Insecticidal quinazoline derivatives with (trifluoromethyl)diazirinyl and azido substituents as NADH:ubiquinone oxidoreductase inhibitors and candidate photoaffinity probes

Article abstract of DOI:10.1021/tx9501602

Two candidate photoaffinity probes are designed from 4-substituted quinazolines known to be potent insecticides/acaricides and NADH:ubiquinone oxidoreductase inhibitors acting at or near the rotenone site. 4-(11- Azidoundecyl-2-amino)quinazoline, based on the undecylamino analog SAN 548A as a prototype, was synthesized in 18% overall yield from ethyl 10- undecenoate by oxidation of the terminal double bond, reductive amination, coupling to 4-chloroquinazoline, and functional group manipulation of the terminal ethyl ester to an alcohol, a mesylate and finally nucleophilic displacement with azide ions. 4-(4-(3-(Trifluoromethyl)-3H-diazirin-3- yl)phenethoxy)quinazoline [the (trifluoromethyl)diazirinyl analog of fenazaquin insecticide/acaricide] was prepared from 4-bromophenethyl alcohol in 31% overall yield by first introducing the trifluoromethylketone moiety followed by its conversion to the (trifluoromethyl)-diazirine and finally coupling to 4-chloroquinazoline as above. Both candidate photoaffinity probes have the inhibitory potency of rotenone (IC₅₀ of 3-4 nM in each case). The azidoundecylamino compound has inadequate photoreactivity whereas that of the (trifluoromethyl)diazirinyl analog is ideal at 350 nm. Radiosynthesis of the latter photoaffinity ligand included introduction of the diazirinyl moiety as the carbene precursor, oxidation of (trifluoromethyl)diazirinylphenethyl alcohol to the corresponding acid with Jones' reagent, and reduction of the phenacetyl chloride intermediate with sodium borotritide to incorporate tritium.

Full text of DOI:10.1021/tx9501602

Chem. Res. Toxicol. 1996, 9, 445-450
445
In secticid a l Qu in a zolin e Der iva tives w ith
(Tr iflu or om eth yl)d ia zir in yl a n d Azid o Su bstitu en ts a s
NADH:Ubiqu in on e Oxid or ed u cta se In h ibitor s a n d
Ca n d id a te P h otoa ffin ity P r obes
Bachir Latli, Edgardo Wood,† and J ohn E. Casida*
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy,
and Management, University of California, Berkeley, California 94720-3112
Received September 15, 1995X
Two candidate photoaffinity probes are designed from 4-substituted quinazolines known to
be potent insecticides/acaricides and NADH:ubiquinone oxidoreductase inhibitors acting at or
near the rotenone site. 4-(11-Azidoundecyl-2-amino)quinazoline, based on the undecylamino
analog SAN 548A as a prototype, was synthesized in 18% overall yield from ethyl 10-
undecenoate by oxidation of the terminal double bond, reductive amination, coupling to
4-chloroquinazoline, and functional group manipulation of the terminal ethyl ester to an alcohol,
a mesylate and finally nucleophilic displacement with azide ions. 4-(4-(3-(Trifluoromethyl)-
3H-diazirin-3-yl)phenethoxy)quinazoline [the (trifluoromethyl)diazirinyl analog of fenazaquin
insecticide/acaricide] was prepared from 4-bromophenethyl alcohol in 31% overall yield by first
introducing the trifluoromethylketone moiety followed by its conversion to the (trifluoromethyl)-
diazirine and finally coupling to 4-chloroquinazoline as above. Both candidate photoaffinity
probes have the inhibitory potency of rotenone (IC50 of 3-4 nM in each case). The
azidoundecylamino compound has inadequate photoreactivity whereas that of the (trifluorom-
ethyl)diazirinyl analog is ideal at 350 nm. Radiosynthesis of the latter photoaffinity ligand
included introduction of the diazirinyl moiety as the carbene precursor, oxidation of (trifluo-
romethyl)diazirinylphenethyl alcohol to the corresponding acid with J ones’ reagent, and
reduction of the phenacetyl chloride intermediate with sodium borotritide to incorporate tritium.
In tr od u ction
Mitochondrial electron transport inhibitors are playing
an increasingly important role in pest control as insec-
ticides, acaricides, and fungicides. The classical inhibitor
is the botanical insecticide rotenone, which was the first
of many compounds shown to act by inhibiting NADH:
ubiquinone oxidoreductase activity (complex I) (EC
1.6.99.3) (1). The inhibition site is partially understood
from radioligand binding studies with [14C]rotenone (2),
[3H]-6′,7′-dihydrorotenone (3), and [14C]piericidin A (4)
F igu r e 1. Structures and designations for alkylaminoquinazo-
lines (1 and 2) and arylethoxyquinazolines (3-5). Compound 1
is SAN 548A, 3 is fenazaquin, and 4 is diazirinylfenazaquin.
and from photoaffinity labeling with [3H]-6′,7′-dihydroro-
tenone (3) and an azidoaryl derivative of 8′-hydroxyro-
tenone (5). These two photoaffinity probes preferentially
label a 33-kDa protein (3, 5), which has not been further
characterized.
Knowledge of complex I with at least 40 protein
subunits is expanding rapidly as to structure and func-
tion (6, 7). There are also a variety of new classes of
NADH:ubiquinone oxidoreductase inhibitors (1, 8-11).
For example, two types of quinazolines have potent
insecticidal activity attributable to the inhibition of
NADH:ubiquinone oxidoreductase activity (1). One is the
candidate alkylaminoquinazoline insecticide 1 (SAN
548A) (1, 9), and the other is the commercial arylethoxy-
quinazoline insecticide/acaricide 3 (fenazaquin) (10, 11)
(Figure 1). They are similar to rotenone in potency as
NADH:ubiquinone oxidoreductase inhibitors and are
proposed to act at the same site in the same way (1).
