Synthesis of N-aryl and N-arylcarbamoylamino derivatives of 1,3-diazinane-5-carboxamide and their activity against glioblastoma LN-229 cell line

Article abstract of DOI:10.1016/j.bmc.2016.09.074

Six structural motifs based on the initial (lead) structure of merbarone were designed, prepared, and tested against the glioblastoma LN-229 cell line. Three different structural moieties were modified in the search for optimal glioblastoma activity: the 1,3-diazinane moiety, the aryl moiety, and the heteroatom linker. Calculated molecular descriptors such as lipophilicity (C log P), acidic strength (calculated pK_a), and polar surface area (PSA) were used to design a diverse structural library of these compounds. From six different structural motifs and 136 compounds, a handful of examples with moderate (100 μg/ml), good (10 μg/ml) and excellent (1 μg/ml) glioblastoma activity were elucidated.

Full text of DOI:10.1016/j.bmc.2016.09.074

Accepted Manuscript
Synthesis of N-aryl and N-arylcarbamoylamino derivatives of 1, 3-diazinane-5-
carboxamide and their activity against glioblastoma LN-229 cell line
Rebecca J. Hron, Branko S. Jursic, Donna M. Neumann
PII:
S0968-0896(16)30860-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.074
BMC 13327
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 August 2016
28 September 2016
30 September 2016
Please cite this article as: Hron, R.J., Jursic, B.S., Neumann, D.M., Synthesis of N-aryl and N-arylcarbamoylamino
derivatives of 1, 3-diazinane-5-carboxamide and their activity against glioblastoma LN-229 cell line, Bioorganic
& Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.09.074
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Revised Manuscript BMC-S-16-01419
Synthesis of N-aryl and N-arylcarbamoylamino derivatives of 1, 3-diazinane-5-
carboxamide and their activity against glioblastoma LN-229 cell line
Rebecca J. Hrona, Branko S. Jursica,b,*, Donna M. Neumannc,d
aDepartment of Chemistry, University of New Orleans, New Orleans, LA 70148, United States
bSTEPHARM, LLC., PO Box 24220, New Orleans, LA 70184, United States
cLouisiana State University Health Sciences Center, Department of Pharmacology and
Experimental Therapeutics, 1901 Perdido St., New Orleans, LA 70112, United States
dDepartment of Ophthalmology, LSUHSC, New Orleans, United States
Abstract
Six structural motifs based on the initial (lead) structure of merbarone were designed,
prepared, and tested against the glioblastoma LN-229 cell line. Three different structural
moieties were modified in the search for optimal glioblastoma activity: the 1, 3-diazinane moiety,
the aryl moiety, and the heteroatom linker. Calculated molecular descriptors such as
lipophilicity (ClogP), acidic strength (calculated pKa), and polar surface area (PSA) were used to
design a diverse structural library of these compounds. From six different structural motifs and
136 compounds, a handful of examples with moderate (100 µg/ml), good (10 µg/ml) and
excellent (1 µg/ml) glioblastoma activity were elucidated.
Keywords: Glioblastoma, anticancer, diazinane, carbamoyl, merbarone

Introduction
Manipulation of the cell cycle is a useful tool for the discovery of novel therapeutics. DNA
topoisomerases, for instance, are popular targets for anti-cancer research due to their
prominent role in the modulation of cellular processes including DNA replication and
transcription.1Topoisomerases are classified as either type I or type II, with type I
topoisomerases initiating single stranded breaks in DNA and type II topoisomerases initiating
double stranded breaks.2 Type I and II topoisomerases are further classified into subcategories
according to their specialized roles in the pathways of DNA replication and transcription among
several others. The intricate and complex nature of topoisomerases and their role in cellular
maintenance strongly lends them to exploitation for therapeutic purposes. Although both type I
and type II topoisomerases have served as targets for anti-cancer drugs, nearly half of all
cancer treatments specifically target topoisomerase II.2
It is widely known that cancer is described as uncontrolled cell division. Since cancer cells
replicate and divide more rapidly than healthy cells, they consequently express higher levels of
topoisomerase II, making them more susceptible to damage by topoisomerase II inhibiting
drugs2. Traditional anti-cancer treatments targeting topoisomerase II typically work by
upregulating topoisomerase II controlled DNA cleavage, thereby creating high steady-state
levels of DNA-topoisomerase complexes which act effectively as cellular toxins.2For this reason,
substances functioning in this manner are referred to as topoisomerase poisons.3An alternative
approach for targeting topoisomerase II activity is to alter the catalytic cycle. This can be
achieved in several ways. One example of this method is to prevent the initial binding of
topoisomerase II to DNA while another avenue is to disrupt ATP hydrolysis, which is necessary
for the completion of the catalytic cycle.3
Compounds acting to this end have a variety of structural features, with some of the most
successful including 5-Phenylcarbamoyl barbituric acids and their sulfur analogues. 5-

Phenylcarbamoylbarbituric acid has exhibited marked inhibition against the rat hepatoma line
AH-130 in vitro as well as activity against Ehrlich carcinoma in vivo.4Sulfur analogues of 5-
pyrimidinecarboxamideshave also been found to have activity against a range of cancer cell
lines including B16 melanoma, L1210 leukemia, M5076 sarcoma and P388 leukemia.5Another
derivative of these potent compounds, merbarone, has undergone extensive investigation as a
topoisomerase II inhibitor.3However, after further examination, it has been suggested that
merbarone may have both genotoxic and cytotoxic effects. Additionally, it has also been
proposed that merbarone promotes endoreduplication, or repeated cellular replication without
division, leading to the accumulation of extra copies of genetic material.1
Results and Discussion
Since merbarone has the unique status of being the only agent that inhibits topoisomerase II
mediated DNA cleavage without affecting protein-DNA binding,3,6we would like to use the key
structural features of merbarone5to construct a series of novel anti-cancer compounds.
Although there are six core structural motifs present, they all share the same characteristic 2, 4,
6-trioxo-1, 3-diazinane-5-carboxamide (barbituric acid), with oxygen replacing sulfur in the 2
position of the 1, 3-diazinane ring (Scheme 1). The first structural motif (Structural Motif A)
varies from merbarone by the inclusion of up to three substituents on the phenyl moiety as well
as different N-substituents on the 1, 3-diazinane-2, 4, 6-trione (barbituric acid) moiety. In
Structural Motifs B and C, in addition to barbituric acid substituents, the phenyl portion is
replaced with larger naphthalenyl and anthracenyl components, respectively. In Structural Motif
D, the phenyl group is replaced with the larger, heterocyclic, benzothiazolyl group. However, in
Structural Motifs E and F, the original structure is extended by replacing the phenyl moiety with
benzamido (NHCOAr) and phenylcarbamoylamino (NHCONHAr) groups, respectively (Scheme
1). With the addition of these two components, additional hydrogen bonding capabilities are
introduced into the target molecules. Calculated partition coefficients (ClogP) were used as a

