Substituted isoquinolines and quinazolines as potential antiinflammatory agents. Synthesis and biological evaluation of inhibitors of tumor necrosis factor α

Article abstract of DOI:10.1021/jm9805900

A series of isoquinolin-1-ones and quinazolin-4-ones and related derivatives were prepared and evaluated for their ability to inhibit tumor necrosis factor α (TNFα) production in human peripheral blood monocytes stimulated with bacterial lipopolysaccharide (LPS). In an effort to optimize the TNFα inhibitory activity, a homologous series of N-alkanoic acid esters was prepared. Several electrophilic and nucleophilic substitutions were also carried out. Alkanoic acid esters of four carbons were found to be optimum for activity in both the isoquinoline and quinazoline series. Ring substituents such as fluoro, bromo, nitro, acetyl, and aminomethyl on the isoquinoline ring resulted in a significant loss of activity. Likewise, similar groups on the quinazoline ring also reduced inhibitory activity. However, the 6- and 7-aminoquinazoline derivatives, 75 and 76, were potent inhibitors, with IC₅₀ values in the TNFα in vitro assay of approximately 5 μM for each. An in vivo mouse model of pulmonary inflammation was then used to evaluate promising candidate compounds identified in the primary in vitro assay. Compound 75 was selected for further study in this inhalation model, and was found to reduce the level of TNFα in brochoalveolar lavage fluid of LPS-treated mice by about 50% that of control mice. Thus, compounds such as 75, which can effectively inhibit proinflammatory cytokines such as TNFα in clinically relevant animal models of inflammation and fibrosis, may have potential as new antiinflammatory agents. Finally, a quinazoline derivative suitable to serve as a photoaffinity radiolabeled compound was prepared to help identify the putative cellular target(s) for these TNFα inhibitors.

Full text of DOI:10.1021/jm9805900

3
860
J . Med. Chem. 1999, 42, 3860-3873
Su bstitu ted Isoqu in olin es a n d Qu in a zolin es a s P oten tia l An tiin fla m m a tor y
Agen ts. Syn th esis a n d Biologica l Eva lu a tion of In h ibitor s of Tu m or Necr osis
F a ctor r
Qi Chao, Lynn Deng, Hsiencheng Shih, Lorenzo M. Leoni, Davide Genini, Dennis A. Carson, and
Howard B. Cottam*
Department of Medicine and The Sam and Rose Stein Institute for Research on Aging, University of California,
San Diego, La J olla, California 92093-0663
Received October 16, 1998
A series of isoquinolin-1-ones and quinazolin-4-ones and related derivatives were prepared
and evaluated for their ability to inhibit tumor necrosis factor R (TNFR) production in human
peripheral blood monocytes stimulated with bacterial lipopolysaccharide (LPS). In an effort to
optimize the TNFR inhibitory activity, a homologous series of N-alkanoic acid esters was
prepared. Several electrophilic and nucleophilic substitutions were also carried out. Alkanoic
acid esters of four carbons were found to be optimum for activity in both the isoquinoline and
quinazoline series. Ring substituents such as fluoro, bromo, nitro, acetyl, and aminomethyl on
the isoquinoline ring resulted in a significant loss of activity. Likewise, similar groups on the
quinazoline ring also reduced inhibitory activity. However, the 6- and 7-aminoquinazoline
derivatives, 75 and 76, were potent inhibitors, with IC50 values in the TNFR in vitro assay of
approximately 5 µM for each. An in vivo mouse model of pulmonary inflammation was then
used to evaluate promising candidate compounds identified in the primary in vitro assay.
Compound 75 was selected for further study in this inhalation model, and was found to reduce
the level of TNFR in brochoalveolar lavage fluid of LPS-treated mice by about 50% that of
control mice. Thus, compounds such as 75, which can effectively inhibit proinflammatory
cytokines such as TNFR in clinically relevant animal models of inflammation and fibrosis,
may have potential as new antiinflammatory agents. Finally, a quinazoline derivative suitable
to serve as a photoaffinity radiolabeled compound was prepared to help identify the putative
cellular target(s) for these TNFR inhibitors.
In tr od u ction
factor kappa B activation, and various agents whose
molecular targets remain undefined, such as gold salts
and penicillamine.
The modulation of the biosynthesis or action of
proinflammatory cytokines (PICs) such as tumor necro-
sis factor R (TNFR) and interleukin 1 (IL-1) has become
an important strategy for pharmacological intervention
in a variety of inflammatory and fibrotic disease states.
Indeed, TNFR and IL-1, among other cytokines, are
believed to be involved in the pathogenesis of such
diverse human chronic diseases as rheumatoid arthri-
Recently, we reported a series of pentoxifylline me-
tabolite analogues, the most active of which were shown
to be potent inhibitors of TNFR production in human
peripheral blood monocytes stimulated with bacterial
5
lipopolysaccharide (LPS). A structure-activity com-
parison of these xanthine-like compounds in four dif-
ferent ring systems revealed that the pteridine system
was the most promising, providing good TNF inhibitory
activity without concomitant PDE inhibition or cyto-
toxicity. The most active derivative in the pteridine
system was found to be the disubstituted pteridinedione
1
2
3
tis, psoriasis, inflammatory bowel disease, chronic
4
bronchitis, and cystic fibrosis. Thus, inhibition of these
PICs may provide the basis for effective therapeutic
treatment of these inflammatory disorders. Reported
strategies for inhibition of these cytokines have included
receptor antagonism, soluble receptor, neutralizing
monoclonal antibodies, antiinflammatory cytokines, and
cytokine synthesis inhibitors. The latter strategy has
received considerable attention recently with regard to
antiinflammatory drug research since this approach is
most amenable to small molecule design. The low
molecular weight PIC biosynthesis inhibitors encompass
such diverse agents as the glucocorticoids, which inhibit
transcription of PICs, the cyclic AMP phosphodiesterase
1
which inhibited production of TNFR at an IC50 of less
than 5 µM in monocytes and was also active in an in
vivo LPS-induced leukopenia model in mice.
(PDE) inhibitors, which elevate cAMP levels in cells, the
cytokine-suppressive antiinflammatory drugs (CSAIDS),
which inhibit stress-activated protein kinases such as
p38 kinase, protease inhibitors, which block the pro-
cessing of TNF and IL-1 precursors, inhibitors of nuclear
In an effort to obtain more potent TNFR inhibitors, a
collection of commercially available heterocycles was
acquired, each having a 6-6 membered fused ring
1
0.1021/jm9805900 CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/27/1999

Synthesis of TNFR Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3861
Sch em e 1a
a
Reagents: (a) Br(CH2)nCo2Et, NaH, DMF; (b) 1 N NaOH, reflux; (c) 1. SOCl2, CH2Cl2: 2. 4-(2-hydroxyethyl)morpholine, CH2Cl2, 3.
HCl.
system containing one nitrogen atom or two nitrogen
demethylation of 13 using the same conditions as
described above to obtain compound 10. Nucleophilic
substitution of the 4-bromo substituent of 13 with a
cyano group was carried out by treatment with NaCN
in N-methyl-2-pyrrolidone at high temperature to give
the 4-cyano derivative 15, which was then reduced by
hydrogenation with Raney Ni as catalyst in saturated
methanolic ammonia to produce the corresponding
aminomethyl derivative 16. Treatment of compound 6
with acetic anhydride in the presence of sulfuric acid
gave the 4-acetyl derivative 17. The 4-nitro derivative
18 was prepared by reaction of compound 6 with nitric
acid and acetic anhydride, and hydrogenation of 18 in
ethanol with palladium on activated carbon as catalyst
produced the corresponding amino derivative, which
was immediately converted to its hydrochloride salt 19
since the free base was subject to rapid decomposition.
Treatment of compound 6 with xenon difluoride in CH2-
Cl2 gave a mixture of the 4,5-difluoroisoquinolinone 20,
the 4-fluoroisoquinolinone 21, and recovered starting
material. The structures of both compounds 20 and 21
atoms in the same ring. The compounds of the collection
were evaluated without modification in the in vitro
TNFR assay system in primary human monocytes using
an ELISA detection method. The initial results revealed
an active member of the isoquinoline series, 6,7-
dimethoxy-1(2H)-isoquinolinone (2), which inhibited
TNFR production at an IC50 of 50 µM. To optimize the
activity in this series, a strategy similar to that which
had been used for the above-mentioned pteridinediones
was employed, wherein a homologous series of N-
alkanoic acid esters was prepared. In addition, several
electrophilic and nucleophilic substitutions were carried
out. These approaches were subsequently extended to
the quinazoline ring system. An in vivo mouse model of
pulmonary inflammation was then used to evaluate
promising candidate compounds identified in the pri-
mary in vitro assay. Finally, a quinazoline derivative
suitable to serve as a photoaffinity radiolabeled com-
pound was prepared in order to facilitate the identifica-
tion of the putative cellular target(s) for these TNFR
inhibitors.
1
were confirmed by H NMR spectra. Thus, for the
difluoro compound 20, the most downfield resonance at
δ 7.69 is assigned to H-8, which is split by F-5. Another
doublet resonance at δ 7.56 is for H-3, which is split by
F-4. There are three resonances in the aromatic region
for the monofluoro compound 21; two singlets at δ 7.80
and δ 7.09 for H-8 and H-5, respectively, and a doublet
at 7.00 for H-3, which is split by F-4. Both esters 6 and
9 were converted to the corresponding amides 22 and
23, respectively, by heating them in a sealed vessel with
40% CH3NH2 in H2O.
Ch em istr y
Isoqu in olin on es. In the isoquinolinone series, 6,7-
dimethoxy-1(2H)-isoquinolinone (2) and its unsubsti-
tuted derivative isocarbostyril (3) served as convenient
commercially available starting materials and required
only a simple alkylation with appropriate alkyl halides.
Thus, treatment of 2 or 3 with the appropriate alkyl
bromide in DMF at 70 °C in the presence of sodium
hydride or potassium carbonate produced the corre-
sponding N-alkylated products, 4-7 (Scheme 1), but the
yields using sodium hydride as base were much higher
than those using potassium carbonate. These alkanoic
ester products, however, were only sparingly soluble in
water. To improve the water solubility, 2-morpholino-
ethyl ester 9 was prepared by reflux of ethyl ester 6 in
1-Su bstitu ted Isoqu in olin es. In this series, we
began with the known compound, 1-chloro-6,7-dimethoxy-
isoquinoline (24, Scheme 3), which was prepared by
7
treatment of compound 2 with refluxing POCl3. How-
ever, this chloro derivative failed to undergo nucleophilic
substitution by aliphatic or aromatic amines under a
variety of conditions. Therefore, compound 24 was
converted to its trifluoroacetic acid salt 25 in order to
decrease the electron density on the aromatic ring and
thereby facilitate nucleophilic substitutions. Subsequent
reaction of 25 with primary amines was then easily
carried out. Thus, reflux of compound 25 with an excess
of the appropriate amine in 2-methyl-2-propanol gave
the corresponding amine derivatives, 26-31.
Qu in a zolin on es. In the quinazolinone series, three
groups of compounds were prepared: the 2-substituted
6,7-dimethoxy-, the 5-substituted 6,7-dimethoxy-, and
various 5-, 6-, and 7-substituted quinazolinones. For the
2-substituted 6,7-dimethoxyquinazolinones, we started
1
N HCl, followed by treatment of the resulting car-
boxylic acid 8 with SOCl2 in CH2Cl2 and then 4-(2-
hydroxyethyl)morpholine. Demethylation of 6 by treat-
ment with BBr3 in CH2Cl2 yielded the dihydroxy
derivative 10 (Scheme 2). Compound 10 was converted
to its dicarbonate (11) and diacetate (12) esters, respec-
tively. Bromination of 6 by treatment with bromine in
acetic acid produced the 4-bromo derivative 13, as
1
confirmed by its H NMR spectrum. It has been re-
6
ported that electrophilic aromatic substitutions, includ-
ing halogenation, acylation, and nitration, of N-alky-
lated isocarbostyril yield exclusively 4-substituted
products. The dihydroxy derivative 14 was produced by

