Cyclobutane carboxamide inhibitors of fungal melanin: Biosynthesis and their evaluation as fungicides

Article abstract of DOI:10.1016/S0968-0896(00)00034-1

A new fungicide lead has been identified by enzyme screening of a focused combinatorial library. The lead compound 4 Inhibitors of scytalone dehydratase (SD)., a potent inhibitor of scytalone dehydratase (SD), exhibits fungicidal activity upon foliar application but does not show systemic activity. The X-ray crystal structure of the enzyme-inhibitor complex and an appreciation for the relationship between physical properties and systemic activity enabled us to rapidly improve upon this initial lead. The geminal halogen-methyl group combination was found to be optimal for interaction with the bounding serine and asparagine side-chain residues. Replacement of CF₃ with methyl was a key discovery, giving inhibitors with slightly diminished enzyme inhibition potency while significantly increasing systemic activity. Amides prepared from amines with 2,4-dichloro substitution on the phenyl ring gave the most potent enzyme inhibitors. Two compounds from this series showed systemic activity comparable to the commercial standard and were selected for outdoor testing in flooded plots which simulate rice paddies. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00034-1

Bioorganic & Medicinal Chemistry 8 (2000) 897±907
Cyclobutane Carboxamide Inhibitors of Fungal Melanin:
Biosynthesis and their Evaluation as Fungicides
Lee D. Jennings,*,y Dennis R. Rayner, Douglas B. Jordan,{ John F. Okonya,
Gregory S. Basarab,x Denise K. Amorose, Beth M. Anaclerio, John K. Lee,
Rand S. Schwartz and Kari Ann Whitmore
DuPont Agricultural Products, Stine Haskell Research Center, PO Box 30, Newark DE, 19714, USA
Received 30 August 1999; accepted 24 November 1999
AbstractÐA new fungicide lead has been identi®ed by enzyme screening of a focused combinatorial library. The lead compound 4,
a potent inhibitor of scytalone dehydratase (SD), exhibits fungicidal activity upon foliar application but does not show systemic
activity. The X-ray crystal structure of the enzyme±inhibitor complex and an appreciation for the relationship between physical
properties and systemic activity enabled us to rapidly improve upon this initial lead. The geminal halogen±methyl group combina-
tion was found to be optimal for interaction with the bounding serine and asparagine side-chain residues. Replacement of CF3 with
methyl was a key discovery, giving inhibitors with slightly diminished enzyme inhibition potency while signi®cantly increasing
systemic activity. Amides prepared from amines with 2,4-dichloro substitution on the phenyl ring gave the most potent enzyme
inhibitors. Two compounds from this series showed systemic activity comparable to the commercial standard and were selected for
outdoor testing in ¯ooded plots which simulate rice paddies. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
pathway are biochemical targets for the design of rice
blasticides.
Blast disease, caused by the pathogen Pyricularia gri-
sea,1 is a major cause of damage to growing rice plants,
resulting in decreased per acre yield of grain. Conse-
quently, the discovery of fungicides able to control blast
disease is an important objective for many agrochemical
businesses. Desirable attributes of rice blasticides are
high intrinsic potency, systemic activity, low toxicity to
non-pathogens, and the absence of growth-stunting
e€ects on the crop plant. Interference in a mechanism
by which a pathogen infects its host, rather than inter-
vention in the biological processes common to all higher
organisms, is an attractive strategy. In order to infect
its host, P. grisea must melanize an infection structure
(the appresorium) to penetrate the leaf surface.2 Con-
sequently, the enzymes of the melanin biosynthetic
The fungal melanin biosynthesis pathway includes a
series of successive reductions and dehydrations of
1,3,6,8-tetrahydroxynaphthalene to yield 1,8-dihydroxy-
napthalene, the last identi®ed precursor of fungal
melanin. Scytalone dehydratase (SD) catalyzes the
dehydration of scytalone and vermelone in this path-
way.3 Carpropamid (1) (Fig. 1), a potent inhibitor of
SD catalytic activity, has recently been commercialized
by Bayer as an agricultural fungicide useful for the
control of rice blast.4 Two additional inhibitors of
SD, diclocymet (2)5 and AC 382042 (3),6 have been
announced as rice fungicides under development. Car-
propamid is characterized by high selectivity toward the
target pathogen and low toxicity to mammals and ®sh.4a
Re®ned, high-resolution structures of SD-inhibitor
complexes have been obtained,7 and the detailed struc-
tural information has enabled structure-based design
of novel inhibitors.8 We have reported the successful
application of combinatorial chemistry and in vitro
screening for the identi®cation of a novel scytalone
dehydratase inhibitor, 4.9 We report here the results of
our e€orts to optimize systemic activity of this potent in
vitro inhibitor.
*Corresponding author. Tel.: +1-302-451-0075; fax: +1-302-366-
5738.
yPresent address: Wyeth-Ayerst Research, 401 N. Middletown Road,
Pearl River, NY 10965, USA.
{Present address: DuPont Pharmaceutical Company, Stine-Haskell
Research Center, 1094 Elkton Road, PO Box 30, Newark, DE 19714,
USA.
xPresent address: DuPont Central Research and Development, Building
328, Experimental Station, Wilmington, DE 19880-0328, USA.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00034-1

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L. D. Jennings et al. / Bioorg. Med. Chem. 8 (2000) 897±907
hydrogen bonding network provides considerable
energy of binding and modi®cations that disrupt this
network are often deleterious.8a The crystal structure
also shows the chlorine and methyl substituents of
the cyclobutane ring oriented towards the Asn131
side chain of SD, apparently making a complimentary
electrostatic interaction. These substituents do not
completely ®ll their respective binding pockets. The tri-
¯uoromethyl group, phenyl group and bromine atom
occupy lipophilic pockets. The tri¯uoromethyl group is
directed towards the p-face of Phe53, which has been
implicated as an important recognition element for SD
inhibitors.7a,8 A Connolly surface10 (Fig. 3) of the inhi-
bitor binding pocket shows that there was an unoccu-
pied region accessible from the 2-position of the
aromatic ring of 4. This pocket could accommodate one
or two heavy atoms. Additionally, examination of the
overlay of crystal structures of SD-inhibitor complexes
from other analogue series demonstrated the potential
for side chains of the protein residues in this region to
move.8c
Figure 1. Inhibitors of scytalone dehydratase (SD).
Results and Discussion
While the lead compound 4 shows strong in vitro
potency and exhibits good in vivo fungicidal activity
upon foliar application, it lacks systemic activity. Thus,
we sought to alter the structure of 4 in such a way as to
impart systemic activity while maintaining SD inhibi-
tion potency. Soil uptake and translocation within the
plant are related to hydrophobicity and our experience
is that a partitioning coecient between octanol and
water (LogP) of 3.0 to 4.0 is optimal for systemic activ-
ity. Thus, our strategy was to use our understanding of
the interaction of the inhibitor with the protein-binding
site to design less lipophilic yet potent inhibitors.
Chemistry
Cyclobutane carboxylic acid 10 was synthesized follow-
ing the procedure of Hall et al.11 with some minor
modi®cations (Scheme 1). Thermolysis of the cyano-
hydrin acetyl ester 7 in a Pyrex tube packed with helices
gave the tri¯uoromethylacrylonitrile 8. Thermal cyclo-
addition of allene with 8 at 200 ꢀC a€orded product in
only 25% yield following distillation. The nitrile was
hydrolyzed with base, and the resulting acid was reacted
with concentrated aqueous HCl to give the acid 10 as a
1:1 mixture of isomers. Alternatively, 9 could be con-
verted directly to 10 by reaction with concentrated HCl
in a pressure vessel heated to 100 ꢀC.
