Synthesis and biological activity of substituted 2,4-diaminopyrimidines that inhibit Bacillus anthracis

Article abstract of DOI:10.1016/j.ejmech.2012.05.018

A series of substituted 2,4-diaminopyrimidines 1 has been prepared and evaluated for activity against Bacillus anthracis using previously reported (±)-3-{5-[(2,4-diamino-5-pyrimidinyl)methyl]-2,3-dimethoxyphenyl} -1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-one (1a), with a minimum inhibitory concentration (MIC) value of 1-3 μg/mL, as the standard. In the current work, the corresponding isobutenyl (1e) and phenyl (1h) derivatives displayed the most significant activity in terms of the lowest MICs with values of 0.5 μg/mL and 0.375-1.5 μg/mL, respectively. It is likely that the S isomers of 1 will bind the substrate-binding pocket of dihydrofolate reductase (DHFR) as in B. anthracis was found for (S)-1a. The final step in the convergent synthesis of target systems 1 from (±)-1-(1-substituted-2(1H)- phthalazinyl)-2-propen-1-ones 6 with 2,4-diamino-5-(5-iodo-3,4-dimethoxybenzyl) pyrimidine (13) was accomplished via a novel Heck coupling reaction under sealed-tube conditions.

Full text of DOI:10.1016/j.ejmech.2012.05.018

European Journal of Medicinal Chemistry 54 (2012) 387e396
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological activity of substituted 2,4-diaminopyrimidines that
inhibit Bacillus anthracis
,
a
b
b
Baskar Nammalwar a, Richard A. Bunce a , K. Darrell Berlin , Christina R. Bourne , Philip C. Bourne ,
*
Esther W. Barrow b, William W. Barrow b
a Department of Chemistry, Oklahoma State University, 107 Physical Sciences, Stillwater, OK 74078, USA
b Department of Veterinary Pathobiology, Oklahoma State University, 250 McElroy Hall, OK 74078, USA
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
< An improved synthesis of 2,4-
NH2
O
R
diaminopyrimidine-based
otics is reported.
antibi-
I
N
N
N
N
+
< Antibiotic synthesis includes a novel
sealed-tube Heck reaction.
< The agents synthesized show
H2N
H2N
N
OCH3
13 OCH3
(Ph3P)2PdCl2
N-ethylpiperidine
DMF, 140 oC
6
activity in the
m
g/mL range.
R
NH2
O
< The size and planarity of the
phthalazine C-1 substituent influ-
ences the activity.
< Steric and electronic factors affect
the binding of the agents to DHFR.
N
N
N
OCH3
OCH3
1
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of substituted 2,4-diaminopyrimidines 1 has been prepared and evaluated for activity against
Bacillus anthracis using previously reported (ꢀ)-3-{5-[(2,4-diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-one (1a), with a minimum inhibitory
Received 15 February 2012
Received in revised form
26 April 2012
Accepted 11 May 2012
Available online 22 May 2012
concentration (MIC) value of 1e3
butenyl (1e) and phenyl (1h) derivatives displayed the most significant activity in terms of the lowest
MICs with values of 0.5 g/mL and 0.375e1.5 g/mL, respectively. It is likely that the S isomers of 1 will
mg/mL, as the standard. In the current work, the corresponding iso-
m
m
bind the substrate-binding pocket of dihydrofolate reductase (DHFR) as in B. anthracis was found for (S)-
1a. The final step in the convergent synthesis of target systems 1 from (ꢀ)-1-(1-substituted-2(1H)-
phthalazinyl)-2-propen-1-ones 6 with 2,4-diamino-5-(5-iodo-3,4-dimethoxybenzyl)pyrimidine (13) was
accomplished via a novel Heck coupling reaction under sealed-tube conditions.
Keywords:
2,4-Diaminopyrimidine
Antibiotic
DHFR inhibitor
Heck coupling
Sealed-tube reaction
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
anthracis, although the minimum inhibitory concentration (MIC)
values were higher than those recorded in this work. Our
Interest has been mounting to develop agents to treat inhalation
anthrax, which is considered an important bioterror threat [1].
Studies by others [2] have revealed the inhibitory capabilities of
several substituted 2,4-diaminopyrimidines against Bacillus
compounds target dihydrofolate reductase (DHFR), a key enzyme in
the folate pathway [2e4]. It is well accepted that DHFR plays
a significant role in DNA synthesis [5,6] and is important in cancer
chemotherapy (methotrexate) [7,8], in antimalarial activity (pyri-
methamine) [9], and in antibacterial activity (trimethoprim)
[10,11]. Other DHFR inhibitors with antibacterial activity include
a related series of dihydrophthalazines [12e14] being pursued by
Basilea Pharmaceutica (Switzerland) as well as Iclaprim [15],
* Corresponding author. Tel.: þ1 405 744 5952; fax: þ1 405 744 6007.
E-mail address: rab@okstate.edu (R.A. Bunce).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.05.018

388
B. Nammalwar et al. / European Journal of Medicinal Chemistry 54 (2012) 387e396
AR-709 [16] under development at Evolva (Switzerland), and
a collection of 7-aryl-2,4-diaminoquinazolines [17] being evaluated
by Trius Therapeutics (San Diego, CA). DHFR inhibitors, also under
investigation at a research and development level, are represented
The requisite aryl iodide 13 for coupling with members of
was synthesized from and 11 (Scheme 2). Conjugate
6
9
addition of morpholine (7) to acrylonitrile (8) [26] generated
3-morpholinopropionitrile (9) [27] in 95% yield. O-Methylation of
5-iodovanillin (10) using potassium carbonate and dimethyl sulfate
in DMF [28] furnished dimethoxyaldehyde 11 (96%). Conversion of
9 and 11 to 13 was then carried out using a procedure improved
from the original synthetic approach [29] (Scheme 3). The
presumed intermediate enaminonitrile 12 was a dark brown oil,
which was treated at once with aniline hydrochloride in boiling
ethanol for 1 h. To the resulting crude, hot mixture was added
guanidine hydrochloride, followed by sodium methoxide, and
heating was continued for 3 h. Isolation and purification of the
product by crystallization afforded 13 (60%), which was character-
ized by 1H NMR and 13C NMR spectral analyses.
Coupling of each member of 6 with 13 was accomplished via
a sealed-tube Heck reaction, promoted by bis(triphenylphosphine)
palladium(II) chloride and N-ethylpiperidine in anhydrous DMF at
140 ꢂC under argon, to afford crude oils containing the desired
product (Scheme 4). Flash chromatography, using CH2Cl2 and
CH3OH (96:4), followed by one recrystallization, then gave racemic
targets 1. Isolated yields ranged from 25 to 71%. Samples of
(ꢀ)-1aeq were characterized by spectral and elemental analyses. A
search of the literature revealed only one previous report of Heck
reactions performed using sealed-tube conditions [30].
by work on
u-(carboxyalkoxy)benzylpyrimidines targeted to
Pneumocystis carinii [18] as well as a number of broad-spectrum
propargyl-based compounds [19]. The common inclusion of
a pyrimidine nucleus in the majority of DHFR inhibitors examined
to date supports the hypothesis that this ring is essential
[2,3,20e22].
The basic structural motif, incorporating a pyrimidine and
a phthalazine ring, led to the preparation of racemic 1a (R ¼ n-
propyl) [3], which was separated into its enantiomers [23].
Complexation of racemic 1a with DHFR in B. anthracis resulted in
co-crystallization where only the S isomer appeared deep in the
active site as determined by X-ray analysis [3]. Additionally, both
R
NH2
O
N
N
N
N
H2N
N
OCH3
OCH3
OCH3
The hydrophilic nature of
1 was clearly revealed by its
1
Pr
NH2
O
propensity to retain water and/or polar solvents, such as methanol,
in the crystal [31]. All elemental analyses of members of 1 con-
tained varying amounts of water and, in the cases of 1n and 1o,
traces of methanol. Exhaustive attempts to remove these polar
contaminants, via extensive drying techniques, were unsuccessful.
Reaction conditions to prepare 6aeq, as well as (ꢀ)-1aeq, varied
for each compound, with the optimum methodology given in the
Experimental section.
N
N
H2N
N
OCH3
(R)-1a (α-propyl)
(S)-1a (β-propyl)
3. Biological activity
Alterations of “R” in 1 serve to modulate interactions at the
interface of the substrate-binding site and the solvent (Fig. 1) [3].
These observations correlate with the measured MIC values, where
shorter and more hydrophobic substitutions [3] exhibit lower MIC
values (Table 1). The protein surfaces complexed by (S)-1a have not
typically been targeted by therapeutics since (S)-1a protrudes
further along and extends to the distal end of the binding site. As
stated previously, the dihydrophthalazine moiety is critical to this
activity and promotes specificity for the binding sites of bacterial
DHFR over human DHFR [3,24]. Prior work [3] clearly demonstrated
that the contacts between the phthalazine moiety in the DHFR-(S)-
1a complex in B. anthracis were hydrophobic, which was found also
for the binding site in S. aureus [24]. Aliphatic portions of Lys33,
Thr36, Leu41, Leu55, Pro56, and Arg58 are located around the
dihydrophthalazine ring [2], with residues Leu29, Glu30, Lys33, and
Arg53 most closely approaching the R group.
(R)- and (S)-1a were potent in inhibiting the growth of Staphylo-
coccus aureus [24]. The focus of the present work was to determine
the antibacterial activity of racemic members of 1 via comparison
with (S)-1a with consideration of the docking orientation of this
enantiomer in B. anthracis DHFR [3]. As stated previously, correla-
tions have been drawn between antibacterial activity and DHFR
inhibition [12e14]. Thus, in the present work, the X-ray analysis on
DHFR-(S)-1a provided a molecular basis for the synthesis of addi-
tional derivatives of the target heterocycles and served as a guide
for the design of related structures with potential antibacterial
activity.
2. Chemistry
We report herein detailed preparations [22] for racemic
members of 1. Phthalazine (2) was treated with selected organo-
metallic reagents 3 in THF to yield dihydrophthalazines 4 as viscous
oils. Subsequent N-acylation of 4 by acryloyl chloride (5) in
dichloromethane in the presence of triethylamine furnished
members of 6, which were chromatographed and used immediately
in the final coupling step to give the desired targets 1. The prepa-
rations leading to 4 utilized Grignard reagents 3 with one exception.
The synthesis of 4c required a lithium-bromine exchange using
methyllithium in ether to convert 1,1,1-trifluoro-3-iodopropane to
the corresponding lithium reagent 3c, which was added to 2 in the
presence of boron trifluoride etherate at ꢁ78 ꢂC [25] (Scheme 1).
4. Results and discussion
Racemic mixtures 1a, 1b, 1c, 1d, and 1e exhibit generally
favorable MIC values (Table 1). Interestingly, 1h, 1l and 1m, which
incorporate large aryl rings, also possess low MIC values. The most
potent derivatives are 1e and 1h, while 1a is the standard for which
the co-crystal structure is available [3]. Smaller differences in MIC
ranges for 1a, 1b, 1c, 1d, 1j, and 1m may be due, in part, to flexibility
around R at C-1 of the dihydrophthalazine ring and/or the unoc-
cupied space around R can accommodate group differences. In
contrast, the maximum activity found for 1e (MIC ꢃ 0.5
mg/mL) may

B. Nammalwar et al. / European Journal of Medicinal Chemistry 54 (2012) 387e396
389
O
R
O
R
Cl
N
N
HN
N
N
N
THF
+
RMgX
Et3N, CH2Cl2, 0 oC
3
4
5
6
R
X
Entry
Time (h)
6
Temp (oC)
Yield (%)
a
b
c
d
e
C3H7
i-C3H7
CF3CH2CH2
i-C4H9
i-C4H7
1-ethylpropyl
c-C6H11
C6H5
2-CH3C6H4
4-CH3C6H4
3,5-(CH3)2C6H3
3-F-C6H4
4-F-C6H4
Br
65
0
−78
0
0
0
65
0
0
0
0
65
65
0
0
0
50
40
65
50
40
55
38
47
45
48
52
38
42
55
50
62
30
Cl
5
a
b
Cl
Br
Br
Cl
Br
Cl
Br
Br
Br
Br
Cl
Cl
Cl
Br
5
2
5
3
6
6
6
6
1
1
3
3
2
8
f
g
h
i
j
k
l
m
n
o
p
q
C6H5CH2
4-CH3C6H4CH2
4-CH3OC6H4CH2
4-CF3OC6H4CH2
0
aThe Li compound was prepared by lithium-bromide exchange at −78 oC.
bThe Li compound was reacted for 30 min in the presence of BF3·Et2O at −78 oC.
