Design, Synthesis, and Molecular Modeling of Novel Pyrido[2,3-d

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Design, synthesis and molecular modeling of novel pyrido[2,3-d]pyrimidine analogs as antifolates: Application of Buchwald-Hartwig aminations of heterocycles Aleem Gangjee, Ojas A. Namjoshi, Sudhir Raghavan, Sherry F. Queener, Roy L. Kisliuk, and Vivian Cody J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400086g • Publication Date (Web): 29 Apr 2013 Downloaded from http://pubs.acs.org on May 1, 2013

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Page 1 of 67

Journal of Medicinal Chemistry

Design, synthesis and molecular modeling of novel 2

1 3 4

pyrido[2,3-d]pyrimidine analogs as antifolates: 6

5 7 9

8

Application of Buchwald-Hartwig aminations of 10 1 13

12

heterocycles 15

14

17

16

Aleem Gangjee,a* Ojas A. Namjoshi,a Sudhir Raghavan,a Sherry F. Queener,b Roy L. Kisliuk,c Vivian 19

18

Codyd 20 21 2 24

23 a

25

Division of Medicinal Chemistry, Graduate School Pharmaceutical Sciences, Duquesne University,

26

600 Forbes Avenue, Pittsburgh, PA 15282. 29

28

27

b

31

30

Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill

Drive, Indianapolis, IN 46202. 32 3 c

34 36

35

Department of Biochemistry, Tufts University Health Science Campus, 136 Harrison Avenue, Boston,

MA 02111. 38

37

d

39

Hauptman-Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, NY 14203.

40 41 43

42

*Corresponding author: [email protected]; 412-396-6070. 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment

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2

1

Abbreviations: PCP, Pneumocystis pneumonia; pj, Pneumocystis jirovecii; pc, Pneumocystis carinii; 3 4

tg, toxoplasma gondii; ma, Mycobacterium avium; rl, rat liver; DHFR, dihydrofolate reductase; TS, 5 7

6

thymidylate synthase; HIV, human immunodeficiency virus; AIDS, acquired immunodeficiency 9

8

syndrome; TMP, trimethoprim; PTX, piritrexim; TMQ, trimetrexate; LiHMDS, lithium 10 1

hexamethyldisilazide; X-Phos, 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl; S-Phos, 214

13

12

Dicyclohexylphosphino-2',6'-dimethoxybiphenyl; rac-BINAP, racemic-2,2'-Bis(diphenylphosphino)16

15

1,1'-binaphthyl; XantPhos, 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene; DCPB, 17 18

dicyclohexylphospho biphenyl; t-Butyl-X-Phos, 2-di-t-butylphosphino-2',4',6'-tri-i-propyl-1,1'-biphenyl; 21

20

19

S-Phos, 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl; Ru-Phos, 2-dicyclohexylphosphino-2',6'-di23

2

i-propoxy-1,1'-biphenyl; DavePhos, 2-(dicyclohexylphosphino)-2'-(N,N-dimethylamino)biphenyl. 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment

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2

1

Abstract: 3 4 6

5

Opportunistic infections caused by Pneumocystis jirovecii (P. jirovecii, pj), Toxoplasma gondii 7 9

8

(T. gondii, tg) and Mycobacterium avium (M. avium, ma) are the principal causes of morbidity and 1

10

mortality in patients with acquired immunodeficiency syndrome (AIDS). The absence of any animal 12 14

13

models for human Pneumocystis jirovecii pneumonia and the lack of crystal structures of pjDHFR and 16

15

tgDHFR make the design of inhibitors challenging. A novel series of pyrido[2,3-d]pyrimidines as 18

17

selective and potent DHFR inhibitors against these opportunistic infections are presented. Buchwald19 20

Hartwig coupling reaction of substituted anilines with pivaloyl protected 2,4-diamino-6-bromo23

2

21

pyrido[2,3-d]pyrimidine was successfully explored to synthesize these analogs. Compound 26 was the 25

24

most selective inhibitor with excellent potency against pjDHFR. Molecular modeling studies with a 26 28

27

pjDHFR homology model explained the potency and selectivity of 26. Structural data are also reported 30

29

for 26 with pcDHFR and 16 and 22 with variants of pcDHFR. 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment

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2

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Introduction: 3 5

4

Dihydrofolate reductase (DHFR) converts dihydrofolate into tetrahydrofolate, a cofactor 7

6

required for the de novo synthesis of purines, thymidylate, and certain amino acids. DHFR has been 9

8

successfully exploited in the design of anticancer and antimicrobial agents.1 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30

Figure 1: Structures of dihydrofolate reductase inhibitors. 32

31 3 35

34

With the advent of highly active antiretroviral therapy (HAART) the incidence of opportunistic 36 38

37

infections has reduced dramatically, but non-compliance, toxicity with current treatments, emergence of 40

39

drug resistant strains, late diagnosis of HIV, and the rise of HIV cases in developing countries all 42

41

contribute to the persistence of opportunistic infections as a clinical problem.2-5 The Centers for Disease 43 45

4

Control report in 2009 has stated that for patients with HIV, under certain circumstances, prophylaxis of 47

46

opportunistic infections may need to be continued for life and that, despite HAART, opportunistic 49

48

infections remain a leading cause of morbidity and mortality in HIV infected patients.6, 7 Pneumocystis 50 52

51

pneumonia (PCP) [caused by the fungus Pneumocystis jirovecii (pj)], toxoplasmosis [caused by the 54

53

protozoan Toxoplasma gondii (tg)] and Mycobacterium avium-intracellulare infection (MAI) [caused 5 57

56

by the bacteria Mycobacterium avium and Mycobacterium intracellular] are the most common 60

59

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opportunistic infections.6 ACS Paragon Plus Environment

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Lipophilic, nonclassical antifolates (Figure 1) like trimethoprim (TMP), pyrimethamine and 2

1

trimetrexate (TMQ) are approved agents for the treatment and prophylaxis of opportunistic infectionsin 3 4

immunocompromised patients, including MAI.8 TMP is a selective, but weak inhibitor of 5 7

6

microbial/fungal DHFR. TMP with sulfamethoxazole (SMX) is the most commonly prescribed regimen 9

8

for opportunistic infections in AIDS patients.6 SMX, an inhibitor of dihydropteroate synthase, is 10 12

1

necessary to compensate for the weak DHFR inhibitory activity of TMP and to decrease the appearance 14

13

of resistant strains. Side effects associated with sulfonamides and dapsone9-11 often leads to 16

15

discontinuation of therapy.12 TMQ is a potent, but non-specific DHFR inhibitor, used in the treatment of 17 18

moderate to severe PCP and causes a high rate of myelosuppression13, 14 and is co-administered with 21

20

19

leucovorin (5-formyltetrahydrofolate) as a rescue agent to prevent host cell toxicity.8, 13, 15 However, this 23

2

dual therapy increases costs and the rescue is not always successful. Piritrexim (PTX) is another potent, 24 26

25

non-selective DHFR inhibitor that has been studied along with leucovorin in clinical trials as an 28

27

antiopportunistic agent in AIDS patients.8 29 30

Given the limitations of both the TMP/sulfonamide combinations, regarding side effects and 31 3

32

resistance due to the sulfa drug component11, 15 and TMQ/leucovorin regimens that do not work under 35

34

several clinical settings, a single DHFR inhibitor that combines the potency of TMQ or PTX with the 36 37

species selectivity of TMP would be highly desirable, in that it would eliminate the need to co40

39

38

administer either a sulfonamide or leucovorin with the DHFR inhibitor. However, to our knowledge, the 42

41

compounds reported thus far in the literature are not both selective and potent enough to achieve the 43 4

goal of producing a clinically viable single therapeutic agent.7 47

46

45

. 49

48

Design of novel inhibitors: 50 52

51

PCP was originally thought to be caused by the fungus Pneumocystis carinii (pc), but, it is now 54

53

known that Pneumocystis jirovecii (pj) is responsible for PCP in humans.16-18 The DHFR versions from 56

5

these two organisms differ by 38% in amino acid sequence and differ in sensitivity to TMP and MTX.16 57

60

59

58

No crystal structure of pjDHFR has been reported thus far. Early analogs in this study were initially ACS Paragon Plus Environment

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designed as pcDHFR inhibitors that served as a surrogate for pjDHFR (albeit not a good surrogate) and 2

1

only in the latter stages of this work, following our isolation of pjDHFR in sufficient quantities,17 was 3 4

the inhibition of pjDHFR included in this study. 5 7

6

Gangjee et al.,19-27 as well as others,28-34 have reported several structural classes of DHFR 9

8

inhibitors in a quest for high potency and high selectivity against microbial/fungal DHFR over human 10 1

DHFR.18 One series of bicyclic pyrido[2,3-d]pyrimidines that is absent in the literature is the N614

13

12

(substituted phenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamines in which the substituted aniline is directly 15 16

attached to the 6-position of the pyrido[2,3-d]pyrimidine ring. These are analogs of PTX (Fig. 1), a 17 19

18

potent but non-selective DHFR inhibitor. The 5-methyl moiety of the 5-deaza-5-methyl classical as well 21

20

as nonclassical antifolates such as PTX and TMQ (Figure 1) make hydrophobic contact with Val115 in 23

2

hDHFR.35 Champness et al.36 indicated that the 5-methyl group of PTX is in van der Waals contact (≤ 4 24 26

25

Å) with Ile123 of pcDHFR. PTX lacks selectivity but is a potent inhibitor of both pcDHFR and 28

27

hDHFR.20 The amino acid sequence of pjDHFR has a corresponding Ile123 as well,17 and results in the 29 31

30

observed high potency but not selectivity (since hDHFR has a Val115). 32 3 34 35 36 37 38 39 40 41 42 43 45

4

Figure 2. Selective pathogen DHFR inhibitors. 46 47 48 49

In 2002, Cody et al.37 showed from X-ray crystal structure that the 5'-methoxy of 1 (Figure 2) 52

51

50

fits deep within the hydrophobic pocket in pcDHFR lined by Ile65, Pro66 and Phe69 (Figure 3). 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment

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1 2 3 4 5 6 7 8 9 10 1 12 13 14 15 16 17 19

18

Figure 3. Stereoview of quinazoline inhibitor 137 in pcDHFR binding site (pdb id: 1LY3); generated using MOE 21

20

2010.10.38 (Hydrogen atoms eliminated for clarity). 2 23 24 25

The corresponding amino acids in hDHFR are Ile60, Pro61 and Asn64, respectively. The 28

27

26

replacement of hydrophobic Phe69 in pcDHFR with the more polar Asn64 allows the design of 30

29

selective agents for pcDHFR with hydrophobic substituents.20, 21, 27 Additionally, the N9-methyl 31 3

32

moiety of 1 is in van der Waals contact (≤ 4 Å) with the side chain of Ile123 of pcDHFR probably 35

34

contributing to its increased potency. 37

36

Gangjee et al.20, 25 have reported pyrido[2,3-d]pyrimidine analogs such as 2a, 2b, and 3 (Figure 38 40

39

2). Compounds 2a and 2b were selective for pcDHFR (selectivity ratios = 15.7 and 13, respectively) 42

41

and tgDHFR (selectivity ratio = 23 and 4.7, respectively) over rlDHFR,25 while compound 3 exhibited 43 4

excellent selectivity for pcDHFR (selectivity ratio = 101) and tgDHFR (selectivity ratio = 303) over 47

46

45

hDHFR.20, 27 Crystallographic data of the ternary complex hDHFR-NADPH and 3 was used to explain 49

48

its selectivity for pathogen DHFR compared to hDHFR.27 50 51

PTX analogs without the 5-methyl groups do not interact with hDHFR as effectively and exibit 54

53

52

lower potency as compared to PTX. If the relative loss of potency of these analogs against hDHFR is 56

5

greater than the loss of potency against pjDHFR (or other microbial DHFR), these compounds would be 57 58 59 60 ACS Paragon Plus Environment

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more selective inhibitors of pjDHFR than hDHFR. This hypothesis has been validated in a previous 2

1

report for pcDHFR and hDHFR.20, 27 3 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28

Figure 4. Novel DHFR inhibitors 4-15 29 30 31 3

32

A similar approach was utilized in the design of analogs 4-15 (Figure 4) of this study in which 35

34

the 5-methyl moiety of PTX was removed and the 6-methylene moiety of PTX was isosterically 36 38

37

replaced by an NH group. Using this hypothesis, variations of electron-donating, electron-withdrawing 40

39

and bulky substituents on the phenyl side chain were implemented to explore the SAR of this novel 42

41

series of compounds. In the previous reports20 involving pyrido[2,3-d]pyrimidines and pyrido[3,243 45

4

d]pyrimidines as DHFR inhibitors against opportunistic infections, it has been shown that compounds 47

46

with electron rich 3',4',5'-trimethoxy and 2',5'-dimethoxy substituted phenyl rings were usually more 48 49

selective (20- to 304-fold for pathogen (tg, E. coli) DHFR. Hence compounds 5-8 were designed as 52

51

50

possible selective inhibitors. These compounds could interact with Phe69 of pcDHFR over Asn64 in 54

53

hDHFR and afford selectivity.37 5 56

In addition, compounds with bulky substituents such as 1-naphthyl and 2-naphthyl rings20, 23 60

59

58

57

were found to be more potent DHFR inhibitors as compared to those with an unsubstituted phenyl ring. ACS Paragon Plus Environment

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Hence compounds 9-11 and 13 were included in this study to explore the possibility of improving the 2

1

overall potency while maintaining selectivity. The naphthyl group can also interact with Phe69 of 3 4

pcDHFR and not with Asn64 in hDHFR, and could afford selective and potent inhibitors of pcDHFR. 5 7

6

In the pyrido[2,3-d]pyrimidine series of compounds Gangjee et al.25 showed that the 2',5'9

8

dichlorophenyl substituted compound 2a (Figure 2) was the most selective inhibitor of both pcDHFR 10 1

(selectivity ratio = 15.7) and tgDHFR (selectivity ratio = 23) versus rlDHFR. Additionally, in a recent 14

13

12

report by Cody et al.17 this pyrido[2,3-d]pyrimidine analog with a 2',5'-dichlorophenyl ring was 16

15

significantly potent and selective for pjDHFR (IC50 = 0.172 µM; selectivity ratio = 33) over rlDHFR. In 17 19

18

the same series of compounds a 3',4',5'-trichlorophenyl substituted pyrido[2,3-d]pyrimidine 2b (Fig. 2) 21

20

also exhibited excellent potency and selectivity for pjDHFR (IC50 = 0.0534 µM; selectivity ratio = 23) 23

2

over rlDHFR. Hence compounds 12, 14 and 15 were included to determine the effect of similar electron 24 26

25

withdrawing substituents on potency and selectivity against pjDHFR. 28

27

The truncated bridge (one N atom vs. the usual two atom C-N bridge) between the heterocycle 30

29

and the aryl ring of these novel series of compounds20-25 has less flexibility as compared to TMQ. This 31 3

32

is expected to decrease the number of possible conformations and hence may increase selectivity and/or 35

34

potency of these compounds. 36 37 38 40

39

Design of compounds 16-26: 42

41

Transposition of the 5-methyl group of PTX and TMQ (Figure 1) to the N9-position in 43 45

4

pyrido[2,3-d]pyrimidine analogs (Figure 2), in general improves potency as well as selectivity against 47

46

pathogen (tg, E. coli) DHFR.20 48 49 50 51 52 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment

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1 2 3 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18

Figure 5. N9-methyl analogs 16-26 19 20 21 23

2

A crystal structure of 3 (Figure 2) complexed with hDHFR showed that the N9-methyl moiety is 25

24

not in hydrophobic contact with any hydrophobic side chain of hDHFR.27 In addition, in the molecular 26 28

27

model of 3 in pcDHFR the N9-methyl moiety of 3 forms hydrophobic contacts with both Ile123 and 30

29

Ile65 of pcDHFR. Additional hydrophobic interactions of the N9-methyl moiety of 3 with pcDHFR 31 32

compared to the lack of hydrophobic interactions with hDHFR was one of the possible reasons for the 35

34

3

increased potency and selectivity of 3 for pcDHFR compared with hDHFR. 37

36

Thus compounds 16-26 (Figure 5) were designed by incorporating a N9-methyl group. This 38 40

39

methyl group was anticipated to interact (Figure 6) with Ile123 of pcDHFR (and also with the 42

41

corresponding Ile123 of pjDHFR) and not with the shorter Val115 in hDHFR in a similar way as that of 4

43

3,27 thus affording both potency and selectivity for pcDHFR and pjDHFR. 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment

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1 2 3 4 5 6 7 8 9 10 1 12 13 14 16

15

Figure 6: Stereoview. Docked pose of 16 in pcDHFR binding pocket (PDB: 1LY337). 17 18 19 21

20

To validate our hypothesis prior to biological evaluation, we docked 16 into the pcDHFR active 23

2

site (PDB: 1LY3) using LeadIT 1.3.0 (details in Experimental).39 Figure 6 shows the docked pose of 16 24 26

25

in the pcDHFR active site. In Figure 6 the N9-methyl group is 3.94 Å from the terminal methyl group of 28

27

Ile123 of pcDHFR. In hDHFR, the corresponding Val115, being shorter by one carbon, may not interact 29 30

with the N9-methyl group. This should improve selectivity as well as potency of these compounds 31 3

32

against pcDHFR (and pjDHFR) over hDHFR. In addition the N9-methyl group of 16 is only 3.8 Å away 35

34

from side chain Ile65 in pcDHFR (Fig. 6), and may improve potency by hydrophobic interactions. 36 37

Further, the N9-methyl group restricts the number of possible conformations of the side chain phenyl 40

39

38

group, thus perhaps contributing to increasing potency and selectivity.20, 40 Compounds with an N942

41

methyl group are more lipophilic than the corresponding N9-H analogs; thus improving hydrophobicity 43 4

and perhaps cell penetration.40 Thus compounds 16-26 with the N9-methyl group and various electron 47

46

45

donating, electron withdrawing and bulky hydrophobic substituents were included to explore the SAR 49

48

and to afford potent and selective inhibitors for pcDHFR and pjDHFR. 50 51 52 54

53

Chemistry: 5 56

The synthesis of target compounds 4-26 were envisioned from the dipivaloyl-protected 657

60

59

58

bromopyrido[2,3-d]pyrimidine intermediate 30 (Scheme 1). ACS Paragon Plus Environment

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Scheme 1: Synthesis of 30 1 2 3 4 5 6 7 8 9 10 1 12 13 15

14

Compound 30 was synthesized from 2,4,6-triaminopyrimidine 27 via the reported method of 17

