Preparation and antitubercular activities in vitro and in vivo of novel Schiff bases of isoniazid

Article abstract of DOI:10.1016/j.ejmech.2009.05.009

Structural modification of the frontline antitubercular isonicotinic acid hydrazide (INH) provides lipophilic adaptations (3-46) of the drug in which the hydrazine moiety of the parent compound has been chemically blocked from the deactivating process of N²-acetylation by N-arylaminoacetyl transferases. As a class, these compounds show high levels of activity against Mycobacterium tuberculosis in vitro and in tuberculosis-infected macrophages. They provide strong protection in tuberculosis-infected mice and have low toxicity. With some representatives of this class achieving early peak plasma concentrations approximately three orders of magnitude above minimum inhibitory concentration, they may serve as tools for improving our understanding of INH-based treatment modalities, particularly for those patients chronically underdosed in conventional INH therapy.

Full text of DOI:10.1016/j.ejmech.2009.05.009

European Journal of Medicinal Chemistry 44 (2009) 4169–4178
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Preparation and antitubercular activities in vitro and in vivo of novel
Schiff bases of isoniazid
,
b
a
a
a
Michael J. Hearn a , Michael H. Cynamon , Michaeline F. Chen , Rebecca Coppins , Jessica Davis ,
Helen Joo-On Kang a, Abigail Noble a, Becky Tu-Sekine a, Marianne S. Terrot a, Daniella Trombino a,
Minh Thai a, Eleanor R. Webster a, Rebecca Wilson a
*
a Department of Chemistry, Wellesley College, 106 Central Street, Wellesley, MA 02481, USA
b Veterans Affairs Medical Center, Syracuse, NY 13210, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Structural modification of the frontline antitubercular isonicotinic acid hydrazide (INH) provides
lipophilic adaptations (3–46) of the drug in which the hydrazine moiety of the parent compound has
been chemically blocked from the deactivating process of N2-acetylation by N-arylaminoacetyl trans-
ferases. As a class, these compounds show high levels of activity against Mycobacterium tuberculosis in
vitro and in tuberculosis-infected macrophages. They provide strong protection in tuberculosis-infected
mice and have low toxicity. With some representatives of this class achieving early peak plasma
concentrations approximately three orders of magnitude above minimum inhibitory concentration, they
may serve as tools for improving our understanding of INH-based treatment modalities, particularly for
those patients chronically underdosed in conventional INH therapy.
Received 17 October 2008
Received in revised form 7 May 2009
Accepted 12 May 2009
Available online 21 May 2009
Keywords:
Tuberculosis
Acetylation
Isoniazid
Ó 2009 Elsevier Masson SAS. All rights reserved.
Schiff base
1. Introduction
Advancement of these important drug characteristics may make it
possible to shorten the current lengthy therapeutic regimens and to
Having been used as the cornerstone of antituberculosis therapy
for more than half a century, much has been learned about the
biochemistry and multifaceted modes of action of the frontline
drug isoniazid (INH, 1, Fig. 1). Indeed, with the enormous global
burden of tuberculosis and the alarming rise in the number of
clinical isolates displaying drug resistance or increased virulence
[1–4], INH has become the single most researched antitubercular
agent [5–9]. The problem of resistance has necessitated combina-
tion regimens for tuberculosis since the earliest days of chemo-
therapy. Researchers have recently begun to better understand the
complex interactions among the pathogen, host and antimicrobial
agent that relate to resistance and thus require combination
therapy [10–13]. It is desirable to maintain drug concentrations
within the mammalian host at levels sufficiently high to restrict
amplification of drug-resistant mutant subpopulations. As a prac-
tical matter, drugs for combination therapy are most efficacious
when their achievable serum concentration levels permit this
restriction of amplification, given concomitant low toxicity.
address the physiologically-controlled underdosing of certain
patient groups, both of which have been identified as pivotal factors
in the rise of acquired and primary drug resistance in the clinic
[14,15].
There is an increasing body of evidence to the effect that many
of the same molecular characteristics determined to enhance
bioavailability of antitubercular drugs also have the potential to
suppress the rise of resistance [16–18]. It has been argued that,
where significant increases can be made in serum concentration
levels, drug efficacy can be enhanced, even against moderately
drug-resistant organisms [19–21]. Persuasive data strongly suggest
that a significant impediment to the implementation of long-
sought intermittent treatment regimens, such as rifapentine and
isoniazid given once per week, is the comparatively low level of
isoniazid that is achievable within the host [22]. With a companion
drug that is more pharmacokinetically suitable, intermittent
rifapentine-based therapy might become a reality [23]. Several
recent experiments indicate that the incorporation of hydrophobic
moieties into the framework of INH can enhance penetration of the
drug into the tissues of the mammalian host and into the waxy cell
wall of the bacterium. This strategy for drug design has been
proposed as a vehicle for controlled study of the growth cycle of the
* Corresponding author. Tel.: þ1 781 283 3127; fax: þ1 781 283 3642.
E-mail address: mhearn@wellesley.edu (M.J. Hearn).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.05.009

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M.J. Hearn et al. / European Journal of Medicinal Chemistry 44 (2009) 4169–4178
2. Results and discussion
CON1HN2H2
CONHN=CR1R2
2.1. Chemistry
Compounds 3–46 are readily prepared in good yields and purity
by functionalization of INH at N2. Treatment of INH with the
appropriate ketone or aldehyde for 1–2 hours in boiling alcohol
typically leads without complication to the condensation product,
the Schiff base, which is generally isolated as a crystalline solid in
analytical purity (Fig. 2). Carbonyl precursors included substituted
aromatic aldehydes, long chain aliphatic aldehydes, palindromic
N
N
2
1
Fig. 1. The antitubercular isonicotinic acid hydrazide (1) and its Schiff base congeners
(2). Position N2 of 1 is subject to deactivating acetylation by the class of enzymes
known as N-arylaminoacetyl transferases (NATs). The deactivation phenomenon is
associated with the rise of resistance. Compound 2 are blocked toward this enzymatic
process.
dialdehydes,
a,b-unsaturated aldehydes, cinnamaldehydes,
aliphatic ketones, aryl alkyl ketones and keto esters. In a represen-
tative example, INH reacted at reflux for 1.25 hours with a molar
equivalent of 2-benzyloxybenzaldehyde in ethanol to produce
pathogen, as well as a means of augmenting fundamental drug
activity [24–27]. Previous work on several members of this class,
particularly the aromatic derivatives, had been carried out some
years ago and had demonstrated good activities by the methods
then available [28–37]. These compounds have continued to attract
the attention of medicinal chemists up to the present day [38–43].
For example, a series of (E)-N0-(monosubstituted-benzylidene)iso-
nicotinoyl-hydrazides were recently evaluated to good effect, with
several of these aromatic compounds showing potent activities in
vitro [44], although structure–activity relationships have not yet
become clear. There is evidence that such structurally-modified
drugs gain efficacy as the result of suppression of xenobiotic
transformation [28–32], and that they obtain noteworthy concen-
tration in several organs of tuberculosis-infected experimental
animals, perhaps most significantly in caseation bodies [33]. These
compounds also possess a vital characteristic of antimycobacterials,
namely, that toxicity is low [32,34–44].
Structurally, the modifications engendering these desirable drug
properties are optimally made at N2 of the INH framework. Such
modifications block the resulting molecule against the action of
N-arylaminoacetyl transferases (NATs). These enzymes are found in
both mycobacteria and their mammalian hosts, and they deactivate
INH by means of reaction at N2. Consistent with a survival strat-
agem in which pathogenic bacteria control their environment
through acetylation [45,46], the NATs have been implicated in the
development of resistance, particularly among those patients
known as the ‘‘fast acetylators,’’ for whom there is an inherent
problem of chronic underdosing of INH under genetic control [47].
