Thrombin inhibitors with lipid peroxidation and lipoxygenase inhibitory activities

Article abstract of DOI:10.1016/j.bmcl.2011.06.089

Vascular oxidative stress, endothelial injury, and thrombosis are intertwined processes that display a synergistic pathological effect in many cardiovascular diseases. Antithrombotic therapy with anticoagulant and/or antiplatelet agents, combined with interventions against vascular oxidative stress and/or inflammation, both boosting endothelial antithrombotic potential, could display a synergistic action in the treatment of thrombosis. Of the compounds 10a-h and 11a-d, shown to possess thrombin inhibitory activity, 11a-d were found to display radical scavenging activity, 10a, 10d, and 10f were demonstrated to inhibit lipid peroxidation of linoleic acid, and 10b and 10h inhibited soybean lipoxygenase. The observed combination of thrombin inhibition with lipid peroxidation and/or lipoxygenase inhibitory activity makes compounds 10 and 11 interesting candidates for further investigations towards multiple antithrombotic drugs.

Full text of DOI:10.1016/j.bmcl.2011.06.089

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4705–4709
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Thrombin inhibitors with lipid peroxidation and lipoxygenase
inhibitory activities
a
b
b
a
a,
⇑
´
Miloš Ilic , Christos Kontogiorgis , Dimitra Hadjipavlou-Litina , Janez Ilaš , Danijel Kikelj
a
ˇ
University of Ljubljana, Faculty of Pharmacy, Aškerceva 7, 1000 Ljubljana, Slovenia
b Aristotle University of Thessaloniki, School of Pharmacy, 54124 Thessaloniki, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Vascular oxidative stress, endothelial injury, and thrombosis are intertwined processes that display a
synergistic pathological effect in many cardiovascular diseases. Antithrombotic therapy with anticoagu-
lant and/or antiplatelet agents, combined with interventions against vascular oxidative stress and/or
inflammation, both boosting endothelial antithrombotic potential, could display a synergistic action in
the treatment of thrombosis. Of the compounds 10a–h and 11a–d, shown to possess thrombin inhibitory
activity, 11a–d were found to display radical scavenging activity, 10a, 10d, and 10f were demonstrated to
inhibit lipid peroxidation of linoleic acid, and 10b and 10h inhibited soybean lipoxygenase. The observed
combination of thrombin inhibition with lipid peroxidation and/or lipoxygenase inhibitory activity
makes compounds 10 and 11 interesting candidates for further investigations towards multiple anti-
thrombotic drugs.
Received 4 May 2011
Revised 16 June 2011
Accepted 19 June 2011
Available online 25 June 2011
Keywords:
Thrombin inhibitor
Antithrombotic
Lipid peroxidation
Lipoxygenase inhibition
Radical scavenging
Ó 2011 Elsevier Ltd. All rights reserved.
Arterial and venous thrombosis are the major causes of morbid-
ity and mortality worldwide.1 In contrast to hemostasis, by which
the body stops blood loss whenever a blood vessel is severed or
ruptured, thrombosis is a pathological process in which hemostatic
mechanisms, that is, blood coagulation and platelet aggregation,
are activated in the absence of bleeding. The blood clots formed
by pathological coagulation and aggregation processes can cause
myocardial infarction, ischemic stroke, limb gangrene, deep vein
thrombosis, and pulmonary embolism, which can be life threaten-
ing. Antithrombotic drugs, including anticoagulants (e.g., warfarin,
heparins, thrombin inhibitors, and factor Xa inhibitors) and anti-
platelet agents such as acetylsalicylic acid, clopidogrel and glyco-
protein IIb/IIIa antagonists, are widely used in clinics, either in
combination or alone for the prevention of thrombosis. However,
due to the limitations of existing drugs, there is a growing need
for antithrombotic agents with a new mode of action to provide
alternatives to existing treatment strategies.2 Multitarget anti-
thrombotic agents,3a designed to modulate two or more targets3b
involved in the development of thrombosis, pose a challenging
emerging strategy for antithrombotic drug discovery.
antithrombotic functions, are vulnerable to oxidative stress initi-
ated by reactive oxygen species (ROS) diffusing from leukocytes
or generated in the endothelium in the context of inflammation.4
The resulting endothelial injury may lead to exposure of thrombo-
genic components of the subendothelial extracellular matrix,
which initiates a vicious cycle of edema, leukocyte adhesion and
thrombosis.5,6 The pathologically altered vasculature involved in
inflammation and oxidative stress is thus an important underlying
cause of thrombosis.
