3-Oxoisoxazole-2(3H)-carboxamides and isoxazol-3-yl carbamates: Resistance-breaking acetylcholinesterase inhibitors targeting the malaria mosquito, Anopheles gambiae

Article abstract of DOI:10.1016/j.bmc.2015.01.026

To identify potential selective and resistance-breaking mosquitocides against the African malaria vector Anopheles gambiae, we investigated the acetylcholinesterase (AChE) inhibitory and mosquitocidal properties of isoxazol-3-yl dimethylcarbamates (15), and the corresponding 3-oxoisoxazole-2(3H)-dimethylcarboxamide isomers (14). In both series, compounds were found with excellent contact toxicity to wild-type susceptible (G3) strain and multiply resistant (Akron) strain mosquitoes that carry the G119S resistance mutation of AChE. Compounds possessing good to excellent toxicity to Akron strain mosquitoes inhibit the G119S mutant of An. gambiae AChE (AgAChE) with k_i values at least 10- to 600-fold higher than that of propoxur, a compound that does not kill Akron mosquitoes at the highest concentration tested. On average, inactivation of WT AgAChE by dimethylcarboxamides 14 was 10-20 fold faster than that of the corresponding isoxazol-3-yl dimethylcarbamates 15. X-ray crystallography of dimethylcarboxamide 14d provided insight into that reactivity, a finding that may explain the inhibitory power of structurally-related inhibitors of hormone-sensitive lipase. Finally, human/An. gambiae AChE inhibition selectivities of these compounds were low, suggesting the need for additional structural modification.

Full text of DOI:10.1016/j.bmc.2015.01.026

Bioorganic & Medicinal Chemistry 23 (2015) 1321–1340
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Bioorganic & Medicinal Chemistry
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3-Oxoisoxazole-2(3H)-carboxamides and isoxazol-3-yl carbamates:
Resistance-breaking acetylcholinesterase inhibitors targeting the
malaria mosquito, Anopheles gambiae
Astha Verma a, Dawn M. Wong a, Rafique Islam b, Fan Tong b, Maryam Ghavami a, James M. Mutunga b,
Carla Slebodnick a, Jianyong Li c, Elisabet Viayna a, Polo C.-H. Lam d, Maxim M. Totrov d,
Jeffrey R. Bloomquist b, Paul R. Carlier a,
⇑
a Department of Chemistry, Virginia Tech, Blacksburg, VA 24061, USA
b Department of Entomology and Nematology, Emerging Pathogens Institute, University of Florida, Gainesville, FL 32610, USA
c Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA
d Molsoft LLC, 11199 Sorrento Valley Road, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
To identify potential selective and resistance-breaking mosquitocides against the African malaria vector
Anopheles gambiae, we investigated the acetylcholinesterase (AChE) inhibitory and mosquitocidal prop-
erties of isoxazol-3-yl dimethylcarbamates (15), and the corresponding 3-oxoisoxazole-2(3H)-dimethyl-
carboxamide isomers (14). In both series, compounds were found with excellent contact toxicity to
wild-type susceptible (G3) strain and multiply resistant (Akron) strain mosquitoes that carry the
G119S resistance mutation of AChE. Compounds possessing good to excellent toxicity to Akron strain
mosquitoes inhibit the G119S mutant of An. gambiae AChE (AgAChE) with ki values at least 10- to 600-fold
higher than that of propoxur, a compound that does not kill Akron mosquitoes at the highest concentra-
tion tested. On average, inactivation of WT AgAChE by dimethylcarboxamides 14 was 10–20 fold faster
than that of the corresponding isoxazol-3-yl dimethylcarbamates 15. X-ray crystallography of dimethyl-
carboxamide 14d provided insight into that reactivity, a finding that may explain the inhibitory power of
structurally-related inhibitors of hormone-sensitive lipase. Finally, human/An. gambiae AChE inhibition
selectivities of these compounds were low, suggesting the need for additional structural modification.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 20 November 2014
Revised 7 January 2015
Accepted 15 January 2015
Available online 22 January 2015
Keywords:
Malaria
Anopheles gambiae
Acetylcholinesterase
Carboxamide
Carbamate
1. Introduction
to their low mammalian toxicity and high insecticidal and
repellant activity against the malaria vector, Anopheles gambiae.
Malaria is one of the deadliest diseases known to mankind.
Recent estimates suggest that approximately 207 million malaria
cases and 625,000 deaths were attributed to this disease alone in
2012.1 This problem is particularly severe in Sub-Saharan Africa
where 90% of the cases occur. The much awaited malaria vaccine
RTS,S/AS01 may help reduce the burden in malaria endemic
regions. However, at present it has shown only partial efficacy in
reducing malaria episodes in vaccinated children.2 Until an effec-
tive vaccine is in place, other effective and economical interven-
tions such as vector control are still needed. Insecticide treated
nets (ITNs) have shown efficacy in reducing malaria transmission
in the past in many malaria endemic regions.3–5 At present, this
method of intervention relies exclusively on pyrethroids, owing
However, due to their widespread use, there has been an upsurge
in mosquito populations resistant to this class of insecticides.6 This
development threatens to compromise the efficacy of ITNs, and has
motivated the development of other classes of insecticides with
different modes of action. Acetylcholinesterase (AChE) inhibitors
appear promising in this regard, in view of their efficacy as an
indoor wall application (‘indoor residual spraying’, IRS) against
adult mosquitoes. However, none of the AChE inhibitors approved
by the World Health Organization (WHO) for IRS (e.g., 1, 2) have
been approved for ITNs, perhaps due to concerns of human
toxicity. Thus, new safe and effective insecticides against the
susceptible and resistant strains of Anopheles gambiae are needed
for deployment on ITNs.
Our recently disclosed series of aryl methylcarbamates (e.g.,
5–7, Fig. 1) has shown high selectivity for An. gambiae AChE
(AgAChE) versus human AChE (hAChE).7,8 These compounds
⇑
Corresponding author. Tel.: +1 540 231 9219.
E-mail address: pcarlier@vt.edu (P.R. Carlier).
http://dx.doi.org/10.1016/j.bmc.2015.01.026
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

1322
A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
Figure 1. Structure of commercial carbamates (1–4, 9), and previously reported
aryl (5–7) and pyrazol-4-yl (8e, 8g, 8h) carbamates.
bearing a c-branched substituent exhibit up to 500-fold selectivity
for AgAChE over hAChE, and are toxic to wild-type (WT) An. gam-
biae (G3 strain, MR4, CDC). This high selectivity must arise from
amino acid substitutions near the active sites of AgAChE and
hAChE.9 Although we cannot yet point to specific residue substitu-
tions that 5–7 exploit to achieve selective inhibition, we have
developed
a
ligand-based selectivity model.8 However, these
compounds, like aryl methylcarbamates 1–4, are not appreciably
toxic to Akron strain An. gambiae (MR4, CDC). Carbamate resistance
of the Akron strain is known to arise from a G119S mutation in the
oxyanion hole of AChE.10,11 To compensate for reduced active site
volume arising from this mutation, we prepared and assayed pyr-
azol-4-yl carbamates (e.g., 8e, 8g, 8h Fig. 1) against susceptible
(G3) and resistant (Akron) strain An. gambiae. Their excellent
contact toxicity towards both strains and inhibition of wild-type
(WT) and G119S AgAChE suggests that a small core structure might
be the key to combat carbamate resistance stemming from the
G119S mutation.12 The excellent Akron toxicity of commercial
insecticide aldicarb (9) can also be explained by this steric
argument.12
In this paper we disclose the synthesis, mosquitocidal and AChE
inhibitory properties of carboxamides and carbamates based on an
isoxazole core. The insecticidal properties of 3-oxoisoxazole-
2(3H)-carboxamides have been documented in the patent
literature,13 but their contact toxicity to An. gambiae was not
described. We will show that compounds from both structural
classes can have high contact toxicity towards both WT and Akron
resistant strain An. gambiae. The high insecticidal activity against
resistant strain An. gambiae is particularly noteworthy.
Scheme 1. Synthesis of 5-substituted 3-oxoisoxazole-2(3H)-carboxamides 14, 16
and isoxazol-3-yl carbamates 15. Reagents and conditions: (i) pyridine, DCM, 0 °C,
15 min; RC(O)Cl, 0 °C, 1.5 h; rt, 1.5 h; (ii) RCOOH, Et3N, DECP, DMF, 0 °C, 30 min; rt,
16 h; (iii) NH(Boc)(OBoc), toluene, 90 °C, 1.5–2 h; (iv) NH(Boc)(OBoc), toluene,
65 °C, 16 h; (v) concd HCl, MeOH, 50 °C, 2.5 h; (vi) ClC(O)NMe2, toluene, reflux,
16 h; (vii) KOt-Bu, THF, 0 °C, 15 min; ClC(O)NMe2, rt, 16 h; (viii) KOt-Bu, THF, 0 °C,
15 min; ClC(O)NHMe, rt, 16 h.
substrates (e.g., 12a, 12f–i, 12l) and the product could be isolated
by column chromatography except in the case of 12d, where we
proceeded to the next step with the crude product. The cyclization
of b-keto hydroxamic acid 12 proceeded in high yields even when
the acid equivalents were reduced to half of that recommended in
the literature.14 The final carboxamide and carbamate products
were obtained regioselectively through judicious use of two
different protocols. The reaction of isoxazol-3-ol 13a–p with
dimethyl-carbamoyl chloride under neutral conditions gave
3-oxoisoxazole-2(3H)-carboxamides 14a–h, j–p as the major
products and isoxazol-3-yl dimethylcarbamate 15a–p as the
minor products (Scheme 1). Under neutral conditions, the more
nucleophilic ring nitrogen attacks the carbonyl carbon of carbamoyl
chloride forming dimethylcarboxamides as the major product.
However, under basic conditions, 13a–p gave isoxazol-3-yl
dimethylcarbamate 15a–p as the major product, with dimethyl-
carboxamides 14a–p formed in 5–17% yield (Scheme 1).
2. Synthesis
Unexpectedly, reaction of 13a–c, e–g, and i–m with methylcar-
bamoyl chloride under basic conditions gave N-methyl-3-oxoisox-
azole-2(3H)-carboxamide 16 with no trace of the corresponding
methylcarbamates (Scheme 1). It is possible that hydrogen bond-
ing between the carbamate NH and the isoxazol-3(2H)-one car-
bonyl in 16 renders the methylcarboxamides considerably more
stable than the methylcarbamate (see Fig. 4A below). In that case,
methylcarbamates initially formed under basic conditions could
undergo O- to N-carbamoyl transfer to yield the observed methyl-
carboxamides. In fact, we noted that methylcarboxamides such as
16 were themselves unstable in aqueous buffer (pH 7.7) over
30 min; it is therefore possible that any traces of methylcarbamate
present in the reaction mixture would be even more unstable and
decomposed during aqueous workup.
Acylated Meldrum’s acids 11a–p were synthesized in moderate
to high yield from 10 and the requisite acid chloride (or acid), using
the published literature procedure (Scheme 1).14 The R substitu-
ents were chosen to investigate the role of branching on inhibition
selectivity, since they will reside at C5 of the isoxazole 13 (cf. 5–7
Fig. 1). Compounds 11b–i feature
a-branched alkyl groups, 11j–l
feature b-branched alkyl groups, and 11m–p feature
c-branched
alkyl groups. The subsequent thermolysis of 11 with N,O-bis-t-
Boc hydroxylamine using the previously reported procedure14
did not proceed smoothly in every case. In some instances (e.g.,
12b, 12c, 12j, 12k), the product was obtained in low yield, whereas
in other cases (12m–o), it could not be purified due to contamina-
tion with residual BocNHOBoc. To optimize the yield for these sub-
strates, we took 1 equiv of the BocNHOBoc and 1.8 equiv of the
compound 12l and heated the reaction to 90 °C for 1.5–2 h. The
reaction time for this step is crucial to avoid loss in yield due to
incomplete conversion and competing side reactions. Using this
modification, we could improve the yield of compound 12 for some
To probe the effect of the N-substituent on mosquitocidal and
enzyme inhibition activities, various N-substituted derivatives of
13g were synthesized as shown in Scheme 2. Compounds 17g,
18g, and 23g were synthesized by reaction of 13g with triphosgene
and corresponding amine hydrogen chloride salt (17g) or amine

A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
1323
predominated under neutral conditions, except in the case of the
carbamoyl chloride derived from pyrrolidine, where the carbamate
25g was obtained as the major product (Scheme 2).
2.1. Characterization of the 3-oxoisoxazole-2(3H)-carboxamides
and isoxazol-3-yl carbamates
The final products were characterized and identified using
1H and 13C NMR, mass spectroscopy, and in two cases (14d, 16j)
X-ray diffraction. Assignments of the carboxamide and carbamate
structures were made in the following way. As illustrated in
Figure 2, dimethylcarboxamides (e.g., 14a) show a broad singlet
for the N-Me protons in the 1H NMR spectrum, whereas dimethylc-
arbamates (e.g., 15a) show two well-resolved singlets for these
protons. The barrier to rotation of the C(O)–N bond in analogous
urea-type dimethylcarboxamide analogues ranges from
8 to
13 kcal/mol,15 whereas in dimethylcarbamates it is higher, typically
ꢀ16 kcal/mol.16 These consistent differences in barriers to rotation
presumably correspond to differences in the C(O)–N double bond
character of ureas and carbamates. Similar behavior is seen in the
13C NMR spectra of these compounds (Fig. 2B), where the N-Me car-
bons of 14a appear as very low intensity, broadened signals, and the
N-Me carbons of 15a appear as two sharp resonances.
Another characteristic difference between 3-oxoisoxazole-
2(3H)-carboxamides (both methyl- and dimethyl), and isoxazol-3-yl
dimethylcarbamates was seen in 1H NMR chemical shifts of H-4
(Fig. 3). This proton appeared between 5.27 ppm and 5.58 ppm for car-
boxamides 14 and 16, whereas for isoxazol-3-yl dimethylcarbamates
Scheme 2. Derivatization of isoxazol-3-ol 13g. Reagents and conditions: (i)
triphosgene, DCM, rt, 2 h; DIPEA, HX or HXÁHCl, rt, 30 min; or (ii) ClC(O)X, toluene,
reflux, 16 h.
(18g, 23g). Compound 22g was obtained in low yield from the
same reaction mixture but appeared to be unstable, and hence
was not assayed. Compounds 19g, 20g, and 21g were synthesized
by refluxing 13g with corresponding carbamoyl chloride in tolu-
ene; the corresponding dimethylcarbamates 24g, 25g, and 26g,
were obtained in the same reactions. As expected, carboxamides
Figure 2. (A) The N-Me regions of the 1H NMR spectra of 3-oxoisoxazole-2(3H)-carboxamide 14a and isoxazol-3-yl carbamate 15a. (B) The N-Me regions of the 13C NMR
spectra of these compounds.

1324
A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
carbamate-resistant) strain An. gambiae, using a modification of
the standard WHO tarsal contact toxicity protocol.18 In the discus-
sion below, contact toxicities will be classified as excellent toxicity
(LC50 <100
lg/mL), good (LC50 = 100–199
lg/mL), moderate
(LC50 = 200–399
lg/mL), and poor (LC50 P400
lg/mL). As men-
tioned previously, commercial carbamates 1–4 bearing a phenyl
core displayed excellent toxicities to the susceptible G3 strain of
An. gambiae (LC50 = 16–39 lg/mL), but no measurable toxicity to
Figure 3. Characteristic chemical shifts of H-4 in 3-oxoisoxazole-2(3H)-carboxam-
ides and isoxazol-3-yl dimethylcarbamates (ppm), as illustrated by 14j, 16j, and
15j, respectively.
resistant Akron strain (Table 1). In contrast pyrazol-4-yl methylc-
arbamate 8e and aldicarb 9 had excellent toxicities to both
strains12 (Table 1). We attributed the low resistance ratios of these
two compounds in part to their smaller core structures, and were
thus interested to learn whether the isoxazol-3-yl core could also
confer high toxicity to Akron strain An. gambiae. As noted above,
isoxazol-3-yl methylcarbamates could not be isolated, and methyl-
carboxamides 16 proved unstable in aqueous buffer. Therefore
our investigations focused on dimethylcarboxamides 14, and
dimethylcarbamates 15. The carboxamide structure represents a
significant departure from the structure of aryl and pyrazole carba-
mates that we previously studied. The possible effects of disubsti-
tution on the carbamate nitrogen on contact toxicity were similarly
unknown. We were pleasantly surprised to find that a number of
dimethylcarboxamides were appreciably toxic to G3 and Akron
strain An. gambiae (Table 1). Compound 14a did not show any con-
15 it appeared in the range 6.07–6.17 ppm (Fig. 3). To correlate this
chemical shift difference to structure, we noted that the H-4
chemical shift of dimethylcarboxamide 14j was similar to that of
methylcarboxamide 16j. The identity of 16j as a methylcarboxa-
mide was confirmed by X-ray crystallography (Fig. 4A).
The identity of 14d (H-4 d = 5.42 ppm) as a dimethyl-carboxam-
ide was also confirmed by X-ray crystallography (Fig. 4B). The
presence of the second methyl group (C9) eliminates the hydrogen
bond seen in 16j, and steric interaction of this group with O2
causes the exocyclic amide moiety to rotate out of the plane of
the isoxazole ring. We will return to this point below in our discus-
sion of inactivation rate constants ki.
tact toxicity at the highest concentration tested (1000
Table 1), perhaps as a consequence of low lipophilicity. However,
-branched dimethylcarboxamides (14b–h) showed appreciable
lg/mL,
3. Tarsal contact toxicity of 3-oxoisoxazole-2(3H)-carboxamides
and isoxazol-3-yl carbamates
a
toxicities towards G3 and Akron strain (Table 2). Compound 14b
(R = c-Pr) showed moderate toxicity towards G3 strain, but was
considerably less toxic against Akron strain. The open-chain analog
Contact toxicity is
that might be deployed on ITNs. Tarsal contact toxicity was
determined towards G3 (WT, susceptible) and Akron (G119S,
a critical property for any insecticide
Table 1
Tarsal contact toxicity of control compounds (1–4, 8e, 9) and N,N-dialkyl-3-oxoisoxazole-2(3H)-carboxamides 14, 17–21 to susceptible (G3) and resistant (Akron) strains of An.
gambiae
Compound
Ra
An. gambiae G3
LC50
An. gambiae Akron
LC50
RRc
b
b
lg/mL
lg/mL
(95% CI)
(95% CI)
1d
NA
NA
NA
NA
NA
NA
Me
39 (32–45)
16 (14–17)
16 (11–25)
37 (14–16)
96 (89–104)
70 (66–74)
>5000d
>5000
>5000
>5000
81 (78–89)
32 (30–35)
ND
>130
>310
>310
>130
0.8
0.5
—
2d
3d
4d
8ed
9d
14a
14b
14c
14d
14e
14f
14g
14h
14j
14k
14l
14m
14n
14o
14p
17g
18g
19g
20g
21g
0% @ 1000 lg/mL
c-Pr
i-Pr
c-Bu
s-Bu
c-C5H11
2-Pentyl
3-Pentyl
i-Bu
2-Methylbutyl
neo-Pentyl
3-Methylbutyl
CH2CH2-c-Pr
CH2CH2-c-Bu
CH2CH2-c-C5H9
2-Pentyl
2-Pentyl
2-Pentyl
2-Pentyl
2-Pentyl
278 (225–345)
316 (22–428)
79 (57–116)
63 (49–81)
105 (78–157)
153 (109–211)
38 (28–53)
10% @ 1000
l
g/mL
—
182 (138–253)
74 (55–105)
129 (91–196)
118 (89–171)
75 (53–111)
40 (31–54)
0.6
0.9
2.0
1.1
0.5
1.1
—
201 (145–284)
25% @ 200
ND
lg/mL
0% @ 1000
lg/mL
80% @ 1000
73 (58–93)
l
g/mL
ND
68 (54–87)
144 (82–229)
ND
ND
ND
—
0.9
1.1
—
—
—
1.5
—
—
133 (71–226)
30% @ 1000
l
g/mL
g/mL
g/mL
0% @ 1000
78% @ 1000
l
l
400 (320–483)
614 (537–684)
15% @ 1000
77% @ 1000
45% @ 1000
lg/mL
lg/mL
lg/mL
ND
ND
ND
—
a
NA signifies not applicable.
b
Mosquitoes were exposed (1 h) to dried filter papers previously treated with ethanolic solutions of carbamates; mortality was recorded after 24 h. LC50 values derive from
the concentrations of inhibitor used to treat the paper. ND designates ‘not determined’; compounds that did not show significant toxicity to susceptible G3 strain (100%
mortality at 1000 g/mL) were generally not tested on the resistant Akron strain.
l
c
Resistance ratio (RR), defined by LC50 (Akron)/LC50 (G3).
Data reported previously.12
d

