Development of substituted pyrido[3,2-d]pyrimidines as potent and selective dihydrofolate reductase inhibitors for pneumocystis pneumonia infection

Article abstract of DOI:10.1016/j.bmcl.2019.06.004

Pneumocystis pneumonia (PCP) caused by Pneumocystis jirovecii (pj) can lead to serious health consequences in patients with an immunocompromised system. Trimethoprim (TMP), used as first-line therapy in combination with sulfamethoxazole, is a selective but only moderately potent pj dihydrofolate reductase (pjDHFR) inhibitor, whereas non-clinical pjDHFR inhibitors, such as, piritrexim and trimetrexate are potent but non-selective pjDHFR inhibitors. To meet the clinical needs for a potent and selective pjDHFR inhibitor for PCP treatment, fourteen 6-substituted pyrido[3,2-d]pyrimidines were developed. Comparison of the amino acid residues in the active site of pjDHFR and human DHFR (hDHFR) revealed prominent amino acid differences which could be exploited to structurally design potent and selective pjDHFR inhibitors. Molecular modeling followed by enzyme assays of the compounds revealed 15 as the best compound of the series with an IC₅₀ of 80 nM and 28-fold selectivity for inhibiting pjDHFR over hDHFR. Compound 15 serves as the lead analog for further structural variations to afford more potent and selective pjDHFR inhibitors.

Full text of DOI:10.1016/j.bmcl.2019.06.004

Accepted Manuscript
Development of substituted pyrido[3,2-d]pyrimidines as potent and selective
dihydrofolate reductase inhibitors for pneumocystis pneumonia infection
Khushbu Shah, Sherry Queener, Vivian Cody, Jim Pace, Aleem Gangjee
PII:
S0960-894X(19)30369-5
https://doi.org/10.1016/j.bmcl.2019.06.004
BMCL 26482
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
12 May 2019
28 May 2019
1 June 2019
Please cite this article as: Shah, K., Queener, S., Cody, V., Pace, J., Gangjee, A., Development of substituted
pyrido[3,2-d]pyrimidines as potent and selective dihydrofolate reductase inhibitors for pneumocystis pneumonia
infection, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.06.004
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Development of substituted pyrido[3,2-d]pyrimidines as potent and
selective dihydrofolate reductase inhibitors for pneumocystis
pneumonia infection
Shah, Khushbua; Queener, Sherryb; Cody, Vivianc; Pace, Jimc, Gangjee, Aleema.
a
Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne
University, Pittsburgh PA 15282, gangjee@duq.edu, Fax: 412-396-5593,
b
Department of Pharmacology and Toxicology, Indiana University School of Medicine,
Indianapolis, IN 46202,
c Hauptman-Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, NY 14203.
Abbreviations:
DHPS, dihydropteroate synthase; PCP, Pneumocystis carinii pneumonia; hDHFR, human
dihydrofolate reductase; pcDHFR, Pneumocystis carinii dihydrofolate reductase; pjDHFR,
Pneumocystis jirovecii dihydrofolate reductase; PTX, Piritrexim; TMP, Trimethoprim; TMQ,
Trimetrexate; SMX, Sulfamethoxazole
1

Abstract
Pneumocystis pneumonia (PCP) caused by Pneumocystis jirovecii (pj) can lead to serious health
consequences in patients with an immunocompromised system. Trimethoprim (TMP), used as
first-line therapy in combination with sulfamethoxazole, is a selective but only moderately potent
pj dihydrofolate reductase (pjDHFR) inhibitor, whereas non-clinical pjDHFR inhibitors, such as,
piritrexim and trimetrexate are potent but non-selective pjDHFR inhibitors. To meet the clinical
needs for a potent and selective pjDHFR inhibitor for PCP treatment, fourteen 6-substituted
pyrido[3,2-d]pyrimidines were developed. Comparison of the amino acid residues in the active
site of pjDHFR and human DHFR (hDHFR) revealed prominent amino acid differences which
could be exploited to structurally design potent and selective pjDHFR inhibitors. Molecular
modeling followed by enzyme assays of the compounds revealed 15 as the best compound of the
series with an IC50 of 80 nM and 28-fold selectivity for inhibiting pjDHFR over hDHFR.
Compound 15 serves as the lead analog for further structural variations to afford more potent and
selective pjDHFR inhibitors.
Keywords
PCP, DHFR, pyrido[3,2-d]pyrimidines, Pneumocystis jirovecii
2