16

Gangjee et al.41, 42 (Scheme 1). Compound 27 was dissolved in absolute ethanol at reflux with a drop 18 20

19

wise addition of concentrated HCl (just sufficient to dissolve 27) followed by the addition of bromo2

21

malonaldehyde 28 at reflux to obtain 29. Both amino groups of 29 were converted to pivaloyl amides by 23 24

reacting 29 with trimethylacetic anhydride in pyridine at reflux to afford 30. Since both amino groups of 25 27

26

30 are protected as pivaloyl amides and since these pivaloyl groups afforded excellent solubility in non29

28

polar solvents, 30 was an excellent substrate for metal-catalyzed coupling reactions for the synthesis of 30 31

the target compounds. 34

3

32

Initial attempts at amination of 30 using a standard Buchwald-Hartwig coupling protocol43, 44 36

35

[Pd(OAc)2, (±)-BINAP, NaOt-Bu, Toluene, reflux] or copper (I) catalyzed aminations45 (CuI, L-Proline, 37 39

38

DMF, Na2CO3, 120 °C) were unsuccessful. Hence a systematic screening approach was undertaken to 41

40

determine if the Buchwald-Hartwig amination of dipivaloyl-protected 6-bromopyrido[2,3-d]pyrimidine 43

42

30 was possible and if so, to determine the optimum conditions. 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment

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Development of a coupling protocol for Buchwald-Hartwig coupling of 30 with substituted 2

1

anilines: 3 4 5 6 7

PPh2 PPh2

8 9

i-Pr

O PPh2

10

13

rac-BINAP

i-Pr

PPh2

1 12

P(C6H11)2

P(C6H11)2

Xantphos

i-Pr X-Phos

DCPB

14 15 16

P[CH3)3]2

17 i-Pr

18

i-Pr

P(C6H11)2 Oi-Pr

P(C6H11)2 i-PrO OMe

MeO

P(C6H11)2 N(Me)2

19 21

20 i-Pr t-ButylX-Phos

2

S-Phos

RuPhos

DavePhos

24

23

Figure 7: Ligands for amination reaction 25 26 28

27

A series (Figure 7) of bidentate [racemic-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (rac29 30

BINAP), 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (XantPhos)] and monodentate-biphenyl 3

32

31

[dicyclohexylphospho biphenyl (DCPB), 2-dicyclohexyl-phosphino-2',4',6'-triisopropylbiphenyl (X35

34

Phos), 2-di-t-butylphosphino-2',4',6'-tri-i-propyl-1,1'-biphenyl (t-Butyl-X-Phos), 236 37

dicyclohexylphosphino-2',6'-dimethoxybiphenyl (S-Phos), 2-dicyclohexylphosphino-2',6'-di-i-propoxy40

39

38

1,1'-biphenyl (Ru-Phos), 2-(dicyclohexylphosphino)-2'-(N,N-dimethylamino)biphenyl (DavePhos)] 42

41

were selected on the basis of previous successful literature reports and to vary the bulk, electronics and 43 4

chelating abilities of the ligands.46-48 47

46

45

Reaction of 30 with p-anisidine 31b to form the coupled product 32b was used as a model 49

48

reaction for ligand-screening (Scheme 2). It was gratifying to note that the biphenyl ligands were 50 52

51

effective and X-Phos was the most suitable ligand for carrying out this conversion. The results of the 54

53

ligand-screening experiments are shown in Chart 1. 5 56 57 58 59 60 ACS Paragon Plus Environment

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1 2 4

3

Scheme 2: Amination reactions of 30 with substituted anilines – Method development 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 34

3

Chart 1: Ligand-screening experiments 36

35

a

37

Isolated yields. [in all cases except with DCPB (resulted in debromination), unreacted starting material was also

38

isolated]. (conditions: 1 equivalent 30; 1.2 equivalent p-anisidine 31b; 2 mol % Pd2dba3; 8 mol % monodentate 39 40

ligand / 4 mol % bidentate ligand; 1.4 equivalents NaOt-Bu; Toluene, 100°C, 20 h). 42

41 43 45

4

For rac-BINAP, XantPhos and DavePhos, unreacted starting material was isolated (90-95 %); 46 47

while when DCPB was used, a majority of the staring material underwent debromination. For X-Phos, 50

49

48

S-Phos, t-ButylX-Phos and Ru-Phos, unreacted starting material was isolated (40-85 %) even after 48 52

51

hours of stirring; which suggests that the catalyst degraded before complete consumption of the starting 53 54

material. 56

5 57 58 59 60

ACS Paragon Plus Environment

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Hence it was decided to optimize the yields by increasing the catalyst loading. Increasing the 2

1

catalyst loading up to 15 mol % (with respect to Pd2dba3) improved the yield from 45 % to 60 %. 3 4

Variation of the base used was attempted next in an effort to improve the reaction yields (Table 1). 5 7

6

Cesium carbonate (Cs2CO3) did not aid in the reaction and starting material was isolated almost 9

8

quantitatively (entries 4 and 5). Addition of 2.2 equivalents of lithium hexamethyldisilazide (LiHMDS) 10 1

led to slight improvement (entry 6) in yield (53%). An additional one equivalent of LiHMDS improved 14

13

12

the yield to 91% (entry 7). 15 16 17 18

Table 1: Selection of a suitable base 2

21

20

19

Entry Base

Equivalents Isolated yield (%)

24

1

NaOt-Bu

1.5

45

2

NaOt-Bu

2.5

48

3

NaOt-Bu

3

43

4

Cs2CO3

1.6

0

5

Cs2CO3

2.6

0

6

LiHMDS

2.2

53

7

LiHMDS

3.2

91

8

LiHMDS

3.7

90

23

29

28

27

26

25

36

35

34

3

32

31

30

41

40

39

38

37

42 43 4

(Conditions: 1 equivalent 30; 1.2 equivalent p-anisidine 31b; 2 mol % Pd2dba3; 8 mol % X-Phos; # of equivalents 47

46

45

of Base; Toluene, 100°C, 20 h). 49

48

Additional NaOt-Bu, however, did not improve the yield significantly (entries 2 and 3; Table 1). 51

50

This also indicated that deprotonation is not the rate determining step of this reaction. These results 52 53

suggested that premature deactivation of the catalyst probably occurs during the reaction and that the 56

5

54

addition of an extra equivalent of LiHMDS prevents this deactivation. Similar observations have been 58

57

reported by Buchwald et al.49 and suggested that the lithiate or a lithium-amide aggregate of the 59 60 ACS Paragon Plus Environment

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deprotonated amide from the substrate functions as a protecting group, thus inhibiting oxygen from 2

1

binding to the palladium center, and preventing its deactivation. 3 4

This strategy was applied for the synthesis of 32a-l (Table 2) in significantly better yields. 5 6 7 9

8

Table 2: Amination with substituted anilines 10 1 12 13 14 15 16 17 18 19 20 21 2 24

23

Entry Comp. No. 25

R1

26

Isolated Yield (%)

27

34

3

32

31

30

29

28

1

32a

H

85

2

32b

4'-OMe

88

3

32c

2'-OMe

56

41

40

39

38

37

36

4

32d

2',5'-diOMe

55

5

32e

3',4',5'-triOMe

78

6

32f

4'-Me

93

7

32g

4'-i-Pr

91

8

32h

3',4'-(C4H4)

75

9

32i

4'-NO2

60

10

32j

2',3'-(C4H4)

65

11

32k

2',5'-diF

78

12

32l

3',4',5'-triF

75

35

46

45

4

43

42

48

47

53

52

51

50

49

56

5

54

57 58 59 60 ACS Paragon Plus Environment

16

Page 17 of 67


Anilines with electron-donating substituents (entries 2, 6-7; Table 2) afforded better yields; 2

1

while those with electron-withdrawing substituents (entries 9, 11-12) led to slightly lower yields. Bulky 3 4

ortho substituents on the anilines (entries 3-4, 10) probably retard the rate of the reaction, leading to 5 7

6

inferior yields. 9

8

In all cases, the 1H NMR spectra of the products showed the appearance of a new broad singlet 10 1

(D2O exchangeable) between δ 5.9- δ 6.4 integrating for 1 proton and corresponding to the 9-NH 14

13

12

proton. Additionally aromatic protons of the coupled arylamine, in the aromatic region, confirmed the 16

15

formation of the products. All the products were confirmed by HRMS analysis. 17 18

Amination of 30 with N-methylanilines using above protocol, however, did not lead to complete 21

20

19

consumption of the starting material even after prolonged reaction time (entries 1 and 2; Table 3). 23

2

Table 3: Amination using N-methyl-4-methoxy-anilines 24 25 26 27 28 29 30 31 32 3 34 35

Entry Ligand 36

Time (h) Results; Isolated

37 38

yield 39 40

1 41

X-Phos

20

Incomplete;

42

Inseparable 43 4 45 46 47 48

2

X-Phos

48a

Incomplete; 57 %

3

S-Phos

20

Complete; 88 %

49 50 52

51 a

54

53

An additional aliquot of Pd2dba3 (2 mol %) and X-Phos (8 mol %) premixed with

toluene was added after 24 h. 5 56 57 58 59 60 ACS Paragon Plus Environment

17


Page 18 of 67

It was hypothesized that the methyl group on the aniline nitrogen may lead to steric hindrance to 2

1

the approach of the palladium complex involving the bulky X-Phos ortho-isopropyl groups. Hence S3 4

Phos with smaller ortho-methoxy groups was employed. Indeed, this led to the complete conversion of 5 7

6

the starting material with excellent isolated yield of the desired 32n. (Table 3, entry 3). 9

8

Table 4: Amination with substituted N-methylanilines 10 1 12 13 14 15 16 17 18 19 20 2

21

Entry Comp. No. 23

R1

Isolated yield

24

(%) 25

30

29

28

27

26

1

32m

H

83

2

32n

4'-OMe

90

35

34

3

32

3

32o

2'-OMe

59

4

32p

2',5'-diOMe

55

5

32q

3',4',5'-triOMe

81

6

32r

4'-Me

96

7

32s

4'-i-Pr

93

8

32t

4'-NO2

63

9

32u

2',3'-(C4H4)

56

10

32v

2',5'-diF

93

11

32w

3',4',5 '-triF

90

31

42

41

40

39

38

37

36

49

48

47

46

45

4

43

52

51

50

53 54 5

This strategy was applied for the amination reactions of 30 with substituted N-methylanilines to 56 58

57

synthesize 32m-w in good to excellent yields (Table 4). The 1H NMR spectra of the products showed 59 60 ACS Paragon Plus Environment

18

Page 19 of 67


the presence of a sharp singlet between δ 3.4 – δ 3.6 integrating for 3 protons of the NCH3 group. All 2

1

the products were confirmed using HRMS analysis. 3 4 5 7

6

Scheme 3: Synthesis of target compounds 4-26 8 9 10 1 12 13 14 15 16 17 18 19 20 2

21

Pivaloyl deprotection of compounds 32a-w was carried out with liquid NH3 in a Parr pressure 24

23

vessel at room temperature to afford target compounds 4-26 (Scheme 3). The 1H NMR spectra of the 25 26

products showed the disappearance of a doublet (~ δ 1.3) corresponding to the pivaloyl groups and the 27 29

28

presence of two broad D2O exchangeable peaks corresponding to the 2,4-diamino groups. 30 31 3

32

Scheme 4: Synthesis of substituted N-methylanilines 34 35 36 37 38 39 40 41 42 43 4 46

45

Substituted N-methylanilines 31p-q, 31s and 31v-w (Scheme 4) were synthesized via a one pot 48

47

reaction in which the corresponding substituted anilines 31d-e, 31g and 31k-l respectively were reacted 49 50

with paraformaldehyde in the presence of sodium methoxide followed by treatment with sodium 53

52

51

borohydride in methanol at reflux.50 The 1H NMR spectra of the products showed the presence of a 5

54

singlet integrating for 3 protons at around δ 2.8 corresponding to the N-methyl moiety. 56 57 58 59 60 ACS Paragon Plus Environment

19


Page 20 of 67

2

1

Biological evaluation and Discussion: 3 4 5 6 7

Compounds 4-26 were evaluated as inhibitors of recombinant pjDHFR, recombinant hDHFR, 9

8

pcDHFR, rlDHFR and maDHFR. Selectivity ratios were determined using recombinant hDHFR or 10 1

using rlDHFR as a mammalian DHFR source. MTX, TMP, PTX, pyrimethamine, and TMQ were used 14

13

12

as positive controls in the assays. 15 16 17 18

Table 5: Inhibitory concentrations (IC50, in µM) against recombinant DHFR from P. jirovecii (pj); human (h) and 19 20

selectivity ratiosa 23

2

21

Compound 24

pjDHFR

hDHFR

µM

µM

IC50

IC50

4

0.30

0.19

0.6

5

0.40

3.65

9.1

6

0.44

0.25

0.6

7

0.18

1.0

5.6

8

0.31

3.9

13

9

0.62

2.1

3.3

10

1.1

5.9

5.6

11

0.40

2.2

5.4

12

3.6

7.2

2.0

13

0.25

2.1

8.2

14

0.95

0.45

0.5

15

0.87

3.1

3.5

25 26

h/pj

27 28

3

32

31

30

29

38

37

36

35

34

45

4

43

42

41

40

39

50

49

48

47

46

5

54

53

52

51

57

56 58 59 60


20

3

2

1

Page 21 of 67


16

0.0022

0.057

26

17

0.0019

0.012

6.6

10

9

8

7

6

5

18

0.0016

0.014

9.3

19

0.0021

0.0047

2.2

20

0.0016

0.0046

2.8

21

0.0024

0.032

13

22

0.0024

0.052

21

23

0.028

0.34

12

24

0.0025

0.0056

2.2

25

0.0034

0.046

14

26

0.0042

0.15

35

TMP

0.12

32.2

268

MTX

0.0051

0.0017

0.34

PTX

0.0016

0.003

1.9

TMQ

0.0021

0.0026

1.2

0.40

3.1

4

15

14

13

12

1

20

19

18

17

16

2

21

27

26

25

24

23

32

31

30

29

28

34

3

36

35

pyrimethamine 0.13 37 38 39 40 41 42 43 45

4 a

46

These assays were carried out at 37 °C under 18 µM dihydrofolic acid concentration. The assay also

48

47

contains 117 µM NADPH, 8.9 mM 2-mercaptoethanol, 150 mM KCl, 41 mM Na phosphate buffer pH 50

49

7.4 and sufficient enzyme to cause a change in OD340 of 0.005/minute. The standard error of the means 51 53

52

for these values are 12% or less than the mean value. 54 5 56 57 58 59 60 ACS Paragon Plus Environment

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Page 22 of 67

The IC50 values and selectivity ratios of compounds 4-26 against recombinant pjDHFR and 2

1

recombinant hDHFR are shown in Table 5. Standard compounds show the properties expected, based on 4

3

prior studies: TMP (IC50 0.12 M) and pyrimethamine are not potent inhibitors of pjDHFR but TMP is 7

6

5

the most selective compound, whereas MTX, PTX and TMQ are all potent but relatively non-selective. 9

8

Experimental compounds with N9-H exhibited submicromolar to micromolar inhibition of pjDHFR and 10 12

1

hDHFR. Compounds with electron-donating substituents at the meta- and/or para- positions on the 14

13

phenyl side chain (5, 7-10) were less potent against hDHFR compared to the unsubstituted phenyl 16

15

compound 4. While against pjDHFR these compounds retained potency (5, 7, 8) or exhibited relatively 17 19

18

less loss of potency than that against hDHFR (9-10) as compared to 4 thus affording improved 21

20

selectivity ratio (8; selectivity ratio = 12.7). 2 23

Compounds with bulky side chains (11 and 13) retained potency against pjDHFR, and exhibited 24 26

25

a 10-fold loss in potency against hDHFR leading to improved selectivity for the pathogen DHFR 28

27

compared to the unsubstituted phenyl compound 4. 29 31

30

Compounds with electron deficient side chains exhibited diminished potency against pjDHFR 3

32

indicating the importance of electronics for potency. Compound 15 (3',4',5'-trifluorophenyl) showed 34 35

slightly better selectivity for pjDHFR (selectivity ratio = 3.5) compared to the unsubstituted phenyl 36 38

37

compound 4. Compound 14 (2',5'-difluorophenyl) showed poor selectivity and 12 (4-nitrophenyl) 40

39

exhibited diminished inhibitory activities against both pjDHFR and hDHFR. Substitution of electron41 42

withdrawing groups on the side chain phenyl ring were not conducive for potency or selectivity as 43 45

4

compared to substitutions with electron-donating groups. 47

46

Inclusion of a methyl group at the N9- position led to a remarkable 100-fold improvement in 48 50

49

potency with superior selectivity. Compound 26 (3',4',5'-trifluorophenyl side chain) was the most 52

51

selective compound of the series. The selectivity may be attributed, in part, to the possible interaction of 53 54

the N9-methyl with the longer side chain of Ile123 of pjDHFR; while interaction with the shorter 5 57

56

Val115 in hDHFR may not be as productive. 58 59 60 ACS Paragon Plus Environment

22

Page 23 of 67


A similar trend in selectivity was observed for compounds 16, 18, 21-23, and 25. However 2

1

compounds 17, 19, 20 and 24 exhibited slightly diminished selectivity compared to the corresponding 3 5

4

N9-H compounds 5, 7, 8 and 11. Thus, the presence of the methyl group at the N9- position perhaps 7

6

dictates the conformation of the bound inhibitor and its binding energy and is different for 18, 21 – 23 9

8

and 25 – 26 compared with 17, 19, 20, and 24. 10 12

1

Compounds with methoxy groups at ortho-, meta- and para- positions of the side chain phenyl 14

13

ring exhibited improved potency against pjDHFR. However, these compounds also showed marked 15 16

improvement in potency against hDHFR leading to diminished overall selectivity for the pathogen 17 19

18

DHFR as compared to the unsubstituted phenyl compound 16. Compound 18 (2'-methoxyphenyl side 21

20

chain) was the most potent compound against pjDHFR with good selectivity (IC50 = 1.55 nM; 2 23

selectivity ratio = 9.3). Compounds with alkyl substituents as well as bulky substituents retained 24 26

25

potency, while those with electron-withdrawing substituents exhibited a slight loss of potency against 28

27

pjDHFR. Compound 26 (3',4',5'-triflurophenyl side chain), exhibited a marked improvement in 29 31

30

selectivity for pjDHFR. 3

32

Analogs 16-26 have high potencies against pjDHFR, similar to the most potent standard 35