Production of ROS is increased in many diseases, such as rheu-
matoid arthritis, ischemic stroke, atheromatosis, and heart attack.
Blood coagulation also stimulates production of reactive oxygen
species by human neutrophils and ROS have emerged as important
modulators of integrins in the blood clotting process through both
outside-in (integrins stimulating ROS production to affect intracel-
lular events) and inside-out signaling.7 The major lipidic proinflam-
matory mediators involved in thrombosis are leukotrienes
generated by 5-lipoxygenase, an enzyme expressed mainly by leu-
kocytes, and it has been demonstrated that leukocyte mediated
vein injury and thrombosis are reduced by 5-lipoxygenase inhibi-
tors.8,9 Besides damaging vascular endothelium, reactive oxygen
species can also initiate lipid peroxidation, which again is linked
to thrombotic events.10 Vascular oxidative stress, inflammation
and thrombosis are thus intertwined processes that display a syn-
ergistic pathological effect in many cardiovascular diseases.4 It
can therefore be anticipated that antithrombotic therapy with anti-
coagulant and/or antiplatelet agents, combined with interventions
directed against vascular oxidative stress and/or inflammation,
both boosting endothelial antithrombotic potential, would display
Under normal circumstances, endothelial cells play a critical
role in preventing intravascular thrombosis (i) by providing a un-
ique surface which prevents activation of coagulation and platelet
aggregation and (ii) by releasing mediators that inhibit prothrom-
botic processes. However, endothelial cells, which normally have
⇑
Corresponding author. Tel.: +386 1 476 9561; fax: +386 1 425 8031.
E-mail address: danijel.kikelj@ffa.uni-lj.si (D. Kikelj).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.089

´
M. Ilic et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4705–4709
4706
a synergistic action in the treatment of thrombosis. New multitar-
get antithrombotic drugs, combining in the same molecule antico-
5-nitrophenol 1 and subsequent N-methylation.17 Reduction of 3
with borane dimethyl sulfide complex afforded alcohols 4 which
were transformed to ethers 5 with 4-cyanophenol under Mitsun-
obu reaction conditions.18 Catalytic reduction of the nitro group
in 5 afforded aromatic amines 6 that were N-alkylated, using var-
ious fluorobenzaldehydes and sodium triacetoxyborohydride as
reducing agent,12,19 to afford secondary amines 7. Alkylation of 7
with ethyl bromoacetate afforded esters 8, and ketoesters 9 were
obtained by acylation of 7 with ethoxalyl chloride. Finally, com-
pounds 10a–h and 11a–d were prepared from nitriles 8 and 9,
using the Pinner reaction.20 Compounds with N-(benzyl)eth-
oxalylamino substituents in position 7 (analogs of 10e–h; struc-
tures not shown) were not tested since they could not be
obtained pure due to their instability.
The results of biological assays performed with compounds 10
and 11 are collected in Table 1. They were tested for thrombin
inhibition,13 as well as for their antioxidant activity and their abil-
ity to inhibit soybean lipoxygenase in order to assess their anti-
thrombotic potential. Taking into account the multifactorial
character of oxidative stress, we have evaluated the in vitro antiox-
idant activity of the synthesized compounds using two different
antioxidant assays: (i) interaction with the stable 1,l-diphenyl-2-
picrylhydrazyl free radical (DPPH)21, and (ii) interaction with the
water-soluble 2,20-azobis(2-amidinopropane) dihydrochloride
(AAPH), which has been used extensively as a clean and controlla-
ble source of thermally produced alkylperoxy free radicals.22 The
results obtained were compared to the antioxidant potency of well
known antioxidant agents, that is, caffeic acid, nordihydroguaiaret-
agulant and anti-inflammatory activity, would present
breakthrough in the therapy of thromboembolic diseases.