Pneumocystis carinii pneumonia (PCP) caused by Pneumocystis jirovecii is a fungal
infection which affects patients with altered host immunity, such as, patients with cancer,
transplant recipients, patients with HIV/AIDS and patients on immunosuppressive medications.
[1-6] In HIV patients, PCP leads to pulmonary inflammation, hospitalization, and death.[7-10] If
left untreated, PCP is almost always fatal. Even when treated, the mortality rate is 10%.[11] In
the USA, 9% of HIV infected patients and 1% of organ transplant recipients develop PCP
infection.[12] Although PCP prophylaxis and highly active antiretroviral therapy (HAART) in
AIDS has decreased the incidences of PCP infection, cases still persist due to non-adherence to
HIV medication, toxicity to the HIV medications, emergence of drug resistant HIV strains, late
diagnosis of HIV and the rise of the number of HIV cases in developing countries along with
resistance to currently used drugs for PCP.[13, 14]
Figure 1. Agents that have been used to treat PCP
3

For past few decades, oral administration of trimethoprim (TMP)-sulfamethoxazole
(SMX) combination (co-trimoxazole) has been the first line choice for both prophylaxis and
treatment of PCP.[15, 16] The combination synergistically blocks folic acid synthesis, since
TMP (Figure 1) is a selective, but weak inhibitor of dihydrofolate reductase (DHFR)[17], while
SMX is an inhibitor of dihydropteroate synthase (DHPS).[18] The low activity of TMP against
DHFR is augmented by SMX in the treatment regimen. This combination is less well tolerated in
patients with HIV/AIDS, where 50-80% experience side-effects due to SMX. The most common
one being a diffuse maculopapular rash, sometimes accompanied with fever which often leads to
discontinuation of treatment.[19, 20] The alternative therapy can be (i) pentamidine (intravenous
- infused over an hour); (ii) TMP-dapsone (oral); or (iii) clindamycin (intravenous) – primaquine
(oral), which are not preferred due to higher prevalence of side-effects and in some cases lower
efficacy.[15, 21-24] Experimental drugs such as piritrexim (PTX) and trimetrexate (TMQ) (Fig.
1) are potent, but non-selective inhibitors of DHFR, which cause dose-limiting toxicities.[15, 25,
26] Thus, for patients who do not respond to first line treatment, development of effective and
safe drugs for the treatment of PCP is critically warranted. Our long-term goal is to provide
analogs with excellent potency along with high selectivity for Pneumocystis jirovecii DHFR
(pjDHFR) over human DHFR (hDHFR). Such agents could be used alone as well as in
combination with sulfonamides and other drugs for PCP infections in humans.
Although it has been widely known that PCP is a host specific infection, drugs designed
for PCP have been evaluated against Pneumocystis carinii DHFR (pcDHFR), and not
pjDHFR.[17, 27] Pneumocystis carinii (pc), the causative species in rats is different from
Pneumocystis jirovecii responsible for human infections. There is a 38% difference in the amino
acid sequence of pcDHFR and pjDHFR which suggests that drugs evaluated against the
4

