Modified 2,4-diaminopyrimidine-based dihydrofolate reductase inhibitors as potential drug scaffolds against Bacillus anthracis

Article abstract of DOI:10.1016/j.bmc.2014.11.009

The current Letter describes the synthesis and biological evaluation of dihydrophthalazine-appended 2,4-diaminopyrimidine (DAP) inhibitors (1) oxidized at the methylene bridge linking the DAP ring to the central aromatic ring and (2) modified at the central ring ether groups. Structures 4a-b incorporating an oxidized methylene bridge showed a decrease in activity, while slightly larger alkyl groups (CH₂CH₃ vs CH₃) on the central ring oxygen atoms (R² and R³) had a minimal impact on the inhibition. Comparison of the potency data for previously reported RAB1 and BN-53 with the most potent of the new derivatives (19b and 20a-b) showed similar values for inhibition of cellular growth and direct enzymatic inhibition (MICs 0.5-2 μg/mL). Compounds 29-34 with larger ester and ether groups containing substituted aromatic rings at R³ exhibited slightly reduced activity (MICs 2-16 μg/mL). One explanation for this attenuated activity could be encroachment of the extended R³ into the neighboring NADPH co-factor. These results indicate that modest additions to the central ring oxygen atoms are well tolerated, while larger modifications have the potential to act as dual-site inhibitors of dihydrofolate reductase (DHFR).

Full text of DOI:10.1016/j.bmc.2014.11.009

Bioorganic & Medicinal Chemistry 23 (2015) 203–211
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Modified 2,4-diaminopyrimidine-based dihydrofolate reductase
inhibitors as potential drug scaffolds against Bacillus anthracis
Baskar Nammalwar a, Christina R. Bourne b, , Nancy Wakeham b, Philip C. Bourne b, , Esther W. Barrow b,
N. Prasad Muddala a, Richard A. Bunce a, , K. Darrell Berlin a, 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, Stillwater, OK 74078, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The current Letter describes the synthesis and biological evaluation of dihydrophthalazine-appended 2,4-
diaminopyrimidine (DAP) inhibitors (1) oxidized at the methylene bridge linking the DAP ring to the
central aromatic ring and (2) modified at the central ring ether groups. Structures 4a–b incorporating
an oxidized methylene bridge showed a decrease in activity, while slightly larger alkyl groups (CH2CH3
vs CH3) on the central ring oxygen atoms (R2 and R3) had a minimal impact on the inhibition. Comparison
of the potency data for previously reported RAB1 and BN-53 with the most potent of the new derivatives
(19b and 20a–b) showed similar values for inhibition of cellular growth and direct enzymatic inhibition
Received 29 August 2014
Revised 29 October 2014
Accepted 5 November 2014
Available online 11 November 2014
Keywords:
Bacillus anthracis inhibition
Dihydrofolate reductase
Antibiotics
Antifolates
Antimicrobial agents
(MICs 0.5–2
lg/mL). Compounds 29–34 with larger ester and ether groups containing substituted
aromatic rings at R3 exhibited slightly reduced activity (MICs 2–16
l
g/mL). One explanation for this
attenuated activity could be encroachment of the extended R3 into the neighboring NADPH co-factor.
These results indicate that modest additions to the central ring oxygen atoms are well tolerated, while
larger modifications have the potential to act as dual-site inhibitors of dihydrofolate reductase (DHFR).
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
antifolates. Targets similar to those reported herein have been stud-
ied as broad-spectrum antibiotics by Basilea Pharmaceutica.11–14
Bacillus anthracis, a Gram-positive bacterium, is the etiological
agent responsible for the acute infectious disease anthrax.1 The
Center for Disease Control and Prevention (CDC) classifies B. anthra-
cis as a Category A potential high-priority bioterror threat agent,
and it is well documented that certain strains of these bacteria have
been modified to produce weapons of mass destruction to humans
and animals.2 It is also well known that these engineered strains
have innate resistance to current commercial drugs.3–6 Thus, there
is a compelling and imminent need to develop new therapeutic
agents to treat these resistant bacteria.
Previous studies from our research group have identified
dihydrophthalazine-appended 2,4-diaminopyrimidine (DAP) deriva-
tives as inhibitors of B. anthracis.7–9 These DAP inhibitors target the
key enzyme dihydrofolate reductase (DHFR) in the folate pathway,
which is intended to respond to intrinsic anti-folate antibiotic
resistance found in this organism.10 Apart from our research group,
there are a number of programs throughout the world developing
Other examples include Iclaprim by Acino Pharma,15 AR-709
by Evolva,16 and 7-aryl-2,4-diaminoquinazolines by Trius
Therapeutics.17 In the current study, potential DAP inhibitors,
incorporating modifications from our potent lead structures RAB1
and BN-53 (Fig. 1), were prepared and evaluated for their biological
activity against B. anthracis.
The basic structural motif of the target is composed of three ring
systems: a 2,4-diaminopyrimidine ring linked via a methylene bridge
to a central 3,4-dimethoxybenzene ring, which is, in turn, tethered
through an acryloyl chain to a substituted dihydrophthalazine
(Fig. 1). Based on previous work by Basilea Pharmaceutica11–14
and Barrow et al.,4 a number of DAP inhibitors were designed, syn-
thesized, and evaluated for their biological activities. The initial
modifications were made at R1 on the dihydrophthalazine ring. A
series of substrates were prepared with different R1 substituents,7,9
and from this set, RAB1 (minimum inhibitory concentration, MIC 1–
4 lg/mL) and BN-53 (MIC 0.5 lg/mL) were identified as potential
drugs.7,10 The second alterations were carried out at R4 on the DAP
ring, but increased activity was not observed.8 In a further quest
for improved potency over RAB1 and BN-53, the current study
investigated analogs resulting from oxidation of the methylene
bridge linking the DAP fragment to the central ring, as well as
⇑
Corresponding author. Tel.: +1 405 744 5952; fax: +1 405 744 6007.
E-mail address: rab@okstate.edu (R.A. Bunce).
Current address: Department of Chemistry and Biochemistry, University of
Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA.
http://dx.doi.org/10.1016/j.bmc.2014.11.009
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

204
B. Nammalwar et al. / Bioorg. Med. Chem. 23 (2015) 203–211
with 3-morpholinopropionitrile (10) using sodium methoxide in
dry DMSO gave adducts 11–14, which were treated sequentially
in dry EtOH with aniline hydrochloride and guanidine hydrochlo-
ride, both in the presence of sodium methoxide, to afford the
substituted 2,4-diamino-5-benzyl-1,3-pyrimidines 15–18 in 72–
80% yields. Heck coupling of 15–18 with 3a and 3b then generated
the target compounds 19a–b, 20a–b, 21a and 22a in 70–78% yields.
Phenol 27, which would allow variation of groups at R3 on the
central ring of RAB1, was synthesized (Scheme 3) from the 3-
methoxymethyl (MOM)-substituted benzaldehyde 23, prepared by
modification of our previously reported procedure.20 Sodium meth-
oxide promoted condensation of 10 with 23 produced 24, which was
cyclocondensed with aniline hydrochloride and guanidine
hydrochloride to give the substituted 2,4-diamino-5-benzyl-1,3-
pyrimidine 25 in 83% yield. Heck coupling of 25 with 3a gave the
MOM-protected catechol 26, which was deprotected using
methanolic hydrogen chloride to give phenol 27 (58%). Finally,
treatment of 27 with DBU at room temperature in dichloromethane,
followed by addition of propyl iodide, benzoyl chloride,
4-methoxybenzoyl chloride, 4-nitrobenzoyl chloride, 4-(trifluoro-
methoxy)benzoyl chloride, 4-fluorobenzoyl chloride, or 4-(trifluoro-
methyl)benzoyl chloride, afforded targets 28–34 in 62–92% yields.
Figure 1. Structural modifications of the DAP-based inhibitor and lead compounds.
2.2. Biology
changes at R2 and R3, and assessed the biological activities of the
resulting compounds.
The biological activities of our new dihydrophthalazine-
appended DAP derivatives are summarized in Table 1. Compounds
incorporating a ketone function between the DAP ring and the cen-
tral dimethoxybenzene ring, as found in 4a (R1 = propyl,
R2 = R3 = CH3) and 4b (R1 = isobutenyl, R2 = R3 = CH3), exhibited
low activity. The impact on direct inhibition of the DHFR enzyme
did display a preference for the isobutenyl moiety at R1, but this
change did not rescue the lack of inhibition of bacterial cell growth.