3
862 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Chao et al.
Sch em e 2a
a
Reagents: (a) BBr3, CH2Cl2; (b) EtOCOCl, pyridine, or Ac2O, pyridine; (c) Br2, AcOH; (d) NaCN, 1-methyl-2-pyrrolidinone; (e) H2,
Raney Ni, EtOH; (f) Ac2O, H2SO4, reflux; (g) HNO3, Ac2O, AcOH; (h) 1. H2, Pd/C, EtOH; 2. HCl; (i) XeF2, CH2Cl2; (j) 40% H2NMe.
Sch em e 3a
in equilibrium with the tetrazole ring form, with the
azide form being favored in strongly acidic medium such
as TFA. Catalytic reduction of the azido group using a
9
palladium catalyst in EtOH and TFA readily provided
the 2-amino derivative 36. The substituted aromatic
amines 37-39 were prepared by nucleophilic substitu-
tion of the 2-chloro function of 35 with the appropriate
anilines in BuOH.
4
(3H)-Quinazolinone (40), unsubstituted at the 2, 6,
and 7 positions, was prepared by heating isatoic anhy-
1
0
dride with formamidine hydrochloride at 200 °C and
3
then was alkylated at N with ethyl 4-bromobutyrate
by the procedure described in method E to provide 42
(Scheme 6). The corresponding 6,7-dimethoxyquinazoli-
none was similarly prepared by using 4,5-dimethoxy-
anthranilic acid with formamidine hydrochloride to form
the known compound 41, and it was alkylated accord-
a
Reagents: (a) TFA, 2-propanol; (b) RNH2, t-BuOH.
1
1
ing to method E with haloalkanoic acid esters of three,
four, and five carbons to give the corresponding N -
with the versatile intermediate 2-chloro-6,7-dimethoxy-
(3H)-quinazolinone (32), which was prepared from 6,7-
dimethoxy-2,4(1H,3H)-quinazolinedione by treatment
3
4
8
alkylated quinazolinone derivatives, 43-45. Demethy-
lation of 44 to the dihydroxy derivative 46 was carried
out by treatment with AlCl3 in the presence of ethaneth-
with POCl3 followed by selective hydrolysis of the
4
-chloro function using 1 N NaOH at room temperature
1
2
iol. Attempts at nitration of the N-alkylated compound
44 with nitric acid and acetic anhydride resulted in no
reaction at room temperature and hydrolysis of the ethyl
ester at higher temperatures. Therefore, the nitration
was carried out on nonalkylated compound 41 with
potassium nitrate in sulfuric acid to produce the 5-nitro
derivative 47. Moreover, for the nitration of 6-substi-
for 5 h. Alkylation of 32 by the general alkylation
procedure for quinazolinones (method E) gave a mixture
of two products (Scheme 4), O-alkylated compound 33
(
14%) and N-alkylated compound 34 (66%). These
1
isomers were readily distinguished in the H NMR since
the chemical shift for the 4-CH2 of 33 (δ 4.62), attached
to oxygen, occurs further downfield than that for N-
alkylated compound 34 (δ 4.35). The 2-azido derivative
1
3
tuted quinazolines, it has been observed that forma-
tion of the 5-nitroquinazoline product is favored over
3
5 was prepared by treatment of 34 with sodium azide
1
4-17
in DMF. As shown in Scheme 5, the azide 35 can exist
that of the 8-nitro isomer.
The NMR data of the

Synthesis of TNFR Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3863
Sch em e 4a
a
Reagents: (a) Br(CH2)3CO2Et, K2CO3, DMF; (b) NaN3, wet DMF; (c) amine, BuOH; (d) H2, Pd/C, EtOH, TFA.
Sch em e 5
notably slower than that of normal aromatic amines,
probably due to the amino group being located between
the methoxy and carbonyl groups. The 5-azido deriva-
tive was prepared by diazotization of compound 49 with
NaNO2in 4 N HCl, followed by treatment with NaN3.
Because the side chain ester of 49 could be hydrolyzed
under Sandmeyer reaction conditions, the 5-chloro
derivative 55 was obtained by hydrogenation of 5-nitro
Sch em e 6a
4
7 to its amino derivative 53, conversion of 53 to the
-chloro derivative 54 by the Sandmeyer reaction, and
5
finally alkylation of 54 by method E.
For the 5-, 6-, and 7-monosubstituted quinazolinones,
N-alkylated derivatives 66-74 were prepared by heat-
ing the mixture of appropriate substituted anthranilic
acids and formamidine hydrochloride at 200 °C, followed
by alkylation of compounds 57-65 using method E
(Scheme 7). Hydrogenation of 6-nitro and 7-nitro de-
rivatives, 73 and 74, by using Pd/C as catalyst in EtOAc
for 30 min, gave the corresponding 6- and 7-amino
derivatives, 75 and 76, respectively. However, extending
the reaction time of hydrogenation of 73 and 74 to 5 h
provided the respective 6- and 7-amino-1,2-dihydro
derivatives 77 and 78.
Finally, a series of 2-substituted O-alkylated quinazo-
lines was prepared to observe any structure-activity
differences in the O- versus N-alkylated isomers. Treat-
ment of 2-chloro-6,7-dimethoxy-4(3H)-quinazolinone (32)
with an appropriate amine in BuOH at elevated tem-
peratures provided the 2-substituted amino derivatives
7
9-81 (Scheme 8). The O-alkylated compounds 82-84
were then obtained in good yield by using method E.
Interestingly, the major products of this alkylation were
O-alkylated isomers, and only trace amounts of the
corresponding N-alkylated derivatives were produced.
The steric effects of the NMe2 or NHAr at the 2-position
of the quinazolinones are likely responsible for the
observed predominant formation of the O- versus the
N-alkylation product. The structures of 82-84 were
1
confirmed by their H NMR spectra.
P h otoa ffin ity Ra d iola beled Der iva tive. The use
of photoaffinity labels to elucidate the target of small
molecule binding has been a valuable technique18 for
gaining information of biological interest. A compound
having some activity in the TNFR in vitro assay and
containing both a photoactivatable group as well as a
radioisotope seemed most suitable for our purposes. The
aromatic azides have been used extensively as photo-
reactive functional groups which produce a highly
reactive nitrene species when activated by light and
form a covalent bond with nearby groups at binding
sites. Compound 35, the 2-azidoquinazolinone, was first
considered as a possible candidate for a photoactivatable
ligand. However, preliminary studies showed that when
a
Reagents: (a) Br(CH2)nCO2Et, K2CO3, DMF; (b) AlCl3, EtSH;
(
(
c) KNO3, H2SO4; (d) H2, Pd/C, EtOH; (e) HCl; (f) Ac2O, pyridine;
g) 1. NaNO2, 4 N HCl; 2. NaN3, or CuCl; (h) H2, Pd/C, AcOH,
MeOH.
product are also consistent with the calculated data for
the 5-nitro derivative. Subsequent alkylation of 47 by
the general procedure gave compound 48, which was
reduced to the 5-amino derivative 49 by catalytic
hydrogenation in EtOH. To increase water solubility,
amino derivative 49 was converted to its hydrochloride
salt 50. Treatment of 49 with acetic anhydride in
pyridine provided the corresponding 5-acetamide de-
rivative 51. Interestingly, the acylation of 49 was

3
864 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Chao et al.
Sch em e 7a
a
Reagents: (a) formamidine hydrochloride, 210 °Cl; (b) Br(CH2)3CO2Et, K2CO3, DMF; (c) H2, Pd/C, EtOAc, 30 min; (d) H2, Pd/C, EtOAc,
5
h.
Sch em e 8a
tive were found to be the most active in this system,
inhibiting the paw edema by over 70% at a dose of 50
2
0
mg/kg i.p. The other report details the antiallergy
properties of a series of 4(3H)-quinazolinones substi-
tuted at 3 by propenoic acid groups determined using a
passive cutaneous anaphylaxis model in rats.11 In
addition, other reports describe the activity of substi-
tuted isoquinoline-1,3-diones as cyclooxygenase 2 (COX-
2
1
2
) inhibitors and substituted quinazoline-2,4-diones as
2
2
inhibitors of nuclear factor of activated T-cells (NFAT)
2
3
and inhibitors of PDE IV. Thus, there is considerable
overlap of activities and structure-activity relationships
in these heterocyclic systems. Whether or not TNFR
inhibition is relevant in these reported series is not
known. It is well known that PDE IV inhibition is one
2
4
a
mechanism by which TNFR may be inhibited. 2 Also,
Reagents: (a) amine, BuOH, heat; (b) Br(CH2)3CO2Et, K2CO3,
5
TNFR is known to mediate COX-2 expression.
DMF.
Table 1 presents a summary of the physicochemical
and in vitro TNFR inhibition data for the isoquinolines
and quinazolines. The pteridinedione 1 was used as a
positive control. Also included in the table are the
results of cell toxicity experiments using an MTT (3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro-
mide) assay in J urkat cells for those compounds of most
interest, i.e., those having TNFR IC50 values of less than
30 µM. Some structure-activity trends are evident
when considering the TNF inhibition data.
a solution of 35 was exposed to ultraviolet light of
appropriate wavelength, no nitrene formation was
observed, as measured by the lack of new product
formation in the presence or absence of an amino acid
such as serine. This resistance to photoactivation may
be due to the propensity of 35 to exist in the tetrazole
form (Scheme 5) as opposed to the azido form. To avoid
this possibility, the 5-azidoquinazolinone 52 was then
studied. This isomer was found to readily undergo
photoactivation, and therefore a dehydro derivative of
Sid e Ch a in Su bstitu en ts. As in the case of the
5
2 was prepared that was suitable for radiolabeling.
previous study of substituted xanthines and related
Thus, compound 53 was treated with sodium azide in
the presence of HCl to obtain the 5-azido derivative 85,
which was then treated with methyl 4-bromocrotonate
to provide the methyl 2-butenoate ester 86 (Scheme 9).
Finally, compound 86 was radiolabeled by catalytic
tritiation to produce the bis-labeled material 87.
5
compounds, a side chain length of four carbons appears
to be optimum for activity in both the isoquinoline and
quinazoline series. However, the quinazoline four-
carbon derivative 44 is more cytotoxic than the iso-
quinoline of corresponding structure (6). Interestingly,
the three-carbon side chain derivative 43 in this ho-
mologous series shows lower cytotoxicity while main-
taining good TNF inhibitory activity. Esters are superior
to the corresponding carboxylic acids at inhibiting TNF
production, probably due to cell permeability differences
Resu lts a n d Discu ssion
A retrospective review of the literature revealed that
there are several reports of 4(3H)-quinazolinones and
(
compare 6 or 9 to 8). Corresponding amides are also
inactive in this model (22 and 23). The water soluble
-morpholinoethyl ester 9 is useful for administration
1
(2H)-isoquinolinones with antiinflammatory and re-
1
9-21
lated activity.
Two reports describe an in vivo rat
2
paw edema model to investigate the aniinflammatory
properties of several series of quinazolinones substituted
but is slightly less active than the ethyl ester 6 and
slowly undergoes hydrolysis to 8 in aqueous solution.
1
9
at the 1 and 3 positions and on the benzo ring of the
quinazolinone.20 Of those prepared and tested, the
Rin g Su bstitu en ts. While the isoquinoline and
3
-methyl-4(3H)-quinazolinone and its 6-chloro deriva-
quinazoline rings are isosteric, a seemingly subtle