guide7to find the so-called “sweet spot” in the drug discovery approach defined by ClogP, pKa,
and polar surface area (PSA).8, 9
H
S
H
N
O
O
O
Polar surface area 87.30
H
N
CLogP 0.79
pKa 4.68
N
Merbarone
(4,6-dioxo-N-phenyl-2-sulfanylidene-1,3-diazinane-5-carboxamide)
R1
R1
N
R1
N
O
N
O
O
O
O
O
O
O
H
N
H
N
H
N
N
X
N
N
R2
R2
R2
O
O
O
O
Structural Motif C
Y
Structural Motif A
Structural Motif B
(anthracenyl)
(phenyl)
(naphthalenyl)
R1
R1
R1
X
Y
O
N
O
O
N
O
O
N
O
H
N
O
H
N
O
H
N
X
N
N
N
N
R2
N
H
N
H
R2
N
H
R2
X
O
O
O
O
S
O
O
Structural Motif F
(phenylcarbamoylamino)
Structural Motif E
Structural Motif D
(benzothiazolyl)
(benzamido)
Scheme 1. Six structural motifs based on merbarone as a lead compound
Synthetic procedures for the C-carboxamidation10 of substituted 1,3-diazinane-2,4,6-trione
(barbituric acid) begin with the preparation of N-arylcarbamates.11The applicability of
carbamates in organic synthesis is well documented for use as a protecting group for amines,
alcohols, and thiols.12Furthermore, the same structural motif is present in many
pharmaceuticals13and agrochemicals.14,15The most common method for the preparation of
carbamates is the reaction of an amine with the corresponding dicarbonate or chloroformate.
Chloroformates are generally avoided as starting materials due to their hazardous nature,
temperature sensitivity and rapid hydrolysis, which necessitate special handling and storage.
For these reasons, several viable alternatives have been developed for the preparation of

carbamates from corresponding amines such as using di-tert-butyldicarbonates16or 1-
alkoxycarbonyl-3-nitro-1,2,4-triazole,17 to name a few.
Despite their moisture sensitivity, we found that chloroformates made excellent reagents for
the preparation of a wide structural array of carbamates. To study the initial C-carboxamidation
of substituted 1, 3-diazinane-2, 4, 6-triones, three different series of carbamates were prepared.
The three different groups were chosen according to their reactivity, with the least reactive being
ethyl carbamate and the most reactive being 4-nitrophenyl carbamate, with the moderately
reactive phenyl carbamate falling in between the two, in terms of reactivity. The carbamates
were prepared from the corresponding chloroformate and chosen aryl amine in basic media.
The most straightforward reaction is between ethyl chloroformate and the corresponding
aromatic amine, since the reaction can be easily carried out in separatory funnel as a bilayer
ethyl acetate – water mixture using sodium carbonate (washing soda) as a base (Scheme 1).
This reaction is completed after several minutes at ambient temperature. The formed ethyl
carbamates are very stable- almost too stable to be suitable for the C-carboxamidation of
substituted 1, 3-diazinane-2, 4, 6-triones. On the other hand, the preparation of 4-nitrophenyl
carbamates from 4-nitrophenyl chloroformate is temperature sensitive and in many instances,
decomposes easily. The preparation of 4-nitrophenyl carbamate starts with an ice-water cooled
mixture of ethyl acetate, water and the desired aryl amine followed by the slow addition of 4-
nitrophenyl chloroformate. In many cases the isolation and purification of these compounds
must be carried out at 0°C, otherwise a substantial amount of symmetric diarylurea byproduct is
formed. If isolated, pure 4-nitrophenyl carbamates are excellent reagents for the C-
carboxamidation of 1, 3-diazinane-2, 4, 6-triones.18Since our goal was to synthesize a library
with an assortment of aryl substituted carbamates, including species of poorly reactive aryl
amines with electron-withdrawing groups, 4-nitrophenyl chloroformate was not our preferred
reagent. Instead, we selected the moderately reactive phenyl chloroformate, which allowed us
to prepare a wide array of corresponding phenyl carbamates.19 Phenyl carbamates have an

intermediate reactivity, and therefore can be easily prepared and stored for long periods of time
regardless of the nature of the substituents on the aromatic amine. The synthesis of these
phenyl carbamates is remarkably simple, beginning with phenyl chloroformate, the
corresponding aryl amine and a base at 0°C with warming to room temperature. Isolated yields
are almost quantitative and in most instances, further purification is not necessary. This
reaction tolerates a variety of aromatic substituents including halogen, hydroxy, carboxylic acid,
ester, amide, nitrile, nitro, alkyl, and alkoxy groups.Due the solubility and reactivity of each aryl
amine, several different methods (Methods I-V) were employed to obtain the desired compound.
Our preferred method for the synthesis of phenyl N-arylcarbamates is carried out initially at 0°C
in a mixture of ethyl acetate/ tetrahydrofuran/ water/ sodium carbonate before being allowed to
warm to room temperature and is isolated via extraction followed by recrystallization from ethyl
acetate/hexane (Scheme 2).
H
O
N
C6H5COCl
Ar
Ar-NH2
CH3CO2C2H5/THF/H2O/Na2CO3
0°C to r.t
O
aryl amine
phenyl N-arylcarbamate
base/solvent
reflux or 130°C
- PhOH
R1
O
N
O
(a)
R1
N
R2
O
N
O
O
H
N
N
O
C
Ar
N
O
(b) HCl/water
R2
Ar
aryl isocyanate
O
N-Aryl-2,4,6-trioxo-1,3-diazinane-5-carboxamide
Method A: Pyridine; Method B: TEA/DMSO;
Method C: NMM/DMF; Method D: Na2CO3/DMF
Scheme 2. Proposed mechanism for direct carboxamidation of 1, 3-diazinane-2, 4, 6-triones

The second step in the C-carbamidation is the preparation of the aryl isocyanate
intermediate,20which is accomplished in situ by the elimination of phenol from phenyl carbamate
(Scheme B). By generating the aryl isocyanate intermediate in situ, contact with toxic aromatic
isocyanates is avoided, an approach commonly used in the preparation of polyamides.21In our
case, we used different combinations of solvents and bases to maximize the formation of the
aromatic isocyanate, which was then immediately captured with the barbituric acid anion. In this
way, the desired urea was formed with only a minimal amount of byproduct. There are several
methods that were applied for the preparation of these compounds. Initially pyridine was used,
acting as both the solvent and base (Method A), to produce the best yield with little to no
formation of the symmetric diaryl urea byproduct. Purification was achieved by simple acid
precipitation of the product from the reaction mixture; if necessary, additional purification may be
done by refluxing the crude material in ethanol. This reaction is carried out overnight and is an
excellent choice for the preparation compounds 1-54 (Table 1-3). In order to improve this
method with the goal of decreasing the reaction time and solubilizing the starting materials, a
polar reaction media was employed. We selected two common polar solvents N, N-
dimethylformamide (DMF) and dimethylsulfoxide (DMSO) in combination with bases such as
pyridine, trimethylamine (TEA), N-methylmorpholine (NMM), and sodium carbonate. Among
these combinations, it was evident that the best reaction media is refluxing TEA/DMSO (Method
B) for the C-carbamidation of 1, 3-diazinane-2, 4, 6-trione (barbituric acid). The reaction is
completed in five hours with little to no formation of byproducts, and in this way compounds 1-22
(Table 1) were prepared in almost quantitative yield. Due to the very low solubility of these
products in ethanol and/or isopropanol, removing any formed byproduct involves the simple
filtration of a refluxing alcohol suspension of the crude product. The simplicity and efficiency of
this method make it our preferred avenue for the preparation of our target compounds lacking
N-substitution on the 1, 3-diazinane-2, 4, 6-trione moiety.