Scheme 1.
reflect the restricted motion in the isobutenyl group. Larger groups,
such as in 1p, are surmised to protrude into solvent space exterior
to the binding site leading to elevated MICs. A similar explanation
can be applied to 1f, 1i, 1k, 1n, 1o, 1p, and 1q where possible critical
steric factors are unfavorable to allow useful activity. The new data
suggest that the double bond of the isobutenyl group in 1e alters its
conformation considerably [3], possibly by centering itself more
evenly between residues Leu29, Gln30, Lys33, and Arg53, with
enhanced activity being the result. Thus, the overall shape and
flexibility (or lack thereof) of the substitution also clearly have an
effect, with 1e displaying the most favorable MIC value regarding
in vitro activity in this assay.
eliciting activity since hydrogen at this position results in loss of
potency [4]. The effect appears to be very subtle since agents 1h
(R ¼ phenyl) and 1j (R ¼ p-tolyl) possess large aryl groups but still
retain reasonably good MICs. Additionally, interchanging hydrogen
and fluorine, as in comparing 1h with 1l and 1m, did not signifi-
cantly improve potency, nor did replacing a fluorine atom with
a methyl group (1m versus 1j). Finally, benzyl substitution, as in 1n,
1o, 1p, and 1q, generally resulted in higher MICs. Thus, refinement
of ligand structures to induce greater activity must take into
account steric considerations and quite possibly electronic differ-
ences in view of the binding site properties [3] of DHFR in
B. anthracis. Since selectivity of (S)-1a for B. anthracis over human
DHFR has been confirmed [3,24], continued investigations of
structureeactivity relationships for inhibiting the bacteria are
warranted.
In summary, 16 substituted 2,4-diaminopyrimidines have been
synthesized using a novel sealed-tube Heck reaction and were
evaluated for activity against B. anthracis, with the previously
described (ꢀ)-(E)-3-{5-[(2,4-diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-
one (1a) serving as the reference [3]. The preparative method may
well have broad scope with promising utility in terms of simplicity
of operation and ease of purifying the products. Agents 1e
(R ¼ isobutenyl) and 1h (R ¼ phenyl) exhibited the best activity
Fig.
1 displays the protein:solvent surface arrangements
involving the complex of B. anthracis with (S)-1a in which the
n-propyl group is somewhat exposed to solvent [3]. This posi-
tioning of the R group is undoubtedly an important factor in
CN
50 oC
N
NH
+
CN
O
O
8
9
7
OHC
I
OHC
I
(CH3O)2SO2
K2CO3/DMF
120 oC
OH
OCH3
(Ph3P)2PdCl2
OCH3
OCH3
6 + 13
1
N-ethylpiperidine, DMF/140 oC
10
11
Scheme 2.
Scheme 4.

390
B. Nammalwar et al. / European Journal of Medicinal Chemistry 54 (2012) 387e396
plates (Analtech No. 21521). Preparative separations were per-
NC
N
I
NaOEt
DMSO/72 oC
PhNH2·HCl
EtOH/78 oC
formed by column chromatography in quartz columns using silica
gel (DavisilÒ grade 62, 60e200 mesh) mixed with UV-active
phosphor (Sorbent Technologies, No. UV-05). Band elution, for all
chromatographic separations, was monitored using a hand-held UV
lamp. Melting points were uncorrected. FT-IR spectra were run as
thin films on NaCl disks. Unless otherwise indicated, 1H and 13C
NMR spectra were measured in CDCl3 on a Varian GEMINI 300
instrument at 300 MHz (1H) and 75 MHz (13C) or a Varian INOVA
400 instrument at 400 MHz (1H) and 100 MHz (13C) and referenced
to internal tetramethylsilane. Elemental analyses were performed
by Atlantic Microlab, Inc.
9 + 11
OCH3
O
OCH3
12
NH2
I
(H2N)2C=NH·HCl
N
NaOMe/EtOH/78 oC
H2N
N
OCH3
OCH3
13
Scheme 3.
5.1.2. Method A. (ꢀ)-1-(1-Propyl-2(1H)-phthalazinyl)-2-propen-1-
one (6a)
with MICs of ꢃ0.5
m
g/mL and 0.375e1.5 mg/mL, respectively. Other
The method of Guerry et al. [22] was modified. Propylmagne-
sium bromide (3a) was generated on a 19.3-mmol scale from 2.38 g
(1.66 mL, 19.3 mmol) of 1-bromopropane and 0.65 g (25.7 mmol) of
magnesium metal in 15 mL of THF. To the resulting Grignard
reagent at room temperature was added dropwise to a stirred
solution of 2.00 g (15.4 mmol) of phthalazine (2) in 8 mL of THF, and
the reaction was gently refluxed for 6 h. The mixture was cooled to
room temperature, and 10 mL of saturated NH4Cl was cautiously
added. The crude mixture was transferred to a separatory funnel
containing 40 mL of additional NH4Cl solution and was extracted
with diethyl ether (3 ꢄ 50 mL). The combined organic extracts were
washed with saturated NaCl, dried (MgSO4), and concentrated
under vacuum for 30 min to give 2.43 g of dihydrophthalazine 4a as
a viscous, brown oil, which was used immediately in the next
reaction.
racemic compounds with activity comparable to that of 1a
(1e3 g/mL) were 1b, 1c, 1d, 1j, and 1m, which showed MICs in the
1e2 g/mL range. It is speculated that the S isomer of each of these
m
m
systems might possess even lower MICs. Interestingly, each
enantiomer of 1a demonstrates distinct binding characteristics
with S. aureus DHFR [24], with the S isomer situating in the normal
binding pocket while the R isomer resides in a shallow surface
cavity. Whether or not the other agents will express similar anti-
bacterial activity and show behavior analogous to (S)-1a with
a specific organism requires additional analysis.
5. Experimental section
5.1. Chemistry
The crude 4a was dissolved in 20 mL of dichloromethane at 0 ꢂC
and 1.82 g (2.50 mL, 18.0 mmol) of triethylamine was added. A
solution of 1.40 g (1.26 mL, 15.5 mmol) of acryloyl chloride (5) in
10 mL of dichloromethane was then added dropwise over 20 min.
The new solution was stirred for 2 h at 0 ꢂC and was then poured
into ice water and extracted with dichloromethane (3 ꢄ 50 mL). The
combined organic extracts were washed with saturated NaCl, dried
(MgSO4), and concentrated under vacuum. The resulting brown oil
was chromatographed on a 50-cm ꢄ 2.5-cm silica gel column
eluted with 5e10% ethyl acetate in hexanes to give 1.75 g (50%) of
6a as a viscous, brown oil. IR: 1664, 1616 cmꢁ1; 1H NMR (300 MHz):
5.1.1. General methods
Commercial anhydrous N,N-dimethylformamide (DMF) and
dimethyl sulfoxide (DMSO) were stored under dry nitrogen and
transferred by syringe into reactions where required. Tetrahydro-
furan (THF) was dried over KOH pellets and distilled from LiAlH4
prior to use. All other commercial reagents were used as received
from various vendors. Commercial Grignard reagents used were
purchased from Aldrich Chemical Co.
Unless otherwise specified, all reactions were run under dry
nitrogen in oven-dried glassware. The saturated NH4Cl and NaCl
used in work-up procedures were aqueous solutions. Reactions
were monitored by thin layer chromatography on silica gel GF
d
7.62 (s, 1H), 7.43 (td, 1H, J ¼ 7.5, 1.5 Hz), 7.35 (td, 1H, J ¼ 7.5, 1.5 Hz),
7.32 (dd, 1H, J ¼ 17.2, 10.4 Hz), 7.27 (d, 1H, J ¼ 7.5 Hz), 7.15 (d, 1H,
J ¼ 7.5 Hz), 6.47 (dd, 1H, J ¼ 17.2, 2.2 Hz), 5.84 (t, 1H, J ¼ 6.7 Hz), 5.77
(dd, 1H, J ¼ 10.4, 2.2 Hz), 1.62 (m, 2H), 1.25 (m, 2H), 0.85 (t, 3H,
Table 1
MIC values for racemic members of 1.
Compound
R
MIC (mg/mL)
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
C3H7
i-C3H7
CF3CH2CH2
i-C4H9
i-C4H7
1-ethylpropyl
c-C6H11
C6H5
2-CH3C6H4
4-CH3C6H4
3,5-(CH3)2C6H3
3-FeC6H4
4-FeC6H4
C6H5CH2
4-CH3C6H4CH2
4-CH3OC6H4CH2
4-CF3OC6H4CH2
1e3
1e2
1e2
1e2
ꢃ0.5
4e8
1e4
0.375e1.5
8
1e2
4e8
2
1e2
8
4e8
8e16
4
Fig. 1. The surface of the B. anthracis DHFR binding site is depicted in gray and cut into
the plane of the page to permit visualization (the backbone of the protein is displayed
in blue). Coordinates were taken from the co-crystal structure [3], which resulted from
complexation with (S)-1a (yellow). (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)

B. Nammalwar et al. / European Journal of Medicinal Chemistry 54 (2012) 387e396
391
J ¼ 7.3 Hz); 13C NMR (75 MHz):
d
166.2, 142.4, 134.1, 131.4, 128.3,
5.1.6. (ꢀ)-1-(1-isobutenyl-2(1H)-phthalazinyl)-2-propen-1-one (6e)
127.9, 127.1, 126.4, 125.6, 123.8, 51.1, 37.2, 18.2, 13.7.
This compound was prepared and purified on the same scale
using Method B (0 ꢂC, 2 h) to give 1.47 g (40%) of 6e as a viscous,
5.1.3. Method B. (ꢀ)-1-(1-Isopropyl-2(1H)-phthalazinyl)-2-
propen-1-one (6b)
brown oil. IR: 1665, 1622, 1613 cmꢁ1 1H NMR (400 MHz):
; d 7.58
(s, 1H), 7.43 (td, 1H, J ¼ 7.4, 1.4 Hz), 7.33 (td, 1H, J ¼ 7.4, 1.1 Hz), 7.30
(dd, 1 H, J ¼ 17.2, 10.5 Hz), 7.27 (dm, 1H, J ¼ 7.4 Hz), 7.14 (d, 1H,
J ¼ 7.5 Hz), 6.51 (d, 1H, J ¼ 10.0 Hz), 6.46 (dd, 1H, J ¼ 17.2, 2.1 Hz),
5.76 (dd, 1H, J ¼ 10.5, 2.1 Hz), 5.27 (dt, 1H, J ¼ 10.0, 1.2 Hz), 2.02
(d, 3H, J ¼ 1.2 Hz), 1.64 (d, 3H, J ¼ 1.2 Hz); 13C NMR (75 MHz):
A solution of 2.00 g (15.0 mmol) of 2 in 20 mL of dry THF was
cooled to 0 ꢂC, and 8.0 mL of 2.0 M isopropylmagnesium chloride
(3b) (16.0 mmol) in ether was slowly added dropwise over 20 min.
