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European	Journal	of	Chemistry	3	(1)	(2012)	106‐111	

European	Journal	of	Chemistry	
ISSN	2153‐2249	(Print)	/	ISSN	2153‐2257	(Online)		2012	EURJCHEM	

DOI:10.5155/eurjchem.3.1.106‐111.489	

	
	

	

	

European	Journal	of	Chemistry	
Journal	homepage:	www.eurjchem.com	

	 	 	

Synthesis,	spectral	characterization	and	biological	evaluation	of		
4H‐1,4‐benzothiazines,	their	sulfones	and	ribofuranosides	

Naveen	Gautam,	Neha	Ajmera*,	Shikha	Gupta	and	Dinesh	Chand	Gautam	

Department	of	Chemistry,	University	of	Rajasthan,	Jaipur‐302004,	India	

*Corresponding	author	at:	Department	of	Chemistry,	University	of	Rajasthan,	Jaipur‐302004,	India.	Tel.:	+91.992.8537300;	fax:	+91.141.4004930.		
E‐mail	address:	ajmneha@yahoo.com	(N.	Ajmera).	

	
	

	 	

	 	 	
ARTICLE	INFORMATION	 	 ABSTRACT
Received:	16	July	2011	
Received	in	revised	form:	04	September	2011	
Accepted:	23	September	2011	
Online:	31	March	2012	

KEYWORDS	

	 Synthesis	 of	 heterocyclic	 compounds	 like	 benzothiazines	 has	 attracted	 attention	 in	 recent
years	 due	 to	 their	 biological	 and	 industrial	 value.	 This	 article	 reflects	 up‐to‐date	 and
comprehensive	 coverage	 of	 biochemical	 aspects	 of	 benzothiazines,	 their	 sulfones	 and
ribofuranosides.	 The	 nitrogen	 and	 sulfur	 containing	 heterocycles	 were	 prepared	 by
condensation	 followed	 by	 oxidative	 cyclization	 of	 2‐aminobenzenethiol	with	 β‐diketones/β‐
ketoesters	 in	 dimethylsulfoxide.	 These	 compounds	 were	 then	 used	 as	 base	 to	 prepare
ribofuranosides	 by	 treating	 them	with	 sugar	 (β‐D‐ribofuranose‐1‐acetate‐2,3,5‐tribenzoate).
On	refluxing	with	hydrogen	peroxide	in	glacial	acetic	acid,	these	substituted	dimethyl	4H‐1,4‐
benzothiazines	 yielded	 4H‐1,4‐benzothiazine‐1,1‐dioxides.	 Antioxidant	 and	 antimicrobial
activity	of	these	compounds	were	carried	out	and	structure	evaluation	was	done	by	spectral
and	elemental	analysis.	

Sulfones	
Heterocycles	
Ribofuranosides	
Antioxidant	activity	
Antimicrobial	activity	
4H‐1,4‐benzothiazines	

	
1.	Introduction	
	

The	 synthesis	 of	 benzothiazines,	 their	 sulfones	 and	
ribofuranosides	has	attracted	tremendous	interest	due	to	wide	
spectrum	of	biological	activities	possessed	by	these	compounds	
such	 as	 antibacterial,	 CNS	 depressants,	 antichloesterolic,	
anticancer,	 antifungal	 etc.	 As	 a	 part	 of	 the	 ongoing	 study,	we	
have	synthesized	some	new	benzothiazines,	their	sulfones	and	
ribofuranosides.	 Substituted	benzothiazines	were	prepared	by	
condensation	 of	 2‐aminobenzenethiols	 with	 β‐diketone/β‐
ketoester	 in	 presence	 of	 dimethylsulfoxide	 through	 oxidative	
cyclization.	 Intermediate	bis‐(2‐aminophenyl)disulfides	under‐
goes	 cyclization	 through	 scission	 of	 S‐S	 bond	 due	 to	 high	
reactivity	of	alpha	position	of	enaminoketone	system	 towards	
nucleophilic	 attack.	 The	 structures	 of	 these	 compounds	were	
determined	 on	 the	 basis	 of	 spectral	 data	 and	 elemental	
analysis.	 These	 compounds	 were	 also	 screened	 for	 biological	
activity	[1‐11].	
	
2.	Experimental	

	
All	 the	 melting	 points	 were	 determined	 in	 open	 capillary	

tubes	but	are	uncorrected.	1H	NMR	and	13C	NMR	were	recorded	
on	 JEOL	AL	 300	 spectrometer	 (300	MHz)	 in	DMSO‐d6	 /	 CDCl3	
using	 TMS	 (tetramethyl	 silane)	 as	 an	 internal	 standard	
(Chemical	 shifts	 are	 measured	 in	 δ,	 ppm).	 IR	 spectra	 were	
recorded	 in	 KBr	 on	 SHIMADZU	 8400	 S	 FTIR	 spectro‐
photometer.	Mass	 spectra	were	 recorded	 on	 JEOL	 SX	102/DA	
600	using	Xenon/Argon	as	FAB	(Fast	Atom	Bombardment)	gas.	
The	 purity	 of	 compounds	 were	 checked	 by	 thin	 layer	
chromatography	 using	 silica	 gel	 "G"	 as	 adsorbent,	 visualizing	
these	by	UV	light	or	in	an	Iodine	chamber.	Elemental	analysis	of	
these	compounds	was	also	done.	

	

2.1.	General	method	of	synthesis	of	substituted	4H‐1,4‐
benzothiazine	(3a‐d)	

	
To	 a	 stirred	 suspension	 of	 0.01	 mole	 of	 β‐diketone/β‐

ketoester	(2a‐d)	in	5	mL	of	dimethylsulfoxide	was	added	0.01	
mole	 of	 2‐aminobenzenethiol	 (1)	 and	 resulting	 mixture	 was	
refluxed	 for	 20	 minutes	 (Table	 1).	 The	 reaction	 mixture	 was	
concentrated,	 cooled	 down	 to	 room	 temperature.	 The	 solid	
separated	 out	was	 filtered,	washed	with	petroleum	ether	 and	
crystallized	 from	methanol	 (Scheme	 1).	 Characterization	 data	
and	spectral	analysis	of	each	compound	(3a‐d)	is	given	as:	