surrogate pcDHFR in vitro or in vivo, may not translate to the treatment of PCP infection in
humans.[28, 29] In the absence of crystal structure information for pjDHFR, a tissue culture or a
PCP animal model for in vitro studies for PCP cause severe impediments in drug discovery
efforts. In fact, since the preliminary evaluation of TMP/SMX there has been no new treatment
for PCP caused by P. jirovecii that could effectively supplant TMP/SMX.
Table 1. Inhibitory Concentrations (IC50, in nM) against Recombinant DHFR from pjDHFR and
human DHFR (hDHFR) and Selectivity Ratios[28]
R1
R2
pjDHFR (IC50,
in nM)
hDHFR (IC50, in
nM)
Selectivity Ratio
[hDHFR/pjDHFR]
1
2
3
4
5
6
H
H
H
H
H
H
H
300
620
400
250
400
870
190
2100
3650
2100
2200
3100
<1
3
9
8
5
4-CH3
4-OCH3
2ˊ,3ˊ-(CH)4
3ˊ,4ˊ-(CH)4
3ˊ,4ˊ,5ˊ -triF
4
TMP
PTX
TMQ
120
1.6
2.1
32200
3.0
2.6
268
2
1
These assays were carried out at 37 °C under 18 μM dihydrofolic acid concentration. The
standard error of the means for these values is 12% or less than the mean value; bTrimethoprim;
cPiritrexim.
Gangjee and coworkers[28] reported pyrido[2,3-d]pyrimidines 1-6 as somewhat selective
and potent pjDHFR inhibitors (Table 1).
5

Figure 2. (A) Docked pose of 1 (magenta) in the homology model of pjDHFR and (B) Docked
pose of 1 (magenta) in the crystal structure of hDHFR (PDB: 4QJC, 1.62 Å)[30]
To compare the binding of 1 in the active sites of pjDHFR and hDHFR, a docking study
of 1 was performed using the refined reported homology model of pjDHFR [32] and the crystal
structure of hDHFR in Schrödinger.[31] In pjDHFR, the pyrido[2,3-d]pyrimidine scaffold is
stabilized by a π-π stacking interaction with Phe36. The protonated N1 and 2-NH2 of 1 form an
ionic bond with Asp32 in the form of a salt bridge. The 4-NH2 forms hydrogen bonding
interaction with the backbone carbonyl of Ile10. The side chain phenyl ring is oriented in a
pocket formed by Leu25, Met33, Ser64, and Leu65. The N9-H group is oriented towards the
Ile123 side chain. In hDHFR, the pyrido[2,3-d]pyrimidine scaffold is also stabilized by a π-π
stacking interaction with Phe34. The protonated N1 and 2-NH2 form an ionic bond with Glu30.
The 4-NH2 forms hydrogen bonding interaction between the backbone carbonyl of Ile7. The side
chain phenyl ring is oriented in a pocket formed by Leu22, Phe31, Ser59, and Ile60. The N9-H
group is oriented towards the Val115 side chain. The docking scores of 1 in the pjDHFR
homology model and hDHFR crystal structure were -7.88 kcal/mol and -9.54 kcal/mol,
6

respectively. The in vitro IC50 values of 1 reflect the docking scores obtained with the inhibition
of hDHFR somewhat better than that for pjDHFR for 1 (Table 1). This provides validation of our
docking protocol in the homology model of pjDHFR compared with docking in hDHFR crystal
structure.
The amino acids that constitute the active site of pjDHFR and hDHFR, as visible from
Figure 2, are quite distinct. The hDHFR active site contains Phe31, Ile60, and Val115, whereas
active site of pjDHFR contains Met33, Leu65, and Ile123. The different amino acids occupying
the same location in the active sites, such as Met33 (in pjDHFR) vs. Phe31 (in hDHFR) and Ile
123 (in pjDHFR) vs. Val115 (in hDHFR), possess varied shapes, sizes and electronic properties.
Such differences can be exploited for the design of inhibitors to provide potency and selectivity
for pjDHFR over hDHFR. We reported our efforts in targeting the amino acid difference of
Met33 (in pjDHFR) and Phe31 (in hDHFR) by varying substitutions on the 7-position of a
truncated pyrrolo[2,3-d]pyrimidine scaffold.[32] The 6-subtituted pyridopyrimidines evaluated
thus far against pjDHFR consist of the pyrido[2,3-d]pyrmidine scaffold. No study of significant
variation of the scaffold has been carried out to our knowledge.
Table 2. Inhibition Concentrations (IC50) against pcDHFR and rlDHFR [33, 34]
O
NH2
X
N
N
O
H
H2N
N
Y
#
7
8
X
N
C
Y
C
N
pcDHFR (nM)
1400
6100
rlDHFR (nM)
430
500
Gangjee et al.[34] reported the pyrido[2,3-d]pyrimidines and their analogs as inhibitors
of pcDHFR. Rosowsky et al.[33] synthesized and evaluated the regioisomeric pyrido[3,2-
7