The crystal structure of 4 bound to SD was solved at
1.8 A resolution and provided an excellent model of
the inhibitor binding pocket.9 Major interactions with
hydrophilic residues are shown in Figure 2. The crystal
structure implies hydrogen bonding interactions
between the inhibitor amide N1 and a bound water
molecule and between the amide O and the hydroxyl
group of the Tyr50 side-chain. Investigations of other
SD inhibitors by our group have shown that this
Replacement of CF3 with other substituents was
achieved by analogous 2+2 cycloadditions. The 3-
chloro-1,3-dimethylcyclobutane carboxylic acid 16 was
synthesized following a similar route (Scheme 2).
Cycloaddition of either methacrylonitrile or ethyl meth-
acrylate with allene a€orded the corresponding methyl-
ene cyclobutanes 12 or 15 in 23±30% yield. 1H NMR of
the crude products prior to distillation indicated that
the major product was octahydronaphthlene 13, which
results from the cascade [4+2] cycloaddition of meth-
acrylonitrile with the dimer of allene, 1,2-dimethylene
cyclobutane.12 Repeating the reaction with an excess of
ethyl methacrylate was operationally simpler and gave
the desired product 15 in 41% yield. Carboxylic acid 19
was prepared from 2-chloroacrylonitrile following a
similar route (Scheme 2).
Fluoromethyl acrylate ester 22 was prepared as shown
in Scheme 3. This intermediate was reacted with allene
following the standard procedure but the crude reaction
mixture contained little or none of the desired cyclo-
butane 23 as judged by 1H NMR. The cyclobutane
carboxylic acid 26 was prepared by alkylation of the
dianion of 3-methylenecyclobutane carboxylic acid
with ethyl iodide followed by hydrolysis with HCl
(Scheme 4).
Figure 2. A schematic drawing of the interactions between 4 and SD.

L. D. Jennings et al. / Bioorg. Med. Chem. 8 (2000) 897±907
899
Figure 3. (A) Cross section of the Connolly surface of the SD binding pocket showing 4. (B) Connolly surface of the SD binding pocket generated
from the crystal structure of the SD-4.
Scheme 1. Synthesis of cyclobutane carboxylic acid 10. (a) KCN, H2SO4, 60%; (b) AcCl, re¯ux, 84%; (c) pyrolysis at 520 ꢀC, 66%; (d) allene (1.6
equiv), hydroquinone (catalytic), benzene, 200 ꢀC, 25%; (e) NaOH, H2O, 100 ꢀC, 96%; (f) concd HCl, rt, 68%.
Scheme 2. Synthesis of cyclobutane carboxylic acids 16 and 19. (a) Allene (1.66 equiv), hydroquinone (catalytic), benzene, 200 ꢀC, 23±30%; (b)
allene (0.25 equiv), hydroquinone (catalytic), benzene, 200 ꢀC, 41%; (c) NaOH, H2O, 100 ꢀC, quantitative; (d) concd HCl, rt, 77%; (e) concd HCl,
100 ꢀC, 40%.

900
L. D. Jennings et al. / Bioorg. Med. Chem. 8 (2000) 897±907
Scheme 3. Attempted preparation of ¯uoromethyl cyclobutane carboxylic acid. (a) Formaldehyde, H3PO4, K2CO3, 69%; (b) DAST, CH2Cl2, 45%;
(c) allene, hydroquinone (catalytic), benzene, 200 ꢀC.
TMS group of 32 was removed by basic hydrolysis. The
3,3-di¯uoro analogue 35 was prepared by reaction of
the cyclobutanone 34 with DAST.
The cyclobutane carboxylic acids were converted to
their corresponding acid chlorides using either thionyl
chloride or oxalyl chloride/DMF for acylation of
amines (Scheme 7). Where possible, cis and trans iso-
mers of the formed amides were separated by chroma-
tography. The identity of the separate isomers was
determined by NOE experiment. Typically, the isomer
with the lower Rf has the halogen substituent cis to the
carboxamide functionality.
Scheme 4. Preparation of cyclobutane carboxylic acid 26. (a) LDA,
THF, then EtI, 68%; (b) concd HCl, 100%.
Biological evaluation
Owing to the ring strain of the cyclobutane ring, the
methylene group of intermediate 27 was highly reactive.
Hydrogen bromide was added across the double bond
at room temperature (Scheme 5). Chlorination of 27
using dichloroiodobenzene gave the chloro-chloro-
methyl cyclobutane carboxylic acid 29. The product 29
Cyclobutane carboxamides were evaluated for activity
in an SD inhibition assay8a and in vivo for their ability
to protect rice plants from infection by P. grisea (Tables
1±3). The soil systemic assay rates disease control of rice
seedlings in ¯ooded culture tubes 7±10 days after
inoculation with P. grisea. The SD inhibiting fungicide
carpropamid is included for comparison (Table 1, entry 6).
1
was shown to be a 1:1 mixture of isomers by H NMR.
Substituents other than methyl were introduced into the
cyclobutane 3-position by the following route (Scheme
6). 3-Methylene cyclobutane carboxylic acid 27 was
reacted with ozone, and the intermediate ozonide was
decomposed with dimethylsul®de to give the cyclobuta-
none 30. The cyclobutanone was reacted with ethyl-
magnesium bromide and lithium TMS acetylide to give
the hydroxy compounds 31 and 32, respectively. The
1-Tri¯uoromethylcyclobutane carboxamides. The cis and
trans isomeric forms of the substituted cyclobutane ring
have a signi®cant e€ect on activity (Table 1, entries 1
and 2). The isomers with the chlorine atom cis to the
carboxamide functionality are typically 10-fold more
potent than the trans isomers. The model of the SD-
inhibitor complex clearly shows the interaction of the
chlorine substituent on the cyclobutane ring with the
Asn131 side-chain carboxamide. Though we cannot
ascertain the orientation of the Asn131 side-chain car-
boxamide from the X-ray crystal structure of the SD-
inhibitor complex, we hypothesize that the observed
signi®cant preference for binding of the cis isomer pairs
the 3-chlorine atom with the NH2 on the Asn131 side-
chain. 2,4-Disubstitution with chlorine on the phenyl
ring increases potency of enzyme inhibition by a small
extent, as anticipated. Extension of the bridge between
the amide and phenyl group from one to three atoms is
tolerated (39). However, with the three atom spacer,
substituents in the 4-position of the phenyl ring are not
tolerated (data not shown). Instead 2- and 2,5-di-
substitution is best.8b,c The R-con®guration of the chiral
Scheme 5. Synthesis of substituted cyclobutanecarboxylic acids 28 and
29. (a) HBr, 66%; (b) dichloroiodobenzene, CH2Cl2, 46%.

L. D. Jennings et al. / Bioorg. Med. Chem. 8 (2000) 897±907
901
Scheme 6. Preparation of cyclobutane carboxylic acids 31, 33 and 35. (a) O3, CH2Cl2, then (CH3)2S, 78%; (b) EtMgBr, THF, 64%, cis and trans
isomers separated by chromatography; (c) lithium TMS acetylide, THF, 85%, cis and trans isomers separated by chromatography; (d) NaOH,
MeOH, 70%; (e) DAST, CH2Cl2; (f) NaOH, H2O, 100 ꢀC, 43% (two steps).
center was found to be critical for good binding and
disease control.
Since the crystal structure showed room in the binding
pocket adjacent to the C3-methyl on the cyclobutane
ring, we sought analogues with larger, lipophilic sub-
stituents here. We reasoned that the decrease in lipo-
philicity made by making the switch from CF3 to methyl
would balance the lipophilicity increase of larger C3
substituents. Modeling indicated that the chloromethyl
group trans to the carboxamide group would be well
accommodated. Surprisingly, both cis and trans isomers
are very potent inhibitors of SD (46 and 47). Perhaps
protein side chains bounding the upper side of the
cyclobutane binding pocket are able to move to accom-
modate the chloromethyl group of 46 in a fashion that
In all cases, 1-tri¯uoromethylcyclobutane carboxamides,
while being potent enzyme inhibitors, and having foliar
disease control, lacked systemic activity. This is con-
sistent with our experience that a LogP higher than 4.0
frequently precludes plant translocation. The LogP of
37 was determined to be 4.25 by high performance
liquid chromatography (HPLC). Therefore, we focused
on making changes to the substituents on the cyclobu-
tane ring which would decrease lipophilicity.
