Synthesis and activity of four (N,N-dimethylamino)benzamide non-steroidal anti-inflammatory drugs based on thiazole and thiazoline

Article abstract of DOI:10.1002/jhet.5570430130

Four compounds derived from 2-aminothiazole and 2-amino-2-thiazoline were prepared by coupling the respective bases with the acid chlorides of either 3- or 4-(N,N-dimethylamino)benzoic acid. Products were identified using infrared spectroscopy. ¹

Full text of DOI:10.1002/jhet.5570430130

Jan-Feb 2006
Synthesis and Activity of Four (N,N-dimethylamino)benzamide Non-
steroidal Anti-inflammatory Drugs Based on Thiazole and Thiazoline.
Daniel E. Lynch, Ravinder Hayer, Samantha Beddows, Joy Howdle and C. Douglas Thake
191
School of Science and the Environment, Coventry University, Coventry CV1 5FB, UK.
Received April 22, 2005
Four compounds derived from 2-aminothiazole and 2-amino-2-thiazoline were prepared by coupling the
respective bases with the acid chlorides of either 3- or 4-(N,N-dimethylamino)benzoic acid. Products were
1
identified using infrared spectroscopy, H NMR spectroscopy and electrospray mass spectroscopy and in
two cases by single-crystal X-ray diffraction. Of the four, N-(thiazol-2-yl)-3-(N,N-dimethylamino)-
benzamide (1), N-(thiazolin-2-yl)-4-(N,N-dimethylamino)benzamide (2), N-(thiazolin-2-yl)-3-(N,N-
dimethylamino)benzamide (3) and N-(thiazolin-2-yl)-4-(N,N-dimethylamino)benzamide (4), the hydro-
-
2
chloride salts of compounds 3 and 4 showed anti-inflammatory activity across a concentration range of 10
–
5 x 10- M while 3 (at a concentration of 10 M) was found to have no adverse effect on myocardial func-
4
-5
tion. The X-ray crystal structure of 2 and the 1:1 adduct structure of 3 with 3-(N,N-dimethylamino)benzoic
acid are reported.
J. Heterocyclic Chem., 43, 191 (2006).
Introduction.
terised by the 4-hydroxybenzothiazine heterocycle.
However, an important characteristic of an oxicam mole-
cule is the presence of the carboxyamide substituent at the
Non-steroidal anti-inflammatory drugs, or NSAIDs, are
one of the most widely used drug classifications in modern
society and include a variety of different chemical agents
with differing chemical classes. The word non-steroidal is
used in the context that the drugs do not resemble corticos-
teroids which themselves are anti-inflammatory com-
pounds. NSAIDs are involved in a variety of clinical situa-
tions such as in the treatment of analgesia, for anti-inflam-
matory purposes (mainly for rheumatoid arthritis and
osteoarthritis), antipyretic and uricosuric effects [1].
NSAIDs have also been associated with a reduction in risk
for developing Alzheimer’s desease [2,3] and in the pre-
vention of ocular inflammation related to cataract surgery
3
-position of the benzothiazine ring, which contributes
towards the acidity of the molecule by stabilising any nega-
tive charge during ionisation. Both Sudoxicam and
Meloxicam [10] are examples of an oxicam with a carbox-
amino-1,3-thiazole attachment, and their anti-inflammatory
and analgesic properties as well as toxicity, have been
reported [11-13]. Sudoxicam is no longer marketed due to
severe side effects but Meloxicam is used medically for the
treatment of arthritic conditions in several countries [14].
Both aminothiazoles [15-17] and carboxamino-1,3-thia-
zoles [18,19] have been investigated for anti-inflammatory
action, while the crystal structures of several carboxamino-
[
(
4]. Common NSAIDs work by inhibiting cyclooxygenase
COX) activity in prostaglandin H synthase, a key enzyme
1,3-thiazoles have also been published [20-24]. In addition
2
to these studies we have prepared four carboxamino-1,3-thi-
azole derivatives containing N,N-dimethylaminophenyl
moieties with the intention of investigating their anti-
inflammatory activity. Anti-inflammatory activity can be
easily appraised by using the bioassay, neutrophil oxidative
activity qualitative nitroblue tetrazolium test (or NBT reduc-
tion test). The use of nitroblue tetrazolium (NBT), 3,3'-
(3,3'-dimethoxy-4,4'-biphenylene)bis-2-(p-nitrophenyl)-5-
phenyl-2H-tetrazolium [25], was introduced in 1968 by
B.H. Park et al. for recognising bacterial infections [26].