Synthesis of TNFR Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3865
Sch em e 9a
a
3
Reagents: (a) 1. NaNO2, HCl, 2. H2O; (b) methyl-4-bromocrotonate, K2CO3, DMF; (c) H2, Pd/C.
difference of a ring nitrogen appears to exert an effect
which results in at least a 10-fold loss of activity going
from the unsubstituted isoquinoline 6 to the unsubsti-
tuted quinazoline 42. In addition, the presence of the
ortho dimethoxy groups are not critical to the bioactivity
in the isoquinoline series (compare 4 and 6) whereas
they are important in the quinazoline series (compare
ment of neutrophils into the bronchoalveolar lavage
fluid (BALF). Thus, LPS was given in the presence or
absence of preadministered aerosolized compound and
then BALF was collected 3 h later (3 h was found to be
2
6
optimum ) and analyzed for TNFR level by an ELISA
method. We selected the 6-aminoquinazoline 75 as a
representative compound having in vitro TNFR inhibi-
tory activity, low cytotoxicity, and sufficient water
solubility (as the acid salt) to allow for aerosolization.
Figure 1 compares the effects of pretreatment using
various aerosol concentrations of 75 with that of dex-
amethasone as a positive control. Compound 75 was
capable of reducing the level of TNFR in BALF of LPS-
treated mice in a dose-dependent manner. The TNFR
levels were reduced by nearly 50% that of control LPS-
treated mice, and this reduction was similar to that
achieved with the dexamethasone positive control.
The mechanism of action of the compounds reported
here is still unknown, and efforts to elucidate their
cellular target(s) are underway. The photoaffinity ra-
diolabeled derivative 87 was successfully prepared and
used in experiments, whereby lysed human THP-1
monocytic leukemia cells were incubated with 87, pho-
toactivated with UV light, and the resulting radiola-
beled macromolecules were then examined by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE). Two competeable bands were clearly observed
(Figure 2), one of about 16 kDa and another of about
30 kDa. Studies utilizing two-dimensional SDS-PAGE,
followed by mass spectral analysis of the proteins and
their digestion fragments, are presently in progress, the
results of which will be reported elsewhere.
4
2 and 44). However, the activity in this substituted
quinazoline series is accompanied by increased toxicity
as well, except for compound 43 as noted above. More-
over, cleavage of the methyl groups to form the ortho
diols resulted in increased activity and toxicity in the
isoquinolines (compare 6 and 10, 13 and 14) but
decreased activity in the quinazoline series (compare 44
and 46). In a prodrug approach, protection of the diol
of 10 by esterification in an effort to decrease the
toxicity, resulted in a 5-fold decrease in activity while
toxicity remained about the same (compounds 11 and
1
2). Substitutions at other positions on the isoquinoline
ring (13-23) by groups such as bromo, aminomethyl,
acetyl, nitro, and fluoro generally resulted in a signifi-
cant decrease in TNFR activity. The 4-bromo and
4
-cyano derivatives, 14 and 15, respectively, however,
maintained good activity but displayed increased cyto-
toxicity. The 1-substituted isoquinolines 27-31 were
found to be weakly active. The unsubstituted aromatic
amino derivative 26, however, was inactive. Of the
2
3
5
-substituted quinazolines, only the 2-azido derivative
5 showed significant activity with low toxicity. The
-substituted derivatives 48-50 and 55 also displayed
good activity with little or no toxicity at doses tested.
Groups on the quinazoline other than 6,7-dimethoxy
were studied and found to render the derivatives
inactive. Thus, 6-fluoro, chloro, bromo, nitro, and 7-flu-
oro, chloro, and nitro derivatives were all devoid of TNF
inhibitory activity. Interestingly, the 6-amino- and
Exp er im en ta l Section
Ch em istr y. Melting points were obtained on a Mel-temp
II capillary melting point apparatus and are uncorrected.
Proton nuclear magnetic resonance spectra were obtained on
a Varian Unity 500 at 499.8 MHz. The chemical shifts are
expressed in δ values (parts per million) relative to tetram-
ethylsilane (TMS) as internal standard. Elemental analyses
were performed by NuMega Resonance Labs, San Diego, CA.
Thin-layer chromatography was performed on silica gel 60
F-254 plates (EM Reagents). E. Merck silica gel (230-400
mesh) was used for flash column chromatography.
7
-aminoquinazolines 75 and 76 were good inhibitors,
although 76 was also toxic. In the course of preparing
these two isomeric aminoquinazolines from their re-
spective nitro precursors, some 6- and 7-amino-1,2-
dihydro products (77 and 78) were obtained and evalu-
ated for activity. Both of these dihydro derivatives were
found to be good inhibitors of TNFR production and to
be nontoxic. Thus, partial reduction of the pyrimidine
ring moiety of 75 had little effect on activity and
rendered 76 much less toxic.
Biologica l Ma ter ia ls. Cells. Peripheral blood mononuclear
cells (PBMC) were isolated from heparinized normal human
blood by ficoll-histopaque density centrifugation. PBMC were
plated in 96-well plates in RPMI 1640 medium (Gibco, Grand
Island, NY) supplemented with 20% autologous plasma and 2
Finally, the 4-O-alkylated 2-substituted quinazolines,
3
3 and 82-84, were prepared and found to have little
5
mmol of L-glutamine at a density of 5 × 10 cells per milliliter.
or no TNF inhibitory activity.
In some experiments, monocytes were purified from PBMC by
In Vivo P u lm on a r y In fla m m a tion Mod el. An in
vivo mouse model of an acute lung inflammatory
response was selected to determine if compounds found
to have in vitro TNF inhibitory properties could be
adherence to gelatin coated flask and used at a density of 5 ×
5
1
0 cells per milliliter.
Mice. Male Balb/c mice (8 weeks old) were purchased from
Harlan Sprague Dawley.
Rea gen ts. Lipopolysaccharides from Escherichia coli sero-
type 026:B6 (LPS), human TNFR, and rabbit anti-human
TNFR were purchased from Sigma Chemical (St. Louis, MO).
Mouse anti-human TNFR was purchased from R & D Systems
2
6
effective in a relevant in vivo setting. In this model,
mice are given aerosolized LPS by inhalation, which
causes increased TNFR production followed by recruit-

3
866 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Chao et al.
Ta ble 1. Isoquinoline and Quinazoline Derivatives’ Physical and TNFR Inhibition Data
TNFR MTT
analysesa IC50b
IC50
c
compd class
R6 & R7
R
X
Y
mp (°C)
formula
1
3
5
50
5
21
>25
>50
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
6
7
8
9
0
1
4
5
6
7
8
9
2
3
4
5
6
8
9
0
1
2
5
6
7
8
9
0
1
2
3
4
5
6
3
7
8
2
3
4
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
II
II
II
H
(CH2)3CO2Et
(CH2)2CO2Et
(CH2)3CO2Et
(CH2)4CO2Et
(CH2)3CO2H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
<40
C15H17NO3
C16H19NO5
C17H21NO5
C18H23NO5
C15H17NO5
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OCO2Et
OCOMe
OCH3
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
125
84-85
43
157-159
116-118
115-116
<50
>50
22
>100
d
C21H28N2O6‚HCl C, H, Ne
(CH2)3CO2M
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CONHMe
10 >100
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
5
5
5
5
6
6
6
6
7
7
7
7
7
7
7
3
7
7
8
8
8
C15H17NO5
C21H25NO9
C19H21NO7
C17H20BrNO5
C15H16BrNO5
C18H20N2O5
C18H24N2O5
C19H23NO6
C17H20N2O7
C, H, N
C, H, N
C, H, N
C, H, Nf
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
2
11
9
51
1
47
40
40
H
58
78-80
Br
Br
CN
CH2NH2
COCH3
NO2
NH2‚HCl
F
F
H
169-172
155-157
204-205
110-112
151-152
>132 (dec.)
112-115
86
162-165
157-160
165-166
167-167
157-158
171-173
151-152
166-167
103-105
123-125
11
10
7
5
13
15
35
37
47
C17H22N2O5‚HCl C, H, Ng
C17H19F2NO5
C17H19F2NO5
C16H20N2O4
C16H19BrN2O4
C17H16N2O2
C17H15ClN2O2
C17H15ClN2O2
C17H15BrN2O2
C17H15BrN2O2
C13H16N2O3
C, H, Nh >100
H
H
H
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
20
>100
>100
>100
35
i
j
(CH2)3CONHMe Br
C6H5
4-Cl-C6H5
3-Cl-C6H5
4-Br-C6H5
3-Br-C6H5
(CH2)2OH
33
38
37
55
48
14
>25
>25
>50
>50
>25
9
3
6
48
11
15 >100
20 >100
>100
>25
22
>25
>25
>25
>25
>25
>25
>25
>25
>25
4
II
II
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
IV
III
III
IV
IV
IV
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)2CO2Et
(CH2)3CO2Et
(CH2)4CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
Cl
N3
NH2
NHC6H5
NHC6H4-4-Br
NHC6H4-4-N3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
HG
H
H
H
H
H
H
Cl
H
H
NMe2
H
H
H
H
H
H
H
H
H
H
H
C16H19ClN2O5
C16H19N5O5
>25
202-5 (dec.) C16H21N3O5
135-136
197-199
>145 (dec.)
40
141-142
125-126
110-112
195-197
105-106
88-89
C22H25N3O5
C22H24BrN3O5
C22H24N6O5
C14H16N2O3
C15H18N2O5
C16H20N2O5
C17H22N2O5
C14H16N2O5
C16H19N3O7
C16H21N3O5
OCH3
OCH3
OCH3
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
F
>25
3
8
NO2
NH2
>25
NH2‚HCl 195-197
C16H21N3O5‚HCl C, H, Nk
C18H23N3O6
C16H19N5O5
NHAc
N3
Cl
H
H
H
Cl
H
H
H
H
104-105
102-104
120
107-108
48
80
76
60
90-91
54-55
88
103-104
64
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C16H19ClN2O5
C14H14F2N2O3
C14H15FN2O3
C14H15FN2O3
C14H15ClN2O3
C14H15ClN2O3
C14H15ClN2O3
C14H15BrN2O3
C14H15N3O5
C14H15N3O5
C14H17N3O3
C14H17N3O3
C16H19ClN2O5
C14H19N3O3
89
6-F, 7-H
6-H, 7-F
H
6-Cl, 7-H
6-H, 7-Cl
6-Br, 7-H
6-NO2, 7-H
6-H, 7-NO2
6-NH2, 7-H
6-H, 7-NH2
OCH3
6-NH2, dihydro (CH2)3CO2Et
7-NH2, dihydro (CH2)3CO2Et
OCH3
OCH3
OCH3
H
H
H
>50
13
9
68
98-99
54-56
109-111
79
94
94
H
H
10 >100
C14H19N3O3
C18H25N3O5
C22H24BrN3O5
C22H24BrN3O5
5
>25
>25
>25
>50
(CH2)3CO2Et
(CH2)3CO2Et
(CH2)3CO2Et
NHC6H4-4-Br
NHC6H4-4-Br
a
All compounds analyzed for C, H, N; results were within (0.4% of theoretical values. b Concentration of compounds in µM which
c
inhibited the production of TNFR by 50% of control. Concentration of compounds in µM which inhibited the growth by 50% of control.
d
h
e
f
g
M ) 2-morpholinoethyl hydrochloride. C: calcd, 57.21; found, 55.19. C: calcd, 51.27; found, 52.24. C: calcd, 55.06; found, 51.10.
C: calcd, 57.46; found, 58.30. i N: calcd, 8.94; found, 9.53. N: calcd, 7.31; found, 7.74. k H: calcd, 5.96; found, 5.48.
j

Synthesis of TNFR Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3867
h, and the plate developed. TNFR levels were determined
1
by extrapolation from a standard curve that was included in
each assay. Samples were run in at least three separate
experiments for compounds whose IC50 was less than 10 µM,
and the variations were less than 10% among samples from
healthy donors. The method for internal variability was
smaller than 1%.
TNF r ELISA. Mou se BALF . A procedure similar to that
described above for the human monocyte ELISA was used in
the BALF model except that rat anti-mouse TNFR and
biotinylated rat anti-mouse TNFR antibodies were used for the
detection system.
In Vivo P u lm on a r y In fla m m a tion TNF r. This model
was used essentially as described by Gon c¸ alves de Moraes et
2
6
al. Briefly, mice (three in each group) were pretreated with
drugs either by inhalation or intraperitoneal injection. One
hour later mice were treated with LPS (E. coli) at 0.3 mg/mL
by inhalation for 10 min in an exposure chamber (Braintree
Scientific, Braintree, MA). The aerosol was generated by an
ultrasonic nebulizer (Ultra-Neb 99, DeVilbiss, particle size
F igu r e 1. Effect of 75 by inhalation on TNFR production in
a mouse pulmonary inflammation model. BALF from LPS-
treated and nontreated mice (three animals in each group) in
the presence or absence of pretreatment with compound 75
or dexamethasone (positive control) was analyzed for TNFR
content by ELISA, expressed as percent inhibition of control
levels. Horizontal bars represent standard error with p < 0.05.
0
.5-5 µm) which was connected to an exposure chamber.
Three hours later animals were sacrificed by CO asphyxiation,
2
and tracheotomy was performed. Bronchoalveolar lavage fluid
was collected and centrifuged at 300g for 10 min. Supernatant
was collected and stored at -70 °C for TNFR ELISA. The pellet
was resuspended in PBS and spun in a Cytospin for 10 min
at 40 speed before cells were stained with Wright-Giemsa stain
(Baxter Scientific). Neutrophil differential counting versus
leukocytes and TNFR level were compared to a control group
and expressed as a percentage inhibition of control levels.
P h otoa ffin ity La belin g. Human monocytic THP-1 cells
6
were grown to a concentration of (1-2) × 10 /mL in RPMI-
1
640 containing 10% fetal bovine serum. The cells were
harvested, washed in PBS, and resuspended in lysing buffer
100 mM Tris pH 7.4, 40 mM NaCl, 0.5% NP-40, 1 mM EDTA,
mM dithiothreotol, 1 mM phenylmethylsulfonyl fluoride
PMSF)). The lysates were ultracentrifuged at 100000g at 4
C for 60 min, and 5-10 µL of the supernatants (100 µg
proteins) were incubated with 10 µL of PBS (pH 7.4) containing
µCi (1 µM) of compound 87. In the competition experiments,
(
1
(
°
1
the “cold” unlabeled compounds were added to the lysates 30
min before the radiolabeled compound 87. Samples were
equilibrated at 4 °C for 30 min in a 96-well plate. Wells of the
plate were aligned with the axis of a UV light at a distance of
F igu r e 2. Photoaffinity labeling of THP-1 cytosolic extracts
with 87: (A) Autoradiogram of 87-labeled proteins separated
by SDS-14% polyacrylamide gel electrophoresis. The extract
was labeled (10 min, 4 °C, 254 nm) in the presence (+) or
absence (-) of a 1000-fold excess of nonlabeled photoaffinity
analogue as the competing ligand. Arrows indicate position of
potential target proteins. (B) Effect of preincubation with a
2
cm between the bottom of the wells and the lamp bulb and
irradiated at 254 nm for 5-15 min at 0 °C. Sample buffer (4
SDS) was added to the reaction mixture wells, and the
×
proteins were separated by SDS-polyacrylamide gel electro-
phoresis on 14% Laemmli precast gels. The proteins were then
electrotransferred to polyvinylidene fluoride (PVDF) mem-
branes, air-dried, and exposed to autoradiography (BioMax
MS) films with BioMax TransScreen-LE intensifying screens
1
000-fold excess of compounds on the 87-labeling of cytosolic
THP-1 extracts. Lane 1: 87 alone (positive control). Lane 2:
nonlabeled 87. Lane 3: 44. Lane 4: 9. Lane 5: 1.
(Eastman Kodak, Rochester, NY) for 3-5 days at -80 °C. No
covalent incorporation of the radiolabel occurred in the absence
of UV irradiation. All of the photolabeling experiments were
performed in at least three independent replicates. The films
were digitized on a UMAX-Astra 1200 scanner using Adobe
Photoshop, and the densities of the bands were determined
by NIH IMAGE 1.59 to find the relative incorporation.
Minneapolis, MN). Peroxidase-conjugated affiniPure goat anti-
rabbit IgG was purchased from J ackson ImmunoResearch
Labs (West Grove, PA). Tetramethylbenzidine came from
Kirkegaard and Perry (Gaithersburg, MD). Other chemicals
were purchased from Aldrich Chemical Co. (Milwaukee, WI).
In Vitr o TNF r. Compounds in RPMI-1640 (2× concentra-
tions) were added in equal volume to the human PBMC in 96-
well microtiter plates, followed by a 60 min incubation. LPS
Meth od A: Gen er a l Alk yla tion P r oced u r e for Iso-
qu in olin on e Com p ou n d s 4-7. To a solution of 1(2H)-
isoquinolinone (3) or 6,7-dimethoxy-1(2H)-isoquinolinone (2,
1 mmol) in 10 mL of dry DMF was added NaH (60% dispersion
(Salmonella, Minnesota) was then added to the cultures at a
final concentration of 10 µg/mL. Eighteen hours later, 50 µL
of each supernatant was collected and assayed for TNFR
2 3
in mineral oil, 1.2 equiv) or K CO (1.2 equiv). After the
5
content by ELISA.
mixture was stirred at room temperature for 20 min, the
appropriate alkyl bromide (1.2 equiv) was added, and the
mixture was then stirred at 70 °C for 8-12 h. The solvent was
evaporated under high vacuum, water was added to the
TNF r ELISA. Hu m a n Mon ocytes. ELISA plates were
coated overnight with 2 µg/mL mouse anti-human TNFR
antibody then blocked for 2 h with 1% bovine serum albumin.
Aliquots of culture (50 µL) supernatant were transferred to
the plate and incubated 2 h at 37 °C and then washed. The
plate was then incubated 1 h with 50 µL of 1:20000 rabbit anti-
TNFR antibody and washed. Peroxidase-conjugated affiniPure
goat anti-rabbit IgG antibody at 1:10000 was then added for
residue, and it was extracted with CH
dried over MgSO and evaporated to yield a crude product.
Crystallization of the crude product in EtOAc or flash silica
2 2
Cl . The extracts were
4
gel chromatography using 0-3% MeOH/CH
analytically pure products 4-7.
2 2
Cl as eluant gave