Ethyl‐3‐trifluoromethyl‐6,8‐dimethyl‐4H‐1,4‐benzothiazine‐
2‐carboxylate	 (3a):	 Yield:	 46%.	M.p.:	 55	 °C.	 IR	 (KBr,	 v,	 cm‐1):	
3260	(N‐H),	1690	(>C=O),	1340,	1160	(CF3	str.),	1255,	1015	(C‐
O‐C	str.),	2885	(‐CH3	str.).	1H	NMR	(DMSO‐d6,	300	MHz,	δ,	ppm):	
8.90	(s,	1H,	N‐H),	8.08‐7.20	(m,	2H,	aromatic‐H),	4.19	(q,	2H,	J	=	
6.9	Hz,	CH2	of	C2H5	at	C2),	1.30	(t,	3H,	J	=	6.2	Hz,	CH3	of	C2H5	at	
C2),	 2.35	 (s,	 3H,	 CH3	 at	 C6),	 2.36	 (s,	 3H,	 CH3	 at	 C8).	 13C	 NMR	
(CDCl3,	300	MHz,	δ,	ppm):	107.2	(C‐2),	138.6	(C‐3),	113.1	(C‐5),	
135.2	(C‐6),	120.2	(C‐7),	139.1	(C‐8),	21.2	(CH3	at	C6),	14.1	(CH3	
at	 C8),	 165	 (C	 of	 CO	 at	 C2),	 114	 (‐CF3	 at	 C3),	 59.2	 (CH2	 of	
COOC2H5	at	C2),	13.8	(CH3	of	COOC2H5	at	C2).	MS	(m/z,	%):	317	
(M+),	 244	 (52),	 202	 (38),	 275	 (76),	 73	 (100).	 Anal.	 calcd.	 for	
C14H14NO2F3S:	 C,	 52.99;	 H,	 4.41;	 N,	 4.41.	 Found:	 C,	 53.25;	 H,	
4.39;	N,	4.35%.	

2‐Trifluoroacetyl‐3‐trifluoromethyl‐6,8‐dimethyl‐4H‐1,4‐
benzothiazine	 (3b):	 Yield:	 68%.	M.p.:	 65	 °C.	 IR	 (KBr,	 v,	 cm‐1):	
3385	 (N‐H),	 1650	 (>C=O),	 1350,	 1180	 (CF3	 str.),	 2895	 (‐CH3	

str.).	 1H	 NMR	 (DMSO‐d6,	 300	MHz,	 δ,	 ppm):	 8.29	 (s,	 1H,	 NH),	
8.26‐6.06	(m,	2H,	aromatic‐H),	2.35	 (s,	3H,	CH3	at	C6),	2.36	(s,	
3H,	‐CH3	at	C8).	13C	NMR	(CDCl3,	300	MHz,	δ,	ppm):	116.2	(C‐2),	
136.3	 (C‐3),	112.2	 (C‐5),	136.4	 (C‐6),	119.2	 (C‐7),	138.2	 (C‐8),	
15.8	 (CH3	at	C6),	20.1	 (CH3	at	C8),	196.5	 (C	of	CO	at	C2),	129.8	
(CF3	at	C2),	114.1	(CF3	at	C3).		

	



Gautam	et	al.	/	European	Journal	of	Chemistry	3	(1)	(2012)	106‐111	 107	
 

	 	
Table	1.	The	exact	reaction	times	and	yields	of	individual	reactions	(3a‐d,	4a‐d,	5a‐b).	
Compound	No	 R3	 R4	 Reaction	times	(min.)	 Yield	(%)	
3a	 CF3	 OC2H5	 22 46	
3b	 CF3	 CF3	 20 68	
3c	 CH2CH3	 OCH3	 23 76	
3d	 CH3	 C6H3(OCH3)2 (o,	p)	 21 80	
4a	 CF3	 OC2H5	 255	 69	
4b	 CF3	 CF3	 275	 72	
4c	 CH2CH3	 OCH3	 295 56	
4d	 CH3	 C6H3(OCH3)2	(o,	p)	 260	 82	
5a	 CF3	 OC2H5	 920	 84	
5b		 CF3	 CF3	 980 68	

 

NH2

SH

R2

R1 NH2

S

R2

R1

OH O

OBzO OCOCH3

BzO OBz

N

S

O

BzO OBz

OBz

C–R4

R3

O

(1) (1´)

S

N R3

C–R4

O

H

N

S C–R4

R3

O

H

O O

N
H

S C

R3

O

R4

S

NH2

Dimethyl sulfoxide

R3–C=CH–C–R4
(2a-d)

(3a-d)

(5a-b)

10 hrs, stirred in
vacuum, 155-160 °C

..

..

(4a-d)

30% H2O2

Glacial acetic acid

1

2

345

6

7
8

1

2

345

6

7
8

1
2

3
4

5
6

7
8

1´
2´ 3´

4´

R1

R2

R2

R1

R1

R2

R1

R2

R2

R1

 
 

 R1 = CH3 
R2 = CH3 
R3 = CF3, CH2CH3, CH3 
R4 = OC2H5, CF3, OCH3, C6H3(OCH3)2 (o, p) 
 
Compound R3

3a, 4a, 5a CF3

3b, 4b, 5b CF3

3c, 4c 
3d, 4d 
 

 

Scheme	1
	
	
MS	 (m/z,	%):	 341	 (M+),	 244	 (58),	 202	 (29),	 327	 (78),	 97	

(100).	 Anal.	 calcd.	 for	 C13H9NOF6S:	 C,	 45.74;	 H,	 2.63;	 N,	 4.10.	
Found:	C,	45.98;	H,	2.65;	N,	4.06%.	