34

compounds MTX, PTX, but have the advantage of significantly enhanced selectivity (16, 22, 26) against 36 38

37

pjDHFR. The most selective compounds are about 10-fold less selective than TMP but are about 50-fold 40

39

more potent (16). Analogs 16, 22 and 26 are lead compounds for the development of potent and 41 42

selective inhibitors of pjDHFR. 43 4 45 47

46

Docking Studies with pjDHFR 48 49 51

50

There is currently no known crystal structure of pjDHFR. A homology model was built using the 52 53

1.60 Å pcDHFR crystal structure (PDB: 2FZI51, chain A) for evaluating the binding of 26 in pjDHFR. 56

5

54

Docking studies were performed using LeadIT 1.3.0.39 The docked poses were exported to MOE 58

57

2010.10,38 rescored using the affinity dG scoring system, refined using the forcefield system and 59 60 ACS Paragon Plus Environment

23


Page 24 of 67

rescored using London dG scoring system. The binding poses were also visualized using the ligplot 2

1

utility in MOE and the Poseview utility in LeadIT 1.3.0. 3 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 24

23

Figure 8: Stereoview. Docked pose of 26 in pjDHFR homology model. 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43

Figure 9: Stereoview. N-Me of 26 interacts with Ile123 (pjDHFR, red) and not as well with Val115 46

45

4

(superimposed hDHFR,35 green). 47 48 50

49

Figure 8 shows the best docked pose of 26 (white) in the folate binding site of the pjDHFR 51 53

52

model. In this pose the protonated N1 and 2-NH2 of 26 interact in an ionic bond with Asp32. This 5

54

bidentate ionic bond with a conserved acid residue has been observed in most DHFR crystal 57

56

structures.52 The 4-NH2 moiety forms hydrogen bonds with the backbone of Ile10. The pyrido[2,358 59 60 ACS Paragon Plus Environment

24

Page 25 of 67


d]pyrimidine scaffold is stabilized by a pi-stacking interaction with Phe36 and with side chain carbon 2

1

atoms of Met33 (not shown) and Leu25. The 3′,4′,5′-trifluorophenyl moiety of 26 can form hydrophobic 3 5

4

interactions with the side chain atoms of Leu25, Thr61, Ser64, and Leu65. The 3′-F of 26 interacts with 7

6

the hydroxyl moiety of Ser64. The N9-Me moiety of 26 is oriented towards the hydrophobic pocket 9

8

formed by Ile123, Leu72 and Leu65 and interacts specifically with Ile123. The docking score of 26 was 10 12

1

-35.98 kJ/mol for pjDHFR compared with -35.41 kJ/mol for hDHFR. The docking scores for TMP 14

13

(-23.23 kJ/mol), MTX (-58.24 kJ/mol) and pyrimethamine (-29.36 kJ/mol) are consistent with the 15 16

corresponding observed inhibitory activities against pjDHFR. Compound 16 makes similar binding 17 19

18

with pjDHFR except for the trifluoro groups in 26. It was of interest to attempt to explain the potency 21

20

and selectivity of 16 and 26 (compared to 4 and 15 respectively) for pjDHFR. From the IC50 values in 2 23

Table 5 for the two pairs, 16 / 4 and 26 / 15, it is evident that the N9-Me moiety makes about a 300- and 24 26

25

a 200-fold difference in potency for pjDHFR respectively. There are two important consequences of 28

27

adding the Me group on the N9. The first one is the interaction of the N9-Me with Ile123 in pjDHFR 29 31

30

(Fig. 9). This interaction is absent in the N9-H analogs, 4 and 15 from modeling studies. The second 3

32

significance from modeling studies (SYBYL x1.3,; around the C6-C9 bond, using 5 increments) is that 34 35

the N9-Me restricts the available low energy conformations (50 conformations, within a 3 k cal/mol cut 36 38

37

off) that the molecule can adopt compared with the unhindered N9-H (94 conformations). Thus it is 40

39

perhaps easier for 16 and 26 to adopt the bound conformation than it is for 4 and 15 and results, in part, 41 42

in the improved IC50 of 16 and 26 over 4 and 15. In addition to potency, the selectivity of 16 and 26 for 43 45

4

pjDHFR over hDHFR are superior to that of 4 and 15. Once again, the N-Me moiety must play a 47

46

significant role in the high selectivity of 16 and 26 for pjDHFR over hDHFR compared to 4 and 15. In 48 50

49

pjDHFR the N9-Me moiety is 3.67 Å away from the longer Ile123 compared to hDHFR where it is 4.64 52

51

Å away from the shorter Val115. These superimpositions of the docked structure of 26 in the pjDHFR 54

53

homology model and hDHFR crystal structure (Fig. 9) and the highly productive interaction of the N95 57

56

Me with Ile123 at 3.67 Å and a somewhat less of a productive interaction of the N9-Me with the shorter 60

59

58

Val115 of hDHFR affords, in part, a molecular explanation of the pjDHFR selectivity of 26 over 15. ACS Paragon Plus Environment

25


Page 26 of 67

Confirmation of these homology model predictions await the X-ray crystal structures of these analogs 2

1

with pjDHFR and hDHFR, currently in progress. 3 4 5 7

6

Table 6: Inhibitory concentrations (IC50, in µM) against DHFR from P. carinii (pc); rat liver (rl); M. 9

8

avium (ma) and selectivity ratiosa 10

13

12

1

Compd.

pcDHFR

rlDHFR

18

17

16

15

#

µM IC50

µM IC50 rl/pc µM IC50

rl/ma

4

2.3

0.42

0.2

1.2

0.3

5

5.4

3.9

0.7

2.5

1.6

6

7.3

0.76

0.1

2.4

0.3

7

3.3

1.7

0.5

1.7

1.0

8

4.6

4.0

0.9

1.5

2.7

9

7.7

4.0

0.5

4.4

0.9

10

4.9

12

2.4

8.7

1.4

11

2.5

3.3

1.3

1.8

1.8

12

16

12

0.8

7.9

1.5

13

1.5

2.9

1.9

1.0

2.9

14

7.7

1.1

0.1

3.3

0.4

15

9.0

5.2

0.6

3.6

1.4

16

0.076

0.12

1.6

0.027

4.6

17

0.0024

0.0059

2.5

0.0021

2.8

18

0.023

0.024

1.0

0.0067

3.5

19

0.0036

0.0037

1.0

0.0022

1.7

20

0.0023

0.0068

3.0

0.00057

12

21

0.025

0.028

1.1

0.027

1.1

14

maDHFR

25

24

23

2

21

20

19

32

31

30

29

28

27

26

37

36

35

34

3

4

43

42

41

40

39

38

51

50

49

48

47

46

45

58

57

56

5

54

53

52

59 60 ACS Paragon Plus Environment

26

3

2

1

Page 27 of 67


22

0.021

0.072

3.4

0.026

2.7

23

0.58

0.61

1.1

0.15

0.9

10

9

8

7

6

5

24

0.0021

0.021

9.9

0.0020

10

25

0.098

0.070

0.7

0.017

3.9

26

0.23

0.43

1.9

0.029

15

TMP

12.5

180

14

0.0015

120000

MTX

0.0013

0.0025

1.9

0.00022

11

PTX

0.013

0.0033

0.3

0.00061

5

TMQ

0.042

0.003

0.07

0.0015

2

PYR

2.4

1.5

0.6

0.15

10

4

17

16

15

14

13

12

1

23

2

21

20

19

18

a

24

These assays were carried out as described for Table 5, except 90 µM dihydrofolic acid was used.

26

25

The standard error of the means for these values are 12% or less than the mean value. 27 28 29 31

30

Compounds 4-26 were also evaluated in an earlier screen against rlDHFR, pcDHFR, and 3

32

maDHFR (Table 6). These data are presented here in order to evaluate the potential of the experimental 34 35

compounds as single selective agents with activity against another important opportunistic pathogen 36 38

37

(MAI) and to demonstrate the differences in the original screen using pcDHFR with rlDHFR, versus the 40

39

modern screen using pjDHFR with hDHFR. 41 42

In the screen using pcDHFR and rlDHFR, compound 24 was the most selective compound 43 45

4

against pcDHFR (selectivity ratio = 9.9). Compounds with bulkier side chains (10, 11, 13, 22 and 24) 47

46

exhibited marginal selectivity for pcDHFR. Inclusion of a methyl group at N9- position improved 48 50

49

potency against both pcDHFR and rlDHFR. However, relative improvement in potency against 52

51

pcDHFR was greater compared to that against rlDHFR leading to a somewhat improved selectivity of 54

53

the N9-methyl compounds 16-26 compared to the corresponding N9-H compounds (4-10, and 12-15). 5 57

56

In the N9-H series of compounds (4-15) substitution of electron-donating as well as electron60

59

58

withdrawing groups on the phenyl side chain led to diminished potency against pcDHFR. Compounds ACS Paragon Plus Environment

27


Page 28 of 67

with bulky substituents on the side chain phenyl ring retained potency against pcDHFR compared to 4. 2

1

Additionally these compounds with bulky substituents exhibited improved selectivity for pcDHFR over 3 5

4

rlDHFR (10, 11, and 13). In the N9-methyl series of compounds (16-26) substitution of electron7

6

donating or bulky groups on the side chain phenyl ring afforded compounds with marked improvement 9

8

in potency (3- to 35-fold) against pcDHFR compared with analogs lacking these substitutions. 10 12

1

Compounds with electron withdrawing groups, however, exhibited decreased inhibitory activity against 14

13

pcDHFR. Generally, compounds with bulkier substituents on the phenyl side chain exhibited a 15 16

combination of superior potency as well as selectivity (13, 22 and 24). This potency and selectivity can 17 19

18

be attributed to the hydrophobic interaction with Phe69 in pcDHFR; while a similar interaction with 21

20

mammalian DHFR is not condusive because of the presence of a corresponding Asn64 in mammalian 2 23

DHFR. Compound 24 (2-naphthyl side chain) was the most potent as well as the most selective 24 26

25

compound against pcDHFR (IC50 = 2.1 nM; selectivity ratio = 9.9). This compound is as selective as 28

27

TMP and is additionally 5952-fold more potent than TMP against pcDHFR (IC50 12.5 M). It is also 629 31

30

fold more potent than PTX against pcDHFR. These differences of potency and selectivity of analogs 3

32

against pjDHFR and pcDHFR are included in this study to further underscore the differences between 34 35

rat derived pathogen DHFR (pc) and the human derived pathogen DHFR (pj). 36 38

37

Against maDHFR, all the compounds from the N9-H series (4-15) exhibited micromolar 40

39

inhibitory potency. Any variation from the unsubstituted phenyl ring led to slight decrease in potency. 41 42

Exception to this trend was 13 substituted with 2',3'-naphthyl side chain, which exhibited slightly 43 45

4

improved inhibitory potency against pcDHFR compared to compound 4 with an unsubstituted phenyl 47

46

ring. Electron-donating as well as electron-withdrawing groups at the ortho position of the side chain 48 50

49

phenyl group led to diminished selectivity for maDHFR (6, 7 and 14). Compounds with substitution on 52

51

the side chain phenyl group at the para and meta positions showed improved selectivity for maDHFR 53 54

(5, 8, 10-12 and 15). Compounds with bulky substituents exhibited good potency as well as selectivity 5 57

56

(13). Compounds from the N9-methyl series (16-26) were potent inhibitors of maDHFR. Compounds 60

59

58

with electron-donating substituents on the side chain phenyl ring (17-20) exhibited marked ACS Paragon Plus Environment

28

Page 29 of 67


improvement (2- to 45-fold) in potency against maDHFR. Compound 23 with an electron-withdrawing 2

1

4-NO2 group on the side chain phenyl ring showed diminished potency against maDHFR as compared 3 5

4

to the unsubstituted compound 16. Compounds with bulkier side chain retained potency. It appears that 7

6

substitution of electron-donating groups at the para- and meta- positions, (17 and 20), on the side chain 9

8

phenyl ring leads to improvement in potency as well as selectivity. In this series, compound 20 (3',4',5'10 12

1

trimethoxyphenyl side chain) was the most potent compound against maDHFR with subnanomolar 14

13

potency (IC50 = 0.573 nM) as well as good selectivity for maDHFR. 15 16

While some similarities were noted in the order of potency observed between the screen using 17 19

18

pjDHFR/hDHFR and the screen using pcDHFR/rlDHFR, there were also clear differences. For 21

20

example while compound 22 rates highly in both screens for selectivity, compound 24 would have been 2 23

selected as a candidate based upon the pcDHFR/rlDHFR screen, but does not rank high in the 24 26

25

pjDHFR/hDHFR screen. These results confirm that the differences in structure between pjDHFR and 28

27

pcDHFR significantly impact drug design and argue for rescreening of earlier libraries of compounds, 29 31

30

an activity that is now underway. 32 3 35

34

X-ray crystal structures (PDB Accession Numbers: pcDHFR-K37S/F69N-#16 (4IXG); pcDHFR36 37

F69N-#22 (4IXF); pcDHFR-#26 (4IXE)) 40

39

38

Structural data were measured for ternary complexes of NADPH and native Pneumocsytis 42

41

carinii (pc) DHFR, as well as their single (F69N) and double (K37S/F69N) variants, with inhibitors 26, 43 45

4

16 and 22, respectively, to validate the binding interactions of these inhibitors in the active site of 47

46

pcDHFR (Figs. 10 and 11 electron density). The overall characteristics of the two variant pcDHFR 49

48

ternary complexes with 16 and 22 are similar to those reported for 1 and preserve the overall pattern of 50 51

contacts with invariant active site residues (Figures 1S and 2S) 37, 52 The overall structure of pcDHFR 54

53

52

with 26 differs in the conformation of the flexible loop regions encompassing residues 43-49 and 82-91 5 56

(Figure 12). Structural data for the majority of pcDHFR inhibitor complexes reveals that loop 83-89 is 57

60

59

58

disordered with poor electron density indicating the high flexibility of this region. In the case of the ACS Paragon Plus Environment

29


Page 30 of 67

pcDHFR-26 complex, this region has a well-defined electron density. In the majority of the pcDHFR 2

1

crystal structures, the conformation of loop 43-49 places Phe46 and Phe49 close to each other, facing 3 4

the outside surface (Figure 13). However, in the crystal structure of the ternary complex with pcDHFR, 5 7

6

NADP+ and folic acid, 53 this loop is inverted, placing these residues toward the inside surface. In the 9

8

pcDHFR-26 structure, this loop is opened, extending the placement of the two phenylalanine residues. 10 1

As illustrated in Figure 12, there is a shift of more than 6Å between the inverted loop of the folic acid 14

13

12

structure and that of 26. 16

15

Analysis of the intermolecular interactions involving the N9-methyl group of the three inhibitors 17 18

shows hydrophobic contacts with the nicotinamide ribose ring (3.7Å/3.9Å/3.8Å), with the methyl of 21

20

19

Thr61 (3.9Å/3.8Å/3.6Å), with CG1 of Ile65 (4.4Å/4.7Å/4.9Å), and with CG1 of Ile123 23

2

(5.2Å/4.6Å/4.8Å), for the inhibitors 16, 22 and 26, respectively (Figure 14). The N9-methyl contacts to 24 26

25

Ile123 observed in these crystal structures are greater than that observed in the crystal structure of 28

27

pcDHFR bound to the quinazoline 137 and are shorter than those predicted from modeling 16 in the 30

29

crystal structure of pcDHFR bound to quinazoline 1 (Fig. 3).37 31 3

32

One factor that may impact the enhanced potency of 26 for pjDHFR compared with pcDHFR (IC50 35

34

0.004 vs 0.228 for pj vs pc, respectively) are the shorter contacts (1-2Å) made by the N9-methyl of 26 with 37

36

the side chains of residues in the binding pocket, e.g., Ile123, Ile65 and the nicotinamide ring , based on the 39

38

homology models for pjDHFR compared with the crystal structure of the pcDHFR-26-NADPH ternary 40 41

complex. 43

42 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60


30

Page 31 of 67


1 2 3 4 5 6 7 8 9 10 1 12 13 14 16

15

Figure 10. Stereoview 2Fo-Fc difference electron density (1.0 ) for the K37S/F69N double mutant of 17 19

18

pcDHFR as a ternary complex with NADPH and 16. Those residues that make hydrophobic contact 21

20

with with the N-methyl or the phenyl ring of 16 are labeled: Ile123, Thr61, Ile65, Asn69. Arg75 is also 2 23

for reference. 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 46

45

Figure 11. Electron density (2Fo-Fc, 1.6) showing the ternary complex of native pcDHFR with 48

47

NADPH and 26 highlighting the contacts to the F atoms of the inhibitor. 49 50 51 52 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment

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Page 32 of 67

1 2 3 4 5 6 7 8 9 10 1 12 13 14 16

15

Figure 12. 17

Stereoview of pcDHFR with 26 (green), 16 (cyan), 22 (vilolet) and folate (yellow). Note

18

the change in conformation of the loop regions near 46-49 and 83-91. 20

19 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43

Figure 13. Close up stereoview of pcDHFR with 26 (green), 16 (cyan), 22 (violet) and folate 46

45

4

(yellow), showing the changes in the conformation of loop 46-49. 47 48 49 50 51 52 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment

32

Page 33 of 67


1 2 3 4 5 6 7 8 9 10 1 12 13 14 16

15

Figure 14. 18

17

Comparison of the active site environment of pcDHFR with 26 (green), 16 (cyan), 22

(violet) and folate (yellow). Residues Glu32, Lys37/Ser37, Phe69/Asn69 are shown. 19 20 21 23

2

In summary, twelve novel N6-(substituted phenyl)-2,4-diaminopyrido[2,3-d]pyrimidines as well 25

24

as their N9-methyl analogs were designed and rationalized with molecular modeling and the binding 26 28

27

interactions of these inhibitors in the active site of pcDHFR were validated using X-ray crystal structure 30

29

data. The compounds were synthesized using Buchwald-Hartwig amination as a key step which was 32

31

optimized for the synthesis of these types of heterocycles. The goal of this study was to develop potent 3 35

34

and selective DHFR inhibitors derived from pathogens that cause opportunistic infections. This was 37

36

achieved as several analogs in this study exhibited potent inhibition of pathogen DHFR with good 38 39

selectivity compared to mammalian DHFR. Compound 26 was the most selective (selectivity ratio = 42

41

40

35.4) inhibitor in this series with excellent potency (IC50 = 4.1 nM) against pjDHFR, the fungal 4

43

infection responsible for PCP in humans. 45

This compound was as potent as PTX and also 18-fold more

46

selective for pjDHFR. Molecular modeling studies using a pjDHFR homology model suggests that the 49