Alterations of the central-ring ether groups to incorporate
slightly larger moieties at either R2 alone, or in addition to R3, had
little impact on the potency. For example, when compared with
unmodified RAB1 (R1 = propyl, R2 = R3 = CH3), 19a (R1 = propyl,
R2 = R3 = CH2CH3) and 20a (R1 = propyl, R2 = CH2CH3, R3 = CH3)
showed similar activity. Substitution of larger groups, such as a
benzyl at R2 in 21a (R1 = propyl, R2 = benzyl, R3 = CH3) or a propyl
at R3 in 28 (R1 = propyl, R2 = CH3, R3 = propyl), exerted a small, but
measurable, affect on the growth of the bacterial cells and on the
purified enzyme. In contrast, compounds having a smaller methy-
lene moiety bridging R2 and R3, as in 22a (R1 = propyl, R2 = R3 =
–CH2–), showed reduced activity. Comparison of the activity data
for unmodified BN-53 (R1 = isobutenyl, R2 = R3 = CH3) with that
for 19b (R1 = isobutenyl, R2 = R3 = CH2CH3) and 20b (R1 = isobute-
nyl, R2 = CH2CH3, R3 = CH3) revealed no measurable change in either
inhibition of cellular growth or direct enzymatic inhibition. When
contextualized within the binding pocket of the DHFR enzyme from
B. anthracis, small extensions (replacing CH3 with CH2CH3) at R2 and
R3 would be readily accommodated without unfavorable steric
interaction with any protein atoms. Finally, the comparable reduc-
tion in activity for the much larger benzyl at R2 in 21a versus the
smaller propyl at R3 in 28 suggests that the R3 position has a greater
impact on potency.
2. Results and discussion
2.1. Chemistry
The molecular targets in Scheme 1 incorporate a ketone
function in the methylene bridge between the DAP moiety and
the central dimethoxybenzene ring, but retain the propyl and
isobutenyl groups at R1 on the dihydrophthalazine. The prepara-
tion of these structures was a two-step process involving benzylic
oxidation of 1 to ketone 2, followed a by Heck coupling to the
acrylamide derivative 3 to yield 4. Benzylic oxidation of 1 was per-
formed in 78% yield using potassium dichromate in acetic acid at
reflux. This was followed by a palladium(II) acetate-mediated Heck
reaction of the resulting ketone 2 with ( )-1-(1-alkyl-2(1H)-
phthalazinyl)-2-propen-1-ones 3a and 3b in DMF at 140 °C to give
the target molecules 4a (79%) and 4b (75%), respectively.7,8,18,19
The synthesis of drug candidates, modified at the central ring
ether groups (R2 and R3), while retaining the propyl and isobutenyl
substitution on the dihydrophthalazine, are shown in Scheme 2.
Benzaldehydes 6–9, derived from 5-iodovanillin, were prepared
using methodology previously described.20 Condensation of 6–9
Further modification in the central ring installed larger groups
at the R3 position to give compounds 29–34 (R1 = propyl,
R2 = CH3, R3 = variable). These compounds exhibited lower efficacy,
and this was revealed more dramatically in the enzyme inhibition
assay. In reactions with purified DHFR protein, four of the six R3
derivatives were unable to achieve at least 50% inhibition at the
limit of compound solubility if the compound was added after
the NADPH. Only two derivatives, 29 and 31, effectively inhibited
the enzyme with this order of addition. Structure 29 contained
the minimum addition of a benzoyl group at R3, although the Ki
Scheme 1. Synthesis of structures incorporating an oxidized methylene bridge.
Reagents and conditions: (a) K2Cr2O7, AcOH, 118 °C; (b) 3a–b [a: R1 = CH2CH2CH3;
b: R1 = CH = C(CH3)2], Pd(OAc)2, 1-ethylpiperidine, DMF, 140 °C.

B. Nammalwar et al. / Bioorg. Med. Chem. 23 (2015) 203–211
205
Scheme 2. Synthesis of structures modified at R2 and R3. Reagents and conditions: (a) Ref. 20; (b) NaOMe, DMSO, 65–90 °C; (c) PhNH2ÁHCl, NaOMe, EtOH, 78 °C; (d)
(H2N2)C = NHÁHCl, NaOMe, EtOH, 78 °C; (e) 3a–b [a: R1 = CH2CH2CH3; b: R1 = CH = C(CH3)2], Pd(OAc)2, 1-ethylpiperidine, DMF, 140 °C.
Scheme 3. Synthesis of analogs of RAB1 modified at R3. Reagents and conditions: (a) NaOMe, DMSO, 65–90 °C; (b) PhNH2ÁHCl, NaOMe, EtOH, 78 °C; (c) (H2N2)C = NHÁHCl,
NaOMe, EtOH, 78 °C; (d) 3a Pd(OAc)2, 1-ethylpiperidine, DMF, 140 °C; (e) HCl, MeOH, 23 °C; (f) R3X, DBU, CH2Cl2, 23 °C.
was barely measurable. When compounds were added prior to the
NADPH co-factor, the inhibition improved remarkably such that all
but one compound had measurable Ki values. Compound 31
(R3 = 4-nitrobenzoyl), the only polarized structure tested, stood
out as remarkably better than the others in this series. Neverthe-
less, it was not as efficacious as RAB1 or BN-53, and the MIC value
did not indicate the same remarkable gain in potency that the Ki
value revealed.
substrate site. These inhibitors are known to dock with the DAP
moiety, which closely mimics the natural folate substrate10,22
Based on our structural data to date, it is likely that each com-
pound within the inhibitor series binds with a relatively conserved
orientation.10 We hypothesize that these larger extensions from R3
are approaching the neighboring NADPH co-factor site. This
hypothesis is supported, in part, by experiments of enzyme
inhibition in which the compounds were instead added prior to
the NADPH co-factor. In this situation, the measurable Ki values
decreased and three additional compounds showed inhibition. It
The compounds containing the larger extensions from R3 pres-
ent an interesting picture when viewed in the context of the DHFR

206
B. Nammalwar et al. / Bioorg. Med. Chem. 23 (2015) 203–211
Table 1
Biological activity of new DAP inhibitors relative to earlier compounds
Compound
MIC (
anthracis Sterne
l
g/mL) B.
Ki (nM) SEM B.
anthracis DHFR
Ki (nM) SEM B.
anthracis NADPH first
TMPa
RAB1b
BN-53b
4a
>2048
1–4
0.5
>128
>128
4
1–2
2
0.5
4
32
4
4–16
8–16
8
8
8–16
8–16
77233
—
—
—
—
—
—
—
—
9.4 0.2
8.3 0.2
83.4 2.3
15.1 0.6
20.2 0.2
9.3 0.2
9.9 0.2
8.8 0.4
38.8 0.6
1300 11
13.8 0.2
2200 20
>2300
4b
19a
19b
20a
20b
21a
22a
28
29
30
31
32
—
14.6 0.4
1090 167
14.6 0.4
720 20
2200 30
260
220
>2400
310
3
6
6
>2100
>2400
>2200
33
34
700 10
a
TMP (trimethoprim) data from Barrow et al.21
RAB1 and BN-53 data from Bunce et al.7
Figure 2. Extension from the R3 position of the DAP inhibitor is predicted to clash
with co-factor binding. The DHFR binding site accommodates an NADPH co-factor,
shown with purple carbon atoms, as well as a folate substrate or inhibitor (the
dihydrophthalazine-appended DAP inhibitor is shown with yellow carbon atoms).
The protein is displayed as a Coulombic-shaded van der Waals surface (red is acidic,
blue is basic) and with a grey backbone ribbon. Modifications of the inhibitor
scaffold are labeled as discussed in the text. Clashes between the protein and
modified inhibitors were calculated and are visualized by green surface shading. Of
note are green shadings proximal to the R3 modification site, the methylene bridge
and the R1 site. The green sphere is Arg-53 of the DHFR enzyme, which can interact
with R1 of the inhibitor.
b
is of note that compound 28 was relatively unchanged by the order
of addition experiment, as it was predicted to not encroach on the
co-factor site. If our hypothesis of dual-site binding is correct, the
Ki values would no longer be reflective of the enzymatic inhibition
as it would now be a double-competitive reaction with both the
folate substrate and with the co-factor. This may contribute to
the immeasurable Ki values while retaining inhibitory activity at
the whole cell level.
area in antifolate development23–25 Future design of DHFR inhibi-
tors involving this important class of 2,4-diaminopyrimidines
should incorporate more restrained linkages and/or more spatially
targeted modifications to assess this possibility.