Methyl‐3‐ethyl‐6,8‐dimethyl‐4H‐1,4‐benzothiazine‐2‐
carboxylate	 (3c):	 Yield:	 76%.	 M.p.:	 115	 °C.	 IR	 (KBr,	 v,	 cm‐1):	
3280	(N‐H),	1700	(>C=O),	1240,	1055	(C‐O‐C	str.),	2875	(‐CH3	

str.).	 1H	NMR	 (DMSO‐d6,	 300	MHz,	 δ,	 ppm):	 8.40	 (s,	 1H,	 NH),	
7.29‐6.38	(m,	2H,	aromatic‐H),	2.35	(s,	3H,	‐CH3	at	C6),	2.36	(s,	
3H,	 ‐CH3	at	C8),	2.00	 (q,	2H,	 J	=	7.01	Hz,	CH2	of	CH2CH3	at	C3),	
1.06	(t,	3H,	 J	=	6	Hz,	CH3	of	CH2CH3	at	C3),	3.76	(s,	3H,	‐CH3	of	
OCH3	 at	 C2).	 13C	 NMR	 (CDCl3,	 300	MHz,	 δ,	 ppm):	 108.2	 (C‐2),	
141.7	 (C‐3),	118.2	 (C‐5),	139.1	 (C‐6),	114.2	 (C‐7),	142.2	 (C‐8),	
19.6	(CH3	at	C6),	14.8	(CH3	at	C8),	24.1	(CH2	of	CH2CH3	at	C3),	8.5	
(CH3	of	CH2CH3	at	C3),	165.0	(C	of	CO	at	C2),	50.1	(C	of	OCH3	at	

C2).	MS	 (m/z,	%):	 263	 (M+),	 204	 (56),	 162	 (31),	 221	 (71),	 59	
(100).	 Anal.	 calcd.	 for	 C14H17NO2S:	 C,	 63.87;	 H,	 6.46;	 N,	 5.32.	
Found:	C,	64.11;	H,	6.40;	N,	5.39%.	

2‐(2´,4´‐Dimethoxybenzoyl)‐3,6,8‐trimethyl‐4H‐1,4‐benzo‐
thiazine	(3d):	Yield:	80%.	M.p.:	53	°C.	IR	(KBr,	v,	cm‐1):	3298	(N‐
H),	 1690	 (>C=O),	 1260,	 1060	 (C‐O‐C	 str.),	 2886	 (CH3	 str.).	 1H	
NMR	(DMSO‐d6,	300	MHz,	δ,	ppm):	9.09	(s,	1H,	NH),	7.16‐6.08	
(m,	2H,	aromatic‐H),	1.71	(s,	3H,	‐CH3	at	C3),	3.73	(s,	3H,	‐OCH3	
at	ortho	position	of	‐COC6H3	(OCH3)2	(o,p)	at	C2),	3.78	(s,	3H,	‐
OCH3	at	para	position	of	‐COC6H3	(OCH3)2	(o,	p)	at	C2),	2.14	(s,	
3H,	 CH3	 at	 C6),	 2.20	 (s,	 3H,	 CH3	 at	 C8),	 7.20‐5.95	 (m,	 3H,	
aromatic‐H	 of	 benzoyl	 group).	 13C	 NMR	 (CDCl3,	 300	 MHz,	 δ,	
ppm):	112.9	(C‐2),	138.6	(C‐3),	111.9	(C‐5),	136.2	(C‐6),	118.2	
(C‐7),	146.2	(C‐8),	16.5	(CH3	at	C3),	187	(C	of	CO	at	C2),	56.8	(C	



108	 Gautam	et	al.	/	European	Journal	of	Chemistry	3	(1)	(2012)	106‐111	
	
of	 OCH3	 at	 ortho	 position	 ‐COC6H3	 (OCH3)2	 (o,	 p)	 at	 C2).	 MS	
(m/z,	%):	355	(M+),	190	(61),	137	(48),	148	(28),	313	(70),	165	
(100).	 Anal.	 calcd.	 for	 C20H21NO3S:	 C,	 67.60;	 H,	 5.91;	 N,	 3.94.	
Found:	C,	67.88;	H,	5.89;	N,	3.89%.	

	
2.2.	General	method	of	synthesis	of	4H‐1,4‐benzothiazine,	
1,1‐dioxides	(sulfones)	(4a‐d)	

	
30%	Hydrogen	peroxide	(5	mL)	was	added	to	a	solution	of	

0.01	mole	of	4H‐1,4‐benzothiazine	 in	20	mL	glacial	acetic	acid	
and	refluxed	for	15	minutes	at	50‐55	°C.	Heating	was	stopped	
and	another	lot	of	5	mL	of	30%.	Hydrogen	peroxide	was	added.	
The	reaction	mixture	was	again	refluxed	for	4‐5	hrs.	The	excess	
of	solvent	was	removed	by	distillation	under	reduced	pressure	
and	the	solution	was	poured	into	a	beaker	containing	crushed	
ice.	 The	 yellow	 residue	 separated	 out	 was	 filtered	 and	 then	
crystallized	 from	 ethanol	 (Scheme	 1).	 Characterization	 and	
spectral	data	of	these	compounds	(4a‐d)	is	given	as:	

Ethyl‐3‐trifluoromethyl‐6,8‐dimethyl‐4H‐1,4‐benzothiazine‐
2‐carboxylate‐1,1‐dioxide	(4a):	Yield:	69%.	M.p.:	70	°C.	IR	(KBr,	
v,	cm‐1):	3270	(N‐H),	1700	(>C=O),	1180,	1140	(SO2	sym.	str.),	
1080	(C‐S	str.).	 1H	NMR	(DMSO‐d6,	300	MHz,	δ,	ppm):	8.95	(s,	
1H,	NH),	8.06‐7.30	(m,	2H,	aromatic‐H),	4.20	(q,	J	=	6.95	Hz,	2H,	
CH2	of	C2H5	a	C2),	1.29	(t,	3H,	J	=	6.3	Hz,	CH3	of	C2H5	at	C2),	2.35	
(s,	3H,	‐CH3	at	C6),	2.36	(s,	3H,	‐CH3	at	C8).	13C	NMR	(CDCl3,	300	
MHz,	δ,	ppm):	96.2	(C‐2),	150.1	(C‐3),	114.9	(C‐5),	143.4	(C‐6),	
120.9	 (C‐7),	 136.4	 (C‐8),	 114.5	 (‐CF3	 at	 C3),	 168	 (C	 of	 CO	 of	
COOC2H5	 at	 C2),	 59.6	 (CH2	 of	 COOC2H5	 at	 C2),	 13.7	 (CH3	 of	
COOC2H5	at	C2).	MS	(m/z,	%):	349	(M+),	276	(51),	234	(29),	307	
(75),	73	(100).	Anal.	calcd.	for	C14H14NO4F3S:	C,	48.13;	H,	4.01;	
N,	4.01.	Found:	C,	48.35;	H,	4.05;	N,	4.03%.	