d]pyrimidine analogs as inhibitors of pcDHFR. The biological evaluation of pyrido[3,2-
d]pyrimidine 7 (Table 2) in pcDHFR and rat liver DHFR (rlDHFR) displayed a 5-fold increase
in potency for pcDHFR, compared to its regio pyrido[2,3-d]pyrimidine analog 8 (Table 2). This
comparison suggested that scaffold replacement of a pyrido[2,3-d]pyrimidine to a pyrido[3,2-
d]pyrimidine improved the potency for pcDHFR and a similar approach could perhaps be useful
to improve the potency for pjDHFR. Gangjee at al. [33] also reported 6-substituted pyrido[3,2-
d]pyrimidines as inhibitors of pcDHFR. However because of the amino acid differences, as
reported in Shah et al.,[31] it has been validated that activity against pcDHFR does not translate
into activity against pjDHFR. Hence, it was important to synthesize and evaluate the pyrido[3,2-
d]pyrimidine series of compounds as selective pjDHFR inhibitors, (Fig. 3).
R1
R2
9
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
H
H
H
H
H
H
H
H
4’-CH3
4’-OCH3
4’-OH
2’,3’-(CH)4
3’,4’-(CH)4
3’,4’-diF
3’,4’,5’-triF
4’-OCF3
H
H
H
H
H
NH2
R1
5
4
9
N
N
3
N
2
R2
6
7
H2N
N
1
8
CH3
CH2CH3
CH2CH2CH3
CH(CH3)2
CH2CH2CH2CH3
Figure 3. Proposed pyrido[3,2-d]pyrmidine compounds
8

Figure 4. (A) Superimposition of docked pose of 1 (magenta) and 9 (cyan) in the homology
model of pjDHFR and (B) Docked pose of 1 (magenta) and 9 (cyan) in the crystal structure of
hDHFR (PDB: 4QJC, 1.62 Å)[30]
To study the probable binding modes of compounds obtained via a scaffold hopping of
pyrido[2,3-d]pyrimidine with pyrido[3,2-d]pyrimidine, 9 (Fig. 3) was docked in the active sites
of pjDHFR and hDHFR using the reported homology model of pjDHFR and crystal structure of
hDHFR, with Schrödinger.[28, 30] (Fig. 4). The pyrido[3,2-d]pyrimidine 9 exhibited a similar
docked pose as 1 suggesting the scaffold binding could be at least as favorable as the pyrido[2,3-
d]pyrimidine scaffold. The docking scores of 9 in the pjDHFR homology model and hDHFR
crystal structure were -8.22 kcal/mol and -8.87 kcal/mol, respectively. Compared to the docking
score of 1 in the pjDHFR homology model (-7.88 kcal/mol), 9 displayed a higher binding
potential to pjDHFR, whereas compared to the docking score of 1 in hDHFR crystal structure (-
9.54 kcal/mol), 9 displayed a lower binding potential to hDHFR. This suggested that a scaffold
change from pyrido[2,3-d]pyrimidine to pyrido[3,2-d]pyrimidine could afford increased potency
and selectivity for pjDHFR.
9