These quinazolines might serve as prototypes for photo-
affinity probes by preparing analogs that combine high
enzyme inhibitory potency and photoreactivity, providing
they are also suitable for radiosynthesis. The compounds
selected for study are an azido analog (2) of SAN 548A
(1) and the (trifluoromethyl)diazirinyl analog (4) of
fenazaquin (3) (Figure 1). This paper describes the
synthesis of these candidate photoaffinity ligands and
compares them as inhibitors of NADH:ubiquinone oxi-
doreductase activity and as toxicants to house flies,
evaluates their photoreactivity, selects the preferred one,
and details the radiosynthesis.
* To whom correspondence should be addressed,at the Environ-
mental Chemistry and Toxicology Laboratory, Department of Envi-
ronmental Science, Policy and Management, 114 Wellman Hall,
University of California, Berkeley, CA 94720-3112; phone: (510) 642-
5424; fax: (510) 642-6497; e-mail address: ectl@nature.berkeley.edu.
† Present address: C.I.P.E.I.N., CITEFA/CONICET, Zufriategui
4380, 1603-Villa Martelli, Buenos Aires, Argentina.
Exp er im en ta l P r oced u r es
Gen er a l. Synthesis procedures were developed for 4-(11-
azidoundecyl-2-amino)quinazoline (2) (Scheme 1), phenethox-
X Abstract published in Advance ACS Abstracts, February 1, 1996.
0893-228x/96/2709-0445$12.00/0 © 1996 American Chemical Society
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446 Chem. Res. Toxicol., Vol. 9, No. 2, 1996
Latli et al.
Sch em e 1a
Sch em e 3a
a
(a) O2, PdCl2, CuCl2, DMF‚H2O (8:1); (b) NH4NO3,NaCNBH3,
CH3OH; (c) 4-chloroquinazoline, Et3N, EtOH; (d) LiAlH4, THF; (e)
CH3SO2Cl, Et3N, CH2Cl2; (f) LiN3, DMF.
Sch em e 2a
a
(a) tert-BuMe2SiCl, DMAP, Et3N, CH2Cl2; (b) n-BuLi, N-
trifluoroacetyl piperidine, Et2O, -55 oC; (c) five steps (12); (d) NaH,
4-chloroquinazoline, THF, DMF; (e) J ones’ reagent, Me2CO; (f)
ClC(O)C(O)Cl, CH2Cl2; (g) NaB*H4, NaOH, EtOH.
a
(a) Mg, Et2O, dry ice; (b) LiAlH4, Et2O; (c) NaH, 4-chloro-
(Aldrich) (2.2 g, 10.3 mmol), PdCl2 (177 mg, 1.0 mmol), and
CuCl2 (0.99 g, 10 mmol) in dimethylformamide (DMF):H2O (8
mL:1 mL) was stirred under an oxygen atmosphere for 20 h.
Ether was then added, and the mixture was filtered through a
short column of Florisil. The liquid was transferred to a
separatory funnel, washed with water, dried (MgSO4), and
concentrated in vacuo (referred to below as the usual workup).
Purification by flash chromatography on silica gel with ethyl
acetate (EtOAc)/hexane gave 2.16 g (91%) of pure 7 as a colorless
oil, Rf on silica TLC ) 0.20 in 10% EtOAc/hexane (analytical
TLC always refers to 0.25 mm gel thickness, 4 × 8 cm plastic
sheets from Macherey-Nagel, Duren, Germany). 1H NMR
(CDCl3) : 4.04 (q, J ) 7.26 Hz, 2H), 2.34 (t, J ) 7.2 Hz, 2H),
2.20 (t, J ) 7.5 Hz, 2H), 2.05 (s, 3H), 1.53 (m, 4H), 1.22 (br s,
8H), 1.17 (t, J ) 7.26 Hz, 3H). 13C NMR (CDCl3) : 173.6, 153.0,
60.0, 43.5, 34.1, 29.7, 29.0, 28.9, 24.7, 23.8, 23.6, 22.5, 14.1. MS-
EI m/z (rel intensity): M+ (2.7), 125 (100), 55 (91).
(B) Eth yl 10-Am in ou n d eca n oa te (8). To a solution of the
above keto ester (0.45 g, 2.0 mmol) and ammonium nitrate (8.0
g, 10 mmol) in methanol (25 mL) was added sodium cyanoboro-
hydride (100 mg, 1.6 mmol) at 25 °C. The resulting solution
was stirred for 3 days before it was concentrated, diluted with
aqueous Na2CO3, and extracted with ether, followed by the usual
workup. The pure amine was obtained by flash chromatography
on silica gel using CH3OH/CHCl3 as eluent (5-100% solution)
to give 400 mg of 8 (89% yield), silica TLC Rf ) 0.26 in 10%
CH3OH/CHCl3. 1H NMR (CDCl3) : 6.70 (br s, 2H), 4.00 (q, J
) 7.10 Hz, 2H), 3.24 (m, 1H), 2.16 (t, J ) 7.50 Hz, 2H), 1.48-
1.64 (m, 4H), 1.20 (d, J ) 6.60 Hz, 3H), 1.17 (br s, 10H), 1.13 (t,
J ) 7.10 Hz, 3H). 13C NMR (CDCl3) : 173.6, 60.0, 48.1, 42.4,
34.0, 28.9, 28.8, 28.7, 28.5, 25.2, 24.6, 18.1, 14.0. MS-EI: M+
(2.7), 55 (100).
quinazoline, THF, DMF.
yquinazoline (5) and its 4-tert-butyl analog (3) (Scheme 2), and
4-((4-(trifluoromethyl)diazirinyl)phenethoxy)quinazoline (4) in
unlabeled form and with 3H at the O-methylene position ([3H]-
4) (Scheme 3) as detailed below. Several of the new compounds
have photosensitive substituents. They must be used in sub-
dued light or semidarkness to minimize photodecomposition
during synthesis and handling. The purity of each of the
compounds was estimated to be >98% on the basis of TLC and
1
of H and 13C NMR integration.
