4-Hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors (HPPD inhibitors) are a class of
herbicides that prevent growth in plants by blocking
4-Hydroxyphenylpyruvate dioxygenase
4-Hydroxyphenylpyruvate dioxygenase (HPPD), also known as α-ketoisocaproate dioxygenase (KIC dioxygenase), is an Fe(II)-containing non-heme oxygenase that catalyzes the second reaction in the catabolism of tyrosine - the conversion of 4-hydro ...
, an
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...
in plants that breaks down the
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha a ...
tyrosine
-Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a non-essential amino acid with a polar side group. The word "tyrosine" is from the G ...
into molecules that are then used by plants to create other molecules that plants need. This process of breakdown, or
catabolism, and making new molecules from the results, or
biosynthesis, is something all living things do. HPPD inhibitors were first brought to market in 1980, although their mechanism of action was not understood until the late 1990s. They were originally used primarily in Japan in rice production, but since the late 1990s have been used in Europe and North America for corn, soybeans, and cereals, and since the 2000s have become more important as weeds have become resistant to
glyphosate
Glyphosate (IUPAC name: ''N''-(phosphonomethyl)glycine) is a broad-spectrum systemic herbicide and crop desiccant. It is an organophosphorus compound, specifically a phosphonate, which acts by inhibiting the plant enzyme 5-enolpyruvylshik ...
and other herbicides.
Genetically modified crops are under development that include resistance to HPPD inhibitors.
There is a
pharmaceutical drug
A medication (also called medicament, medicine, pharmaceutical drug, medicinal drug or simply drug) is a drug used to diagnose, cure, treat, or prevent disease. Drug therapy (pharmacotherapy) is an important part of the medical field an ...
on the market,
nitisinone, that was originally under development as an herbicide as a member of this class, and is used to treat an
orphan disease
A rare disease is any disease that affects a small percentage of the population. In some parts of the world, an orphan disease is a rare disease whose rarity means there is a lack of a market large enough to gain support and resources for discove ...
,
type I tyrosinemia.
HPPD inhibitors can be classified into three fundamental
chemical frameworks: pyrazolones, triketones, and diketonitriles. The triketone class is based on a chemical that certain plants make in self-defense called
leptospermone; the class was developed by scientists at companies that eventually became part of
Syngenta.
Bayer CropScience
Bayer AG (, commonly pronounced ; ) is a German multinational pharmaceutical and biotechnology company and one of the largest pharmaceutical companies in the world. Headquartered in Leverkusen, Bayer's areas of business include pharmaceutica ...
has also been active in developing new HPPD inhibitors.
Mechanism of action
The mechanism of action for HPPD inhibitors was misunderstood for the first twenty years that these products were sold, starting in 1980.
They were originally thought to be inhibitors of
protoporphyrinogen oxidase
Protoporphyrinogen oxidase or protox is an enzyme that in humans is encoded by the ''PPOX'' gene.
Protoporphyrinogen oxidase is responsible for the seventh step in biosynthesis of protoporphyrin IX. This porphyrin is the precursor to hemoglobin, ...
(protox).
4-Hydroxyphenylpyruvate dioxygenase
4-Hydroxyphenylpyruvate dioxygenase (HPPD), also known as α-ketoisocaproate dioxygenase (KIC dioxygenase), is an Fe(II)-containing non-heme oxygenase that catalyzes the second reaction in the catabolism of tyrosine - the conversion of 4-hydro ...
(HPPD) is an
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...
found in both plants and animals which catalyzes the
catabolism of the
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha a ...
tyrosine
-Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a non-essential amino acid with a polar side group. The word "tyrosine" is from the G ...
.
Preventing the breakdown of tyrosine has three negative consequences: the excess of tyrosine stunts growth; the plant suffers
oxidative damage
Oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Disturbances in the normal r ...
due to lack of
tocopherols
Tocopherols (; TCP) are a class of organic chemical compounds (more precisely, various methylated phenols), many of which have vitamin E activity. Because the vitamin activity was first identified in 1936 from a dietary fertility factor in rats, ...
(vitamin E); and
chlorophyll is destroyed due to lack of
carotenoids
Carotenoids (), also called tetraterpenoids, are yellow, orange, and red organic pigments that are produced by plants and algae, as well as several bacteria, and fungi. Carotenoids give the characteristic color to pumpkins, carrots, parsnips, co ...
that protect it.