3
868 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Chao et al.
Meth od B: Gen er a l P r oced u r e for Dem eth yla tion of
Isoqu in olin on e Com p ou n d s 6 a n d 13. To a solution of an
appropriate compound (6 or 13, 1 mmol) in 10 mL of dried
60 °C to yield the corresponding quinazolinone compounds,
which were used directly without further purification.
Eth yl 4-(1,2-Dih yd r o-1-oxoisoqu in olin -2-yl)bu ta n oa te
(4). Compound 4 was prepared from 3 by method A using NaH
CH
2
Cl
2
at -78 °C was added dropwise 5 equiv of 1.0 M solution
1
of boron tribromide in CH
2
Cl . The reaction mixture was
2
as base in 75% yield as a waxy solid (269 mg): mp <40 °C; H
stirred for 1 h, during which time it was brought to room
temperature, and another 2 h at room temperature. Ice-water
3
NMR (CDCl ) δ 8.42 (d, 1H), 7.63 (t, 1H), 7.46-7.51 (m, 2H),
7.08 (d, 1H), 6.50 (d, 1H), 4.11 (q, 2H), 4.06 (t, 2H), 2.39 (t,
2H), 2.11 (m, 2H), 1.23 (t, 3H). Anal. C15 ‚0.5H O.
was added, and the mixture was extracted with CH
2
Cl
and evaporated to yield a
crude product, which was purified by flash silica gel chroma-
2
. The
H
17NO
3
2
extracts were dried over MgSO
4
Eth yl 3-(1,2-Dih yd r o-6,7-d im eth oxy-1-oxoisoqu in olin -
2-yl)p r op a n oa te (5). Compound 5 was prepared from 2 by
method A using K CO as base to yield 4 as white solid in 32%
tography using 0-5% MeOH in CH
analytical pure products, 10 and 14.
2 2
Cl as eluant to yield
2
3
1
yield (183 mg): mp 125 °C; H NMR (DMSO-d ) δ 7.55 (s, 1H),
7.31 (d, 1H), 7.12 (s, 1H), 6.5 (d, 1H), 4.14 (t, 2H), 4.01 (q, 2H),
6
Meth od C: Gen er a l P r oced u r e for Tr a n sfor m a tion of
Ester 6 a n d 9 to Am id e 22 a n d 23. A mixture of an
appropriate compound (6 or 9, 1 mmol) in 10 mL of methy-
lamine (40% in water) was placed in a sealed steel reaction
vessel and heated at 100 °C for 20 h. After most of the
remaining methylamine and water was evaporated, 20 mL of
3.86 (s, 3H), 3.84 (s, 3H), 2.74 (t, 2H), 1.11 (t, 3H). Anal. C16
NO
19
H -
5
.
Eth yl 4-(1,2-Dih yd r o-6,7-d im eth oxy-1-oxoisoqu in olin -
2-yl)bu ta n oa te (6). Compound 6 was made from 2 by method
A using NaH as base in 72% yield as a white solid (1.36 g):
1
5
% NaHCO
with CH Cl
rated to yield a crude product, which was purified by flash
3
solution was added and the mixture was extracted
mp 84-85 °C; H NMR (DMSO-d
6
) δ 7.59 (s, 1H), 7.32 (d, 1H),
. The extracts were dried over MgSO and evapo-
4
7.15 (s, 1H), 6.54 (d, 1H), 4.02 (q, 2H), 3.97 (t, 2H), 3.89 (s,
3H), 3.86 (s, 3H), 2.32 (t, 2H), 1.93 (m, 2H), 1.51 (t, 3H). Anal.
2
2
silica gel chromatography using 0-6% MeOH in CH
eluant to yield analytically pure products, 22 or 23.
2
Cl
2
as
17 5
C H21NO .
Eth yl 5-(1,2-Dih yd r o-6,7-d im eth oxy-1-oxoisoqu in olin -
-yl)p en ta n oa te (7). Compound 7 was made from 2 by
method A using K CO as base to yield the white solid product
in 24% yield (116 mg): mp 43 °C; H NMR (DMSO-d
(d, 1H), 7.40 (s, 1H), 7.30 (s, 1H), 7.22 (d, 1H), 4.44 (t, 2H),
2
Meth od D: Gen er a l P r oced u r e for Su bstitu tion of
-Ch lor o-6,7-d im eth oxyisoqu in olin e (24) w ith Am in es to
1
2
3
1
6
) δ 7.82
Com p ou n d s 26-31. A solution of 1-chloro-6,7-dimethoxyiso-
6
quinoline (24, 1 mmol) in 5 mL of 2-propanol was treated with
4
2
.02 (q, 2H), 3.88 (s, 6H), 2.40 (t, 2H), 1.82 (m, 2H), 1.73 (m,
H), 1.14 (t, 3H). Anal. C18
-(1,2-Dih yd r o-6,7-d im et h oxy-1-oxoisoq u in olin -2-yl)-
trifluoroacetic acid (1.2 equiv). The solvent was evaporated to
give trifluoroacetate 25 as a white solid. The trifluoroacetate
5 and the appropriate amine (2.2 mmol) were dissolved in
0 mL of 2-methyl-2-propanol, and the reaction mixture was
H
5
23NO .
4
2
1
bu ta n oic Acid (8). A solution of 6 (50 mg, 0.16 mmol) in 1 N
NaOH (3 mL) was heated at reflux for 90 min, cooled, and
neutralized with H+ ion-exchange resin. The white solid which
brought to reflux. After reflux for 16-20 h, the mixture was
cooled to room temperature, quenched with saturated NaHCO
solution, and extracted with CH Cl . The extracts were dried
over MgSO and evaporated to yield a crude product, which
was purified by flash silica gel chromatography using 0-2%
MeOH in CH Cl as eluant. Crystallization of the pure
products from ethyl ether yielded analytically pure compounds
3
formed was extracted into ethyl acetate, dried over Na
and evaporated to yield 27 mg (59%): mp 157-159 °C;
NMR (DMSO-d ) δ 7.59 (s, 1H), 7.32 (d, 1H), 7.15 (s, 1H), 6.54
d, 1H), 3.97 (t, 2H), 3.89 (s, 3H), 3.86 (s, 3H), 2.32 (t, 2H),
.93 (m, 2H). Anal. C15
-Mor ph olin oeth yl 4-(1,2-Dih ydr o-6,7-dim eth oxy-1-oxo-
isoqu in olin -2-yl)bu ta n oa te, Hyd r och lor id e (9). Compound
(1.0 g, 3.4 mmol) was dissolved with warming in dichlo-
2 4
SO ,
2
2
1
H
4
6
(
2
2
1
5
H17NO .
2
2
6-31.
Meth od E: Gen er a l Alk yla tion P r oced u r e for Qu in -
8
a zolin on e Com p ou n d s. A suspension of the appropriate
quinazolinone compound (1 mmol), alkyl bromide (1.3 equiva-
lent), and potassium carbonate (2 equivalent) in 10 mL of DMF
was stirred at 70 °C for 8-10 h. The solvent was evaporated
under high vacuum, water was added to the residue, and the
romethane (25 mL). Thionyl chloride (1 mL, 13.7 mmol) was
added followed by a few drops of DMF. After a few minutes a
white solid formed, assumed to be the acid chloride. The
reaction mixture was evaporated to dryness. The residue was
suspended in dry acetonitrile (5 mL), and morpholinoethyl
alcohol (1.24 mL, 10.2 mmol) was added. The mixture was
heated at 80 °C for 5 min, cooled, and absorbed onto silica gel
mixture was extracted with CH
2 2
Cl . The extracts were dried
over MgSO and evaporated to yield a crude product. Crystal-
4
lization of the crude product in EtOAc or flash silica gel
chromatography using the appropriate solvent system yielded
analytically pure compounds 33, 34, 42-45, 48, 55, 66-74,
and 82-84.
2 2
and chromatographed using 5% MeOH-CH Cl . A small
amount of 1 N HCl was added to the residue and the residue,
was placed in the freezer overnight, yielding 930 mg (62%):
1
mp 116-118 °C; H NMR (DMSO-d
6
) δ 7.77 (s, 1H), 7.01 (d,
Meth od F : Gen er a l P r oced u r e for Su bstitu tion of
1
H), 6.84 (s, 1H), 6.40 (d, 1H), 4.18 (t, 2H), 4.05 (t, 2H), 3.98
E t h yl
4-(2-Ch lor o-3,4-d ih yd r o-6,7-d im et h oxy-4-oxo-
(s, 3H), 3.96 (s, 3H), 3.67 (t, 4H), 2.58 (t, 2H), 2.46 (t, 4H),
qu in azolin -3-yl)bu tan oate (34) or 2-Ch lor o-6,7-dim eth oxy-
(3H)-qu in a zolin on e (32) w ith Am in es to Com p ou n d s
7-39 a n d 77-79. A solution of compound 34 or 32 and an
appropriate amine (2 equiv) in 10 mL of 1-butanol was refluxed
for 10-18 h. For compounds 37-39, the solvent was evapo-
rated to yield a crude product, which was purified by flash
2
28 2 6
.39 (t, 2H), 2.10 (m, 2)H). Anal. C21H N O ‚HCl.
4
3
Eth yl 4-(1,2-Dih yd r o-6,7-d ih yd r oxy-1-oxoisoqu in olin -
2
-yl)bu ta n oa te (10). Compound 10 was prepared from 6 by
using method B as needles in 76% yield (686 mg): mp 115-
1
1
6
4
3
16 °C; H NMR (CDCl ) δ 9.99 (br, 1H, OH), 8.25 (s, 1H),
.93 (d, 1H), 6.89 (s, 1H), 6.69 (br, 1H, OH), 6.44 (d, 1H), 4.07-
.14 (m, 4H), 2.39 (t, 2H), 2.10 (m, 2H), 1.21 (t, 3H). Anal.
silica gel chromatography using 0-4% MeOH in CH
2 2
Cl as
eluant to produce analytically pure compounds. For compounds
15 5
C H17NO .
7
2
7-79, the reaction mixture was filtered and washed with
-propanol to yield the products which were used directly in
the next step without further purification.
Eth yl 4-[6,7-Bis(eth oxycar bon yloxy)-1,2-dih ydr o-1-oxo-
isoqu in olin -2-yl]bu ta n oa te (11). To a solution of compound
0 (291 mg, 1.0 mmol) in 5 mL of dry pyridine under argon
1
Meth od G: Gen er a l P r oced u r e for P r ep a r a tion of
Qu in a zolin on e Com p ou n d s 57-65. An appropriate substi-
tuted 2-aminobenzoic acid (3.0 mmol) and formamidine hy-
drochloride (1.5 equiv) were ground and mixed very well and
then spread around the bottom of a round-bottom flask. After
heating at 210 °C for 15 min, the reaction mixture was allowed
to cool to room temperature. To the mixture was added a dilute
NaOH solution (0.3 N, 10 mL). The resulting solid was
collected, washed with water, and dried under vacuum at 50-
was added dropwise neat ethyl chloroformate (271 mg, 2.5
mmol). The reaction mixture was stirred at room temperature
for 24 h, poured into ice-water, and extracted with CH Cl .
2
2
The extracts were dried over MgSO and evaporated to yield
4
a crude product which was purified by flash silica gel chro-
matography using 0-3% MeOH in CH
2 2
Cl as eluant to yield
compound 11 as a waxy solid in 78% yield (339 g): mp < 50
1
°C; H NMR (CDCl
3
) δ 8.29 (s, 1H), 7.46 (s, 1H), 7.10 (d, 1H),
6.45 (d, 1H), 4.34 (q, 2H), 4.33 (q, 2H), 4.12 (q, 2H), 4.04 (t,

