Relationships between the chemical structure of substances and their antimycobacterial activity against atypical strains. Part 18. 3-phenyl-2H-1,3-benzoxazine-2,4(3H)-diones and isosteric 3-phenylquinazoline-2,4(1H,3H)-diones

Article abstract of DOI:10.1135/cccc19991902

A series of 3-phenyl-2H-1,3-benzoxazine-2,4(3H)-diones 2 and 3-phenylquinazoline-2,4(1H,3H)-diones 5 substituted on the phenyl rings were synthesized. The target compounds as well as the intermediates were tested against Mycobacterium tuberculosis, M. kansasii, and M. avium. The replacement of the oxygen atom by nitrogen resulted in a decrease or loss of antimycobacterial activity. 2-[(Ethoxycarbonyl)amino]benzanilides 4 appeared to be inactive. Salicylanilides 1 and 3-phenyl-2H-1,3-benzoxazine-2,4(3H)-diones 2 exhibit significant activity against both M. tuberculosis and nontuberculous mycobacteria (the MICs within the range of 4-250 μmol/l for all compounds). The antimycobacterial activity of the compounds increases with increasing both electron-withdrawing properties and hydrophobicity of the substituent(s) on the phenyl moiety. The antimycobacterial profile of the compounds was analyzed according to the criteria based on vector algebra, such as cosine coefficients. Moreover, salicylanilides 1 exhibit activity against other microorganisms tested by the agar diffusion method.

Full text of DOI:10.1135/cccc19991902

1902
Waisser et al.:
RELATIONSHIPS BETWEEN THE CHEMICAL STRUCTURE OF
SUBSTANCES AND THEIR ANTIMYCOBACTERIAL ACTIVITY AGAINST
ATYPICAL STRAINS. PART 18. 3-PHENYL-2H-1,3-BENZOXAZINE-2,4(3H)-
DIONES AND ISOSTERIC 3-PHENYLQUINAZOLINE-2,4(1H,3H)-DIONES
a4
Karel WAISSERa1,*, Miloš MACHÁČEKa2, Hynek DOSTÁLa3, Jiří GREGOR
,
Lenka KUBICOVÁa5, Věra KLIMEŠOVÁa6, Jiří KUNEŠa7,b, Karel PALÁTa8, Jr.,
a
c
d
Jana HLADŮVKOVÁ , Jarmila KAUSTOVÁ and Ute MÖLLMANN
a
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,
500 05 Hradec Králové, Czech Republic; e-mail: waisser@faf.cuni.cz, machacek@faf.cuni.cz,
dostal@faf.cuni.cz, gregor@faf.cuni.cz, kubicova@faf.cuni.cz, klimesova@faf.cuni.cz,
1
2
3
7
4
5
6
8
kunes@faf.cuni.cz, palat@faf.cuni.cz
b
c
Laboratory of Structure and Interactions of Biologically Active Molecules, Faculty of Pharmacy,
Charles University, 500 05 Hradec Králové, Czech Republic
National Reference Laboratory for Mycobacterium kansasii, Regional Institute of Hygiene,
728 92 Ostrava, Czech Republic; e-mail: khsova@ha-vel.cz
d
Hans Knöll Institute of Natural Products Research, 07745 Jena, Germany
Received August 5, 1999
Accepted October 1, 1999
Dedicated to Professor Otto Exner on the occasion of his 75th birthday.
A
series of 3-ph en yl-2H-1,3-ben zoxazin e-2,4(3H)-dion es 2 an d 3-ph en ylquin azolin e-
2,4(1H,3H)-dion es 5 substituted on th e ph en yl rin gs were syn th esized. Th e target com -
poun ds as well as th e in term ediates were tested again st Mycobacterium tuberculosis, M.
kansasii, an d M. avium. Th e replacem en t of th e oxygen atom by n itrogen resulted in a de-
crease or loss of an tim ycobacterial activity. 2-[(Eth oxycarbon yl)am in o]ben zan ilides 4 ap-
peared to be in active. Salicylan ilides 1 an d 3-ph en yl-2H-1,3-ben zoxazin e-2,4(3H)-dion es 2
exh ibit sign ifican t activity again st both M. tuberculosis an d n on tuberculous m ycobacteria
(th e MICs with in th e ran ge of 4–250 µm ol/l for all com poun ds). Th e an tim ycobacterial ac-
tivity of th e com poun ds in creases with in creasin g both electron -with drawin g properties an d
h ydroph obicity of th e substituen t(s) on th e ph en yl m oiety. Th e an tim ycobacterial profile of
th e com poun ds was an alyzed accordin g to th e criteria based on vector algebra, such as co-
sin e coefficien ts. Moreover, salicylan ilides 1 exh ibit activity again st oth er m icroorgan ism s
tested by th e agar diffusion m eth od.
Key w ord s: Tuberculostatics; An tim icrobial activity; QSAR; 1,3-Ben zoxazin es; Quin azolin es;
Salicylan ilides; An th ran ilan ilides; Cosin e coefficien t.
+ For Part 17, see ref.1
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

Antimycobacterial Agents
1903
Th e search for substan ces active again st Mycobacterium avium is presen tly
on e of th e prim ary tasks of m edicin al ch em istry. Dissem in ated in fection
with th e M. avium com plex is th e m ost com m on system atic bacterial in fec-
tion com plicatin g AIDS (ref.2). In th e Czech Republic, an in crease in th e oc-
curren ce of M. kansasii h as been observed in som e areas3.
We h ave reported on a series of 3-ph en yl-2H-1,3-ben zoxazin e-2,4(3H)-
dion es an d th eir 6-m on o- an d 6,8-dih alogen o derivatives as a n ovel group
of an tim ycobacterially active com poun ds4. Recen tly, in itial studies of struc-
ture–activity relation sh ips of 6-h alogen o derivatives h ave been publish ed5.
Besides th e in fluen ce of th e h alogen substituen t, th e studies proved th at
th e activity of th e com poun ds again st M. tuberculosis an d M. kansasii in -
creases with in creasin g electron -acceptor properties of substituen t(s) on th e
ph en yl rin g. On th e con trary, for an adequate explan ation of ph ysico-
ch em ical in fluen ces on in h ibition of M. tuberculosis an d n on tuberculous
m ycobacteria, both electron ic an d h ydroph obic term s m ust be con sidered
in 6,8-dih alogen o com poun ds4b. Oth er biological properties of 3-ph en yl-
2H-1,3-ben zoxazin e-2,4(3H)-dion es were also reviewed6.
As an exten sion of our syn th etic study of 3-ph en yl-2H-1,3-ben zoxazin e-
2,4(3H)-dion es, a group of isosteric 3-ph en ylquin azolin e-2,4(1H,3H)-dion es
substituted on th e 3-ph en yl rin g was also in vestigated. Th e presen t report
describes th eir syn th esis an d th e activity of th ese com poun ds, as well as of
oth er syn th esized in term ediates, again st Mycobacterium tuberculosis, M.
kansasii, an d M. avium. With n ew an tim ycobacterial data at h an d, th e
quan titative structure–activity relation sh ips were rein vestigated. Furth er-
m ore, th e results of an tim icrobial screen in g of som e of th ese com poun ds
are reported. In addition , th e com poun ds were evaluated with regard to th e
con sidered th erapeutical use, i.e., as broad-spectrum an tim ycobacterial
agen ts. For th e sake of clarity, a very brief outlin e of th is procedure is also
provided.
No drug produces on ly a sin gle effect: a drug is adequately described on ly
by m ean s of th e full profile of effects. As a m easure of i-th biological activ-
ity, th e logarith m of th e reciprocal value of m olar con cen tration Ci of th e
evaluated com poun d required to produce i-th effect in th e predeterm in ed
in ten sity is used:
Ai = log 1/Ci = – log Ci ,
i = 1, …, n .
(1)
Each com poun d is th en represen ted by an activity vector,
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