Park and co-workers reported that the NBT test might be
useful in differentiating patients with bacterial infection
in the biosynthesis of prostoglandins [5], although other
modes of action are known. Other actions that are related
to the treatment of inflammation, such as the inhibition of
lipoxygenases [6], can be used to distinguish between indi-
vidual NSAIDs. NSAIDs are also being investigated for
application in combination therapy for the stimulation of
hair growth [7] and photo-protection compositions [8] (i.e.
sunscreens). NSAIDs consist of several subcategories,
with the main ones being the salicylates, propionic acids
(profens), the aryl and heteroaryl acetic acids, the anthrani-
lates (fenamates), the anilides, the phenylpyrazolones and
oxicams (enolic acids) [9], with oxicams being charac-

1
92
D. E. Lynch, R. Hayer, S. Beddows, J. Howdle and C. D. Thake
Vol. 43
from individuals with non-bacterial disease. The NBT test
theory is based on the hypothesis that a metabolic change
and an increased reduction of NBT dye would take place
when leukocytes are involved in phagocytosis in-vivo dur-
ing a natural infection [27]. NBT is a clear yellow dye that
is absorbed by the neutrophils during phagocytosis. As a
result of the superoxide produced by the activated neu-
trophil [in this study induced by N-formylme-
synthesis and chemical characterisation of N-(thiazol-2-yl)-
3-(N,N-dimethylamino)benzamide (1), N-(thiazol-2-yl)-4-
(N,N-dimethylamino)benzamide (2), N-(thiazolin-2-yl)-3-
(N,N-dimethylamino)benzamide (3) and N-(thiazolin-2-yl)-
4-(N,N-dimethylamino)benzamide (4) (Figure 1). All com-
1
pounds were characterised using infrared spectroscopy, H
NMR and electrospray mass spectrometry. The single crys-
tal X-ray structures of compound 2 and the 1:1 adduct of 3
with (N,N-dimethylamino)benzoic acid 3a are also reported.
NBT tests were performed on the hydrochloride salts of 3
and 4, while 3 was investigated for effect on myocardial
function.
+
thionylleucylphenylalanine (fMLP)], NBT accepts H and
is reduced to formazan but is observed as deep purple pre-
cipitates inside the phagocytes when stained. Thus the pres-
ence of these coloured zones within each neutrophil is a
visual indication of inflammatory activation. However, the
percentage of activated neutrophils is suppressed if the sam-
ple has been treated with an anti-inflammatory compound.
As a prequel to their anti-inflammatory experiments,
Andreani et al. tested for and reported that none of their
compounds were chemotaxins for human neutrophils, secre-
tagogue agents or were able to trigger superoxide anion pro-
duction [19]. In their anti-inflammatory tests, Andreani et
al. reported that all compounds investigated showed effects,
Results and Discussion
Carboxamide linkages are commonly produced by the
coupling of an acid chloride with an amine group with the
loss of hydrochloric acid, thus an equimolar amount of tri-
ethylamine should be present to form the hydrochloride salt
which can be easily removed by aqueous extraction in the
work-up. Although an inert atmosphere is not required dry
conditions are still necessary else some of the acid chloride
hydrolyses back to the carboxylic acid and then both react to
form the anhydride [30]. In our procedure, completion of the
addition of the acid chloride to the amine was followed by
removal of the solvent under reduced pressure to yield an
expected mixture of compounds including the water soluble
salt and, assuming that some acid chloride had been hydrol-
ysed, the benzoic acid derivative. The removal of both of
these side-products was easily achieved by washing the
resultant mass with dilute aqueous base. However in the case
of 3 removal of the reaction solvent yielded crystals that re-
dissolved in an organic solvent, indicating that they were
organic and not triethylammonium chloride. Subsequent
analysis of crystals of this unknown material yielded the
structure of 3a, proving the presence of some benzoic acid
derivative in the final reaction mixture. Thus, washing with
dilute aqueous base instead of just water was an essential part
of the work-up process. Dissolution in minimal ethyl acetate
and then precipitation in light petroleum was found to be an
effective procedure for the removal of small amounts of the
aminoheterocycle and any anhydride that had formed. As a
final process, and a precursor to preparing the compounds for
pharmacological testing, the products were dissolved in acid,
