Synthesis, biological evaluation, and quantitative structure -activity relationship analysis of new Schiff bases of hydroxysemicarbazide as potential antitumor agents

Article abstract of DOI:10.1021/jm010252q

Thirty Schiff bases of hydroxysemicarbazide (Ar-CH=NNHCONHOH) have been synthesized and tested against L1210 murine leukemia cells. The IC₅₀ values were found to be in a range from 2.7 × 10^-6 to 9.4 × 10^-4 M. A total of 17

Full text of DOI:10.1021/jm010252q

410
J . Med. Chem. 2002, 45, 410-419
Syn th esis, Biologica l Eva lu a tion , a n d Qu a n tita tive Str u ctu r e-Activity
Rela tion sh ip An a lysis of New Sch iff Ba ses of Hyd r oxysem ica r ba zid e a s
P oten tia l An titu m or Agen ts†
Shijun Ren,‡ Rubin Wang,‡,§ Kenichi Komatsu,‡,| Patricia Bonaz-Krause, Yegor Zyrianov,
Charles E. McKenna, Csaba Csipke,# Zoltan A. Tokes,# and Eric J . Lien*,‡
Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue,
Los Angeles, CA 90089-9121, Department of Chemistry, University of Southern California, Los Angeles, CA 90089-0744, and
Department of Biochemistry and Molecular Biology and the Norris Comprehensive Cancer Center, Keck School of Medicine,
University of Southern California, Los Angeles, CA 90033
Received J une 6, 2001
Thirty Schiff bases of hydroxysemicarbazide (Ar-CHdNNHCONHOH) have been synthesized
and tested against L1210 murine leukemia cells. The IC50 values were found to be in a range
from 2.7 × 10-6 to 9.4 × 10-4 M. A total of 17 out of the 30 compounds had higher inhibitory
activities than hydroxyurea (an anticancer drug currently used for the treatment of melanoma,
leukemia, and ovarian cancer) against L1210 cells. Six compounds with IC50 values in
micromolar range were 11- to 30-fold more potent than hydroxyurea (IC50 ) 8.2 × 10-5 M).
The partition coefficient (log P) and ionization constants (pKa) of a model compound [1-(3-
trifluoromethylbenzylidene)-4-hydroxysemicarbazide, 1] were measured by the shake-flask
method, and the measured log P was used to derive Hansch-Fujita π constant of -CHd
NNHCONHOH. On the basis of the newly derived π and those of other moieties, the partition
coefficients (SlogP) of the other 29 compounds were calculated by the summation of π values.
Quantitative structure-activity relationship (QSAR) analysis showed that, besides the essential
pharmacophore (-NHCONHOH), hydrophobicity (SlogP), molecular size/polarizability (calcu-
lated molar refractivity), and the presence of an oxygen-containing group at the ortho position
(I) were important determinants for the antitumor activities. In conclusion, the results obtained
in this study show that several Schiff bases of hydroxysemicarbazide are potent inhibitors of
tumor cells and warrant further investigation as cancer chemotherapeutic agents.
In tr od u ction
against various tumor cell lines and viruses for several
compounds. Some of the active compounds have shown
in vivo anticancer activities and clinical potential.5,12 On
the basis of previous studies, an essential pharmacoph-
ore [-C(dX)NHOH; X ) O, NH] has been identified for
antitumor/antiviral activities.2,10
Ribonucleotide reductase (RR) is a key enzyme that
catalyzes the reduction of ribonucleotides to deoxyribo-
nucleotides and provides the building blocks for the de
novo DNA synthesis in all dividing cells. Previous
studies have clearly indicated that RR plays a funda-
mental role in the critical early events involved in tumor
promotion, and the RR activity is tightly linked to the
neoplastic expression state.1 Therefore, RR is a promis-
ing target for the design and development of anticancer
drugs.
Several different classes of compounds such as hy-
droxyguanidine, thiosemicarbazone, and substituted
benzohydroxamic acid and their derivatives have been
shown to be RR inhibitors by Lien’s group,2-10 Sartorel-
li’s group,11,12 and others.1,13-15 Submicromolar median
inhibitory concentrations (IC50) have been observed
Among all RR inhibitors investigated, only hydrox-
yurea (HU) has been marketed and used for the treat-
ment of melanoma, chronic myelocytic leukemia, and
recurrent, metastatic, or inoperable ovarian cancer. HU
is also used in the local control of primary squamous
cell carcinomas of the head and neck.16 Investigation of
HU has gained new momentum in the wake of success
stories about combination therapy in cancer and AIDS
treatment.17-19 At the present time, HU is the first-line
chemotherapeutic agent in the chronic phase of myel-
ogenous leukemia. Clinical data have clearly shown the
superiority of HU over busulfan.18 HU has also been
used in the treatment of AIDS in combination with
didanosine, showing no viral rebound after 1 year’s
treatment.19 HU causes an immediate inhibition of DNA
synthesis by acting on the R2 subunit of the RR enzyme
by one-electron transfer from HU to the tyrosyl free
radical and by destabilizing the iron center.20,21 Thera-
peutic application of HU has several disadvantages such
as short half-life (1.9-3.9 h) in patients due to its small
molecular size (MW ) 76.06) and extremely polar
nature (log Po/w ) -1.80), the necessity of using high
† Taken in part from the Ph.D. dissertation of S. Ren, School of
Pharmacy, University of Southern California, 2001. Parts of this work
have been presented at the 1999 and 2000 AAPS Annual Meetings
and the 2000 AAPS Western Regional Meeting as poster/podium
presentations.
* To whom correspondence should be addressed. Phone: 323-442-
1400. Fax: 323-442-1390. E-mail: elien@hsc.usc.edu and sren@maxim.
com.
‡ School of Pharmacy, University of Southern California.
§ Current address: College of Pharmacy, Shandong University,
J inan, China.
| Current address: Hokkaido College of Pharmacy, Otaru, J apan.
Department of Chemistry, University of Southern California.
#
Keck School of Medicine, University of Southern California.
10.1021/jm010252q CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/07/2001

Schiff Bases of Hydroxysemicarbazide
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 411
Sch em e 1
dosage (80 mg/kg every third day or 20-30 mg/kg daily),
and the rapid development of resistance.16,22,23
To obtain more potent and selective RR inhibitors, a
series of Schiff bases of hydroxysemicarbazide (SB-HSC)
with the essential pharmacophore (-NHCONHOH) and
diverse hydrophobic, electronic, and steric properties are
designed. We now report the synthesis, in vitro antitu-
mor activity against murine leukemia L1210 cells, and
quantitative structure-activity relationship (QSAR)
analysis of the SB-HSCs.
be due to breakage of the Schiff base bond (-CHdN-)
in aqueous solution at higher pH.
Biologica l Stu d y. The IC50 values of the newly
synthesized compounds were determined against mu-
rine leukemia L1210 cells using MTS/PES [MTS )
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium, PES ) phenazine etho-
sulfate] microculture tetrazolium assay in 96-well
plates.26-29 HU (H2NCONHOH), HSC (H2NHNCON-
HOH), and semicarbazide (SC, H2NHNCONH) were
used as reference compounds for comparison. The IC50
values of the compounds (see Table 2) range from 2.7 ×
10-6 to 9.4 × 10-4 M. Compounds 2, 4, 7, 21, 29, and
30 are the most active compounds with IC50 values in
the micromolar range. According to the chemical struc-
tures, the six most active compounds can be divided into
three groups. Compounds 2, 4, and 7 containing 2-OH-
3,5-dihalogen-substituted phenyl rings consist of the
first group. Compound 21 with a 2,3,4-OH-substituted
phenyl ring represents the second group. Compounds
29 and 30 belong to the third group because both of
them have substituted anthryl moieties. The IC50 ratios
of HU over compounds 2, 4, 7, 21, 29, and 30 range from
11 to 30, indicating that the six compounds are 11- to
30-fold more potent than HU against L1210 cells. It is
interesting to note that compound 24 with a nitrothienyl
group has an IC50 value of 1.1 × 10-5 M. Compounds
10, 20, and 23 with IC50 values greater than 8.0 × 10-4
M are the least active of the 30 Schiff bases. The ranking
order (SC . HSC > HU) of the IC50 values of the three
reference compounds reveals that the (N)-OH function
is essential for antitumor activity (see Table 2).