Synthesis of TNFR Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3869
2
(
H), 2.37 (t, 2H), 2.09 (m, 2H), 1.38 (t, 3H), 1.379 (t, 3H), 1.24
t, 3H). Anal. C21
Eth yl 4-(6,7-Bis(a cetoxy)-1,2-d ih yd r o-1-oxoisoqu in o-
The residue was purified by flash silica gel chromatography
H
25NO
9
.
using 0-5% MeOH in CH
as a yellow solid in 58% yield (98 mg): mp 110-112 °C; H
NMR (CDCl
2 2
Cl as eluant to yield compound 17
1
3
) δ 8.58 (s, 1H), 7.97 (s, 1H), 7.89 (s, 1H), 4.11-
.16 (m, 4H), 4.03 (s, 3H), 3.99 (s, 3H), 2.57 (s, 3H), 2.41 (t,
H), 2.15 (m, 2H), 1.24 (t, 3H). Anal. C19
Eth yl 4-(1,2-Dih yd r o-6,7-d im eth oxy-4-n itr o-1-oxoiso-
lin -2-yl)bu ta n oa te (12). To a solution of compound 10 (200
mg, 0.68 mmol) in 9 mL of dry pyridine under argon was added
dropewise acetic anhydride (1 mL). The reaction mixture was
stirred at room temperature for 18 h, poured into ice-water
4
2
H
6
23NO .
and extracted with CH
MgSO and evaporated to yield a crude product. The crude
product was purified by flash silica gel column chromatography
using 0-3% MeOH in CH Cl as eluant to yield compound 12
as a white solid in 81% yield (207 mg): mp 58 °C; H NMR
CDCl ) δ 8.19 (s, 1H), 7.37 (s, 1H), 7.08 (d, 1H), 6.44 (d, 1H),
.11 (q, 2H), 4.04 (t, 2H), 2.32 (s, 3H), 2.31 (s, 3H), 2.08 (m,
H), 1.23 (t, 3H). Anal. C19
Eth yl 4-(4-Br om o-1,2-d ih yd r o-6,7-d im eth oxy-1-oxoiso-
qu in olin -2-yl)bu ta n oa te (13). To a solution of compound 6
2.13 g, 6.7 mmol) in 10 mL of acetic acid was added dropwise
2
Cl
2
. The extracts were dried over
qu in olin -2-yl)bu ta n oa te (18). To a solution of compound 6
(1.00 g, 3.1 mmol) in 10 mL of acetic anhydride at 0 °C was
added dropwise a solution of nitric acid (3.1 mmol) in 1 mL of
acetic acid. The reaction mixture was stirred at room temper-
ature for 1 h. Ice was added to the mixture and stirred for
4
2
2
1
another 20 min, and then the mixture was extracted with CH
Cl . The extracts were dried over MgSO , and solvent was
evaporated. The residue was purified by flash silica gel
chromatography using 0-2% MeOH in CH Cl as eluant to
yield compound 18 as a pale yellow solid in 55% yield (624
2
-
(
4
2
3
2
4
H
7
21NO .
2
2
1
mg): mp 151-152 °C; H NMR (CDCl
H), 7.83 (s, 1H), 4.18 (t, 2H), 4.14 (q, 2H), 4.07 (s, 3H), 4.02
s, 3H), 2.42 (t, 2H), 2.16 (m, 2H), 1.25 (t, 3H). Anal.
3
) δ 8.60 (s, 1H), 8.20 (s,
(
1
(
a solution of bromine (1.07 g, 6.7 mmol) in 5 mL of acetic acid.
After being stirred at room temperature for 1 h, the reaction
17 20 2 7
C H N O .
mixture was poured into ice-water and extracted with CH
Cl . The extracts were dried over MgSO and evaporated to
dryness. The crude product was purified by flash silica gel
Cl as eluant.
2
-
Eth yl 4-(4-Am in o-1,2-d ih yd r o-6,7-d im eth oxy-1-oxoiso-
qu in olin -2-yl)bu ta n oa te Hyd r och lor id e (19). To a solution
of compound 18 (100 mg, 0.27 mmol) in 5 mL of ethanol and
2
4
chromatography using 0-3% MeOH in CH
2
2
5
mL of EtOAc was added palladium on activated carbon
Crystallization of the pure product fraction from ethyl ether
(palladium content 10%, 10 mg). After the mixture was shaken
yielded compound 13 as needles in 61% yield (1.62 g): mp 78-
8
1
1
under 30 psi of hydrogen for 2 h, 0.5 mL of 1 N HCl was added
to the reaction mixture. The mixture was then filtered with
Celite and the filter cake washed with ethanol. The filtrate
was evaporated, and the residue was crystallized from 2-pro-
0 °C; H NMR (CDCl
3
) δ 7.80 (s, 1H), 7.29 (s, 1H), 7.15 (s,
H), 4.12 (q, 2H), 4.04 (t, 2H), 4.03 (s, 3H), 4.00 (s, 3H), 2.38
20BrNO
Eth yl 4-(4-Br om o-1,2-d ih yd r o-6,7-d ih yd r oxy-1-oxoiso-
(t, 2H), 2.10 (m, 2H), 1.24 (t, 3H). Anal. C17
H
5
.
panol to yield compound 19 as a white solid in 56% yield (56
qu in olin -2-yl)bu ta n oa te (14). Compound 14 was prepared
from 13 by using method B as needles in 81% yield (224 mg):
1
mg): mp > 200 °C (dec); H NMR (DMSO-d
6
) δ 9.90 (br, 3H),
7
.65 (s, 1H), 7.42 (s, 1H), 7.29 (s, 1H), 3.95-4.02 (m, 4H), 3.92
s, 3H), 3.88 (s, 3H), 2.35 (t, 2H), 1.91 (m, 2H), 1.14 (t, 3H).
Anal. C17 ‚HCl.
1
mp 169-172 °C; H NMR (CDCl
3
) δ 9.30 (br, 1H, OH), 8.26 (s,
(
1
H), 7.28 (s, 1H), 7.25 (s, 1H), 6.50 (br, 1H, OH), 4.14 (q, 2H),
.07 (t, 2H), 2.41 (t, 2H), 2.11 (m, 2H), 1.25 (t, 3H). Anal.
16BrNO
Eth yl 4-(4-Cya n o-1,2-d ih yd r o-6,7-d im eth oxy-1-oxoiso-
qu in olin -2-yl)bu ta n oa te (15). A solution of compound 13
500 mg, 1.2 mmol) and CuCN (202 mg, 2.2 mmol) in 10 mL
22 2 5
H N O
4
C
Eth yl 4-(4,5-Diflu or o-1,2-d ih yd r o-6,7-d im eth oxy-1-oxo-
isoqu in olin -2-yl)bu ta n oa te (20) a n d Eth yl 4-(1,2-Dih y-
d r o-6,7-d im e t h oxy-4-flu or o-1-oxoisoq u in olin -2-yl)b u -
ta n oa te (21). To a solution of compound 5 (300 mg, 0.9 mmol)
15
H
5
.
(
2 2
in 10 mL of dry CH Cl at -78 °C under argon was added
of N-methyl-2-pyrrolidone was heated at 200 °C for 2 h. To
the reaction mixture was added 50 mL of water, and the
mixture was filtered. The filtered material was washed with
xenon difluoride (304 mg, 1.8 mmol). The reaction mixture was
stirred overnight, and the temperature was gradually raised
to room temperature. To the mixture was added 10 mL of 5%
sodium bicarbonate solution, and the mixture was extracted
with CH Cl . The extracts were dried over MgSO , and the
CH
2
Cl
2
, and the aqueous layer was extracted with CH
2
Cl
2
. The
, the
combined washings and extracts were dried over MgSO
4
2
2
4
and solvent was evaporated. The residue was purified by flash
chromatography using 50% EtOAc in hexane as eluant.
Crystallization of the pure product fraction from EtOAc yielded
solvent was evaporated. The residue was purified by flash
silica gel chromatography using 50-75% EtOAc in hexane as
eluant. Difluoride compound 20 eluted first from the column
and was obtained as a pale yellow solid (61 mg, 18%). The
second product to elute was monofluoride compound 21, as a
pale yellow solid in 35% yield (119 mg). Finally, starting
material 3 was recovered (about 20%).
compound 15 as needles in 73% yield (314 mg): mp 155-157
1
°
C; H NMR (CDCl
3
) δ 7.77 (s, 1H), 7.66 (s, 1H), 7.11 (s, 1H),
4
2
1
1
.13 (q, 2H), 4.10 (t, 2H), 4.04 (s, 3H), 4.00 (s, 3H), 2.38 (t,
H), 2.11 (m, 2H), 1.25 (t, 3H); C NMR (CDCl ) δ 172.61,
60.59, 154.60, 150.49, 139.00, 128.98, 119.19, 116.13, 108.08,
04.20, 90.51, 60.70, 56.40, 56.28, 49.14, 30.73, 24.30, 14.08.
1
3
3
1
Physical data for compound 20: mp 112-115 °C; H NMR
(
(
3
CDCl ) δ 7.69 (d, 1H), 7.56 (d, 1H), 4.07-4.11 (m, 4H), 4.06
Anal. C18
20 2 5
H N O .
s, 3H), 4.02 (s, 3H), 2.38 (t, 2H), 2.01 (m, 2H), 1.22 (t, 3H).
NO
Eth yl 4-(4-Am in om eth yl-1,2-d ih yd r o-6,7-d im eth oxy-1-
oxoisoqu in olin -2-yl)bu ta n oa te (16). To a solution of com-
pound 15 (340 mg, 0.99 mmol) in 20 mL of saturated ethanolic
Anal. C17
H
19
F
2
5
.
1
Physical data for compound 21: mp 86 °C; H NMR (CDCl )-
3
δ 7.80 (s, 1H), 7.09 (s, 1H), 7.00 (d, 1H), 4.12 (q, 2H), 4.03 (t,
2H), 4.02 (s, 3H), 4.01 (s, 3H), 2.39 (t, 2H), 2.10 (m, 2H), 1.24
(t, 3H). Anal. C H20FNO5.
3
NH was added Raney Ni as catalyst, and the mixture was
shaken under 25 psi hydrogen for 7 h. The reaction mixture
was filtered and washed with ethanol. The filtrate was
evaporated to yield an oil that was triturated with EtOAc. The
1
7
N-Meth yl 4-(1,2-Dih yd r o-6,7-d im eth oxy-1-oxoisoqu in -
olin -2-yl)bu ta n a m id e (22). Compound 22 was prepared from
analytically pure 16 was formed as a white solid in 88% yield
6
using method C and was obtained as a white solid in 65%
1
(
302 mg): mp 204-206 °C; H NMR (CDCl
3
) δ 7.83 (s, 1H),
1
yield (45 mg): mp 162-165 °C; H NMR (CDCl ) δ 7.74 (s, 1H),
3
7
3
2
.084 (s, 1H), 7.078 (s, 1H), 4.08 (q, 2H), 4.03 (t, 2H), 4.01 (s,
H), 3.98 (s, 3H), 2.60 (br, 2H, NH ), 2.38 (t, 2H), 2.09 (m,
H), 1.21 (t, 3H); Anal. C18
Eth yl 4-(4-Acetyl-1,2-d ih yd r o-6,7-d im eth oxy-1-oxoiso-
qu in olin -2-yl)bu ta n oa te (17). To a solution of compound 6
150 mg, 0.47 mmol) in 8 mL of acetic anhydride was added 8
7.00 (d, 1H), 6.85 (d, 1H), 6.79 (br, 1H, NH), 6.44 (s, 1H), 4.04
(t, 2H), 3.97 (s, 3H), 3.95 (s, 3H), 2.77 (d, 3H), 2.20 (t, 2H),
2.06 (m, 2H). Anal. C H N O ‚0.5H O.
2
24 2 5
H N O .
1
6
20
2
4
2
N-Meth yl 4-(4-Br om o-1,2-dih yd r o-6,7-dim eth oxy-1-oxo-
isoqu in olin -2-yl)bu ta n a m id e (23). Compound 23 was pre-
(
pared from 9 using method C and was obtained as a white
1
drops of concentrated sulfuric acid, and the reaction mixture
was brought to reflux for 1 h. The mixture was poured into
solid in 59% yield (168 mg): mp 157-160 °C; H NMR (CDCl
3
)
δ 7.79 (s, 1H), 7.32 (s, 1H), 6.54 (s, 1H), 4.05 (t, 2H), 4.03 (s,
3H), 4.01 (s, 3H), 2.78 (d, 3H), 2.22 (t, 2H), 2.09 (m, 2H). Anal.
ice-water, stirred 30 min, and extracted with CH
2
Cl
2
. The
extracts were dried over MgSO , and solvent was evaporated.
4
16 2 4
C H19BrN O .