48

47

N9-Me moiety of 26 selectively interacts with Ile123 in pjDHFR over the corresponding shorter Val115 51

50

in hDHFR and, in addition, induces conformational restriction of the side chain phenyl ring that 52 54

53

contributes, in part, to the potency and selectivity of 26 for pjDHFR. Compound 24 was the most potent 56

5

(IC50 = 2.1 nM) and selective (selectivity ratio = 9.9) inhibitor of pcDHFR, and displays good selectivity 58

57

(selectivity ratio = 10.3) against maDHFR as well. In addition, the N9-Me analogs were hightly potent 59 60 ACS Paragon Plus Environment

33


Page 34 of 67

against maDHFR with 20 exhibiting a subnanomolar IC50 value. In general compounds with the N92

1

methyl group exhibited improved potency against pathogen DHFR (IC50 values in nanomolar range) as 3 4

well as much better selectivity. 5 7

6

This is the first report of a systematic structure-activity study against pjDHFR, the pathogen that 9

8

causes the PCP infection in humans. In addition, compound 26 was found to have potent and selective 10 1

inhibition of pjDHFR and maDHFR, indicating that single agents against multiple infections including 14

13

12

opportunistic infections are a real possibility. For example, compound 26 is nearly 30-fold more potent 16

15

than the clinically used TMP; compound 26 is also 30-fold more selective than the clinically used TMQ 17 18

with only 50% less potency. Other compounds discovered in this study such as 20 also have potent and 21

20

19

selective inhibition against multiple pathogen DHFR and serve along with 26 as lead analogs for 23

2

development and for future compounds. 24 25 26 28

27

Experimental Section: 29 30

All evaporations were carried out in vacuo with a rotary evaporator. Analytical samples were 31 3

32

dried in vacuo (0.2 mm Hg) in a CHEM-DRY drying apparatus over P2O5 at 70 °C. Melting points were 35

34

determined on a MEL-TEMP II melting point apparatus with FLUKE 51 K/J electronic thermometer 37

36

and are uncorrected. Nuclear magnetic resonance spectra for proton (1H NMR) and carbon (13C NMR) 40

39

38

were recorded on a Bruker 400 MHz NMR spectrometer. The chemical shift values are expressed in 42

41

ppm (parts per million) relative to tetramethylsilane as an internal standard: s, singlet; d, doublet; t, 43 4

triplet; q, quartet; m, multiplet; br, broad singlet. The relative integrals of peak areas agreed with those 47

46

45

expected for the assigned structures. High-resolution mass spectra (HRMS) were recorded on a 49

48

MICROMASS AUTOSPEC (EBE Geometry) double focusing mass spectrometer (Electron Impact – 50 52

51

EI) or Waters Q-TOF (quadrupole/time-of-flight tandem instrument) mass spectrometer (Electro-Spray 54

53

Ionization – ESI). Elemental analyses were performed by AtlanticMicrolab, Inc., Norcross, GA. 5 56

Element compositions are within 0.4% of the calculated values. Fractional moles of water or organic 57

60

59

58

solvents frequently found in some analytical samples of antifolates could not be prevented in spite of ACS Paragon Plus Environment

34

Page 35 of 67


24-48 h of drying in vacuo and were confirmed where possible by their presence in the 1H NMR 2

1

spectra. On the basis of elemental analysis, the final compounds, 4-26, were of > 95% purity. Thin3 4

layer chromatography (TLC) was performed on WHATMAN UV254 silica gel plates with a fluorescent 5 7

6

indicator, and the spots were visualized under 254 and/or 365 nm illumination. Proportions of solvents 9

8

used for TLC are by volume. Column chromatography was performed on a 230–400 mesh silica gel 10 1

purchased from Fisher Scientific. All solvents and chemicals were purchased from Strem Chemicals 14

13

12

Inc., Sigma-Aldrich Chemical Co. or Fisher Scientific. All of the chemicals and the solvents were used 16

15

as received. In some cases solvents were degassed using freeze-thaw cycle and will be indicated in the 17 18

procedure. 20

19 21 23

2

6.1. General Procedure for the Synthesis of Compounds 27a-w 24 26

25

General Procedure for the Synthesis of Compounds 32a-w. In an oven-dried vial (5 mL or 28

27

20 mL, sealable with Teflon-faced silicone septa and aluminum crimp cap) with a stir bar were added 29 30

30, an appropriate aniline, Pd2dba3 (2 mol %) and an appropriate ligand (8 mol %). The vial was sealed 3

32

31

with a crimp-cap septum and evacuated and back-filled with argon (repeated three times). Freshly 35

34

degassed (three freeze-thaw cycles) toluene (2 mL or 8 mL) was added to the tube through silicone 36 37

septa to obtain a purple suspension. LiHMDS (1M solution in toluene, 3.2 equivalents) was added and 40

39

38

the tube was placed into a pre-heated oil bath (100 °C). The reaction mixture was stirred vigorously at 42

41

100 °C for an appropriate time. At the end of the reaction (monitored with TLC) the tubes were cooled 43 4

to ambient temperature. Dilute HCl (1N, 2 mL) was added to quench the reactions. The mixture was 47

46

45

then poured into saturated NaHCO3 solution (20 mL) and was extracted with EtOAc (25 mL x 2). 49

48

Organic extracts were separated, washed with water (20 mL) and brine (20 mL), dried over anhydrous 50 52

51

Na2SO4. Silica gel (500 mg) was added and the solvent was evaporated under reduced pressure to afford 54

53

a plug. The plug was loaded on a silica column (2 cm x 15 cm) and eluted with 1 % MeOH in CH2Cl2 5 56

or 50 % EtOAc in Hexanes. The fractions containing the product spot (TLC) were pooled and 57

60

59

58

evaporated under reduced pressure to afford the product. ACS Paragon Plus Environment

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Page 36 of 67

N,N'-(6-anilinopyrido[2,3-d]pyrimidine-2,4-diyl)bis(2,2-dimethylpropanamide) (32a): 2

1

Reaction of 30 (102 mg, 0.25 mmol) with Aniline 31a (28mg, 0.3 mmol) in the presence of Pd2dba3 (4.6 3 4

mg, 0.005 mmol), X-Phos (9.5 mg, 0.02 mmol) and LiHMDS (1M solution in toluene; 0.8 mL, 0.8 5 7

6

mmol) using a general procedure described above, gave 32a (89 mg; 85 %) as a yellow solid: TLC Rf 9

8

0.37 (MeOH/CHCl3, 1:10); mp: 149.5-151 °C; 1H NMR (400 MHz) (CDCl3): δ 1.30-1.35 (d, 18 H, 10 1

C(CH3)3), 6.03 (br, 1H, N9-H, exch), 7.05-7.09 (t, 1 H, C6H5), 7.17-7.19 (d, 2H, C6H5), 7.34-7.38 (t, 2 14

13

12

H, C6H5), 8.43 (br, 1H, NH Piv, exch), 8.43-8.43 (d, 1 H, C5-H, J = 3Hz), 8.73-8.74 (d, 1 H, C7-H, J = 16

15

3Hz), 15.69 (br, 1H, NHPiv, exch); 13C NMR (100 MHz) (CDCl3): δ194.4, 179.1, 155.6, 151.9, 148.4, 17 18

145.0, 141.2, 138.0, 129.6, 122.8, 118.8, 118.4, 115.3, 42.5, 40.3, 27.6, 27.1; HRMS (EI) calculated for 21

20

19

C23H28N6O2 : 420.2273, found : 420.2268. 23

2

N,N'-{6-[(4-methoxyphenyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl)bis(2,224 26

25

dimethylpropanamide) (32b): Reaction of 30 (102 mg, 0.25 mmol) with p-anisole 31b (37mg, 0.3 28

27

mmol) in the presence of Pd2dba3 (4.6 mg, 0.005 mmol), X-Phos (9.5 mg, 0.02 mmol) and LiHMDS 29 30

(1M solution in toluene; 0.8 mL, 0.8 mmol) using a general procedure described above, gave 32b (101 31 3

32

mg; 88 %) as a yellow solid: TLC Rf 0.42 (MeOH/CHCl3, 1:10); mp: 161.2-163.6 °C; 1H NMR (400 35

34

MHz) (CDCl3): δ 1.29-1.34 (d, 18 H, C(CH3)3), 3.84 (s, 3H, OCH3), 5.85 (s, 1H, N9-H, exch), 6.9136 37

6.95 (d, 2 H, C6H4, J = 8.9Hz), 7.14-7.18 (d, 2H, C6H4, J = 8.9Hz), 8.12 (br, 1H, NH Piv, exch), 8.2240

39

38

8.23 (d, 1 H, C5-H, J = 3.1Hz), 8.61-8.61 (d, 1 H, C7-H, J = 3.1Hz), 15.6 (br, 1H, NHPiv, exch); 13C 42

41

NMR (100 MHz) (CDCl3): δ 194.3, 179.0, 156.3, 155.6, 151.1, 146.9, 144.5, 139.9, 133.6, 122.5, 116.5, 43 4

115.3, 114.9, 55.6, 42.5, 40.2, 27.6, 27.1,; HRMS (ESI) calculated for C24H31N6O3 [M+H]+ : 451.2458, 47

46

45

found : 451.2456. 49

48

N,N'-{6-[(2-methoxyphenyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl)bis(2,250 52

51

dimethylpropanamide) (32c): 54

53

Reaction of 30 (102 mg, 0.25 mmol) with o-anisole 31c (37mg, 0.3

mmol) in the presence of Pd2dba3 (4.6 mg, 0.005 mmol), X-Phos (9.5 mg, 0.02 mmol) and LiHMDS 5 56

(1M solution in toluene; 0.8 mL, 0.8 mmol) using a general procedure described above, gave 32c (63 57

60

59

58

mg; 56 %) as orange-brown crystals: TLC Rf 0.42 (MeOH/CHCl3, 1:10); mp: 141.1-143.5 °C; 1H NMR ACS Paragon Plus Environment

36

Page 37 of 67


(400 MHz) (CDCl3): δ 1.30-1.35 (d, 18 H, C(CH3)3), 3.93 (s, 3H, OCH3), 6.42 (s, 1H, N9-H, exch), 2

1

6.96-7.01 (m, 3 H, C6H5), 7.39 (d, 2H, C6H5), 8.24 (br, 1H, NH Piv, exch), 8.50-8.50 (d, 1 H, C5-H, J = 4

3

2.8 Hz), 8.76-8.77 (d, 1 H, C7-H, J = 2.8 Hz), 15.69 (br, 1H, NHPiv, exch); 13C NMR (100 MHz) 5 7

6

(CDCl3): δ 194.4, 179.4, 155.6, 151.9, 149.0, 148.8, 145.0, 137.7, 130.8, 122.0, 120.8, 118.9, 115.5, 9

8

115.2, 110.8, 55.6, 42.5, 40.3, 27.6, 27.1; HRMS (ESI) calculated for C24H31N6O3 [M+H]+: 451.2458, 10 1

found : 451.2455. 13

12 14

N,N'-{6-[(2,5-dimethoxyphenyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl)bis(2,216

15

dimethylpropanamide) (32d): 17

Reaction of 30 (408 mg, 1 mmol) with 2,5-dimethoxyaniline 31d

18

(183 mg, 1.2 mmol) in the presence of Pd2dba3 (18.3 mg, 0.02 mmol), X-Phos (38 mg, 0.08 mmol) and 21

20

19

LiHMDS (1M solution in toluene; 3.2 mL, 3.2 mmol) using a general procedure described above, gave 23

2

32d (264 mg; 55 %) as a dark yellow solid; part of it was recrystallized from hexanes-dichloromethane 24 25

as fine yellow-orange needles: TLC Rf 0.47 (MeOH/CHCl3, 1:10); mp: 144.5-146 °C; 1H NMR (400 28

27

26

MHz) (CDCl3): δ 1.34-1.35 (d, 18 H, C(CH3)3), 3.78 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 6.48 (br, 1H, 29 30

N9-H, exch), 6.49-6.52 (dd, 1 H, C6H3, J1 = 2.9Hz, J2 = 8.8Hz), 6.85-6.88 (d, 1H, C6H3, J = 8.8Hz), 3

32

31

6.98-6.99 (d, 2H, C6H4, J = 2.9Hz), 8.25 (s, 1H, NHPiv, exch), 8.55-8.55 (d, 1 H, C5-H, J = 3.1Hz), 35

34

8.77-8.78 (d, 1 H, C7-H, J = 3.1Hz), 15.71 (br, 1H, NHPiv, exch); 13C NMR (100 MHz) (CDCl3): δ 36 37

194.3, 179.2, 154.1, 149.3, 142.8, 137.1, 119.2, 115.2, 111.6, 105.9, 101.6, 56.2, 55.8, 42.5, 40.3, 27.6, 40

39

38

27.0 ; HRMS (ESI) calculated for C25H33N6O4 [M+H]+ : 481.2563, found : 481.2564. 41 42

N,N'-{6-[(3,4,5-trimethoxyphenyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl)bis(2,243 4

dimethylpropanamide) (32e): Reaction of 30 (408 mg, 1 mmol) with 3,4,5-trimethoxyaniline 31e 47

46

45

(220 mg, 1.2 mmol) in the presence of Pd2dba3 (18.3 mg, 0.02 mmol), X-Phos (38 mg, 0.08 mmol) and 49

48

LiHMDS (1M solution in toluene; 3.2 mL, 3.2 mmol) using a general procedure described above gave 50 52

51

32d (398 mg; 78 %) as a dark yellow solid: TLC Rf 0.425 (MeOH/CHCl3, 1:10); mp: 149.0-151.5 °C; 54

53

1

5

H NMR (400 MHz) (CDCl3): δ 1.29-1.35 (d, 18 H, C(CH3)3), 3.85 (s, 9H, OCH3), 5.97 (br, 1H, N9-H,

56

exch), 6.43 (s, 2 H, C6H2), 8.23 (br, 1H, NHPiv, exch), 8.44-8.45 (d, 1 H, C5-H, J = 3.1Hz), 8.66-8.67 60

59

58

57

(d, 1 H, C7-H, J = 3.1Hz), 15.72 (br, 1H, NHPiv, exch); 13C NMR (100 MHz) (CDCl3): δ194.3, 179.2, ACS Paragon Plus Environment

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Page 38 of 67

155.7, 154.0, 151.8, 144.9, 138.3, 137.1, 115.2, 96.5, 61.0, 56.1, 42.5, 40.3, 27.6, 27.1; HRMS (EI) 2

1

calculated for C26H34N6O5: 510.2590 , found : 510.2580. 3 4

N,N'-{6-[(4-methylphenyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl)bis(2,25 7

6

dimethylpropanamide) (32f): 9

8

Reaction of 30 (102 mg, 0.25 mmol) with p-toluedine 31f (32.15 mg,

0.3 mmol) in the presence of Pd2dba3 (4.6 mg, 0.005 mmol), X-Phos (9.5 mg, 0.02 mmol) and LiHMDS 10 1

(1M solution in toluene; 0.8 mL, 0.8 mmol) using a general procedure described above, gave 32f (101 14

13

12

mg; 93 %) as a dark yellow solid; part of which was recrystallized from hexanes-dichloromethane as 16

15

fine yellow-orange needles: TLC Rf 0.385 (MeOH/CHCl3, 1:10); mp: 203.9-204.5 °C; 1H NMR (400 17 18

MHz) (CDCl3): δ 1.32-1.35 (d, 18 H, C(CH3)3), 2.36 (s, 3H, CH3), 5.92 (br, 1H, N9-H, exch), 7.08-7.10 21

20

19

(d, 2 H, C6H4, J = 8.3 Hz), 7.17-7.19 (d, 2 H, C6H4, J = 8.3 Hz), 8.21 (br, 1H, NHPiv, exch), 8.35-8.36 23

2

(d, 1 H, C5-H, J = 3.1 Hz), 8.68-8.69 (d, 1 H, C7-H, J = 3.1 Hz), 15.69 (br, 1H, NHPiv, exch); 13C 24 26

25

NMR (100 MHz) (CDCl3): δ193.9, 179.1, 155.7, 148.0, 139.8, 138.2, 132.5, 130.0, 118.9, 117.0, 115.1, 28

27

42.4, 39.8, 27.2, 20.2; HRMS (ESI) calculated for C24H30N6O2Na [M+Na]+ : 457.2328 , found : 29 30

457.2300. 31 3

32

N,N'-{6-[(4-isopropylphenyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl)bis(2,235

34

dimethylpropanamide) (32g): 36

Reaction of 30 (102 mg, 0.25 mmol) with p-isopropylaniline 31g

37

(40.6 mg, 0.3 mmol) in the presence of Pd2dba3 (4.6 mg, 0.005 mmol), X-Phos (9.5 mg, 0.02 mmol) and 40

39

38


41

32g (105 mg; 91 %) as yellow crystals: TLC Rf 0.38 (MeOH/CHCl3, 1:10); mp: 217.0-218.0 °C; 1H 43 4

NMR (400 MHz) (CDCl3): δ 1.26-1.28 (d, 6H, CH(CH3)2),1.30-1.35 (d, 18 H, C(CH3)3), 2.89-2.95 (m, 47

46

45

1H, CH(CH3)2), 5.94 (br, 1H, N9-H, exch), 7.11-7.13 (d, 2 H, C6H4, J = 8.3 Hz), 7.22-7.24 (d, 2H, 49

48

C6H4, J = 8.3 Hz), 8.19 (br, 1H, NH Piv, exch), 8.34-8.35 (d, 1 H, C5-H, J = 3.2 Hz), 8.70-8.71 (d, 1 H, 50 51

C7-H, J = 3.2 Hz), 15.67 (br, 1H, NHPiv, exch); 13C NMR (100 MHz) (CDCl3): δ 194.3, 179.0, 155.6, 54

53

52

151.6, 147.8, 144.7, 144.0, 138.8, 138.7, 127.5, 119.4, 118.0, 115.3, 42.5, 40.3, 33.5, 27.6, 27.1, 24.0; 56

5

HRMS (ESI) calculated for C26H35N6O2 [M+H]+ : 463.2821, found : 463.2796. 58

57 59 60


38

Page 39 of 67


N,N'-[6-(2-naphthylamino)pyrido[2,3-d]pyrimidine-2,4-diyl]-bis(2,22

1

dimethylpropanamide) (32h): Reaction of 30 (408 mg, 1 mmol) with 2-naphthylamine 31h (172 mg, 3 4

1.2 mmol) in the presence of Pd2dba3 (18.3 mg, 0.02 mmol), X-Phos (38 mg, 0.08 mmol) and LiHMDS 7