filtered, and then re-precipitated with excess base. This last
step ensured that the entire final product was capable of
being made water-soluble by production of the hydrochlo-
ride salt. Many drugs containing base units are stored and
administered as protonated salts. These salts are of course
more water-soluble and are mainly chlorides, sulfates and
hydrogen sulfates, although very insoluble compounds are
complexed with citrates or tartrates [31]. The preparation of
the hydrochloride salts of 1 – 4 consisted of a measured
amount of each compound being dissolved in an aqueous
solution containing a stoichiometric equivalent of hydrochlo-
ric acid, filtering and evaporation to dryness. Although
-5
-9
at concentrations 10 – 10 M, but not all were statistically
significant. For this reason we chose to examine higher con-
-2
-4
centrations (10 – 5 x 10 M) to be sure of any effect. In
addition to our own compounds we also tested the commer-
cial anti-inflammatory drug, Diclofenac, so that some com-
parative strengths of effect could be evaluated. For this pur-
pose Andreani et al. used Indomethacin and although
Meloxicam may have been a more structurally similar drug
for us to use (Diclofenac being a fenamate), Meloxicam’s
mode of action is preferentially COX-2 inhibition whereas
Diclofenac is known to act through both COX inhibition and
the down-regulation of L-selectin adhesion molecules thus
making it a more effective anti-inflammatory agent [28,29].
The testing of compounds for their own potential toxic
affect is important and the results of Andreani et al. indicate
that our compounds will be passive to neutrophil activity.
However, as an addition to the results of Andreani et al. we
decided to investigate the effects, if any, that our compounds
would have on myocardial function. Reported here is the
Figure 1. Chemical diagrams for the compounds prepared in this study.

Jan-Feb 2006
Synthesis and Activity of Four (N,N-dimethylamino)benzamide
193
initially soluble, aqueous solutions of 1 and 2 over time
encountered problems with precipitation of the compound
salt. The additional use of both citric and tartaric acids did
not improve the long-term solubility of 1 and 2 at the con-
centrations needed to match 3 and 4 so 1 and 2 were not
tested for anti-inflammatory activity.
for their thiazoline derivatives were lower than those of the
thiazoles [19]. In the H NMR spectra of 1 – 4, none dis-
1
played distinct N-H peaks, observed around δ = 12.5 ppm
in previous reports [19]. Interestingly, the aromatic peaks
in the 1H NMR of 2 show the possible existence of a second
conformation in solution with minor peaks neighbouring
the main aromatic proton doublets between δ = 6.6 – 8.1
ppm (Figure 2). Integration across both minor and major
doublets in both cases gives the whole proton value, thus
the existence of the minor peaks is potentially due to a sec-
ond conformation that is most likely to be one where the
phenyl ring is inclined to the approximate plane of the rest
of the molecule. This second conformation is not observed
in the solid-state crystal structure. The electrospray mass
Two characteristic bands are commonly observed in the
infrared spectra of amides, or carboxamides, these are the
-1
carbonyl stretching vibration between 1690 – 1640 cm
and the N-H stretching absorption around 3200 – 3300
-1
cm . The frequency of the carbonyl vibration is lower for
-1
amide C=O than for esters or ketones (≥ 1700 cm ) and can
-1
be reduced by as much as 20 – 60 cm [32,33]. In the case
-1
of 4 the carbonyl stretch is lowered to 1600 cm , becoming
essentially a shoulder on the side of the strong aromatic
peak typically observed at that wavenumber. Similarly, the
carbonyl stretch for 3 is also lower than expected for a sec-
ondary amide. In support of these observations, Andreani
et al. also reported that the carbonyl stretching frequencies
spectrometry spectra show a variety of results in addition to
MH+ with compounds 2 – 4 also assembling with Na as
either MNa+ or 2MNa+.
The single crystal structure of 2 comprises a slightly
twisted molecule with a dihedral angle of 30.00(6)° between
Figure 3. Molecular configuration and atom numbering scheme for 2,
showing 50% probability ellipsoids.
the phenyl and thiazole rings (Figure 3). One hydrogen-
bonding association exists between the carboxamide NH
and the thiazole nitrogen atom; listed in Table 1 as well
as selected torsion angles for the carboxamide
1
Figure 2. The aromatic region of the H nmr for 2.