2‐Trifluoroaceyl‐3‐trifluoromethyl‐6,8‐dimethyl‐4H‐1,4‐
benzothiazine‐‐1,1‐dioxide	 (4b):	 Yield:	 72%.	 M.p.:	 220	 °C.	 IR	
(KBr,	v,	cm‐1):	3390	(N‐H),	1680	(>C=O),	1172,	1150	(SO2	sym.	
str.),	1085	(C‐S	str.).	1H	NMR	(DMSO‐d6,	300	MHz,	δ,	ppm):	8.30	
(s,	1H,	NH),	8.16‐7.08	(m,	2H,	aromatic‐H),	2.35	(s,	3H,	‐CH3	at	
C6),	2.36	(s,	3H,	‐CH3	at	C8).	13C	NMR	(CDCl3,	300	MHz,	δ,	ppm):	
101	(C‐2),	154	(C‐3),	118	(C‐5),	144	(C‐6),	121	(C‐7),	146	(C‐8),	
21.2	(CH3	at	C6),	13.7	(‐CH3	at	C3),	114	(‐CF3	at	C3),	196.8	(C	of	
CO	at	C2),	128.8	 (CF3	at	C2).	MS	(m/z,	%):	373	(M+),	276	 (48),	
234	 (28),	 331	 (72),	 97	 (100).	 Anal.	 calcd.	 for	 C13H9NO3F6S:	 C,	
41.82;	H,	2.41;	N,	3.75.	Found:	C,	42.09;	H,	2.39;	N,	3.78%.	

Methyl‐3‐Ethyl‐6,8‐dimethyl‐4H‐1,4‐benzothiazine‐2‐
carboxylate‐1,1‐dioxide	 (4c):	Yield:	56%.	M.p.:	240	°C.	 IR	 (KBr,	
v,	cm‐1):	3290	(N‐H),	1710	(>C=O),	1165,	1160	(SO2	sym.	str.),	
1095	(C‐S	str.).	 1H	NMR	(DMSO‐d6,	300	MHz,	δ,	ppm):	8.80	(s,	
1H,	NH),	7.30‐6.40	(m,	2H,	aromatic‐H),	2.16	(q,	2H,	J	=	7.2	Hz,	
CH2	of	CH2CH3	at	C3),	1.09	(t,	3H,	 J	=	6.4	Hz,	CH3	of	CH2CH3	at	
C3),	3.79	(s,	3H,	‐CH3	of	OCH3	at	C2),	1.86	(s,	3H,	CH3	at	C6),	2.31	
(s,	3H,	CH3	at	C8).	13C	NMR	(CDCl3,	300	MHz,	δ,	ppm):	99.2	(C‐2),	
144	(C‐3),	113.8	(C‐5),	145.1	(C‐6),	120.9	(C‐7),	143	(C‐8),	25.2	
(CH2	of	CH2CH3	at	C3),	8.9	(CH3	at	CH2CH3	at	C3).	MS	(m/z,	%):	
295	 (M+),	 236	 (38),	 194	 (30),	 253	 (76),	 59	 (100).	Anal.	 calcd.	
for	 C14H17NO4S:	 C,	 56.94;	H,	 5.76;	N,	 4.74.	 Found:	 C,	 57.22;	H,	
5.70;	N,	4.78%.	

2‐(2´,4´‐Dimethoxybenzoyl)‐3,6‐8‐trimethyl‐4H‐1,4‐benzo‐
thiazine‐1,1‐dioxide	(4d):	Yield:	82%.	M.p.:	81	°C.	IR	(KBr,	v,	cm‐

1):	3320	(N‐H),	1700	(>C=O),	1177,	1150	(SO2	sym.	str.),	1077	
(C‐S	 str.).	 1H	 NMR	 (DMSO‐d6,	 300	MHz,	 δ,	 ppm):	 9.12	 (s,	 1H,	
NH),	 7.23‐6.09	 (m,	 2H,	 aromatic‐H),	 1.92	 (s,	 3H,	 ‐CH3	 at	 C3),	
3.92	(s,	3H,	‐OCH3	at	ortho	position	of	‐COC6H3(OCH3)2	(o,	p)	at	
C2),	3.79	(s,	3H,	‐OCH3	at	para	position	of	COC6H3(OCH3)2	(o,	p)	
at	C2).	13C	NMR	(CDCl3,	300	MHz,	δ,	ppm):	97	(C‐2),	153	(C‐3),	
113.8	 (C‐5),	 138	 (C‐6),	 123	 (C‐7),	 145	 (C‐8),	 16.2	 (CH3	 at	 C3),	
189	(C	of	CO	of	COC6H3(OCH3)2	(o,	p)	at	C2),	56.9	(C	of	OCH3	at	
ortho	position	COC6H3(OCH3)2	 (o,	 p)	 at	 C2).	MS	 (m/z,	%):	 387	
(M+),	222	 (50),	180	 (21),	345	 (70),	165	 (100).	Anal.	 calcd.	 for	
C20H21NO5S:	C,	62.01;	H,	5.42;	N,	3.61.	Found:	C,	62.26;	H,	5.38;	
N,	3.56%.	
	
	

2.3.	General	method	of	synthesis	of	substituted	N‐(2´,3´,5´‐tri‐
O‐benzoyl‐β‐D‐ribofuranosyl)benzothiazine	(5a‐b)	

	
To	 a	 concentrated	 solution	 of	 synthesized	 benzothiazines	

(3a‐d),	 (0.002)	 mole	 in	 toluene,	 β‐D‐ribofuranose‐1‐acetate‐
2,3,5‐tribenzoate	 (0.002)	 mole	 was	 added	 and	 stirred,	 in	
vacuuo,	 on	 an	 oil	 bath,	 at	 155‐160	 °C,	 for	 15	 minutes.	 The	
vacuum	 was	 broken	 and	 the	 reaction	 was	 protected	 from	
moisture	by	using	a	guard	tube.	Stirring	was	further	continued	
for	 10‐15	 hours	 with	 application	 of	 vacuum	 for	 15	 minutes	
after	 every	 hour.	 The	melt	was	 dissolved	 in	methanol,	 boiled	
for	 10	 minutes	 and	 cooled	 to	 room	 temperature.	 The	
precipitate	 was	 filtered	 and	 the	 filtrate	 was	 evaporated	 to	
dryness.	 The	 viscous	 residue,	 thus	 obtained	was	 dissolved	 in	
ether,	filtered,	concentrated	and	kept	in	refrigerator	overnight	
to	 get	 the	 crystalline	 compound	 (5a‐b)	 (Scheme	 1).	
Characterization	and	spectral	data	of	these	compounds	(5a‐b)	
is	given	as:		