Sp ectr oscop y. NMR spectra were recorded for CDCl3
solutions with a Bruker AM-300 spectrometer. Chemical shifts
( ) are reported for 1H at 300 MHz and for 13C at 75 MHz
relative to internal tetramethylsilane and CDCl3, respectively.
Mass spectra were acquired by GC/MS with the Hewlett-
Packard 5971A or 5985B instrument in the electron impact (EI)1
mode (70 eV, 200 °C). Fast atom bombardment (FAB)-MS
(both low and high resolution) was conducted with the Fisons
ZAB2-EQ spectrometer.
Syn th esis of 4-(11-Azid ou n d ecyl-2-a m in o)qu in a zolin e
(2) via In ter m ed ia tes 6-11 (Sch em e 1). (A) Eth yl 10-
Oxou n d eca n oa te (7). A mixture of ethyl 10-undecenoate (6)
1
Abbreviations: DMAP, 4-dimethylaminopyridine; DMF, dimeth-
ylformamide; EI, electron impact; EtOAc, ethyl acetate; FAB, fast atom
bombardment; LAH, lithium aluminum hydride; PB, piperonyl butox-
ide; THF, tetrahydrofuran.
2
Although not detailed here, bovine heart mitochondrial electron
transport particles with specifically bound [3H]-4 were irradiated at
350 nm and then fractionated by sodium dodecyl sulfate-polyacryla-
mide gel electrophoresis to give specifically labeled protein(s) that
appeared exclusively in the 22-24-kDa region. Tryptic digestion, HPLC
of the peptides, and amino acid sequence analysis of the single 3H-
labeled peak revealed two peptides, subunit d and the oligomycin
sensitivity conferral protein, both of the ATP synthase stalk (unpub-
lished results of E. Wood, B. Latli, and J . E. Casida).
(C) 4-(10-(E t h oxyca r b on yl)d ecyl-2-a m in o)q u in a zolin e
(9). A solution of 4-chloroquinazoline (10) (361 mg, 2.2 mmol),
the above amine (502 mg, 2.2 mmol), and triethylamine (0.5 mL)
in ethanol (10 mL) was stirred overnight at room temperature.
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Insecticidal Quinazoline Photoaffinity Probes
Chem. Res. Toxicol., Vol. 9, No. 2, 1996 447
Then it was diluted with water and extracted with EtOAc with
the usual workup. The crude product was purified by prepara-
tive silica TLC to give 0.72 g of a white solid (99% yield), silica
TLC Rf ) 0.73 in 10% CH3OH/CHCl3. 1H NMR (CDCl3) : 8.60
(s, 1H), 7.77 (d, J ) 8.42 Hz, 1H), 7.76 (t, J ) 7.67 Hz, 1H),
7.60 (d, J ) 8.42 Hz, 1H), 7.36 (t, J ) 7.67 Hz, 1H), 5.90 (d, J
) 7.40 Hz, 1H), 4.40 (m, 1H), 4.10 (q, J ) 7.10 Hz, 2H), 2.20 (t,
J ) 7.40 Hz, 2H), 1.55 (m, 4H), 1.24 (d, J ) 6.50 Hz, 3H), 1.20
(br s, 10H), 1.17 (t, J ) 7.10 Hz, 3H). 13C NMR (CDCl3) : 173.7,
158.9, 155.3, 149.2, 132.2, 128.2, 125.6, 120.6, 114.9, 60.0, 46.6,
36.7, 34.2, 29.3, 29.2, 29.0, 28.9, 25.9, 24.8, 20.6, 14.1. MS-EI:
M+ (14.1), 145 (100).
J ) 8.21 Hz, 2H), 3.16 (s, 2H), 1.30 (s, 9H). 13C NMR (CDCl3)
: 176.9, 149.6, 128.8, 125.2, 40.4, 34.2, 31.1.
(B) 4-ter t-Bu tylp h en eth yl Alcoh ol (14). To a suspension
of LAH (156 mg, 4.0 mmol) in dry ether (15 mL), the above acid
(0.50 g, 2.6 mmol) was added dropwise in dry ether (10 mL).
The reaction was warmed to 25 °C and stirred for 4 h. Water
(0.16 mL), NaOH (15% solution, 0.16 mL), and water (0.48 mL)
were added successively, and the precipitate was filtered,
washed with ether, and concentrated in vacuo to give 0.45 g of
crude material. Purification by silica flash chromatography (as
for 7) gave 0.33 g of pure alcohol (72% yield), silica TLC Rf )
0.20 in 20% EtOAc/hexane. 1H NMR (CDCl3) : 7.31 (d, J )
8.21 Hz, 2H), 7.13 (d, J ) 8.21 Hz, 2H), 3.77 (t, J ) 6.70 Hz,
2H), 2.79 (t, J ) 6.70 Hz, 2H), 1.30 (s, 9H). 13C NMR (CDCl3)
: 149.1, 135.3, 128.6, 125.3, 63.5, 38.5, 34.3, 31.3.