3
870 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
,7-Dim eth oxy-1-p h en yla m in oisoqu in olin e (26). Com-
Chao et al.
1
6
205 °C; H NMR (CDCl
3
) δ 11.15 (br, 1H, NH), 8.15 (br, 1H,
NH), 7.43 (s, 1H), 6.95 (s, 1H), 4.21 (q, 2H), 4.03 (m, 5H), 3.94
(s, 3H), 2.53 (t, 2H), 1.97 (m, 2H), 1.30 (t, 3H). Anal.
pound 26 was prepared from 25 and aniline by method D and
was obtained as a white solid in 85% yield (154 mg): mp 165-
1
1
2
1
66 °C; H NMR (CDCl
3
) δ 8.00 (d, 1H), 7.48 (d, 2H), 7.30 (m,
16 21 3 5
C H N O .
H), 7.13 (s, 1H), 7.05 (d, 1H), 6.98-7.01 (m, 2H), 6.91 (br,
H, NH), 3.99 (s, 3H), 3.91 (s, 3H). Anal. C17
-(4-Ch lor op h en yl)a m in o-6,7-d im et h oxyisoqu in olin e
27). Compound 27 was made from 25 and 4-chloroaniline as
described in method D and was obtained as a white solid in
Eth yl 4-(3,4-Dih yd r o-6,7-d im eth oxy-2-p h en yla m in o-4-
H
16
N
2
O
2
.
oxoqu in azolin -3-yl)bu tan oate (37). Compound 37 was made
from 34 and aniline by using method F and was obtained as
1
1
(
a white solid in 89% yield (102 mg): mp 135-136 °C; H NMR
(CDCl ) δ 8.36 (br, 1H, NH), 8.86 (d, 2H), 7.49 (s, 1H), 7.39 (t,
3
1
7
4% yield (189 mg): mp 157-158 °C; H NMR (CDCl
d, 1H), 7.46-7.49 (m, 2H), 7.28-7.31 (m, 2H), 7.09-7.10 (m,
H), 7.05 (s, 1H), 6.75 (br, 1H, NH), 4.02 (s, 3H), 4.00 (s, 3H).
Anal. C17
-(3-Ch lor op h en yl)a m in o-6,7-d im et h oxyisoqu in olin e
28). Compound 28 was prepared from 25 and 3-chloroaniline
by using method D and was obtained as a white solid in 90%
3
) δ 8.00
2H), 7.11 (t, 1H), 6.90 (s, 1H), 4.27 (q, 2H), 4.19 (m, 2H), 3.97
(s, 3H), 3.95 (s, 3H), 2.56 (t, 2H), 2.08 (m, 2H), 1.31 (t, 3H).
(
2
25 3 5
Anal. C22H N O .
H
15ClN
2
O
2
.
E t h yl 2-(4-Br om op h en yla m in o)-3,4-d ih yd r o-6,7-d im -
eth yoxy-4-oxoqu in a zolin -3-yl)bu tyr a te (38). Compound 38
was prepared from 34 and 4-bromoaniline by using method F
1
(
1
as white solid in 66% yield (136 mg): mp 197-199 °C; H NMR
1
yield (156 mg): mp 166-167 °C; H NMR (CDCl
H), 7.64 (s, 1H), 7.38 (m, 1H), 7.26 (s, 1H), 7.23 (d, 1H), 7.12
d, 1H), 7.09 (s, 1H), 7.06 (s, 1H), 6.99 (br, 1H, NH), 3.99 (s,
H), 3.91 (s, 3H). Anal. C17
-(4-Br om op h en yl)a m in o-6,7-d im et h oxyisoq u in olin e
29). Compound 29 was made from 25 and 4-bromoaniline by
using method D and was obtained as a white solid in 74% yield
3
) δ 8.04 (d,
3
(CDCl ) δ 8.47 (br, 1H, NH), 7.79 (d, 2H), 7.47-7.49 (s and d,
1
(
3
3H), 6.88 (s, 1H), 4.26 (q, 2H), 4.16 (m, 2H), 3.97 (s, 3H), 3.95
(s, 3H), 2.56 (t, 2H), 2.05 (m, 2H), 1.32 (t, 3H). Anal. C22
BrN
Eth yl
24
H -
H
15ClN
2
O
2
.
3 5
O .
1
4-(2-[(4-Azid op h en yl)a m in o]-3,4-d ih yd r o-6,7-
(
d im et h oxy-4-oxoq u in a zolin -3-yl)b u t a n oa t e (39). Com-
pound 39 was made from 34 and 4-azidoaniline by using
method F and was obtained as a yellow solid in 63% yield (119
) δ 8.46 (br, 1H,
NH), 7.89 (d, 2H), 7.49 (s, 1H), 7.06 (d, 2H), 6.88 (s, 1H), 4.27
(q, 2H), 3.97 (s, 3H), 3.95 (s, 3H), 3.92 (m, 2H), 2.56 (t, 2H),
1
(
7
(
178 mg): mp 171-173 °C; H NMR (CDCl
.43 (s, 5H), 7.10 (d, 1H), 7.05 (s, 1H), 6.86 (br, 1H, NH), 4.02
s, 3H), 3.99 (s, 3H). Anal. C17
-(3-Br om op h en yl)a m in o-6,7-d im et h oxyisoq u in olin e
30). Compound 30 was prepared from 25 and 3-bromoaniline
3
) δ 7.99 (d, 1H),
1
mg): mp 133-135 °C (dec); H NMR (CDCl
3
H
15BrN
2
O
2
.
1
(
24 6 5
2.06 (m, 2H), 1.32 (t, 3H). Anal. C22H N O .
by using method D and was obtained as a white solid in 80%
Eth yl 4-(3,4-Dih yd r o-4-oxoqu in a zolin -3-yl)bu ta n oa te
(42). Compound 42 was prepared from 40 by using method E
1
yield (192 mg): mp 151-152 °C; H NMR (CDCl
3
) δ 8.03 (d,
H), 7.76 (s, 1H), 7.44 (d, 1H), 7.05-7.20 (m, 5H), 6.78 (br,
H, NH), 4.02 (s, 3H), 3.99 (s, 3H). Anal. C17
,7-Dim et h oxy-1-(2-h yd r oxyet h yl)a m in oisoqu in olin e
31). Compound 31 was made from 25 and ethanolamine by
using method D and was obtained as a white solid in 75% yield
1
1
and was obtained as a white solid in 72% yield (256 mg): mp
1
H
15BrN
2
O
2
.
40 °C; H NMR (CDCl
3
) δ 8.31 (d, 1H), 8.05 (s, 1H), 7.76 (t,
6
1H), 7.71 (d, 1H), 7.51 (t, 1H), 4.12 (q, 2H), 4.08 (t, 2H), 2.40
(t, 2H), 2.12 (m, 2H), 1.24 (t, 3H). Anal. C14
(
16 2 3
H N O .
Eth yl 3-(3,4-Dih yd r o-6,7-d im eth oxy-4-oxoqu in a zolin -
3-yl)p r op a n oa te (43). Compound 43 was prepared from 41
1
(
7
6
125 mg): mp 166-167 °C; H NMR (CDCl
3
) δ 7.81 (d, 1H),
.26 (s, 2H), 6.99 (br, 1H), 6.88 (d, 1H), 5.53 (br, 1H), 4.00 (s,
H), 3.90 (t, 2H), 3.77 (t, 2H). Anal. C13
E t h yl 4-(2-Ch lor o-6,7-d im et h oxyq u in a zol-4-yloxy)-
by using method E and was obtained as a white solid in 82%
1
H
16
N
2
O
3
.
yield (424 mg): mp 141-142 °C; H NMR (CDCl
3
) δ 8.14 (s,
1H), 7.63 (s, 1H), 7.11 (s, 1H), 4.25 (t, 2H), 4.13 (q, 2H), 3.99
(s, 6H), 2.88 (t, 2H), 1.22 (t, 3H). Anal. C15
bu ta n oa te (33) a n d Eth yl 2-Ch lor o-3,4-d ih yd r o-6,7-
d im eth oxy-4-oxy-3-qu in a zolin ebu ta n oa te (34). Alkylation
of 32 by using method E produced O-alkylated compound 33
in 14% yield (126 mg) and N-alkylated compound 34 in 66%
yield (605 mg).
18 2 5
H N O .
Eth yl 4-(3,4-Dih yd r o-6,7-d im eth oxy-4-oxoqu in a zolin -
3-yl)bu ta n oa te (44). Compound 44 was made from 41 by
using method E and was obtained as a white solid in 87% yield
1
(531 mg): mp 125-126 °C; H NMR (CDCl ) δ 7.96 (s, 1H),
3
Physical data for compound 33: mp 98-99 °C; 1H NMR
7.62 (s, 1H), 7.10 (s, 1H), 4.12 (q, 2H), 4.07 (t, 2H), 3.99 (s,
(
4
3
CDCl
3
) δ 7.29 (s, 1H), 7.20 (s, 1H), 4.62 (t, 2H), 4.13 (q, 2H),
20 2 5
6H), 2.40 (t, 2H), 2.12 (m, 2H), 1.24 (t, 3H). Anal. C16H N O .
Eth yl 5-(3,4-Dih yd r o-6,7-d im eth oxy-4-oxoqu in a zolin -
3-yl)p en ta n oa te (45). Compound 45 was made from 41 by
.01 (s, 3H), 4.00 (s, 3H), 2.52 (t, 2H), 2.24 (m, 2H), 1.23 (t,
H). Anal. C16
Physical data for compound 34: mp 103-105 °C; H NMR
CDCl ) δ 7.53 (s, 1H), 7.03 (s, 1H), 4.35 (t, 2H), 4.12 (q, 2H),
.98 (s, 3H), 3.97 (s, 3H), 2.44 (t, 2H), 2.12 (m, 2H), 1.24 (t,
H). Anal. C16
E t h yl 4-(2-Azid o-3,4-d ih yd r o-6,7-d im et h oxy-4-oxo-
qu in a zolin -3-yl)bu ta n oa te (35). To a solution of compound
4 (200 mg, 0.56 mmol) in 10 mL of wet DMF was added
H
19ClN
2
O
5
.
1
using method E and was obtained as a white solid in 61% yield
1
(
3
3
3
(491 mg): mp 110-112 °C; H NMR (CDCl
3
) δ 7.95 (s, 1H),
7.62 (s, 1H), 7.09 (s, 1H), 4.11 (q, 2H), 4.02 (t, 2H), 3.99 (s,
6H), 2.35 (t, 2H), 1.84 (m, 2H), 1.71 (m, 2H), 1.23 (t, 3H). Anal.
H
2 5
19ClN O .
17 22 2 5
C H N O .
E t h yl 4-(3,4-Dih yd r o-6,7-d ih yd r oxy-4-oxoqu in a zolin -
3-yl)bu ta n oa te (46). To a solution of compound 44 (200 mg,
0.62 mmol) and ethanethiol (0.7 mL) in 10 mL of dried CH
Cl under argon was added aluminum chloride (416 mg, 3.12
mmol). After the reaction mixture was stirred at room tem-
perature for 6 h, K CO and water were carefully added, and
the mixture was stirred for another 20 min and then extracted
with CH Cl . The extracts were dried over MgSO and solvent
was evaporated to yield a crude product, which was purified
by flash silica gel chromatography using 0-5% MeOH in CH
Cl as eluant to yield compound 46 as white solid in 57% yield
(104 mg): mp 195-197 °C; H NMR (DMSO-d
3
sodium azide (55 mg, 0.84 mmol), and the mixture was stirred
at 100 °C for 16 h. The reaction mixture was quenched with
water and extracted with EtOAc. The extracts were dried over
2
-
2
MgSO
4
and evaporated to yield a crude product which was
2
3
purified by flash silica gel chromatography using 35-50%
EtOAc in hexane as eluant to yield compound 35 in 85% yield
2
2
4
1
(
7
3
172 mg): mp 123-125 °C; H NMR (CDCl
3
) δ 7.72 (s, 1H),
.68 (s, 1H), 4.46 (t, 2H), 4.11 (s, 3H), 4.08 (q, 2H), 4.03 (s,
H), 2.45 (t, 2H), 2.23 (m, 2H), 1.22 (t, 3H). Anal. C16
Eth yl 4-(2-Am in o-3,4-d ih yd r o-6,7-d im eth oxy-4-oxo-
qu in a zolin -3-yl)bu ta n oa te (36). To a solution of compound
5 (140 mg, 0.4 mmol) in 10 mL of ethanol and 1 mL of
trifluoroacetic acid was added palladium on activated carbon
palladium content 10%, 20 mg). After being shaken under 50
2
-
H
19
N
5
O
5
.
2
1
6
) δ 10.00 (br,
2H, OH), 8.08 (s, 1H), 7.38 (s, 1H), 6.92 (s, 1H), 3.97 (q, 2H),
3.91 (t, 2H), 2.30 (t, 2H), 1.90 (m, 2H), 1.11 (t, 3H). Anal.
3
14 16 2 5
C H N O .
(
E t h yl
qu in a zolin -3-yl)bu ta n oa te (48). To a solution of compound
41 (1.00 g, 4.8 mmol) in 15 mL of concentrated H SO was
4-(3,4-Dih yd r o-6,7-d im et h oxy-5-n it r o-4-oxo-
psi of hydrogen for 24 h, the mixture was filtered with Celite,
the filter cake was washed with EtOH, and the filtrate was
basified to pH 8 with 2 N NaOH. The solvent was evaporated
to yield crude product which was crystalled from EtOH to yield
compound 36 as white solid in 94% yield (122 mg): mp 202-
2
4
added potassium nitrate (728 mg, 7.2 mmol) in several
portions. After it was stirred at room temperature for 6 h, ice
and 10 N NaOH were added to the mixture to a pH of 2-3 to