N‐(2´,3´,5´‐tri‐O‐benzoyl‐β‐D‐ribofuranosyl)‐ethyl‐3‐trifluoro	
methyl‐6,8‐dimethyl‐4H‐1,4‐benzothiazine‐2‐carboxylate	 (5a):	
Yield:	 84%.	 M.p.:	 80	 °C.	 IR	 (KBr,	 v,	 cm‐1):	 1700	 (C=O),	 1350,	
1170	(‐CF3	str.),	1170	(C‐O‐C	str.).	1H	NMR	(DMSO‐d6,	300	MHz,	
δ,	ppm):	8.52‐7.10	(m,	2H,	aromatic‐H),	4.21	(q,	2H,	J	=	6.8	Hz,	
CH2	of	C2H5	at	C2),	1.40	(t,	3H,	J	=	6.86	Hz,	`CH3	of	C2H5	at	C2).	13C	
NMR	 (CDCl3,	 300	MHz,	 δ,	 ppm):	 108.2	 (C‐2),	 139	 (C‐3),	 113.2	
(C‐5),	136.8	(C‐6),	121.2	(C‐7),	140.2	(C‐8),	66.8	(C‐1´),	78.5	(C‐
2´),	 72.5	 (C‐3´),	 71.6	 (C‐4´).	MS	 (m/z,	%):	 761	 (M+),	 688	 (39),	
646	 (31),	 73	 (100).	Anal.	 calcd.	 for	C40H34NO9F3S:	 C,	 63.07;	H,	
4.46;	 N,	 1.83.	 Found:	 C,	 63.28;	 H,	 4.40;	 N,	 1.80%.	 Optical	
rotation	[α]D	23=	‐17.28	°C.	

N‐(2´,3´,5´‐Tri‐O‐benzoyl‐β‐D‐ribofuranosyl)‐2‐trifluoro	
acetyl‐3‐trifluoromethyl‐6,8‐dimethyl‐4H‐1,4‐benzothiazine	
(5b):	 Yield:	 68%.	 M.p.:	 82	 °C.	 IR	 (KBr,	 v,	 cm‐1):	 1680	 (C=O),	
1355,	1185	(‐CF3	str.),	1165	(C‐O‐C	str.).	1H	NMR	(DMSO‐d6,	300	
MHz,	δ,	ppm):	8.28‐7.01	(m,	2H,	aromatic‐H),	2.38	(s,	2H,	CH3	at	
C6),	2.37	(s,	3H,	‐CH3	at	C8).	13C	NMR	(CDCl3,	300	MHz,	δ,	ppm):	
117.2	 (C‐2),	 173.3	 (C‐3),	 113.6	 (C‐5),	 136.4	 (C‐6),	 120	 (C‐7),	
136	 (C‐8),	 69.2	 (C‐1´),	 75.2	 (C‐2´),	 76.1	 (C‐3´),	 72.1	 (C‐4´).	MS	
(m/z,	%):	785	 (M+),	688	 (37),	646	 (27),	97	 (100).	Anal.	 calcd.	
for	C39H29NO8F6S:	C,	59.61;	H,	3.69;	N,	1.78.	Found:	C,	59.83;	H,	
3.63;	N,	1.76%.	Optical	rotation	[α]D	23=	‐20.18	°C.	

	

2.4.	Biological	activity	
	

2.4.1.	Antioxidant	activity	
	

2.4.1.1.	DPPH	radical	scavenging	assay	
	
Radical	 scavenging	 activity	 of	 the	 synthesized	 compounds	

against	 stable	 1,1‐diphenyl‐2‐picrylhydrazyl	 (DPPH)	 radical	
was	 determined	 spectrophotometrically	 as	 described	 by	
Cuendet	 et	 al.	 [12].	 A	 stock	 solution	 of	 1	 mg/mL	 of	 the	
compound	 was	 prepared	 in	 methanol.	 50	 μL	 of	 compounds	
were	 added	 to	5	mL	of	 a	 0.004%	methanol	 solution	of	DPPH.	
After	 30	 minutes	 incubation	 in	 dark	 at	 room	 temperature,	
absorbance	was	read	against	a	blank	at	517	nm	(Table	2).	Since	
IC50	 Value	 is	 inversely	 related	 to	 the	 antioxidant	 activity;	
ascorbic	acid,	which	is	a	very	good	antioxidant,	shows	a	lower	
IC50	 value	 of	 17.8	 µg/mL.	 Among	 all	 the	 synthesized	
compounds;	compound	3c	shows	a	value	of	23.56	µg/mL	which	
shows	its	good	antioxidant	nature.	

The	assay	was	carried	out	 in	triplicate	and	the	percentage	
of	inhibition	was	calculated	by	using	the	following	formula.	

	

%
( )

 Inhibition
AB AA

AB



100

	 	 	 (1)	
	

where	 AB	 =	 Absorption	 of	 blank,	 AA	 =	 Absorption	 of	 test,	
Ascorbic	acid	(shows	antioxidant	activity)	as	a	positive	control	
and	methanol	(no	antioxidant	activity)	as	a	negative	control	has	
been	used	in	this	assay.	

	



Gautam	et	al.	/	European	Journal	of	Chemistry	3	(1)	(2012)	106‐111	 109	
 

	 	
Table	2.	Antioxidant	activity	of	the	synthesized	compounds	(DPPH)	assay	(3a‐d,	4a‐d,	5a‐b).	
Compound	No	 R3	 R4	 DPPH	%	inhibition	of	1	mg/mL	of	compound	 IC50	(µg/mL)	
3a	 CF3	 OC2H5	 42.74	±	0.03 823.78	
3b	 CF3	 CF3	 65.13	±	0.02 500.8	
3c	 CH2CH3	 OCH3	 90.76	±	0.07 23.56	
3d	 CH3	 C6H3(OCH3)2	(o,	p)	 70.79	±	0.08 79.12	
4a	 CF3	 OC2H5	 23.48	±	0.09	 ‐	
4b	 CF3	 CF3	 67.49	±	1.10	 432.90	
4c	 CH2CH3	 OCH3	 11.88	±	0.05 ‐	
4d	 CH3	 C6H3(OCH3)2	(o,	p)	 34.10	±	1.20	 ‐	
5a	 CF3	 OC2H5	 73.27	±	0.06	 62.70	
5b	 CF3	 CF3	 75.37	±	0.08 58.90	
Ascorbic	acid	 	 	 92.96	±	00.9 17.82	
	
Table	3.	Antioxidant	activity	f	the	synthesized	compounds*	(ABTS•+	assay)	(3a‐d,	4a‐d,	5a‐b).	