A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
1325
Table 2
Amongst the b-branched dimethylcarbamates examined
(15j–l), only 15j with an isobutyl side chain proved moderately
toxic towards G3. Interestingly, this compound proved even more
toxic to Akron strain than it was to G3 (cf. LC50 = 151 and
253 lg/mL, respectively). Amongst c-branched compounds, unlike
corresponding carboxamides, no clear trend was observed in G3
Tarsal contact toxicity of isoxazol-3-yl dialkylcarbamates to G3 and Akron strain of
An. gambiae
Compound
R
An. gambiae
An. gambiae
RRb
G3
Akron
LC50alg/mL (95%
LC50
a
CI)
lg/mL (95%
CI)
and Akron toxicity. The optimal LC50 was achieved for carbamates
15n (R = CH2CH2-c-Pr, G3 LC50 = 191 lg/mL). 15m, 15o, and 15p
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
15m
15n
15o
15p
23g
25g
26g
Me
c-Pr
i-Pr
c-Bu
0% @1000
0% @1000
84 (30–124)
349 (266–456)
41 (28–58)
60% @ 1000
234 (163–307)
70% @ 1000 g/mL
0% @ 1000 g/mL
253 (184–367)
30% @ 1000 g/mL
0% @ 1000 g/mL
415 (306–582)
191 (109–343)
40% @ 1000
674 (575–715)
410 (304–590)
l
l
g/mL
g/mL
ND
ND
—
—
exhibited poor toxicity towards both strains of An. gambiae. Finally,
as was seen for the carboxamides, variation of the N-substituent
reduced toxicity. Only 23g (ethylmethylcarbamate) showed mod-
erate or better toxicity to G3 strain, but had reduced toxicity for
Akron strain. Compound 25g (pyrrolidinylcarbamate), and 26g
(morpholinocarbamate) did not show any toxicity to G3 strain
An. gambiae.
Looking at both series, three conclusions can be drawn. First,
for a given C5-substituent, there is no general trend for dimethyl-
carboxamide versus dimethylcarbamate toxicity. In some cases
(b, d, f, g, h, j, l, m) the dimethylcarboxamide 14 is more toxic,
and in others (c, e, h, p) the dimethylcarbamate 15 is more toxic.
Second, among the exocyclic N-substituents explored, dimethyl
proved optimum, though ethylmethyl analogs 18g and 23g did
116 (79–151)
392 (281–593)
58 (42–92)
ND
296 (210–381)
ND
ND
151 (112–211)
ND
ND
429 (331–563)
198 (119–321)
ND
ND
1.4
1.1
1.4
-
1.3
—
—
0.6
—
s-Bu
c-Pentyl
2-Pentyl
3-Pentyl
3-Heptyl
i-Bu
2-Methylbutyl
neo-Pentyl
3-Methyl-butyl
CH2CH2-c-Pr
CH2CH2-c-Bu
CH2CH2-c-C5H9
2-Pentyl
2-Pentyl
2-Pentyl
l
g/mL
l
l
l
l
—
1.0
1.0
l
g/mL
—
1.4
—
574 (513–633)
ND
ND
0% @ 1000
10% @ 1000
l
g/mL
show significant toxicity. Third, both in the
a- and c-branched
l
g/mL
—
series, excellent G3 toxicity and low cross-resistance can be
attained. The resistance ratios (RR) for 14c, 14g and 15j are all
less than 1.0, which signifies greater susceptibility of resistant
strain An. gambiae towards these novel insecticides. This property
would certainly be favorable for a new anticholinesterase-based
mosquitocide.
a
Mosquitoes were exposed (1 h) to dried filter papers previously treated with
ethanolic solutions of carbamates; mortality was recorded after 24 h. LC50 values
derive from the concentrations of inhibitor used to treat the paper. ND designates
‘not determined’; compounds that did not show significant toxicity to susceptible
G3 strain (100% mortality at 1000 lg/mL) were generally not tested on the resistant
Akron strain.
b
Resistance ratio (RR), defined by LC50 (Akron)/LC50 (G3).
4. Inhibition of WT and G119S AgAChE by 3-oxoisoxazole-2(3H)-
carboxamide and isoxazol-3-yl carbamates
carboxamides 14c (R = i-Pr), and 14g (R = 2-pentyl) showed moder-
ate and good G3 toxicity, respectively, and were even more toxic to
the Akron strain. Compound 14d (R = c-Bu) exhibited excellent
toxicity towards both strains of An. gambiae. The compounds with
Carbamates are pseudo-irreversible inhibitors of AChE; they
inhibit the enzyme by carbamoylating the active site serine. We
have previously used the Ellman Assay to monitor the time-depen-
dent inhibitory activity of carbamates.7,8,12,19 We employed the
same method to determine the inhibition of AChE for our heterocy-
highest G3 toxicity in the
a-branched series were 14e (R = s-Bu,
LC50 = 63 g/mL) and 14h (R = 3-pentyl, LC50 = 38
l
lg/mL), and they
showed good and excellent Akron toxicity, respectively. Amongst
the b-branched dimethylcarboxamides explored (14j–l), only 14j
(R = i-Bu) showed moderate or better G3 toxicity, but Akron
clic carbamates and carboxamides. Enzyme velocities (
fixed inhibitor concentration were measured as a function of time
t. Plots of ln( 0) versus incubation time t were constructed and
the slope provided the pseudo first-order rate constant kobs (minÀ1
m/m0) at a
m/m
toxicity was lower. Among the
c-branched compounds explored
)
(14m–p), the carboxamide 14m (R = i-pentyl) was the most toxic
and showed equivalent LC50 values towards G3 and Akron strain.
A sharp decrease in G3 and Akron toxicity was observed from
carboxamides 14n (R = CH2CH2-c-Pr) to 14p (R = CH2CH2-c-C5H9),
concomitant with the increase in the size of the pendant carbocy-
clic ring. Finally, a range of compounds (17g–21g) were examined
to assess the effect of varying the substitution on the exocyclic
nitrogen. Only 18g (ethylmethylcarboxamide) showed poor or
better toxicity to G3 strain, and it was 2–3 fold less toxic than
the dimethylcarboxamide 14g. These data may suggest that the
N-substituents of 17g–21g are too big to inhibit AgAChE well,
and we address this point below. The low toxicity of azetidine
carboxamide 17g relative to dimethylcarboxamide 14g may be
due to instability of the azetidine ring, as discussed below.
for inactivation. For each inhibitor, kobs values were determined at
three or more inhibitor concentrations [I]. Finally, the linear fit
slope of plots of kobs versus [I] provided the second order rate con-
stant ki (mMÀ1 minÀ1) for inactivation. Note that non-saturating [I]
were chosen to give linear kobs versus [I] plots.7,8,12 The inhibition
data and the sensitivity ratios for commercial carbamates are given
in Table 3. As expected from their high toxicological resistance
ratios (RR), the commercial aryl carbamates 1–4 inhibited the sus-
ceptible enzyme (WT AChE) much more rapidly than the resistant
enzyme (G119S AChE), giving enzymatic sensitivity ratios (SR)
greater than 3800-fold. In contrast, the SR values for pyrazol-4-yl
methylcarbamate 8e and aldicarb (9) were only 32- and 4.2-fold,
which correlate well with their low toxicological resistance ratios
(0.8 and 0.5, respectively).
On the basis of low toxicological resistance ratios seen for many
isoxazol-3-yl dimethylcarbamates and dimethylcarboxamides
(Tables 1 and 2), we predicted low SR values for these compounds.
However, as will be seen below, this expectation was only partially
realized. The ki values for N,N-alkyl-3-oxoisoxazole-2(3H)-carbox-
amides are presented in Table 3. Compound 14a (R = Me) showed
very slow inactivation of AgAChE-WT (0.46 mMÀ1 minÀ1), which
could be a consequence of high desolvation penalty (low lipophil-
Turning to the corresponding dimethylcarbamates, 15a bearing
a C5-methyl group was not toxic to G3 strain at the highest con-
centration tested (Table 2). In contrast
a-branched analogs 15b–i
displayed G3 toxicities ranging from excellent (15c, e) to poor
(15b, f, h, i, Table 2). Compounds 15c (R = i-Pr) and 15e (R = s-Bu)
also demonstrated good and excellent toxicities towards Akron
strain An. gambiae, respectively. One interesting trend is that
open-chain analogs typically showed greater G3 toxicity than their
cyclic analogs (cf. 15c vs 15b, 15e vs 15d, and 15g vs 15f). Finally
15i (R = 3-heptyl) was not toxic to G3 strain at the highest concen-
icity). Dimethylcarboxamides bearing larger
R groups at C5
showed much greater WT AgAChE ki values, ranging from 24.3 to
tration tested (1000 lg/mL).

1326
A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
Table 3
Inactivation rate constants (ki) of control compounds (1–4, 8e, 9) and carboxamides 14a–p, 17g–21g for rAgAChE (WT and G119S) and rhAChE (h)
Compound
Ra
WT
ki
G119S
h
ki
WT/G119S
SRc
ki
(mMÀ1 minÀ1
)
(mMÀ1 minÀ1
)
(mMÀ1 minÀ1
)
b
b
b
1d
NA
NA
NA
NA
NA
NA
Me
c-Pr
i-Pr
c-Bu
s-Bu
c-C5H11
2-Pentyl
3-Pentyl
i-Bu
2-Methylbutyl
neo-Pentyl
3-Methylbutyl
CH2CH2-c-Pr
CH2CH2-c-Bu
CH2CH2-c-C5H9
2-Pentyl
2-Pentyl
2-Pentyl
2-Pentyl
2-Pentyl
266
9
<0.037 0.007
<0.055 0.007
<0.044 0.020
0.40 0.03
17.0 0.4
7200 1400
15,000 2000
60,000 27,000
3800 400
2d
839 22
2620 150
1510 100
9140 260
13.3 0.3
0.46 0.04
50.5 1.4
500 10
111
428 12
126
5
3d
4d
3
8ed
9d
290
7
805 36
6.5 0.3
1.22 0.04
32
1
3.15 0.08
4.2 0.1
4.7 1.4
250 30
270 10
480 40
220 10
400 40
360 10
380 30
450 20
480 20
440 80
670 200
390 30
460 130
60 4.1
14a
14b
14c
14d
14e
14f
14g
14h
14j
14k
14l
14m
14n
14o
14p
17g
18g
19g
20g
21g
0.098 0.029
0.20 0.03
1.85 0.09
0.62 0.05
10.6 0.5
2.58 0.22
14.7 0.3
22.5 1.1
0.55 0.02
0.99 0.05
0.37 0.06
0.40 0.12
1.07 0.06
0.31 0.09
0.41 0.27
36.7 2.7
3.84 0.20
1.24 0.05
1.90 0.11
2.15 0.27
103
5
561 21
233 13
2170 40
677 26
3110 120
1990 70
30.9 2.6
293
9
2290 80
1020 50
5240 140
8530 420
252
477
161 12
266
420 15
141
24.3 0.1
2240 80
4
7
112
5
26.2 1.1
80.9 2.0
9
144
7
4
74.5 4.4
92.0 4.0
1860 40
1080 50
61
41
5
3
156
7.30 0.09c
134
39.0 2.1
6
c
180
3
5.9 0.3
8
379 24
26.0 0.3
71
18
6
3
a
b
c
NA signifies not applicable.
Measured at 23 1 °C, pH 7.7, 0.1% (v/v) DMSO. Recombinant sources of AgAChE are rAgAChE-WT and rAgAChE-G119S.
Enzymatic sensitivity ratio is calculated as ki(WT)/ki(G119S). Standard error in the ratio is calculated according to a standard propagation of error formula.20
Data reported previously.12
d
8,530 mMÀ1 minÀ1 (14p and 14h, respectively). In general, the
largest AgAChE inactivation rate constants were realized with
dimethylamino group of 14g with an azetidine moiety (17g)
reduced WT AgAChE ki value by 50%, but more than doubled the
G119S AgAChE ki value. Replacement of one of the methyl groups
of 14g with an ethyl group (18g) reduced the WT AgAChE ki value
30-fold, and the G119S AgAChE ki value nearly 4-fold, suggesting
steric inhibition of binding or carbamoylation in the acyl pocket
of AgAChE. Consistent with this hypothesis, pyrrolidino- and mor-
pholino-carboxamides 20g and 21g offered more rapid inactivation
than the diethylcarboxamide 19g, but all three were much slower
than dimethyl carboxamide 14g.
a-branched alkyl substitution, in particular 14e–h. These dimeth-
ylcarboxamides also exhibited the largest G119S inactivation
constants (2.58–22.5 mMÀ1 minÀ1); the rate constants for 14g
and 14h were approximately 5- and 7-fold higher than that of
aldicarb (9). Although the WT/G119S enzymatic sensitivity ratios
for these compounds are still very large (360- to 380-fold), they
are 10-fold lower than that of 1–4.
Comparison of cycloalkyl analogs 14b, d, f with the correspond-
ing open chain
a
-branched analogs 14c, e, g, h shows that in each
Turning to the ki values of the dimethylcarbamates, compound
15a (like its carboxamide counterpart 14a) exhibited extremely
slow inactivation of all three enzymes; again low lipophilicity
case, the open chain compounds offered significantly more rapid
inactivation of WT or G119S AgAChE than the cycloalkyl analogs
(cf 14c vs 14b, 14e vs 14d, and 14g or 14h vs 14f). It is possible that
the flexibility of the side chain gives 14c, 14e, 14g, and 14h a better
fit in the active site of the enzyme as compared to 14b, 14d, and
may play a role (Table 4). Among
arbamates, the highest WT and G119S AgAChE values were seen for
the -branched compounds (e.g., 15e–h), as was the case for
a-, b-, and c-branched dimethylc-
a
14f. With regard to AgAChE/hAChE selectivity, the
carboxamides did not offer more than 4-fold selectivity. The three
b-branched dimethylcarboxamides investigated (14j–l) exhibited
a
-branched
dimethylcarboxamides 14 (Table 3). The highest G119S AgAChE
ki values in the dimethylcarbamate series are in the range of the
dimethylcarboxamides; in particular 15h, 15e, and 15g have
G119S ki values ranging from 3- to 10-fold of that of aldicarb (9).
reduced ki values compared to those with
a-branching. Unfortu-
nately, selectivity for AgAChE over hAChE within this series
However, the a-branched dimethylcarbamates 15e–h exhibited
remained much lower than desired, reaching a maximum of 8-fold
at least 10-fold slower inactivation of WT AgAChE than the
corresponding dimethylcarboxamides 14e–h. Consequently, the
enzymatic sensitivity ratios of the dimethylcarbamates are roughly
10-fold lower than that of the dimethylcarboxamides (cf. SR values
in Tables 3 and 4). As was seen for dimethylcarboxamides, WT and
G119S AgAChE ki values for open chain a-branched dimethylcarba-
mates were higher than those of their cycloalkyl homologs (cf. 15c
vs 15b, 15e vs 15d, 15h, g vs 15f).
for 14j. By analogy to compounds 5 to 7, it was hoped that a
branched alkyl group would confer high inactivation selectivity.
However within the -branched dimethylcarboxamide series
c-
c
(14m–p), the maximum AgAChE inactivation selectivity achieved
was 3-fold (14n). With regard to WT and G119S inactivation, an
approximate two-fold increase in ki was seen for 14n (R = CH2CH2-
c-Pr) compared to 14m (R = CH2CH2-i-Pr). However, increasing size
of the carbocycle in the series 14n, 14o, 14p reduced both WT and
G119S ki values.
Variation of the exocyclic nitrogen substituents, while retaining
a 2-pentyl group at C5, had dramatic effects on both WT and G119S
AgAChE ki values. The relatively conservative replacement of the
Among the b-branched dimethylcarbamates, the bulky neopen-
tyl compound 15l was much less inhibitory at all three enzymes
than the isobutyl (15j) and 2-methylbutyl (15k) compounds. The
c-branched dimethylcarbamates 15m–p were again examined
in the hope that they (like 5–7) would exhibit good WT Ag/h

A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
1327
Table 4
Inactivation rate constants (ki) of isoxazol-3-yl dialkylcarbamates for rAgAChE (WT and G119S), and rhAChE (h)
Compound
R
WT ki
G119S ki
h ki
WT/G119S SRb
a
a
a
(mMÀ1 minÀ1
)
(mMÀ1 minÀ1
)
(mMÀ1 minÀ1
)
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
15m
15n
15o
15p
23g
25g
26g
Me
c-Pr
i-Pr
c-Bu
0.0323 0.0082c
3.02 0.13
7.71 0.20
0.0224 0.0125
0.30 0.03
0.50 0.04
0.44 0.04
20.4 0.6
1.94 0.04
30.6 0.5
10.5 0.7
0.26 0.01
0.87 0.06
0.50 0.05
0.05 0.01
0.37 0.03
2.01 0.05
0.62 0.03
0.12 0.03
3.37 0.20
2.24 0.08c
2.30 0.10c
0.118 0.019
2.02 0.27
4.46 0.12
1.86 0.10
60.1 1.8
1.4 0.9
10
15
16
16
33
14
21
11
12
15
1
1
2
1
2
1
2
1
1
2
6.84 0.20
s-Bu
323
6
c-Pentyl
2-Pentyl
3-Pentyl
3-Heptyl
i-Bu
2-Methylbutyl
neo-Pentyl
3-Methylbutyl
CH2CH2-c-Pr
CH2CH2-c-Bu
CH2CH2-c-C5H9
2-Pentyl
2-Pentyl
2-Pentyl
64.7 2.7
4.62 0.07
50.9 1.0
416
221
7
7
9.04 0.31
2.13 0.09
1.59 0.08
1.70 0.09
0.44 0.04
1.91 0.04
8.26 0.26
7.24 0.90
13.1 0.4
2.85 0.11
10.6 0.5
7.64 0.21
0.12 0.03
3.12 0.16
23.5 1.1
5.08 0.23
0.60 0.05
24.5 0.5
12.4 0.1c
18.6 0.3c
2.4 0.7
8.3 0.8
12
1
8.2 0.6
5.2 1.2
7.3 0.5
5.5 0.2
8.1 0.4
9.14 1.97
1.62 0.07c
2.68 0.06c
a
b
c
Measured at 23 1 °C, pH 7.7, 0.1% (v/v) DMSO. Recombinant sources of AgAChE are rAgAChE-WT and rAgAChE-G119S.
Enzymatic sensitivity ratio (SR) is calculated as ki(WT)/ki(G119S). Standard error in the ratio is calculated according to a standard propagation of error formula.20
ki values extrapolated from single point incubation at various inhibitor concentration, for example, t = 10 min.
selectivity. However, as can be seen by inspection of the AgAChE
and hAChE ki values in Table 4, the highest selectivity (24-fold)
site of mouse AChE, and compared it to the experimental X-ray
structure of mouse AChE (mAChE) covalently bound by 27, a potent
trifluoromethylketone (TFK) inhibitor (Fig. 5).21
was obtained for the
a-branched compound 15h. Within the
c
-branched series 15m–p, the highest values of WT and G119S
Flexible ligand docking of the dimethylcarboxamide tetrahedral
covalent intermediate adduct derived from 14c and mAChE was
performed in ICM using default settings for ‘covalent’ docking
mode (ICM-Docking module, Molsoft).24,25 The choice of mAChE
was motivated by three factors: (1) availability of a high-resolution
crystal structure of its complex with 27 (PDB ID 2H9Y);26 (2) high
sequence identity (88%) of mAChE and hAChE;27 and (3) our obser-
vation of fast inactivation of hAChE by the dimethylcarboxamides
(cf. Table 3). The choice to model the tetrahedral intermediate
rather than the carbamoylated AChE reflects our assumption that
formation of the tetrahedral intermediate is likely the rate-limiting
AgAChE ki were again seen for R = CH2CH2-c-Pr (15n). Finally, with
regard to variation of the exocyclic N-substituents, increasing steric
bulk reduced ki values at all three enzymes (cf 23g, 25g, 26g vs 15g)
again suggesting steric crowding in the acyl pocket of AChE.
5. Discussion
To gain insight into the unexpectedly high inactivation rates
constants (ki) of the dimethylcarboxamides relative to the
dimethylcarbamates, we modeled inhibitor 14c in the active
Figure 4. (A) Anisotropic displacement ellipsoid drawings (50%) of X-ray structure of compound 16j. Hydrogen atoms were omitted for clarity, except at the exocyclic N. A
predicted hydrogen bond is shown with a dotted black line (H1–O2 distance 2.11 Å). (B) Anisotropic displacement ellipsoid drawings (50%) of X-ray structure of compound
14d. These structures were deposited at the Cambridge Structural Database (CCDC-1034990 and CCDC-1034991) and were visualized using OLEX2.17