[ Plants turn white without deformation due to a complete loss of chlorophyll, which has led compounds of this class to be classified as "bleaching herbicides", as are protox inhibitors.][
More specifically, inhibition of HPPD prevents the formation of a breakdown product, ]homogentisic acid
Homogentisic acid (2,5-dihydroxyphenylacetic acid) is a phenolic acid usually found in ''Arbutus unedo'' (strawberry-tree) honey. It is also present in the bacterial plant pathogen ''Xanthomonas campestris'' pv. ''phaseoli'' as well as in the ye ...
, which in turn is a key precursor for the biosynthesis of both tocopherols and plastoquinone
Plastoquinone (PQ) is an isoprenoid quinone molecule involved in the electron transport chain in the light-dependent reactions of photosynthesis. The most common form of plastoquinone, known as PQ-A or PQ-9, is a 2,3-dimethyl-1,4-benzoquinone m ...
. Plastoquinone is in turn a critical co-factor in the formation of carotenoids
Carotenoids (), also called tetraterpenoids, are yellow, orange, and red organic pigments that are produced by plants and algae, as well as several bacteria, and fungi. Carotenoids give the characteristic color to pumpkins, carrots, parsnips, co ...
, which protect chlorophyll in plants from being destroyed by sunlight.[
This class of herbicides represents one of the last discoveries of a new herbicide mode of action in the wave of discovery that ended in late 1990s.][
]
Resistance in weeds
very little resistance was known: In 2010 Waterhemp (''Amaranthus tuberculatus
''Amaranthus tuberculatus'', commonly known as roughfruit amaranth, rough-fruited water-hemp, tall waterhemp, or common waterhemp, is a species of flowering plant. It is a summer annual broadleaf with a germination period that lasts several mont ...
'') in two different locations - Iowa
Iowa () is a state in the Midwestern region of the United States, bordered by the Mississippi River to the east and the Missouri River and Big Sioux River to the west. It is bordered by six states: Wisconsin to the northeast, Illinois to th ...
and Illinois
Illinois ( ) is a state in the Midwestern United States. Its largest metropolitan areas include the Chicago metropolitan area, and the Metro East section, of Greater St. Louis. Other smaller metropolitan areas include, Peoria and Rockf ...
- developed resistance.
Products and economics
As of 2009, HPPD inhibitors had three fundamental chemical frameworks:[
* ]pyrazolones Pyrazolone is 5-membered heterocycle containing two adjacent nitrogen atoms. It can be viewed as a derivative of pyrazole possessing an additional carbonyl (C=O) group. Compounds containing this functional group are useful commercially in analgesic ...
: pyrazolate (Sanbird, introduced by Sankyo, 1980); pyrazoxyfen (Paicer, introduced by Ishihara, 1985); benzofenap (Yukawide, introduced by Mitsubishi Chemical Corp and Rhône-Poulenc Agro, 1987, as of 2009 sold by Bayer CropScience as Taipan); pyrasulfotole (Huskie and Infinity, introduced by Bayer CropScience, 2007); and topramezone (Clio and Impact, introduced by BASF, 2006)
* triketones
In organic chemistry, a triketone or trione is an organic compound containing three ketone () groups. The simplest triketones, such as cyclopropanetrione and 2,3,4-pentanetrione, are only of occasional theoretical interest. More pertinent are ...
: sulcotrione (Mikado, introduced by Zeneca, 1990 and as of 2009 sold by Bayer CropScience)); mesotrione (Callisto and Lumax, introduced by Zeneca, 2002 and as of 2009 sold by Syngenta); benzobicyclon (ShowAce, introduced by SDS Biotech and Sandoz Crop Protection (now Syngenta), 2001); tembotrione (Laudis introduced by Bayer CropScience, 2007)
* diketonitriles: isoxaflutole (aka IFT) (Balance and Merlin, introduced by Rhône-Poulenc Agro, 1996 and as of 2009 sold by Bayer CropScience)
Agricultural use
Pyrazolate, pyrazoxyfen and benzofenap were first commercialized in the Japanese rice market starting in 1980, but became less important when sulfonylurea
Sulfonylureas (UK: sulphonylurea) are a class of organic compounds used in medicine and agriculture, for example as antidiabetic drugs widely used in the management of diabetes mellitus type 2. They act by increasing insulin release from the bet ...
herbicides were introduced. In 1990 sulcotrione was introduced for post- emergence weed control in corn. Isoxaflutole opened the market more broadly for HPPD inhibitors when it was introduced in 1996 for corn and sugarcane, and for use as a pre-emergence herbicide that could control broadleaf weeds Broadleaf weeds are unwanted tough plants that may grow in lawns, gardens or yards. They can be easy to spot when growing among grasses. They multiply with ease and can be very hard to eradicate.
