06 and D07, containing substituents at the para position, were inactive against RKN.
Compounds D08-D11 of Table 1 compare a series of substitution patterns for dichloro-arylsulfonamides. The 2,5-
and 2,6-analogs, D09 and D10 respectively, were similar in nematicidal efficacy (EC50 = 4.7-8.3 ppm). However,
phytoxicity was observed with the 2,6-analog but not with the 2,5-analog. This improvement in plant safety was
observed with other 2,5-disubstituted analogs. Nematicidal activity was not observed for the 3,5-dichloro analog
D11.
Table 1. Root-knot nematode (Meloidogyne incognita, RKN) activity of 8-chloro-N-phenylsulfonyl-6-(trifluoromethyl)
imidazo[1,2a]pyridine-2-carboxamides.
Entry
R1
R2
RKNa
EC50 ppm
3.9
RKNa
EC90 ppm
19.3
D01
D02
D03
D04
D05
D06
D07
D08
D09
D10
D11
2-Cl
2-Me
3-Me
3-OMe
3-CF3
4-Me
4-Cl
2-Cl
2-Cl
2-Cl
3-Cl
H
H
H
H
H
H
H
3-Cl
5-Cl
6-Cl
5-Cl
4.3
16.2
38.0
57.3
>250
>250
25.5
4.7
8.3
71.6
63.4
149.1
>250
>250
>250
185.7
40.4
55.7
>250
>250
a. Mortality values were obtained for multiple test rates, each tested in replicate. EC50 and EC90 calculations were determined by Probit analysis.
Confidence intervals are contained within the supplementary information.
The compounds of Table 2 represent a series of the 2,5-disubstituted arylsulfonamides containing both 2-Me (D12-
D21) and 2-Cl (D22-D32) substituents. Examination of the EC50 and EC90 values show a wide range of efficacy with
the most active analogs containing 5-Me, 5-OMe and 5-OEt substituents, i.e. compounds D17, D22, D25 and D26
(EC50 = 2.0-4.2 ppm). Analogs containing other 5-substituents such as SMe, SO2Me, OCF2H, OCF3, cyano, nitro,

carbomethoxy, and dimethylamino generally showed a modest to significant reduction in RKN activity. Among the
most active compounds D17 and D22 showed some plant phytotoxicity, whereas the 2-chloro-5-alkoxy analogs D25
(5-OMe) and D26 (5-OEt) provided the highest margins of plant safety combined with exceptional nematicidal
activity.
Table 2. Root-knot nematode (Meloidogyne incognita, RKN) activity of 8-chloro-N-phenylsulfonyl-6-(trifluoromethyl)
imidazo[1,2a]pyridine-2-carboxamides.
Entry
R1
R2
RKNa
EC50 ppm
7.0
RKNa
EC90 ppm
34.8
56.9
91.0
237.7
53.4
10.1
93.1
58.9
>250
>250
6.0
64.0
42.5
14.9
15.8
D12
D13
D14
D15
D16
D17
D18
D19
D20
D21
D22
D23
D24
D25
D26
D27
D28
D29
D30
D31
D32
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Me
F
Cl
Br
CF3
OMe
OCF2H
C(O)Me
SO2Me
NO2
Me
Br
CF3
OMe
OEt
O-iPr
OCF3
NMe2
SMe
CO2Me
CN
20.9
23.1
14.8
5.7
2.6
12.3
6.1
>250
21.2
2.0
17.3
4.5
3.1
4.2
19.2
32.6
18.2
>250
91.1
17.2
40.5
>250
114.3
>250
>250
>250
Cl
Cl
a. Mortality values were obtained for multiple test rates, each tested in replicate. EC50 and EC90 calculations were determined by Probit analysis.
Confidence intervals are contained within the supplementary information.
The compounds of Table 3 compare modifications to the 8-chloro-6-trifluoromethylimidazopyridine-2-carboxamide
group. Removal of the 8-chloro-6-trifluoromethyl substituents, i.e. the unsubstituted analog D33, resulted in a loss
of activity. Replacement of the 6-trifluoromethyl group with 6-Cl, D34, also resulted in a loss of activity. The
dibromo analogs, D35 and D36, similarly resulted in a significant loss of nematicidal activity with D35 showing
only modest RKN control. In contrast, compounds D37 through D42, which all retain the 6-trifluoromethyl
substituent and replace the 8-chloro group with H, F, Br or Me show moderate to good levels of RKN control
indicating the importance of the 6-trifluoromethyl substituent. However, none of these compounds were more potent
than the analogous 8-chloro analogs D24 or D25. Beyond testing compounds in the RKN assay for comparison of
intrinsic potency the most preferred analogs were evaluated in field trials against a broad spectrum of nematodes and
in a variety of studies to evaluate toxicological and environmental attributes. From this group D25 (fluazaindolizine)
was selected for development.