a
Recently we reported a new type of antithrombotic compound
possessing both thrombin inhibitory and GPIIb/IIIa receptor antag-
onistic activities,11,12 and later systematically varied them in the P3
part to improve their thrombin inhibitory activity and fibrinogen
receptor binding affinity. Whereas the resulting compounds 10
and 11 (Scheme 1, Table 1) were all found to be submicromolar
thrombin inhibitors, their IC50 values for antagonizing binding of
fibrinogen to GPIIb/IIIa were higher than 100 lM. Based on the
well known involvement of reactive oxygen species, lipid peroxi-
dation and 5-lipoxygenase mediated production of proinflamma-
tory leukotrienes in the pathology of thrombosis,4,14 as well as
the known antioxidant activities of some benzoxazine15 and mor-
pholine16 derivatives, we screened thrombin inhibitors 10 and 11
for their radical scavenging potential, inhibition of linoleic acid
peroxidation, and inhibition of soybean lipoxygenase, to assess
their multiple antithrombotic potential. The results demonstrate,
surprisingly, that some of our compounds, in addition to thrombin
inhibition, display antioxidant, and anti-lipid peroxidation activity
together with soybean lipoxygenase inhibition, which all might
contribute synergistically to their antithrombotic potential and
pave the way for a novel concept of antithrombotic therapy.
The synthesis of the target compounds is given in Scheme 1.
Ethyl 2,4-dimethyl-6/7-nitro-3-oxo-3,4-dihydro-2H-1,4-benzoxa-
zine-2-carboxylates 3 were prepared by cyclization of 2-amino-4/
CH3
CH3
CH3
OH
O
O
O
c
d
COOEt
COOEt
a
b
O2N
OH
O2N
O2N
O2N
NH2
N
N
H
O
N
O
CH3
CH3
1
2
3
4
CH3
O
N
CH3
CH3
O
O
e
HN
f
O
CN
O2N
H2N
O
CN
O
CN
N
N
CH3
CH3
CH3
R
5
6
7
CH3
O
CH3
EtOOC
NH2
NH
O
N
EtOOC
i
N
O
g
N
O
CN
N
CH3
CH3
8
10
R
R
O
N
O
CH3
CH3
O
O
EtOOC
EtOOC
NH2
NH
i
h
N
O
CN
O
N
N
CH3
CH3
R
R = 3-F; 4-F; 3,5-difluoro; 4,5-difluoro
R
9
11
Scheme 1. Reagents and conditions: (a) Br(CH3)C(COOEt)2, KF, DMF, 60 °C, 6 h; (b) MeI, KF, DMF, rt, 12 h; (c) Me2S ꢀ BH3, THF, reflux, overnight; (d) 4-cyanophenol, PPh3,
DIAD, THF, reflux, 48 h; (e) H2, Pd/C, THF, rt, 2 h; (f) fluorobenzaldehyde, NaBH(AcO)3, ClCH2CH2Cl, rt, 6 h; (g) BrCH2COOEt, BTEAC, K2CO3, CH3CN, 60 °C, 24 h; (h) ethoxalyl
chloride, Et3N, THF, rt, overnight; (i) HCl(g), EtOH, 0 °C, 30 min, then NH4OAc, EtOH, rt, 24 h.

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M. Ilic et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4705–4709
Table 1
Thrombin inhibition, interaction with DPPH, inhibition of lipid peroxidation (LP) and in vitro inhibition of soybean lipoxygenase (LO) by compounds 10 and 11.g
No.