1904
Waisser et al.:
A = (A1, …, An) ,
(2)
wh ere n is th e n um ber of th e activities studied.
Th e evaluation of th e com poun ds with regard to th e con sidered th era-
peutical use is based on th e idea of th e com parison with an “ideal” drug of
predefin ed profile of activities m akin g full use of th e vector represen tation .
Accordin gly, th e “ideal” drug is represen ted by vector B,
B = (B1, …, Bn) ,
or, m ore con ven ien tly, by th e un it vector U (g(U) = 1),
U = (U1, …, Un) = B/g(B) .
(3)
(4)
(5)
Th e Euclidean n orm of vector, e.g. A,
1/ 2
n
g(A) =
Ai2
,
∑
i=1
th en ch aracterizes th e size of th e vector.
For th e purpose of an evaluation , a com plex criterion S was proposed7 as
equal to scalar (dot) product of vectors represen tin g both com poun ds:
S = (A, U) .
(6)
Th e term “com plex” is given by th e fact th at th e criterion S can be decom -
posed in to two com pon en ts,
S = g(A) k(A, U) ,
(7)
of wh ich th e first con cern s th e evaluated com poun d itself an d th e oth er re-
flects its sim ilarity to th e “ideal” drug. Th e form er is th e above m en tion ed
Euclidean n orm of th e activity vector, g(A), as a m easure of th e overall po-
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Antimycobacterial Agents
1905
ten cy of th e evaluated com poun d an d th e latter is th e cosin e coefficien t
k(A, U),
k(A, U) = S/g(A) ,
(8)
as a m easure of th e relative sim ilarity of th e evaluated com poun d to th e
“ideal” drug.
Th e followin g two situation s are of great im portan ce. In th e case of
broad-spectrum drugs, th e “ideal” drug is represen ted by th e un it vector U0;
its com pon en ts are given by
U0 i = 1 n ,
i = 1,…, n
(9)
so th at th e form ula for calculatin g th e com plex criterion S0 can be written
as
n
1
S0 =
Ai .
(10)
∑
n
i=1
In th e case of th e selectivity of th e s-th effect, th e “ideal” drug is repre-
sen ted by th e un it vector Us, th e com pon en ts of wh ich are given by
(n – 1) / n n – 1
for i = s ,
(
)
)
Usi
=
(11)
– 1 / n n – 1
for i ≠ s , i = 1,…, n .
(
Th e n egative sign attributed to all but th e s-th effect reflects th e fact th at
th ey are con sidered to be un desired. Th e form ula for calculatin g th e com -
plex criteria Ss (com plex selectivities) can be written as
n
1
Ss =
nAs −
A
,
wh ere s = 1,…, n .
(12)
∑
i
n n – 1
i=1
(
)
Th e respective cosin e coefficien ts k0 an d ks are th en calculated accordin g to
Eq. (8).
Th e application of Han sch or Free–Wilson approach to th e com plex crite-
ria S is eviden t7.
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Waisser et al.:
EXPERIMENTAL
Th e m eltin g poin ts were determ in ed on a Kofler apparatus an d are un corrected. Th e sam ples
for an alysis an d an tim ycobacterial tests were dried over P4O10 at 61 °C an d 66 Pa for 24 h .
Elem en tal an alyses were perform ed on a CHNS-O CE elem en tal an alyser (Fision s EA 1110,
Milan o). Th e IR spectra were m easured in KBr pellets on a Nicolet Im pact 400 apparatus; th e
waven um bers are given in cm –1. Th e 1H NMR spectra of n ew com poun ds were recorded in
CDCl3 solution s (with th e exception of com poun ds 1j, 2k, 5c–5e, an d 5f wh ich were m ea-
sured in ((CD3)2SO) at am bien t tem perature on a Varian Mercury-Vx BB 300 spectrom eter
operatin g at 300 MHz. Ch em ical sh ifts were recorded as δ values an d were in directly refer-
en ced to tetram eth ylsilan e (for 1H 7.26 in CDCl3 an d 2.49 in ((CD3)2SO). Th e purity of th e
com poun ds was ch ecked by TLC (Silufol UV254, Kavalier, Votice, Czech Republic) in ben -
zen e–aceton e (1 : 3) for com poun ds 1, in cycloh exan e–aceton e (1 : 3) for com poun ds 2, an d
in ch loroform –aceton e (1 : 3 an d 1 : 9) for com poun ds 3–5, usin g UV detection .
Salicylan ilides 1a–1l
Th e title com poun ds were syn th esized from salicylic acid an d th e respective an ilin e as de-
scribed previously4b
.
Salicylanilide (1a). M.p. l33–135 °C (ref.8 m .p. 136–137 °C an d ref.9 m .p. 134–135 °C).
IR: 1 612.
4′-Methylsalicylanilide (1b). M.p. 154–156 °C (ref.8 m .p. 156–158 °C). IR: 1 603.
4′-Bromosalicylanilide (1c). M.p. 168–170 °C (ref.8 m .p. 169–172 °C). IR: 1 614.
4′-Methoxysalicylanilide (1d ). M.p. 158–160 °C (ref.10 m .p. 159–160 °C). IR: 1 628.
4′-Chlorosalicylanilide (1e). M.p. 165–167 °C (ref.8 m .p. 168–170 °C). IR: 1 615.
3′,4′-Dichlorosalicylanilide (1f). M.p. 213–215 °C (ref.11 m .p. 213–216 °C). IR: 1 614.
3′-Chlorosalicylanilide (1g). M.p. 173–175 °C (ref.8 m .p. 175–177 °C). IR: 1 616.
3′-Fluorosalicylanilide (1h ). M.p. 151–153 °C. For
C13H10FNO2 (231.2) calculated:
67.53% C, 4.36% H, 6.06% N; foun d: 67.53% C, 4.60% H, 6.11% N. IR: 1 615. 1H NMR
(CDCl3): 11.81 bs, 1 H (OH); 8.00 bs, 1 H (NH); 7.59–7.42 m , 3 H (H-4, H-6, H-2´); 7.39–7.29 m ,
1 H (H-5′); 7.26–7.21 m , 1 H (H-6′); 7.07–7.01 m , 1 H (H-3); 6.97–6.86 m , 2 H (H-5, H-4′).
4′-Fluorosalicylanilide (1i). M.p. 158–160 °C (ref.12 m .p. 160–161 °C). IR: 1 619.
3′-Nitrosalicylanilide (1j). M.p. 220–221 °C. For C13H10N2O4 (258.2) calculated: 60.47% C,
3.90% H, 10.85% N; foun d: 60.47% C, 4.00% H, 10.87% N. IR: 1 643. 1H NMR ((CD3)2SO):
10.70 bs, 1 H (OH); 8.80–8.70 m , 1 H (H-2′); 8.10–8.01 m , 1 H (H-4′); 8.00–7.92 m , 1 H
(H-6′); 7.92–7.86 m , 1 H (H-6); 7.62 t, 1 H, J = 8.0 (H-5′); 7.48–7.39 m , 1 H (H-4); 7.03–6.92 m ,
2 H (H-3, H-5).
4′-Nitrosalicylanilide (1k). M.p. 233–235 °C (ref.13 230 °C). IR: 1 616.
4′-(Dimethylamino)salicylanilide (1l). M.p. 150–152 °C. For C14H16N2O2 (256.3) calculated:
70.29% C, 6.29% H, 10.93% N; foun d: 69.97% C, 6.46% H, 11.14% N. IR: 1 615. 1H NMR
(CDCl3): 12.23 s, 1 H (OH); 7.83 bs, 1 H (NH); 7.53–7.47 m , 1 H (H-6); 7.47–7.41 m , 1 H
(H-4); 7.41–7.34 m (AA′BB′), 2 H (H-2′, H-6′); 7.05–6.99 m , 1 H (H-3); 6.94–6.86 m , 1 H
(H-5); 6.78–6.71 m (AA′BB′), 2 H (H-3′, H-5′); 2.95 s, 6 H (2 × (CH3).
3-Ph en yl-2H-1,3-ben zoxazin e-2,4(3H)-dion es 2a–2l
Th e title com poun ds were syn th esized from salicylan ilides 1 as described previously4b
.
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Antimycobacterial Agents
1907
3-Phenyl-2H-1,3-benzoxazine-2,4(3H)-dione (2a). M.p. 246–247 °C (ref.8 m .p. 247–248 °C).
IR: 1 770, 1 693.
3-(4-Methylphenyl)-2H-1,3-benzoxazine-2,4(3H)-dione (2b ). M.p. 224–225 °C (ref.8 m .p.
226–227 °C). IR: 1 761, 1 697.
3-(4-Bromophenyl)-2H-1,3-benzoxazine-2,4(3H)-dione (2c). M.p. 250–252 °C (ref.8 m .p.
247–248 °C). IR: 1 772, 1 695.
3-(4-Methoxyphenyl)-2H-1,3-benzoxazine-2,4(3H)-dione (2d ). M.p. 206–207 °C (ref.4a m .p.
207–208 °C). IR: 1 763, 1 701.
3-(4-Chlorophenyl)-2H-1,3-benzoxazine-2,4(3H)-dione (2e). M.p. 242–244 °C (ref.8 m .p.
243–244 °C). IR: 1 772, 1 708.
3-(3,4-Dichlorophenyl)-2H-1,3-benzoxazine-2,4(3H)-dione (2f). M.p. 212–214 °C (ref.14 m .p.
192–195 °C). For C14H7Cl2NO3 (308.1) calculated: 54.57% C, 2.29% H, 4.55% N; foun d:
54.55% C, 2.36% H, 4.34% N. IR: 1 774, 1 712.
3-(3-Chlorophenyl)-2H-1,3-benzoxazine-2,4(3H)-dione (2g). M.p. 196–198 °C (ref.8 m .p.
198–200 °C). IR: 1 756, 1 709.
3-(3-Fluorophenyl)-2H-1,3-benzoxazine-2,4(3H)-dione (2h ). M.p. 192–193 °C. For C14H8FNO3
(257.2) calculated: 65.37% C, 3.13% H, 5.45% N; foun d: 65.72% C, 3.27% H, 5.55% N. IR:
1 769, 1 697. 1H NMR (CDCl3): 8.16–8.08 m , 1 H (H-5); 7.82–7.72 m , 1 H (H-7); 7.56–7.32 m ,
3 H (H-6, H-8, H-5´); 7.25–7.16 m , 1 H (H-4´); 7.16–7.02 m , 2 H (H-2´, H-6´).
3-(4-Fluorophenyl)-2H-1,3-benzoxazine-2,4(3H)-dione (2i). M.p. 237–238 °C. For C14H8FNO3
(257.2) calculated: 65.37% C, 3.13% H, 5.45% N; foun d: 65.33% C, 3.34% H, 5.48% N. IR:
1 772, 1 709. 1H NMR (CDCl3): 8.17–8.09 m , 1 H (H-5); 7.82–7.73 m , 1 H (H-7); 7.46–7.34 m ,
2 H (H-6, H-8); 7.34–7.17 m , 4 H (H-2′, H-3′, H-5′, H-6′).
3-(3-Nitrophenyl)-2H-1,3-benzoxazine-2,4(3H)-dione (2j). M.p. 204–206 °C. For C14H8N2O5
(284.2) calculated: 59.16% C, 2.84% H, 9.86% N; foun d: 58.72% C, 2.81% H, 9.76% N. IR:
1 768, 1 705. 1H NMR (CDCl3): 8.41–8.32 m , 1 H (H-4′); 8.28–8.21 m , 1 H (H-2′); 8.17–8.10 m ,
1 H (H-5); 7.85–7.64 m , 3 H (H-7, H-5′, H-6′); 7.51–7.35 m , 2 H (H-6, H-8).
3-(4-Nitrophenyl)-2H-1,3-benzoxazine-2,4(3H)-dione (2k). M.p. 250–252 °C. For C14H8N2O5
(284.2) calculated: 59.16% C, 2.84% H, 9.86% N; foun d: 58.73% C, 3.09% H, 9.97% N. IR:
1 767, 1 707. 1H NMR ((CD3)2SO): 8.43–8.33 m (AA′BB′), 2 H (H-3′, H-5′); 8.04–7.97 m , 1 H
(H-5); 7.92–7.83 m , 1 H (H-7); 7.80–7.72 m (AA′BB′), 2 H (H-2′, H-6′); 7.55–7.44 m , 2 H (H-6,
H-8).
3-(4-Dimethylaminophenyl)-2H-1,3-benzoxazine-2,4(3H)-dione (2l). M.p. 302–303 °C. For
C
16H14N2O3 (282.3) calculated: 68.08% C, 5.00% H, 9.92% N; foun d: 68.22% C, 5.08% H,
9.93% N. IR: 1 760, 1 706. 1H NMR (CDCl3): 8.17–8.11 m , 1 H (H-5); 7.78–7.69 m , 1 H
(H-7); 7.44–7.31 m , 2 H (H-6, H-8); 7.18–7.10 m (AA′BB′), 2 H (H-2′, H-6′); 6.85–6.77 m
(AA′BB′), 2 H (H-3′, H-5′); 3.01 s, 6 H (2 × CH3).
Preparation of An th ran ilan ilides 3a–3h . Gen eral Procedure
A suspen sion of 2-(carboxyam in o)ben zoic an h ydride (7.2 g, 44.2 m m ol) in glacial acetic acid
(300 m l) was added gradually to a stirred solution of th e correspon din g an ilin e (44.2 m m ol)
in th e sam e solven t (100 m l). Th e stirred m ixture was slowly h eated to its reflux tem pera-
ture an d th en poured in to water (2 l). After 12 h , th e product was filtered off an d crystal-
lized from aqueous eth an ol (yield 70–85%).
Anthranilanilide (3a). M.p. 123–125 °C (ref.15 m .p. 125–126.5 °C). IR: 1 644.
4′-Methylanthranilanilide (3b). M.p. 149–150 °C (ref.15 m .p. 150–151 °C). IR: 1 635.
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