Resu lts a n d Discu ssion
Ch em istr y. The SB-HSCs were prepared by a dehy-
dration reaction of hydroxysemicarbazide (HSC) with
different aromatic and heterocyclic aldehydes. HSC,
which was not commercially available, was synthesized
in two steps using modified procedures of Grobner and
Steinberg (see Scheme 1).24,25 The percent yields and
corrected melting points are summarized in Table 1.
The logarithm of partition coefficient (log P) in 1-oc-
tanol/phosphate buffer (pH 7.3) and ionization constant
(pKa) values of compound 1 were measured. The log P
value of compound 1 was found to be 1.64. There are
two pKa values for compound 1, with a basic group (d
N-, pKa ) 1.87) and an acidic group (-OH, pKa
10.16).
)
Hansch-Fujita π substituent constants of dNNH-
CONHOH and -CHdNNHCONHOH were derived
from the measured log P of compound 1 and other π
constants as the following:
πdNNHCONHOH ) log P - π-C6H4 - π-CF3 - π-CHd
)
1.64 - 1.96 - 0.88 - 0.33 ) -1.53
QSAR. The CQSAR program of BioByte Corp. was
utilized to calculate ClogP (calculated log P) and CMR
(calculated molar refractivity) and to derive all regres-
sion equations.30 SlogP was calculated by taking the
sum of the π of -CHdNNHCONHOH derived from the
experimental log P value of compound 1 and those of
other moieties.31-33
π-CHdNNHCONHOH ) log P - π-C6H4 - π-CF3 ) 1.64 -
1.96 - 0.88 ) -1.20
From these, the SlogP values of other 29 compounds
were calculated by taking the sum of π values of
constituent groups and were used in subsequent QSAR
analysis as well as ClogP from CQSAR program (vide
infra).
The correlation between the inhibitory activities (log-
(1/IC )) against L1210 cells and the physicochemical
50
properties of the 30 SB-HSCs and two reference com-
pounds (HU, HSC) was analyzed by a nonweighted
least-squares method. Another compound, SC, was not
included in the QSAR analysis because it lacked the
common (N)-OH group and the IC50 value was greater
than 2.2 × 10-3 M. The physicochemical parameters
examined included hydrophobic (ClogP, SlogP), steric
(CMR), and electronic (e.g., dipole moment) parameters,
as well as hydrogen bonding capacity (Hb). An indicator
The stability study in phosphate buffers demonstrated
that there was no change in UV absorbance in 72 h at
pH 3 for compound 1. At higher pH, 2.7-21.3% changes
in UV absorbance were observed. At 25 °C 97.3% of
compound 1 remained at pH 7 and 78.7% remained at
pH 11 at the end of 72 h. The data indicated that less
than 3% of compound 1 was degraded in 72 h at neutral
pH at room temperature. Since the formation of Schiff
base is reversible, the change in the UV absorbance may

412 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Ren et al.
Ta ble 1. Percent Yields and Melting Points of Schiff Bases of Hydroxysemicarbazide Synthesized (Ar-CHdNNHCONHOH)
a
Compound 20 is a known compound.24
variable I was used for the presence (I )1) or absence
(I ) 0) of an oxygen-containing group (-OH, -OCH3,
keto) at the ortho position of -CHdNNHCONHOH.
The antitumor activities and physicochemical param-
eters used in the regression analysis are summarized
in Table 2. The quantitative relationship between the
The statistical parameters describing the regression
are n, the number of data points upon which the
equation is based; R, the correlation coefficient; Q2, a
measure of the predictive power of the equation (also
called cross-validated R2);34 and s, the standard devia-
tion from the regression. The level of significance of
single- or multiple-variable equations and that of ad-
dition of a single variable were examined by overall and
stepwise F tests, respectively. The probability of being
a chance correlation is designated as p. The number in
antitumor activities (represented by log(1/IC )) and the
50
structures (represented by the physicochemical proper-
ties) can be described by the stepwise regression equa-
tions in Table 3.

Schiff Bases of Hydroxysemicarbazide
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 413
Ta ble 2. Inhibitory Activities and Physicochemical Properties of HU, SC, HSC, and SB-HSCs Analyzed in the Regression Analysis
log(1/IC ) (M)
IC50 ( sda
(×10-6 M)
deviation
(obsd - calcd)
50
c
d
e
compd
HU
obsd
calcdb
ClogP
SlogP
CMRc
1.57
I
82.0 ( 6.0
4.086
3.856
0.230
-1.80g
-1.80g
0
(60.6)f
SCh
HSC
1
>2192
-2.75g
-2.91
1.94
-2.75g
-3.26
1.64i
1.61
1.78
1.94
5.55
6.17
281.6 ( 21.4
39.5 ( 2.7
6.5 ( 0.6
3.550
4.403
5.187
3.680
4.332
5.092
-0.130
0.071
0.095
0
0
1
2
1.88
(1.75)j
1.55
(1.48)j
1.16
3
4
10.1 ( 1.0
7.2 ( 1.7
4.996
5.143
4.905
5.191
0.091
-0.048
5.97
6.75
1
1
2.23
1.97
(2.03)j
1.32
(1.77)j
1.01
5
6
7
37.7 ( 5.7
80.4 ( 3.5
4.7 ( 1.5
4.424
4.095
5.328
4.536
4.271
5.250
-0.112
-0.176
0.078
6.59
6.34
7.80
1
0
1
2.18
1.91
2.68
2.39
(2.48)j
0.49
(2.19)j
0.19
8
208.5 ( 1.0
446.3 ( 44.0
820.6 ( 73.1
3.681
3.350
3.086
3.357
3.540
4.214
(3.117)l
3.494
3.596
3.596
4.465
4.274
4.087
4.173
4.570
4.056
2.862
3.786
3.148
3.212
3.399
3.477
4.530
4.398
3.461
5.158
5.394
0.324
-0.190
-1.128k
(-0.031)l
-0.095
-0.163
-0.097
0.056
5.52
6.33
6.41
0
0
1
9
1.22
0.94
10k
0.73
-0.47
(-3.88)j
0.48
0.74
0.74
0.72
0.07
0.05
2.42
(-2.68)j
0.80
11
12
13
14
15
16
17
18
19
20
21k
22
23
24k
25
26
27
28
29
30
399.3 ( 88.1
368.8 ( 1.5
316.7 ( 13.1
30.1 ( 5.0
60.2 ( 2.4
91.4 ( 0.9
48.1 ( 9.9
42.7 ( 7.9
130.8 ( 12.3
944.1 ( 78.7
2.7 ( 0.6
3.399
3.433
3.499
4.521
4.220
4.039
4.318
4.370
3.883
3.025
5.565
3.217
3.050
4.975
3.322
4.550
4.492
3.749
5.357
5.268
5.65
5.66
5.66
6.27
5.81
6.43
8.17
7.55
5.34
6.37
5.50
4.83
5.29
5.46
6.16
7.50
7.90
6.09
8.88
8.91
0
0
0
1
1
1
0
0
1
0
1
0
0
0
0
1
1
0
0
0
0.97
0.97
1.06
0.49
0.52
2.74
2.94
-0.28
0.07
-0.88
-0.44
0.06
0.53
1.04
1.72
1.72
-0.054
-0.048
0.145
-0.200
-0.173
0.163
2.72
-0.58
-0.21
-1.25
-0.70
-0.20
0.13
0.71
1.38
1.38
0.58
1.779k
0.069
607.0 ( 69.5
891.0 ( 128.3
10.6 ( 0.3
476.6 ( 37.3
28.2 ( 2.6
32.2 ( 0.4
178.3 ( 4.5
4.4 ( 0.0
-0.162
1.576k
-0.155
0.020
0.094
0.288
0.98
3.90
4.11
0.199
-0.126
3.64
3.84
5.4 ( 0.4
a
b
The average median inhibitory concentration obtained from two or three measurements. Calculated from eq 5. c Calculated by using
CQSAR program of BioByte.30 Calculated by taking the sum of the π of -CHdNNHCONHOH derived from the experimental log P
d
value of compound 1 and other constants.31,32 e An indicator variable indicating the presence (I ) 1) or absence (I ) 0) of an oxygen-
containing substituent at the ortho position of the Ar group. f From ref 4. Measured log P values obtained from the CQSAR program.30
g
Not included in the regression analysis. Experimental log P value from this study. Calculated distribution coefficients (log D).32
h
k
i
j
l
Statistical outliers, excluded from eqs 5 and 7. Calculated from eq 9.
parentheses is the 95% confidence interval of each
coefficient in the equations.