(D) 4-(11-Hyd r oxyu n d ecyl-2-a m in o)qu in a zolin e (10). To
a suspension of lithium aluminum hydride (LAH) (10 mg, 0.26
mmol) in dry THF (1.0 mL) at 0 °C was added the above ester
(45 mg, 0.13 mmol) in THF (1.0 mL). The resulting mixture
was stirred for 1 h, then warmed to room temperature, and
stirred for an additional 2 h. A solution of NaOH (1.0 N, 2 mL)
was added, followed by extraction with CH2Cl2 and workup in
the usual manner, and then purification by preparative silica
TLC to give 25 mg of 10 (59% yield), Rf ) 0.33 in 10% CH3OH/
CHCl3. 1H NMR (CDCl3) : 8.60 (s, 1H), 7.70 (m, 3H), 7.40 (t,
J ) 7.67 Hz, 1H), 5.80 (d, J ) 7.40 Hz, 1H), 4.40 (m, 1H), 3.60
(t, J ) 7.20 Hz, 2H), 1.60 (m, 6H), 1.23 (d, J ) 6.50 Hz, 3H),
1.20 (br s, 10H). 13C NMR (CDCl3) : 158.9, 155.2, 148.9, 132.5,
128.0, 125.8, 120.6, 115.1, 62.4, 36.7, 32.6, 29.8, 29.5, 29.3, 28.6,
26.0, 25.6, 24.7, 21.0.
(C) 4-(4-ter t-Bu tylp h en eth oxy)qu in a zolin e (3). To a
suspension of NaH (97%, 56 mg, 2.25 mmol) in dry THF (2.0
mL) was added the above alcohol (200 mg, 1.12 mmol) dropwise
in THF (2.0 mL), and the mixture was stirred at room temper-
ature for 1 h. 4-Chloroquinazoline (195 mg, 1.12 mmol) in DMF
(3.0 mL) was added dropwise at 25 °C. The resulting yellow
mixture was stirred overnight under nitrogen. Water was added
followed by extraction with CH2Cl2 and the usual workup to
give 250 mg of crude product. Purification by preparative TLC
on silica gel gave 200 mg of pure product (58% yield), silica TLC
Rf ) 0.27 in 20% EtOAc/hexane. 1H NMR (CDCl3) : 8.78 (s,
1H), 8.13 (d, J ) 8.20 Hz, 1H), 7.91 (d, J ) 8.20 Hz, 1H), 7.78
(t, J ) 7.60 Hz, 1H), 7.52 (t, J ) 7.60 Hz, 1H), 7.35 (d, J ) 8.20
Hz, 2H), 7.26 (d, J ) 8.20 Hz, 2H), 4.76 (t, J ) 6.80 Hz, 2H),
(E) 4-(11-Mesyloxyu n d ecyl-2-a m in o)qu in a zolin e (11).
To a solution of 10 (60 mg, 0.19 mmol) and triethylamine (50
mg, 0.5 mmol) in dry CH2Cl2 (3.0 mL), stirred at 0 °C under
nitrogen, was added methanesulfonyl chloride (20 L, 0.26
mmol). The resulting mixture was stirred for 1 h and then
warmed to 25 °C. Water was added, and the mixture was
extracted with CH2Cl2, followed by the usual workup and
preparative TLC on silica gel to give 50 mg of 11 (67% yield),
Rf ) 0.50 in 10% CH3OH/CHCl3. 1H NMR (CDCl3) : 8.60 (s,
1H), 7.70 (m, 3H), 7.40 (t, J ) 7.67 Hz, 1H), 5.80 (d, J ) 7.40
Hz, 1H), 4.46 (m, 1H), 4.16 (t, J ) 6.57 Hz, 2H), 1.60 (m, 6H),
3.77 (t, J ) 6.80 Hz, 2H), 1.31 (s, 9H). 13C NMR (CDCl3)
:
166.5, 154.3, 150.8, 149.3, 134.7, 133.3, 128.6, 127.5, 126.8,
125.3, 123.4, 116.6, 67.6, 34.6, 34.3, 31.3. MS: low resolution
FAB: MH+ (100); high resolution FAB: MH+ calcd 307.18104;
found 307.18077.
(D) 4-(P h en eth oxy)qu in a zolin e (5). Phenethyl alcohol
(15) (Aldrich) (100 mg, 0.82 mmol) was added dropwise in dry
THF (1.0 mL) to a suspension of NaH (41 mg, 1.64 mmol) in
THF (1.0 mL) at 0 °C. After stirring for 1 h at room tempera-
ture, 4-chloroquinazoline (135 mg, 0.82 mmol) in dry DMF (2.0
mL) was added dropwise. The reaction mixture was stirred
overnight with workup as for 3 to give 100 mg (49% yield) of
the desired product, silica TLC Rf ) 0.83 in 10% CH3OH/CHCl3.
1H NMR (CDCl3) : 8.68 (s, 1H), 8.02 (d, J ) 8.31 Hz, 1H), 7.81
(d, J ) 8.31 Hz, 1H), 7.70 (m, 1H), 7.46 (m, 1H), 7.12-7.24 (m,
5H), 4.68 (t, J ) 6.91 Hz, 2H), 3.11 (t, J ) 6.91 Hz, 2H). 13C
NMR (CDCl3) : 166.5, 154.2, 150.8, 137.8, 133.4, 128.9, 128.4,
127.6, 126.9, 126.5, 123.4, 116.6, 67.5, 35.2. MS: low resolution
FAB: MH+ (100); high resolution FAB: MH+ calcd 251.11844;
found 251.11841.
1.30 (d, J ) 6.50 Hz, 3H), 1.26 (br s, 10H). 13C NMR (CDCl3)
:
158.8, 155.3, 148.9, 132.2, 128.2, 125.6, 120.3, 110.7, 70.2, 46.6,
36.7, 32.8, 29.3, 29.1, 28.9, 28.7, 25.9, 25.2, 24.3, 20.5.