1328
A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
Figure 5. Serine hydrolase inhibitors related to dimethylcarboxamides 14.13,22,23
step of enzyme inactivation, which then leads quickly to the carba-
moylated enzyme. Figure 6 shows the tetrahedral adduct formed
after the attack of active site serine (S203) on the carbonyl of
TFK 27 (Fig. 6A) and 14c (Fig. 6B). In both cases, the oxyanion
formed is stabilized through hydrogen bonding with oxyanion hole
residues G121, G122 and A204. Note that the corresponding active
site serine and oxyanion residues in AgAChE are S199, G118, G119,
and A200, respectively, (see Supplementary data Fig. S1). The over-
lay of the two structures in Figure 6C highlights the similar pose of
the two molecules in the active site of AChE: the NMe2 group of
14c overlays the trifluoromethyl group of 27 in the acylpocket. It
is well known that the CF3 group is similar in volume to an isopro-
pyl group;28 as Figure 6C illustrates, it is also similar in size to
NMe2. Lastly, the C5-i-Pr substituent of 14c occupies the same
locus as the trimethylammonium group of 27, in the choline-
binding site. It is possible that the C5-alkyl substituents of 14c
and analogs could have van der Waals interactions with W86, in
place of the cation–p interaction experienced by 27. Given these
similarities in the predicted binding pose of 14c and 27 to mouse
AChE, the high ki values seen at hAChE (and by extension AgAChE)
are more readily understood.
As mentioned earlier, the X-ray crystal structure of 14d (Fig. 4B)
also provides some insight into the reactivity of the dimethylcarb-
oxamides. The extent of resonance between the endocyclic N (N1)
and the amide carbonyl carbon (C8) appears to be low based on the
out-of-plane twisting of the dimethylcarbamoyl group. The magni-
tude of amide twist can be expressed as the
s
value29,30 along a
given N–C bond. As expected, the endocyclic amide bond N1–C7
has a low value (À12.6°), as does the dimethylamino amide bond
N2–C8 (8.6°). However, the bond from N1 to the reactive carbonyl
carbon C8 has a much larger twist, with a
s value of 47.5°. As men-
tioned earlier, this twist appears to be caused by steric interaction
of C9 and O2, and the ensuing lack of resonance donation of N1
into the C8 carbonyl should serve to increase the electrophilicity
of the dimethylcarboxamides. We note that N1 in 14d is also quite
pyramidalized (the sum of angles at N1 is only 346°). Additional
details on this structure, and for that of 16j are provided in the
Supplementary material. Close analogs of these carboxamides have
been cited in the literature as effective serine hydrolase inhibitors.
Isoxazolonyl carboxamide22 28 and 1,2,4-triazole carboxamide23
29 are potent hormone-sensitive lipase (HSL) inhibitors (Fig. 5).
Like vertebrate AChE, HSL belongs to the family of serine hydro-
Figure 6. Experimental and modeled tetrahedral adducts of mouse AChE active site
serine S203 with electrophilic carbonyl derivatives. (A) X-ray structure of the
tetrahedral adduct of 27 with S203 (PDB ID 2H9Y).26 (B) Computational modeling of
the tetrahedral adduct of dimethylcarboxamide 14c with S203 of mouse AChE. (C)
Overlay of structures (A) and (B). In both cases the backbone NH moieties of
oxyanion hole residues G121, G122 and A204 are within hydrogen bonding range of
the anionic oxygen. Image created with Molsoft Browser Pro version 3.7-a.
Finally, it would seem obvious to expect that G3 toxicity and
WT AgAChE ki values would be highly correlated. However, as we
have shown for aryl methylcarbamates, ADME can be very influen-
tial,7 and no general correlation is seen between G3 LC50 and
AgAChE ki values in either the isoxazole carboxamide or carbamate
lases with a
a
/b hydrolase fold.31 Since they originated from the
same ancestor, the catalytic residues are preserved in these
enzymes. Triazamate 30, a carbamoyl triazole AChE inhibitor
aphicide, has been approved for plant protection in many European
countries.32 Based on the structural similarity of these compounds
to 14d, it seems likely that the serine hydrolase inhibitory power of
28–30 derives in part from a similar out-of-plane twist of the exo-
cyclic amide moiety.
series. For example, the
almost equal toxicities towards G3 An. gambiae (38 and 41
a
-branched compounds 14h and 15e have
g/mL,
l
respectively). Despite the similar toxicities, 15e is 26-fold less
inhibitory than 14h (323 and 8530 mMÀ1 minÀ1, respectively).
Similarly, 14j and 14m have similar AgAChE (WT) ki values (252

A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
1329
and 266 mMÀ1 minÀ1, respectively), but the G3 LC50 value of 14j is
2.4 fold higher than of 14m (201 and 73 g/mL, respectively). Fur-
stirred for 1.5 h at 0 °C, and for an additional 1.5 h at room temper-
ature. The reaction was quenched with 2 M hydrochloric acid, and
extracted with dichloromethane. The combined organic layers
were dried over sodium sulfate, filtered, and concentrated in
vacuo. The residue was purified by silica gel chromatography using
a gradient from 5% to 10% ethyl acetate in hexane, 1% acetic acid to
yield acyl Meldrum’s acids (11a–e, j, l, p).
l
ther, despite the fact that these two compounds have similar
G119S AgAChE ki values, 14m is considerably more toxic to Akron
strain than 14j. In all these cases, differential pharmacokinetics and
metabolism may be dominant factors in toxicity. Nevertheless, we
can offer the following observations. Firstly, C5-methyl dimethyl-
carboxamide 14a and dimethylcarbamate 15a had very poor toxic-
ity and very slow enzyme inactivation rate constants, likely as a
consequence of low lipophilicity. The calculated CLogP values for
these compounds are 0.70 and 0.20, as compared to 2.2 and 1.7
for the s-Bu substituted compounds 14e and 15e.33 It is also possi-
ble that if the C5-substituent is insufficiently large, the carbamoyl
group will not be positioned properly to react with the active site
Method B: Adapted from the procedure of Sørensen et al..14 Mel-
drum’s acid (1 equiv) and corresponding acid (1 equiv) were dis-
solved in DMF, and cooled to 0 °C. To this solution was added
diethyl cyanophosphonate (1.1 equiv) and triethylamine
(3.1 equiv) drop wise. The mixture was allowed to stir at 0 °C for
30 min followed by 16 h at room temperature. The reaction was
quenched with 2 M hydrochloric acid and extracted twice with
ethyl acetate. The combined organic extracts were washed with
brine, and dried over sodium sulfate. The solution was filtered,
and concentrated in vacuo. The residue was purified by silica gel
chromatography using a gradient from 5% to 10% ethyl acetate in
hexane, 1% acetic acid to yield acyl Meldrum’s acids (11f–i, k, m,
n, o).
serine. Secondly, as previously mentioned, open chain a-branched
dimethylcarbamates were more toxic to G3 strain than the
corresponding cycloalkyl analogs (cf 15c vs 15b, 15e vs 15d and
15g vs 15f), and this trend correlates well with differences in
enzyme inactivation ki values. Third, increasing the size of the exo-
cyclic N-substituent reduced toxicity in both dimethylcarboxamide
(14g vs 17–21g) and dimethylcarbamate (15g vs 23, 25g, 26g), and
decreased enzyme inactivation ki values accompany these
decreased toxicities. Fourth, for those compounds having excellent
(14d, 14g, 14h, 14m, and 15e) and good (14c, 14e, 14f, 14n, 15c,
15j, and 15n) toxicity to Akron strain An. gambiae, we examined
the corresponding G119S AgAChE ki values. All these compound
give measurable ki values at the G119S enzyme, ranging from
13% (14m) to 700% (14h) of the ki value of aldicarb (9). The fact
that Akron/G3 toxicological resistance ratios (RR) for these
compounds are much smaller than the WT/G119S enzyme
sensitivity ratios (SR) may reflect enhanced bioactivation or
compromised detoxification in Akron, relative to G3. Nevertheless
it seems likely that the Akron-toxic properties of these compounds
indeed stem from engagement of the G119S AgAChE target. This
clearly advantageous property would be even more valuable, if it
could be achieved in compound class that offers excellent
selectivity against inhibition of hAChE. Work to achieve this goal
is in progress.
6.2.1. 5-(1-Hydroxyethylidene)-2,2-dimethyl-1,3-dioxane-4,6-
dione (11a)
Prepared using the general procedure above (Method A) from
Meldrum’s acid (500 mg, 3.47 mmol), acetyl chloride (0.25 mL,
3.47 mmol), pyridine (0.71 mL, 6.94 mmol), and CH2Cl2 (4.2 mL).
Aqueous workup, and silica gel chromatography (5–10% ethyl ace-
tate in hexane, 1% acetic acid) afforded 11a as a white solid
(536 mg, 83%). 1H NMR (500 MHz, CDCl3) d 15.12 (s, 1H), 2.67 (s,
3H), 1.73 (s, 6H). 13C NMR (126 MHz, CDCl3) d 194.8, 170.3,
160.6, 105.1, 92.0, 27.0, 23.7.
6.2.2. 5-(Cyclopropyl(hydroxy)methylene)-2,2-dimethyl-1,3-
dioxane-4,6-dione (11b)
Prepared using the general procedure above (Method A) from
Meldrum’s acid (500 mg, 3.46 mmol), cyclopropyl chloride
(0.32 mL, 3.46 mmol), pyridine (0.56 mL, 6.93 mmol), and CH2Cl2
(5.0 mL). Aqueous work up, and silica gel chromatography
(5–10% ethyl acetate in hexane, 1% acetic acid) afforded 11b as a
yellowish oil (462 mg, 63%). 1H NMR (500 MHz, CDCl3) d 15.41 (s,
1H), 3.54–3.44 (m, 1H), 1.74 (s, 6H), 1.49–1.41 (m, 2H),
1.32–1.20 (m, 2H). 13C NMR (126 MHz, CDCl3) d 198.1, 170.7,
161.4, 104.8, 91.2, 26.9, 15.7, 14.3.
6. Experimental
6.1. Chemistry
NMR spectra were obtained on JEOL Eclipse-plus 500 MHz spec-
trometer at 500 (1H) and 126 (13C) MHz or Unity-plus 400 at 400
(1H) and 101 (13C) MHz. The chemical shifts are reported in d
(ppm), and coupling constants are given in Hz. High-resolution
ESI mass spectra were obtained on an Agilent 6220 accurate mass
TOF LC/MS. X-ray data collection routine, unit cell refinement,
and data processing were carried out with the program CrysAlisPro.
The structure was solved using SHELXS-2013 and refined using
SHELXL-2013 via OLEX2. THF for moisture sensitive reactions was
distilled from sodium-benzophenone. Other dry solvents were
purchased from EMD Millipore and were used without any further
purification. Column chromatography was performed using Silica
6.2.3. 5-(1-Hydroxy-2-methylpropylidene)-2,2-dimethyl-1,3-
dioxane-4,6-dione (11c)
Prepared using the general procedure above (Method A) from
Meldrum’s acid (2.00 g, 13.9 mmol), isobutyryl chloride (1.46 mL,
13.9 mmol), pyridine (2.24 mL, 27.7 mmol), and CH2Cl2 (17.0 mL).
Aqueous workup, and silica gel chromatography (5–10% ethyl ace-
tate in hexane, 1% acetic acid) afforded 11c as a yellowish oil
(1.77 g, 60%). 1H NMR (500 MHz, CDCl3) d 15.53 (s, 1H), 4.08 (hept,
J = 6.8 Hz, 1H), 1.73 (s, 6H), 1.23 (d, J = 6.8 Hz, 6H). 13C NMR
(126 MHz, CDCl3) d 202.6, 171.1, 160.1, 104.8, 90.3, 33.2, 26.9, 19.2.
6.2.4. 5-(Cyclobutyl(hydroxy)methylene)-2,2-dimethyl-1,3-
dioxane-4,6-dione (11d)
gel (ZEOprep 60 ECO 40–63 l) was purchased from AIC. Reagents
were purchased mainly from Sigma Aldrich and were used as
received.
Prepared using the general procedure above (Method A) from
Meldrum’s acid (500 mg, 3.47 mmol), cyclobutane carbonyl chlo-
ride (0.40 mL, 3.47 mmol), pyridine (0.71 mL, 6.94 mmol), and
CH2Cl2 (4.2 mL). Aqueous work up and silica gel chromatography
(5–10% ethyl acetate in hexane, 1% acetic acid) afforded 11d as a
yellowish oil (604 mg, 77%). 1H NMR (500 MHz, CDCl3) d 15.53 (s,
1H), 4.47 (quintet, J = 8.5 Hz, 1H), 2.42–2.27 (m, 4H), 2.12–2.01
(m, 1H), 1.96–1.86 (m, 1H), 1.72 (s, 6H). 13C NMR (126 MHz, CDCl3)
d 198.3, 170.8, 160.0, 104.8, 89.8, 39.3, 26.8, 25.4, 17.9. HRMS (ESI)
calcd for C11H14O5 [MÀH]À 225.0768, found 225.0759.
6.2. General procedure for the synthesis of acyl Meldrum’s acids
Method A: Adapted from the procedure of Sørensen et al.14 To a
solution of Meldrum’s acid (1 equiv) in dichloromethane at 0 °C
was added pyridine (2 equiv) drop wise, and the resulting solution
was stirred for 15 min. The corresponding acid chloride (1 equiv)
was added to this reaction mixture. Thereafter, the reaction was

1330
A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
6.2.5. 5-(1-Hydroxy-2-methylbutylidene)-2,2-dimethyl-1,3-
dioxane-4,6-dione (11e)
(5–10% ethyl acetate in hexane, 1% acetic acid) afforded 11i as a
yellowish oil (2.82 g, 75%). 1H NMR (500 MHz, CDCl3) d 15.42
(s, 1H), 4.04 (tt, J = 8.7, 5.6 Hz, 1H), 1.73 (s, 6H), 1.72–1.52 (m,
4H), 1.34–1.18 (m, 4H), 0.91 (t, J = 7.5 Hz, 3H), 0.87 (t, J = 7.1 Hz,
3H). 13C NMR (126 MHz, CDCl3) d 201.8, 170.8, 160.5, 104.7, 92.8,
44.6, 32.5, 29.6, 26.9, 26.9, 26.4, 22.9, 14.0, 11.8. HRMS (ESI) calcd
for C14H22O5 [MÀH]À 269.1394, found 269.1421.
Prepared using the general procedure above (Method A) from
Meldrum’s acid (3.00 g, 20.8 mmol), 2-methyl butyryl chloride
(2.58 mL, 20.8 mmol), pyridine (3.36 mL, 41.6 mmol), and CH2Cl2
(25.0 mL). Aqueous work up, and silica gel chromatography (5–
10% ethyl acetate in hexane, 1% acetic acid) afforded 11e as a yel-
lowish oil (2.44 g, 52%). 1H NMR (500 MHz, CDCl3) d 15.48 (s, 1H),
3.97 (hextet, J = 6.9 Hz, 1H), 1.81–1.74 (m, 1H), 1.73 (s, 6H), 1.54
(doublet of quintet, J = 14.6, 7.3 Hz, 1H), 1.21 (d, J = 6.8 Hz, 3H),
0.93 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, CDCl3) d 202.2, 170.9,
160.3, 104.8, 91.3, 39.5, 27.3, 27.0, 26.8, 17.1, 11.9. HRMS (ESI)
calcd for C11H16O5 [M+Na]+ 251.0895, found 251.1618.
6.2.10. 5-(1-Hydroxy-3-methylbutylidene)-2,2-dimethyl-1,3-
dioxane-4,6-dione (11j)
Prepared using the general procedure above (Method A) from
Meldrum’s acid (300 mg, 2.08 mmol), isovaleryl chloride (0.25 mL,
2.08 mmol), pyridine (0.34 mL, 4.16 mmol), and CH2Cl2 (2.5 mL).
Aqueous work up, and silica gel chromatography (5–10% ethyl ace-
tate in hexane, 1% acetic acid) afforded 11j as a yellowish oil
(274 mg, 58%). 1H NMR (500 MHz, CDCl3) d 15.30 (s, 1H), 2.98 (d,
J = 7.1 Hz, 2H), 2.26–2.14 (m, 1H), 1.73 (s, 6H), 1.01 (d, J = 6.7 Hz,
6H). 13C NMR (126 MHz, CDCl3) d 197.7, 170.7, 160.4, 104.8, 92.0,
44.0, 27.5, 27.0, 22.7. HRMS (ESI) calcd for C11H16O5 [MÀH]À
227.0925, found 227.0939.
6.2.6. 5-(Cyclopentyl(hydroxy)methylene)-2,2-dimethyl-1,3-
dioxane-4,6-dione (11f)
Prepared using the general procedure above (Method B) from
Meldrum’s acid (500 mg, 3.47 mmol), cyclopentanoic acid
(0.38 mL, 3.47 mmol), diethyl cyanophosphonate (0.58 mL,
3.82 mmol), triethylamine (1.52 mL, 10.7 mmol), and DMF
(7.2 mL). Aqueous work up, and silica gel chromatography (5–
10% ethyl acetate in hexane, 1% acetic acid) afforded 11f as an off
white solid (721 mg, 86%); mp 68.8–70.7 °C. 1H NMR (500 MHz,
CDCl3) d 15.51 (s, 1H), 4.24–4.17 (m, 1H), 2.08–1.98 (m, 2H),
1.88–1.75 (m, 5H), 1.73 (s, 7H), 1.71–1.66 (m, 1H). 13C NMR
(126 MHz, CDCl3) d 201.6, 171.0, 160.4, 104.8, 90.8, 43.9, 31.1,
26.9, 26.6. HRMS (ESI) calcd for C12H16O5 [MÀH]À 239.0925, found
239.0935.
6.2.11. 5-(1-Hydroxy-3-methylpentylidene)-2,2-dimethyl-1,3-
dioxane-4,6-dione (11k)
Prepared using the general procedure above (Method B) from
Meldrum’s acid (300 mg, 2.08 mmol), 3-methylvaleric acid
(0.26 mL, 2.08 mmol), diethyl cyanophosphonate (0.35 mL,
2.29 mmol), triethylamine (0.86 mL, 6.45 mmol), and DMF
(4.3 mL). Aqueous work up, and silica gel chromatography (5–10%
ethyl acetate in hexane, 1% acetic acid) afforded 11k as a yellowish
oil (393 mg, 78%). 1H NMR (500 MHz, CDCl3) d 15.31 (s, 1H), 3.08
(dd, J = 13.4, 7.2 Hz, 1H), 2.91 (dd, J = 13.4, 7.2 Hz, 1H), 2.04–1.93
(m, 1H), 1.72 (s, 6H), 1.44 (doublet of quintet, J = 14.8, 7.4, 1H),
1.30 (doublet of quintet, J = 14.8, 7.4 Hz, 1H), 0.96 (d, J = 6.7 Hz,
3H), 0.92 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) d 198.1,
170.7, 160.4, 104.8, 92.1, 42.2, 33.7, 29.8, 26.9, 19.2, 11.4. HRMS
(ESI) calcd for C12H18O5 [MÀH]À 241.1081, found 241.1093.
6.2.7. 5-(1-Hydroxy-2-methylpentylidene)-2,2-dimethyl-1,3-
dioxane-4,6-dione (11g)
Prepared using the general procedure above (Method B) from
Meldrum’s acid (3.00 g, 20.8 mmol), 2-methyl valeric acid
(2.60 mL, 20.8 mmol), diethyl cyanophosphonate (3.47 mL,
22.9 mmol), triethylamine (8.99 mL, 64.6 mmol), and DMF
(25.0 mL). Aqueous work up, and silica gel chromatography (5–
10% ethyl acetate in hexane, 1% acetic acid) afforded 11g as a yel-
lowish oil (3.96 g, 79%). 1H NMR (500 MHz, CDCl3) d 15.46 (s, 1H),
4.05 (hextet, J = 6.8 Hz, 1H), 1.71 (s, 6H), 1.50–1.24 (m, 4H), 1.20
(d, J = 6.8 Hz, 3H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, CDCl3)
d 202.2, 170.9, 160.2, 104.7, 91.1, 37.8, 36.2, 26.9, 26.7, 20.6, 17.5,
14.1. HRMS (ESI) calcd for C12H18O5 [MÀH]À 241.1081, found
241.1091.
6.2.12. 5-(1-Hydroxy-3,3-dimethylbutylidene)-2,2-dimethyl-
1,3-dioxane-4,6-dione (11l)
Prepared using the general procedure above (Method A) from
Meldrum’s acid (500 mg, 3.47 mmol), 3,3-dimethylbutanoyl
chloride (0.49 mL, 3.47 mmol), pyridine (0.71 mL, 6.94 mmol), and
CH2Cl2 (4.0 mL). Aqueous work up, and silica gel chromatography
(5–10% ethyl acetate in hexane, 1% acetic acid) afforded 11l as an
off white solid (377 mg, 45%). 1H NMR (500 MHz, CDCl3) d 15.38
(s, 1H), 3.12 (s, 2H), 1.73 (s, 6H), 1.07 (s, 9H). 13C NMR (126 MHz,
CDCl3) d 196.9, 170.6, 160.5, 104.4, 93.0, 46.4, 34.0, 30.0, 26.8.
6.2.8. 5-(2-Ethyl-1-hydroxybutylidene)-2,2-dimethyl-1,3-
dioxane-4,6-dione (11h)
Prepared using the general procedure above (Method B) from
Meldrum’s acid (2.00 g, 13.9 mmol), 2-ethylbutyric acid (1.75 mL,
13.9 mmol), diethyl cyanophosphonate (2.32 mL, 15.3 mmol), tri-
ethylamine (5.99 mL, 43.0 mmol), and DMF (28.8 mL). Aqueous
workup and silica gel chromatography (5–10% ethyl acetate in hex-
ane, 1% acetic acid) afforded 11h as a yellowish oil (1.65 g, 49%). 1H
NMR (500 MHz, CDCl3) d 15.43 (s, 1H), 4.02–3.93 (m, 1H), 1.73 (s,
6H), 1.77–1.68 (m, 2H), 1.68–1.58 (m, 2H), 0.92 (t, J = 7.5 Hz, 6H).
13C NMR (126 MHz, CDCl3) d 201.6, 170.7, 160.5, 104.7, 93.0,
46.1, 26.9, 25.9, 11.8. HRMS (ESI) calcd for C12H18O5 [MÀH]À
242.1116, found 242.1096.
6.2.13. 5-(1-Hydroxy-4-methylpentylidene)-2,2-dimethyl-1,3-
dioxane-4,6-dione (11m)
Prepared using the general procedure above (Method B)
from Meldrum’s acid (500 mg, 3.47 mmol), 4-methylvaleric acid
(0.44 mL, 3.47 mmol), diethyl cyanophosphonate (0.58 mL,
3.82 mmol), triethylamine (1.52 mL, 10.7 mmol), and DMF
(7.2 mL). Aqueous work up, and silica gel chromatography (5–10%
ethyl acetate in hexane, 1% acetic acid) afforded 11m as a yellowish
oil (646 mg, 77%). 1H NMR (500 MHz, CDCl3) d 15.29 (s, 1H), 3.11–
3.03 (m, 2H), 1.74 (s, 6H), 1.72–1.62 (m, 1H), 1.61–1.55 (m, 2H),
0.95 (d, J = 6.5 Hz, 6H). 13C NMR (126 MHz, CDCl3) d 198.8, 170.7,
160.3, 104.9, 91.3, 35.1, 34.1, 28.3, 26.9, 22.4. HRMS (ESI) calcd
for C12H18O5 [MÀH]À 241.1154, found 241.073.
6.2.9. 5-(2-Ethyl-1-hydroxyhexylidene)-2,2-dimethyl-1,3-
dioxane-4,6-dione (11i)
Prepared using the general procedure above (Method B)
from Meldrum’s acid (2.00 g, 13.9 mmol), 2-ethylhexanoic acid
(2.21 mL, 13.9 mmol), diethyl cyanophosphonate (2.32 mL,
15.3 mmol), triethylamine (5.99 mL, 43.0 mmol), and DMF
(28.8 mL). Aqueous work up, and silica gel chromatography
6.2.14. 5-(3-Cyclopropyl-1-hydroxypropylidene)-2,2-dimethyl-
1,3-dioxane-4,6-dione (11n)
Adapted from the general procedure above (Method B) from
Meldrum’s acid (350 mg, 2.40 mmol), 3-cyclobutylpropanoic