Compound	No	 R3	 R4	
ABTS•+	activity	at	different	intervals	(min.)

0	 1	 2	 4		 6	
3a	 CF3	 OC2H5	 0.697	 0.696	 0.691	 0.687	 0.482	
3b	 CF3	 CF3	 0.698 0.303 0.264 0.097	 0.025
3c	 CH2CH3	 OCH3	 0.688	 0.109	 0.09	 0.002	 0.002	
3d	 CH3	 C6H3(OCH3)2	(o,	p)	 0.695	 0.087	 0.039	 0.025	 0.025	
4a	 CF3	 OC2H5	 0.699	 0.525	 0.406	 0.365	 0.339	
4b	 CF3	 CF3	 0.686 0.392 0.367 0.294	 0.262
4c	 CH2CH3	 OCH3	 0.693 0.682 0.609 0.382	 0.329
4d	 CH3	 C6H3(OCH3)2	(o,	p)	 0.700 0.276 0.162 0.027	 0.021
5a	 CF3	 OC2H5	 0.686 0.194 0.095 0.032	 0.016
5b	 CF3	 CF3	 0.714 0.228 0.195 0.069	 0.023
Ascorbic	acid	 	 	 0.694 0.040 0.003 0.003	 0.003
*Ascorbic	acid	is	used	as	a	reference	compound.	

Table	4.	Antimicrobial	activity	of	the	synthesized	compounds	(3a‐d,	4a‐d,	5a‐b).		

Compound	No	 R3	 R4	
Antibacterial	activity	a Antifungal	activity	b	

Coagulase	negative	
staphylococci	

Coagulase	positive	
staphylococci	

Enterobacter Candida	albicans	

3a	 CF3	 OC2H5	 23	 10 10 18	
3b	 CF3	 CF3	 11	 11 13 22	
3c	 CH2CH3	 OCH3	 15	 13 ‐ 18	
3d	 CH3	 C6H3(OCH3)2(o,	p)	 16	 12 12 15	
4a	 CF3	 OC2H5	 12	 10 ‐ ‐	
4b	 CF3	 CF3	 11	 10 11 11	
4c	 CH2CH3	 OCH3	 ‐	 ‐ ‐ 11	
4d	 CH3	 C6H3(OCH3)2(o,	p)	 10	 11	 ‐	 18	
5a	 CF3	 OC2H5	 10	 ‐	 11	 14	
5b	 CF3	 CF3	 20	 10 ‐ 12	
Gatifloxacin	 	 ‐	 ‐	 17	 ‐	
Vancomycin		 15	 15 ‐ ‐	
Flucanazole		 ‐	 ‐ ‐ 25	
a	Zone	of	inhibition	in	mm;	<7	mm	inactive;	7‐9	mm	weakly	active;	10‐12	mm,	moderately	active;	>12	mm,	active.	
b	Zone	of	inhibition	in	mm;	<7	mm	inactive;	7‐11	mm	weakly	active;	12‐17	mm,	moderately	active;	>17	mm,	active.	

	
	

2.4.1.2.	ABTS	radical	cation	decolorization	assay	
	
The	 2,2‐azinobis(3‐ethybenzothiazoline‐6‐sulphonic	 acid)	

radical	 cation	 (ABTS•+)	 decolorization	 test	 was	 carried	 out	
using	an	 improved	assay	of	Re	et	al.	 [13].	 In	brief,	ABTS•+	was	
generated	 by	 oxidation	 of	 ABTS	 with	 potassium	 persulphate.	
For	 this	 purpose,	 ABTS	 was	 dissolved	 in	 ionized	 water	 at	
concentration	of	7	mM,	and	potassium	persulphate	was	added	
to	a	concentration	of	2.45	mM.	The	reaction	mixture	was	left	at	
room	temperature	overnight	(12‐15	hours),	in	the	dark	before	
use;	 the	 ABTS	 solution	 then	 was	 diluted	 with	 ethanol	 to	 an	
absorbance	of	0.700	±	0.020	at	734	nm.	After	addition	of	1	ml	of	
the	 diluted	 ABTS	 solution	 to	 10	 μL	 of	 compound	 and	mixing,	
absorbance	readings	were	taken	at	30	°C	at	intervals	of	exactly	
1‐6	min.	The	experiment	was	carried	out	in	triplicate	(Table	3	
and	Figure	1).	We	have	used	ascorbic	acid	as	a	positive	control	
and	ethanol	as	a	negative	control	in	this	assay.	

	
2.4.2.	Antimicrobial	activity	

	
The	antimicrobial	assay	of	the	synthesized	compounds	was	

carried	 out	 by	 using	 paper	 disc	 method	 of	 Gould	 et	 al.	 [13]	
against	 some	 bacteria	 and	 fungi	 at	 100	 μg	 per	 disc	
concentration	 using	 Vancomycin,	 Gatifloxacin	 as	 reference	
compounds	against	bacteria	(Coagulase	negative	Staphylococci,	
Coagulase	positive	Staphylococci,	Enterobacter)	and	Flucanazole	
against	 fungus	(Candida	albicans).	Paper	disc	method	 includes	

preparation	 of	 plates	 by	 pouring	 molten	 media	 into	 sterile	
petriplates	 which	was	 then	 allowed	 to	 solidify	 for	 5	 minutes	
and	 0.1%	 inoculum	 suspension	 was	 swabbed	 uniformly	 and	
allowed	 to	dry	 for	5	min.	The	compound	discs	prepared	were	
then	placed	over	the	plates	and	incubated	for	37	oC	for	24	hrs.	
At	 the	 end	 inhibition	 zones	 were	 measured	 with	 ruler	 in	
millimetre.	 These	 microorganisms	 were	 obtained	 from	
Microbiology	 Department,	 Swai	 Man	 Singh	 Medical	 College,	
Jaipur	(Table	4).	