(F ) 4-(11-Azid ou n d ecyl-2-a m in o)qu in a zolin e (2). The
above mesylate (40 mg, 0.10 mmol) in dry DMF (2.0 mL) was
stirred with LiN3 (25 mg, 0.50 mmol) at room temperature under
nitrogen overnight. Water was added, and the organic phase
was extracted with CH2Cl2 followed by the usual workup and
preparative TLC to give 20 mg of pure azide (58% yield), silica
TLC Rf ) 0.68 in 10% CH3OH/CHCl3. 1H NMR (CDCl3) : 8.65
(s, 1H), 7.83 (d, J ) 8.42 Hz, 1H), 7.74 (t, J ) 7.67 Hz, 1H),
7.67 (d, J ) 8.42 Hz, 1H), 7.46 (t, J ) 7.67 Hz, 1H), 5.70 (d, J
) 7.40 Hz, 1H), 4.47 (m, 1H), 3.23 (t, J ) 6.91 Hz, 2H), 1.60 (m,
4H), 1.30 (d, J ) 6.56 Hz, 3H), 1.28 (br s, 10H). 13C NMR
(CDCl3) : 158.9, 155.2, 148.8, 132.5, 128.1, 125.9, 120.5, 58.3,
51.4, 46.8, 36.8, 32.2, 29.5, 29.3, 29.0, 28.8, 26.6, 26.0, 20.7. FT-
IR (KBr pellet) (cm-1): 3407, 3300, 2931, 2858, 2092 (N3), 1574,
Syn t h esis of 4-((4-(Tr iflu or om et h yl)d ia zir in yl)p h en -
eth oxy)qu in a zolin e (4) via In ter m ed ia tes 16-19 (Sch em e
3). (A) 4-Br om op h en eth oxy-ter t-bu tyld im eth ylsila n e (17).
A solution of 2-(4-bromophenyl)ethanol (16) (Aldrich) (7.0 g, 35
mmol), 4-dimethylaminopyridine (DMAP) (117 mg, 0.95 mmol),
and triethylamine (7.0 g, 70 mmol) in anhydrous CH2Cl2 (50
mL) was stirred at 0 °C under nitrogen. tert-Butyldimethyl-
chlorosilane (5.23 g, 35 mmol) was added in portions, and the
reaction mixture was stirred overnight. Hexane was added, and
the precipitate was filtered and washed thoroughly with hexane,
and the filtrate was concentrated in vacuo to give a colorless
oil that was purified by flash chromatography (silica gel treated
with triethylamine and developed with EtOAc/hexane) to afford
9.8 g (89% yield) of pure product, silica TLC Rf ) 0.66 in 10%
EtOAc/hexane. 1H NMR (CDCl3) : 7.40 (d, J ) 7.80 Hz, 2H),
7.10 (d, J ) 7.80 Hz, 2H), 3.80 (t, J ) 6.74 Hz, 2H), 2.78 (t, J )
1534, 1498, 1256, 1324, 1121. UV (ethanol)
) 218, ꢀ )
max
9445. MS-EI: M+ (0.5), 145 (100). Low resolution FAB: MH+
(40), 146 (100); high resolution FAB: MH+ calcd 341.24528;
found 341.24537.
Syn th esis of 4-P h en eth oxyqu in a zolin e (5) a n d Its 4′-
ter t-Bu tyl An a log (3) via 12-15 (Sch em e 2). (A) 4-ter t-
Bu tylp h en yla cetic Acid (13). tert-Butylbenzyl bromide (12)
(Aldrich) (5.0 g, 22 mmol) in dry ether (20 mL) was added
dropwise to Mg turnings (0.54 g, 22 mmol). The Grignard
reaction was initiated with a few iodine crystals. After the
addition was completed, ca. 3 h, the mixture was refluxed
overnight under nitrogen atmosphere. The cooled solution was
then added dropwise to a suspension of dry ice in ether. Stirring
was continued for 3 h and then water was added followed by
extraction with ether and the usual workup to give 3.4 g of crude
material. Purification by silica column chromatography gave
1.2 g (28% yield) of 13, silica TLC Rf ) 0.17 in 20% EtOAc/
hexane. 1H NMR (CDCl3) : 7.34 (d, J ) 8.21 Hz, 2H), 7.21 (d,
6.74 Hz, 2H), 0.89 (s, 9H), 0.1 (s, 6H). 13C NMR (CDCl3)
:
152.3, 143.3, 138.3, 131.2, 130.9, 119.9, 64.0, 38.9, 25.9, 18.3,
-5.4.
(B) 4-(Tr iflu or oa cetyl)p h en eth oxy-ter t-bu tyld im eth yl-
sila n e (18) a n d 4-((Tr iflu or om eth yl)d ia zir in yl)p h en eth yl
Alcoh ol (19). The above compound 17 (0.95 g, 3.0 mmol) in
dry ether (20 mL) was stirred at -55 to -50 °C (acetonitrile/
dry ice bath) under nitrogen. n-Butyl lithium (1.6 M solution
+
+

448 Chem. Res. Toxicol., Vol. 9, No. 2, 1996
Latli et al.