The reported pyrido[2,3-d]pyrimidine compounds 1-6 (Table 1) displayed moderate
potencies for pjDHFR, but none were significantly selective for pjDHFR over hDHFR.
Compounds 9-17 (Figure 3), were designed as pyrido[3,2-d]pyrimidine analogs of 1-6. The
shape of the pjDHFR active site and hDHFR active site differ due to presence of Met33 (flexible
residue) and Leu65 in pjDHFR and Phe31 (bulkier residue) and Ile60 in hDHFR. Compounds 9-
12 (Fig. 3) were designed as analogs of 1-3 (Table 1) to determine the influence of the
pyrido[3,2-d]pyrimidine system and the electron donating substitutions on the side chain aryl
group on activity against pjDHFR and hDHFR. These compounds were anticipated to provide a
structure activity relationship through electron donating groups (inductive and resonance).
Compounds 13 and 14 (Table 3) were designed as pyrido[3,2-d]pyrimidine analogs of 4 and 5
(Table 1). Due to the bulkier size of the naphthyl group, the side chains of these proposed
compounds were expected to sterically clash with the side chain Phe31 in hDHFR. This clash is
absent in pjDHFR, since the pjDHFR active site is composed of a flexible Met33 residue instead
of Phe31 (in hDHFR) at this position. Compounds 15-17 (Table 3) were designed as analogs of 6
(Table 1). These compounds contain electron withdrawing groups to evaluate their influence on
DHFR inhibitory activity.
10

Figure 5. (A) Superimposition of docked pose of 9 (cyan) and 18 (magenta) in the homology
model of pjDHFR and (B) Docked pose of 9 (cyan) and 18 (magenta) in the crystal structure of
hDHFR (PDB: 4QJC, 1.62 Å)[30]
As observed from the docking of 9 and 18 in Figure 5, the N-CH3 of 18 is oriented
towards the Ile123 side chain in the pjDHFR active site. An appropriate alkyl substitution on the
nitrogen lead to compounds that can interact with the longer Ile123 (in pjDHFR) and not with
shorter Val115 (in hDHFR). Hence, the N-CH3 analog 18 would be expected to afford an
improvement in pjDHFR potency and selectivity over hDHFR. Docking of 18 in the active site
of pjDHFR using the reported homology model of pjDHFR and superimposition of the docked
pose of 18 in the active site of pjDHFR over the docked pose of 9 in the active site of pjDHFR is
shown in Figure 5a. The docking study revealed that both compounds display a bidentate ionic
bond between the protonated N1 and the 2-NH2 with Asp32. The 4-NH2 moiety for both 9 and 18
forms hydrogen bonds with the backbone of Ile10 and the pyrrolo[3,2-d]pyrimidine scaffold is
stabilized by π-π -stacking interaction with Phe36. The phenyl moiety is oriented in the pocket
formed by Leu25, Met33, Ser64 and Leu65. The distance between Ile123 side chain and the N9-
11

of the pyrido[3,2-d]pyrimidine 9 was 4.47 Å, whereas the distance between the Ile123 side chain
and N9-methyl group of the pyrido[3,2-d]pyrimidine 18 was 3.43 Å. Since all other interactions
are similar for 9 and 18, the additional N9-CH3 on 18 can potentially have stronger hydrophobic
interactions with pjDHFR, than 9. The best docked poses for 9 and 18 in the pjDHFR homology
model generated docking scores of -8.22 kcal/mol and -8.02 kcal/mol, respectively. Figure 5b
displays the best docked conformation of 9 and 18 in hDHFR crystal structure (PDB: 4QJC, 1.61
Å).[30] The analogs exhibit a bidentate ionic interaction of the protonated N1 and 2-NH2 with
Glu30. The 4-NH2 hydrogen bonds with the backbone of Ile7. The phenyl moiety is oriented in
the pocket formed by Leu22, Phe31 and Ser59. The scaffold is stabilized by π-π-stacking
interactions with Phe34. The distance between Val115 side chain and nitrogen substituted at the
6-position of the pyrido[3,2-d]pyrimidine 9 was 5.83 Å, whereas the distance between Val115
side chain and N9-CH3 of the pyrido[3,2-d]pyrimidine 18 as 4.36 Å. The best docked poses for 9
and 18 in hDHFR crystal structure generated docking scores of -8.87 kcal/mol and -9.39
kcal/mol, respectively. To further evaluate the optimum length of the alkyl moiety required to
interact with Ile123 (in pjDHFR) and not the shorter Val115 (in hDHFR), longer and branched
chain alkyl groups were designed for evaluation for substitution at the N9-position. Thus, it was
of interest to synthesize N9-alkyl pyrido[3,2-d]pyrimidines 18-22. All the proposed compounds
9-22 displayed better docking scores for pjDHFR over hDHFR, compared to the lead compound
1 (docking scores are included in the supporting information) indicating possible increase in
potency and selectivity for pjDHFR over hDHFR.
12