	
3.	Results	and	discussion	

	
2‐Aminobenzenethiol	 (1)	 and	 β‐diketones	 /	 β‐ketoesters	

(2a‐d)	 were	 refluxed	 in	 dimethyl	 sulfoxide	 which	 involves	
condensation	and	oxidative	cyclization.	A	bis‐(2‐aminophenyl)	
disulfide	 (1´)	 was	 obtained	 by	 oxidation	 of	 2‐aminobenzene	
thiol	 which	 cyclizes	 to	 form	 4H‐1,4‐benzothiazines	 (3a‐d)	 by	
cleavage	 of	 sulfur‐sulfur	 bond	 due	 to	 high	 reactivity	 of	 α‐
position	 of	 enaminoketone	 system	 towards	 nucleophillic	
attack.	 Compound	 (3a‐d)	 on	 treatment	 with	 30%	 hydrogen	
peroxide	 in	 glacial	 acetic	 acid	 were	 converted	 into	 their	
corresponding	sulfones	(4a‐d).	Treatment	of	(3a‐b)	in	toluene	
with	 β‐D‐ribofuranose‐1‐acetate‐2,3,5‐tribenzoate	 in	 vacuum	
gave	 the	 corresponding	 ribofuranosides	 (5a‐b)	 (Scheme	 1).	
The	 structures	 of	 synthesized	 compounds	 are	well	 supported	
by	spectral	data	and	elemental	analysis.	

	



110	 Gautam	et	al.	/	European	Journal	of	Chemistry	3	(1)	(2012)	106‐111	
	

	
ABTS•+	activity	of	4H‐1,4‐benzothiazines	(3a‐d)	

	
(a)	

	
ABTS•+	activity	of	4H‐1,4‐benzothiazine	sulfones	(4a‐d)	

	
(b)	

	
ABTS•+	activity	of	4H‐1,4‐benzothiazine	ribofuranosides	(5a‐b)	

	
(c)	

	
Figure	1.	The	effect	of	time	on	the	suppression	of	absorbance	of	ABTS	by	
synthesized	compounds.	After	addition	of	1	mL	of	diluted	ABTS	solution	
(A	 734	 nm	 =	 0.700±0.020)	 to	 10	 μL	 of	 the	 compound	 the	 absorbance	
reading	was	taken	at	30	°C	exactly	1	min.,	after	initial	mixing	and	up	to	6	
min.	All	determinations	were	carried	out	in	triplicates.	

	
3.1.	Spectral	analysis	

	
3.1.1.	IR	spectra	

	
Compounds	(3a‐d)	showed	peaks	in	region	3385‐3260	cm‐1	

due	 to	 N‐H	 stretching	 vibrations	 and	 1700‐1650	 cm‐1	 due	 to	
>C=O	 stretching	 vibrations	 which	 gets	 shifted	 to	 higher	
frequencies	 to	 3390‐3270	 cm‐1	 and	 1710‐1680	 cm‐1,	
respectively,	 in	 compounds	 (4a‐d).	 Compounds	 (4a‐d)	 also	
exhibited	 two	 intense	 peaks	 in	 region	 1360‐1340	 cm‐1	 and	
1180‐1140	 cm‐1	 due	 to	 asymmetric	 and	 symmetric	 stretching	
vibrations	of	sulfonyl	group.	Compounds	(4a‐d)	also	showed	C‐
S	stretching	vibrations	in	region	1095‐1077	cm‐1.	

Absence	 of	 stretching	 vibrations	 due	 to	 >N‐H	 group	 in	
compounds	 (5a‐b)	 showed	 site	 of	 ribosylation,	 further	 in	
compounds	 (5a‐b)	 bands	 due	 to	 C‐O‐C	 linkage	 of	 sugar	
appeared	in	the	region	1170‐1165	cm‐1.	

	
3.1.2.	1H	NMR	spectra	

	
All	compounds	showed	multiplet	in	region	δ	8.52‐6.06	ppm	

due	to	aromatic	protons	and	compounds	(3a‐d)	and	(4a‐d)	also	

showed	a	 singlet	 due	 to	N‐H	proton	 in	 the	 region	δ	9.12‐8.29	
ppm.	 Peak	 due	 to	 N‐H	 proton	 was	 found	 to	 be	 absent	 in	
compounds	(5a‐b)	due	to	ribosylation.	In	ribofuranosides,	C´4‐
H	 proton	 showed	 multiplet	 in	 region	 δ	 4.43‐4.80	 ppm,	 C2´‐H	
and	 C3´‐H	 protons	 appeared	 in	 region	 δ	 4.51‐5.90	 ppm	 as	
multiplet	and	C1´‐H	proton	appeared	as	doublet	at	δ	6.30‐6.40	
ppm.	

	
3.1.3.	Mass	spectra	

	
The	molecular	 ion	peaks	of	4H‐1,4‐benzothiazines	were	 in	

accordance	with	 their	molecular	weights.	 In	 all	 the	 cases	 side	
chain	 at	 C2	 appears	 as	 a	 base	 peak	 which	 is	 obtained	 by	 its	
fission	(Scheme	2).	

	

 
	

Scheme	2	
	

3.2.	Biological	activity	(Antioxidant	and	Antimicrobial)	
	
All	 the	 synthesized	 compounds	 (3a‐d),	 (4a‐d),	 and	 (5a‐b)	

were	 screened	 for	 their	 antioxidant	 activity	 by	 DPPH	 radical	
scavenging	 assay	 and	 (ABTS•+)	 radical	 cation	 decolorization	
assay.	 The	 synthesized	 compounds	 were	 also	 screened	 for	
antimicrobial	 activity	 (antibacterial	 and	 antifungal)	 by	 paper	
disc	 method.	 The	 present	 study	 demonstrated	 that	 these	
compounds	showed	mixed	activity	in	DPPH	and	ABTS•+	assay.	

(1)	 Compounds	 (3b,	 3c,	 3d,	 4b,	 5a,	 5b)	 showed	 strong	
radical	 scavenging	 activity	 in	 DPPH	 assay	 that	 have	 DPPH	%	
inhibition	>50.	

(2)	Compounds	(3a,	4d)	showed	moderate	activity	in	DPPH	
assay	that	have	DPPH	%	inhibition	>30.	

(3)	 Compounds	 (4a,	 4c)	 showed	 mild	 activity	 (<30)	 in	
DPPH	assay.	

(4)	Compounds	(3b,	3c,	3d,	4b,	4d,	5a,	5b)	were	active	in	
ABTS•+	assay.	