Compound
MW
ClogP Thrombin
calcd Ki ( M)
% Reduction of DPPH activitya
50 10
20 min 60 min 20 min 60 min
LP Inhibition
IC50
M)b
LO Inhibition
(percentd/IC50 M))e
(l
l
(
l
lM
lM
NH
NH2
O
N
O
O
10a
10b
10c
10d
10e
10f
520.6 5.64
520.6 5.64
538.6 5.71
538.6 5.71
534.6 4.97
534.6 4.97
552.6 5.04
552.6 5.51
520.6 5.64
0.90 0.21 11
14
14
16
10
14
12
28
28
63
10
11
8
23
24
21
18
24
21
46
36
85
59
9.7d
73e
>0d
41.7d
>0d
>0d
>0d
99e
>0d
F
N
N
O
OEt
NH
NH2
O
N
0.69 0.21 11
77
68
36
90
51
61.5
69
81
O
F
OEt
NH
NH2
O
N
O
0.66 0.14
11
F
F
N
N
N
O
OEt
NH
NH2
O
N
O
0.95 0.20
8
8
F
O
F
OEt
NH
NH2
O
N
O
O
0.30 0.11 11
12
10
35
25
71
F
O
O
OEt
NH
NH2
O
N
0.33 0.08 10
N
N
N
O
F
O
O
O
OEt
NH
NH2
O
N
O
10g
10h
11a
0.37 0.07
23
F
F
O
OEt
NH
NH2
O
N
O
0.29 0.08 23
F
O
F
F
OEt
NH
NH2
0.35 0.08 48
N
O
O
N
O
EtO
(continued on next page)

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M. Ilic et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4705–4709
4708
Table 1 (continued)
No.
Compound
MW
ClogP Thrombin
calcd Ki ( M)
% Reduction of DPPH activitya
50 10
20 min 60 min 20 min 60 min
LP Inhibition
LO Inhibition
(percentd/IC50 M))e
(l
l
IC50
(
l
M)b
lM
lM
F
NH
NH2
11b
11c
520.6 5.64
0.31 0.08 62
70
81
88
84
93
90
94.5
>0d
N
O
N
O
EtO
F
O
NH
NH2
F
538.6 5.78
0.43 0.09 70
70
69
>0d
N
O
N
O
EtO
O
F
NH
NH2
F
11d
538.6 5.78
0.48 0.08 54
68
81
75
86
83
>0d
N
O
O
N
O
EtO
NDGAf
302.4 3.92
180.2 0.82
250.3 4.34
n.d.
n.d.
n.d.
68
n.d.
n.d.
83
n.d.
n.d.
n.d.
63% @ 100
n.d.
Caffeic acid
600e
TroloxÒ
l
Mc n.d.
a
b
c
d
e
f
Percent reduction of DPPH optical absorption at 517 nm measured after 20 and 60 min at 50 and 10
IC50 for inhibition of linoleic acid peroxidation.
Percent inhibition of linoleic acid peroxidation at 0.1 mM inhibitor concentration,
Percent inhibition of soybean lipoxygenase.
IC50 for inhibition of soybean lipoxygenase.
NDGA: nordihydroguairetic acid.
lM DPPH final concentration.
g
Values are means (SD < 10%) of three or four different determinations; n.d. = not determined.
ic acid (NDGA) and 6-hydroxy-2,5,7,8-tetramethychroman-2-car-
boxylic acid (TroloxÒ), a water-soluble vitamin E derivative used
in biological and biochemical applications to reduce oxidative
stress damage.
oxidation of linoleic acid to conjugated diene hydroperoxide by UV
spectrophotometry. The use of AAPH is recommended as more
appropriate for measuring radical-scavenging activity in vitro, be-
cause the activity of the peroxyl radicals produced by the action of
AAPH shows a greater similarity to cellular activities such as lipid
peroxidation. The results in Table 1 suggest that inhibition of lipid
peroxidation is inherent to 6-substituted derivatives 10, among
which compounds 10a, 10d, and 10f showed higher inhibition of li-
pid peroxidation values than TroloxÒ, which was used as a reference
inhibitor. Trolox exerts its inhibitory effect on lipid peroxidation
mainly through the ability of its 6-hydroxy-5,7,8-trimethylchro-
mane moiety to break the radical chain.25 Due to the similarity of
thestructuresof compounds 10 to thatof TroloxÒ (Fig. 1), the 6-ami-
no-dihydro-1,4-benzoxazine moiety could be made responsible for
generation of radicals effectively stabilized through resonance to
break the radical chain reaction.