3. Conclusions
The current investigation describes the synthesis and biological
evaluation of dihydrophthalazine-appended DAP inhibitors oxi-
dized at the methylene bridge linking the DAP ring to the structure
and modified at the ether groups of the central aromatic ring. The
indication from activity studies of 4a and 4b is a requirement for
flexibility in the methylene linkage between the DAP group and
the central dialkoxy-substituted ring. Alteration of this tetrahedral
geometry to a trigonal planar arrangement, as found in the ketone-
derivatized structures, abolished all cellular growth inhibition
(Table 1). Alterations at R2 and R3 are well tolerated when the
added group is small and conservative, such as the addition of
ethyl groups in compounds 19a–b and 20a–b. This is also true
when a larger and hydrophobic benzyl moiety is added at R2, as
in 21a, or propyl at R3, as in 22a (Fig. 2). This is particularly striking
when viewed versus the ketone modifications in compounds 4a
and 4b, and strongly suggests locations in the inhibitory com-
pounds that can accept substitutions while maintaining potency.
However, larger additions at R3 (viz. 29–34) are less well tolerated
at the level of enzymatic inhibition, while those modifications have
a more tempered effect on the inhibition of whole cell growth. Fur-
ther experimentation is warranted to completely explore the rea-
son for the reduced potency and possible mechanisms for dual-
site inhibition. Our working hypothesis is that this effect results
from extension of the compound beyond the targeted folate sub-
strate pocket. If this occurs, it is sub-optimal due to the strained
conformation the compound would have to adopt to fill this
pocket. It is reasonable that this happens in only a fraction of the
interactions, and in the remaining binding region, this added com-
pound bulk would promote unfavorable interplay with the solvent
surrounding the substrate-binding site. While we cannot rule out
cross-reaction with bacterial targets aside from DHFR in this set-
ting, the concept of creating a dual-pocket inhibitor is a promising
4. Experimental section
Commercial anhydrous N,N-dimethylformamide (DMF) and
dimethyl sulfoxide (DMSO) were stored under dry nitrogen and
transferred by syringe into reactions when needed. Tetrahydrofu-
ran (THF) was dried over potassium hydroxide pellets and distilled
from lithium aluminum hydride prior to use. All other commercial
reagents were used as received.
Unless otherwise specified, all reactions were run under dry
nitrogen in oven-dried glassware. Reactions were monitored by
thin layer chromatography on silica gel GF plates (Analtech, No.
21521). Preparative separations were performed by column chro-
matography on silica gel (Davisil,Ògrade 62, 60–200 mesh) mixed
with UV-active phosphor (Sorbent Technologies, No. UV-05); band
elution was monitored using a hand held UV lamp. Saturated
NaHCO3, NaCl and NH4Cl used in work-up procedures refer to
aqueous solutions. Melting points were uncorrected. FT-IR spectra
were run as thin films on sodium chloride disks. 1H and 13C NMR
spectra were measured on a Varian GEMINI 300 instrument at
300 MHz and 75 MHz, respectively, and referenced to internal tet-
ramethylsilane. Elemental analyses were performed by Atlantic
Microlab, Inc., Norcross, GA 30071.
4.1. Synthesis of structures incorporating an oxidized
methylene bridge (Scheme 1)
4.1.1. (2,4-Diaminopyrimidin-5-yl)(3-iodo-4,5-dimethoxyphen-
yl)methanone (2)
To a stirred solution of 1 (10.0 g, 25.9 mmol) in AcOH (80 mL)
was added K2Cr2O7 (30.5 g, 0.104 mol, 4 equiv), and the solution
was placed in a preheated oil bath at 120 °C and refluxed for a

B. Nammalwar et al. / Bioorg. Med. Chem. 23 (2015) 203–211
207
period of 6 h. The reaction mixture was cooled to 0 °C and poured
4.2. Synthesis of structures modified at R2 and R3 (Scheme 2)
into ice cold water with stirring. The mixture was then extracted
with EtOAc (3 Â 200 mL). The organic extracts were washed with
saturated NaHCO3 (3 Â 100 mL), water, saturated NaCl, dried
(MgSO4), filtered, and concentrated under vacuum to give a thick
brown liquid. The compound was purified using a silica gel column
and eluted with CH2Cl2/MeOH/Et3N (10:4:1). Evaporation of the
solvent yielded product 2 (8.08 g, 78%) as an off-white solid, mp
4.2.1. Benzaldehydes 6–9 and 23
These compounds were prepared on a 54-mmol scale via a two-
step literature procedure20 in the following yields: 6 (80%), 7 (92%),
8 (92%), 9 (78%) and 23 (88%). The spectral data matched those
reported.20
206–208 °C. IR: 3448, 3382, 3326, 1598 cmÀ1
;
1H NMR
4.2.2. 3-Morpholinopropionitrile (10)
(DMSO-d6): d 8.28 (br s, 1H), 8.14 (s, 1H), 7.54 (br s, 1H), 7.45 (d,
J = 1.6 Hz, 1H), 7.20 (d, J = 1.6 Hz, 1H), 7.13 (br s, 2H), 3.85 (s,
3H), 3.78 (s, 3H); 13C NMR (DMSO-d6): d 191.5, 164.3, 163.8,
163.6, 152.0, 150.1, 137.2, 129.8, 113.4, 103.3, 92.4, 60.0, 56.1.
This compound was prepared on a 0.28-mol scale according to a
literature procedure.26 The crude product was vacuum distilled to
give 10 (37.2 g, 95%) as a colorless liquid, bp 88–90 °C (0.5 mmHg)
[lit.26 bp 149 °C (20 mmHg)].
4.2.3. 5-(3,4-Diethoxy-5-iodobenzyl)pyrimidine-2,4-diamine
(15)
4.1.2. ( )-(E)-3-[5-(2,4-Diaminopyrimidine-5-carbonyl)-2,3-
dimethoxyphenyl]-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-
1-one (4a)
To a stirred solution of NaOMe (1.69 g, 31.3 mmol, 1.0 equiv) in
DMSO (25 mL) was added dropwise 3-morpholinopropionitrile 10
(5.25 g, 37.5 mmol, 1.2 equiv) at 65 °C. The reaction mixture was
heated to 90 °C, followed by the addition of a warm solution of 6
(10.0 g, 31.3 mmol) in DMSO (15 mL) over 20 min. Stirring was
continued at 90 °C for 45 min. The crude reaction mixture was then
cooled in an ice bath, poured into ice-cold water, and extracted
with CH2Cl2 (3 Â 100 mL). The combined organic extracts were
washed with saturated NaCl (1 Â 100 mL), dried (MgSO4), filtered,
and concentrated under vacuum to give crude 11 as dark red oil.
The crude product 11 was re-dissolved in dry ethanol (150 mL),
aniline hydrochloride (4.46 g, 34.4 mol, 1.1 equiv) was added, and
the reaction mixture was heated under reflux for 1 h. Guanidine
hydrochloride (7.16 g, 75.0 mmol, 2.4 equiv), followed by NaOMe
(6.75 g, 125 mmol, 4 equiv), was then added to the reaction mix-
ture under hot conditions, and refluxing was continued for 4 h.
The reaction mixture was concentrated to 1/4 of the volume under
vacuum, and ice-cold water was added, resulting in the formation
of a pale yellow solid. The solid was collected, washed with ice-
cold ethanol, water, and finally with Et2O to give an off-white solid.
The product was recrystallized using ethanol/water (4:1) to give 15
(10.4 g, 80%) as a white solid, mp 173–175 °C. IR: 3472, 3327,
To a stirred solution of 2 (1.00 g, 2.50 mmol) in dry DMF
(7 mL) was added
a solution of ( )-3a (657 mg, 2.88 mmol,
1.15 equiv)7 in DMF (1 mL), followed by N-ethylpiperidine
(340 mg, 0.41 mL, 3.00 mmol 1.2 equiv) and Pd(OAc)2 (20 mg,
0.089 mmol). The reaction mixture was heated at 140 °C for
20 h and then gradually cooled to 0 °C. Isolation of the product
was achieved by pouring the cooled reaction mixture directly
onto a 50-cm  2.5-cm silica gel flash chromatography column
packed in CH2Cl2. Impurities were eluted using CH2Cl2, and the
final product was collected using CH2Cl2/MeOH/Et3N (95:5:1).