R1
R4
R2
R3
O
O
O
H
O
N
N
H
Structural Motif A
N
H
1-22
Table 1. Isolated yields (Method B) and glioblastoma LN-229 cell line activity of phenyl
substituted2, 4, 6-trioxo-N-phenyl-1, 3-diazinane-5-carboxamides
Compound
R1
H
OH
H
CH3
H
OCH3
OCH3
H
H
H
H
Cl
H
Cl
H
H
H
H
R2
H
H
OH
CH3
OCH3
OCH3
OCH3
F
Cl
H
Cl
H
Cl
H
Br
H
R3
H
H
H
H
H
H
OCH3
H
R4 Yield (%)
ClogP
-0.10
-0.41
-041
0.99
-0.26
-0.42
-0.58
0.07
0.50
1.11
1.11
1.11
1.11
1.11
0.67
0.67
0.83
-0.44
0.26
-1.25
-0.16
-0.16
pKa
4.20
4.00
3.99
4.37
3.92
3.85
3.79
3.71
3.71
3.34
3.34
3.34
3.34
3.34
3.69
3.69
3.68
3.64
3.79
3.89
3.08
2.95
PSA
GA (µg/ml)
NA
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
92
87
91
88
89
94
92
91
94
77
85
78
92
95
98
85
87
89
83
98
81
75
104.37
124.60
124.60
104.37
113.60
122.83
132.06
104.37
104.37
104.37
104.37
104.37
104.37
104.37
104.37
104.37
104.37
141.67
130.67
147.46
147.51
147.51
NA
100
100
NA
NA
100
NA
NA
NA
NA
NA
NA
NA
NA
100
NA
NA
100
NA
100
NA
H
Cl
H
H
Cl
Cl
H
Br
H
H
H
H
H
I
CO2H
CO2Et
CONH2
NO2
H
H
H
H
H
NO2
PSA = polar surface area in square angstroms (Ų); GA = glioblastoma activity
R1
O
O
O
H3C
O
N
N
H
R2
Structural Motif A
R3
N
H
23-30
Table 2.Isolated yields (Method D) and glioblastoma LN-229 cell line activity of phenyl
substituted 1-methyl-2, 4, 6-trioxo-N-phenyl-1, 3-diazinane-5-carboxamides
Compound
R1
R2
R3
Yield (%)
ClogP
pKa
4.22
4.02
4.02
3.94
3.74
3.38
3.38
PSA
95.58
115.81
115.81
104.81
95.58
95.58
95.58
GA (µg/ml)
23
24
25
26
27
28
29
H
H
OH
OCH3
Cl
H
OH
H
H
H
H
H
H
H
H
Cl
Cl
92
88
86
93
91
88
82
0.12
NA
100
NA
100
100
NA
NA
-0.18
-0.18
-0.04
0.73
1.33
1.33
H
Cl
Cl
H

30
CO2H
H
H
89
-0.22
3.65
132.88
NA
PSA = polar surface area in square angstroms (Ų); GA = glioblastoma activity
R1
R2
O
O
O
H3C
O
N
N
H
R3
Structural Motif A
R4
N
31-54
CH3
Table 3.Isolated yields (method D) and glioblastoma LN-229 cell line activity of phenyl
substituted 1, 3-dimethyl-2, 4, 6-trioxo-N-phenyl-1, 3-diazinane-5-carboxamides
Compound
R1
H
H
H
H
H
H
H
OCH3
H
H
H
Cl
H
Cl
H
H
H
H
R2
H
H
H
OH
CH3
OCH3
OCH3
OCH3
Cl
H
Cl
H
Cl
H
Br
H
I
R3
H
H
OH
H
CH3
H
OCH3
OCH3
H
Cl
H
H
Cl
Cl
H
Br
H
H
R4
H
OH
H
H
H
H
H
H
Yield (%)
ClogP
0.35
0.04
0.04
0.04
1.37
0.19
0.03
-0.13
0.95
1.55
1.55
1.55
1.55
1.55
1.11
1.11
1.27
0.00
0.71
-0.80
0.29
0.29
1.99
pKa
5.52
5.34
5.33
5.32
5.67
5.25
5.17
5.10
5.06
4.70
4.70
4.70
4.70
4.70
5.03
5.03
5.01
4.11
5.13
5.22
5.00
5.01
5.71
PSA
86.79
107.02
107.02
107.02
86.79
96.02
105.25
114.48
86.79
86.79
86.79
86.79
86.79
86.79
86.79
86.79
86.79
124.09
113.09
129.88
129.93
129.93
86.79
GA (µg/ml)
100
100
100
100
NA
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
89
71
83
87
91
94
92
95
91
76
83
81
89
90
95
86
89
83
95
98
86
82
85
NA
NA
NA
10
H
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
Phen
yl
100
100
100
100
NA
100
100
100
100
100
NA
CO2H
CO2Et
CONH2
NO2
H
H
H
H
H
H
H
H
NO2
H
100
NA
100
H
H
54
H
Phenyl
H
H
92
1.99
5.71
86.79
100
PSA = polar surface area in square angstroms (Ų); GA = glioblastoma activity
Similarly, N-substituted and N, N-disubstituted 1, 3-diazinane-2, 4, 6-triones can also
undergo C-carboxamidation using pyridine, TEA/DMSO, and NMM/DMSO as reaction media.
However, our method of choice for these compounds is Na2CO3/DMF (Method D) because it is
a two step, one pot reaction. In the first step, the sodium salt of the nitrogen mono and
disubstituted 1, 3-diazinane-2, 4, 6-trione is formed under elevated temperature (100°C) with
sonication. In the second step, the corresponding phenyl carbamate is added and sonication

with heating continued for an additional four hours. Using this approach, the conversion to our
target compounds 23-80 was maximized (Tables 2-5). Most of these compounds are partially
soluble in organic solvents and can be recrystallized from either ethanol or isopropanol. Due to
their unique physical properties, carboxamides 81-100 (Tables 6-8) were also prepared in DMF
with sodium carbonate as a base using the procedure described in Method D. The isolated
yields for the C-carboxamidation of N-substituted 3-diazinane-2, 4, 6-triones is almost
quantitative and purification again involves simple washing of the crude product with hot alcohol.
R1
R2
O
O
O
N
N
H
R3
Structural Motif A
R4
O
N
H
55-74
Table 4. Isolated yields (Method D) and glioblastoma LN-229 cell line activity of phenyl
substituted 1-phenyl-2, 4, 6-trioxo-N-phenyl-1, 3-diazinane-5-carboxamides
Compou
pKa
R1
R2
R3
R4 Yield (%) ClogP
PSA
GA (µg/ml)
nd
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
H
H
H
H
H
OCH3
H
H
H
H
Cl
H
Cl
H
H
H
H
H
H
H
H
OH
CH3
OCH3
OCH3
OCH3
F
Cl
H
Cl
H
H
H
CH3
H
OCH3
OCH3
H
H
Cl
H
H
Cl
Cl
H
H
H
H
H
NO2
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
1.78
1.48
2.81
1.62
1.46
1.31
1.92
2.38
2.99
2.99
2.99
2.99
2.99
1.11
2.71
1.44
0.63
1.72
1.72
3.43
100
NA
NA
100
NA
100
NA
NA
NA
100
NA
NA
NA
100
100
100
NA
NA
NA
NA
87
83
90
92
91
85
86
92
88
91
87
88
93
96
91
85
94
88
83
95
3.75
3.58
3.88
3.53
3.48
3.40
3.37
3.36
3.05
3.05
3.05
3.05
3.05
3.33
3.31
3.35
3.50
3.82
3.79
3.79
95.58
115.81
95.58
104.81
114.04
123.27
95.58
95.58
95.58
95.58
95.58
95.58
95.58
95.58
95.58
132.88
138.67
138.72
138.72
95.58
Cl
H
Br
I
CO2H
CONH2
NO2
H
71
72
73
74
Phenyl
PSA = polar surface area in square angstroms (Ų); GA = glioblastoma activity