The reaction was allowed to stir at 0 ꢂC for 5 h and was then
quenched with 50 mL of saturated NH4Cl and extracted with ethyl
acetate (3 ꢄ 50 mL). The combined organic extracts were washed
with saturated NaCl, dried (MgSO4), and concentrated under
vacuum to give dihydrophthalazine 4b as a viscous, brown oil. This
oil was dried under high vacuum for 30 min and was used without
further purification.
d
165.9, 141.4, 134.3, 134.0, 131.7, 128.0, 127.7, 127.2, 126.0, 125.6,
123.0, 121.9, 49.7, 25.5, 18.4.
5.1.7. (ꢀ)-1-[1-(1-Ethylpropyl)-2(1H)-phthalazinyl]-2-propen-1-
one (6f)
This compound was prepared and purified on the same scale
The crude 4b in dichloromethane at 0 ꢂC was treated, as above,
with 1.82 g (2.50 mL, 18.0 mmol) of triethylamine and 1.40 g
(1.26 mL, 15.5 mmol) of 5, and the reaction was stirred for 2 h.
Work-up and chromatography gave 1.83 g (40%) of 6b as a viscous,
using Method B (0 ꢂC, 5 h) to give 2.16 g (55%) of 6f as a viscous,
brown oil. IR: 1664, 1611 cmꢁ1; 1H NMR (400 MHz):
d 7.65 (s, 1H),
7.44 (td, 1H, J ¼ 7.4, 1.5 Hz), 7.36 (td, 1H, J ¼ 7.4, 1.5 Hz), 7.32 (dd, 1H,
J ¼ 17.2, 10.3 Hz), 7.28 (d, 1H, J ¼ 7.2 Hz), 7.18 (d, 1H, J ¼ 7.5 Hz), 6.46
(dd, 1H, J ¼ 17.2, 2.1 Hz), 5.88 (d, 1H, J ¼ 7.0 Hz), 5.77 (dd, 1H,
J ¼ 10.3, 2.1 Hz), 1.56 (m, 1H), 1.42e1.24 (complex m, 3H), 1.10
(m, 1H), 0.96 (t, 3H, J ¼ 7.4 Hz), 0.80 (t, 3H, J ¼ 7.1 Hz); 13C NMR
brown oil. IR: 1664, 1615 cmꢁ1; 1H NMR (300 MHz):
d 7.63 (s, 1H),
7.45 (td, 1H, J ¼ 7.7, 1.6 Hz), 7.37 (t, 1H, J ¼ 7.7 Hz), 7.34 (dd, 1H,
J ¼ 17.0, 10.4 Hz), 7.28 (d, 1H, J ¼ 7.7 Hz), 7.17 (d, 1H, J ¼ 7.7 Hz), 6.46
(dd, 1H, J ¼ 17.0, 2.2 Hz), 5.78 (dd, 1H, J ¼ 10.4, 2.2 Hz), 5.67 (d, 1H,
J ¼ 7.1 Hz), 1.99 (septet, 1H, J ¼ 6.8 Hz), 0.92 (d, 3H, J ¼ 6.6 Hz), 0.77
(100 MHz):
d 166.3, 143.6, 132.7, 131.2, 128.3, 127.9, 127.21, 127.20,
125.5, 124.5, 52.7, 45.4, 21.0, 20.6, 11.1, 10.5.
(d, 3H, J ¼ 6.6 Hz); 13C NMR (75 MHz):
d 166.5, 143.2, 132.0, 131.1,
128.3, 128.0, 127.5, 127.2, 125.4, 124.6, 56.3, 33.4, 19.1, 18.0.
5.1.8. (ꢀ)-1-(1-Cyclohexyl-2(1H)-phthalazinyl)-2-propen-1-one (6g)
This compound was prepared and purified on the same scale
using Method A (65 ꢂC, 3 h) to give 1.56 g (38%) of 6g as a viscous,
5.1.4. Method C. (ꢀ)-1-[1-(3,3,3-Trifluoropropyl)-2(1H)-
phthalazinyl]-2-propen-1-one (6c)
brown oil. IR: 1663, 1614 cmꢁ1; 1H NMR (400 MHz):
d 7.64 (s, 1H),
The procedure of Uno et al. [25] was modified. A solution of
2.00 g (15.0 mmol) of 2 and 3.60 mL (30.0 mmol) of 1,1,1 trifluoro-3-
iodopropane in 20 mL of ether was cooled to ꢁ78 ꢂC, and 3.90 mL
(30.0 mmol) of boron trifluoride etherate was added dropwise over
10 min. The solution was stirred for 5 min before 19.2 mL of 1.6 M
methyllithium in ether (30.0 mmol) was added dropwise. The
reaction mixture was stirred at ꢁ78 ꢂC for 30 min and was then
poured into 50 mL of saturated NH4Cl and extracted with ether
(3 ꢄ 50 mL). The combined ether layers were washed with satu-
rated NaCl, dried (MgSO4), and concentrated under vacuum to give
dihydrophthalazine 4c as a viscous, yellow oil. This oil was dried
under high vacuum for 30 min and was used without further
purification.
7.44 (td, 1H, J ¼ 7.4, 1.1 Hz) 7.37 (td, 1H, J ¼ 7.4, 1.1 Hz), 7.33 (dd, 1H,
J ¼ 17.2, 10.5 Hz), 7.29 (dd, 1H, J ¼ 7.4, 0.8 Hz), 7.15 (d, 1H, J ¼ 7.4 Hz),
6.46 (dd, 1H, J ¼ 17.2, 2.1 Hz), 5.78 (dd, 1H, J ¼ 10.5, 2.1 Hz), 5.67
(d, 1H, J ¼ 7.2 Hz), 1.80e1.50 (complex m, 6H), 1.20e0.97 (complex
m, 3H), 0.87 (m, 2H); 13C NMR (75 MHz):
d 166.5, 143.6, 132.3, 131.0,
128.4, 128.0, 127.6, 127.1, 125.4, 124.5, 55.8, 42.7, 29.2, 28.5, 26.01,
25.98, 25.9.
5.1.9. (ꢀ)-1-(1-Phenyl-2(1H)-phthalazinyl)-2-propen-1-one (6h)
This compound was prepared and purified on the same scale
using Method B (0 ꢂC, 6 h) to give 1.90 g (47%) of 6h as a viscous,
brown oil. IR: 1664, 1615 cmꢁ1; 1H NMR (400 MHz):
d 7.65 (s, 1H),
7.44 (td, 1H, J ¼ 7.4, 1.6 Hz), 7.38 (td, 1H, J ¼ 7.4, 1.1 Hz), 7.32 (dd, 1H,
J ¼ 17.2, 10.5 Hz), 7.32 (d, 1H, J ¼ 7.4 Hz), 7.27 (d, 1H, J ¼ 7.4 Hz),
7.25e7.28 (complex m, 5H), 6.98 (s, 1 H), 6.48 (dd, 1H, J ¼ 17.2,
The crude 4c in dichloromethane at 0 ꢂC was treated, as above,
with 1.82 g (2.50 mL, 18.0 mmol) of triethylamine and 1.34 g
(1.20 mL, 15.0 mmol) of 5, and the reaction mixture was stirred for
2 h. Work-up and chromatography gave 2.82 g (65%) of 6c as
2.1 Hz), 5.79 (dd, 1H, J ¼ 10.5, 2.1 Hz); 13C NMR (75 MHz):
d 166.4,
141.7,141.0, 132.9, 132.0, 128.8, 128.5, 128.3, 127.7,127.2,127.0, 126.9,
125.9, 123.5, 54.2.
a yellow liquid. IR: 1666, 1622, 1325 cmꢁ1 1H NMR (400 MHz):
;
d
7.67 (s, 1H), 7.50 (td, 1H, J ¼ 7.4, 1.4 Hz), 7.41 (t, 1H, J ¼ 7.4 Hz), 7.33
(dd, 1H, J ¼ 7.4, 1.4 Hz), 7.30 (dd, 1H, J ¼ 17.2, 10.5 Hz), 7.19 (dd, 1H,
J ¼ 7.4, 0.4 Hz), 6.50 (dd, 1H, J ¼ 17.2, 2.1 Hz), 5.96 (t, 1H, J ¼ 6.6 Hz),
5.83 (dd, 1H, J ¼ 10.5, 2.1 Hz), 2.18e1.98 (complex m, 2H), 1.90
5.1.10. (ꢀ)-1-[1-(2-Methylphenyl)-2(1H)-phthalazinyl]-2-propen-
1-one (6i)
This compound was prepared and purified on the same scale
(m, 2H); 13C NMR (100 MHz):
d 166.4, 142.6, 132.6, 132.1, 129.1,
using Method B (0 ꢂC, 6 h) to give 1.90 g (45%) of 6i as a viscous,
128.7, 126.7, 126.7 (q, JCeF ¼ 276.3 Hz), 126.3, 126.0, 123.7, 49.8, 30.0
brown oil. IR: 1666, 1613 cmꢁ1; 1H NMR (400 MHz):
d 7.58 (s, 1H),
(q, JCeF ¼ 29.2 Hz), 27.8 (q, JCeF ¼ 2.6 Hz).
7.35e7.26 (complex m, 3H), 7.30 (dd, 1H, J ¼ 17.2, 10.5 Hz), 7.19 (dd,
1H, J ¼ 7.4, 1.6 Hz), 7.15e7.00 (complex m, 4H), 6.96 (s, 1H), 6.39 (dd,
1H, J ¼ 17.2, 2.1 Hz), 5.72 (dd, 1H, J ¼ 10.5, 2.0 Hz), 2.74 (s, 3H); 13C
5.1.5. (ꢀ)-1-(1-Isobutyl-2(1H)-phthalazinyl)-2-propen-1-one (6d)
This compound was prepared and purified on the same scale
using Method B (0 ꢂC, 5 h) to give 1.83 g (50%) of 6d as a viscous,
NMR (100 MHz):
d 166.4, 142.5, 139.8, 134.4, 133.9, 132.1, 130.6,
128.4, 128.0, 127.6, 127.4, 127.3, 126.7, 126.5, 126.4, 122.2, 52.1, 20.0.
brown oil. IR: 1663, 1616 cmꢁ1; 1H NMR (300 MHz):
d 7.68 (s, 1H),
7.44 (td, 1H, J ¼ 7.7, 1.6 Hz), 7.36 (td, 1H, J ¼ 7.1, 1.1 Hz), 7.30 (dd, 1H),
7.29 (d, 1H, J ¼ 6.6 Hz), 7.17 (d, 1H, J ¼ 7.1 Hz), 6.47 (dd, 1H, J ¼ 17.0,
2.2 Hz), 5.92 (t, 1H, J ¼ 7.1 Hz), 5.68 (dd, 1H, J ¼ 10.4, 2.2 Hz),
1.58e1.36 (complex m, 3H), 0.98 (d, 3H, J ¼ 6.0 Hz), 0.92 (d, 3H,
5.1.11. (ꢀ)-1-[1-(4-Methylphenyl)-2(1H)-phthalazinyl]-2-propen-
1-one (6j)
This compound was prepared and purified on the same scale
using Method B (0 ꢂC, 6 h) to give 2.00 g (48%) of 6j as a viscous,
J ¼ 6.0 Hz); 13C NMR (75 MHz):
d
166.1, 143.0, 134.6, 131.4, 128.4,
brown oil. IR: 1665, 1615 cmꢁ1; 1H NMR (300 MHz):
d
7.64 (s, 1H),
128.0, 127.1, 126.2, 125.8, 123.8, 49.5, 43.7, 23.8, 23.1, 22.2.