Evaporation of the solvent gave a pale yellow solid, which was
further purified using a 15-cm  2-cm silica gel column, packed
with CH2Cl2/MeOH/Et3N (95:5:1). This second chromatography
removed colored impurities as well as minor contaminants. Evap-
oration of the solvent gave 4a as an off-white solid. The com-
pound was further purified by recrystallization from MeOH to
give a white solid (990 mg, 79%), mp 212–214 °C. IR: 3377,
3143, 1660, 1593 cmÀ1 1H NMR (DMSO-d6): d 8.33 (br s, 2H),
;
8.18 (s, 1H), 7.93 (s, 1H), 7.89 (d, J = 16.5 Hz, 1H), 7.68 (d,
J = 16.5 Hz, 1H), 7.58–7.37 (complex m, 5H), 7.24 (s, 1H), 7.12
(br s, 2H), 5.84 (t, J = 6.6 Hz, 1H), 3.88 (s, 3H), 3.87 (s, 3H), 1.54
(m, 2H), 1.17 (sextet, J = 7.1 Hz, 2H), 0.82 (t, J = 7.1 Hz, 3H); 13C
NMR (DMSO-d6): d 192.8, 165.4, 164.4, 163.9, 163.6, 152.5,
149.3, 143.0, 135.9, 135.5, 133.6, 131.7, 128.3, 128.0, 126.5,
126.1, 123.6, 119.2, 119.0, 11.7, 103.5, 60.9, 56.1, 50.4, 36.9,
17.8, 13.7. Anal. Calcd for C27H28N6O4; C, 64.79; H, 5.64; N,
16.79. Found: C, 64.63; H, 5.75; N, 16.51.
3176 cmÀ1 1H NMR (DMSO-d6): d 7.57 (s, 1H), 7.15 (s, 1H), 6.95
;
(s, 1H), 6.19 (br s, 2H), 5.82 (br s, 2H), 4.01 (q, J = 7.1 Hz, 2H),
3.93 (q, J = 7.1 Hz, 2H), 3.53 (s, 2H), 1.32 (coincident t, J = 7.1 Hz,
6H); 13C NMR (DMSO-d6): d 162.4, 162.2, 156.0, 151.1, 145.8,
138.5, 129.1, 114.6, 105.3, 93.2, 68.2, 63.9, 31.7, 15.7, 14.6.
4.2.4. 5-(4-Ethoxy-3-iodo-5-methoxybenzyl)pyrimidine-2,4-
diamine (16)
4.1.3. ( )-(E)-3-[5-(2,4-Diaminopyrimidine-5-carbonyl)-2,3-
dimethoxyphenyl]-1-(1-isobutenyl-2(1H)-phthalazinyl)-2-
propen-1-one (4b)
This compound was prepared as described above for compound
15 using 7 (10.0 g, 32.7 mmol), 10 (5.49 g, 39.2 mmol, 1.2 equiv),
and NaOMe (1.77 g, 32.7 mmol, 1.0 equiv) in DMSO (40 mL) to give
crude 12 as a dark red oil. This oil in ethanol (150 mL) was further
treated with PhNH2ÁHCl (5.08 g, 39.2 mmol, 1.2 equiv), guani-
dineÁHCl (7.50 g, 78.5 mmol, 2.4 equiv), and NaOMe (7.06 g,
130.7 mmol, 4.0 equiv) to give 16 (10.2 g, 78%) as a white solid,
This compound was prepared as above using
2 (1.00 g,
2.50 mmol), ( )-3b (691 mg, 2.88 mmol, 1.15 equiv),7 N-ethylpi-
peridine (340 mg, 0.41 mL, 3.00 mmol 1.2 equiv), and Pd(OAc)2
(20 mg, 0.089 mmol) in dry DMF (8 mL) under N2 to give 4b
(960 mg, 75%) as an off-white solid, mp 138–140 °C. IR: 3320,
mp 170–172 °C. IR: 3477, 3323, 3174 cmÀ1 1H NMR (DMSO-d6):
;
3179, 1655, 1594, cmÀ1 1H NMR (DMSO-d6): d 8.65 (br s, 1H),
;
d 7.57 (s, 1H); 7.14 (d, J = 1.8 Hz, 1H), 6.97 (d, J = 1.8 Hz, 1H), 6.16
(br s, 2H), 5.77 (br s, 2H), 3.90 (q, J = 7.0 Hz, 2H), 3.76 (s, 3H),
3.53 (s, 2H), 1.31 (t, J = 7.0 Hz, 3H); 13C NMR (DMSO-d6): d 162.4,
162.1, 156.1, 152.0, 145.5, 138.7, 129.1, 113.7, 105.3, 93.2, 68.2,
55.8, 31.7, 15.6.
8.39 (br s, 1H), 8.11 (d, J = 16.5 Hz, 1H), 7.67 (d, J = 16.5 Hz, 1H),
7.62 (s, 1H), 7.49 (d, J = 1.6 Hz, 1H), 7.43 (td, J = 7.1, 1.6 Hz, 1H),
7.33 (t, J = 7.1 Hz, 1H), 7.27 (d, J = 6.6 Hz, 1H), 7.16 (d, J = 6.6 Hz,
1H), 7.15 (d, J = 1.6 Hz, 1H), 6.58 (d, J = 9.9 Hz, 1H), 5.67 (br s,
1H), 5.47 (br s, 2H), 5.30 (d, J = 9.9 Hz, 1H), 3.93 (s, 3H), 3.91 (s,
3H), 2.05 (s, 3H), 1.65 (s, 3H); 13C NMR (DMSO-d6): d 194.3,
166.2, 164.6, 164.4, 163.4, 153.1, 150.7, 141.5, 136.3, 134.6,
134.5, 134.2, 131.9, 129.5, 127.9, 126.2, 125.8, 123.3, 122.1,
120.1, 119.7, 113.2, 105.2, 61.5, 56.0, 50.0, 25.7, 18.6. Anal. Calcd
for C28H28N6O4Á0.4 H2O: C, 64.70; H, 5.58; N, 16.17. Found: C,
64.67; H, 5.63; N, 15.83.
4.2.5. 5-[4-(Benzyloxy)-3-iodo-5-methoxybenzyl]pyrimidine-
2,4-diamine (17)
This compound was prepared as described above for compound
15 using 8 (10.0 g, 27.2 mmol), 10 (4.57 g, 32.6 mmol, 1.2 equiv),
and NaOMe (1.47 g, 27.2 mmol, 1.0 equiv) in DMSO (40 mL) to give
crude 13 as a dark brown oil. This oil in ethanol (150 mL) was

208
B. Nammalwar et al. / Bioorg. Med. Chem. 23 (2015) 203–211
further treated with PhNH2ÁHCl (4.22 g, 32.6 mmol, 1.2 equiv),
guanidineÁHCl (6.23 g, 65.2 mmol, 2.4 equiv), and NaOMe (5.87 g,
109 mol, 4.0 equiv) to afford the product. The work-up procedure
was altered for this compound. After completion, the reaction
mixture was concentrated to dryness under vacuum, 100 mL of
ice-cold water was added, and the compound was extracted with
EtOAc (3 Â 150 mL). The combined organic extracts were washed
with saturated NaCl (1 Â 100 mL), dried (MgSO4), filtered, and con-
centrated under vacuum. The crude mixture was then purified by
column chromatography using a 30-cm  2.5-cm silica gel column
eluted with CH2Cl2/MeOH/Et3N (98:2:1) to afford 17 (9.03 g, 72%)
1.31 (t, J = 7.1, 3H); 13C NMR (DMSO-d6): d 165.3, 163.3, 156.9,
151.9, 145.5, 142.0, 137.2, 134.4, 133.8, 133.5, 132.2, 128.3,
128.2, 126.2 (2C), 123.1, 122.1, 119.0, 117.9, 115.8, 107.8, 68.7,
63.9, 49.2, 31.8, 25.3, 18.4, 15.5, 14.7 (one aromatic C unresolved).
Anal. Calcd for C30H34N6O3Á5.7 H2O: C, 57.26; H, 7.27; N, 13.35.
Found: C, 57.12; H, 7.00; N, 13.27.