6

5

(1M solution in toluene; 3.2 mL, 3.2 mmol) using a general procedure described above gave 32h (352 9

8

mg; 75 %) as an orange solid: TLC Rf 0.36 (MeOH/CHCl3, 1:10); mp: 159.9-162.0 °C; 1H NMR (400 10 1

MHz) (CDCl3): δ 1.36 (d, 18 H, C(CH3)3), 6.22 (br, 1H, N9-H, exch), 7.28-7.31 (dd, 1 H, C10H7), 7.3714

13

12

7.41 (t, 1H, C10H7), 7.45-7.49 (t, 1H, C10H7), 7.60 (s, 1H, C10H7), 7.69-7.71 (d, 1H, C10H7), 7.79-7.85 16

15

(m, 2H, C10H7), 8.30 (br, 1H, NHPiv, exch), 8.58-8.59 (d, 1 H, C5-H, J = 2.8 Hz), 8.81-8.82 (d, 1 H, 17 18

C7-H, J = 2.8 Hz), 15.71 (br, 1H, NHPiv, exch); 13C NMR (100 MHz) (CDCl3): δ 194.3, 179.4, 171.5, 21

20

19

155.6, 134.3, 129.8, 129.6, 127.7, 126.8, 126.7, 124.3, 119.6, 112.6, 42.5, 40.3, 27.6, 27.1; HRMS (EI) 23

2

calculated for C27H30N6O5: 470.2430 , found : 470.2422. 24 26

25

N,N'-{6-[(4-nitrophenyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl)bis(2,228

27

dimethylpropanamide) (32i): 29

Reaction of 30 (102 mg, 0.25 mmol) with 4-nitroaniline 31i (34.5 mg,

30

0.3 mmol) in the presence of Pd2dba3 (4.6 mg, 0.005 mmol), X-Phos (9.5 mg, 0.02 mmol) and LiHMDS 3

32

31

(1M solution in toluene; 0.8 mL, 0.8 mmol) using a general procedure described above, gave 32i (70 35

34

mg; 60 %) as a yellow solid: TLC Rf 0.37 (MeOH/CHCl3, 1:10); mp: 166.3-168.0 °C; 1H NMR (400 36 37

MHz) (CDCl3): δ 1.31-1.36 (d, 18 H, C(CH3)3), 6.55 (s, 1H, N9-H, exch), 7.07-7.09 (d, 2 H, C6H4, J = 40

39

38

9.2 Hz), 8.20-8.23 (d, 2 H, C6H4, J = 9.2 Hz), 8.29 (br, 1H, NH Piv, exch), 8.61-8.62 (d, 1 H, C5-H, J = 42

41

2.8 Hz), 8.84-8.85 (d, 1 H, C7-H, J = 2.8 Hz), 15.69 (br, 1H, NHPiv, exch); 13C NMR (100 MHz) 43 4

(CDCl3): δ 194.5, 179.3, 155.1, 151.1, 148.5, 141.1, 134.3, 126.3, 115.3, 114.3, 42.6, 40.4, 27.5, 27.0; 47

46

45

HRMS (ESI) calculated for C23H28N7O4 [M+H]+: 466.2203, found : 466.2179. 49

48

N,N'-[6-(1-naphthylamino)pyrido[2,3-d]pyrimidine-2,4-diyl]-bis(2,250 52

51

dimethylpropanamide) (32j): 54

53

Reaction of 30 (102 mg, 0.25 mmol) with 1-naphthylamine 31j (43

mg, 0.3 mmol) in the presence of Pd2dba3 (4.6 mg, 0.005 mmol), X-Phos (9.5 mg, 0.02 mmol) and 5 56


60

59

58

32j (76 mg; 65 %) as an orange solid; part of which was recrystallized from dichloromethane-hexanes ACS Paragon Plus Environment

39


Page 40 of 67

(1:1) to obtain fluffy orange crystals: TLC Rf 0.36 (MeOH/CHCl3, 1:10); mp: 162.0-163.9 °C; 1H NMR 2

1

(400 MHz) (CDCl3): δ 1.25-1.35 (d, 18 H, C(CH3)3), 6.24 (s, 1H, N9-H, exch), 7.46-7.56 (m, 4H, 3 4

C10H7), 7.70-7.72 (dd, 1H, C10H7), 7.91-7.94 (m, 1 H, C10H7), 8.01-8.03 (m, 1H , C10H7), 8.19 (br, 1H, 7

6

5

NH Piv, exch), 8.25-8.26 (d, 1 H, C5-H, J = 3.2 Hz), 8.69-8.70 (d, 1 H, C7-H, J = 3.2 Hz), 15.67 (br, 9

8

1H, NHPiv, exch); 13C NMR (100 MHz) (CDCl3): δ194.3, 179.0, 155.6, 147.8, 144.8, 139.7, 136.8, 10 1

134.7, 128.7, 127.9, 126.5, 125.8, 124.8, 121.5, 118.7, 117.1, 115.4, 42.4, 40.3, 27.5, 27.1; HRMS (EI) 14

13

12

calculated for C23H28N7O4 : 470.2430, found : 470.2423. 15 16

N,N'-{6-[(2,5-diflurophenyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl)bis(2,217 18

dimethylpropanamide) (32k): 21

20

19

Reaction of 30 (204 mg, 0.5 mmol) with 2,5-difluoroaniline 31k (77.5

mg, 0.6 mmol) in the presence of Pd2dba3 (9.2 mg, 0.01 mmol), X-Phos (19 mg, 0.04 mmol) and 23

2

LiHMDS (1M solution in toluene; 1.6 mL, 1.6 mmol) using a general procedure described above, gave 24 26

25

32k (178 mg; 78 %) as dark yellow crystals: TLC Rf 0.41 (MeOH/CHCl3, 1:10); mp: 210.8-211.5 °C; 28

27

1

29

H NMR (400 MHz) (CDCl3): δ 1.31-1.36 (d, 18 H, C(CH3)3), 6.14 (s, 1H, N9-H, exch), 6.60-6.66 (m,

30

1H, C6H3), 7.03-7.13 (m, 2H, C6H3), 8.23 (br, 1H, NHPiv, exch), 8.53-8.54 (d, 1 H, C5-H, J = 3.2 Hz), 3

32

31

8.79-8.80 (d, 1 H, C7-H, J = 3.2 Hz), 15.69 (br, 1H, NHPiv, exch); 13C NMR (100 MHz) (CDCl3): δ 35

34

194.4, 179.2, 155.3, 153.2, 149.6, 145.8, 135.6, 121.8, 116.7, 115.2, 107.7, 103.6, 42.6, 40.3, 27.5, 27.1. 36 37

N,N'-{6-[(3,4,5-triflurophenyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl)bis(2,240

39

38

dimethylpropanamide) (32l): 42

41

Reaction of 30 (102 mg, 0.25 mmol) with 3,4,5-trifluoroaniline 31l (44

mg, 0.3 mmol) in the presence of Pd2dba3 (4.6 mg, 0.005 mmol), X-Phos (9.5 mg, 0.02 mmol) and 43 4


46

45

32l (89 mg; 75 %) as a yellow solid; part of which was recrystallized from methylene chloride-hexanes 49

48

to obtain fluffy yellow crystals: TLC Rf 0.44 (MeOH/CHCl3, 1:10); mp: 221.3-223.2 °C; 1H NMR (400 50 52

51

MHz) (CDCl3): δ 1.30-1.35 (d, 18 H, C(CH3)3), 6.04 (br, 1H, N9-H, exch), 6.72-6.75 (q, 2H, C6H2), 54

53

8.22 (br, 1H, NH Piv, exch), 8.45-8.46 (d, 1 H, C5-H, J = 3.2 Hz), 8.72-8.73 (d, 1 H, C7-H, J = 3.2 Hz), 56

5

15.68 (br, 1H, NHPiv, exch); 13C NMR (100 MHz) (CDCl3): δ 194.4, 179.2, 155.4, 153.1, 149.4, 145.8, 60

59

58

57

136.1, 121.3, 115.3, 101.7 (d), 42.6, 40.3, 27.5, 27.0. ACS Paragon Plus Environment

40

Page 41 of 67


N,N'-{6-[methyl(phenyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl}bis(2,22

1

dimethylpropanamide) (32m): 3

Reaction of 30 (204 mg, 0.5 mmol) with N-methylaniline 31m (64

4

mg, 0.6 mmol) in the presence of Pd2dba3 (9.2 mg, 0.01 mmol), S-Phos (16.4 mg, 0.04 mmol) and 7

6

5


8

32m (190 mg; 87 %) as a lemon yellow solid; part of which was recrystallized from methylene chloride10 1

hexanes to obtain lemon yellow lustrous crystals: TLC Rf 0.45 (MeOH/CHCl3, 1:10); mp: 104.1-105.5 14

13

12

°C; 1H NMR (400 MHz) (CDCl3): δ 1.32-1.35 (d, 18 H, C(CH3)3), 3.44 (s, 3H, -NCH3), 7.19-7.21 (d, 3 16

15

H, C6H5), 7.38-7.41 (t, 2H, C6H5), 8.15-8.16 (d, 1 H, C5-H, J = 3.2 Hz), 8.22 (br, 1H, NHPiv, exch), 17 18


20

19

194.6, 179.1, 155.7, 151.0, 147.2, 144.6, 143.3, 129.9, 124.6, 123.7, 114.9, 42.5, 40.3 (d), 27.7, 27.1; 23

2

HRMS (ESI) calculated for C24H30N6O2Na [M+Na]+: 457.2328, found : 457.2314. 24 26

25

N,N'-{6-[(4-methoxyphenyl)(methyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl}bis(2,228

27

dimethylpropanamide) (32n): 29

Reaction of 30 (204 mg, 0.5 mmol) with 4-methoxy-N-methylaniline

30

31n (82 mg, 0.6 mmol) in the presence of Pd2dba3 (9.2 mg, 0.01 mmol), S-Phos (16.4 mg, 0.04 mmol) 3

32

31

and LiHMDS (1M solution in toluene; 1.6 mL, 1.6 mmol) using a general procedure described above, 35

34

gave 32n (227 mg; 98 %) as a lemon yellow solid; part of which was recrystallized from methylene 36 37

chloride-hexanes to obtain fluffy lemon yellow crystals: TLC Rf 0.51 (MeOH/CHCl3, 1:10); mp: 189.540

39

38

191.1 °C; 1H NMR (400 MHz) (CDCl3): δ 1.34-1.37 (d, 18 H, C(CH3)3), 3.38 (s, 3H, NCH3), 3.85 (s, 42

41

3H, OCH3), 6.95-6.96 (d, 2 H, C6H4, J = 8.8 Hz), 7.16-7.18 (d, 2H, C6H4, J = 8.8 Hz), 7.99-8.00 (d, 1 H, 43 4

C5-H, J = 3.2 Hz), 8.19 (br, 1H, NHPiv, exch), 8.43-8.44 (d, 1 H, C7-H, J = 3.2 Hz), 15.74 (br, 1H, 47

46

45

NHPiv, exch); 13C NMR (100 MHz) (CDCl3): δ 194.1, 179.0, 157.6, 155.5, 149.9, 146.9, 144.9, 144.6, 49

48

144.0, 139.9, 127.1, 115.3, 55.5, 42.5, 40.5, 40.2, 27.7, 27.4, 27.3, 27.0; HRMS (ESI) calculated for 50 51

C24H30N6O2Na [M+H]+: 465.2614, found : 465.2578. 54

53

52

N,N'-{6-[(2-methoxyphenyl)(methyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl}bis(2,25 56

dimethylpropanamide) (32o): 57

Reaction of 30 (204 mg, 0.5 mmol) with 2-methoxy-N-methylaniline

60

59

58

31o (82 mg, 0.6 mmol) in the presence of Pd2dba3 (9.2 mg, 0.01 mmol), S-Phos (16.4 mg, 0.04 mmol) ACS Paragon Plus Environment

41


Page 42 of 67


1

gave 32o (137 mg; 59 %) as a dark yellow solid: TLC Rf 0.54 (MeOH/CHCl3, 1:10); mp: 185.3-186.8 4

3

°C; 1H NMR (400 MHz) (CDCl3): δ 1.31-1.33 (d, 18 H, C(CH3)3), 3.36 (s, 3H, NCH3), 3.76 (s, 3H, 7

6

5

OCH3), 7.01-7.05 (t, 2 H, C6H4), 7.23-7.25 (dd, 1H, C6H4), 7.29-7.33 (t, 1H, C6H4), 7.92-7.93 (d, 1 H, 9

8

C5-H, J = 3.2 Hz), 8.24-8.25 (d, 1 H, C7-H, J = 3.2 Hz), 8.26 (br, 1H, NHPiv, exch), 15.72 (br, 1H, 1

10

NHPiv, exch); 13C NMR (100 MHz) (CDCl3): δ 194.2, 178.9, 155.9, 155.5, 149.6, 145.8, 143.8, 143.5, 14

13

12

134.2, 128.7, 128.1, 121.6, 114.9, 113.2, 112.4, 55.4, 42.4, 40.2, 39.4, 36.6, 27.7, 27.1. 16

15

N,N'-{6-[(2,5-dimethoxyphenyl)(methyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl}bis(2,217 18

dimethylpropanamide) (32p): Reaction of 30 (204 mg, 0.5 mmol) with 2,5-dimethoxy-N21

20

19

methylaniline 31p (100.3 mg, 0.6 mmol) in the presence of Pd2dba3 (9.2 mg, 0.01 mmol), S-Phos (16.4 23

2

mg, 0.04 mmol) and LiHMDS (1M solution in toluene; 1.6 mL, 1.6 mmol) using a general procedure 24 26

25

described above, gave 32p (190 mg; 77 %) as a yellow-brown oil; part of which was recrystallized from 28

27

methylene chloride-hexanes to obtain yellow crystals : TLC Rf 0.55 (MeOH/CHCl3, 1:10); mp: 130.630

29

132.3 °C; 1H NMR (400 MHz) (CDCl3): δ 1.30-1.34 (d, 18 H, C(CH3)3), 3.37 (s, 3H, NCH3), 3.72-3.78 3

32

31

(d, 6H, OCH3), 6.81-6.86 (m, 2 H, C6H3), 6.94-6.96 (d, 1H, C6H3), 7.95-7.96 (d, 1 H, C5-H, J = 3.2 Hz), 35

34

8.16 (br, 1H, NHPiv, exch), 8.28-8.29 (d, 1 H, C7-H, J = 3.2 Hz), 15.74 (br, 1H, NHPiv, exch); 13C 36 37

NMR (100 MHz) (CDCl3): δ 194.2, 178.8, 156.0, 154.2, 149.8, 149.6, 146.1, 143.8, 143.3, 134.9, 114.9, 40

39

38

114.4, 113.5, 113.3, 112.4, 55.9, 55.8, 42.4, 40.2, 39.4, 27.7, 27.1; HRMS (ESI) calculated for 42

41

C26H35N6O4 [M+H]+: 495.2720, found : 495.2749. 43 45

4

N,N'-{6-[(3,4,5-trimethoxyphenyl)(methyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl}bis(2,247

46

dimethylpropanamide) (32q): 49

48

Reaction of 30 (204 mg, 0.5 mmol) with 3,4,5-trimethoxy-N-

methylaniline 31q (118 mg, 0.6 mmol) in the presence of Pd2dba3 (9.2 mg, 0.01 mmol), S-Phos (16.4 50 52

51

mg, 0.04 mmol) and LiHMDS (1M solution in toluene; 1.6 mL, 1.6 mmol) using a general procedure 54

53

described above, gave 32q (233 mg; 89 %) as a yellow solid; part of which was recrystallized from 5 56

methylene chloride-hexanes to obtain lemon yellow crystals : TLC Rf 0.55 (MeOH/CHCl3, 1:10); mp: 60

59

58

57

187.9-189 °C; 1H NMR (400 MHz) (CDCl3): δ 1.32-1.34 (d, 18 H, C(CH3)3), 3.41 (s, 3H, NCH3), 3.82ACS Paragon Plus Environment

42

Page 43 of 67


3.87 (d, 9H, OCH3), 6.43 (d, 2 H, C6H2), 8.09-8.10 (d, 1 H, C5-H, J = 3.2 Hz), 8.18 (br, 1H, NHPiv, 2

1

exch), 8.54-8.55 (d, 1 H, C7-H, J = 3.2 Hz), 15.73 (br, 1H, NHPiv, exch); 13C NMR (100 MHz) 3 4

(CDCl3): δ 194.3, 179.0, 155.8, 154.2, 150.6, 148.2, 144.4, 143.6, 143.2, 116.3, 114.9, 102.2, 61.0, 42.5, 7

6

5

40.5, 40.2, 27.7, 27.1; HRMS (ESI) calculated for C27H36N6O5 [M+Na]+: 547.2645, found : 547.2615. 8 9

N,N'-{6-[methyl(4-methylphenyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl}bis(2,210 1

dimethylpropanamide) (32r): Reaction of 30 (204 mg, 0.5 mmol) with N-methyltoluidine 31r (73 14

13

12

mg, 0.6 mmol) in the presence of Pd2dba3 (9.2 mg, 0.01 mmol), S-Phos (16.4 mg, 0.04 mmol) and 16

15

LiHMDS (1M solution in toluene; 1.6 mL, 1.6 mmol) using a general procedure described above, gave 17 18

32r (215 mg; 96 %) as a yellow solid: TLC Rf 0.59 (MeOH/CHCl3, 1:10); mp: 120.0-121.5 °C; 1H 21

20

19

NMR (400 MHz) (CDCl3): δ 1.32-1.35 (d, 18 H, C(CH3)3), 2.38 (s, 3H, ArCH3), 3.40 (s, 3H, NCH3), 23

2

7.10-7.12 (d, 2 H, C6H4, J = 8.2 Hz), 7.21-7.23 (d, 2 H, C6H4, J = 8.2 Hz), 8.07-8.08 (d, 1 H, C5-H, J = 24 26

25

3.1 Hz), 8.18 (br, 1H, NHPiv, exch), 8.52-8.53 (d, 1 H, C7-H, J = 3.1 Hz), 15.73 (br, 1H, NHPiv, exch); 28

27

13

29

C NMR (100 MHz) (CDCl3): δ 194.3, 178.9, 155.8, 144.5, 143.7, 135.0, 130.6, 124.6, 116.0, 42.5,

30

40.4, 40.2, 27.7, 27.1, 20.9. 31 32 3

N,N'-{6-[methyl(4-isopropylphenyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl}bis(2,235