in hexane, 2.25 mL, 3.6 mmol) was added dropwise, and the
mixture was warmed gradually to 0 °C and stirred for 2 h, then
cooled again to -50 °C, and N-trifluoroacetylpiperidine (made
freshly from piperidine and trifluoroacetic anhydride) (0.71 g,
3.9 mmol) in dry ether (4.0 mL) was added dropwise. The
reaction was stirred for 3 h before it was quenched at -50 °C
with a saturated solution of NH4Cl (15 mL). The mixture was
diluted with EtOAc (20 mL) and washed with aqueous NH4Cl
(20 mL twice and H2O, dried (MgSO4), filtered, and concentrated
in vacuo to give 1.35 g of crude oil. Purification by column
chromatography (silica gel treated with triethylamine) using
10-50% EtOAc/hexane as eluent gave 0.96 g (96% yield) of a
yellowish oil, silica with triethylamine TLC Rf ) 0.53 in 10%
EtOAc/hexane. 1H NMR (CDCl3) : 8.02 (d, J ) 8.01 Hz, 2H),
7.42 (d, J ) 8.01 Hz, 2H), 3.89 (t, J ) 6.42 Hz, 2H), 2.93 (t, J )
6.42 Hz, 2H), 0.91 (s, 9H), 0.10 (s, 6H). 13C NMR (CDCl3)
:
152.1, 149.8, 130.0, 129.1, 128.1, 127.0, 126.0, 63.4, 39.6, 25.8,
18.2, -5.6. Silane 18 was converted to the corresponding
phenethyl alcohol (19) in a five-step procedure according to the
literature (12).
(C)
4-((4-(Tr iflu or om et h yl)d ia zir in yl)p h en et h oxy)-
qu in a zolin e (4). A suspension of NaH (6.5 mg, 0.26 mmol) in
dry THF (2.0 mL) was stirred at 0 °C. Phenethyl alcohol
derivative 19 (30 mg, 0.13 mmol) in dry THF (1.0 mL) was added
dropwise. The resulting mixture was stirred for 1 h before
4-chloroquinazoline (26 mg, 0.15 mmol) was added in dry DMF
(1.0 mL). Stirring was continued overnight and then water was
added and extracted with CH2Cl2, dried (MgSO4), filtered, and
concentrated in vacuo. Preparative TLC purification on silica
gel gave 34 mg of a solid product (73% yield), silica TLC Rf )
0.70 in 10% CH3OH/CHCl3. 1H NMR (CDCl3) : 8.78 (s, 1H),
8.09 (d, J ) 8.27 Hz, 1H), 7.93 (d, J ) 8.27 Hz, 1H), 7.84 (m,
1H), 7.57 (m, 1H), 7.37 (d, J ) 8.10 Hz, 2H), 7.16 (d, J ) 8.10
F igu r e 2. Tritium NMR of [3H]-4. IBM AF 300 spectrometer
at 320 MHz using CDCl3 and 5-mm probe. No other signals are
observed.
Filtration through glass wool packed in a pasteur pipet and
lyophilization gave a yellowish oil that was dissolved in dry THF
(1.0 mL). NaH (10 mg) was added, resulting in gas production.
The mixture was stirred for 1 h, then 4-chloroquinazoline (12
mg) dissolved in DMF (1.0 mL) was added dropwise, and the
dark mixture was stirred overnight. Water was then added,
and the organics were extracted with CH2Cl2, dried (MgSO4),
filtered, frozen at liquid nitrogen temperature, and lyophilized
to dryness. The solid residue was dissolved in CH2Cl2 and
purified by preparative silica gel TLC, Rf ) 0.70 in 10% CH3-
OH/CHCl3. The band corresponding to the product was ex-
tracted with CH3OH and lyophilized as above. A second
purification by HPLC involved the HP 1040A instrument
equipped with diode array UV and -ram 3H detectors and a
reverse-phase preparative column (Supelco 0.5 × 20 cm, LC 18
DB) monitoring at 260 nm, resulting in 8 mCi of pure product
(>98% radiochemical purity) with a specific activity of 4.7 Ci/
mmol as calculated from HPLC using a standard solution of cold
product, tR ) 6.5 min, 8% H2O/92% CH3OH with a flow of 3.0
mL/min. The purity of the product was confirmed again by silica
gel 2D TLC, Rf ) 0.70 in 10% CH3OH/CHCl3 and Rf ) 0.40 in
Hz, 2H), 4.78 (t, J ) 6.72 Hz, 2H), 3.23 (t, J ) 6.72 Hz, 2H). 13
C
NMR (CDCl3) : 166.4, 154.2, 151.0, 140.0, 133.6, 129.5, 127.7,
127.5, 127.1, 126.9, 126.7, 124.0, 123.3, 116.5, 67.0, 34.9. UV
(ethanol):
and ꢀ, respectively, 360 and 398, 310 and 3023,
max
298 and 2992, 264 and 4990, 236 and 6488. MS: low resolution
FAB: MH+ (100); high resolution FAB: MH+ calcd 359.11133;
found 359.11197.
Syn t h esis of [O-3H-m eth ylen e]4-((4-(Tr iflu or om et h yl)-
d ia zir in yl)p h en eth oxy)qu in a zolin e ([3H]-4) via In ter m e-
d ia t es 19-22. (A) 4-((Tr iflu or om et h yl)d ia zir in yl)p h e-
n yla cetic a cid (20). To a solution of the alcohol (19) (200 mg,
0.87 mmol) in acetone (15 mL) was added dropwise a solution
of J ones’ reagent (1.0 mL, 5.0 M solution), and the mixture was
stirred for 30 min at room temperature. Ethanol (2.0 mL) was
then added, and the resulting green mixture was concentrated
in vacuo. The residue was dissolved in water and extracted with
EtOAc, dried (MgSO4), filtered, and concentrated to give 184
mg of pure acid as a white solid (87% yield), silica TLC Rf )
0.18 in 20% EtOAc/hexane. 1H NMR (CDCl3) : 10.54 (s, 1H),
7.30 (d, J ) 8.21 Hz, 2H), 7.14 (d, J ) 8.21 Hz, 2H), 3.65 (s,
1
50% THF/hexane. 3H NMR (CD3OD) H-decoupled : 4.82 (d,
J ) 8.53 Hz); 3H NMR (CD3OD) 1H-coupled : 4.82 (m) (Figure
2).