4.2.9. ( )-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2-etho-
xy-3-methoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-prop-
en-1-one (20a)
This compound was prepared as described above for 4a using
16 (1.00 g, 2.50 mmol), ( )-3a (656 mg, 2.88 mmol, 1.15 equiv),
N-ethylpiperidine (339 mg, 0.41 mL, 3.00 mmol 1.2 equiv), and
Pd(OAc)2 (20 mg, 0.089 mmol) in dry DMF (8 mL) under N2 to give
20a (925 mg, 74%) as an off-white solid, mp 145–147 °C. IR: 3334,
3196, 1650, 1603 cmÀ1; 1H NMR (DMSO-d6): d 7.94 (s, 1H), 7.89 (d,
J = 15.9 Hz, 1H), 7.67 (d, J = 15.9 Hz, 1H), 7.56 (s, 1H), 7.53 (m, 2H),
7.46 (d, J = 6.8 Hz, 1H), 7.40 (t, J = 7.1 Hz, 1H), 7.27 (s, 1H), 7.07 (br
s, 2H), 7.02 (s, 1H), 6.57 (br s, 2H), 5.84 (t, J = 6.6 Hz, 1H), 3.97 (q,
J = 7.1 Hz, 2H), 3.79 (s, 3H), 3.63 (s, 2H), 1.54 (m, 2H), 1.30 (t,
J = 7.1 Hz, 3H), 1.17 (sextet, J = 7.1 Hz, 2H), 0.82 (t, J = 7.1 Hz, 3H);
13C NMR (DMSO-d6): d 165.6, 163.0, 158.4, 152.7, 148.4, 145.2,
142.7, 137.1, 135.0, 133.6, 131.9, 131.7, 128.2, 126.5, 126.1,
123.6, 118.9, 117.9, 114.8, 107.2, 68.7, 55.8, 50.3, 36.8, 32.0, 17.8,
15.4, 13.6. Anal. Calcd for C28H32N6O3Á4.5 H2O: C, 57.82; H, 7.10;
N, 14.45. Found: C, 57.89; H, 6.94; N, 14.49.
as a yellow solid, mp 158–160 °C. IR: 3330, 3178 cmÀ1 1H NMR
;
(DMSO-d6): d 7.59 (s, 1H), 7.53 (m, 2H), 7.38 (m, 3H), 7.19 (s,
1H), 7.03 (s, 1H), 6.21 (br s, 2H), 5.82 (br s, 2H), 4.89 (s, 2H), 3.82
(s, 3H), 3.57 (s, 2H); 13C NMR (DMSO-d6): d 162.4, 162.2, 156.0,
152.1, 145.1, 139.0, 137.1, 129.3, 128.2 (2C), 128.0, 113.8, 105.3,
92.9, 73.7, 55.9, 31.8.
4.2.6. 5-(3-Iodo-4,5-methylenedioxybenzyl)pyrimidine-2,4-
diamine (18)
This compound was prepared as described above for compound
15 using 9 (9.03 g, 32.7 mmol), 10 (5.49 g, 39.2 mmol, 1.2 equiv),
and NaOMe (1.77 g, 32.7 mmol, 1.0 equiv) in DMSO (40 mL) to give
crude 14 as a dark red oil. This oil in ethanol (150 mL) was further
treated with PhNH2ÁHCl (5.08 g, 39.2 mmol, 1.2 equiv), guani-
dineÁHCl (7.49 g, 78.5 mmol, 2.4 equiv), and NaOMe (7.06 g,
130.7 mmol, 4.0 equiv) to give 18 (9.07 g, 75%) as a white solid,
mp 218–220 °C. IR: 3440, 3322, 3214 cmÀ1
;
1H NMR (CDCl3): d
4.2.10. ( )-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2-eth-
oxy-3-methoxyphenyl}-1-(1-isobutenyl-2(1H)-phthalazinyl)-2-
propen-1-one (20b)
7.54 (s, 1H), 7.02 (s, 1H), 6.78 (s, 1H), 6.14 (br s, 2H), 6.03 (s, 2H),
5.76 (br s, 2H), 3.49 (s, 2H); 13C NMR (CDCl3): d 162.2, 162.1,
155.9, 147.3, 146.2, 136.5, 128.9, 108.9, 105.6, 100.5, 71.3, 31.7.
This compound was prepared as described above for 4a using
16 (1.00 g, 2.50 mmol), ( )-3b (690 mg, 2.88 mmol, 1.15 equiv),
N-ethylpiperidine (339 mg, 0.41 mL, 3.00 mmol 1.2 equiv), and
Pd(OAc)2 (20 mg, 0.089 mmol) in dry DMF (8 mL) under N2 to give
20b (896 mg, 70%) as an off-white solid, mp 140–142 °C. IR: 3354,
3146, 1662, 1591 cmÀ1; 1H NMR (DMSO-d6): d 7.93 (s, 1H), 7.89 (d,
J = 15.9 Hz, 1H), 7.64 (d, J = 15.9 Hz, 1H), 7.60 (s, 1H), 7.52 (m, 2H),
7.43 (t, J = 7.1 Hz, 1H), 7.31 (d, J = 7.1 Hz, 1H), 7.25 (s, 1H), 7.01 (s,
1H), 6.66 (br s, 2H), 6.51 (d, J = 9.9 Hz, 1H), 6.19 (br s, 2H), 5.25 (d,
J = 9.9 Hz, 1H), 3.97 (q, J = 7.1 Hz, 2H), 3.80 (s, 3H), 3.63 (s, 2H), 1.97
(s, 3H), 1.61 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H); 13C NMR (DMSO-d6): d
165.3, 162.6, 160.3, 152.6, 152.0, 145.1, 142.0, 137.2, 135.7, 133.8,
133.5, 132.2, 128.2 (2C), 126.2, 126.1, 123.1, 122.2, 118.8, 117.9,
114.7, 106.5, 68.7, 55.8, 49.2, 32.2, 25.3, 18.4, 15.4. Anal. Calcd
for C29H32N6O3Á2.4 H2O: C, 62.65; H, 6.38; N, 15.10. Found: C,
62.52; H, 6.10; N, 14.75.
4.2.7. ( )-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
diethoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-one
(19a)
This compound was prepared as described above for 4a using
15 (1.00 g, 2.42 mmol), ( )-3a (634 mg, 2.78 mmol, 1.15 equiv),
N-ethylpiperidine (328 mg, 0.40 mL, 2.90 mmol 1.2 equiv), and
Pd(OAc)2 (20 mg, 0.089 mmol) in dry DMF (8 mL) under N2 to give
19a (968 mg, 78%) as an off-white solid, mp 118–120 °C. IR: 3334,
1652, 1630 cmÀ1 1H NMR (DMSO-d6): d 7.93 (s, 1H), 7.89 (d,
;
J = 15.9 Hz, 1H), 7.64 (d, J = 15.9 Hz, 1H), 7.57 (s, 1H), 7.52 (m,
2H), 7.45 (d, J = 6.8 Hz, 1H), 7.39 (t, J = 7.1 Hz, 1H), 7.23 (s, 1H),
6.97 (s, 1H), 6.53 (br s, 2H), 6.06 (br s, 2H), 5.84 (t, J = 6.6 Hz,
1H), 4.01 (overlapping q, J = 7.1 Hz, 4H), 3.59 (s, 2H), 1.54 (m,
2H), 1.34 (t, J = 6.6 Hz, 3H), 1.30 (t, J = 7.1 Hz, 3H), 1.17 (sextet,
J = 7.1 Hz, 2H), 0.82 (t, J = 7.1 Hz, 3H); 13C NMR (DMSO-d6): d
165.6, 162.5, 161.0, 153.4, 151.8, 145.3, 142.8, 137.2, 135.9,
133.6, 131.7, 128.3, 128.2, 126.5, 126.1, 123.7, 118.7, 117.7,
115.6, 106.3, 68.7, 63.7, 50.4, 36.8, 32.3, 17.8, 15.5, 14.7, 13.7. Anal.
Calcd for C29H34N6O3Á2.5 H2O: C, 62.24; H, 7.06; N, 15.02. Found: C,
62.46; H, 7.26; N, 15.05.
4.2.11. ( )-(E)-3-{2-(Benzyloxy)-5-[(2,4-diamino-5-pyrim-idinyl)
methyl]-3-methoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-
propen-1-one (21a)
This compound was prepared as described above for 4a using
17 (1.00 g, 2.16 mmol), ( )-3a (568 mg, 2.49 mmol, 1.15 equiv),
N-ethylpiperidine (294 mg, 0.36 mL, 2.60 mmol 1.2 equiv), and
Pd(OAc)2 (20 mg, 0.089 mmol) in dry DMF (8 mL) under N2 to give
21a (850 mg, 70%) as an off-white solid, mp 123–125 °C. IR: 3474,
4.2.8. ( )-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2,3-
diethoxyphenyl}-1-(1-isobutenyl-2(1H)-phthalazinyl)-2-
propen-1-one (19b)
3342, 3181, 1654, 1605 cmÀ1 1H NMR (DMSO-d6): d 8.06 (d,
;
This compound was prepared as described above for 4a using
15 (1.00 g, 2.42 mmol), ( )-3b (667 mg, 2.78 mmol, 1.15 equiv),
N-ethylpiperidine (328 mg, 0.40 mL, 2.90 mmol 1.2 equiv), and
Pd(OAc)2 (20 mg, 0.089 mmol) in dry DMF (8 mL) under N2 to give
19b (915 mg, 72%) as an off-white solid, mp 251–252 °C. IR: 3325,
3147, 1661, 1590 cmÀ1; 1H NMR (DMSO-d6): d 7.91 (s, 1H), 7.87 (d,
J = 15.9 Hz, 1H), 7.63 (d, J = 15.9 Hz, 1H), 7.54 (s, 1H), 7.51 (m, 2H),
7.42 (m, 3H), 7.31 (d, J = 7.1 Hz, 1H), 7.26 (s, 1H), 7.00 (s, 1H), 6.89
(br s, 2H), 6.50 (d, J = 9.9 Hz, 1H), 5.24 (d, J = 9.9 Hz, 1H), 4.02 (m,
4H), 3.63 (s, 2H), 1.96 (s, 3H), 1.60 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H),
J = 15.9 Hz, 1H), 7.80 (s, 1H), 7.64 (d, J = 15.9 Hz, 1H), 7.62 (s, 1H),
7.46 (m, 3H), 7.40–7.25 (complex m, 5H), 7.18 (d, J = 7.1 Hz, 1H),
7.11 (s, 1H), 6.67 (s, 1H), 5.90 (t, J = 6.6 Hz, 1H), 5.00 (s, 2H), 4.77
(br s, 2H), 4.60 (br s, 2H), 3.81 (s, 3H), 3.68 (s, 2H), 1.65 (m, 2H),
1.27 (m, 2H), 0.86 (t, J = 7.1 Hz, 3H); 13C NMR (DMSO-d6): d
166.5, 162.6, 162.2, 156.7, 153.7, 145.9, 142.2, 137.4, 137.0,
134.2, 134.1, 131.4 130.2, 128.6, 128.3, 128.00, 127.95, 126.5,
125.6, 124.0, 118.9, 118.8, 112.9, 106.4, 75.4, 55.9, 51.3, 37.3,
34.4, 18.3, 13.8. Anal. Calcd for C33H34N6O3Á0.3 H2O: C, 69.77; H,
6.14; N, 14.79. Found: C, 69.70; H, 6.14; N, 14.44.