Scheme 1. Synthesis of intermediate 26
a) 0% HNO3, H2SO4, reflux, 3 h; b) CuCN, 180 °C, 15 min; c) aniline, monoethyl glycol,
pyridine, 140 °C, 12 h; d) aniline, LDA, THF, -78 °C, 12 h
Intermediate 25 (Scheme 1) was synthesized following a reported procedure.[35, 36] The
2,6-dichloropyridine 23 was nitrated with 90% nitric acid and sulfuric acid, followed by
substitution of the 2-Cl moiety with cuprous cyanide at 180 °C to afford 2-cyano-3-nitro-6-
chloropyridine 25. The structure of 25 was confirmed from the coupling constants (J) of the
protons [1H-NMR (400 Hz) δ 7.80 (d, J = 7.2 Hz, 1 H), 8.58 (d, J = 7.2 Hz, 1 H)]. The coupling
constants match the characteristic J constants for ortho coupling between aromatic protons.[37]
Following the reported procedure,[38] synthesis of 26 (Scheme 1) was attempted.
Compound 25 was reacted with the 3,4,5-trifluoroaniline in monoethylglycol in the presence of
pyridine at 140 °C. The reaction did not yield any product after 12 h. The reason for the failure
of the reaction could be the poor nucleophilicity of the 3,4,5-trifluoroaniline.
In order to carry out the displacement with a weak nucleophile such as 3,4,5-
trifluoroaniline on 25, the alternate methodology reported by Gangjee et al.[38] was followed
(Scheme 2). The 3,4,5-trifluoroaniline was first treated with LDA at -78 °C to afford a more
13

reactive nitrogen anion, followed by displacement of the 6-chloro group of 25 to afford 26 in
10% yield.
Scheme 2. Synthesis of target compounds 9-22
a) substituted aniline, isopropanol, 130 ⁰C, 3-16 h; b) conc. HCl, Fe powder, reflux, 0.5-2 h; c)
chlorformamidine HCl, dimethyl sulfone, 140 ⁰C, 3-16 h
The syntheses of 9-22 were accomplished from 25 as shown in Scheme 2. To a solution
of 6-chloro-3-nitro-2-pyridinecarbonitrile (25) in isopropanol, the appropriate aniline was added
and the mixture was heated at 130 °C (Scheme 1). After 3-16 h, the solution was concentrated
under reduced pressure to afford 26-38. This step using isopropanol afforded increased yields
and provided safer conditions, compared to the displacement using LDA. The mixture was not
14

separated and was advanced to the next step. The nitro compounds 26-38 were reduced using
iron powder in concentrated hydrochloric acid to afford 39-52.[38] The formation of the amino
compounds 39-52 is indicated by a blue fluorescence on TLC at 254 nm. These amino
compounds 39-52 were not separated and advanced to the cyclization step. During the reduction
of 29 to 41 (R2= 4-OCH3), the methoxy group undergoes partial ether cleavage in the work up
step and afforded two very close spots on the TLC (possibly for 41 and 42). To obtain the target
compounds, the reported method[38] was followed for the cyclization of 39-52 in dimethyl
sulfone at 140 ⁰C to afford 9-22.
Table 3. Inhibition Concentrations (IC50) of 9-22 against pjDHFR and hDHFR and Selectivity
Ratios
R1
R2
pjDHFR (IC50, hDHFR (IC50, Selectivity
Ratio
#
in nM)
122
174
239
150
112
275
80
155
194
96
150
123
201
66
in nM)
1526
2626
2459
1576
1098
3185
2253
1808
4125
942
[hDHFR/pjDHFR]
9
10
11
12
13
14
15
16
17
18
29
20
21
22
TMPb
PTXc
H
H
H
H
H
H
H
H
H
13
15
10
10
10
12
28
12
21
10
10
11
7
4ˊ-CH3
4ˊ-OCH3
4ˊ-OH
2ˊ,3ˊ-(CH)4
3ˊ,4ˊ-(CH)4
3ˊ,4ˊ,5ˊ-triF
3ˊ,4ˊ-diF
4ˊ-OCF3
H
H
H
H
H
H
CH3
CH2CH3
CH2CH2CH3
CH(CH3)2
CH2CH2CH2CH3
1571
1338
1373
903
14
266
0.05
92
41
24500
2
15