1908
Waisser et al.:
4′-Bromoanthranilanilide (3c). M.p. 161–163 °C (ref.15 m .p. 148–149 °C). For C13H11BrN2O
(267.1) calculated: 53.63% C, 3.81% H, 9.62% N; foun d: 53.15% C, 3.97% H, 9.42% N. IR:
1 637. 1H NMR (CDCl3): 7.79 bs, 1 H (NH); 7.50–7.40 m , 5 H (H-6, H-2′, H-3′, H-5′, H-6′);
7.30–7.22 m , 1 H (H-4); 6.75–6.67 m , 2 H (H-3, H-5); 5.49 bs, 2 H (NH2).
4′-Methoxyanthranilanilide (3d ). M.p. 123–125 °C (ref.15 m .p. 125–126 °C). IR: 1 635.
3′,4′-Dichloroanthranilanilide (3e). M.p. 146–147 °C (ref.16 m .p. 142–143 °C). For
C
13H10Cl2N2O (281.1) calculated: 55.54% C, 3.59% H, 9.96% N; foun d: 55.87% C, 3.20% H,
9.43 % N. IR: 1 638.
4′-Fluoroanthranilanilide (3f). M.p. 131–132 °C. For
C13H11FN2O (216.2) calculated:
67.82% C, 4.82% H, 12.17% N; foun d: 67.39% C, 4.97% H, 11.97% N. IR: 1 630. 1H NMR
(CDCl3): 7.78 bs, 1 H (NH); 7.55–7.48 m , 2 H (H-2′, H-6′), 7.48–7.43 m , 1 H (H-6); 7.30–7.22 m ,
1 H (H-4); 7.11–7.01 m , 2 H (H-3′, H-5′); 6.75–6.65 m , 2 H (H-3, H-5); 5.49 bs, 2 H (NH2).
3′-Nitroanthranilanilide (3g). M.p. 189–190 °C. For
C13H11N3O3 (257.2) calculated:
4.31% C, 60.70% H, 16.33% N; foun d: 60.25% C, 4.49% H, 16.22% N. IR: 1 633.
3′,4´-Dimethylanthranilanilide (3h ). M.p. 114–116 °C. For C15H16N2O (240.3) calculated:
74.97% C, 6.71% H, 11.66% N; foun d: 75.1% C, 6.58% H, 11.86% N. IR: 1 636. 1H NMR
(CDCl3): 7.66 s, 1 H (NH); 7.49–7.42 m , 1 H (H-6); 7.40–7.33 m , 1 H (H-2′); 7.31–7.20 m ,
2 H (H-4, H-6′); 7.15–7.08 m , 1 H (H-5′); 6.75–6.65 m , 2 H (H-3, H-5); 5.50 bs, 2 H (NH2);
2.27 s, 3 H (CH3); 2.25 s, 3 H (CH3).
Preparation of 2-[(Eth oxycarbon yl)am in o]ben zan ilides 4a–4h . Gen eral Procedure
Eth yl ch loroform ate (1 g, 9.24 m m ol) was added dropwise to a stirred solution of an appro-
priate an ilide 3 (7.7 m m ol) in dry pyridin e (15 m l) at 0 °C. Th e m ixture was h eated on a wa-
ter bath for 2 h an d th en poured in to water (100 m l). After 12 h , th e product was filtered
off an d crystallized from aqueous eth an ol (yield 75–85%).
2-[(Ethoxycarbonyl)amino]benzanilide (4a). M.p. 155–157 °C (ref.17 m .p. 153–154 °C). IR:
1 738, 1 636. 1H NMR (CDCl3): 9.99 s, 1 H (NH); 8.18 bs, 1 H (NH); 8.32–8.25 m , 1 H (H-6);
7.66–7.59 m , 2 H (H-2′, H-6′); 7.56–7.50 m , 1 H (H-3); 7.47–7.36 m , 3 H (H-5, H-3′, H-5′);
7.24–7.15 m , 1 H (H-4′); 7.07–6.98 m , 1 H (H-4); 4.21 q, 2 H, J = 14.16, J = 7.14 (CH2); 1.31 t,
3 H, J = 7.14 (CH3).
4′-Methyl-2-[(ethoxycarbonyl)amino]benzanilide (4b ). M.p. 162–164 °C. For C17H18N2O3
(298.3) calculated: 68.44% C, 6.08% H, 9.39% N; foun d: 68.65% C, 5.59% H, 9.81% N. IR:
1 706, 1 659. 1H NMR (CDCl3): 10.00 s, 1 H (NH); 8.30 bs, 1 H (NH); 8.30–8.15 m , 1 H
(H-6); 7.55–7.45 m , 3 H (H-3, H-2′, H-6′); 7.44–7.32 m , 1 H (H-5); 7.24–7.15 m (AA′BB′), 2 H
(H-3′, H-5′); 7.05–6.93 m , 1 H (H-4); 4.20 q, 2 H, J = 14.28, J = 7.15 (CH2); 2.35 s, 3 H (CH3);
1.30 t, 3 H, J = 7.15 (CH3).
4′-Bromo-2-[(ethoxycarbonyl)amino]benzanilide (4c). M.p. 156–158 °C. For C16H14BrN2O3
(363.2) calculated: 52.91% C, 4.16% H, 7.71% N; foun d: 53.14% C, 4.45% H, 7.41% N. IR:
1 702, 1 655. 1H NMR (CDCl3): 9.86 s, 1 H (NH); 8.40 bs, 1 H (NH); 8.22–8.14 m , 1 H (H-6);
7.65–7.43 m , 5 H (H-3, H-2′, H-3′, H-5′, H-6′); 7.42–7.31 m , 1 H (H-5); 7.05–6.93 m , 1 H
(H-4); 4.23 q, 2 H, J = 14.29, J = 7.15 (CH2); 1.33 t, 3 H, J = 7.15 (CH3).
4′-Methoxy-2-[(ethoxycarbonyl)amino]benzanilide (4d ). M.p. 164–165 °C. For C17H18N2O4
(314.3) calculated: 64.96% C, 5.77% H, 8.91% N; foun d: 64.91% C, 5.77% H, 8.84% N. IR:
1 739, 1 635. 1H NMR (CDCl3): 10.08 s, 1 H (NH); 8.03 bs, 1 H (NH); 8.35–8.25 m , 1 H
(H-6); 7.60–7.47 m , 1 H (H-3); 7.53–7.48 m (AA′BB′), 2 H (H-2′, H-6′); 7.47–7.37 m , 1 H
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