A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
1331
acid34
(0.27 g,
2.40 mmol),
diethyl
cyanophosphonate
competing side reactions. After concentration in vacuo, the residue
was purified on silica gel chromatography using 5% ethyl acetate in
hexane to yield b-keto hydroxamic acid (12a, d, f–i, l, p) as color-
less oil.
(0.45 mL, 2.64 mmol), triethylamine (1.04 mL, 7.44 mmol), and
DMF (4.0 mL). Aqueous work up, and silica gel chromatography
(5–10% ethyl acetate in hexane, 1% acetic acid) afforded 11n as a
yellowish oil (38 mg, 65%). 1H NMR (400 MHz, CDCl3) d 15.26 (s,
1H), 3.15 (t, J = 7.3 Hz, 2H), 1.70 (s, 6H), 1.58 (q, J = 7.3 Hz, 2H),
0.48–0.39 (m, 3H), 0.08 (q, J = 5.7, 5.0 Hz, 2H). 13C NMR
(101 MHz, CDCl3) d 198.1, 170.6, 160.2, 104.8, 91.4, 36.0, 31.4,
26.9, 10.8, 4.7. HRMS (ESI) calcd for C12H16O5 [MÀH]À 239.0925,
found 239.0917.
6.3.1. tert-Butyl tert-butoxycarbonyloxy(3-
oxobutanoyl)carbamate (12a)
Prepared using the general procedure above (Method D) from
acyl Meldrum’s acid (11a) (314 mg, 1.69 mmol), N,O-bis(tert-
butoxycarbonyl)-hydroxylamine (219 mg, 0.94 mmol), and toluene
(4.5 mL). Concentration of the reaction mixture in vacuo followed
by silica gel flash chromatography (5% ethyl acetate in hexane)
yielded 12a as a clear oil (212 mg, 72%). 1H NMR (500 MHz, CDCl3)
of keto tautomer d 4.11–3.85 (m, 2H), 2.26 (s, 3H), 1.54 (s, 9H), 1.50
(s, 9H). 13C NMR (126 MHz, CDCl3) of keto tautomer d199.9, 163.3,
151.0, 149.7, 86.5, 86.1, 52.6, 30.1, 27.9, 27.6. The keto/enol ratio
for this compound was approximately 6:1.
6.2.15. 5-(3-Cyclobutyl-1-hydroxypropylidene)-2,2-dimethyl-
1,3-dioxane-4,6-dione (11o)
Prepared using the general procedure above (Method B) from
Meldrum’s acid (1.05 g, 7.26 mmol), 3-cyclobutylpropanoic acid34
(939 mg, 7.26 mmol), diethyl cyanophosphonate (1.2 mL,
7.99 mmol), triethylamine (3.14 mL, 22.51 mmol), and DMF
(13.0 mL). Aqueous work up, and silica gel chromatography (5–
10% ethyl acetate in hexane, 1% acetic acid) afforded 11o as a yel-
lowish oil (1.31 g, 70%). 1H NMR (400 MHz, CDCl3) d 15.24 (s, 1H),
2.94 (m, 2H), 2.33 (hept, J = 7.9 Hz, 1H), 2.11–1.98 (m, 2H), 1.90–
1.70 (m, 4H), 1.71 (s, 6H), 1.63 (pd, J = 9.1, 1.8 Hz, 2H). 13C NMR
(101 MHz, CDCl3) d 198.4, 170.6, 160.2, 104.8, 91.3, 35.8, 33.9,
33.3, 28.0, 26.9, 18.3. HRMS (ESI) calcd for C13H18O5 [MÀH]À
253.1081, found 253.1077.
6.3.2. tert-Butyl tert-butoxycarbonyloxy(3-cyclopropyl-3-
oxopropanoyl)carbamate (12b)
Prepared using the general procedure above (Method C) from
acyl Meldrum’s acid (11b) (600 mg, 2.82 mmol), N,O-bis(tert-
butoxycarbonyl)-hydroxylamine (659 mg, 2.82 mmol), and tolu-
ene (26.0 mL). Concentration of the reaction mixture in vacuo
followed by silica gel flash chromatography (5% ethyl acetate in
hexane) yielded 12b as a clear oil (503 mg, 52%). 1H NMR of keto
tautomer (500 MHz, CDCl3) d 4.27–3.98 (m, 2H), 2.02–1.96 (m,
1H), 1.53 (s, 9H), 1.50 (s, 9H), 1.15–1.06 (m, 2H), 0.98–0.90 (m,
2H). 13C NMR of keto tautomer (126 MHz, CDCl3) d 202.2, 163.6,
151.0, 149.5, 86.3, 85.9, 52.5, 27.9, 27.6, 20.8, 11.6. The keto/enol
ratio for this compound was approximately 33:1.
6.2.16. 5-(3-Cyclopentyl-1-hydroxypropylidene)-2,2-dimethyl-
1,3-dioxane-4,6-dione (11p)
Prepared using the general procedure above (Method A) from
Meldrum’s acid (1.50 g, 10.4 mmol), cyclopentane propionyl chlo-
ride (1.59 mL, 10.4 mmol), pyridine (2.12 mL, 20.8 mmol), and
CH2Cl2 (13.0 mL). However, the reaction was stirred at room tem-
perature for 3.5 h instead of 1.5 h for this particular substrate.
Aqueous workup and silica gel chromatography (5–10% ethyl ace-
tate in hexane, 1% acetic acid) afforded 11p as a yellowish oil
(2.42 mg, 87%). 1H NMR (500 MHz, CDCl3) d 15.27 (s, 1H), 3.09–
3.04 (m, 2H), 1.91–1.75 (m, 3H), 1.73 (s, 6H), 1.72–1.67 (m, 2H),
1.66–1.57 (m, 2H), 1.57–1.47 (m, 2H), 1.20–1.08 (m, 2H). 13C
NMR (126 MHz, CDCl3) d 198.6, 170.7, 160.3, 104.9, 91.3, 40.1,
35.4, 32.6, 27.0, 25.3. HRMS (ESI) calcd for C14H20O5 [MÀH]À
267.1238, found 267.1228.
6.3.3. tert-Butyl tert-butoxycarbonyloxy(4-methyl-3-
oxopentanoyl)carbamate (12c)
Prepared using the general procedure above (Method C) from
acyl Meldrum’s acid (11c) (196 mg, 0.92 mmol), N,O-bis
(tert-butoxycarbonyl)-hydroxylamine (213 mg, 0.92 mmol), and
toluene (8.5 mL). Concentration of the reaction mixture in vacuo
followed by silica gel flash chromatography (5% ethyl acetate in
hexane) yielded 12c as a clear oil (100 mg, 32%). 1H NMR
(500 MHz, CDCl3) of keto tautomer d 4.23–3.89 (m, 2H), 2.73
(hept, J = 6.9 Hz, 1H), 1.54 (s, 9H), 1.49 (s, 9H), 1.13 (d,
J = 6.9 Hz, 6H). 13C NMR (126 MHz, CDCl3) for keto/enol tauto-
meric mixture d 206.0, 187.8, 168.7, 163.8, 151.5, 151.0, 149.7,
87.6, 86.3, 86.2, 85.8, 85.3, 50.1, 41.1, 34.9, 28.0, 27.9, 27.7,
27.6, 19.9, 18.1. The keto/enol ratio for this compound was
approximately 4:1.
6.3. General procedure for the synthesis of N,O-diBoc-protected
b-keto hydroxamic acids
Method C: Adapted from the procedure of Sørensen et al.14 To a
solution of acyl Meldrum’s acid (0.915 mmol) in toluene (8.5 mL)
was
added
N,O-bis(tert-butoxycarbonyl)hydroxylamine
(0.915 mmol). The resulting reaction mixture was stirred at 65 °C
for 16 h, and then cooled to room temperature. The solvent was
concentrated in vacuo, and the residue was purified on silica gel
chromatography using 5% ethyl acetate in hexane to yield the
intermediate b-keto hydroxamic acid (12b, c, e, j, k, m–o) as color-
less oil. In the case of 12e, and 12m–o, this procedure gave the
desired hydroxamic acids as roughly 9:1 mixtures with residual
BocNHOBoc. Rather than achieve analytical purity for these com-
pounds, the crude products were used directly in the next step.
Thus synthetic details and characterization data for 12e, and
12m–o are not reported below.
6.3.4. tert-Butyl tert-butoxycarbonyloxy(3-cyclobutyl-3-
oxopropanoyl)carbamate (12d)
Prepared using the general procedure above (Method D) from
acyl Meldrum’s acid (11d) (522 mg, 2.30 mmol), N,O-bis
(tert-butoxycarbonyl)-hydroxylamine (298 mg, 1.28 mmol), and
toluene (6.4 mL). Concentration of the reaction mixture in vacuo
followed by silica gel flash chromatography (5% ethyl acetate in
hexane) yielded 12d as a clear oil (crude) contaminated with
residual N,O-bis(tert-butoxycarbonyl)hydroxylamine as the
impurity (389 mg, 85% (crude)). 1H NMR (500 MHz, CDCl3) of
keto tautomer
d 4.13–3.77 (m, 2H), 3.37 (p, J = 9.0 Hz, 1H),
Method D: To a solution of acyl Meldrum’s acid (1.69 mmol,
1.8 equiv) in dry toluene (4.5 mL) at room temperature was added
N,O-bis(tert-butoxycarbonyl)hydroxylamine (219 mg, 0.938 mmol,
1.0 equiv), and the reaction mixture was immersed in an oil bath
preheated to 90 °C. The reaction was monitored for the complete
2.37–2.22 (m, 2H), 2.21–2.10 (m, 2H), 2.01–1.90 (m, 1H),
1.88–1.77 (m, 1H), 1.54 (s, 9H), 1.49 (s, 9H). 13C NMR
(126 MHz, CDCl3) d 203.2, 184.7, 168.6, 163.6, 151.5, 151.0,
149.6, 149.4, 88.2, 86.4, 86.2, 85.9, 85.3, 53.6, 49.6, 45.6, 40.1,
27.9, 27.7, 26.1, 24.5, 18.3, 17.9. HRMS (ESI) calcd for
consumption
of
N,O-bis(tert-butoxycarbonyl)hydroxylamine.
C
17H27NO7 [M+Na]+ 380.1680, found 380.1706. The keto/enol
Leaving the reaction for longer time can lead to loss in yield and
ratio for this compound was approximately 5:1.