Synthesis of TNFR Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3871
precipitate the product. The product was filtered, washed with
water, and dried under vacuum to yield compound 47 as little
yellow solid in 60% (724 mg): mp 261-263 °C (dec), which
E t h yl 4-(5-Ch lor o-3,4-d ih yd r o-6,7-d im et h oxy-4-oxo-
qu in a zolin -3-yl)bu ta n oa te (55). To a solution of compound
52 (1.00 g, 4.5 mmol) in 15 mL of 6 N HCl solution at -15 °C
was added dropwise a solution of sodium nitrite (407 mg, 5.9
mmol) in 2 mL of water over 20 min. After the mixture was
stirred for 10 min, a cold suspension of CuCl (584 mg, 5.9
mmol) in 5 mL of 6 N HCl was added. The temperature of the
reaction was gradually raised to room temperature over 1 h,
and then the mixture was stirred at 60 °C for another hour.
After the pH of the mixture was adjusted to 5-6 with 10 N
NaOH solution, the mixture was filtered and the resulting
solid washed with EtOH to produce compound 54 as pale
yellow solid in 74% (804 mg), which was used directly in next
was used directly in next step without further purification;
1
H NMR (DMSO-d ) δ 12.40 (br, 1H, NH), 8.13 (s, 1H), 7.41
6
(
s, 1H), 4.02 (s, 3H), 3.84 (s, 3H).
Compound 48 was made from 47 by using method E and
was obtained as a white solid in 74% yield (2.62 g): mp 105-
1
1
2
06 °C; H NMR (CDCl
3
) δ 8.02 (s, 1H), 7.21 (s, 1H), 4.12 (q,
H), 4.03 (t, 2H), 4.02 (s, 3H), 3.95 (s, 3H), 2.37 (t, 2H), 2.08
(
m, 2H), 1.25 (t, 3H). Anal. C16
Eth yl 4-(5-Am in o-3,4-d ih yd r o-6,7-d im eth oxy-4-oxo-
qu in a zolin -3-yl)bu ta n oa te (49). To a solution of compound
8 (502 mg, 1.3 mmol) in 15 mL of ethanol was added
19 3 7
H N O .
1
step without further purification: mp >230 °C (dec); H NMR
4
(
3
DMSO-d
.94 (s, 3H), 3.75 (s, 3H).
Compound 55 was made from 54 by using method E and
was obtained as a white solid in 73% yield (742 mg): mp 120
6
) δ 12.13 (br, 1H, NH), 8.01 (s, 1H), 7.16 (s, 1H),
palladium on activated carbon (palladium content 10%, 60 mg).
After being shaken under 35 psi of hydrogen for 24 h, the
mixture was filtered with Celite, the filter cake washed with
EtOH, and the solvent was evaporated. Crystallization of the
crude product from EtOAc-hexane yielded compound 49 as
1
°
C; H NMR (CDCl
4.02 (t, 2H), 3.98 (s, 3H), 3.89 (s, 3H), 2.39 (t, 2H), 2.11 (m,
H), 1.25 (t, 3H). Anal. C16
Eth yl 4-(6,7-Diflu or o-4-oxoqu in a zolin -3-yl)bu ta n oa te
3
) δ 7.98 (s, 1H), 7.06 (s, 1H), 4.12 (q, 2H),
1
white solid in 89% yield (386 mg): mp 88-89 °C; H NMR
2
2 5
H19ClN O .
(
(
2
CDCl
3
2
) δ 7.84 (s, 1H), 6.49 (s, 1H), 6.30 (br, 2H, NH ), 4.12
q, 2H), 3.97 (t, 2H), 3.92 (s, 3H), 3.82 (s, 3H), 2.38 (t, 2H),
.08 (m, 2H), 1.24 (t, 3H). Anal. C16
Eth yl 4-(5-Am in o-3,4-d ih yd r o-6,7-d im eth oxy-4-oxo-
H
21
N
3
O
5
.
(66). Compound 57 was made from 56 by using method G and
was obtained as a pale yellow solid in 84% (456 mg), mp 265-
2
67 °C, which was used directly: 1H NMR (DMSO-d
6
) δ 12.48
qu in a zolin -3-yl)bu ta n oa te Hyd r och lor id e (50). To a solu-
tion of compound 49 (100 mg, 0.3 mmol) in 5 mL of 2-propanol
was added 1 equiv of 1 N HCl solution. The solvent was
evaporated, and ethanol was added and evaporated, repeated
(br, 1H, NH), 8.13 (s, 1H), 8.02 (dd, 1H), 7.74 (dd, 1H).
Compound 66 was obtained from 57 by using method E and
was obtained as a white solid in 92% (602 mg): mp 120 °C;
1
H NMR (CDCl
4.13 (q, 2H), 4.07 (t, 2H), 2.40 (t, 2H), 2.11 (m, 2H), 1.25 (t,
H). Anal. C14
Eth yl 4-(6-F lu or o-4-oxoqu in a zolin -3-yl)bu ta n oa te (67).
3
) δ 8.05 (dd, 1H), 8.03 (s, 1H), 7.49 (dd, 1H),
several times. Compound 50 was obtained as a white solid in
1
quantitative yield: mp 195-196 °C; H NMR (DMSO-d
6
) δ 8.74
3
14 2 2 3
H F N O .
(
s, 1H), 6.51 (s, 1H), 5.20 (br, 3H), 3.98 (q, 2H), 3.94 (t, 2H),
3
.87 (s, 3H), 3.66 (s, 3H), 2.37 (t, 2H), 1.94 (m, 2H), 1.13 (t,
H). Anal. C16 ‚HCl.
E t h yl 4-(5-Acet a m id o-3,4-d ih yd r o-6,7-d im et h oxy-4-
oxoqu in a zolin -3-yl)bu ta n oa te (51). A solution of compound
9 (200 mg, 0.6 mmol) and acetic anhydride (2 mL) in 8 mL of
3
H
21
N
3
O
5
Compound 58 was prepared from 56 by using method G and
was obtained as a white solid in 82% (404 mg), mp 255-257
°
C, which was used directly: 1H NMR (DMSO-d
6
) δ 12.37 (br,
1
1
H, NH), 8.08 (s, 1H), 7.78 (dd, 1H), 7.74 (dd, 1H), 7.69 (dd,
H).
Compound 67 was made from 58 by using method E and
4
dry pyridine was stirred at room temperature for 48 h, at
which time TLC showed the reaction was complete. The
solvent was evaporated, water was added, and the mixture
1
was obtained as a white solid in 80% (537 mg): mp 48 °C; H
NMR (CDCl
3
) δ 8.01 (s, 1H), 7.93 (dd, 1H), 7.72 (dd, 1H), 7.47
ddd, 1H), 4.12 (q, 2H), 4.07 (t, 2H), 2.40 (t, 2H), 2.12 (m, 2H),
was extracted with CH
MgSO and the solvent was evaporated to yield a crude
product, which was purified by flash silica gel chromatography
using 0-3% MeOH in CH Cl as eluant to yield compound 51
as white solid in 60% yield (136 mg): mp 104-105 °C; H NMR
CDCl ) δ 9.65 (br, 1H, NH), 7.88 (s, 1H), 6.93 (s, 1H), 4.08 (q,
H), 3.94 (t, 2H), 3.91 (s, 3H), 3.87 (s, 3H), 2.33 (t, 2H), 2.19
s, 3H), 2.02 (m, 2H), 1.19 (t, 3H). Anal. C18
E t h yl 4-(5-Azid o-3,4-d ih yd r o-6,7-d im et h oxy-4-oxo-
qu in a zolin -3-yl)bu ta n oa te (52). To a solution of compound
9 (450 mg, 1.3 mmol) in 10 mL of 4 N HCl at -15 °C was
2
2
Cl . The extracts were dried over
(
4
1
.24 (t, 3H). Anal. C14
2 3
H15FN O .
Eth yl 4-(7-F lu or o-4-oxoqu in a zolin -3-yl)bu ta n oa te (68).
2
2
1
Compound 59 was made from 56 by using method G and was
obtained as a white solid in 62% (305 mg), mp 247-248 °C,
(
3
which was used directly: 1H NMR (DMSO-d
NH), 8.17 (dd, 1H), 8.13 (s, 1H), 7.44 (dd, 1H), 7.38 (dd, 1H).
) δ 12.32 (br, 1H,
2
6
(
H
23
N
3
O
6
.
Compound 68 was prepared from 59 by using method E and
1
was obtained as a white solid in 91% (379 mg): mp 80 °C; H
NMR (CDCl
3
) δ 8.31 (dd, 1H), 8.05 (s, 1H), 7.35 (dd, 1H), 7.22
4
(dt, 1H), 4.13 (q, 2H), 4.06 (t, 2H), 2.40 (t, 2H), 2.12 (m, 2H),
added dropwise a solution of sodium nitrite (120 mg, 1.74
mmol) in 2 mL of water over 20 min. After the mixture was
stirred for 10 min, a solution of sodium azide (174 mg, 2.7
mmol) in 2 mL of water was added. The reaction mixture was
stirred at room temperature for 1 h, quenched with water, and
1
.24 (t, 3H). Anal. C14
2 3
H15FN O .
Eth yl 4-(5-Ch lor o-4-oxoqu in a zolin -3-yl)bu ta n oa te (69).
Compound 60 was prepared from 56 by using method G and
was obtained as a brown solid in 76% (546 mg), mp 270-272
°C, which was used directly: 1H NMR (DMSO-d
1H, NH), 8.07 (s, 1H), 7.71 (t, 1H), 7.59 (d, 1H), 7.51 (d, 1H).
) δ 12.30 (br,
extracted with CH
2
Cl
2
. The extracts were dried over MgSO
4
6
and the solvent was evaporated to yield a crude product, which
was purified by flash silica gel chromatography with 0-3%
Compound 69 was made from 60 by using method E as
1
MeOH in CH
2
Cl
2
as eluant. Crystallization of pure product
white needles in 80% (606 mg): mp 76 °C; H NMR (CDCl
3
) δ
fraction from ethyl ether yielded compound 52 as white needles
8.04 (d, 1H), 7.59-7.61 (m, 2H), 7.49 (dd, 1H), 4.13 (q, 2H),
4.04 (t, 2H), 2.40 (t, 2H), 2.12 (m, 2H), 1.24 (t, 3H). Anal.
1
in 73% yield (353 mg): mp 102-104 °C; H NMR (CDCl
3
) δ
.96 (s, 1H), 6.95 (s, 1H), 4.12 (q, 2H), 4.01 (t, 2H), 3.97 (s,
H), 3.92 (s, 3H), 2.38 (t, 2H), 2.10 (m, 2H), 1.24 (t, 3H). Anal.
7
3
14 2 3
C H15ClN O .
Eth yl 4-(6-Ch lor o-4-oxoqu in a zolin -3-yl)bu ta n oa te (70).
C
16
H
19
N
5
O
5
.
Compound 61 was made from 56 by using method G and was
obtained as a yellow solid in 85% (612 mg), mp 268-271 °C,
5
-Am in o-6,7-d im eth oxy-4(3H)-qu in a zolin on e (53). To a
which was used directly: 1H NMR (DMSO-d
) δ 12.44 (br, 1H,
solution of compound 47 (2.51 g, 10 mmol) in 80 mL of
methanol was added palladium on activated carbon (palladium
content 10%, 250 mg). After being shaken under 50 psi of
hydrogen for 20 h, the mixture was filtered with Celite and
the filter cake washed with EtOH. The solvent was evaporated
6
NH), 8.12 (s, 1H), 8.04 (d, 1H), 7.84 (dd, 1H), 7.69 (d, 1H).
Compound 70 was obtained from 61 by using method E and
1
was obtained as a white solid in 83% (624 mg): mp 83 °C; H
NMR (CDCl
(d, 1H), 4.12 (q, 2H), 4.06 (t, 2H), 2.39 (t, 2H), 2.11 (m, 2H),
1.24 (t, 3H). Anal. C14
3
) δ 8.25 (d, 1H), 8.03 (s, 1H), 7.68 (dd, 1H), 7.65
to yield compound 52 as pale yellow solid in 85% yield (1.87
1
g): mp >250 °C (dec); H NMR (DMSO-d
6
) δ 11.74 (br, 1H,
), 6.39 (s, 1H), 3.84 (s, 3H),
‚0.25H O.
2 3
H15ClN O .
NH), 7.82 (s, 1H), 6.72 (br, 2H, NH
.64 (s, 3H). Anal. C10
2
Eth yl 4-(7-Ch lor o-4-oxoqu in a zolin -3-yl)bu ta n oa te (71).
Compound 62 was made from 56 by using method G and was
3
H
11
N
3
O
3
2