Table 1
Hydrogen bonding geometries and selected torsion angles for compounds 2 and 3a.
.
..
D--H A
D–H(Å)
0.85(2)
H…A(Å)
2.16(2)
D…A(Å)
3.000(2)
D–H…A(°)
.
..
2
3
N(21)--H N(3)
170(2)
.
..
a
N(21A)--H O(10B)
0.89(2)
1.26(2)
1.94(2)
1.28(2)
2.830(2)
2.540(1)
173(3)
173(3)
.
..
O(11B)--H N(3A)
Torsion angles
2
3a
S(1)-C(2)-N(21)-C(6)
C(2)-N(21)-C(6)-C(7)
N(21)-C(6)-C(7)-C(12)
C(2)-N(21)-C(6)-O(6)
O(6)-C(6)-C(7)-C(8)
0.5(2)
S(1A)-C(2A)-N(21A)-C(6A)
-4.7(2)
165.4(1)
163.4(1)
-13.6(2)
157.6(1)
C(2A)-N(21A)-C(6A)-C(7A)
N(21A)-C(6A)-C(7A)-C(12A)
C(2A)-N(21A)-C(6A)-O(6A)
O(6A)-C(6A)-C(7A)-C(8A)
-163.4(1)
-173.1(1)
15.9(2)
-167.7(1)

1
94
D. E. Lynch, R. Hayer, S. Beddows, J. Howdle and C. D. Thake
Vol. 43
et al. undertook in vivo experiments on rats and reported
their data as dose responses (i.e. LD50 and ED ) [18]. For
50
this study, results are given in percentage inhibition com-
pared to the number of activated neutrophils in physiologi-
cal saline (baseline). Results for the hydrochloride salts of 3
and 4, and for Diclofenac are listed in Table 2 with the
results for each run being the average of three slides (twice
counted) prepared from the same mixture. For 3, the 0.5
mM concentration exhibited an average range of inhibition
of neutrophil activity of 8 – 33% with the higher value aris-
ing due to the second of the three runs with a difference of
>20%. The 1 mM concentration gave higher, and more con-
sistent, results with an average of 41 – 54% inhibition of
neutrophil activity. The 5 mM and 10 mM had 15 – 38%
and 27 – 39% inhibition of neutrophil activity with the third
run at 5 mM being c. 20% lower than the other two runs.
The results for 4 are more consistent and inhibition slowly
increases with concentration. The 0.5 mM concentration of
4
had an average range of neutrophil inhibition of 24 – 28%.
The 1 mM and 5 mM concentration gave slightly higher
results with an average of 29 – 37% and 32 – 36% inhibition
of neutrophil activity while the 10 mM concentration had
Figure 4. Molecular configuration and atom numbering scheme for 3a,
showing 50% probability ellipsoids. Hydrogen-bonding associations are
shown as dotted lines.
5
0% inhibition of neutrophil activity for all three runs.
Results for Diclofenac were expected to be much higher and
for the higher concentrations (5 and 10 mM) a range of 71 –
7
4% inhibition of neutrophil activity is observed but the
Table 2
lower concentrations (0.5 and 1 mM) give results that are
not too different from 3 and 4 with percentages ranging from
Percentage inhibition of neutrophil activity for each compound, concen-
tration and run (normalized against saline solution).
33 – 43% inhibition and 37 – 49% inhibition, respectively.
Unfortunately, Andreani et al. did not examine the anti-
Compound
Conc. (mM)
Run 1
Run 2
Run 3
inflammatory activity of their pre-cursor compounds that 1
4 are different analogues of. Instead the compounds they
did examine gave similar anti-inflammatory responses (35
55% inhibition) compared to 3 and 4 but at much weaker
3
0.5
8
33
54
35
39
29
37
33
50
33
49
73
73
11
41
15
27
24
29
32
50
34
37
71
74
–
1
5
.0
.0
50
38
32
28
31
36
50
43
47
74
71
–
1
0.0
-5
-9
-2
-4
4
0.5
concentrations [10 – 10 M as opposed to 10 – 5 x 10
1
5
.0
.0
M for 3 and 4]. At their most effective concentrations,
compounds 3 and 4 both show approximately 50% inhibi-
tion of neutrophil activation, which directly relates to a
1
0.0
Diclofenac
0.5
2
0% reduction in the best anti-inflammatory activity of
1
5
.0
.0
Diclofenac, across the same concentration range although
at a concentration of 1 mM, 3 shows slightly more inhibi-
tion activity than Diclofenac. Testing the effects of 3 on
myocardial function added to the studies of Andreani et al.