Table 3. Root-knot nematode (Meloidogyne incognita, RKN) activity of imidazopyridine-2-carboxylic-N-(2-
chlorophenyl)sulfonamides.
Entry
R2
R3
R4
RKNa
EC50 ppm
>250
>250
58.2
>250
15.6
3.5
RKNa
EC90 ppm
>250
>250
>250
>250
127.4
11.5
48.9
47.0
132.6
>250
D33
D34
D35
D36
D37
D38
D39
D40
D41
D42
OMe
CF3
OMe
CF3
OMe
OMe
CF3
OMe
CF3
H
Cl
Br
Br
H
Br
Br
F
H
Cl
Br
Br
CF3
CF3
CF3
CF3
CF3
CF3
8.2
5.2
9.9
12.9
F
Me
CF3
a. Mortality values were obtained for multiple test rates, each tested in replicate. EC50 and EC90 calculations were determined by Probit analysis.
Confidence intervals are contained within the supplementary information.
In aqueous testing, juvenile RKN (J2) treated with fluazaindolizine (5-50 ppm) exhibit increasing immobility and
ultimately death between 24-96 hours without significant symptoms being observed in the first hours of exposure.
However, even at lower doses (1-5 ppm) and with short exposure periods the RKN fitness was reduced to an extent
that they were unable to detect and infect roots. Adult C. elegans (a model non-plant parasitic nematode) treated
with fluazaindolizine (300 ppm) showed no mortality or significant effect on motility over a 120 hour period.
Further, the life cycle (egg to adult) of the fruit fly, Drosophila melanogaster, was unaffected when grown in diet
containing fluazaindolizine (200 ppm). The nematicide was tested against in vitro assays for acetylcholinesterase
(Diabrotica undecimpunctata), mitochondrial electron transport (C. elegans) nicotinic acetylcholine receptors (C.
elegans), and glutamate-gated chloride channels (P. americana) with no significant activity observed at
concentrations up to 30 uM. Specificity for plant parasitic nematodes coupled with absence of activity against
targets of commercial nematicides suggests that fluazaindolizine has a novel mode of action.
Fluazaindolizine has been extensively tested in laboratory, greenhouse, micro-plot and field trials in North America,
Latin America, Europe and in the Asia-Pacific region. In these trials fluazaindolizine has proven extremely effective
against a wide range of root-knot nematode species (Meloidogyne spp.), and other important plant parasitic
nematodes, such as reniform (Rotylenchulus reniformis), dagger (Xiphinema spp.), spiral (Helicotylenchus spp.) and
some lesion nematode species (Pratylenchus spp.), as well as other important plant parasitic nematode species in
certain circumstances.
Global development of fluazaindolizine is primarily as a suspension concentrate (500SC) liquid formulation, with
granular formulations also under development for certain markets. The physico-chemical properties of
fluazaindolizine lend it to be well balanced in terms of soil mobility (average Kfoc of 128) and residual properties
(DT50 ~ 35 days) in the soil root zone. As such it is compatible with a variety of grower application methods; such
as drip irrigation, bed sprays, micro-jets, pre-plant hole drench, in furrow applications and soil incorporation. Thus,
agronomically, it has a fit in a range of crops including fruiting and cucurbit vegetables, root vegetables (carrot,
sweet potato, potato), soybean, sugarcane, coffee, corn, citrus, tree nuts, stone fruit and grapes. Application rates
vary by crop and application method and range typically from 0.25 to 2 kg ai/ha. The formulated products will be