O2N
R1
O
O
O
N
N
H
Structural Motif A
O
N
H
75-80
Table 5.Isolated yields (Method D) and glioblastoma LN-229 cell line activity of phenyl
substituted 1-(4-nitrophenyl)-2, 4, 6-trioxo-N-phenyl-1, 3-diazinane-5-carboxamides
R1
H
OCH3
F
Cl
ClogP
1.72
1.56
1.86
2.32
0.57
1.66
pKa
3.32
3.17
3.02
3.02
3.15
3.48
PSA
Compound
GA (µg/ml)
Yield (%)
75
76
77
78
79
80
138.72
147.95
138.72
138.72
181.81
181.86
NA
NA
NA
NA
NA
NA
84
88
87
90
91
75
CONH2
NO2
PSA = polar surface area in square angstroms (Ų); GA = glioblastoma activity
O
O
O
R1
O
R3
N
N
H
81-90
Structural Motifs B and C
N
R2
Table 6. Isolated yields (Method D) and glioblastoma LN-229 cell line activity of substituted 2, 4,
6-trioxo-1, 3-diazinane-5-carboxamide
Compound
R1
H
H
H
CH3
CH3
CH3
CH3
Phenyl
Phenyl
Phenyl
R2
H
H
H
R3
Yield (%)
Clog P
0.89
0.89
1.88
2.10
1.33
1.33
2.32
2.77
2.77
3.76
pKa
PSA
GA (µg/ml)
81
82
83
84
85
86
87
88
89
90
1-napththyl
2-napththyl
2-anthracenyl
2-anthracenyl
1-napththyl
2-napththyl
2-anthracenyl
1-napththyl
2-napththyl
2-anthracenyl
91
96
93
94
97
96
92
94
4.41 104.37
4.35 104.37
4.43 104.37
4.42
5.69
5.64
5.70
3.90
3.86
3.92
100
100
100
100
100
100
100
100
H
95.58
86.79
86.79
86.79
95.58
95.58
95.58
CH3
CH3
CH3
H
H
H
91
93
NA
100
PSA = polar surface area in square angstroms (Ų); GA = glioblastoma activity
R3
O
O
O
N
R1
O
S
N
N
H
91-96
N
Structural Motif D
R2
Table 7.Isolated yields (Method D) and glioblastoma LN-229 cell line activity of substituted N-(1,
3-benzothiazol-2-yl)-2, 4, 6-trioxo-1,3-diazinane-5-carboxamide
Compound
R1
H
H
CH3
CH3
Phenyl
Phenyl
R2
H
H
CH3
CH3
H
R3
OCH3
NO2
OCH3
NO2
OCH3
NO2
Yield (%)
ClogP
0.56
0.66
1.01
1.11
2.45
2.54
pKa
3.73
2.89
5.08
4.82
3.38
3.65
PSA
GA (µg/ml)
91
92
93
94
95
96
91
84
94
83
87
84
126.49
160.40
108.91
142.82
117.70
151.61
NA
NA
NA
NA
100
100
H
PSA = polar surface area in square angstroms (Ų); GA = glioblastoma activity

O
O
O
H
N
R1
O
N
N
H
Structural Motif E
N
R2
97-100
R1
Table 8. Isolated yields (Method D) and glioblastoma LN-229 cell line activity of substituted 2, 4,
6-trioxo-N'-phenyl-1, 3-diazinane-5-carbohydrazide
Compound
R1
H
H
CH3
CH3
R2
H
NO2
H
NO2
Yield (%)
ClogP
-0.24
-0.30
0.21
pKa
5.52
3.17
7.22
6.72
PSA
GA (µg/ml)
97
98
99
95
91
97
93
116.40
159.54
98.82
NA
NA
NA
100
100
0.15
141.96
PSA = polar surface area in square angstroms (Ų); GA = glioblastoma activity
Unsubstituted carbamoylamino derivatives 101-109 (Table 9) are even less soluble in
organic solvents, including DMSO, than carboxamides 1-22 (Table 1), making it difficult not only
to prepare but also to analyze these compounds. Several approaches were used, but the
combination of TEA and DMSO (Method B) is the most reproducible. Although solubility is a
concern, here the extremely low solubility of these compounds simplifies their purification, which
is done by refluxing the crude material in a large amount of alcohol and filtering off the insoluble
product. Another interesting finding in terms of the solubility of these compounds is that while
they have low solubility in chloroform and dimethylsulfoxide, they have surprisingly reasonable
solubility in trifluroacetic acid (TFA) and various combinations of CHCl3/TFA and DMSO/TFA.In
light of the TFA solubility of these compounds, they can be further purified by recrystallization
from tetrachloroethylene/TFA. The combination of CDCl3/TFA and DMSO-d6/TFA was also
used for recording the NMR spectra. Due to fact that several rotamers are present in the
solution, a simple 1H-NMR spectrum of these compounds becomes very complex.22The ratio of
signals representing different rotamers varies with the concentration of TFA in the NMR solvent,
which can be delineated from nonpolar tetrachloroethylene, less polar chloroform, and more
polar DMSO. More simplified spectra were obtained by electrospray mass spectroscopy with
methanol as a solvent. Despite the obstacles associated with handling these compounds,
isolated yields are good to excellent.

O
O
O
H
N
H
N
R1
H
O
N
N
H
O
N
H
R2
101-109
Structural Motif F
Table 9. Isolated yields (Method B) and glioblastoma LN-229 cell line activity of phenyl
substituted 2, 4, 6-trioxo-N-[(phenylcarbamoyl) amino]-1, 3-diazinane-5-carboxamide
Compound
R1
H
OH
H
H
H
Cl
H
H
NO2
R2
H
H
OH
F
Cl
Cl
CN
NO2
OH
Yield (%)
ClogP
-1.03
-1.33
-1.33
-0.88
-0.42
0.18
-1.17
-1.09
-1.39
pKa
5.22
5.13
5.16
5.09
5.13
5.05
5.10
3.25
3.28
PSA
GA (µg/ml)
100
NA
101
102
103
104
105
106
107
108
91
82
86
89
91
96
90
83
145.50
165.73
165.73
145.50
145.50
145.50
169.29
188.64
208.87
NA
NA
100
NA
NA
NA
NA
109
87
PSA = polar surface area in square angstroms (Ų); GA = glioblastoma activity
Although all described C-carboxamidation methods may be used for N-substituted and N, N-
disubstituted carboxamides 110-136 (Tables 10-12), the best results are obtained with DMSO
as the solvent and N-methylmorpholine as the base (Method C). This method produces the
most consistency in terms of both yield and purity. Again, the solubility in common organic
solvents is very low so removing any impurity involves the simple washing of the crude product
with hot ethanol or isopropanol.
O
O
H
N
H
N
R1
H3C
O
N
N
H
O
N
H
O
R2
110-118
Structural Motif F
Table 10. Isolated yields (Method C) and glioblastoma LN-229 cell line activity of phenyl
substituted 1-methyl-2, 4, 6-trioxo-N-[(phenylcarbamoyl) amino]-1, 3-diazinane-5-carboxamide
Compound
R1
H
OH
H
H
H
Cl
H
H
NO2
R2
H
H
OH
F
Cl
Cl
CN
NO2
OH
Yield (%)
ClogP
-0.80
-1.10
-1.10
-0.66
-0.20
-0.41
-0.95
-0.86
-1.16
pKa
5.25
5.17
5.19
5.13
5.17
5.09
5.14
3.30
3.31
PSA
GA (µg/ml)
110
111
112
113
114
115
116
117
88
84
82
87
85
89
91
83
136.71
156.94
156.94
136.71
136.71
136.71
160.50
179.85
200.08
NA
NA
NA
NA
100
NA
NA
100
NA
118
84
PSA = polar surface area in square angstroms (Ų); GA = glioblastoma activity