7.43 (td, 1H, J ¼ 7.1, 1.6 Hz), 7.39e7.27 (complex m, 3H), 7.24 (d, 1H,

392
B. Nammalwar et al. / European Journal of Medicinal Chemistry 54 (2012) 387e396
J ¼ 7.7 Hz), 7.10 (d, 2H, J ¼ 8.2 Hz), 7.03 (d, 2H, J ¼ 8.2 Hz), 6.94 (s,1H),
6.47 (dd, 1H, J ¼ 17.0, 1.6 Hz), 5.77 (dd, 1H, J ¼ 10.4, 1.6 Hz), 2.24
brown oil. IR: 1665, 1613 cmꢁ1; 1H NMR (400 MHz):
d 7.48 (s, 1H),
7.30 (dd, 1H, J ¼ 17.2, 10.3 Hz), 7.30e7.19 (complex m, 3H), 6.97
(d, 2H, J ¼ 7.6 Hz), 6.76 (d, 2H, J ¼ 7.6 Hz), 6.61 (d, 1H, J ¼ 7.4 Hz),
6.48 (dd, 1H, J ¼ 17.2, 2.1 Hz), 5.96 (dd, 1H, J ¼ 8.6, 4.7 Hz), 5.78 (dd,
1H, J ¼ 10.3, 2.1 Hz), 2.92 (dd, 1H, J ¼ 12.9, 4.7 Hz), 2.85 (dd, 1H,
(s, 3H); 13C NMR (75 MHz):
d 166.4, 141.7, 138.2, 137.5, 133.1, 132.0,
129.2, 128.7, 128.2, 127.14, 127.11, 126.9, 125.9, 123.5, 54.0, 21.0.
5.1.12. (ꢀ)-1-[1-(3,5-Dimethylphenyl)-2(1H)-phthalazinyl]-2-
propen-1-one (6k)
J ¼ 12.9, 8.6 Hz), 2.28 (s, 3H); 13C NMR (100 MHz):
d 166.2, 141.9,
136.0, 133.0, 132.6, 130.9, 129.7, 128.7, 128.4, 128.0, 127.1, 127.0,
125.5, 123.8, 53.1, 40.4, 21.0.
This compound was prepared and purified on the same scale
using Method B (0 ꢂC, 6 h) to give 2.32 g (52%) of 6k as a viscous,
brown oil. IR: 1665, 1610 cmꢁ1; 1H NMR (400 MHz):
d
7.65 (s, 1H),
5.1.17. (ꢀ)-1-[1-(4-Methoxybenzyl)-2(1H)-phthalazinyl]-2-
propen-1-one (6p)
7.42 (td, 1H, J ¼ 7.4, 1.6 Hz), 7.34 (td, 1H, J ¼ 7.4, 1.2 Hz), 7.34 (dd, 1H,
J ¼ 17.2, 10.3 Hz), 7.32 (dd, 1H, J ¼ 7.4, 1.6 Hz), 7.25 (d, 1H, J ¼ 7.4 Hz),
6.87 (s, 1H), 6.81 (s, 1H), 6.82 (s, 2H), 6.46 (dd, 1H, J ¼ 17.2, 2.1 Hz),
5.77 (dd, 1H, J ¼ 10.3, 2.1 Hz), 2.20 (s, 6H); 13C NMR (100 MHz):
This compound was prepared and purified on the same scale
using Method B (0 ꢂC, 2 h) to give 2.90 g (62%) of 6p as a viscous,
brown oil. IR: 2835, 1663, 1611 cmꢁ1 1H NMR (400 MHz):
; d 7.49
d
166.3, 141.7, 141.2, 138.1, 133.3, 132.0, 129.5, 128.7, 128.2, 127.15,
(s, 1H), 7.35e7.22 (complex m, 4H), 7.31 (dd, 1H, J ¼ 17.2, 10.3 Hz),
6.78 (d, 2H, J ¼ 8.6 Hz), 6.71 (d, 2H, J ¼ 8.6 Hz), 6.48 (dd, 1H, J ¼ 17.2,
2.1 Hz), 5.94 (dd, 1H, J ¼ 8.5, 4.8 Hz), 5.80 (dd, 1H, J ¼ 10.3, 2.1 Hz),
3.77 (s, 3H), 2.90 (dd, 1H, J ¼ 13.1, 4.8 Hz), 2.84 (dd, 1H, J ¼ 13.1,
127.13, 125.9, 124.5, 123.7, 54.3, 21.3.
5.1.13. (ꢀ)-1-[1-(3-Fluorophenyl)-2(1H)-phthalazinyl]-2-propen-
1-one (6l)
8.5 Hz); 13C NMR (100 MHz):
d 166.2, 158.4, 141.9, 132.7, 130.93,
A mixture of 2.80 g (16.0 mmol) of 1-bromo-3-fluorobenzene,
25 mL of THF and 0.49 g (19.0 mmol) of magnesium metal was
stirred vigorously at room temperature for 5 min and then refluxed
for 1.5 h to give a yellow solution. Heating was discontinued, and
a solution of 2.00 g (15.0 mmol) of 2 in 8 mL of THF was added
dropwise with stirring over a period of 10 min. The reaction was
then refluxed for 1 h and carried forward as in Method A (65 ꢂC,1 h)
130.89, 128.5, 128.2, 128.1, 127.14, 127.06, 125.5, 123.9, 113.5, 55.1,
53.2, 40.0.
5.1.18. (ꢀ)-1-[1-(4-Trifluoromethoxybenzyl)-2(1H)-phthalazinyl]-
2-propen-1-one (6q)
This compound was prepared and purified on the same scale
using Method B (0 ꢂC, 8 h) to give 1.66 g (30%) of 6q as a viscous,
to give 1.63 g (38%) of 6l as a viscous, brown oil. IR: 1666,1611 cmꢁ1
;
brown oil. IR: 1665, 1612 cmꢁ1; 1H NMR (400 MHz):
d 7.47 (s, 1H),
1H NMR (400 MHz):
d
7.66 (s, 1H), 7.45 (td, 1H, J ¼ 7.4, 1.6 Hz), 7.38
7.36e7.23 (complex m, 3H), 7.29 (dd, 1H, J ¼ 17.2, 10.5 Hz), 7.02
(d, 2H, J ¼ 8.4 Hz), 6.88 (d, 2H, J ¼ 8.4 Hz), 6.65 (d, 1H, J ¼ 7.4 Hz),
6.48 (dd, 1H, J ¼ 17.2, 1.6 Hz), 5.99 (apparent t, 1H, J ¼ 7.0 Hz),
5.80 (dd, 1H, J ¼ 10.5, 1.6 Hz), 2.98e2.86 (complex m, 2H); 13C
(td, 1H, J ¼ 7.4, 1.1 Hz), 7.33 (dd, 1H, J ¼ 7.4, 1.6 Hz), 7.32 (dd, 1H,
J ¼ 17.2, 10.5 Hz), 7.25 (dt, 1H, J ¼ 7.4, 0.6 Hz), 7.18 (m, 1H), 7.03
(dm, 1H, J ¼ 7.4 Hz), 6.96 (s, 1H), 6.89 (dd, 2H, J ¼ 9.2, 1.2 Hz), 6.49
(dd, 1H, J ¼ 17.2, 2.1 Hz), 5.80 (dd, 1H, J ¼ 10.5, 2.1 Hz); 13C NMR
NMR (100 MHz):
131.2, 131.1, 128.7, 128.3, 126.9, 126.8, 125.7, 123.9, 120.5, 120.4
d
166.2, 148.1 (q, JCeF ¼ 1.5 Hz), 141.7, 135.0, 132.1,
(100 MHz):
d
166.4, 162.7 (d, JCeF
¼
246.6 Hz), 143.3
(d, JCeF ¼ 5.9 Hz), 141.7, 132.3, 132.1, 130.1 (d, JCeF ¼ 8.1 Hz), 129.1,
128.6, 127.1, 126.8, 126.1, 123.4, 122.5 (d, JCeF ¼ 2.9 Hz), 114.7
(d, JCeF ¼ 21.4 Hz), 114.0 (d, JCeF ¼ 22.1 Hz), 53.7 (d, JCeF ¼ 1.5 Hz).
(q, JCeF ¼ 257.0 Hz), 52.8, 40.2.
5.1.19. 3-Morpholinopropionitrile (9)
This compound was prepared on a 0.47-mol scale according to
the literature procedures [26,27]. The crude product was distilled at
88e90 ꢂC/0.5 mm Hg (lit [27] bp 149 ꢂC/20 mm Hg) to give 38.2 g
(95%) of 9 as a colorless liquid. IR: 2253 cmꢁ1; 1H NMR (300 MHz):
5.1.14. (ꢀ)-1-[1-(4-Fluorophenyl)-2(1H)-phthalazinyl]-2-propen-
1-one (6m)
Using 4-fluorophenylmagnesium bromide, generated as above,
this compound was prepared and purified on the same scale using
Method A (65 ꢂC, 1 h) to give 1.80 g (42%) of 6m as a viscous, brown
d
3.72 (t, 4H, J ¼ 4.7 Hz), 2.68 (t, 2H, J ¼ 6.8 Hz), 2.52 (t, 2H,
J ¼ 7.0 Hz), 2.50 (t, 4H, J ¼ 4.7 Hz); 13C NMR (75 MHz):
d 118.6, 66.7,
oil. IR: 1666, 1604 cmꢁ1
;
1H NMR (400 MHz):
d
7.67 (s, 1H), 7.47
53.6, 53.0, 15.7.
(td, 1H, J ¼ 7.1, 1.6 Hz), 7.40 (td, 1H, J ¼ 7.4, 1.4 Hz), 7.36 (dd, 1H,
J ¼ 7.4, 1.4 Hz), 7.31 (dd, 1H, J ¼ 17.2, 10.5 Hz), 7.24 (d, 1H, J ¼ 7.4 Hz),
7.18 (m, 2H), 6.96 (s, 1H), 6.92 (m, 2H), 6.49 (dd, 1H, J ¼ 17.2, 1.6 Hz),
5.1.20. 5-Iodo-3,4-dimethoxybenzaldehyde (11)
This compound was prepared on a 0.27-mol scale using the
method of Nimgirawath [28]. The crude product was recrystallized
(4:1 ethanol:water) to give 25.2 g (96%) of 11 as a white solid, mp
5.78 (dd, 1H, J ¼ 10.5, 1.6 Hz); 13C NMR (75 MHz):
d 166.4, 162.1
(d, JCeF ¼ 246.8 Hz), 141.7, 136.9, 132.6, 132.0, 128.9, 128.8, 128.4,
127.1, 126.9, 126.0, 123.4, 115.3 (d, JCeF ¼ 21.5 Hz), 53.4.