Antimycobacterial Agents
1909
(H-5); 7.10–6.98 m , 1 H (H-4); 6.95–6.90 m (AA′BB′), 2 H (H-3′, H-5′); 4.20 q, 2 H, J = 14.10,
J = 7.0 (CH2); 3.82 s, 3 H (OCH3); 1.29 t, 3 H, J = 7.0 (CH3).
3′,4′-Dichloro-2-[(ethoxycarbonyl)amino]benzanilide (4e). M.p. 174–176
°C. For
C
16H14Cl2N2O3 (353.2) calculated: 54.41% C, 4.00% H, 7.93% N; foun d: 54.53% C,
3.80% H, 7.94% N. IR: 1 727, 1 673. 1H NMR (CDCl3): 9.80 s, 1 H (NH); 8.45 bs, 1 H (NH);
8.20–8.13 m , 1 H (H-6); 7.98–7.93 m , 1 H (H-2′); 7.50–7.41 m , 3 H (H-3, H-5′, H-6′);
7.42–7.32 m , 1 H (H-5); 7.05–6.95 m , 1 H (H-4); 4.25 q, 2 H, J = 14.28, J = 7.15 (CH2); 1.34 t,
3 H, J = 7.15 (CH3).
4′-Fluoro-2-[(ethoxycarbonyl)amino]benzanilide (4f). M.p. 163–165 °C. For C16H14FN2O3
(302.3) calculated: 63.57% C, 5.00% H, 9.27% N; foun d: 63.30% C, 4.98% H, 9.24% N. IR:
1 707, 1 656. 1H NMR (CDCl3): 9.95 s, 1 H (NH); 8.22 bs, 1 H (NH); 8.28–8.23 m , 1 H (H-6);
7.65–7.55 m , 2 H (H-2′, H-6′); 7.55–7.47 m , 1 H (H-3); 7.45–7.35 m , 1 H (H-5); 7.15–6.97 m ,
3 H (H-4, H-3′, H-5′); 4.22 q, 2 H, J = 14.29, J = 7.14 (CH2); 1.31 t, 3 H, J = 7.14 (CH3).
3′-Nitro-2-[(ethoxycarbonyl)amino]benzanilide (4g). M.p. 183–185 °C. For
C16H15N3O5
(329.3) calculated: 58.36% C, 4.59% H, 8.97% N; foun d: 57.86% C, 4.39% H, 12.78% N. IR:
1 725, 1 659.
3′,4′-Dimethyl-2-[(ethoxycarbonyl)amino]benzanilide (4h ). M.p. 133–135 °C. For C18H20N2O3
(312.4) calculated: 69.21% C, 6.45% H, 8.97% N; foun d: 69.56% C, 6.22% H, 8.63% N. IR:
1 717, 1 663. 1H NMR (CDCl3): 10.06 s, 1 H (NH); 8.02 bs, 1 H (NH); 8.36–8.26 m , 1 H
(H-6); 7.56–7.48 m , 1 H (H-3); 7.48–7.35 m , 2 H (H-5, H-2′); 7.35–7.27 m , 1 H (H-6′);
7.18–7.10 m , 1 H (H-5′); 7.07–6.96 m , 1 H (H-4); 4.20 q, 2 H, J = 14.28, J = 7.0 (CH2); 2.28 s,
3 H (CH3); 2.26 s, 3 H (CH3); 1.30 t, 3 H, J = 7.0 (CH3).
Preparation of 3-Ph en ylquin azolin e-2,4(1H,3H)-dion es 5a–5h . Gen eral Procedure
An appropriate ben zan ilide 4 (0.5 g) was h eated at 250–270 °C for 10–20 m in . Th e product
was crystallized from aqueous aceton e (yield 75–85%).
3-Phenylquinazoline-2,4(1H,3H)-dione (5a). M.p. 280–281 °C (ref.18 m .p. 295–296 °C an d
ref.19 m .p. 272 °C). For C14H10N2O2 (238.2) calculated: 70.58% C, 4.23% H, 11.75% N;
foun d: 70.18% C, 4.18% H, 11.85% N. IR: 1 724, 1 672. 1H NMR (CDCl3): 8.17–8.13 m , 1 H
(H-5), 7.60–7.46 m , 4 H (H-7, H-3′, H-4′, H-5′); 7.35–7.30 m , 2 H (H-2′, H-6′); 7.28–7.20 m , 1 H
(H-6); 6.94–6.89 m , 1 H (H-8).
3-(4-Methylphenyl)quinazoline-2,4(1H,3H)-dione (5b). M.p. 275–277 °C (ref.20 m .p. 263–265 °C).
For C14H12N2O2 (252.3) calculated: 71.42% C, 4.79% H, 11.10% N; foun d: 71.34% C,
4.85% H, 11.17% N. IR: 1 720, 1 672. 1H NMR (CDCl3): 8.17–8.08 m , 1 H (H-5); 7.53–7.43 m ,
1 H (H-7); 7.38–7.31 m (AA′BB′), 2 H (H-2′, H-6′); 7.24–7.16 m , 3 H (H-6, H-3′, H-5′);
6.85–6.78 m , 1 H (H-8); 10.19 s, 1 H (NH); 2.45 s, 3 H (CH3).
3-(4-Bromophenyl)quinazoline-2,4(1H,3H)-dione (5c). M.p. 329–330 °C (ref.21 m .p. 297 °C).
For C14H9BrN2O2 (317.1) calculated: 53.02% C, 2.85% H, 8.83% N; foun d: 52.85% C,
2.83% H, 8.83% N. IR: 1 723, 1 672. 1H NMR ((CD3)2SO): 7.94–7.87 m , 1 H (H-5); 7.71–7.62 m ,
3 H (H-7, H-3′, H-5′); 7.32–7.25 m , 2 H (H-2′, H-6′); 7.24–7.15 m , 2 H (H-6, H-8).
3-(4-Methoxyphenyl)quinazoline-2,4(1H,3H)-dione (5d ). M.p. 311–312 °C (ref.20 m .p.
306–308 °C). For C15H12N2O3 (268.3) calculated: 67.16% C, 4.51% H, 10.44% N; foun d:
66.99% C, 4.44% H, 10.43% N. IR: 1 720, 1 669. 1H NMR ((CD3)2SO): 7.95–7.88 m , 1 H
(H-5); 7.72–7.63 m , 1 H (H-7); 7.25–7.16 m , 4 H (H-6, H-8, H-2′, H-6′); 7.04–6.96 m , 2 H
(H-3′, H-5′).
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