Assa y a s In h ibitor s of NADH:Ubiqu in on e Oxid or ed u c-
ta se Activity (13). NADH:ubiquinone oxidoreductase activity
was assayed in UV semimicro-cuvettes (1.5 mL) by adding buffer
(0.25 M sucrose and 50 mM potassium phosphate at pH 7.6)
(976 L) followed by electron transport particles [prepared by
the standard procedure of Crane et al. (14)] (40 g of protein
(15) in 4 L of buffer) and then ethanol (10 L) alone (control)
or with a candidate inhibitor at various concentrations. After
5-min preincubation at 25 °C, residual enzyme activity was
determined by adding a solution of NADH in buffer (10 L, final
concentration 0.28 mM) and immediately and continuously
monitoring the loss of absorbance at 340 nm for 3 min at 25 °C
with a Hewlett Packard 8452A diode array spectrophotometer.
The slope of the line was compared for a sample with inhibitor
to that for the control without inhibitor to obtain the percent
inhibition. At least five concentrations were used in each case,
which gave 10-90% inhibition of activity.
Assa y for Toxicity to Hou se F lies. Adult female house
flies (Musca domestica L.) were treated topically on the ventrum
of the abdomen with the test compound in acetone (0.22 L).
They were also assayed with the microsomal oxidase inhibitor
piperonyl butoxide (PB) applied topically at 250 g/g to the
abdomen 1 h prior to the test compound. Mortality was
determined 24 h after treatment.
2H). 13C NMR (CDCl3)
: 176.9, 135.0, 130.4, 129.8, 126.7,
126.0, 123.9, 120.2, 40.6, 28.0 (m, CF3).
(B) 4-((Tr iflu or om eth yl)d ia zir in yl)p h en a cetyl Ch lor id e
(21). The above acid (184 mg, 0.75 mmol) was dissolved in dry
CH2Cl2 (10 mL), and oxalyl chloride (1.3 mL, 15 mmol) was
added at 25 °C. Stirring was continued overnight under
nitrogen before it was concentrated in vacuo to give 200 mg of
an oil (100% yield), silica TLC Rf ) 0.66 in 20% EtOAc/hexane.
1H NMR (CDCl3) : 7.30 (d, J ) 7.21 Hz, 2H), 7.19 (d, J ) 8.21
Hz, 2H), 4.15 (s, 2H). 13C NMR (CDCl3) : 171.3, 132.9, 130.0,
129.3, 127.0, 126.6, 123.8, 120.2, 52.4, 28 (m, CF3).
(C) [3H]-4 via [O-3H-m eth ylen e]4-((Tr iflu or om eth yl)d ia z-
ir in yl)p h en eth yl Alcoh ol (22). To a suspension of NaB3H4
(1 Ci, 70 Ci/mmol, Amersham) in absolute ethanol (1.0 mL) and
NaOH (0.01 N solution, 50 L) was added dropwise, via a
syringe, acyl chloride 21 (20 L, ≈0.08 mmol). The mixture was
stirred in the dark for 3 h before the addition of EtOAc (5 mL)
followed by a solution of NaH2PO4 (1.0 M solution, 0.3 mL).
Stirring was continued for 10 min, then MgSO4 (1.5 g) was
added, and the mixture was stirred vigorously for 15 min.
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Insecticidal Quinazoline Photoaffinity Probes
Chem. Res. Toxicol., Vol. 9, No. 2, 1996 449
Ta ble 1. P oten cy of 4-Su bstitu ted Qu in a zolin es a n d
Roten on e a s In h ibitor s of NADH:Ubiqu in on e
Oxid or ed u cta se a n d Toxica n ts to Hou se F lies Alon e a n d
Syn er gized w ith P B
Resu lts a n d Discu ssion
Syn th esis. Azidoalkylaminoquinazoline 2 was mod-
eled on 4-(undecyl-2-amino)quinazoline (1) as a potent
NADH:ubiquinone oxidoreductase inhibitor (16) and on
5′-azido-∆8-tetrahydrocannabinol with a 5-azidopentyl
substituent as a photoaffinity probe (17). Azide 2 was
prepared in a six-step synthesis (Scheme 1) in 18%
overall yield starting from ethyl 10-undecenoate (6),
which was oxidized to ethyl 10-oxoundecanoate (7) by
oxygen, PdCl2, and CuCl2 in DMF:water mixture (18).
The amine (8) was obtained from treatment of the ketone
(7) with ammonium nitrate and sodium cyanoborohy-
dride in methanol (19) and finally coupled to 4-chloro-
quinazoline to obtain 9. LAH reduction to alcohol 10
followed by mesylation to 11 and displacement with azide
in DMF gave the azido compound (2).
house fly LD50 ( g/g)
compd
IC50 (nM ( SD)
-PB
+PB
1
2
3
4
5
3.8 ( 0.0
3.9 ( 0.1
4.0 ( 0.0
2.8 ( 0.3
32 ( 1
38
25
500
34
44
193
565
>500 (20%)a
420
>500 (20%)a
375
>500 (6%)
rotenone
4.4 ( 0.4
a
Percent mortality at 500 g/g.
Diazirinylfenazaquin (4) was modeled on fenazaquin
(3), which has high enzyme inhibitory potency (16), by
replacing the tert-butyl moiety with (trifluoromethyl)-
diazirinyl, a preferred photoreactive substituent (20, 21).