O
O
O
H
N
H
N
R1
H3C
O
N
N
H
O
N
R2
119-127
CH3
Structural Motif F
Table 11. Isolated yields (Method C) and glioblastoma LN-229 cell line activity of phenyl
substituted 1, 3-dimethyl-2, 4, 6-trioxo-N-[(phenylcarbamoyl) amino]-1, 3-diazinane-5-
carboxamide
Compound
119
R1
H
OH
H
H
H
Cl
H
H
NO2
R2
H
H
OH
F
Cl
Cl
CN
NO2
OH
Yield (%)
ClogP
-0.58
-0.88
-0.88
-0.44
0.03
pKa
6.93
6.85
6.87
6.81
6.85
6.77
6.82
6.69
6.89
PSA
GA (µg/ml)
89
81
82
88
89
91
93
83
127.92
148.15
148.15
127.92
127.92
127.92
151.71
171.06
191.29
NA
NA
10
100
10
1
100
10
120
121
122
123
124
125
126
127
0.63
-0.73
-0.64
-0.94
77
10
PSA = polar surface area in square angstroms (Ų); GA = glioblastoma activity
O
O
O
H
N
H
N
R1
N
N
H
O
O
N
H
R2
128-136
Structural Motif F
Table 12. Isolated yields (Method C) and glioblastoma LN-229 cell line activity of phenyl
substituted 1-phenyl-2, 4, 6-trioxo-N-[(phenylcarbamoyl) amino]-1, 3-diazinane-5-carboxamide
Compound
128
R1
H
OH
H
H
H
Cl
H
H
R2
H
H
OH
F
Cl
Yield (%)
87
ClogP
0.86
0.55
0.55
1.00
1.46
2.06
0.71
0.80
0.49
pKa
PSA
GA (µg/ml)
NA
3.80 136.71
3.74 156.94
3.76 156.94
3.71 136.71
3.74 136.71
3.76 136.71
3.71 160.50
4.48 179.85
4.48 200.08
129
130
131
132
133
134
135
136
90
83
87
88
92
91
81
78
NA
NA
NA
100
100
100
100
100
Cl
CN
NO2
OH
NO2
PSA = polar surface area in square angstroms (Ų); GA = glioblastoma activity
For the anti-cancer screening of our target compound we selected the glioblastoma LN-229
cell line because the treatment of glioblastoma remains challenging, with no curative modern
treatment available.23The standard treatment for glioblastoma consists of surgery and radiation,

followed by chemotherapy with the drug temozolomide.24The survival rates for those affected
with glioblastoma after surgery, radiation and chemotherapy using nitrosourea-based regimens
are 1-, 3-, and 5-year survival rates of 46%, 18%, and 18%, respectively. A major hindrance to
the use of chemotherapeutic agents for brain tumors is the reality that the blood-brain barrier
(BBB) excludes many agents from the central nervous system. This fact necessitates the
development of new anti-cancer compounds with both higher activity and the possibility to be
transferred into prodrugs that have a more favorable probability to cross the BBB.25 There are
indications that merbarone, besides being active against leukemia, shows promising activity
against astrocystoma cell lines at concentrations of 20 µg/ml.26
Merbarone itself is a non-polar, acidic compound with an acceptable polar surface area
(PSA). Molecules with a PSA value greater than 140 Å2 are believed to have poor cell
membrane permeability.27A PSA value less than 90 Å2 is required for the molecule to penetrate
the BBB.28The first group of our compounds that was tested against glioblastoma belongs to
Structural Motif A, with R1 = R2 = H (Table 1, structures 1-22). The difference between our first
structure 1 and merbarone is the replacement of the sulfur atom with oxygen. This minor
structural modification is reflected in the substantial change in the calculated lipophilicity, acidity,
polarizability, and PSA. All computed factors indicate that the molecule is far too polar to cross
the BBB and should have little to no activity (Table 1). The introduction of the hydroxy group
makes the molecule even more polar and thus less desirable as a potential drug candidate.
However, the low solubility in both water and organic solvents of this class of compounds makes
proper evaluation of their anti-cancer activity difficult. Therefore, it is not a surprise that
compound 3 shows moderate activity, despite the fact that the calculated physical properties are
unfavorable for crossing the BBB, due to the hydroxy group increasing water solubility. The
introduction of two methyl groups changes the calculated properties in the desired direction,
indicating moderate anti-cancer activity for compound 4, which has a ClogP value of 0.99 and a
PSA value of 104.37 Å2 (Table 1). Trimethoxy bearing structures 7 also have all of the physical

predictors pointing in an unfavorable direction, but most likely due to increased solubility there is
still moderate activity. All predictors for the halogen substituted compounds (8-17) indicate
improved physical properties in regards to unsubstituted 1, but only bromo substituted
compound 16 shows moderate activity at a concentration of 100 µg/ml. One of the limitations of
computational methods is that the calculated polar descriptors are identical for identical
substituents regardless of their position in the phenyl ring. Thus, for dichloro compounds 10-14
all calculated physical descriptors are identical (Table 1). All other compounds, including 4-
nitrophenyl derivatives 21, also show moderate activity.
Furthermore, we have explored the change in polarity of our lead compound by substituting
one of the nitrogen hydrogens on the 1, 3-diazinane moiety with a methyl group. The calculated
physical descriptors suggest that these new compounds will have enhanced lipophilicity, lower
acidity, and a smaller polar surface area. For instance, if we compare compound 1 (Table 1)
with compound 23 (Table 1), the ClogP increases by 0.22, the pKa increases by 0.02, and the
PSA decreases by 8.79 by adding a single N-methyl group. All of these physical descriptors
indicate that compound 23 has a higher probability to pass BBB than compound 1. However,
there is no observed anti-cancer activity for either 1 or 23 at concentrations of 100 μg/ml. From
the limited number of examples with an N-methyl modification and Structural Motif A, only
compounds with hydroxy, methoxy, and chloro substituents on the phenyl moiety 24, 26, and 27
respectively, show moderate anti-cancer activity at concentrations of 100 µg/ml (Table 2).
It is now reasonable to assume that analogues with N, N-dimethyl substitution on the 1, 3-
diazinane moiety will be more lipophilic than their corresponding unsubstituted analogues.
Accordingly, a series of N, N- dimethyl derivatives was prepared and tested for anti-cancer
activity (Table 3). Computed physical descriptors for first N, N- dimethyl derivative 1, an
unsubstituted phenyl ring, come very close to the computed physical descriptors for merbarone.
Although unsubstituted 1 and N-methyl substituted 23 do not show activity, N, N- dimethyl
substituted 31 is almost as active as merbarone (Table 3). The ClogP increases by 0.22 for the

N-methyl and an additional 0.23 for the N, N- dimethyl. This group of compounds has far
superior activity than the corresponding N, N- dimethyl analogs in Tables 1 and 2. It seems that
the presence of a hydroxy group on the phenyl moiety results in a slight increase in lipophilicity,
leading to compounds 32, 33, and 34 showing moderate activity at 100 µg/ml concentrations.
Compounds with monohalogenated phenyl groups show moderate activity, with 4-chlorophenyl
39 being the most potent (10 µg/ml) of this category (Table 3) and dichloro derivatives such as
40, 41, 42, and 43 having moderate (100 μg/ml) activity. This is also true for carboxylic acid
(48), ester (49), 4-nitro (52), and additional phenyl (53 and 54) compounds (Table 3). The
monochloro compound 39 is the superior candidate from this group, having anti-cancer activity
slightly better than merbarone.
Considering the phenyl contribution to the overall lipophilicity of the molecule, one can
assume that the replacement of the methyl group in position 1 (Table 2) will result with new
compounds that will now have a higher ClogP value, lower molecular surface area and higher
acidity (Table 4).The major problem with this class of compounds is their inherently low
solubility, which makes it challenging to accurately evaluate their glioblastoma activity. Although
ClogP values and PSA values are close to the optimal values for almost all compounds 55-74
(Table 4), there is only moderate activity for a handful of them. This group contains the
unsubstituted phenyl group 55, 4-methyoxy 58, trimethoxy 60, dichloro 64, 4-bromo 68, 4-iodo
69, and 4-carboxyl 70, all showing activity at concentrations of 100 µg/ml. However, none of
these compounds show activity at 10 µg/ml concentrations (Table 4). If a nitro group is added
on the 1-phenyl group, a new group of compounds with Structural Motif A is formed with higher
polarity and a larger molecular surface area (Table 5). Unfortunately, this modification did not
produce any drug candidates with activity at concentrations of 100 μg/ml or lower. Therefore,
the best candidate from Structural Motif A (Scheme 1) is 1, 3-dimethyl diazinane and p-
chlorophenyl substituted compound 39, which has good activity at 10 μg/ml (Table 3).