Cyclobutane modi®cation. A series of cyclobutane car-
boxylic acids were synthesized and coupled to (R)-1-(4-
bromophenyl)ethylamine (Table 2). Deletion of sub-
stituents on C1 or C3 has a deleterious e€ect on enzyme
inhibitory potency (40 and 41). Replacement of CF3
with halogen gives good enzyme inhibitors which lacked
systemic activity (42 and 44), presumably due to higher
lipophilicity. Replacement of CF3 with methyl gives
an inhibitor with slightly diminished potency (45).
However, this modi®cation signi®cantly improved its
systemic activity. 45 protected rice plants from P. grisea
infection in the systemic test at 3 ppm concentration
whereas 4 failed to protect the plants from infection at
that rate.
Table 1. Inhibition of SD and control of blast disease in systemic
assays by 1-tri¯uoromethyl cyclobutane carboxamides
Compd no.
Structure
Ki (nM)
Systemic assay
4
0.026‹0.002
68 at 3 ppm
50 at 0.8 ppm
0 at 0.2 ppm
36
37
38
39
1
0.43‹0.03
0.067‹0.002
0.011‹0.001
0.350‹0.030
0.041‹0.001
13
13
0
82
35
31
0
0
0
64
0
0
98
78
27
Scheme 7. Preparation of cyclobutane carboxamides (a) (i) (COCl)2,
cat. DMF, CH2Cl2; (ii) RNH2, Et3N; (b) (for XˆOH) EDC, RNH2,
Et3N; (c) separate isomers by chromatography.

902
L. D. Jennings et al. / Bioorg. Med. Chem. 8 (2000) 897±907
Table 2. Inhibition of SD and control of blast disease in systemic
assays by cyclobutane carboxamides
Table 3. Inhibition of SD and control of blast disease in systemic
assay by 1-methylcyclobutane carboxamides
Compd no.
Structure
Ki (nM)
Systemic assay
Not tested
Compd no.
Structure
Ki (nM)
Systemic assay
40
520‹50
53
0.012‹0.003
98 at 3 ppm
100 at 0.8 ppm
48 at 0.2 ppm
41
42
44‹1
Not tested
54
55
56
57
58
3.2‹0.2
0
0
0
0.45‹0.009
17 at 3 ppm
0 at 0.8 ppm
0 at 0.2 ppm
0.016‹0.002
0.030‹0.002
0.420‹0.02
0.057‹0.008
93
0
25
43
44
45
46
47
6‹0.3
93
0
0
100
100
98
0.310‹0.02
0.100‹0.005
0.027‹0.001
0.016‹0.002
46
0
0
99
65
16
99
38
22
21
0
0
96
0
31
93
79
0
substitution patterns tolerated in this region of the
enzyme active site. Accordingly, we synthesized a small
set of amines that included those known to a€ord high
inhibitory potency and those that addressed opportu-
nities presented by the steric demands of the binding
pocket Connolly surface.
48
49
50
51
52
0.021‹0.003
480‹40
16‹1
Not tested
Not tested
Not tested
Not tested
Not tested
As anticipated, for hydrophobic substituents that are
shaped correctly, potency increases with increased bur-
ied hydrophobic surface area (53, 55 and 56) (Table 3).
Lower activity of the inhibitor 54 prepared from 1-(a-
naphyl)ethylamine demonstrates that the hydrophobic
pocket cannot accommodate this substituent. Amides
prepared from phenoxypropylamines with halogen sub-
stitution in a 2,5-con®guration gave potent inhibitors
that were also found to have good systemic activity (57,
58).
28‹0.5
3.2‹0.1
was not anticipated by modeling experiments. Alter-
natively, the inhibitor may adopt a new orientation that
shifts the position of the cyclobutane relative to the
binding pocket.
Conclusions
The available model of the enzyme±inhibitor complex
and an appreciation for the relationship between physi-
cal properties and systemic activity enabled us to focus
our synthetic e€ort on modi®cations likely to improve
activity of our initial lead 4. The geminal halogen±
methyl group combination seemed optimal for electro-
static interaction with the asparagine sidechain bound-
ing the end of the cyclobutane binding pocket. No
modi®cation otherwise at the cyclobutane 3-position is
superior. Replacement of CF3 of 4 with methyl was a
key discovery, giving inhibitors with slightly diminished
enzyme potency but superior systemic activity. Amides
prepared from amines with 2,4-dichloro substitution on
the phenyl ring gave the most potent enzyme inhibitors.
Two compounds from this series (47 and 55) were iden-
ti®ed as being on par with carpropamid and were selec-
ted for outdoor testing in ¯ooded plots which simulate
real rice paddies.
Although we observed that hydrogen accepting sub-
stituents on the cyclobutane ring are making favorable
contacts with the hydrogen donating carboxamide NH2
of Asn131 (Fig. 2), we were interested to see if hydrogen
donating substituents might donate a hydrogen to the
carboxamide carbonyl. We were able to make a number
of cyclobutane carboxamides substituted with hydroxyl
groups (49±52). However, in all cases potency was
adversely a€ected. Presumably the increased cost to
desolvate the more hydrophilic inhibitors contributed to
the poor inhibition constants observed.
Amine modi®cation. The phenyl group occupies a
hydrophobic region of the SD inhibitor binding pocket.
A large number of analogues made on other series
of SD inhibitors8b,c delineates the modi®cations and

L. D. Jennings et al. / Bioorg. Med. Chem. 8 (2000) 897±907
903
Experimental
Methods and materials
added and the mixture was stirred at rt for 30 min. To
the resulting LDA solution was added 3-methylene-
cyclobutanecarboxylic acid (2.24 g, 20 mmol) in 10 mL
of THF and the mixture was heated at 50ꢀC for 1 h. The
reaction mixture was then cooled to 0 ꢀC, iodoethane
(3.10 g, 1.6 mL, 20 mmol) was added, and the resulting
mixture was stirred at rt for 2 h. Upon completion of the
reaction, THF was removed by evaporation in vacuo,
aq NaOH (1M, 100 mL) was added to the residue and
excess amine was extracted from the basic aq layer with
CH2Cl2 (3Â50 mL). The aq layer was acidi®ed with
concd HCl (ꢁ10 mL) and extracted with CH2Cl2
(3Â50 mL). The combined CH2Cl2 extracts were dried
(MgSO4), concentrated in vacuo and puri®ed by chro-
matography (EtOAc:hexanes, 1:4) to provide a yellow
oil (1.90 g, 68%): 1H NMR (CDCl3, 300 MHz) d 0.91 (t,
J=7.4 Hz, 3H), 1.85 (q, J=7.4 Hz, 2H), 2.56 (m, 2H),
3.14 (m, 2H), 4.86 (m, 2H); MS (APCI ) 139 (M-1).
Thin-layer chromatography (TLC) and ¯ash chromato-
graphy were performed with E. Merck silica gel (230±
400 mesh). Compounds were visualized on TLC using
10% phosphomolybdic acid in EtOH. Except where
indicated, anhydrous solvents and reagents were used as
1
received. All melting points are uncorrected. H NMR
spectra, except where noted, were recorded at 300 MHz
in CDCl3. 19F NMR spectra were recorded at 282 or
376 MHz in CDCl3. Mass spectra were obtained on a
Micromass Platform 2 using atmospheric pressure che-
mical ionization detection at 3.5 kV positive ion and
2.5 kV negative ion. Accurate mass elemental composi-
tion measurements were performed on a Finnigan
MAT900XL mass spectrometer using electron multi-
plier detection at 1.80 kV positive ion and 1.90 kV
negative ion.
cis- and trans-1-Ethyl-3-chloro-3-methylcyclobutanecar-
boxylic acid (26). 1-Ethyl-3-methylenecyclobutanecar-
boxylic acid (25) (1.50 g, 10.7 mmol) was added
dropwise to a stirred solution of concd HCl (20 mL).