Basic characteristics
Broadleaf weeds can emerge ...
as did sulcotrione, but also additional grass weeds. Benzobicyclon, was introduced in 2001 for control of broadleaf weeds and some sedges
The Cyperaceae are a family of graminoid (grass-like), monocotyledonous flowering plants known as sedges. The family is large, with some 5,500 known species described in about 90 genera, the largest being the "true sedges" genus ''Carex'' wit ...
that are problems in rice, that had become resistant to sulfonylurea herbicides. Mesotrione was introduced in 2002 and like sulcotrione is a triketone, so it is effective on the same weeds and crops, but is more potent, making it more useful in mixes with other herbicides - an important factor for fully controlling weeds and preventing the development of resistance. It has become the biggest selling member of the HPPD class.[
Topramezone was introduced in 2006 for corn and soy, and is the most potent HPPD inhibitor, but has serious carry-over issues especially for soybean in US, where the minimum time from application to planting is 18 months. Tembotrione was introduced in 2007 for corn, and works against key grass species and importantly, kills broadleaf weeds, including glyphosate-, ALS- and dicamba-resistant weeds. Used with safeners there are no crop-rotation restrictions. Pyrasulfotole was also introduced in 2007 for cereals in North America, and was the first new class of herbicide in cereals in many years, and an important advance against weeds that had become resistant to existing herbicides. It remains active in the soil during the growing season and when used with safeners, it does not damage crops and there are no crop-rotation restrictions.][
]
Herbicide risks and toxicities
Tembotrione has low acute toxicity via the oral, dermal and inhalation routes of exposure (Toxicity category III or IV). It is a dermal sensitizer but not an eye or dermal irritant
Genetically modified crops
To deal with rising resistance to existing herbicides, Bayer CropScience
Bayer AG (, commonly pronounced ; ) is a German multinational pharmaceutical and biotechnology company and one of the largest pharmaceutical companies in the world. Headquartered in Leverkusen, Bayer's areas of business include pharmaceutica ...
has been developing various genetically modified crops resistant to HPPD inhibitors: in one version, the crops are resistant to both HPPD inhibitors and glyphosate
Glyphosate (IUPAC name: ''N''-(phosphonomethyl)glycine) is a broad-spectrum systemic herbicide and crop desiccant. It is an organophosphorus compound, specifically a phosphonate, which acts by inhibiting the plant enzyme 5-enolpyruvylshik ...
, and in collaboration with Syngenta, crops that are resistant to HPPD inhibitors and glufosinate
Glufosinate (also known as phosphinothricin and often sold as an ammonium salt) is a naturally occurring broad-spectrum herbicide produced by several species of ''Streptomyces'' soil bacteria. Glufosinate is a non-selective, contact herbicide, w ...
.
The collaboration to develop the stacked HPPD inhibitor/glyphosate resistant products was first announced in 2007.
Medical use
In Type I tyrosinemia, a different enzyme involved in the breakdown of tyrosine, fumarylacetoacetate hydrolase
Fumarylacetoacetase is an enzyme that in humans is encoded by the ''FAH'' gene located on chromosome 15. The FAH gene is thought to be involved in the catabolism of the amino acid phenylalanine in humans.
Function
Fumarylacetoacetate hydrolase ( ...
is mutated and doesn't work, leading to very harmful products building up in the body.[National Organization for Rare Disorders]
Physician’s Guide to Tyrosinemia Type 1
Fumarylacetoacetate hydrolase acts on tyrosine after HPPD does, so scientists working on making these inhibitors hypothesized that inhibiting HPPD and controlling tyrosine in the diet could treat this disease. A series of small clinical trials were attempted with one of their compounds, nitisinone and were successful, leading to nitisinone being brought to market as an orphan drug.
History of the discovery of the triketone class of HPPD inhibitors
The origin of the triketone family of HPPD inhibitors had its beginnings in the curiosity of a biologist about allelopathic weed control of weeds in the back yard of his house. This curiosity led to the discovery and development of the triketone class of herbicides. Investigation of the mode of action of this class of compounds led to the discovery that it could be used to treat patients with tyrosinaemia type 1, a treatment which has been said to “…. transform the natural history of tyrosinaemia.”