34

dimethylpropanamide) (32s): Reaction of 30 (102 mg, 0.25 mmol) with 4-isopropyl-N-methylaniline 36 37

31s (45 mg, 0.3 mmol) in the presence of Pd2dba3 (4.6 mg, 0.005 mmol), S-Phos (8.2 mg, 0.02 mmol) 40

39

38


41

gave 32s (118 mg; 98 %) as a yellow solid; part of which was recrystallized from methylene chloride43 4

hexanes to obtain fluffy lemon-yellow crystals: TLC Rf 0.6 (MeOH/CHCl3, 1:10); mp: 209.9-211.2 °C; 47

46

45

1

49

48

H NMR (400 MHz) (CDCl3): δ 1.27-1.29 (d, 6H, CH(CH3)2), 1.31-1.34 (d, 18 H, C(CH3)3), 2.29-2.97

(m, 1H, CH(CH3)2), 3.42 (S, 3H, NCH3), 7.13-7.15 (d, 2 H, C6H4, J = 8. Hz), 7.26-7.28 (d, 2 H, C6H4, J 50 52

51

= 8 Hz), 8.04-8.05 (d, 1 H, C5-H, J = 3.2 Hz), 8.18 (br, 1H, NHPiv, exch), 8.55-8.56 (d, 1 H, C7-H, J = 54

53

3.2 Hz), 15.72 (br, 1H, NHPiv, exch); 13C NMR (100 MHz) (CDCl3): δ 194.2, 179.9, 155.8, 150.4, 5 56

147.8, 146.1, 144.7, 143.7, 127.9, 124.7, 116.1, 115.0, 42.5, 40.3, 40.2, 33.6, 27.7, 27.1, 24.0; HRMS 57

60

59

58

(ESI) calculated for C27H37N6O2 [M+H]+: 477.2978, found: 477.2979. ACS Paragon Plus Environment

43


Page 44 of 67

N,N'-{6-[methyl(4-nitrophenyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl}bis(2,22

1

dimethylpropanamide) (32t): Reaction of 30 (204 mg, 0.5 mmol) with 4-nitro-N-methylaniline 31t 3 4

(92 mg, 0.6 mmol) in the presence of Pd2dba3 (9.2 mg, 0.01 mmol), S-Phos (16.4 mg, 0.04 mmol) and 7

6

5


8

32t (151 mg; 63 %) as a yellow solid: TLC Rf 0.67 (MeOH/CHCl3, 1:10); mp: 175.0-177.2 °C; 1H NMR 10 1

(400 MHz) (CDCl3): δ 1.37 (d, 18 H, C(CH3)3), 3.55 (s, 3H, NCH3), 6.84-6.87 (d, 2 H, C6H4, J = 9.3 14

13

12

Hz), 8.14-8.17 (d, 2 H, C6H4, J = 9.3 Hz), 8.36 (br, 1H, NHPiv, exch), 8.59-8.60 (d, 1 H, C5-H, J = 2.8 16

15

Hz), 8.82-8.83 (d, 1 H, C7-H, J = 2.8 Hz), 15.73 (br, 1H, NHPiv, exch); 13C NMR (100 MHz) (CDCl3): 17 18

δ 194.6, 179.4, 155.1, 152.8, 147.3, 140.6, 137.6, 130.9, 128.6, 125.9, 115.6, 113.9, 42.5, 40.7, 40.4, 21

20

19

27.5, 27.0; HRMS (ESI) calculated for C24H29N7O4Na [M+Na]+: 502.2179, found: 502.2181. 23

2

N,N'-{6-[methyl(1-naphthyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl}bis(2,224 26

25

dimethylpropanamide) (32u): Reaction of 30 (204 mg, 0.5 mmol) with N-methyl-128

27

naphthylaminehydrochloride 31u (116 mg, 0.6 mmol) in the presence of Pd2dba3 (9.2 mg, 0.01 mmol), 29 30

S-Phos (16.4 mg, 0.04 mmol) and LiHMDS (1M solution in toluene; 2.2 mL, 2.2 mmol) using a general 31 3

32

procedure described above, gave 32u (160 mg; 66 %) as a yellow solid: TLC Rf 0.63 (MeOH/CHCl3, 35

34

1:10); mp: 149.0-150.6 °C; 1H NMR (400 MHz) (CDCl3): δ 1.32-1.34 (d, 18 H, C(CH3)3), 3.53 (s, 3H, 36 37

NCH3), 7.40-7.42 (dd, 1 H, C10H7), 7.47-7.57 (m, 3 H, C10H7), 7.83-7.90 (q, 2 H, C10H7), 7.96-7.98 (d, 1 40

39

38

H, C10H7), 8.04-8.05 (d, 1 H, C5-H, J = 3.2 Hz), 8.14-8.15 (d, 1 H, C7-H, J = 3.2 Hz), 8.16 (br, 1H, 42

41

NHPiv, exch), 15.74 (br, 1H, NHPiv, exch); 13C NMR (100 MHz) (CDCl3): δ 194.3, 178.9, 155.9, 43 4

148.7, 144.4, 143.1, 130.6, 127.0, 126.6, 125.2, 122.9, 115.0, 42.6, 40.2, 27.7, 27.1; HRMS (EI) 47

46

45

calculated for C28H32N6O2: 484.2586, found: 484.2594. 49

48

N,N'-{6-[(2,5-difluorophenyl)(methyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl}bis(2,250 52

51

dimethylpropanamide) (32v): Reaction of 30 (204 mg, 0.5 mmol) with 2,5-difluoro-N-methylaniline 54

53

31v (86 mg, 0.6 mmol) in the presence of Pd2dba3 (9.2 mg, 0.01 mmol), S-Phos (16.4 mg, 0.04 mmol) 5 56


60

59

58

gave 32v (218 mg; 93 %) as a yellow solid: TLC Rf 0.66 (MeOH/CHCl3, 1:10); mp: 93.8-95.6 °C; 1H ACS Paragon Plus Environment

44

Page 45 of 67


NMR (400 MHz) (CDCl3): δ 1.31-1.36 (d, 18 H, C(CH3)3), 3.43 (s, 3H, NCH3), 6.94-7.05 (m, 2 H, 2

1

C6H3), 7.13-7.19 (m, 1 H, C6H3), 8.08-8.09 (d, 1 H, C5-H, J = 3.2 Hz), 8.21 (br, 1H, NHPiv, exch), 4

3


6

5

194.1, 179.2, 160.1 (d), 157.7, 155.6, 152.9(d), 150.9, 146.6, 144.8, 142.2, 134.7(d), 117.9 (t), 116.1, 9

8

114.8, 53.4, 42.4, 40.2, 39.6 (d), 27.6, 27.0; HRMS (ESI) calculated for C24H28N6O2F2Na [M+Na]+: 10 1

493.2140, found: 493.2178. 14

13

12

N,N'-{6-[(3,4,5-trifluorophenyl)(methyl)amino]pyrido[2,3-d]pyrimidine-2,4-diyl}bis(2,216

15

dimethylpropanamide) (32w): Reaction of 30 (204 mg, 0.5 mmol) with 3,4,5-trifluoro-N17 18

methylaniline 31w (96.7 mg, 0.6 mmol) in the presence of Pd2dba3 (9.2 mg, 0.01 mmol), S-Phos (16.4 21

20

19

mg, 0.04 mmol) and LiHMDS (1M solution in toluene; 1.6 mL, 1.6 mmol) using a general procedure 23

2

described above, gave 32w (220 mg; 90 %) as a yellow solid; part of which was recrystallized from 24 26

25

methylene chloride-hexanes to obtain fluffy lemon-yellow crystals: TLC Rf 0.62 (MeOH/CHCl3, 1:10); 28

27

mp: 119.0-121.5 °C; 1H NMR (400 MHz) (CDCl3): δ 1.32-1.36 (d, 18 H, C(CH3)3), 3.40 (s, 3H, NCH3), 29 30

6.61-6.65 (q, 2 H, C6H2), 8.24 (br, 1H, NHPiv, exch), 8.36-8.37 (d, 1 H, C5-H, J = 3.2 Hz), 8.68-8.69 3

32

31

(d, 1 H, C7-H, J = 3.2 Hz), 15.72 (br, 1H, NHPiv, exch); 13C NMR (100 MHz) (CDCl3): δ 194.4, 179.2, 35

34

155.3, 153.3, 152.0, 146.0, 141.8, 124.5, 115.2, 104.0, 103.7, 42.6, 40.5, 40.3, 27.6, 27.0; HRMS (ESI) 37

36

calculated for C24H28N6O2F3 [M+H]+: 489.2226, found: 489.2234. 40

39

38

General procedure for the synthesis of compounds 4-26: 42

41

A solution of 32a-w (0.2-0.3

mmol) in dichloromethane-MeOH (3:1, 20 mL) was taken in a Parr acid-digestion pressure vessel 43 4

followed by the addition of liquid ammonia (15 mL). The vessel was sealed maintained at room 47

46

45

temperature for 72 days with stirring. At the end of this period, all liquids were allowed to evaporate 49

48

overnight at room temperature. The residue thus obtained was stirred with water (25 mL) for 30 min and 50 52

51

then filtered with additional washings with water (25 mL). The solid was air-dried and then extracted 54

53

with a hot mixture MeOH-acetone (1:1, 200 mL). The warm extracts were further filtered through 5 56

sintered glass filter and were evaporated under reduced pressure to obtain 4-26. 57 58 59 60 ACS Paragon Plus Environment

45


Page 46 of 67

N6-phenylpyrido[2,3-d]pyrimidine-2,4,6-triamine (4): Reaction of 32a (130 mg, 0.3 mmol) 2

1

using a general procedure described above, afforded 4 (35 mg; 45 %) as a yellow solid: TLC Rf 0.255 4

3

(MeOH/CHCl3/NH4OH, 1:5:0.5); mp: 322.5-324 °C; 1H NMR (400 MHz) (DMSO-d6): δ 6.08 (br, 2H, 7

6

5

NH2, exch), 6.73-6.77 (t, 1H, C6H5, J = 8 Hz), 6.94-6.96 (d, 2H, C6H5, J = 8 Hz), 7.17-7.21 (t, 2H, 9

8

C6H5, J = 8 Hz), 7.38-7.40 (br, 2H, -NH2 - exch), 8.11-8.12 (br, 2 H, C5-H and N9-H, exch), 8.46 (d, 1 10 1

H, C7-H); HRMS (EI) calculated for C13H12N6: 252.1123, found: 252.1125; Anal. (C13H12N6 · 0.11 14

13

12

H2O) C, H, N. 16

15

N6-(4-methoxyphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (5): Reaction of 32b (100 mg, 17 18

0.22 mmol) using a general procedure described above, afforded 5 (52 mg; 69 %) as a light brown solid: 21

20

19

TLC Rf 0.24 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: 250.2-252.3 °C; 1H NMR (400 MHz) (DMSO-d6): δ 23

2

3.70 (s, 3H, OCH3), 6.86-6.88 (m, 4H, C6H4 and -NH2 - exch), 7.03-7.05 (d, 2H, C6H4), 7.99-8.00 (d, 1 24 26

25

H, C5-H, J = 2.8 Hz), 8.15 (br, 3H, -NH2 and N9-H exch), 8.42-8.43 (d, 1 H, C7-H, J = 2.8 Hz); HRMS 28

27

(EI) calculated for C14H14N6O: 282.1229, found: 282.1228; Anal. (C14H14N6O · 0.34 H2O) C, H, N. 30

29

N6-(2-methoxyphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (6): Reaction of 32c (113 mg, 31 3

32

0.25 mmol) using a general procedure described above, afforded 6 (50.5 mg; 71 %) as a dark orange 35

34

solid: TLC Rf 0.35 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: > 255 °C (dec); 1H NMR (400 MHz) (DMSO36 37

d6): δ 3.80 (s, 3H, OCH3), 6.88-7.19 (m, 4H, C6H4), 8.01-8.04 (m, 3H, C5-H and -NH2 - exch), 8.5240

39

38

8.53 (d, 1H, C7-H), 8.83 (br, 2H, -NH2), 9.08 (br, 1H, N9-H, exch); HRMS (EI) calculated for 42

41

C14H14N6O: 282.1229, found: 282.1225; Anal. (C14H14N6O · 0.57 H2O) C, H, N. 43 4

N6-(2,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (7): Reaction of 32d (96 47

46

45

mg, 0.20 mmol) using a general procedure described above, afforded 7 (40 mg; 65 %) as a yellowish 49

48

brown solid: TLC Rf 0.36 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: 202.2-204.3 °C; 1H NMR (400 MHz) 50 52

51

(DMSO-d6): δ 3.62 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 6.33-6.36 (dd, 1H, C6H3, J1 = 2.8 Hz, J2 = 8.8 54

53

Hz), 6.46 (br, 2H, -NH2, exch), 6.52-6.53 (d, 1H, C6H3, J = 2.8 Hz), 6.87-6.89 (d, 1H, C6H3, J = 8.8 Hz), 5 56

7.52 (s, 1H, N9-H exch), 7.72 (br, 2H, -NH2, exch), 8.13-8.14 (d, 1 H, C5-H, J = 2.7 Hz), 8.51-8.52 (d, 58

57 59 60


46

Page 47 of 67


1 H, C7-H, J = 2.7 Hz); HRMS (ESI) calculated for C15H17N6O2 (M+H)+: 313.1413, found: 313.1403; 2

1

Anal. (C15H16N6O2 · 0.20 H2O) C, H, N. 3 4

N6-(3,4,5-trimethoxyphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (8): Reaction of 32e 5 7

6

(120 mg, 0.235 mmol) using a general procedure described above, afforded 8 (66 mg; 82 %) as an 9

8

orange powder: TLC Rf 0.26 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: > 260 °C (dec); 1H NMR (400 10 1

MHz) (DMSO-d6): δ 3.59 (s, 3H, OCH3), 3.71 (s, 6H, OCH3), 6.30 (s, 2H, C6H2), 6.84-6.87 (br, 2H, 14

13

12

NH2, exch), 8.11-8.14 (br, 2H, -NH2, exch), 8.22-8.23 (d, 1 H, C5-H, J = 2.8 Hz), 8.28 (s, 1H, N9-H 16

15

exch), 8.44-8.45 (d, 1 H, C7-H, J = 2.8 Hz); HRMS (EI) calculated for C16H18N6O3: 342.1440, found: 17 18

342.1445; Anal. (C16H18N6O3 · 0.37 H2O) C, H, N. 20

19 21

N6-(4-methylphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (9): Reaction of 32f (100 mg, 23

2

0.23 mmol) using a general procedure described above, afforded 9 (46.5 mg; 76 %) as an orange 24 25

powder: TLC Rf 0.28 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: > 285 °C (dec); 1H NMR (DMSO-d6): δ 28

27

26

2.22 (s, 3H, CH3), 6.92 (br, 2H, -NH2, exch), 6.95-6.97 (d, 2H, C6H4, J = 8.3 Hz), 7.05-7.07 (d, 2H, 29 30

C6H4, J = 8.3 Hz), 8.12-8.13 (d, 1 H, C5-H, J = 2.6 Hz), 8.17 (br, 2H, -NH2, exch), 8.28 (s, 1H, N9-H 3

32

31

exch), 8.46-8.46 (d, 1 H, C7-H, J = 2.6 Hz); HRMS (ESI) calculated for C14H15N6 (M+H)+: 267.1358, 35

34

found: 267.1372; Anal. (C14H14N6) C, H, N. 36 37

N6-(4-isopropylphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (10): Reaction of 32g (154 38 40

39

mg, 0.33 mmol) using a general procedure described above, afforded 10 (67.5 mg; 69 %) as an orange42

41

yellow solid: TLC Rf 0.3 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: 271.0-272.5 °C; 1H NMR (400 MHz) 43 4

(DMSO-d6): δ 1.16-1.18 (d, 6H, CH(CH3)2), 2.77-2.84 (m, 1H, CH(CH3)2, 6.94 (br, 2H, -NH2, exch), 47

46

45

6.98-7.0 (d, 2H, C6H4, J = 8.4 Hz), 7.11-7.13 (d, 2H, C6H4, J = 8.4 Hz), 8.16-8.17 (d, 1 H, C5-H, J = 2.5 49

48

Hz), 8.23 (br, 2H, -NH2, exch), 8.32 (br, 1H, N9-H, exch), 8.46-8.47 (d, 1 H, C7-H, J = 2.5 Hz); HRMS 50 51

(ESI) calculated for C16H19N6 (M+H)+: 295.1671, found: 295.1656; Anal. (C16H18N6 · 0.11 H2O) C, H, 54

53

52

N. 56

5

N6-2-naphthylpyrido[2,3-d]pyrimidine-2,4,6-triamine (11): Reaction of 32h (117.5 mg, 0.25 57

60

59

58

mmol) using a general procedure described above, afforded 11 (32 mg; 43 %) as a dark yellow solid: ACS Paragon Plus Environment

47


Page 48 of 67

TLC Rf 0.26 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: 379.0-382.0 °C; 1H NMR (400 MHz) (DMSO-d6): δ 2

1

6.98 (br, 2H, NH2, exch), 7.23-7.27 (t, 2H, C10H7), 7.35-7.38 (d, 2H, C10H7), 7.67-7.69 (d, 1H, C10H7), 3 4

7.74-7.80 (q, 2H, C10H7), 8.21 (br, 2H, -NH2, exch), 8.25 (s, 1 H, C5-H), 8.58 (s, 1H, N9-H, exch), 8.64 7

6

5

(s, 1 H, C7-H); HRMS (EI) calculated for C17H14N6: 302.1279, found: 302.1285; Anal. (C17H14N6) C, 9

8

H, N. 1

10

N6-(4-nitrophenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (12): Reaction of 32i (116 mg, 14

13

12

0.25 mmol) using a general procedure described above, afforded 12 (45 mg; 60.5 %) as a bright orange 16

15

solid: TLC Rf 0.24 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: > 300 °C (dec.); 1H NMR (400 MHz) 17 18

(DMSO-d6): δ 7.0-7.02 (d, 2H, C6H4, J = 8.0 Hz), 7.02 (br, 2H, -NH2, exch), 8.09-8.11 (d, 2H, C6H4, J 21

20

19

= 8.0 Hz), 8.20 (br, 2H, -NH2, exch), 8.42 (s, 1 H, C5-H), 8.58 (s, 1 H, C7-H), 9.42 (s, 1H, N9-H, exch); 23

2

HRMS (EI) calculated for C13H11N7O2: 297.0974, found: 297.0966; Anal. (C13H11N7O2) C, H, N. 24 25