Evaluations of pharmacokinetic diversity among patients treated
with INH indicate that its sources are complex and that the problem
of underdosing is more widespread than once believed, under-
scoring the need for clinicians to know the acetylator status of their
patients [48]. Underdosing typically places fast acetylators in
jeopardy for selective amplification of drug-resistant organisms
within a mutant selection range of serum drug concentrations.
Improving serum drug concentrations should narrow this range
[49–51], and structurally blocking INH toward the actions of NATs
at N2 may thus combat the rise of resistance. We previously
reported results on one of these lipophilic compounds modified at
N2 (46) that combined low toxicity with serum concentration levels
as much as three orders of magnitude above minimum inhibitory
concentration (MIC) [32]. We now report findings from our further
experiments in vitro and in vivo on Schiff base derivatives of INH
(2, Fig. 1), including results from a new short-course therapy model
in mice. In line with the above discussion, our rationale has been to
prepare Schiff bases with enhanced lipophilicity and to examine
their efficacy against Mycobacterium tuberculosis with up-to-date
methods. Our objective is the development of these compounds as
tools for probing interactions among pathogen, host and drugs in
INH-based treatment modalities.
compound
crystalline solid, 90%, m.p.: 185 ꢀC. FT-IR:
NH), 1653 (acylhydrazone carbonyl). 1H NMR:
2
(R1 ¼ H, R2 ¼ 2-(OCH2C6H5)C6H4, 3) as
(cmꢁ1) 3227 (hydrazone
(ppm) 8.85
a white
n
d
(1H, singlet, azomethine), 8.75 (2H, doublet, pyridine alpha-
protons), 7.98–7.01 (9H, multiplets, aromatics and pyridine beta-
protons), 5.20 (2H, singlet, benzyl protons). 13C NMR:
d (ppm)
161.89 and 157.36 (azomethine and carbonyl). HRMS (FAB MHþ):
(C20H18N3O2) Calc 332.1399. Found: 332.1414. Anal. (C20H17N3O2)
Calc C 72.49, H 5.17. Found: C 72.59, H 5.35. The calculated value of
the logarithm of the octanol/water partition coefficient (C log P) for
this compound (4.57) is greater than that of INH (0.49), consistent
with the improved solubility observed for this compound in
a
number of organic solvents and indicative of enhanced
lipophilicity [52].
Our results on the preparation of the Schiff bases included in
this study are summarized in Table 1. The Schiff bases may be
conveniently divided into aromatic compounds (3–22) and
aliphatic derivatives (23–46). Using the methods described,
recrystallization was generally not required. In some cases, it was
necessary to concentrate the reaction mixture to half volume and to
precipitate the product with ether. As alternative solvents, both
water and toluene produced inferior results in synthesis. The
compounds were stable for prolonged periods on the shelf when
not stored in direct light. All the Schiff bases had C log P values
considerably greater (more lipophilic) than did INH itself (Table 1).
In laboratory practice, experimental estimates of partitioning
between aqueous and organic phases for a number of these
compounds (see Experimental protocols) also demonstrated their
organic solubilities to be several orders of magnitude greater than
that of INH. During our investigations, we found that there were
some structural limits on the procedure. Thus we observed that aryl
alkyl ketones, such as 4 with alkyl groups larger than methyl,
required strenuous conditions for reaction, namely, refluxing in
dimethyl sulfoxide (DMSO). We attribute this lower reactivity to
a sterically congested approach (Fig. 3) for nucleophilic attack in
which the lone pair on N2 must stand orthogonal to the conjugated
p
system of the remainder of the INH molecule. With alkyl groups
larger than methyl in the ketone component of the transition state,
the conformational preference for the lone pair on N2 renders this
CONHN=CR1R2
CONHNH2
N
R1COR2
+
N
2
Fig. 2. Preparation of Schiff bases from carbonyl precursors.

M.J. Hearn et al. / European Journal of Medicinal Chemistry 44 (2009) 4169–4178
4171
Table 1
Preparation of Schiff base 2.
Entry
Compound
R1
R2
% Yield
mp, ꢀ
185
116–117
218–219
218–221
195–196
243
234
221
215–216
241–244
239
C
C log Pa
MIC,
m
g/mLb
1.
2.
3.
4.
5.
6.
7.
8.
3
4
5
6
7
H
2–(OCH2C6H5)C6H4
C6H5
4-BrC6H4
2-ClC6H4
3,4-F2C6H3
90
64
91
66
86
92
90
87
94
88
100
98
97
97
100
92
87
85
98
91
54
73
93
84
59
29
48
90
76
4.57
4.49
3.84
3.56
3.32
4.30
3.80
3.76
3.56
4.08
3.32
4.08
4.08
1.06
1.58
2.68
5.49
1.06
3.04
4.00
2.24
1.85
2.64
3.03
2.97
3.58
1.70
3.78
3.94
0.06c
0.39
3.13
0.78
1.56
0.20
0.20
0.39
0.20
0.20
0.10
0.78
<6.25
0.39
0.39
(CH2)4CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
8
9
4-IC6H4
3-I-4,5-(OCH3)2C6H2
3-I-4-OH-5-OCH3C6H2
4-ClC6H5
3,4-Cl2C6H3
2,6-F2C6H3
2,3-Cl2C6H3
2,6-Cl2C6H3
2-NO2C6H4
4-Cl-3-NO2C6H3
C6H5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
227
Semisolid
230
231
169–170
135–138
225
<6.25
0.39
4-CH3(CH2)5OC6H4
3-NO2C6H4
C6H5
0.125c
0.05
197
4-CH3(CH2)3OC6H4
t-CH]CHCH2CH3
t-CH]CHCH3
t-CH]CHCH2CH2CH3
t-CH]CH(CH2)3CH3
C]CCH3CH2CH2CH]CH2
CH2CHCH3CH2CH2CH]C(CH3)2
CH3
148–149
161–164
198–201
152–154
152–154
124–125
78–80
161–162
104
0.10
0.05
<6.25
<6.25
<6.25
0.10
0.20
0.10
0.10
<0.025
H
CH3
CH2Ph
R1R2]CCH2CH
CH2CO2CH2CH3
149–151
(CH3)CH2C(CH3)2CH2
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
H
H
H
H
H
H
H
H
CH]C(C6H5)2
86
89
94
84
99
92
90
90
98
85
90
90
73
73
81
223
218
204
121
129
206
200
4.43
2.73
1.08
4.10
3.97
2.81
3.34
1.58
2.96
1.93
4.04
4.24
5.73
5.33
2.85
0.10
0.20
0.25c
0.50
0.06c
0.10
0.50
0.10
0.39
0.20
0.05
0.10
0.20
0.20
0.03
t-CH]CH-4-OCH3C6H4
t-CH]CH-2-NO2C6H4
C(n-C3H7)]CH(CH2)3CH3
C(i-C3H7)]CHCH2CH(CH3)2
t-CH]CH-2-OCH3C6H4
t-CCH3]CHC6H5
CH]NNHCOC5H4N
CH2CO2CH2CH3
CH2CO2CH2CH3
>300 (d)d
89–90
99
CH2CH2CH3
CH3
H
CH2CH2C6H5
H
H
4-(CH]NNHCOC5H4N)C6H4
CO2CH2CH3
CH3(CH2)11CH2
>300 (d)d
104
89–91
92–93
167–168
CH3(CH2)10CH2
R1R2]C(CH2CH2)2CH2
a
Calculated using HyperChem Pro, Version 7.5. For INH, C log P is 0.49.
MIC90 against M. tuberculosis strain H37Rv, unless otherwise noted. MIC INH control 0.03–0.06 mg/mL.
MIC90 against M. tuberculosis strain Erdman. MIC INH control 0.03–0.06 mg/mL. MIC PAS control 0.03–0.06 mg/mL.