1332
A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
6.3.5. tert-Butyl tert-butoxycarbonylox(3-cyclopentyl-3-
oxopropanoyl)carbamate (12f)
0.91–0.84 (m, 9H). 13C NMR of keto/enol tautomer (126 MHz, CDCl3)
d 205.5, 186.1, 168.5, 163.7, 151.5, 151.0, 149.6, 149.3, 90.3, 86.3,
86.2, 85.7, 85.3, 53.6, 51.3, 48.6, 32.5, 30.2, 29.7, 29.3, 28.1, 27.9,
27.7, 27.7, 26.1, 23.9, 23.0, 22.9, 14.1, 14.1, 12.1, 11.6. HRMS (ESI)
calcd for C20H35NO7 [M+Na]+ 424.2306, found 424.2343. The
keto/enol ratio for this compound was approximately 2:1.
Prepared using the general procedure above (Method D) from
acyl Meldrum’s acid (11f) (93.3 mg, 0.39 mmol), N,O-bis(tert-
butoxycarbonyl)-hydroxylamine (50.0 mg, 0.22 mmol), and tolu-
ene (1.0 mL). Concentration of the reaction mixture in vacuo fol-
lowed by silica gel flash chromatography (5% ethyl acetate in
hexane) yielded 12f as a clear oil (59.0 mg, 73%). 1H NMR
(500 MHz, CDCl3) of keto tautomer d 4.22–3.89 (m, 2H), 2.98 (p,
J = 8.0 Hz, 1H), 1.91–1.76 (m, 4H), 1.70–1.63 (m, 2H), 1.62–1.56
(m, 2H), 1.54 (s, 9H), 1.49 (s, 9H). 13C NMR (126 MHz, CDCl3) of
keto/enol tautomeric mixture d 204.7, 186.6, 168.5, 163.7, 151.5,
151.0, 149.6, 149.4, 88.4, 86.3, 86.2, 85.8, 85.3, 51.7, 51.2, 45.9,
30.9, 28.9, 28.0, 27.9, 27.7, 27.6, 26.0, 26.0. HRMS (ESI) calcd for
6.3.9. tert-Butyl tert-butoxycarbonyloxy(5-methyl-3-
oxohexanoyl)carbamate (12j)
Prepared using the general procedure above (Method C) from
acyl Meldrum’s acid (11j) (239 mg, 1.05 mmol), N,O-bis(tert-butox-
ycarbonyl)-hydroxylamine (244 mg, 1.05 mmol), and toluene
(9.7 mL). Concentration of the reaction mixture in vacuo followed
by silica gel flash chromatography (5% ethyl acetate in hexane)
yielded 12j as a clear oil (125 mg, 33%). 1H NMR (500 MHz, CDCl3)
of keto tautomer d 4.16–3.74 (m, 2H), 2.41 (d, J = 6.8 Hz, 2H), 2.22–
2.11 (m, 1H), 1.54 (s, 9H), 1.49 (s, 9H), 0.93 (d, J = 6.7 Hz, 6H). 13C
NMR (126 MHz, CDCl3) for keto/enol tautomeric mixture d 201.6,
182.3, 168.3, 163.3, 151.3, 150.8, 149.5, 149.2, 90.5, 86.2, 86.0,
85.7, 85.2, 52.2, 51.5, 45.2, 27.9, 27.8, 27.5, 27.5, 26.6, 24.1, 22.4.
C
18H29NO7 [M+Na]+ 394.1836, found 394.1868. The keto/enol ratio
for this compound was approximately 6:1.
6.3.6. tert-Butyl tert-butoxycarbonyloxy(4-methyl-3-
oxoheptanoyl)carbamate (12g)
Prepared using the general procedure above (Method D)
from acyl Meldrum’s acid (11g) (278 mg, 1.15 mmol), N,O-bis-
(tert-butoxycarbonyl)-hydroxylamine (149 mg, 0.64 mmol), and
toluene (3.1 mL). Concentration of the reaction mixture in vacuo
followed by silica gel flash chromatography (5% ethyl acetate in
hexane) yielded 12g as a clear oil (163 mg, 68%). 1H NMR
(500 MHz, CDCl3) of keto tautomer d 4.22–3.88 (m, 2H), 2.65 (h,
J = 6.7 Hz, 1H), 1.73–1.62 (m, 2H), 1.54 (s, 9H), 1.49 (s, 9H),
1.40–1.26 (m, 2H), 1.11 (d, J = 7.0 Hz, 3H), 0.89 (t, J = 7.2 Hz,
3H). 13C NMR (126 MHz, CDCl3) of keto/enol tautomer d205.9,
187.3, 168.6, 163.7, 151.5, 151.0, 149.7, 149.4, 88.7, 86.3, 86.2,
85.8, 85.3, 50.6, 46.2, 40.4, 36.6, 34.8, 28.0, 27.9, 27.7, 27.6,
20.6, 20.3, 18.2, 15.9, 14.2, 14.1. HRMS (ESI) calcd for
HRMS (ESI) calcd for
C
17H29NO7 [M+Na]+ 382.1836, found
382.869. The keto/enol ratio for this compound was approximately
4:1.
6.3.10. tert-Butyl tert-butoxycarbonyloxy(5-methyl-3-
oxoheptanoyl)carbamate (12k)
Prepared using the general procedure above (Method C) from
acyl Meldrum’s acid (11k) (380 mg, 1.57 mmol), N,O-bis(tert-
butoxycarbonyl)-hydroxylamine (366 mg, 1.57 mmol), and toluene
(14.5 mL). Concentration of the reaction mixture in vacuo followed
by silica gel flash chromatography (5% ethyl acetate in hexane)
yielded 12k as a clear oil (191 mg, 33%). 1H NMR (500 MHz, CDCl3)
of keto tautomer d 4.25–3.66 (m, 2H), 2.59–2.44 (m, 1H), 2.39–2.18
(m, 1H), 1.95 (m, J = 13.4, 6.7 Hz, 1H), 1.54 (s, 9H), 1.49 (s, 9H), 1.42–
1.27 (m, 1H), 1.26–1.12 (m, 1H), 0.90 (d, J = 6.7 Hz, 3H), 0.87 (t,
J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) for keto/enol tautomeric
mixture d 201.8, 182.6, 168.2, 163.4, 151.3, 150.8, 149.5, 149.2,
90.6, 86.2, 86.1, 85.7, 85.2, 52.2, 49.7, 43.3, 32.8, 30.2, 29.4, 29.3,
27.9, 27.8, 27.5, 27.5, 19.3, 19.0, 11.3, 11.2. HRMS (ESI) calcd for
C18H31NO7 [M+H2O] 391.21, found 391.25. The keto/enol ratio
for this compound was approximately 6:1.
6.3.7. tert-Butyl tert-butoxycarbonyloxy(4-ethyl-3-
oxohexanoyl)carbamate (12h)
Prepared using the general procedure above (Method D) from acyl
Meldrum’s acid (11h) (942 mg, 3.9 mmol), N,O-bis(tert-butoxycar-
bonyl)-hydroxylamine (504 mg, 2.16 mmol), and toluene (11.0 mL).
Concentration of the reaction mixture in vacuo followed by silica
gel flash chromatography (5% ethyl acetate in hexane) yielded 12h
as a clear oil (536 mg, 66%). 1H NMR of keto/enol tautomeric mixture
(500 MHz, CDCl3) d 13.13 (s, 1H), 6.14 (s, 1H), 4.29–3.80 (m, 2H),
2.49–2.42 (m, 1H), 2.00–1.92 (m, 1H), 1.75–1.55 (m, 4H), 1.55 (s,
9H), 1.54 (s, 9H), 1.53 (s, 9H), 1.49 (s, 9H), 0.91–0.84 (m, 6H). 13C
NMR of keto/enol tautomer (126 MHz, CDCl3) d 205.4, 185.9, 168.4,
163.7, 151.5, 151.0, 149.6, 149.3, 90.4, 86.3, 86.2, 85.7, 85.3, 55.0,
51.3, 50.2, 28.0, 27.9, 27.7, 27.6, 25.7, 23.3, 12.1, 11.5. HRMS (ESI)
calcd for C18H31NO7 [M+Na]+ 396.1993, found 396.197. The keto/enol
ratio for this compound was approximately 2:1.
C
18H31NO7 [M+Na]+ 396.1993, found 396.2007. The keto/enol ratio
for this compound was approximately 4:1.
6.3.11. tert-Butyl tert-butoxycarbonyloxy(5,5-dimethyl-3-
oxohexanoyl)carbamate (12l)
Prepared using the general procedure above (Method D) from
acyl Meldrum’s acid (11l) (362 mg, 1.49 mmol), N,O-bis(tert-butox-
ycarbonyl)-hydroxylamine (194 mg, 0.83 mmol), and toluene
(4.2 mL). Concentration of the reaction mixture in vacuo followed
by silica gel flash chromatography (5% ethyl acetate in hexane)
yielded 12l as a clear oil (242 mg, 78%). 1H NMR (500 MHz, CDCl3)
of keto tautomer d 4.14–3.67 (m, 2H), 2.44 (s, 2H), 1.54 (s, 9H), 1.49
(s, 9H), 1.03 (s, 9H). 13C NMR (126 MHz, CDCl3) of keto/enol tauto-
meric mixture d 201.4, 182.0, 168.4, 163.5, 151.5, 151.0, 149.7,
149.3, 91.8, 86.3, 86.2, 85.8, 85.3, 54.9, 53.6, 49.9, 31.9, 30.9,
30.0, 29.6, 28.0, 27.9, 27.7, 27.7. The keto/enol ratio for this com-
pound was approximately 2:1.
6.3.8. tert-Butyl tert-butoxycarbonyloxy(4-ethyl-3-
oxooctanoyl)carbamate (12i)
Prepared using the general procedure above (Method D) from
acyl Meldrum’s acid (11i) (54.6 mg, 0.20 mmol), N,O-bis(tert-
butoxycarbonyl)-hydroxylamine (26.1 mg, 0.11 mmol), and tolu-
ene (0.9 mL). Concentration of the reaction mixture in vacuo
followed by silica gel flash chromatography (5% ethyl acetate in
hexane) yielded 12i as a clear oil (30.2 mg, 66%). 1H NMR of keto/
enol tautomeric mixture (500 MHz, CDCl3) d 13.14 (s, 0.5H), 6.13
(s, 0.5H), 4.41–3.63 (m, 2H), 2.50 (p, J = 6.5, 5.9 Hz, 1H), 2.07–
2.00 (m, 0.5H), 1.80–1.57 (m, 4H), 1.55 (s, 4.5H), 1.54 (s, 9H),
1.53 (s, 4.5H), 1.49 (s, 9H), 1.48–1.35 (m, 2H), 1.34–1.15 (m, 7H),
6.3.12. tert-Butyl tert-butoxycarbonyloxy(5-cyclopentyl-3-
oxopentanoyl)carbamate (12p)
Prepared using the general procedure above (Method D) from
acyl Meldrum’s acid (11p) (2.32 g, 8.65 mmol), N,O-bis(tert-butox-
ycarbonyl)-hydroxylamine (2.02 g, 8.65 mmol), and toluene
(80.0 mL). Concentration of the reaction mixture in vacuo followed
by silica gel flash chromatography (5% ethyl acetate in hexane)
yielded 12p as a clear oil (1.50 g, 43%). 1H NMR (500 MHz, CDCl3)
of keto tautomer
d 4.26–3.54 (m, 2H), 2.57–2.50 (m, 2H),

A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
1333
1.81–1.70 (m, 3H), 1.65–1.57 (m, 6H), 1.54 (s, 9H), 1.49 (s, 9H),
1.15–1.00 (m, 2H). 13C NMR (126 MHz, CDCl3) d of keto tautomer
202.2, 163.4, 150.8, 149.5, 86.2, 85.8, 51.8, 41.9, 39.4, 32.4, 29.4,
27.8, 27.5, 25.1. HRMS (ESI) calcd for
422.2149, found 422.2172. The keto/enol ratio for this compound
6.4.5. 5-sec-Butylisoxazol-3-ol (13e)
Prepared using the general procedure above (Method E) from
crude b-keto hydroxamic acid (12e) (1.96 g, 5.53 mmol), concen-
trated HCl (20.0 mL), and methanol (15.0 mL). Concentration of
the reaction mixture in vacuo followed by an aqueous work up,
and silica gel chromatography (5–10% ethyl acetate in hexane, 1%
acetic acid) afforded 13e as an off white solid (620 mg, 77%); mp
37.0–38.0 °C. 1H NMR (500 MHz, CDCl3) d 11.41 (s, 1H), 5.64 (s,
1H), 2.74 (h, J = 7.0 Hz, 1H), 1.74–1.64 (m, 1H), 1.58 (doublet of
quintet, J = 14.1, 7.2 Hz, 1H), 1.24 (d, J = 7.0 Hz, 3H), 0.90 (t,
J = 7.2 Hz, 3H). 13C NMR (126 MHz, CDCl3) d 178.8, 171.2, 92.1,
34.5, 28.2, 18.1, 11.4. HRMS (ESI) calcd for C7H11NO2 [M+H]+
142.0863, found 142.086.
C
20H33NO7 [M+Na]+
was approximately 6:1.
6.4. General procedure for the synthesis of 5-substituted
isoxazol-3-ols 13a–p
Method E: Adapted from the procedure of Sørensen et al.14 b-
keto hydroxamic acid (1.33 mmol) was dissolved in methanol
(3.0 mL), and added drop wise to concentrated HCl (5.0 mL) pre-
heated to 50 °C. The reaction mixture was stirred for 2.5 h at
50 °C prior to being brought to room temperature. The reaction
mixture was concentrated in vacuo. The residual reaction mixture
was partitioned between water and dichloromethane. The aqueous
layer was extracted twice with dichloromethane and the combined
organic layers were dried with sodium sulfate. The solvent was
evaporated, and the subsequent residue was purified on silica gel
chromatography using a gradient from 5% to 10% ethyl acetate in
hexane, 1% acetic acid to yield isoxazol-3-ols.
6.4.6. 5-Cyclopentylisoxazol-3-ol (13f)
Prepared using the general procedure above (Method E) from
b-keto hydroxamic acid (12f) (1.77 g, 4.76 mmol), concentrated
HCl (25.0 mL), and methanol (10.0 mL). Concentration of the
reaction mixture in vacuo followed by an aqueous work up, and
silica gel chromatography (5–10% ethyl acetate in hexane, 1%
acetic acid) afforded 13f as a white solid (508 mg, 70%); mp
71.3–73.4 °C. 1H NMR (500 MHz, CDCl3) d 10.42 (s, 1H), 5.63 (s,
1H), 3.06 (quintet, J = 7.9 Hz, 1H), 2.09–1.96 (m, 2H), 1.80–1.61
(m, 6H). 13C NMR (126 MHz, CDCl3) d 178.4, 171.2, 91.8, 38.0,
31.6, 25.4. HRMS (ESI) calcd for C8H11NO2 [M+H]+ 154.0863,
found 154.0855.
6.4.1. 5-Methylisoxazol-3-ol (13a)
Prepared using the general procedure above (Method E) from b-
keto hydroxamic acid (12a) (66.5 mg, 0.21 mmol), concentrated
HCl (1.0 mL), and methanol (0.5 mL). Concentration of the reaction
mixture in vacuo followed by an aqueous work up, and silica gel
chromatography (5–10% ethyl acetate in hexane, 1% acetic acid)
afforded 13a as a white solid (15.5 mg, 75%). 1H NMR (500 MHz,
CDCl3) 11.57 (s, 1H), 5.67 (s, 1H), 2.33 (s, 3H). 13C NMR
(126 MHz, CDCl3) d 171.4, 170.4, 94.0, 13.0.
6.4.7. 5-(Pentan-2-yl)isoxazol-3-ol (13g)
Prepared using the general procedure above (Method E) from b-
keto hydroxamic acid (12g) (2.85 g, 7.63 mmol), concentrated HCl
(15.0 mL), and methanol (16.0 mL). Concentration of the reaction
mixture in vacuo followed by an aqueous work up, and silica gel
chromatography (5–10% ethyl acetate in hexane, 1% acetic acid)
afforded 13g as a colorless oil (889 mg, 75%). 1H NMR (500 MHz,
CDCl3) d 11.20 (s, 1H), 5.62 (s, 1H), 2.82 (h, J = 7.0 Hz, 1H), 1.64
(ddt, J = 13.5, 9.4, 6.6 Hz, 1H), 1.50 (ddt, J = 13.5, 9.4, 6.6 Hz, 1H),
1.38–1.26 (m, 2H), 1.23 (d, J = 7.1 Hz, 3H), 0.89 (t, J = 7.4 Hz, 3H).
13C NMR (126 MHz, CDCl3) d 179.0, 171.2, 91.8, 37.4, 32.7, 20.2,
18.6, 13.9. HRMS (ESI) calcd for C8H13NO2 [M+H]+ 156.0946, found
156.1485.
6.4.2. 5-Cyclopropylisoxazol-3-ol (13b)
Prepared using the general procedure above (Method E) from b-
keto hydroxamic acid (12b) (2.69 g, 7.83 mmol), concentrated HCl
(25.0 mL), and methanol (16.0 mL). Concentration of the reaction
mixture in vacuo followed by an aqueous work up, and silica gel
chromatography (5–10% ethyl acetate in hexane, 1% acetic acid)
afforded 13b as an off white solid (464 mg, 47%). 1H NMR
(500 MHz, CDCl3) d 10.94 (s, 1H), 5.57 (s, 1H), 1.95–1.86 (m, 1H),
1.08–1.00 (m, 2H), 0.97–0.91 (m, 2H). 13C NMR (126 MHz, CDCl3)
d 175.6, 171.5, 91.1, 8.6, 8.3.
6.4.8. 5-(Pentan-3-yl)isoxazol-3-ol (13h)
Prepared using the general procedure above (Method E) from
b-keto hydroxamic acid (12h) (496 mg, 7.63 mmol), concentrated
HCl (5.0 mL), and methanol (3.0 mL). Concentration of the reac-
tion mixture in vacuo followed by an aqueous work up, and silica
gel chromatography (5–10% ethyl acetate in hexane, 1% acetic
acid) afforded 13h an off white solid (115 mg, 81%); mp 49.0–
50.7 °C. 1H NMR (500 MHz, CDCl3) d 10.00 (s, 1H), 5.65 (s, 1H),
2.55 (tt, J = 8.3, 5.8 Hz, 1H), 1.72–1.56 (m, 4H), 0.87 (t, J = 7.4 Hz,
6H). 13C NMR (126 MHz, CDCl3) d 177.7, 171.1, 93.0, 42.2, 26.3,
11.7. HRMS (ESI) calcd for C8H13NO2 [M+H]+ 156.1019, found
156.1013.
6.4.3. 5-Isopropylisoxazol-3-ol (13c)
Prepared using the general procedure above (Method E) from b-
keto hydroxamic acid (12b) (973 mg, 2.82 mmol), concentrated
HCl (12.0 mL), and methanol (6.0 mL). Concentration of the reac-
tion mixture in vacuo followed by an aqueous work up, and silica
gel chromatography (5–10% ethyl acetate in hexane, 1% acetic acid)
afforded 13c as a white solid (276 mg, 77%). 1H NMR (500 MHz,
CDCl3) d 10.17 (s, 1H), 5.64 (s, 1H), 2.94 (hept, J = 6.9 Hz, 1H),
1.27 (d, J = 7.0 Hz, 6H). 13C NMR (126 MHz, CDCl3) d 179.7, 171.2,
91.5, 27.7, 20.6.
6.4.9. 5-(Heptan-3-yl)isoxazol-3-ol (13i)
6.4.4. 5-Cyclobutylisoxazol-3-ol (13d)
Prepared using the general procedure above (Method E) from
b-keto hydroxamic acid (12i) (2.29 g, 5.70 mmol), concentrated
HCl (22.0 mL), and methanol (12.0 mL). Concentration of the reac-
tion mixture in vacuo followed by an aqueous work up, and silica
gel chromatography (5–10% ethyl acetate in hexane, 1% acetic
acid) afforded 13i as a colorless oil (396 mg, 40%). 1H NMR
(500 MHz, CDCl3) d 5.66 (s, 1H), 2.62 (quintet, J = 7.4 Hz, 1H),
1.74–1.53 (m, 4H), 1.39–1.12 (m, 6H), 0.95–0.81 (m, 6H). 13C
NMR (126 MHz, CDCl3) d 178.1, 171.2, 93.0, 40.6, 33.2, 29.4,
26.8, 22.7, 14.1, 11.7. HRMS (ESI) calcd for C10H17NO2 [M+H]+
184.1332, found 184.1339.
Prepared using the general procedure above (Method E) from b-
keto hydroxamic acid (12d) (318 mg, 0.89 mmol), concentrated
HCl (3.1 mL), and methanol (2.0 mL). Concentration of the reaction
mixture in vacuo followed by an aqueous work up, and silica gel
chromatography (5–10% ethyl acetate in hexane, 1% acetic acid)
afforded 13d as a semi-solid (75.0 mg, 61%). 1H NMR (500 MHz,
CDCl3) d 5.68 (s, 1H), 3.50 (p, J = 8.5 Hz, 1H), 2.40–2.31 (m, 2H),
2.30–2.20 (m, 2H), 2.10–2.01 (m, 1H), 2.01–1.91 (m, 1H). 13C
NMR (126 MHz, CDCl3) d177.4, 171.2, 91.9, 32.6, 27.8, 18.9. HRMS
(ESI) calcd for C7H9NO2 [M+H]+ 140.0706, found 140.0693.