All of the herbicidal and pharmaceutical triketone HPPD inhibitors including mesotrione (Callisto)(I), sulcotrione (Mikado)(II) and nitisinone (Orfadin)(III) had their common origin in the observation in 1977 by Reed Gray, a biologist at Stauffer Chemical
Stauffer Chemical Company was an American chemical company which manufactured herbicides and pesticides for various agricultural crops. It was acquired by Imperial Chemical Industries from Chesebrough-Pond's Inc. in 1987. In 1987, Stauffer's head ...
’s Western Research Center (WRC) in California, that few weeds emerged under bottlebrush plants ('' Callistemon citrinus'') in the back yard of his house.
To investigate this effect, he took soil from underneath these plants and extracted and fractionated it. The resulting extracts were applied to soil flats containing watergrass (''Echinochloa crus-galli
''Echinochloa crus-galli'' is a type of wild grass originating from tropical Asia that was formerly classified as a type of panicum grass. It is commonly known as cockspur (or cockspur grass), barnyard millet, Japanese millet, water grass, common ...
'') as an indicator species at the very high application rate of . There was a herbicidal effect from this test, so the extracts were developed on a preparative thin layer chromatographic sheet. The same ''Echinochloa crus-galli'' seeds were placed on this sheet and germinated. The active ingredient was identified by bleaching symptoms on the test species.
The area where the herbicidal activity was seen was extracted and he submitted the isolated active ingredient to Ken Cheng at the Western Research Center who, using proton NMR
Proton nuclear magnetic resonance (proton NMR, hydrogen-1 NMR, or 1H NMR) is the application of nuclear magnetic resonance in NMR spectroscopy with respect to hydrogen-1 nuclei within the molecules of a substance, in order to determine the struct ...
, IR and mass spectrometry identified the structure as being that of Leptospermone (IV) which was a known natural product that had been derived from the steam-volatile oils of some Australian plants, but which had never been cited as having any biological activity.
He approached a chemist, Ron Rusay, at the Western Research Center who independently synthesized the compound and submitted it for further greenhouse testing. These tests showed that it had modest herbicidal activity against grass weeds at a very high application rate of . He prepared a series of analogs in which the alkanoyl group was modified and a patent was obtained on this series of compounds. These compounds had similar, weak herbicidal activity similar to that found with the lead compound. Because of the weak herbicidal activity, work on other analogs was not pursued.
Shortly after this, another WRC chemist, Bill Michaely, synthesized an aroyl triketone (V) as an unexpected byproduct when attempting to synthesize a sethoxydim (VI) analog.
While this compound did not have any herbicidal activity, it did show activity in a screen designed to show antidotal activity toward other herbicides. When attempting to optimize the antidote activity, several aryl substituted analogs were prepared. What was discovered was that those compounds with an '' ortho'' substituent had herbicidal activity but no antidote activity. This observation coupled with the knowledge of the herbicidal activity of the earlier leptospermone analogs was pivotal in formulating an idea of a potential toxophore
A toxophore is the chemical group that produces the toxic effect in a toxin molecule: commonly used in pharmaceutical
A medication (also called medicament, medicine, pharmaceutical drug, medicinal drug or simply drug) is a drug used to d ...
for this class of herbicides.
A small task force consisting of David Lee, Bill Michaely and Don James prepared a number of substituted triketones with chloro-, bromo- and methyl-substituents in the ''ortho'' position. Biological activity remained modest and the task force was disbanded after a short while.
One working hypothesis was that the active ingredient in these triketones was the cyclized tetrahydroxanthenones(VII). Bill Michaely prepared several of these, but the herbicidal activity remained modest and all work in the area was terminated. It was not until David Lee was able to show that these compounds were in equilibrium with the 2-hydroxy triketones(VIII) by trapping the intermediate with methyl iodide that the reason for the biological activity was understood.
David Lee had a strong background in quantitative structure–activity relationship
Quantitative structure–activity relationship models (QSAR models) are regression or classification models used in the chemical and biological sciences and engineering. Like other regression models, QSAR regression models relate a set of "predic ...
s (QSAR) after a post-doctoral year with Professors Manfred Wolff and Peter Kollman where he used the Prophet system. Looking over the QSAR (quantitative structure–activity relationship
Quantitative structure–activity relationship models (QSAR models) are regression or classification models used in the chemical and biological sciences and engineering. Like other regression models, QSAR regression models relate a set of "predic ...
s) of the triketones, David Lee saw a potential discrepancy in the existing structure-activity analysis. Other than the 2-chloro-4-nitro substitution pattern, no other triketones with electron-withdrawing substituents in the 4-position had ever been prepared. He hypothesized that the activity of the triketones could be correlated to the electron withdrawing ability of the substituents. The activity of the 2-chloro-4-nitro analog was an outlier, and it was theorized that perhaps the nitro group was being reduced ''in vivo''. The 4-methylsulfonyl group was then prepared to test this hypothesis, and what was to become the commercial herbicide Mikado (II) was prepared. The prospect of improving the biological activity with new aromatic substitution patterns totally rejuvenated work on the triketones.