commercialized under the brand DuPont™ Salibro™ nematode control, and the active ingredient (fluazaindolizine)
will be branded as Vellozine™ nematode control. Use of the formulated product in the field, under the proposed
directions for use, has demonstrated consistent root protection and the potential for increased yields in the crops
tested.
Intrinsic sensitivities differ to fluazaindolizine, with plant parasitic nematodes being of higher sensitivity than other
groups of the soil nematode community. Fluazaindolizine works by contact with nematodes in the soil pore water,
and is not considered systemic in plants by soil application. It has a highly favourable profile for operators and also
for non-target organisms making it an ideal product for use in integrated pest management (IPM) systems. In
particular, fluazaindolizine has been shown to be highly compatible with a broad range of naturally occurring or
introduced biological control agents, such as beneficial fungi and nematodes, bacteria and other important non target
organisms that inhabit the soil rhizosphere and help sustain crop and soil health.
In summary fluazaindolizine has demonstrated excellent control of plant parasitic nematodes and the damage they
cause to plant roots, resulting in higher quality crops and increased potential in crop yields. Specificity for
nematodes, coupled with the absence of activity against target sites of commercial nematicides, suggests that it has a
novel mode of action. Fluazaindolizine promises to offer farmers around the world a valuable new tool for crop
protection and plant parasitic nematode management.
References and notes
1) Lahm, G.P., Lett, R.M., Smith, B.T., Smith, B.K., Tyler, A.C. World Patent 10/129500, 2010. Chem. Abstr.
2010, 153, 609287.
2) Sasser, J.N., Freckman, D.W. A World Prospective in Nematology. The Role of Society. In: Veech, J.A.,
Dickson, D.W., eds., Vista on Nematology, Society of Nematologists, Hyattsville, MD, 1987: 7-14.
3) Decraemer W., Hunt D.J. Structure and classification. In: Perry R.N., Moens M., eds. Plant nematology. CAB
International, Wallingford, Cambridge 2006: 3–32. http://dx.doi.org/10.1079/9781845930561.0392 accessed
14.11.16.
4) Haydock, P.J., Woods, S.R., Grove, G., Hare, M.C. Chemical control of nematodes. In: Perry R.N., Moens M.,
eds.
Plant
nematology.
CAB
International,
Wallingford,
Cambridge
2006:
392-408.
http://dx.doi.org/10.1079/9781845930561.0392 accessed 14.11.16.
5) Crop Protection Manufacturers Report 2010: A Strategic Market Analysis of the U.S. Crop Protection Industry.
Report #P0110 | © 2011 Kline & Company, Inc./Prochaska & Company
6) Greul, J.N., Mansfield, D., Fuesslein, M., Rieck, H., Riedrich, M., Rodefeld, L., Kather, K., Malsam, O.,
Loesel, P., Voerste, A., Schwarz, H.G., Ilg, K., Goergens, U., Carles, L., Coqueron, P.Y., Desbordes, P.,
Meresse, P., World Patent 13/064460, 2013. Chem. Abstr. 2013, 158, 633200.
7) Watanabe, Y., Ishikawa, K., Otsu, Y., Shibuya, K., Abe, T., World Patent 01/002378, 2001. Chem. Abstr. 2001,
134, 100860.
8) Oka, Y.; Shuker, S.; Tkachi, N. Pest Manag Sci. 2012; 68(2): 268-275.
9) Synthesis and characterization of compound D25: To 8-chloro-6-(trifluoromethyl)-imidazo[1,2-a]pyridine-2-
carboxylic acid (243 mg, 0.92 mmol) was added a solution of 4-(dimethylamino)pyridine (340 mg, 2.76 mmol)
and 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide hydrochloride (232 mg, 2.3 mmol) in t-butanol (5 mL)
and dichloromethane (5 mL). The reaction mixture was stirred for 15 min and then 2-chloro-5-
methoxybenzenesulfonamide (190 mg, 0.86 mmol) was added, and the mixture was left to stir at room
temperature overnight. Dichloromethane (200 mL) was then added and the mixture was extracted with 1 N
hydrochloric acid (3 x 100 mL). The organic phase was dried over magnesium sulfate and concentrated under
reduced pressure to afford a solid. The solid was rinsed with diethyl ether to afford 240 mg of D25 as a white
1
solid, m.p. 211-212 °C. HNMR (CDCl3) δ10.10 (br s, 1H), 8.46 (s, 1H), 8.27 (s, 1H), 7.86 (d, 1H), 7.54 (s,
1H), 7.38 (d, 1H), 7.09 (dd, 1H), 3.91 (s, 3H); 13C NMR (100 MHz, DMSO) δ56.57, 116.45 (q, JC-F 34.5 Hz),

117.99, 120.89, 121.49, 121.92, 122.01, 123.41 (q, JC-F 271.6 Hz), 124.10, 127.98 (q, JC-F 5.9 Hz), 133.23,
135.67, 137.87, 138.28, 142.07, 158.28, 160.48; HRMS (APESI, M+) C21H19ClF3N5O2: m/z calcd 466.9721,
m/z found 466.9724.
10) Control of the southern root-knot nematode (Meloidogyne incognita) through contact and/or systemic means
was evaluated in test units consisting of small open containers filled with approximately 10 ml of a sandy soil
mixture and cucumber seedlings. Test compounds were formulated using a solution containing 50% acetone
and 50% water. Test compounds (330 μl) were applied directly to the soil of the test units at concentrations of
250, 50, 10 and 2 ppm active ingredient. Each test was replicated 3 times. After treatment, the test units were
allowed to dry for 1 hour, after which time about 250 second-stage juvenile (J2) larvae were pipetted into the
soil. The test units were held at 27° C. and watered as needed for 7 days. Nematicidal efficacy was determined
by the amount of root gall formation observed when compared to an untreated control. No gall formation was
indicative of 100% nematode control. Gall formation equivalent to that found in the untreated control was
indicative of 0% control. No nematode control rating was given to compounds showing significant
phytotoxicity.

Graphical abstract
Fluazaindolizine