that reported that this series of compounds were not chemo-
taxins for human neutrophils. Compound 3, at a lower con-
1
0.0
linkage. The single crystal structure of the 1:1 adduct of N-
thiazolin-2-yl)-3-(N,N-dimethylamino)benzamide with
N,N-dimethylamino)benzoic acid 3a comprises the two con-
(
(
-5
centration of 0.01 mM or 10 M, was introduced after an
ischemic insult was induced in the heart (i.e. a heart attack)
but before reperfusion began. The haemodynamic proper-
ties of the heart (i.e. left ventricular developed pressure,
coronary flow and heart rate) remained unaffected with the
inclusion of 3 and continuing throughout the reperfusion
process. In addition to this, the percent necrosis (infarc-
tion), calculated by evaluating slices of the stained heart
and determining the percentage of stained or healthy tissue
against non-stained or dead tissue, was found to be 36.7%,
2
stituent molecules associated via a hydrogen-bonded R (8)
graph set motif involving the carboxylic acid group across the
N(21A) / N(3A) site of the aminoheterocyclic moiety (Figure
2
4
). Hydrogen-bonding geometries of this association and
selected torsion angles are also listed in Table 1. The dihedral
angle in molecule A between the phenyl ring and the calcu-
lated median plane of the thiazoline ring is 26.65(7)°.
Andreani et al. reported their anti-inflammatory results as
percentage inhibition of human neutrophils [19] but Viallet

Jan-Feb 2006
Synthesis and Activity of Four (N,N-dimethylamino)benzamide
195
Table 3
dine. After further stirring for 30 minutes the solvent was
removed under reduced pressure to yield an off-white powder,
Crystallographic details for 2 and 3a.
1
.06 g (96%), mp 135 – 136º (lit. 135 – 136º) [35].
N-(Thiazol-2-yl)-3-(N,N-dimethylamino)benzamide (1).
-(N,N-Dimethylamino)benzoic chloride (1.00 g, 5.5 mmol)
Compound 2
Compound 3a
CCDC reference
Formula
Mr
Crystal class
Space group
a (Å)
211677
C12H13N OS
247.31
monoclinic
211678
C21H26N O S
414.52
3
3
4 3
was slowly added to a stirred solution of 2-aminothiazole (0.55 g,
5.5 mmol) and triethylamine (0.56 g, 5.5 mmol) in dry
dichloromethane (20 mL). After further stirring for 30 minutes,
the solvent was removed under reduced pressure to yield an off-
white gelatinous material. The crude product was then washed
with dilute sodium hydroxide solution, filtered, dissolved in min-
imal ethyl acetate, filtered and then precipitated by adding to 50
mL of light petroleum (40 - 60 °C). The solid was redissolved in
dilute hydrochloric acid solution and reprecipitated by again
adding to dilute sodium hydroxide solution in excess. The prod-
monoclinic
P2 /c
P2 /c
1
1
7.34590(10)
11.3488(2)
14.3981(3)
103.8536(8)
1165.41(4)
1.410
15.1552(12)
8.3453(7)
16.7265(13)
108.0190(10)
2011.7(3)
1.369
b (Å)
c (Å)
β (°)
3
V(Å )
-
3
D (g cm )
c
Z
4
4
-
1
µ(Mo-K ) (mm )
0.264
0.192
α
Tmin, Tmax
Colour
Crystal size (mm)
Total data
Unique data
Rint
0.897, 0.920
colourless
0.42 x 0.34 x 0.32
5188
2668
0.014
0.945, 0.987
colourless
0.30 x 0.27 x 0.07
11179
4115
0.018
uct was collected in vacuo to afford a white powder, 0.71 g
1
(
52%), mp 134 – 136°; ir (KBr): 3140m (NH), 1680 (CO); H
nmr (250 MHz; CDCl ; Me Si): δ 2.99 (s, 6 H, NCH ), 6.92 (d, 1
3
4
3
H, J = 3.5, ThH), 6.95 (d, 1 H, J = 8.3, ArH), 7.06 (d, 1 H, J = 3.5,
+
ThH), 7.28 – 7.38 (m, 3 H, ArH); es-ms: m/z 248 (100) (MH ),
+
4
95 (8) (2MH ).