1910
Waisser et al.:
3-(3,4-Dichlorophenyl)quinazoline-2,4(1H,3H)-dione (5e). M.p. 328–329 °C (ref.21 m .p.
317 °C). For C14H8Cl2N2O2 (307.1) calculated: 54.57% C, 2.94% H, 9.09% N; foun d: 54.76% C,
2.69% H, 8.94% N. IR: 1 725, 1 674. 1H NMR ((CD3)2SO): 7.95–7.88 m , 1 H (H-5); 7.78–7.66 m ,
3 H (H-7, H-2′, H-5′); 7.48–7.36 m , 1 H (H-6′); 7.26–7.18 m , 2 H (H-6, H-8).
3-(4-Fluorophenyl)quinazoline-2,4(1H,3H)-dione (5f). M.p. 280–281 °C (ref.21 m .p. 272–274 °C).
For C14H9FN2O2 (256.2) calculated: 65.62% C, 3.54% H, 10.93% N; foun d: 65.22% C, 3.49% H,
10.95% N. IR: 1 724, 1 672. 1H NMR ((CD3)2SO): 7.92 dd, 1 H, J = 8.1, J = 1.5 (H-5);
7.73–7.64 m , 1 H (H-7); 7.41–7.17 m , 6 H (H-6, H-8 + C6H4F).
3-(3-Nitrophenyl)quinazoline-2,4(1H,3H)-dione (5g). M.p. 352–353 °C (ref.21 m .p. >350 °C).
For C14H9N3O4 (283.2) calculated: 59.37% C, 3.20% H, 14.84% N; foun d: 59.02% C, 3.25% H,
14.64% N. IR: 1 724, 1 676.
3-(3,4-Dimethylphenyl)quinazoline-2,4(1H,3H)-dione (5h ). M.p. 262 °C. For C16H14N2O2
(266.3) calculated: 72.17% C, 2.86% H, 10.52% N; foun d: 72.15% C, 2.85% H, 10.56% N. IR:
1 727, 1 674. 1H NMR (CDCl3): 8.17–8.10 m , 1 H (H-5); 7.52–7.43 m , 1 H (H-7); 7.33–7.27 m ,
1 H (H-5′); 7.25–7.15 m , 1 H (H-6); 7.11–7.02 m , 2 H (H-2′, H-6′); 6.88–6.80 m , 1 H (H-8);
10.05 s, 1 H (NH); 2.33 s, 3 H (CH3); 2.29 s, 3 H (CH3).
Microbiological Assays
Antimycobacterial Activity
For th e evaluation of th e an tim ycobacterial activity of th e substan ces in vitro, th e followin g
strain s were used: Mycobacterium tuberculosis CNCTC My 331/88, M. kansasii CNCTC My
235/80, an d M. avium CNCTC My 330/88, obtain ed from th e Czech Nation al Collection of
Type Cultures (CNCTC), Nation al In stitute of Public Health , Prague, an d a clin ic isolate of
M. kansasii 6509/96. An tim ycobacterial activity of th e com poun ds again st th ese strain s was
determ in ed in th e Šula sem isyn th etic m edium (SEVAC, Prague). Each strain was sim ulta-
n eously in oculated in to a Petri dish con tain in g th e Löwen stein –Jen sen m edium for th e con -
trol of th e sterility of th e in oculum an d its growth . Th e com poun ds were added to th e
m edium in dim eth yl sulfoxide solution s. Th e followin g con cen tration s were used: 1 000,
500, 250, 125, 62, 31, 16, 8, an d 4 µm ol/l. Th e m in im um in h ibitory con cen tration s (MICs)
were determ in ed after in cubation at 37 °C for 14 an d 21 days. MIC was th e lowest con cen -
tration of a substan ce, at wh ich th e in h ibition of th e growth occurred. Ison iazid was used as
stan dard.
General Antimicrobial Activity
An tim icrobial activity was determ in ed by th e agar diffusion test22. Th e Gram -positive an d
Gram -n egative bacteria, as well as th e fun gi used were from th e culture collection s (Bacillus
subtilis ATCC 6633, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 1031, an d Pseu-
domonas aeruginosa ATCC 27922) or from th e stock of th e Han s Kn öll In stitute of Natural
Products Research (Mycobacterium smegmatis SG 987, Staphylococcus aureus SG 511, P.
aeruginosa SG 137, Serratia marcescens SG 621, an d Sporobolomyces salmonicolor 549). Pen etra-
tion m utan t DC 2 an d its paren t strain E. coli DC 0 were described by Rich m on d et al.23
Pseudomonas aeruginosa K799/WT an d its pen etration m utan t K799/61 by Zim m erm an n 24
,
.
Stenotrophomas meltophilia GN 12873 was kin dly provided by Prof. S. Mitsuh ash i, Eisom e In -
stitute, Gun m a, Japan . Azlocillin e was used as stan dard.
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

Antimycobacterial Agents
1911
Calculation s
Log P Calculations
Th e calculation s of log P values were carried out with th e HyperCh em Suite for Win dows
(release 5.1), m odule Ch em Plus 1.6 usin g atom ic param eters derived by Gh ose et al.25 an d
later exten ded26
.
Quantitative Structure–Activity Relationship Study (QSARs)
All regression equation s were calculated with th e use of th e Multireg H program (Prof. P.
Klem era) for Microsoft Excel. Th e values of Ham m ett con stan ts σ an d h ydroph obic
substituen t con stan ts π were taken from th e literature27
.
Antimycobacterial Profile Evaluation
For th is purpose, logarith m s of reciprocal values of m in im um in h ibitory con cen tration s
were calculated as if expressed in m m ol/l to obtain on ly positive values. Activities again st
Mycobacterium tuberculosis CNCTC My 331/88, M. kansasii CNCTC My 235/80, an d M. avium
CNCTC My 330/88 form th e com pon en ts of vector A (in th e given order) represen tin g th e
evaluated com poun d. Subscripts 1, 2, an d 3 are used to furth er in dicate th e respective strain .
Com plex criteria S0 (Eq. (10)), S1 to S3 (Eq. (12)) an d th e n orm g(A) (Eq. (5)) were used for
th e calculation of th e respective cosin e coefficien ts k0 to k3 (Eq. (8)).
RESULTS AND DISCUSSION
Th e startin g salicylan ilides 1 were prepared from salicylic acid an d an ap-
propriate an ilin e in ch loroben zen e. Treatm en t of
1
with eth yl
ch loroform ate yielded th e correspon din g 3-ph en yl-2H-1,3-ben zoxazin e-
2,4(3H)-dion es 2 (Sch em e 1). All salicylan ilides 1 sh owed IR ban ds at
1 603–1 643 cm –1, wh ich are ch aracteristic of th e am ide C=O group, an d
1,3-ben zoxazin e-2,4(3H)-dion es 2 sh owed ch aracteristic absorption m axim a
of two C=O groups at 1 770–1 774 cm –1 an d 1 693–1 712 cm –1. Th e startin g
OH
OH
O
O
(i)
(ii)
H
N
N
COOH
R
R
O
O
1
2
1,2
a
b
c
d
R
H
1,2
i
j
k
l
R
4-F
3-NO2
4-NO2
1,2
e
f
g
h
R
4-Cl
3,4-Cl2
3-Cl
3-F
4-CH3
4-Br
4-OCH3
4-N(CH3)2
(i) substituted aniline, PCl3/chlorobenzene; (ii) ethyl chloroformate/pyridine
SCHEME 1
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

1912
Waisser et al.:
an th ran ilan ilides 3 were prepared from 2-(carboxyam in o)ben zoic an h y-
dride an d appropriate an ilin es in glacial acetic acid. Treatm en t of 3 with
eth yl ch loroform ate yielded 2-[(eth oxycarbon yl)am in o]ben zan ilides 4,
wh ich were th erm ally cyclized to 3-ph en ylquin azolin e-2,4(1H,3H)-dion es 5
(Sch em e 2). Th is m eth od gives products of h igh er purity an d in h igh er
yields as com pared with oth er m eth ods. An th ran ilan ilides 3 sh owed
characteristic bands of C=O groups at 1 630–1 644 cm –1, 2-[(ethoxycarbonyl)-
am in o]ben zan ilides 4 at 1 635–1 673 cm –1 an d 1 702–1 738 cm –1, an d
3-ph en ylquin azolin e-2,4(1H,3H)-dion es 5 at 1 669–1 676 cm –1 an d 1 720–
1 727 cm –1. Most of th e products h ave already been reported in th e litera-
ture8–21. Th e spectroscopic data of th e n ew com poun ds (1h , 1j, 1l, 2h –2l,
3f–3h , 4b–4h , an d 5h ) as well as of com poun ds with m eltin g poin ts differ-
en t from th e literature on es, are in agreem en t with th e assign ed structures.
13C NMR spectra of 4′-substituted com pounds 2 will be published separately28.
H
NH2
H
N
N
O
NHCOOC2H5
H
N
(i)
(ii)
O
R
R
R
O
O
O
4
3
(iii)
3,4,5
R
H
4-CH3
4-Br
3,4,5
R
H
a
b
c
d
e
f
3,4-Cl2
4-F
3-NO2
3,4-(CH3)2
O
N
N
g
h
4-OCH3
O
5
(i) substituted aniline/glacial acetic acid; (ii) ethyl chloroformate/pyridine; (iii) heating
SCHEME 2
All th e com poun ds prepared (with th e exception of 2h ) were tested in vi-
tro again st m ycobacterial strain s Mycobacterium tuberculosis, M. kansasii, an d
M. avium obtain ed from th e Czech Nation al Collection of Type Cultures
an d a clin ic isolate of M. kansasii 6509/96 (Tables I–IV). A com parison of
th e m in im um in h ibitory con cen tration s for an th ran ilan ilides 3 an d
3-ph en ylquin azolin e-2,4(1H,3H)-dion es 5 (Tables III an d IV) with th eir
h ydroxy/oxa an alogues (Tables I an d II) in dicates th at replacem en t of th e
oxygen atom by n itrogen resulted in
a decrease or loss of th e
an tim ycobacterial activity. 2-[(Eth oxycarbon yl)am in o]ben zan ilides 4 ap-
peared to be com pletely in active. It is worth n otin g th at 3′,4′-dich loro-
an th ran ilan ilide 3e displays th e lowest MICs of th e groups of com poun ds 3,
4, an d 5.
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