Decomposed without melting.
b
c
d
nitrogen poorly poised for nucleophilic attack. Such stereo-
electronic effects have been observed previously in the reactions of
acylhydrazines as nucleophiles [53].
against the pathogen (strain Erdman) with an MIC of 0.06
(INH control 0.06 g/mL, para-aminosalicylic acid (PAS) control
0.06 g/mL).
mg/mL
m
m
Thirty-one of the more active compounds were evaluated for
cytotoxicity in VERO cells at concentrations ten times the MIC
(Table 2) [54]. After 72 hours exposure, viability was assessed using
a standard cell proliferation assay, in which the selection criterion
was a selectivity index (SI, IC50/MIC) greater than 10. Nearly all of
the Schiff bases met this criterion, giving an acceptable range
between their active and cytotoxic concentrations. For the eleven
compounds with outstanding SI values exceeding 1000, the mean
C log P was 3.2.
For eleven of the compounds, efficacy in vitro was further
explored in a tuberculosis-infected macrophage model [54]. This
assay measures killing of M. tuberculosis strain Erdman in mono-
layers of mouse bone marrow macrophages. In Table 3, EC90 and
EC99 show the lowest concentrations giving 90% and 99% reduction
in colony-forming units at 7 days (compared to drug-free controls)
at four-fold concentrations equivalent to 0.25, 1, 4 and 16 times the
MIC. In this model, EC90 represents bacteriostatic activity and
2.2. Biological assays
In a standard primary screen against M. tuberculosis strain
H37Rv, all the compounds in this study (3–46) were active and
displayed minimum inhibitory concentrations necessary to inhibit
growth of the organism by 90% of less than 6.25 mg/mL [54].
Activities in vitro were also determined against M. tuberculosis
strain Erdman using a broth dilution method, referred to known
antituberculars as standards [32]. Individual MIC values are
reported in Table 1. The mean of the MIC values for the forty-four
compounds tested was 1
values considerably less than this. Compounds displaying MICs of
0.06 g/mL or less were derived from cyclohexanones (31, 46),
mg/mL, with most having individual
m
benzaldehydes (3, 21) and enals (23, 24), having an average C log P
value of 3.1. Compound 3 provides a representative example of the
biological activities of the Schiff bases, demonstrating good potency

4172
M.J. Hearn et al. / European Journal of Medicinal Chemistry 44 (2009) 4169–4178
Table 3
H
H
H
Efficacies within tuberculosis-infected macrophages.a
Entry
Compound
EC90
,
m
g/mL
EC99
,
m
g/mL
EC90/MIC
O
1.
4
11
12
13
14
17
23
30
31
32
46
0.050
0.04
0.25
0.14
0.596
0.15
0.30
0.30
0.529
0.211
0.15
0.13
0.20
0.53
0.37
0.09
0.20
0.56
0.10
2.52
0.28
2.7
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
0.106
0.037
0.071
0.078
0.028
0.010
0.063
0.028
0.08
..
N
0.121
0.42
CH2(CH2)2CH3
H
a
Data from TAACF [54]. EC90 INH control 0.03 mg/mL. EC99 INH control 0.42 mg/
mL. EC90/MIC INH control 1. The ratio provides a measure of bioavailability and
metabolism of the active agent within the living host cell; whenever the ratio is less
than 16, it is said to indicate effective reduction in residual mycobacterial growth.
O
it lives within the host. For INH, the ratio is unity. Whenever the
ratio is less than 16, it is concluded that there has been an effective
reduction in residual mycobacterial growth. This condition is
fulfilled by all eleven of the compounds assessed. The data thus
indicate that the compounds are active against both extracellular
and intracellular organisms. For the eleven compounds displaying
excellent efficacies within the tuberculosis-infected macrophage
model, the mean value of C log P was 3.5.
H
Fig. 3. With alkyl groups larger than methyl in the ketone component, nucleophilic
attack by the hydrazide moiety is hindered.
EC99 indicates bactericidal activity. For INH, EC90 is 0.03 mg/mL and
EC99 is 0.42 mg/mL. Most of the Schiff bases gave EC90 and
EC99 values equal to or less than those of INH. The ratio EC90/MIC
(Table 3) gives a measure of bioavailability and metabolism of the
compound within the living host cell, comparing the in vitro
activity against the bacillus to the activity against the bacillus while
For our studies in vivo, we used a short-course treatment
method that has recently been described [55]. This method
provides a demanding test of the activities of candidate drugs and
has the advantage of cutting in half the time required to produce
results in animal studies, compared to the traditional four-week
treatment model (Table 4). In brief, C57BL/6 mice are infected
intranasally with approximately one million viable M. tuberculosis
organisms. Treatment begins one day post-infection and is
administered for two days. Mice are sacrificed three days post-
infection, their right lungs are removed aseptically, and cell counts
are determined. Mice are only able to present a strong immune
response to control the growth of M. tuberculosis about ten to
fourteen days after initial infection, so the reduction in mycobac-
teria using the two-day treatment regimen is entirely drug related.
Three aspects of the model have been established: (1) If a test
compound shows activity after two days of treatment, then the
compound must have reached the lung after oral delivery, it being
known that there is no detectable circulation of the pathogen
within the first four days of infection after intranasal deposition.
Bacterial reduction observed in the lung after treatment is thus due
to mycobacterial killing within the lung itself. (2) If a test drug has
activity in the model, it has entered the macrophage in which the
bacteria reside. (3) Activity indicates that the drug is killing the
bacteria within the phagosome of the macrophage. The short-
course murine model thus provides a rigorous and robust indica-
tion of the efficacy of an antimycobacterial agent. In the short-
course therapy model, representative compound 3 and INH were
dosed at 25 mg/kg of body weight. No ill effects or overt toxicity
was noted in the animals, in keeping with our observations on
Schiff base 46, for which the maximally tolerated dose in mice was
determined to be 1000 mg/kg [32]. Compared to early untreated
controls, compound 3 displayed strong activity, giving 1.01 log10
reduction in colony-forming units (CFU). This was somewhat more
protection than provided by INH, which gave 0.92 log10 CFU
reduction. Results in vivo for selected Schiff bases are provided in
Table 2. These compounds generally appeared to have strong
activities in murine models. For the Schiff bases examined in vivo,
the mean C log P value was 3.7.
Table 2
Selectivity indices of Schiff base 2.a
Entry
Compound
SI (IC50/MIC)
1.
2.
3.
4.
5.
6.
7.
8.
4
5
6
8
9
>25
12
>128
>50
297
>160
>50
>50
>1250
>32
>64
1.5
10
11
12
13
14
17
19
20
21
22
23
28
29
30
31
32
33
35
36
38
40
41
42
43
45
46
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
8
4000
>2000
712
>1000
>10,000
>1250
>2000
68
>50
1.60
>10
>250
>2631
>5000
>126
>1250
374
>40,000
a
Data from TAACF [54]. Evaluation for cytotoxicity in VERO cells at concentrations
10 times the MIC for M. tuberculosis H37Rv. SI INH control >40,000. The activity
criterion for this assay is an SI > 10.

M.J. Hearn et al. / European Journal of Medicinal Chemistry 44 (2009) 4169–4178
4173
Table 4
melting points. A representative procedure for this method is
provided for compound 29. Our data for activities in vitro and in
vivo for compound 46 (2, R1–R2 ¼ (CH2)5) have been previously
reported [32]. Nuclear magnetic resonance (NMR) spectra were
taken on a Bruker 300 Fourier transform instrument in dimethyl
sulfoxide-d6, recorded at 300 MHz (1H NMR) or 75 MHz (13C NMR)
In vivo activities of Schiff bases.
Compound
3a
Group
log CFU/lung
Early controls
Late controls
Isoniazid
6.50 ꢃ 0.14
6.37 ꢃ 0.24
5.58 ꢃ 0.28
5.49 ꢃ 0.63
Schiff base 3
and are reported in parts per million delta (d) downfield from
36b
38b
Early controls
Late controls
Isoniazid
6.38 ꢃ 0.14
6.51 ꢃ 0.13
4.96 ꢃ 0.13
5.49 ꢃ 0.26
internal tetramethylsilane as reference, with coupling constants
given in cycles per second (cps). In some proton spectra, only
signals in the region 0–10 ppm are reported. High resolution mass
spectra (HRMS, fast atom bombardment method) and low resolu-
tion mass spectra were determined at the NIH Mass Spectrometry
Facility at Michigan State University, East Lansing, Michigan, USA,
unless otherwise noted. Values for the calculated logarithm of the
octanol–water partitioning coefficient (C log P) were obtained
using the QSAR properties function of HyperChem Pro, Version 7.5.