The mixture was stirred at rt for 2 h under N2. The
product was extracted with CH2Cl2 (3Â20 mL) and the
combined CH2Cl2 extracts were washed with H2O
(20 mL), dried (MgSO4) and evaporated in vacuo to
Starting materials
(R)-(+)-1-(4-Bromophenyl)ethylamine was purchased
from Fluka Chemie AG, (R)-(+)-1-(4-chlorophenyl)-
ethylamine was purchased from Celgene, (R)-(+)-1-(1-
naphthyl)ethylamine was purchased from the Aldrich
Chemical Company, and (R)-(+)-1-(2-naphthyl)ethyl-
amine was purchased from Fluka Chemie AG. cis- and
trans-3-Chloro-3-methyl-1-tri¯uoromethylcyclobutane
carboxylic acid (10), cis- and trans-3-chloro-1,3-dimeth-
ylcyclobutanecarboxylic acid (16), 3-methylenecyclo-
butane carboxylic acid (24) and 3-methylene-1-methyl-
cyclobutane carboxylic acid (27) were made following
the published procedures11 with minor modi®cations as
described in the text. (R)-3-[(2-¯uorophenyl)oxy]propyl-
2-amine, (R)-3-[(2,5-di¯uorophenyl)oxy]propyl-2-amine,
and (R)-3-[(2-chloro-5-¯uorophenyl)oxy]propyl-2-amine
were made following our published procedure.8b,13 (R)-
1-(2,4-Dichlorophenyl)ethylamine14 was prepared from
(R)-1-(4-chlorophenyl)ethylamine by the method of
Polniaszek and Lichti.15 The known ‹1-(4-bromo-2-
methoxyphenyl)ethylamine16 was prepared by Fries
rearrangement17 of 1-acetoxy-3-bromobenzene followed
by methylation of the hydroxy group and reductive
amination of the resulting ketone.18
provide a white solid (1.89 g, 100%): mp 54±56 ꢀC; H
1
NMR (CDCl3, 300 MHz, mixture of cis and trans iso-
mers) d 0.89 (t, J=7.4 Hz, 3H), 1.73 and 1.74 (two s,
3H), 1.81 and 2.01 (two q, J=7.4 Hz, 2H), 2.35 and 2.57
(two d, J=13.7 and 14.3 Hz, 2H), 2.87 and 3.09 (two d,
J=13.7 and 14.3 Hz, 2H).
cis- and trans-3-Chloro-3-(chloromethyl)-1-methylcyclo-
butanecarboxylic acid (29). To iodobenzenedichloride20
(2.4 g, 9.72 mmol) in 9 mL CH2Cl2 was added 3-methyl-
ene-1-methylcyclobutane carboxylic acid (27) (1.0 g,
7.93 mmol) in 2 mL CH2Cl2. The solution was stirred at
ambient temperature for 1 h, re¯uxed for 1 h, and
allowed to cool. The solution was extracted once with
1 M NaOH and the aq phase was acidi®ed with concd
HCl. The product precipitated as an oil upon acidi®ca-
tion. The product was extracted with CH2Cl2, dried and
concentrated to provide the product as a colorless oil
(0.71 g, 46%) as an equal mixture of cis and trans iso-
1
cis- and trans-1,3-Dichloro-3-methylcyclobutanecarboxylic
acid (19). 3-Chloro-3-cyanomethylene cyclobutane19
(18) (5.53 g, 43.35 mmole) was dissolved in concentrated
aqueous HCl (45 mL). The solution was stirred at rt
overnight and then re¯uxed for another 30 min. The
product was extracted from the aqueous layer with
CH2Cl2. The combined organic extracts were dried
(MgSO4) and concentrated. 40% Of the desired car-
boxylic acid was obtained as a 1:1 ratio of isomers
which were not separated. 1H NMR (300 MHz, d6-
DMSO) d 1.64 (s, 3H), 1.86 (s, 3H), 2.90±3.05 (m, 4H),
3.30±3.37 (m, 4H), 13.75 (br s, 2H).
mers which were not separated: H NMR (CHCl3) d:
1.46 (s, 3H), 1.65 (s, 3H), 2.48 (d, J=15 Hz, 2H), 2.61
(d, J=15 Hz, 2H), 3.05 (d, J=19 Hz, 2H), 3.10 (d,
J=19 Hz, 2H), 3.78 (s, 2H), 3.83 (s, 2H).
1-Methyl-3-oxocyclobutanecarboxylic acid (30). 1-Methyl-
3-methylenecyclobutane carboxylic acid (27) (3.0 g,
19 mmol) was dissolved in CH2Cl2 (150 mL) and cooled
to 78 ꢀC. Ozone from an ozone generator was bubbled
through the solution while stirring for 30 min, in which
time the reaction mixture turned blue. Oxygen was
bubbled through the solution followed by N2 until
the blue color disappeared. Dimethyl sul®de (4.3 mL,
58 mmol) was added to the clear solution and the mix-
ture was stirred overnight at rt. The reaction mixture
was washed with H2O, dried (MgSO4) and evaporated
in vacuo to provide the ketone as an o€-white solid
1-Ethyl-3-methylenecyclobutanecarboxylic acid (25). Di-
isopropyl amine (4.44 g, 6.08mL, 44mmol) was dissolved
in THF (100mL) under N2 and cooled to 78ꢀC. n-
BuLi (2.5 M in hexanes, 20 mL, 50 mmol) was carefully

904
L. D. Jennings et al. / Bioorg. Med. Chem. 8 (2000) 897±907
(3.8 g, 75%): mp 62±65 ꢀC; 1H NMR (CDCl3, 300 MHz)
d 1.48 (s, 3H), 2.95 (d, J=19.6 Hz, 2H), 3.39 (d, J=
19.6 Hz, 2H), 12.65 (s,1H); MS(APCI ) 127 (M-1).
oil (0.40 g, 76%). Saponi®cation of the ester provided a
brown oil quantitatively: 1H NMR (CDCl3, 300 MHz) d
1.53 (s, 3H), 2.47 (m, 2H), 3.09 (m, 2H); 19F NMR
(CDCl3, 282 MHz) d 92.5 (d), 88.2 (d).
cis- and trans-3-Ethyl-3-hydroxy-1-methylcyclobutanecar-
boxylic acid (31). 1-Methyl-3-oxocyclobutanecarboxylic
acid (1.00 g, 7.8 mmol) was dissolved in 15 mL of anhyd
THF and cooled to 0 ꢀC under N2. EtMgBr (1 M in
THF, 17.0 mL, 17.0 mmol) was added to the solution
dropwise. The mixture was warmed to rt and stirred for
2 h. The reaction mixture was then treated with 1 M
HCl to pH 2 and extracted with EtOAc. The combined
EtOAc extracts were dried (MgSO4) and evaporated in
vacuo to provide the crude product. Puri®cation by
column chromatography (EtOAc:hexanes 1:1) provided
the cis and trans isomers: trans-isomer, light yellow oil,
0.43 g, 35%; 1H NMR (CDCl3, 300 MHz) d 0.89 (t,
J=7.2 Hz, 3H), 1.55 (s, 3H), 1.63 (q, J=7.2 Hz, 2H),
1.97 (d, J=13.9 Hz, 2H), 2.64 (d, J=13.9 Hz, 2H). cis-
Standard conditions for the coupling of cyclobutane
carboxylic acids to amines were as follows.
Method A. A solution of 0.240 g (1.11 mmol) of 3-
chloro-3-methyl-1-tri¯uoromethylcyclobutane carboxy-
lic acid in 5 mL SOCl2 was heated at re¯ux for 1 h.
Solvent was removed in vacuo. The residue was added
to a solution of Na2CO3 (0.176 g, 1.66 mmol) and (R)-
(+)-1-(4-bromophenyl)ethylamine (0.172 g, 1.11 mmol)
in 6 mL of 1:1 dioxane:water, pre-cooled to 0 ꢀC. After
stirring at rt overnight, the mixture was poured into
50 mL water and extracted twice with 50 mL EtOAc.