Antimycobacterial Agents
1913
Th e data in Tables I an d II sh ow th at th e values of MICs are with in th e
ran ge of 4–250 µm ol/l both for salicylan ilides 1 an d 3-ph en yl-2H-1,3-
ben zoxazin e-2,4(3H)-dion es 2. By com parin g th eir MICs with th at of
ison iazid (INH), th e m ost active 3,4-dich loro an alogues 2f an d 1f h ave ac-
tivity again st M. tuberculosis com parable to th at of INH (MIC = 4 µm ol/l).
However, all th e tested com poun ds 1 an d 2 possess activity again st atypical
m ycobacteria greater th an INH (MIC = 500 µm ol/l). Wh ile n on tuberculous
m ycobacteria are m oderately susceptible towards INH, th e n ewly prepared
com poun ds display virtually th e sam e activity again st all th e tested strain s.
A sim ilar profile of an tim ycobacterial activity h as recen tly been described
for 4-(ben zylsulfan yl)pyridin e derivatives29.
TABLE I
In vitro an tim ycobacterial activity of salicylan ilides 1 expressed as MIC (µm ol/l)
M. tuberculosis
My 331/88
M. kansasii
My 235/80
M. kansasii
6509/96
M. avium
My 330/88
Com poun d
14 days 21 days
14 days 21 days
14 days 21 days
14 days 21 days
1a
1b
1c
1d
1e
1f
62
62
16
62
31
8
62
62
31
62
31
8
125
62
16
250
31
4
250
125
31
62
62
16
125
16
4
125
125
16
125
31
4
62
31
125
62
31
31
250
31
62
125
31
31
8
16
31
1g
1h
1i
16
31
62
16
8
16
31
62
16
16
250
4
8
8
8
8
31
31
62
62
62
16
250
500
62
62
62
16
62
62
31
62
62
125
>62
16
31
62
1j
31
>31
31
1k
1l
16
16
31
a
a
250
4
>250
500
125
500
250
500
INHb
a
a
a
b
Not determ in ed. Ison iazid.
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

1914
Waisser et al.:
For th e detailed evaluation of th e differen ces in th e an tim ycobacterial
profiles of in dividual com poun ds (Tables V an d VI), a com plex criterion S0
ch aracterizin g equipoten cy of th e th ree com pared activities, com plex
selectivities S1–S3 towards th e in dividual m ycobacterial strain s an d th eir de-
com position in to respective cosin e coefficien ts k0–k3, an d th e n orm of ac-
tivity vector represen tin g given com poun d, g(A), were used. Th is treatm en t
is som ewh at m ore com plex th an th at in previous papers7 wh ere th e crite-
rion S0 an d its decom position were sufficien t. MICs for Mycobacterium tuber-
culosis, M. kansasii, an d M. avium obtain ed from CNCTC were used for th e
calculation . For better orien tation , th e clin ic isolate of M. kansasii was om it-
ted, as it exh ibits th e sam e or even better susceptibility th an M. kansasii
from th e CNCTC.
TABLE II
In vitro an tim ycobacterial activity of 3-ph en yl-2H-1,3-ben zoxazin e-2,4(3H)-dion es 2 expressed
as MIC (µm ol/l)
M. tuberculosis
My 331/88
M. kansasii
My 235/80
M. kansasii
6509/96
M. avium
My 330/88
Com poun d
14 days 21 days
14 days 21 days
14 days 21 days
14 days 21 days
2a
2b
2c
2d
2e
2f
2g
2h
2i
125
62
31
62
16
8
125
62
31
125
31
8
125
125
16
250
125
31
62
62
16
125
16
4
125
125
31
62
31
31
31
62
8
125
62
31
31
62
16
125
16
250
16
125
16
4
4
4
31
31
8
8
4
8
31
31
a
a
a
a
a
a
a
a
125
16
125
31
62
31
62
62
62
16
62
31
62
16
125
31
2j
2k
2l
16
16
16
16
16
16
16
31
a
a
>125
>125
>125
>125
>125
>125
a
Not determ in ed.
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

Antimycobacterial Agents
1915
TABLE III
In vitro an tim ycobacterial activity of an th ran ilan ilides 3 expressed as MIC (µm ol/l)
M. tuberculosis
M. kansasii
M. kansasii
M. avium
Com poun d
My 331/88
My 235/80
6509/96
My 330/88
14 days 21 days
>1 000 >1 000
14 days 21 days
>1 000 >1 000
14 days 21 days
>1 000 >1 000
14 days 21 days
500
500
3a
3b
3c
3d
3e
3f
>250
125
500
62
250
125
500
62
>250
125
>250
125
>250
62
>250
125
500
31
250
>125
>250
>62
>250
>125
>250
>62
>500
31
>500
62
500
31
>250
500
>125
>500
500
>125
>500
>500
>250
>500
>125
>125
125
>250
>250
125
3g
3h
>1 000 >1 000
>125 >125
>1 000 >1 000
>125 >125
TABLE IV
In vitro an tim ycobacterial activity of 3-ph en ylquin azolin e-2,4(1H,3H)-dion es 5 expressed as
MIC (µm ol/l)
M. tuberculosis
M. kansasii
M. avium
Com poun d
My 331/88
My 235/80
My 330/88
14 days
21 days
14 days
21 days
>250
14 days
21 days
>500
500
>500
250
>250
5a
5b
5c
5d
5e
5f
500
250
>500
>500
>500
>250
>250
200
250
>250
>125
>125
>125
>125
>250
500
>500
>250
>250
>250
>250
500
500
>125
>125
>125
>125
>62
>500
>250
>500
>125
>125
>125
250
>125
>62
>125
250
5g
5h
250
500
250
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

1916
Waisser et al.:
Th e h igh est values of th e n orm g(A) correspon d to th e m ost poten t
3,4-dich loro derivatives, 2f an d 1f. Th e sam e is true for th e values of com -
plex criterion S0, h owever, th e n on -zero values of th e correspon din g com -
TABLE V
Com plex criteria S, n orm g(A), cosin e coefficien ts k (see th e text), an d log P for salicyl-
an ilides 1
S0
S1
S2
S3
g(A)
k0
k1
k2
k3
log P
Com poun d
14 days / 21 days of in cubation
1a
1b
1c
1d
1.916
1.566
0.125 –0.249 0.125 1.932 0.992
0.372 –0.370 –0.002 1.624 0.965
0.064 –0.129 0.064 2.997
0.229 –0.228 –0.001
2.266 –0.123 –0.123 0.246 2.279 0.994 –0.054 –0.054 0.108 3.464
1.916
2.945
2.614
1.742
1.566
2.614
3.632
3.293
0.125 –0.249 0.125 1.932 0.992
0.117 0.117 –0.234 2.954 0.997
0.000 0.000 0.000 2.614 1.000
0.247 –0.495 0.247 1.811 0.962
0.372 –0.370 –0.002 1.624 0.965
0.000 0.000 0.000 2.614 1.000
0.000 0.369 –0.369 3.657 0.993
0.240 0.240 –0.480 3.327 0.990
0.064 –0.129 0.064
0.040 0.040 –0.079 3.788
0.000 0.000 0.000
0.137 –0.273 0.137 2.744
0.229 –0.228 –0.001
1ea
1f
0.000 0.000 0.000 3.515
0.000 0.101 –0.101 4.033
0.072 0.072 –0.144
1ga
1h a
1i
3.119 –0.006 0.363 –0.357 3.146 0.991 –0.002 0.115 –0.114 3.515
2.266 0.246 –0.123 –0.123 2.279 0.994 0.108 –0.054 –0.054 3.136
2.266 –0.123 –0.123 0.246 2.279 0.994 –0.054 –0.054 0.108 3.136
1.916
2.606
3.119
2.945
0.125 –0.249 0.125 1.932 0.992
0.357 –0.363 0.006 2.639 0.988
0.363 –0.006 –0.357 3.146 0.991
0.117 0.117 –0.234 2.954 0.997
0.064 –0.129 0.064
1jb
1k
0.135 –0.138 0.002 2.950
0.115 –0.002 –0.114 2.950
0.040 0.040 –0.079
1lb
1.216 –0.123 –0.123 0.246 1.241 0.980 –0.099 –0.099 0.198 3.261
INHa,c
1.732
1.712 –0.856 –0.856 2.435 0.711
0.703 –0.352 –0.352
–
a
b
No differen ce between th e values after 14 an d 21 days of in cubation . After 14 days of in -
c
cubation .
Ison iazid.
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