All	 these	 compounds	were	 found	 to	 be	moderately	 active	
against	 various	 bacteria	 such	 as	 (Coagulase	 negative	
Staphylococci,	 Coagulase	 positive	 Staphylococci,	 Enterobacter)	
and	 fungi	 (Candida	 albicans).	 Compounds	 (3a,	 3c,	 3d,	 5b)	
showed	good	activity	against	Coagulase	negative	Staphylococci	
and	 compounds	 (3a,	 3b,	 3c	 and	 4d)	 shows	 good	 activity	
against	Candida	albicans.	

	
4.	Conclusion	

	
The	 structures	 of	 synthesized	 compounds	 are	 well	

supported	 by	 spectral	 data	 and	 elemental	 analysis.	 The	
synthesized	 compounds	 were	 also	 screened	 for	 antioxidant	
activity	 (DPPH	 assay	 and	 ABTS•+	 assay)	 and	 antimicrobial	
activity	 (antibacterial	 and	 antifungal).	 The	 present	 study	
demonstrated	that	these	compounds	showed	mixed	activity	in	
DPPH	 and	ABTS•+	 assay.	 Compounds	3c,	3d	 showed	 excellent	
antioxidant	activity	in	DPPH	assay.	Compounds	5a,	5b	showed	
much	 better	 activity	 in	 DPPH	 assay	 than	 compounds	 3a,	 3b	
which	 are	 the	 precursors	 of	 5a	 and	 5b.	 It	 shows	 that	 the	
ribofuranosides	 5a	 and	 5b	 showed	 much	 better	 antioxidant	
activity	than	their	phenothiazine	bases	(3a,	3b).	This	is	due	to	
the	 replacement	 of	 H	 by	 the	 sugar	 moiety	 (ribosylation).	
Compounds	3b,	3c,	3d,	4b,	5a,	5b	showed	good	activity	in	both	
DPPH	and	ABTS	assays.	

The	 present	 paper	 is	 focused	 on	 the	 synthesis	 of	 novel	
heterocyclic	 compounds	 as	 possible	 antibacterial	 and	
antifungal	agents.	Compounds	3c	and	3d	showed	antibacterial	
activity	 against	 Coagulase	 negative	 Staphylococci	 which	 is	
comparable	to	vancomycin.	Compound	3a	 is	much	better	than	



Gautam	et	al.	/	European	Journal	of	Chemistry	3	(1)	(2012)	106‐111	 111	
 

vancomycin	in	antibacterial	activity	against	Coagulase	negative	
Staphylococci.	 All	 these	 compounds	 were	 found	 to	 be	
moderately	 active	 against	 various	bacteria	 such	as	 (Coagulase	
negative	 Staphylococci,	 Coagulase	 positive	 Staphylococci,	
Enterobacter)	 and	 fungi	 (Candida	 albicans).	 Compounds	 (3a,	
3c,	 3d,	 5b)	 showed	 good	 activity	 against	 Coagulase	 negative	
Staphylococci	and	compounds	(3a,	3b,	3c	and	4d)	shows	good	
activity	against	Candida	albicans.	

	
Acknowledgements	

	
The	 authors	 are	 grateful	 to	 Department	 of	 Chemistry,	

University	 of	 Rajasthan,	 Jaipur,	 India	 for	 providing	 necessary	
facilities.	The	University	Grants	Commission	 (Research	Award	
Scheme)	and	Council	of	Scientific	and	Industrial	Research,	New	
Delhi,	 India	 are	 duly	 acknowledged	 for	 financial	 support.	We	
are	 also	 thankful	 to	 Department	 of	 Zoology,	 University	 of	
Rajasthan	 and	 Sawai	 ManSingh	 Medical	 College,	 Jaipur,	 India	
for	assistance	in	carrying	out	the	biological	activity.	

	
References	

	
[1]. Gautam,	V.;	Sharma,	M.;	Panwar,	M.;	Gautam,	N.;	Kumar,	A.;	Sharma,	I.	

K.;	Gautam,	D.	C.	Phosphorus	Sulfur	2009,	184(11),	3090‐3109.		
[2]. Gupta,	S.;	Ajmera,	N.;	Meena,	P.;	Gautam,	N.;	Kumar,	A.;	Gautam,	D.	C.	

Jordan	J.	Chem.	2009,	4(3),	209‐221.		
[3]. Gupta,	 V.;	 Gautam,	 R.	 K.;	 Jain,	 S.	 K.;	 Gupta,	 R.	 R.	 Phosphorus	 Sulfur	

1990,	47(1)	,	225‐228.		
[4]. Clercq,	E.	D.	Nucleos.	Nucleot.	Nucl.	1985,	4(1),	3‐11.		
[5]. Gupta,	A.;	Saraswat,	V.;	Mukherji,	S.	K.;	Gupta,	R.	R.	Phosphorus	Sulfur	

1993,	85(1),	101‐106.		
[6]. Kachee,	T.	L.;	Gupta,	V.;	Gautam,	D.	C.;	Gupta,	R.	R.	Phosphorus	Sulfur	

2005,	180(10),	2225‐2234.		
[7]. Gautam,	N.;	Hans,	D.;	Gautam,	D.	C.	Oriental	J.	Chem.	2005,	21(2),	299‐

302.		
[8]. Kumar,	N.;	Singh,	G.;	Yadav,	A.	K.	Heteroatom	Chem.	2001,	12(1),	52‐

56.		
[9]. Gupta,	 R.	 R.	 Phenothiazines	 and	 1,4‐benzothiazines	 Chemical	 and	

Biomedical	aspects,	Elsevier,	Amsterdam,	1988	pp.	160‐210.		
[10]. Sharma,	P.	R.;	Gupta,	V.;	Gautam,	D.	C.;	Gupta,	R.	R.	Phosphorus	Sulfur	

2003,	178(7),	1483‐1488.		
[11]. Gautam,	N.;	Sharma,	M.;	Gautam,	V.	Asian	J.	Chem.	2010,	22(7),	5380‐

5388.		
[12]. Cuendet,	M.;	Hostettmann,	 K.;	 Potterat,	 O.;	Dyatmiko,	W.	Helv.	Chim.	

Acta	1997,	80(4),	1144‐1152.		
[13]. Re,	R.;	Pellegrini,	N.;	Proteggente,	A.;	Pannala,	A.;	Yang,	M.;	Rice‐Evans,	

C.	Free	Radic.	Biol.	Med.	1999,	26,	1231‐1237.		
[14]. Gould,	J.	C.;	Browie,	J.	H.	Edinb.	Med.	J.	1952,	59(4),	178‐199.