In exploring the structural modifications which will result in higher anticancer potency, the
phenyl group of the original merbarone was replaced with larger, polycyclic, aromatic
hydrocarbons such as naphthalene and anthracene resulting in a new class of compounds with
Structural Motifs B and C (Table 6). Our initial calculations suggest that these compounds have
similar electronic properties to merbarone (Scheme 1). As we predicted, nearly all of these
compounds show moderate activity at 100 μg/ml concentrations (Table 6), although it is lower
than we expected. Due to the low anti-cancer activity of these compounds, we did not proceed
with any further structural modifications of compounds with Motifs B and C. Another structural
derivative we wished to explore was the introduction of an additional heterocycle by replacing
the phenyl group with 1, 3-benzothiazol-2-yl, forming a new set of compounds with Structural
Motif D (Table 7). Unfortunately, introducing this heterocycle also changed the computed polar
parameters away from the desired optimal values, with only compounds 95 and 96 showing
moderate activity at concentrations of 100 µg/ml (Table 7).
The next structural modification was to increase the distance between the diazinane and
aryl moieties by inserting an NH group, forming Structural Motif E. For our initial studies, only
four examples from this class were prepared (Table 8). Unfortunately almost all of these
compounds do not show any activity except for compound 100, which has a 4-nitrophenyl group
and moderate activity at 100 μg/ml concentrations. The most promising structural modification
of all of these compounds is the insertion of the urea (NHCONH) structural unit into our original
structure, resulting in a new group of compounds called Structural Motif F (Scheme 1). There
are four additional subgroups with this structural motif: unsubstituted (Table 9), with one methyl
(Table 10), with two methyls (Table 11), and with one phenyl (Table 12) substituent on the
diazinane moiety.
The first group of compounds with Structural Motif F does not have any substituents on the
1, 3-diazine moiety. As expected from the studies performed above, computed physical
parameters indicate even higher polarity, lower solubility, and high molecular surface area

(Table 9). This is attributed to the insertion of the polar urea moiety into 1, 3-diazone – aryl
linker, which moves these molecules further away from the desired optimal values for computed
polar parameters. Therefore, it is not surprising that only two of the nine compounds from this
group show moderate activity: unsubstituted 101 and p-chlorophenyl substituted 105 (Table 1).
These results indicate that this group of compounds must be modified by a decrease in polarity.
As in the case above, introducing one methyl group into a 1, 3-diazine moiety will substantially
alter the polarity and polar distribution in the new molecule (Table 10). The calculated partition
coefficient is slightly higher, and the molecular polar surface slightly lower. Since these changes
are not substantial enough to move these molecules towards the targeted optimal values, it is
not unexpected that only chloro 114 and nitro 117 derivatives have modest anti-cancer activity
at 100 µg/ml concentrations. Introducing a second methyl group gives new physical molecular
descriptors that are closer to the desired values, although the ClogP value is still very low.
These groups of new compounds contain several very interesting anti-cancer drug
candidates (Table 11). Fluoro 122 and cyano 125 substituted phenyl compounds show
moderate glioblastoma activity at concentrations of 100 µg/ml. Hydroxy 121, chloro 123, nitro
126, and a combination of hydroxy and nitro 127 show good glioblastoma activity at even lower
concentrations of 10 µg/ml, while 3, 4-dichloro 124 shows excellent glioblastoma activity at a
very low concentration of 1 µg/ml (Table 11). One may argue that replacing the two methyl
groups with one phenyl group should further alter physical properties in such a way that will
bring the calculated molecular descriptors closer to the desired values (Table 11). In fact, the
calculated partition coefficients for these new molecules are much closer to the desired value;
however, the calculated pKa is far too low and the polar surface area is far too high. Therefore
it does not come as a surprise that in these groups chloro 132 and 133 and nitro 135 and 136
compounds show moderate glioblastoma activity at concentrations of 100 µg/ml (Table 12). In
order to compare the computed partition coefficients to the experimental values, we used the

distribution between 1-octanol and water for four of our selected compounds (31, 39, 112 and
124). The experimental logP values of compounds 31, 39, 112 and 124 are 0.40, 1.18, 1.08
and 0.73, and are in good agreement with the ClogP values of 0.35, 0.95, 1.10, and 0.63,
respectively. As mentioned throughout this paper, the poor solubility of these compounds in
both water and organic solvents complicates their anticancer evaluation. One solution to this
dilemma is to increase the concentration of DMSO to 1% when testing compounds in
concentrations of 100 mg/ml. However, some precipitation of the compounds was still observed,
most likely due to their poor water solubility, with the four chosen compounds having
experimental water solubility values of 0.14, 0.13, 0.07, and 0.5 mg/1ml respectively.
Conclusion
There are several merbarone derived compounds that have similar or better glioblastoma
activity than the original structure. From our studies it is clear that the combination of three key
molecular descriptors (ClogP, pKa, and PSA) can be used as a guide for the design of new
potent structural derivatives of merbarone. It was estimated that the so-called “sweet spot” in
terms of these three descriptors are ~1.0 for CLogP, ~6.0 for pKa, and ~100 Å2 for PSA. The
most effective of these compounds share structural variations of 1, 3-dimethyl-2, 4, 6-trioxo-N-
[(phenylcarbamoyl)amino]-1,3-diazinane-5-carboxamide (119) with additions of hydroxy, chloro,
or nitro to the phenyl group further enhancing activity and with the dichloro derivative 124 having
the highest activity of all (1 μg/ml). Given these promising results, additional structural
modifications to these compounds to optimize their efficacy against glioblastoma is currently
under extensive investigation.
Experimental Section
Chemistry

General remarks
All starting materials were reagent grade purchased from Sigma-Aldrich or Ark Pharm. 1H-NMR
spectra were recorded on Varian Unity 400 MHz, Varian Inova 500 MHz, Varian Inova 600 MHz,
Bruker 300 MHz, and Agilent 400 MHz NMRs in CDCl3, DMSO-d6, CDCl3-TFA, DMSO-d6-TFA,
and in TFA with internal D2O tube as NMR solvents using the solvent chemical shifts as an
internal standard. Electrospray Mass Spectroscopy (EMS) was performed on MDS Sciex 3200
Qtrap LC/MS/MS system in positive or negative mode with methanol or acetonitrile as solvents.
1-Methylbarbituric acid was prepared from methylurea and malonic acid in acetic anhydride
under microwave conditions.291-Phenylbarbituric acid and 1-(4- nitrophenyl) barbituric acid were
prepared from phenylurea (or 4- nitrophenylurea) and diethyl malonate in sodium
ethoxide/ethanol reaction media.30The partition coefficient (clogP) was calculated using Marvin
Sketch software. The calculation method is based on atomic and fragment method
contribution.31
General Procedure for preparation of phenyl arylcarbamates:
Method I (ethyl acetate/THF/ aqueous Na2CO3).Preparation of phenyl 4-
carbamoylphenylcarbamate (20 CF). Into a stirring mixture of ethyl acetate (300 ml),
tetrahydrofuran (100 ml), water (100 ml), sodium carbonate (6.4 g; 0.06 mol),and 4-
aminobenzamide (13.6 g; 0.1 mol), phenyl chloroformate (17.1 g; 0.11 mol) was added
dropwise at 0ºC. The reaction was stirred at 0°C for 1 hour and at room temperature for an
additional hour. The organic layer was separated, washed with water (3x20 ml), followed by 5%
hydrochloric acid (3x20 ml), and again with water (3x20 ml). After drying over anhydrous
sodium sulfate, the solvent was evaporated to dryness (22.1 g; 86%). If necessary, the product
can be further purified by crystallization from ethyl acetate/ hexane. 1H-NMR (DMSO-d6, Varian
Unity 400 MHz) δ 10.47 (1H, s), 7.90 (1H, broad s), 7.85 (2H, d, J = 8.4 Hz), 7.56 (2H, d, J = 8.4