Fenazaquin (3) and des-tert-butylfenazaquin (5) were also
prepared for method development and structure-activity
considerations. Fenazaquin was produced in three steps
in 12% overall yield from 4-tert-butylbenzyl bromide (12)
(Scheme 2) via the Grignard reagent to give the acid (13),
using commercial dry ice, followed by LAH reduction to
alcohol 14 and coupling to 4-chloroquinazoline using NaH
in THF/DMF mixture. Phenethyl alcohol (15) was used
in a similar coupling reaction to obtain des-tert-bu-
tylfenazaquin (5) (Scheme 2).
An alternative procedure (Scheme 3) was used for
synthesis of the candidate photoaffinity analog (4) in
unlabeled form in eight steps and 31% overall yield
starting from 4-bromophenethyl alcohol (16), which was
first protected as the silyl ether (17) via the silyl chloride
in the presence of DMAP. The trifluoromethylketone
moiety was then introduced (18) (20) followed by conver-
sion in five steps to the (trifluoromethyl)diazirine (19)
(12), which was coupled to 4-chloroquinazoline as before.
To prepare [3H]-4 using sodium borotritide (22), com-
pound 19 was oxidized with J ones’ reagent in acetone to
the acid (20) and then converted to the acyl chloride
derivative (21). The (trifluoromethyl)diazirinyl substitu-
ent is stable to relatively harsh conditions (23-30), which
fortuitously includes treatment with J ones’ reagent at
room temperature. The acyl chloride was then reduced
with sodium borotritide (specific activity ) ∼70 Ci/mmol,
Amersham) in ethanol. However, this approach gave 22
in disappointingly low yield and specific activity probably
due to inadequate exclusion of moisture from the reac-
tion. Nevertheless, the alcohol obtained (22) was coupled
to 4-chloroquinazoline and purified by preparative TLC
first and then by HPLC to give the desired product at
4.7 Ci/mmol with >98% radioactive purity.
F igu r e 3. Photodecomposition of 4 in ethanol (0.5 mM).
activity can be associated with a higher IC50 in the case
of arylethoxyquinazoline 5, lacking the important tert-
butyl group, but rotenone is of lower toxicity despite its
high inhibitory activity. This type of correlation is
limited by comparisons of data for an insect with a
mammalian enzyme, i.e., a different organism and in vivo
versus in vitro. In any case, although there are obvious
spatial and electronic differences between azidomethyl
versus methyl and (trifluoromethyl)diazirinyl versus tert-
butyl substituents, it is clear that the candidate photo-
probes probably fit the binding site in the same way as
their optimized insecticidal counterparts.
P h ot or ea ct ivit y of Ca n d id a t e P h ot oa ffin it y
P r obes. Azidoalkylaminoquinazoline 2 at 0.15 mM in
ethanol is surprisingly stable with a half-life of more than
30 min on exposure at a distance of 3 cm from four 254-
nm lamps in a Rayonet photochemical reactor (The
Southern New England Ultraviolet Co., Hamden, CT).
(Trifluoromethyl)diazirinyl compound 4 is ideal in its
photoreactivity with a half-life at 0.5 mM in ethanol of
less than 1 min on comparable exposure at 350 nm
(Figure 3). The (trifluoromethyl)diazirinyl moiety is not
only reactive under conditions that minimize photodam-
age to proteins when irradiating but also is ideally located
in the para position for covalent reaction with an amino
acid(s) in the binding site. Another advantage of the
(trifluoromethyl)diazirinyl moiety is that the resulting
carbene can insert in even very hydrophobic regions of a
peptide chain, in contrast to many photolabels that
require a biological nucleophile in close proximity.
NADH:Ubiqu in on e Oxidor edu ctase In h ibition an d
In secticid a l Activity (Ta ble 1). The candidate pho-
toaffinity probes 2 and 4 are almost identical in potency
to their prototypes 1 and 3, respectively, and to rotenone
as inhibitors of NADH:ubiquinone oxidoreductase with
IC50 values of 3-4 nM in all cases. The alkylamino-
quinazoline 1 and the arylethoxyquinazolines 3 and 4 are
of essentially the same toxicity to house flies in the
presence of PB as a cytochrome P450-dependent oxidase
inhibitor, but only the latter two compounds appear to
undergo rapid oxidative detoxification, i.e., greatly in-
creased in toxicity with the synergist. Lower insecticidal
Ack n ow led gm en t. We thank our laboratory col-
leagues Gary Quistad, Richard Staub, Pauline Yu, Nor-
man Birchfield, and J eong-Han Kim for advice and
assistance. The radiosynthesis was carried out in col-
laboration with Hiromi Morimoto and Phillip G. Williams
of the National Tritium Labelling Facility (Lawrence
Berkeley National Laboratory, University of California,
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450 Chem. Res. Toxicol., Vol. 9, No. 2, 1996
Latli et al.
Methods of Biochemical Analysis, Vol. 22 (Glick, D., Ed.), pp 123-
175, J ohn Wiley, New York.
(14) Crane, F. L., Glenn, J . L., and Green, D. E. (1956) Studies on the
electron transfer system. IV. The electron transfer particle.
Biochim. Biophys. Acta 22, 475-487.
(15) Bradford, M. M. (1976) A rapid and sensitive method for the
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Biochem. Soc. Trans. 22, 230-234.
Berkeley), which is supported by NIH Grant RR 01237,
Division of Research Resources. We thank Matthew
Krueger (Molecular Biology Division, Veterans Admin-
istration Medical Center, San Francisco, CA) for the
initial assay on the first candidate photoaffinity probe.
The project described was supported by grants from
CITEFA and CONICET, Argentina, to E.W. and by Grant
P01 ES00049 from the National Institute of Environ-
mental Health Sciences, NIH, to J .E.C.
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