Drying (MgSO4) and removal of the solvent gave an oil
which was chromatographed on silica gel, eluting with
25% hexane±CH2Cl2, to a€ord the isomeric cyclobu-
tane carboxamides 4 and 36.
1
isomer, light yellow oil, 0.29 g, 24%; H NMR (CDCl3,
300 MHz) d 0.92 (t, J=7.2 Hz, 3H), 1.40 (s, 3H), 1.63
(q, J=7.2 Hz, 2H), 2.10 (d, J=14.2 Hz, 2H), 2.52 (d,
J=14.2 Hz, 2H).
Method B. To 0.30 g (2.0 mmol) of 3,3-di¯uoro-1-
methylcyclobutane carboxylic acid (36) in CH2Cl2
(8 mL) under N2 was added 1 drop DMF and oxalyl
chloride (0.21 mL, 2.4 mmol). The mixture was stirred
overnight at rt under N2. A solution of (R)-(+)-(4-bro-
mophenyl)ethylamine hydrochloride (0.47 g, 2.0 mmol)
and triethylamine (0.61 mL, 4.4 mmol) in CH2Cl2
(5 mL) was added to the reaction mixture and stirring
was continued overnight at rt. The reaction mixture was
diluted with CH2Cl2 and washed with water, 1 M HCl
and 1 M NaOH. The organic phase was dried (MgSO4)
and evaporated in vacuo to yield 43 (0.49 g, 74%).
cis- and trans-3-Hydroxy-1-methyl-3-trimethylsilylethyn-
ylcyclobutanecarboxylic acid (32). n-BuLi (2.5 M in
hexanes, 6.9 mL, 17.2 mmol) was added dropwise to a
solution of TMS acetylene (1.70 g, 2.4 mL, 17.2 mmol)
in 20 mL of THF under N2 at 78 ꢀC. The mixture was
stirred for 30 min. A solution of 1-methyl-3-oxocyclo-
butanecarboxylic acid (1.00 g, 7.8 mmol) in THF (5 mL)
was added dropwise and the mixture was warmed to rt
and stirred for 2 h. The reaction mixture was then trea-
ted with 1 M HCl to pH 2 and extracted with EtOAc.
The combined EtOAc extracts were dried (MgSO4) and
evaporated in vacuo to provide a brown oil (1.48 g,
Method C. To a mixture of 3-ethynyl-3-hydroxy-1-
methyl cyclobutane carboxylic acid (33) (0.30 g,
1.9 mmol), (R)-(+)-1-(4-bromophenyl)ethylamine hydro-
chloride (0.46 g, 1.9 mmol) and 1-(3-dimethylaminopro-
pyl)-3-ethylcarbodiimide hydrochloride (EDC) (0.41 g,
2.1 mmol) in CH2Cl2 under N2 was added Et3N
(0.54 mL, 3.9 mmol). The mixture was stirred at rt for
24 h. The reaction mixture was then diluted with
CH2Cl2, washed with 0.5 M HCl, NaHCO3 and H2O,
dried (MgSO4) and the solvent was removed by eva-
poration under reduced pressure. The residue was
chromatographed on a silica gel column eluting with
EtOAc:hexanes, 1:1, to obtain 51 and its trans isomer.
1
84%): H NMR (CDCl3, 300 MHz, mixture of cis and
trans isomers) d 0.16 (two s, 9H), 1.55 (br s, 3H),
2.52 (overlapping d, 2H), 2.82 (overlapping d, 2H);
MS(APCI ) 225 (M-1).
cis- and trans-3-Ethynyl-3-hydroxy-1-methylcyclobutane-
carboxylic acid (33). A mixture of 3-hydroxy-1-methyl-
3-trimethylsilylethynylcyclobutanecarboxylic acid (2.50g,
11.0 mmol) and aq NaOH (1 M, 11.5 mL, 11.5 mmol) in
15 mL of MeOH was stirred overnight at rt. The solvent
was removed by evaporation under reduced pressure
and the residue was dissolved in Et2O, washed with H2O
and brine, dried (MgSO4), and evaporated in vacuo to
provide an orange solid (1.2 g, 70%): mp 88±91 ꢀC; H
Products 4, 36±58 were prepared following these proce-
dures. Characterization data for these compounds are
as follows:
1
NMR (CDCl3, 300 MHz, mixture of cis and trans iso-
mers) d 1.55 (s, 3H), 2.26 and 2.50 (two d, J=13.8 Hz,
2H), 2.60 and 2.63 ( two s, 1H), 2.82 and 3.06 (two d,
J=13.8 Hz, 2H); HRMS m/e calcd for C8 H9 O3 (M-1)
153.0552. Found: 153.0557.
cis- and trans-(R)-3-Chloro-3-methyl-1-tri¯uoromethy-N-
[1-(4-bromophenyl)ethyl] cyclobutanecarboxamide (4 and
36). Method A, cis isomer 36 isolated as a white solid
(38% yield): mp 128±129 ꢀC; 1H NMR (400 MHz,
CDCl3) d 1.49 (d, J=6.8 Hz, 3H), 1.54 (s, 3H), 2.86±
2.95 (m, 2H), 2.97±3.02 (m, 2H), 5.05±5.12 (m, 1H), 5.85
(br s, 1H), 7.15 (d, J=8.0 Hz, 2H), 7.46 (d, J=8.0 Hz,
2H); 19F NMR (376MHz, CDCl3) d 73.13 (s); irradia-
tion at 73.13 ppm showed no NOE enhancement on
C-3 methyl. HRMS m/e calcd for C15H17Br Cl F3 N O:
3,3-Di¯uoro-1-methylcyclobutanecarboxylic acid (35). A
solution of ethyl 1-methyl-3-oxocyclobutane carboxy-
late (34) in 15 mL CH2Cl2 under N2 was treated with
DAST (1.15 mL, 8.92 mmol) and stirred overnight at rt.
The reaction mixture was poured over ice and extracted
with CH2Cl2, dried (MgSO4) and evaporated in vacuo
to yield the corresponding di¯uoro ester as an orange

L. D. Jennings et al. / Bioorg. Med. Chem. 8 (2000) 897±907
905
398.0134. Found 398.0148. trans Isomer 4 isolated as a
2H), 8.10 (d, J=7.5 Hz, 1H); HRMS m/e calcd for C13
H17BrNO: 282.0494. Found: 282.0494.
white solid (39%): mp 142±144 ꢀC; H NMR (CDCl3) d
1
1.52 (d, J=6.8 Hz, 3H), 1.74 (s, 3H), 2.80±2.88 (m,
2H), 3.10±3.16 (m, 2H), 5.15 (m, 1H), 5.85 (br s, 1H),
7.19 (d, J=8.0 Hz, 2H), 7.31 (d, J=8.0 Hz, 2H); 19F
NMR (376 MHz, CDCl3) d 73.71 (s); irradiation at
73.71 ppm showed positive NOE enhancement on
C-3 methyl; HRMS m/e calcd for C15 H17BrClF3NO:
398.0134. Found. 398.0147.
cis-(R)-3-Chloro-3-methyl-N-[1-(4-bromophenyl)ethyl]-
cyclobutanecarboxamide (41). Method A, isolated as a
ꢀ
1
white solid (7%): mp 110±131 C; H NMR (300 MHz,
d6-DMSO) d 1.30 (d, J=7.13 Hz, 3H), 1.72 (s, 3H),
2.46±2.70 (m, 4H), 2.85±2.95 (m, 1H), 4.90±4.95 (m,
1H), 7.22 (d, J=8.4 Hz, 2H), 7.50 ppm (d, J=8.4 Hz,
2H), 8.30 (d,J=7.5 Hz, 1H); HRMS m/e calcd for
C14H18ClNO: 330.0260. Found: 330.0251.
trans-(R)-3-Chloro-3-methyl-1-tri¯uoromethy-N-[1-(4-
chlorophenyl)ethyl]cyclobutane carboxamide (37).