Compounds 10a–h and 11a–d displayed moderate to good lev-
els of thrombin inhibition in vitro, with Ki values between 290 and
900 nM. DPPH has been used for measuring radical scavenging
ability21 of the tested compounds. Because of its unpaired electron,
DPPH has a strong absorption maximum at 517 nm, which is de-
creased when the unpaired electron of the radical becomes paired
in the presence of a hydrogen donor. The interaction of the exam-
ined compounds with the stable free radical DPPH (measured as
percent reduction in the optical absorption at 517 nm)22 indicates
their radical scavenging ability in an iron free system. Comparing
the radical scavenging effectiveness of the tested compounds with
that of NDGA, only 7-substituted derivatives (compounds 11a–d),
possessing a 1,4-diaminophenyl moiety, displayed high interaction
values that increased in a time and concentration dependent man-
ner. Compounds 10a–h showed low radical scavenging ability,
which could be attributed mainly to the absence of easily oxidized
functionalities like those present in 11a–d and NDGA (two catechol
moieties). Comparing the interaction values for the couples of m-
and p-isomers, it appears that p-F compound 11b is more potent
than 11a that possesses a m-F substituent. The presence of fluorine
in the para-position appears to have a favorable effect on the inter-
action with DPPH (11b and 11c). Of the 6-substituted oxo deriva-
tives 10e–h, the difluoro derivatives were more potent radical
scavengers than their monofluoro analogs (cf. 10e–f vs 10g–h).
Lipophilicity, expressed as theoretically calculated ClogP values,23
does not appear to account for the observed activity.
In the meta-fluoro analogs, the presence of an oxo group next
to the ethyl ester moiety decreased the lipid peroxidation inhib-
itory potency [cf. compound 10a (IC50 = 59
10e (IC50 = 90 M) and 10d (IC50 = 36 M) compared to 10h
(IC50 = 69 M)]. In contrast, in the the p-F analog 10b exhibiting
an IC50 of 77 M, introduction of an oxo goup in the position
to the ester carbonyl to give compound 10f increased the lipid
lM) compared to
l
l
l
l
a
peroxidation inhibitory potency to IC50 of 51
again does not seem to influence the results.
lM. Lipophilicity
The synthesized compounds were evaluated further for their
ability to inhibit soybean lipoxygenase (LO), using a UV absorbance
based enzyme assay.25 Although the results (Table 1) cannot be di-
rectly extrapolated to the inhibition of mammalian 5-lipoxygenase,
it has been shown that inhibition of plant lipoxygenase activity by
non-steroidal anti-inflammatory agents is qualitatively similar to
their inhibition of the rat mast cell lipoxygenase.25 The soybean
In an assay for lipid peroxidation inhibition,24 a water-soluble
azo compound AAPH was used as free radical initiator to follow

´
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M. Ilic et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4705–4709
CH3
lipoxygenase activity. Compounds that combine in the same mole-
cule thrombin inhibitory activity with lipid peroxidation and
lipoxygenase inhibition would be expected to display a synergistic
action in the treatment of thrombosis. This makes compounds 10
and 11 interesting candidates for further investigations towards
multiple antithrombotic drugs.
CH3
H3C
HO
O
COOH
CH3
Trolox
Acknowledgments
CH3
O
NH2
NH
This work was supported by Slovenian Research Agency Grant
No. P1-208. We wish to thank Dr. C. Hansch and Biobyte Corp.,
201 West 4th Str., Suite 204, Claremont, CA 91711, USA, for free ac-
cess to the C-QSAR program and Dr. Roger Pain from J. Stefan Insti-
tute, Ljubljana for critical reading of the manuscript.
X
N
O
EtOOC
N
CH3
R = 3-F;4-F; 3,5-difluoro; 4,5-difluoro
References and notes
R
10a-d: X = H, H
10e-h: X = O
1. (a) Lopez, A. D.; Mathers, C. D.; Ezzati, M.; Jamison, D. T.; Murray, C. J. L. Lancet
2006, 367, 1747; (b) http://www.who.int/topics/cardiovascular_diseases/en/.
2. Jeffrey, I.; Weitz, J. I.; Hirsh, J.; Samama, M. M. Chest 2008, 133, 234S.
Figure 1. Structures of TroloxÒ and compounds 10a–h.
´
3. (a) Ilic, M.; Kikelj, D.; Ilaš, J. Curr. Top. Med. Chem. in press.; (b) Morphy, J. R.;
Rankovic, Z. J. Med. Chem. 2005, 48, 6523.