B. Nammalwar et al. / Bioorg. Med. Chem. 23 (2015) 203–211
209
4.2.12. ( )-(E)-3-{5-[(2,4-Diaminopyrimidin-5-yl)methyl]-2,3-
methylenedioxyphenyl}-1-(1-propylphthalazin-2(1H)-yl)-2-
propen-1-one (22a)
was removed under vacuum. This process was repeated 5–6 times
to produce an off-white solid. This solid was triturated with Et2O,
and the product was collected to give 27 (0.86 g, 94%) as a white
This compound was prepared as described above for 4a using
18 (1.00 g, 2.70 mmol), ( )-3a (709 mg, 3.11 mmol, 1.15 equiv),
N-ethylpiperidine (366 mg, 0.44 mL, 3.24 mmol 1.2 equiv), and
Pd(OAc)2 (20 mg, 0.089 mmol) in dry DMF (8 mL) under N2 to give
22a (901 mg, 71%) as an off-white solid, mp 123–125 °C. IR: 3424,
solid, mp 135–137 °C. IR: 3481, 3345, 3204, 1652, 1617 cmÀ1 1H
;
NMR (DMSO-d6):
d 9.50 (br s, 1H), 7.94 (s, 1H), 7.86 (d,
J = 15.9 Hz, 1H), 7.63 (d, J = 15.9 Hz, 1H), 7.58–7.48 (complex m,
3H), 7.46 (d, J = 7.1 Hz, 1H), 7.39 (d, J = 7.1 Hz, 1H), 7.15 (s, 1H),
6.70 (s, 1H), 6.16 (br s, 2H), 5.85 (t, J = 6.6 Hz, 1H), 5.79 (br s,
2H), 3.73 (s, 3H), 3.54 (s, 2H), 1.54 (m, 2H), 1.17 (sextet,
J = 7.1 Hz, 2H), 0.82 (t, J = 7.1 Hz, 3H); 13C NMR (DMSO-d6): d
165.6, 162.3, 162.2, 155.6, 150.2, 145.0, 142.7, 137.1, 136.3,
133.6, 131.7, 128.2, 127.8, 126.5, 126.0, 123.7, 118.1, 117.3,
105,5, 60.5, 50.3, 36.8, 32.0, 17.8, 14.0, 13.7. Anal. Calcd for C26H28-
N6O3Á0.5 H2O: C, 64.85; H, 6.07; N, 17.45. Found: C, 64.93; H, 5.94;
N, 17.15.
3312, 3100, 1635, 1599 cmÀ1 1H NMR (DMSO-d6): d 7.96 (s, 1H),
;
7.74 (d, J = 16.0 Hz, 1H), 7.55 (s, 1H), 7.54–7.41 (complex, 3H),
7.50 (d, J = 16.0 Hz, 1H), 7.38 (d, J = 7.4 Hz, 1H), 7.06 (s, 1H), 6.82
(s, 1H), 6.13 (2s, 4H), 5.85 (t, J = 6.7 Hz, 1H), 5.73 (br s, 2H), 3.53
(s, 2H), 1.52 (m, 2H), 1.17 (sextet, J = 7.4 Hz, 2H), 0.81 (t,
J = 7.4 Hz, 3H); 13C NMR (DMSO-d6): d 165.5, 162.3, 162.1, 155.7,
147.8, 144.4, 143.0, 136.8, 134.5, 133.6, 131.7, 128.3, 126.5,
126.1, 123.6, 122.0, 119.2, 116.7, 110.0, 105.8, 101.7, 50.4, 36.8,
32.2, 17.8, 13.7. Anal. Calcd for C26H26N6O3Á0.6 EtOH: C, 65.58; H,
5.99; N, 16.87. Found: C, 65.40; H, 5.85; N, 16.76.
4.3.4. ( )-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2-met-
hoxy-3-propoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-pro-
pen-1-one (28)
4.3. Synthesis of compounds modified at R3 (Scheme 3)
To a stirred solution of 27 (150 mg, 0.318 mmol) in CH2Cl2
(8 mL) was added DBU (53 mg, 0.052 mL, 0.350 mmol, 1.1 equiv),
and the solution was stirred for 30 min. To the reaction mixture
was added dropwise propyl bromide (43 mg, 0.032 mL,
0.350 mmol, 1.1 equiv), and stirring was continued for 20 h. The
reaction mixture was evaporated to dryness and purified on a
20-cm  20-cm silica gel, preparative thin layer chromatography
plate eluted with CH2Cl2/MeOH/Et3N (95:5:1). The product band
was washed with CH2Cl2/MeOH/Et3N (95:5:1) to afford an off-
white solid. This solid was triturated with Et2O to remove Et3N,
and the compound was filtered and dried under vacuum to afford
28 (144 mg, 88%) as a white solid, mp 194–195 °C. IR: 3342, 3157,
4.3.1. 5-[3-Iodo-4-methoxy-5-(methoxymethoxy)benzyl]-
pyrimidine-2,4-diamine (25)
This compound was prepared as described above for compound
15 using 2320 (10.0 g, 24.0 mmol), 10 (4.03 g, 28.8 mmol, 1.2 equiv)
and NaOMe (1.30 g, 24.0 mmol, 1.0 equiv) in DMSO (40 mL) to give
intermediate 24 (9.24 g, 90%) as a dark brown oil. This oil in etha-
nol (150 mL) was treated with PhNH2ÁHCl (3.10 g, 24.0 mmol,
1.0 equiv), guanidineÁHCl (5.50 g, 57.6 mmol, 2.4 equiv), and
NaOMe (5.18 g, 96.0 mmol, 4.0 equiv) to afford a dark yellow solu-
tion. The work-up procedure was altered for this compound. The
crude reaction mixture was concentrated to dryness under vac-
uum, 100 mL of ice-cold water was added, and the compound
was extracted with EtOAc (3 Â 150 mL). The combined organic
extracts were washed with saturated NaCl (1 Â 100 mL), dried
(MgSO4), filtered, and concentrated under vacuum. The crude mix-
ture was then purified by column chromatography using 30-
cm  2.5-cm silica gel column eluted with CH2Cl2/MeOH/Et3N
(98:2:1) to afford 25 (8.29 g, 83%) as a white solid, mp 123–
1659, 1637 cmÀ1 1H NMR (DMSO-d6): d 7.94 (s, 1H), 7.87 (d,
;
J = 15.9 Hz, 1H), 7.64 (d, J = 15.9 Hz, 1H), 7.53 (m, 3H), 7.46 (d,
J = 7.1 Hz, 1H), 7.40 (d, J = 7.1 Hz, 1H), 7.28 (s, 1H), 7.09 (br s,
2H), 7.01 (s, 1H), 6.59 (br s, 2H), 5.85 (t, J = 6.3 Hz, 1H), 3.94 (t,
J = 6.0 Hz, 2H), 3.78 (s, 3H), 3.62 (s, 2H), 1.77 (sextet, J = 6.6 Hz,
2H), 1.54 (m, 2H), 1.17 (sextet, J = 7.1 Hz, 2H), 1.01 (t, J = 7.1 Hz,
3H), 0.82 (t, J = 7.1 Hz, 3H); 13C NMR (DMSO-d6): d 165.6, 163.0,
158.4, 151.9, 148.3, 146.3, 142.8, 136.7, 135.1, 133.6, 131.7,
128.3, 127.9, 126.5, 126.1, 123.6, 118.6, 117.9, 115.7, 107.3, 69.9,
60.7, 50.3, 36.8, 32.0, 22.1, 17.8, 13.7, 10.6. Anal. Calcd for C29H34-
N6O3Á3.7 H2O: C, 59.92; H, 7.18; N, 14.46. Found: C, 59.83; H, 7.19;
N, 14.39.