Safety Notes: Gloves were worn during the chemical synthesis, and
the reactions were carried out in the hood. In general, any scale-up
of preparations of compounds with relatively high proportions of
nitrogen and oxygen was done with due caution. No specific safety
problems were encountered with the methods given below. No
attempt was made to optimize yields.
Schiff base 36
Early controls
Late controls
Isoniazid
6.38 ꢃ 0.14
6.51 ꢃ 0.13
4.96 ꢃ 0.13
5.22 ꢃ 0.36
Schiff base 38
46c
Early controls
Late controls
Isoniazid
6.74 ꢃ 0.98
8.80 ꢃ 0.18
4.71 ꢃ 0.89
4.65 ꢃ 0.27
Schiff base 46
a
Groups of six mice, short-course therapy model.
Groups of five mice, short-course therapy model.
Groups of eight mice, traditional four-week therapy model.
b
c
3. Conclusions
4.1.2. Representative example of Schiff base formation.
N2-(2-benzyloxy)benzylidenyl isonicotinic acid hydrazide (3)
INH (1.37 g, 10.0 mmol) was mixed with absolute ethanol
(15 mL) and the mixture brought to the boil, producing a slurry.
Barely sufficient additional ethanol was then added to give
The Schiff bases of INH were readily prepared for evaluation
against M. tuberculosis in good yield and purity using inexpensive
commonly available reagents. The structural modification of the
INH framework described here provides a lipophilic adaptation of
isoniazid in which the hydrazine unit has been chemically blocked
from the deactivating process of N2-acetylation by NATs. Across the
class, these compounds show strong levels of activity in vitro and in
experimental animals. In each of the biological assays, high potency
was observed for compounds with values of C log P considerably
greater than that for INH itself, and this appears to be the most
significant structure–activity relationship. The exploration of more
subtle structure–activity relationships is the subject of ongoing
work in our laboratory. With some representatives of this class
achieving early peak plasma concentrations approximately three
orders of magnitude above MIC [32], the Schiff bases may serve as
discovery tools in probing INH-based treatment modalities,
particularly for those patients chronically underdosed in conven-
tional INH therapy.
a
homogeneous solution at reflux. 2-Benzyloxybenzaldehyde
(2.12 g, 10.0 mmol) was added dropwise over 5 minutes and
washed with 3 mL of ethanol. The reaction mixture was refluxed for
1.25 hours, then allowed to cool slowly and to stand over night,
producing a white crystalline solid, which was filtered off and
dried. Yield: 2.88 g, (90%), m.p.: 185 ꢀC. FT-IR:
n
(cmꢁ1) 3227, 3077,
1653,1598,1549,1371,1249. 1H NMR:
d
(ppm) 8.85 (1H, s), 8.75 (2H,
d, J ¼ 7 cps), 7.98–7.01 (9H, m) from which emerged at 7.88 (2H, d,
J ¼ 7 cps), 5.20 (2H, s). 13C NMR:
d (ppm) 161.89, 157.36, 150.61,
144.51, 140.85, 137.02, 132.15, 128.90, 128.38, 127.88, 126.14, 123.46,
122.69, 121.91, 121.36, 113.48, 70.07. HRMS (FAB MHþ):
(C20H18N3O2) Calc 332.1399. Found: 332.1414. Anal. (C20H17N3O2)
Calc C 72.49, H 5.17. Found: C 72.59, H 5.35.
4.1.3. Isonicotinoylhydrazone of n-hexanophenone (4)
4. Experimental protocols
To a homogeneous solution of INH (0.45 g, 3.30 mmol) in boiling
DMSO (11 mL) was added n-hexanophenone (1.0 mL, 5.40 mmol).
The mixture was refluxed for an hour, the solution allowed to cool
for 20 minutes, and water was (9 mL) added. After several minutes
a solid began to form. The solid was filtered off and washed with
ether (3 ꢂ 10 mL) to give the title compound as a white solid. Yield:
4.1. Chemistry
4.1.1. General
Elemental analyses were carried out by Galbraith Laboratories,
Knoxville, Tennessee, USA. Melting points (m.p., ꢀC) were taken in
open capillary tubes using a Mel-Temp apparatus (Laboratory
Devices, Cambridge, MA, USA), and are corrected. Infrared (FT-IR)
spectra were recorded on a Perkin–Elmer Spectrum One Fourier
transform spectrophotometer fitted with a universal attenuated
total reflectance sampling accessory, reported in wavenumbers
0.62 g (64%), m.p.: 116–117 ꢀC. FT-IR:
n
(cmꢁ1) 3166, 1649, 1602,
(ppm) 11.30
1542,1347,1325,1284,1145,1090,1068, 834. 1H NMR:
d
(1H, br s), 8.78 (2H, d, J ¼ 7 cps), 7.91 (2H, d, J ¼ 7 cps), 7.65–7.21
(5H, m), 2.89 (m, 2H), 1.53–1.20 (m, 6H), 0.81 (m, 3H). Anal.
(C18H21N3O) Calc C 73.20, H 7.10. Found: C 73.23, H 7.16.
(n
, cmꢁ1). Most reactants, reagents and solvents were obtained
4.1.4. N2-4-bromobenzylidenyl isonicotinic acid hydrazide (5)
from Aldrich Chemical Company (Milwaukee, Wisconsin, USA) and
Lancaster Synthesis Incorporated (Windham, New Hampshire,
USA) and were used as received. All preparations were carried out
in a manner similar to that for compound 3. In some cases, it was
necessary to concentrate the reaction mixture to half volume and
add ether to precipitate the product. A number of the Schiff bases
were further characterized by their exchange reactions with
2,4-dinitrophenylhydrazine to produce the corresponding highly
insoluble 2,4-dinitrophenylhydrazones, readily identified by their
Yield: 1.65 g (91%), m.p.: 218–219 ꢀC. FT-IR:
n
(cmꢁ1) 3252, 3079,
(ppm) 12.18 (1H,
1657, 1603, 1589, 1551, 1290, 1067, 822. 1H NMR:
d
br s), 8.79 (2H, d, J ¼ 7 cps), 8.49 (1H, s), 7.86 (2H, d, J ¼ 7 cps), 7.70
(4H, aromatic pseudoquartet). Anal. (C13H10N3OBr) Calc C 51.34,
H 3.31. Found: C 51.32, H 3.30.
4.1.5. N2-2-chlorobenzylidenyl isonicotinic acid hydrazide (6)
Yield: 1.08 g (66%), m.p.: 218–221 ꢀC. FT-IR: (cmꢁ1) 3060, 1673,
n
1600, 1552, 1412, 1341, 1281, 1216, 1156, 1047, 1027, 1001, 842.

4174
M.J. Hearn et al. / European Journal of Medicinal Chemistry 44 (2009) 4169–4178
1H NMR:
d
(ppm) 8.95 (1H, s), 8.80 (2H, d, J ¼ 7 cps), 8.05 (1H, m),
1096, 1060, 1042, 997, 972, 940, 848, 783, 742, 708. Anal.
(C13H9N3OCl2) C 53.08, H 3.08. Found: C 53.16, H 3.20.
7.88 (2H, d, J ¼ 7 cps), 7.48 (4H, m). Anal. (C13H10N3OCl) Calc C 60.13,
H 3.88. Found: C 60.11, H 3.86.