Hz), 7.41 (2H, t, J = 8.0 Hz), and 7.22 (4H multiplet = 1 broad s + 1t + 1d) ppm. 13C-NMR
(DMSO-d6, Varian Unity 400 MHz) δ 152.4, 151.0, 142.1, 130.2, 129.3, 129.2, 126.3, 122.6 and
118.3 ppm. EMS- (CH3OH) m/z 264.5 (M-1), 363.5 (M-2), EMS MS2 (265) 265.1, 178.2, 153.0,
1
and 134.8. Phenyl 4-hydroxy-3-nitrophenylcarbamate (89a) (22 g; 80%). H-NMR (DMSO-d6,
Varian Inova 500 MHz) δ 10.35 (1H, s), 10.00 (1H, broad s), 7.45 (3H, t + s), 7.34 (1H, d, J =
2.5 Hz), 7.16 (2H, t, J = 7.5 Hz), and 7.15 (1H, d, J = 10 Hz) ppm. 13C-NMR (DMSO-d6, Varian
Inova 500 MHz) δ 155.8, 153.2, 151.3, 130.5, 130.2, 128.7, 126.2, 123.1, 122.38, 121.9, and
111.6 ppm.
Method II (ethyl acetate/triethylamine): Preparation of phenyl 4-fluorophenylcarbamate (8 CF).
Into an ice cooled solution of ethyl acetate (50 ml) and triethylamine (5 ml), 4-fluoroaniline (1.11
g; 10 mmol) and phenyl chloroformate (1.7 g; 11 mmol) were slowly added. The reaction
mixture was stirred for 1 hour at 0°C on ice followed by 2 hours at room temperature. The ethyl
acetate solution was then washed with water (5x15 ml), followed by 5% hydrochloric acid (5x15
ml), again with water (5x15ml) and dried over anhydrous sodium sulfate. The drying material
was removed via gravity filtration and the ethyl acetate filtrate evaporated to dryness under
reduced pressure. The solid material was mixed with hexane (15 ml) and sonicated at room
temperature for 30 minutes, resulting in a white suspension which was then filtered, washed
1
with hexane (3x5 ml) and dried at room temperature to give pure product (1.9 g; 82%). H-NMR
(DMSO-d6, Varian Unity 400 MHz) δ10.30 (1H, s) 7.55 (2H, d of d, J1 = 6.4 Hz, J2 = 3.2 Hz),
7.44 (2H, t, J = 6.4 Hz), 7.27 (1H, t, J = 6.4 Hz), 7.23 (2H, d, J =6.4 Hz), and 7.18 (2H, t, J = 6.4
Hz) ppm. 13C-NMR (DMSO-d6, Varian Inova 500 MHz) δ 158.7 (d, J = 952.5 Hz), 152.6, 151.2,
135.7, 130.1, 126.2, 122.6, 120.9, and 116.2 (d, J = 90 Hz) ppm.
Method III (tetrahydrofuran/pyridine). For highly THF soluble anilines. Preparation of phenyl 2,
3-dichlorophenylcarbamate (40 CF). Phenyl chloroformate (1.71 g; 11 mmol) was slowly added

into a stirring solution of tetrahydrofuran (30 ml), 2, 3-dichloroaniline (1.62 g; 10 mmol) and
pyridine (0.9 ml; 0.9 g; 11 mmol) which had been previously cooled on ice for 30 minutes. The
reaction continued stirring at 0°C for 30 minutes and then room temperature for one hour. The
solvent was evaporated under reduced pressure with the temperature not exceeding 20°C. The
formed white residue was mixed with dichloromethane (50 ml), washed with 3% hydrochloric
acid (3x10 ml), water (3x10 ml), and dried over anhydrous sodium sulfate. The drying material
was removed via gravity filtration and the volume of the dichloromethane filtrate was reduced to
~ 10 ml, to which hexane (20 ml) was added. The resulting precipitate was isolated by vacuum
1
filtration, washed with hexane and dried on air (2.5 g; 88%). H-NMR (CDCl3, Varian Inova 600
MHz) δ 8.14 (1H, d, J = 6.6 Hz), 7.56 (1H, broad s), 7.40 (2H, t, J = 7.8 Hz), 7.26 (1H, t, J =7.8
Hz), 7.24 (1H, d, J = 6.6 Hz), and 7.21 (2H, t, J = 7.8 Hz) ppm. EMS+ (CH3CN) m/z 283 (M+1),
305 (M+Na).
Method IV (ethyl acetate/tetrahydrofuran/pyridine).For low THF soluble and less reactive
anilines.Preparation of phenyl 4-nitrophenylcarbamate (51 CF). A pyridine (20 ml) and
tetrahydrofuran (30 ml) suspension of 4-nitroaniline (1.38 g; 10 mmol) was stirred with
sonication at room temperature until all nitroaniline was dissolved (~ 1hr). This solution was
diluted with ethyl acetate (200 ml) and cooled to 0ºC. An ethyl Acetate (30 ml) solution of phenyl
chloroformate (1.71 g; 11 mmol) was slowly added and the reaction mixture allowed to stir at
0°C for 1 hour and then at room temperature overnight. The reaction mixture was washed with
water (3x50 ml), followed by 5% hydrochloric acid (3x50 ml), again with water (3x50 ml), dried
over anhydrous sodium sulfate and evaporated to afford a pure, dry, yellow product (2.2 g;
1
85%). H-NMR (CDCl3, Varian Unity 400 MHz) δ 8.23 (2H, d, J = 9.2 Hz), 7.62 (2H, d, J = 9.2
Hz), 7.41 (2H, t, J = 7.6 Hz), 7.33 (1H, broad s), 7.28 (1H, t, J = 7.6 Hz), and 7.19 (2H, d, J = 7.6
Hz) ppm.