N [I>2.0 (I)]
R1
2334
0.040
3389
0.035
Anal. Calcd. for C H N OS: C, 58.3; H, 5.3; N, 17.0. Found:
12 13 3
C, 58.0; H, 5.1; N, 17.1.
N-(Thiazolin-2-yl)-4-(N,N-dimethylamino)benzamide (2).
-(N,N-Dimethylamino)benzoic chloride (1.00 g, 5.5 mmol)
was slowly added to a stirred solution of 2-aminothiazole (0.55 g,
.5 mmol) and triethylamine (0.56 g, 5.5 mmol) in dry
wR2
S
A, B [a]
0.090
1.05
0.0506, 0.4053
0.095
1.04
0.0627, 0.0000
4
2
a] w = [σ (Fo2) + (AP) + BP] [where P = (F 2 + 2F 2)/3]
2
-1
[
o
c
5
dichloromethane (20 mL). After further stirring for 30 minutes,
the solvent was removed under reduced pressure to yield an off-
white powder. The crude product was then washed with dilute
sodium hydroxide solution, filtered, dissolved in minimal ethyl
acetate, filtered and then precipitated by adding to 50 mL of light
petroleum (40 - 60 °C). The solid was redissolved in dilute
hydrochloric acid solution and reprecipitated by again adding to
dilute sodium hydroxide solution in excess. The product was col-
which is a minimal variation from the control value of 35%
34]. Thus, compounds 3 and 4 display anti-inflammatory
activity and compound 3 has no adverse effect on myocar-
dial function which is a good basis for further studies on
phenylcarboxamides based on derivatives of 1,3-thiazoles.
[
EXPERIMENTAL
lected in vacuo to afford a white powder, 0.68 g (50%), mp 138-
1
1
40°; ir (KBr): 3165w (NH), 1654m (CO); H nmr (400 MHz;
CDCl ; Me Si): δ 3.10 (s, 6 H, CH ), 6.69 (25%) & 6.74 (75%)
All chemicals were purchased from Sigma-Aldrich.
Triethylamine was distilled before use. Pyridine was distilled, dried
and stored over potassium hydroxide. Dichloromethane was dried
using molecular sieves. Infrared spectra were recorded as pressed
3
4
3
(
d, 2 H, J = 9, ArH), 6.97 (d, 1 H, J = 3, ThH), 7.37 (d, 1 H, J = 4,
ThH), 7.94 (75%) & 8.02 (25%) (d, 2 H, J = 9, ArH); es-ms: m/z
+
+
2
48 (55) (MH ), 470 (100) (MNa ).
Anal. Calcd. for C H N OS: C, 58.3; H, 5.3; N, 17.0. Found:
1
KBr discs on a Nicolet 205 FT-IR spectrometer. H NMR data were
12 13 3
C, 58.2; H, 5.2; N, 17.3.
N-(Thiazolin-2-yl)-3-(N,N-dimethylamino)benzamide (3).
-(N,N-Dimethylamino)benzoic chloride (1.00 g, 5.5 mmol)
was slowly added to a stirred solution of 2-amino-2-thiazoline
0.56 g, 5.5 mmol) and triethylamine (0.56 g, 5.5 mmol) in dry
recorded on a Bruker AC250 NMR spectrometer for 1 and 3 and a
Bruker Spectrospin 400 NMR spectrometer for 2 and 4.
Electrospray mass spectra were recorded in positive ion mode on a
Micromass Platform mass spectrometer (Southampton University).
3
3
-(N,N-Dimethylamino)benzoic Chloride.
(
dichloromethane (20 mL). After further stirring for 30 minutes, the
solvent was removed under reduced pressure to yield an off-white
crystalline material. The crude product was redissolved in minimal
ethyl acetate, filtered and then precipitated by adding to 50 mL of
light petroleum (40 - 60 °C). A small amount of the product was
then crystallized from ethyl acetate and analysed using single crys-
tal X-ray diffraction techniques to give the 1:1 adduct structure of
N-(thiazolin-2-yl)-3-(N,N-dimethylamino)benzamide with 3-(N,N-
dimethylamino)benzoic acid (3a), mp 110 – 112°.