Compounds 10, 21, and 24 were statistical outliers
with deviations greater than 2s. Upon deletion of the
three outliers, better equations with higher R2 values
of 0.955 and 0.948 and lower s values of 0.166 and 0.177
were obtained for eqs 5 and 7, respectively. By these
two equations, 95.5% and 94.8% of the variances in the
data can be accounted for. Here, the QSAR analysis
reveals that compounds 21 and 24 might have different
limiting steps from other compounds as inhibitors of the
tumor cells. Besides the common pharmacophore of
-NHCONHOH, three phenolic OH groups of compound
21 may act as additional free radical scavengers and/or
chelator of the Fe3+ ions, which are required for the RR
enzyme activity. This explanation is consistent with the
findings of van’t Riet,35,36 Tihan,37 and Szekeres.14
Among the different benzohydroxamic acid derivatives
investigated, hydroxy-substituted benzohydroxamic acid
derivatives turned out to be more effective RR inhibitors
than HU. Further structure-activity analysis revealed
that addition of at least two hydroxyls on the benzene
ring could dramatically increase the enzyme inhibitory
capacity of the compound. Addition of a third hydroxyl
group could even further enhance the enzyme inhibitory
Nonweighted least-squares regression analysis of the
data reveals three parameters to be the best predictors
of the inhibitory activities of the compounds against
L1210 cells. These parameters are ClogP (or SlogP),
CMR, and the indicator variable I. First, ClogP (or
SlogP), a measure of the hydrophobicity of the com-
pounds, makes a positive contribution to the inhibitory
activities of the compounds tested. The inhibitory activi-
ties (log(1/IC )) nonlinearly depend on ClogP (or SlogP).
50
Second, CMR, a measure of molecular size and polar-
izability, makes a negative contribution to the inhibitory
activities against L1210 cells, suggesting that as CMR
increases, the inhibitory activity decreases after cor-
recting for the contribution of hydrophobicity (ClogP or
SlogP). Third, the indicator variable (I) makes a positive
contribution to the inhibitory activities. This indicates
that the presence of an oxygen-containing substituent
at the ortho position is beneficial to the inhibitory
activity. The OH groups on the phenyl ring act as free
radical scavengers and thus improve the antitumor
activity.

414 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Ta ble 3. Stepwise Regression Equations
Ren et al.
no.
1
equation
n
R2
Q2
s
overall F test
stepwise F test
log(1/IC )) 0.252(0.169) ClogP + 3.903(0.305)
32
0.231
0.122
0.685
F1,30 ) 9.02,
p < 0.01
50
2
3
log(1/IC ) ) 0.245(0.153) ClogP + 0.647(0.450)I +
32
0.407
0.585
0.616
0.955
0.644
0.948
0.684
0.952
0.784
0.981
0.245
0.399
0.399
0.921
0.440
0.915
0.548
0.929
0.653
0.978
0.612
0.521
0.510
0.166
0.492
0.177
0.463
0.172
0.354
0.166
F2,29 ) 9.96
p < 0.0005
F3,28 ) 13.14
p < 0.0005
F4,27 ) 10.81
p < 0.0005
F4,24 ) 126.08
p < 0.0005
F4,27 ) 12.20
p < 0.0005
F4,24 ) 109.59
p < 0.0005
F4,27 ) 14.62
p < 0.0005
F4,25 ) 124.75
p < 0.0005
F3,25 ) 30.20
p < 0.0005
F1,28 ) 1454.8
p < 0.0005
F1,29 ) 8.62
p < 0.01
F1,28 ) 11.96
p < 0.005
F1,27 ) 2.18
p < 0.25
3.64570(0.328)
log(1/IC ) ) 0.082(0.162) ClogP +
32
50
0.097(0.058)(ClogP)2 + 0.832(0.400)I + 3.431(0.308)
log(1/IC ) ) 0.296(0.337) ClogP + 0.092(0.057)(ClogP)2 +
0.93530(0.417)I - 0.217(0.302) CMR + 4.512(1.532)
log(1/IC ) ) 0.465(0.120) ClogP + 0.088(0.019)(ClogP)2 +
1.01520(0.142)I - 0.329(0.104) CMR + 4.924(0.524)
log(1/IC ) ) 0.489(0.282) SlogP + 0.084(0.054)(SlogP)2 +
0.98570(0.408)I - 0.248(0.264) CMR + 4.779(1.417)
log(1/IC ) ) 0.499(0.117) SlogP + 0.084(0.020)(SlogP)2 +
1.03550(0.153)I - 0.311(0.104) CMR + 4.978(0.555)
4
32
5
29a
32
6
7
29a
32
8b
9b
10
11
log(1/IC ) ) 0.431(0.196) ClogP + 0.075(0.047)(ClogP)2
1.05550(0.389)I - 0.312(0.212) CMR + 4.996(1.079)
log(1/IC ) ) 0.500(0.075) ClogP + 0.088(0.018)(ClogP)2
1.05590(0.149)I - 0.355(0.080) CMR + 5.042(0.404)
log(1/IC ) ) 0.698(0.232) ClogP + 0.856(0.294)I -
0.41530(0.218) CMR + 5.541(1.082)
+
+
30c
29a
30
ClogP ) 0.954(0.051) SlogP + 0.386(0.079)
a
b
Compounds 10, 21, and 24 were deleted as statistical outliers with deviations greater than 2s. The calculated log D values of
compounds 2, 4, 7, and 10 were used in the equations. c Compounds 21 and 24 were deleted as statistical outliers with deviations greater
than 2s.
Ta ble 4. Squared Correlation Matrix (R2) of the
potency of the compound. A good correlation has been
Physicochemical Parameters Used in the Regression Analysis
for HU, HSC, and SB-HSCs (n ) 32)
demonstrated between the ability to scavenge the free
radical and inhibition of the RR enzyme activity.38 It
has been reported that resveratrol (3,5,4′-trihydroxystil-
bene) and p-propoxyphenol are active RR inhibitors with
IC50 values of 1.0 × 10-4 and 3.0 × 10-4 M in soluble
extracts of murine leukemia L1210-R2 cells.39,40 Further
study is needed to clarify the outlier behavior of
compound 24, which contains a 5-nitrothienyl as Ar
group.
ClogP
1.000
SlogP
CMR
I
ClogP
SlogP
CMR
I
0.986
1.000
0.806
0.758
1.000
0.001
0.000
0.038
1.000
mechanism of action as inhibitors of the tumor cells.
Here, the QSAR analysis provides an insight for the
action of the different compounds against tumor cells.
The squared correlation matrix of the physicochemical
properties used in the regression analysis is shown in
Table 4. From Table 4, one can see that ClogP (or SlogP)
and CMR are highly interdependent, and there is no
covariance between ClogP (or SlogP) and I and between
CMR and I (R2 < 0.04).
The outlier behavior (observed log(1/IC ) < calculated
50
log(1/IC ), deviation ) -1.128) of compound 10 probably
50
resulted from the ionization of the phenolic OH group.
The phenolic OH group with a pKa of 3.99 (obtained
from ref 28) is 99.96% ionized at the physiological
condition (pH ) 7.4) because of the strong electron-
withdrawing effect of 3,5-dinitro groups. The degrees
of ionization of compounds 2, 4, and 7 range from 26%
to 36%. The distribution coefficients (log D) of these
ionized compounds were calculated according to the
relationship of log D and log P,32 and then they were
used in the regression analysis. Compound 10 with a
deviation of -0.031 was no longer an outlier in eq 9
when its log D was used in the regression analysis
because of the considerable difference between log P
(0.73) and log D (-2.68). However, the use of the log D
values of compounds 2, 4, and 7 did not significantly
improve the correlation (see eq 9) simply because only
0.13-0.20 log unit differences were observed between
the log D and log P values for compounds 2, 4, and 7.