Method A, cis isomer isolated as a white solid (11%):
(R)-1,3,3-Trichloro-N-[1-(4-bromophenyl)ethyl]cyclobut-
anecarboxamide (42). Method A, isolated as a white
mp 129±130 ꢀC; H NMR (300 MHz, CDCl3) d 1.49 (d,
1
solid (42%): mp 149±151 ꢀC; H NMR (300 MHz, d6-
1
J=6.9 Hz, 3H), 1.67 (s, 3H), 2.82±3.05 (m, 4H), 5.05-
5.15 (m, 1H), 5.85 (br s, 1H), 7.20 (d, J=8.5 Hz, 2H),
7.32 (d, J=8.5 Hz, 2H); HRMS m/e calcd for C15H17Cl2
F3NO: 354.0639. Found: 354.0627. trans Isomer 37
isolated as a white solid (19%): mp 145±146 ꢀC; 1H
NMR (300 MHz, CDCl3) d 1.54 (d, J=6.9 Hz, 3H), 1.74
(s, 3H), 2.79±2.86 (m, 2H), 3.05±3.15 (m, 2H), 5.10±5.20
(m, 1H), 5.85 (br s, 1H), 7.23 (d, J=8.5 Hz, 2H), 7.31
(d, J=8.5 Hz, 2H); HRMS m/e calcd for C15H17
Cl2F3NO: 354.0639. Found: 354.0623.
DMSO) d 1.40 (d, J=6.9 Hz, 3H), 3.45±3.55 (m, 2H),
3.85±3.95 (m, 2H), 4.85±4.95 (m, 1H), 7.30 (d, J=
8.4 Hz, 2H), 7.50 (d, J=8.4 Hz, 2H), 8.85 (d, J=7.5 Hz,
1H); HRMS m/e calcd for C13H14Cl3BrNO: 383.9324.
Found: 383.9339.
(R)-3,3-Di¯uoro-1-methyl-N-[1-(4-bromophenyl)ethyl]-
cyclobutanecarboxamide (43). Method B, isolated as an
orange solid (72%): mp 120±122 ꢀC; H NMR (CDCl3,
1
300 MHz) d 1.48 (d, J=7.2Hz, 3H), 1.58 (s, 3H), 2.40 (m,
2H), 3.01 (m, 2H), 5.06 (m, 1H), 5.66 (d, J=7.2Hz, 1H),
7.17 (d, J=8.1Hz, 2H), 7.47 (d, J=8.1Hz, 2H); 19F
NMR (CDCl3, 282 MHz) d 93.3 (s), 87.4 (s); HRMS
m/e calcd for C14H17BrF2NO: 332.0462. Found: 332.0476.
trans-(R)-3-Chloro-3-methyl-1-tri¯uoromethy-N-[1-(2,4-
dichlorophenyl)ethyl]cyclobutanecarboxamide
(38).
Method A, cis isomer isolated as a white solid (9%):
mp 149±150 ꢀC; 1H NMR (CDCl3) d 1.54 (d, J=7.5 Hz,
3H), 1.70 (s, 3H), 2.82±3.05 (m, 4H), 5.30±5.40 (m, 1H),
6.05 (br s, 1H), 7.18±7.28 (m, 3H); HRMS m/e calcd for
C15H16Cl3F3NO: 388.0250. Found: 388.0253. trans
Isomer 38 isolated as a white solid (9%): mp 180±
cis- and trans-(R)-1,3-Dichloro-3-methyl-N-[1-(4-bromo-
phenyl)ethyl]cyclobutanecarboxamide (44). Method A,
isolated in 13% yield as a 1:1 mixture of two isomers
181 ꢀC; H NMR (CDCl3) d 1.52 (d, J=7.5 Hz, 3H),
which were not separated: mp 109±113 ꢀC; H NMR
1
1
1.74 (s, 3H), 2.80±2.90 (m, 2H), 3.10±3.20 (m, 2H),
5.30±5.40 (m, 1H), 6.05 (br s, 1H), 7.22 (s, 2H), 7.40 (s,
1H); HRMS m/e calcd for C15H16Cl3F3NO: 388.0250.
Found: 388.0268.
(CDCl3, 300 MHz) d 1.40 (d, J=7.0 Hz, 3H), 1.80 (s,
3H), 2.85±2.95 (m, 2H), 3.30±3.40 (m, 2H), 4.90±5.00
(m, 1H), 7.30 (d, J=8.5 Hz, 2H), 7.55 (d, J=8.5 Hz,
2H), 8.70 (d, J=7.5 Hz, 1H).
trans-(R)-3-Chloro-3-methyl-1-tri¯uoromethy-N-[3-(2-
¯uorophenyl)oxy-2-propyl] cyclobutanecarboxamide (39).
Method A, cis isomer isolated as a white solid (27%):
cis-(R)-3-Chloro-1,3-dimethyl-N-[1-(4-bromophenyl)ethyl]-
cyclobutanecarboxamide (45). Method A, isolated as an
orange solid (3%): mp 128±139 ꢀC; 1H NMR (300 MHz,
d6-DMSO) d 1.30 (overlapping doublet and singlet, 6H),
1.75 (s, 3H), 2.25±2.35 (m, 2H), 2.90±3.00 (m, 2H),
4.85±4.95 (m, 1H), 7.25 (d, J=8.5 Hz, 2H), 7.45 (d, J=
8.4 Hz, 2H), 8.09 (d, J=7.5 Hz, 1H).
mp 70±72 ꢀC, H NMR (300 MHz, CDCl3) d 1.37 (d,
1
J=7.2 Hz, 3H), 1.67 (s, 3H), 2.83±2.90 (m, 1H), 2.97±
3.05 (m, 3H), 4.0±4.10 (m, 2H), 4.35±4.45 (m, 1H), 6.05
(br s, 1H), 6.90±6.98 (m, 2H), 7.01±7.12 (m, 2H), 19F
NMR (282 MHz, CDCl3) d 73.59(s), 135.08 (s);
HRMS m/e calcd for C16H19ClF4NO2: 368.1040.
Found: 368.1033. trans isomer 39 isolated as a white
solid (35%): mp 145±146 ꢀC; 1H NMR (400 MHz,
CDCl3) d 1.40 (d, J=7.2 Hz, 3H), 1.75 (s, 3H), 2.80±
2.85 (m, 2H), 3.05±3.20 (m, 2H), 4.0±4.12 (m, 2H), 4.42±
4.52 (m, 1H), 6.10 (br s, 1H), 6.90±7.0 (m, 2H), 7.01±
7.12 (m, 2H), 19F NMR (376 MHz, CDCl3) d 73.98
(s), 135.195 (s), Irradiation at 73.98 ppm showed
positive NOE enhancement on C-3 methyl. HRMS m/e
calcd for C16H19ClF4NO2: 368.1040. Found: 368.1034.
cis- and trans-(R)-3-Chloro-3-(chloromethyl)-1-methyl-
N-[1-(4-bromophenyl)ethyl] cyclobutanecarboxamide (46
and 47). Method A, trans isomer 46 isolated as a white
solid (24%): mp 96±103 ꢀC; 1H NMR (CDCl3,
300 MHz) d 1.47 (d, J=7.0 Hz, 3H), 1.61 (s, 3H), 2.38±
2.44 (m, 2H), 3.00±3.06 (m, 2H), 3.80 (d, J=3.75 Hz,
2H), 4.97±5.07 (m, 1H), 5.61 (br s, 1H), 7.18 (d,
J=8.5 Hz, 2H), 7.48 (d, J=8.5 Hz, 2H); irradiation at
3.80 ppm showed no NOE enhancement on C-1 methyl;
HRMS m/e calcd for C15H19Cl2BrNO: 378.0027.