4.1.14. N2-2,6-dichlorobenzylidenyl isonicotinic acid hydrazide (15)
4.1.6. N2-3,4-difluorobenzylidenyl isonicotinic acid hydrazide (7)
Yield: 97%, semisolid. FT-IR: n
(cmꢁ1) 3150, 1681, 1605, 1592,
Yield: 1.46 g (86%), m.p.: 195–196 ꢀC. FT-IR:
n
(cmꢁ1) 3159, 1679,
1555, 1416, 1354, 1274, 1222, 1192, 1148, 1000, 925, 842, 791, 778,
752, 720. Anal. (C13H9N3OCl2) C 53.08, H 3.08. Found: C 53.28,
H 3.21.
1567,1512,1376,1280,1218,1160,1134,1071, 850, 814, 757. 1H NMR:
d
(ppm) 12.20 (1H, br s), 8.80 (2H, d, J ¼ 7 cps), 8.44 (1H, s),
8.00–7.36 (5H, m, from which emerged at 7.82 a doublet, J ¼ 7 cps).
Anal. (C13H9N3OF2) Calc C 59.77, H 3.47. Found: C 59.44, H 3.56.
4.1.15. N2-2-nitrobenzylidenyl isonicotinic acid hydrazide (16)
Yield: 97%, m.p.: 230 ꢀC (uncorr). FT-IR:
n
(cmꢁ1) 3188, 1678,
4.1.7. N2-4-iodobenzylidenyl isonicotinic acid hydrazide (8)
1602, 1556, 1515, 1413, 1315, 1287, 1273, 1214, 1149, 1139, 1062, 998,
964, 932, 920, 880, 856, 848, 836, 788, 744. Anal. (C13H10N4O3)
C 57.78, H 3.73. Found: C 57.51, H 3.65.
Yield: 1.39 g (92%), m.p.: 243 ꢀC. FT-IR:
n
(cmꢁ1) 3229, 3063,
1654, 1606, 1583, 1543, 1478, 1404, 1393, 1359, 1290, 1218, 1148,
1066, 1057, 996, 959, 936, 922, 881, 846, 828, 809. 1H NMR:
d
(ppm)
8.80 (2H, d, J ¼ 7 cps), 8.40 (1H, s), 7.82 (4H, m, aromatic pseudo-
4.1.16. N2-4-chloro-3-nitrobenzylidenyl isonicotinic acid
quartet), 7.54 (2H, d, J ¼ 7 cps). 13C NMR:
d
(ppm) 162.08, 150.67,
hydrazide (17)
148.39, 140.72, 138.08, 133.91, 129.40, 121.89, 97.64. HRMS (FAB
MHþ): (C13H11N3OI) Calc 351.9947. Found: 351.9946. Anal.
(C13H10N3OI) Calc C 44.47, H 2.87. Found: C 44.49, H 3.03.
Yield: 100%, m.p.: 231 ꢀC (uncorr). FT-IR: (cmꢁ1) 3189, 1685,
n
1599, 1558, 1530, 1412, 1351, 1277, 1252, 1214, 1154, 1125, 1074, 1061,
1049, 998, 962, 942, 896, 846, 823, 748, 721. Anal. (C13H9N4O3Cl)
C 51.25, H 2.98. Found: C 51.20, H 2.90.
4.1.8. N2-3-iodo-4,5-dimethoxybenzylidenyl isonicotinic acid
hydrazide (9)
4.1.17. Isonicotinoylhydrazone of acetophenone (18)
Yield: 3.15 g (90%), m.p.: 234 ꢀC. FT-IR:
n
(cmꢁ1) 3200, 2980,
Yield: 92%, m.p.: 169–170 ꢀC. FT-IR: (cmꢁ1) 3173, 1652, 1599,
n
2929, 1673, 1599, 1585, 1538, 1494, 1474, 1455, 1408, 1358, 1273,
1235, 1216, 1190, 1173, 1150, 1063, 1046, 994, 960, 898, 860, 843.
1540, 1288, 1150, 1103, 975, 835, 757, 722. Anal. (C14H13N3O)
C 70.27, H 5.48. Found: C 70.18, H 5.51.
1H NMR:
d
(ppm) 8.58 (2H, d, J ¼ 7 cps), 8.13 (1H, s), 7.61 (2H, d,
J ¼ 7 cps), 7.51 (1H, s), 7.22 (1H, s), 3.69 (3H, s), 3.54 (3H, s).
4.1.18. N2-4-hexyloxybenzylidenyl isonicotinic acid hydrazide (19)
13C NMR:
d
(ppm) 162.08, 152.88, 150.67, 150.29, 147.43, 140.72,
Yield: 87%, m.p.: 135–138 ꢀC. FT-IR: (cmꢁ1) 3234, 3062, 1650,
n
132.56, 129.68, 121.90, 111.18, 93.37, 60.35, 56.36. HRMS (FAB MHþ):
(C15H15N3O3I) Calc 412.0158. Found: 412.0158. Anal. (C15H14N3O3I)
Calc C 43.81, H 3.43. Found: C 43.87, H 3.53.
1608, 1572, 1549, 1513, 1415, 1398, 1297, 1240, 1179, 1154, 1127, 1112,
1070, 1032, 993, 976, 961, 941, 922, 863, 846, 832, 810, 752, 725.
Anal. (C19H23N3O2) C 70.06, H 7.07. Found: C 70.07, H 7.07.
4.1.9. N2-3-iodo-4-hydroxy-5-methoxybenzylidenyl isonicotinic
4.1.19. N2-3-nitrobenzylidenyl isonicotinic acid hydrazide (20)
acid hydrazide (10)
Yield: 85%, m.p.: 225 ꢀC (uncorr). FT-IR: (cmꢁ1) 3232, 1691,
n
Yield: 3.46 g (87%), m.p.: 221 ꢀC. FT-IR:
n
(cmꢁ1) 3192, 3035,
1609, 1600, 1548, 1524, 1412, 1352, 1314, 1271, 1141, 1101, 1062, 996,
961, 948, 887, 843, 827, 815, 750, 739, 710. Anal. (C13H10N4O3)
C 57.78, H 3.73. Found: C 57.69, H 3.77.
2866, 1648, 1585, 1572, 1550, 1493, 1464, 1413, 1379, 1343, 1324,
1305, 1290, 1197, 1182, 1168, 1146, 1063, 1050, 1007, 986, 956, 905,
856, 833, 796, 762, 730. 1H NMR:
d
(ppm) 10.20 (1H, br), 8.75 (2H, d,
J ¼ 7 cps), 8.28 (1H, s), 7.82 (2H, d, J ¼ 7 cps), 7.62 (1H, s), 7.36
(1H, s), 3.92 (3H, s). 13C NMR:
(ppm) 161.91, 150.63, 148.94, 148.23,
4.1.20. N2-benzylidenyl isonicotinic acid hydrazide (21)
d
Yield: 98%, m.p.: 197 ꢀC, lit m.p. [59] 194–195 ꢀC. FT-IR: (cmꢁ1
n )
147.63, 140.86, 130.82, 127.59, 121.88, 109.38, 84.71, 56.45. HRMS
(FAB MHþ): (C14H13N3O3I) Calc 398.0002. Found: 398.0007. Anal.
(C14H12N3O3I) Calc C 42.34, H 3.05. Found: C 42.21, H 3.22.
3192, 1691, 1598, 1565, 1412, 1354, 1284, 1150, 1081, 1058, 998, 952,
920, 845, 767, 724.
4.1.21. N2-4-butoxylbenzylidenyl isonicotinic acid hydrazide (22)
4.1.10. N2-4-chlorobenzylidenyl isonicotinic acid hydrazide (11)
Yield: 91%, m.p.: 148–149 ꢀC. FT-IR: (cmꢁ1) 3272, 1650, 1260.
n
Yield: 94%, m.p.: 215–216 ꢀC. FT-IR:
n
(cmꢁ1) 3167, 1660, 1611,
Anal. (C17H19N3O2) C 68.68, H 6.44. Found: C 68.84, H 6.61.