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5. Doerschuk, C. M.; Quinlan, W. M.; Doyle, N. A.; Bullard, D. C.; Vestweber, D.;
Jones, M. L.; Takei, F.; Ward, P. A.; Beaudet, A. L. J. Immunol. 1996, 157, 4609.
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9. Schaub, R. G.; Yamashita, A. Exp. Mol. Pathol. 1986, 45, 343.
lipoxygenase inhibition assay may therefore be used as a simple
qualitative screen for lipoxygenase inhibitory activity. Although
most of the LO inhibitors are antioxidants or free radical scaveng-
ers,26 the relationship between LO inhibition and the ability of the
inhibitors to chelate and reduce the active site Fe3+, or compete
with arachidonic acid for binding to the enzyme active site, is well
known.14 Perusal of the soybean LO inhibition values (Table 1)
shows that, with the exception of 10b, 10d, and 10h, which display
better inhibition than caffeic acid, the standard inhibitor, other
compounds were devoid of activity. The active compounds were
characterized by moderate values of interaction with AAPH, but
low interaction values with DPPH, showing that lipid peroxidation
activity is not always accompanied by DPPH radical scavenging
ability and vice versa.27 This can be attributed to the different
chemical reactions involved in the two assays. Thus, although com-
pounds 11a–d interacted strongly with DPPH, they did not inhibit
soybean lipoxygenase. Although it appears that LO inhibition is
connected to the 6-substitution pattern, further experiments are
needed in order to explain the absence of activity in other deriva-
tives of the 6-substituted series.
10. Cabre, A.; Girona, J.; Vallve, J. C.; Masana, L. J. Cell. Physiol. 2004, 198, 230.
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11. Štefanic Anderluh, P.; Anderluh, M.; Ilaš, J.; Mravljak, J.; Sollner Dolenc, M.;
Stegnar, M.; Kikelj, D. J. Med. Chem. 2005, 48, 3110.
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12. Ilaš, J.; Jakopin, Z.; Borštnar, T.; Stegnar, M.; Kikelj, D. J. Med. Chem. 2008, 51,
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13. For experimental protocols see Ref. 12
14. Pontiki, E.; Hadjipavlou-Litina, D. Curr. Enzyme Inhib. 2005, 1, 11.
15. Largeron, M.; Lockhart, B.; Pfeiffer, B.; Fleury, M. B. J. Med. Chem. 1999, 42, 5043.
16. Blattes, E.; Lockhart, B.; Lestage, P.; Schwendimann, L.; Gressens, P.; Fleury, M.
B.; Largeron, M. J. Med. Chem. 2005, 48, 1282.
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17. Kikelj, D.; Suhadolc, E.; Urleb, U.; Zbontar, U. J. Heterocyclic Chem. 1993, 30, 597.
18. Mitsunobu, O. Synthesis 1981, 1.
19. Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah, R. D. J.
Org. Chem. 1996, 61, 3849.
20. Roger, R.; Neilson, D. G. Chem. Rev. 1961, 61, 179.
21. Blois, M. S. Nature 1958, 181, 1199.
22. Kontogiorgis, C.; Hadjipavlou-Litina, D. J. Enzyme Inhib. Med. Ch. 2003, 18, 63.
23. Biobyte Corp., C-QSAR Database, 201 West 4th Str., Suite 204, Claremont CA,
California 91711, USA.
24. Liegois, C.; Lermusieau, G.; Colin, S. J. Agr. Food Chem. 2000, 48, 1129.
25. Taraborewala, R. B.; Kauffman, J. M. J. Pharm. Sci. 1990, 79, 173.
26. Muller, K. Arch. Pharm. 1994, 327, 3.
27. Curini, M.; Epifano, F.; Genovese, S.; Menghini, L.; Ricci, D.; Fraternale, D.;
Giamperi, L.; Bucchini, A.; Bellacchio, E. Nat. Prod. Commun. 2006, 1, 1141.
In conclusion, of the compounds 10a–h and 11a–d possessing
thrombin inhibition activity, 11a–d were found to possess good
radical scavenging activity, 10a, 10d, and 10f inhibited lipid perox-
idation of linoleic acid, and 10b, 10d, and 10h inhibited soybean