Because both log P and (log P)2 terms have positive
coefficients, this is different from the traditional para-
bolic or reversed parabolic dependence of biological
activity on log P. Upon deletion of the (log P)2 term, eq
10 was obtained with a correlation coefficient of 0.885,
which was still statistically highly significant (see Table
3).
The ClogP values of the 30 SB-HSCs are highly
correlated with the SlogP values, with a mean difference
of 0.34(ClogP - SlogP), as shown by eq 11 (see Table
3). It should be noted that in a ClogP calculation many
correction factors are used (e.g., 3,5-dinitro), while in
our SlogP calculation no correction factors are used.
In conclusion, on the basis of the pharmacophore of
-NHCONHOH and the nature of RR enzyme, we have
designed and synthesized a series of HU-based RR
inhibitors with in vitro antitumor activity. A total of 17
compounds have been shown to have higher in vitro
inhibitory activities than HU against L1210 murine
leukemia cells. A total of 6 of the 17 compounds with
IC50 values in micromolar range are 11- to 30-fold more
potent than HU. The QSAR study suggests that, besides
the essential pharmacophore (-NHCONHOH), both
hydrophobicity and molecular size/polarizability are
important determinants for the inhibitory activity against
L1210 cells. The presence of an oxygen-containing group
at the ortho position also makes a positive contribution
to the antitumor activity. From the chemical structure
point of view, the SB-HSCs most likely target the same
enzyme as HU. This hypothesis is further supported by
It is interesting to note that HU and HSC fit eqs 5,
7, and 9 very well, suggesting that HU, HSC, and SB-
HSCs most likely have the same rate-limiting step and

Schiff Bases of Hydroxysemicarbazide
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 415
the QSAR analysis in which HU fits the regression
equations very well.
chelating and free radical scavenging properties as
shown by the studies on HU.1,20,21 We expect that the
SB-HSCs may inhibit the RR enzyme more effectively.
A series of Schiff bases of N-hydroxy-N′-aminoguanidine
tosylate (SB-HAG.T) have been synthesized and tested
for antitumor activities. Micromolar and submicromolar
IC50 values have been observed, but only limited in vivo
activity has been obtained on one of the series of
compounds.2,49
Selassie et al.50 revealed that anticancer drugs at the
extreme ends of the molecular weight and hydrophobic-
ity scales showed minimal cross-resistance with each
other in a QSAR analysis of cross-resistant anticancer
drugs. For example, small drugs (such as HU) exhibited
the least cross-resistance with large drug molecules (like
mithramycin). This interesting finding provides insight
into better understanding of the lack of cross-resistance
between HU and compound 29 (large and hydrophobic
molecule).
Testing results of compounds 4, 21, and 29 against
human leukemia CCRF-CEM cells have shown that
compounds 4, 21, and 29 have IC50 values comparable
to those observed in murine leukemia L1210 cells.41 The
IC50 values ranging from 2.7 × 10-6 to 1.2 × 10-5
M
have been observed for compound 29 against four
different solid tumor cell lines.41 Compounds 2 and 29
have no cross-resistance with HU in HU-resistant
human oropharyngeal carcinoma KB cells.41 These in
vitro data as first-line evidence indicate that compounds
2 and 29 may have potential in the treatment of solid
tumors and HU-resistant cancers. It has been shown
that in a variety of tumor and nontumor cell lines
compound 29 has more favorable selectivity than HU
against tumor cells. The relative selectivity indices span
from 8 to 1350 for the eight tumor cell lines tested vs a
nontumor cell line (3T3 Swiss mouse embryo fibro-
blasts),41 suggesting that the SB-HSCs have better
selectivity against tumor cell growth and thus may have
lower toxicity in vivo. The enzyme inhibition capacity
at a single concentration (5 × 10-5 M) has been
correlated with the cell growth inhibition potency by
Zhou et al. in a preliminary study.42
To compare the title compounds with other com-
pounds currently being explored,1-15 further in vivo
activity and toxicity studies are needed to assess the
potentials of these new compounds as cancer chemo-
therapeutic agents. The results obtained from this study
can be used as guidelines for further improvement of
RR inhibitors as anticancer agents.
The disadvantages associated with HU’s physico-
chemical properties, e.g., very high hydrophilicity (log P
) -1.80) and small molecular size, may be overcome
by the structural modification employed in this study.
The most active SB-HSCs possess more suitable phys-
icochemical properties, namely, higher hydrophobicity
(log P) and molecular size (MR or MW) than HU. We
thus expect that the most active compounds should have
better pharmacokinetic and pharmacodynamic profiles,
such as longer half-lives, higher permeability through
various biological membranes, and higher binding af-
finity to the RR enzyme. The SB-HSC compounds
synthesized are structurally related to HU, a well-
known anticancer drug that is currently used in the
treatment of different cancers and has been shown to
have clinical efficacy and tolerance in patients. The
efficacies of the SB-HSC compounds against both leu-
kemia and solid tumor cells have been significantly
improved by the structural modifications compared with
HU.
Exp er im en ta l Section
Ch em istr y. Melting points were determined in melting
point capillary tubes with a MEL-TEMP melting point ap-
paratus and were corrected. All elemental analyses, conducted
by Galbraith Laboratories, Tennessee, were within 0.4% of the
theoretical values for C, H, and N after taking solvent of
crystallization into account for compounds 5, 12, 18, 25, 29,
and 30. To further establish the correct structures of these
six compounds, mass spectra were obtained from Mass Spec-
trometry Laboratory of University of California Riverside,
using the DCI (desorption chemical ionization) method on a
VG 7070 high-resolution mass spectrometer. IR spectra were
recorded with a Beckman IR-4210 spectrophotometer using
KBr pellets. The new compounds were characterized by 1H,
13C {1H}, and 13C NMR on a Bruker AMX 500 MHz FT NMR
spectrometer. Some compounds were further characterized by
1
1H, H correlation spectroscopy (COSY) NMR (compounds 1,
11), and 13C,1H correlated NMR spectroscopy (compounds 1,
6, 7, 11, 12).
N-Hyd r oxyp h en yl Ca r ba m a te. N-hydroxyphenyl car-
bamate was synthesized by using modified procedures of
Grobner and Steinberg.24,25 Yield 18.6 g (89%); mp 105-107
°C (reported mp 105-107 °C24).
Hyd r oxysem ica r ba zid e. A modified procedure of Grobner
and Steinberg24,25 was used in the synthesis of hydroxysemi-
carbazide. Yield 7.8 g (86%); mp 129-131 °C (reported mp 126
°C24). 1H NMR: δ 3.94, 7.51, 8.36, 8.40. 13C NMR: δ 162.78
(CdO). IR, cm-1: 3340, 3200, 1675, 1620, 1520.
Gen er a l P r oced u r e for Syn th eses of Com p ou n d s 1-30.
A mixture of an aldehyde (0.0098-0.0308 mol) and an equimo-
lar quantity of hydroxysemicarbazide in 100-200 mL of
anhydrous methanol was refluxed for 24 h. After checking for
product formation via TLC in methanol/ethyl acetate/chloro-
form (2:3:4), the mixture was concentrated to 5-20 mL in
vacuo. The precipitate was collected by filtration and recrys-
tallized in methanol (or ethanol) to give compounds 1-30.
The most active hydroxybenzohydroxamic acid de-
rivatives, didox (3,4-dihydroxybenzohydroxamic acid)
and trimidox (3,4,5-trihydroxybenzohydroxamidoxime),
inhibited growth of wild-type L1210 cells with IC50
values of 6.0 × 10-5 and 3.3 × 10-5 M, respectively.37
However, it is commonly known that mono- or polyphe-
nols are not very stable in an aqueous solution and can
be oxidized by the air readily. A phase II clinical trial
using didox in advanced breast cancer revealed no
therapeutic responses.43 Heterocyclic carboxaldehyde
thiosemicarbazones have been reported to be potent RR
and tumor cell inhibitors, but because of the low efficacy
in patients, metabolic instability, and high toxicity,
there are no further clinical applications at this point.44-48
Thiosemicarbozones exert an RR inhibitory effect only
through the chelating of the non-heme iron of the R2
subunit because they have no (N)-OH groups in the
pharmacophore.48 Our compounds possess the essential
pharmacophore of -NHCONHOH, which has both iron
1-(3-Tr iflu or om eth ylben zylid en e)-4-h yd r oxysem ica r -
1
3
ba zid e (1). H NMR: δ 7.59 (t, J ) 7.3 Hz, 1H, 5-ArH), 7.64
3
3
(d, J ) 7.1 Hz, 1H, 4-ArH), 7.92 (d, J ) 7.5 Hz, 1H, 6-ArH),
8.05 (s, 1H, -CHd), 8.10 (s, 1H, 2-ArH), 8.53, 9.48, 10.46 (NH,
NH, OH). 13C NMR: δ 122.65 (2-ArC), 124.07 (-CF3), 125.17
(4-ArC), 129.47 (5-ArC), 129.69 (3-ArC), 130.68 (6-ArC), 135.90

416 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Ren et al.