1597,1412,1219,1158,1118,1087,1012, 952,1000, 879, 838, 818, 751,
722. Anal. (C13H10N3OCl) C, 60.13; H, 3.88. Found: C, 60.05; H, 3.89.
4.1.22. N2-2-trans-pentenylidenyl isonicotinic acid hydrazide (23)
The reaction of trans-2-pentenal (0.84 g, 10.0 mmol) with INH
(0.822 g, 6 mmol) in ethanol (20 mL) for 2 hours was followed by
concentration to half volume and the addition of ether (25 mL).
After standing over night, the title compound was obtained by
filtration from the reaction mixture and drying. Yield: 0.71 g (54%),
4.1.11. N2-3,4-dichlorobenzylidenyl isonicotinic acid hydrazide (12)
Yield: 88%, m.p.: 241–244 ꢀC. FT-IR:
n
(cmꢁ1) 3178, 1682, 1590,
1552, 1413, 1352, 1278, 1215, 1147, 1121, 1078, 1062, 1028, 999, 952,
937, 886, 846, 813, 747, 722. Anal. (C13H9N3OCl2) C 53.08, H 3.08.
Found: C 52.85, H 3.20.
m.p.: 161–164 ꢀC. FT-IR:
n
(cmꢁ1) 3237, 2725, 1654, 1638, 1546,
(ppm) 11.75 (1H, br s), 8.78 (2H, br d,
1293, 1001. 1H NMR:
d
4.1.12. N2-2,6-difluorobenzylidenyl isonicotinic acid hydrazide (13)
J ¼ 7 cps), 8.07 (1H, d, J ¼ 9 cps), 7.88 (2H, br d, J ¼ 7 cps), 6.35
Yield: 100%, m.p.: 239 ꢀC (uncorr). FT-IR:
n
(cmꢁ1) 3161, 1654,
(2H, m), 2.25 (2H, m), 1.05 (3H, t, J ¼ 6 cps). 13C NMR:
d (ppm)
1624,1608,1569,1550,1412,1305,1238,1154,1076,1066,1000, 960,
926, 840, 782, 758, 728. Anal. (C13H9N3OF2) C 59.77, H 3.47. Found:
C 59.92, H 3.70.
161.68, 151.55, 150.62, 146.12, 140.85, 126,70, 121.81, 25.69, 13.03.
HRMS (FAB MHþ): (C11H14N3O) Calc 204.1137. Found: 204.1139.
Anal. (C11H13N3O) Calc C 65.01, H 6.45. Found: C 65.18, H 6.42.
4.1.13. N2-2,3-dichlorobenzylidenyl isonicotinic acid hydrazide (14)
4.1.23. N2-2-trans-butenylidenyl isonicotinic acid hydrazide (24)
Yield: 98%, m.p.: 227 ꢀC (uncorr). FT-IR:
n
(cmꢁ1) 3188, 1687,
Yield: 0.83 g (73%), m.p.: 198–201 ꢀC. FT-IR: (cmꢁ1) 3178, 2725,
n
1602, 1585, 1547, 1497, 1411, 1350, 1278, 1248, 1214, 1189, 1155, 1141,
1664, 1637, 1578, 1547, 1299, 983. 1H NMR:
d (ppm) 11.78 (1H, br s),

M.J. Hearn et al. / European Journal of Medicinal Chemistry 44 (2009) 4169–4178
4175
8.75 (2H, br d, J ¼ 7 cps), 8.05 (1H, m), 7.78 (2H, d, J ¼ 7 cps), 6.28
2,4-dinitrophenylhydrazine by the same method) 127–128 ꢀC; lit
m.p. 128 ꢀC [56–58].
(2H, m), 1.88 (m, 3H). 13C NMR:
d (ppm) 161.71, 151.49, 150.61,
140.85,139.83,128.95,121.80,18.73. HRMS (FAB MHþ): (C10H12N3O)
Calc 190.0980. Found: 190.0983. Anal. (C10H11N3O) Calc C 63.48,
H 5.86. Found: C 63.79, H 5.94.
4.1.29. N2-1-ethoxycarbonyl-3-phenylpropylidenyl isonicotinic acid
hydrazide (30)
Yield: 1.72 g (90% in two crops), m.p.: 104 ꢀC. FT-IR:
n )
(cmꢁ1
4.1.24. N2-2-trans-hexenylidenyl isonicotinic acid hydrazide (25)
3250, 1702, 1664, 1592, 1553, 1510, 1399, 1245, 1143, 1074, 1022, 941,
843. Anal. (C18H19N3O3) Calc C 66.45, H 5.89. Found: C 66.10, H 5.96.
Yield: 1.21 g (in two crops, 93%), m.p.: 152–154 ꢀC. FT-IR:
n
d
(cmꢁ1) 3223, 2724, 1654, 1638, 1545, 1299, 994. 1H NMR:
(ppm) 11.78 (1H, br s), 8.76 (2H, d, J ¼ 7 cps), 8.06 (1H, m),
4.1.30. N2-3,5,5-trimethylcyclohexylidenyl isonicotinic acid
7.78 (2H, d, J ¼ 7 cps), 6.26 (2H, m), 2.22 (2H, m), 1.45 (2H,
hydrazide (31)
sextet, J ¼ 6 cps), 0.90 (3H, t, J ¼ 6 cps). 13C NMR:
d
(ppm)
Yield: 1.55 g (76%), m.p.: 149–151 ꢀC. FT-IR:
n
(cmꢁ1) 3174, 1649,
161.71, 151.50, 150.62, 144.49, 140.85, 127.82, 121.81, 34.62,
21.66, 13.86. HRMS (FAB MHþ): (C12H16N3O) Calc 218.1284.
Found: 218.1284. Anal. (C12H15N3O) Calc C 66.34, H 6.96. Found:
C 66.33, H 7.13.
1627, 1552, 1533, 1338, 1292. 1031. 1H NMR:
d (ppm) 10.91 and 10.08
(1H, br s), 8.69 (2H, d, J ¼ 7 cps), 7.71 (2H, d, J ¼ 7 cps), 2.94–0.78
(16H, m). Anal. (C15H21N3O)CalcC 69.47, H 8.16. Found:C 69.42, H8.17.
4.1.31. N2-
Yield: 0.83 g (86%), m.p.: 223 ꢀC. FT-IR:
b
-phenylcinnamylidenyl isonicotinic acid hydrazide (32)
4.1.25. N2-2-trans-heptenylidenyl isonicotinic acid hydrazide (26)
n
(cmꢁ1) 3186, 1648,
Yield: 1.16 g (in two crops, 84%), m.p.: 152–154 ꢀC. FT-IR:
1603, 1564, 1546, 1522, 1298, 1284, 1155, 1132, 1062, 1016, 842, 770.
Anal. (C21H17N3O) Calc C 77.04, H 5.23. Found: C 76.78, H 5.36.
n
d
(cmꢁ1) 3245, 2726, 1656, 1633, 1545, 1296, 1006, 962. 1H NMR:
(ppm) 11.78 (1H, br s), 8.76 (2H, d, J ¼ 7 cps), 8.08 (1H, m), 7.78
(2H, d, J ¼ 7 cps), 6.27 (2H, m), 2.20 (2H, m), 1.37 (4H, m), 0.85 (3H, t,
4.1.32. N2-4-methoxycinnamylidenyl isonicotinic acid hydrazide
J ¼ 6 cps). 13C NMR:
d
(ppm) 161.69, 151.50, 150.62, 144.73, 140.84,
(33)
127.66, 121.81, 32.25, 30.54, 21.99, 14.07. HRMS (FAB MHþ):
(C13H18N3O) Calc 232.1450. Found: 232.1443. Anal. (C13H17N3O)
Calc C 67.51, H 7.41. Found: C 67.38, H 7.53.