Antimycobacterial Agents
1917
plex are in dicative of differen t MICs for th e tested m ycobacterial strain s. To
avoid th is discrepan cy, cosin e coefficien ts k0 an d ks obtain ed as th e ratios
S0/g(A) an d Ss/g(A) sh ould be taken in to con sideration . Gen erally, th e m ore
th e value of th e cosin e coefficien t is close to 1, th e m ore th e profile of th e
evaluated com poun d relatively agrees with th at of th e “ideal” drug. As fol-
lows from Tables V an d VI, th e values of th e cosin e coefficien t k0
TABLE VI
Com plex criteria S, n orm g(A), cosin e coefficien ts
3-ph en yl-2H-1,3-ben z- oxazin e-2,4(3H)-dion es 2
k (see th e text), an d log P for
S0
S1
S2
S3
g(A)
k0
k1
k2
k3
log P
Com poun d
14 days / 21 days of in cubation
1.740 –0.125 –0.125 0.249 1.758 0.990 –0.071 –0.071 0.142 3.988
2a
2b
2c
2d
2e
2f
1.390
2.090
1.916
0.123 –0.246 0.123 1.412 0.985
0.002 –0.372 0.370 2.133 0.980
0.125 –0.249 0.125 1.932 0.992
0.087 –0.174 0.087
0.001 –0.174 0.174 4.455
0.064 –0.129 0.064
2.779 –0.117 0.234 –0.117 2.789 0.996 –0.042 0.084 –0.042 4.779
2.614
2.090
0.000 0.000 0.000 2.614 1.000
0.002 –0.372 0.370 2.133 0.980
0.000 0.000 0.000
0.001 –0.174 0.174 3.735
1.740 –0.125 –0.493 0.618 1.859 0.936 –0.067 –0.265 0.332
2.771
2.606
0.240 0.240 –0.480 2.813 0.985
0.006 0.357 –0.363 2.639 0.988
0.085 0.085 –0.171 4.506
0.002 0.135 –0.138
3.806 –0.123 0.246 –0.123 3.814 0.998 –0.032 0.064 –0.032 5.024
3.632 0.000 0.369 –0.369 3.657 0.993 0.000 0.101 –0.101
2ga
2i
2.953 –0.240 0.480 –0.240 2.992 0.987 –0.080 0.160 –0.080 4.506
1.916 –0.249 0.125 0.125 1.932 0.992 –0.129 0.064 0.064 4.127
1.740 –0.125 0.249 –0.125 1.758 0.990 –0.071 0.142 –0.071
2j
2.945
2.440
3.111
2.945
0.117 –0.234 0.117 2.954 0.997
0.123 –0.246 0.123 2.452 0.995
0.000 0.000 0.000 3.111 1.000
0.117 0.117 –0.234 2.954 0.997
0.040 –0.079 0.040 3.941
0.050 –0.100 0.050
2k
0.000 0.000 0.000 3.941
0.040 0.040 –0.079
a
No differen ce between th e values after 14 an d 21 days of in cubation .
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

1918
Waisser et al.:
TABLE VII
An tim icrobial activity of salicylan ilides 1
In h ibition zon e, m m
Microorgan ism
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l AZLa
B. subtilis
ATCC 663
A
15p 13/
16p
0
13/ 11
15p
12.5 12P 12P A13 17
0
31
S. aureus
SG 511
16P
A
23
10
0
0
0
0
0
0
0
0
20
17.5 23
17
0
13P 15
21
11
0
0
0
0
0
0
0
0
33
P. aeruginosa
K799/61
0
0
0
19
10
0
0
0
12
0
0
12
36
P. aeruginosa
K799/WT
14p 10
15p 11
10
18p 18p
13P 10
32
P. aeruginosa
SG 137
10.5
11.5
0
0
10.5 11
19
S. marcescens
SG 621
10P 11P 11.5
11.5 10P 11.5 11.5 10.5P 11.5p 12
18p
24
P. aeruginosa
ATCC 27853
0
0
0
0
0
A
0
16p 10.5
12p 14
13.5 10
10.5
14
0
10.5
0
0
0
10.5
K. pneumoniae
ATCC 10031
10.5P 13.5 11P
0
12P 14
16p
12
E. coli
ATCC 25922
10.5
A
0
10
A
10.5
A
0
10
0
11
0
S. meltophilia
GN 12873
0
11
11
0
11
0
11.5 10p
E. coli
DC 0
12P 10.5
0
0
10
0
10.5
0
11P 11
0
0
15
29
E. coli
DC 2
14/
21P
14/
13P
19
0
14
20
A
13.5 13P
A
A
25P
M. smegmatis
SG 987
0
0
20
17
17
18
17
16P
16P 22
13P 17
0
0
0
0
S. salmonicolor 14p 13P 15.5
A
16
13
549
a
Azlocillin e. A, In dication of in h ibition ; p, colon ies in th e in h ibition zon e; P, a n um ber of
colon ies in th e in h ibition zon e.
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

Antimycobacterial Agents
1919
(0.936–1.000) justify th e con clusion th at th e com poun ds represen t
broad-spectrum an tim ycobacterial agen ts. As can be seen for com poun ds
1c, 1e, an d 2c (results after 21 days of in cubation ), com poun ds with iden ti-
cal susceptibility to all con sidered strain s h ave k0 = 1 an d, sim ultan eously,
k1 = k2 = k3 = 0. Ran kin g of th e values of th e cosin e coefficien ts k1–k3 gives
th e order of susceptibilities to in dividual m ycobacterial strain s, wh ere th e
m axim um value in dicates th e m ost susceptible strain . For exam ple, values
of k2 after 21 days of in cubation in dicate th at com poun ds 1g, 2e, 2f, 2g,
an d 2i exh ibit a better efficacy again st M. kansasii th an again st M. tuberculo-
sis an d M. avium. If two strain s are equally susceptible, th e respective cosin e
coefficien ts h ave th e sam e value, wh ich is positive if th e oth er strain is less
susceptible, or n egative if th e oth er strain is m ore susceptible. It is worth
n otin g th at th e sum of all but on e cosin e coefficien t ks taken with th e op-
posite sign is equal to th e rem ain in g cosin e coeficien t ks.
Salicylan ilides 1 an d 3-ph en yl-2H-1,3-ben zoxazin e-2,4(3H)-dion es 2 were
also assessed for th eir ability to in h ibit th e growth of oth er m icroogan ism s
usin g th e agar diffusion m eth od, specified in DAB 9, at 100 µg/m l (ref.22)
(Tables VII an d VIII). In form ation about th e in fluen ce of th e pen etrability
of th e com poun ds in to th e bacterial cell on th eir an tibacterial activity is ob-
tain ed by a com parison of th e activity of “wild” type strain E. coli DC 0 an d
P. aeruginosa K799/WT with th eir pen etration m utan ts DC 2 an d K799/61
in an tim icrobial assays. Stron ger in h ibition of th e pen etration m utan ts
m ean s h in dered pen etrability. A sm all differen ce between th e in h ibition of
th e pen etration m utan t an d th e “wild” type in dicates good pen etrability.
TABLE VIII
An tim icrobial activity of 3-ph en yl-2H-1,3-ben zoxazin e-2,4(3H)-dion es 2
In h ibition zon e, m m
Microorgan ism
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
S. aureus
SG 511
0
0
0
0
A
0
0
14P 18.5 22
16
0
0
14
17
17
0
0
M. smegmatis
17P
16P 16
16p 18P
18P
SG 987
A, In dication of in h ibition ; p, colon ies in th e in h ibition zon e; P, a n um ber of colon ies in
th e in h ibition zon e.
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

1920
Waisser et al.:
TABLE IX
QSAR for salicylan ilides 1. MICs after 14 days (1) or 21 days (2) of in cubation ;
log 1/MIC(Mycobacterium) = a σ + b π + c
log 1/MIC(Mycobacterium) = a σ + b log P + c
(a),
(b)
Rela-
tion sh ip
Mycobacterium
a
b
c
r
s
F
n
Eq.
M. tuberculosis
My 331/88
1a
0.840 0.212 –1.732 0.970 0.119 73.37
±0.082 ±0.070 ±0.045
12
(13)
1b
2a
2b
1a
1b
2a
2b
1a
1b
2a
2b
0.886 0.245 –2.459 0.962 0.134 56.10
±0.072 ±0.099 ±0.328
12
12
12
12
12
10
10
11
11
11
11
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
0.757 0.198 –1.762 0.971 0.107 73.14
±0.074 ±0.063 ±0.04
0.800 0.230 –2.445 0.962 0.121 56.38
±0.081 ±0.096 ±0.318
M. kansasii
My 235/80
0.655 0.670 –2.017 0.939 0.215 33.46
±0.149 ±0.127 ±0.081
0.783 0.927 –4.809 0.949 0.197 40.62
±0.132 ±0.158 ±0.519
1.226 0.457 –2.181 0.937 0.226 25.04
±0.244 ±0.149 ±0.106
1.230 0.573 –3.853 0.927 0.243 21.26
±0.263 ±0.213 ±0.697
M. kansasii
6509/96
0.907 0.507 –1.891 0.946 0.169 34.23
±0.157 ±0.101 ±0.077
0.886 0.666 –3.842 0.937 0.182 28.96
±0.170 ±0.146 ±0.479
0.965 0.562 –2.041 0.938 0.198 29.23
±0.184 ±0.118 ±0.090
0.949 0.703 –4.091 0.913 0.233 19.92
±0.218 ±0.187 ±0.614
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