4
4
(1-ArC), 139.96 (-CHd, 156.96 (CdO). IR, cm-1: 3430, 3200,
3120, 2980, 1705, 1660, 1530, 1330. Anal. (C9H8F3N3O2) C, H,
N.
) 8.5 Hz, J ) 2.3 Hz,4J ) 1.1 Hz, 1H, 4-ArH), 8.54 (t, J )
2.0 Hz 1H, 2-ArH), 8.53, 9.51, 10.55 (NH, NH, OH). 13C NMR:
δ 120.56 (2-ArC), 123.21 (4-ArC), 130.01 (5-ArC), 132.87 (6-
ArC), 136.72 (1-ArC), 139.09 (-CHd), 148.38 (3-ArC), 156.65
(CdO). IR, cm-1: 1705, 1660, 1525, 1350. Anal. (C8H8N4O4)
C, H, N.
1-(2-H yd r oxy-3,5-d ich lor ob en zylid en e)-4-h yd r oxyse-
1
4
m ica r ba zid e (2). H NMR: δ 7.45, 7.52 (d, J ) 2.5 Hz, 1H;
4
d, J ) 2.5 Hz, 1H; 4,6-ArH), 8.33 (s, 1H, -CHd), 8.78, 9.33,
10.96 11.98 (NH, NH, OH, 2-ArOH). 13C NMR: δ 121.47,
121.85 (1,3-ArC), 122.93 (5-ArC), 127.21 (6-ArC), 129.16 (4-
1-(3-Met h oxyb en zylid en e)-4-h yd r oxysem ica r b a zid e
(12). 1H NMR: δ 3.79 (s, 3H, -OCH3), 6.91 (ddd, 3J ) 8.2 Hz,
ArC), 142.06 (-CHd), 151.46 (2-ArC), 156.70 (CdO). IR, cm-1
:
4J ) 2.6 Hz, J ) 1.0 Hz, 1H, 4-ArH), 7.19 (dt, J ) 7.6 Hz, J
) 1.1 Hz, 1H, 6-ArH), 7.28 (t, 3J ) 7.8 Hz, 1H, 5-ArH), 7.31 (t,
4J ) 1.5 Hz, 1H, 2-ArH), 7.94 (s, 1H, -CHd), 8.44, 9.24, 10.25
(NH, NH, OH). 13C NMR δ 55.03 (-OCH3), 110.71 (2-ArC),
115.31, 119.54 (4,6-ArC), 129.34 (5-ArC), 136.00 (1-ArC),
141.36 (-CHd), 156.74 (CdO), 159.41 (3-ArC). IR, cm-1: 1690,
1670, 1525, 1275, 1140. MS (DCI, m/e): 210 (M + H)+. Anal.
(C9H11N3O3‚0.135CH3OH) C, H, N.
4
3
4
3360, 1695, 1665, 1540. Anal. (C8H7Cl2N3O3) C, H, N.
1-(2-Hyd r oxy-5-br om oben zylid en e)-4-h yd r oxysem ica r -
ba zid e (3). 1H NMR: δ 6.82 (d, 3J ) 8.7 Hz, 1H, 3-ArH), 7.31
(dd, 3J ) 8.7 Hz, 4J ) 2.5 Hz, 1H, 4-ArH), 7.81 (d, 4J ) 2.5
Hz, 1H, 6-ArH), 8.25 (s, 1H, -CHd), 8.58, 9.32, 10.58, 10.75
(NH, NH, OH, 2-ArOH). 13C NMR: δ 110.50 (1-ArC), 118.30
(3-ArC), 122.19 (5-ArC), 129.66 (6-ArC), 132.47 (4-ArC), 139.50
(-CHd), 155.55 (2-ArC), 156.86 (CdO). IR, cm-1: 3360, 1695,
1660, 1525. Anal. (C8H8BrN3O3) C, H, N.
1-(4-Met h oxyb en zylid en e)-4-h yd r oxysem ica r b a zid e
(13). 1H NMR: δ 3.76 (s, 3H, -OCH3), 6.92 (d, 3J ) 8.8 Hz,
3
2H, 3,5-ArH), 7.62 (d, J ) 8.8 Hz, 2H, 2,6-ArH), 7.95 (s, 1H,
1-(2-H yd r oxy-3,5-d ib r om ob en zylid en e)-4-h yd r oxyse-
-CHd), 8.51, 9.16, 10.18 (NH, NH, OH). 13C NMR: δ 55.18
(-OCH3), 114.07 (3,5-ArC), 127.34 (1-ArC), 128.17 (2,6-ArC),
141.86 (-CHd), 157.27 (CdO), 160.22 (4-ArC). IR, cm-1: 1670,
1615, 1500, 1250, 1130, 1035. Anal. (C9H11N3O3) C, H, N.
1-(2,5-Dim et h oxyb en zylid en e)-4-h yd r oxysem ica r b a -
zid e (14). 1H NMR: δ 3.74 (s, 3H, -OCH3), 3.76 (s, 3H,
1
4
m ica r ba zid e (4). H NMR: δ 7.63, 7.68 (d, J ) 2.4 Hz, 1H;
4
d, J ) 2.4 Hz, 1H, 4,6-ArH), 8.31 (s, 1H, -CHd), 8.81, 9.31,
11.01, 12.24 (NH, NH, OH, 2-ArOH). 13C NMR: δ 110.30 (5-
ArC), 111.18, 121.93 (1,3-ArC), 130.98 (6-ArC), 134.52 (4-ArC),
142.45 (-CHd), 152.94 (2-ArC), 156.65 (CdO). IR, cm-1: 3350,
1700, 1670, 1525. Anal. (C8H7Br2N3O3) C, H, N.
3
4
-OCH3), 6.89 (dd, J ) 9.0 Hz, J ) 3.0 Hz, 1H, 4-ArH), 6.95
1-(2-H yd r oxy-3-m et h oxy-5-b r om ob en zylid en e)-4-h y-
3
4
1
(d, J ) 9.0 Hz, 1H, 3-ArH), 7.52 (d, J ) 2.9 Hz, 1H, 6-ArH),
8.26 (s, 1H, -CHd), 8.41, 9.30, 10.29 (NH, NH, OH). 13C
NMR: δ 55.50, 56.20 (-OCH3), 109.93, 113.00, 116.67 (3,4,6-
ArC), 123.51 (1-ArC), 151.72, 153.38 (2,5-ArC), 136.88 (-CHd
d r oxysem ica r ba zid e (5). H NMR: δ 3.81 (s, 3H, -OCH3),
7.05 (d, 4J ) 2.2 Hz, 1H, 4-ArH), 7.43 (d, 4J ) 2.2 Hz, 1H,
6-ArH), 8.27 (s, 1H, -CHd), 8.65, 9.43, 10.25, 10.66 (NH, NH,
OH, 2-ArOH). 13C NMR δ 56.21 (-OCH3), 109.93 (5-ArC),
115.33 (4-ArC), 121.22 (6-ArC), 121.66 (1-ArC), 139.86 (-CHd
), 156.89 (CdO). IR, cm-1
(C10H13N3O4) C, H, N.
: 1675, 1535, 1225, 1045. Anal.
), 145.32 (2-ArC), 148.87 (3-ArC), 156.66 (CdO). IR, cm-1
:
3360, 1695, 1670, 1525, 1105. MS (DCI, m/e): 304 (M + H)+.