A key discovery in the preparation of these compounds was the finding by Jim Heather in the WRC Process Development group that acetone cyanohydrin
Acetone cyanohydrin (ACH) is an organic compound used in the production of methyl methacrylate, the monomer of the transparent plastic polymethyl methacrylate (PMMA), also known as acrylic. It liberates hydrogen cyanide easily, so it is used as a s ...
was a good catalyst for the preparation of the o-chloro analogs. Use of this catalyst allowed for the first time production of the o-nitro triketones.
At this point a very large effort on the synthesis of analogs was commenced with David Lee coordinating the effort. Key chemists participating in this were Charles Carter, Bill Michaely, Hsiao-ling Chin, Nhan Nguyen and Chris Knudsen, although at one time almost every synthesis chemist at the WRC worked on this project. Within a relatively short time, major progress was made in optimizing certain substitution combinations. SC-0051 (sulcotrione) was synthesized and tested in Sept. – Oct. 1983, and SC-1296 (mesotrione) and SC-0735 (nitisinone) were both synthesized and tested in early 1984. Triketones were in widespread university field trials in 1985. The first of the triketone patents was published in 1986.
With a long history of working with bleaching herbicides which inhibit phytoene desaturase including the commercial herbicide flurochloridone(IX), there was some considerable interest to find that these compounds do not inhibit phytoene desaturase ''in vitro''. The fact that phytoene desaturase inhibitors typically have a high log P, whereas the triketones do not, further suggested a different mode of action.
There had been some early toxicological concern about corneal and paw lesions which were observed with rats that had repeatedly dosed with a triketone. Several chemists and toxicologists came upon a paper describing very similar ocular, but not skin, lesions with inhibitors of tyrosine hydroxylase
Tyrosine hydroxylase or tyrosine 3-monooxygenase is the enzyme responsible for catalyzing the conversion of the amino acid L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA). It does so using molecular oxygen (O2), as well as iron (Fe2+) and t ...
. Linda Mutter in the WRC Toxicology Section used a spot test for tyrosine on the urine of treated rats and had positive results. Plasma tyrosine analysis further confirmed the buildup of tyrosine in treated rats.[(14) M.P. Prisbylla, B.C. Onisko ,J.M. Shribbs, D.O. Adams, Y. Liu, M.K. Ellis, T.R Hawkes, L.C. Mutter, The Novel Mechanism of Action of the Herbicidal Triketones, Brighton Crop Protection Conference – Weeds (1993.) 731-8]
Work on the mode of action and toxicology of the triketones took on a broader range of interactions when Stauffer Chemical was purchased by ICI in June 1987. ICI then split off the pharmaceutical and agrochemical businesses as Zeneca and then Syngenta was formed in 2000 by the merger of Novartis Agribusiness and Zeneca Agrochemicals.
As part of toxicology
Toxicology is a scientific discipline, overlapping with biology, chemistry, pharmacology, and medicine, that involves the study of the adverse effects of chemical substances on living organisms and the practice of diagnosing and treating e ...
studies, Martin Ellis at the ICI Central Toxicology Laboratory identified triketone inhibition of tyrosine catabolism in rat liver and also found that tyrosine hydroxylase
Tyrosine hydroxylase or tyrosine 3-monooxygenase is the enzyme responsible for catalyzing the conversion of the amino acid L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA). It does so using molecular oxygen (O2), as well as iron (Fe2+) and t ...
was not inhibited by the triketones. He furthermore found that the urine of rats treated with III showed elevated levels of both ''p''-hydroxyphenylpyruvate and ''p''-hydoxyphenyllactic acids. These results suggested that ''p''-hydroxyphenylpyruvate dioxygenase (HPPD) was the enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...
that was inhibited, a fact which was confirmed by S. Lindstedt.[Lindsted, W. and B. Odelhög , 4-Hydroxyphenylpyruvate dioxygenase from human liver (1987) Methods Enzymol. 142;139-142] Further tests established that HPPD was the enzyme inhibited in plants as well as mammals.
References
{{Herbicides
Enzymes
*