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A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
6.4.10. 5-Isobutylisoxazol-3-ol (13j)
chromatography (5–10% ethyl acetate in hexane, 1% acetic acid)
afforded 13o as a white crystalline solid (340 mg, 42% over 2
steps). 1H NMR (500 MHz, CDCl3) d 11.81 (s, 1H), 5.63 (s, 1H),
2.52 (t, J = 7.7 Hz, 2H), 2.28 (hept, J = 8.1 Hz, 1H), 2.10–1.98 (m,
2H), 1.92–1.76 (m, 2H), 1.73 (q, J = 7.6 Hz, 2H), 1.60 (pd, J = 9.0,
2.4 Hz, 2H). 13C NMR (126 MHz, CDCl3) d 174.6, 171.3, 93.0, 35.4,
34.3, 28.0, 25.1, 18.4. HRMS (ESI) calcd for C9H13NO2 [M+H]+
168.1019, found 168.1022.
Prepared using the general procedure above (Method E) from b-
keto hydroxamic acid (12j) (2.98 g, 8.67 mmol), concentrated HCl
(25.0 mL), and methanol (18.0 mL). Concentration of the reaction
mixture in vacuo followed by an aqueous work up, and silica gel
chromatography (5–10% ethyl acetate in hexane, 1% acetic acid)
afforded 13j as an off white solid (804 mg, 66%). 1H NMR
(500 MHz, CDCl3) d 11.80 (s, 1H), 5.66 (s, 1H), 2.50 (d, J = 7.1 Hz,
2H), 2.06–1.94 (m, 1H), 0.95 (d, J = 6.7 Hz, 6H). 13C NMR
(126 MHz, CDCl3) d 173.8, 171.3, 93.8, 36.3, 27.6, 22.3.
6.4.16. 5-(2-Cyclopentylethyl)isoxazol-3-ol (13p)
Prepared using the general procedure above (Method E) from b-
keto hydroxamic acid (12p) (1.50 g, 3.75 mmol), concentrated HCl
(13.0 mL), and methanol (8.0 mL). Concentration of the reaction
mixture in vacuo followed by an aqueous work up, and silica gel
chromatography (5–10% ethyl acetate in hexane, 1% acetic acid)
afforded 13p as a white solid (521 mg, 77%); mp 67.0–68.0 °C. 1H
NMR (500 MHz, CDCl3) d 5.65 (s, 1H), 2.67–2.59 (m, 2H), 1.84–
1.75 (m, 3H), 1.70–1.64 (m, 2H), 1.64–1.49 (m, 4H), 1.17–1.01
(m, 2H). 13C NMR (126 MHz, CDCl3) d 174.9, 171.3, 93.0, 39.6,
33.6, 32.6, 26.7, 25.3. HRMS (ESI) calcd for C10H15NO2 [M+H]+
182.1176, found 182.1182.
6.4.11. 5-(2-Methylbutyl)isoxazol-3-ol (13k)
Prepared using the general procedure above (Method E) from b-
keto hydroxamic acid (12k) (3.09 g, 8.27 mmol), concentrated HCl
(30.0 mL), and methanol (17.0 mL). Concentration of the reaction
mixture in vacuo followed by an aqueous work up, and silica gel
chromatography (5–10% ethyl acetate in hexane, 1% acetic acid)
afforded 13k as an oil (871 mg, 68%). 1H NMR (500 MHz, CDCl3) d
10.87 (s, 1H), 5.66 (s, 1H), 2.62 (dd, J = 14.9, 6.1 Hz, 1H), 2.45 (dd,
J = 14.9, 7.9 Hz, 1H), 1.84–1.71 (m, J = 6.6 Hz, 1H), 1.46–1.33 (m,
1H), 1.23 (dq, J = 21.1, 7.5 Hz, 2H), 0.92 (d, J = 6.7 Hz, 3H), 0.91 (t,
J = 7.5 Hz, 3H). 13C NMR (126 MHz, CDCl3) d 173.9, 171.3, 94.0,
34.4, 33.9, 29.2, 19.2, 11.4. HRMS (ESI) calcd for C8H13NO2
[M+H]+ 156.1019, found 156.1013.
6.5. General procedure for the synthesis of N,N-dimethyl-3-
oxoisoxazole-2(3H)-carboxamide 14a–h, j–p
Method F: To solution of isoxazol-3-ol (0.40 mmol) in toluene
(2.0 mL) was added N,N-dimethylcarbamoyl chloride (0.19 mL,
2.00 mmol) under nitrogen atmosphere. The resulting reaction
mixture was refluxed overnight. The reaction mixture was cooled
to room temperature and solvent was evaporated in vacuo. The
resulting residue was purified by flash column chromatography
on silica gel using a gradient of 20–30% ethyl acetate in hexane
to yield carboxamide as the major product and carbamate as the
minor product.
6.4.12. 5-Neopentylisoxazol-3-ol (13l)
Prepared using the general procedure above (Method E) from b-
keto hydroxamic acid (12l) (2.29 g, 6.40 mmol), concentrated HCl
(20.0 mL), and methanol (14.0 mL). Concentration of the reaction
mixture in vacuo followed by an aqueous work up, and silica gel
chromatography (5–10% ethyl acetate in hexane, 1% acetic acid)
afforded 13l as an off white solid (664 mg, 74%). 1H NMR
(500 MHz, CDCl3) d 5.67 (s, 1H), 2.52 (s, 2H), 0.98 (s, 9H). 13C
NMR (126 MHz, CDCl3) d 173.1, 171.1, 94.9, 41.6, 31.7, 29.6. HRMS
(ESI) calcd for C8H13NO2 [M+H]+ 156.1019, found 156.1021.
6.5.1. N,N,5-Trimethyl-3-oxoisoxazole-2(3H)-carboxamide (14a)
Prepared using the general procedure above (Method F) from
5-methylisoxazol-3-ol (13a) (45.7 mg, 0.46 mmol), N,N-dimethyl-
carbamoyl chloride (0.21 mL, 2.31 mmol), and toluene (2.5 mL).
Concentration of the reaction mixture in vacuo followed by silica
gel flash chromatography (20–30% ethyl acetate in hexane)
yielded 14a as an off white solid (66.3 mg, 84%). 1H NMR
(500 MHz, CDCl3) d 5.41 (s, 1H), 3.10 (br s, 6H), 2.31 (s, 3H). 13C
NMR (126 MHz, CDCl3) d 174.5, 167.9, 150.2, 96.9, 38.8, 37.0,
13.9. HRMS (ESI) calcd for C7H10N2O3 [M+H]+ 171.0691, found
171.0525.
6.4.13. 5-Isopentylisoxazol-3-ol (13m)
Prepared using the general procedure above (Method E) from
crude b-keto hydroxamic acid (12m) (1.64 g, 4.39 mmol), concen-
trated HCl (11.0 mL), and methanol (9.0 mL). Concentration of
the reaction mixture in vacuo followed by an aqueous work up,
and silica gel chromatography (5–10% ethyl acetate in hexane, 1%
acetic acid) afforded 13m as a white solid (629 mg, 92%); mp
53.0–54.8. 1H NMR (500 MHz, CDCl3) d 9.64 (s, 1H), 5.65 (s, 1H),
2.65–2.61 (m, 2H), 1.66–1.49 (m, 3H), 0.93 (d, J = 6.5 Hz, 6H). 13C
NMR (126 MHz, CDCl3) d 175.0, 171.3, 93.0, 36.1, 27.7, 25.4, 22.3.
HRMS (ESI) calcd for C8H13NO2 [M+H]+ 156.1019, found 156.1011.
6.5.2. 5-Cyclopropyl-N,N-dimethyl-3-oxoisoxazole-2(3H)-
carboxamide (14b)
6.4.14. 5-(2-Cyclopropylethyl)isoxazol-3-ol (13n)
Prepared using the general procedure above (Method F) from 5-
cyclopropylisoxazol-3-ol (13b) (20.5 mg, 0.16 mmol), N,N-dimeth-
ylcarbamoyl chloride (0.08 mL, 0.82 mmol), and toluene (1.0 mL).
Concentration of the reaction mixture in vacuo followed by silica
gel flash chromatography (20–30% ethyl acetate in hexane) yielded
14b as a clear oil (4.60 mg, 14%). 1H NMR (500 MHz, CDCl3) d 5.27
(s, 1H), 3.07 (br s, 6H), 1.91–1.81 (m, 1H), 1.19–1.00 (m, 4H). 13C
NMR (126 MHz, CDCl3) d 180.0, 168.3, 150.4, 92.9, 38.8, 36.9, 9.3,
9.3. HRMS (ESI) calcd for C9H12N2O3 [M+H]+ 197.0921, found
197.0933.
Prepared using the general procedure above (Method E) from
crude b-keto hydroxamic acid (1.45 g), concentrated HCl
(21.0 mL), and methanol (9.5 mL). Concentration of the reaction
mixture in vacuo followed by an aqueous work up, and silica gel
chromatography (5–10% ethyl acetate in hexane, 1% acetic acid)
afforded 13n as a slight yellow crystalline solid (290 mg, 34% over
2 steps). 1H NMR (400 MHz, CDCl3) d 11.73 (s, 1H), 5.67 (s, 1H),
2.73 (t, J = 7.6 Hz, 2H), 1.56 (q, J = 7.3 Hz, 2H), 0.79–0.63 (m, 1H),
0.54–0.35 (m, 2H), 0.06 (dt, J = 5.9, 4.4 Hz, 2H). 13C NMR (101 MHz,
CDCl3) d 174.5, 171.3, 93.2, 32.5, 27.6, 10.6, 4.6. HRMS (ESI) calcd
for C8H11NO2 [M+H]+ 154.0863, found 154.0856.
6.5.3. 5-Isopropyl-N,N-dimethyl-3-oxoisoxazole-2(3H)-
carboxamide (14c)
Prepared using the general procedure above (Method F) from 5-
isopropylisoxazol-3-ol (13c) (136 mg, 1.07 mmol), N,N-dimethyl-
carbamoyl chloride (0.49 mL, 5.34 mmol), and toluene (6.0 mL).
Concentration of the reaction mixture in vacuo followed by silica
gel flash chromatography (20–30% ethyl acetate in hexane) yielded
6.4.15. 5-(2-Cyclobutylethyl)isoxazol-3-ol (13o)
Prepared using the general procedure above (Method E)
from crude b-keto hydroxamic acid (1.18 g), concentrated HCl
(7.5 mL), and methanol (16.5 mL). Concentration of the reaction
mixture in vacuo followed by an aqueous work up, and silica gel

A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
1335
14c as a white solid (129 mg, 61%); mp 94.0–95.5 °C. 1H NMR
(500 MHz, CDCl3) d 5.37 (s, 1H), 3.10 (br s, 6H), 2.88 (hept,
J = 6.7 Hz, 1H), 1.29 (d, J = 7.0 Hz, 6H). 13C NMR (126 MHz, CDCl3)
d 183.2, 168.0, 150.3, 94.1, 38.8, 37.1, 28.2, 19.9. HRMS (ESI) calcd
for C9H14N2O3 [M+H]+ 198.1004, found 199.1083.
they were clearly visible in the 1H NMR spectrum (see Fig. 2 for
explanation).
6.5.8. N,N-Dimethyl-3-oxo-5-(pentan-3-yl)isoxazole-2(3H)-
carboxamide (14h)
Prepared using the general procedure above (Method F) from 5-
(pentan-3-yl)isoxazol-3-ol (13h) (62.1 mg, 0.40 mmol), N,N-
dimethylcarbamoyl chloride (0.18 mL, 2.00 mmol), and toluene
(2.0 mL). Concentration of the reaction mixture in vacuo followed
by silica gel flash chromatography (20–30% ethyl acetate in hex-
ane) yielded 14h as a clear oil (73.4 mg, 81%). 1H NMR (500 MHz,
CDCl3) d 5.36 (s, 1H), 3.10 (br s, 6H), 2.52–2.44 (m, 1H), 1.74–
1.57 (m, 4H), 0.94 (t, J = 7.4 Hz, 6H). 13C NMR (126 MHz, CDCl3) d
181.8, 168.2, 150.3, 95.7, 42.6, 37.0, 25.6, 11.6. HRMS (ESI) calcd
for C11H18N2O3 [M+Na]+ 249.121, found 249.1213.
6.5.4. 5-Cyclobutyl-N,N-dimethyl-3-oxoisoxazole-2(3H)-
carboxamide (14d)
Prepared using the general procedure above (Method F) from 5-
cyclobutylisoxazol-3-ol (13d) (63.0 mg, 0.45 mmol), N,N-dimethyl-
carbamoyl chloride (0.21 mL, 2.26 mmol), and toluene (2.2 mL).
Concentration of the reaction mixture in vacuo followed by silica
gel flash chromatography (20–30% ethyl acetate in hexane) yielded
14d as a white solid (75.0 mg, 79%); mp 62.5–64.3 °C. 1H NMR
(500 MHz, CDCl3) d 5.42 (s, 1H), 3.44 (quintet, J = 8.6 Hz, 1H),
3.10 (br s, 6H), 2.42–2.33 (m, 2H), 2.32–2.22 (m, 2H), 2.13–2.02
(m, 1H), 2.02–1.90 (m, 1H). 13C NMR (126 MHz, CDCl3) d 180.9,
167.9, 150.3, 94.5, 38.8, 37.0, 32.8, 27.1, 18.9. HRMS (ESI) calcd
for C10H14N2O3 [M+H]+ 211.1077, found 211.1068.
6.5.9. 5-Isobutyl-N,N-dimethyl-3-oxoisoxazole-2(3H)-
carboxamide (14j)
Prepared using the general procedure above (Method F) from 5-
isobutylisoxazol-3-ol (13j) (49.0 mg, 0.35 mmol), N,N-dimethylcar-
bamoyl chloride (0.16 mL, 1.74 mmol), and toluene (1.7 mL).
Concentration of the reaction mixture in vacuo followed by silica
gel flash chromatography (20–30% ethyl acetate in hexane) yielded
14j as a clear oil (40.0 mg, 54%). 1H NMR (500 MHz, CDCl3) d 5.39
(s, 1H), 3.10 (br s, 6H), 2.47 (d, J = 7.1 Hz, 2H), 2.12–1.97 (m, 1H),
1.00 (d, J = 6.7 Hz, 6H). 13C NMR (126 MHz, CDCl3) d 177.7, 168.0,
150.3, 96.7, 36.9, 27.1, 22.4. Due to signal broadening (slow chem-
ical exchange), the carboxamide methyls could not be detected by
13C NMR, though they were clearly visible in the 1H NMR spectrum
(see Fig. 2 for explanation). HRMS (ESI) calcd for C10H16N2O3
[M+H]+ 213.1509, found 213.1261.
6.5.5. 5-sec-Butyl-N,N-dimethyl-3-oxoisoxazole-2(3H)-
carboxamide (14e)
Prepared using the general procedure above (Method F) from 5-
sec-butylisoxazol-3-ol (13e) (96.0 mg, 0.68 mmol), N,N-dimethyl-
carbamoyl chloride (0.31 mL, 3.38 mmol), and toluene (3.0 mL).
Concentration of the reaction mixture in vacuo followed by silica
gel flash chromatography (20–30% ethyl acetate in hexane) yielded
14e as clear oil (104 mg, 73%). 1H NMR (500 MHz, CDCl3) d 5.35 (s,
1H), 3.09 (br s, 6H), 2.67 (hextet, J = 6.9 Hz, 1H), 1.71 (doublet of
quintets, J = 14.3, 7.3 Hz, 1H), 1.59 (doublet of quintets, J = 14.3,
7.3 Hz, 1H), 1.25 (d, J = 7.0 Hz, 8H), 0.95 (t, J = 7.3 Hz, 3H). 13C
NMR (126 MHz, CDCl3) d 182.7, 168.1, 150.3, 94.7, 38.8, 35.0,
27.5, 17.3, 11.3. HRMS (ESI) calcd for
213.1234, found 213.1238.
C
10H16N2O3 [M+H]+
6.5.10. N,N-Dimethyl-5-(2-methylbutyl)-3-oxoisoxazole-2(3H)-
carboxamide (14k)
Prepared using the general procedure above (Method F) from 5-
(2-methylbutyl)isoxazol-3-ol (13k) (29.0 mg, 0.19 mmol), N,N-
dimethylcarbamoyl chloride (0.09 mL, 0.94 mmol), and toluene
(1.0 mL). Concentration of the reaction mixture in vacuo followed
by silica gel flash chromatography (20–30% ethyl acetate in hex-
ane) yielded 14k as a clear oil (19.3 mg, 46%). 1H NMR (500 MHz,
CDCl3) d 5.38 (s, 1H), 3.10 (br s, 6H), 2.58 (dd, J = 15.0, 7.0 Hz,
1H), 2.39 (dd, J = 15.0, 7.0 Hz, 1H), 1.80 (doublet of quintets,
J = 13.5, 6.7 Hz, 1H), 1.48–1.35 (m, 1H), 1.32–1.18 (m, 1H), 0.97
(d, J = 6.7 Hz, 3H), 0.91 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz,
CDCl3) d 177.9, 168.0, 150.3, 96.7, 39.0, 37.1, 35.0, 33.3, 29.3,
19.2, 11.3. HRMS (ESI) calcd for C11H18N2O3 [M+H]+ 227.139, found
227.1403.
6.5.6. 5-Cyclopentyl-N,N-dimethyl-3-oxoisoxazole-2(3H)-
carboxamide (14f)
Prepared using the general procedure above (Method F) from 5-
cyclopentylisoxazol-3-ol (13f) (21.0 mg, 0.14 mmol), N,N-dimeth-
ylcarbamoyl chloride (0.06 mL, 0.69 mmol), and toluene (1.0 mL).
Concentration of the reaction mixture in vacuo followed by silica
gel flash chromatography (20–30% ethyl acetate in hexane) yielded
14f as a white solid (21.0 mg, 67%); mp 69.0–71.8 °C. 1H NMR
(500 MHz, CDCl3) d 5.36 (s, 1H), 3.10 (br s, 6H), 3.04–2.96 (m,
1H), 2.14–1.99 (m, 2H), 1.86–1.60 (m, 6H). 13C NMR (126 MHz,
CDCl3) d 182.3, 168.0, 150.3, 94.4, 38.4, 31.2, 25.5. HRMS (ESI) calcd
for C11H16N2O3 [M+Na]+ 225.1234, found 225.1212. Due to signal
broadening (slow chemical exchange), the carboxamide methyls
could not be detected by 13C NMR, though they were clearly visible
in the 1H NMR spectrum (see Fig. 2 for explanation).
6.5.11. N,N-Dimethyl-5-neopentyl-3-oxoisoxazole-2(3H)-
carboxamide (14l)
Prepared using the general procedure above (Method F) from 5-
neopentylisoxazol-3-ol (13l) (40.0 mg, 0.28 mmol), N,N-dimethyl-
carbamoyl chloride (0.13 mL, 1.41 mmol), and toluene (1.4 mL).
Concentration of the reaction mixture in vacuo followed by silica
gel flash chromatography (20–30% ethyl acetate in hexane) yielded
14l as a white solid (52.0 mg, 81%); mp 70.7–72.0. 1H NMR
(500 MHz, CDCl3) d 5.39 (s, 1H), 3.11 (br s, 6H), 2.48 (s, 2H), 1.03
(s, 9H). 13C NMR (126 MHz, CDCl3) d 177.0, 168.2, 150.3, 97.7,
42.0, 38.8, 36.8, 31.8, 29.6. HRMS (ESI) calcd for C11H18N2O3
[M+H]+ 227.139, found 227.1383.
6.5.7. N,N-Dimethyl-3-oxo-5-(pentan-2-yl)isoxazole-2(3H)-
carboxamide (14g)
Prepared using the general procedure above (Method F) from
5-(pentan-2-yl)isoxazol-3-ol
(13g)
(98.0 mg,
0.63 mmol),
N,N-dimethylcarbamoyl chloride (0.17 mL, 1.59 mmol), and
toluene (3.0 mL). Concentration of the reaction mixture in vacuo
followed by silica gel flash chromatography (20–30% ethyl acetate
in hexane) yielded 14g as a clear oil (110 mg, 78%). 1H NMR
(500 MHz, CDCl3) d 5.35 (s, 1H), 3.10 (br s, 6H), 2.75 (hextet,
J = 6.9 Hz, 1H), 1.71–1.64 (m, 1H), 1.56–1.47 (m, 1H), 1.44–1.31
(m, 2H), 1.26 (d, J = 7.0 Hz, 3H), 0.92 (t, J = 7.3 Hz, 3H). 13C NMR
(126 MHz, CDCl3) d 182.9, 168.1, 150.3, 94.6, 36.7, 33.3, 20.2,
17.8, 14.0. HRMS (ESI) calcd for C11H18N2O3 [M+H]+ 227.139, found
227.1385. Due to signal broadening (slow chemical exchange), the
carboxamide methyls could not be detected by 13C NMR, though
6.5.12. 5-Isopentyl-N,N-dimethyl-3-oxoisoxazole-2(3H)-
carboxamide (14m)
Prepared using the general procedure above (Method F) from 5-
isopentylisoxazol-3-ol (13m) (100 mg, 0.64 mmol), N,N-dimethyl-