Anal. (C9H10BrN3O4‚0.2CH3OH) C, H, N.
1-(2-Hyd r oxy-4-m eth oxyben zylid en e)-4-h yd r oxysem i-
1
4
ca r ba zid e (15). H NMR: δ 3.74 (s, 3H, -OCH3), 6.43 (d, J
1-(3-Iod oben zylid en e)-4-h yd r oxysem ica r ba zid e (6). 1H
NMR: δ 7.17 (t, 3J ) 7.8 Hz, 1H, 5-ArH), 7.62 (dt, 3J ) 7.8
) 2.5 Hz, 1H, 3-ArH), 6.45 (dd, J ) 8.5 Hz, J ) 2.5 Hz, 1H,
3
4
3
5-ArH), 7.31 (d, J ) 8.6 Hz, 1H, 6-ArH), 8.29 (s, 1H, -CHd),
4
3
4
Hz, J ) 1.3 Hz, 1H, 6-ArH), 7.68 (ddd, J ) 7.9 Hz, J ) 1.7
8.66, 9.03, 10.46 11.28 (NH, NH, OH, 2-ArOH). 13C NMR: δ
55.16 (-OCH3), 101.21 (3-ArC), 106.02 (5-ArC), 112.38 (1-ArC),
130.30 (6-ArC), 144.63 (-CHd), 157.17 (CdO), 158.59 (2-ArC),
4
4
Hz, J ) 1.0 Hz, 1H, 4-ArH), 8.16 (t, J ) 1.6 Hz, 1H, 2-ArH),
7.88 (s, 1H, -CHd), 8.48, 9.40, 10.38 (NH, NH, OH). 13C
NMR: δ 94.97 (3-ArC), 126.44 (6-ArC), 130.56 (5-ArC), 134.33
(2-ArC), 137.06 (1-ArC), 137.45 (4-ArC), 139.62 (-CHd),
156.76 (CdO). IR, cm-1: 3380, 1700, 1670, 1525. Anal. (C8H8-
IN3O2) C, H, N.
161.37 (4-ArC). IR, cm-1
: 1630, 1565, 1250, 1140. Anal.
(C9H11N3O4) C, H, N.
1-(2-Hyd r oxy-4,6-d im eth oxyben zylid en e)-4-h yd r oxyse-
1
m ica r ba zid e (16). H NMR: δ 3.75 (s, 3H, -OCH3), 3.79 (s,
3H, -OCH3), 6.07, 6.09 (d, 4J ) 2.3 Hz, 1H; d, 4J ) 2.2 Hz,
1H; 3,5-ArH), 8.65 (s, 1H, -CHd), 8.67, 8.93, 10.61, 12.17 (NH,
NH, OH, 2-ArOH). 13C NMR: δ 55.27, 55.84 (-OCH3), 90.27,
93.90 (3,5-ArC), 101.06 (1-ArC), 159.09, 160.20, 162.30 (2,4,6-
ArC), 142.62 (-CHd), 157.17 (CdO). IR, cm-1: 1635, 1610,
1575, 1215, 1115. Anal. (C10H13N3O5) C, H, N.
1-(2-Hyd r oxy-3,5-d iiod oben zylid en e)-4-h yd r oxysem i-
1
4
ca r ba zid e (7). H NMR: δ 7.68 (d, J ) 2.4 Hz, 1H, 6-ArH),
7.95 (d, 4J ) 2.1 Hz, 1H, 4-ArH), 8.24 (s, 1H, -CHd), 8.82,
9.27, 11.01, 12.55 (NH, NH, OH, 2-ArOH). 13C NMR: δ 81.65,
87.43 (3,5-ArC), 121.11 (1-ArC), 137.84 (6-ArC), 143.01 (-CHd
), 145.55 (4-ArC), 156.01 (2-ArC), 156.68 (CdO). IR, cm-1
:
1675, 1540. Anal. (C8H7I2N3O3) C, H, N.
1-(4-Be n zyloxyb e n zylid e n e )-4-h yd r oxyse m ica r b a -
zid e (17). 1H NMR: δ 5.13 (s, 2H, -CH2O-), 7.02 (d, 3J ) 8.8
Hz, 2H, 3,5-ArH), 7.32 (tt, 3J ) 7.3 Hz, 4J ) 1.4 Hz, 1H, 4′-
1-(4-Cya n oben zylid en e)-4-h yd r oxysem ica r ba zid e (8).
3
3
1H NMR: δ 7.79, 7.88 (d, J ) 8.3 Hz, 2H, Ar-H; d, J ) 8.4
Hz, 2H, ArH), 8.00 (s, 1H, -CHd), 8.65, 9.60, 10.70 (NH, NH,
OH). 13C NMR: δ 110.82 (4-ArC), 118.47 (-CN), 127.00 (2,6-
ArC), 132.17 (3,5-ArC), 139.11 (1-ArC), 139.29 (-CHd), 156.36
(CdO). IR, cm-1: 2230, 1675, 1550. Anal. (C9H8N4O2‚1H2O)
C, H, N.
3
4
ArH), 7.38 (td, J ) 7.4 Hz, J ) 1.4 Hz, 2H, 3′,5′-ArH), 7.44
(dd, 3J ) 7.5 Hz, 4J ) 1.4 Hz, 2H, 2′,6′-ArH), 7.62 (d, 3J ) 8.7,
2H, 2,6-ArH), 7.93 (s, 1H, -CHd), 8.44, 9.12, 10.15 (NH, NH,
OH). 13C NMR: δ 69.39 (-CH2O-), 114.99 (3,5-ArC), 127.55,
127.67, 127.82, 128.13, 128.38 (1, 2, 6, 2′-6′-ArC), 136.89 (1′-
ArC), 141.58 (-CHd), 157.14 (CdO), 159.28 (4-ArC). IR, cm-1
1685, 1505, 1225, 1000. Anal. (C15H15N3O3) C, H, N.
:
1-(4-Dim et h yla m in ob en zylid en e)-4-h yd r oxysem ica r -
1
3
ba zid e (9). H NMR: δ 2.93 [s, 6H, -N(CH3)2], 6.69 (d, J )
3
9.0 Hz, 2H, 3,5-ArH), 7.48 (d, J ) 8.9 Hz, 2H, 2,6-ArH), 7.86
1-(4-P h en ylben zyliden e)-4-h ydr oxysem icar bazide (18).
(s, 1H, -CHd), 8.41, 8.96, 9.97 (NH, NH, OH). 13C NMR: δ
39.76 [-N(CH3)2], 111.77 (3,5-ArC), 122.34 (1-ArC), 127.85
(2,6-ArC), 142.74 (-CHd), 151.04 (4-ArC), 157.33 (CdO). IR,
cm-1: 1680, 1610, 1520, 1360. Anal. (C10H14N4O2) C, H, N.
1-(2-Hyd r oxy-3,5-d in itr oben zylid en e)-4-h yd r oxysem i-
1H NMR: δ 7.35 (tt, 3J ) 7.3 Hz, 4J ) 1.1 Hz, 1H, 4′-ArH),
3
4
7.45 (td, J ) 7.4 Hz, J ) 1.8 Hz, 2H, 3′,5′-ArH), 7.67, 7.78
(d, 3J ) 8.4 Hz, 4H; d, 3J ) 8.3 Hz, 2H, 2,3,5,6,2′,6′-ArH), 8.06
(s, 1H, -CHd), 8.51, 9.24, 10.33 (NH, NH, OH). 13C NMR: δ
126.34 (3′,5′-ArH), 126.55, 127.05, 128.69 (2,6,3,5,2′,6′-ArC),
127.40 (4′-ArC), 133.71, 139.33, 140.58 (1,1′,4-ArC), 141.27
(-CHd), 156.86 (CdO). IR, cm-1: 1650, 1555. MS (DCI, m/e):
256 (M + H)+. Anal. (C14H13N3O2‚0.2CH3OH) C, H, N.