71e72 ꢂC (lit [28] mp 71e72 ꢂC). IR: 2832, 2730, 2693 cmꢁ1 1H
;
NMR (300 MHz):
d
9.83 (s, 1H), 7.85 (d, 1H, J ¼ 1.7 Hz), 7.41 (d, 1H,
5.1.15. (ꢀ)-1-(1-Benzyl-2(1H)-phthalazinyl)-2-propen-1-one (6n)
This compound was prepared and purified on the same scale
using Method B (0 ꢂC, 3 h) to give 2.35 g (55%) of 6n as a viscous,
J ¼ 1.7 Hz), 3.93 (s, 3H), 3.92 (s, 3H); 13C NMR (75 MHz):
d 189.7,
154.2, 153.0, 134.7, 133.9, 111.0, 92.1, 60.7, 56.1.
brown oil. IR: 1663, 1612 cmꢁ1; 1H NMR (400 MHz):
d
7.49 (s, 1H),
5.1.21. 2,4-Diamino-5-(5-iodo-3,4-dimethoxybenzyl)pyrimidine (13)
The general method of Roth et al. [29] was modified. A solution
of 6.92 g (54.1 mmol) of 9 in 20 mL of anhydrous DMSO was treated
with 0.29 g (5.40 mmol) of sodium methoxide, and heated at
70e72 ꢂC. A warm solution of 12.2 g (41.8 mmol) of 11 in 15 mL of
DMSO was added to the solution of 9 over 15 min, and stirring was
continued at 72 ꢂC for 45 min. The crude reaction mixture was
cooled in an ice bath, and 40 mL of ice-cold water was added. The
mixture was extracted with dichloromethane (3 ꢄ 100 mL), and the
combined organic layers were washed with saturated NaCl, dried
(MgSO4), and concentrated under vacuum to give 3-morpholino-2-
(5-iodo-3,4-dimethoxybenzyl)acrylonitrile (12) as dark red oil. This
7.31 (dd,1H, J ¼ 17.2, 10.4 Hz), 7.34e7.21 (complex m, 3H), 7.20e7.14
(complex m, 3H), 6.88 (m, 2H), 6.60 (d, 1H, J ¼ 7.4 Hz), 6.48 (dd, 1H,
J ¼ 17.2, 2.1 Hz), 5.99 (dd, 1H, J ¼ 8.6, 4.9 Hz), 5.79 (dd, 1H, J ¼ 10.3,
2.1 Hz), 2.97 (dd, 1H, J ¼ 12.9, 4.9 Hz), 2.89 (dd, 1H, J ¼ 12.9, 8.6 Hz);
13C NMR (100 MHz):
d 166.2, 141.9, 136.1, 132.5, 130.9, 129.9, 128.5,
128.1, 128.0, 127.1, 127.0, 126.6, 125.5, 123.9, 53.1, 40.8.
5.1.16. (ꢀ)-1-[1-(4-Methylbenzyl)-2(1H)-phthalazinyl]-2-propen-
1-one (6o)
This compound was prepared and purified on the same scale
using Method B (0 ꢂC, 3 h) to give 2.25 g (50%) of 6o as a viscous,

B. Nammalwar et al. / European Journal of Medicinal Chemistry 54 (2012) 387e396
393
crude material was dissolved in dry ethanol, 6.76 g (52.2 mmol) of
aniline hydrochloride was added, and the mixture was refluxed for
1 h. While the mixture was still hot, 9.55 g (100 mmol) of guanidine
hydrochloride was added, followed by 9.00 g (167 mmol) of sodium
methoxide (Caution! The initial addition must be done very slowly).
The reaction mixture was then heated under reflux for 3 h and
concentrated under vacuum to one-third volume. The mixture was
cooled to 0 ꢂC for 30 min, and 40 mL of ice-cold water was added.
The resulting crude product was filtered, washed with water and
recrystallized twice (4:1 ethanol:water) to give 13 (9.68 g, 6ꢁ01%) as
152.4, 145.9, 143.6, 136.6 (2C), 131.4, 131.3, 128.3, 127.8, 127.5, 125.9,
124.3, 118.3, 117.9, 114.7, 105.8, 60.8, 55.7, 55.5, 32.9, 32.4, 18.9, 17.9.
Anal. Calcd for C27H30N6O3$1.5 H2O: C, 63.16; H, 6.43; N, 16.37.
Found: C, 63.14; H, 6.48; N, 16.36.
5.1.24. (ꢀ)-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-[1-(3,3,3-trifluoropropyl)-2(1H)-phthala
zinyl]-2-propen-1-one (1c)
This compound was prepared and purified on the same scale as
described above. Work-up, chromatography and recrystallization
(methanol) gave 790 mg (71%) of 1c as an off-white powder, mp
118e120 ꢂC. IR: 3467, 3341, 3186, 1649,1609 cmꢁ1; 1H NMR (DMSO-
a tan solid, mp 217e218 ꢂC. IR: 3467, 3315, 3140, 1638 cm
;
1H
NMR (DMSO-d6, 300 MHz):
6.98 (d, 1H, J ¼ 1.8 Hz), 6.16 (br s, 2H), 5.77 (br s, 2H), 3.77 (s, 3H),
3.66 (s, 3H), 3.54 (s, 2H); 13C NMR (DMSO-d6, 75 MHz):
162.4,
d
7.57 (s, 1H), 7.14 (d, 1H, J ¼ 1.8 Hz),
d6, 300 MHz):
d
8.00 (s, 1H), 7.89 (d, 1H, J ¼ 16.2 Hz), 7.63e7.53
d
(complex m, 3H), 7.58 (d, 1H, J ¼ 16.2 Hz), 7.50 (t, 2H, J ¼ 6.6 Hz),
7.26 (s, 1H), 7.02 (s, 1H), 6.36 (br s, 2H), 6.01 (t, 1H, J ¼ 6.6 Hz), 5.91
(br s, 2H), 3.80 (s, 3H), 3.75 (s, 3H), 3.60 (s, 2H), 2.18 (m, 2H), 1.79
162.1, 156.0, 152.0, 146.3, 138.9, 129.1, 113.8, 105.2, 92.4, 59.8, 55.8,
31.7.
(q, 2H, J ¼ 7.7 Hz); 13C NMR (DMSO-d6, 75 MHz):
d 165.9, 162.3,
5.1.22. (ꢀ)-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-
one (1a)
161.5,154.3, 152.5, 146.1,143.0,136.9,136.3, 132.2, 132.1,128.8,127.7,
127.0 (q, JCeF ¼ 274.0 Hz), 126.6, 126.4, 123.5, 118.4, 117.6, 114.9,
106.0, 60.8, 55.8, 49.1, 32.3, 29.0 (q, JCeF ¼ 28.3 Hz), 26.8. Anal. Calcd
for C27H27F3N6O3$1.5 H2O: C, 57.14; H, 5.29; N, 14.81. Found: C,
57.39; H, 4.98; N, 14.88.
A procedure of Guerry et al. [22] was significantly modified. A
solution of 800 mg (2.07 mmol) of 13, 472 mg (2.07 mmol) of 6a,
18 mg (0.026 mmol) of bis(triphenylphosphine)palladium(II)
dichloride [(Ph3P)2PdCl2] and 257 mg (0.30 mL, 2.27 mmol) of
N-ethylpiperidine in 4 mL of anhydrous DMF was added to a 15-mL
Pyrex pressure vessel (Chemglass CG-1880-01with O-ring CG-309-
210) equipped with a magnetic stirrer. The solution was purged
with argon for 2 min, and the tube was tightly closed. The reaction
was heated at 140 ꢂC for 18 h, during which time the mixture
turned a bright red color. After cooling, the crude reaction mixture
was transferred directly to a 30 cm ꢄ 2 cm silica gel column slurry
packed in dichloromethane. Impurities were eluted using
dichloromethane, and the final product was eluted using 4%
methanol in dichloromethane. Evaporation of the solvent gave
a pale yellow solid, which was rechromatographed using a 15-
cm ꢄ 2-cm silica gel column packed with 5% triethylamine in
dichloromethane and eluted with 4% methanol in dichloro-
methane. Recrystallization of the isolated material (methanol) gave
422 mg (42%) of 1a as a white powder, mp 121e124 ꢂC (shrinks to
5.1.25. (ꢀ)-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-(1-isobutyl-2(1H)-phthalazinyl)-2-propen-1-
one (1d)
This compound was prepared and purified on the same scale as
described above. Work-up, chromatography and recrystallization
(methanol) gave 670 mg (65%) of 1d as an off-whiteꢁp1owder, mp
127e129 ꢂC. IR: 3443, 3352, 3173, 1644, 1596 cm
;
1H NMR
(DMSO-d6, 300 MHz):
d
8.00 (s, 1H), 7.86 (d, 1H, J ¼ 16.5 Hz), 7.58
(d, 1H, J ¼ 16.5 Hz), 7.57 (s, 1H), 7.55 (overlapping d and t, 2H,
J z 7.7 Hz), 7.46 (t,1H, J ¼ 7.4 Hz), 7.38 (d,1H, J ¼ 7.1 Hz), 7.27 (s,1H),
7.02 (s, 1H), 6.87 (br s, 2H), 6.38 (br s, 2H), 5.89 (t, 1H, J ¼ 6.6 Hz),
3.80 (s, 3H), 3.74 (s, 3H), 3.62 (s, 2H), 1.38 (m, 3H), 0.93 (d, 3H,
J ¼ 4.9 Hz), 0.88 (d, 3H, J ¼ 4.9 Hz); 13C NMR (DMSO-d6, 75 MHz):
d
165.4, 162.8, 159.3, 152.5, 150.1, 146.1, 143.3, 136.6, 135.5, 134.0,
131.8, 128.3, 127.9, 126.2 (2C), 123.6, 118.6, 117.9, 114.9, 106.9, 60.8,
55.8, 48.8, 43.3, 32.1, 23.5, 22.9, 22.2. Anal. Calcd for
C28H32N6O3$1.5 H2O: C, 63.76; H, 6.64; N, 15.94. Found: C, 63.85; H,
6.44; N, 15.94.
a glass-like bead). IR: 3474, 3341, 3182, 1645, 1608 cmꢁ1
;
1H NMR
8.07 (d, 1H, J ¼ 16.1 Hz), 7.80 (s, 1H), 7.66
(DMSO-d6, 300 MHz):
d
(s, 1H), 7.64 (d, 1H, J ¼ 16.1 Hz), 7.45 (td, 1H, J ¼ 7.3, 1.5 Hz), 7.36 (td,
1H, J ¼ 7.5, 1.3 Hz), 7.28 (dd, 1H, J ¼ 7.5, 1.5 Hz), 7.18 (dd, 1H, J ¼ 7.3,
1.5 Hz), 7.13 (d, 1H, J ¼ 1.9 Hz), 6.66 (d, 1H, J ¼ 1.9 Hz), 5.91 (t, 1H,
J ¼ 6.8 Hz), 4.84 (br s, 2H), 4.66 (br s, 2H), 3.84 (s, 3H), 3.80 (s, 3H),
3.68 (s, 2H), 1.64 (m, 2H), 1.27 (m, 2H), 0.87 (t, 3H, J ¼ 7.1 Hz); 13C
5.1.26. (ꢀ)-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-(1-isobutenyl-2(1H)-phthalazinyl)-2-propen-
1-one (1e)
This compound was prepared and purified on the same scale as
described above. Work-up, chromatography and recrystallization
(methanol) gave 671 mg (65%) of 1e as an off-whiteꢁp1owder, mp
;
1H NMR
NMR (DMSO-d6, 75 MHz):
d 166.5, 162.6, 162.3, 156.8, 153.5, 147.3,
142.3,137.1, 134.2, 134.0, 131.4,129.7,128.0, 126.5,125.6,124.0,118.7,
118.6, 112.9, 106.3, 61.4, 55.9, 51.3, 37.3, 34.4, 18.3, 13.8. Anal. Calcd
for C27H30N6O3$2.0 H2O$0.5 CH3OH: C, 61.34; H, 6.69; N, 15.61.
Found: C, 61.56; H, 6.42; N, 15.81.
130e132 ꢂC. IR: 3474, 3339, 3172, 1642, 1603 cm
(DMSO-d6, 400 MHz):
d
7.91 (s, 1H), 7.86 (d, 1H, J ¼ 16.0 Hz), 7.59
(d, 1H, J ¼ 16.0 Hz), 7.57 (s, 1H), 7.52 (m, 2H), 7.42 (t, 1H, J ¼ 7.4 Hz),
7.31 (d, 1H, J ¼ 7.7 Hz), 7.25 (s, 1H), 7.01 (s, 1H), 6.72 (br s, 2H), 6.50
(d,1H, J ¼ 8.8 Hz), 6.24 (br s, 2H), 5.24 (d,1H, J ¼ 8.8 Hz), 3.80 (s, 3H),
3.74 (s, 3H), 3.61 (s, 2H), 1.96 (s, 3H), 1.60 (s, 3H); 13C NMR (DMSO-
5.1.23. (ꢀ)-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-(1-isopropyl-2(1H)-phthalazinyl)-2-propen-1-
one (1b)
d6, 100 MHz):
d 165.3, 162.7, 159.9, 155.9, 152.5, 151.2, 146.1, 142.0,
This compound was prepared and purified on the same scale as
described above. Work-up, chromatography and recrystallization
(methanol) gave 400 mg (40%) of 1b as an off-whiteꢁp1owder, mp
136.7, 135.7, 133.8, 133.5, 132.1, 128.2, 127.9, 126.1 (2C), 123.0, 122.1,
118.5, 118.0, 114.8, 106.6, 60.8, 55.8, 49.2, 25.2, 18.4. Anal. Calcd for
C28H30N6O3$1.5 H2O: C, 64.00; H, 6.29; N, 16.00. Found: C, 64.37; H,
6.35; N, 16.07.