Found: 378.0036. cis Isomer 47 isolated as a white solid
ꢀ
1
(R)-N-[1-(4-Bromophenyl)ethyl]cyclobutanecarboxamide
(40). Method A, isolated as a white solid (83%): mp
(24%): mp 102±117 C; H NMR (CDCl3, 300 MHz) d
1.39 (s, 3H), 1.50 (d, J=7.0 Hz, 3H), 2.57±2.62 (m, 2H),
2.88±2.95 (m, 2H), 3.77 (s, 2H), 5.07±5.17 (m, 1H), 5.81
(br s, 1H), 7.20 (d, J=8.5 Hz, 2H), 7.44 (d, J=8.5 Hz,
2H); irradiation at 3.77 ppm showed positive NOE
140±150 ꢀC; H NMR (300 MHz, d6-DMSO) d 1.30 (d,
1
J=6.9 Hz, 3H), 1.75±2.18 (m, 6H), 3.05 (m, 1H), 4.85
(m, 1H), 7.23 (d, J=8.4 Hz, 2H), 7.49 (d, J=8.4 Hz,

906
L. D. Jennings et al. / Bioorg. Med. Chem. 8 (2000) 897±907
enhancement on C-1 methyl. HRMS m/e calcd for
C15H15Cl2BrNO: 378.0027. Found: 378.0042.
7.79±7.88 (m, 2H), 8.04±8.10 (m, 1H); HRMS m/e calcd
for C19 H23 Cl N O: 316.1468. Found: 316.1464.
cis-(R)-3-Chloro-1-ethyl-3-methyl-N-[1-(4-bromophenyl)-
ethyl]cyclobutanecarboxamide (48). Method B, cis iso-
mer isolated as a white solid (29%): mp 127.5±129 ꢀC;
1H NMR (CDCl3, 300 MHz) d 0.76 (t, J=7.4 Hz, 3H),
1.49 (d, J=7.2 Hz, 3H), 1.72 (q, J=7.4 Hz, 2H), 1.73 (s,
3H), 2.34 (d, J=15 Hz, 2H), 2.93 (d, J=15 Hz, 2H),
5.13 (m, J=7.2 Hz, 1H), 5.75 (d, J=7.2 Hz, 1H), 7.21
(d, J=8.7 Hz, 2H), 7.46 (d, J=8.7 Hz, 2H); HRMS m/e
calcd for C16H22ClBrNO: 358.0573. Found: 358.0570.
cis-(R)-3-Chloro-1,3-dimethyl-N-[1-(2-napthyl)ethyl]cyclo-
butanecarboxamide (55). Method A, cis isomer isolated
as a white solid (33%): mp 116±126 ꢀC; 1H NMR
(CDCl3, 300 MHz) d 1.41 (s, 3H), 1.61 (d, J=6.75 Hz,
3H), 1.77 (s, 3H), 2.25±2.37 (m, 2H), 3.0±3.11 (m, 2H),
5.30±5.35 (m, 1H), 5.90 (br s, 1H) 7.43±7.50 (m, 3H),
7.77±7.84 (m, 4H); HRMS m/e calcd for C19H23ClNO:
316.1468. Found: 316.1458.
cis-(‹)-3-Chloro-1,3-dimethyl-N-[1-(4-bromo-2-methoxy-
phenyl)ethyl]cyclobutanecarboxamide (56). Method B,
cis isomer isolated as a white solid (43%): mp 167±
168 ꢀC; 1H NMR (CDCl3, 300 MHz) d 1.37 (s, 3H), 1.44
(d, J=6.9 Hz, 3H), 1.77 (s, 3H), 2.33 (m, 2H), 3.01 (m,
2H), 3.88 (s, 3H), 5.20 (m, 1H), 6.49 (d, J=8.4 Hz, 1H),
7.01±7.10 (m, 3H); HRMS m/e calcd for C16H22
ClBrNO2: 374.0522. Found: 374.0539.
cis- and trans-(R)-1,3-Dimethyl-3-hydroxy-N-[1-(4-bromo-
phenyl)ethyl]cyclobutanecarboxamide (49 and 50).
Method C, trans isomer 49 isolated as a white solid
(22%): mp 103±105 ꢀC; H NMR (CDCl3, 300 MHz) d
1
1.31 (s, 3H), 1.46 (d, J=6.9 Hz, 3H), 1.48 (s, 3H), 1.63
(br s, 1H), 1.99 (m, 1H), 2.03 (m, 1H), 2.54 (m, 1H),
2.58 (m, 1H), 5.06 (m, 1H), 5.62 (d, J=7.5 Hz, 1H), 7.17
(d, J=8.4 Hz, 2H), 7.46 (d, J=8.7 Hz, 2H); HRMS m/e
calcd for C15H21BrNO2: 326.0756. Found: 326.0763.
cis Isomer 50 isolated in 13% yield as a yellow solid:
cis-(R)-3-Chloro-1,3-dimethyl-N-[3-(2,5-di¯uorophenyl)-
oxy-2-propyl]cyclobutanecarboxamide (57). Method A,
mp 129±131.5 ꢀC; H NMR (CDCl3, 300 MHz) d 1.36
cis isomer isolated as a white solid (18%): H NMR
1
1
(s, 3H), 1.38 (s, 3H), 1.47 (d, J=6.9 Hz, 3H), 2.06 (m,
2H), 2.45 (m, 2H), 4.24 (br s, 1H), 5.01 (m, J=6.9 Hz,
1H), 6.20 (d, J=6.9 Hz, 1H), 7.17 (d, J=8.3 Hz, 2H),
7.46 (d, J=8.3 Hz, 2H); HRMS m/e calcd for
C15H21BrNO2: 326.0756. Found: 326.0761.
(300 MHz, d6-DMSO) d 1.19 (d, J=6.76 Hz, 3H), 1.30
(s, 3H), 1.75 (s, 3H), 2.20±2.30 (m, 2H), 2.90±3.00 (m,
2H), 3.95±4.05 (m, 2H), 4.05±4.20 (m, 1H), 6.75±6.80
(m, 1H), 7.10±7.30 (m, 2H), 7.62 (d, J=7.5 Hz, 1H).
cis-R-3-Chloro-1,3-dimethyl-N-[3-(2-chloro-5-¯uorophen-
yl)oxy-2-propyl] cyclobutanecarboxamide (58). Method
A, cis isomer isolated as a white solid (29%): mp 77±
84 ꢀC; 1H NMR (300 MHz, d6-DMSO) d 1.16 (d,
J=6.3 Hz, 3H), 1.31 (s, 3H), 1.73 (s, 3H), 2.20±2.30 (m,
2H), 2.90±3.00 (m, 2H), 3.94±4.05 (m, 2H), 4.15±4.22
(m, 1H), 6.78±6.83 (m, 1H), 7.16±7.20 (m, 1H), 7.40±
7.45 (m, 1H), 7.62 (d, J=7.5 Hz, 1H); HRMS m/e calcd
for C16H21Cl2FNO2: 348.0933. Found: 348.0923.
cis-(R)-3-Ethynyl-3-hydroxy-1-methyl-N-[1-(4-bromophen-
yl)ethyl]cyclobutanecarboxamide (51). Method C, cis
isomer isolated as a yellow oil (11%): 1H NMR (CDCl3,
300 MHz) d 1.48 (d, J=6.9 Hz, 3H); 1.50 (s, 3H), 2.23
(d, J=13.2 Hz, 1H), 2.53 (s, 1H), 2.95 (d, J=13.2 Hz,
2H), 5.09 (q, J=6.9 Hz, 1H), 5.70 (br s, 1H), 7.19 (d,
J=8.4 Hz, 2H), 7.19 (d, J=8.4 Hz, 2H); HRMS m/e
calcd for C16H19BrNO2: 336.0599. Found: 336.0600.
cis-(R)-3-Ethyl-3-hydroxy-1-methyl-N-[1-(4-bromophenyl)-
ethyl]cyclobutane carboxamide (52). Method C, cis iso-
Rice soil systemic protocol
ꢀ
1
mer isolated as a white solid (16%): mp 100±103 C; H
NMR (CDCl3, 300 MHz) d 0.90 (t, J=7.2 Hz, 3H), 1.36
(s, 3H), 1.47 (d, J=6.6 Hz, 3H), 1.59 (q, J=7.2 Hz, 2H),
2.01 (m, 1H), 2.05 (m, 1H), 2.30±2.40 (m, 2H), 3.99 (s,
1H), 5.08 (m, 1H), 6.31 (d, J=7.2 Hz, 1H), 7.18 (d,
J=8.4 Hz, 2H), 7.46 (d, J=8.4 Hz, 2H); HRMS m/e
calcd for C16H23BrNO2: 340.0912. Found: 340.0920.