Yield: 2.51 g (89%), m.p.: 218 ꢀC. FT-IR: (cmꢁ1) 3221, 3044,
n
2840, 1649, 1622, 1603, 1576, 1537, 1509, 1456, 1442, 1415, 1362,
1318,1305,1291, 1259,1242, 1213,1173,1143, 1110,1086,1069, 1048,
1026, 982, 949, 934, 909, 841, 824, 812, 754. 1H NMR:
d (ppm) 8.54
4.1.26. N2-3,7-dimethyl-2,6-octadienylidenyl isonicotinic acid
hydrazide (27)
(2H, d, J ¼ 7 cps), 8.20 (1H, d, J ¼ 9 cps), 7.80 (2H, d, J ¼ 7 cps), 7.57
(2H, d, J ¼ 9 cps), 6.98 (4H, m), 3.78 (3H, s). 13C NMR:
d (ppm)
Compound 27 was prepared from INH (0.83 g, 6.06 mmol) and
citral (2 mL). The citral employed was the commercial mixture of
geranial and neral (D ¼ 0.888 g/mL). Yield: 0.97 g (59%), m.p.:
161.70, 160.35, 151.75, 150.62, 140.84, 140.17, 129.13, 128.80, 123.32,
121.85, 114.65, 55.55. HRMS (FAB Mþ): (C16H16N3O2) Calc 282.1243.
Found: 282.1241. Anal. (C16H16N3O2) Calc C 68.31, H 5.37. Found:
C 68.43, H 5.38.
124–125 ꢀC. FT-IR:
n
(cmꢁ1) 3178, 3028, 1638, 1548, 1297. Anal.
(C16H21N3O) Calc C 70.82, H 7.80. Found: C 70.78, H 7.99.
4.1.33. N2-2-nitrocinnamylidenyl isonicotinic acid hydrazide (34)
4.1.27. N2-3,7-dimethyl-6-octenylidenyl isonicotinic acid
hydrazide (28)
Yield: 2.77 (94%), m.p.: 204 ꢀC. FT-IR: (cmꢁ1) 3243, 3044, 1655,
n
1624, 1606, 1595, 1569, 1552, 1536, 1510, 1472, 1444, 1408, 1363,
1344, 1290, 1248, 1216, 1180, 1169, 1134, 1070, 1056, 975, 942, 910,
Schiff base 28 was formed from INH (0.85 g, 6.20 mmol) and
citronellal (2 mL). Yield: 0.49 g (29%), m.p.: 78–80 ꢀC. FT-IR:
859, 840, 828, 780, 755, 717. 1H NMR:
d
(ppm) 8.84 (2H, d, J ¼ 7 cps),
n
(cmꢁ1) 3226, 3060, 1651, 1619, 1544, 1410, 1296, 1215, 1135, 1067,
8.28 (1H, d, J ¼ 9 cps), 8.02 (2H, m), 7.85 (2H, d, J ¼ 7 cps), 7.75 (1H, t,
J ¼ 9 cps), 7.60 (1H, t, J ¼ 9 cps), 7.38 (1H, d, J ¼ 12 cps), 7.15 (1H, dd,
1032, 975, 906, 844, 757, 722. Anal. (C16H23N3O) Calc C 70.30,
H 8.48. Found: C 70.35, H 8.78.
J ¼ 12, 9 cps). 13C NMR:
d (ppm) 161.98, 150.68, 150.55, 148.17,
140.59, 134.09, 133.94, 133.36, 130.68, 130.23, 130.05, 128.73, 121.87.
Anal. (C15H12N4O3) Calc C 60.80, H 4.08. Found: C 60.68, H 4.12.
4.1.28. N2-isopropylidenyl isonicotinic acid hydrazide (29)
The Schiff base was produced from the reaction of INH (0.82 g,
6.00 mmol) with acetone (2 mL). It was necessary to concentrate
the reaction mixture to half volume and then to precipitate the
product with ether. Yield: 0.51 g (48%), m.p.: 161–162 ꢀC. FT-IR:
4.1.34. N2-2-propyl-2-heptenylidenyl isonicotinic acid
hydrazide (35)
The aldehyde used was the commercial mixture of cis and trans
n
(cmꢁ1) 3181, 3060, 1654, 1632, 1534, 1298. 1H NMR:
d
(ppm) 10.74
isomers. Yield: 2.29 g (84%), m.p.: 121 ꢀC. FT-IR: (cmꢁ1) 3210,
n
(1H, br s), 8.72 (2H, d, J ¼ 7 cps), 7.78 (2H, d, J ¼ 7 cps), 2.01 (3H, s),
1.91 (3H, s). Anal. (C9H11N3O) Calc C 61.00, H 6.25. Found: C 60.95,
H 6.21. This material was further characterized by its exchange
reaction with 2,4-dinitrophenylhydrazine to produce the 2,4-dini-
trophenylhydrazone of acetone, in a procedure used for a number
of compounds described in this work but only exemplified here.
Thus compound 29 (0.135 g) was dissolved in the minimum volume
(5 mL) of absolute ethanol. To this solution was then added stan-
dard 2,4-dinitrophenylhydrazine reagent (10 mL) [55], at room
temperature. Formation of a yellow precipitate was instantaneous.
The mixture was warmed to just below the boiling point and
swirled continuously. The mixture was then cooled, and the
resulting yellow-orange 2,4-dinitrophenylhydrazone of acetone
(72%) was allowed to stand for a few hours, then filtered off by
gravity, m.p. 127–128 ꢀC (from ethanol); mixed m.p. with an
authentic specimen (prepared directly from acetone and
3075, 2956, 2927, 2870, 1651, 1629, 1566, 1551, 1455, 1401, 1367,
1304, 1218, 1160, 1061, 1048, 976, 872, 838, 756, 723. 1H NMR:
d
(ppm) 11.68 (1H, br s), 8.78 (2H, d, J ¼ 7 cps), 7.99 (1H, s), 7.78 (2H,
d, J ¼ 7 cps), 5.89 (1H, t, J ¼ 7 cps), 2.39–1.99 (4H, m), 1.53–1.28 (6H,
m), 0.90 (6H, t, J ¼ 7 cps). 13C NMR:
d (ppm) 161.65, 154.00, 150.57,
141.91, 141.09, 137.53, 121.79, 31.25, 28.01, 27.33, 22.22, 21.89, 14.30,
14.13. Anal. (C16H23N3O) Calc C 70.30, H 8.48. Found: C 70.32, H 8.62.
4.1.35. N2-2-isopropyl-5-methyl-2-hexenylidenyl isonicotinic acid
hydrazide (36)
The aldehyde used was the commercial mixture of cis and
trans isomers. Yield: 2.71 g (99%), m.p.: 129 ꢀC. FT-IR:
n )
(cmꢁ1
3250, 3060, 2955, 2869, 1650, 1549, 1462, 1408, 1362, 1301, 1216,
1149, 1066, 993, 965, 892, 843, 754. 1H NMR:
d
(ppm) 11.75–11.50
(1H, br m), 8.78 (2H, m), 8.51 and 7.90 (1H, m), 7.78 (2H, m), 5.91–
5.62 (1H, m), 3.05 (1H, septet, J ¼ 6 cps), 2.12 (2H, m), 1.65 (1H, m),

4176
M.J. Hearn et al. / European Journal of Medicinal Chemistry 44 (2009) 4169–4178
1.19–0.78 (12H, m). 13C NMR:
d
(ppm) 168.43, 161.66, 150.57, 149.44,
4.1.44. N2-4-tridecanylidenyl isonicotinic acid hydrazide (45)
Yield: 73%, m.p.: 92–93 ꢀC. FT-IR: (cmꢁ1) 3260, 3066, 1654,
1624, 1597, 1548, 1412, 1294, 1041, 846, 754, 727. Anal. (C19H31N3O)
C 71.88, H 9.84. Found: C 71.68, H 10.14.