1336
A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
carbamoyl chloride (0.30 mL, 3.22 mmol), and toluene (3.0 mL).
Concentration of the reaction mixture in vacuo followed by silica
gel flash chromatography (20–30% ethyl acetate in hexane) yielded
14m as a clear oil (122 mg, 71%). 1H NMR (500 MHz, CDCl3) d 5.38
(s, 1H), 3.08 (br s, 6H), 2.57 (t, J = 7.9 Hz, 2H), 1.70–1.50 (m, 3H),
0.92 (d, J = 6.5 Hz, 6H). 13C NMR (126 MHz, CDCl3) d 178.9, 168.0,
150.3, 95.7, 38.8, 37.0, 35.1, 27.7, 26.0, 22.2. HRMS (ESI) calcd for
acid, and then brine, and dried over sodium sulfate. The solvent
was evaporated and the residue was purified by silica gel column
chromatography using 20% ethyl acetate in hexane to afford the
dimethyl carbamate as the major product.
6.6.1. 5-Methylisoxazol-3-yl dimethylcarbamate (15a)
Prepared using the general procedure above (Method G) from 5-
methylisoxazol-3-ol (13a) (200 mg, 2.01 mmol), potassium tert-
butoxide (2.62 mL, 2.62 mmol), N,N-dimethylcarbamoyl chloride
(0.46 mL, 5.04 mmol), and THF (7.0 mL). Aqueous work up fol-
lowed by silica gel flash chromatography (20% ethyl acetate in hex-
ane) of the residue yielded 15a as a clear oil (256 mg, 75%). 1H NMR
(500 MHz, CDCl3) d 6.09 (s, 1H), 3.04 (s, 3H), 2.95 (s, 3H), 2.34 (s,
3H). 13C NMR (126 MHz, CDCl3) d 170.9, 166.7, 151.8, 96.2, 36.8,
36.6, 13.1. HRMS (ESI) calcd for C7H10N2O3 [M+H]+ 171.0691, found
171.0771.
C
11H18N2O3 [M+H]+ 227.1390, found 227.1397.
6.5.13. 5-(2-Cyclopropylethyl)-N,N-dimethyl-3-oxoisoxazole-
2(3H)-carboxamide (14n)
Prepared using the general procedure above (Method F) from 5-
(2-cyclopropylethyl)isoxazol-3-ol (13n) (74 mg, 0.48 mmol), N,N-
dimethylcarbamoyl chloride (0.23 mL, 2.41 mmol), and toluene
(2.5 mL). Concentration of the reaction mixture in vacuo followed
by silica gel flash chromatography (20–30% ethyl acetate in hex-
ane) yielded 14n as
a
clear liquid (78 mg, 72%). 1H NMR
(400 MHz, CDCl3) d 5.39 (s, 1H), 3.08 (br s, 6H), 2.67 (t, J = 7.9 Hz,
2H), 1.56 (q, J = 7.3 Hz, 2H), 0.80–0.65 (m, 1H), 0.53–0.40 (dq,
J = 8.3, 2.67 Hz, 3H), 0.07 (q, J = 4.6 Hz, 2H). 13C NMR (101 MHz,
CDCl3) d 178.4, 167.9, 150.3, 96.0, 38.7, 37.1, 31.5, 28.2, 10.5, 4.7.
HRMS (ESI) calcd for
225.1231.
6.6.2. 5-Cyclopropylisoxazol-3-yl dimethylcarbamate (15b)
Prepared using the general procedure above (Method F) from 5-
cyclopropylisoxazol-3-ol (13b) (20.5 mg, 0.16 mmol), N,N-dimeth-
ylcarbamoyl chloride (0.08 mL, 0.82 mmol), and toluene (1.0 mL).
Concentration of the reaction mixture in vacuo followed by silica
gel flash chromatography (20–30% ethyl acetate in hexane) yielded
15b as a clear oil (25.0 mg, 78%). 1H NMR (500 MHz, CDCl3) d 6.07
(s, 1H), 3.08 (s, 3H), 3.00 (s, 3H), 1.98 (tt, J = 8.5, 5.1 Hz, 1H), 1.07–
1.01 (m, 2H), 1.01–0.95 (m, 2H). 13C NMR (126 MHz, CDCl3) d176.2,
166.8, 151.9, 93.3, 37.0, 36.7, 8.8, 8.3. HRMS (ESI) calcd for
C9H12N2O3 [M+H]+ 197.0921, found 197.0921.
C
11H16N2O3 [M+H]+ 225.1234, found
6.5.14. 5-(2-Cyclobutylethyl)-N,N-dimethyl-3-]oxoisoxazole-
2(3H)-carboxamide (14o)
Prepared using the general procedure above (Method F) from 5-
(2-cyclobutylethyl)isoxazol-3-ol (13o) (90 mg, 0.54 mmol), N,N-
dimethylcarbamoyl chloride (0.25 mL, 2.72 mmol), and toluene
(3.5 mL). Concentration of the reaction mixture in vacuo followed
by silica gel flash chromatography (20–30% ethyl acetate in hex-
6.6.3. 5-Isopropylisoxazol-3-yl dimethylcarbamate (15c)
Prepared using the general procedure above (Method G) from
5-isopropylisoxazol-3-ol (13c) (122 mg, 0.96 mmol), potassium
tert-butoxide (1.24 mL, 1.25 mmol), N,N-dimethylcarbamoyl
chloride (0.22 mL, 2.39 mmol), and THF (7.0 mL). Aqueous work
up followed by silica gel flash chromatography (20% ethyl
acetate in hexane) of the residue yielded 15c as a clear oil
(170 mg, 80%). 1H NMR (500 MHz, CDCl3) d 6.12 (s, 1H), 3.09 (s,
3H), 3.06–2.96 (m, 1H), 3.00 (s, 3H), 1.29 (d, J = 7.0 Hz, 6H). 13C
NMR (126 MHz, CDCl3) d 180.2, 166.7, 152.0, 93.7, 37.0, 36.7,
27.9, 20.6. HRMS (ESI) calcd for C9H14N2O3 [M+H]+ 198.1004,
found 199.1079.
ane) yielded 14o as
a
clear liquid (87 mg, 67%). 1H NMR
(400 MHz, CDCl3) d 5.33 (s, 1H), 3.05 (br s, 6H), 2.44 (td, J = 7.8,
0.7 Hz, 3H), 2.27 (hept, J = 7.8 Hz, 1H), 2.11–1.95 (m, 2H), 1.89–
1.74 (m, 2H), 1.71 (q, J = 7.7 Hz, 2H), 1.64–1.51 (m, 2H). 13C NMR
(101 MHz, CDCl3) d 178.5, 167.8, 150.2, 95.7, 38.7, 36.9, 35.2,
33.2, 27.9, 27.9, 25.7, 18.2. HRMS (ESI) calcd for C12H18N2O3
[M+H]+ 239.1390, found 239.1387.
6.5.15. 5-(2-Cyclopentylethyl)-N,N-dimethyl-3-oxoisoxazole-
2(3H)-carboxamide (14p)
Prepared using the general procedure above (Method F) from
5-(2-cyclopentylethyl)isoxazol-3-ol (13p) (77.8 mg, 0.43 mmol),
N,N-dimethylcarbamoyl chloride (0.20 mL, 2.15 mmol), and
toluene (3.0 mL). Concentration of the reaction mixture in vacuo
followed by silica gel flash chromatography (20–30% ethyl acetate
in hexane) yielded 14p as a white solid (85.0 mg, 79%); mp 47.8–
47.9 °C. 1H NMR (500 MHz, CDCl3) d 5.39 (s, 1H), 3.11 (br s, 6H),
2.59 (t, J = 7.9 Hz, 2H), 1.87–1.74 (m, 3H), 1.71–1.65 (m, 2H),
1.65–1.61 (m, 2H), 1.58–1.49 (m, 2H), 1.18–1.01 (m, 2H). 13C
NMR (126 MHz, CDCl3) d 178.8, 167.9, 150.3, 95.8, 39.6, 38.8,
36.9, 32.6, 32.5, 27.3, 25.2. HRMS (ESI) calcd for C13H20N2O3
[M+H]+ 253.1547, found 253.1547.
6.6.4. 5-Cyclobutylisoxazol-3-yl dimethylcarbamate (15d)
Prepared using the general procedure above (Method G) from
5-cyclobutylisoxazol-3-ol (13d) (39.0 mg, 0.28 mmol), potassium
tert-butoxide (0.42 mL, 0.42 mmol), N,N-dimethylcarbamoyl chlo-
ride (0.06 mL, 0.70 mmol), and THF (2.0 mL). Aqueous work up fol-
lowed by silica gel flash chromatography (20% ethyl acetate in
hexane) of the residue yielded 15d as a clear oil (52.0 mg, 89%).
1H NMR (500 MHz, CDCl3) d 6.17 (s, 1H), 3.57 (quintet, J = 8.5 Hz,
1H), 3.09 (s, 3H), 3.01 (s, 3H), 2.43–2.21 (m, 4H), 2.11–1.87 (m,
2H). 13C NMR (126 MHz, CDCl3) d 178.0, 166.8, 152.0, 94.2, 37.0,
36.8, 32.8, 27.8, 18.8. HRMS (ESI) calcd for C10H14N2O3 [M+H]+
211.1077, found 211.1072.
6.6. General procedure for the synthesis of synthesis of isoxazol-
3-yl dimethylcarbamate 15a–p
6.6.5. 5-sec-Butylisoxazol-3-yl dimethylcarbamate (15e)
Prepared using the general procedure above (Method G) from
5-sec-butylisoxazol-3-ol (13e) (45.1 mg, 0.32 mmol), potassium
tert-butoxide (0.48 mL, 0.48 mmol), N,N-dimethylcarbamoyl
chloride (0.07 mL, 0.80 mmol), and THF (2.0 mL). Aqueous work
up followed by silica gel flash chromatography (20% ethyl acetate
in hexane) of the residue yielded 15e as a clear oil (55.5 mg,
82%). 1H NMR (500 MHz, CDCl3) d 6.14 (s, 1H), 3.09 (s, 3H), 3.01
(s, 3H), 2.82 (sextet, J = 7.0 Hz, 1H), 1.72 (doublet of quintet,
J = 14.3, 7.2 Hz, 1H), 1.61 (doublet of quintet, J = 14.3, 7.2 Hz, 1H),
1.27 (d, J = 7.0 Hz, 3H), 0.91 (t, J = 7.2 Hz, 3H). 13C NMR (126 MHz,
Method G: To a dry round bottom flask was added isoxazol-3-ol
(0.47 mmol) and dry tetrahydrofuran (3.0 mL) prior to cooling it to
0 °C. To this solution was added potassium tert-butoxide (1 M in
THF, 0.71 mL, 0.71 mmol) at 0 °C and subsequently the reaction
mixture was stirred for 20 min at rt. To this solution was added
N,N-dimethyl carbamoyl chloride (0.11 mL, 1.18 mmol), and the
resulting solution was stirred at room temperature for additional
16 h. The solvent was concentrated and the residue was taken up
in dichloromethane and washed with 0.25 M HCl hydrochloric

A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
1337
CDCl3) d 179.4, 166.7, 152.0, 94.3, 37.0, 36.8, 34.7, 28.2, 18.1, 11.5.
HRMS (ESI) calcd for
213.1243.
lowed by silica gel flash chromatography (20% ethyl acetate in hex-
ane) of the residue yielded 15j as a clear oil (248 mg, 66%). 1H NMR
(500 MHz, CDCl3) d 6.16 (s, 1H), 3.09 (s, 3H), 3.01 (s, 3H), 2.58 (d,
J = 7.1 Hz, 2H), 2.09–1.97 (m, 1H), 0.96 (d, J = 6.7 Hz, 6H). 13C
NMR (126 MHz, CDCl3) d174.4, 166.8, 152.0, 96.1, 37.0, 36.8,
C
10H16N2O3 [M+H]+ 213.1234, found
6.6.6. 5-Cyclopentylisoxazol-3-yl dimethylcarbamate (15f)
Prepared using the general procedure above (Method G) from
5-cyclopentylisoxazol-3-ol (13f) (250 mg, 1.63 mmol), potassium
tert-butoxide (2.12 mL, 2.12 mmol), N,N-dimethylcarbamoyl chlo-
ride (0.37 mL, 4.08 mmol), and THF (10.0 mL). Aqueous work up
followed by silica gel flash chromatography (20% ethyl acetate
in hexane) of the residue yielded 15f as a clear oil (300 mg,
82%). 1H NMR (500 MHz, CDCl3) d 6.13 (d, J = 0.8 Hz, 1H), 3.14
(quintet, J = 7.5 Hz, 1H), 3.09 (s, 3H), 3.01 (s, 3H), 2.12–1.97 (m,
2H), 1.80–1.70 (m, 4H), 1.70–1.60 (m, 2H). 13C NMR (126 MHz,
CDCl3) d 178.9, 166.6, 151.9, 93.9, 38.1, 36.9, 36.6, 31.5, 25.2.
36.6, 27.6, 22.4. HRMS (ESI) calcd for
213.1509, found 213.1261.
C
10H16N2O3 [M+H]+
6.6.11. 5-(2-Methylbutyl)isoxazol-3-yl dimethylcarbamate (15k)
Prepared using the general procedure above (Method G) from 5-
(2-methylbutyl)isoxazol-3-ol (13k) (300 mg, 1.93 mmol), potas-
sium tert-butoxide (2.5 mL, 2.5 mmol), N,N-dimethylcarbamoyl
chloride (0.44 mL, 4.83 mmol), and THF (10 mL). Aqueous work
up followed by silica gel flash chromatography (20% ethyl acetate
in hexane) of the residue yielded 15k as a clear oil (359 mg,
82%). 1H NMR (500 MHz, CDCl3) d 6.15 (s, 1H), 3.09 (s, 3H), 3.00
(s, 3H), 2.69 (dd, J = 15.0, 7.0 Hz, 1H), 2.52 (dd, J = 15.0, 7.0 Hz,
1H), 1.79 (doublet of quintet, J = 13.5, 7.2 Hz, 1H), 1.47–1.32 (m,
1H), 1.27–1.16 (m, 1H), 0.92 (d, J = 6.7 Hz, 3H), 0.90 (t, J = 7.2 Hz,
3H). 13C NMR (126 MHz, CDCl3) d 174.5, 166.8, 152.0, 96.2, 37.0,
36.7, 34.6, 33.9, 29.2, 19.2, 11.4. HRMS (ESI) calcd for C11H18N2O3
[M+Na]+ 249.121, found 249.1201.
HRMS (ESI) calcd for
247.1036.
C
11H16N2O3 [M+Na]+ 247.1053, found
6.6.7. 5-(Pentan-2-yl)isoxazol-3-yl dimethylcarbamate (15g)
Prepared using the general procedure above (Method G)
from 5-(pentan-2-yl)isoxazol-3-ol (13g) (170 mg, 1.09 mmol),
potassium tert-butoxide (1.42 mL, 1.42 mmol), N,N-dimethyl-
carbamoyl chloride (0.25 mL, 2.74 mmol), and THF (7.0 mL).
Aqueous work up followed by silica gel flash chromatography
(20% ethyl acetate in hexane) of the residue yielded 15g as a
clear oil (207 mg, 84%). 1H NMR (500 MHz, CDCl3) d 6.11 (s,
1H), 3.06 (s, 3H), 2.98 (s, 3H), 2.88 (sextet, J = 7.0 Hz, 1H),
1.66 (ddt, J = 13.4, 9.5, 6.5 Hz, 1H), 1.50 (ddt, J = 13.4, 9.5,
6.5 Hz, 1H), 1.38–1.26 (m, 2H), 1.24 (d, J = 7.0 Hz, 3H), 0.87
6.6.12. 5-Neopentylisoxazol-3-yl dimethylcarbamate (15l)
Prepared using the general procedure above (Method G) from
5-neopentylisoxazol-3-ol (13l) (230 mg, 1.63 mmol), potassium
tert-butoxide (2.12 mL, 2.12 mmol), N,N-dimethylcarbamoyl
chloride (0.37 mL, 4.07 mmol), and THF (7.0 mL). Aqueous work
up followed by silica gel flash chromatography (20% ethyl acetate
in hexane) of the residue yielded 15l as a clear oil (262 mg,
71%). 1H NMR (500 MHz, CDCl3) d 6.13 (s, 1H), 3.04 (s, 3H),
2.95 (s, 3H), 2.54 (s, 2H), 0.93 (s, 9H). 13C NMR (126 MHz,
CDCl3) d 173.3, 166.5, 151.8, 97.0, 41.5, 36.8, 36.6, 31.6, 29.3.
HRMS (ESI) calcd for C11H18N2O3 [M+NH4]+ 244.1656, found
244.1668.
(t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, CDCl3)
166.6, 151.9, 94.1, 37.4, 36.9, 36.7, 32.9, 20.1, 18.5, 13.9.
d
179.5,
HRMS (ESI) calcd for
227.1677.
C
11H18N2O3 [M+H]+ 227.1317, found
6.6.8. 5-(Pentan-3-yl)isoxazol-3-yl dimethylcarbamate (15h)
Prepared using the general procedure above (Method G) from 5-
(pentan-3-yl)isoxazol-3-ol (13h) (73.5 mg, 0.47 mmol), potassium
tert-butoxide (0.71 mL, 0.71 mmol), N,N-dimethylcarbamoyl chlo-
ride (0.11 mL, 1.18 mmol), and THF (3.0 mL). Aqueous work up fol-
lowed by silica gel flash chromatography (20% ethyl acetate in
hexane) of the residue yielded 15h as a clear oil (89.3 mg, 83%).
1H NMR (500 MHz, CDCl3) d 6.17 (s, 1H), 3.10 (s, 3H), 3.01 (s,
3H), 2.67–2.59 (m, 1H), 1.78–1.50 (m, 4H), 0.87 (t, J = 7.4 Hz, 6H).
13C NMR (126 MHz, CDCl3) d 178.3, 166.7, 152.0, 95.1, 42.3, 37.0,
6.6.13. 5-Isopentylisoxazol-3-yl dimethylcarbamate (15m)
Prepared using the general procedure above (Method G) from
5-isopentylisoxazol-3-ol (13m) (300 mg, 1.93 mmol), potassium
tert-butoxide (2.5 mL, 2.5 mmol), N,N-dimethylcarbamoyl chlo-
ride (0.44 mL, 4.83 mmol), and THF (10.0 mL). Aqueous work
up followed by silica gel flash chromatography (20% ethyl ace-
tate in hexane) of the residue yielded 15m as
a clear oil
(342 mg, 80%). 1H NMR (500 MHz, CDCl3) d 6.15 (s, 1H), 3.10
(s, 3H), 3.01 (s, 3H), 2.71 (t, J = 8.0, 7.6 Hz, 2H), 1.68–1.54 (m,
36.8, 26.3, 11.7. HRMS (ESI) calcd for
227.139, found 227.1392.
C
11H18N2O3 [M+H]+
3H), 0.93 (d, J = 6.3 Hz, 6H). 13C NMR (126 MHz, CDCl3)
d
175.4, 166.7, 151.9, 95.1, 36.9, 36.6, 35.9, 27.5, 25.4, 22.2.
HRMS (ESI) calcd for
227.1398.
6.6.9. 5-(Heptan-3-yl)isoxazol-3-yl dimethylcarbamate (15i)
Prepared using the general procedure above (Method G) from 5-
(heptan-3-yl)isoxazol-3-ol (13i) (230 mg, 1.25 mmol), potassium
tert-butoxide (1.63 mL, 1.63 mmol), N,N-dimethylcarbamoyl chlo-
ride (0.29 mL, 3.13 mmol), and THF (8.0 mL). Aqueous work up fol-
lowed by silica gel flash chromatography (20% ethyl acetate in
hexane) of the residue yielded 15i as a clear oil (194 mg, 61%).
1H NMR (500 MHz, CDCl3) d 6.15 (s, 1H), 3.08 (s, 3H), 2.99 (s,
3H), 2.68 (quintet, J = 7.2 Hz, 1H), 1.70–1.53 (m, 4H), 1.34–1.12
(m, 4H), 0.88–0.80 (m, 6H). 13C NMR (126 MHz, CDCl3) d 178.5,
166.7, 151.9, 95.0, 40.7, 36.9, 36.7, 33.0, 29.3, 26.7, 22.7, 14.0,
11.6. HRMS (ESI) calcd for C13H22N2O3 [M+H]+ 255.1427, found
255.1630.
C
11H18N2O3 [M+H]+ 227.139, found
6.6.14. 5-(2-Cyclopropylethyl)isoxazol-3-yl dimethylcarbamate
(15n)
Prepared using the general procedure above (Method G) from
5-(2-cyclopropylethyl)isoxazol-3-ol (13n) (80 mg, 0.54 mmol),
potassium tert-butoxide (1 M in THF, 0.81 mL, 0.81 mmol), N,N-
dimethylcarbamoyl chloride (0.125 mL, 1.35 mmol), and THF
(5.0 mL). Aqueous work up followed by silica gel flash chroma-
tography (20% ethyl acetate in hexane) of the residue yielded
15n as a clear liquid (90 mg, 75%). 1H NMR (400 MHz, CDCl3)
d
11.73 (s, 1H), 5.67 (s, 1H), 2.73 (t, J = 7.6 Hz, 2H), 1.56
(q, J = 7.3 Hz, 2H), 0.79–0.63 (m, 1H), 0.54–0.35 (dq, J = 8.5,
4.2 Hz 2H), 0.06 (dt, J = 5.9, 4.4 Hz, 2H). 13C NMR (126 MHz,
CDCl3) d 175.1, 166.8, 152.0, 95.5, 37.0, 36.8, 32.5, 27.8, 10.6,
4.6. HRMS (ESI) calcd for C11H16N2O3 [M+H]+ 225.1234, found
225.1229.
6.6.10. 5-Isobutylisoxazol-3-yl dimethylcarbamate (15j)
Prepared using the general procedure above (Method G) from 5-
isobutylisoxazol-3-ol (13j) (252 mg, 1.78 mmol), potassium tert-
butoxide (2.32 mL, 2.32 mmol), N,N-dimethylcarbamoyl chloride
(0.41 mL, 4.45 mmol), and THF (10.0 mL). Aqueous work up fol-