Antimycobacterial Agents
1921
TABLE IX
(Continued)
Rela-
Mycobacterium
tion sh ip
a
b
c
r
s
F
n
Eq.
M. avium
My 330/88
1a
1b
2a
2b
0.300 0.224 –1.745 0.885 0.12
±0.084 ±0.071 ±0.045
16.19
12
12
11
11
(25)
(26)
(27)
(28)
0.346 0.280 –2.580 0.865 0.129 13.40
±0.087 ±0.104 ±0.341
0.492 0.225 –1.936 0.935 0.124 27.79
±0.100 ±0.082 ±0.051
0.549 0.278 –2.762 0.929 0.13
±0.097 ±0.112 ±0.369
25.10
Com plex
criteria S0
1a
1.033 0.633 2.034 0.975 0.167 84.89
±0.116 ±0.099 ±0.063
12
(29)
a
1b
2a
2b
1.160 0.830 –0.455 0.968 0.187 67.00
±0.125 ±0.149 ±0.492
12
10
10
(30)
(31)
(32)
1.386 0.517 1.815 0.956 0.209 37.47
±0.225 ±0.138 ±0.098
1.390 0.649 –0.078 0.946 0.231
±0.251 ±0.203 ±0.664
29.96
a
Th e values followin g ± sign are stan dard errors.
See Table V.
In con trast to salicylan ilides 1, 3-ph en yl-2H-1,3-ben zoxazin e-2,4(3H)-
dion es 2 were essen tially in active in th e sam e an tim icrobial assays. On ly
two out of fourteen strain s, n am ely Staphylococcus aureus SG 511 an d, as ex-
pected, M. smegmatis SG 987, were susceptible to com poun ds 2c, 2e–2h , 2j,
an d 2k (Table VIII). Moreover, com poun ds 2g an d 2k in h ibited th e growth
of B. subtilis ATCC 6633 (in h ibition zon e 12 m m in diam eter with a n um -
ber of colon ies) an d th e m utan t strain of E. coli DC 2 gave an in dication of
in h ibition by th e sam e com poun ds (in h ibition zon e of 13.5 m m ). As iron
depletion is on e of th e defen se m ech an ism s foun d in th e in fected h ost,
growth in h ibition screen in g h as also been perform ed again st Mycobacterium
smegmatis SG 987 cultured in th e assay m edium supplem en ted with
2,2′-bis(2-h ydroxyph en yl)-2,2′-(eth ylen ediim in o)diacetic acid (EDDHA, 50
µm ol/l) as an iron ch elator. In terestin gly, som e com poun ds, e.g. 1c, 1e, an d
1k, as well as 2g, 2h , and 2k, showed increased activity under these conditions.
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

1922
Waisser et al.:
TABLE X
QSAR for 3-ph en yl-2H-1,3-ben zoxazin e-2,4(3H)-dion es 2. MICs after 14 days (1) or 21 days
(2) of in cubation ;
log 1/MIC(Mycobacterium) = a σ + b π + c
(a),
log 1/MIC(Mycobacterium) = a σ + b log P + c
(b)
Rela-
tion sh ip
Mycobacterium
a
b
c
r
s
F
n
Eq.
M. tuberculosis
My 331/88
1a
0.826 0.273 –1.873 0.865 0.234 10.38
±0.219 ±0.142 ±0.110
10
(33)
1b
2a
2b
1a
1b
2a
2b
1a
1b
2a
2b
1a
1b
2a
2b
0.826 0.313 –3.084 0.844 0.25
±0.235 ±0.203 ±0.869
8.64
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
(34)
(35)
(36)
(37)
(38)
(39)
(40)
(41)
(42)
(43)
(44)
(45)
(46)
(47)
(48)
0.829 0.360 –2.006 0.952 0.141 33.80
±0.131 ±0.085 ±0.066
0.820 0.451 –3.768 0.936 0.162 24.67
±0.152 ±0.132 ±0.562
M. kansasii
My 235/80
0.973 0.567 –1.994 0.945 0.199 29.34
±0.186 ±0.120 ±0.094
0.958 0.715 –4.787 0.924 0.233 20.38
±0.219 ±0.190 ±0.811
1.041 0.651 –2.172 0.910 0.290 16.89
±0.271 ±0.176 ±0.137
1.018 0.850 –5.505 0.901 0.304 15.01
±0.286 ±0.247 ±1.057
M. kansasii
6509/96
0.962 0.500 –1.839 0.929 0.216 22.17
±0.201 ±0.13 ±0.101
0.943 0.658 –4.418 0.923 0.225 20.11
±0.211 ±0.183 ±0.781
0.998 0.489 –1.992 0.916 0.240 18.31
±0.224 ±0.146 ±0.113
0.984 0.619 –4.411 0.900 0.261 14.96
±0.245 ±0.212 ±0.907
Com plex
criteria S0
1.322 0.553 2.014 0.942 0.245 27.74
±0.229 ±0.148 ±0.115
a
1.318 0.655 –0.532 0.915 0.295 18.08
±0.276 ±0.240 ±1.025
1.295 0.702 1.726 0.944 0.259 28.75
±0.242 ±0.157 ±0.122
1.284 0.860 –1.627 0.916 0.316 18.15
±0.297 ±0.258 ±1.101
a
Th e values followin g ± sign are stan dard errors.
See Table V.
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

Antimycobacterial Agents
1923
For th e quan titative evaluation of th e structure–an tim ycobacterial activ-
ity relation sh ips, two-param eter regression equation s were em ployed. Th e
Ham m ett con stan t σ of th e substituen t on th e ph en yl rin g served as on e
param eter, wh ile th e secon d param eter was th e h ydroph obic con stan t π of
th e sam e substituen t or th e calculated log P value. On ly th ose equation s for
salicylan ilides 1 an d 3-ph en yl-2H-1,3-ben zoxazin e-2,4(3H)-dion es 2 th at
are statistically sign ifican t at th e 95% level or better are listed in Tables IX
an d X, respectively. No sign ifican t correlation could be foun d for activity of
3-ph en yl-2H-1,3-ben zoxazin e-2,4(3H)-dion es 2 again st M. avium. Regardless
of th at, h igh ly sign ifican t correlation equation s were obtain ed between th e
sam e param eters an d th e com plex criteria S0 wh ich are lin ear com bin ation s
of th ree an tim ycobacterial activities n ot on ly for th e salicylan ilides 1
(Eqs (29)–(32)) but also for 3-ph en yl-2H-1,3-ben z- oxazin e-2,4(3H)-dion es 2
(Eqs (45)–(48)). Th e an tim ycobacterial activities of both groups of com -
poun ds, 1 an d 2, in crease with in creasin g electron -with drawin g properties
an d with in creasin g h ydroph obicity of th e substituen ts on th e ph en yl rin g
(relation sh ips (a)) or calculated log P (relation sh ips (b)). Sin ce th e relation -
sh ips (a) h ave h igh er correlation coefficien ts, th ey are con sidered to be m ore
sign ifican t th an th e relation sh ips (b). Th e structure–an tim ycobacterial activ-
ity relation sh ips are sim ilar n ot on ly in both groups of com poun ds, 1 an d
2, but th e Eqs (33) an d (37) are also con sisten t30 with th e relation sh ips for
th e 6,8-dih alogen o-3-ph en yl- 2H-1,3-ben zoxazin e-2,4(3H)-dion es4b. Th e
discrepan cy arisin g from th e com parison of th e previous results4b,5 can be
explain ed by th e fact th at th e MICs obtain ed for differen t m ycobacterial
strain s (an d in differen t laboratories) were com pared.
The authors wish to thank Ms J. Žižková from the Department of Inorganic and Organic Chemistry
for measurements of IR spectra, Ms D. Karlíčková from the Department of Pharmaceutical Chemistry
and Drug Control, Faculty of Pharmacy, Charles University, for elemental analyses, Ms B. Janíčková
and Ms M. Malíková from National Reference Laboratory for Mycobacterium kansasii, Regional Insti-
tute of Hygiene, Ostrava, for their assistance in antimycobacterial testing. The work was supported by
the Grant Agency of the Czech Republic (grant No. 203/99/0030) and by the Higher Education Devel-
opment Fund (grant No. 1273/99). Laboratory of Structure and Interactions of Biologically Active Mol-
ecules is supported by the Ministry of Education of the Czech Republic (project No. VS97124).
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Collect. Czech. Chem. Commun. (Vol. 64) (1999)