1-(2,4-Dih yd r oxyb en zylid en e)-4-h yd r oxysem ica r b a -
1
ca r ba zid e (10). H NMR: δ 8.50 (s, 1H, -CHd), 8.68, 8.71
4
4
(d, J ) 2.8 Hz, 1H; d, J ) 2.5 Hz, 1H, ArH), 8.50, 8.71, 9.46,
11.19 (NH, NH, OH, ArOH). 13C NMR: δ 121.22 (4-ArC),
123.65 (1-ArC), 127.44 (6-ArC), 137.12, 137.59 (3,5-ArC),
140.50 (-CHd), 156.43 (CdO), 156.92 (2-ArC). IR, cm-1: 1640,
1615, 1530, 1345. Anal. (C8H7N5O7) C, H, N.
1
3
zid e (19). H NMR: δ 6.28 (d, J ) 2.4 Hz, 1H, 3-ArH), 6.31
(dd, 3J ) 8.4 Hz, 4J ) 2.4 Hz, 1H, 5-ArH), 7.19 (d, 3J ) 8.4
Hz, 1H, 6-ArH), 8.25 (s, 1H, -CHd), 8.66, 8.99, 9.75, 10.37,
11.02 (NH, NH, OH, 2,4-ArOH). 13C NMR: δ 102.66, 107.40
(3,5-ArC), 111.06 (1-ArC), 130.49 (6-ArC), 145.23 (-CHd),
1-(3-Nitr oben zylid en e)-4-h yd r oxysem ica r ba zid e (11).
1H NMR: δ 7.66 (t, 3J ) 8.0 Hz, 1H, 5-ArH), 8.08 (s, 1H, -CHd
3
4
3
), 8.10 (dt, J ) 7.8 Hz, J ) 1.1 Hz, 1H, 6-ArH), 8.15 (ddd, J

Schiff Bases of Hydroxysemicarbazide
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 417
157.28 (CdO), 158.66, 159.86 (2,4-ArC). IR, cm-1: 1655, 1630,
1575. Anal. (C8H9N3O4) C, H, N.
1-(4-Ace t a m id ob e n zylid e n e )-4-h yd r oxyse m ica r b a -
1H, 5-ArH), 7.84 (s, 1H, -CHd), 8.42, 9.17, 10.14 (NH, NH,
OH). 13C NMR: δ 63.99, 64.22 (2,3-C), 114.77, 117.04, 120.34
(5,7,8-ArC), 128.21 (6-ArC), 141.34 (-CHd), 143.54, 144.53 (9,-
10-ArC), 157.06 (CdO). IR, cm-1
:
1680, 1580, 1125, 1070.
1
3
zid e (20). H NMR: δ 2.04 (-NHCOCH3), 7.59, 7.60 (d, J )
8.7 Hz, 2H; d, 3J ) 8.7 Hz, 2H, ArH), 7.92 (s, 1H, -CHd),
8.47, 9.14, 9.94, 10.21 (NH, NH, OH, -NHCO-). 13C NMR: δ
23.94 (-NHCOCH3), 118.86, 127.17 (2,3,5,6-ArC), 129.38 (1-
ArC), 140.17 (4-ArC), 141.55 (-CHd), 157.11 (CdO), 168.33
Anal. (C10H11N3O4) C, H, N.
1-[9-(10-Meth yla n th r yl)m eth ylen e]-4-h yd r oxysem ica r -
1
ba zid e (29). H NMR: δ 3.10 (s, 3H, -CH3), 7.58, 8.40, 8.59
(m, 8H, ArH), 8.63 (s, 1H, -CHd), 8.97, 9.25, 10.62 (NH, NH,
OH). 13C NMR: δ 13.99 (-CH3), 124.95, 125.17, 125.45, 125.86
(ArCH), 124.63, 129.07, 129.19, 132.15 (Ar-C), 142.17 (-CHd
), 157.13 (CdO). IR, cm-1: 1660, 1530, 1125. MS (DCI, m/e):
294 (M + H)+. Anal. (C17H15N3O2‚0.2CH3OH) C, H, N.
1-[9-(10-Ch lor oa n th r yl)m eth ylen e]-4-h yd r oxysem ica r -
(-NHCOCH3). IR, cm-1
(C10H12N4O3) C, H, N.
: 1680, 1640, 1535, 1335. Anal.
1-(2,3,4-Tr ih yd r oxyben zylid en e)-4-h yd r oxysem ica r ba -
1
3
3
zid e (21). H NMR: δ 6.32, 6.65 (d, J ) 8.4 Hz, 1H; d, J )
8.6 Hz, 1H, 5,6-ArH), 8.24 (s, 1H, -CHd), 8.35, 8.76, 9.06,
9.29, 10.58, 11.21 (NH, NH, OH, 2,3,4-ArOH). 13C NMR: δ
107.35 (5-ArC), 111.45 (1-ArC), 120.17 (6-ArC), 132.68 (3-ArC),
146.25 (-CHd), 146.83, 147.85 (2,4-ArC), 157.22 (CdO). IR,
cm-1: 1665, 1630, 1565, 1365, 1195. Anal. (C8H9N3O5) C, H,
N.
1
3
4
ba zid e (30). H NMR: δ 7.67, 7.73 (td, J ) 7.5 Hz, J ) 1.2
3
4
Hz, 2H; td, J ) 7.5 Hz, J ) 1.2 Hz, 2H, 2,3,6,7-ArH), 8.48,
8.64 (d, 3J ) 8.7 Hz, 2H; d, 3J ) 8.7, 2H, 1,4,5,8-ArH), 9.06 (s,
1H, -CHd), 8.67, 9.25, 10.72 (NH, NH, OH). 13C NMR: δ
124.43, 125.76, 127.02, 127.47 (Ar-CH), 126.60, 127.79, 128.88,
129.70 (Ar-C), 141.29 (-CHd), 157.23 (CdO). IR, cm-1: 1685,
1-(3-P yr id ylm eth ylen e)-4-h yd r oxysem ica r ba zid e (22).
1H NMR: δ 7.38 (dd, 3J ) 8.0 Hz, 4J ) 4.9 Hz, 1H, 5-ArH),
8.00 (s, 1H, -CHd), 8.12 (dt, 3J ) 8.0 Hz, 4J ) 1.9 Hz, 1H,
1660, 1555. MS (DCI, m/e): 314 (M + H)+. Anal. (C16H12
ClN3O2‚0.3CH3OH) C, H, N.
-
Mea su r em en ts of P a r tition Coefficien t (log P ) a n d
Ion iza tion Con sta n ts (p Ka ). Measurements of log P and pKa
values of one representative compound were performed using
the shake-flask method with two immiscible solvents (1-
octanol/phosphate buffer).33,51-53 Compound 1 was first dis-
solved in methanol (2 mg/mL). To 38.4 mL of phosphate buffers
(pH 2.2, 7.3, 11.4) saturated with 1-octanol, 1.6 mL of
3
4
4-ArH), 8.51 (dd, J ) 4.9 Hz, J ) 1.9 Hz, 1H, 6-ArH), 8.82
4
(d, J ) 1.8 Hz, 1H, 2-ArH), 8.54, 9.40, 10.48 (NH, NH, OH).
13C NMR: δ 123.60 (5-ArC), 130.57 (3-ArC), 133.18 (4-ArC),
138.60 (-CHd), 148.17, 149.70 (2,6-ArC), 156.78 (CdO). IR,
cm-1: 1685, 1650, 1545, 1145. Anal. (C7H8N4O2) C, H, N.
1-[2-(6-Meth ylp yr id yl)m eth ylen e]-4-h yd r oxysem ica r -
1
3
ba zid e (23). H NMR: δ 2.48 (s, 3H, -CH3), 7.16, 7.86 (d, J
compound
1 methanol solution and 40 mL of 1-octanol
3
3
) 7.6 Hz, 1H; d, J ) 7.7 Hz, 1H, 3,5-ArH), 7.66 (t, J ) 7.6
Hz, 1H, 4-ArH), 7.97 (s, 1H, -CHd), 8.55, 9.35, 10.55 (NH,
NH, OH). 13C NMR δ 23.76 (-CH3), 116.78, 122.74 (3,5-ArC),
136.57 (4-ArC), 142.31 (-CHd), 153.10, 156.68, 157.36 (2,6-
ArC, CdO). IR, cm-1: 1675, 1640, 1520, 1450. Anal. (C8H10N4O2)
C, H, N.
saturated with phosphate buffers were added. The mixtures
were shaken 4 h and centrifuged 3 min at 1000 rpm (IEC
Clinical Centrifuge, International Equipment Company). The
concentrations of compound 1 in phosphate buffer and that in
the 1-octanol phase were measured with a Hitachi U-2000 UV
spectrophotometer at λmax. The standard curves were obtained
for 1-octanol and pH 2, pH 7, and pH 11 buffers and then used
for measurement of concentrations of compound 1.