Nine day old rice seedlings grown in 25 mmÂ125 mm
culture tubes containing 28 mL total water and
approximately 30 cc of sifted tama soil are treated with
acetone solutions of the test compound at the desired
concentration. Seven days after treatment, plants are
sprayed with an aqueous suspension of Pyricularia gri-
sea conidia (isolate BV0-184) at 50 000/mL. They are
then incubated in a dew chamber at 24 ꢀC, 100% rela-
tive humidity, for 24 h. After incubation they are moved
back to the growth chamber. Infected plants are scored
7±10 days after inoculation by estimating the amount of
infected area of the last fully extended leaves at the time
of inoculation. Using the scores from the untreated
check plants, percent disease control is calculated using
the formula [1 (% disease in treatmentÄ% disease in
check)]Â100.
cis-(R)-3-Chloro-1,3-dimethyl-N-[1-(2,4-dichlorophenyl)-
ethyl]cyclobutanecarboxamide (53). Method A, cis iso-
ꢀ
1
mer isolated as a white solid (25%): mp 182±192 C, H
NMR (300 MHz, d6-DMSO) d 1.33 (overlapping s and
d, J=6.9 Hz, 6H), 1.75 (s, 3H), 2.25±2.35 (m, 2H), 2.85±
3.00 (m, 2H), 5.10±5.20 (m, 1H), 7.43 (s, 2H), 7.56 (s,
1H), 8.24 (d, J=7.5 Hz, 1H).
cis-(R)-3-Chloro-1,3-dimethyl-N-[1-(1-napthyl)ethyl]cyclo-
butanecarboxamide (54). Method A, cis isomer isolated
as a white solid (14%): mp 153±158 ꢀC; 1H NMR
(CDCl3, 300 MHz) d 1.35 (s, 3H), 1.70 (d, J=6.75 Hz,
3H), 1.73 (s, 3H), 2.26±2.31 (m, 2H), 3.0±3.06 (m, 2H),
5.76 (br s, 1H), 5.91±5.96 (m, 1H), 7.43±7.59 (m, 4H),
Acknowledgements
We are indebted to G. Shawn Smith, Jennifer A. Leet
and J. Mike Bonman for creating the rice blast soil

L. D. Jennings et al. / Bioorg. Med. Chem. 8 (2000) 897±907
907
systemic assay and for compound testing; Gina L.
Blankenship and John C. Groce for performing NOE
experiments and to Kent Woudstra and Arthur J.
Fogiel for HRMS analysis of compounds.
8. (a) Chen, J. M.; Xu, S. L.; Wawrzak, Z.; Basarab, G. S.;
Jordan, D. B. Biochemistry 1998, 37(51), 17735. (b) Jordan, D.
B.; Lessen, T.; Wawrzak, Z., Bisaha, J. J.; Gehret, T. C.;
Hansen, S. L.; Schwartz, R. S.; Basarab, G. S. Bioorg. Med.
Chem. Lett. 1999, 9, 1607. (c) Basarab, G. S.; Jordan, D. B.;
Gehret, T. C.; Schwartz, R. S.; Wawrzak, Z. Bioorg. Med.
Chem. Lett. 1999, 9, 1613.
9. Jennings, L. D.; Wawrzak, Z.; Amorose, D.; Schwartz, R.
S.; Jordan, D. B. Bioorg. Med. Chem. Lett. 1999, 9, 2509.
10. Molecular modeling was carried out using the Sybyl
molecular modeling package from Tripos, Inc. (St Louis, MO)
on a Silicon Graphics Inc. Indigo. A Connolly solvent-acces-
sible surface was generated with the MOLCAD algorithm
from Tripos, Inc. using a 1.3 A probe while selecting residues
that line the binding pocket and removing the inhibitor and
the two associated water molecules.
References and Notes
1. The labroratory strains are known as Magnapothe grisea.
2. Howard, R. J.; Ferrari, M. A. Exp. Mycol. 1989, 13, 403.
3. (a) Bell, A. A.; Wheeler, M. H. Annu. Rev. Phytopath. 1986,
24, 411. (b) Chumley, F. G.; Valent, B. Mol. Plant-Microbe
Interact. 1990, 3, 135.
4. (a) Kurahashi, Y.; Sakawa, S.; Kinbara, T.; Tanaka, K.;
Kagabu, S. J. Pesticide Sci. 1997, 22, 108. (b) Tsuji, G.;
Takeda, T.; Furusawa, I.; Horino, O.; Kubo, Y. Pesticide
Biochemistry and Physiology 1997, 57, 211.
11. Hall, H. K., Jr.; Blanchard, E. P., Jr.; Martin, E. L. Mac-
romolecules 1971, 4, 142.
5. (a) Manabe, A.; Mizutani, M.; Maeda, K.; Ooishi, T.;
Takamo, H.; Kirime, O. US Patent 4,946,867 (1990). (b)
Manabe, A.; Mizutani, M.; Maeda, K.; Takano, J. Japan.
Patent Application 89-293394. (c) Agrow, PJP Publications
Ltd.: UK, 1997; Vol. 287, pp 21±22.
6. Sieverding, E.; Hirooka, T.; Nishiguchi, T.; Yamamoto, Y.;
Spadafora, V. J.; Hasui, H. In Proc., The 1998 Brighton Con-
ferenceÐPests and Diseases; British Crop Protection Council:
Brighton, England, 1998; Vol. 2, pp 359±366.
7. (a) Lundqvist, T.; Rice, J.; Hodge, C. N.; Basarab, G. S.;
Pierce, J.; Lindqvist, Y. Structure (London) 1994, 2(10), 937.
(b) Nakasako, M.; Motoyama, T.; Kurahashi, Y.; Yamaguchi,
I. Biochemistry 1998, 37(28), 9931. (c) Wawrzak, Z; Sanda-
lova, T.; Ste€ens, J. J.; Basarab, G. S.; Lundqvist, T.; Lindq-
vist, Y.; Jordan, D. B. Proteins: Struct., Funct., Genet. 1999,
35(4), 425.
12. Cripps, H. N.; Williams, J. K.; Sharkey, W. H. J. Am.
Chem. Soc. 1959, 81, 2723.
13. Basarab, G. S.; Hansen, S. L.; Jordan, D. B.; Lessen, T. A.
PCT Int. Appl. WO 9833765, 1998.
14. Masumoto, K.; Hagitani, K; Harada, E. Japan Kokai
Tokkyo Koho JP 09012515, 1997.
15. Polniaszek, R. P.; Lichti, C. F. Synthetic Commun. 1992,
22 (1), 171.
16. Manabe, A.; Mizutani, M.; Maeda, K; Takano, J. Japan
Kokai Tokkyo Koho JP 03086855, 1991.
17. Martin, R. Org. Prep. and Procedures Int. 1992, 24, 369.
18. Tokes, A. L. Synth. Commun. 1989, 19, 2081.
19. Bienfait, B.; Coppe-Motte, G.; Merenyi, R.; Viehe, H. G.;
Sicking, W.; Sustmann, R. Tetrahedron 1991, 47 (38), 8167.
20. Lucas, H. J.; Kennedy, E. R. Org. Synth. Coll. 1955, 3,
482.