N6-1-naphthylpyrido[2,3-d]pyrimidine-2,4,6-triamine (13): 28

27

26

Reaction of 32j (141 mg, 0.3

mmol) using a general procedure described above, afforded 13 (43.5 mg; 48 %) as a brick-red solid: 30

29

TLC Rf 0.39 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: > 300 °C (dec.); 1H NMR (400 MHz) (DMSO-d6): δ 3

32

31

6.99 (br, 2H, -NH2, exch), 7.23-7.27 (m, 2H, C10H7), 7.36-7.39 (d, 2H, C10H7), 7.66-7.69 (d, 1H, C10H7), 35

34

7.75-7.79 (m, 2H, C10H7), 8.22 (br, 2H, -NH2, exch), 8.26 (s, 1 H, C5-H), 8.63 (s, 1H, N9-H, exch), 8.64 37

36

(s, 1 H, C7-H); HRMS (ESI) calculated for C17H15N6 (M+H)+: 303.1358, found: 303.1352; Anal. 40

39

38

(C17H14N6 · 0.38 H2O) C, H, N. 42

41

N6-(2,5-difluorophenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (14): Reaction of 32k (182 43 4

mg, 0.40 mmol) using a general procedure described above, afforded 14 (90 mg; 78 %) as a brown 47

46

45

solid: TLC Rf 0.37 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: > 300 °C (dec.); 1H NMR (400 MHz) 49

48

(DMSO-d6): δ 6.65-6.69 (t, 1H, C6H3), 6.91-6.96 (m, 1H, C6H3), 7.21-7.27 (m, 3H, C6H3 and -NH2 50 52

51

exch), 8.25 (s, 1 H, C5-H), 8.40 (br, 2H, -NH2, exch), 8.46 (s, 1H, N9-H exch), 8.55-8.55 (d, 1 H, C754

53

H); HRMS (EI) calculated for C13H10N6F2: 288.0935, found: 288.0931; Anal. (C13H10N6F2 · 0.55 H2O) 5 56

C, H, N, F. 57 58 59 60 ACS Paragon Plus Environment

48

Page 49 of 67


N6-(3,4,5-trifluorophenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (15): Reaction of 32l (142 2

1

mg, 0.3 mmol) using a general procedure described above, afforded 15 (75 mg; 82 %) as a greenish 4

3

yellow powder: TLC Rf 0.25 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: > 300 °C (dec.); 1H NMR (400 7

6

5

MHz) (DMSO-d6): δ 6.78 (m, 2H, C6H2), 6.99 (br, 2H, -NH2, exch), 8.19 (br, 2H, -NH2, exch), 8.26 (br, 9

8

1H, N9-H exch), 8.25-8.26 (d, 1 H, C5-H, J = 2.8 Hz), 8.49 (d, 1 H, C7-H, J = 2.8 Hz); HRMS (ESI) 1

10

calculated for C13H10N6F3 (M+H)+: 307.0919, found: 307.0927; Anal. (C13H9N6F3 · 0.44 H2O) C, H, N, 14

13

12

F. 16

15

N6-methyl-N6-phenylpyrido[2,3-d]pyrimidine-2,4,6-triamine (16): Reaction of 32m (100 17 18

mg, 0.23 mmol) using a general procedure described above, afforded 16 (42 mg; 68.5 %) as a yellow21

20

19

green solid: TLC Rf 0.4 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: 288.2-290.2 °C; 1H NMR (400 MHz) 23

2

(DMSO-d6): δ 3.29 (s, 3H, NCH3), 6.91-6.97 (m, 3H, C6H5), 7.14 (br, 2H, -NH2, exch), 7.25-7.29 (t, 2H, 24 26

25

C6H5), 8.25 (d, 1 H, C5-H, J = 2.8 Hz), 8.31 (br, 2H, -NH2 - exch), 8.42-8.43 (d, 1 H, C7-H, J = 2.8 Hz); 28

27

HRMS (ESI) calculated for C14H15N6 (M+H)+: 267.1358, found: 267.1375; Anal. (C14H14N6 · 0.25 H2O) 29 30

C, H, N. 31 3

32

N6-methyl-N6-(4-methoxyphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (17): Reaction of 35

34

32n (120 mg, 0.26 mmol) using a general procedure described above, afforded 17 (70 mg; 91.5 %) as a 37

36

light brown solid: TLC Rf 0.48 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: 290.4-291.8 °C; 1H NMR (400 40

39

38

MHz) (DMSO-d6): δ 3.22 (s, 3H, NCH3), 3.73 (s, 3H, OCH3), 6.98 (br, 2H, -NH2, exch), 6.92-6.94 (d, 42

41

2H, C6H4, J = 8.8 Hz), 7.05-7.07 (d, 2H, C6H4, J = 8.8 Hz), 7.98-7.99 (d, 1 H, C5-H, J = 2.8 Hz), 8.2143 4

8.22 (d, 1 H, C7-H, J = 2.8 Hz), 8.26 (br, 2H, -NH2, exch); HRMS (EI) calculated for C15H16N6O: 47

46

45

296.1468, found: 294.1452; Anal. (C15H16N6O ·0.29 H2O) C, H, N. 49

48

N6-methyl-N6-(2-methoxyphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (18): Reaction of 50 52

51

32o (120 mg, 0.26 mmol) using a general procedure described above, afforded 18 (45 mg; 59 %) as a 54

53

yellow-brown solid: TLC Rf 0.48 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: 283-284 °C ; 1H NMR (400 5 56

MHz) (DMSO-d6): δ 3.22 (s, 3H, NCH3), 3.70 (s, 3H, OCH3), 7.0-7.03 (t, 1H, C6H4), 7.12-7.14 (d, 1H, 60

59

58

57

C6H4), 7.23-7.27 (br and m, 4H, C6H4 and -NH2 - exch), 7.85-7.88 (d, 2 H, C5-H and C7-H); HRMS ACS Paragon Plus Environment

49


Page 50 of 67

(ESI) calculated for C15H17N6O (M+H)+: 297.1464, found: 297.1469; Anal. (C15H16N6O ·0.36 H2O) C, 2

1

H, N. 3 4

N6-methyl-N6-(2,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (19): 5 7

6

Reaction of 32p (175 mg, 0.35 mmol) using a general procedure described above, afforded 19 (100 mg; 9

8

86 %) as a lemon yellow solid: TLC Rf 0.5 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: > 280 °C (dec.); 1H 10 1

NMR (400 MHz) (DMSO-d6): δ 3.21 (s, 3H, NCH3), 3.62 (s, 3H, OCH3), 3.69 (s, 3H, OCH3), 6.81-6.86 14

13

12

(m, 4H, C6H3 and NH2 - exch ), 7.03-7.05 (d, 1H, C6H3), 7.78 (s, 1 H, C5-H), 7.92-7.93 (d, 1 H, C7-H), 16

15

8.21 (br, 2H, -NH2, exch); HRMS (ESI) calculated for C16H19N6O2 (M+H)+: 327.1569, found: 17 18

327.1557; Anal. (C16H18N6O2) C, H, N. 20

19 21

N6-methyl-N6-(3,4,5-trimethoxyphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (20): 23

2

Reaction of 32q (160 mg, 0.3 mmol) using a general procedure described above, afforded 20 (87 mg; 81 24 25

%) as a yellow solid: TLC Rf 0.49 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: 218-221 °C; 1H NMR (400 28

27

26

MHz) (DMSO-d6): δ 3.28 (s, 3H, NCH3), 3.60 (s, 3H, OCH3), 3.67 (s, 6H, OCH3), 6.28 (s, 2H, C6H2), 29 30

6.89 (br, 2H, -NH2, exch), 8.09 (br, 2H, -NH2, exch), 8.13 (d, 1 H, C5-H, J = 2.8 Hz), 8.38-8.39 (d, 1 H, 3

32

31

C7-H, J = 2.8 Hz); HRMS (ESI) calculated for C17H21N6O3 (M+H)+: 357.1675, found: 357.1680; Anal. 35

34

(C17H20N6O3 ·0.16 H2O) C, H, N. 36 37

N6-methyl-N6-(4-methylphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (21): 38 40

39

Reaction of

32r (160 mg, 0.35 mmol) using a general procedure described above, afforded 21 (90 mg; 90.2 %) as a 42

41

yellow solid: TLC Rf 0.47 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: 288.0-289.4 °C (dec); 1H NMR (400 43 4

MHz) (DMSO-d6): δ 2.23 (s, 3H, CH3), 3.25 (s, 3H, NCH3), 6.89-6.91 (d, 2H, C6H4, J = 8 Hz), 7.02 (br, 47

46

45

2H, -NH2, exch), 7.08-7.10 (d, 2H, C6H4, J = 8 Hz), 8.16 (br, 3H, C5-H, -NH2 - exch), 8.33 (s, 1 H, C749

48

H); HRMS (ESI) calculated for C15H17N6 (M+H)+: 281.1515, found: 281.1520; Anal. (C15H16N6) C, H, 50 52

51

N. 54

53

N6-methyl-N6-(4-isopropylphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (22): Reaction of 5 56

32s (140 mg, 0.30 mmol) using a general procedure described above, afforded 22 (70 mg; 77 %) as a 57

60

59

58

yellow solid: TLC Rf 0.5 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: > 320 °C (dec.); 1H NMR (400 MHz) ACS Paragon Plus Environment

50

Page 51 of 67


(DMSO-d6): δ 1.16-1.18 (d, 6H, CH(CH3)2), 2.79-2.86 (m, 1H, CH(CH3)2, 3.26 (s, 3H, NCH3), 6.922

1

6.94 (d, 2H, C6H4, J = 8.4 Hz), 7.03 (br, 2H, -NH2, exch), 7.15-7.17 (d, 2H, C6H4, J = 8.4 Hz), 8.17-8.18 3 4

(d, 1 H, C5-H, J = 2.5 Hz), 8.23-8.24 (br, 2H, -NH2, exch), 8.36 (d, 1 H, C7-H, J = 2.5 Hz); HRMS 7

6

5

(ESI) calculated for C17H21N6 (M+H)+: 309.1828, found: 309.1807; Anal. (C17H20N6) C, H, N. 9

8

N6-methyl-N6-(4-nitrophenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (23): Reaction of 32t 10 1

(45 mg, 0.09 mmol) using a general procedure described above, afforded 23 (26 mg; 89 %) as a yellow14

13

12

brown solid: TLC Rf 0.47 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: > 250 °C (dec.); 1H NMR (400 MHz) 16

15

(DMSO-d6): δ 3.43 (s, 3H, NCH3), 6.86-6.88 (d, 2H, C6H4, J = 9.2 Hz), 7.26-7.28 (br, 2H, -NH2, exch), 17 18

8.06-8.09 (d, 2H, C6H4, J = 9.2 Hz), 8.62 (d, 1 H, C5-H, J = 2.4 Hz), 8.67 (br, 2H, -NH2, exch), 8.7221

20

19

8.72 (d, 1 H, C7-H, J = 2.4 Hz); HRMS (ESI) calculated for C14H14N7O2 (M+H)+: 312.1209, found: 23

2

312.1237; Anal. (C14H13N7O2 · 0.39 H2O) C, H, N. 24 25

N6-methyl-N6-1-naphthylpyrido[2,3-d]pyrimidine-2,4,6-triamine (24): Reaction of 32u 28

27

26

(115 mg, 0.2375 mmol) using a general procedure described above, afforded 24 (45 mg; 60 %) as a 30

29

yellow solid: TLC Rf 0.39 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: > 250 °C (dec.); 1H NMR (400 MHz) 3

32

31

(DMSO-d6): δ 3.38 (s, 3H, NCH3), 6.84 (br, 2H, NH2, exch), 7.41-7.43 (d, 1H, C10H7), 7.49-7.58 (m, 35

34

3H, C10H7), 7.76-7.77 (d, 1 H, C5-H, J = 2.8 Hz), 7.78-7.81 (d, 1H, C10H7), 7.86-7.88 (d, 1H, C10H7), 36 37

7.94-7.95 (d, 1 H, C7-H, J = 2.8 Hz), 7.98-8.0 (d, 1H, C10H7), 8.17 (br, 2H, NH2, exch); HRMS (ESI) 40

39

38

calculated for C18H17N6 (M+H)+: 317.1515, found: 317.1542; Anal. (C18H16N6) C, H, N. 42

41

N6-methyl-N6-(2,5-difluorophenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (25): Reaction of 43 4

32v (188 mg, 0.40 mmol) using a general procedure described above, afforded 25 (110 mg; 91 %) as a 47

46

45

yellow solid: TLC Rf 0.47 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: 297.5-299.6 °C; 1H NMR (400 MHz) 49

48

(DMSO-d6): δ 3.30 (s, 3H, NCH3), 6.99-7.04 (m, 3H, C6H3 and -NH2 - exch), 7.21-7.33 (m, 2H, C6H3), 50 52

51

8.04-8.05 (d, 1 H, C5-H, J = 2.4 Hz), 8.24 (br, 2H, -NH2, exch), 8.31 (d, 1 H, C7-H, J = 2.4 Hz); HRMS 54

53

(ESI) calculated for C14H13N6F2 (M+H)+: 303.1170, found: 303.1154; Anal. (C14H12N6F2) C, H, N, F. 56

5

N6-methyl-N6-(3,4,5-trifluorophenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (26): Reaction 57

60

59

58

of 32w (170 mg, 0.35 mmol) using a general procedure described above, afforded 26 (93 mg; 83 %) as a ACS Paragon Plus Environment

51


Page 52 of 67

light yellow-brown solid: TLC Rf 0.43 (MeOH/CHCl3/NH4OH, 1:5:0.5); mp: > 325 °C (dec.); 1H NMR 2

1

(400 MHz) (DMSO-d6): δ 3.24 (s, 3H, NCH3), 6.45 (br, 2H, NH2, exch), 6.55-6.59 (q, 2H, C6H2), 7.54 3 4

(br, 2H, -NH2, exch), 8.28 (d, 1 H, C5-H, J = 2.8 Hz), 8.51 (d, 1 H, C7-H, J = 2.8 Hz); HRMS (ESI) 7

6

5

calculated for C14H12N6F3 (M+H)+: 321.1076, found: 321.1068; Anal. (C14H11N6F3 · 0.16 H2O) C, H, N. 9

8

General procedure for the synthesis of methylanilines 31p-q, 31s and 31v-w: According to 1

10

Teichert et al.50 substituted aniline (5-10 mmol) was added to a suspension of NaOMe (5 equivalents) in 14

13

12

MeOH (8-15 mL). The resulting solution was poured into a suspension of paraformaldehyde (1.4 16

15

equivalents) in MeOH (5-10 mL). The reaction mixture was stirred for 5 h at RT and then sodium 17 18

borohydride (1 equivalent) was added. The solution was heated to reflux for 2 h. After evaporating part 21

20

19

of the solvent, the reaction mixture was treated with 1 M KOH. The product was extracted with diethyl 23

2

ether, dried (anhydrous MgSO4). Silica gel (3 - 5 g) was added followed by evaporation of the solvent 24 26

25

under reduced pressure to afford a plug which was loaded on a silica column and eluted with 28

27

Hexanes/EtOAc 2:1. The fractions containing the product spot (TLC) were pooled and evaporated under 29 30

reduced pressure to afford substituted-N-methylanilines. 31 3

32

2,5-dimethoxy-N-methylaniline (31p): Reaction of 31d (1.24 gm, 8.12 mmol) using the 35

34

general procedure described above afforded 31p (0.45 gm, 33 %) as a brown liquid: TLC Rf 0.73 37

36

(EtOAc/Hexanes, 2:1); 1H NMR (400 MHz) (CDCl3): δ 2.86 (s, 3H, NHCH3), 3.78-3.81 (d, 6H, OCH3), 40

39

38

4.28 (br, 1H, -NH, exch), 6.14-6.17 (dd, 1H, C6H3, J1 = 2.8 Hz, J2 = 8.5 Hz), 6.22 (d, 1H, C6H3, J = 2.8 42

41

Hz), 6.66-6.68 (d, 1H, C6H3, J = 8.5 Hz); HRMS (EI) calculated for C9H13NO2: 167.0946, found: 43 4

167.0947. 47

46

45

3,4,5-trimethoxy-N-methylaniline (31q): Reaction of 31e (1.48 gm, 8.12 mmol) using the 49

48

general procedure described above afforded 31q (1.5 gm, 94 %) as an off-white solid: TLC Rf 0.44 50 51

(EtOAc/Hexanes, 2:1). Melting point and spectral data matched with that from the literature reference.54 54

53

52

4-isopropyl-N-methylaniline (31s): Reaction of 31g (1.35 gm, 10 mmol) using the general 5 56

procedure described above afforded 31s (700 mg, 87 %) as a pale yellow liquid: TLC Rf 0.66 (Hexanes 58

57 59 60


52

Page 53 of 67


/EtOAc, 2:1); 1H NMR (400 MHz) (CDCl3): δ 2.77-2.79 (d, 3H, NHCH3), 3.76 (br, 1H, NHCH3, exch), 2

1

6.12-6.16 (m, 2H, C6H2); HRMS (EI) calculated for C10H15N: 149.1204, found: 149.1220. 3 4

2,5-difluoro-N-methylaniline (31v): Reaction of 31k (1.29 gm, 10 mmol) using the general 5 7

6

procedure described above afforded 31v (0.96 gm, 67 %) as a pale yellow liquid: TLC Rf 0.76 (Hexanes 9

8

/EtOAc, 2:1); 1H NMR (400 MHz) (CDCl3): δ 2.86-2.87 (d, 3H, NHCH3), 4.05 (br, 1H, NHCH3, exch), 10 1

6.23-6.29 (m, 1H, C6H3), 6.34-6.39 (m, 1H, C6H3), 6.84-6.90 (m, 1H, C6H3); HRMS (EI) calculated for 14

13

12

C7H7NF2: 143.0546, found: 143.0545. 16

15

3,4,5-trifluoro-N-methylaniline (31w): Reaction of 31l (735 mg, 5 mmol) using the general 17 18

procedure described above afforded 31w (700 mg, 87 %) as a pale yellow liquid: TLC Rf 0.66 (Hexanes 21

20

19

/EtOAc, 2:1); 1H NMR (400 MHz) (CDCl3): δ 2.77-2.79 (d, 3H, NHCH3), 3.76 (br, 1H, NHCH3, exch), 23

2

6.12-6.16 (m, 2H, C6H2); HRMS (EI) calculated for C7H6NF3: 161.0452, found: 161.0444. 24 25 26 28

27

Molecular Modeling 29 30

Docking Studies with pcDHFR. 31

Docking studies were performed for 16 using the 1.90 Å

3

32

crystal structure of pcDHFR (PDB: 1LY337) complexed with 2,4-diamino-6-[N-(2',5'35