130e132 ꢂC. IR: 3474, 3337, 3183, 1648, 1604 cm
;
1H NMR
(DMSO-d6, 300 MHz):
d
7.95 (s, 1H), 7.86 (d, 1H, J ¼ 16.2 Hz), 7.66
(d, 1H, J ¼ 16.2 Hz), 7.60 (s, 1H), 7.59e7.42 (complex m, 3H), 7.38
(d, 1H, J ¼ 7.1 Hz), 7.26 (s, 1H), 6.99 (s, 1H), 6.21 (br s, 2H), 5.76 (br s,
2H), 5.66 (d, 1H, J ¼ 7.1 Hz), 3.79 (s, 3H), 3.73 (s, 3H), 3.59 (s, 2H),
1.89 (octet, 1H, J ¼ 6.6 Hz), 0.84 (d, 3H, J ¼ 6.6 Hz), 0.72 (d, 3H,
5.1.27. (ꢀ)-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-[1-(1-ethylpropyl)-2(1H)-phthalazinyl]-2-
propen-1-one (1f)
This compound was prepared and purified on the same scale as
described above. Work-up, chromatography and recrystallization
J ¼ 6.6 Hz); 13C NMR (DMSO-d6, 75 MHz):
d 165.8, 162.2 (2C), 155.5,

394
B. Nammalwar et al. / European Journal of Medicinal Chemistry 54 (2012) 387e396
(methanol) gave 575 mg (54%) of 1f as an off-whiteꢁp1owder, mp
;
1H NMR
118.2, 117.8, 114.8, 105.7, 60.8, 55.7, 51.4, 32.4, 19.6. Anal. Calcd for
C31H30N6O3$1.0 H2O: C, 67.39; H, 5.80; N, 15.22. Found: C, 67.17; H,
5.53; N, 15.17.
134e136 ꢂC. IR: 3479, 3332, 3184, 1656, 1605 cm
(DMSO-d6, 300 MHz):
d
8.00 (s, 1H), 7.85 (d, 1H, J ¼ 16.0 Hz), 7.65
(d, 1H, J ¼ 16.0 Hz), 7.59 (s, 1H), 7.56 (d, 1H, J ¼ 7.4 Hz), 7.53 (t, 1H,
J ¼ 7.2 Hz), 7.46 (t, 1H, J ¼ 7.4 Hz), 7.36 (d, 1H, J ¼ 7.4 Hz), 7.27 (s, 1H),
7.01 (d, 1H, J ¼ 1.2 Hz), 6.57 (br s, 2H), 6.11 (br s, 2H), 5.83 (d, 1H,
J ¼ 7.4 Hz), 3.79 (s, 3H), 3.74 (s, 3H), 3.61 (s, 2H), 1.48 (m, 1H), 1.26
(m, 3H), 1.01 (m 1H), 0.89 (t, 3H, J ¼ 7.2 Hz), 0.74 (t, 3H, J ¼ 7.2 Hz);
5.1.31. (ꢀ)-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-[1-(4-methylphenyl)-2(1H)-phthalazinyl]-2-
propen-1-one (1j)
This compound was prepared and purified on the same scale as
described above. Work-up, chromatography and recrystallization
(methanol) gave 740 mg (67%) of 1j as a light pink powder, mp
178e180 ꢂC. IR: 3442, 3329, 3149,1652,1603 cmꢁ1; 1H NMR (DMSO-
13C NMR (DMSO-d6, 75 MHz):
d 165.6, 162.5, 160.6, 152.5 (2C),146.0,
144.0, 136.6, 136.0, 132.1, 131.5, 128.2, 127.8, 127.2, 126.0, 124.3,
118.5, 117.9, 114.8, 106.4, 60.8, 55.8, 52.1, 45.4, 32.2, 20.6, 20.1, 10.8,
10.4. Anal. Calcd for C29H34N6O3$1.5 H2O: C, 64.32; H, 6.84; N, 15.52.
Found: C, 63.95; H, 7.10; N, 15.27.
d6, 300 MHz):
d
7.97 (s, 1H), 7.86 (d, 1H, J ¼ 15.9 Hz), 7.69 (d, 1H,
J ¼ 15.9 Hz), 7.60 (s, 1H), 7.56 (m, 3H), 7.44 (m, 1H), 7.27 (s, 1H), 7.11
(d, 2H, J ¼ 8.8 Hz), 7.07 (d, 2H, J ¼ 8.8 Hz), 6.99 (s,1H), 6.94 (s,1H), 6.19
(br s, 2H), 5.74 (br s, 2H), 3.78 (s, 3H), 3.72 (s, 3H), 3.59 (s, 2H), 2.20
5.1.28. (ꢀ)-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-(1-cyclohexyl-2(1H)-phthalazinyl)-2-propen-
1-one (1g)
(s, 3H); 13C NMR (DMSO-d6, 75 MHz):
d 165.8, 162.3, 162.1, 155.8,
152.4, 146.0, 142.0, 138.8, 137.1, 136.8, 136.6, 133.1, 132.2, 129.1, 128.5,
127.7, 127.1, 126.4, 126.0, 123.0, 118.4, 117.7, 114.8, 105,7, 60.8, 55.7,
53.5, 32.4, 20.5. Anal. Calcd for C31H30N6O3$0.5 H2O: C, 68.51; H,
5.71; N, 15.47. Found: C, 68.55; H, 5.58; N, 15.48.
This compound was prepared and purified on the same scale as
described above. Work-up, chromatography and recrystallization
(methanol) gave 510 mg (47%) of 1g as an off-whiteꢁp1owder, mp
;
1H NMR
177e178 ꢂC. IR: 3480, 3340, 3180, 1645, 1604 cm
(DMSO-d6, 300 MHz):
d
7.96 (s, 1H), 7.85 (d, 1H, J ¼ 16.2 Hz), 7.66
5.1.32. (ꢀ)-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-[1-(3,5-dimethylphenyl)-2(1H)-phthalazinyl]-
2-propen-1-one (1k)
(d, 1H, J ¼ 16.2 Hz), 7.59 (s, 1H), 7.59e7.42 (complex m, 3H), 7.36 (d,
1H, J ¼ 7.7 Hz), 7.26 (s, 1H), 7.00 (d, 1H, J ¼ 1.6 Hz), 6.48 (br s, 2H),
6.01 (br s, 2H), 5.67 (d, 1H, J ¼ 7.1 Hz), 3.79 (s, 3H), 3.74 (s, 3H), 3.60
(s, 2H), 1.74e1.37 (complex m, 6H), 1.10e0.84 (complex m, 5H); 13C
This compound was prepared and purified on the same scale as
described above. Work-up, chromatography and recrystallization
(methanol) gave 480 mg (42%) of 1k as a light pinkꢁp1owder, mp
NMR (DMSO-d6, 75 MHz):
d 165.8, 162.5, 160.9, 153.1, 152.5, 146.0,
143.8, 136.6, 136.1, 131.7, 131.3, 128.3, 127.8, 127.5, 125.9, 124.2, 118.5,
117.9, 114.8, 106.3, 60.8, 55.8, 54.9, 42.1, 32.3, 28.8, 28.1, 25.6, 25.5,
25.3. Anal. Calcd for C30H34N6O3$2.5 H2O: C, 63.05; H, 6.83; N, 14.71.
Found: C, 63.06; H, 6.53; N, 14.47.
215e217 ꢂC. IR: 3440, 3336, 3169, 1658, 1601 cm
;
1H NMR
(DMSO-d6, 400 MHz):
d
7.96 (s, 1H), 7.85 (d, 1H, J ¼ 16.1 Hz), 7.70
(d, 1H, J ¼ 16.1 Hz), 7.59 (s, 1H), 7.54 (complex m, 1H), 7.52 (m, 2H),
7.43 (m, 1H), 7.29 (d, 1H, J ¼ 1.6 Hz), 7.00 (d, 1H, J ¼ 1.6 Hz), 6.87
(s, 1H), 6.84 (s, 2H), 6.45 (br s, 2H), 5.98 (br s, 2H), 3.79 (s, 3H), 3.72
(s, 3H), 3.60 (s, 2H), 2.18 (s, 6H); 13C NMR (DMSO-d6, 100 MHz):
5.1.29. (ꢀ)-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-(1-phenyl-2(1H)-phthalazinyl)-2-propen-1-
one (1h)
d
165.7, 162.4, 161.1, 153.5, 152.5, 146.1, 142.0, 141.9, 137.6, 137.0,
136.2, 133.1, 132.2, 129.0, 128.4, 127.7, 127.1, 126.4, 125.6, 122.7, 118.4,
117.7, 114.9, 106.2, 60.8, 55.8, 54.0, 32.3, 21.0. Anal. Calcd for
C32H32N6O3$1.75 H2O: C, 66.26; H, 6.13; N,14.50. Found: C, 66.22; H,
6.00; N, 14.46.
This compound was prepared and purified on the same scale as
described above. Work-up, chromatography and recrystallization
(methanol) gave 647 mg (60%) of 1h as a light pinkꢁp1owder, mp
;
1H NMR
142e144 ꢂC. IR: 3478, 3340, 3178, 1653, 1607 cm
(DMSO-d6, 300 MHz):
d
7.98 (s, 1H), 7.87 (d, 1H, J ¼ 15.9 Hz), 7.70
5.1.33. (ꢀ)-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-[1-(3-fluorophenyl)-2(1H)-phthalazinyl]-2-
propen-1-one (1l)
This compound was prepared and purified on the same scale as
described above. Work-up, chromatography and recrystallization
(methanol) gave 667 mg (60%) of 1l as an off-white powder, mp
153e155 ꢂC. IR: 3495, 3433, 3375, 3165, 1647, 1600 cmꢁ1; 1H NMR
(d, 1H, J ¼ 15.9 Hz), 7.64e7.50 (complex m, 3H), 7.60 (s, 1H), 7.45
(t, 1H, J ¼ 7.1 Hz), 7.33e7.18 (complex m, 7H), 6.99 (s, 1H), 6.19 (br s,
2H), 5.74 (br s, 2H), 3.78 (s, 3H), 3.72 (s, 3H), 3.59 (s, 2H); 13C NMR
(DMSO-d6, 75 MHz):
d 165.8, 162.3, 162.1, 155.8, 152.4, 146.0, 142.0,
141.7, 137.1, 136.6, 132.9, 132.2, 128.58, 128.51, 127.6, 127.5, 127.2,
126.4, 126.0, 122.9, 118.4, 117.6, 114.9, 105.7, 60.8, 55.7, 53.8, 32.4.
Anal. Calcd for C30H28N6O3$0.5 H2O: C, 68.05; H, 5.48; N, 15.87.
Found: C, 68.41; H, 5.49; N, 15.93.