Method V (ethyl acetate/N-methylmorpholine) for active arylamines.Preparation of phenyl 3, 4,
5-trimethoxyphenylcarbamate (38 CF). An ethyl acetate (15 ml) solution of phenyl
chloroformate (3.42 g; 22 mmol) was slowly added into an ice cooled ethyl acetate (150 ml)
solution of N-methylmorpholine (15 ml) and 3, 4, 5-trimethoxyaniline (3.66 g, 20 mmol). After
stirring for 1 hour at room temperature, the ethyl acetate reaction mixture was washed with
water (3x50 ml), 5% hydrochloric acid (3x50 ml), water (3x50 ml), dried over anhydrous sodium
1
sulfate, and evaporated to dryness to give the pure product (2.6 g; 86%). H-NMR (CDCl3,
Varian Inova 600 MHz) δ 7.39 (2H, t, J = 7.8 Hz), 7.34 (1H, t, J = 7.8 Hz), 7.17 (2H, d, J = 7.8
Hz), 6.92 (1H, broad s), 6.74 (2H, s), 3.83 (6H, s), and 3.81 (3H, s) ppm. EMS+ (CH3CN) m/z
304 (M+1), 326 (M+Na).
General Procedure for Preparation of N-arylhydrazinecarboxamide.
Preparation of N-(4-hydroxy-3-nitrophenyl)hydrazinecarboxamide (109b CF). An ethanol
(500 ml) solution of the desired carbamate 109a CF (20 g; 73 mmol) and hydrazine
monohydrate (20g; 0.4 mol) was refluxed for 1 hour. The volume of the reaction mixture was
reduced to ~10 ml, yielding a deep red suspension which was then mixed with water (300 ml)
and cooled on ice for 2 hours. The insoluble product which formed was isolated by vacuum
filtration, washed with ice cold water (3x30 ml) and dried at 110ºC for 30 minutes (12.5 g; 81%).
1H-NMR (DMSO-d6, Varian Inova 600 MHz) δ 10.70 (1H, s), 9.99 (1H, s), 8.41 (1H, s, 1H, s),
7.46 (1H, s), 7.15 (1H, d, J = 10.8 Hz), and 4.61 (2H, broad s) ppm. 13C-NMR (DMSO-d6 Varian
Inova 600 MHz)δ 157.6, 152.0, 137.0, 129.1, 124.6, 123.2, and 110.6 ppm.
General Procedure for Preparation of 2, 4, 6-trioxo-N-phenyl-1, 3-diazinane-5-
carboxamide
Method A (Pyridine):

Preparation of N-(4-fluorophenyl)-2, 4, 6-trioxo-1, 3-diazinane-5-carboxamide (8). A pyridine
(5 ml) solution of barbituric acid (128 mg; 1 mmol) and phenyl 4-fluorophenylcarbamate (231
mg; 1 mmol) was refluxed overnight (~17 hours). Pyridine was evaporated, resulting in a solid
residue which was then mixed with a solution of water (10 ml) and concentrated hydrochloric
acid (5 ml) before being sonicated at 60°C for 30 minutes. The insoluble white crystalline
material was isolated via vacuum filtration, washed with water (3x5 ml), and dried at 110⁰C.
The crude white solid material was mixed with ethanol (15 ml), refluxed for 30 minutes and left
at room temperature for 2 hours. The purified material was then separated by vacuum filtration
and washed with cold ethanol to give the pure product (240 mg; 90%). 1H-NMR (DMSO-d6
Varian Inova 600 MHz) δ 12.06 (1H, broad s), 11.43 (1H, s), 11.35 (1H, broad s), 7.53 (2H, m,
J1 = 8.4 Hz, J2 = 4.8 Hz), and 7.21 (2H, t, J = 8.4 Hz) ppm. EMS- (CH3OH) m/z 264.5 (M-1),
363.5 (M-2), EMS MS2 (265) 265.1, 178.2, 153.0, and 134.8.
Method B (TEA/DMSO):
Preparation of N-(3, 4-dichlorophenyl)-2, 4, 6-trioxo-1, 3-diazinane-5-carboxamide (13). A
dimethyl sulfoxide (3 ml) and trimethylamine (3ml) mixture of barbituric acid (422 mg; 3.3 mmol)
and phenyl 3, 4-dichlorophenylcarbamate (840 mg; 3 mmol) was refluxed with stirring for 3
hours. The trimethylamine was evaporated at reduced pressure and the colorless reaction
mixture mixed with 5% hydrochloric acid (15 ml). The suspension which formed was cooled in
an ice bath to yield the crude product which was then isolated by vacuum filtration, washed with
water (5x5 ml) and dried at 110⁰C. The crude material was purified by sonication with refluxing
in ethanol (20 ml) for one hour. The insoluble white solid was isolated by vacuum filtration to
give the pure product (870 mg; 92%). 1H-NMR (DMSO-d6, Varian Inova 500 MHz) δ 11.77 (2H,
broad s), 11.52 (1H, s), 7.90 (1H, d, J = 1.5 Hz), 7.59 (1H, d, J = 7 Hz), and 7.45 (1H, d of d, J1
= 7 Hz, J2 = 1.5 Hz) ppm. EMS-(CH3OH) m/z 315 (M-1).

Method C (NMM/DMSO):
Preparation of 4-(2, 4, 6-trioxo-1, 3-diazinane-5-amido) benzoic acid (18). An N-
methylmorpholine (10 ml) - DMSO (5ml) mixture of barbituric acid (128 mg; 1 mmol) and 4-
(phenoxycarbonylamino) benzoic acid (257 mg; 1 mmol) was refluxed for 5 hours. The volume
of the reaction mixture was reduced by 2/3, mixed with 5% hydrochloric acid (20 ml) and left at
room temperature with stirring for several hours. The formed insoluble white material was
separated by vacuum filtration, washed with water (5x10 ml) and further purified by refluxing in
80% ethanol (15 ml) with a few drops of concentrated hydrochloric acid. The ethanolic
suspension was cooled in an ice bath and the precipitate isolated by vacuum filtration, washing
1
with water (3x10ml) and then dried at 110°C for 30 minutes(260 mg; 89%). H-NMR (DMSO-d6,
Varian Inova 500 MHz) δ 12.84 (1H, s), 11.71 (1H, s), 11.70 (1H, broad s), 7.91 (2H, d, J = 7.0
Hz), and 7.73 (2H, d, J = 7.0 Hz) ppm. EMS-(CH3OH) m/z 290 (M-1). MS2 (291) m/z 291, 204,
153, 152, 136, 126, and 66.
Method D (Na2CO3/DMF):
Preparation of N-{[(4-hydroxy-3-nitrophenyl) carbamoyl] amino}-2, 4, 6-trioxo-1, 3-diazinane-
5-carboxamide (109). A mixture of sodium bicarbonate (130 mg; 1.2 mmol), N,N-
dimethylformamide (3 ml) and barbituric acid (256 mg 2.2 mmol) was sonicated for 1 hour at
60ºC. Into this solution the desired phenyl carbamate 109b CF (603 mg; 2.2 mmol) was added.
The reaction mixture was sonicated at 100ºC for 2 hours followed by heating at 130ºC for 2
additional hours. The reaction mixture was mixed with 5% hydrochloric acid (30 ml) and left to
stir at room temperature for 1 hour. The formed precipitate was isolated via vacuum filtration,
washed with water (3x30 ml), refluxed in ethanol (15 ml) for 15 minutes, and then filtered to give
the pure product (89%). 1H-NMR (DMSO-d6, Agilent 400 MHz) δ 11.15 (3H, broad s), 10.59 (1H,
s), 9.42 (1H, broad s), 9.30 (1H, s), 7.87 (1H, d, J = 8 Hz), 7.36 (1H, d, J = 4 Hz), and 7.08 (1H,
d of d, J1 =8 Hz, J2 = 4H) ppm. EMS- (CH3OH) m/z 365 (M-1); M2 (366) m/z 366, 185 and 186.

Biological Evaluation: LN-229 cells (obtained from ATCC) were used for all in vitro cell death
assays. Dilutions of each drug were made to 100 µg/ml, 10 µg/ml or 1 µg/ml in a maximum of
1% DMSO. 1% DMSO was used in all control wells. LN-229 cells were plated in 24-well plates
until a confluent monolayer of cells was present (10% DMEM at 5% CO2 and 37°C). The media
was removed and replaced with DMEM containing antibiotic/antimycotic but no serum and drug
dilutions added to each well. The cells were incubated for 24 hours and then assayed by light
microscopy, where 100% cell death was used to determine the efficacy of each compound. To
confirm the disruption of cells, the plates were fixed in 10% formalin for 30 minutes (following
the removal of media) and then stained with 0.01% crystal violet in sterile water.32
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