1338
A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
6.6.15. 5-(2-Cyclobutylethyl)isoxazol-3-yl dimethylcarbamate
(15o)
1.14 mmol, 1.8 equiv) in dichloromethane (1.0 mL) was added drop
wise, and the reaction was stirred for an additional 30 min. The sub-
sequent reaction mixture was washed with aqueous solution of
ammonium chloride, and the organic phase was dried over sodium
sulfate and concentrated. The residue was purified by silica gel
chromatography eluting with 20% ethyl acetate in hexanes to yield
the desired compound 18g as clear oil (66.1 mg, 44% yield).
1H NMR (500 MHz, CDCl3) d 5.35 (s, 1H), 3.47 (br s, 2H), 3.09 (br
s, 3H), 2.75 (hextet, J = 7.0 Hz, 1H), 1.72–1.61 (m, 1H), 1.56–1.47
(m, 1H), 1.45–1.31 (m, 2H), 1.26 (d, J = 7.0 Hz, 3H), 1.23 (t,
J = 7.2 Hz, 3H), 0.92 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, CDCl3)
d 182.91, 168.23, 150.03, 94.58, 44.80, 36.68, 35.97, 33.31, 20.14,
17.84, 13.96, 12.24. HRMS (ESI) calcd for C12H20N2O3 [M+H]+
241.1547, found 241.1527.
Prepared using the general procedure above (Method G) from
5-(2-cyclobutylethyl)isoxazol-3-ol (13o) (50 mg, 0.33 mmol),
potassium tert-butoxide (1 M in THF, 0.5 mL, 0.5 mmol), N,N-
dimethylcarbamoyl chloride (0.08 mL, 0.83 mmol), and THF
(3.5 mL). Aqueous work up followed by silica gel flash chromatog-
raphy (20% ethyl acetate in hexane) of the residue yielded 15o as a
clear liquid (50 mg, 64%). 1H NMR (500 MHz, CDCl3) d 6.12 (s, 1H),
3.08 (s, 3H), 2.99 (s, 3H), 2.59 (t, J = 7.7 Hz, 2H), 2.28 (h, J = 7.7 Hz,
1H), 2.08–1.98 (m, 2H), 1.90–1.78 (m, 2H), 1.75 (q, J = 7.6 Hz, 2H),
1.59 (pd, J = 9.0, 2.5 Hz, 2H). 13C NMR (126 MHz, CDCl3) d 175.1,
166.6, 151.8, 95.2, 36.8, 36.6, 35.2, 34.1, 27.9, 25.2, 18.3. HRMS
(ESI) calcd for C12H18N2O3 [M+H]+ 239.1390, found 239.1389.
6.6.16. 5-(2-Cyclopentylethyl)isoxazol-3-yl dimethylcarbamate
(15p)
6.7.3. N,N-Diethyl-3-oxo-5-(pentan-2-yl)isoxazole-2(3H)-
carboxamide (19g)
Prepared using the general procedure above (Method G) from
5-(2-cyclopentylethyl)isoxazol-3-ol (13p) (58.4 mg, 0.32 mmol),
potassium tert-butoxide (0.48 mL, 0.48 mmol), N,N-dimethylcar-
bamoyl chloride (0.074 mL, 0.81 mmol), and THF (1.0 mL). Aque-
ous work up followed by silica gel flash chromatography (20%
ethyl acetate in hexane) of the residue yielded 15p as a clear
oil (73.0 mg, 90%). 1H NMR (500 MHz, CDCl3) d 6.15 (s, 1H),
3.09 (s, 3H), 3.01 (s, 3H), 2.71 (t, J = 7.9 Hz, 2H), 1.86–1.73
(m, 3H), 1.69 (q, J = 7.2 Hz, 2H), 1.65–1.57 (m, 2H), 1.56–1.48
Prepared using the general procedure above (Method F) from
5-(pentan-2-yl)isoxazol-3-ol (13g) (85.0 mg, 0.55 mmol), N,N-
diethylcarbamoyl chloride (0.35 mL, 2.74 mmol), and toluene
(3.0 mL). Concentration of the reaction mixture in vacuo followed
by silica gel flash chromatography (20–30% ethyl acetate in hex-
ane) yielded 19g as a clear oil (96.0 mg, 69%). 1H NMR (500 MHz,
CDCl3) d 5.34 (s, 1H), 3.49 (q, J = 6.5 Hz, 4H), 2.74 (hextet,
J = 7.0 Hz, 1H), 1.74–1.61 (m, 1H), 1.56–1.45 (m, 1H), 1.45–1.29
(m, 2H), 1.27–1.21 (m, 9H), 0.91 (t, J = 7.3 Hz, 3H). 13C NMR
(126 MHz, CDCl3) d 182.81, 168.36, 149.62, 94.31, 42.47, 36.45,
33.07, 19.89, 17.59, 13.71, 13.30. Due to signal broadening (slow
chemical exchange) in carboxamide the two CH2 and two CH3 of
the N,N-diethyl overlap in 13C NMR. This is consistent with simi-
lar observations in seen 1H NMR (see Fig. 2 for explanation).
(m, 2H), 1.20–0.99 (m, 2H). 13C NMR (126 MHz, CDCl3)
d
175.5, 166.8, 152.0, 95.3, 39.6, 37.0, 36.8, 33.5, 32.5, 26.9,
25.3. HRMS (ESI) calcd for C13H20N2O3 [M+H]+ 253.1547, found
253.1525.
HRMS (ESI) calcd for
255.1687.
C
13H22N2O3 [M+H]+ 255.1703, found
6.7. Synthesis of N-substituted derivatives of 13g (17g–26g)
6.7.1. 2-(Azetidine-1-carbonyl)-5-(pentan-2-yl)isoxazol-3(2H)-
one (17g)
6.7.4. 5-(Pentan-2-yl)-2-(pyrrolidine-1-carbonyl)isoxazol-
3(2H)-one (20g)
To a solution of triphosgene (63.5 mg, 0.21 mmol, 0.35 equiv)
in DCM (1.0 mL) was added a solution of 5-(pentan-2-yl)iso-
xazol-3-ol (13g) (94.0 mg, 0.61 mmol, 1 equiv) in DCM (2.0 mL),
and the resulting mixture was allowed to stir at room tempera-
ture for 5.3 h. To the reaction mixture, a solution of azetidine
hydrochloride (85.0 mg, 0.91 mmol, 1.5 equiv) and diisopropyl-
ethylamine (0.35 mL, 1.99 mmol, 3.3 equiv) in dichloromethane
(3 mL) was added drop wise, and the reaction was stirred for an
additional 30 min. The subsequent reaction mixture was washed
with aqueous solution of ammonium chloride, and the organic
phase was dried over sodium sulfate and concentrated. The resi-
due was purified by silica gel chromatography eluting with 20%
ethyl acetate in hexanes to yield the desired compound 17g as
clear oil (59.3 mg, 41% yield). Starting material recovered
(18.0 mg). 1H NMR (500 MHz, CDCl3) d 5.39 (s, 1H), 4.37 (s, 2H),
4.19 (s, 2H), 2.73 (sextet, J = 7.0 Hz, 1H), 2.36 (quintet, J = 7.8 Hz,
2H), 1.71–1.59 (m, 1H), 1.56–1.45 (m, 1H), 1.43–1.29 (m, 2H),
1.25 (d, J = 7.0 Hz, 3H), 0.92 (t, J = 7.3 Hz, 3H). 13C NMR
(126 MHz, CDCl3) d 181.06, 165.57, 148.98, 95.50, 53.01, 49.54,
36.63, 33.18, 20.14, 17.84, 16.57, 13.94. HRMS (ESI) calcd for
Prepared using the general procedure above (Method F) from 5-
(pentan-2-yl)isoxazol-3-ol (13g) (65.1 mg, 0.42 mmol), 1-pyrroli-
dinecarbonyl chloride (0.23 mL, 2.10 mmol), and toluene
(2.0 mL). Concentration of the reaction mixture in vacuo followed
by silica gel flash chromatography (20–30% ethyl acetate in hex-
ane) yielded 20g as a clear oil (38.7 mg, 37%). 1H NMR (500 MHz,
CDCl3) d 5.36 (s, 1H), 3.73 (t, J = 6.3 Hz, 2H), 3.57 (t, J = 6.4 Hz,
2H), 2.75 (sextet, J = 7.0 Hz, 1H), 1.94 (s, 4H), 1.72–1.61 (m, 1H),
1.56–1.47 (m, 1H), 1.45–1.31 (m, 2H), 1.26 (d, J = 7.0 Hz, 3H),
0.92 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, CDCl3) d 182.14,
167.78, 148.43, 94.91, 48.37, 47.31, 36.68, 33.24, 25.89, 24.48,
20.14, 17.83, 13.96. HRMS (ESI) calcd for C13H20N2O3 [M+H]+
253.1547, found 253.1547.
6.7.5. 2-(Morpholine-4-carbonyl)-5-(pentan-2-yl)isoxazol-
3(2H)-one (21g)
Prepared using the general procedure above (Method F) from
5-(pentan-2-yl)isoxazol-3-ol (13g) (99.4 mg, 0.64 mmol), 4-mor-
pholinecarbamoyl chloride (0.37 mL, 3.20 mmol), and toluene
(3.0 mL). Concentration of the reaction mixture in vacuo fol-
lowed by silica gel flash chromatography (20–30% ethyl acetate
in hexane) yielded 21g as a clear oil (133 mg, 78%). 1H NMR
(500 MHz, CDCl3) d 5.36 (s, 1H), 3.80–3.75 (m, 4H), 3.64–3.56
(m, 4H), 2.77 (sextet, J = 7.0 Hz, 1H), 1.73–1.61 (m, 1H), 1.57–
1.48 (m, 1H), 1.45–1.30 (m, 2H), 1.27 (d, J = 7.0 Hz, 3H), 0.93
(t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, CDCl3) d 183.23, 167.55,
148.92, 94.29, 66.66, 47.73, 45.19, 36.53, 33.22, 20.00, 17.68,
13.80. HRMS (ESI) calcd for C13H20N2O4 [M+H]+ 269.1496, found
269.1476.
C
12H18N2O3 [M+H]+ 239.139, found 239.1371.
6.7.2. N-Ethyl-N-methyl-3-oxo-5-(pentan-2-yl)isoxazole-2(3H)-
carboxamide (18g)
To a solution of triphosgene (66.2 mg, 0.22 mmol, 0.35 equiv) in
DCM (1.0 mL) was added a solution of 5-(pentan-2-yl)isoxazol-3-ol
(13g) (98.0 mg, 0.63 mmol, 1 equiv) in DCM (2.0 mL), and the
resulting mixture was allowed to stir at room temperature for
2 h. To the reaction mixture, a solution of N-ethylmethyl amine
(0.8 mL, 0.95 mmol, 1.5 equiv) and diisopropylethylamine (0.2 mL,

A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
1339
6.7.6. 5-(Pentan-2-yl)isoxazol-3-yl ethyl(methyl)carbamate
(23g)
3.59–3.51 (m, 2H), 2.91 (h, J = 7.0 Hz, 1H), 1.74–1.64 (m, 1H),
1.58–1.48 (m, 1H), 1.39–1.29 (m, 2H), 1.28 (d, J = 7.0 Hz, 3H),
0.90 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, CDCl3) d 179.86,
166.47, 150.75, 94.11, 66.59, 66.48, 45.20, 44.42, 37.46, 33.01,
20.22, 18.61, 14.03. HRMS (ESI) calcd for C13H20N2O4 [M+H]+
269.1496, found 269.1478.
To a solution of triphosgene (66.2 mg, 0.22 mmol, 0.35 equiv) in
DCM (1.0 mL) was added a solution of 5-(pentan-2-yl)isoxazol-3-ol
(13g) (98.0 mg, 0.63 mmol, 1 equiv) in DCM (2.0 mL), and the
resulting mixture was allowed to stir at room temperature for
2 h. To the reaction mixture, a solution of N-ethylmethyl amine
(0.8 mL, 0.95 mmol, 1.5 equiv) and diisopropylethylamine (0.2 mL,
1.14 mmol, 1.8 equiv) in dichloromethane (1.0 mL) was added drop
wise, and the reaction was stirred for an additional 30 min. The sub-
sequent reaction mixture was washed with aqueous solution of
ammonium chloride, and the organic phase was dried over sodium
sulfate and concentrated. The residue was purified by silica gel
chromatography eluting with 20% ethyl acetate in hexanes to yield
the desired compound 23g as clear oil (30.0 mg, 20% yield).
(Observed (Z)- and (E)-amide rotamers in 1:1 ratio, the following inte-
gral ratios correspond to the 1:1 mixture) 1H NMR (500 MHz, CDCl3) d
6.14 (s, 2H, both (Z)- and (E)-), 3.46 (q, J = 7.2 Hz, 2H), 3.39 (q,
J = 7.2 Hz, 2H), 3.05 (s, 3H), 2.98 (s, 3H), 2.90 (sextet, J = 7.0 Hz,
2H), 1.74–1.62 (m, 2H), 1.58–1.46 (m, 2H), 1.39–1.29 (m, 4H),
1.27 (d, J = 7.0 Hz, 6H), 1.21 (t, J = 7.2 Hz, 3H), 1.18 (t, J = 7.2 Hz,
3H), 0.89 (t, J = 7.3 Hz, 6H). 13C NMR (126 MHz, CDCl3) d 179.57,
166.75, 166.72, 151.71, 151.57, 94.21, 94.16, 44.59, 44.45, 37.47,
34.51, 34.18, 32.98, 20.21, 18.62, 14.02, 13.28, 12.37. HRMS (ESI)
calcd for C12H20N2O3 [M+H]+ 241.1547, found 241.1532.
6.8. General procedure for the synthesis of N-methyl-3-
oxoisoxazole-2(3H)-carboxamide
6.8.1. 5-Isobutyl-N-methyl-3-oxoisoxazole-2(3H)-carboxamide
(16j)
To a dry round bottom flask was added 5-isobutylisoxazol-3-ol
(13j) (165 mg, 1.17 mmol) and dry tetrahydrofuran (7.0 mL) prior
to cooling it to 0 °C. To this solution was added potassium tert-
butoxide (1 M in THF, 1.52 mL, 1.52 mmol) at 0 °C and subse-
quently the reaction mixture was stirred for 20 min at RT. To this
solution was added N-methylcarbamoyl chloride (273 mg,
2.92 mmol) and the resulting solution was stirred at room temper-
ature for additional 16 h. The solvent was concentrated and the
residue was taken up in dichloromethane and washed with
0.25 M HCl hydrochloric acid, and then brine, and dried over
sodium sulfate. The solvent was evaporated and the residue was
purified using silica gel column chromatography (20% ethyl acetate
in hexane) to yield 16j as a white semi-solid (207 mg, 90%). 1H
NMR (500 MHz, chloroform-d) d 7.83 (s, 1H), 5.56 (s, 1H), 2.96 (d,
J = 4.8 Hz, 3H), 2.49 (d, J = 7.2 Hz, 2H), 2.14–1.98 (m, 1H), 0.99 (d,
6.7.7. 5-(Pentan-2-yl)isoxazol-3-yl diethylcarbamate (24g)
Prepared using the general procedure above (Method F) from 5-
(pentan-2-yl)isoxazol-3-ol (13g) (85.0 mg, 0.55 mmol), N,N-dieth-
ylcarbamoyl chloride (0.35 mL, 2.74 mmol), and toluene (3.0 mL).
Concentration of the reaction mixture in vacuo followed by silica
gel flash chromatography (20–30% ethyl acetate in hexane) yielded
24g as a clear oil (23.0 mg, 16%). 1H NMR (500 MHz, CDCl3) d 6.17
(s, 1H), 3.43 (q, J = 7.1 Hz, 2H), 3.37 (q, J = 7.2 Hz, 2H), 2.91 (sextet,
J = 7.0 Hz, 1H), 1.69 (ddt, J = 13.6, 9.2, 6.5 Hz, 1H), 1.59–1.47 (m,
1H), 1.41–1.28 (m, 2H), 1.27 (d, J = 7.0 Hz, 3H), 1.23 (t, J = 7.2 Hz,
3H), 1.20 (t, J = 7.2 Hz, 3H), 0.90 (t, J = 7.3 Hz, 3H). 13C NMR
(126 MHz, CDCl3) d 179.52, 166.75, 151.39, 94.21, 42.70, 42.34,
37.48, 32.98, 20.22, 18.64, 14.24, 14.02, 13.24. HRMS (ESI) calcd
for C13H22N2O3 [M+H]+ 255.1703, found 255.1687.
J = 6.7 Hz, 6H). 13C NMR (126 MHz, Chloroform-d)
d 174.44,
165.47, 148.45, 98.12, 36.55, 27.16, 26.48, 22.35. HRMS (ESI) calcd
for C9H14N2O3 [M+H]+ 199.1077, found 199.1086.
6.9. Bioassays
6.9.1. Enzyme inhibition assays
Recombinant WT and G119S AgAChE were prepared as previ-
ously described.12 Recombinant hAChE (C1682) were purchased
from Sigma–Aldrich (St. Louis, MO, USA). Inhibition potency of car-
bamate and carboxamide insecticides was assessed by measuring
apparent second-order rate constants ki (mMÀ1 minÀ1) for inacti-
vation of the enzymes. We followed our previous progressive inac-
tivation approach,8,12 in which enzymes were incubated with
different concentrations of carbamates or carboxamides for differ-
ing times before measuring enzyme residual activity (v/v0).
Enzymes were incubated with typically five concentrations of
inhibitors (and an inhibitor-free control) for up to 6 min at approx-
imately 1 min intervals. Each concentration was present in the
microplate in duplicate, and each experiment was repeated. Note
that for the G119S enzyme inhibition was typically very low after
6.7.8. 5-(Pentan-2-yl)isoxazol-3-yl pyrrolidine-1-carboxylate
(25g)
Prepared using the general procedure above (Method F) from
5-(pentan-2-yl)isoxazol-3-ol (13g) (65.1 mg, 0.42 mmol), 1-pyrr-
olidinecarbonyl chloride (0.23 mL, 2.10 mmol), and toluene
(2.0 mL). Concentration of the reaction mixture in vacuo followed
by silica gel flash chromatography (20–30% ethyl acetate in hex-
ane) yielded 25g as a clear oil (60.4 mg, 57%). 1H NMR (500 MHz,
CDCl3) d 6.17 (s, 1H), 3.56 (t, J = 6.6 Hz, 2H), 3.46 (t, J = 6.6 Hz,
2H), 2.90 (sextet, J = 7.0 Hz, 1H), 2.01–1.84 (m, 4H), 1.74–1.63 (m,
1H), 1.58–1.47 (m, 1H), 1.41–1.28 (m, 2H), 1.26 (d, J = 7.0 Hz,
3H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, CDCl3) d 179.51,
166.67, 150.20, 94.11, 46.80, 46.71, 37.46, 32.97, 25.84, 25.01,
20.21, 18.62, 14.02. HRMS (ESI) calcd for C13H20N2O3 [M+H]+
253.1547, found 253.1528.
10 min at 10 lM, and so incubations with these enzymes were
often extended to 60 min (10 min intervals). Residual activities v/
v0 are the ratio of the rate in the presence of inhibitor to a time-
matched inhibitor-free control and thus correct for slow thermal
inactivation of the enzyme. At each inhibitor concentration [I],
plots of ln(v/v0) versus time (t) were constructed; the slopes of
these unconstrained linear fits represent the pseudo first-order
rate constants (kobs) associated with those inhibitor concentrations
(see Supplementary material, Fig. S2). These kobs values (obtained
in at least two separate experiments) were then plotted against
[I]; in each case the slope of the unconstrained linear fit is the
apparent second-order rate constant ki (mMÀ1 minÀ1) for inactiva-
tion (see Supplementary material, Fig. S3). Note that the error in
kobs is typically smaller than the symbol in these plots (see
Fig. S3). The error in ki is estimated as the standard error in the
slope of this plot. Note that inhibitor concentrations [I] were cho-
sen to be low enough to remain in the domain where a plot of kobs
versus [I] was linear. For fast-inactivating carbamates, such as
6.7.9. 5-(Pentan-2-yl)isoxazol-3-yl morpholine-4-carboxylate
(26g)
Prepared using the general procedure above (Method F) from 5-
(pentan-2-yl)isoxazol-3-ol (13g) (99.4 mg, 0.64 mmol), 4-mor-
pholinecarbamoyl chloride (0.37 mL, 3.20 mmol), and toluene
(3.0 mL). Concentration of the reaction mixture in vacuo followed
by silica gel flash chromatography (20–30% ethyl acetate in hex-
ane) yielded 26g as a clear oil (20.0 mg, 12%). 1H NMR (500 MHz,
CDCl3) d 6.14 (s, 1H), 3.77–3.69 (m, 4H), 3.68–3.64 (m, 2H),

1340
A. Verma et al. / Bioorg. Med. Chem. 23 (2015) 1321–1340
terbam (4) on rAgAChE-WT, we saw signs of saturation behavior
bmc.2015.01.026. These data include MOL files and InChiKeys of
the most important compounds described in this article.
above [I] = 0.2 lM. Saturation behavior is expected for a two-step
mechanism of inhibition.35 Enzymatic sensitivity ratios (SR) and
Ag/h selectivity for inhibition (Tables 3 and 4) were calculated from
the measured ki values; the error in these ratios was determined
using a standard propagation of error method.20
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Acknowledgments
We thank the MR4 as part of the BEI Resources Repository,
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by M. Akogbeto. We thank the NIH for financial support (AI082581).
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Supplementary data (enzyme alignments, details of X-ray crys-
tallography, and calculation of
can be found, in the online version, at http://dx.doi.org/10.1016/j.
s values) associated with this article