125 °C. IR: 3461, 3153, 1625 cmÀ1 1H NMR (DMSO-d6): d 7.54 (s,
;
1H), 7.22 (d, J = 1.6 Hz, 1H), 7.05 (d, J = 1.6 Hz, 1H), 6.14 (br s,
2H), 5.76 (br s, 2H), 5.9 (s, 2H), 3.70 (s, 3H), 3.51 (s, 2H), 3.40 (s,
3H); 13C NMR (DMSO-d6): d 162.4, 162.2, 156.1, 149.3, 147.3,
138.9, 130.7, 117.2, 105.1, 94.6, 92.6, 60.0, 56.0, 31.6.
4.3.5. ( )-(E)-5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2-methoxy-
3-[3-oxo-3-(1-propyl-2(1H)-phthalazinyl)-1-propen-1-yl]phenyl
benzoate (29)
4.3.2. ( )-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2-met-
hoxy-3-(methoxymethoxy)-phenyl]-1-(1-propyl-2(1H)-phthala-
zinyl)-2-propen-1-one (26)
This compound was prepared as described above for compound
28 using 27 (150 mg, 0.318 mmol), DBU (53 mg, 0.052 mL,
0.350 mmol, 1.1 equiv), and benzoyl chloride (49 mg, 0.041 mL,
0.350 mmol, 1.1 equiv) in CH2Cl2 (8 mL) to give 29 (169 mg, 92%)
as an off-white solid, mp 125–127 °C. IR: 3335, 3194, 1739, 1654,
This compound was prepared as described above for 4a using
25 (1.00 g, 2.40 mmol), with ( )-3a (630 mg, 2.76 mmol,
1.15 equiv), N-ethylpiperidine (326 mg, 0.40 mL, 2.88 mmol
1.2 equiv), and Pd(OAc)2 (20 mg, 0.089 mmol) in dry DMF (8 mL)
under N2 to give 26 (719 mg, 58%) as a brown solid. 1H NMR
revealed the product 26 contained compound 27 (hydroxy deriva-
tive) in the ratio of (8:2) as a mixture. The product was taken to the
next step without further purification or analysis.
1604 cmÀ1 1H NMR (DMSO-d6): d 8.20 (d, J = 7.7 Hz, 2H), 8.01 (d,
;
J = 15.9 Hz, 1H), 7.78 (s, 1H), 7.71 (d, J = 15.9 Hz, 1H), 7.66 (m,
2H), 7.52 (t, J = 7.7 Hz, 2H), 7.44 (obscured, 1H), 7.42 (s, 1H), 7.36
(t, J = 7.1 Hz, 1H), 7.29 (d, J = 7.1 Hz, 1H), 7.18 (d, J = 7.1 Hz, 1H),
6.99 (s, 1H), 5.90 (t, J = 6.6 Hz, 1H), 5.05 (br s, 2H), 4.76 (br s,
2H), 3.81 (s, 3H), 3.72 (s, 2H), 1.64 (m, 2H), 1.26 (m, 2H), 0.87 (t,
J = 7.1 Hz, 3H); 13C NMR (DMSO-d6): d 166.3, 164.7, 163.6, 161.8,
155.9, 144.9, 144.6, 142.5, 136.4, 134.2, 134.1, 133.8, 131.4,
130.9, 130.3, 128.9, 128.7, 128.0, 126.5, 125.7, 125.0, 124.2,
123.9, 119.6, 105.8, 62.1, 51.4, 37.3, 33.6, 18.3, 13.8. Anal. Calcd
for C33H32N6O4Á5.3 H2O: C, 58.97; H, 6.39; N, 12.50. Found: C,
58.98; H, 6.52; N, 12.50.
4.3.3. ( )-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-3-hyd-
roxy-2-methoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-
propen-1-one (27)
To a stirred solution of crude 26 (1.00 g, 1.94 mmol) in CH2Cl2
(35 mL) was added MeOHÁHCl (35 mL), and the reaction mixture
was stirred for 24 h at room temperature. Evaporation of the sol-
vent gave a dark brown liquid. To remove all traces of MeOHÁHCl,
a 50-mL portion of Et2O/CH2Cl2 (1:1) was added, and the solvent

210
B. Nammalwar et al. / Bioorg. Med. Chem. 23 (2015) 203–211
4.3.6. ( )-(E)-5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2-methoxy-
3-[3-oxo-3-(1-propyl-2(1H)-phthalazinyl)-1-propen-1-yl]phenyl
4-methoxybenzoate (30)
0.350 mmol, 1.1 equiv), and 4-fluorobenzyl bromide (66 mg,
0.044 mL, 0.350 mmol, 1.1 equiv) in CH2Cl2 (8 mL) to give 33
(160 mg, 87%) as a yellow solid, mp 115–117 °C. IR: 3473, 3338,
This compound was prepared as described above for compound
28 using 27 (150 mg, 0.318 mmol), DBU (53 mg, 0.052 mL,
0.350 mmol, 1.1 equiv), and 4-methoxybenzoyl chloride (60 mg,
0.047 mL, 0.350 mmol, 1.1 equiv) in CH2Cl2 (8 mL) to give 30
(168 mg, 87%) as an off-white solid, mp 124–126 °C. IR: 3474,
3183, 1649, 1605 cmÀ1 1H NMR (CDCl3): d 8.07 (d, J = 15.9 Hz,
;
1H), 7.78 (s, 1H), 7.67 (s, 1H), 7.66 (d, J = 15.9 Hz, 1H), 7.45 (t,
J = 7.1 Hz, 1H), 7.40–7.26 (complex m, 4H), 7.19 (d, J = 7.1 Hz,
1H), 7.16 (s, 1H), 7.05 (t, J = 8.8 Hz, 2H), 6.66 (d, J = 1.1 Hz, 1H),
5.91 (t, J = 6.6 Hz, 1H), 5.00 (s, 2H), 4.78 (br s, 2H), 4.52 (br s,
2H), 3.87 (s, 3H), 3.65 (s, 2H), 1.65 (m, 2H), 1.28 (m, 2H), 0.87 (t,
J = 7.1 Hz, 3H); 13C NMR (CDCl3): d 166.5, 162.55, 162.50 (d,
J = 245.0 Hz), 162.2, 156.7, 152.3, 147.9, 142.4, 137.1, 134.3,
133.9, 132.3, 131.4, 130.0, 129.3 (J = 7.7 Hz), 128.0, 126.5, 125.7,
124.0, 119.4, 118.9, 115.5 (J = 21.3 Hz), 115.1, 106.3, 70.2, 61.4,
51.3, 37.3, 34.2, 18.3, 13.8. Anal. Calcd for C33H33FN6O3Á0.5 H2O:
C, 67.22; H, 5.81; N, 14.25. Found: C, 67.04; H, 5.99; N, 14.00.
3344, 3188, 1732, 1605 cmÀ1 1H NMR (DMSO-d6): d 8.14 (d,
;
J = 8.8 Hz, 2H); 8.01 (d, J = 15.9 Hz, 1H); 7.81 (s, 1H), 7.70 (d,
J = 15.9 Hz, 1H), 7.67 (s, 1H), 7.48–7.40 (complex m, 2H), 7.36 (t,
J = 7.1 Hz, 1H), 7.30 (d, J = 7.1 Hz, 1H), 7.18 (d, J = 7.1 Hz, 1H),
6.99 (complex m, 3H), 5.90 (t, J = 6.6 Hz, 1H), 4.77 (br s, 2H), 4.65
(br s, 2H), 3.90 (s, 3H), 3.80 (s, 3H), 3.72 (s, 2H), 1.65 (m, 2H),
1.28 (m, 2H), 0.87 (t, J = 7.1 Hz, 3H); 13C NMR (DMSO-d6): d
166.4, 164.4, 164.1, 162.5, 162.3, 157.1, 150.0, 144.7, 142.5,
136.6, 134.3, 134.2, 132.0, 131.4, 130.8, 128.0, 126.5, 125.7,
124.9, 124.4, 124.0, 121.2, 119.5, 113.9, 105.8, 62.0, 55.5, 51.4,
37.3, 33.7, 18.3, 13.8. Anal. Calcd for C34H34N6O5ÁH2O: C, 65.37;
H, 5.81; N, 13.45. Found: C, 65.15; H, 5.65; N, 13.10.