1-[2-(5-Nitr oth ien yl)m eth ylen e]-4-h yd r oxysem ica r ba -
1
3
zid e (24). H NMR: δ 7.37 (d, J ) 4.3 Hz, 1H, 3-ArH), 8.02
(d, 3J ) 4.3 Hz, 1H, 4-ArH), 8.23 (s, 1H, -CHd), 8.64, 9.35,
10.80 (NH, NH, OH). 13C NMR: δ 127.57, 130.26 (3,4-ArC),
135.31 (-CHd), 147.51, 150.07 (2,5-ArC), 156.17 (CdO). IR,
cm-1: 1680, 1560, 1325. Anal. (C6H6N4O4S‚1H2O) C, H, N.
1-(3-In d olylm eth ylen e)-4-h yd r oxysem ica r ba zid e (25).
The pKa values were calculated with the following equations
derived from the buffer equation.
[D]pb
at pH 11, pKa(acid) ) pH - log P
- 1
3
3
3
1H NMR: δ 7.10, 7.16 (td, J ) 7.8 Hz, J ) 1.3 Hz, 1H; td, J
) 7.1 Hz, 4J ) 1.3 Hz, 1H, 5,6-ArH), 7.40, 8.22 (d, 3J ) 8.1
Hz, 1H; d, 3J ) 7.9 Hz, 1H, 4,7-ArH), 7.64 (s, 1H, 2-ArH), 8.24
(s, 1H, -CHd), 8.45, 8.75, 9.92, 11.31 (NH, NH, OH, indole-
NH). 13C NMR: δ 111.39 (7-ArC), 111.58 (3-ArC), 119.89,
121.63, 122.13 (4,5,6-ArC), 124.15 (9-ArC), 128.53 (2-ArC),
136.78 (8-ArC), 139.84 (-CHd), 157.50 (CdO). IR, cm-1: 1690,
1655, 1540, 1625, 1450. MS (DCI, m/e): 219 (M + H)+. Anal.
(C10H10N4O2‚0.2CH3OH) C, H, N.
(
)
[D]o
[for (N)-OH group]
[D]pb
at pH 2, pKa(base) ) pH + log P
- 1
(
)
[D]o
(for the imino -Nd nitrogen)
where P is partition coefficient, [D]pb the drug concentration
in phosphate buffer, and [D]o is the drug concentration in
1-octanal.
The stability of compound 1 was studied in pH 3, pH 7, and
pH 11 phosphate buffers at room temperature up to 72 h. The
concentrations at different time intervals were determined by
using a Hitachi U-2000 UV spectrophotometer.
1-[3-(6,8-Dich lor o-4-oxo-4H-1-ben zop yr a n )m eth ylen e]-
4-h yd r oxysem ica r ba zid e (26). 1H NMR: δ 7.94, 8.11 (large
4
4
peak, J ) 2.3 Hz, 1H; large peak, J ) 2.4 Hz, 1H, 5,7-ArH),
7.99 (s, 1H, -CHd), 8.50 (s, 1H, 2-ArH), 9.04, 9.39, 10.51 (NH,
NH, OH). 13C NMR δ 119.18 (3-ArC), 123.28 (5-ArC), 123.96
(8-ArC), 125.41 (10-ArC), 130.01 (6-ArC), 131.81 (-CHd),
133.68 (7-ArC), 150.24 (9-ArC), 154.20 (2-ArC), 156.53 (Cd
O), 173.24 (4-ArC). IR, cm-1: 1670, 1640, 1555, 1150. Anal.
(C11H7Cl2N3O4) C, H, N.
In Vitr o An titu m or Activity. The lymphocytic murine
leukemia cell line L1210 was obtained from Cell Culture Core
Facility of the Norris Comprehensive Cancer Center at the
Keck School of Medicine, University of Southern California.
The cells were maintained in a 5% CO2 humidified atmosphere
at 37 °C in RPMI-1640 medium containing penicillin (170 IU/
mL), streptomycin (170 µg/mL) (Mediatech-Cellgro, Herndon,
VA), and 10% fetal bovine serum (FBS) (Omega Scientific,
Tarzana, CA). The cell culture was passaged every 3 days
using an initial density of 4 × 104 cells/mL.
1-[3-(6-Isop r op yl-4-oxo-4H-1-ben zop yr a n )m eth ylen e]-
4-h yd r oxysem ica r ba zid e (27). 1H NMR: δ 1.24, 1.26 [s, 6H,
3
-CH(CH3)2], 3.04 [sep, J ) 7.0 Hz, 1H, -CH(CH3)2], 7.61 (d,
3
4
3J ) 8.7 Hz, 1H, 8-ArH), 7.72 (dd, J ) 8.7 Hz, J ) 2.0 Hz,
1H, 7-ArH), 7.92 (d, 4J ) 2.3 Hz, 1H, 5-ArH), 8.07 (s, 1H,
-CHd), 8.46 (d, 4J ) 1.0 Hz, 1H, 2-ArH), 8.93, 9.35, 10.42
(NH, NH, OH). 13C NMR: δ 23.60 [-CH(CH3)2], 32.87 [-CH-
(CH3)2], 118.51 (3-ArC), 118.55 (8-ArC), 121.54, 121.58 (5,7-
ArC), 123.12 (10-ArC), 133.05 (-CHd), 146.17 (6-ArC), 154.05
(2-ArC), 154.23 (9-ArC), 156.70 (CdO), 174.83 (4-ArC). IR,
cm-1: 1640, 1620, 1545, 1355, 1135. Anal. (C14H15N3O4) C, H,
N.
The IC50 values of the test compounds against L1210 cells
were determined using MTS/PES microculture tetrazolium
assay in 96-well microtiter plates.26-29 For each experiment,
the cells were suspended in RPMI-1640 medium with 10% FBS
at 1 × 105/mL and seeded in 96-well plates at 50 µL/well. Each
compound was initially dissolved in 100% DMSO immediately
before use and further diluted into RPMI-1640 medium to
obtain six gradual concentrations ranging from 2 × 10-3 to 1
1-(1,4-Ben zod ioxa n -6-ylm eth ylen e)-4-h yd r oxysem ica r -
ba zid e (28). 1H NMR: δ 4.24 [s, 4H, 2,3-CH2-], 6.83 (d, 3J )
3
8.5 Hz, 1H, 8-ArH), 7.11 (d, J ) 8.5 Hz, 1H, 7-ArH), 7.24 (s,

418 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Ren et al.
× 10-8 M. Each dilution was added to the wells at 50 µL/well
in triplicates. The final concentration of DMSO in the cell
cultures was 1% for the highest drug concentration, and in
the corresponding controls, all others had <0.25% to avoid the
toxicity of DMSO. Hydroxyurea (Aldrich Chemical Co., St.
Louis, MO) was used as a positive control. Negative control,
containing only DMSO at identical dilutions, was run with
each experiment. All experiments were repeated at least twice.
The plate was incubated in a 5% CO2 humidified atmosphere
at 37 °C for 65 h. A total of 20 µL/well of MTS/PES CellTiter
96 Aqueous One Solution reagent (Promega Corp., Madison,
WI) was added to each well, and the plate was incubated for
3 more hours. The absorbance at 490 nm was recorded by using
a microplate reader (Dynatech Laboratories, Revelation, ver-
sion 3.04, Chantilly, VA).
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Ack n ow led gm en t. This work was supported in part
by H & L Charitable Foundation (S. Ren, R. Wang, K.
Komatsu, E. J . Lien) and Grants NIH EY12356 (P.
Bonaz-Krause, Y. Zyrianov, C. E. McKenna) and DHEW-
NCI CA-14089 (C. Csipke, Z. A. Tokes). The authors
thank Dr. Corwin Hansch of Pomona College and Dr.
Albert Leo of BioByte for providing us with access to
the CQSAR program, and Mr. Zhenghong Fu for per-
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