3
872 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Chao et al.
obtained as a yellow solid in 81% (584 mg), mp 249-252 °C,
and the solvent was evaporated. The residue was purified by
1
which was used directly: H NMR (DMSO-d
6
) δ 12.41 (br, 1H,
flash silica gel chromatography with 2-4% MeOH/CH
2 2
Cl as
NH), 8.13 (s, 1H), 8.10 (d, 1H), 7.72 (m, 1H), 7.55 (m, 1H).
Compound 71 was made from 62 by using method E and
eluant to produce compound 78 as a white solid in 60% yield
1
(150 mg): mp 109-111 °C; H NMR (CDCl ) δ 7.73 (s, 1H),
3
was obtained as a white needles in 97% (728 mg): mp 90-91
6.19 (dd, 1H), 5.89 (d, 1H), 4.56 (s, 2H), 4.09 (br, q, 3H), 3.90
1
°
C; H NMR (CDCl
3
) δ 8.23 (d, 1H), 8.05 (s, 1H), 7.70 (d, 1H),
(br, 2H), 3.50 (t, 2H), 2.38 (t, 2H), 1.92 (m, 2H), 1.21 (t, 3H).
1
7
2
.45 (dd, 1H), 4.12 (q, 2H), 4.06 (t, 2H), 2.40 (t, 2H), 2.11 (m,
H), 1.24 (t, 3H). Anal. C14
Eth yl 4-(6-Br om o-4-oxoqu in a zolin -3-yl)bu ta n oa te (72).
Anal. C14
H
19
N
3
O
3
‚ /
8
H
2
O.
H
2 3
15ClN O .
Eth yl 4-(2-Dim eth yla m in o-6,7-d im eth oxyqu in a zol-4-
yloxy)bu ta n oa te (82). Compound 79 was made by using
method F and was obtained as a white solid in 82% yield (203
Compound 63 was prepared from 56 by using method G as
brown solid in 78% (524 mg), mp 267-269 °C, which was used
mg), which was used directly without further purification: mp
1
directly: H NMR (DMSO-d
1
6
) δ 12.44 (br, 1H, NH), 8.18 (d,
1
2
(
38-240 °C; H NMR (DMSO-d
6
) δ 10.83 (br, 1H, NH), 7.24
H), 8.13 (s, 1H), 7.95 (dd, 1H), 7.61 (d, 1H).
Compound 72 was made from 63 by using method E and
was obtained as a white solid in 86% (647 mg): mp 54-55 °C;
s, 1H), 6.73 (s, 1H), 3.82 (s, 3H), 3.77 (s, 3H), 3.04 (s, 6H).
Compound 82 was made from 79 by using method E and
was obtained as a white solid in 84% yield (152 mg): mp 79
1
H NMR (CDCl
3
) δ 8.43 (d, 1H), 8.05 (s, 1H), 7.83 (dd, 1H),
.58 (d, 1H), 4.12 (q, 2H), 4.07 (t, 2H), 2.40 (t, 2H), 2.11 (m,
H), 1.24 (t, 3H). Anal. C14
Eth yl 4-(6-Nitr o-4-oxoqu in a zolin -3-yl)bu ta n oa te (73).
1
°
4
2
C; H NMR (CDCl
3
) δ 7.17 (s, 1H), 6.80 (s, 1H), 4.55 (t, 2H),
.12 (q, 2H), 3.98 (s, 3H), 3.94 (s, 3H), 3.24 (s, 6H), 2.52 (t,
H), 2.21 (m, 2H), 1.22 (t, 3H). Anal. C18
Eth yl 4-(6,7-Dim eth oxy-2-p h en yla m in oqu in a zol-4-yl-
7
2
H
2 3
15BrN O .
25 3 5
H N O .
Compound 64 was made from 56 by using method G and was
oxy)bu ta n oa te (83). Compound 80 was made by using
method F and was obtained as a white solid in 73% yield (218
obtained as a yellow solid in 81% (465 mg), mp 287-288 °C,
1
which was used directly: H NMR (DMSO-d
6
) δ 12.77 (br, 1H,
mg), which was used directly without further purification: mp
NH), 8.80 (d, 1H), 8.54 (dd, 1H), 8.04 (s, 1H), 7.86 (d, 1H).
1
2
58-261 °C; H NMR (DMSO-d
6
) δ 10.65 (br, 1H, NH), 8.55
Compound 73 was obtained from 64 by using method E as
1
(br, 1H, NH), 7.72 (d, 2H), 7.31-7.35 (m, 3H), 7.01 (m, 1H),
.89 (s, 1H), 3.87 (s, 3H), 3.80 (s, 3H).
plates in 84% (512 mg): mp 88 °C; H NMR (CDCl
3
) δ 9.17 (d,
6
1
4
H), 8.54 (dd, 1H), 8.19 (s, 1H), 7.84 (d, 1H), 4.10-4.16 (m,
H), 2.42 (t, 2H), 2.14 (m, 2H), 1.25 (t, 3H). Anal. C14
Eth yl 4-(7-Nitr o-4-oxoqu in a zolin -3-yl)bu ta n oa te (74).
H
15
N
3
O
5
.
Compound 83 was made from 80 by using method E and
was obtained as a white solid in 77% yield (159 mg): mp 87
1
°
7
C; H NMR (CDCl
.02-6.98 (m, 3H), 4.57 (t, 2H), 4.13 (q, 2H), 4.00 (s, 3H), 3.97
(s, 3H), 2.54 (t, 2H), 2.24 (m, 2H), 1.23 (t, 3H). Anal.
3
) δ 7.74 (d, 2H), 7.35 (t, 2H), 7.24 (s, 1H),
Compound 65 was prepared from 56 by using method G and
was obtained as a brown solid in 79% (452 mg), mp 269-270
1
°
1
C, which was used directly: H NMR (DMSO-d
6
) δ 12.67 (br,
H, NH), 8.37 (s, 1H), 8.34 (d, 1H), 8.22-8.26 (m, 2H).
22 25 3 5
C H N O .
Compound 74 was made from 65 by using method E as
E t h yl
4-[2-(4-Br om op h en yl)a m in o-6,7-d im et h oxy-
1
plates in 78% (476 mg): mp 103-104 °C; H NMR (CDCl
3
) δ
.55 (d, 1H), 8.46 (d, 1H), 8.26 (dd, 1H), 8.16 (s, 1H), 4.12 (q,
H), 4.11 (t, 2H), 2.42 (t, 2H), 2.14 (m, 2H), 1.25 (t, 3H). Anal.
qu in a zol-4-yloxy]bu ta n oa te (84). Compound 81 was made
by using method F and was obtained as a white solid in 68%
yield (256 mg), which was used directly without further
purification: mp 238-240 °C; H NMR (DMSO-d ) δ 9.59 (br,
2H, NH), 7.65 (d, 2H), 7.55 (d, 2H), 7.33 (s, 1H), 6.59 (s, 1H),
8
2
C
1
14
H
15
N
3
O
5
.
6
Eth yl 4-(6-Am in o-4-oxoqu in a zolin -3-yl)bu ta n oa te (75).
To a solution of compound 73 (305 mg, 1.0 mmol) in 20 mL of
ethyl acetate was added palladium on activated carbon (pal-
ladium content 10%, 40 mg). After being shaken under 20 psi
of hydrogen for 30 min, the mixture was filtered with Celite,
3 3
3.86 (s, 3H), 3.81 (s, 3H). Anal. C16H14BrN O .
Compound 84 was made from 81 by using method E and
was obtained as a white solid in 82% yield (161 mg): mp 94
1
°
7
(
C; H NMR (CDCl
3
) δ 7.65 (d, 2H), 7.43 (d, 2H), 7.24 (s, 1H),
the filter cake was washed with CH
evaporated. The residue was purified by flash silica gel
chromatography with 0-3% MeOH/CH Cl as eluant to pro-
duce compound 75 as a white solid in 92% yield (345 mg): mp
2 2
Cl , and the solvent was
.03 (s, 1H), 6.96 (br, 1H, NH), 4.55 (t, 2H), 4.13 (q, 2H), 4.01
s, 3H), 3.98 (s, 3H), 2.54 (t, 2H), 2.23 (m, 2H), 1.23 (t, 3H).
Anal. C22
2
2
H
3 5
24BrN O .
1
Met h yl 4-[5-Azid o-6,7-d im et h oxy-4-oxoq u in a zolin -3-
yl]-2-bu ten oa te (86). To a solution of compound 53 (800 mg,
6
1
2
4 °C; H NMR (CDCl
H), 7.10 (dd, 1H), 4.12 (q, 2H), 4.04 (t, 2H), 3.98 (br, 2H),
.39 (t, 2H), 2.10 (m, 2H), 1.24 (t, 3H). Anal. C14
Eth yl 4-(7-Am in o-4-oxoqu in a zolin -3-yl)bu ta n oa te (76).
3
) δ 7.85 (s, 1H), 7.53 (d, 1H), 7.46 (d,
3
.6 mmol) in concentrated HCl (7 mL) and H
ice-salt bath (-15 °C) were dropped a solution of NaNO
mG, 4.0 mmol) in H O (2 mL) over 20 min and then added a
solution of NaN (468 mg, 7.2 mmol) in H O (2 mL). After the
2
O (3 mL) in an
17 3 3
H N O .
2
(276
2
The compound 76 was produced from 74, by the same
3
2
procedure used for compound 75, as syrup in 79% (296 mg):
1
cold bath was removed, the reaction mixture was stirred at
room temperature overnight, during which time a precipitate
was formed. The mixture was filtered and washed with H O.
2
The collected precipitate was dried to give compound 85 as
white solid in 75% yield (668 mg): mp >190 °C (dec); H NMR
H NMR (CDCl
dd, 1H), 6.65 (br, 2H), 4.12 (q, 2H), 4.03 (t, 2H), 2.39 (t, 2H),
.10 (m, 2H), 1.24 (t, 3H). Anal. C14
Eth yl 4-(6-Am in o-1,2-d ih yd r o-4-oxoqu in a zolin -3-yl)bu -
3
) δ 8.14 (d, 1H), 8.01 (s, 1H), 7.34 (d, 1H), 6.99
(
2
17 3 3
H N O .
1
ta n oa te (77). To a solution of compound 73 (180 mg, 0.6 mmol)
in 20 mL of ethyl acetate was added palladium on activated
carbon (palladium content 10%, 40 mg). After being shaken
under 20 psi of hydrogen for 3 h, the mixture was filtered with
(
3
6
DMSO-d ) δ 12.00 (br, 1H), 8.25 (s, 1H), 7.08 (s, 1H), 3.94 (s,
H), 3.82 (s, 3H). Without further purification, compound 85
was used in the next step.
Celite, the filter cake was washed with CH
was evaporated. The residue was purified by flash silica gel
chromatography with 2-4% MeOH/CH Cl as eluant to pro-
duce compound 77 as a pale yellow solid in 76% yield (140
2
Cl
2
, and the solvent
A solution of compound 85 (200 mg, 0.8 mmol), methyl
4-bromocrotonate (85%, 202 mg, 0.96 mmol), and K
mg, 2 mmol) in 10 mL of DMF was stirred at 70 °C for 8 h.
After DMF was evaporated by high vacuum, 15 mL of H
was added to the residue and the mixture extracted with CH
Cl . The extracts were dried over M SO and the solvent was
2 3
CO (276
2
2
2
O
1
mg): mp 54-56 °C; H NMR (CDCl
H), 7.46 (d, 1H), 7.10 (dd, 1H), 4.12 (q, 2H), 4.04 (t, 2H), 3.98
br, 2H), 2.39 (t, 2H), 2.10 (m, 2H), 1.24 (t, 3H). Anal.
3
) δ 7.85 (s, 1H), 7.53 (d,
2
-
1
(
2
2
4
evaporated. The crude product was purified by silica gel
1
C
14
H
19
N
3
O
3
‚ /
4
CH
2
Cl
2
.
chromatography using 50-100 EtOAc/hexane as eluant to give
Eth yl 4-(7-Am in o-1,2-d ih yd r o-4-oxoqu in a zolin -3-yl)bu -
compound 86 as a white solid in 73% yield (202 mg): mp 125-
1
ta n oa te (78). To a solution of compound 74 (250 mg, 0.82
mmol) in 20 mL of ethyl acetate was added palladium on
activated carbon (palladium content 10%, 40 mg). After being
shaken under 20 psi of hydrogen for 3 h, the mixture was
127 °C; H NMR (CDCl
3
) δ 7.88 (s, 1H), 6.99 (dt, 1H), 6.96 (s,
1H), 5.88 (dt, 1H), 4.70 (dd, 2H), 3.99 (s, 3H), 3.93 (s, 3H),
3.72 (s, 3H). Compound 86 was submitted to New England
Nulclear Products for tritiation and was catalytically reduced
to provide 87.
2 2
filtered with Celite, the filter cake was washed with CH Cl ,

Synthesis of TNFR Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3873
Ack n ow led gm en t. This work was supported in part
by Grant AR44850 from the National Institutes of
Health.
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