4.3.10. ( )-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2-met-
hoxy-3-[(4-(trifluoromethyl)benzyl)oxy]phenyl}-1-(1-propyl-
2(1H)-phthalazinyl)-2-propen-1-one (34)
This compound was prepared as described above for compound
28 using 27 (150 mg, 0.318 mmol), DBU (53 mg, 0.052 mL,
0.350 mmol, 1.1 equiv), and 4-(trifluoromethyl)benzyl bromide
(84 mg, 0.054 mL, 0.350 mmol, 1.1 equiv) in CH2Cl2 (8 mL) to give
34 (124 mg, 62%) as a yellow solid, mp 145–147 °C. IR: 3328,
4.3.7. ( )-(E)-5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2-methoxy-
3-[3-oxo-3-(1-propyl-2(1H)-phthalazinyl)-1-propen-1-yl]phenyl
4-nitrobenzoate (31)
This compound was prepared as described above for compound
28 using 27 (150 mg, 0.318 mmol), DBU (53 mg, 0.052 mL,
0.350 mmol, 1.1 equiv), and 4-nitrobenzoyl chloride (65 mg,
0.350 mmol, 1.1 equiv) in CH2Cl2 (8 mL) to give 31 (154 mg, 78%)
as a yellow solid, mp 130–132 °C. IR: 3480, 3344, 3176, 1744,
3175, 1659, 1620 cmÀ1 1H NMR (CDCl3): d 8.01 (d, J = 15.9 Hz,
;
1H), 7,68 (d, J = 15.9 Hz, 1H), 7.66 (s, 1H), 7.60 (d, J = 8.0 Hz, 2H),
7.54 (d, J = 8.0 Hz, 2H), 7.42 (t, J = 7.1 Hz, 1H), 7.33 (m, 3H), 7.27
(s, 1H), 7.27 (d, J = 7.1 Hz, 1H), 7.14 (d, J = 7.1 Hz, 1H), 7.13 (s,
1H), 6.80 (s, 1H), 6.69 (br s, 4H), 5.86 (t, J = 6.6 Hz, 1H), 5.12 (s,
2H), 3.86 (s, 1H), 3.66 (s, 2H), 1.61 (m, 2H), 1.33 (m, 2H), 0.82 (t,
J = 7.1 Hz, 3H); 13C NMR (CDCl3): d 166.6, 163.6, 157.1, 152.3,
147.9, 142.7, 140.6, 136.8, 134.1, 132.2, 131.5, 130.2, 130.0,
129.8, 128.1, 127.5, 126.5, 125.8, 125.3, 124.1 (d, J = 270.9 Hz),
123.9, 120.2, 119.4, 115.4, 108.3, 70.1, 61.4, 51.4, 37.3, 33.5, 18.3,
13.8. Anal. Calcd for C34H33F3N6O3Á3.0 H2O: C, 59.64; H, 5.74; N,
12.27. Found: C, 59.70; H, 5.95; N, 12.37.
1607 cmÀ1
; d 8.38 (s, 2H), 8.00 (d,
1H NMR (DMSO-d6):
J = 15.9 Hz, 1H), 7.81 (s, 1H), 7.72 (d, J = 15.9 Hz, 1H), 7.68 (s, 1H),
7.46 (complex m, 4H), 7.37 (t, J = 7.1 Hz, 1H), 7.30 (d, J = 6.6 Hz,
1H), 7.29 (d, J = 7.1 Hz, 1H), 6.98 (d, J = 1.6 Hz, 1H), 5.90 (t,
J = 6.6 Hz, 1H), 4.78 (br s, 2H), 4.63 (br s, 2H), 3.80 (s, 3H), 3.74
(s, 2H), 1.65 (m, 2H), 1.28 (m, 2H), 0.87 (t, J = 7.1 Hz, 3H); 13C
NMR (DMSO-d6): d 166.2, 162.9, 162.4, 162.3, 157.1, 151.0, 149.6,
144.3, 142.6, 136.1, 134.7, 134.3, 134.2, 131.5, 131.4, 131.1,
128.0, 126.5, 125.7, 125.6, 123.9, 123.8, 123.6, 120.0, 105.6, 62.3,
51.4, 37.3, 33.6, 18.3, 13.8. Anal. Calcd for C33H31N7O6Á0.8 H2O: C,
62.31; H, 5.17; N, 15.41. Found: C, 62.28; H, 4.86; N, 15.53.
4.4. Biological measurements
The methods used to assess potency have been previously pub-
lished.7,8 Briefly, the minimum inhibitory concentration (MIC) was
the lowest concentration of compound needed to block growth of a
standardized culture of B. anthracis Sterne as measured by turbid-
ity at 600 nm and by visual inspection.27 Enzymatic activity was
reconstituted in vitro with saturating concentrations of co-factor
and dihydrofolate reductase. The amount of tetrahydrofolate pro-
duced was monitored, and the concentration of compound that
inhibited 50% of this reaction was then determined. This value,
an IC50, was converted to an inhibition constant (Ki) by incorporat-
ing the strength of binding to dihydrofolate (Km) through the for-
malism of Cheng-Prusoff.28
4.3.8. ( )-(E)-5-[(2,4-Diamino-5-pyrimidinyl)methyl]-2-methoxy-
3-[3-oxo-3-(1-propyl-2(1H)-phthalazinyl)-1-propen-1-yl]phenyl
4-(trifluoromethoxy)benzoate (32)
This compound was prepared as described above for compound
28 using 27 (150 mg, 0.318 mmol), DBU (53 mg, 0.052 mL,
0.350 mmol, 1.1 equiv), and 4-(trifluoromethoxy)benzoyl chloride
(79 mg, 0.055 mL, 0.350 mmol, 1.1 equiv) in CH2Cl2 (8 mL) to give
32 (172 mg, 82%) as an off-white solid, mp 118–120 °C. IR: 3339,
3182, 1743, 1656, 1606 cmÀ1 1H NMR (DMSO-d6): d 8.25 (d,
;
J = 8.8 Hz, 2H), 8.00 (d, J = 16.5 Hz, 1H), 7.81 (s, 1H), 7.70 (d,
J = 16.5 Hz, 1H), 7.68 (s, 1H), 7.45 (m, 2H), 7.41–7.28 (complex m,
4H), 7.19 (d, J = 7.7 Hz, 1H), 6.97 (d, J = 1.6 Hz, 1H), 5.90 (t, J = 6.6,
1H), 4.79 (br s, 2H), 4.62 (br s, 2H), 3.80 (s, 3H), 3.73 (s, 2H), 1.65
(m, 2H), 1.28 (m, 2H), 0.87 (t, J = 7.1 Hz, 3H); 13C NMR (DMSO-d6):
d 166.3, 163.5, 162.5, 162.2, 156.9, 153.2, 149.8, 144.5, 142.6,
136.3, 134.4, 134.2, 132.3, 131.5, 131.0, 128.0, 127.2, 126.5, 125.7,
125.3, 124.0, 123.9, 123.7 (q, J = 258.1 Hz), 120.5, 119.8, 105.7,
62.2, 51.4, 37.3, 33.6, 18.3, 13.8. Anal. Calcd for C34H31F3N6O5Á5.5
H2O: C 53.75; H 5.57; N 11.06. Found: C, 53.96; H, 5.57; N, 11.04.
Acknowledgments
We gratefully acknowledge support of this work by the
National Institutes of Allergy and Infectious Diseases (R01-
AI090685) of the NIH/NIAID and the Sitlington Chair in Infec-
tious Diseases, both to W.W.B. NSF (BIR-9512269), the Oklahoma
State Regents for Higher Education, the W.M. Keck Foundation,
and Conoco, Inc, provided funding for 300 MHz and 400 MHz
NMR spectrometers of the Oklahoma Statewide Shared NMR
Facility. The authors also wish to thank the OSU College of Arts
and Sciences for funds to upgrade our departmental FT-IR
instruments.
4.3.9. ( )-(E)-3-{5-[(2,4-Diamino-5-pyrimidinyl)methyl]-3-[(4-
fluorobenzyl)oxy]-2-methoxyphenyl}-1-(1-propyl-2(1H)-phth-
alazinyl)-2-propen-1-one (33)
This compound was prepared as described above for compound
28 using 27 (150 mg, 0.318 mmol), DBU (53 mg, 0.052 mL,

B. Nammalwar et al. / Bioorg. Med. Chem. 23 (2015) 203–211
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