Oxalyl chloride (3.10 g, 24 mmol) was drop-wise added to a
stirred solution of 3-(N,N-dimethylamino)benzoic acid (1.00 g, 6
mmol) in dry dichloromethane (20 mL) with 3 drops of dry pyri-
dine. After further stirring for 30 minutes the solvent was
removed under reduced pressure to yield an off-white powder,
1
.05 g (95%), mp 138 – 140º (lit. 138 – 140º) [35].
4
-(N,N-Dimethylamino)benzoic Chloride.
Oxalyl chloride (3.10 g, 24 mmol) was drop-wise added to a
stirred solution of 4-(N,N-dimethylamino)benzoic acid (1.00 g, 6
Anal. Calcd. for C H N O S: C, 60.85; H, 6.3; N, 13.5.
21 26 4 3
mmol) in dry dichloromethane (20 mL) with 3 drops of dry pyri-
Found: C, 61.1; H, 6.5; N, 13.3.

196
D. E. Lynch, R. Hayer, S. Beddows, J. Howdle and C. D. Thake
Vol. 43
The remaining solid product was washed with dilute sodium
phosphate-buffered saline (PBS), and 5 µL of 10-3 M fMLP in 1:9
hydroxide solution, filtered, redissolved in dilute hydrochloric acid
solution and reprecipitated by again adding to dilute sodium
hydroxide solution in excess. The product was collected in vacuo
DMSO:0.9% PBS, followed by the respective addition of 50 µL of
blood to each solution. The samples were gently mixed and placed
in a water bath and incubated at 37 ºC for 10 minutes. Following
this time they were removed and allowed to cool to room tempera-
ture for another 10 minutes completed with further gentle agitation.
3 x 40 µL of each mixture were then gravity spread respectively
over 3 separate glass slides. Each smear was air dried and then
stained using 1 mL of ACCUSTAIN Wright Stain, allowing it to
flood the sample for 90 seconds. To the flooded smears 1 mL of
deionised water was added and the slides were again allowed to
stand for another 90 seconds. The stain was then rinsed off with
deionised water and the slides left to air dry. Each slide was exam-
ined twice on separate days by counting 100 neutrophils and
recording the number that were activated. All slides were blind
counted (i.e. labels covered) and randomly ordered. In total, for
every concentration of every compound there were three runs per-
formed and each run consisted of treating and twice measuring
three slides whose results were averaged to obtain a result for each
run. The background activation percentage was obtained by aver-
aging three slides (twice counted) treated with the saline solution.
to afford a white powder, 0.44 g (32%), mp 123 – 125°, ir (KBr):
1
3
2
8
154w (NH), 1623s (CO); H nmr (250 MHz; CDCl ; Me Si) δ
3
4
.98 (s, 6 H, NCH ), 3.20 (t, 2 H, J = 8.0, CH ), 3.59 (t, 2 H, J =
3
2
.0, CH ), 6.66 (d, 1 H, J = 8.0, ArH), 7.20 – 7.47 (m, 3 H, ArH);
2
+
+
+
es-ms: m/z 250 (100) (MH ), 499 (12) (2MH ), 521 (15) (2MNa ).
Anal. Calcd. for C H N OS: C, 57.8; H, 6.1; N, 16.9. Found:
1
2 15 3
C, 58.0; H, 6.2; N, 16.7.
N-(Thiazolin-2-yl)-4-(N,N-dimethylamino)benzamide (4).
-(N,N-Dimethylamino)benzoic chloride (1.00 g, 5.5 mmol)
4
was slowly added to a stirred solution of 2-amino-2-thiazoline
0.56 g, 5.4 mmol) and triethylamine (0.56 g, 5.5 mmol) in dry
(
dichloromethane (20 mL). After further stirring for 30 minutes,
the solvent was removed under reduced pressure to yield an off-
white powder. The crude product was then washed with dilute
sodium hydroxide solution, filtered, dissolved in minimal ethyl
acetate with a few drops of added chloroform (to enhance disso-
lution), filtered and then precipitated by adding to 50 mL of light
petroleum (40 - 60 °C). The solid was redissolved in dilute
hydrochloric acid solution and reprecipitated by again adding to
dilute sodium hydroxide solution in excess. The product was col-
Myocardial Ischaemia/Reperfusion [34].