(DMSO-d6, 400 MHz):
d
7.99 (s, 1H), 7.88 (d, 1H, J ¼ 16.1 Hz), 7.70
(d, 1H, J ¼ 16.1 Hz), 7.65 (d, 1H, J ¼ 7.4 Hz), 7.60 (s, 1H), 7.57 (m, 1H),
7.56 (d, 1H, J ¼ 7.2 Hz), 7.47 (m, 1H), 7.34 (m, 1H), 7.29 (d, 1H,
J ¼ 1.6 Hz), 7.07 (m, 3H), 7.02 (s, 1H), 7.01 (d, 1H, J ¼ 1.6 Hz), 6.31 (br
s, 2H), 5.85 (br s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 3.60 (s, 2H); 13C NMR
5.1.30. (ꢀ)-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-[1-(2-methylphenyl)-2(1H)-phthalazinyl]-2-
prop-2-en-1-one (1i)
(DMSO-d6,100 MHz):
d
165.9,162.3,162.1 (d, JCeF ¼ 244.9 Hz),161.8,
This compound was prepared and purified on the same scale as
described above. Work-up, chromatography and recrystallization
(methanol) gave 638 mg (58%) of 1i as an off-whiꢁte1 solid, mp
;
1H NMR
154.7, 152.5, 146.1, 144.5 (d, JCeF ¼ 6.6 Hz), 142.0, 137.4, 136.4, 132.41,
132.38, 130.8 (d, JCeF ¼ 8.1 Hz), 128.8, 127.6, 127.2, 126.6, 122.8, 121.9
(d, JCeF ¼ 2.2 Hz), 118.4, 117.5, 114.9, 114.4 (d, JCeF ¼ 20.6 Hz), 112.9
(d, JCeF ¼ 22.8 Hz), 105.9, 60.8, 55.8, 53.4, 32.4. Anal. Calcd for
C30H27FN6O3$0.7 H2O: C, 65.38; H, 5.16; N,15.26. Found: C, 65.21; H,
5.21; N, 15.32.
143e145 ꢂC. IR: 3474, 3344, 3178, 1650, 1607 cm
(DMSO-d6, 400 MHz):
d
7.92 (s, 1H), 7.79 (d, 1H, J ¼ 16.1 Hz), 7.65
(d, 1H, J ¼ 16.1 Hz), 7.60 (s, 1H), 7.54 (dd, 1H, J ¼ 7.4, 1.2 Hz), 7.43 (td,
1H, J ¼ 7.4, 1.6 Hz), 7.39 (td, 1H, J ¼ 7.4, 1.2 Hz), 7.25 (d, 1H,
J ¼ 1.6 Hz), 7.23 (d, 1H, J ¼ 7.6 Hz), 7.15 (m, 2H), 7.09 (td, 1H, J ¼ 7.2,
1.8 Hz), 7.05 (td, 1H, J ¼ 7.6, 1.6 Hz), 6.98 (d, 1H, J ¼ 1.6 Hz), 6.94
(s, 1H), 6.18 (br s, 2H), 5.74 (br s, 2H), 3.77 (s, 3H), 3.70 (s, 3H), 3.59
5.1.34. (ꢀ)-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-[1-(4-fluorophenyl)-2(1H)-phthalazinyl]-2-
propen-1-one (1m)
This compound was prepared and purified on the same scale as
described above. Work-up, chromatography and recrystallization
(methanol) gave 608 mg (55%) of 1m as an off-white powder, mp
(s, 2H), 2.71 (s, 3H); 13C NMR (DMSO-d6, 100 MHz):
d
165.6, 162.3,
162.2, 155.8, 152.4, 146.0, 143.2, 140.2, 136.8, 136.6, 133.9, 133.7,
132.3, 130.3, 128.3, 127.7, 127.4, 127.0, 126.83, 126.78, 126.6, 121.9,

B. Nammalwar et al. / European Journal of Medicinal Chemistry 54 (2012) 387e396
395
143e145 ꢂC. IR: 3479, 3347, 3180, 1649, 1600 cmꢁ1
(DMSO-d6, 300 MHz):
;
1H NMR
113.4, 105.7, 60.8, 55.7, 54.9 (2C), 52.4, 32.4. Anal. Calcd for
C32H32N6O4$0.5 H2O: C, 67.02; H, 5.76; N, 14.66. Found: C, 67.15; H,
5.69; N, 14.66.
d
8.00 (s, 1H), 7.88 (d, 1H, J ¼ 15.9 Hz), 7.69
(d, 1H, J ¼ 15.9 Hz), 7.61e7.56 (complex m, 3H), 7.60 (s, 1H), 7.46
(m, 1H), 7.27 (complex m, 3H), 7.12 (t, 2H, J ¼ 8.8 Hz), 7.02 (s, 2H),
6.19 (br s, 2H), 5.75 (br s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 3.59 (s, 2H);
5.1.38. (ꢀ)-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-[1-(4-trifluoromethoxybenzyl)-2(1H)-
phthalazinyl]-2-propen-1-one (1q)
This compound was prepared and purified on the same scale as
described above. Work-up, chromatography and recrystallization
(methanol) gave 320 mg (25%) of 1q as an off-white powder, mp
125e127 ꢂC. IR: 3481, 3335, 3183,1657,1607 cmꢁ1; 1H NMR (DMSO-
13C NMR (DMSO-d6, 75 MHz):
d 165.8, 162.3, 162.2, 161.4
(d, JCeF ¼ 243.9 Hz),155.8, 152.5, 146.0, 142.1,137.9 (d, JCeF ¼ 2.9 Hz),
137.3, 136.6, 132.7, 132.3, 128.7, 128.3 (d, JCeF ¼ 8.3 Hz), 127.6, 127.2,
126.5, 122.9, 118.4, 117.5, 115.4 (d, JCeF ¼ 21.5 Hz), 114.4, 105.7, 60.8,
55.7, 53.0, 32.4. Anal. Calcd for C30H27FN6O3$0.5 H2O: C, 65.81; H,
5.12; N, 15.36. Found: C, 65.88; H, 5.14; N, 15.24.
d6, 400 MHz):
d
7.81 (d,1H, J ¼ 16.1 Hz), 7.80 (s,1H), 7.60 (s,1H), 7.58
5.1.35. (ꢀ)-(E)-1-(1-Benzyl-2(1H)-phthalazinyl)-3-{5-[(2,4-
diamino-5-pyrimidinyl)methyl]-2,3-dimethoxyphenyl}-2-propen-
1-one (1n)
(d, 1H, J ¼ 16.1 Hz), 7.44 (m, 3H), 7.24 (s, 1H), 7.17 (d, 2H, J ¼ 8.0 Hz),
7.08 (m, 1H), 6.99 (s, 1H), 6.98 (d, 2H, J ¼ 8.0 Hz), 6.18 (br s, 2H), 6.08
(t, 1H, J ¼ 6.3 Hz), 5.73 (br s, 2H), 3.79 (s 3H), 3.73 (s, 3H), 3.59
(s, 2H), 2.95 (dd, 1H, J ¼ 13.2, 7.0 Hz), 2.85 (dd, 1H, J ¼ 13.2, 6.1 Hz);
This compound was prepared and purified on the same scale as
described above. Work-up, chromatography and recrystallization
(methanol) gave 620 mg (56%) of 1n as an off-whiteꢁp1owder, mp
13C NMR (DMSO-d6, 100 MHz):
d 165.6, 162.3, 162.2, 155.7, 152.5,
147.1, 146.0, 142.4, 136.6, 135.8, 132.3, 131.5, 131.4, 128.5, 127.7, 126.6,
126.0, 123.8, 120.5, 120.0 (q, JCeF ¼ 256.2 Hz), 118.8, 118.3, 117.7,
114.8, 105.7, 60.8, 55.7 (2C), 52.0, 32.4. Anal. Calcd for
C32H29F3N6O4$2.5 H2O: C, 57.92; H, 5.13; N, 12.67. Found: C, 57.74;
H, 4.91; N, 12.37.
143e145 ꢂC. IR: 3479, 3336, 3179, 1658, 1604 cm
;
1H NMR
(DMSO-d6, 400 MHz):
d
7.83 (d, 1H, J ¼ 16.0 Hz), 7.78 (s, 1H), 7.61
(s, 1H), 7.60 (d, 1H, J ¼ 16.0 Hz), 7.47e7.37 (complex m, 3H), 7.24
(s, 1H), 7.17 (m, 3H), 7.00 (d, 1H, J ¼ 1.2 Hz), 6.96 (m, 1H), 6.86
(m, 2H), 6.19 (br s, 2H), 6.04 (t, 1H, J ¼ 6.4 Hz), 5.73 (br s, 2H), 3.79
(s, 3H), 3.74 (s, 3H), 3.60 (s, 2H), 2.90 (dd, 1H, J ¼ 13.0, 7.6 Hz), 2.83
5.2. Biology
(dd, 1H, J ¼ 13.0, 5.7 Hz); 13C NMR (DMSO-d6, 100 MHz):
d 165.6,
162.3, 162.2, 155.8, 152.5, 146.0, 142.3, 136.64, 136.57, 136.2, 132.4,
131.3, 129.6, 128.4, 127.9, 127.8, 126.6, 126.5, 125.9, 123.8, 118.3,
117.8, 114.7, 105.7, 60.8, 55.7, 52.3, 48.6, 32.4. Anal. Calcd for
C31H30N6O3$1.0 CH3OH: C, 67.84; H, 6.01; N, 14.84. Found: C, 68.13;
H, 5.86; N, 15.12.
5.2.1. Biological assay-determinations of the MICs
The minimum inhibitory concentration (MIC in
mg/mL) was
evaluated for each agent against B. anthracis Sterne strain using the
broth microdilution method following CLSI protocols [4,32].
Compounds were screened using two-fold dilutions, typically
starting with 16
which were also screened with 12
m
g/mL, except for racemic compounds 1a and 1h,
5.1.36. (ꢀ)-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-[1-(4-methylbenzyl)-2(1H)-phthalazinyl]-2-
propen-1-one (1o)
This compound was prepared and purified on the same scale as
described above. Work-up, chromatography and recrystallization
(methanol) gave 545 mg (48%) of 1o as an off-white powder, mp
135e137 ꢂC. IR: 3477, 3338, 3176, 1657, 1605 cmꢁ1; 1H NMR (DMSO-
mg/mL as a starting concentra-
tion. The MIC values were determined as the nearest dilution that
inhibited growth to 80% of the positive growth control, as evaluated
visually and by optical density (turbidity) at 600 nm. The data were
derived from two independent experiments performed in duplicate
and are presented in Table 1.
d6, 400 MHz):
d
7.83 (d,1H, J ¼ 16.1 Hz), 7.78 (s,1H), 7.61 (s, 1H), 7.60
Acknowledgments
(d, 1H J ¼ 16.1 Hz), 7.44 (m, 1H), 7.41 (dd, 2H, J ¼ 5.6, 3.4 Hz), 7.24
(d, 1H J ¼ 1.6 Hz), 6.99 (s, 1H), 6.98 (d, 2H, J ¼ 8.0 Hz), 6.96 (m, 1H),
6.73 (d, 2H, J ¼ 8.0 Hz), 6.18 (br s, 2H), 6.00 (t, 1H, J ¼ 6.4 Hz), 5.73
(br s, 2H), 3.79 (s, 3H), 3.74 (s, 3H), 3.60 (s, 2H), 2.85 (dd,1H, J ¼ 13.2,
7.6 Hz), 2.78 (dd, 1H, J ¼ 13.2, 5.5 Hz), 2.23 (s, 3H); 13C NMR (DMSO-
We gratefully acknowledge support of this work by the National
Institutes of Allergy and Infectious Diseases [1-R01-AI090685-01]
of the NIH/NIAID to WWB. We are also pleased to acknowledge
funding for the Oklahoma Statewide NMR Facility by the National
Science Foundation (BIR-9512269), the Oklahoma State Regents for
Higher Education, the W. M. Keck Foundation, and Conoco, Inc.