34

dimethoxybenzyl)- N-methylamino]quinazoline. The active site was defined by a sphere of 6.5 Å near 36 37

the ligand. Protein preparation prior to docking was performed using the LigX functionality in MOE 40

39

38

2010.10. LigX is a graphical interface and collection of procedures for conducting interactive ligand 42

41

modification and energy minimization in the active site of a flexible receptor. In LigX calculations, the 43 4

receptor atoms far from the ligand are constrained and not allowed to move while receptor atoms in the 47

46

45

active site of the protein are allowed to move but are subject to tether restraints that discourage gross 49

48

movement. The procedure was performed with the default settings. The process of protein preparation 50 52

51

using LigX involves addition of hydrogen atoms according to the ionization state of the atoms of the 54

53

molecule/protein loaded. The heavy atoms are then fixed and a brief energy minimization is carried out 5 56

to refine the positions of the added hydrogen atoms. The receptor atoms are then tethered during 57

60

59

58

geometry optimization so that they do not deviate too much from their initial coordinates and then ACS Paragon Plus Environment

53


Page 54 of 67

energy minimized using the Amber99 forcefield. Ligands used for docking were sketched in MOE, 2

1

minimized and exported as an SDF file. 3 4

Docking of ligands into the pcDHFR active site was performed using LeadIT 1.3.0. Polar 5 7

6

hydrogen atoms of amino acids with a polar side chain (Asn23, Ser24, Tyr35, Thr61, Ser64, Tyr129 and 9

8

Thr144) were not constrained, thereby permitting free rotation. Base placement of fragments for 10 1

docking was carried out using triangle matching. Default parameters were used for scoring and clash 14

13

12

handling. The maximum number of solutions per iteration and the maximum number of solution per 16

15

fragmentation were set to 500. Ten poses were obtained per molecule. Docking processes were repeated 17 18

to ensure reproducibility of the docked conformations. The docked poses were exported to MOE 21

20

19

2010.11, rescored using the affinity dG scoring system, refined using the forcefield system and rescored 23

2

using London dG scoring system. The binding poses were also visualized using the ligplot utility in 24 26

25

MOE and the Poseview utility in LeadIT 1.3.0. 28

27

The native crystal structure from 1LY3, 2,4-diamino-6-[N-(2',5'-dimethoxybenzyl)-N29 30

methylamino]quinazoline, was sketched, prepared and docked into the pcDHFR active site as described 31 3

32

above. The best docked pose displayed and RMSD of 1.07 Å compared to the crystal structure ligand, 35

34

thereby validating LeadIT 1.3.0 for our docking purposes. Docking studies with 16 were performed 36 37

similarly. The docked score of 16 in pcDHFR was -42.4161 kJ/mol. 40

39

38

Docking Studies with pjDHFR. There is currently no known crystal structure of pjDHFR. A 42

41

homology model was hence built for evaluating the binding of 15, 16, and 26 in pjDHFR. The 206 43 4

amino acid sequence of dihydrofolate domain was obtained from the UniProt database (ID: 47

46

45

Q9UUP5_PNEJI [Q9UUP5]). 49

48

A BLAST search for the pjDHFR sequence showed high sequence identity with pcDHFR, (61% 50 52

51

sequence identity). 54

53

Homology model building and validation. The high sequence identity between pjDHFR and 5 56

pcDHFR and the availability of high quality crystal structures of pcDHFR in the PDB makes it a valid 57

60

59

58

template for building the pjDHFR homology models. The homology model was built with MOE ACS Paragon Plus Environment

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Page 55 of 67


2010.10 using the 1.60Å crystal structure of pcDHFR as a template (PDB: 2FZI, chain A). A 2

1

Ramachandran plot generated showed that with the exception of Asp2 all the residues have acceptable 3 4

geometries. Since Asp2 is distant from the active site, and was not expected to influence the docking 5 7

6

studies, modeling studies were performed without any additional refinements. Additional validation of 9

8

the model using Procheck55 indicated 87.5% of residues in the most favored regions, 11.5% residues in 10 1

additional allowed regions, 1% in the generously allowed regions and 0% residues in the disallowed 14

13

12

region. The validated model was used for docking studies with 15, 16, and 26. 16

15

Active site definition and docking to the pjDHFR model. The pjDHFR homology model 17 18

prepared in MOE was superimposed on the X-ray crystal structure of pcDHFR (PDB: 2FZI, chain A) 21

20

19

and NADPH and DH3 (2,4-diamino-5-[3',4'-dimethoxy-5'-(5-carboxyl-1-pentynyl)]benzyl pyrimidine), 23

2

the co-crystallized ligand in 2FZI, were added to the model. The active site was defined by a sphere of 24 26

25

6.5 Å near the ligand. Docking of ligands to the pjDHFR model was performed using LeadIT 1.3.0. as 28

27

described above. 29 30

Validation of the docking system. The pjDHFR structure was obtained by means of 31 3

32

homology modeling using pcDHFR as template. Hence, the validation of LeadIT 1.3.0 as suitable 35

34

docking systems for pjDHFR was carried out by redocking DH3 (2,4-diamino-5-[3',4'-dimethoxy-5'-(536 37

carboxyl-1-pentynyl)]benzyl pyrimidine), the native ligand in the X-ray crystal structure of pcDHFR 40

39

38

(PDB: 2FZI). The protein was prepared as mentioned above. The ligand was built and minimized in 42

41

MOE. Docking was carried out with LeadIT 1.3.0 as described above. The best docked pose of the 43 4

ligand had an RMSD of 0.941 compared to the crystal structure. Thus, LeadIT 1.3.0 was validated for 47

46

45

docking studies with the proposed analogs. 49

48

Conformational analysis. 50

Low energy conformers of 4, 15, 16, and 26 within 3 kcal/mol

51

were generated using the Systematic Search option in Sybyl X 1.356 around the C6-N9 bond using 5° 54

53

52

increments. 5 56

Crystallization and X-ray Data Collection and Refinement. Expression and purification of 57

60

59

58

variant pcDHFR were carried out as previously described for the human DHFR variants.57, 58 ACS Paragon Plus Environment

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Page 56 of 67

Mutations at positions 37 and 69 of pcDHFR were introduced into the cDNA of pcDHFR and the entire 2

1

coding sequence was verified by Roswell Park Cancer Center (Buffalo, NY). DNA oligonucleotides 3 4

were obtained from Integrated DNA Technologies (Coralville,IA) and used without further purification. 5 7

6

Plasmid DNA was purified using the Qiaspin mini prep kit (Qiagen). Mutagenesis was performed using 9

8

the QuikChange site-directed mutagenesis kit (Agilent Technologies). Primers were designed according 10 1

to manufacturer’s recommendations as were the PCR reactions, with adjustments made for the Tm of 14

13

12

the respective primers. 16

15

Recombinant mutant and native pcDHFR was washed in a Centricon-10 with 50 mM MES 17 18

buffer, pH 6.0 and 100 mM KCl and concentrated to 11.4 – 14.1 mg/mL for these samples. All three 21

20

19

pcDHFR proteins were incubated for 1 hr over ice with a 10-fold excess of NADPH and of compounds 23

2

16, 22 and 26, respectively, prior to crystallization using the hanging drop vapor diffusion method. The 24 26

25

reservoir solution for compounds 16 and 22 contained 35% PEG 2K, 49 mM MES, pH 6.0, 100 mM 28

27

KCl while compound 26 contained ProComplex (Qiagen) #11 ( 0.1 M HEPES, pH 7.5, 25% PEG 2000 30

29

MME). Crystals of all three pcDHFR complexes grew over several days at 14 °C and were monoclinic, 31 3

32

space group P21. Data were collected to 1.70Å resolution for both mutant complexes using the remote 35

34

access robot at liquid N2 temperatures on beamline 9-2 at the Stanford Synchrotron Research Laboratory 37

36

(SSRL) imaging plate system, and on beamline 11-1 for native pcDHFR-26 crystals 59-61 . The data for 40

39

38

the two pcDHFR mutant complexes were processed using Mosflm62 , while the XDS program package63 42

41

was used to process the pcDHFR 26 complex. The diffraction statistics are shown in Table 7 for the 43 4

three complexes. 47

46

45

The three structures were solved by molecular replacement methods using the coordinates for 49

48

pcDHFR (3cd2)53 in the program Molref62 . Inspection of the resulting difference electron density 50 51

maps made using the program COOT 64 running on an iMac workstation revealed density for the ternary 54

53

52

complex in all three crystals. The final cycles of refinement were carried out using the program 56

5

Refmac5 in the CCP4 suite of programs62 . The Ramachandran conformational parameters from the last 57

60

59

58

cycle of refinement generated by RAMPAGE65 showed that more than 98% of the residues refined have ACS Paragon Plus Environment

56

Page 57 of 67


the most favored conformation and none are in the disallowed regions. Coordinates for these structures 2

1

have been deposited with the Protein Data Bank. 3 4 5 7

6

Table 7. Data collection and refinement statistics for native and variant pcDHFR-NADPH inhibitor 9

8

complexes. 10 1

Data collection 13

12 14 15 16

pcDHFR-

pcDHFR-F69N pcDHFR-26

K37S/F69N

NADPH-22

NADPH-16 17 18

PDB accession # 21

20

19

pcDHFR-K37S/F69N-#16 (4IXG); pcDHFR-F69N-#22 (4IXF); pcDHFR-#26 (4IXE)

Space group 23

2

P21

P21

P21

a

37.2

37.4

36.7

b

42.6

42.8

42.7

c

60.5

60.9

58.5



94.8

95.5

93.6

Beamline

SSRL 9-2

SSRL 9-2

SSRL 11-1

Resolution (Ǻ)

1.70 (1.80)

1.70 (1.80)

1.54 (1.62)

Wavelength (Ǻ)

0.975

0.975

0.975

Rmerge (%) a,b

0.068 (0.39)

0.080 (0.61)

0.021 (0.06)

Completeness (%) a

98.9 (98.1)

99.5 (97.6)

95.6 (83.0)

Observed reflections

52462

77032

85164 (9052)

Unique reflections

20874

21146

25934 (3254)

6.3 (1.6)

7.2 (1.4)

38.0 (14.3)

2.5 (2.4)

3.6 (3.5)

3.3 (2.8)

34.8 – 1.70

37.2 – 1.70

36.6 – 1.54

Cell dimensions (Ǻ) 24

29

28

27

26

25

31

30

36

35

34

3

32

43

42

41

40

39

38

37

48

47

46

45

4

50

49 51 52 54

53

Multiplicity a 5 56

Refinement and model quality 60

59

58

57

Resolution range (Ǻ)


57


Page 58 of 67

No. of reflections

19287

20020

24614

R-factor c

22.8

19.9

17.7

Rfree-factor d

28.0

24.8

22.0

Total protein atoms

1782

1842

1871

Total water atoms

34

79

114

Average B-factor (Ǻ2)

30.4

29.9

15.6

Bond lengths (Ǻ)

0.019

0.020

0.023

Bond angles (o)

2.26

2.14

2.39

Luzzati

0.246

0.208

0.154

Most favored regions (%)

91.2

92.6

99.0

Additional allowed regions (%)

6.4

4.9

1.0

Generously allowed regions (%)

2.5

2.5

0.0

1 2 3 4 5 6 7 8 9 10 1 12 13 14 16

15

Rms deviation from ideal 17 18 19 20 21 2 23 24 26

25

Ramachandran plot 29

28

27

31

30

35

34

3

32

Disallowed regions (%) 38

37

36

b

Rsym = ΣhΣi|Ih,i - | / ΣhΣi|Ih,i|, where is the mean intensity of a set of equivalent reflections.

c

43

0.0

The values in parentheses refer to data in the highest resolution shell.

41 42

0.0

a

39 40

0.0

R-factor = Σ|Fobs – Fcalc| / ΣFobs, where Fobs and Fcalc are observed and calculated structure factor

45

4

amplitudes. 47

46

d

48

Rfree-factor was calculated for R-factor for a random 5% subset of all reflections.

49 50 52

51

Acknowledgement: 54

53

This was supported, in part, by the National Institute of Health and

National Institute of Allergy and Infectious Diseases grant AI069966 (AG), the Duquesne University 5 56

Adrian Van Kaam Chair in Scholarly Excellence (AG) and the National Science Foundation equipment 60

59

58

57

grant (NMR: CHE 0614785). ACS Paragon Plus Environment

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1 2

Supporting Information Available: 4

3

Results from elemental analysis for the target

compounds 4-26 and high-resolution mass spectrometry data for the new compounds. This material is 5 6

available free of charge via the internet at http://pubs.acs.org. 7 8 9 1

10

References: 12 13

1. 15

14 16

Roth, B., Design of dihydrofolate reductase inhibitors from X-ray crystal structures. Fed. Proc. 1986, 45, 2765-2772.

18

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2. 19

Kelly, M. N.; Shellito, J. E. Current understanding of Pneumocystis immunology. Future

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Microbiol. 2010, 5, 43-65. 23

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3. 24 25

Catherinot, E.; Lanternier, F.; Bougnoux, M.-E.; Lecuit, M. Couderc, L.-J.; Lortholary, O. Pneumocystis jirovecii pneumonia. Infect. Dis. Clin. N. Am. 2010, 24, 107-138.

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4. 29 30

Huang, L.; Crothers, K. HIV-Associated Opportunistic Pneumonias. Respirology, 2009, 14, 474485.

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Foucauld, J.; Uphouse, W.; Berenberg, J., Dapsone and aplastic anemia. Ann. Intern. Med. 1985,

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Sattler, F. R.; Frame, P.; Davis, R.; Nichols, L.; Shelton, B.; Akil, B.; Baughman, R.; Hughlett,

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C.; Weiss, W.; Boylen, C. T.; van der Horst, C.; Black, J.; Powderly, W.; Steigbigel, R. T.; 31 32 3

Leedom, J. M.; Masur, H. and Feinberg, J. Trimetrexate with leucovorin versus trimethoprim34 35

sulfamethoxazole for moderate to severe episodes of Pneumocystis carinii pneumonia in patients 36 37

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Masur, H.; Polis, M. A.; Tuazon, C. U.; Ogata-Arakaki, D.; Kovacs, J. A.; Katz, D.; Hilt, D.;

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Simmons, T.; Feuerstein, I.; Lundgren, B.; Lane, H. C.; Chabner, B. A. and Allegra, C. J. 45 46 47

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Ma, L.; Kovacs, J. A., Expression and characterization of recombinant human-derived Pneumocystis carinii dihydrofolate reductase. Antimicrob. Agents Chemother. 2000, 44, 3092-

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Cody, V.; Chisum, K.; Pope, C.; Queener, S. F., Purification and characterization of humanderived Pneumocystis jirovecii dihydrofolate reductase expressed in Sf21 insect cells and in

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Escherichia coli. Protein Expr. Purif. 2005, 40, 417-423. 5 7

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18. 8 9

Gangjee, A.; Kurup, S.; Namjoshi, O., Dihydrofolate reductase as a target for chemotherapy in parasites. Curr. Pharm. Des. 2007, 13, 609-639.

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inhibitors of dihydrofolate reductases. J. Med. Chem. 1997, 40, 479-485. 17 18

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Gangjee, A.; Vasudevan, A.; Queener, S. F.; Kisliuk, R. L., 2,4-diamino-5-deaza-6-substituted pyrido[2,3-d]pyrimidine antifolates as potent and selective nonclassical inhibitors of

2 23

dihydrofolate reductases. J. Med. Chem. 1996, 39, 1438-1446. 24 26

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21. 27 28

Gangjee, A.; Vasudevan, A.; Queener, S. F.; Kisliuk, R. L., 6-substituted 2,4-diamino-5methylpyrido[2,3-d]pyrimidines as inhibitors of dihydrofolate reductases from Pneumocystis

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carinii and Toxoplasma gondii and as antitumor agents. J. Med. Chem. 1995, 38, 1778-1785. 31 3

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22. 34 35

Gangjee, A.; Mavandadi, F.; Queener, S. F., Effect of N9-methylation and bridge atom variation on the activity of 5-substituted 2,4-diaminopyrrolo[2,3-d]pyrimidines against dihydrofolate

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reductases from Pneumocystis carinii and Toxoplasma gondii. J. Med. Chem. 1997, 40, 117338 39 40

1177. 42

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Gangjee, A.; Zhu, Y.; Queener, S. F., 6-Substituted 2,4-diaminopyrido[3,2-d]pyrimidine

4

analogues of piritrexim as inhibitors of dihydrofolate reductase from rat liver, Pneumocystis 45 46 47

carinii, and Toxoplasma gondii and as antitumor agents. J. Med. Chem. 1998, 41, 4533-4541. 49

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Gangjee, A.; Adair, O.; Queener, S. F., Pneumocystis carinii and Toxoplasma gondii

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dihydrofolate reductase inhibitors and antitumor agents: synthesis and biological activities of 54

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3 4

Toxoplasma gondii dihydrofolate reductase and as antiopportunistic infection and antitumor 5 6 7

agents. J. Med. Chem. 2003, 46, 5074-5082. 9

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Gangjee, A.; Adair, O.; Pagley, M.; Queener, S. F., N9-substituted 2,4-diaminoquinazolines:

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synthesis and biological evaluation of lipophilic inhibitors of Pneumocystis carinii and 12 13 14

Toxoplasma gondii dihydrofolate reductase. J. Med. Chem. 2008, 51, 6195-6200. 16

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Gangjee, A.; Vidwans, A. P.; Vasudevan, A.; Queener, S. F.; Kisliuk, R. L.; Cody, V.; Li, R.;

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Galitsky, N.; Luft, J. R.; Pangborn, W., Structure-based design and synthesis of lipophilic 2,419 20 21

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Rosowsky, A.; Mota, C. E.; Queener, S. F.; Waltham, M.; Ercikan-Abali, E.; Bertino, J. R., 2,4Diamino-5-substituted-quinazolines as inhibitors of a human dihydrofolate reductase with a site-

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and Toxoplasma gondii. J. Med. Chem. 1995, 38, 745-752. 42

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Design, synthesis and molecular modeling of novel pyrido[2,3-d]pyrimidine analogs as antifolates: Application of Buchwald-Hartwig aminations of heterocycles 7

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Aleem Gangjee,a* Ojas A. Namjoshi,a Sudhir Raghavan,a Sherry F. Queener,b Roy L. Kisliuk,c Vivian Codyd 9

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TOC Graphic 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment

Design, Synthesis, and Molecular Modeling of Novel Pyrido[2,3-d

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