147.74, 142.72, 142.60, 142.23, 141.07, 140.94, 139.42, 139.21, 123.25,
121.79, 37.28, 35.94, 28.75, 28.55, 27.85, 26.76, 22.63, 22.56, 22.41,
20.89. Anal. (C16H23N3O) Calc C 70.30, H 8.48. Found: C 70.23,
H 8.48.
n
4.1.45. Experimental estimates of relative lipophilicities
of Schiff bases
4.1.36. N2-2-methoxycinnamylidenyl isonicotinic acid
hydrazide (37)
To a weighed amount of the Schiff base of interest (0.75 mmol)
in a Florence flask containing a stirring bar was delivered by pipet
distilled water (5 mL) and also by pipet chloroform (5 mL). The flask
was sealed with a cork and paraffin, and the contents were
magnetically stirred for 30 minutes. The cork, paraffin and stirring
bar were carefully removed, and the contents of the flask were
transferred to a separatory funnel and separated. The individual
layers were allowed to evaporate to dryness over night on a pre-
weighed watchglass. The ratio of amounts of dry compound
remaining after evaporation of the layers was obtained, then
compared to the same ratio for INH as standard, expressing the
comparison as follows:
Yield: 2.59 g (92%), m.p.: 206 ꢀC. FT-IR:
n
(cmꢁ1) 3246, 3023,
2994, 2839, 1646, 1615, 1595, 1581, 1540, 1487, 1465, 1436, 1408,
1368, 1314, 1291, 1245, 1216, 1186, 1172, 1141, 1107, 1067, 1044, 1025,
1001, 956, 909, 838, 748. 1H NMR:
d
(ppm) 11.95 (1H, br s), 8.82 (2H,
d, J ¼ 7 cps), 8.23 (1H, d, J ¼ 9 cps), 7.80 (2H, d, J ¼ 7 cps), 7.72–6.98
(6H, m), 3.82 (3H, s).13C NMR:
(ppm) 161.83,157.30,152.00,150.64,
d
140.82, 135.18, 130.84, 128.07, 126.29, 123.30, 121.86, 111.92, 55.90.
Anal. (C16H15N3O2) Calc C 68.31, H 5.37. Found: C 67.92, H 5.39.
4.1.37. N2-2-trans-2-methylcinnamylidenyl isonicotinic acid
hydrazide (38)
Yield: 2.38 g (90%), m.p.: 200 ꢀC. FT-IR:
n
(cmꢁ1) 3198, 3015,
AcmpdðCHCl3Þ=AcompdðH2OÞ : AINHðCHCl3Þ=AINHðH2OÞ
2863, 1650, 1620, 1598, 1572, 1550, 1493, 1439, 1402, 1362, 1325,
1305, 1212, 1159, 1074, 1015, 989, 966, 931, 877, 862, 839, 748, 724.
where Acmpd(solvent) refers to the amount of compound in
chloroform or water, as appropriate, and AINH(solvent) refers to the
amount of INH in chloroform or water, as appropriate. Represen-
tative examples of these estimates are shown in Fig. 4, in which
results are plotted against C log P [51] of the carbonyl precursor to
the Schiff base. In this way, we observed that functionalization at N2
led to lipophilicities as much as several orders of magnitude greater
than that of INH itself.
1H NMR:
d
(ppm) 8.78 (2H, d, J ¼ 7 cps), 8.24 (1H, s), 7.92 (2H, d,
J ¼ 7 cps), 7.58–7.28 (5H, m), 6.89 (1H, s), 2.14 (3H, s). 13C NMR:
d
(ppm) 160.02, 152.68, 148.79, 139.11, 136.17, 134.65, 132.75, 127.90,
126.96, 126.38, 120.03, 11.31. Anal. (C16H15N3O) Calc C 72.43, H 5.70.
Found: C 72.19, H 5.67.
4.1.38. Glyoxal di-isonicotinoylhydrazone (39)
This compound was prepared from glyoxal and isonicotinic acid
hydrazide (2 equiv). Yield: 90%, m.p.: >300 ꢀC (dec). FT-IR: (cmꢁ1
n )
3198, 3052, 1666, 1579, 1535, 1407, 1310, 1288, 1213, 1156, 1063, 963,
915, 842, 763, 730. Anal. (C14H12N6O2) Calc C 56.75, H 4.08. Found:
C 56.85, H 4.10.
4.2. Biological methods
M. tuberculosis ATCC 35801 (strain Erdman) was obtained from
the American Type Culture Collection (ATCC, Manassas, VA, USA).
INH was purchased from Sigma Chemical Company (St. Louis, MO,
USA). For testing, a given Schiff base was dissolved in dimethyl
sulfoxide and subsequently diluted in distilled water. INH was
dissolved in distilled water. Stock solutions were filter-sterilized by
4.1.39. Ethyl butyryl acetate isonicotinoylhydrazone (40)
Yield: 98%, m.p.: 89–90 ꢀC. FT-IR:
n
(cmꢁ1) 3163, 1733, 1675,
1655, 1627, 1599, 1558, 1548, 1426, 1406, 1336, 1298, 1261, 1196,
1162, 1095, 1044, 1030, 992, 974, 918, 886, 842, 762, 722. Anal.
(C14H19N3O3) Calc C 60.64, H 6.91. Found: C 60.26, H 7.02.
passage through a 0.22 mm-pore-size membrane filter and stored at
ꢁ20 ꢀC until use. The drugs were prepared each morning, before
experimentation. With respect to testing against this isolate, the
MICs of all antimicrobial agents were determined in modified 7H10
broth (7H10 agar formulation with agar and malachite green
omitted; pH 6.6) supplemented with 10% Middlebrook oleic
4.1.40. Ethyl acetoacetate isonicotinoylhydrazone (41)
Yield: 85%, m.p.: 99 ꢀC. FT-IR:
n
(cmꢁ1) 3200, 1733, 1690, 1655,
1635, 1598, 1554, 1535, 1406, 1336, 1301, 1268, 1182, 1142, 1067,
1037, 840, 756, 721, 672. Anal. (C12H15N3O3) Calc C 57.82, H 6.07.
Found: C 57.88, H 6.26.
4.1.41. Terephthalaldehyde di-isonicotinoylhydrazone (42)
Modifications of Lipophilicity
This compound was prepared from terephthalaldehyde and
isonicotinic acid hydrazide (2 equiv). Yield: 90%, m.p.: >300 ꢀC
4500
4000
3500
3000
2500
2000
1500
1000
500
(dec). FT-IR: n
(cmꢁ1) 3246, 3067, 1653, 1600, 1543, 1507, 1408, 1293,
1215, 1154, 1107, 1068, 969, 923, 839, 812, 755, 719. Anal.
(C20H16N6O2) Calc C 64.51, H 4.33. Found: C 64.59, H 4.44.
4.1.42. Isonicotinoylhydrazone of ethyl 2-oxo-4-phenylbutyrate (43)
Yield: 90%, m.p.: 104 ꢀC. FT-IR:
n
(cmꢁ1) 3251, 1702, 1685, 1592,
1554, 1511, 1417, 1403, 1301, 1246, 1213, 1143, 1112, 1074, 1022, 994,
942, 875, 843, 798, 764, 754, 721, 706. Anal. (C18H19N3O3) C 66.45,
H 5.89. Found: C 66.10, H 5.96.
0
-0.89
(inh)
1.64
3.48
5.69
(tridecanal)
4.1.43. N2-tetradecanylidenyl isonicotinic acid hydrazide (44)
(benzaldehyde) (citronellal)
Yield: 73%, m.p.: 89–91 ꢀC. FT-IR:
n
(cmꢁ1) 3258, 1654, 1624,
C log P (carbonyl precursor)
1553, 1410, 1295, 1221, 1158, 1120, 1099, 1065, 1042, 962, 846,
753, 718. Anal. (C20H33N3O) C 72.46, H 10.04. Found: C 72.20,
H 10.33.
Fig. 4. Bar graph depicting the relative lipophilicities of INH and its Schiff bases of
benzaldehyde, citronellal and tridecanal.

M.J. Hearn et al. / European Journal of Medicinal Chemistry 44 (2009) 4169–4178
4177
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