Finally, molecular graphics images were produced using the UCSF
Chimera package from the Resource for Biocomputing, Visualiza-
tion, and Informatics at the University of California-San Francisco
[supported by NIH P41 RR001081].
d6, 100 MHz):
d 165.5, 162.3, 162.2, 155.8, 152.5, 146.0, 142.2, 136.6,
136.5, 135.4, 133.1, 132.5, 131.3, 129.5, 128.6, 128.3, 127.8, 126.7,
125.9,123.8,118.3,117.8,114.7,105.7, 60.8, 55.7, 52.4, 48.6, 32.4, 20.7.
Anal. Calcd for C31H30N6O3$1.0 CH3OH: C, 67.84; H, 6.01; N, 14.84.
Found: C, 68.17; H, 6.11; N, 14.78.
5.1.37. (ꢀ)-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
dimethoxyphenyl}-1-[1-(4-methoxybenzyl)-2(1H)-phthalazinyl]-2-
propen-1-one (1p)
References
This compound was prepared and purified on the same scale as
described above. Work-up, chromatography and recrystallization
(methanol) gave 595 mg (51%) of 1p as an off-whiteꢁp1owder, mp
[1] T.V. Inglesby, D.A. Henderson, J.G. Bartlett, M.S. Ascher, E. Eitzen,
A.M. Friedlander, J. Hauer, J. McDade, M.T. Osterholm, T. O’Toole, G. Parker,
T.M. Perl, P.K. Russell, K. Tonat, Anthrax as a biological weapon: medical and
public health management. Working group on civilian biodefense, JAMA 281
(1999) 1735e1745.
[2] J.M. Beierlien, K.M. Frey, D.B. Bolstad, P.M. Pelphey, T.M. Joska, A.E. Smith,
N.D. Priestley, D.L. Wright, A.C. Anderson, Synthetic and crystallographic
studies of a new inhibitor series targeting Bacillus anthracis dihydrofolate
reductase, J. Med. Chem. 51 (2008) 7532e7540.
[3] C.R. Bourne, R.A. Bunce, P.C. Bourne, K.D. Berlin, E.W. Barrow, W.W. Barrow,
Crystal structure of Bacillus anthracis dihydrofolate reductase with the
dihydrophthalazine-based trimethoprim derivative RAB1 provides a struc-
tural explanation of potency and selectivity, Antimicrob. Agents Chemother.
53 (2009) 3065e3073.
253e255 ꢂC. IR: 3478, 3333, 3174, 1657, 1605 cm
;
1H NMR
(DMSO-d6, 400 MHz):
d
7.83 (d, 1H, J ¼ 16.1 Hz), 7.78 (s, 1H), 7.60
(s,1H), 7.60 (d,1H, J ¼ 16.1 Hz), 7.42 (m, 3H), 7.24 (s,1H), 6.99 (s,1H),
6.97 (m, 1H), 6.76 (d, 2H, J ¼ 9.1 Hz), 6.74 (d, 2H, J ¼ 9.1 Hz), 6.18
(br s, 2H), 5.97 (t, 1H, J ¼ 6.2 Hz), 5.73 (br s, 2H), 3.79 (s, 3H), 3.73
(s, 3H), 3.68 (s, 3H), 3.59 (s, 2H), 2.83 (dd, 1H, J ¼ 13.3, 7.4 Hz), 2.76
(dd, 1H, J ¼ 13.3, 5.5 Hz); 13C NMR (DMSO-d6, 100 MHz):
d 165.5,
162.3, 162.2, 157.9, 155.8, 152.5, 146.0, 142.2, 136.6, 136.5, 132.5,
131.3,130.6, 128.3, 128.0, 127.8, 126.7, 125.9, 123.8, 118.3,117.8, 114.2,

396
B. Nammalwar et al. / European Journal of Medicinal Chemistry 54 (2012) 387e396
[4] E.W. Barrow, J. Drier, S. Reinelt, P.C. Bourne, W.W. Barrow, In vitro efficacy of
[20] A. Gangjee, J. Yu, J.J. McGuire, V. Cody, N. Galitsky, R.L. Kisliuk, S.F. Queener,
Design, synthesis and X-ray crystal structure of a potent dual inhibitor of
thymidine synthase and dihydrofolate reductase as an antitumor agent,
J. Med. Chem. 43 (2000) 3837e3851.
[21] A. Gangjee, A. Viwdans, E. Elzein, J.J. McGuire, S.F. Queener, R.L. Kiliuk,
Synthesis, antifolate, and antitumor activities of classical and nonclassical
2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyridmidines, J. Med. Chem. 44
(2001) 1993e2003.
[22] A patent in this area contained citations to 2, but little experimental detail and
very few properties were revealed; see P. Guerry, C. Hubschwerlen, S. Jolidon,
J.-L. Specklin, P.C. Wyss, Substituted 2,4-diaminopyrimidines, World Patent
WO9839328 (1998) Chem. Abstr. 129 (1998) 230736.
[23] R.A. Bunce, K.D. Berlin, C.R. Bourne, P.C. Bourne, E.W. Barrow, W.W. Barrow,
Identification and enantiomeric separation of (þ)- and (ꢁ)-(E)-3-[5-(2,4-
diaminopyrimidin-5-ylmethyl)-2,3-dimethoxyphenyl]-1-(1-propyl-1H-
phthalazin-2-yl)propenone by supercritical fluid chromatography, Proc. Okla.
Acad. Sci. 90 (2010) 139e142.
[24] C.R. Bourne, R.A. Bunce, E.W. Barrow, P.C. Bourne, K.D. Berlin, W.W. Barrow,
Inhibition of antibiotic resistant Staphylococcus aureus by the broad-spectrum
dihydrofolate reductase inhibitor RAB1, Antimicrob. Agents Chemother. 54
(2010) 3825e3833.
[25] H. Uno, S. Okada, Y. Shiraishi, K. Shimokawa, H. Suzuki, Boron trifluoride-
assisted ZieglereZeiser reaction of perfluoroalkyllithiums. An efficient
synthesis of perfluoroalkylated heterocycles, Chem. Lett. (1988) 1165e1168.
[26] W.F. Holcomb, C.S. Hamilton, Derivatives of 4-amino-6-methoxyquinaldine,
J. Am. Chem. Soc. 64 (1942) 1309e1311.
new antifolates against trimethoprim-resistant Bacillus anthracis, Antimicrob.
Agents Chemother. 51 (2007) 4447e4452.
[5] R.C. Jackson, in: A.L. Jackman (Ed.), Antifolate Drugs in Cancer Therapy,
Humana Press, Totowa, NJ, 1999.
[6] E.M. Berman, L.M. Werbel, The renewed potential for folate antagonists in
contemporary cancer chemotherapy, J. Med. Chem. 34 (1991) 479e485.
[7] C.H. Takimoto, Antifolates in clinical development, Semin. Oncol. 24 (1997)
S18-40eS18-51.
[8] W.A. Bleyer, The clinical pharmacology of methotrexate. New applications of
an old drug, Cancer Treat. Rev. 41 (1978) 36e51.
[9] L. Adane, P.V. Bharatam, Modelling and informatics in the analysis of
P. falciparum DHFR enzyme inhibitors, Curr. Med. Chem. 15 (2008) 1552e1569.
[10] M. Finland, S.F. Kass, Symposium on trimethoprim-sulfmethoxazole, J. Infect.
Dis. 128 (1973) S425eS816.
[11] W.J. Suling, L.E. Seitz, V. Pathak, L. Westbrook, E.W. Barrow, S. Zywno-Van-
Ginkel, R.C. Reynolds, J.R. Piper, W.W. Barrow, Antimycobacterial activities of
2,4-diamino-5-deazapteridine derivatives and effects on mycobacterial
dihydrofolate reductase, Antimicrob. Agents Chemother. 44 (2000)
2784e2793.
[12] C. Clark, L.M. Ednie, G. Lin, K. Smith, K. Kosowska-Shick, P. McGhee,
B. Dewasse, L. Beachel, P. Caspers, B. Gaucher, G. Mert, S. Shapiro,
P.C. Appelbaum, Antistaphylococcal activity of dihydrophthalazine antifolates,
a family of novel antibacterial drugs, Antimicrob. Agents Chemother. 53
(2009) 1353e1361.
[13] P. Caspers, L. Bury, B. Gaucher, J. Heim, S. Shapiro, S. Siegrist, A. Schmitt-
Hoffmann, L. Thenoz, H. Urwyler, In vitro and in vivo properties of dihy-
drophthalazine antifolates, a novel family of antibacterial drugs, Antimicrob.
Agents Chemother. 53 (2009) 3620e3627.
[27] F.C. Whitmore, H.S. Mosher, R.R. Adams, R.B. Taylor, E.C. Chapin, C. Weisel,
W. Yanko, Basically substituted aliphatic nitriles and their catalytic reduction
to amines, J. Am. Chem. Soc. 66 (1944) 725e731.
[14] K.E. Bowker, P. Caspers, B. Gaucher, A.P. MacGowan, In vitro activities of three
new dihydrofolate reductase inhibitors against clinical isolates of gram-
positive bacteria, Antimicrob. Agents Chemother. 53 (2009) 4949e4952.
[15] S. Hawser, S. Lociuro, K. Islam, Dihydrofolate reductase inhibitors as anti-
bacterial agents, Biochem. Pharm. 71 (2006) 941e948.
[16] K. Smith, L.M. Ednie, P.C. Appelbaum, S. Hawser, S. Lociuro, Antistreptococcal
activity of AR-709 compared to that of other agents, Antimicrob. Agents
Chemother. 52 (2008) 2279e2282.
[28] S. Nimgirawath, Synthesis of (ꢀ)-isoautumnaline and (ꢀ)-dysoxylin, Aust. J.
Chem. 47 (1994) 957e962.
[29] A. Stuart, T. Paterson, B. Roth, E. Aig, 2,4-Diamino-5-benzylpyrimidine and
analogues as antibacterial Agents. 6. A one-step synthesis of trimethoprim
derivatives and activity analysis by molecular modeling, J. Med. Chem. 26
(1983) 667e673.
[30] No examples were found in a recent comprehensive review M. Oestreich (Ed.),
The Mizoroki-Heck Reaction, Wiley & Sons, Chichester, West Sussex, UK, 2009
However, a report has recently appeared using sealed tube Heck reactions, see
K. Geoghegan, S. Kelleher, P. Evans, J. Org. Chem. 76 (2011) 2187e2194.
[31] X-ray data on related compounds shows the 2,4-diaminopyrimidine ring
H-bonded with water in the crystal structure K.M. Frey, M.N. Lombardo,
D.L. Wright, A.C. Anderson, Towards the understanding of resistance mecha-
nisms in clinically isolated trimethoprim-resistant, methicillin-resistant
Staphylococcus aureus dihydrofolate reductase, J. Struc. Biol. 170 (2010) 93e97.
[32] Wayne, PA Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria
that Grow Aerobically; Approved Standard, eighth ed., vol. 29, (2009) pp.
1e65.
[17] X. Li, M. Hilgers, M. Cunningham, Z. Chen, M. Trzoss, J. Zhang, L. Kohnen, T. Lam,
C. Creighton, G.C. Kedar, K. Nelson, B. Kwan, M. Stidham, V. Brown-Driver,
K.J. Shaw, J. Finn, Structure-based designofnewDHFR-basedantibacterialagents:
7-aryl-2,4-diaminoquinazolines, Bioorg. Med. Chem. Lett. 21 (2011) 5171e5176.
[18] V. Cody, J. Pace, Structural analysis of Pneumocystis carinii and human DHFR
complexes with NADPH and a series of five potent 6-[50-(
u-carboxyalkoxy)
benzyl]pyrido[2,3-d]-pyrimidine derivatives, Acta Cryst. D67 (2011) 1e7.
[19] J.M. Beierlein, N.G. Karri, A.C. Anderson, Targeted mutations of Bacillus
anthracis dihydrofolate reductase condense complex structure-activity rela-
tionships, J. Med. Chem. 53 (2010) 7327e7336.