aThese assays were carried out at 37 °C under 9 μM dihydrofolic acid concentration. The
standard error of the means for these values is 12% or less than the mean value; bTrimethoprim;
cPiritrexim.
Table 3 displays the IC50 values of compounds 9-22 for pjDHFR and hDHFR.
Comparison of activities of pyrido[3,2-d]pyrimidines vs. pyrido[2,3-d]pyrimidines for pjDHFR,
hDHFR and selectivity ratios, indicates that the scaffold replacement strategy was favorable.
Biological evaluation of 9 against pjDHFR and hDHFR displayed an improvement of greater
than 2-fold in pjDHFR potency, compared to its regio isomer 1 (Table 3).[28] Additionally, the
selectivity for pjDHFR over hDHFR for 9 also improved significantly (>13-fold), compared to 1.
As observed for pyrido[3,2-d]pyrimidine 9, a considerable improvement in selectivity
was seen for compounds 10 (vs. 2), 11 (vs. 3), 13 (vs. 4), 14 (vs. 5), and 15 and 16 (vs. 6),
validating the rationale of the scaffold hopping strategy. The trifluoro-substituted analog 15
showed the highest selectivity for pjDHFR over hDHFR in the series, which is 560-fold greater
than the selectivity ratio of PTX. Compared to its pyrido[2,3-d]pyrimidine analog 6, 15 showed
an 11-fold improvement in the potency towards pjDHFR and a 7-fold improvement in the
selectivity ratio for pjDHFR over hDHFR. The improved potency and selectivity of 15 for
pjDHFR could be due to a better binding of 15 with the active site of pjDHFR compared to that
of other ligands.
The alkyl analogs 18-22 in the N9-substituted pyrido[3,2-d]pyrimidine-2,4,6-triamines
were designed to obtain better hydrophobic interactions with Ile123 (in pjDHFR) vs. Val115 (in
hDHFR). The N9-alkyl analogs 18-21 did not show a significant improvement in pjDHFR
potency and selectivity, compared to the N9-H analog 9. The absence of improvement in potency
and/or selectivity for pjDHFR could be due to the smaller size of the alkyl groups, which are not
able to effectively interact with Ile123 in the pjDHFR active site. The N9-butyl analog 22
16

demonstrated the best potency for pjDHFR of the series which could be attributed to the length
of the butyl substitution which can interact with Ile123 (pjDHFR) better than the smaller alkyl
groups of 18-21. Compound 22 with the best potency (IC50, 66 nM) against pjDHFR
demonstrated a 14-fold selectivity for pjDHFR, whereas 15 despite a comparatively lower
potency for pjDHFR (IC50, 80 nM) showed a significantly higher selectivity. Thus, in the N9-
substituted pyrido[3,2-d]pyrimidine-2,4,6-triamine series, 15 remained the best in terms of its
potency and selectivity for pjDHFR. Though the selectivities of some of these compounds fall
short of that for TMP for pjDHFR, the potency is equivalent or slightly better than TMP.
Utilizing the information provided by this study, the design and synthesis of potentially more
potent and selective compounds are currently underway and will be the subject of future reports.
ACKNOWLEDGEMENT:
This work was supported, in part, by grants from the National Institute of Health (NIH), National
Institute of Allergy and Infectious Diseases (NIAID), RO1AI098458, Adrian van Kaam Chair in
Scholarly Excellence (AG) Duquesne University, and NSF for NMRs.
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