A male Sprague-Dawley rat heart, in a classic Langendorff
heart set up, was perfused retrogradely with modified Krebs-
Hensleit bicarbonate buffer that contained (in mM) NaCl 118.5,
lected in vacuo to afford a white powder, 0.75 g (55%); mp 128-
1
1
31°; ir (KBr): 3216m (NH), 1600s (CO); H nmr (400 MHz;
NaHCO 25.0, KCl 4.8, MgSO 1.2, KH PO 1.2, CaCl 1.7, and
3
4
2
4
2
CDCl ; Me Si) δ 3.07 (s, 6 H, CH ), 3.31 (t, 2 H, J = 8, CH ),
3
4
3
2
glucose 12.0. The buffer was gassed with a mixture consisting of
95% O and 5% CO . The heart was allowed to stabilise for 15
3
=
.89 (t, 2 H, J = 8, CH ), 6.69 (d, 2 H, J = 9, ArH), 8.04 (d, 2 H, J
2
2
2
+
+
9, ArH); es-ms: m/z 250 (100) (MH ), 272 (65) (MNa ).
Anal. Calcd. for C H N OS: C, 57.8; H, 6.1; N, 16.9. Found:
minutes prior to being subjected to 30 minutes of regional
ischemia followed by 100 minutes of reperfusion. The heart tem-
perature was maintained between 36.5 and 37.5 ºC. Regional
ischemia was induced by tightening a suture around the left main
coronary artery and reperfusion initiated by releasing the ends of
the suture. An aqueous solution containing the hydrochloride of
1
2 15 3
C, 57.9; H, 6.0; N, 17.0.
X-ray Structure Analysis.
General crystallographic details for compounds 2 and 3a are
listed in Table 3. Crystals were grown from ethyl acetate solu-
tions. Crystallographic data was collected on a Bruker Nonius
Kappa CCD area diffractometer using monochromatised Mo-Kα
x-ray radiation (λ = 0.71073Å) equipped with an Oxford
Cryosystems low temperature device (Southampton University).
Lattice parameters were calculated using 7564 2 and 8432 3a
reflections with 2.91º < θ < 27.48º. Intensity data were collected
at a temperature of 120 K 2 and 150 K 3a using ϕ-ω scans to a
maximum 2θ value of 50° 2 and 53º 3a. Multi-scan absorption
corrections were applied to all data sets using the program SOR-
TAV [36]. Structures were solved by direct methods and refined
using the SHELX-97 package [37]. All hydrogen atoms not
involved in the strong hydrogen-bonding associations were
included in the refinement at calculated positions as riding mod-
3
was administered to the Krebs buffer, resulting in an overall
concentration of 0.01 mM of 3, 10 minutes before reperfusion
commenced. Throughout the duration of the experiment record-
ings were taken every 5 minutes of left ventricle developed pres-
sure, coronary flow and heart rate. Left ventricle developed pres-
sure was monitored by inserting and inflating a latex balloon in
the left ventricle resulting in an end-diastolic pressure of 8 – 10
mmHg. The heart was then stained with triphenyltetrazolium and
frozen for 24 hours after which it was sliced and placed into
formaldehyde for a further 24 hours. From the slices the percent-
age of necrosis was determined.
Acknowledgements.
The authors thank the School of Science and the Environment
els with C-H set to 0.95 Å (Ar-H) and 0.98 Å (CH ). The NH and
3
(
Coventry) for financial assistance, the EPSRC National
Crystallography Service (Southampton) and Dr G. J. Langley
Southampton) for the collection of es-ms data. Dr H. Maddock
OH hydrogen atoms were located by difference methods and both
positional and thermal parameters were refined.
(
Nitroblue Tetrazolium (NBT) Reduction Test.
and Mr B. Harb (Coventry) are thanked for performing the
myocardial experiment.
All tests were performed using venous blood from resting
healthy male subjects aged 20 – 30 years. The hydrochlorides of 3
and 4, and diclofenac acid sodium salt were prepared in 10.0, 5.0,
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